common-pile/pubmed · Datasets at Hugging Face

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PMC547904	Background ========== Infection with Chlamydia pneumoniae(Cpn) usually causes acute respiratory tract infections \[[@B1]\]. Chronical infection with Cpn may also contribute to formation and progression of atherosclerotic lesions apart from the classical risk factors such as hypertension, hypercholesterolemia and hyperlipidemia \[[@B2]\]. Cpn has been extensively studied in the context of atherosclerosis \[[@B3]\] because atherosclerosis and cardiovascular disease are the leading causes of death in the United States, Europe and much of Asia \[[@B4],[@B5]\]. The obligate intracellular bacterium has been detected in atherosclerotic lesions by immunohistochemistry, polymerase chain reaction and electron microscopy \[[@B6]-[@B8]\] and has also been cultured from atheromatous plaques \[[@B9],[@B10]\]. On the cellular level smooth muscle cells and macrophages in the intima have been found to be infected with Cpn \[[@B11],[@B12]\]. In general terms, if the inflammatory response does not effectively neutralize or remove the offending agents, such as Cpn, it can continue indefinitely resulting in the progression of the disease \[[@B2]\]. Chlamydiaeexhibit a unique developmental cycle with two morphological distinct infectious and reproductive forms: the elementary- and the reticulate body. The life cycle proceeds for 48 -- 72 h and ends with pathogen release that may damage the host cells \[[@B13]\]. Infection with this pathogen is accompanied by cytoplasmic alterations and damage of the host cells \[[@B12]\]. A balance between pro- and anti-apoptotic influences by Chlamydiacan be postulated. On one hand it has been shown that Chlamydia psittaciand Chlamydia trachomatiscan induce apoptosis in vitro\[[@B14],[@B15]\]. On the other hand established cell lines infected with Cpn or Chlamydia trachomatiswere protected from apoptotic cell death induced by various stimuli \[[@B16],[@B17]\]. Most of the studies investigating pro- and anti-apoptotic activity of Cpn were performed in tumor cells or established cell lines. Since the character of cell death is affected by host cell type and Chlamydiastrain it is of relevance to determine cell death in Chlamydiainfected primary cells \[[@B18]\]. As regards the role of Cpn in atherosclerosis we need to understand death promoting and inhibiting capacities of Cpn in primary cultures of vascular cells. Human aortic smooth muscle cells (HASMC) play an important role in the development of atherosclerotic lesions \[[@B19]\]. Therefore, we sought to clarify the nature of cell death induced by Cpn in HASMC. Apoptosis and necrosis represent two extreme morphologically defined forms of cell death \[[@B20]-[@B23]\]. Recently the hybrid term aponecrosis was introduced describing the incomplete execution of the internal apoptotic pathway and the following necrotic degeneration \[[@B24]\]. Whether smooth muscle cell death is apoptotic, necrotic or even aponecrotic in nature would predictably influence the inflammatory response in the plaque. Necrotic cells releasing their contents provoke inflammation, whereas in apoptosis dead cells are ingested by neighboring cells and, therefore, do not provoke an inflammatory response \[[@B25]\]. In order to characterize the fate of HASMC following infection with Cpn we analyzed cell death by morphological and biochemical methods. Results ======= Various morphological characteristics of bacterial infection in HASMC --------------------------------------------------------------------- In order to examine the morphological characteristics of Cpn infection HASMC as well as the tumor cell line HEp-2 were infected with Cpn-K6 and Cpn-VR1310, respectively, and stained after 24 h, 48 h and 72 h with anti-Cpn-MOMP antibody. According to the staining protocol intra- and extracellular localization of Cpn could be distinguished (Figure [1](#F1){ref-type="fig"}). Already after 24 h, but also after 48 h and 72 h, HASMC as well as HEp-2 cells were infected with numerous bacteria. Round or oval shaped smooth bordered intracellular inclusions were found in tumor cells (Figure [1E](#F1){ref-type="fig"}) as well as HASMC (Figure [1B,D](#F1){ref-type="fig"}). In contrast to tumor cells in primary HASMC Cpn were found also as single or rarely as irregular shaped, aggregated immunoreactive spots (Figure [1A, C](#F1){ref-type="fig"}, [4D](#F4){ref-type="fig"}). Quantification of morphology in HASMC revealed that after 72 h all cells were infected with at least one chlamydial spot (100% infection rate). Approximately 25% of the cells carried one or multiple inclusions. The different morphological characteristics in HASMC (inclusions, single and aggregated spots) could also be discriminated 48 h post infection. The ratio of cells carrying inclusions versus cells carrying spots was much higher in HEp-2 cells compared to HASMC irrespective of the infectious dose (Figure [2A](#F2){ref-type="fig"}). This ratio did not considerably vary between different Cpn strains K6 and VR1310 or as a function of the number of bacteria used for infection. Spots were usually much lower in number and not as prominently observed in HEp-2 cells compared to HASMC (Figure [2C](#F2){ref-type="fig"}). In HASMC the number of spots increased with higher infectious doses while at the same time the number of inclusions remained constant (Figure [2B](#F2){ref-type="fig"}) in contrast to Cpn infections of HEp-2 cells where more inclusions were formed at higher infectious doses without an increase of spots. Cpn lyse HASMC in a dose-dependent manner ----------------------------------------- To compare the lytic capacity of different Cpn isolates and to identify a possible time course of host cell death during the chlamydial infectious cycle, LDH release of HASMC was analyzed at 24, 48 and 72 h post infection. HASMC were inoculated with two different Cpn isolates: Cpn-VR1310 and Cpn-K6 at serial 2-fold dilutions ranging from 1 to 32 inclusion forming unit (IFU)/cell (Figure [3](#F3){ref-type="fig"}). Both isolates were found to be free of Mycoplasma spec. contamination as assessed by PCR. LDH release of mock treated HASMC or of cells inoculated with heat inactivated bacteria was comparable to the spontaneous LDH release of uninfected cells (data not shown). Both Cpn isolates lysed HASMC in a strictly dose dependent fashion at all time points analyzed. Cpn mediated host cell lysis at 48 h post infection is shown in figure [3A](#F3){ref-type="fig"}. Data obtained from 24 h and 72 h post infection were similar and consistent with the displayed data subset. The levels of HASMC lysis induced by infection with 8 IFU/cell of Cpn-K6 (0.9% ± 3) and Cpn-VR1310 (18.6% ± 8) indicate large differences in the lytic capabilities of both Cpn isolates. Comparison of Cpn-induced LDH release at different time points (24, 48 and 72 h) demonstrated a constant increase in Chlamydia-specific HASMC lysis indicating continuous triggering of cell death throughout the whole infectious cycle (Figure [3B](#F3){ref-type="fig"}). In order to reach the same amount of HASMC lysis (27% ± 2) 4 IFU/cell of Cpn-VR1310 and 32 IFU/cell of Cpn-K6 respectively were required. This suggests that for a given degree of HASMC lysis either high amounts of a lytically less active isolate or lower amounts of a lytically more active isolate are required. Cpn infection of HASMC causes nuclear chromatin condensation as well as extensive damage of organelles and cell membrane ------------------------------------------------------------------------------------------------------------------------ In order to discriminate between apoptotic and necrotic cell death of infected HASMC ultrastructural analysis by transmission electron microscopy (TEM) was carried out. As a control apoptosis was induced by treatment with the well described kinase inhibitor staurosporin. Untreated HASMC had an elongated nucleus rich in euchromatin with one or two well-defined nucleoli (Figure [4A](#F4){ref-type="fig"}). The cytoplasm was scattered by numerous narrow-spaced profiles of rough endoplasmic reticulum (rER), a small Golgi complex and single elongated mitochondria. In Cpn infected cultures a variable number of HASMC harbored large inclusions containing many infectious elementary bodies and metabolically active reticular bodies (Figure [4B](#F4){ref-type="fig"}). The nucleus of these cells was slightly rounded but chromatin structure as well as morphology of organelles was unchanged. A large number of infected HASMC without Cpn inclusions were destroyed (Figure [4C](#F4){ref-type="fig"}). The round shaped nuclei showed a distinct condensation of heterochromatin and condensed nucleoli. Cell organelles including mitochondria and rER profiles were dilated and cell membranes were completely disrupted. Detection of single bacteria by TEM was possible only on rare occasions since distorted organelles in dying cells were barely distinguishable from Cpn. Apoptotic HASMC treated with staurosporin showed a distinct condensation of chromatin (Figure [4D](#F4){ref-type="fig"}) and nuclei were fragmented frequently; damage of cell membranes and swelling of organelles appeared to a lower extent than in Cpn infected HASMC. In general the overall ultrastructural morphology of HASMC infected with spot-like Cpn infection suggests a hybrid form of cell death: it shares chromatin condensation with apoptosis and damage of organelles and membranes with necrosis. Spot-like infection induces aponecrotic morphology in HASMC ----------------------------------------------------------- The combination of chromatin staining by DAPI with labeling of DNA strand breaks via TUNEL staining and assessment of membrane integrity by NHS-biotin staining allows a distinction between necrosis as well as early and late phases of apoptosis using confocal laser scanning microscopy \[[@B23]\]. Cells dying by necrosis exhibit NHS-biotin labeling of the cytoplasm but not chromatin condensation or TUNEL staining of nuclei. Cells in early phases of apoptosis display nuclei with condensed or fragmented chromatin, with or without TUNEL staining, and are surrounded by NHS-biotin negative cytoplasm due to intact cell membranes. In late phases of the apoptotic cell death TUNEL positive nuclei occur together with a NHS-biotin positive cytoplasm. HASMC were infected with Cpn for chosen periods of time or were inoculated with mock isolates or treated with staurosporin. Cells infected with Cpn-VR1310 (Figure [5E, F](#F5){ref-type="fig"}) showed an identical staining pattern as cells infected with Cpn-K6 (Figure [5A -- D](#F5){ref-type="fig"}). Mock inoculated cells displayed round or oval shaped nuclei with finely dispersed chromatin visualized by DAPI staining and were always TUNEL negative. NHS-biotin was bound exclusively to the cell surface indicating intact membranes (Figure [5G](#F5){ref-type="fig"}). In Cpn infected cultures a variable labeling pattern of HASMC was observed. Cells with Cpn inclusions always displayed normal chromatin staining and no TUNEL or NHS-biotin labeling (Figure [5A](#F5){ref-type="fig"}). HASMC with spot-like infection either contained an unaltered nucleus and NHS-biotin negative cytoplasm (Figure [5B](#F5){ref-type="fig"}) or showed a TUNEL positive nucleus with condensed chromatin embedded in NHS-biotin stained cytoplasm (Figure [5C, E](#F5){ref-type="fig"}). In HASMC displaying both Cpn spots and aggregates the nucleus was always labeled by TUNEL and the cytoplasm by NHS-biotin (Figure [5E, F](#F5){ref-type="fig"}). Cpn-infected HASMC never showed membrane damage in combination with an unaltered nucleus, representing necrotic cell death. Cpn-infected HASMC displaying TUNEL positive nuclei and damaged membranes could be detected at 24, 48 and 72 h post infection. 8 h post infection was the earliest time point when TUNEL positive nuclei could be detected, suggesting induction of apoptosis during the whole life cycle of Cpn (not shown). TUNEL-positive HASMC were found even at infectious doses of 1 IFU / cell for Cpn-VR1310 and 8 IFU / cell for Cpn-K6 and the amount of TUNEL-positive HASMC increased strictly dependent on the chlamydial dose used for infection (Figure [5I](#F5){ref-type="fig"}). Again, both Cpn strains used in this study elicited the same features of cell death in HASMC according to the infection morphology. Identical to the lytic capacity (Figure [3A](#F3){ref-type="fig"}) of these strains, K6 had to be used at higher IFU compared to VR1310 to generate a similar number of cells with TUNEL-positive nuclei and NHS-biotin positive cytoplasm. In contrast to HASMC neither Cpn-K6 nor Cpn-VR1310 induced a concentration dependent increase in TUNEL positive nuclei in HEp-2 cells and numbers of TUNEL positive nuclei remained below 10% (not shown). In staurosporin treated cells the rounded nuclei were fragmented or exhibited chromatin that was condensed marginally, whereas NHS-biotin labeling was predominantly negative (Figure [5H](#F5){ref-type="fig"}). Just a minority of the cells revealed a condensed TUNEL-positive nucleus and at the same time were also NHS-biotin positive reflecting late stages of apoptotic cell death. Cpn infection induces phosphatidylserine externalization in HASMC ----------------------------------------------------------------- HASMC were infected for 48 h with Cpn-K6 or -VR1310 in 2-fold dilutions ranging from 8 to 386 IFU/cell or from 1 to 32 IFU/cell, respectively. As controls cultures were inoculated with mock isolates or with heat inactivated bacteria (data not shown) and analyzed for annexin-V binding and propidium iodide uptake. Uninfected as well as Na-azide treated HASMC served as negative and staurosporin-treated cells as positive controls for apoptosis (Figure [6C](#F6){ref-type="fig"} upper right and upper left panel). Control cultures inoculated with mock lysate (Figure [6](#F6){ref-type="fig"}) or heat inactivated bacteria and untreated cells showed always less than 20% annexin-V single positive cells. This proportion of annexin-V positive cells represented background staining, probably due to the experimental procedure. Upon infection with 128 IFU/cell of Cpn-K6 for 72 h the number of single annexin-V positive cells increased to more than 50% (Figure [6B](#F6){ref-type="fig"}). Upon Cpn infection annexin-V and propidium iodide double-positive staining reached 10% as compared with 4% in mock-inoculated and 3% in untreated HASMC (not shown). In staurosporin treated cultures the amount of both annexin-V single and double positive cells increased strongly (Figure [6](#F6){ref-type="fig"} upper left panel) indicating a continued induction of classical apoptotic cell death. The amount of annexin-V single positive HASMC infected with Cpn increased linearly in a dose- (Figure [6A](#F6){ref-type="fig"}) and time- (Figure [6B](#F6){ref-type="fig"}) dependent manner. Cpn-VR1310 again induced phosphatidylserine externalization at much lower doses compared to Cpn-K6. Neither mock inoculation (Figure [5A, B](#F5){ref-type="fig"}[6C](#F6){ref-type="fig"} lower left panel), nor inoculation with heat inactivated bacteria (data not shown), induced apoptosis. This indicates that infection with living bacteria is required for induction of cell-death. Taken together, the dose- and time dependence between infection and phosphatidylserine externalization were consistent with induction of early apoptotic events. Chlamydiainfection in HASMC induces caspase-3 independent cell death by dissipation of mitochondrial membrane potential ------------------------------------------------------------------------------------------------------------------------- After induction of apoptosis an enzymatic cascade is activated leading to the disassembly of the cell \[[@B23]\]. Key enzyme in this cascade is caspase-3. However, in some cases apoptosis can proceed without involvement of caspase-3 \[[@B26]\]. In order to investigate the role of caspase-3 HASMC were infected with Cpn-K6 128 IFU/cell or Cpn-VR1310 32 IFU/cell, respectively, for 48 h (24 h and 72 h not shown) or treated with 1 μM staurosporin for 12 h to induce apoptosis. Samples were analyzed for caspase-3 activation by measuring enzyme activity after lysing the cells. No activity of caspase-3 in Cpn infected HASMC (Figure [7A](#F7){ref-type="fig"}) could be detected. In contrast distinct caspase-3 activity was found in staurosporin-treated cells. A major role in the induction and regulation of cell death is played by mitochondria. Pro-apoptotic factors result at mitochondria in the dissipation of the mitochondrial membrane potential and release of cytochrome c. After infection of HASMC with Cpn-K6 or Cpn-VR1310 cells were loaded with TMRE (tetramethylrhodamineethylester) \[[@B27]\], a dye, that is only taken up into mitochondria with an intact mitochondrial potential \[[@B28]\]. Cells with intact membranes excluding propidium iodide were analyzed by flow cytometry. The number of TMRE negative cells strictly increased with the chlamydial dose whereas blocking of chlamydial protein synthesis by addition of chloramphenicol completely abrogated this effect (Figure [7B](#F7){ref-type="fig"}). Moreover, labeling of Cpn-K6 infected or mock treated cells for cytochrome c and mitotracker red revealed distinct loss of cytochrome c staining of the mitochondria in the infected population (Figure [7D](#F7){ref-type="fig"}) but not in mock treated cells (Figure [7C](#F7){ref-type="fig"}). Taken together, the data suggest Chlamydia pneumoniaeinduce caspase-independent apoptosis-like cell death in infected HASMC by involving mitochondrial membrane dissipation resulting in the release of cytochrome c. Discussion ========== In our in vitro study cell death was only induced in HASMC by infection with viable Cpn but not after incubation with heat inactivated or chloramphenicol treated bacteria. Additionally, different Cpn isolates showed various lytic activities towards host cells that correlated to different reproduction rates in tumor cells as measured by real time PCR \[[@B29]\]. A reproductive infection would seem to be a prerequisite for cell death induction in HASMC. The observation that heat inactivated or chloramphenicol treated bacteria never caused changes of the host cells underlines that cell death induction is not a bystander effect of a potential cytopathic chlamydial component such as the heat stable LPS. It can be concluded that HASMC death depends on internalization of the Chlamydiaand is associated with the suppression of bacterial replication and productive infection. The necessity for a reproductive infection was also shown for Chlamydia psittaciwhich induced caspase 3 independent apoptosis in macrophages and epithelial cells only after reproductive infection along with bacterial protein synthesis \[[@B15]\]. Cell death induction was never found in cells harbouring inclusions which normally are viewed as the manifestation of a reproductive infection. HASMC formed inclusions with a relatively low frequency compared to tumor cells. In contrast, spots resulting presumably from single bacteria were found frequently illustrating a different infection morphology of Cpn in these primary vascular cells. Currently no data are available regarding metabolism, protein synthesis and reproduction or functional relation with host cells of these single, spot-like bacteria. From our data it only can be concluded that these single bacteria are sensitive to chloramphenicol which inhibits protein synthesis in Cpn and abrogates cell death induction. Cell death seems to result from an interaction of the host cell with these single bacteria as it is ongoing for extended times. Cpn induced cell death in HASMC did not fulfill all criteria for apoptosis or classical necrosis. Condensation of chromatin, positive TUNEL staining, externalization of phosphatidylserine, inhibition of TMRE labeling and loss of cytochrome c immunoreactivity in mitochondria were clear markers for apoptosis. But early damage of cell membrane and organelles as found by TEM and NHS-biotin labeling indicated necrotic type of cell death in Cpn infected cells. According to previous classifications Cpn mediated cell death belongs to apoptosis-like cell death \[[@B30]\] or can be termed aponecrosis due to characteristic ultrastructural features \[[@B24]\]. Cpn induced cell death in HASMC having features of necrosis and apoptosis is in agreement with a study employing mouse embryonic fibroblasts infected with C. trachomatiswhich died through a combination of necrosis and apoptosis \[[@B18]\]. If cell death induction by Cpn in HASMC is caspase-3 independent the question arises about alternative pathways that are involved. Mitochondria are important regulators of cell death and loss of mitochondrial permeability transition is an important indication for apoptosis \[[@B31]\]. Here we show by TMRE labeling dissipation of the mitochondrial potential of Chlamydiainfected cells. This effect could be inhibited by blocking bacterial protein synthesis using chloramphenicol. Moreover, cytochrome c was released from mitochondria of infected HASMC as indicated by loss of immunorecactivity. The important role of mitochondria in Chlamydiainduced cell death has also been shown in tumor cell lines infected with Chlamydia psittaci\[[@B14]\]. Pro-apoptotic Bcl-2 family members like Bax were activated and apoptosis induction occurred during the whole chlamydial life cycle. Apoptosis induced by Chlamydia psittaciwas blocked in cells over expressing Bax inhibitor and anti-apoptotic Bcl-2 \[[@B14]\]. Bax^-/-^mouse embryonic fibroblasts were shown to be resistant to Chlamydia muridarum-induced apoptosis and fewer bacteria were recovered after two infection cycles. This suggests that Bax-dependent apoptosis may be used to initiate a new round of infection \[[@B32]\]. In a second study, Chlamydia trachomatisinfected mouse fibroblasts showed a higher level of apoptosis than Chlamydiainfected HeLa cells as measured by annexin V / propidium iodide double labeling \[[@B18]\]. This not only shows that Bax activation could reflect a stress response in infected cells \[[@B33]\] but that primary cells show a different stress response to Chlamydiainfection than tumor cells. Beside Bax other factors like apoptosis-inducing factor (AIF) may be involved in caspase-independent cell death \[[@B34]\] but they have never been investigated in Chlamydia-induced cell death. AIF which is upregulated and translocated from the mitochondria to the cytosol may play an important role in induction of caspase-3 independent cell death \[[@B34]-[@B36]\]. Injection of AIF into the cytoplasm induces similar chromatin condensation coupled to phosphatidylserine exposure as we found in Cpn infected HASMC \[[@B37]\]. Future experiments will have to show if AIF, Bax or other Bcl-2 family members are involved in Cpn induced HASMC death. There exist numerous studies showing that Cpn and Chlamydia trachomatisprevent cells in vitro from undergoing apoptosis \[[@B16],[@B17],[@B38]-[@B44]\]. However, these experiments were performed on established cell lines. Our study shows that in primary cultures of cells such as HASMC various types of Cpn infection occur. Spot like infection resulted in aponecrosis whereas cells with Cpn inclusions appeared to be prevented from cell death. Apparently Cpn in inclusions have evolved strategies to inhibit cell death presumably to complete the developmental cycle. About possible mechanisms can be speculated: it has been shown that Cpn inserts components of a type-III secretion system into the inclusion membrane \[[@B45]\] and releases factors into the host cell \[[@B46]-[@B48]\] that affect host cell metabolism. Cpn secretes proteins that translocate from the inclusion to the cytoplasm \[[@B47],[@B49]\]. CADD, a secreted protein from Chlamydia trachomatis, has been shown to interact with death domains and co-localizes with Fas. Recombinant CADD has been shown to induce caspase dependent apoptosis in several tumor cell lines \[[@B50]\]. Therefore, other yet unidentified proteins from Chlamydiacould interact with the host cell death machinery and inhibit or promote cell death. Currently, it is unclear how the observed morphology of Cpn in HASMC and the induced aponecrotic cell death affects the progression of atherosclerosis. Cpn infection of HASMC was accompanied by phosphatidylserine externalization which might result in the ingestion of dying cells by macrophages. Macrophages will then either stop further progress of Cpn mediated damage or in turn become activated and thereby contribute to inflammation in the atherosclerotic vessel \[[@B51],[@B52]\]. Early membrane damage found in the aponecrotic HASMC may provoke or assist inflammation in the atherosclerotic vessel due to release of cellular constituents. Finally damage of vascular smooth muscle cells by Cpn infection might lead to plaque instability \[[@B19]\]. Conclusions =========== Cpn infection of cultured HASMC results in cell death that can be termed aponecrosis due to the sharing of apoptotic and necrotic features. This form of cell death occurs just in cells bearing single or aggregated bacteria but not in cells with inclusions. Aponecrotic HASMC may in vivo assist chronic infection in the vascular wall supporting the progression of atherosclerosis. Methods ======= Chlamydial organisms, cells, antibodies and reagents ---------------------------------------------------- Cpn-VR1310 and Cpn isolate Kajaani 6 (Cpn-K6) were kindly provided by G. Christiansen (Institute of Microbiology and Immunology, University Aarhus, Denmark) and M. Puolakkainen (Haartman Institute, University of Helsinki, Finland), respectively. HEp-2 cells obtained from ATCC (Manassas, USA) were cultivated in MEM-Eagle\'s (Gibco BRL, Invitrogen, Basel, Switzerland) and HASMC purchased from Clonetics (Verviers, Belgium) were maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, all from Gibco BRL, Invitrogen, and 10 μg/ml neomycin (Sigma Chemicals, Buchs Switzerland). Cells and chlamydial organisms were tested for Mycoplasma spec. contamination by PCR \[[@B53]\] and 4\',6-Diamidin-2\'-phenylindol-dihydrochloride (DAPI) (Roche, Mannheim, Germany) staining and were mycoplasma free. Anti-Cpn-MOMP monoclonal antibody was from DAKO (Zug, Switzerland); anti-cytochrome c antibody raised in sheep was from Sigma, FITC-labeled monoclonal anti-Chlamydia-LPS antibody from Biorad (Redmont, USA). Donkey anti-mouse polyclonal antibody was obtained from Jackson Immuno Research (Baltimore, USA) and staurosporin from Sigma Chemicals. Chlamydial culture and infection of HASMC ----------------------------------------- Cpn was cultured according to an established protocol \[[@B54]\]. Briefly, confluent HEp-2 cells were inoculated with Cpn at 1 IFU/cell in medium, centrifuged at 1000 × g for 1 h and grown for 72 h in medium containing 10% FCS, 2 mM glutamine, 10 μg/ml neomycin and 2 μg/ml cycloheximide (Sigma). Subsequently, cells were harvested, disrupted by sonification and bacteria were purified on a discontinuous renografin (Sigma Chemicals) gradient as previously described \[[@B55]\]. Non infected HEp-2 cells were subjected to the same harvesting / purification procedure and referred to as mock controls. In order to determine the chlamydial titer (IFU/ml), confluent HEp-2 cells grown on cover slips were infected with serial 2-fold dilutions of purified bacteria. After 72 h cells were fixed, stained with anti-Chlamydia-LPS antibody, inclusions were counted under a fluorescence microscope and the titer was calculated. HASMC were seeded into 6-well plates (Nunc, Roskilde, Denmark) at a density of 2.4 × 10^4^cells/cm^2^one day prior to infection. Infection with chlamydial organisms was performed in DMEM using titers between 8 and 386 IFU/cell which were added to HASMC, centrifuged onto the cells for 1 h at 1000 × g. Subsequently, the supernatant was replaced by DMEM containing 10% FCS, 2 mM glutamine and 10 μg/ml neomycin. In some experiments chloramphenicol 0.1 mg/ml was added analysis or cells were treated with 1 μM staurosporin for 14 h. Cells were grown at 37°C in the presence of 5% CO~2~until they were analyzed. Determination of infection morphology, intra- versus extracellular staining of Cpn ---------------------------------------------------------------------------------- Live HASMC and HEp-2 cells were incubated with anti-Cpn-MOMP antibody (1:50) in PBS + 2% BSA for 1 h at room temperature (RT). After washing and fixation with 2% paraformaldehyde + 3% sucrose in PBS at RT, cells were incubated for 1 h with donkey anti-mouse-FITC antibody, diluted 1:100 in PBS + 2% BSA. Bacteria located extracellularly are stained exclusively green by this step. Subsequently, cells were lysed with 0.1% Triton-X 100 for 1 min at RT, rinsed with PBS + 2% BSA for 20 min, and incubated with anti-Cpn-MOMP antibody (1:50) followed by Texas Red-labeled donkey anti-mouse antibody (1:200) in 2% BSA in PBS. The second staining step identifies both intracellular and extracellular bacteria. Counting of cells with inclusions or spots as well as the number of inclusions and spots were performed on volume data obtained using a laser scanning microscope (SP2, Leica, Heidelberg, Germany) followed by image analysis using the software package Imaris (Bitplane, Zurich, Switzerland). Determination of cytotoxicity by lactate dehydrogenase release (LDH) -------------------------------------------------------------------- LDH release of HASMC was assessed using a cytotoxicity detection kit (Roche) following the manufacturer\'s instruction. In brief, HASMC were seeded in a 96-well plate (NUNC) at a density of 2.4 × 10^4^cells/cm^2^, infected with Cpn between 2 and 32 IFU/cell and cultivated for 24 h, 48 h and 72 h. For cell death analysis 100 μl of cell-free supernatant was harvested, mixed with dye solution, incubated for 20 min and absorption was measured at 490 nm. Percent Chlamydia-specific lysis was calculated according to the formula: ((experimental value -- spontaneous release)/(maximum release -- spontaneous release) × 100. Spontaneous release corresponded to untreated HASMC and infected cells lysed additionally with Triton-X 100 showed the maximal release. Transmission electron microscopy (TEM) -------------------------------------- Cells were fixed in 50 mM sodium cacodylate buffer pH 7.3 containing 2% glutaraldehyde and 0.8% paraformaldehyde and postfixed with 1% OsO~4~in 50 mM sodium cacodylate buffer, pH 7.3 dehydrated in an ethanol series and embedded into epon (Catalys). Ultrathin sections of 50 nm were contrasted with uranyl acetate and lead citrate and studied with a CM 100 transmission electron microscope (Phillips, Leiden, Netherlands). TUNEL-, NHS-biotin-, Cpn- and DAPI-staining for analysis by confocal microscopy ------------------------------------------------------------------------------- Cells were trypsinized, washed in PBS and incubated with 0.1 mg/ml NHS-biotin (Pierce, Rockford, USA) in PBS for 30 min on ice, fixed with 3% paraformaldehyde + 2% sucrose in PBS and cytospinned onto glass slides. Samples were permeabilized with 0.1% Triton-X 100 in PBS for 1 min at RT and stained for DNA strand breaks using terminal transferase (Roche), incorporating dUTP-FITC (Roche) overnight at 37°C as previously described \[[@B56]\]. Subsequently, cells were incubated with anti-Cpn-MOMP antibody (1:50) and detected with donkey anti-mouse Texas Red antibody (1:200) in 2% BSA in PBS for 1 h, followed for 45 min by 0.5 μg/ml streptavidin-Cy5 (Jackson Immuno Research) detection of NHS-biotin. Nuclei and DNA of bacteria were stained with 1 μg/ml DAPI in PBS for 10 min. Samples were then embedded in fluorescence mounting medium (Dako) and analyzed using a confocal laser scanning microscope (SP2, Leica, Heidelberg, Germany). Annexin-V/propidium iodide staining for flow cytometry ------------------------------------------------------ Phosphatidylserine exposure and propidium iodide incorporation was assessed using the annexin-V-FLUOS staining kit (Roche). In brief, supernatants of HASMC cultures were collected and mixed with trypsinized cells. Samples were washed in PBS followed by 20 min staining with annexin-V / propidium iodide staining solution and analyzed by flow cytometry (Becton Dickinson, Basel, Switzerland). Determination of caspase-3 activity ----------------------------------- 0.2 × 10^6^HASMC consisting of floating and adherent cells were lysed at 4°C in cell lysis buffer (20 mM PIPES, pH 7.2, 100 mM NaCl, 1 mM EDTA, 10 mM DTT, 0.1% CHAPS, 10% sucrose), subjected to three freeze-thaw cycles and centrifuged at 14000 × g for 10 min. Protein content of supernatants was measured using the micro BCA protein assay reagent kit (Pierce). Cell lysates containing 40 μg protein were incubated with DEVD-p-nitroanilide (0.8 mM) in lysis buffer and absorption was measured every 5 min in a spectrophotometer at 405 nm for 6 h at 37°C. Caspase-3 activity was calculated as the initial ascending slope of absorption depicted as arbitrary units. TMRE and propidium iodide staining for analysis of mitochondrial membrane potential ----------------------------------------------------------------------------------- Cells were stained with TMRE 100 nM and propidium iodide 2 μg/ml in DMEM with 10% FCS for 30 min at 37°C and 5% CO~2~, collected by trypsinization, combined with detached cells at time of trypsinization and analysed by flow cytometry (Becton Dickinson). Cytochrome c and mitotracker red staining for confocal microscopy analysis -------------------------------------------------------------------------- Cells were incubated in the presence of 50 nM mitotracker red for 30 min, subsequently fixed with 3% paraformaldehyde + 2% sucrose and were cytospinned onto glass slides. Samples were permeabilized with 0.1% Triton-X 100 in PBS for 1 min at RT. Subsequently stained with sheep anti cytochrome c (1 : 2000) in 3 % BSA in PBS for 1 h at 37°C followed by detetion with donkey anti sheep biotin antibody (1:200) in 0.5 % BSA in PBS for 1 h at RT. Cells were labeled with streptavidin Cy-5 (1 : 200) for 1 h at RT and analysed under a confocal laser scanning microscope. Authors\' contributions ======================= CD performed all infections and analyzed morphological markers for cell death. CFM and MKJS performed FACS experiments. DG initiated the study and performed the first infections of vascular cells. MW analyzed viability experiments and provided input for exeriments. PG provided critical intellectual input to the study and organized financial support. UZ established assays for the detection of caspase 3, morphological cell death markers and was leading the study. Acknowledgements ================ We would like to thank to Miriam Erni, Gery Barmettler and Marina Balzer for excellent technical assistance, Michel Le Hir and Jörg D. Seebach for fruitful discussions and Lloyd Vaughan for carefully reading the manuscript. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Diverse morphology of Cpn infection.HASMC (A, B, C and D) and HEp-2 cells (E) were infected for 72 h with Cpn-K6 at 128 IFU/cell (HASMC; A, B), Cpn-VR1310 at 8 IFU/cell (HASMC; C, D) and Cpn-K6 at 5 IFU/cell (HEp-2; E), respectively. Bacteria localized on the cell surface are stained with FITC (green), Chlamydiainternally localized are stained with Texas Red (red) and FITC (for detail see material and methods). DNA of bacteria and host cells is stained with DAPI (blue). N = nucleus. Left row: extracellular Cpn. Middle row: extra- and intracellular Cpn. Right row: overlay of left- and middle row and DAPI staining. A, C: spot like infection of a HASMC; note that the majority of bacteria are localized intracellularly B, D: HASMC with a Cpn inclusion. E: Hep-2 cells with Cpn inclusions. ::: ![](1471-2180-5-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Ratio of cells with inclusions versus cells with spotsHASMC and HEp-2 were infected with Cpn-K6 or Cpn-VR1310. Cells carrying inclusions or spots, respectively, were counted 48 h post infection. A: Depicted is the ratio of cells with inclusions versus cells with spots; -◆-: HASMC Cpn-K6 infected, -■-: HASMC Cpn-VR1310 infected, -◇-: HEp-2 Cpn-K6 infected, -□-: HEp-2 Cpn VR1310 infected B: inclusions / cell, C: spots / cell; black bars: HASMC, open bars: HEp-2 cells. The number of inclusions / cell in HASMC remained constant in contrast to the number of spots / cell when increasing the infectious dose, whereas in HEp-2 cells the number of inclusions / cell increases and the spots / cell remain constant. ::: ![](1471-2180-5-2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Lytic capability of different Cpn isolates.HASMC were infected with two different Cpn isolates. LDH release was measured in triplicates 24 h, 48 h and 72 h post infection and Cpn specific lysis was calculated. The average and standard deviation is plotted. One representative result out of three independent experiments is shown. A: Dose dependent Cpn specific lysis after 48 h revealed different capacities of the isolates tested to lyse HASMC. B: Different amounts of the various Cpn isolates leading to a comparable lysis over time of HASMC. Isolates were used as follows: Cpn-VR1310, 4 IFU/cell -◆-, Cpn-K6, 32 IFU/cell -■- ::: ![](1471-2180-5-2-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Ultrastructure of Cpn infected HASMC.Cells were either infected with 128 IFU/cell Cpn-K6 for 72 h (B,C) or incubated with staurosporin 1 μM for 16 h (D) or were left untreated (A). A: Untreated HASMC with normal ultrastructure, note the elongated nucleus with finely dispersed euchromatin. B: HASMC with a typical Cpn inclusion. The membrane bound vesicle contains dark (= elementary bodies) and pale (= reticular bodies) stained bacteria. The chromatin structure in the nucleus is unchanged. C: Cpn infected HASMC undergoing cell death. The nucleus is characterized by highly condensed heterochromatin (arrow head) and a compact nucleolus and is surrounded by dilate organelles (arrow). The cell membrane is completely disrupted. D: Staurosporin treated HASMC. The nucleus is partially fragmented containing patchy condensed heterochromatin (arrow heads). ::: ![](1471-2180-5-2-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Light microscopic analysis of the effect of Cpn infection on HASMC.HASMC were infected with Cpn-K6 128 IFU/cell (A, B, C, D), Cpn-VR1310 32 IFU/cell (E, F) for 72 h or were inoculated with mock lysate (G) or treated with staurosporin 1 μM for 14 h (H). The cells were subsequently stained for TUNEL (green), NHS-biotin (brown), anti-Cpn-MOMP (red) and DNA (blue). A: HASMC bearing a Cpn-K6 inclusion. DAPI stained chromatin structure is normal and TUNEL as well as NHS-biotin labeling are negative. B: Cpn-K6 infected HASMC with multiple chlamydial spots (arrow heads) and normal nuclear structure. C, E: Cpn-K6 (C) or Cpn-VR1310 (E) infected HASMC with spot like infection (arrow heads). The condensed nucleus is TUNEL positive, the cytoplasm is diffusely labeled with NHS-biotin. D, F: HASMC with Cpn-K6 (D) or Cpn-VR1310 (F) in aggregated (arrow) spots and single spots (arrow heads). Note positive TUNEL labeling of the shrunken nucleus and NHS-biotin staining of the cytoplasm. G: Mock inoculated HASMC. Note the fine granular chromatin structure and lack of TUNEL- and cytoplasmic NHS-biotin staining. H: HASMC incubated with staurosporin. The nucleus exhibits a distinct nuclear fragmentation (DAPI staining) but no TUNEL labeling. The cytoplasm is NHS-biotin negative indicating an intact cell membrane. I: Quantification of TUNEL positive nuclei depending on the chlamydial infectious dose 72 h post infection reveals a dose dependent increase of TUNEL positive cells. Cells in 5 random fields with an area of 62\'500 μm^2^were counted and the percentage of TUNEL positive cells was calculated. -▲-: Cpn-K6; -■-: Cpn-VR1310 ::: ![](1471-2180-5-2-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Dose response relationship between bacterial titer and phosphatidylserine exposure in Cpn infected HASMC.Cells infected with Cpn-K6 in doses between 8 -- 386 IFU/cell or with Cpn-VR1310 in doses between 1 -- 32 IFU/cell were stained with annexin-V / propidium iodide and analyzed by flow cytometry 48 h post infection. Mock inoculated cells are shown as controls. In figure A and B pooled data out of three independent experiments are displayed. A: The amount of single annexin-V positive cells increased dependent on the chlamydial infectious dose (-◆ -: Cpn-K6; -●-: Cpn-VR1310) but not in mock treated (-○-) cells. (\: significant compared to mock treated cells with p \< 0.001) B: the number of single annexin-V positive cells increased over the time when cells were infected with Cpn-K6 128 IFU/cell (-●-) but not if they were mock treated (-○-) (significant compared to mock treated cells with p \< 0.001)(\#)). C: Dot plots of one representative experiment are depicted to illustrate the amount of annexin V / propidium iodide double labeled cells. Upper left panel: HASMC treated with staurosporin (apoptosis). Upper right panel: HASMC treated with Na-azide (necrosis). Middle left panel: HASMC infected with Cpn-VR1310 at 32 IFU / cell 48 h post infection. Middle right panel: HASMC infected with Cpn-K6 at 128 IFU / cell 48 h post infection. Lower left panel: mock treated HASMC. ::: ![](1471-2180-5-2-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Caspase 3 activity and mitochondrial membrane permeability*A: Caspase-3 activity was assessed in HASMC infected with 128 IFU/cell Cpn-K6 ± Chloramphenicol (CA), loaded with mock isolate or treated with 1 μM staurosporin for 14 h. No caspase-3 activity can be detected in Cpn infected or mock inoculated cells in contrast to staurosporin treated cells. B: HASMC were infected with 4 -- 128 Cpn-K6 (-●-) or 4 -- 32 IFU/cell Cpn-VR1310 (-◆-) and stained by TMRE for detection of mitochondrial membrane potential in combination with propidium iodide. Propidium iodide negative cells were analyzed by flow cytometry for TMRE staining. Infected HASMC treated with 0.1 μg/ml chloramphenicol served as controls (open symbols). Loss of TMRE staining is induced in HASMC infected with viable Cpn but not in cells infected with Cpn treated with chloramphenicol. Mean values with standard error of three independent experiments are shown. C, D: HASMC were infected with Cpn-K6 (IFU 128 IFU/cell, 48 h) and stained for cytochrome c (white). Mitochondria were labeled with mitotracker red (red). Loss of cytochrome c staining is found in mitochondria of HASMC infected with Cpn (D) but not in mock treated cells (C). ::: ![](1471-2180-5-2-7) :::	PubMed Central	2024-06-05T03:55:52.407707	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547904/", "journal": "BMC Microbiol. 2005 Jan 21; 5:2", "authors": [ { "first": "Claudia", "last": "Dumrese" }, { "first": "Christine F", "last": "Maurus" }, { "first": "Daniel", "last": "Gygi" }, { "first": "Mårten KJ", "last": "Schneider" }, { "first": "Michael", "last": "Walch" }, { "first": "Peter", "last": "Groscurth" }, { "first": "Urs", "last": "Ziegler" } ] }
PMC547905	Background ========== Epidemiological studies have shown a significant correlation of blood pressure levels in close relatives and higher concordance values for occurrence of hypertension in monozygotic vs. dizygotic twins, and thus support the idea that genetic factors influence susceptibility to essential hypertension \[[@B1]\]. While recent molecular genetic studies have provided compelling evidence for mutations in at least seven different genes underlying rare forms of monogenic hypertension \[[@B1],[@B2]\], progress in the understanding of the molecular basis of human essential hypertension has been much slower. Hundreds of case-control studies have suggested hypertension-related genetic variants of which only a few if any have tolerated replication analyses; it is possible that common variants of angiotensinogen \[[@B3]\], α-adducin \[[@B4]\] and the G-protein β subunit \[[@B5]\] confer susceptibility to elevated blood pressure in at least some populations. Since 1999, a number of genome-wide linkage studies in families with multiple affected hypertensive members have been published with highly varying results (for review, see \[[@B6]\]). Recent large-scale searches for genes predisposing to hypertension, published as a recent series of articles \[[@B7]-[@B11]\], failed to identify definite linkage of hypertension to any chromosomal locus, although some DNA regions showing suggestive linkage were disclosed. Reasons for these disappointing data were put on the account of the unsuitability of using a single-locus linkage strategy for a multifactorial genetic disease, inherent genetic heterogeneity of essential hypertension, and complex interplay of genetic and environmental factors underlying regulation of blood pressure variation \[[@B12]\]. Disappointments in the previous strategies justify alternative approaches in which a better phenotyping of the study individuals is connected to their targeted molecular genetic characterization. There are several features that collectively make the genes encoding the beta (βENaC) and gamma (γENaC) subunits of the kidney tubular epithelial sodium channel as serious candidates for susceptibility genes of low-renin human essential hypertension. First, gain of function mutations in β and γ ENaC subunits cause Liddle\'s syndrome, a well-known monogenic form of human hypertension associated with low renin activity and low plasma aldosterone level \[[@B13]-[@B15]\]. Second, common βENaC variants occur in increased frequency in hypertensive black individuals \[[@B16]-[@B18]\]. Third, an extensive locus-targeted study on hypertensive family members demonstrated a significant linkage of hypertension to chromosome 16q region harboring both the βENaC and γENaC genes \[[@B19]\]. These data prompted us to carry out a search for common variants of these two genes in Finnish hypertensive patients who were admitted to a special center because of treatment-resistant hypertension and whose renin-aldosterone system was systematically examined. These circumstances provided a group of hypertensive patients, in which secondary forms of hypertension were effectively excluded and who originated from a genetic isolate. Our data suggest that common variants of the ENaC subunits confer susceptibility to human essential hypertension. Methods ======= Patients with hypertension -------------------------- The clinical records of all consecutive patients with hypertension (n = 615) referred to the Hypertension Outpatient Ward, Helsinki University Central Hospital, between 1992--96 were reviewed. Moderate-to-severe hypertension, suspicion of secondary forms of hypertension, or hypertension resistant to drug treatment were causes to the admittance. A letter with request to donate a blood sample for genetic studies on hypertension was sent to those 598 individuals whose address became available in 1998. A total of 399 individuals (67%) of these responded and were subsequently examined at the Hypertension Outpatient Ward in 1998 to 1999. Clinical and family histories were recorded, and venous blood samples taken for DNA analysis. Based on the previous documents and current examinations, altogether 52 individuals were excluded from the present study: clinical records were missing or insufficient in four cases, 22 subjects were considered as normotensive, while 26 were judged to have a secondary form of hypertension. The latter group consisted of the following cases: renal artery stenosis (n = 12), adrenal cortical adenoma (n = 3), hydronephrosis (n = 2), pheochromocytoma (n = 1), IgA glomerulonephritis (n = 1), non-specific chronic glomerulonephritis (n = 1), LED nephritis (n = 1), diabetic nephropathia (n = 1), chronic pyelonephritis (n = 1), hypernephroma (n = 1), fibromuscular dysplasia (n = 1), unspecified renal failure (n = 1). The remaining 347 patients (186 females and 161 males, mean age 49.3 years, SD ± 10.0) comprised our final cohort of patients with moderate-to-severe essential hypertension. Antihypertensive drug treatment was in use in 283 (82%) of the patients (diuretics, 19%; beta-blocking agents, 35%; calcium-channel blockers, 21%; ACE-inhibitors, 33%; angiotensin receptor antagonists, 1%). At least two concomitant drugs were used by 24% of the patients. A flow-chart of the study design is illustrated in Fig. [1](#F1){ref-type="fig"}. Control individuals ------------------- ### Blood donors DNA was extracted from 301 randomly selected healthy blood donors aged 40--50 years (mean, 45 years) visiting the Finnish Red Cross Blood Transfusion Service. Their residences represent the same capital area from which the hypertensive patients originated. ### Normotensive controls These individuals were selected from the participants in the Alpha-Tocopherol, Beta Carotene (ATBC) study \[[@B20]\] using the criteria described previously \[[@B21]\]. In brief, a total of 27271 male smokers (aged 50 to 69 years) with no previous history of myocardial infarction were initially recruited for a cancer prevention trial. DNA samples were available from 70% of the original participants. We picked up all the available blood samples from those fulfilling the following criteria: no known hypertensive disorder, no antihypertensive drugs ever in use, systolic and diastolic blood pressure values ≤ 128 and ≤ 84 mmHg, respectively, at each blood pressure measurement, repeated five times at one-year intervals during a five-year follow-up. We ended up with 175 normotensive men whose mean systolic and diastolic blood pressures were 114.9 (SD ± 5.4) and 73.7 (SD ± 4.3) mmHg, respectively, during this five-year follow-up. The Ethics Review Committee of the Helsinki University Central Hospital approved this study, and all patients and controls gave their informed consent. Laboratory measurements in the hypertensive patients ---------------------------------------------------- The patients were advised to stop using estrogens and spironolactone at least 4 weeks before the tests, diuretics and prostaglandin inhibitors at least two weeks before the tests, and β-adrenergic antagonists and ACE inhibitors at least one week before the test. The only antihypertensive agents permitted at the time of the test were calcium channel blockers. Some of the patients were on oral potassium supplementation because of hypokalemia. The mean baseline blood pressure level at the time of captopril test was 139 ± 16/94 ± 10 mmHg in those without any drugs (n = 79), and 142 ± 16/95 ± 11 mmHg in those with calcium channel blockers (n = 234). Fasting blood samples were taken for determination of serum creatinine, uric acid, cholesterol, potassium, sodium and blood glucose concentrations. Urine samples for determination of the daily (24 h) excretion of potassium and sodium were collected. Most hypertensive patients (n = 298) underwent a test for the responsiveness of serum aldosterone level and plasma renin activity to postural change. To this end, the first blood sample was taken after at least 60 minutes of rest in supine position. After 2 hours of standing and moderate walking, a second blood sample was taken. This test was carried out at the inpatient ward in 220 cases and at the outpatient ward in 78 cases. Urinary electrolyte excretion rates were analyzed in 262 patients (26 ENaC variant carriers and 236 non-carriers) who did not use potassium supplementation. One to three days later, a captopril challenge test (CCT) was carried out as described earlier \[[@B22]\]. This test was carried out in a total of 313 patients, and was performed at the inpatient ward in 229 cases and at the outpatient ward in 84 cases. CCT was started by sitting for at least 30 minutes, followed by oral administration of 50 mg captopril. Blood pressure in the non-dominant arm was measured at 15-minute intervals. Blood samples for the determination of plasma renin activity and serum aldosterone concentration were drawn immediately before and 60 minutes after captopril administration. DNA analysis ------------ Genomic DNA was extracted from peripheral venous blood using standard techniques. For targeted search for ENaC variants postulated to be associated with increased channel activity, we chose to sequence the exons 13 coding for the carboxyterminal domains of βENaC (amino acids 515--640) and γENaC (amino acids 524--649), as well as the 5\'-flanking intronic regions, using oligonucleotide primers, PCR (polymerase chain reaction) conditions and sequencing instruments described previously by us \[[@B21]\]. DNA samples of 27 patients of those 399 initially visiting the Hypertension Outpatient Ward showing the lowest plasma renin activities (median 0.7 μg/L/h at 0 minutes and 0.9 at 60 minutes) and serum aldosterone concentrations (median 236 pmol/L at 0 minutes and 212 at 60 minutes) during CCT were selected for this initial step. Specific PCR-based methods were set up for assaying the three ENaC variants detected during the present study. After PCR of the βENaC fragment, the βENaC -i12 -17CT and βENaC G589S variants could be assayed simultaneously. An aliquot (8 μl) of the PCR product was digested with 3.0 U of AluI (New England Biolabs, Beverly, Massachusetts, USA), followed by analysis of the cleavage products on a 12% polyacrylamide gel. The wild-type (wt) allele results in longest fragments of 266 and 137 bp, while the variant allele produces fragments of 266 and 147 bp for βENaC -i12 -17CT, and 240 and 137 bp for βENaC G589S. For the γENaC V546I variant, 2.0 U of SfaNI (New England Biolabs) was used and the cleavage products were analyzed on a 2% agarose gel. The resulting fragment sizes were 357 and 77 bp for the wild-type allele and 279, 78 and 77 bp for the variant allele. For studies on the possible splicing errors brought about by the βENaC i12-17 variant, we collected lymphocytes from two subjects heterozygous for this variant and one control subject. Total lymphocytic RNA was isolated using Qiagen RNeasy kit (Qiagen, Valencia, California, USA), and first strand synthesis was performed using Superscript system for RT-PCR (Invitrogen Corporation, Carlsbad, California, USA). For gene-specific PCR, we used two sets of primers amplifying a fragment extending from exon 12, either to exon 13 (180 bp) or 141 bp downstream of exon 13 (551 bp). The amplified products were run on a 12 % polyacrylamide gel and visualized by ethidium bromide. The amplified fragments were also sequenced to exclude presence of any splicing defects. Additionally, possible splicing differences between the wild-type and i12-17 variant of βENaC were studied in silicousing GrailEXP v3.3 (Perceval) exon prediction program \[[@B23]\]. Site-directed mutagenesis and functional characterization of the ENaC variants ------------------------------------------------------------------------------ The human βENaC cDNA and γENaC cDNA cloned into the pBSK-SP6-globin vector were used in construction of the βENaC G589S and γENaC V546I mutations, respectively. Site-directed mutagenesis was performed using the Transformer site-directed mutagenesis kit (Clontech Laboratories, East Meadow Circle, California, USA). Mutagenic primers used were 5\'-cacaccaactttAgcttccagcctg-3\' and 5\'-gctgctctgttgtctgcAtcatcgagatcatcgagg-3\' for the G589S and V546I mutations, respectively. The primer 5\'-ccctcgctcgTgtgatctggt-3\', which mutates the XhoI restriction enzyme site in the pBSK-SP6-globin vector, was used as the selection primer in the mutagenesis reactions. The mutagenic clones were sequenced to confirm the presence of the mutations and to exclude undesired errors during mutagenesis. Healthy stage V and VI Xenopusoocytes were injected with mRNAs encoding the β human (h)ENaC wild-type or βG589S hENaC mutant, the γhENaC wt or γV546I hENaC mutant together with the mRNA encoding the αhENaC wt. The total amount of mRNA encoding the three αβγ ENaC subunits was 10 ng. Electrophysiological measurements were taken at 16--24 hours after injection. ENaC activity was assessed by measurement of the amiloride-sensitive current (I~Na~in μA) recorded at -100 mV with a two-electrode voltage clamp amplifier (TEV-200, Dagan Corp.) in a standard solution containing 110 mmol/L NaCl, 1.8 mmol/L CaCl~2~, 10 mmol/L HEPES-NaOH, pH 7.35. The amiloride concentration used was 5 μmol/l in the bath solution. Four batches of oocytes were obtained from different Xenopusfrogs in which 5 to 7 oocytes were tested for each αβγ ENaC wt and ENaC variants. Statistical analysis -------------------- The renin and aldosterone values were nonnormally distributed, as analyzed using skewness, kurtosis and Kolmogorov-Smirnov tests. Therefore, nonparametric tests (Mann-Whitney\'s U) were used in the statistical analyses, and interquartile (IQ) range and median are used to describe the distributions of target variables. When covariates were included in the analyses, ANCOVA with ranks or logarithm-transformed values of the variables was used. Chi square test, or Fisher\'s exact test if observed frequency in any cell was less than five, were used for the frequency analysis of the variants. Logistic regression was used to obtain age and gender adjusted odds ratios for hypertension in ENaC variant carriers vs. non-carriers. All data was analyzed using statistical SPSS program (version 11.0). Because of relatively small variant group sizes, the primary analyses were performed with all variant groups combined. Secondarily, the variant groups were also compared separately with the wild-type ENaC group. Results ======= Identification of three common ENaC variants and screening for their presence in the three different study groups ----------------------------------------------------------------------------------------------------------------- DNA samples of 27 hypertensive patients with lowest renin activities and aldosterone concentrations were initially selected for targeted search for ENaC variants. The sequencing strategy chosen permits detection of mutations and polymorphisms in the entire coding parts of exons 13 of β and γENaC genes, as well as 26 or 43 nucleotides at the 3\'-ends of introns 12. Three different single-nucleotide substitutions were detected, two in the βENaC and one in the γENaC subunit (Fig. [2](#F2){ref-type="fig"}). Four out of the 27 samples showed a previously unreported substitution of T for C in intron 12 of the βENaC gene (i12-17CT), located 17 nucleotides upstream of the 5\'-end of exon 13. In one DNA sample a single G to A substitution changed the codon 589 of βENaC from GGC to AGC, predicted to result in a substitution of serine for glycine (G589S). This variant has been described previously \[[@B24],[@B25]\]. Upon screening of exon 13 of the γENaC gene for mutations, one sample was detected with a novel point mutation changing codon 546 from GTC to ATC, which results in a substitution of isoleucine for valine (V546I). We next conducted a search for these three ENaC variants in our whole material of patients with essential hypertension (n = 347), normotensive males (n = 175) and randomly chosen blood donors (n = 301) (Table [1](#T1){ref-type="table"}). Altogether, we identified 46 heterozygous carriers of these variant alleles, but no homozygous or compound heterozygous individuals. Their prevalence was significantly different in the three study groups (χ^2^= 15.0, p = 0.0006). Subanalysis of the three groups indicated that the variant allele frequency was higher among the hypertensive patients (9.2%) than in normotensive males (2.9%; p = 0.007) or blood donors (3.0%; p = 0.001), while in the latter two groups it was similar (Table [1](#T1){ref-type="table"}). When frequencies of the individual gene variants in the hypertensive patients were compared to those in the two other groups (normotensive males and blood donors) combined, the βENaC i12-17CT variant was found to occur significantly more often among the hypertensive patients than in other groups (p = 0.001) whereas the differences in the prevalences of βENaC G589S (p = 0.15) and γENaC V546I (p = 0.14) did not reach statistical significance. Clinical characteristics of the variant allele carriers and non-carriers ------------------------------------------------------------------------ Clinical and laboratory data of the hypertensive patients grouped according to their carrier status of the three ENaC variants detected are summarized in Table [2](#T2){ref-type="table"}. There were no significant differences, associated with carrying a variant allele, in the sex, age or BMI of the hypertensive patients, nor their serum creatinine, lipid, potassium or sodium levels. Variant alleles did not seem to associate with cerebrovascular events or diabetes among the hypertensive patients, but small numbers prevent definitive conclusions (Table [2](#T2){ref-type="table"}). Our original study protocol was not designed to disclose health information of the two reference groups (normotensive males and healthy blood donors), and a similar comparison of variant allele carriers and non-carriers in these groups is therefore not feasible. Relation of the variant ENaC alleles to the activity of the renin-aldosterone system ------------------------------------------------------------------------------------ The dynamics of the circulating renin and aldosterone levels in most hypertensive individuals were studied during two challenge tests: during a supine-upright postural test and in response to captopril administration. Baseline plasma renin activity was very similar in the patients with and without variant alleles, whether investigated during the postural test or captopril administration (Table [3](#T3){ref-type="table"}). Plasma renin levels after attainment of upright posture (p = 0.11) and captopril administration (p = 0.12) were not significantly different among carriers and non-carriers of the ENaC variants (Table [3](#T3){ref-type="table"}, Fig. [3](#F3){ref-type="fig"}). Plasma aldosterone levels did not significantly vary according to the ENaC variant carrier status (Table [3](#T3){ref-type="table"}). We also analyzed renin responses (stimulated value minus baseline value) in the two tests according to the ENaC variant carrier status. We found some evidence of a blunted renin response to both postural (p = 0.21) and captopril (p = 0.087) challenge tests in carriers of variant alleles compared to non-carriers, but there was wide interindividual variation in the test results (Fig. [4](#F4){ref-type="fig"}). Use of covariates (urinary sodium excretion, age and BMI) did not cause significant changes in the results of these analyses. We next related the activity of circulating renin-aldosterone system to sodium-potassium homeostasis in ENaC variant carriers and non-carriers. Serum sodium and potassium concentrations in these two groups of hypertensive patients were similar (Table [2](#T2){ref-type="table"}). Urinary sodium excretion rate was not associated with the ENaC polymorphisms studied (data not shown). Urinary potassium excretion rates were not statistically significantly different in patients with (median, 83 mmol/day) and without (median, 79 mmol/day, p = 0.23) ENaC variants (Table [4](#T4){ref-type="table"}). However, when daily potassium excretion (dU-K, in mmol/day) was related to plasma renin activity (in μg/L/h), as mirrored by the renin levels during the postural challenge test, a significant difference was noticed: the median dU-K/renin ratios in the variant carriers vs. non-carriers were 114 vs. 92 when supine (p = 0.29) and 56 vs. 38 when upright (p = 0.034) (Table [4](#T4){ref-type="table"}); the corresponding values for the average (mean of supine and upright) dU-K/renin ratios were 74 and 51, respectively (p = 0.048) (Table [4](#T4){ref-type="table"}). A similar analysis of dU-K/plasma aldosterone ratios demonstrated higher ratios in female variant carriers vs. non-carriers for supine (p = 0.16), upright (p = 0.014) and the average values (p = 0.012), while no significant differences were seen in males. Collectively, these data suggests that hypertensive individuals carrying the ENaC variants tend to excrete increased amounts of potassium in relation to prevailing plasma renin and aldosterone levels. Molecular characterization of the ENaC variants ----------------------------------------------- We tested whether the βG589S or γV546I have any functional impact on ENaC expressed in Xenopusoocytes, the most commonly used expression system for ENaC functional studies. When αβγ ENaC subunits were co-expressed to obtain maximal channel activity, neither the βG589S nor γV546I affected ENaC activity as measured by the amiloride-sensitive Na^+^currents. In other words, these data indicate that the current carried by Na^+^ions through ENaC channels present at the cell surface is similar for ENaC wild-type and mutant channels, indicating that the βG589S and γV546I mutations have no detectable functional consequences on ENaC activity, at least when expressed in Xenopusoocytes. In order to clarify whether the C-T substitution at position -17 of the intron 12 of the βENaC could affect mRNA splicing, cDNA was synthesized from an RNA fraction prepared from lymphocytes of two hypertensive patients heterozygous for the βENaC i12-17CT mutation and a control individual without this gene variant. Primer pairs for reverse transcription were designed in a way permitting identification of a possible failure to splice intron 12 properly. Regardless of the primer pairs used, similar DNA fragments were generated from the samples of the βENaC i12-17CT carriers and control subject (data not shown). Furthermore, sequence analysis of the amplified DNA fragments revealed the presence of only normally spliced DNA sequence in the βENaC i12-17CT carriers. Furthermore, in silicoanalysis of the βENaC wild-type and variant DNA sequences suggested no differences in exon splicing. However, this analysis is only of predictive value and does not exclude a splicing defect introduced by the variant nucleotide in renal tissue. Discussion ========== The present study indicates that three common variants of the kidney epithelial sodium channel ENaC occur approximately three times more often in patients with moderate-to-severe essential hypertension compared to normotensive males and While direct in vitrostudies have failed to demonstrate a gain-of-function for these ENaC variants, their association with an increased urinary potassium loss in relation to existing plasma renin activity suggests that in the long run in vivothey may result in sodium retention, suppression of renin and aldosterone levels and hypertension. A large number of common and rare polymorphisms of the α-, β-and γENaC have been described in different populations (reviewed in \[[@B26]\] and \[[@B27]\]), but their pathophysiologic role, if any, has remained obscure, at least in the White populations. A systematic search in approximately 500 hypertensive probands, mostly of Caucasian origin, disclosed seven variants of the βENaC and six variants of the γENaC subunit, but no variant, with the possible exception of the βENaC G589S substitution, showed an increased ENaC activity in vitro, nor showed cosegregation with hypertension \[[@B24],[@B28]\]. The G589S variant was also identified in a Swedish hypertensive patient \[[@B25]\]. Two amino acid variants, βENaC G589S and γENaC V546I, both occurred with a frequency of about 2% in the hypertensive patients but in only 1% of the background population or normotensive males. The G589 is located in the poorly conserved cytoplasmic carboxyterminal portion of βENaC, 27 amino acids upstream of the functionally important PY motif. Persu et al. \[[@B24]\] identified the same substitution in a hypertensive female with mild hypokalemia and suppressed plasma renin activity. Using measurements of sodium channel activity and amiloride-sensitive sodium flux in Xenopusoocytes, these investigators were able to show a borderline 1.3 to 1.5-fold increase in activity for the G589S variant compared with the wild-type subunit. A similar trend was noticed in our experiments (Fig. [5](#F5){ref-type="fig"}). It remains possible that the functional expression of ENaC in Xenopusoocytes is not sensitive enough to detect subtle increases in ENaC activity, as it could well be the case for βG589S ENaC variant, and only mutations leading to large changes in ENaC activity are liable to be detected. On the other hand, even minute changes in ENaC may result in significant in vivoeffects when operating for decades under the influence of unfavorable living habits or variants of other modifier genes promoting salt reabsorption. Accordingly, the βENaC G589S could confer some susceptibility to low-renin hypertension, but more data on untreated patients and families are needed. The γENaC V546I substitution is located in the second transmembrane domain of the ENaC subunit, and it has not been described previously. Seven out of the eight carriers were females, and their renin and aldosterone levels were very similar to those in non-carriers. When expressed in vitroin Xenopusoocytes, this substitution did not result in an increase in sodium current (Fig. [5](#F5){ref-type="fig"}). It is not possible at present to deduce whether the V546I variant constitutes a pathophysiologically significant allele by itself or merely a genetic marker conferring susceptibility to hypertension. The C→T variant of the nucleotide -17 of intron 12 of βENaC is a novel one and, interestingly, it was present in 4.6% of the hypertensive patients but in only 1% of the 301 random blood donors (p = 0.009) and 175 normotensive males (p = 0.043). Patients with this variant allele displayed the lowest plasma renin levels and responses of all the subgroups examined (Table [3](#T3){ref-type="table"}, Fig. [4](#F4){ref-type="fig"}), but due to large interindividual variation the differences were not statistically significant. This βENaC variant may have remained undetected in earlier studies as they have mostly employed 5\'-PCR primers annealing at the region containing this substitution. Theoretically, a mutation at this site of an intron could affect RNA splicing. We explored this possibility by reverse transcription-PCR experiments of RNA samples from two variant carriers and a control individual, prepared from peripheral lymphocytes known to express βENaC \[[@B29]\]. We could not demonstrate a splicing error, but since homozygous individuals were not available for studies, we may have missed subtle changes. Furthermore, it is not known how well βENaC mRNA splicing in lymphocytes reflects the mechanism in kidney epithelial cells. Another possibility is that the DNA region around the variant nucleotide -17 of intron 12 contains interaction site for regulatory factors affecting transcription of βENaC in tubular cells, or the i12-17CT variant may be in linkage disequilibrium with some yet unidentified mutation present elsewhere in the βENaC or in the closely linked γENaC gene. The fact that we did not find hypokalemia or statistically significant suppression of renin levels in our patients with variant ENaC alleles does not abandon the hypothesis that they act as subtle genes conferring liability to sodium retention and hypertension during lifetime. In fact, even in cases with unequivocal Liddle\'s syndrome due to activating ENaC mutations the penetrance of disease phenotype is variable, with inconstant occurrence of hypertension, hypokalemia and suppressed renin levels from patient to patient \[[@B13],[@B30]-[@B32]\]. This suggests that Liddle\'s syndrome may represent an intermediate between single-gene and complex genetic diseases, necessitating the effect of extrinsic factors, such as substantial salt intake or other modifier genes, to complete the spectrum of syndrome manifestations. It is of particular note that molecular variants resulting in increased ENaC activity may occur outside the cytoplasmic PY motif that long was considered as a critical domain to be affected in Liddle patients \[[@B21],[@B33],[@B34]\]. Our present data are supported by findings of Rayner et al. \[[@B18]\] who recently discovered another βENaC variant (R563Q), which is located in the cytoplasmic domain just adjacent of the cell membrane and was found to be strongly associated with low-renin, low-aldosterone hypertension in a South African black population. Unfortunately, functional characterization of the R563Q variant was not carried out. Previously, another βENaC variant (T594M) was identified in the African Americans \[[@B35]\]. Although initially not linked to elevated blood pressure in the Blacks \[[@B35]\], subsequent studies in a London black population suggested a positive association with hypertension \[[@B16],[@B36]\]. The T594M substitution was reported to result in an increased responsiveness to a cAMP analog due to loss of protein kinase C inhibition of the ENaC \[[@B35],[@B37]\], but other studies have failed to show increased sodium currents in transfected cells \[[@B24]\]. An additional βENaC variant (G442V) present almost exclusively in Blacks has also been suggested to be associated with biochemical alterations compatible with increased ENaC activity in vivo \[[@B17]\]. Our present results and the previous data summarized above suggest that subtle β and γENaC variants do exist in the population that may variably result in elevated ENaC activity, suppression of plasma renin and aldosterone levels, urinary loss of potassium, and elevated blood pressure levels. Individual patients may variably manifest either only one or several of these features, and in some of the variant carriers these parameters may be entirely normal. It will be of interest to test the effectiveness of amiloride in our patients with the βG589S, i12-17CT and γV546I variants as an antihypertensive drug as this specific ENaC antagonist was shown to control blood pressure as well as increase plasma renin, aldosterone and potassium levels in black hypertensive individuals carrying the βT594M allele \[[@B38]\]. There are certain limitations in our study. First, our hypertensive patients represent a highly selected type of patients, since they were recruited by admittance to a specific center focusing on problems in conventional treatment. The clinical study protocol was initially designed for studies on screening for renovascular hypertension in a population, which explains the use of captopril test in the test panel. Unfortunately, urinary aldosterone levels, integrating aldosterone secretion rate over a longer observation period and serving as a valuable marker of Liddle\'s syndrome \[[@B13],[@B31]\], in particular when related to urinary potassium excretion levels \[[@B30]\],were not studied systematically. Our single-point plasma renin and aldosterone measurements may have been liable to incidental variations in their plasma levels, and prevent direct comparison to previous studies relying on urinary aldosterone assays. Second, our normotensive reference population comprised of male patients only. However, we had the advantage of picking up the extreme lowest end, as regards systolic and diastolic blood pressure levels in the absence of any antihypertensive drugs, from a very large material of more than 27000 individuals \[[@B20]\]. Third, due to ethical limitations of the study design, we did not have access to the clinical data of the subjects in the two reference groups (normotensive males and healthy blood donors); it would have been of interest to review the health data of ENaC variant carriers in these two groups. Fourth, our study had limited statistical power for several of the questions asked, particularly when either genetic variant was analyzed alone in carriers versus non-carriers. Therefore, for several of the questions asked in this study these variants may well have only modest effects, too small to be detected using the parameters of the present study. Fifth, pooling of the three genetic variants may represent an oversimplification, as it is uncertain whether these three variants exert similar effects on the various endpoints studied. Finally, our statistical analyses were not corrected for multiple comparisons and therefore some of the results observed in this study could represent chance findings rather than real phenomena. However, this is probably not the case for the observed increased frequency of ENaC genetic variants in hypertensive patients versus normotensive males and blood donors, because this was the primary hypothesis tested and the p values for these comparisons were 0.007 and 0.001, respectively. Conclusions =========== We have demonstrated that almost 9% of Finnish patients with hypertension admitted to a specialized center carry genetic variants of β and γ subunits of the kidney epithelial sodium channel ENaC, a percentage three times higher than that in the normotensive individuals or random healthy controls. Patients with the variant alleles tended to have suppressed renin levels and renin responsiveness to challenging stimuli, and they showed a significantly increased urinary potassium excretion in relation to their renin levels. It will be important to study whether carriers of ENaC variants respond favorably to ENaC blockers (amiloride and triamterene). Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= TH-H collected the clinical material, and participated in the DNA analyses and drafting of the manuscript. KK and TPH designed the study and drafted the manuscript. IT, TT, FF, KH designed the clinical chemical and hormonal assays, and participated in collection of the patient material. HF, HEM and KP participated in DNA analyses and bioinformatics. JV and TK collected the control populations and designed their studies. SS consulted in the statistical analyses and assisted in data handling. IG and LS carried out the electrophysiological studies and participated in drafting of the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2350/6/4/prepub> Acknowledgements ================ We thank Ms Tuula Soppela, Saara Nyqvist, Tarja Pajunen and Susanna Tverin for expert technical assistance. This work was supported by grants from the Finnish Academy, the Finnish Foundation for Cardiovascular Research, the Sigrid Juselius Foundation (to K.K.), and by a grant (3100-059217) from the Swiss National Science Foundation (to L.S.). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Flow diagram of the recruitment of the patients with primary hypertension. ::: ![](1471-2350-6-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Sequence analysis of the variant β and γENaC alleles.Chromatograms from both sequencing directions, nucleotide substitutions and predicted amino acid changes from three different hypertensive patients are shown. ::: ![](1471-2350-6-4-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Renin values in postural test and after captopril administration.Individual plasma renin activities at supine, upright, and in response to captopril administration (CCT; 60-minute values) in carriers and non-carriers of the three ENaC variant alleles. The horizontal bars indicate the median renin values in each group. ::: ![](1471-2350-6-4-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Renin responses in postural and captopril tests.Plasma renin responses (median and interquartile ranges) during the postural and captopril challenge tests (stimulated values minus baseline values in both cases) in carriers and non-carriers of the variant ENaC alleles. ::: ![](1471-2350-6-4-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Channel activity of βENaC G589S and γENaC V546I variants in vitro.Comparison of channel activity of hENaC wild-type, and the βhENaC G589S and γhENaC V546I variants, when expressed in Xenopusoocytes. ENaC activity was measured as amiloride-sensitive Na^+^current. Absolute currents were 5.09 ± 0.98 and 6.75 ± 1.23 μA for ENaC wt in the two series of experiments (number of oocytes given in parentheses). ::: ![](1471-2350-6-4-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### β and γ ENaC variants identified among the three study groups ::: Hypertension n (%) Normotensive males n (%) Blood donors n (%) Adjusted OR (95% CI)^1^ ------------------ -------------------- -------------------------- -------------------- ------------------------- All variants 32 (9.2) 5 (2.9)\\ 9 (3.0)\\ 3.1 (1.6--6.0) βENaC i12-17CT 16 (4.6) 2 (1.1)\* 3 (1.0)\\ 4.6 (1.6--13.0) βENaC G589S 8 (2.3) 2 (1.1) 3 (1.0) 2.4 (0.77--7.7) γENaC V546I 8 (2.3) 1 (0.6) 3 (1.0) 2.2 (0.63--7.5) Non-carriers 315 (90.8) 170 (97.1) 292 (97.0) \*P\<0.05 and \\P\<0.01 vs. Hypertension group. ^1^OR for hypertension (versus combined control groups) in ENaC variant carriers vs. non-carriers, adjusted for age and gender. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Demographic and clinical features of the hypertensive subjects, according to their ENaC variant status ::: Carriers of the ENaC variants Non-carriers -------------------------------- ----------------------------------- --------------------- --------------------- -------------- ------------- βENaC i12-17CT (n = 16) βENaC G589S (n = 8) γENaC V546I (n = 8) All (n = 32) (n = 315) Female/male (n) 9/7 4/4 7/1 20/12 166/149 Age (y) 49.4 ± 8.5 49.3 ± 5.8 50.5 ± 9.0 49.7 ± 7.8 49.3 ± 10.2 BMI (kg/m^2^) 28.0 ± 4.9 28.2 ± 4.8 27.9 ± 3.8 28.0 ± 4.5 27.4 ± 4.9 Serum creatinine (μmol/L) 88 ± 18.9 91 ± 12.8 85 ± 17.1 88 ± 6.7 88 ± 15.1 Serum uric acid (μmol/L) 329 ± 95.3 335 ± 65.6 386 ± 173.2 338 ± 87.1 340 ± 91.2 Fasting blood glucose (mmol/L) 5.3 ± 0.9 5.7 ± 1.1 5.5 ± 0.9 5.4 ± 0.9 5.6 ± 1.1 Serum cholesterol (mmol/L) 5.5 ± 0.8 6.0 ± 1.2 5.3 ± 0.7 5.6 ± 0.9 5.6 ± 1.0 Serum potassium (mmol/L) 4.1 ± 0.3 4.2 ± 0.4 4.2 ± 0.5 4.2 ± 0.4 4.1 ± 0.3 Serum sodium (mmol/L) 141 ± 2.4 140 ± 3.5 138 ± 2.2 140 ± 2.8 140 ± 0.3 Potassium supplementation (n) 1 0 2 3 (9.4%) 31 (9.8%) Cerebrovascular disorder (n) 1 0 0 1 (3.1%) 17 (5.4%) Diabetes (n) 1 1 1 3 (9.4%) 36 (11.4%) Gestational hypertension (n) 3 1 2 6 (18.8%) 44 (14.0%) Data for age, creatinine, uric acid, glucose, cholesterol, potassium and sodium is given as mean ± SD. Carriers of the ENaC variants did not differ significantly from non-carriers. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Plasma renin activity and serum aldosterone concentration during postural and captopril challenge tests ::: Carriers of the ENaC variants Non-carriers P-values ------------------------ ----------------------------------- ------------------ ------------------ ----------------- ----------------- ---------------------------------- βENaC i12-17CT βENaC G589S γENaC V546I All All variants versus non-carriers Postural test (n) 15 8 7 30 268 PRA, supine 0.7 (0.4--1.7) 0.7 (0.4--1.0) 0.6 (0.3--1.8) 0.7 (0.4--1.2) 0.8 (0.5--1.4) 0.37 PRA, upright 1.2 (0.8--2.3) 1.5 (0.7--2.9) 1.6 (0.3--2.4) 1.5 (0.7--2.4) 1.9 (1.0--3.5) 0.11 Aldosterone, supine 343 (225--398) 457 (251--592) 358 (238--622) 368 (243--482) 369 (273--474) 0.76 Aldosterone, upright 663 (353--1073) 725 (552--960) 1036 (585--1643) 761 (484--1129) 939 (584--1255) 0.21 Captopril test (n) 15 8 8 31 282 PRA, 0 min 1.2 (0.8--2.4) 1.3 (0.5--2.9) 1.3 (0.2--3.4) 1.2 (0.6--2.6) 1.4 (0.7--2.5) 0.53 PRA, 60 min 1.7 (1.0--6.2) 3.0 (0.5--5.7) 2.3 (0.5--8.2) 1.9 (0.8--5.7) 3.6 (1.3--7.7) 0.12 Aldosterone, 0min 554 (302--820) 608 (474--806) 645 (361--1312) 599 (340--845) 618 (431--877) 0.47 Aldosterone, 60min 400 (245--513) 400 (336--472) 356 (261--803) 392 (250--513) 397 (311--539) 0.41 Data is given as median (interquartile range). PRA, plasma renin activity (μg/L/h); Aldosterone, serum aldosterone concentration (pmol/L). Reference values: renin 0.9--2.0 (supine) and 2.0--5.0 (upright), and aldosterone 85--470 (supine) and 220--1000 (upright). ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Urinary potassium excretion and relation to plasma renin and aldosterone levels ::: Carriers of the ENaC variants Non-carriers P-values ------------------- ----------------------------------- --------------------- --------------------- ------------------- ------------------- ---------------------------------- βENaC i12-17CT (n = 14) βENaC G589S (n = 7) γENaC V546I (n = 5) All (n = 26) (n = 236) All variants versus non-carriers dU-K (mmol) 86 (68--112) 85 (66--120) 80 (70--96) 83 (68--102) 79 (65--94) 0.23 dU-K/PRA upright 54 (31--122) 57 (38--180) 97 (43--274) 56 (35--129) 38 (23--84) 0.034 dU-K/PRA mean 67 (37--168) 77 (55--252) 107 (59--321) 74 (46--170) 51 (31--118) 0.048 dU-K/Aldo upright 0.15 (0.06--0.25) 0.10 (0.07--0.15) 0.08 (0.06--0.25) 0.10 (0.07--0.21) 0.08 (0.06--0.14) 0.108 Data is given as median (interquartile range). dU-K, daily urinary potassium excretion; PRA, plasma renin activity (μg/L/h); Aldo, serum aldosterone concentration (pmol/L); PRA mean, average of supine and upright PRA. :::	PubMed Central	2024-06-05T03:55:52.410884	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547905/", "journal": "BMC Med Genet. 2005 Jan 20; 6:4", "authors": [ { "first": "Tuula", "last": "Hannila-Handelberg" }, { "first": "Kimmo", "last": "Kontula" }, { "first": "Ilkka", "last": "Tikkanen" }, { "first": "Tuula", "last": "Tikkanen" }, { "first": "Frej", "last": "Fyhrquist" }, { "first": "Karri", "last": "Helin" }, { "first": "Heidi", "last": "Fodstad" }, { "first": "Kirsi", "last": "Piippo" }, { "first": "Helena E", "last": "Miettinen" }, { "first": "Jarmo", "last": "Virtamo" }, { "first": "Tom", "last": "Krusius" }, { "first": "Seppo", "last": "Sarna" }, { "first": "Ivan", "last": "Gautschi" }, { "first": "Laurent", "last": "Schild" }, { "first": "Timo P", "last": "Hiltunen" } ] }
PMC547906	Background ========== TRAIL ([T]{.underline}umour necrosis factor [r]{.underline}elated [a]{.underline}poptosis [i]{.underline}nducing ligand) is one of the most promising anti-cancer agent being currently under investigation (for review see \[[@B1]-[@B5]\]). Initially it was shown that TRAIL specifically induces tumour cell apoptosis and tumour regression in nude mice, even when applied as single agent \[[@B6],[@B7]\]. In the meantime rare reports questioned the tumour cell specificity of TRAIL since it was shown that TRAIL induced apoptosis also occurred in normal human liver cells \[[@B8],[@B9]\]. However, subsequentially it was shown that the biochemical preparation of TRAIL rather than TRAIL itself was responsible for the observed toxic effect on hepatocytes \[[@B10]\]. This is strongly supported by the lack of toxicity of TRAIL receptor agonists in humans in first phase I studies \[[@B11]\]. One possible explanation for the tumour specifity of TRAIL could lie in its potential role as a mediator of tumour immune surveillance in vivo. In this regard it has been shown that mice lacking TRAIL display a significantly reduced capacity to eliminate syngenic tumour cells in the liver \[[@B12],[@B13]\]. Although TRAIL is a member of the death receptor ligand family certain differences exist to the well characterised ligands TNF ([T]{.underline}umour necrosis factor) or CD95. Most important in this regard is the fact that at least five, instead of only two resp. one, different TRAIL receptors have been identified: The proapoptotic DR4/Trail-R1 \[[@B14]\] and DR5/TRAIL-R2/TRICK2 \[[@B15]-[@B18]\] as well as TRAIL-R3/DcR1/TRID \[[@B16],[@B33],[@B20]\] and TRAIL-R4/DcR2/TRUNDD \[[@B21],[@B22]\], which lack any pro-apoptotic function. The latter were shown to protect cells from TRAIL induced apoptosis by competing with the agonistic receptors for TRAIL binding. In addition, TRAIL-R4/DcR2 is able to induce NF-κB activation, which might upregulate a wide array of anti-apoptotic proteins \[[@B19]-[@B21],[@B23],[@B24]\]. The role of the fifth TRAIL binding protein, the soluble osteoprotegerin (OPG) is still unclear, since it displays only low binding affinity to TRAIL at physiological temperatures \[[@B25],[@B26]\]. In analogy with the signalling events triggered by CD95, multimerisation of the agonistic TRAIL receptors induces the recruitment of the FADD adapter molecule to the receptor, leading to a subsequent autoproteolytic activation of initiator caspase-8 \[[@B27],[@B28]\]. Active caspase-8 in turn triggers the proteolytic activation of downstream caspases including caspase-3. Downstream caspases ultimately degrade a broad range of cellular proteins and apoptosis is finalized (for review see \[[@B29]\]). Up to now, caspase-8 was shown to be the most crucial mediator of TRAIL induced apoptosis \[[@B30],[@B31]\]. However, it has been shown that caspase-10 may act as a surrogate for caspase-8 in some cell systems \[[@B32],[@B33]\]. In contrast to receptor mediated apoptosis, DNA damage triggers apoptosis mainly via mitochondrial death pathways (for review see \[[@B34],[@B35]\]). Key step of mitochondrial apoptosis pathways is the mitochondrial release of pro-apoptotic mediators including cytochrome c. This release is generally controlled by a complex interplay of pro-apoptotic members of the Bcl-2 family namely Bax, Bak, Noxa and Puma. Activation of either of those molecules may occur directly via conformational changes \[[@B36]\] or transcriptional upregulation \[[@B37]-[@B39]\]. Cytochrome c released from the mitochondria triggering the activation of caspase-9 by association with APAF-1 in an ATP dependent manner \[[@B39],[@B40]\]. Caspase-9 subsequently activates the downstream effector caspase cascade including caspase-3 and, in analogy to receptor mediated apoptosis, cell death is finalised. With only very few exceptions \[[@B41]\], apoptosis induction via mitochondrial death pathways is abrogated by anti-apoptotic members of the Bcl-2 family. Anti-apoptotic proteins of the Bcl-2 family interfere with the cytochrome c release from mitochondria on multiple stages \[[@B42]-[@B44]\]. Interestingly, both (death receptor mediated and mitochondrial) pathways are interconnected on several levels. In case of death receptor activation, the propagation of the apoptotic signal is enhanced by caspase-8 mediated activation of Bid \[[@B46]\]. Bid like other BH3-only molecules triggers the release of cytochrome c from mitochondria ultimately resulting in activation of caspase-9 \[[@B45],[@B40]\]. Thus, receptor mediated death pathways are directly connected to mitochondrial death pathways. Vice versa, activation of caspase-9 via the mitochondrial pathway results in secondary activation of caspase-8 and Bid also leading to an amplification of the intracellular death signal \[[@B47]-[@B49]\]. Although TRAIL induces apoptosis when given alone, it has been shown that combination of TRAIL with cytotoxic drugs as 5-fluorouracil, etoposide, paclitaxel, actinomycin C and cisplatin has an even higher apoptotic efficacy \[[@B50]-[@B59],[@B6]\]. Whereas abundant data therefore support the combination of TRAIL with cytotoxic drugs, only limited studies of TRAIL combined with ionising radiation have been performed \[[@B31],[@B33],[@B60]-[@B62]\]. Except for breast and renal cancer, no data supporting the application of TRAIL in the field of radiation oncology are available. Up to now, statistical analysis of a synergistic efficacy have been presented rarely. In addition, potentially harmful effects of a combination on normal cells have not sufficiently ruled out. Methods ======= Chemicals --------- All biochemicals were obtained from Sigma-Aldrich chemicals (Deisenhofen, Germany) unless otherwise specified. Hoechst 33342 was purchased from Calbiochem and dissolved in distilled water as 1.5 mM stock solution. Cell culture ------------ The tumour cell lines MDA-MB 231, HCT-15, Colo 205, NCI H460, FaDu DD and SCC-4 cells were purchased from ATCC (Bethesda, MD, USA). HSF6/ HSF7 fibroblasts, HUVEC and SMC were kindly provided from H-P. Rodemann and R. Kehlbach (Tübingen, Germany). Respectively. All cells were grown in RPMI 1640 medium (Gibco Life Technologies, Eggenstein, Germany) and maintained in a humidified incubator at 37°C and 5% CO~2~. Normal human epithelial cells (HMEC, PrEC, RPTEC, SAEC) and hepatocytes were obtained from Clonetics/Cambrex (Taufkirchen, Germany). Cell culture was performed according to the manufacturer\'s protocols. TRAIL stimulation ----------------- TRAIL induced apoptosis was induced with recombinant human TRAIL/TNFSF10 (R&D Systems, Wiesbaden-Nordenstadt, Germany) in concomitant or sequential application with irradiation. Irradiation ----------- Cells were irradiated with 6 MV Photons using a Siemens Mevatron linear accelerator with a dose rate of 4 Gy per min at room temperature. Quantification of apoptosis induction ------------------------------------- Apoptosis induction was quantified by counting of cells with a characteristic apoptotic morphology after DNA staining with Hoechst 33342. Cells were stained by incubation with Hoechst 33342 at a final concentration of 1.5 μM for 15 min. Microscopy was performed using a Zeiss Axiovert 200 microscope (Carl Zeiss, Jena, Germany) using an excitation wavelength filter of 380 nm. All apoptotic rates were means of at least three independent experiments. The given error bars represent the standard error of the mean from independent measurements of the same cell batch. Westernblotting --------------- Cells (1 × 10^6^) were lysed for 30 min in a lysis buffer containing 25 mM HEPES, 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100, 10 mM EDTA, 10 mM NaF and 125 mM NaCl on ice. After removing insoluble material by centrifugation for 10 min at 12.000 g, 20 μg lysate was separated by SDS-PAGE. Blotting was performed employing a tank blotting apparatus (Biorad, Munich, Germany) onto Hybond C membranes (Amersham, Braunschweig, Germany). Equal protein loading was confirmed by Ponceau S staining (Sigma). Blots were blocked in PBS buffer containing 0.05 % Tween 20 and 5% bovine serum albumin at 4°C over night. Primary antibodies were detected after repeated washings with PBS/Tween 20 (0.05%) of the membrane, using a secondary antibody (anti IgG-AP 1:10.000, Santa-Cruz-Biotech, Heidelberg, Germany) diluted in PBS/Tween and incubated for 3 hours at room temperature and washed three times with PBS/Tween. Detection of antibody binding was performed employing enhanced chemoluminescence (CSPD^®^-Solution Tropix, Applied Biosystems, MA, USA). PARP cleavage was tested using a polyclonal antibodies for cleaved and uncleaved PARP from Boehringer (Mannheim, Germany) in a 1: 1000 dilution. Monoclonal antibodies for caspase 8 were a gently gift from Prof. K. Schultze-Osthoff and used in a 1:45 dilution. β-Actin (Santa Cruz, Heidelberg, Germany) antibody was used in a 1: 5000 dilution. Receptor expression ------------------- Cells (0,2 × 10^6^) were washed twice with PBS and incubated for 30 min with PE-labeled anti-R1/DR4 or -R2/DR5-antibody (R&D Systems, Wiesbaden-Nordenstadt, Germany) at a dilution of 1: 400 in 0,5% FCS/PBS. FACS analyses of superficial receptor expression was performed according to manufacturer\'s protocol with the Quantibrite™ kit from BD (Heidelberg, Germany). Statistical analysis -------------------- Efficacy of the combined modalities were evaluated by the isobolic method \[[@B63]\]. Results ======= Ionising radiation sensitises solid tumour cells to TRAIL induced apoptosis --------------------------------------------------------------------------- Rates of apoptosis induction in response to ionising radiation or TRAIL alone and after combination were determined. Since previous studies on Jurkat T cells or breast cancer cells demonstrated an upregulation of TRAIL receptor R2/DR5 after combined treatment with TRAIL and irradiation \[[@B54],[@B31]\] two different application schedules were tested. TRAIL was either applied directly after cell irradiation or 12 hours later, to allow for receptor upregulation. As shown in figure [1](#F1){ref-type="fig"}, ionising radiation alone (10 Gy) at 48 h induced apoptosis in all cell systems from e.g. 16,0 % in FaDu cells and 34,1% in NCI H460 up to 58,0 % in Colo 205 cells, whereas 0.1 ng/ml TRAIL had a very limited activity in FaDu (3,0%) and Colo 205 cells (14,5%) and up to 30,7 % in NCI H460 tumour cells. In contrast, combination of irradiation (10 Gy) with immediate TRAIL application (0,1 ng/ml) was associated with a much higher apoptotic response (e.g. FaDu 23,3 %, NCI H460 51,9% and Colo 205 80,3%). This effect was even more pronounced when TRAIL was applied 12 hours after irradiation (e.g. FaDu 30,0 %, NCI H460 65,2% and Colo 205 88,0%). In order to test whether the effect of TRAIL and radiation was additive or synergistic an isobologram analysis was performed. Even when applied concomitantly the interaction was synergistic in some cell systems (figure [2a](#F2){ref-type="fig"}, Colo205, HCT 15 and FaDu) but additive in others (MDA MB 231 and SCC-4). When TRAIL was applied 12 hours after irradiation the interaction was synergistic in all but one cell system (figure [2b](#F2){ref-type="fig"}). Processing of caspase 8 and PARP -------------------------------- In order to substantiate the findings on apoptosis induction, caspase activation was verified by analysis of caspase-8 and the processing of the caspase-3 substrate PARP 24 hours after TRAIL application. In keeping with the above results, the most prominent effects were found for Colo 205 and NCI H460 cells with strongly increased caspase-8 and PARP processing after combined treatment. Colo 205 cells were particularly sensitive to sequential application of irradiation and TRAIL, whereas less intensive caspase-8 and PARP processing was found for MDA MB231, SCC4 and FaDu (fig. [3](#F3){ref-type="fig"}). These data correlate well with the kinetics of apoptosis induction as determined by fluorescence microscopy of Hoechst stained cells (fig. [1](#F1){ref-type="fig"}). Irradiation induced regulation of TRAIL receptors ------------------------------------------------- In order to analyse the role of TRAIL receptor regulation in combined therapy with irradiation and TRAIL receptor expression was quantified by flow cytometry using the Quantibrite™ kit system from Becton Dickinson (Heidelberg, Germany). No upregulation of DR4/R1 was found in all tested tumour cell lines. Instead, a subtle downregulation of DR4/R1 after irradiation was observed (fig. [4A](#F4){ref-type="fig"}). Fig. [4B](#F4){ref-type="fig"} demonstrates upregulation of TRAIL receptor DR5/R2 12--18 h after irradiation with 10 Gy in four of six tested cell lines. Colo 205 cells showed the most pronounced receptor upregulation of 196,8%. In HCT-15 and NCI H460 cells an upregulation of R2/DR5 of 118,0% resp. 96,4% could be measured. In MDA MB231 cells and SCC-4 cells no significant upregulation of receptors was found. FaDu cells do not express TRAIL-receptor R2/DR5 and therefore no upregulation of R2/DR5 could be observed after treatment. Combined treatment of TRAIL and ionising radiation do not damage normal tissue ------------------------------------------------------------------------------ As stated above, hardly any data on normal tissue toxicity after combined treatment are available. We therefore analysed the effect of irradiation plus TRAIL on human hepatocytes, fibroblasts (HSF6 and 7), epithelial cells from prostata (PrEC), kidney (RPTEC), breast (HMEC) and small airways (SAEC), endothelial cells from umbilical cord (HUVEC) and small muscle cells (SMC). Normal tissue cells were treated with 10 Gy and a tenfold higher dosage of TRAIL (1 ng/ml) as used for tumour cells. For evaluation of apoptosis strict morphologic criteria as condensation of chromatin and nuclear fragmentation were used. 48 h after combined treatment no relevant sensitisation of normal tissue cells to TRAIL induced apoptosis could be detected in all cultures. Moreover, even preirradiation did not sensitise normal cells to TRAIL induced apoptosis as observed in tumour cells (table [1](#T1){ref-type="table"} and fig. [5](#F5){ref-type="fig"}). Discussion ========== Based on the rationale that radiation and TRAIL induce cell death via distinct but overlapping cell death pathways, tumour cell lines and normal tissue cultures were subjected to either radiation or TRAIL alone or combined with varying application schedules. Our data show that combining radiation with TRAIL induces apoptosis in a significantly higher percentage than either treatment alone. It is important to note that for TRAIL stimulation in our experiments on tumour cells very low concentrations of TRAIL were used (0.1 ng/ml). The pharmacodynamic properties of TRAIL, especially the peak plasma levels in humans, are not known. Since it is likely that toxicity rises with higher doses of TRAIL, the observation of pronounced effects on tumour cell kill at such low doses is particularly intriguing. Any combination of radiation with TRAIL proved to be more effective than either one alone; however depending on dose level and schedule of stimulation less than additive, additive and synergistic effects were detectable. When TRAIL was applied simultaneously with irradiation three of the six cell systems reacted synergistically. In contrast, five of the six cell systems reacted with synergistic effects when TRAIL was given sequentially 12 hours after irradiation. One of the cell lines displayed only less than additive effects. Statistical analysis confirmed, that synergistic effects are more pronounced and occur with greater likelihood after sequential application of TRAIL after irradiation when compared to concomitant treatment schedules. Possible explanations for the positive interaction of TRAIL and DNA damaging agents including ionising radiation are being disputed. Since the sensitisation was associated with upregulation of the DR5 receptor in some experimental settings \[[@B31],[@B54],[@B64],[@B68]-[@B70]\], it is thought that the synergy is based on the increased surface density of the death receptors. Our data support the notion that DNA damage leads to an increased surface expression of the DR5 receptor. However, no tight correlation between receptor upregulation and magnitude of cell kill was observed. It has been suggested, that intact p53 is essential for upregulation of R2/DR5 death receptor expression by ionising radiation \[[@B64],[@B68]\]. However, at least in one of our cell lines (HCT-15) known to harbour a non-function p53 DR5 upregulation was clearly upregulated. Thus, alternative p53 independent pathways for the upregulation of DR5 may exist. This finding is in accordance with data on p53 ^-/-^HCT-116 cells and mouse embryonic fibroblasts showing that NF-κB may also be important for a irradiation induced upregulation of death receptors \[[@B65]\]. Recently, it was shown in overexpression experiments of NF-κB that its subunits c-Rel and RelA regulate expression of cell death molecules in a differential manner. This suggests that RelA, in contrast to c-Rel, acts as a survival factor by inhibiting expression of DR4/DR5 and caspase-8 and up-regulating cIAP1 and cIAP2 \[[@B71]\]. This process depends also on cell type and microenvironment \[[@B54],[@B65]\]. Therefore NF-κB subunits seem to play an ambiguous role in regulation of apoptotic pathways. To know its exact role in radiation induced cell death further research is necessary. Additionally, conflicting data regarding the role of Bcl-2 in death receptor-mediated apoptosis have been provided in the past few years. Interestingly, new data point to a complex relationship between Bcl-2-mediated inhibition of apoptosis and the Bcl-2 protein expression level, the strength and the duration of the death receptor stimulus \[[@B66]\]. Therefore, Bcl-2 expression levels might play a critical role in the modulation of TRAIL sensitivity of tumour cells. Recently, it has been shown that the interaction of 5-FU with TRAIL is strictly Bax but not Bak dependent in HCT 116 cells \[[@B67]\]. Thus, these experiments suggest also a critical role for the proapoptotic Bcl-2 homolog Bax in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signalling cascade. However, the general mechanism of the positive interaction of TRAIL with irradiation remains unclear. It may ultimately turn out, that manifold mechanisms exist and only some mechanisms will be operative in a single cell system \[[@B68]\]. The second part of our experiments the toxicity of TRAIL combined with radiation on normal tissues. In order to be able to draw relevant conclusions for normal tissue cells \[[@B8]\], 10 fold higher doses of TRAIL were used in these experiments. The first important finding is that TRAIL did not induce apoptosis in any of our cell systems including human liver cells. Thus, our experiments confirm the high tumour cell specificity of TRAIL \[[@B6],[@B7]\]. Prior to embarking upon a phase I trial combining TRAIL with irradiation we wished to show a lack of sensitisation to TRAIL by pre-irradiation in normal tissues, compared to tumour cells. Using a wide array of normal cell systems we could not detect any effects of a pre-irradiation on TRAIL sensitivity. Combination of TRAIL with irradiation showed no increase in toxicity over radiation alone. These results are in good accordance with data from Shankar and coworkers showing that TRAIL induced apoptosis rates are only mildly enhanced by a preirradiation of non-malignant human prostate epithelial cells \[[@B68]\]. Conclusions =========== Our data suggest that TRAIL has great potential in cancer treatment, especially in sequential combination with radiotherapy. We did not observe any sensitising effect of the sequential treatment on TRAIL sensitivity in normal tissue cells. Xenograft experiments designed to answer the questions regarding the short and long term efficacy of a combination of radiation with either TRAIL or TRAIL specific antibodies are underway in our laboratory. Abbreviations ============= APAF, apoptosis protease activating factor; FADD, Fas-associated death domain protein; NFκB, nuclear factor κB; PARP, poly-ADP-ribosyl polymerase; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis inducing ligand. HMEC: human mammary epithelial cells, PrEC: human prostate epithelial cells, RPTEC: epithelial cells of renal proximal tubule, SAEC: small airways epithelial cells, HUVEC: human epithelial cells of umbilical vein, SMC: smooth muscle cells, HSF 6,7: human fibroblasts Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= PM performed FACS-analysis and microscopic evaluation of Hoechst stained cells, analyzed the data, participated in the conception of the trial and participated in the preparation of the manuscript. AS participated in microscopic evaluation of tumour cell apoptosis and accomplished analysis of the resulting data. VJ participated in receptor analysis and microscopic evaluation of Hoechst stained cells. HF carried out Western blotting. PTD participated on the preparation of the manuscript. WB performed isobologramm analysis. CB participated in the conception, design of the study, coordination of the study as well as preparation of the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/5/prepub> Acknowledgements ================ We thank the \"Deutsche Krebshilfe\" for continuous support; grant (10-1764 BeI) to C.B, P.M. W.B. Special thanks to Dr. Stephanie Halene for final revision of and helpful comments on the english manuscript. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Time course of induction of apoptosis in six solid tumour cell lines.Apoptosis was determined by microscopic evaluation of Hoechst stained cell nuclei 12 to 48 h after treatment with TRAIL 0,1 ng/ml and 10 Gy alone, and after simultaneous and sequential application of combined therapy. Data represent means of three independent experiments; bars ± SD ::: ![](1471-2407-5-5-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Isobolographic analysis of apoptosis induction after combined treatment.Apoptosis was determined by microscopic evaluation of Hoechst stained cell nuclei 48 h after combined treatment. Envelopes of additivity were calculated with data of 3 independently performed experiments. Datapoints below the curves resemble a synergistic effect, datapoints between the curves demonstrate an additive effect and above the curves a subadditive response of combined treatment. A : tumour cells were treated simultaneously with 10 Gy and 0,1 ng/ml TRAIL. B: cells were irradiated with 10 Gy 12 h prior to treatment with 0,1 ng/ml TRAIL. ::: ![](1471-2407-5-5-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Westernblot analysis of Caspase-8 activation and PARP-cleavage.Lysates were prepared as described in methods. In six tumour cell lines(Colo 205, NCI H460,, HCT-15, MDA MB 231, FaDu and SCC-4) caspase-8 and PARP cleavage was analysed in untreated cells (lane 1), 24 h after treatment with TRAIL 0,1 ng/ml (lane 2) and 10 Gy alone (lane 3), after simultaneous (lane 4) and sequential application (lane 4) of combined therapy. Every cell line shows a different cleavage pattern according to the rate of apoptosis induction. β-Actin staining was used as loading control. The mapped blots represent each one of three independently performed immunoblots. ::: ![](1471-2407-5-5-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Surface expression of TRAIL receptors after irradiation with 10 Gy in six tumour cell lines. Quantification of receptor expression was performed 6 to 48 h after irradiation by FACS analyis using the Quantibrite™ evalution system from BD(Heidelberg, Germany) according to manufacturer\'s instructions. Data shown are from one representative experiment (n ≥ 3). A: Cell surface expression of R1/DR4 B: Cell surface expression R2/DR5 ::: ![](1471-2407-5-5-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Microscopic evaluation of normal tissue cells.Lack of apoptosis was determined by microscopic evaluation of Hoechst stained cell nuclei. Micrographs depict cells of eight different normal tissues, 48 h after irradiation alone and after sequential treatment with 10 Gy 12 h previously to application of 1,0 ng/ml TRAIL. ::: ![](1471-2407-5-5-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Normal tissue toxicity. ::: Hepatocytes HMEC PrEC RPTEC SAEC HUVEC SMC HSF6 HSF7 ----------------------------------- ----------------- ----------- ----------- ----------- ----------- ----------- ----------- ---------- ---------- Control 0,3 ± 0,5 0,3 ± 0,5 0,3 ± 0,5 0 0,3 ± 0,5 0,2 ± 0,2 0,5 ± 0,2 0 0 TRAIL 1,0 ng/ml 0,3 ± 0,5 0,5 ± 0,4 0 0 0 0,2 ± 0,1 0,1 ± 0,1 0 0 10 Gy 0 0,5 ± 0,5 0 0 0,1 ± 0,2 0,3 ± 0,2 1,5 ± 0,5 0 0 Simultaneous combined therapy 0 1,6 ± 0,5 0,7 ± 0,5 0 0 0,3 ± 0,1 1,3 ± 0,9 0 0 Sequential combined Therapy 0,3 ± 0,5 0,8 ± 0,5 1,5 ± 1,0 0 0,3 ± 0,5 0,2 ± 0,2 1,2 ± 1,0 0 0 Cells were treated with 1,0 ng/ml TRAIL, 10 Gy, simultaneous combined therapy and with 10 Gy 12 h prior to TRAIL application. Microscopic evaluation of Hoechst stained cell nuclei with strict apoptotic criteria as chromatin condensation and nuclear fragmentation was performed 48 hours after treatment. Table shows percentage of apoptotic cells ± SD (n = 3). :::	PubMed Central	2024-06-05T03:55:52.415486	2005-1-14	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547906/", "journal": "BMC Cancer. 2005 Jan 14; 5:5", "authors": [ { "first": "Patrizia", "last": "Marini" }, { "first": "Angelika", "last": "Schmid" }, { "first": "Verena", "last": "Jendrossek" }, { "first": "Heidrun", "last": "Faltin" }, { "first": "Peter T", "last": "Daniel" }, { "first": "Wilfried", "last": "Budach" }, { "first": "Claus", "last": "Belka" } ] }
PMC547907	Background ========== Obsessive-compulsive disorder (OCD) is a prevalent, chronic and disabling disorder \[[@B1]\]. Controlled pharmacotherapy studies have established superiority of serotonin re-uptake inhibitors (SRI\'s) over noradrenaline reuptake inhibitors and over placebo in OCD and these currently form the cornerstone of pharmacotherapy management \[[@B2]\]. Despite the considerable advances made with the introduction of the SRI\'s into clinical practice, 40--60% of subjects still fail to respond adequately to initial therapy \[[@B3],[@B4]\]. From this it is clear that a need exists to pursue more effective treatments for those with OCD who fail to respond or respond inadequately to SRI\'s. To this end, preliminary evidence supports a role for the addition of atypical antipsychotics to SRIs in OCD. These agents combine serotonin-dopamine antagonism with the advantage of being well tolerated including a low potential for inducing motor side-effects. To date a number of open-label studies have suggested that augmenting SRI\'s with atypical antipsychotics is an effective strategy for treatment-refractory OCD. These include support for risperidone \[[@B5]-[@B7]\], olanzapine \[[@B8]-[@B13]\], and more recently amisulpride \[[@B14]\] and quetiapine \[[@B15]-[@B18]\]. A single open-label study using quetiapine as augmentation showed lack of effect in a small sample using low doses \[[@B19]\]. The outcome of the first controlled study in this area with the antipsychotic haloperidol demonstrated preferential benefit for refractory OCD subjects with co-morbid tic disorder \[[@B20]\]. In two subsequent studies the efficacy of risperidone in SRI refractory OCD has also been reported \[[@B21],[@B22]\]. Interestingly the former study \[[@B21]\], did not replicate the particular advantage for subjects with co-morbid tic disorder. Efficacy has also been shown for quetiapine \[[@B23]\] and olanzapine \[[@B24]\] using similar designs, but the effects on co-morbid tic disorders were not reported. In contrast a recent controlled study using olanzapine failed to demonstrate efficacy over placebo in a six week study \[[@B25]\]. Despite some mixed evidence in this area, in general the available literature appears to support the use of relatively short trials with low doses of antipsychotic agents as augmentation to SRIs. Quetiapine has a particularly interesting profile in that it is the only available antipsychotic with significant 5-HT1D effects and this serotonin receptor subtype has been implicated in OCD \[[@B26],[@B27]\]. Our objective was to examine the effects of quetiapine augmentation in subjects with OCD who had failed to respond adequately to a 12 week trial of an SRI, employing a double-blind, placebo-controlled, six week study design. Methods ======= Patients -------- Forty two subjects aged 18--65 years inclusive were recruited in our multi-centre study comprising five sites in South Africa and one in Canada. Recruitment took place between May 2002 and November 2003. Prior to commencement, all sites in the study received approval from their relevant Research Ethics committees/Institutional review boards and regulatory authorities. All subjects provided written informed consent prior to the commencement of any study-related procedures. Diagnosis was confirmed using the MINI Neuropsychiatric Interview (Version 5.00, 1998) \[[@B28]\] to ensure compatibility with the Diagnostic and Statistical Manual for Mental Disorders, Fourth Edition (DSM-IVTR)\[[@B29]\] criteria for OCD. Subjects with any co-existing Axis I disorder were excluded unless the co-morbid condition was deemed to be secondary to the OCD. Female subjects of childbearing potential were required to use adequate contraception and were not permitted to breastfeed while on the study. Subjects were excluded if they suffered from unstable medical conditions including renal or hepatic insufficiency, epilepsy or had suffered previous brain injury or undergone brain surgery. Taking medication that was deemed likely to interact with quetiapine or any other psychoactive substance was grounds for exclusion. Study Design ------------ All subjects were treated and monitored by investigators for the minimum twelve week duration of SRI-alone treatment phase before inclusion into this study. This was to ensure that patients met criteria for duration of SRI treatment which included at least 6 weeks on the maximum tolerated dose of the relevant SRI. Sample size calculations were based conservatively on similar work in this area \[[@B20],[@B21]\]. Accordingly, for the primary outcome variable (YBOCS), clinically meaningful differences between treatment groups of 6.67 with a standard deviation (SD) of 6 would be detected with a power of 80% at a 5% significance level with a sample size of 14 in each of the treatment groups. The larger sample recruited reflects the anticipation of a 33% drop-out rate in the double-blind treatment phase. A double-blind, randomised, parallel-group six-week augmentation with quetiapine or matching placebo of the SRI to which participants had not responded adequately, was undertaken. Specific SRI\'s, mean doses and dose range are provided in Table [1](#T1){ref-type="table"}. Non-responsiveness to an SRI was defined as either an improvement score on the clinical global impression scale of minimally improved (3) or worse (4,5,6), or less than 25% reduction in Yale Brown Obsessive-compulsive score following twelve weeks of treatment. Inadequate response, as defined above, to at least one SRI administered for a minimum of 12 weeks of which 6 weeks was either at the maximum tolerated dose or alternatively the manufacturer\'s recommended maximum daily dose. SRI doses were maintained at the same level throughout the double-blind treatment phase. For assignment to either quetiapine or placebo groups, we used a computer generated randomization schedule supplied by the sponsoring pharmaceutical company which also packaged the medication. This procedure ensured blinded, balanced allocation to each treatment group across all the study sites. All investigators remained blind to this schedule until closure of the study. No incidents requiring investigators to break the blind occurred through the course of the study. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### SRI\'s used by subjects for failed treatment trial prior to inclusion in the study. ::: Drug N Mean maximum tolerated dose (mg/day) Median daily dose (mg/day) Range -------------- ------- ------------------------------------------ -------------------------------- ----------- Fluoxetine 13 60 60 60 Citalopram 10 61 60 10 Paroxetine 4 65 60 20 Fluvoxamine 10 290 300 100 Sertraline 1 200 200 0 Clomipramine 2 250 250 0 ::: Treatment --------- At baseline participants were randomly allocated to receive treatment with either quetiapine or placebo using a computer generated schedule and numbered dispensing wallets. A flexible dosing schedule was initiated at 25 mg per day for one week and then doubled weekly to the start of week 4. Based on Clinical Global Impression of Improvement (CGI-I) scores of minimally improved or worse, clinicians were permitted to increase the dose to a maximum of 300 mg per day for the final two weeks of the study. In addition to clinical measures of improvement, clinicians also considered patient tolerability in their decision to adjust doses. Following completion of the treatment phase, subjects were withdrawn from study medication while continuing their SRIs. All subjects were then followed up for any adverse effects. Ratings ------- Patients were assessed by clinicians at baseline and on completion of weeks 2, 4 and 6. Telephonic assessments were performed on completion of weeks 1 and 3. Symptoms of obsessive-compulsive disorder were measured by the same clinician where possible at all study visits using the Yale Brown Obsessive Compulsive Scale YBOCS) \[[@B30],[@B31]\]. A global assessment of severity and improvement was made by clinicians at all assessment points using the Clinical Global Impressions scale of Severity (CGI-S) and Improvement (CGI-I) \[[@B32]\]. Depression was rated using the 10-item Montgomery-Asberg Depression rating scale (MADRS) \[[@B33]\]. For a measure of patient-rated disability we used the Sheehan Disability scale (SDS) \[[@B34]\]. For subjects with tics, frequency and severity were rated using the Yale Global Tic Severity Scale (YGTSS) \[[@B35]\]. Our primary outcome measures for OCD symptoms were (1) the change in YBOCS score from baseline to endpoint and (2) the clinical global impression of improvement (CGI-I) at endpoint. In the final analysis, treatment response was defined as a 25% or greater reduction in YBOCS score and a CGI-I of 1 (very much improved) or 2 (much improved) from baseline to endpoint. Secondary outcome measures included the MADRS, SDS and YGTSS (in subjects with co-morbid tics). Statistical analysis -------------------- Thirty-nine of the forty-two randomised subjects successfully completed the six-week treatment phase. Two subjects withdrew from the study prematurely (Week 1 and Week 4) due to severe levels of sedation. In both of these cases at least one week of study medication had been taken and at least one post-baseline clinical assessment was completed. Both of these subjects were included in the final analysis using data from the last observation carried forward (LOCF). The single subject not included in the efficacy analysis completed the study, but was found not to have correctly fulfilled the study definition of treatment refractoriness and was excluded. Twenty subjects were allocated to the quetiapine arm and twenty-one to the placebo arm. Student\'s t-tests were used to determine any baseline differences in the groups for age, gender, number of previous trials of SSRI\'s, severity of symptoms in relation to OCD, depressive symptoms, and CGI-S. Analysis of variance was undertaken with group and tics as factors. All tests were two-tailed with p-values of less than 0.05 considered significant. Results ======= Study sample characteristics ---------------------------- For the final analysis, our sample comprised 19 men and 22 women. Baseline characteristics of two treatment groups did not differ with respect to age (years) (quetiapine group 33.8(SD 9.66), placebo group 31.81 (SD 12.14); p = 0.57), gender (p = 0.29), number of previous adequate SRI trials (quetiapine 1.55 (SD 1), placebo 1.62 (SD 1.02) (p = 0.83), baseline severity of OCD (CGI-severity, p = 0.47; YBOCS, p = 0.33), depressive symptoms (p = 0.91), patient-rated disability (p = 0.28) or the presence (n = 11, p = 0.66 and severity (p = 0.87) of co-morbid tics. Treatment outcomes ------------------ For the primary outcome measure of severity (YBOCS), quetiapine (p \< 0.0001) and placebo (p = 0.001) augmentation of an SRI significantly improved symptoms of OCD. However quetiapine did not demonstrate significant benefit over placebo at the end of the six-week treatment period (F = .19; p = .636) (Figure [1](#F1){ref-type="fig"}). The mean reduction in YBOCS scores for the combined group was 7.15 points (quetiapine = 7.10; placebo 7.19). Forty percent (n = 8/20) of subjects on quetiapine were classified as responders (YBOCS reduction of \>25% from baseline and CGI-improvement score of 1 or 2) while 47.6% (n = 10/21) of subjects on placebo were classified as responders. A higher number of previous SRI trials for the each treatment group did not correlate with the degree of change on the YBOCS or the response status. Table [2](#T2){ref-type="table"} provides details of individual subject SRI doses, baseline clinical severity ratings and response status. Table [3](#T3){ref-type="table"} provides a summary of baseline and change scores for each of the primary and secondary outcome variables. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### YBOCS change for treatment groupsQuetiapine and placebo groups improved significantly, without significant between group differences (F = 0.19; p = 0.636) ::: ![](1471-244X-5-5-1) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Baseline characteristics of treatment groups ::: Treatment Group SRI baseline SRI Dose baseline (mg/day) Previous SRI trials Total YBOCS -- Baseline Total YBOCS -- Week 6 Endpoint dose (mg/day) % CHANGE CGI -- Improvement Response status ----------------- ---- ------------------ -------------------------------- ------------------------- ----------------------------- --------------------------- ---------------------------- -------------- ------------------------ --------------------- Quetiapine 1 Paroxetine 60 1 33 29 50 -12.00 3 N/R 2 Citalopram 60 2 25 23 200 -8.00 4 N/R 3 Fluvoxamine 300 1 32 27 300 -16.00 3 N/R 4 Citalopram 70 1 27 25 25 (E/W)\* -7.00 4 N/R 5 Clomipramine 250 2 22 27 100 23.00 5 N/R 6 Paroxetine 60 5 35 32 300 -9.00 4 N/R 7 Fluoxetine 80 3 25 16 300 -36.00 2 R 8 Sertraline 200 1 18 3 50 -83.00 1 R 9 Fluoxetine 20 1 21 20 300 -5.00 4 N/R 10 Fluoxetine 60 1 22 17 300 -23.00 2 N/R 11 Citalopram 60 1 32 17 300 -47.00 1 R 12 Fluoxetine 80 1 32 14 300 -56.00 1 R 13 Fluvoxamine 300 1 30 12 50 -60.00 1 R 14 Fluoxetine 60 1 25 7 50 -72.00 1 R 15 Citalopram 60 1 27 24 200 -11.00 4 N/R 16 Fluvoxamine 300 1 24 12 150 -50.00 2 R 17 Fluvoxamine 200 2 22 22 50 .00 4 N/R 18 Fluvoxamine 300 2 25 22 25 -12.00 4 N/R 19 Fluoxetine 60 2 24 25 25 (E/W)\* 4.00 4 N/R 20 Clomipramine 250 2 27 12 300 -56.00 2 R Placebo 1 Fluvoxamine 300 2 32 25 300 -22.00 3 N/R 2 Fluvoxamine 300 1 30 28 300 -7.00 3 N/R 3 Fluoxetine 80 2 34 38 300 12.00 4 N/R 4 Paroxetine 60 1 22 18 300 -18.00 1 N/R 5 Citalopram 60 1 28 26 300 -7.00 4 N/R 6 Fluoxetine 60 5 26 24 300 -8.00 3 N/R 7 Fluoxetine 60 1 23 23 300 .00 4 N/R 8 Fluoxetine 60 1 27 10 300 -63.00 1 R 9 Fluoxetine 40 1 32 18 300 -44.00 2 R 10 Citalopram 60 2 24 23 300 -4.00 4 N/R 11 Citalopram 60 1 35 23 300 -34.00 2 R 12 Fluvoxamine 300 3 22 14 300 -36.00 2 R 13 Citalopram 60 1 28 10 50 -65.00 2 R 14 Fluoxetine 60 1 28 4 50 -86.00 2 R 15 Citalopram 60 1 26 19 100 -27.00 2 R 16 Fluoxetine 60 1 27 12 100 -56.00 1 R 17 Fluoxetine 20 1 26 18 200 -31.00 2 R 18 Fluvoxamine 300 2 23 35 200 52.00 6 N/R 19 Citalopram 60 2 26 9 100 -65.00 2 R 20 Paroxetine 80 1 32 29 300 -9.00 3 N/R 21 Fluvoxamine 300 3 31 25 100 -19.00 2 N/R \*E/W = Early withdrawal ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Summary scores (baseline) and change scores for primary and secondary outcome variables. ::: Quetiapine Placebo -------------------------- ---------------- ---------------- YBOCS (baseline) 26.4 (SD4.6) 27.7(SD3.9) YBOCS (change at week 6) -7.1(SD7.2) -7.2(SD8.4) YBOCS % change -26.9% -26% CGI-Severity (baseline) 5.2 (SD0.8) 5.3 (SD0.8) CGI-Severity (week 6) 4.1 (SD1.4) 4.1(SD1.5) MADRS (baseline) 10.6 (SD 4.8) 10.71 (SD 9.8) MADRS (change at week 6) -2.6 (SD 6.5) -3 (SD 8.3) SDS (baseline) 17.9 (SD 5.3) 19.6(SD4.7) SDS (change at week 6) -5.3(SD5.6) -6.1 (SD4.8) YGTSS (baseline) 24.7 (SD 19.3) 22.6(SD 22.3) YGTSS (change at week 6) -4.5(SD 5.1) -9.4 (SD 14.6) YGTSS % change -18.2% -41.6% ::: Of the 11 subjects with co-morbid tics, six were randomised to quetiapine. Endpoint data was missing for one subject on quetiapine. Of the remaining 10 subjects, 3 (quetiapine n = 2 (33%); placebo n = 1(20%)) were classified as YBOCS responders. The reduction in the YGTSS did not differ significantly between treatment groups with tics (quetiapine -4.5, placebo -9.4; F = 2.8, p = .46). Severity ratings for depressive symptoms (MADRS) were low at baseline (mean 10.6, SD 4.8), showed little change over the study period, and at week 6 remained similar for both groups (quetiapine = 8.2, SD 4.8; placebo = 7.7, SD 6.1). The mean daily dose at week 6 for the quetiapine group was 168.75 mg (SD 120.82) compared to 228.57 mg (SD 99.46) per day for those on placebo. Quetiapine responders (187.5 mg, SD 124.6) did not differ significantly from quetiapine non-responders (156.25 mg, SD 122.1) in their mean daily dose at Week 6 (p = .585). Furthermore, within the quetiapine group, participants receiving ≥ 200 mg/day (10/20 at week 6 demonstrated non-significant differences (F = 6.837, p = .988) and a marginally lower percentage reduction in YBOCS at endpoint (26.7%, SD 20.34) compared to those receiving a dose ≤ 200 mg/day (26.9%, SD 36.24) at endpoint. Tolerability ------------ Quetiapine was generally well tolerated and no serious adverse events (SAE\'s) were reported through the course of the study period. Two patients on quetiapine withdrew from the study due to severe sedation (Week 1 and Week 4) that was judged to be drug related. Otherwise adverse events were in the mild to moderate range and were mostly self-limiting. No subjects on placebo withdrew from the study. Table [4](#T4){ref-type="table"} provides a list of the adverse events and their frequencies in the respective study groups. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Percentage of subjects for each treatment group reporting adverse events ::: Adverse event Quetiapine (%, n) Placebo (%, n) ------------------------ ------------------- ---------------- Sedation 75% (15) 33.3%(7) Dry mouth 15% (3) 0 Headache 15% (3) 38% (8) Fatigue 15% (3) 19% (4) Irritability 10% (2) 4.7% (1) Impaired concentration 10% (2) 0 Dizziness 5% (1) 14.3% (3) Nausea 5% (1) 9.5% (2) Increased appetite 5% (1) 9.5% (2) Delayed ejaculation 5% (1) 0 Weight gain 5% (1) 0 Worsening mood 5%(1) 4.7%(1) Memory difficulties 5%(1) 0 Muscle aches 5%(1) 0 Abdominal tenderness 5%(1) 0 Slurred speech 5%(1) 0 ::: Discussion ========== Our findings indicate that both quetiapine and placebo significantly reduced symptoms in subjects with OCD who had failed to respond adequately to 12 weeks of an SSRI and, that the difference between groups was not significant. Similarly in the subgroup with co-morbid tics, no preferential benefit was noted for quetiapine. Interestingly, the high placebo response was similar to that seen in a recent failed controlled trial of olanzapine \[[@B25]\], but stands in contrast to the positive studies in this area in which low placebo response rates were seen when demonstrating efficacy of quetiapine \[[@B23]\], risperidone \[[@B21]\], and olanzapine \[[@B24]\]. It is likely that features of study design or specific study population characteristics may have contributed to this finding and these are discussed below. First, the duration of a therapeutic trial of an SRI prior to augmentation with an antipsychotic should be of adequate dose and duration. In our study the majority of participants had failed only the single trial of an SRI on which they continued during the study (63.4% mean 1.59). Notably only six weeks of this treatment was required at the maximum tolerated dose. Despite the notion that an optimum trial of pharmacotherapy in OCD is 12 weeks, it may be argued that higher and ultimately effective doses of an SRI had not been maintained for an optimum duration prior to randomization. Given that therapeutic doses of SRIs in OCD are usually on the upper end of the dose range, it seems feasible that the high placebo response rate may reflect a response to SRI\'s once they had been administered at these higher doses for the additional six weeks of the study. It seems possible that the recent study by Shapira et al \[[@B25]\] may have been impacted by similar factors. In a second and related point; the number of previous SRI trials in the subgroup receiving quetiapine did not predict a poorer response to treatment. This effect is probably related to the lack of statistical power to detect these differences in a group in which the low number of previous SRI trials was a distinguishing characteristic. Certainly, previous positive studies in this area have used relatively more refractory groups based on the number of previously failed SRI trials. Taken together with the first point above, we suggest that future work in this area should consider longer periods at maximum tolerated doses of SRI\'s prior to categorisation of subjects as treatment refractory. Third, the use of a slow up-titration resulted in a relatively low mean daily dose being administered for the majority of the study. For instance, a mean daily (week 6) dose of 168 mg/day in the quetiapine group (median 175 mg) had only been achieved for the final two weeks of the study. These doses are comparably low to those used in the negative single-blind study using low dose quetiapine by Sevincok et al \[[@B19]\]. In contrast the positive study using quetiapine by Denys et al \[[@B23]\] employed a more rapid up-titration and a fixed-dose design. This meant that subjects were exposed to 200 mg daily doses that were generally well tolerated, from the start of week 3. The authors of this study were able to show significant YBOCS differences between groups from the end of week 4. Similarly Mc Dougle et al \[[@B21]\], using risperidone, began treatment on 1 mg per day for one week and permitted weekly 1 mg incremental increases for 6 weeks. They found that by the beginning of week 2, most subjects were on or around the mean daily dose for treatment responders (2.2 mg). Despite the significant improvement in the quetiapine group demonstrated in our study, the apparent lack of benefit of doses higher than 200 mg per day may seem surprising, however, we cannot rule out the possibility that administering these higher doses for an adequate duration would have changed the outcome. In addition it must be noted that in our study the quetiapine group reported high rates of sedation (n = 15, 75%) and a 10% (n = 2) rate of premature withdrawal was experienced. As such it seems likely that a more aggressive up-titration schedule might have resulted in even higher rates of withdrawal. By comparison, rates of sedation were equally high, but did not appear to restrict use of the more rapid up-titration in the study by Denys et al \[[@B23]\]. Certainly evidence of efficacy using lower doses has been demonstrated in studies of 6 and 8 weeks duration \[[@B21],[@B23],[@B24]\], and it seems that therapeutically adequate doses should probably be reached earlier than week 4 in a 6 week study. Fourth, the impact of repeated clinical assessments and rating of relatively small changes in clinical severity combined with regular dose increases, may conceivably have increased the placebo response rates resulting from increased optimism, a tendency to over-report improvements and belief that higher doses are more likely to be more effective than lower doses. This may be particularly true for the placebo-treated group that were considerably less likely to report sedation as an adverse event and as such were more likely to have their treatment dose increased at each visit. Our results differ, with respect to placebo response, from a considerable literature that suggests a consistently lower placebo response rate in treatment trials in OCD than in other mood and anxiety disorders. While we believe that the reasons (1--3) discussed above probably provide the main reasons for our finding, the impact of repeated assessments and the potential effect thereof cannot be entirely discounted. Conclusions =========== Despite significant improvement in each of the study groups, response to quetiapine augmentation in SRI non-responders, failed to separate from placebo treated subjects at the end of the six week treatment phase. A number of limitations in study design make comparisons with previous studies in this area difficult and probably contributed to our negative findings. Future work in this important clinical area should address these limitations. Competing interests =================== Dr. Stein has received research grants and/or consultancy honoraria from Astrazeneca, Eli-Lilly, GlaxoSmithKline, Lundbeck, Orion, Pfizer, Pharmacia, Roche, Servier, Solvay, Sumitomo, and Wyeth. Authors\' contributions ======================= PDC drafted manuscript and analysed data, BV protocol design, SS protocol design, JEM drafting of manuscript, statistical design, data acquisition, MVM protocol design, DJS principal investigator, protocol design, drafting of manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-244X/5/5/prepub> Acknowledgements ================ This study was funded by Astra Zeneca The authors wish to acknowledge the contribution of the participating investigators: Dr\'s, Maud, Mohr, Nel, Potgieter, Russouw and Verster	PubMed Central	2024-06-05T03:55:52.417324	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547907/", "journal": "BMC Psychiatry. 2005 Jan 24; 5:5", "authors": [ { "first": "Paul D", "last": "Carey" }, { "first": "Bavanisha", "last": "Vythilingum" }, { "first": "Soraya", "last": "Seedat" }, { "first": "Jacqueline E", "last": "Muller" }, { "first": "Michael", "last": "van Ameringen" }, { "first": "Dan J", "last": "Stein" } ] }
PMC547908	Background ========== Sprains of the lateral ligaments of the ankle joint account for between 3 and 5% of all emergency department (ED) attendances in the UK \[[@B1]\], with approximately 5600 injuries each day \[[@B2]\]. The injury is painful and incapacitating, and, for all but the most minor injuries, weight bearing is difficult to tolerate. Activities of daily living can be significantly compromised in the first few weeks, and although acute symptoms resolve, persistent symptoms are reported to occur in between 30 to 50% of people \[[@B3]-[@B6]\]. Persistent symptoms include recurrent sprains, instability, swelling, unsightly appearance and pain. Crichton has developed a classification of severity of ankle injuries \[[@B7]\]. • GRADE I -- the ligament is stretched but not torn and the anterior talofibular ligament is usually involved. The anterior draw test is negative. • GRADE II -- the ligaments are partially torn; laxity may be present and there is moderate swelling. • GRADE III -- complete rupture of the ligament resulting in joint instability. The anterior draw test is positive. The focus of this trial is Grade II and III sprains (referred to as severe sprains from this point). Severe sprains have poorer prognosis, and necessitate more intensive management than Grade I sprains (minor sprains). Minor sprains are considered to be self-limiting and require minimal treatment. Recent systematic reviews have highlighted the lack of high quality evidence to support clinicians in managing severe sprains \[[@B2],[@B7]\]. There are few reliable studies describing long-term outcome. Most trials required x-ray evidence of talar tilt or an arthrogram for inclusion and are, therefore, not generalisable to clinical practice in the ED. Current practice ranges from no intervention, physiotherapy, and different types of mechanical support to surgical repair of the ligaments. A recent UK survey of 83 EDs demonstrated the most popular treatments were ice, elevation, Tubigrip^®^support stocking and exercise, each of which was reported as being used in most cases by over 70% of respondents \[[@B8]\]. In addition, over half of responding departments reported that crutches, early weight bearing, and non-steroidal anti-inflammatory drugs were used in most cases. Follow up was used only in selected cases. Practice has been gradually changing from immobilisation of the injured joint to early mobilisation. To aid mobilisation the use of mechanical supports has been suggested. These supports vary in the amount of ankle movement they allow, but all encourage ankle flexion/extension and aim to minimise inversion/eversion that theoretically reduces the risk of further ligament injury. Movement is also proposed as a way of retraining the ankle proprioception which may reduce recurrent injury rate \[[@B9]\]. Other hypothesised benefits of mechanical supports are early restoration of functional mobility, rapid return to usual activities, reduction of pain because the joint is stabilised and protected, and improved quality of life. However, there is also the possibility of discomfort and inconvenience owing to restriction of joint range, and delayed healing, skin and circulatory problems. The present study has two aims 1\. To estimate the clinical effectiveness of three different methods of mechanical support (below knee cast, Aircast^®^ankle support and Bledsoe^®^boot) in comparison to Tubigrip^®^in a\. The recovery of mobility (primary outcome) b\. The recovery of usual occupation c\. Avoidance of residual symptoms including recurrent instability, lasting limitation of physical activity, and need for further medical, rehabilitation or surgical treatment. 2\. To measure the cost of each strategy, including treatment and subsequent health care costs. The NHS National Co-ordinating Centre for Health Technology Assessment has guided the selection of treatments. Tubigrip^®^has been chosen as the reference treatment because it is the cheapest and one of the most commonly used \[[@B10]\]. The Bledsoe^®^boot is lightweight and incorporates a novel design to promote ease of walking. However it is considerably more expensive, and it\'s clinical and cost effectiveness is yet to be proven. The below knee cast is Scotch^®^Cast is commonly used for casting in the NHS \[[@B9]\]. There is a range of lightweight mechanical supports available, and we have selected the Aircast^®^Brace. Methods ======= This pragmatic randomised controlled trial is being run in 6 trusts (covering eight hospitals) across the UK. The trusts are: Birmingham Heartlands and Solihull NHS Trust, North Bristol NHS Trust, Oxford Radcliffe Hospitals NHS Trust, South Warwickshire General Hospitals NHS Trust, University Hospitals Coventry and Warwickshire NHS Trust and the Worcestershire Acute Hospitals NHS Trust. Figure [1](#F1){ref-type="fig"} provides an overview of the trial method and patient journey. Multi-centre Research Ethics Committee approval has been awarded by the Northern and Yorkshire Regional Office and local ethical and research governance approvals have been obtained. Informed consent to participate in the trial and allowing researchers to access hospital records is obtained from all participants. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Study design ::: ![](1471-2474-6-1-1) ::: Study population ---------------- The target population is people attending the ED with a severe sprain of the lateral ligament complex of the ankle. Inclusion criteria ------------------ People who attend with sprain of the ankle who are unable to weight bear, aged 16 years and older, and give informed consent. Weight bearing is used as a proxy for severe sprains as clinical grading is not possible in the acute phase \[[@B7]\]. Flake fractures (\<=2 mm) of the lateral malleolus are included as they are normally treated as soft tissue injuries \[[@B11]\]. Exclusion criteria ------------------ Age less than 16 years old, ankle or other fracture sustained in addition to the sprain (such as to the wrist, head etc). An x-ray is used to exclude fracture where this is clinically indicated and as guided by the Ottawa guidelines \[[@B12]\]. People who can weight bear and those with fractures are ineligible for the trial, and are managed in accordance with normal local practices. Age is used as an exclusion criterion because of the complications involved in the management of epiphyseal injuries (growth plate injuries). Growth plate injuries would not normally be managed using the treatments being tested \[[@B13]\]. Patients are also excluded if they have a contra-indication to any of the four arms of the trial. This is most likely to occur if a patient has a DVT, high risk of DVT or other circulatory disturbance. Other contra-indications include poor skin viability preventing splinting or casting. The decision to exclude on these criteria is made by the attending clinician. The process of identifying participants --------------------------------------- A standard approach to identifying potential participants has been instituted across all participating centres. The approach was designed in consultation with the departments to dovetail with current procedures, and eradicate duplication of clinical and research data collection. The attending clinician (nurse practitioner, physiotherapist or doctor) assesses weight-bearing status. These data are recorded on a form that includes details of the remainder of the clinical examination, including if indicated, x-ray results. At this stage, people able to weight bear are excluded. The attending clinician gives a brief explanation of the trial, an information pack, and arranges an appointment at a follow-up clinic two to three days later. Delay is an accepted practice in the application of mechanical supports, allowing for the resolution of acute swelling. It also filters out patients in whom the diagnosis of severe sprain at initial contact was incorrect, and gives participants sufficient time to consider whether they wish to participate in the trial. A physiotherapist who staffs the follow-up clinic is responsible for checking eligibility and providing a further opportunity for patients to ask questions. If appropriate, the physiotherapist recruits the patients, contacts the randomisation centre, and arranges application of the treatments. Reasons for declining to participate in the trial are recorded. Assessing the generalisability of the results --------------------------------------------- Data on all ankle sprains attending the participating EDs during the recruitment period are collected using a standardised proforma. A copy of this proforma, which is entered into the medical record, is anonymised and passed to the research team. It provides descriptive data on the injury pattern, occupation, age, and sex of patients, including those who are ineligible or decline trial participation at various time points. Completed proformas are audited against ED attendance records to check whether attending clinicians have referred appropriate patients to the follow-up trial clinics. This allows clinicians who are unfamiliar with the trial to be identified and approached individually to be briefed on trial procedures. Treatment allocation -------------------- Randomisation is stratified by centre and provided via a phone-in service, which utilises a computer generated random allocation. Allocation concealment, which shields people who enter patients into a trial from knowing future allocations, is thereby ensured. The randomisation service is independently administered and quality controlled by the Birmingham Cancer Trials Unit. Treatment protocol ------------------ The mechanical supports are fitted to each individual by a trained health professional (physiotherapist, nurse or plaster technician) in the follow-up clinic, to ensure comfort and correct fit. Participants are provided with standardised written and verbal instructions including when to remove the support, encouragement of normal walking with limits of tolerance, simple exercise advice, what to do in the event of experiencing difficulties with the support, and washing instructions. The protocols for duration of support, weight-bearing status, and activity have been determined by the manufacturers recommendations and the results of a national survey of practice completed in the planning phase of the trial \[[@B8]\]. Treatments are applied within three days of the injury, and within an hour of randomisation. Other treatments ---------------- All other treatments are standardised and include the provision of crutches, advice to elevate and pain relieving medications if needed. Withdrawal of the latter treatments would be inappropriate as they constitute normal and accepted care. Physiotherapy is not provided as part of the trial treatment protocol, and is not part of routine care provided in the participating centres \[[@B9]\]. If the participant receives these types of treatment in addition to the trial protocol, this is recorded as an outcome. Baseline and outcome measures ----------------------------- Clinical status is measured at baseline, 4 weeks, 12 weeks and 9 months. The baseline assessment also includes date of birth, sex, body mass index, ethnicity, an assessment of pre-injury abilities including usual levels of mobility, engagement in sporting activity, usual occupation and employment (including hours worked and type of work undertaken) and completion of the baseline version of the outcome questionnaires. The selection of outcome measures is based on a systematic review completed during the preparatory stage \[[@B14]\], and are shown in Table [1](#T1){ref-type="table"}. There is a paucity of information on the psychometric properties of self-completed questionnaires for ankle conditions. The Foot and Ankle Outcome Score (FAOS) is a questionnaire that ascertains functional limitations (including mobility) and the severity of other symptoms after ligament sprains, and has evidence of validity and reliability \[[@B10]\]. Previous versions have been validated against objective tests of ankle function \[[@B15]\]. The Functional Limitations Profile (FLP) is the British version \[[@B16]\] of the Sickness Impact Profile \[[@B17]\]. We are using the FLP occupation and mobility sub-scales to provide more detailed information on the impact of the injuries and treatments including adaptations that occur after the injury. The FLP has not been used in studies of ankle injuries previously. Health related quality of life will be measured using the SF-12 version 1 and EQ-5D \[[@B18]\]. Return to normal occupation and leisure activities will be recorded as the date that people return to work and normal activities. Patients are asked to make a record of significant dates on a calendar to aid recall. The effectiveness of these calendars is being tested in a separate study. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Outcome measures ::: Outcome Outcome measure --------------------------------------------------------------------------------- --------------------------------------------------------------------------- Functional limitations and severity of symptoms after ligament sprains Foot and Ankle Outcome Score (FAOS) Impact of injuries and treatments including adaptations that occur after injury Functional Limitations Profile (FLP) occupation and mobility sub scales Health related quality of life Short Form 12 version 1 (SF-12 v1) Health related quality of life EuroQol (EQ-5D) Health economics Resource Use Questionnaire Return to normal occupation and leisure activities Date of return to work and normal activities recorded on a trial calendar ::: Timing of follow-up ------------------- The mechanical supports are likely to have maximal impact and benefit during the first three months of recovery, and this time period is defined as the primary time point. We anticipate that some participants will still be wearing the mechanical support at 4 weeks. The natural time course of recovery of ankle sprains is for functional limitations to stabilise between 3 and 9 months and it is expected that the difference between treatments will narrow in the longer term as the majority of people will recover \[[@B3],[@B5],[@B6]\]. Participants will be followed to 9 months to ensure that there are no longer-term complications from injury or their treatments. Follow-up and masking --------------------- Follow-up data are collected by postal questionnaire. We have implemented an intensive approach to follow-up. Participants are mailed a follow-up questionnaire, and if a response is not received within one week, they are telephoned to ensure they have received the questionnaire. A second questionnaire is mailed if necessary. If there is still no response, the participant is contacted one week later, and a core set of outcomes are collected over the telephone. All follow-up data are collected and analysed by individuals who are independent of the recruitment, randomisation, baseline assessment and are masked from the treatment provision. This level of masking will be maintained until final analysis of the data has been completed. The only exception to this rule will be if the data monitoring committee require unmasked data, and in this circumstance, only the independent Chairperson will be aware of assignments. Quality assurance ----------------- Trial procedures are audited at regular intervals to ensure compliance with the protocol. Maintaining trial profile and competence in trial procedures is challenging in the ED, because of a high turn-over of staff and a diversity in the staff groups dealing with injuries in different hospitals. To combat these difficulties, training is provided on cycles that coincide with staff rotation, as well as various other time points (e.g. in-service training). Data analyses ------------- The analysis will be conducted as intention to treat. An analysis of all people who completed the trial will be undertaken, and in addition, a sensitivity analysis will be undertaken to assess the range of potential biases that could result from loss to follow up or withdrawal. Numerical and graphical summaries of all the data will be compiled, including a detailed description of missing data at the clinic visit, questionnaire and individual level. Logistic and log-linear multinomial regression models will be used to provide estimates of the recovery rates and the prevalence of residual symptoms, with confidence intervals. As there are multiple centres and repeat assessment, random effect (or hierarchical) models will be used to investigate the components of variation. The common current and cheapest therapy is Tubigrip^®^, so each of the three other treatments will first be compared with it. Any treatments found to be more effective than Tubigrip^®^will be compared with each other. Regression modelling will allow an assessment of factors that might indicate the appropriate choice of treatment. As these analyses are pre-specified, issues of multiple comparisons are minimal. The sensitivity of the above analyses to missing data at various levels will be assessed and quantified using modern statistical methods for incomplete multivariate data \[[@B19]\]. Economic analysis ----------------- Severe sprains may have a range of direct cost consequences across primary and secondary health care, they may also have cost consequences for patients themselves in terms of their personal expenditure and return to work. The costing study will seek to adopt a broader societal perspective, including patient costs, when estimating differences in the cost of resources used in the four arms of the trial. The economic analysis will compare resource use (costs) with any measurable changes in health outcomes (benefits). The health outcomes of the four technologies will be measured in terms of changes in specific disease parameters and validated measures of health-related quality of life (HRQL). Any uncertainties in the cost and outcomes data will be incorporated into a sensitivity analysis. The cost of each mechanical support protocol will be determined through observation and will include staff time, overheads, equipment and transport; plus follow-up visits to hospital, GP surgeries, physiotherapists or others. The consequences for patients in terms of GP and hospital visits, including travel expenses and time off work, and expenditure on aids or private practitioner input are obtained from structured resource use questions added to patient follow-up questionnaires. Further treatments recorded include pain relieving medications, anti-inflammatory and other topical agents, bandages, supports or footwear. Patients are also asked to distinguish whether these are NHS-funded or private treatment paid for by the individual or private provider. Patient self-reported information on service use has been shown to be accurate in terms of intensity of use of different services \[[@B20]\]. Hospital notes and records will also be audited for information on service use. The cost of primary and hospital services will be estimated from a variety of sources, including the finance departments of the trial hospitals and services concerned and national sources \[[@B21],[@B22]\]. Differences in resource use and outcomes are ascertained in follow-up questionnaires at 12 weeks and 9 months. The appropriate technique of economic evaluation will depend on the results of the study \[[@B23]\]. The simplest eventuality would be where the least expensive intervention is found to be better on at least one outcome measure and no worse on any other i.e. dominant. Another is where two interventions have the same outcomes in which case cost-minimisation analysis will be used to compare the two. However, where an intervention is clearly better in terms of outcome but is also more costly, a different approach is required. One accepted method is to compare the different interventions in terms a single outcome measure identified as clinically important for the condition being treated. The primary outcome meets this requirement. Therefore, the costs per unit improvement in mobility will be used to provide an estimate of overall cost-effectiveness; average and incremental (relative to reference treatment) cost-effectiveness ratios will be estimate for the different treatments used. However, this approach does not allow comparison with other types of intervention/condition combinations and also does not consider the value (utility) of differences in improvement in health status. In the present study, the EQ-5D will be used to generate such utility scores that can be compared to costs. A cost-utility analysis will estimate outcomes in terms of differences in quality-adjusted life years (QALYs), representing the period of life subsequent to a health care intervention weighted or adjusted for the quality of life experienced by the patient during that period, and compare these with cost for the four interventions \[[@B23]\]. The cost-utility analysis will present the incremental cost of the extra benefit gained both in summary form in terms of incremental cost per QALY, and also using a \'disaggregated\' approach where the extra costs are presented alongside the HRQOL dimensions such as pain. Sample size ----------- Although there was a paucity of published data available to inform the sample size estimate at the outset of the trial, the preliminary phase of the study has enabled us to make a more accurate estimation of the sample size required. The presented estimate is based on a standard sample size calculation for a two-sample t-test with equal variances and a significance level of 0.05, using the variance estimated from an ANOVA of the 4-week data (n = 100). The minimal clinically important difference is set at 10 percent, which represents a small to moderate effect size. A target of 600--650 participants will allow us to detect clinically important outcomes at 4 and 12 weeks. We are powered \>90% to detect differences of 10 percent in the primary outcomes, and have sufficient power to detect differences in a range of secondary outcomes at 80% power. To account for the possibility of loss to follow-up, the estimate includes an inflation of 20%. However, the trial should be able to report whether any of the treatments have a sustained negative effect on outcome at 9 months. We are taking a pragmatic approach to estimating the sample size, and the Data Monitoring Committee are reviewing assumptions underlying the calculation at 6-monthly intervals. Qualitative sub-study --------------------- A purposive sample of randomised patients will be interviewed to obtain qualitative data on the patients\' experiences of the various treatments in a semi-structured interview. Patients will also be asked for their views on their willingness to pay a deposit for the boots/splints to encourage their return. Up to 20 patients will be interviewed using a semi-structured format. The data will be analysed using thematic content analysis. Conclusions =========== We have described the protocol and conduct of a large scale UK randomised controlled trial of mechanical supports for the management of acute severe ankle sprains. There has been a paucity of good quality research conducted to date, which may be partly explained by the challenges implicit to studying acute events within EDs \[[@B24],[@B25]\]. These include a very short window of opportunity in which patients satisfy the criteria of acute injury and reliance on attending clinical staff to identify and approach for inclusion potential participants who are often in a state of discomfort. The trial is currently in the middle of the recruitment phase and is running well. Trial procedures have been well received by both patients and clinicians. Much effort has gone into maintaining the profile of the trial and disseminating trial procedures across all disciplines of ED staff (e.g. doctors, nurses, reception staff and plaster technicians). This has been paramount to the success of the trial. By referring potential patients to the follow-up trial clinics ED personnel, with no direct involvement in the trial, play a vital role in the trial process. The intense follow-up has produced good completion rates. Using a pragmatic approach to sample size estimation was useful, in particular, re-estimation after an adequate run in period, and is recommended where little prior data exists. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= SEL wrote the original protocol, secured funding, act as co-principal investigator and lead in the writing of this manuscript. MWC wrote the original protocol, secured funding and act as co-principal investigator. JRD and SW contributed to the development of the protocol and securing funding. JLH and AS contributed to the development of the protocol and securing funding and wrote the statistical and economic aspects of the protocol respectively. RAN is responsible for the day to day co-ordination of the trial, has contributed to refining the protocol and outcome measure package. JLM revised the protocol and the sample size calculations. MC refined the economics section of the protocol and is providing economic input. EJW is responsible for the data management and trial support. All authors have contributed to and approved the final manuscript. CAST research team ================== Lamb SE, Cooke MW, Nakash RA, Withers EJ, Hutton JL, Marsh JL, Szczepura A, Wilson S, Clark M, Dale JR, MacNamara A, Kendall J, Skinner D, Sakr M, Kelly C, O\'Byrne G, Morrell R, Dunn M, Bateman S, Kempson S, Hooker F, Parker N, Noakes E, Vaux N, Doughty G, Nichols V, Ritchie C, Sabine E. Trial Steering Committee ------------------------ Professor Bill Gillespie, Professor Sallie Lamb, Dr Matthew Cooke, Professor Jeremy Dale, Professor Jane Hutton, Dr Jen Marsh, Professor Ala Szczepura, Dr Sue Wilson, Rachel Nakash, Vicki Staples. Data Monitoring Committee ------------------------- Dr Janet Dunn, Professor Damian Griffin, Pat Overton-Brown. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2474/6/1/prepub> Acknowledgements ================ This project is funded by the NHS R&D Programme Health Technology Assessment Programme (project number 01/14/10). The views and opinions expressed herein are those of the authors and do not necessarily reflect those of the Department of Health. Lisa Craven for data entry and patient follow-up. Ellen Jørstad for assisting with preparing this manuscript for publication. Thanks to Braun Medical, Sheffield and Aircast Ltd Partnership for their product supply. Thanks to clinicians, reception staff and plaster technicians at all trial centre sites.	PubMed Central	2024-06-05T03:55:52.421033	2005-1-13	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547908/", "journal": "BMC Musculoskelet Disord. 2005 Jan 13; 6:1", "authors": [ { "first": "SE", "last": "Lamb" }, { "first": "RA", "last": "Nakash" }, { "first": "EJ", "last": "Withers" }, { "first": "M", "last": "Clark" }, { "first": "JL", "last": "Marsh" }, { "first": "S", "last": "Wilson" }, { "first": "JL", "last": "Hutton" }, { "first": "A", "last": "Szczepura" }, { "first": "JR", "last": "Dale" }, { "first": "MW", "last": "Cooke" } ] }
PMC547909	Background ========== Nicotinic acetylcholine receptors (nAChRs) are the most studied members of the cys-loop family of ligand-gated ion channels (LGICs) which also contains γ-aminobutyric acid (GABA) receptors, glycine receptors and 5-HT~3~receptors \[[@B1]\]. Distinct subtypes of nAChRs mediate, for example, fast synaptic transmission in the brain and at neuromuscular junctions \[[@B2]\]. All are believed to be pentameric assemblies of various combinations of different subunits (α, β, γ/ε, δ), some of which exist as multiple isoforms \[[@B3]\]. nAChRs are important targets for novel analgesics as well as new drugs being devised for Alzheimer\'s disease and schizophrenia \[[@B4],[@B5]\]. Mutations in nAChRs are associated with certain forms of epilepsy \[[@B6],[@B7]\] and several congenital myasthenias \[[@B8]\]. There is a substantial and growing body of physiological, pharmacological, genomic structural and modeling data on receptors formed from these subunits. The volume and diversity of these data present severe challenges for their efficient storage and interpretation. Here we describe a relational database, initially relating to the α7 subunit, whose aim is to provide an easy to use, extensible web-based interface to access functional data and relate it back, wherever possible, to structural data. Construction and content ======================== The database is currently limited to nAChR α7 subunits. This makes the initial task of populating the database manageable. To allow 3^rd^level normalization, the principal information prototype stored in the database is referred to as an \"experiment event\". An experiment event is a collection of simultaneous measurements and their associated measurement conditions. For example, two drugs tested against the wild type α7 and a mutant under the same experimental conditions would be described by 4 (2 drugs × 2 sequences) experiment events. If the identical measurements were repeated in, say, calcium-free saline, this would yield 8 experiment events. Using this prototype a unique set of experimental conditions is associated with a single set of data. Therefore, the database tables (see Figure [1](#F1){ref-type="fig"}) have been designed around the central experiment table \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. Quite often, several real experiments are performed within any one publication. We wanted to capture all of that data. Thus, all the data for one experiment that was reported have been stored (non-redundantly) as a separate entity. The database is managed by the MySQL relational database management system. PHP scripts query the MySQL system and transform the data into HTML pages for serving to the client. The database currently aims to store molecular information rather than whole-organism genetic information which can be sourced from elsewhere, see for example \[[@B9],[@B10]\]. A typical set of experiments reported in one paper might concern the properties of one mutation at a particular position and its associated changes in physiology and/or pharmacology. All of this data is held in the database. By use of appropriate alignments (either stored within the server or uploaded) the positions of these data can be highlighted on a homology model based on the acetylcholine binding protein AChBP \[[@B11]\]. The model is viewable by either using the Chime plugin, or by setting the browser to use Rasmol as a helper application. User instructions for configuring different browsers are provided on the server, as well as the list of combinations of operating system and browser that we have tested to date. For any database, data integrity will ultimately determine its usefulness. The initial population of the database has been carried out by the authors, but it is desirable and practical in the long term to have a procedure whereby any laboratory can submit data. To help ensure reliable deposition procedure, each depositor will have a unique identifier (thus making the data accountable). A depositor will submit their data which is then tabulated and presented as a form which the depositor is then asked to confirm as being correct. Only when this confirmation is received is the data committed to the database. Although this will reduce some entry-based mistakes, the database will nonetheless need to be curated to maintain integrity. The database does not attempt to duplicate the functionality of other databases. For example we store a local alignment of α7 subunits taken directly from pfam \[[@B12]\], but we do not perform any alignments ourselves. Such tools are already available on numerous web-servers and in any case the user may wish to upload their own alignment. Wherever possible, we link to a well-established resource, as is the practice in for example, SWISS-PROT \[[@B13]\]. Utility and discussion ====================== Access to the database is through various routes. The first route is by a very simple search string (e.g. citation = Smith\). The user is then presented with a page informing him of the number of hits and asking what information should be displayed from those hits. The second is a simple incremental search in which the user applies criteria sequentially until he chooses to examine the results. As the query is built, the number of hits returned by the query in its current state is shown. The third search method is also incremental but via the use of a set of tabbed pages which divide the information into intuitive categories such as pharmacology and physiology. Additionally, the user can make choices about alignments used and even upload their own. As the query is built, summary information of its status is also displayed. The fourth method to access the data is provided by a \'fast lane\' route which provides more experienced users with a quicker and more direct route to the data of interest. The user formulates and constructs a query offline and then uploads it as a simple ASCII file. A sample query form and details of the format are presented in the supplementary information. The result of a query is presented in tabular form. Only the information requested is actually presented with the option for further or refined searches. In addition to this, the results of any position matches can be displayed via the homologous 3-dimensional structure of the AChBP. This is automatically available if the user selects the alignments available from the server. If the user uploads their own alignment the model will only be produced if AChBP was included. The server streams a Rasmol script or spawns a Chime window depending on browser configuration. In addition to these query routes, the user is also able to browse the contents of the database. The current database has been designed such that it could easily be extended to include other nAChR subunits and then on to other members of the cys-loop ligand-gated ion channel family. We are currently pursuing this aspect. Such expansion will allow trends that link structure to function in this receptor family to be more readily identified. The database is very complementary to some existing resources, in particular the LGICdb \[[@B14],[@B15]\]. This database contains sequence, phylogenetic data and sets of coordinates, but does not attempt to store all aspects of individual experiments as we report here. However, this could be used in conjunction with our database to for example explore equivalent positions in related receptors using sequence and phylogenetic information stored therein. Our approach is somewhat similar to that employed by the ProTherm database \[[@B16]\] which aims to store thermodynamic data for mutant and wildtype proteins \[[@B17]\]. Although currently there are no entries common to both databases, it is envisaged that thermodynamic data reported for either the α7 nAChR receptor or related channels will appear in the ProTherm database and can thus be used in conjunction with the A7DB to help illustrate for example how loss of function might be related to protein stability. Furthermore, the similarity in design of these databases should make cross-interrogation possible in the future. We should state that the main difference between A7DB and ProTherm is that instead of thermodynamic data we store pharmacological and physiological data and in that sense it is closer to the voltage-gated potassium channel database \[[@B18],[@B19]\]. Finally, the A7DB is also complementary to the Protein Mutant Database \[[@B20]\] where mutations across many proteins are stored but the searching and data tabulation is primarily text-based. This might be particularly useful if another mutation had been reported in a different protein that bound a similar ligand, for example in acetylcholine also binds to acetylcholinesterase, which we do not store data for. Conclusions =========== We have shown here how the collation and careful storage of experimental data pertaining to one sub-family (the α7 nAChR sub-family) of the ligand-gated ion channels can be assembled into a useful resource. However, the real power of the database will be when it is combined with machine-learning technologies to explore complex relationships between sequence, structure and function \[[@B21]\]. We believe that this resource will grow in usefulness as the amount of data increases. For example, during the construction of this database, atomic coordinates were released for the transmembrane region of the nAChR from Torpedo marmorata*\[[@B22]\]. Its development is particularly timely given these recent structural developments which allow three-dimensional information to be correlated with function derived from an ever-increasing body of experimental data. Availability and requirements ============================= The database is freely available at: <http://www.lgics.org/a7db/> Authors\' contributions ======================= The project was conceived and technical aspects managed by PCB. The coding and design was done by SB. MSPS and DBS provided critical evaluation of the design and construction process. DBS, LP, AKJ and LB populated the database. All authors have approved the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Supplementary information. This doc file provides a complete description of the table entries and also provides a description of the uploadable query file. Further information about the online help is also provided. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ SDB, MSPS and PCB are supported by the Wellcome Trust. AKJ, LB, LP and DBS acknowledge support from The Medical Research Council of the UK. AKJ also acknowledges support of a grant to DBS from Dupont. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The A7DB database scheme. All the information pertaining to an experiment is stored within a single table called \"experiment\". The sequence table stores all the wildtype sequences for the different species contained within the database. The third table is the admin table which makes entries and changes traceable. A full description of each of the entries is supplied as supplementary information. ::: ![](1471-2202-6-2-1) :::	PubMed Central	2024-06-05T03:55:52.423613	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547909/", "journal": "BMC Neurosci. 2005 Jan 20; 6:2", "authors": [ { "first": "Steven D", "last": "Buckingham" }, { "first": "Luanda", "last": "Pym" }, { "first": "Andrew K", "last": "Jones" }, { "first": "Laurence", "last": "Brown" }, { "first": "Mark SP", "last": "Sansom" }, { "first": "David B", "last": "Sattelle" }, { "first": "Philip C", "last": "Biggin" } ] }
PMC547910	Background ========== The worldwide spread of the problem of infections due to Gram-negative bacteria such us Pseudomonas aeruginosaand Acinetobacter baumanniiresistant to most classes of antimicrobial agents has made the medical community to rethink of colistin, an old, basically abandoned for the last two decades antibiotic. Intravenous colistin (polymyxin E) has been used in the industrialized countries during the last years mainly in patients with cystic fibrosis infected with Pseudomonas aeruginosaresistant to other available antimicrobial classes \[[@B1]\]. Excluding this population, the agent has been infrequently used during the last two decades because of major concerns about nephrotoxicity and neurotoxicity \[[@B2]\]. Several reports during the early years of use of the medication, mainly in the decade 1960 to 1969 left the medical community with the impression that the medication is very toxic \[[@B3],[@B4]\]. However, the recent experience of several clinicians worldwide with the use of the medication has not verified the old reports about the serious or common toxicity of colistin \[[@B5]-[@B8]\]. We analysed data of a group of patients who received colistin for more than four weeks to investigate further the concerns about the toxicity of the medication. Methods ======= Design of the study-patient selection ------------------------------------- Patients who received intravenous colistin from 1/October/2000 to 31/January/2004 at \"Henry Dunant\" Hospital, a 450-bed tertiary care center in Athens, Greece, were identified by the pharmacy electronic databases. All patients who were found to have received intravenous colistin for more than four weeks continuously, or in two courses of colistin but with no more than seven days interruption between the courses were included in this study. Administration of intravenous colistin in two courses for more than four weeks in each course were studied as two different units in the analysis of toxicity, if there was more than one month colistin-free period between the two courses. One milligram of the colistin formulations used is approximately equal to 12.500 IU (Colomycin, Forest Laboratories^®^, Kent, UK or Colistin, Norma^®^, Athens, Greece). The study was approved by the Institutional Review Board (IRB) of the hospital. Definitions of infections ------------------------- Diagnosis of pneumonia required two or more serial chest radiographs with at least one of the following: new or progressive and persistent infiltrate, consolidation, cavitation, or pleural effusion. In addition, patients must have had fever \>38°C with no other recognized cause, or abnormal white blood cell count \[leukopenia (\< 4000 WBC/mm^3^) or leukocytosis (= 12.000 WBC/mm^3^)\], and at least two of the following: new onset of purulent sputum or change in character of sputum, increased respiratory secretions or increased suctioning requirements, new onset or worsening of cough or dyspnea or tachypnea, rales or bronchial breath sounds, or worsening gas exchange \[[@B9]\]. Bacteremia required either growth of a recognized pathogen from one or more blood specimen cultures or at least one of the following signs or symptoms: fever (\>38°C), chills, or hypotension andat least one of the following: a) common skin contaminant (e.g., diphtheroids, Bacillussp., Propionibacteriumsp., coagulasenegative staphylococci, or micrococci) grown from two or more blood cultures drawn on separate occasions or b) common skin contaminant (e.g., diphtheroids, Bacillussp., Propionibactαiumsp., coagulase-negative staphylococci, or micrococci) grown from at least one blood culture from a patient with an intravascular line and physician- instituted antimicrobial therapy \[[@B9]\]. Infections at other body sites or fluids, such as urinary tract infections and surgical site infections were defined based on guidelines from the Centers for Disease Control and Prevention \[[@B9]\]. Clinical specimens of all body sites were considered when defining the etiologic microbiologic agent of the infection. Data collection-entry --------------------- Data for several variables including demographic and clinical information as well as results of laboratory and imaging tests were collected and entered in a computer database. All available results of renal function tests (serum creatinine, urea, creatinine clearance, urinalysis), liver function tests (SGPT, SGOT, alkaline phosphatase, γ-GT, bilirubin), creatine phosphokinase (CPK) during the course of colistin treatment and at hospital discharge were collected. Data analysis ------------- Creatinine values at the beginning of colistin treatment were compared with the maximum value of creatinine during therapy as well as with the creatinine value at the end of treatment using a non-parametric test (Wilcoxon). The data analysis was performed using SPSS and S-plus software. Results ======= Study population ---------------- During the period from 1/October/2000 to 31/January/2004, 152 patients received intravenous colistin. Data about the efficacy of the medication were analysed and reported elsewhere \[[@B5]\]. In this report, we present detailed data regarding the toxicity of colistin in a subgroup of 17 patients who received more than four weeks of intravenous colistin. Table [1](#T1){ref-type="table"} describes various characteristics of this group of patients including demographic and clinical data, such as comorbidity, site of infection, and responsible pathogens. Two patients received two prolonged courses of intravenous colistin each with more than one month colistin-free period between the courses. Subsequently, there were 19 courses of prolonged colistin for the analysis of toxicity. Among these 19 courses, the administration of intravenous colistin was without interruption in 15 courses, while there was interruption of colistin administration in 2 courses for two days, interruption in 1 course for 3 days, and interruption in 1 course for 4 days. The cause for the interruption of colistin in these cases was the attending physicians\' attempts to obtain cultures without the confounding influence of antimicrobial treatment in patients with puzzling continuing symptoms of infection. The mean duration of colistin administration (± SD), for each course of colistin therapy was 43,4 days (± 14,6 days). The mean daily dosage ± SD for each course was 4.4 million IU (352 mg) ± 2.1 million IU (168 mg) and the mean cumulative dosage of colistin ± SD for each course was 190.4 million IU (15.232 mg) ± 91.0 million IU (7.280 mg). The mean daily dosage for a patient with body weight of 70 kg was 62.857 IU/kg (5 mg/kg). For patients with impaired renal function, dosage adjustments were done mainly after consulting the ICU director or the Infectious Disease specialists of the hospital, based on the following protocol: if serum creatinine level was 1.3--1.5 mg/dl, 1.6--2.5 mg/dl or = 2.6 mg/dl, the dosage of colistin administered was 2 million IU (160 mg) every 12 hours, 24 hours, or 36 hours, respectively. Patients who were on dialysis treatment received 1 million IU (80 mg) of colistin after dialysis. Colistin was given for long duration in patients mainly in the ICU setting, because of persistence of infections due to Gram-negative bacteria sensitive only to colistin or bacteria sensitive also to antibiotics of other classes that were previously administered (based on in-vitro susceptibility test results) but failed. Subsequently, no other therapeutic strategy was considered better than the continuation of colistin (as combination therapy or monotherapy) because of failure of previous antibiotic regimens and the lack of other therapeutic options. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographic and clinical features of patients managed with prolonged intravenous colistin for infections caused by multidrug-resistant Gram-negative bacteria. ::: Characteristics of patients Median (range) n (%) ---------------------------------------------------------- -------------------------- Demographic Age, years \[median (range)\] 51 (18 -- 79) Sex, male 12/17 (70%) APACHE II score On admission to ICU \[median (range)\] 14 (7 -- 35) On 1^st^day of colistin treatment \[median (range)\] 14 (6 -- 22) Comorbidity Malignancy 2/17 (11%) Hemodialysis 2/17 (11%) Urogenital disorders 3/17 (18%) Heart dysfunction 5/17 (29%) Diabetes mellitus 5/17 (29%) Lung dysfunction 5/17 (29%) Liver failure 1/17 (6%) Hematological disorders 3/17 (18%) Neurological disorders 8/17 (47%) Neuropathy/myopathy 4/17 (23%) Trauma 7/17 (41%) Transfusion 15/17 (88%) Prior hospitalization 12/17 (70%) Prior surgery 14/17 (82%) Elective 9/14 (64%) Emergency 5/14 (36%) Prosthetic material 11/17 (64%) Catheters/Invasive devices Tracheostomy 14/17 (82%) CSF drainage 5/17 (29%) Prior medications Prior antibiotic use 17/17 (100%) Prior antifungal use 8/17 (47%) Anti-tumor treatment 0/17 (0%) Cortisone treatment 9/17 (53%) Duration of hospitalization (days)\[median (range)\] 152 (29 -- 591) Duration of stay in ICU (days)\[median (range)\] 70 (22 -- 134) Site of infection\* Pneumonia 13/19 (68%) Bacteremia 1/19 (5%) Urinary tract infection 2/19 (11%) Meningitis 2/19 (11%) Surgical site infection 1/19 (5%) Pathogens† Pseudomonas aeruginosa 12/20 (60%) Acinetobacter baumannii 5/20 (25%) Klebsiella pneumoniae 2/20 (10%) Enterobacter cloacae 1/20 (5%) Mortality 7/17 (41%) \* The analysis of the site of infection was based on 19 episodes of infection in 17 patients. †The analysis of the responsible pathogens was based on 20 isolates from 19 episodes of infection in 17 patients \[two pathogens were isolated in one episode of pneumonia (Klebsiella pneumoniae + Acinetobacter baumanniimixed infection)\]. ::: The analysis of the responsible pathogens was based on 20 isolates from 19 episodes of infection in 17 patients (two pathogens were isolated in one episode). In 2 episodes of infection, both caused by Pseudomonas aeruginosastrains, the minimum inhibitory concentrations (MICs) were not available. In the remaining 10 Pseudomonas aeruginosaisolates, the MICs to colistin ranged from = 0.5 mg/l to 2 mg/l. In vitro susceptibility testing for the 5 isolated Acinetobacter baumanniistrains revealed MICs to colistin = 0.5 mg/l, for the 2 Klebsiella pneumoniaestrains MICs were 0.5 mg/l and 2 mg/l, and for the Enterobacter cloacaestrain MIC was 1 mg/l. The all-cause in-hospital mortality was 41,2% (7 out of 17 patients). Clinical cure and improvement, defined as resolution and partial resolution, respectively, of presenting symptoms and signs of the infection by the end of colistin treatment was observed in 14 of the 19 episodes of infection (73.7%) \[cure 10/19 episodes (52.6%), improvement 4/19 episodes (21.1%)\]. Unresponsiveness, defined as persistence or worsening of presenting symptoms and/or signs of the infection during intravenous colistin administration, was observed in 5 of the 19 episodes of infection (26.3%). All patients had been admitted to the ICU at some time during their hospitalisation. In 12 courses out of the 19 analyzed the whole colistin therapy was administered in the ICU, in 1 course it was administered in a hospital ward, and in 6 courses it was given both in the ICU and a hospital ward. Renal toxicity -------------- Figure [1](#F1){ref-type="fig"} depicts the box plots of serum creatinine and blood urea values of patients on the first day of colistin, the maximum values during the course of administration of colistin, and the values at the end of the treatment with colistin (one patient, who was on hemodialysis treatment prior to and during the colistin course, was removed from the analysis of the comparisons of the creatinine and urea values). The median value of baseline creatinine at the beginning of colistin therapy for the 18 prolonged courses of colistin was 0,6 mg/dl. Compared to this value, there was a slight increase of the median of values of creatinine at the end of the colistin course by 0,1 mg/dl, without statistical significance (p = 0,67). The median of the maximum values of creatinine observed was 0,85 mg/dl and was found to be statistically different from both the median of start (p \< 0,001) and end of treatment values (p \< 0,001). The maximum absolute increase of creatinine, compared to baseline, observed in an episode was 1,4 mg/dl. Only one patient had an increase of more than 50% of the baseline creatinine level to a value higher than 1.3 mg/dl at the end of colistin treatment. For the subgroup of 12 patients with 14 courses of prolonged colistin administration, with normal serum creatinine at the initiation of treatment, the median serum creatinine values at baseline and at the end of colistin treatment were 0,60 mg/dl and 0,55 mg/dl, respectively. In addition, the median maximum value of serum creatinine during the course of colistin treatment was 0,80 mg/dl among this group of patients. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The distribution of serum creatinine and blood urea levels on the 1^st^day of colistin treatment (start), at the peak value (max), and the end of colistin treatment (end) in all studied courses of prolonged treatment with colistin. (The horizontal line within the boxes represents the median creatinine or blood urea baseline value at the 1^st^day of colistin treatment. \"Shaded\" boxes in the figures represent the distribution of the laboratory values contained between the 25^th^and 75^th^percentiles (the interquartile range). Dotted lines (whisker lines) extend from the box, down and up, to the minimum and maximum values of the distributions that are not outliers (i.e. that are within 1.5 times the interquartile range); these values of the distribution are shown as \"brackets\". Any value, which is an outlier, is drawn as a \"horizontal line\"). ::: ![](1471-2334-5-1-1) ::: At the initiation of colistin administration the median value of baseline blood urea for the 18 courses was 42 mg/dl. During the 18 prolonged courses of colistin administration there was a slight increase in blood urea values; the median maximum value was 60 mg/dl. However, at the completion of colistin therapy median blood urea value returned to 41 mg/dl. During the 19 courses of colistin administration, other potentially nephrotoxic drugs were given. Specifically, aminoglycosides were co-administered in 12 courses \[median duration of co-administration (± SD) in these 12 courses 12,5 (± 16,8) days\], teicoplanin in 10 \[median duration of co-administration 16,0 (± 9,0) days\], vancomycin in 8 \[median duration of co-administration 23,0 (± 11,2) days\], amphotericin in 4 \[median duration of co-administration 8,0 (± 9,0) days\]. Four patients had history of chronic renal dysfunction, with only one of them receiving hemodialysis treatment prior to admission. This patient continued the hemodialysis during his treatment with colistin and died due to sepsis. For the other 3 patients the baseline, the maximum, and the end-of-therapy creatinine values (mg/dl), were 1,8/2,1/2,1 for the first patient, 3,7/3,7/2,4 for the second patient and 2,4/3,8/2,3 for the third patient. Only one patient had acute renal failure prior to colistin use, which was due to severe haemorrhage during surgery. This patient received 13 courses of hemodialysis. One day after the last course, colistin therapy had to be initiated for the management of Acinetobacter baumanniipneumonia, sensitive only to colistin and gentamicin. Apart from a slight elevation of serum creatinine (from 2,3 mg/dl to 2,7 mg/dl) on the 5^th^day of treatment no other sign of nephrotoxicity was observed during a 34-day administration of colistin (the 9^th^day creatinine returned to 0,7 mg/dl). Neuromuscular toxicity ---------------------- No apnea or other evidence of neuromuscular blockade was noted in any of these patients who received prolonged treatment with colistin. Four patients were clinically diagnosed to have polyneuropathy and/or myopathy before or during colistin treatment. In the first patient the polyneuropathy symptoms appeared while she was on her 25^th^day of colistin treatment and became worse on the 27^th^day. From then on, and although colistin was continued for 11 more days, the symptoms gradually subsided. No confirmatory electromyographic testing was performed. The second patient was transferred from another ICU where he was diagnosed as having myopathy due to sepsis. Fifteen days after his admission, and while his myopathy was improving, therapy with colistin was initiated; the treatment did not affect the gradual improvement. An electromyography performed on his 21^st^day in our ICU, showed mild axial sensory-motor polyneuropathy and myopathy. He received colistin for a total of 52 days and made full recovery from his neuropathy/myopathy during the course of colistin administration. The third patient developed ICU polyneuropathy one week prior to colistin administration and, again, recovered gradually from it while he was on colistin treatment. The fourth patient was transferred from another ICU, where he had developed ICU polyneuropathy. Only this patient had moderate worsening of the neuropathy while he was receiving colistin. Despite that, he received colistin for a total of 35 days for a respiratory infection with persisting Pseudomonas aeruginosasensitive only to colistin. His neuropathy improved gradually after the end of colistin treatment. Thus, our assessment regarding these 4 patients who experienced polyneuropathy and/or myopathy is that colistin therapy was probably associated with the development of neurotoxicity only in one of the 4 patients. Liver and biliary tree toxicity ------------------------------- Data for the possible hepatobiliary toxicity of colistin were collected. Both laboratory and clinical findings were taken into consideration. In subgroup analyses of patients for whom data were available, no substantial changes on liver function tests was found, as described in Figure [2](#F2){ref-type="fig"} (data for SGPT, alkaline phosphatase, direct and indirect bilirubin are not shown). Three of our patients were found to have increased levels of hepatic and cholestatic enzymes during colistin administration. One of them was found to have acute cholecystitis, the second had severe inflammatory systemic reaction (SIRS), and the third had transient elevation of transaminases while he was receiving anti-epileptic medications together with colistin. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The distribution of liver function tests \[SGOT (AST = aspartate aminotransferase), γ-GT, and total bilirubin\] on the 1^st^day of colistin treatment (start), at the peak value (max), and at the end of colistin treatment (end) in studied courses of prolonged treatment with colistin. (The horizontal line within the boxes represents the median creatinine or blood urea baseline value at the 1^st^day of colistin treatment. \"Shaded\" boxes in the figures represent the distribution of the laboratory values contained between the 25^th^and 75^th^percentiles (the interquartile range). Dotted lines (whisker lines) extend from the box, down and up, to the minimum and maximum values of the distributions that are not outliers (i.e. that are within 1.5 times the interquartile range); these values of the distribution are shown as \"brackets\". Any value, which is an outlier, is drawn as a \"horizontal line\"). ::: ![](1471-2334-5-1-2) ::: Conclusions =========== The main finding of the study is that no significant deterioration of renal function was found in patients with baseline normal serum creatinine value during prolonged administration of intravenous colistin. In addition, in the group of patients with pre-existing chronic renal dysfunction (not on hemodialysis) the administration of the medication for a long duration did not influence further their renal function. The observed slight increase in the serum creatinine values at the end of treatment (0,1 mg/dl increase in the median creatinine values from the start to end of colistin administration) cannot be attributed only to the prolonged colistin administration as other factors such as concomitantly administered potential nephrotoxic agents, and sepsis per ce may have had an effect. In addition, apnea or other evidence of neuromuscular blockade was not observed in any of the 17 patients who received prolonged treatment with colistin. Early experience with colistin revealed a high incidence of toxicity, mainly nephrotoxicity. In 1969, Ryan et al. reported the fatal case of a previously healthy 4-year-old child with persistent fever after appendectomy who died due to cardiac arrest \[[@B10]\]. The child had received colistin intramuscularly due to the growth of Pseudomonas aeruginosaand E. colistrains from the excised specimen. This patient developed acute tubular necrosis (confirmed by autopsy) and apnea requiring intubation and mechanical ventilation. However, in this case the dosage of colistin used was 30 mg/kg of body weight every six hours (120 mg/kg/day) whereas the recommended dose is 2,5 -- 5 mg/kg/day. One year later, several reports showed high renal adverse reaction rates after colistin administration \[[@B2],[@B4],[@B11]\]. In many of them the dosage used was also higher compared to the recommended, i.e. 6,3 mg/kg/day in one report, and 26 mega units in another administered as 10 mega units by intramuscular injection, 10 mega units by intravenous injection and 6 mega units by inhalation (no details are provided in the report about the ratio of IU of colistin per mg). In addition, dosage adjustments in patients with renal dysfunction were not always followed, as well as there were also other factors which perhaps contributed to toxicity, such as the co-administration of other medications with potential toxicity, including anesthetic drugs and muscle relaxants. However, recent data from published reports do not corroborate this finding \[[@B14]\]. Notably, one study conducted in 35 patients with ventilator-associated pneumonia due to multidrug-resistant Acinetobacter baumannii, who received either intravenous colistin (21 patients) or imipenem (14 patients), showed an almost double increase in nephrotoxicity rates among the patients in the imipenem group compared to colistin group \[[@B12]\]. In addition, Stein et al. recently reported their experience from 3 patients with orthopedic infections who received colistin for 3 -- 6 months without developing any adverse effect requiring discontinuation of the treatment \[[@B13]\]. The conclusion in the majority of the published studies during 1960 -- 1970 was that colistin must be used in serious infections where other antimicrobial agents have failed, always with close monitor of renal function, and with precaution in the co-administration of other possible nephrotoxic or neutotoxic agents \[[@B2],[@B4]\]. Subsequently, the considerable difference regarding reporting toxicity of colistin between old and recent studies deserves an explanation. First, the formulations of currently used colistin may be more purified compared with the old ones. The experience that was accumulated from the use of vancomycin may support this possibility. It is known that the concerns about serious and/or common nephrotoxicity of vancomycin overwhelmed the older literature. Second, fluid supplementation and supportive treatment of severely ill patients has been improved nowadays. Third, higher doses of colistin were administered to patients during the first years of its use. Our study is not without limitations. It is a retrospective study with the inherited problems related to this study design, including difficulties in the determination of colistin associated sensory neurotoxicity. In addition, the number of studied patients is relatively small and there is no control group. However, our study offers insight related to interesting experience from a group of patients who received prolonged treatment with colistin for various infections, mainly as a salvage treatment of persisting and serious infections. In conclusion, intravenous colistin seems to be a safe therapeutic intervention for serious infections due to multidrug-resistant Gram-negative bacteria even when it is administered for a prolonged duration (more than four weeks). More studies will be needed to further clarify the correct use of this old antibiotic in the 21^st^century. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contribution ====================== MEF and AM conceived the idea; MR, IAB, KR, and SKK collected the data; all authors contributed in the writing and preparation of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/5/1/prepub>	PubMed Central	2024-06-05T03:55:52.425243	2005-1-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547910/", "journal": "BMC Infect Dis. 2005 Jan 10; 5:1", "authors": [ { "first": "Matthew E", "last": "Falagas" }, { "first": "Michael", "last": "Rizos" }, { "first": "Ioannis A", "last": "Bliziotis" }, { "first": "Kostas", "last": "Rellos" }, { "first": "Sofia K", "last": "Kasiakou" }, { "first": "Argyris", "last": "Michalopoulos" } ] }
PMC547911	Background ========== Enterococcal meningitis is an uncommon disease accounting for only 0.3% to 4% of cases of bacterial meningitis which is nevertheless associated with a high mortality rate. It has been described most frequently in patients with neurosurgical conditions (i.e. head trauma, shunt devices, or cerebrospinal fluid leakage), although it can also occur as a \"spontaneous\" infection complicating remote enterococcal infections such as endocarditis or pyelonephritis \[[@B1]\]. Enterococcus faecalisand Enterococcus faeciumare the two species most frequently isolated during the course of meningitis (76%--90% and 9--22% respectively). Enterococcus casseliflavus, first considered as a subspecies of E. faecium, is a motile enterococcus that produces a yellow pigment in agar and often has a VanC phenotype determining an intrinsic low level resistance to vancomycin. It has been implicated in a wide variety of infections in humans, especially immunocompromised hosts, but to the best of our knowledge it has never been associated to meningitis \[[@B2]-[@B5]\]. We describe here a case of enterococcal meningitis caused by E. casseliflavusthat was believed to originate from the gut in an old patient with bowel erosions. Case presentation ================= A 77-year-old Italian female presented for evaluation of fever, stupor, diarrhea and vomiting of 3 days duration. She had no urinary symptoms. There was no history of head injury nor of previous surgical procedures. She had been suffering from rheumatoid arthritis for 30 years, for which she was being treated with steroids and methotrexate; other medical problems were insulin-dependent diabetes and moderate renal failure. On admission, she was febrile (temperature, 38.0°C), alert but not oriented to time and place. Her neck was stiff, and she had a positive Kernig\'s sign. A CT brain scan showed an increase in subarachnoid space and in the volume of the ventricular system. Laboratory examinations revealed a white blood cell count of 15,100/μL with 70% neutrophils and 23% lymphocytes. Results of urinalysis were unremarkable. Cultures of blood and urine were drawn and subsequently resulted negative. The patient\'s cerebrospinal fluid (CSF) was opalescent with a glucose concentration of 14 mg/dl, a protein level of 472 mg/dl, and a white cell count of 200/μL with 95% polymorphonuclear leukocytes and 5% lymphocytes. Gram staining of CSF revealed no organisms. Pending the culture results the patient was empirically treated with intravenous meropenem, cotrimoxazole, acyclovir and dexamethasone. Culture of CSF yielded E. casseliflavusthat was identified using the Vitek-2 system (bioMérieux-Vitek) on the basis of 6.5% NaCl tolerance, bile-esculin hydrolysis, and growth rate at 45°C, arginine hydrolysis, methyl-a-D-glucopyranoside testing, and acid production from ribose, motility testing, and yellow pigmentation testing. The isolate was sensitive to penicillin, ampicillin, ampicillin-sulbactam, imipenem, teicoplanin, tetracyclines and linezolid; it exhibited intermediate sensitivity to vancomycin (MIC, ≥8 μg/mL), trimethoprim-sulfamethoxazole (MIC, ≥10 μg/mL), levofloxacin (MIC, 4 μg/mL), norfloxacin (MIC, 8 μg/mL), ciprofloxacin (MIC, 2 μg/mL) and quinupristin-dalfopristin (MIC, 2 μg/mL); it was resistant to clindamycin (MIC, 4 μg/mL) and showed high resistance to gentamicin, streptomycin and kanamycin (MIC, ≥2000 μg/mL). The patient became afebrile 48 hours after the beginning of antibiotic therapy with rapid improvement of her mental status and disappearance of meningeal signs (within 36 hours). Once the organism was identified (4 days later), trimethoprim-sulfamethoxazole and acyclovir were discontinued and ampicillin-sulbactam (3 g every 6 hours) was added. After 2 weeks of antibiotic therapy the patient was discharged in good health with sterilization of the CSF culture. An echocardiogram revealed no vegetations whereas a colonoscopy examination showed two ulcerative lesions associated with two polyps, oedema and multiple punctuate erosions. Enterococcal infections of the central nervous system are quite rare and according to a MEDLINE search of the English literature only three cases of CNS infection by a motile Enterococcusidentified as E. gallinarumhave been previously documented; they occurred in patients with ventriculoperitoneal shunts for hydrocephalus \[[@B6],[@B7]\]. E. casseliflavusand E. gallinarumare responsible for 1--2% of all enterococcal infections and are characterized by the fact that they possess intrinsic low-level vancomycin resistance \[[@B8]\]. The VanC-1 ligase is specific for E. gallinarum, and the VanC-2/3 ligase is specific for E. casseliflavus\[[@B9]\]. Organisms with resistance to VanC remain susceptible to teicoplanin. This naturally occurring vancomycin resistance has not been shown to be transferable, and the related genes are chromosomally encoded in the members of these species \[[@B4],[@B9],[@B10]\]. Despite the intrinsic low-level vancomycin resistance exhibited by E. casseliflavusit is important to remember that most strains are susceptible to penicillin and ampicillin. Combination therapy of ampicillin with an aminoglycoside such as gentamicin or streptomycin is considered the standard therapy of enterococcal meningitis due to ampicillin-susceptible strains \[[@B1]\]. Meropenem is not superior to ampicillin for therapy of enterococcal infections and most species of E. casseliflavusare beta-lactamase negative. There was not therefore a clear indication for the use of combination therapy with meropenem and a beta-lactamase inhibitor in the case reported. Our patient had gastrointestinal signs and symptoms, and colonoscopy revealed multiple erosions, which probably were the portal of entry of E. casseliflavus. In fact, enterococcal meningitis may appear as a complication of diverse gastrointestinal diseases such as enterocolitis, peritonitis, abdominal surgery, or bowel carcinoma. A case of bowel erosions as the portal of entry of enterococcal meningitis has been previously reported \[[@B11]\]. Several studies have demonstrated that E. gallinarumand E. casseliflavuscolonize the gastrointestinal tracts of both hospitalized individuals and nonhospitalized healthy ones \[[@B8],[@B12],[@B13]\]. Therapy with various antimicrobial agents, including cephalosporins and vancomycin, may play a role in increasing colonization with these organisms. Edlund et al. reported a significant increase in the emergence of E. gallinarumand E. casseliflavusin healthy subjects who were administered oral vancomycin \[[@B14]\]. Our patient was not taking any antimicrobials before hospital admission. E. gallinarumand E. casseliflavus/flavescensare part of the normal stool flora of the general population; this has perhaps impacted the ability of researchers to detect specific risk factors \[[@B8]\]. Although our patient had significant underlying conditions this case of meningitis was mild and had most of the typical features of \"spontaneous\" enterococcal meningitis (community-acquired infection, severe underlying diseases, and immunodepression). The clinical significance of the enterococci that are intrinsically resistant to vancomycin has not been fully established yet. Infection with E. gallinarumand E. casseliflavushas been associated with high mortality, but it is difficult to attribute their mortality directly to infection or to the underlying conditions of the patient. Conclusions =========== This case is the first report of E. casseliflavusmeningitis and it is the fourth documented so far with a motile Enterococcusspecies. Awareness of infection of central nervous system with Enterococcusspecies that possess an intrinsic vancomycin resistance should be increased. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= IC and RDL carried out the clinical study of the patient and conceived of the study. GS and GBC carried out the microbiologic studies. AT and AC carried out the clinical study of the patient, conceived of the study and drafted the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/5/3/prepub> Acknowledgements ================ Written consent was obtained from the patient for publication of study	PubMed Central	2024-06-05T03:55:52.427698	2005-1-14	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547911/", "journal": "BMC Infect Dis. 2005 Jan 14; 5:3", "authors": [ { "first": "Chiara", "last": "Iaria" }, { "first": "Giovanna", "last": "Stassi" }, { "first": "Gaetano Bruno", "last": "Costa" }, { "first": "Rita", "last": "Di Leo" }, { "first": "Antonio", "last": "Toscano" }, { "first": "Antonio", "last": "Cascio" } ] }
PMC547912	Background ========== Unilateral vocal fold paralysis is symptomatic when it results in failure of the mobile vocal fold to approximate the paralyzed vocal fold during adduction. Despite the lack of movement, the paralyzed vocal fold will often contact the contra lateral mobile vocal fold permitting adequate glottic closure \[[@B1]\]. Medialization laryngoplasty is a common procedure used to restore glottic competence. This procedure was popularized by Ishiki and initially used in patients with vocal fold paralysis. In recent years, the indications for this procedure have expanded to include most forms of glottic incompetence, including the use of bilateral medialization in mobile vocal folds for vocal fold bowing and atrophy \[[@B2]\]. Although medialization is now widely accepted, the choice of implant material is still a subject of controversy. The ideal vocal fold injection material would be readily available, have excellent biointegration with no or minimal immunologic response, has an excellent biomechanical in vivo match to the injection site tissues, and is deliverable through a fine-gauge needle. Such a material does not presently exist. Teflon (Polytetrafluoroethylene) was the first modern material used for vocal fold injection. Long-term results, unfortunately, have shown an unacceptably high rate of granuloma formation associated with Teflon injection. This results in a gradual degradation of the injected material by foreign body giant cells and fibroblastic proliferation in the location of the implant \[[@B3]\]. Autologous tissues such as fat or fascia are well described. These tissues have the benefit of medializing the vocal fold with minimal tissue reaction \[[@B4],[@B5]\]. Collagen injection has been used with some success, its major drawback is the need for serial injections in some cases \[[@B2]\]. Recently, an expanded polytetrafluroethelyne implant (e PTFE, Gore-Tex^®^) has been advocated as an implant material for medialization of the vocal fold \[[@B6]\]. This material has been widely used in cardiac- and vascular surgery and soft tissue augmentation with minimal complications \[[@B2]\]. Cashman et al published a histological study of changes in the rabbit larynx in response to Gore-Tex implantation. They found evidence of a foreign body giant cell reaction and a thin rim of fibrous tissue surrounding the implant. Although the fibrous capsule invaginated between the folds of the implant, it did not appear to grow into the implant itself. The implant was secure in the soft tissue with no migration or spontaneous extrusion noted \[[@B2]\]. Case presentation ================= A 64-year-old female patient underwent transcervical excision of a right vagal neurofibroma. This was complicated by right vocal fold paralysis with dysphonia and aspiration. Six months later, the patient underwent fat injection for medialization of the right vocal fold. After an initial improvement, the patient suffered a recurrence of the aspiration, chest infections and dysphonia. The patient subsequently underwent right medialization using Gore-Tex implant (Gore Thyroplasty Device, Medtronics, Xomed^®^) with cricothyroid approximation. Videofluroscopy and Speech assessment confirmed an improvement in aspiration and speech. Six months later, her symptoms recurred with subsequent aspiration pneumonia. A decision was made after lengthy counseling to proceed with a laryngectomy 13 months following the implantation of Gore-Tex. Figure [1](#F1){ref-type="fig"} shows the Gore-Tex in the laryngectomy specimen. Histopathology -------------- Light microscopy revealed a mild foreign-body giant cell granulomatous reaction with some associated fibrosis (Figures [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}). The granulomatous response was limited to the periphery of the Gore-Tex and although it closely followed the profile of the material it did not encroach into or significantly break up the material. However, some fibrosis penetrated a short way into the Gore-Tex in a few areas. The giant cells were present mainly in a multinucleated form. Figure [4](#F4){ref-type="fig"} shows the Gore-Tex strip under crossed Polaroids showing birefringence of the implanted material. Birefringent fragments were only very occasionally seen within isolated giant cells. The giant cell response was mainly seen in areas where the end of a Gore-Tex strip formed a sharp corner. In one area a very small focus of metaplastic bone formation was seen. There was no significant lymphocytic, neutrophilic inflammation or vascular ingrowth. Discussion ========== Gore-Tex has been used as an effective implant for medialization laryngoplasty in the management of paralytic dysphonia and to a lesser extent aspiration \[[@B6],[@B7]\]. Animal studies involving rabbits and a porcine model have demonstrated host tolerance of the implant, with no evidence of granuloma formation, resorption, extrusion or migration of the implant \[[@B2],[@B8]\]. However, adverse responses to Gore-Tex in humans requiring implant removal from the lips, face, nose and larynx have been reported \[[@B9]-[@B12]\]. To date, there have been no reports describing the histological changes in a human laryngectomy specimen with a Gore-Tex implant. Conclusions =========== Our findings are consistent with the animal models confirming that Gore-Tex implantation does not result in a significant granulomatous reaction in the human larynx over a 13-month period. Moreover, there is no evidence of resorption or infection. Further, the lack of lymphocytes in association with the granulomas indicates that there is no significant immunological hypersensitivity. Histologically, the slight permeation by connective tissue is similar to that seen in Gore-Tex vascular and cardiac implants. The degree of the slight giant cell response appears to be dependent on the profile of the material; a sharp edge incited more of a response than a flat surface. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= All authors equally contributed towards the background research and writing of the paper. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6815/5/1/prepub> Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Laryngectomy specimen showing Gore-Tex (arrow) as a pale nodule in the paraglottic space lying beneath an intact mucosa. ::: ![](1472-6815-5-1-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Low power magnification showing broad interlacing bands of Gortex material (white arrow) surrounded by mild foreign-body giant cell granulomatous reaction with some associated fibrosis (black arrow). ::: ![](1472-6815-5-1-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### High power magnification showing only a small number of multinucleated foreign-body type macrophage giant cells (arrowhead) associated with the periphery of the Gortex bands (arrow). There is little evidence of breakdown of Gore-Tex. ::: ![](1472-6815-5-1-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Gore-Tex under crossed polaroids showing birefringence (arrow). ::: ![](1472-6815-5-1-4) :::	PubMed Central	2024-06-05T03:55:52.428587	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547912/", "journal": "BMC Ear Nose Throat Disord. 2005 Jan 21; 5:1", "authors": [ { "first": "K", "last": "Rajkumar" }, { "first": "HS", "last": "Khalil" }, { "first": "M", "last": "Elloy" }, { "first": "E", "last": "Sheffield" }, { "first": "DL", "last": "Baldwin" } ] }
PMC547913	Background ========== In the post-genomic era, biologists encounter a flood of information derived mainly from microarray experiments. The blessing of this wealth of information is accompanied by a great difficulty in identifying the biologically significant findings, which are often embedded in irrelevant information. Currently, there are several approaches to deal with this problem. One approach is to identify a category of genes which is overrepresented in the microarray output. This approach can be carried out using the Gene Ontology project (GO) which describes gene products in terms of their associated biological processes, cellular components and molecular functions \[[@B1]\]. The advantage of this approach is that it can be easily automated and thus can be used for quick screening of large outputs. On the other hand, this approach limits the analysis to the structure of the GO project and thus does not support the desire of many researchers to customize their analysis. A second approach involves searching the literature for information about each of the genes on the list. Although this approach is comprehensive, it suffers from many downsides: it is time consuming; there is no systematic way to integrate the information learned about each gene; usually one gets distracted with seemingly interesting comparisons early on during the literature search and thus does not give the genes at the end of the list the same weight that was given to genes that appear at the top of the list; there are multiple names and symbols for each gene and thus it is hard to extract the literature information for any particular gene since each author may refer to it differently. A third approach entails curated databases that have gathered all the known information pertaining to each gene. This approach is limited by the quality of the curation process. For example for studying the yeast Saccharomyces cerevisiae, there are excellent curated databases, such as the Yeast Proteome Database \[[@B2]\] and the Saccharomyces Genome Database \[[@B3]\], which contain all the known information about each gene. On the other hand in other organisms the curation procedure is at a less advanced stage and thus the information contained in the curated databases is still partial. We have developed an analysis tool that combines the advantages of all the mentioned approaches and overcomes some of the disadvantages. Our tool (MILANO -- Microarray Literature-based Annotation) uses an automatic search of literature databases for performing custom annotation of the list of genes obtained from a microarray output. This is done by generating dynamic annotations for genes, built according to terms provided by the researcher. The program receives as input a list of gene identifiers obtained from any microarray experiment and a set of custom search terms. The program expands each gene identifier to its informative synonyms and searches literature databases for co- occurrences of every gene on the list with each of the custom terms. The program\'s output is an annotation table with the numbers of publications for each gene-term combination (hit-counts). This novel annotation format can be easily used within a web browser or a spreadsheet program to quickly identify genes within the list that are related to the terms provided by the researcher, and may be easily extended, as every hit-count in the annotation is a hyperlink to the query\'s results. The great advantage achieved by this method over standard static annotations, such as Gene Ontology (GO) annotations, is that the annotations are generated based on terms provided by the researcher, and therefore help in addressing the specific scientific question the researcher is pursuing. The program is able to search two literature databases, GeneRIF \[[@B4]\] and Medline \[[@B5]\]. GeneRIF contains \~90,000 short summaries of curated articles relevant to known genes. An initial search of the microarray results against the GeneRIF database provides results within minutes and is easily evaluated, thereby providing immediate insights to the microarray results. This search is followed by a comprehensive Medline search via Pubmed, allowing the identification of more subtle biological insights. To demonstrate the power of this strategy, we have analyzed a list of 148 genes affected by over-expression of p53 \[[@B6]\]. Our analysis assisted in retrieving from the list 11 known p53 targets, which are all the known targets in the list, and in identifying within the p53-affected genes a subset of putative p53 target genes that are known to be involved in apoptosis (43 genes), in cell cycle arrest (21 genes), and in Cancer (48 genes) as shown in Figure [3](#F3){ref-type="fig"}. This example demonstrates the usefulness of our tool in narrowing down microarray results to a small list of genes involved in a specific biological activity. Implementation ============== Web Interface ------------- MILANO is accessed through a familiar web form (Fig [1A](#F1){ref-type="fig"}). A CGI (Common Gateway Interface)-based Perl \[[@B7]\] program is executed on submission, which creates the combined Boolean searches for the requested databases. The user can decide whether to provide gene symbols directly, or provide LocusLink/Gene numbers, which are expanded to synonyms as described below. Results, formatted as an HTML table, are displayed immediately on-screen for GeneRIF searches and sent by e-mail for Pubmed searches. Synonym expansion ----------------- Gene aliases are collected from the LocusLink database file, downloaded from the NCBI ftp server \[[@B8]\]. We use an awk \[[@B9]\] program to extract gene symbols, aliases and product names. The alias collection is then processed by a Perl program that removes symbols that are shorter then three characters or that appear in a 23,000-word English dictionary, enhanced for scientific terms. This database is stored in a fashion than enables us to extract processed aliases for a gene by its LocusLink number. Pubmed searches --------------- Pubmed searches are performed by a Perl program which uses the NCBI eutilities esearch web service for accessing the Pubmed database \[[@B10]\]. There are limitations on when and how often we can query the NCBI server, so we integrated into the program a mechanism that makes sure that is does not make more than one query every three seconds. The Generic NQS (Network Queuing System) \[[@B11]\] ensures that jobs that include more than 100 queries run only between 9 p.m. and 5 a.m. ET. GeneRIF searches ---------------- The GeneRIF collection is automatically downloaded weekly from the NCBI ftp server \[[@B12]\], and processed by a Perl program to include gene symbols from the synonym expansion database into every GeneRIF. The database is then indexed by a database server (SRS 7.1.3, Lion Bioscience AG), which provides a query interface for counting and displaying GeneRIF entries. Results ======= Expanding the search terms -------------------------- One of the major problems in the automation of literature searches is the ambiguity in gene names \[[@B13]\]. Multiple names are used in the literature for any specific gene and thus it is not straightforward to define the Medline query that will find most of the relevant information on a gene. In order to overcome this problem we used the LocusLink database \[[@B14]\] to expand any gene symbol to all its synonyms. We also included in the expanded form the gene product name since many genes are mentioned in the literature by their product name and not by one of their symbols (for example most of the citations for the beta actin gene can be found by searching the Medline with the term \"beta actin\" and not with its official symbol \"ACTB\"). Although expansion of the search terms is a useful tool to increase the number of articles retrieved for each gene it also adds many irrelevant articles due to the fact that some of the gene symbols are not informative as Medline query terms. For example one of the aliases of the gene aquaporin\_1 is CO, a term that is mostly mentioned as an abbreviation for Carbon mono-oxide, and one of the aliases of the gene CD36\_antigen is FAT, which is found in over 100,000 articles, unrelated to CD36. In order to diminish this problem we filtered out from the list of gene symbols any term that was shorter than three characters and any term that is an English word. In order to check our name expansion strategy we conducted a Medline search for 16862 well-known human genes (all the genes that have an NM number indicating the identification of their full length mRNA), using three search strategies: using only the official symbol for each gene (Symbol), using the official symbol together with all its aliases and the gene product (Expanded) and using only the informative terms (Filtered). Using the Expanded search allowed the identification of literature information on about \~1900 additional genes over a query using the official symbol only (Table [1](#T1){ref-type="table"}). Using the Filtered search terms allowed this addition without adding significantly to the number of queries that returned non-reasonable results. In addition to expanding the number of genes that were found in the literature, the Filtered search terms also increased the number of articles found per gene (from an average of 198 articles per gene found by searching with the symbol alone to an average of 451 articles per gene when searching with the filtered terms). These results indicate that our gene name expansion strategy achieves a higher percentage of relevant literature for each gene while limiting the addition of irrelevant information. Conducting automatic literature searches ---------------------------------------- After expanding the search terms, MILANO performs an automatic search of literature databases, and retrieves the number of hits each query returned. MILANO performs Boolean searches in which one can search for co-occurrence of each of the primary terms (the expanded gene name) with user defined secondary terms (Figure [1](#F1){ref-type="fig"}). The program\'s output is a table (Figure [2](#F2){ref-type="fig"}) containing the number of publications for each gene-term combination (hit-counts). This table could serve as an annotation table, because the number of publications reflects the relationship between the genes to the secondary term used. For example a gene that has a role in DNA damage will appear in more articles about \"DNA damage\" or \"gamma irradiation\" than unrelated genes. In order to assist in further evaluation of the results, we have built the annotation table such that each number in the table is a hyper-link to the literature database and thus clicking on it will perform this specific search again and will open a window containing the actual abstracts found by this combination of search terms. Literature databases supported by the program --------------------------------------------- The MILANO program can search two databases (Figure [1](#F1){ref-type="fig"}) -- the full Medline database, currently containing more than 12,000,000 references, and the GeneRIF database that contains more than 90,000 short summaries of curated articles relevant to known genes. There are several advantages in using the GeneRIF database over the full Medline: the searches are quick and the results are obtained within minutes; each article is summarized by a sentence or two, reducing the amount of information that needs to be read; the curation procedure extracts from the papers only the information relevant to the gene, minimizing the cases in which two terms appear in the same abstract but are not related to each other; the GeneRIF entries are based on the full text of the articles and not only on the abstracts. However, since the curation procedure is an on-going process, the coverage of this database is only partial and thus information is missing and can be found only by performing a Medline search. For that reason our tool allows a combined search strategy in which both databases can be searched simultaneously. The GeneRIF database provides results within minutes and is easily evaluated, thereby providing immediate insights to the microarray results. In parallel a comprehensive Medline search can be done. Although this search takes longer and its results obtained by email, it allows the identification of more subtle biological insights. P53 --- To demonstrate the power of our literature-based annotation strategy, we analyzed a list of 148 genes affected by over-expression of p53 \[[@B6]\]. This list of genes was obtained by microarray experiments and nicely demonstrates the difficulty of microarray analysis since it contains many putative p53 target genes and their relevance to p53 cellular activity is not clear. Our first aim was to identify the known p53 target genes that were affected by p53 overproduction in this experiment. By using specific secondary terms, we were able to trim down the list of 148 genes to a much shorter list of genes highly enriched for known p53 target genes (Figure [3A](#F3){ref-type="fig"}). In order to evaluate the number of target genes that were missed by our annotation strategy, we manually compiled a list of all known p53 target genes, \~60 genes. Eleven of these 60 genes were represented in the list of genes affected by over-expression. Our automatic annotation strategy found all of them. Moreover, the use of MILANO reduced the amount of articles per gene from an average of 2088 articles per gene in the initial list to 56 articles per gene in the limited list (Figure [3B](#F3){ref-type="fig"}). The p53 example also demonstrates the usefulness of searching the GeneRIF curated database in which the use of the secondary term p53 allows filtering out most of the irrelevant genes without losing any known target gene (Figure [3A](#F3){ref-type="fig"}). P53 is involved in apoptosis, cell cycle arrest and cancer. It is interesting to find out which of the genes affected by p53 is involved in these processes. Using MILANO we easily identified genes known to be involved in these processes (Figure [3C](#F3){ref-type="fig"}), which helped the process of analyzing the microarray data. Comparison with other tools --------------------------- Recently, few literature mining tools has been developed, using a similar approach to the one presented here \[[@B15]-[@B17]\], however all of them suffer from the problem of inappropriate use of primary search terms. In order to demonstrate the advantage of using MILANO over the other tools, we have performed a comparative analysis of all these tools by looking at their performance on the 11 known p53 target genes described above. The software were run with these 11 genes as the primary search terms and \"P53\" as the secondary term and reported the number of occurrences of those terms. The results that are summarized in table [2](#T2){ref-type="table"} demonstrate that MILANO-GeneRIF search was the only method that revealed connections between all 11 genes and p53 and that the MILANO-Medline search gave the most comprehensive search results. PubMatrix \[[@B15]\] does not expand the primary search terms and thus it misses many literature occurrences. This problem is best demonstrated by its poor performance on the CDKN1A gene which is one of the most studied targets of p53. The synonym expansion methods used by MicroGENIE \[[@B16]\] improved the results regarding the CDKN1A gene, but missed the SFN gene completely, and gave non-informative synonyms to XRCC5 and TRAF4 (\"Ku\" and \"TNF\" respectively). BEAR GeneInfo \[[@B17]\] did not perform synonym expansion correctly for CDKN1A, and gave non-informative synonyms for PCNA and TRAF4 (\"cyclin\" and \"h. mln62 mrna\" respectively). When we attempted to analyze the full data set of 148 genes, some of the compared tools failed to give results due to errors. Discussion ========== MILANO is a simple and intuitive literature search tool. It allows automatic Medline and GeneRIF searches followed by a quick survey of the results. Using this tool dramatically reduces the time needed to query literature databases. Moreover, due to its systematic nature, it assists in treating the 1^st^and the 100^th^query in an unbiased manner. The MILANO program uses all the published information for the annotation of each gene according to its co-occurrence in the literature with a user defined secondary search term. These features of MILANO makes it especially suitable for analyzing microarray results, since it can be used to annotate the results with terms defined by the user and not limited by preset terms such as the GO terms based annotation. We have demonstrated the power of our program by the analysis of a list of 148 genes that were deregulated in cells that overproduced the p53 tumor suppressor gene \[[@B6]\]. Frequently one of the first tasks in microarray data analysis is to determine the overlap between new results and results expected based on the literature. For example in analyzing the list of genes induced by over expression of p53 one expects to find known p53 target genes. Thus, we applied our automatic literature search tool in order to answer this question. We found that use of this tool dramatically shortens the time needed for such an analysis by allowing the researcher to focus on a relatively small subset of potential target genes and by reducing the amount of literature relevant to each gene (Figure [3](#F3){ref-type="fig"}). Our tool was also found useful in automatically sorting the target genes into functional groups. Based on the knowledge of p53 cellular functions we defined secondary search terms that fit p53\'s main activities -- apoptosis and cell cycle arrest \[[@B18]\]. Using these terms allowed the quick identification, from the primary list, of a subset of genes that were not known to be involved in those processes and thus may be interesting for further research (Figure [3C](#F3){ref-type="fig"}). Several literature mining approaches have been developed to integrate multiplex biological datasets into the context of published medical literature. A good example of such an approach is the PubGene program \[[@B19]\], which searches for literature co-occurrences of gene names in order to build a network among the genes. PubGene is useful for quickly realizing and viewing known relationships between genes, but it does not assist in annotating gene lists. To this end one needs an automatic literature searching tool that allows the use of flexible secondary terms with which co-occurrences are counted. Recently such tools have been built. PubMatrix \[[@B15]\] allows automatic Boolean searches to be performed on Pubmed using any list of primary and secondary terms. This tool carries out the search on the exact terms entered by the user thus in order to apply it to the analysis of microarray data, one has to translate each of the enriched spots to a name suitable for a Medline search. Two other tools -- microGENIE \[[@B16]\] and BEAR GeneInfo \[[@B17]\] uses a very similar approach but in order to make it more compatible to microarray analysis, they allow the use of gene identifiers as input and provides the needed translation to gene names. During the translation the gene name is expanded to include its synonyms. All of these tools have improved the ability of researchers to quickly use the published literature to annotate lists of genes. However, they suffer from the limitations of any literature data search tool; the ambiguity of gene names and the partial information that can be retrieved by limiting the literature searches to abstracts \[[@B13]\]. MILANO\'s aim is to further improve the literature based automatic annotation approach by adding two essential features that address these limitations: Smart synonyms -------------- Each gene symbol is expanded to all its aliases, while removing non-informative terms, and the gene product name is added to the query. This addresses the synonym problem, while omitting many of the irrelevant results, thus reducing the polysemy problem (words with multiple meanings). The advantage of our synonym expansion scheme over the existing tools is demonstrated by the comparison presented in table [2](#T2){ref-type="table"}. The GeneRIF database -------------------- In contrast to the existing tools, MILANO is able to search not only the Medline database, but also the GeneRIF database, which contains short summaries of articles relevant to known genes. The curation of GeneRIF is done by the National Library of Medicine\'s MeSH indexing staff, who have advanced degrees in the life sciences and use the full text of articles for the indexing process \[[@B4]\]. Using this database reduces the limitations of relying only on abstracts and aids in finding only relevant information about each gene. Nevertheless, the GeneRIF database suffers from the problems of all manually curated databases; it is partial and contains mistakes and biases introduced by the curation team. However, our ability to identify all of the p53 target genes within a group of p53-affected genes by using the GeneRIF database alone (Figure [3](#F3){ref-type="fig"}) demonstrates that, at least for well annotated genes, using such a database may be the ideal solution for annotating microarrays results. The quality of GeneRIF-based annotation depends on the amount of information entered for each gene in the GeneRIF database, which for many genes is insufficient (data not shown). However, its performance will improve as more information is incorporated into this database and we believe that in the future it will become the preferred annotation tool. Meanwhile, we recommend using MILANO for performing combined searches; searching the GeneRIF database provides quick results and searching the full Medline database allows a broader view that is not limited by the curation procedure. Conclusions =========== We present MILANO <http://milano.md.huji.ac.il>, a literature mining tool that can help in annotating microarray results in light of all available literature using experiment-specific terms. In designing MILANO we focused on the accuracy of the search results by providing two novel features: i) Expansion of gene names to include in the literature searches all their informative synonyms, while removing non-informative synonyms; ii) Searching two literature databases -- Medline and GeneRIF. While Medline encompasses all the literature and provides the most comprehensive results, it also contains many irrelevant articles. GeneRIF provides a subset of Medline articles that are relevant to known genes and thus avoids most of the irrelevant results often found in Medline searches. The usefulness of MILANO is demonstrated by the automatic analysis of a list of 148 p53 target genes. The use of literature mining dramatically reduced the time and effort required for a task such as identifying the known p53 target genes within this list. A search in GeneRIF immediately discovered the full list of target genes, with no false hits. Availability ============ All software and databases are freely available and may be executed online at our web site: <http://milano.md.huji.ac.il>. The author will provide data, scripts and programs used on demand. We encourage users to install the software on their own servers, as we provide no assurance to the privacy or accuracy of the results. Authors\' contributions ======================= RR designed and programmed the software. IS managed the project and drafted the manuscript. Acknowledgments =============== We would like to thank Zahava Siegfried for critical readings of the manuscript. R.R. was supported by a fellowship from the Sudarsky Center for Computational Biology <http://www.cbc.huji.ac.il>. This research was supported by grants from the Association for International Cancer Research (AICR) and by the F.I.R.S.T. (Bikura) program of the Israel Science Foundation (Grant No. 4103/03). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The MILANO data input page <http://milano.md.huji.ac.il>. ::: ![](1471-2105-6-12-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### An example of a result of MILANO search using a short list of gene symbols that were expanded by the program to include all their informative synonyms versus p53 related terms. All reported numbers are hyperlinked and will initiate a new search for that specific term combination. ::: ![](1471-2105-6-12-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Analysis of a list of genes affected by p53 overproduction.A. The number of genes remaining after filtering the p53-affected genes with terms intended to reveal known p53 targets. B. Average number of articles per gene in the different queries. C. Venn diagram depicting the different functions of p53 affected genes as reflected by a GeneRIF search. ^1^Search term is \"p53 AND (target OR transcriptional OR activation OR repression)\" ::: ![](1471-2105-6-12-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Summary of Medline hit counts for all the full length mRNA genes (16,862 genes) using different search strategies. ::: Type of primary term^a^ Positive results^b^ Non reasonable results^c^ Articles per gene^d^ ------------------------- --------------------- --------------------------- ---------------------- Symbol 10,045 20 198 Expanded 12,028 140 817 Filtered 11,910 22 451 ^a^The Medline search was conducted using three searching strategies: Symbol refers to a search in which each gene was represented by its official symbol; Expanded refers to searches in which each gene was represented by the gene symbol, all its synonyms and the official gene product name; Filtered refers to searches in which non informative names were filtered out of the expanded list. ^b^Number of queries that returned at least one result. ^c^Number of queries that returned more than 33,000 results. We used 33,000 as a rough estimate of non reasonable results based on the fact that some of the most investigated genes, like p53, appear in less than 33,000 abstracts. ^d^The average number of abstracts per gene counting only genes that appeared at least once and did not appear in more than 33,000 abstracts. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Comparative analysis of literature mining tools. Eleven known p53 target genes were analyzed using five methods. The numbers represent the number of reoccurrences of each gene with the term \"P53\". ::: Gene Id Primary Symbol MILANO -- GeneRIF^a^ MILANO -- Medline^a^ PubMatrix^b^ BEAR GeneInfo^a^\[17\] MicroGenie^c^ -------------------- -------------------- -------------------------- -------------------------- -------------------- ---------------------------- -------------------- [1026]{.underline} CDKN1A [74]{.underline} [4180]{.underline} [49]{.underline} [50]{.underline} [3058]{.underline} [1647]{.underline} GADD45A [7]{.underline} [281]{.underline} [21]{.underline} [45]{.underline} [313]{.underline} [2810]{.underline} SFN [2]{.underline} [25]{.underline} [25]{.underline} [25]{.underline} 0 [3486]{.underline} IGFBP3 [1]{.underline} [42]{.underline} [36]{.underline} [42]{.underline} [36]{.underline} [355]{.underline} TNFRSF6 [25]{.underline} [740]{.underline} [418]{.underline} [43]{.underline} [707]{.underline} [4583]{.underline} MUC2 [2]{.underline} [29]{.underline} [29]{.underline} [29]{.underline} [29]{.underline} [4609]{.underline} MYC [21]{.underline} [1715]{.underline} [1715]{.underline} [1715]{.underline} [1715]{.underline} [5111]{.underline} PCNA [13]{.underline} [1269]{.underline} [1173]{.underline} [3671]{.underline} [1173]{.underline} [59]{.underline} ACTA2 [1]{.underline} 0 0 0 0 [7520]{.underline} XRCC5 [1]{.underline} [52]{.underline} [42]{.underline} [42]{.underline} [583]{.underline} [9618]{.underline} TRAF4 [2]{.underline} [1]{.underline} [1]{.underline} [1570]{.underline} [326]{.underline} ^a^The search was performed with LocusLink ids as the primary search terms. ^b^The search was performed with the primary gene symbols as the primary search terms. ^c^The search was performed with UniGene ids as the primary search terms. :::	PubMed Central	2024-06-05T03:55:52.429338	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547913/", "journal": "BMC Bioinformatics. 2005 Jan 20; 6:12", "authors": [ { "first": "Ran", "last": "Rubinstein" }, { "first": "Itamar", "last": "Simon" } ] }
PMC547914	Background ========== In the last quarter century, more than five hundred published manuscripts contained experiments using the popular rat crypt cell lines IEC-6 and IEC-18 originally established by Quaroni and Isselbacher in 1978 and 1981 \[[@B1],[@B2]\]. These lineages have stood the test of time as being two of the most reliable and phenotypically well-preserved gastrointestinal cell lines to date. In our lab, we utilize the IEC-18 lineage because it is more robust in serum-restricted conditions and, in our experience, it is more suitable for correlation with distal intestinal crypt and enterocyte physiology than cell lineages derived from colonic tumors. One of the more exciting aspects of IEC-18 cells is their capacity to retain the crypt cell phenotype as well as the continued potential for differentiation/maturation into enterocytes \[[@B3]-[@B5]\]. However, as we have worked with this lineage over the years, the rare but unmistakable morphology of an occasional neuroendocrine-like cell, the suspicious frothiness of a possible paneth-like cell and the rare giant vacuole of a goblet cell precursor have prompted us to wonder how prevalent spontaneous differentiation towards other lineages and/or transformants might be (especially in serum-restricted conditions). This is a fairly crucial experimental question because serum-restricted conditions allow the tightest experimental rigor but may drive IECs towards altered cell fates, potentially introducing uncontrolled experimental confounders. Until recently, we did not have the necessary tools with which to determine more subtle epigenetic divergences in IECs. Our current investigation uses a novel strategy to demonstrate that spontaneous cell fate divergence is not only highly prevalent in IEC-18 cells, but it is fairly uniform and therefore a predictable confounder for gene array studies in this cell line. Results ======= Initial characterization of IEC-18 culture heterotypy using immunolocalization ------------------------------------------------------------------------------ We have recently discovered a novel antigen localization pattern by using anti-carboxyl IGF binding protein-2 (IGFBP-2) antibody (to an antigen which we call C2) to demonstrate a multivesicular pattern in some IEC-18 cells but not in others that are immediately adjacent (illustrated in Figure [1A](#F1){ref-type="fig"}). We note that anti-amino IGFBP-2 showed no staining in IEC-18 cells and that pretreatment with synthetic antigen abolished C2 staining (data not shown), confirming that C2 is a carboxyl fragment of IGFBP-2. For the purpose of this study, we wanted to determine whether or not this heterotypy represented cell fate divergence or systemic pleiomorphism. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Immuno-characterization of IEC-18 cell heterotypy. A: Carboxyl IGFBP-2 immunostaining of the sequestered C2 fragment in cells with a preserved crypt cell phenotype but not in others. B: F-actin immunostaining (performed without antigen retrieval to visualize dynamic actin filaments) demonstrates intense staining in crypt cells but not in others. C: Cells plated at variable density with 10% FBS start out as weakly C2 positive (i) but have a progressive loss of C2 immunostaining prior to confluence (ii-iv). D: Immunolocalization of the C2 antigen demonstrates that both C2 positive (i) and C2 negative (ii) cells preserve their phenotypes during proliferation. E-F: Control immunolocalization using the IGF type 2 receptor (E) as a prelysosome-localized antigen and villin (F) as a cytoplasmic-localized antigen to demonstrate that both intravesicular and cytoplasmic antigens can be evenly detected throughout IEC culture. ::: ![](1471-2121-6-2-1) ::: To further characterize the observed heterotypy, we examined IEC-18 cells with anti-actin antibody in IEC-18 cells without performing antigen retrieval and allowing the detection reaction to proceed until only a limited amount of staining is seen. When used with the right antibody, this technique is a means to visualize dynamic portions of actin filaments, such as stress fibers and the proximal ends of microvilli, because they lack the actin binding proteins that obscure the antigen. If the reaction is allowed to continue, eventually all actin filaments will stain. In the version that we use, the assay is a qualitative assessment of cytoskeletal turnover when we are comparing two adjacent cells (because both cells had exactly the same conditions for detection of actin). Both stress fibers and microvilli are heavily stained and appear to have high turnover rates in cells that have C2 staining, but not in those without it (Figure [1B](#F1){ref-type="fig"}). We note that the punctate dots of filamentous-actin corresponded with small microvilli in C2 positive cells and that the C2 negative cells had a paucity of microvilli on their surface -- which was confirmed by focusing up and down the shafts of the microvilli (data not shown). IEC-18 cells spontaneously lose C2 staining prior to confluence, despite 10% FBS -------------------------------------------------------------------------------- When IEC-18 cells were plated at variable densities in 10% fetal bovine serum (FBS), we saw that all crypt cells were initially positive for C2 staining, but with increasing density there were increasing numbers of C2 negative cells (Figure [1C](#F1){ref-type="fig"}). We also saw that C2 positive cells had increased staining intensity as they approached confluence. This experiment demonstrates two important points: first, that loss of C2 staining begins before confluence and second, that it can occur with comparable prevalence in the presence of FBS as it does in serum free media (SFM). C2 positive cells and C2 negative cells are both capable of phenotype preservation during proliferation ------------------------------------------------------------------------------------------------------- To determine if the C2 positive and negative IEC-18 cells were each capable of phenotype preservation during proliferation, IGFBP-2 stained cells on coverslips were searched for mitotic cells in the stage of cytokinesis and their images were captured by digital photomicroscopy (n = 6 cell pairs for each phenotype). In each case, all daughter cells had the same phenotype as their sister, confirming that both the C2 positive and C2 negative phenotypes are conserved in subsequent cycles of mitosis (representative examples in Figure [1D](#F1){ref-type="fig"}). C2 positive cell abundance is increased in proportion to the efficacy of IGF agonist treatment ---------------------------------------------------------------------------------------------- Because IEC-18 cells grow best in the presence of high dose insulin, we suspected that crypt cell proliferation was dependent upon IGF receptor stimulation. In general, differentiated and benignly transformed epithelial cells are less likely to proliferate upon reaching confluence, so we sought to preferentially drive crypt cell proliferation in a graded fashion using different IGF receptor agonists (Figure [2](#F2){ref-type="fig"}). IGF-II analog, a weak agonist, reduced the crypt cell abundance while NBI 31772 (an agent that displaces IGFs from IGFBPs) increased it significantly and R^3^-IGF-I doubled the number of crypt cells per 10X field (also highly significant) while none of the treatments significantly altered the abundance of C2 negative cells. This strategy allowed us to systematically skew the cell composition and use gene array analysis to determine whether C2 positive cells were epigenetically divergent. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Mean number of C2 positive and C2 negative cells for each treatment condition. C2 positive cells are boxed in gray and C2 negative cells in stripes. Note that IGF-II analog reduces C2 positive cells when compared to SFM, whereas both NBI 31772 and R^3^-IGF-I significantly increase C2 positive and total cell abundance when compared to SFM (\\\* = p \< 0.01, \\ = p \< 0.05, \* = p \< 0.1). In contrast, no treatment significantly altered C2 negative cell abundance. ::: ![](1471-2121-6-2-2) ::: Our gene array methodology identified four candidate genes as potential markers of cell fate divergence in IEC-18 culture ------------------------------------------------------------------------------------------------------------------------- Eleven genes met our criteria for a significant fold change, four were positively correlated with crypt cell abundance and seven were inversely correlated (Figure [3](#F3){ref-type="fig"}). Of these first pass candidates, only six showed the appropriate fold trends across all treatment conditions, consistent with our hypothesis of constitutive expression that could reflect divergent cell fates. Of these six, one was found to have a significant difference between IGF-II analog and SFM, suggesting a direct treatment effect by IGF agonists but not by NBI 31772 (dithiolethione-inducible gene 1). Enzymatic glycosylation-regulating gene is known to be an insulin responsive gene and was also excluded because of the high probability of a direct effect by our IGF agonists \[[@B6]\]. Of the four remaining candidates, one (brain acyl CoA hydrolase) had absolute values that bordered on background levels in the SFM, IGF-II analog and NBI 31772 treatment conditions (defined as 10 arbitrary fluorescent units) and has had a relatively limited characterization in the literature \[[@B7]-[@B9]\]. We saw no obvious means for obtaining or generating an antibody to it and thought it unlikely to be a robust marker at the protein level, in large part because its message has only been found in brain thus far. Another had high homology with the EGF family and is currently a predicted protein based on genomic sequence \[[@B10]\]. However, the two remaining candidate genes we pulled out were well-characterized gut-related proteins (APC and 5-HT2A). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Gene array screen using skewed IEC-18 cell composition to find potential phenotypic markers. Affymetrix rat gene chips were used to compare RNA from SFM and R^3^-IGF-I treated cells to find individual genes with significant fold changes -- defined as greater than two fold more (A) or two fold less (B) with a p-value of less than 0.1 for the purpose of this screen. These were then compared with the fold changes from IGF-II analog and NBI 31772 to look for fold change trends that paralleled the changing cell composition (summarized in C as the percentage of crypt cells). The results are presented as upward arrows (strong positive correlations), upward dashed arrows (weak positive correlations), downward arrows (strong inverse correlations) and downward dashed arrows (weak inverse correlations). The large asterisk points to an example where both direct receptor agonists resulted in a significant difference but NBI 31772 did not, suggesting a direct drug effect rather than an effect of cell composition -- making this gene a less likely candidate. For smaller asterisks, \\\* = p \< 0.01, \\ = p \< 0.05, and \* = p \< 0.1. ::: ![](1471-2121-6-2-3) ::: Immunolocalization for 5-HT2A and APC revealed 2 divergent phenotypes within IEC-18 cell culture ------------------------------------------------------------------------------------------------ Western blots using antibodies against APC and 5-HT2A in IEC-18 cell lysates revealed staining of the appropriate sized band for each (Figure [4](#F4){ref-type="fig"}). In the case of APC, there were several discrete smaller bands which were inversely proportional in abundance to the 300 kD full-sized protein (consistent with proteolytic fragments) when compared across multiple samples (data not shown). In the case of 5-HT2A, there was a single 28 kD band that was faint, suggesting relatively low abundance. Both antibodies were deemed suitable for immunolocalization. Immunolocalization for APC revealed that C2 positive cells also had intense APC staining whereas C2 negative cells either had limited or no APC staining (Figure [5A](#F5){ref-type="fig"}). This finding is in keeping with our gene array analysis and suggests that there is substantial and wide spread divergence of IEC-18 crypt cells away from the crypt cell phenotype and towards an adenoma-like transformation. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Immunolocalization of APC and 5-HT2A in IEC-18 culture. A: APC immunostaining parallels that of C2 and is found in crypt cell strands but is absent or scant in adjacent cells. B-D: 5-HT2A immunostaining is absent in the majority of cells but is found in a few rare cells. On closer inspection, we found that there seemed to be a progressive increase in staining intensity that correlated with a morphologic transition away from IEC-18 cell morphology and towards that of a neuroendocrine-like cell (illustrated by the numbered circles). ::: ![](1471-2121-6-2-5) ::: Immunolocalization for 5-HT2A demonstrated a second cell phenotype (Figure [5B,C](#F5){ref-type="fig"} and [5D](#F5){ref-type="fig"}). We had previously noted rare neuroendocrine-like morphology characterized by spindle-shaped cells with bipolar, dendritic arbors (unpublished findings), however with 5-HT2A immunostaining we found a mean of 5 positively stained cells per coverslip of confluent cells (n = 6 coverslips of confluent cells in SFM). 5-HT2A is present in high abundance within paneth and neuroendocrine cell types but is absent within small intestine crypt cells in vivo\[[@B11]\]. The cells that we observed appeared to be in transition, going from IECs to neuroendocrine-like cell morphology -- with corresponding increases in staining intensity. While still a very low prevalence, the staining was quite intense in this small subset and was not detected in any of the adjacent cells. Double labeling confirms that C2 negative cells have diminished APC abundance ----------------------------------------------------------------------------- Double immunolabeling with overlay technique (using C2 and actin antibodies) demonstrated that C2 negative cells have a paucity of microvilli in comparison to C2 positive cells (Figure [6A](#F6){ref-type="fig"}). Additionally, double labeling with C2 and APC demonstrated that C2 positive cells have uniform APC staining whereas C2 negative cell have variable and overall diminished staining in comparison (Figure [6B](#F6){ref-type="fig"}). These experiments provide an objective demonstration that C2 positive cells retain the IEC phenotype whereas C2 negative cell are undergoing transformation (which is an obligatory step associated with the loss of APC). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Double labeling overlay immunolocalization of C2 with either actin or APC. *Ai*. C2 immunostaining of wet-prepped cells. Aii. C2 immunostaining in the same cells, overlaid with f-actin immunostaining. The proximal cores of actin-stained microvilli bundles located on C2 positive cells are encircled, whereas there are either no or few microvilli on C2 negative cells. Bi. C2 immunostaining of wet-prepped cells. Bii*. C2 immunostaining in the same cells, overlaid with APC immunostaining. The dotted line divides C2 positive cells (below) from C2 negative cells (above). There is consistent APC staining in C2 positive cells, whereas there is variable and comparably reduced APC staining in C2 negative cells. ::: ![](1471-2121-6-2-6) ::: Discussion ========== In this manuscript we have taken advantage of an IEC immunolocalization marker that we call C2 to demonstrate two forms of cell fate divergence within IEC-18 culture. Using a combination of gene array screening and immunolocalization, we found that C2 is lost in over half the cells by the time of confluence and its loss is also associated with a down regulation of the APC gene, decreased APC protein abundance, decreased actin filament turn over, and reduced microvillar density. In short, there is an adenoma-like phenotype that fits with this genotype and this fits well with what is known about the function of the APC protein \[[@B12]-[@B15]\]. In addition, another cell genotype-phenotype correlate was detected by screening out the 5-HT2A gene and visualizing the cells that express its protein, i.e. neuroendocrine-like cells. These findings also fit well with cell phenotypes known to express 5-HT2A in the gut endoderm \[[@B11],[@B16],[@B17]\] and strongly argue against the dogma that IECs persist as a single lineage prior to reaching confluence. While we think our findings have important implications for the existing IEC literature, the more important aspect of this manuscript may be in the methodology we have piloted. In many ways, cell culture, whether primary or immortalized, transformed or not, is by definition a model in flux. The progenitor lineage that a researcher starts with is rarely the hodge podge they end up with after a limited number of passages and it is common, if not expected that epigenetic drift will occur with each cell culture passage. However, what we describe in this manuscript is different in that IEC-18 cells are displaying a uniform trend in cell fate divergence -- a trend that can be modulated with IGF. Many if not most gut epithelial cell lines are IGF (or high dose insulin) dependent for proliferation and thus are potentially vulnerable to this biological confounder. It will remain to be seen if other epithelial cell types have similar behaviors when examined in this fashion. Conversely, there is also a positive light to our findings; IEC-18 cells could be a compelling model for spontaneous adenomatous transformation because these adenoma-like cells are arising from a genetically competent progenitor prior to reaching confluence. To our knowledge, no such model with this property has been previously defined. Our study has notable weaknesses and strengths. First, we have identified two divergent cell fates but have only partially characterized them because we were focused on developing a viable screening methodology (hence the ubiquitous use of the word \"like\" in this manuscript). Second, we are using a rat gene array chip that has approximately 9000 non-EST genes per chip. This is not an exhaustive survey of the rat genome and it is possible that there are other cell fates present in IEC-18 culture that we did not detect. Third, our phenotype assays are based on immunolocalization, which is a semi-quantitative technique with regards to assessing protein abundance. However, in this case we are actually combining cell-to-cell differences in protein abundance with distinguishing morphologic characteristics (e.g. loss of microvilli, flattening, bipolar shape, dendritic arbors, etc.) to delineate the phenotypes between adjacent cells. In short, what we are quantifying, in the case of adenoma-like cells, is the percentage of cells with a given phenotype. For this purpose, blinded immunolocalization is simple, quantitative and exceedingly efficient. The combined methodologies we chose result in a highly accurate technique for assessing divergence and their specificity can be bolstered by comparative studies of co-divergent markers (as we demonstrated with C2 and APC). As for other positives, the methodology is relatively rapid and can detect low prevalence phenotypes (as demonstrated by the anti-5-HT2A antibody). Additionally, we have demonstrated that a transcriptional marker is not required to create an effective screen. What is required, and what should probably prompt a researcher to employ this methodology, is a probe, a phenotype, or a pleiotropism that results in a consistently heterogeneous and quantifiable pattern (as C2 proved to be for us). In closing, we point out one last caveat. Gene array investigation is an evolving science but there remain three potential pitfalls for every new application: experimental design flaws, data integrity issues and biological misassumptions \[[@B18]-[@B20]\]. In this study, we used a well-accepted screening principle (i.e. significant fold changes within individual genes in response to a treatment); we included a paired reference standard for each treatment condition (the SFM control); we increased the screening stringency by adding a requirement for parity in fold trend in accordance with changing cell compositions; and then confirmed our findings by phenotype assays. However, we demonstrated that a small minority of neuroendocrine-like cells were still able to significantly alter the outcome of our gene array screen (a possibility that we had thought to be remote, given our assay\'s stringency). We conclude that even low frequency epigenetic events can be a serious biologic confounder of gene array studies in cell culture. Conclusions =========== We have demonstrated a novel methodology for detecting and characterizing cell fate divergence in cell cultures derived from a common progenitor. The majority of IEC-18 cells are transformed into adenoma-like cells in SFM. IGF agonists reduce the rate of transformation by driving proliferation of the progenitor phenotype but do not prevent it. We also detected a very low incidence of differentiation toward a neuroendocrine-like cell type in these same cultures. Methods ======= Cell culture ------------ Rat ileal epithelial cells (IEC-18 -- American Type Culture Collection, Rockville, MD) from aliquots of passage numbers 6--8 were grown to confluence in DMEM with 10% fetal bovine serum (FBS) and .1% insulin. The time period for complete epithelial cell confluence was 24--48 hours. Confluent cells were incubated for 72 hours in DMEM to establish a serum free period either alone or with 10^-6^M NBI 31772 (Calbiochem, San Diego, CA \[[@B21]\]), 0.5 × 10^-6^M IGF-II analog, or 0.5 × 10^-6^M R^3^-IGF-I (Sigma, St. Louis MO), as a means of boosting crypt cell proliferation. We point out that crypt cells can be driven to proliferate despite confluence whereas more differentiated epithelial cell types are resistant to IGF-driven proliferation upon reaching confluence. Alternatively, IEC-18 cells were diluted to 1/8, 1/4 and 1/2 the original seeding density and plated in DMEM with 10% FBS for 24 hours as a means to evaluate cell fate divergence in the presence of serum. Western blots ------------- Cell lysate samples were collected from 150 mm dishes, washed with PBS and recovered in 700 uL of lysis buffer by scraping the dish. Cells were spun down for 10 minutes in a pre-chilled centrifuge. The supernatant was diluted 4:1 with loading buffer and run on 12 or 15% acrylamide gels. After the gel had been transferred onto a nitrocellulose membrane, 0.5% Ponceau S stain was applied to confirm equitable protein transfer across all lanes. Western blot membranes were incubated in 5% milk block for 1 hour at RT. Goat polyclonal primary antibody for APC and 5-HT2A were applied for 1 hour at RT. Primary antibody was washed off with TBS-Tween and secondary antibody was placed on the membranes for one hour, then washed again. ABC (Elite Series, Vector Labs) was placed on the membrane for an hour and then washed and visualized with SuperSignal chemiluminescent substrate (Pierce, Rockford, IL), each was prepared per the manufacturer\'s instructions. Gene array analysis ------------------- IEC-18 cells were treated and processed as described in Cell Culturemethods and then utilized to screen for specific cell fate markers. Total RNA was harvested for each treatment condition in triplicate experiments and then used for gene chip analysis per the manufacturer\'s protocol (rat gene chip \# 230A, Affymetrix, Santa Clara, CA). All available non-EST tags were searched within the R^3^-IGF-I data set and those with significantly different fold changes when compared to SFM cells were selected as potential candidates for further analysis (for the purpose of a first pass screen, a significant fold change was defined as a p value \< 0.1 and a greater than two fold increase or decrease). To further refine the list of potential candidates, the fold changes for NBI 31772 and IGF-II analog, which had step-wise reductions in effects upon crypt cell proliferation, were used to determine if there was a positive or inverse trend between a selected gene\'s mRNA abundance and crypt cell abundance. In this way, we sought to select constitutively expressed genes, whose primary differences in abundance would be due to differences in cell composition. Our statistical test for determining significance was a two-tailed paired t-test (provided as part of the standard analysis by the University of Virginia Biomolecular Research Facility and the Dept. of Health Evaluation Sciences \[[@B22]\]). Immunolocalization experiments ------------------------------ Immunolocalization in IEC-18 cells was performed for IGFBP-2 (C2), f-actin, IGF receptor type 2 and villin as well as two proteins that were screened out by our gene array analysis (APC and 5-HT2A) in a minimum of three separate experiments for each. In brief, the cells were fixed in formalin overnight, washed in DIG buffer (4% 1M Tris Base, 6% 5M NaCl, 16% 1M Tris HCl) 5 times then put in blocking solution (1% wt/vol BSA in DIG buffer) for 1 hour at room temperature. This was followed by a one-hour incubation with goat polyclonal primary antibody (all were obtained from Santa Cruz Biotechnology, CA; specifically for the anti-actin antibody the product number was sc-1615) in antibody diluent solution (5% 1M Tris HCl, 1% 1 M Tris Base, 9% NaCl, 3.3% Triton-X 100). Primary antibody was washed off with DIG × 5 and secondary antibody (donkey anti-goat \[Jackson Immunoresearch Labs, West Grove, PA\]) was placed on the slides for one hour. ABC (Elite Series, Vector Labs) was prepared and applied per the manufacturer\'s instructions, washed as above and visualized with DAB solution (Sigma, St. Louis, MO) in parallel. The slides were counterstained with hematoxylin, cover-slipped, photomicrographed and representative images were chosen for publication. With respect to the actin visualization, detection was performed such that colored precipitate was observed under the microscope and stopped when stress fibers and microvillus cores were evident and before the high background of the total cell f-actin began to stain efficiently. In the crypt cell quantification experiments, six 10X images were taken at random, printed on color paper at maximum size, blinded, and all cells were assessed as C2 positive or C2 negative and their means and standard deviations calculated for each treatment condition. C2 double labeling immunolocalization experiments ------------------------------------------------- To objectively test our observations that C2 positive cells have a distinctive colocalization pattern when compared to C2 negative cells, C2 double labeling experiments with f-actin and with APC were performed as overlay experiments using digital microscopy. In brief, C2 immunolocalization was performed as described above except that a grid was drawn on the wet mount slide, allowing digital photomicrographs to be taken at 40X of C2 staining while still covered with stop solution and then a second antibody, either for f-actin or APC was applied and the same process repeated before being counter-stained with hematoxylin and cover-slipped. The same exact images were then re-mapped using the grid and the stored digital images were used for precise position verification. The overlaid double-labeled image was then taken and contrasted to the original image to allow comparison of differential localization of the two antigens. Abbreviations ============= IGF, insulin-like growth factor; IGFBP-2, IGF binding protein-2; IGF-II analog, synthetic truncated IGF-II; R^3^-IGF-I, synthetic long arginine IGF-I; NBI 31772, an alpha-numeric designation for a non-biologic compound that best displaced IGFs from IGF binding proteins in a large bioassay screen; SFM, serum-free media; APC, adenomatous polyposis coli; 5-HT2A, serotonin receptor 2A; C2*, IGFBP-2 carboxyl fragment Authors\' contributions ======================= PG designed the study, participated in the immunolocalization studies, and performed data analyses. JP carried out the immunolocalization, cell count, and Western blot studies. NF maintained the IEC cells lines and expertly provided uniformly confluent cells. PG and JP produced the figures and drafted the manuscript. All authors read and approved the final manuscript. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Western blots of APC and 5-HT2A in IEC-18 cell lysates. Visualization of the appropriate sized band (and break down products) for APC is shown in lane A and 5-HT2A is shown in lane B. ::: ![](1471-2121-6-2-4) :::	PubMed Central	2024-06-05T03:55:52.431908	2005-1-18	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547914/", "journal": "BMC Cell Biol. 2005 Jan 18; 6:2", "authors": [ { "first": "Phillip V", "last": "Gordon" }, { "first": "Jessica B", "last": "Paxton" }, { "first": "Nena S", "last": "Fox" } ] }
PMC547915	Background ========== There is a large number of pharmacological strategies for treating serotonin-related disorders like depression and anxiety. Well-known antidepressant drug effects are blockade of the serotonin (5-HT) and/or norepinephrine (NE) reuptake pumps, direct effects on the 5-HT- and/or NE receptors and inhibition of the monoamine oxidase A (MAO-A) enzyme. Irrespective of which drug or combination of drugs that is used, there is a delay of a few weeks before any therapeutic effect is noticed. This initial lag-period usually is associated with several side-effects, many of which fade away with the appearance of the therapeutic effect. Considering the lag-period needed for the antidepressant effect to emerge, it has been suggested that the antidepressant mechanisms involve secondary molecular neuronal adaptations, rather than being a result of the primary actions of the drugs \[[@B1],[@B2]\]. Some attempts to elucidate the molecular mechanisms involved in such lag-period have been reported, for example both SSRIs and MAO-Is cause desensitization of somatodendritic 5-HT~1A~autoreceptors \[[@B3],[@B4]\]. Furthermore, SSRIs have also, during the initial lag period, been shown to cause desensitization of terminal 5-HT~1B/1D~autoreceptors \[[@B5],[@B6]\]. Gaining more knowledge about molecular mechanisms involved in the initial lag-period may prove important for the discovery of new antidepressant drug targets and also for the knowledge of the mechanisms of action of antidepressants. Transcription factors, with their specific ability to regulate gene expression, have been suggested as prominent novel drug targets \[[@B7]-[@B11]\]. Transcription factor AP-2 is a critical regulatory factor for neuronal gene expression and neuronal development, e.g., in the brainstem \[[@B12]-[@B14]\]. Five different AP-2 genes have been identified, i.e., AP-2α, AP-2β, AP-2γ, AP-2δ and AP-2ε \[[@B15]-[@B19]\]. The isoforms are expressed from different genes and have a molecular weight of around 50 kD. The cis-acting DNA sequences 5\'-(G/C)CCCA(G/C)(G/C)(G/C)-3\' and the palindromic sequence 5\'-GCCNNNGGC-3\' are considered as consensus AP-2 binding sites for all AP-2 proteins \[[@B20]\]. Several genes encoding proteins involved in the brainstem monoaminergic systems have multiple AP-2 binding sites in their regulatory regions \[[@B20]-[@B26]\] indicating an involvement of AP-2 in the expression of these genes. We have recently reported positive correlations between brainstem AP-2α and AP-2β levels and monoaminergic activity in rat frontal cortex \[[@B27]\], indicating a regulatory function of AP-2α and AP-2β not only for neuronal development, but also for neuronal adaptive mechanisms in the adult brain. In two independent studies it was shown that the AP-2β genotype is associated with anxiety-related personality traits \[[@B28],[@B29]\]. The AP-2β genotype has also been linked to binge-eating disorder \[[@B30]\] and to platelet MAO activity \[[@B31]\], which the latter is associated with personality traits. Furthermore, the AP-2β genotype has been associated to CSF-levels of homovanillic acid (HVA) in women \[[@B32]\]. In a previous study, we reported that the levels of AP-2α and AP-2β were decreased in rat whole brain after treatment for 10 days with citalopram, imipramine and lithium, respectively \[[@B33]\]. We have also reported that citalopram changes the levels of AP-2α and AP-2β in rat whole brain in a time-dependent manner, i.e., AP-2 levels were decreased after 7 days of treatment but returned to control levels after 21 days of treatment \[[@B34]\]. Furthermore, AP-2α and AP-2β levels were shown to be increased in rat whole brain after 10 days of treatment with phenelzine \[[@B35]\]. In the present study, we report that neither treatment with citalopram for 1, 7 nor 21 days affect the AP-2α and AP-2β levels in the rat brainstem. Treatment with phenelzine, however, increased the levels of both AP-2α and AP-2β in the rat brainstem after 7 days of treatment, but after 21 days of treatment the levels had returned to control levels. Results ======= The mean relative amounts of AP-2α and AP-2β protein ± standard deviation (SD) for each of the citalopram-, phenelzine- and saline treated animal groups are shown in tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}, respectively. No significant differences in the amounts of AP-2α and AP-2β were found between any of the citalopram and saline treated groups. With regard to phenelzine treatment, there was a significant increase in AP-2α levels after 7 days of treatment (2.74 ± 0.57 vs 2.16 ± 0.14: mean relative amount of AP-2α ± SD, phenelzine vs saline, p = 0.037), which returned to control levels after 21 days. A similar result was obtained with regard to AP-2β levels with a significant increase only after 7 days of treatment (2.25 ± 0.25 vs 1.86 ± 0.22: mean relative amount of AP-2β ± SD, phenelzine vs saline, p = 0.017). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Relative amount of AP-2α protein ± SD, for the different animal treatment groups. ::: day 1 day 7 day 21 ---------------- ------------- ---------------- ------------- saline 2.29 ± 0.43 2.16 ± 0.14 2.45 ± 0.33 phenelzine 2.28 ± 0.61 2.74 ± 0.57 \* 2.54 ± 0.43 citalopram 2.35 ± 0.31 2.33 ± 0.49 2.49 ± 0.47 Values are means ± SD, for each group of animals, n = 6. \p \< 0.05 as compared to animals treated with saline for the same time-period. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Relative amount of AP-2β protein ± SD, for the different animal treatment groups. ::: day 1* day 7 day 21 ---------------- ------------- ---------------- ------------- saline 2.00 ± 0.44 1.86 ± 0.22 2.10 ± 0.14 phenelzine 2.14 ± 0.61 2.25 ± 0.25 \* 2.00 ± 0.28 citalopram 2.16 ± 0.34 2.07 ± 0.47 2.05 ± 0.28 Values are means ± SD, for each group of animals, n = 6. \*p \< 0.05 as compared to animals treated with saline for the same time-period. ::: When comparing the untreated naive animals with the treated animal groups no differences with regard to the levels of AP-2α (untreated animals: 2.49 ± 0.71, mean relative amount AP-2α ± SD) or AP-2β (untreated animals: 2.04 ± 0.64, mean relative amount AP-2β ± SD) were found. Moreover, no differences in the levels of AP-2α and AP-2β were observed between the groups of animals treated with saline for the different time periods. Discussion ========== In two independent studies, we have shown that the levels of AP-2α and AP-2β in rat whole brain were decreased after subchronic (7 or 10 days) treatment with citalopram \[[@B33],[@B34]\]. We have also shown that the AP-2α and AP-2β levels in rat whole brain were increased after treatment with phenelzine for 10 days \[[@B35]\]. For several reasons, we hypothesized that the brainstem should be of particular importance in this regard. Thus, many genes encoding proteins involved in the brainstem monoaminergic systems have binding sites for AP-2 in their regulatory regions. Furthermore, we have previously observed correlations between brainstem AP-2 levels and cortical monoamine activity \[[@B27]\]. The lag-period initially seen before the antidepressant therapeutic effect is obtained also made it interesting to study possible changes in AP-2 levels over time. In the present study, we found that treatment with citalopram for 1, 7 or 21 days did not have any effect on the brainstem levels of AP-2α and AP-2β. Treatment with phenelzine for 7 days, on the other hand, increased the brainstem levels of AP-2α and AP-2β, but after 21 days of treatment the levels had returned to control levels. The phenelzine data presented here, are in line with our previous study showing an increase in AP-2α and AP-2β levels in rat whole brain after 10 days of treatment with phenelzine \[[@B35]\]. The different responses on AP-2 of citalopram and phenelzine are likely to be explained by the different molecular mechanisms of the two drugs. Considerering the specific drug targets for citalopram and phenelzine, respectively, the target for citalopram, 5-HTT, is specific for membranes of the serotonergic system, while the target for phenelzine, MAO-A, is not located in the serononergic neurons \[[@B36]\]. It has been shown that some SSRIs, to some extent, are also able to inhibit MAO-activity \[[@B37]\]. However, all SSRIs, including citalopram, tested had a much higher selectivity for MAO-B than MAO-A and MAO-A, and not MAO-B, is the enzyme considered to be involved in the antidepressant effect \[[@B38]\]. Thus, a MAO-inhibiting effect of citalopram should not be a confounding factor with regard to interpretation of the present results. The fact that citalopram treatment did not affect AP-2 levels in rat brainstem indicates that the decrease in AP-2 levels previously seen in rat whole brain after citalopram treatment, takes place in some other brain region. It has been shown that postsynaptic 5-HT~1A~receptors in hippocampus get an enhanced response to 5-HT after treatment with TCA (that blocks both 5-HT and NE reuptake) for a time-period that corresponds to the time required for initiation of the antidepressant therapeutic effect \[[@B39]\]. An increased 5-HT responsiveness has also been demonstrated for other serotonin receptors than 5-HT~1A~and in other projection areas than hippocampus \[[@B40]\]. Thus, there are reasons to presume that the effect we have seen on AP-2 levels after subchronic citalopram treatment occurs in the 5-HT projection areas rather than in the 5-HT cell bodies in the brainstem. With regard to the increase in brainstem levels of AP-2 after 7 days of phenelzine treatment, it is in line with previous reports that MAO-Is partially enhance the 5-HT transmission by increasing the amount of 5-HT released per action potential \[[@B38]\], an effect which is likely to be regulated by presynaptic mechanisms located in the brainstem. The temporary changes in AP-2 levels after administration of antidepressants (present data and \[[@B34]\]), coinciding in time with the appearance of side-effects, makes it tempting to speculate that those two phenomena are somehow interrelated. In a previous study, we reported higher levels of AP-2α and AP-2β in rat whole brain in untreated naive animals compared to animals treated with citalopram or saline for different time periods \[[@B34]\]. In the present study, however, we did not see any differences in the brainstem levels of AP-2α and AP-2β between naive untreated animals and treated animal groups. This indicates that the changes in AP-2α and AP-2β levels in rat whole brain previously seen in animals treated with citalopram or saline compared to naive untreated animals are located in some other AP-2 containing brain region than the brainstem. As mentioned earlier, we have previously shown that the AP-2β genotype is associated with platelet MAO activity \[[@B31]\]. Thus, it is seems likely that AP-2 is involved in the regulation of the expression of the MAO enzyme. A possible explanation for the elevated AP-2α and AP-2β levels during subchronic phenelzine treatment could be that they are part of a feedback mechanism to counteract the reduction in MAO activity. Conclusions =========== Unraveling of the molecular mechanisms involved in the initial phase of antidepressant treatment is essential for the development of new efficient antidepressant drugs with less side-effects. We find transcription factors, such as AP-2, with ability to regulate expression of specific genes involved in the monoaminergic mechanisms, to be interesting candidates as novel antidepressant drug targets. Methods ======= Animals and treatment paradigms ------------------------------- Adult male Sprague-Dawley rats (10 weeks of age, B&K Universal AB, Sollentuna, Sweden) were housed in groups of three and maintained on a 12 hour light /dark cycle with food and water freely available. Animals were administered phenelzine (n = 18, 10 mg/kg, Sigma, Sweden), or citalopram (n = 18, 10 mg/kg, Lundbeck AB, Helsingborg, Sweden) subcutaneously with daily injections. All drugs were dissolved in saline (NaCl, 9 mg/kg). Saline treated animals (n = 18) received saline injections in the same volume as that given the citalopram and phenelzine treated animals. Each group of animals was treated for 1, 7 or 21 days, respectively. All animals were sacrificed by CO~2~inhalation 24 hours after their last injection. A group of untreated naive animals (n = 6) was sacrificed after 21 days. After sacrifice the brainstem was dissected and nuclear extracts were prepared for measurement of AP-2 levels by Enzyme-Linked Immunosorbent Assay(ELISA). This study was carried out with permission from the local animal ethics committee in Uppsala, Sweden. Extraction of nuclear extracts ------------------------------ Rat brainstem was homogenized in 3 ml buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, pH 7.9). The homogenate was incubated on ice for 15 minutes. To this 125 μl Nonidet P40 was added, and the homogenate was centrifuged for 30 seconds at 14000 rpm in 4°C. The pellet was resuspended in 500 μl buffer C 20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, pH 7.9). Thereafter the tubes were put on a shaker for 15 minutes and centrifuged at 14000 rpm for 5 minutes (4°C). The supernatant i.e. the nuclear protein were aliquoted and stored at -80°C. The protein concentration for all nuclear extracts were determined by the method by Lowry at al. (1951) \[[@B41]\]. The concentration of nuclear extracts were \~8 μg/μl. ELISA measurements ------------------ 96-well microtiter plates were coated (50 μl/ well) with nuclear extracts (10 μg/ml) diluted in 50 mM Carbonate-Bicarbonate buffer pH 9.0. The plates were covered with parafilm and incubated overnight at 4°C. Antigen solution was then removed and 200 μl blocking buffer (PBS, 1 % BSA) was added to each well and the plates were incubated for two hours in room temperature. Following this the blocking buffer was removed and the plates were washed with PBS. Primary antibody (50 μl, goat polyclonal AP-2α and AP-2β, 15 μg/ml respectively, SDS Biosciences, Falkenberg, Sweden) diluted in blocking buffer was then added and the plates incubated overnight at 4°C. After incubation the antibody was removed and the plates were washed three times with Wash buffer I (PBS, 0.05 % Tween-20). Secondary antibody (Donkey anti-goat IgG AP conjugated, SDS Biosciences, Falkenberg, Sweden) diluted 1:350 in blocking buffer, was then added (50 μl) to each well and the plates were incubated for two hours in room temperature. After removal of the secondary antibody the plates were washed three times with Wash buffer I, and once with Wash buffer II (10 mM diethanolamine, 0.5 mM MgCl~2~, pH 9.5). Thereafter, 50 μl substrate (Phosphatase substrate, 5 mg tablets, Sigma, Sweden, diluted in 5 ml Wash buffer II) was added to each well. The reaction continued for 30 minutes and was terminated by adding 50 μl of 0.1 M EDTA, pH 7.5. The plates were analysed in an ELISA reader (Molecular Devices, Thermo Max) at optical density (OD) 405/490. The OD of the AP-2 isoforms for each rat was correlated to a value in a standard curve, where known concentrations of antibody were plotted against optical density. The value form the standard curve was then divided with the concentration of total protein in the nuclear extracts. The quota was used as a value of the relative amount of AP-2α and AP-2β protein. Each rat were analysed twice for accuracy. Statistical analyses -------------------- The statistical comparisons between drug treated and saline treated animals for each time-point were analysed using unpaired t-test. When comparing the groups of untreated animals with all treatment groups we used analysis of variance (ANOVA) and Fisher\'s Protected Least Significant Difference (PLSD). To test if any of the groups of saline treated animals differed in the amounts of AP-2α and AP-2β protein ANOVA and PLSD test were used. All calculations were performed using Stat View 5.0 software (SAS Institute Inc., Cary, NC, USA). Results have been considered statistically significant when p \< 0.05. Authors\' contributions ======================= CB planned the experiments, carried out the molecular and statistical analyses and drafted the manuscript, MD participated in planning the experiments and writing of the manuscript, LO conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Acknowledgements ================ This study was supported by grants from the Swedish Medical Research Council (\#4145), the Swedish Brain Foundation, the AFA foundation, Svenska Lundbecksstiftelsen and Organon.	PubMed Central	2024-06-05T03:55:52.434649	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547915/", "journal": "BMC Pharmacol. 2005 Jan 21; 5:1", "authors": [ { "first": "Cecilia", "last": "Berggard" }, { "first": "Mattias", "last": "Damberg" }, { "first": "Lars", "last": "Oreland" } ] }
PMC547916	Background ========== Brain arteriovenous malformations (AVM) represent a relatively infrequent but devastating source of neurological morbidity in relatively young adults \[[@B1]\]. Prevention of new or recurrent intracranial hemorrhage (ICH) is the primary rationale for treating AVMs. The optimal management of AVMs is not well defined, and the risk of aggressive surgical therapy can be significantly high. There is a subset of AVM patients that are considered to be inoperable due to the location and size of their lesions \[[@B2]\]. To date, there is no clinically available pharmacological treatment of inoperable AVMs to decrease the rate of spontaneous intracranial hemorrhage. Matrix metalloproteinases (MMPs), a family of proteolytic enzymes, degrade extracellular matrix proteins, cell surface molecules, and other peri-cellular substances \[[@B3]\]. Excessive degradation of the vascular matrix by MMPs may result in the destabilization of the blood vessel that potentially leads to weakening of the vessel wall, passive dilatation, and rupture \[[@B4]\]. Previously, we reported increased levels of MMP-9 activity in AVMs that may result in vascular instability associated with growth and bleeding. There is an increasing interest in utilizing MMP inhibitors in treating vascular diseases including abdominal aortic aneurysms. Doxycycline is a clinically available antibiotic agent that possesses non-specific inhibitory effects on various MMPs, and for years it has had a well-established safety record in treating infectious diseases. The primary aim of this study is to demonstrate the feasibility of utilizing doxycycline as an MMP inhibitor to decrease MMP-9 activity in AVMs and potentially decrease the rate of spontaneous hemorrhage. This exploratory investigation supports the concept that further studies be conducted to document the ability of tetracycline and its derivatives to decrease MMP-9 levels in AVM nidal tissue. Such a demonstration could provide a firm rationale for proceeding with clinical trials to test the hypothesis that tetracycline and its derivatives are useful to decrease the rate of spontaneous hemorrhage from AVMs in otherwise untreatable patients or in those awaiting interventional treatment. First, we demonstrate that doxycycline can decrease MMP-9 activity in cultured AVM tissues as a proof of the concept. Further, we present results from a pilot clinical study demonstrating effects of oral doxycycline treatment on MMP-9 expression in AVM tissues. Methods ======= Ex-vivo treatment of cultured AVM tissues ----------------------------------------- After institutional review and informed consent, we obtained AVM specimens after microsurgical resection. AVM nidus was dissected away from any adjacent brain tissue in the operating room and a representative portion of nidus tissue was used for ex-vivo treatment. Ex-vivo culture of AVM tissue was performed using previously described method with modifications \[[@B5]\]. AVM tissues were minced into 1--2 mm fragments and vigorously washed with phosphate buffered saline (PBS). Blood cells dissociated from the tissues were removed by filtration. AVM tissue fragments were placed onto cell culture inserts and immersed in cell culture medium. During the first 24 hours, AVM tissues were incubated in DME H-21 medium containing 10% fetal bovine serum (FBS) to aid in tissue recovery. Following this recovery period, tissue debris and dissociated cells were removed, and AVM tissues were incubated in DME H-21 medium containing 1% FBS for 4 hours. The tissue fragments were equally divided into 12--24 wells. Then AVM tissues were incubated in the medium containing PBS (control) or doxycycline (1, 5, 10 and 100 μg/ml). Each group included 3--5 wells. Size of the tissues dictated a number of treatment groups in each specimen. Medium from each well was collected after 48 hours. Active and total MMP-9 were measured using substrate zymography. Medium was mixed with SDS sample buffer (Invitrogen, Carlsbad, CA, U.S.A.) and separated under non-reducing conditions in a 10% zymogram gel (Invitrogen) containing 0.1% gelatin incorporated as a substrate. Recombinant MMP-2 and MMP-9 proteins (R&D systems) were used as positive controls. After running, the gel was incubated with renaturing buffer (Invitrogen). The gel was then incubated with developing buffer (Invitrogen) overnight at 37°C. The gel was then stained with colloidal blue stain (Invitrogen). Proteolytic bands in the zymogram gels were quantified using Image J Software (NIH). Tissue viability was assessed by measuring the amount of LDH (lactate dehydrogenase) released in the medium according to the manufacturer\'s instructions (Roche, Penzburg, Germany). Some of the tissue fragments were embedded in paraffin for histological assessment. Pilot clinical study -------------------- Fourteen AVM patients received either 100 mg of doxycycline or placebo twice a day for one week, prior to elective AVM resection. During the AVM resection, AVM tissues were collected, and nidus tissues were frozen in liquid nitrogen. Frozen tissues were stored at -80°C until analysis. Clinical data were collected as previously described \[[@B6]\]. The specimens were homogenized and insoluble materials were removed by centrifugation at 3000 rpm for 5 minutes. We used total MMP-9 ELISA kit (R&D) and active MMP-9 ELISA kit (Amersham). Statistical analysis -------------------- Data are presented as mean ± standard deviation. The data for total MMP-9 and active MMP-9 from ex-vivo AVM tissues are presented as a relative expression with control brain samples as 100%. We used ANOVA for comparison, and statistical significance was taken at P\< .05. Results ======= Ex-vivo treatment of cultured AVM tissues ----------------------------------------- We collected two AVM tissues for ex-vivo treatment with doxycycline. These patients were not enrolled in the pilot clinical study. AVM-I ----- AVM tissue-I was collected from a 28 y.o. patient with a 29 mm AVM (Spetzler-Martin score 3). The patient had an AVM hemorrhage 236 days prior to the AVM resection. The patient did not receive embolization treatment or radiosurgery. AVM tissue-I was divided into 9 wells and treated with 0, 10, and 100 ng/ml of doxycycline (3 wells for each). Two doxycycline doses were selected to test (1) whether an average doxycycline concentration (10 ng/ml) achieved by standard clinical treatment has any effects on MMP-9 in AVM tissue, and (2) whether a high concentration of doxycycline (100 ng/ml) has any effects of viability of AVM tissues in vitro. There was a gradual increase in LDH release over the course of 8 days, indicating continuous cellular death and a gradual decrease in tissue viability (Figure [1](#F1){ref-type="fig"}). However, there was no difference among different treatment groups. To avoid effects from ongoing cellular death and to ensure tissue viability during the treatment, we chose the first 48 hours to assess the effects of doxycycline on MMP-9 levels in AVM tissues. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Tissue viability assessed by LDH (lactate dehydrogenase) release.There was a gradual increase in LDH release (mean ± SD) over the course of 8 days, indicating continuous cellular death and a gradual decrease in tissue viability. However, there was no difference among different treatment groups. ::: ![](1471-2377-5-1-1) ::: Tissue integrity was further assessed by examining H&E staining of ex-vivo cultured tissues (Figure [2](#F2){ref-type="fig"}). On day 0 (before the treatment), although cutting and mincing of the AVM tissues had caused minor tissue injury and distortion, a majority of blood vessels were intact and viable. On day 2 (after 48 hours of treatment), blood vessels were still intact and viable, and there was no apparent necrosis or hyalinization of the tissues. However, on day 7, a major part of the tissues, especially tissues surrounding blood vessels were anuclear and hyalinized, indicating that tissues were not intact or viable anymore. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### H&E staining of ex-vivo cultured AVM tissues.On day 0 (before the treatment), although cutting and mincing of the AVM tissues appeared to cause minor tissue injury and distortion, a majority of blood vessels was intact and viable. On day 2 (after 48 hours of treatment), blood vessels were still intact and viable, and there was no apparent necrosis or hyalinization of the tissues. However, on day 7, a major part of the tissues, especially tissues surrounding blood vessels were anuclear and hyalinized, indicating that tissues were not intact or viable anymore. ::: ![](1471-2377-5-1-2) ::: Doxycycline 10 μg/ml and 100 μg/ml significantly decreased active MMP-9 (control vs doxycycline 10 μg/ml: 100 ± 8 vs 48 ± 16%-control, P \< .05; control vs doxycycline 100 μg/ml: 100 ± 8 vs 59 ± 10%-control, P \< .05) (Figure [4A](#F4){ref-type="fig"}). In addition, there was a significant reduction of total MMP-9 by doxycycline at 10 and 100 μg/ml (control vs doxycycline 10 μg/ml: 100 ± 6 vs 60 ± 16%-control, P \< .05; control vs doxycycline 100 μg/ml: 100 ± 6 vs 61 ± 9%-control, P \< .05). (Figure [4B](#F4){ref-type="fig"}) There was no difference in active MMP-9 and total MMP-9 between doxycycline at 10 μg/ml and 100 μg/ml. A representative zymogram is shown in Figure [3](#F3){ref-type="fig"}. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Representative zymogram showing MMP-9 and MMP-2 standard, control AVM tissues, AVM tissues treated with 10 μg/ml doxycycline, and AVM tissues treated with 100 μg/ml doxycycline.In AVM tissues, there were proteolytic bands corresponding to pro-MMP-9 (≈97 kDa) and active-MMP-9 (≈88 kDa). ::: ![](1471-2377-5-1-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Active and total MMP-9 levels after 48 hours of ex-vivo doxycycline treatment.AVM-I:Doxycycline 10 μg/ml and 100 μg/ml significantly decreased active MMP-9 There was a significant reduction of total MMP-9 by doxycycline at 10 and 100 μg/ml There was no difference in active MMP-9 and total MMP-9 between doxycycline at 10 μg/ml and 100 μg/ml. AVM-II:Doxycycline 10 μg/ml significantly decreased active and total MMP-9. There was a trend for doxycycline 1 and 5 μg/ml to decrease active and total MMP-9. (mean ± SD) ::: ![](1471-2377-5-1-4) ::: AVM-II ------ AVM tissue-II was collected from a 63 y.o. patient with a 22 mm AVM (Spetzler-Martin score 2). The patients had a history of AVM hemorrhage 167 days prior to the AVM resection. The patient received an embolization treatment 1 day before the AVM resection. The patient did not receive radiosurgery. Using this tissue, we aimed to study effects of doxycycline at more clinically relevant concentrations of doxycycline (1--10 μg/ml). Therefore, AVM tissue-II was treated with 0, 1, 5, and 10 μg/ml of doxycycline for 48 hours. Since the results from AVM tissue-I showed a relatively wide variation in MMP-9 levels in each well among the same treatment group, we increased wells per treatment group from 3 to 4. There was no difference in tissue viability indicated by LDH release among different treatment groups at 48 hours (data not shown). Similar to the AVM-I, doxycycline 10 μg/ml significantly decreased active and total MMP-9 (active MMP-9: control vs doxycycline 10 μg/ml: 100 ± 54 vs 43 ± 15%-control, P \< .05; total MMP-9: control vs doxycycline 10 μg/ml: 100 ± 41 vs 59 ± 21%-control, P \< .05) (Figure [4C](#F4){ref-type="fig"} &[4D](#F4){ref-type="fig"}). In addition, there was a trend for doxycycline 1 and 5 μg/ml to decrease active and total MMP-9. Pilot clinical trial -------------------- 14 AVM patients were enrolled. The trial was originally started as a double blinded randomized trial, and 9 patients were randomized. In an effort to improve recruitment for this feasibility study, we converted to an open label drug design. 10 patients received doxycycline, and 4 patients received placebo. Clinical data are shown in Table [1](#T1){ref-type="table"}. Subjects were informed regarding possible side effects including gastrointestinal symptoms, cutaneous photosensitivity, skin pigmentation, teeth discoloration, and vestibular side effects. To monitor compliance and possible side effects, study subjects were interviewed by phone three times during one-week treatment period. One patient had nausea that was relieved by taking food with the drug. Other side effects were not noted. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of AVM patients enrolled in a pilot clinical study to test effects of oral doxycycline treatment on MMP-9 in AVM lesions. ::: Group Age Gender S-M score\* AVM size\\ Draining veins History of hemorrhage Number of embolization treatments --------- ----- -------- ------------- -------------- -------------------- ----------------------- ----------------------------------- Dox 50 M 4 38 Superficial & Deep No 1 Dox 48 F 4 30 Deep Yes 0 Dox 52 M 4 42 Superficial & Deep Yes 2 Dox 43 M 3 29 Superficial & Deep No 1 Dox 23 M 4 34 Superficial & Deep No 2 Dox 28 M 4 40 Superficial Yes 2 Dox 56 M 2 10 Superficial No Dox 41 F 3 30 Superficial No 1 Dox 62 M 2 41 Superficial No 1 Dox 42 M 2 2 Superficial Yes 0 Placebo 53 M 2 28 Superficial & Deep No 1 Placebo 17 M 1 18 Superficial No 1 Placebo 48 F 2 13 Deep No 0 Placebo 22 M 3 19 Deep Yes 0 \* Spetzler-Martin score (15), \\AVM maximum diameter (10). Dox = doxycycline ::: There was a trend for both total MMP-9 and active MMP-9 levels to be lower in the doxycycline group than in the placebo group (total MMP-9: 2.18 ± 1.94 vs 3.26 ± 3.58 ng/100 μg protein, P = .50; active MMP-9: 0.48 ± 0.48 vs 0.95 ± 1.01 ng/100 μg protein, P = .25) (Figure [5](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Effects of oral doxycycline treatment on MMP-9 in AVM tissues.There was a trend for both total MMP-9 and active MMP-9 levels to be lower in the doxycycline group than in the placebo group. (mean ± SD) ::: ![](1471-2377-5-1-5) ::: Discussion ========== In this study, we demonstrated the feasibility of doxycycline, a tetracycline derivative, to decrease MMP-9 activity in AVM tissues. First, we demonstrated that a clinically relevant concentration of doxycycline decreased MMP-9 without affecting tissue viability in ex-vivo AVM tissues. Second, there was a trend that oral administration of doxycycline for as short as one week in AVM patients resulted in a decrease in MMP-9 at the target site -- in the AVM nidus. By decreasing MMP-9 activity in AVM tissues, doxycycline may be able to restore the structural stability of AVM blood vessels and modify the clinical course of AVMs. Our data will provide the basis to conduct a clinical study to assess effects of tetracycline derivative treatment on the prevention of hemorrhage from AVMs. MMP-9 is a major enzyme that degrades the vascular extracellular matrix and has been implicated in a number of vascular diseases that involve abnormal angiogenesis and vascular remodeling \[[@B7]\]. MMPs are reported to be increased in cerebral aneurysms, atherosclerotic carotid plaque, and abdominal aortic aneurysm \[[@B8]-[@B10]\]. MMPs are emerging as a potentially new therapeutic target to treat vascular diseases. It has been proposed that pharmacological inhibition of MMPs may stabilize the unstable blood vessels and prevent complications such as vessel rupture \[[@B7]\]. In patients with abdominal aortic aneurysm, doxycycline treatment for one week prior to the repair surgery resulted in decreased MMP-9 and MMP-2 in the wall of the aneurysms \[[@B10]\]. Similar results have been reported in patients with atherosclerotic carotid plaques who received doxycycline for 2--8 weeks \[[@B11]\]. Our data using ex-vivo AVM tissues showed that abnormally high levels of MMP-9 expression in AVM tissues could be reduced by a clinically relevant concentration of doxycycline (i.e. 1--10 μg/ml). In patients with abdominal aortic aneurysm, doxycycline treatment at a conventional dose, 200 mg/day, resulted in a mean plasma concentration of doxycycline at 4.6 μg/ml with a range of 1.3 to 14.4 μg/ml in humans, and there was a corresponding reduction in plasma MMP-9 levels \[[@B12]\]. Similar plasma levels of doxycycline in mice successfully inhibited growth of experimental abdominal aortic aneurysms \[[@B13]\]. We were able to show that doxycycline at 10 μg/ml for as short as 48 hours can reduce MMP-9 expression in AVM tissues. A longer duration of treatment often used in clinical settings may result in a more pronounced reduction in MMP-9 expression in AVM tissues. One of the limitations of the ex-vivo study was the relatively short duration of doxycycline treatment. We chose 48 hours treatment to ensure tissue viability and integrity. Although we chose the doxycycline concentrations based on plasma doxycycline levels achieved by a commonly used therapeutic regimen, it is possible that tissue levels of doxycycline might be significantly lower than those of plasma. Furthermore, functional consequences of the reduction of MMP-9 in AVMs need to be examined in animal models or clinical studies. However, at the present time, there is no animal model that approximates recurrent intracranial hemorrhage from AVMs. There are models that can mimic certain aspects of the AVM phenotype. Hyperstimulation of mouse brain with VEGF using adenoviral transduction causes an increase in capillary density, increased MMP-9 activity, and may, in the appropriate genetic background, result in small vascular malformations \[[@B14]\]. Doxycycline can reduce capillary density and MMP-9 activity in this model \[[@B15]\]. Doxycycline has been shown in other human vascular diseases to reduce MMP levels. For example, Axisa et al. treated patients undergoing carotid endarterectomy with doxycycline or placebo for 2--8 weeks. Although MMP-1, MMP-3, and MMP-9 were reported to be increased, only MMP-1 had a statistically significant reduction by doxycycline; there was a trend for MMP-9 to be decreased by 22%. Baxter et al. used a much longer treatment period, 6 months, in patients with abdominal aortic aneurysm. They reported a gradual reduction of plasma MMP-9 levels by oral doxycycline over 6 month treatment period (baseline vs 3 months vs 6 months: 119 ± 38 vs 84 ± 33 vs 66 ± 24 ng/ml) \[[@B12]\]. To further explore the feasibility of a clinical application of doxycycline in modifying the clinical course of AVMs, we conducted a pilot clinical trial. This trial was primarily designed to test the effects of a short-term treatment with oral doxycycline on the expression of MMP-9 in AVM tissues. Despite the small sample size and short duration of treatment used in this study, there was a clear trend for one week of oral doxycycline treatment prior to surgical resection to reduce expression of both active and total MMP-9 in AVM tissues. These results indicate the feasibility of oral treatment with tetracycline derivatives in reducing MMP-9 activity in AVM tissues. Despite a large difference in the mean values of MMP levels in our pilot data, variance of MMP levels was high. Accordingly, a sample size of 46 in each group would be needed to demonstrate a difference in active MMP-9, assuming an alpha of 0.05 and power of 80%. However, variance may be reduced by utilizing a longer duration of treatment, as suggested by studies in other diseases \[[@B11],[@B12]\]. A clinical study with a modestly larger sample size and longer treatment duration should suffice to verify effects of oral doxycycline on reducing MMP-9 activity in AVMs. There are some further considerations that may account for our high variance that could be avoided in future studies. Surgical AVM tissues consist mainly from nidal tissues, but they may contain intervening astroglial or neuronal tissues that may not express angiogenic factors to the same extent as nidal tissue. Histological examination of each AVM tissue fragments to confirm a predominance of nidal tissues may yield more homogenous tissue and decrease variability in MMP-9 levels. Some of the homogenized samples underwent a several \"thaw-freeze\" processes during the preliminary phase of the experiments, which may have increased variability in MMP-9 levels. Embolization treatment may affect production and activation of MMPs. Our previous study, however, showed no association between MMP-9 and embolization treatment in 37 AVM patients \[[@B16]\]. Based on the experience from this pilot study, future studies can be performed in a more standardized fashion to decrease tissue-to-tissue variability. We used different methods to measure MMP-9 in the medium collected from ex-vivo AVM tissues and homogenized AVM tissues from the pilot clinical trial. We chose zymography, a gel based method detecting enzymatic activity of MMPs, for ex-vivo AVM tissues, because the medium volume that can be used for MMP-9 assay was extremely limited, and our preliminary experiments showed higher sensitivity of zymography compared to ELISA. One of the disadvantages of using zymography is that \"gel-to-gel\" variability in sensitivity makes it difficult to combine the data obtained from multiple gels. On the other hand, ELISA method allows researchers to assay approximately 40 samples at a time when duplicates are used. In addition, establishing a standard curve, ELISA method can express MMP-9 expression values by the absolute unit such as μg/ml or ng/100 μg protein. Therefore, for the clinical pilot trial, in order to quantitatively compare MMP-9 expression among a large number of samples originally planned, we used ELISA to MMP-9 expression. Conclusions =========== In summary, results suggest that doxycycline may have a therapeutic potential to reduce MMP-9 activity in AVM tissues and stabilize blood vessels that are prone to rupture. Our findings may provide a basis for a larger clinical trial to study effects of tetracycline derivatives in preventing AVM hemorrhage. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= TH, a corresponding author, participated in all aspects of the study and manuscript preparation. MMM participated in study design, experiments, analysis, interpretation of data, and manuscript preparation. JFL participated in experiments, analysis and interpretation of data. MTL participated in study design, analysis, interpretation of data, and manuscript preparation. WLY participated in study design, analysis, interpretation of data, and manuscript preparation. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2377/5/1/prepub> Acknowledgments =============== The authors wish to thank Broderick Belenson and Carroll Schreibman for assistance in preparation of the manuscript; Nancy Quinnine, RN and Manju Chopra, MD, for technical assistance; and the members of Center for Cerebrovascular Research and the UCSF AVM Study Project for their continued support. Portions of this work were supported by NIH grants RO1-27713 and K24-NS02091 (WLY), P01-NS44155 (WLY, TH), and American Heart Association Beginning Grant-in-Aid (TH).	PubMed Central	2024-06-05T03:55:52.436360	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547916/", "journal": "BMC Neurol. 2005 Jan 24; 5:1", "authors": [ { "first": "Tomoki", "last": "Hashimoto" }, { "first": "Melissa M", "last": "Matsumoto" }, { "first": "Jenny F", "last": "Li" }, { "first": "Michael T", "last": "Lawton" }, { "first": "William L", "last": "Young" } ] }
PMC547917	Background ========== Stress can be described as the sum total of all the reactions of the body, which disturb the normal physiological condition and result in a state of threatened homeostasis. Stress is an internationally recognized phenomenon fortified by advancement of industrialization in a demanding civilization. Thus, every individual is likely to face stressful situations in day-to-day life. Stress represents a reaction of the body to a stimulus that tends to alter its normal physiological equilibrium or homeostasis and has been defined as a nonspecific response of the body to any demand imposed on it \[[@B1]\]. Since the introduction of adaptogens, several plants have been investigated, which were once used as tonics due to their adaptogenic and rejuvenating properties in traditional medicine \[[@B2]\]. The drugs of plant origin are gaining increasing popularity and are being investigated for remedies of a number of disorders including antistress (adaptogenic) activity \[[@B3]\]. The initial studies on Ocimum sanctum\[[@B4]\], Withania somnifera\[[@B5]\] opened a vast area of research and substantial work has been carried out on plants such as Eleuthrococcus senticosusand Panax ginseng\[[@B6]\]. Vitis vinifera(Linn.) (Family: Vitaceae) also called as common grape or wine grape or European grape is one of the fruit crops most widely grown throughout the world \[[@B7]\]. In the indigenous Indian system of medicine (Ayurveda), the aerial parts of V. viniferahave been widely used to treat a variety of common and stress related disorders \[[@B8]\]. The composition and properties of grape seeds have been extensively investigated, and reported to have many favourable effects on human health such as the lowering of low-density lipoprotein \[[@B9],[@B10]\], reduction of cardiovascular diseases and cancer \[[@B11]\]. In addition, the seed extracts of V. viniferaare reported to have antimicrobial and free radical scavenging properties \[[@B12]\]. In the present investigation, the antistress activity of Vitis viniferawas evaluated in-vivo, in normal and stress induced rats following a biochemical approach. The antioxidant potential of the extract was evaluated in-vitroto support the antistress activity. The plant extract was further evaluated for nootropic activity using conditioned avoidance response in rats. Methods ======= Preparation of extract ---------------------- The riped fruits of Vitis viniferawere collected from the Chittor district of Andhra Pradesh, India in the month of October and the seeds were separated from the pulp and shade dried. The dried powdered seed material of Vitis vinifera(5 kg) was extracted with boiling water (25 L) for 45 minutes and the filtrate was evaporated under vacuum below 70°C in a vacuum drier to give a final yield of 50 gm. Chemicals used -------------- Vanillylmandelic acid (VMA) and scopolamine butylbromide (SBB) were purchased from Sigma-Aldrich, St. Louis, USA, while ascorbic acid was purchased from Loba Chemie, Mumbai. All other reagents used were of analytical grade. Animals ------- All animal experiments were performed in accordance with our Institutional Animal Ethics Committee and by the animal regulatory body of the government (Regd. No. 516/01/A/CPCSEA). Albino rats of either sex obtained from Ghosh Enterprises, Kolkata were used in the study. They were housed six per cage at a temperature of 22 ± 2°C with 12 h light/ dark cycle under controlled environment. Rats were fed with standard pellet diet (Ratan Brothers, Hyderabad), and water ad libitum. Animals were kept for seven days in laboratory for habituation. Antistress activity ------------------- Rats of either sex weighing between 120--150 gm were divided into four groups (I, II, III, IV) each containing six animals. The 24 h urine sample from each group was collected into two different beakers, one containing 5 ml of 10% oxalic acid for the spectrophotometric determination of ascorbic acid at 550 nm \[[@B13]\] and the other containing 0.5 ml of 6 Nhydrochloric acid for the determination of vanillylmandellic acid (VMA) spectrophotometrically at 360 nm \[[@B14]\]. The experimental protocol was divided into four phases. In the first phase of the experiment, 24 h urine samples were collected in all the four groups and subjected to analysis for both VMA and ascorbic acid and the normal values were recorded for seven consecutive days. In the second phase, the animals in each group were subjected to fresh water swimming stress individually. In this method, rats were forced to swim until exhausted (three to four minutes) in a cylindrical vessel of 60 cm height and 45 cm diameter containing water at room temperature (28°C). Water depth was always maintained at 40 cm. The 24 h urinary levels of VMA and ascorbic acid under stressed conditions were determined again as described above daily for seven consecutive days. The third phase of the experiment consists of administration of V. viniferaextract to the same groups of animals after having recovered completely to normal condition. Groups II, III and IV were administered orally with V. vinifera(suspended in 2% gum acacia) at daily doses of 100, 200 and 300 mg/kg body weight respectively for seven consecutive days while group I serving as control. The 24 h urine samples were collected and the levels of both VMA and ascorbic acid were determined. The final phase of the experiment consisted of administration of V. viniferaextract to the same groups of animals after a recovery period of one week. Groups II, III and IV were administered orally with V. viniferaat doses of 100, 200 and 300 mg/kg body weight respectively, one hour prior to the daily induction of stress for seven consecutive days while group I serving as control. The 24 h urine samples were collected and analyzed for VMA and ascorbic acid for seven consecutive days to study the influence of the extract on the stress induced biochemical changes. Nootropic activity ------------------ The Nootropic activity of V. viniferawas evaluated by using the conditioned avoidance response (CAR) in rats as described by Cook and Weidley \[[@B15]\]. Rats were divided into 4 groups each containing six animals. Groups II, III and IV were administered orally with 100, 200 and 300 mg/kg body weight respectively of V. vinifera(suspended in 2% gum acacia) while animals in group I were served as control. After 60 minutes, all the animals were subjected to a training schedule individually by placing inside the perspex chamber of the apparatus. After an accustomed period of five minutes to the chamber, a buzzer was given followed by a shock through the grid floor. The rat had to jump on the pole to avoid foot shock. Jumping on the pole functionally terminates the shock and this was classified as an escape while such jumping prior to the onset of the shock was considered as avoidance. The session was terminated after completion of 60 trials with an interval of 20--30 seconds given for each trial. This procedure was repeated at 24 h intervals until all groups reach 95 to 99% avoidance. After attaining complete training of a particular group, the animals were treated with a single dose of scopolamine butyl bromide (1 mg/kg body weight, i.p.), thirty minutes before the next day dosing. The training schedule was continued further with the daily doses of the extract and vehicle until they returned to normal level from scopolamine induced amnesia. Antioxidant activity -------------------- The antioxidant activity of V. viniferawas determined based on its ability to scavenge the hydroxyl radicals \[[@B16]\]. Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and the extract for hydroxyl radicals generated from the Fe^3+^-ascorbate-EDTA-H~2~O~2~system. The hydroxyl radicals attacks deoxyribose and eventually results in the formation of thiobarbituric acid reacting substances (TBARS). The reaction mixture containing deoxyribose (2.8 mM), ferric chloride (0.1 mM), EDTA (0.1 mM), H~2~O~2~(1 mM), ascorbate (0.1 mM) phosphate buffer (20 mM, pH 7.4) and various quantities of the extracts in a final volume of 1 mL was incubated for 1 h at 37°C. Deoxyribose degradation was measured as TBARS by the method of Ohkawa et al., 1979 and the percentage free radical inhibition was calculated from control. Data and statistical analysis ----------------------------- The results are expressed as means ± standard error of means. Statistical analysis was done using Student\'s paired t-test. In all the cases, p \< 0.05 was considered statistically significant. Results ======= Antistress activity ------------------- The urinary data of VMA and ascorbic acid observed in various phases of the experiment are shown in Fig. [1](#F1){ref-type="fig"} and Fig. [2](#F2){ref-type="fig"} respectively. Induction of forced swim stress to the animals produced a significant increase in VMA and decrease in ascorbic acid excretion compared to their respective basal excretion in normal condition. Both the parameters were found to return to their normal levels in three to four days after withdrawal of stress. Daily treatment of V. viniferato the animals under normal condition produced no change in the excretion of VMA and ascorbic acid compared to normal basal levels indicating that V. viniferadid not alter excretion of VMA and ascorbic acid in normal condition. Daily administration of V. viniferaone hour prior to the induction of stress inhibited the increase in VMA and decrease in ascorbic acid excretion which was manifested during stress alone. The inhibition was found to be significant at all dose levels in a dose dependent manner. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *Influence of Vitis viniferaseed extract on the 24 h urinary levels of VMA in normal and stress induced rats. Each bar indicates the mean excretion of six animals. Significant difference from normal condition of the corresponding groups: \p \< 0.05 Significant difference from stress condition of the corresponding groups: \#p \< 0.05 NS- No significant difference from normal condition of the corresponding groups. §No significant difference from stress condition. ::: ![](1472-6882-5-1-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### *Effect of Vitis viniferaseed extract on the 24 h urinary levels of ascorbic acid in normal and stress induced rats. Each bar indicates the mean excretion of six animals. Significant difference from normal condition of the corresponding groups: \p \< 0.05 Significant difference from stress condition of the corresponding groups: \#p \< 0.05 NS- No significant difference from normal condition of the corresponding groups. §No significant difference from stress condition. ::: ![](1472-6882-5-1-2) ::: Nootropic activity ------------------ The CARof rats administered with the extract of Vitis viniferaor vehicle increased gradually to 95% over seven to ten days. The percent avoidance was always higher in the extract treated groups compared to vehicle treated control group. The acquisition (time to achieve 95% CAR) for the extract treated groups was quicker and found to be dose dependent. Animals receiving 300 mg/kg body weight of the extract have taken seven days whereas, groups treated with 200 and 100 mg/ kg doses of the extract required eight and nine days respectively to reach the point of acquisition (Fig. [3](#F3){ref-type="fig"}). Administration of scopolamine produced amnesia as seen from reduction in the observed CAR. However, continued treatment of V. viniferaproduced better retention and recovery in a dose dependent manner than the vehicle treated animals. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### *Effect of Vitis viniferaseed extract on the mean percent of conditioned avoidance response after oral administration in rats. Scopolamine butylbromide (SBB) was administered thirty minutes before the next day dosing with the extract after attaining complete acquisition. ::: ![](1472-6882-5-1-3) ::: Antioxidant activity -------------------- Degradation of deoxyribose mediated by hydroxyl radicals generated by Fe^3+^/ascorbate/EDTA/H~2~O~2~system was found to be inhibited by Vitis vinifera. The extract at quantities of 100, 200, 400 and 800 μg levels scavenged the hydroxyl radicals in a dose dependent manner. Ascorbic acid at concentration of 2500, 5000 and 10000 μg was also found to produce dose dependent inhibition of hydroxyl radicals. The quantity of the extract needed for 50% inhibition was found to be 610 μg (Fig. [4](#F4){ref-type="fig"}). Similar effect was produced by ascorbic acid at a concentration of 4875 μg. (Fig. [5](#F5){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Hydroxyl radical scavenging activity of Vitis viniferain in-vitrosystems. Graphical representation of the concentration of Vitis viniferarequired to inhibit 50 percent of hydroxyl radicals. Each point represents the mean percentage inhibition of six experiments. ::: ![](1472-6882-5-1-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Hydroxyl radical scavenging activity of ascorbic acid in in-vitrosystems. Graphical representation of the concentration of ascorbic acid required to inhibit 50 percent of hydroxyl radicals. Each point represents the mean percentage inhibition of six experiments. ::: ![](1472-6882-5-1-5) ::: Discussion ========== Advancement in the understanding of processes leading to explore the reason for stress induced disorders cannot obscure the simple fact that the exhaustion of energy supply still forms the basis that triggers the disorders and collapse of energy metabolism following glucose deprivation in circulation \[[@B17]\]. The desire to control the coping mechanism has led to emergence of science of adaptation, focusing to elucidate the mechanism that may help in modification so that insufficient, excessive and unnecessary responses can be prevented. Literature reports indicate that noradrenaline is released during stressful conditions \[[@B18]\] and metabolized to vanillyl mandelic acid(VMA) peripherally and 3-methoxy 4-hydroxyphenyl glycol (MOPEG) centrally. In the light of such reports, VMA, the major metabolite of sympathetic amines, was taken as indirect biochemical index to represent the increase in peripheral sympathetic activity during stress. In the present study, the increase in the urinary VMA excretion during stress was used as a non-invasive biochemical marker to study the antistress activity of V. vinifera. Several studies also indicated that the tissue levels of ascorbic acid decreased on application of stress \[[@B19]\]. Ascorbic acid being a free radical scavenger \[[@B20]\], it is more likely utilized in scavenging the free radicals involved in stress resulting in its decreased urinary concentration and also it has role in the biosynthesis of noradrenaline \[[@B21]\]i.e., as a cofactor in the conversion of dopamine to noradrenaline \[[@B22]\]. Based on the above studies ascorbic acid excretion in urine was taken as an indirect biochemical index to indicate the influence of stress on catecholamine synthesis in rats and antistress (adaptogenic) activity of the Vitis viniferaextract on prior administration of stress induction. Treatment with Vitis viniferaextract along with stress reversed the stress induced biochemical changes i.e., increase in urinary VMA levels and decrease in urinary ascorbic acid levels, in a dose dependent manner. A number of Indian medicinal plants like Ocimum sanctum, Withania somnifera, Panax ginsengetc have been identified for their antistress activity. It was concluded that the antioxidant activity of these plants was partly responsible for their antistress activity \[[@B23]\]. Based on these reports the antioxidant activity of Vitis viniferaextract was also done using hydroxyl radical assay method. It was found that Vitis viniferaextract has significant good antioxidant activity which was 8 fold more than that of ascorbic acid. The antistress and antioxidant activities were correlated with the nootropic activity of the extract since the role of stress and free radicals have been implicated in the loss of memory, concentration and also in Alzheimer\'s disease \[[@B24],[@B25]\]. The process of nootropic activity involves acquisition, retention and retrieval and is measured using conditioned avoidance response. The acquisition was quicker in the extract treated rats (100, 200, 300 mg/kg body weight) in comparision to control, indicating the involvement of antistress activity of the extract. When challenged with scopolamine butylbromide (1 mg/kg body weight), the amnesia was less in treated group showing better retention and recovery than control group and the Vitis viniferaextract was shown to decrease memory loss which could be due to its central cholinomimetic activitiy apart from its free radical scavenging mechanisms. Furthermore, the antioxidant activity of the seed extract provide mechanistic basis in relieving stress by way of combating oxidative damage. Conclusion ========== In conclusion, the present study provides scientific support for the antistress (adaptogenic), antioxidant and nootropic activities of V. vinifera*seed extract and substantiate the traditional claims for the usage of grape fruits and seeds in stress induced disorders. Further investigations are required to characterize the active constituent(s) responsible for observed activities of the seed extract. Abbreviations ============= VMA: Vanillylmandelic acid CAR: Conditioned avoidance response SBB: Scopolamine butylbromide EDTA: Ethylene diamine tetra acetic acid H~2~O~2~: Hydrogen peroxide Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= SS conceived the study, made substantial contributions in data analysis, data interpretation, writing of the manuscript and in coordination of the experiments. SN made substantial contributions in conceptualization of statistical analyses, drafting the final manuscript and designing the illustrations. RK and SK helped to conceptualize the work. KMB made significant contribution in designing the studies, conducting the experiments, interpretation of the data, and drafting the final manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6882/5/1/prepub> Acknowledgements ================ The Authors are indebted to the All India Council for Technical Education AICTE), Government of India, New Delhi for providing a major financial support to carryout the research work. The authors are also grateful to the Department of Pharmaceutical Sciences Andhra University, Visakhapatnam, India for providing all the facilities to carryout the studies.	PubMed Central	2024-06-05T03:55:52.438202	2005-1-19	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547917/", "journal": "BMC Complement Altern Med. 2005 Jan 19; 5:1", "authors": [ { "first": "Satyanarayana", "last": "Sreemantula" }, { "first": "Srinivas", "last": "Nammi" }, { "first": "Rajabhanu", "last": "Kolanukonda" }, { "first": "Sushruta", "last": "Koppula" }, { "first": "Krishna M", "last": "Boini" } ] }
PMC547972	Introduction {#s1} ============ Due to their ability to interact with fatty acids and other lipids, nuclear hormone receptors (NHRs), including peroxisome proliferator-activated receptors (PPARs), liver X receptor, and farnesoid X receptor, are important regulators of mammalian fat metabolism; accordingly, these small-molecule-gated transcription factors have been favored targets for therapies designed to combat diabetes, obesity, and atherosclerosis \[[@pbio-0030053-b01],[@pbio-0030053-b02],[@pbio-0030053-b03]\]. Although the molecular mechanisms employed by NHRs have been intensively probed, their pleiotropic effects on overall animal physiology are only partially understood. Thus, developing a broader range of experimental systems would be useful for characterizing the fat regulatory networks integrated by NHRs and for revealing the breadth of their physiological influence. The study of invertebrates, such as Caenorhabditis elegans, has facilitated the elucidation and interpretation of biological networks, particularly in the context of the whole animal. However, despite the fact that C. elegans contains 284 NHR genes, compared to only 48 in mammals, there is a notable absence of worm NHRs orthologous to mammalian receptors involved in fat metabolism \[[@pbio-0030053-b04],[@pbio-0030053-b05]\]. In fact, it has been suggested that fat-regulating NHRs, along with several other NHR types, were lost from the C. elegans and Drosophila lineages as these organisms simplified their body plans \[[@pbio-0030053-b06]\]. Conceivably, then, the biological activities associated with these NHRs may also have vanished. Therefore, it is still not clear if worm receptors mediate control over fat metabolism comparable to that of mammalian NHRs. Whether or not C. elegans employs NHRs to regulate fat metabolism, the evolution of the C. elegans NHR family is intrinsically interesting. Only 15 of the 284 worm NHRs are members of the broadly conserved subfamilies found in mammals and other metazoans \[[@pbio-0030053-b04],[@pbio-0030053-b05]\]. The biological activities of most of these "conserved" NHRs have been broadly defined, revealing roles in development, molting, dauer formation, and sex determination \[[@pbio-0030053-b07],[@pbio-0030053-b08]\]. The remaining 269 "divergent" NHRs have thus far been found only in nematodes and are predicted to have originated from repeated duplication of an ancestral gene that also gave rise to the hepatocyte nuclear factor 4 (HNF4) family of receptors \[[@pbio-0030053-b06]\]. Although mammalian HNF4 receptors have been implicated in liver development and glucose homeostasis \[[@pbio-0030053-b09]\], virtually nothing is known about the functions of these 269 C. elegans "nematode-specific" HNF4-like receptors. It will be interesting to determine if these divergent NHRs have adopted novel regulatory functions or whether they carry out physiological tasks similar to those of other metazoan NHRs. Elucidating the physiological responsibilities of even one of the worm HNF4-like receptors will likely advance our understanding of NHR-regulated gene networks and provide insight into the evolution and diversification of the NHR family in nematodes. To this end, we have been systematically investigating the function of HNF4-like receptors in C. elegans. In this study, we present a physiological characterization of nhr-49 (K10C3.6), one of the C. elegans receptor genes most closely related to the mammalian HNF4. Our findings reveal a surprising role for this HNF4-like receptor in the regulation of fat storage and metabolism. Results {#s2} ======= nhr-49 Is Necessary for Normal Life Span {#s2a} ------------------------------------------ In an RNA interference (RNAi) screen designed to identify the role of C. elegans HNF4-like receptors in worm development and longevity, we found that interference of nhr-49 resulted in dramatically reduced life span ([Figure 1](#pbio-0030053-g001){ref-type="fig"}A). For further characterization, we obtained a C. elegans strain, nhr-49(nr2041), which harbors a deletion in the nhr-49 gene encompassing part of the DNA binding domain and more than half of the ligand binding domain (LBD); this deletion likely results in complete loss of function \[[@pbio-0030053-b10]\]. At 23 °C, nhr-49(nr2041) worms lived only 6--8 d as adults, significantly shorter than the 15 to 18-d life span of N2 wild-type (WT) animals ([Figure 1](#pbio-0030053-g001){ref-type="fig"}A). Although nhr-49 deletion did not noticeably affect development or fertility, nhr-49(nr2041) worms experienced rapid decline in function beginning around day 3 of adulthood, when vacuoles appeared in the intestine and gonad ([Figure 1](#pbio-0030053-g001){ref-type="fig"}B). By days 4 and 5, nhr-49(nr2041) animals were significantly smaller than WT, vacuoles were ubiquitous, and there was widespread gonadal necrosis. By days 5--7, the gonad had completely deteriorated, and the worms died shortly thereafter. Thus, nhr-49 function is not required for development or fertility, but is clearly essential for normal longevity. Even though the increased vacuole formation was consistent with reported aging characteristics \[[@pbio-0030053-b11]\], we have not determined whether the shortened life span of nhr-49(nr2041) reflects accelerated aging or an unrelated pathology. ::: {#pbio-0030053-g001 .fig} Figure 1 ::: {.caption} ###### nhr-49(nr2041) Animals Have Reduced Life Span and Higher Fat Content \(A) Adult life span of WT (black squares, solid line), nhr-49(nr2041) (black circles, dotted line), and nhr-49 RNAi animals (black diamonds, dashed line). \(B) Nomarksi images of nhr-49(nr2041) at days 3, 5, and 7 of adulthood. The arrow in the day 3 image points to a gonadal vacuole that is typical of day 3 worms; the arrow in the day 5 image shows the continued deterioration of oocytes in the gonad, and the arrow in the day 7 image points to the clearing that results from complete gonadal necrosis and collapse. \(C) Nile Red intestinal fat staining of WT and nhr-49(nr2041). Each image displays two representative worms from a population of L4 animals. ::: ![](pbio.0030053.g001) ::: nhr-49(nr2041) Animals Display Abnormally High Fat Content {#s2b} ------------------------------------------------------------ nhr-49 was recently identified in a C. elegans genomewide RNAi screen as one of 112 genes that, when knocked down by RNAi, resulted in abnormally high Nile Red fat staining \[[@pbio-0030053-b12]\]. To determine whether deletion of nhr-49 also yielded a high-fat phenotype, we used a similar Nile Red assay to visualize fat content in nhr-49(nr2041). Indeed, we found that nhr-49(nr2041) animals stained more brightly with Nile Red than did WT worms ([Figure 1](#pbio-0030053-g001){ref-type="fig"}C). The difference in fat content between WT and nhr-49(nr2041) animals was most pronounced in the L3 and L4 stages of larval development; by day 2 of adulthood there was only a slight change in Nile Red staining, suggesting that the effects of nhr-49 on visible fat content may vary during development (data not shown). nhr-49 Regulates Genes Involved in Energy Metabolism {#s2c} ------------------------------------------------------ The high-fat phenotype of the nhr-49(nr2041) mutant led us to suspect that nhr-49 might regulate genes involved in energy metabolism. To test this hypothesis, we identified from a survey of the C. elegans genome 65 genes predicted to participate in fatty acid synthesis, β-oxidation, desaturation, elongation, and binding/transport, in addition to 16 genes expected to function in glycolysis, gluconeogenesis, and glucose transport, and eight genes involved in the glyoxylate pathway ([Figure 2](#pbio-0030053-g002){ref-type="fig"}). We then employed quantitative RT-PCR (QRT-PCR) to measure the expression of all 89 of these genes in WT and nhr-49(nr2041) mutant worms. For the purpose of our survey, gene expression was measured in all four larval stages (for a complete dataset, see [Table S1](#st001){ref-type="supplementary-material"}). ::: {#pbio-0030053-g002 .fig} Figure 2 ::: {.caption} ###### nhr-49 Deletion Affects the Expression of 13 Energy Metabolism Genes Putative C. elegans fatty acid, glucose, and glyoxylate metabolism pathways are shown here; each box represents a gene predicted by sequence homology to code for the enzyme indicated in the figure. The fatty acid desaturation elongation pathway is adapted from previously published studies \[[@pbio-0030053-b14],[@pbio-0030053-b17]\]. Mitochondrial localization of β-oxidation enzymes was predicted by TargetP and PSORT (see [Materials and Methods](#s4){ref-type="sec"}); peroxisomal β-oxidation enzymes all contain a carboxy-terminal peroxisomal localization signal. QRT-PCR was used to measure the expression of these 89 genes in WT and nhr-49(nr2041). Genes expressed at lower levels in nhr-49(nr2041) are shown in red (\>32-fold lower); orange (8- to 32-fold lower); or yellow (2- to 8-fold lower); and genes expressed at higher levels are shown in light green (2- to 8-fold higher) or dark green (\>8-fold higher). Online QRT-PCR data for this analysis are available in [Table S1](#st001){ref-type="supplementary-material"}. ::: ![](pbio.0030053.g002) ::: Our screen revealed that deletion of nhr-49 significantly altered the expression of 13 genes, including six genes predicted to be involved in fatty acid β-oxidation, three genes involved in fatty acid desaturation, two genes involved in fatty acid binding/transport, and two genes involved in the glyoxylate pathway ([Figure 2](#pbio-0030053-g002){ref-type="fig"}). Similar changes in gene expression were observed when nhr-49 was knocked down in WT animals using RNAi (data not shown). Overall, nhr-49 deletion displayed the most dramatic effects on two metabolic pathways, mitochondrial β-oxidation and fatty acid desaturation, as the expression of multiple genes in each of these pathways was significantly compromised by loss of nhr-49 function ([Figure 2](#pbio-0030053-g002){ref-type="fig"}). In contrast, nhr-49 deletion had smaller and mixed consequences for genes that participate in peroxisomal β-oxidation and lipid binding/transport. nhr-49 knockout also marginally reduced expression of two enzymes involved in the glyoxylate pathway, a pathway that facilitates the conversion of fatty acid β-oxidation products to glucose ([Figure 2](#pbio-0030053-g002){ref-type="fig"}). nhr-49 deletion did not, however, significantly affect the expression of any genes predicted to be involved in glucose metabolism. Thus, it is clear that nhr-49 is extensively involved in the control of fatty acid metabolism, with a pronounced role in the promotion of mitochondrial β-oxidation and fatty acid desaturation. nhr-49(nr2041) Animals are High in Fat Due to Reduced Expression of β-Oxidation Genes {#s2d} --------------------------------------------------------------------------------------- Three genes predicted to participate in mitochondrial β-oxidation, ech-1 (C29F3.1), F09F3.9, and an acyl-CoA synthetase gene (acs-2) (F28F8.2), were expressed at significantly lower levels in nhr-49(nr2041) animals throughout development ([Figure 3](#pbio-0030053-g003){ref-type="fig"}A). acs-2 is predicted to encode a mitochondrial acyl-CoA synthetase, whereas F09F3.9 likely codes for a carnitine palmitoyl transferase, and ech-1 appears to encode a mitochondrial β-oxidation trifunctional enzyme. Because mitochondrial β-oxidation facilitates the degradation of stored fats for the production of energy, we suspected that nhr-49(nr2041) worms might be high in fat due to the reduced expression of these key β-oxidation enzymes. To test this hypothesis, we used RNAi to reduce separately the expression of acs-2, F09F3.9, and ech-1 in WT animals. Indeed, we found that RNAi of either acs-2 or ech-1 resulted in worms with elevated Nile Red fat staining ([Figure 3](#pbio-0030053-g003){ref-type="fig"}B). acs-2 was previously identified for a similar effect on fat storage \[[@pbio-0030053-b12]\]. We conclude that nhr-49 promotes the expression of three β-oxidation genes, two of which, in WT worms, reduce overall fat storage. Thus, nhr-49(nr2041) mutant worms are likely to be high in fat due to the impaired expression of these genes. ::: {#pbio-0030053-g003 .fig} Figure 3 ::: {.caption} ###### Regulation of β-Oxidation Gene Expression by nhr-49 \(A) QRT-PCR measurement of acs-2, F09F9.3, and ech-1 expression in WT (gray bars) and nhr-49(nr2041) animals (blue bars). Expression was measured in all four stages of larval development. Error bars represent standard error of measurement. \(B) RNAi of nhr-49, acs-2, or ech-1 resulted in increased Nile Red fat staining. Each image shows 3 or 4 representative worms from a population of L4 animals grown on plates containing RNAi bacteria and Nile Red fat-staining dye. \(C) acs-2::gfp is expressed in many tissues including hypodermis (hyp), intestine (int), body wall muscle (bwm), neurons (neu), and pharynx (pha). ACS-2::GFP localizes to subcellular structures in a pattern similar to what has been reported for mitochondrial proteins \[[@pbio-0030053-b13]\]. \(D) Expression of acs-2::gfp was lower in nhr-49(nr2041) mutant animals. \(E) Expression of P~nhr-49~::gfp promoter fusion in WT animals revealed that nhr-49 is expressed in multiple tissues, including hypodermis (hyp), body wall muscle (bwm), pharynx (pha), and intestine (int). The animals shown here are genetically mosaic, harboring the P~nhr-49~::gfp construct in only a fraction of total cells. \(F) Ectopic expression of acs-2::gfp in nhr-49(nr2041) was sufficient to reduce Nile Red staining to WT levels. ::: ![](pbio.0030053.g003) ::: ACS-2 Is Expressed in Multiple Tissues and Localizes to Mitochondria {#s2e} -------------------------------------------------------------------- Out of five predicted mitochondrial acs genes in C. elegans, nhr-49 affected only the expression of acs-2, suggesting that nhr-49 may only influence a subset of mitochondrial β-oxidation pathways in worms. To determine if acs-2 was expressed in a specific set of tissues, we fused the full-length acs-2 gene and promoter to gfp (acs-2::gfp); injection of this construct into WT worms revealed that the ACS-2::green fluorescent protein (GFP) was widely expressed in many cell types, including intestine, hypodermis, pharynx, body wall muscle, and several neurons ([Figure 3](#pbio-0030053-g003){ref-type="fig"}C). Moreover, ACS-2::GFP expression was reduced in all of these tissues in nhr-49(nr2041) worms, indicating that the effects of nhr-49 on acs-2 expression were widespread ([Figure 3](#pbio-0030053-g003){ref-type="fig"}D). Notably, the expression pattern of acs-2 overlapped extensively with that of an nhr-49 promoter/GFP fusion, consistent with the idea that nhr-49 could control acs-2 through direct transcriptional activation ([Figure 3](#pbio-0030053-g003){ref-type="fig"}E). As predicted, ACS-2::GFP localized to subcellular structures in patterns similar to those reported for other mitochondrial proteins \[[@pbio-0030053-b13]\] ([Figure 3](#pbio-0030053-g003){ref-type="fig"}C). Overexpression of ACS-2::GFP Is Sufficient to Suppress the High-Fat Phenotype of nhr-49(nr2041) {#s2f} ------------------------------------------------------------------------------------------------- Strikingly, overexpression of the ACS-2::GFP fusion was able to suppress the high-fat phenotype of nhr-49(nr2041) animals ([Figure 3](#pbio-0030053-g003){ref-type="fig"}F). These data suggest that acs-2 expression is reduced in nhr-49(nr2041) such that it becomes a rate-limiting factor in the consumption of fat; thus, high fat levels can be overcome simply by increasing acs-2 expression. Overexpression of acs-2 in WT animals did not affect fat content, however, indicating that WT levels of acs-2 expression are not rate limiting. These data further support the conclusion that reduced expression of acs-2 is a significant contributor to the high-fat phenotype of the nhr-49(nr2041) mutant. nhr-49 Modulates Fatty Acid Composition by Promoting Expression of Δ9-Desaturases {#s2g} ----------------------------------------------------------------------------------- In addition to stimulating genes in the mitochondrial β-oxidation pathway, nhr-49 also promoted the expression of three C. elegans fatty acid desaturases (see [Figure 2](#pbio-0030053-g002){ref-type="fig"}). fat-5 (W06D12.3) and fat-7 (F10D2.9) expression was dramatically lowered in nhr-49(nr2041) worms (\>30-fold) in all four larval stages, whereas fat-6 (VZK8221.1) expression was marginally reduced (approximately 2-fold) only in L3 and L4 animals ([Figure 4](#pbio-0030053-g004){ref-type="fig"}A). ::: {#pbio-0030053-g004 .fig} Figure 4 ::: {.caption} ###### Deletion of nhr-49 Hinders Fatty Acid Desaturation \(A) QRT-PCR measurement of fat-5, fat-6, and fat-7 expression in WT (gray bars) and in nhr-49(nr2041) mutant worms (blue bars). Expression was measured in all four stages of larval development. Error bars represent standard error of measurement. \(B) Relative abundance of individual fatty acid species expressed as percentage of total measured fatty acid. Fatty acids included in total fatty acid measurement but not shown in the figure include C14:0, C15:0, and C17:Δ. Fatty acids were isolated and quantified by GC/MS from WT (dark gray bars), nhr-49(nr2041) (blue bars), fat-7 RNAi animals (red bars), and nhr-49 RNAi animals (light gray bars). Error bars represent standard error. The fatty acid desaturation/elongation pathway, along with enzymes involved in desaturation and elongation, is shown in the inset; this pathway was adapted from previous studies \[[@pbio-0030053-b14],[@pbio-0030053-b17]\]. ::: ![](pbio.0030053.g004) ::: Studies in yeast have shown that the C. elegans fat-5, fat-6, and fat-7 genes encode Δ9-desaturases, which preferentially convert saturated C16:0 and C18:0 fatty acids to monounsaturated C16:1 and C18:1 fatty acids \[[@pbio-0030053-b14]\]. fat-5 is a palmitoyl-CoA desaturase, specifically acting on palmitic acid (C16:0), and fat-6 and fat-7 are stearoyl-CoA desaturases (SCDs), preferentially functioning to desaturate stearic acid (C18:0). Mammalian SCDs have been shown to be important for maintaining an appropriate level of fatty acid desaturation, vital for modulating membrane fluidity and for controlling lipid metabolism \[[@pbio-0030053-b15],[@pbio-0030053-b16]\]. Furthermore, in C. elegans, SCDs are also involved in catalyzing the first step in the synthesis of polyunsaturated fatty acids (PUFAs) \[[@pbio-0030053-b17]\] (see [Figure 4](#pbio-0030053-g004){ref-type="fig"}B inset). Because the expression of these three C. elegans Δ9-desaturases was significantly impaired by nhr-49 deletion, we used gas chromatography/mass spectrometry (GC/MS) to determine whether overall fatty acid desaturation and PUFA synthesis were altered in nhr-49(nr2041) animals. The most significant consequence of nhr-49 deletion on fatty acid composition was a marked increase in the abundance of stearic acid (C18:0) and a corresponding decrease in the level of oleic acid (C18:1n9) ([Figure 4](#pbio-0030053-g004){ref-type="fig"}B). This result is consistent with a reduction in SCD activity. In mammals, a primary role of SCD is to control the ratio of fully saturated stearic acid to monounsaturated oleic acid (C18:0/C18:1n9), and this ratio has been employed as an indicator of the activity of the mammalian SCD1 \[[@pbio-0030053-b15],[@pbio-0030053-b16]\]. In C. elegans we found that the ratio of stearic acid to oleic acid (C18:0/C18:1n9) was increased from 1.9 in WT animals to 4.3 in nhr-49(nr2041) ([Table 1](#pbio-0030053-t001){ref-type="table"}). A similar but less pronounced alteration in the ratio of stearic acid to oleic acid was observed when nhr-49 was knocked down in WT animals using RNAi ([Figure 4](#pbio-0030053-g004){ref-type="fig"}B and [Table 1](#pbio-0030053-t001){ref-type="table"}). This smaller influence on fatty acid desaturation likely resulted from the fact that nhr-49 RNAi did not reduce Δ9-desaturase expression as dramatically as nhr-49 knockout (data not shown). ::: {#pbio-0030053-t001 .table-wrap} Table 1 ::: {.caption} ###### Life Span, Fat Content, and Relative C18:0 Fatty Acid Abundance ::: ![](pbio.0030053.t001) Fat content was determined by Nile Red staining. Life span indicated is the day when half of the worms in a given population expired. Fatty acid abundance is indicated by percent of total fatty acid measured by GC/MS ::: C. elegans SCDs are also important for the synthesis of PUFAs; however, we did not observe any measurable changes in PUFA abundance in nhr-49(nr2041) animals ([Figure 4](#pbio-0030053-g004){ref-type="fig"}B). Therefore, despite the dramatic effect of nhr-49 on SCD activity, nhr-49 does not appear to play an appreciable role in overall PUFA synthesis. To determine which of the Δ9-desaturases accounted for the influence of nhr-49 deletion on fatty acid composition, we used RNAi to individually knock down expression of fat-5 and fat-7 (because fat-6 expression is only reduced by approximately 2-fold in nhr-49\[nr2041\], we did not examine fat-6 RNAi animals). Although we did not observe a significant consequence of fat-5 RNAi on fatty acid desaturation, fat-7 RNAi altered fatty acid composition in a manner very similar to that of nhr-49 deletion ([Figure 4](#pbio-0030053-g004){ref-type="fig"}B and [Table 1](#pbio-0030053-t001){ref-type="table"}). fat-7 RNAi animals displayed a significant increase in the abundance of C18:0 fatty acid, as well as a decrease in the levels of C18:1n9 and C18:2n6 fatty acids. The influence of fat-7 RNAi on fatty acid composition was stronger than that of nhr-49 deletion, increasing the ratio of stearic to oleic acid (C18:0/C18:1n9) to 8.0. fat-7 RNAi animals also exhibited a slight lowering of PUFA abundance. The stronger effect of fat-7 RNAi on fatty acid composition suggests that fat-7 expression was reduced to levels even lower than those observed in nhr-49(nr2041). Because fat-7 shares significant homology (approximately 85% nucleotide identity) with fat-6, it was possible that fat-7 RNAi also partially interfered with fat-6 expression; however, we found by QRT-PCR that the overall levels of fat-6 mRNA were not noticeably reduced by fat-7 RNAi. (data not shown). Therefore, we conclude that by strongly promoting SCD expression, nhr-49 influences fatty acid composition in C. elegans. Although it appears that the primary effect of nhr-49 deletion is a modulation of stearic and oleic acid levels, not PUFA abundance, we cannot rule out the possibility that nhr-49 influences PUFA synthesis in a more localized manner, such that the changes in PUFA composition are not detectable by our whole-worm analysis. Interference of fat-7 Expression Reproduces Shortened Life-Span Phenotype {#s2h} --------------------------------------------------------------------------- Using RNAi to knock down the nhr-49-dependent genes identified in this study, we found that interference of only fat-7 resulted in a shortened life-span phenotype similar to that of nhr-49(nr2041) (see [Table 1](#pbio-0030053-t001){ref-type="table"}). fat-7 RNAi animals displayed many of the same characteristics of the nhr-49(nr2041) mutant, including widespread vacuole formation and germ line necrosis ([Figure 5](#pbio-0030053-g005){ref-type="fig"}). ::: {#pbio-0030053-g005 .fig} Figure 5 ::: {.caption} ###### fat-7 Is Necessary for Normal Life Span and Inhibits β-Oxidation \(A) Nomarski images of WT worms subjected to fat-7 RNAi at days 1 and 3 of adulthood. The arrow in the day 1 image points to vacuole formation in the intestine, and the arrow in the day 3 image points to clearing that results from collapse of the gonad. These characteristics are nearly identical to those observed for nhr-49(nr2041) worms (see [Figure 1](#pbio-0030053-g001){ref-type="fig"}B). \(B) QRT-PCR measurement of acs-2 and ech-1 expression in WT and nhr-49(nr2041) L4 animals grown on control RNAi bacteria (dark gray bars) or on fat-7 RNAi bacteria (blue bars). Error bars represent standard error of measurement. \(C) RNAi knockdown of fat-7 expression in WT animals reduced Nile Red fat staining \(D) RNAi of fat-7 in nhr-49(nr2041) also decreased fat staining. ::: ![](pbio.0030053.g005) ::: Interestingly, the effect of fat-7 RNAi on life span was even more potent than nhr-49 deletion, reducing adult life span to 3--5 d, as opposed to the 5- to 7-d life span of nhr-49(nr2041) (see [Table 1](#pbio-0030053-t001){ref-type="table"}). This is consistent with the finding that SCD activity is more dramatically compromised in fat-7 RNAi animals than it is in the nhr-49(nr2041) mutant. In fact, we observed a striking correlation between fat-7 activity, as determined by the ratio of stearic to oleic acid in our various mutants, and life span (see [Table 1](#pbio-0030053-t001){ref-type="table"}). WT animals (stearic/oleic = 1.9) lived approximately 16--18 d, nhr-49 RNAi animals (stearic/oleic = 2.3) lived approximately 10--12 d, nhr-49(nr2041) animals (stearic/oleic = 4.3) lived approximately 6--8 d, and fat-7 RNAi animals (stearic/oleic = 8.0) lived approximately 3--5 d. Because fat-7 expression is significantly compromised by nhr-49 deletion, and because lowered fat-7 expression can lead to shortened life span, we suggest that the reduced life-span phenotype of nhr-49(nr2041) likely reflects, at least in part, diminished fat-7 expression. Stimulation of fat-7 by nhr-49 Feeds Back to Inhibit β-Oxidation {#s2i} -------------------------------------------------------------------- By promoting β-oxidation gene expression, nhr-49 stimulates fat consumption, and by enhancing fat-7 expression, nhr-49 modulates fat desaturation and ensures normal longevity. We next set out to determine whether nhr-49 regulates these two pathways independently. Although RNAi of acs-2 or ech-1 was sufficient to cause a high-fat phenotype, acs-2 or ech-1 interference did not significantly impact life span, fatty acid composition, or desaturase gene expression, demonstrating that the effects of nhr-49 deletion on fat-7 expression and life span were not a downstream consequence of compromised β-oxidation or increased fat content (see [Table 1](#pbio-0030053-t001){ref-type="table"}). In contrast, RNAi of fat-7 significantly impacted fat storage and β-oxidation. The effect was paradoxical, however, as fat-7 interference produced the opposite result of nhr-49 deletion, causing reduced fat content and increased expression of genes in β-oxidation, including acs-2 and ech-1 ([Figure 5](#pbio-0030053-g005){ref-type="fig"}). As the influence of fat-7 on the expression of β-oxidation genes is opposite that of nhr-49, it is clear that the stimulation of β-oxidation by nhr-49 does not result from promotion of fat-7 expression. In fact, by enhancing fat-7 expression, nhr-49 indirectly causes inhibition of the very same β-oxidation genes that it is independently stimulating ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). ::: {#pbio-0030053-g006 .fig} Figure 6 ::: {.caption} ###### Model for nhr-49 Regulation of Fat Storage and Composition By stimulating expression of genes predicted to be involved in fatty acid β-oxidation, nhr-49 promotes fat consumption; likely by facilitating the flow of fatty acids (FA) into the mitochondrial matrix. Through an independent pathway, nhr-49 modulates fatty acid desaturation by enhancing the expression of the fat-7 SCD. Stimulation of fat-7 is necessary for normal adult life span. Finally, fat-7 also feeds back to partially inhibit expression of β-oxidation genes. ::: ![](pbio.0030053.g006) ::: Because fat-7 targets some of the same β-oxidation genes as nhr-49, we wondered if the effects of fat-7 on fat storage might be dependent on nhr-49; for example, perhaps fat-7 represses ech-1 and acs-2 expression by blocking the nhr-49 mediated induction of these genes. However, we found that both ech-1 and acs-2 were induced when fat-7 was knocked down even further in the nhr-49(nr2041) mutant using RNAi, indicating that fat-7 hinders the expression of ech-1 and acs-2 through a mechanism that is independent of nhr-49 (see [Figure 5](#pbio-0030053-g005){ref-type="fig"}B). Furthermore, RNAi of fat-7 reduced fat content in nhr-49(nr2041) worms, demonstrating that the stimulation of fat storage by fat-7 is also not dependent upon nhr-49 (see [Figure 5](#pbio-0030053-g005){ref-type="fig"}D). Taken together, our findings demonstrate that nhr-49 controls two distinct circuits within a gene network: The stimulation of β-oxidation gene expression is independent of fat-7, and the promotion of fat-7 expression is independent of nhr-49\'s effects on β-oxidation. However, because fat-7 expression inhibits the same β-oxidation genes induced by nhr-49, the regulation of fat-7 by nhr-49 connects these two circuits into a single regulatory network, serving to limit β-oxidation through an apparent feedback mechanism ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). Discussion {#s3} ========== Here we demonstrated that deletion of the C. elegans nhr-49 gene produced two global phenotypes: shortened life span and high fat content. Using a QRT-PCR strategy, we found 13 genes involved in energy metabolism that depend upon nhr-49 for expression. The phenotypes of nhr-49(nr2041) were attributable to deficiencies in two different metabolic pathways, fatty acid β-oxidation and fatty acid desaturation. The high-fat phenotype is explained by lowered expression of β-oxidation enzymes, and the shortened life-span phenotype results from reduced expression of the fat-7 SCD. Interestingly, these pathways are linked by an apparent feedback mechanism, as fat-7 also serves to inhibit the expression of fatty acid β-oxidation genes ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). These results represent the first detailed characterization of a divergent HNF4-like receptor in C. elegans and provide the first description of an NHR-controlled fat-regulatory network in invertebrates. Due to their evolutionary relationship to nhr-49, it seems conceivable that many of the C. elegans HNF4-like NHRs might participate in the regulation of fat metabolism. However, we have found that several other worm HNF4-like receptors, including those previously implicated for affecting overall fat storage \[[@pbio-0030053-b12]\], do not significantly impact the expression of fatty acid metabolism genes (M. R. Van Gilst and K. R. Yamamoto, unpublished data). Thus, the regulation of fat metabolism does not appear to be a general mechanism common to all of the divergent NHRs; whether or not nhr-49 is unique in its control of C. elegans fat metabolism remains to be determined. The mechanism by which nhr-49 influences fat storage is likely to be through modulation of β-oxidation gene expression ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). Overexpression of acs-2 alone was sufficient to suppress the high-fat phenotype of nhr-49(nr2041), indicating that acs-2 expression is rate-limiting in nhr-49 deletion mutants, thus leading to reduced fat breakdown and excessive fat accumulation. Acyl-CoA synthetases activate fatty acids for many different metabolic pathways; in particular, mitochondrial acyl-CoA synthetases are likely to activate fatty acids for transport into the mitochondrial matrix, where the fatty acids are then subject to β-oxidation \[[@pbio-0030053-b18],[@pbio-0030053-b19]\]. Notably, mitochondrial acyl-CoA synthetases are commonly regulatory targets for factors that control fat consumption \[[@pbio-0030053-b18]\]. We propose that by stimulating acs-2 expression, nhr-49 likely acts by increasing the flow of fatty acids into the mitochondria for β-oxidation ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). Out of 11 acs genes in C. elegans, five of which are predicted to be mitochondrial, nhr-49 specifically affected the expression of only acs-2, suggesting that nhr-49 may be promoting transport of a specific subset of fatty acids into mitochondria. These subsets may differ by fatty acid chain length or tissue distribution. As both acs-2 and nhr-49 are expressed in numerous C. elegans tissues, regulation of fat consumption by nhr-49 may occur throughout the animal. Although acs-2 is related (with approximately 25% identity) to multiple mammalian acyl-CoA synthetases, mammals contain a particular homolog with approximately 41% identity to the C. elegans ACS-2 protein; this mammalian ACS protein is yet to be characterized. It will be interesting to determine whether the mammalian counterpart of acs-2 also serves as an important focal point for gene networks that regulate fat utilization in mammals. Interestingly, we also found that nhr-49 stimulates expression of a carnitine palmitoyl transferase (F09F3.9). Carnitine palmitoyl transferases execute the step immediately downstream of acyl-CoA synthetase in mitochondrial β-oxidation, directly shuttling activated acyl-CoAs into the mitochondrial matrix. Thus, nhr-49 appears to target multiple enzymes involved in transporting fatty acids across the mitochondrial membrane for β-oxidation ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). RNAi of F09F3.9 did not detectably alter overall fat content, however, suggesting that interference of F09F3.9 expression alone is not sufficient to affect overall fat consumption under the conditions surveyed in our study. In contrast, we found that RNAi of ech-1, another β-oxidation gene targeted by nhr-49, was sufficient to increase overall fat content; thus it is likely that the reduced expression of ech-1 also contributes, in some fashion, to the high-fat phenotype of nhr-49(nr2041). Thus, we have demonstrated that nhr-49 strongly promotes the expression of three separate enzymes predicted to participate in mitochondrial β-oxidation. Consequently, we suggest that nhr-49 plays a prominent role in promoting the breakdown of fatty acids in mitochondria, and that, through control of β-oxidation, nhr-49 influences overall fat storage. In addition to its effect on β-oxidation, we found that nhr-49 helps to maintain the balance of saturated and monounsaturated fatty acids. By stimulating the expression of three fatty acid Δ9-desaturase genes, nhr-49 facilitates the conversion of saturated fatty acids to monounsaturated fatty acids, and consequently, nhr-49(nr2041) mutant animals are high in saturated fat and lower in monounsaturated fat. In C. elegans, the FAT-6 and FAT-7 SCDs may also be involved in the synthesis of PUFAs \[[@pbio-0030053-b17]\]. However, a dramatic reduction in fat-7 expression, either by nhr-49(nr2041) deletion or by fat-7 RNAi, did not significantly affect PUFA synthesis. This result suggests that a normal level of FAT-7 expression is not necessary for PUFA synthesis, likely because FAT-6 is the principle enzyme in this pathway, or because FAT-6 is capable of substituting for reduced FAT-7 activity. In either event, we propose that the primary effect of nhr-49 on fatty acid composition is a modulation of the ratio of C18:0 to C18:1n9 fatty acid ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). However, we cannot rule out the possibility that nhr-49 and fat-7 also affect PUFA synthesis in a more localized manner, for example, specific cell types, such that changes in PUFA abundance are not detected in our whole-worm analysis. Our results also indicate that the short life-span phenotype of nhr-49(nr2041) is due, at least in part, to reduced expression of fat-7. The strong correlation between the increase in C18:0 saturated fatty acid and the shortening of life span is compelling. Several reports in mammals have described changes in fatty acid composition during aging, and it has been argued that an alteration of fatty acid saturation ratios in mitochondrial membranes may affect the rate of aging \[[@pbio-0030053-b20]\]. Additionally, imbalance of fatty acid saturation has also been linked to numerous pathological conditions \[[@pbio-0030053-b15],[@pbio-0030053-b16]\]. Although SCDs have not yet been linked to life span in mammals, studies in C. elegans have identified fat-7 as one of the targets of the mechanisms that extend longevity through the insulin pathway \[[@pbio-0030053-b21]\]. Thus, it will be interesting to determine if the shortened life-span phenotype of nhr-49(nr2041) and fat-7 RNAi animals results from an improper ratio of saturated and monounsaturated fats, and if the mechanistic cause of the reduced life span is related to accelerated aging or to another pathology. Although it is clear that nhr-49 independently promotes the expression of genes in fatty acid β-oxidation and desaturation, we found that downstream components of these pathways do indeed communicate with each other. By inhibiting the expression of genes involved in β-oxidation, including the nhr-49-dependent genes acs-2 and ech-1, fat-7 works against the actions of nhr-49, acting to hinder fat consumption ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). Thus, our results reveal an elegant feedback mechanism: nhr-49 induces the expression of β-oxidation enzymes---thus promoting fat consumption---yet by independently stimulating fat-7 expression, nhr-49 tempers the expression of the very same β-oxidation genes ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). Thus, we suggest that nhr-49 serves as a key regulator of fat usage, governing pathways that consume fat for energy and partition fat for storage, potentially shifting the balance in response to changing energy needs. Indeed, we have found that in response to starvation, nhr-49 selectively modulates the expression of these two pathways in order to increase fat consumption (M. R. Van Gilst and K. R. Yamamoto, unpublished data). As the predominant function of fat-7 is to convert C18:0 to C18:1n9 fatty acid, it is tempting to speculate that one of these two fatty acid species serves as a signal to modulate the feedback effect of fat-7 on fatty acid β-oxidation ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). In this scenario, C18:0 could be serving as an activator of β-oxidation gene expression, C18:1n9 could be functioning as a repressor, or both ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). Although it seems reasonable to propose that an NHR would communicate this fatty acid signal, we found that the effects of fat-7 on fatty acid β-oxidation were not dependent on nhr-49; hence, one of the other 283 C. elegans NHRs may mediate this effect. Despite its strong sequence similarity with the mammalian HNF4 receptors, the overall biological effects of nhr-49 on metabolism, fat storage, and life span are remarkably similar to those of the mammalian PPARs. Perhaps most striking is the finding that nhr-49 and the PPARs, particularly PPARα and PPARδ, positively influence similar genes in multiple metabolic processes, including fatty acid β-oxidation, fatty acid desaturation, and fatty acid binding/transport \[[@pbio-0030053-b22],[@pbio-0030053-b23]\]. Moreover, knockout of PPARα or PPARδ can lead to high-fat phenotypes \[[@pbio-0030053-b23],[@pbio-0030053-b24]\], demonstrating that the physiological effects of these receptors on fat storage are similar to those of nhr-49. The regulation of SCDs by nhr-49 also parallels the regulation of the SCD1 gene by PPARα. In mammals, the Δ9-desaturase SCD1 is a lipogenic enzyme that is controlled by several key regulators of fat storage, including PPARα, leptin, and sterol response element-binding protein \[[@pbio-0030053-b15],[@pbio-0030053-b25],[@pbio-0030053-b26]\]. When SCD1 expression is left unchecked, as occurs in leptin knockout mice, severe obesity can result. Similar to what we found for the C. elegans fat-7 gene, mammalian SCD1 affects fat storage by inhibiting β-oxidation \[[@pbio-0030053-b27]\]. Thus, because PPARα also promotes fat consumption by stimulating β-oxidation gene expression\[[@pbio-0030053-b28]\], it is clear that PPARα is independently regulating pathways that promote fat consumption and stimulate fat storage. In fact, a similar feedback mechanism to what we have described here for nhr-49 has been proposed for PPARα and SCD1 \[[@pbio-0030053-b27],[@pbio-0030053-b29]\]. Taken together, these results demonstrate that the multicircuited regulatory networks governing fat usage appear well conserved between C. elegans nhr-49 and the mammalian PPARα. Although we have not yet determined whether or not the nhr-49-dependent genes identified in this study are direct transcriptional targets, each is homologous to known transcriptional targets of the PPARs \[[@pbio-0030053-b22],[@pbio-0030053-b23]\]. Because NHR-49 and the PPARs are clearly regulating similar physiological processes and gene networks, we believe that they are likely to be using similar molecular mechanisms, including direct transcriptional regulation. However, it is possible that nhr-49 controls fatty acid β-oxidation and desaturation through an indirect mechanism, functioning upstream of the transcription factors that directly act on the nhr-49-dependent genes identified in this study. The latter possibility would also be intriguing, as it would demonstrate NHR involvement in fat regulation at a point upstream of a PPAR-like factor. As the physiological functions and regulatory targets of nhr-49 are highly similar to those of the mammalian PPARs, it seems reasonable to speculate that, like the PPARs, NHR-49 may be regulated by fatty acid ligands. By binding directly to ingested lipids and/or their metabolic products, NHR-49 could serve both as a sensor of intracellular fatty acid levels and an effector, responding to changes in fatty acid composition through modulation of fatty acid β-oxidation and desaturation. Interestingly, the mammalian HNF4 receptors, whose LBDs share 33% identity with the NHR-49 LBD, can bind to several saturated and monounsaturated fatty acids, including stearic and oleic acid \[[@pbio-0030053-b30],[@pbio-0030053-b31]\]; the mammalian PPARs also bind to many types of fatty acids, although they interact preferentially with PUFAs \[[@pbio-0030053-b22]\]. It will be interesting to determine if mammalian HNF4 receptors harbor an undiscovered capacity to regulate fat metabolism in mammals, or if PPARs have adopted their regulatory functions by evolving from an ancestral HNF4 gene that had properties similar to nhr-49 and the modern PPARs. Alternatively, the PPARs and nhr-49 may have acquired their activities through convergent evolution. Precise regulation of fatty acid β-oxidation and desaturation is crucial for physiological homeostasis; imbalance in these metabolic pathways in humans can lead to disorders such as diabetes, obesity, atherosclerosis, and accelerated aging. However, although NHRs are important targets for drugs that moderate these metabolic disorders, the NHR-mediated regulatory networks that govern fat usage are notably complex and poorly understood. We showed here that many features of C. elegans nhr-49 regulation of fat metabolism are closely analogous to those of the mammalian PPARs, key targets for the treatment of metabolic disease. Moreover, our C. elegans studies revealed that nhr-49, like PPARα, oversees a branch point for two separate circuits within a network that controls fatty acid consumption and composition. The extent of the functional homology between nhr-49 and PPARα remains to be determined, that is, whether or not nhr-49 also functions by binding to fatty acid ligands and directly regulating gene transcription. Nevertheless, our finding that nhr-49 is a key regulator of fat metabolism in C. elegans represents a considerable advance in our understanding of NHR control of fat storage and composition in invertebrates. Consequently, we suggest that these and future studies in C. elegans will continue to enhance our understanding of the complex roles of NHRs and their ligands on gene networks governing fat physiology. Materials and Methods {#s4} ===================== {#s4a} ### RNAi constructs {#s4a1} All of the RNAi constructs used in the NHR screen were created by cloning full-length NHR cDNAs into the vector L4440 (Andy Fire, Stanford University). The fat-7 RNAi vector was a gift from Jennifer Watts (Washington State University). All of the other RNAi constructs were obtained from the Ahringer RNAi library \[[@pbio-0030053-b32]\]. ### Life span assays {#s4a2} Approximately 30--40 starved L1 worms were transferred to RNAi bacteria (HT115 transformed with RNAi clone) and life-span assays were carried out at 23 °C as described previously \[[@pbio-0030053-b33]\]. nhr-49(nr2041) and WT life-span assays were carried out at 23 °C on OP50 bacteria. ### Nile Red assays {#s4a3} Nile Red fat-staining assays were carried out as described previously \[[@pbio-0030053-b12]\]. ### Selection of fatty acid and glucose metabolism genes {#s4a4} Most of the fatty acid and glucose metabolism candidate genes examined in this study were initially identified using the KEGG pathway database (<http://www.genome.ad.jp/kegg/pathway.html>). Other genes were identified using BLAST searches designed to find C. elegans open reading frames that were highly related to known mammalian glucose and fat metabolism enzymes, or to plant glyoxylate cycle genes. For prediction of β-oxidation enzyme subcellular localization, we used the following online prediction tools. TargetP server version 1.01 (<http://www.cbs.dtu.dk/services/TargetP/>) \[[@pbio-0030053-b34]\] and PSORT (<http://psort.nibb.ac.jp/form.html>). ### Preparation of nematode total RNA {#s4a5} Both C. elegans N2-Bristol (WT) and nhr-49(nr2041) were grown at 23 °C on high-growth plates seeded with OP50 bacteria. Gravid adults from 30 10-cm plates were bleached, and embryos were dispersed onto 15-cm nematode growth media (NGM)-lite plates seeded with OP50. The worms were distributed as follows: for L1 harvest, 80,000 embryos/plate; for L2 harvest, 40,000 embryos/plate; for L3 harvest, 20,000 embryos/plate; and for L4 harvest, 10,000 embryos/plate. The remaining embryos were saved for embryonic RNA preparation. Worms were harvested at the appropriate stage, washed twice with M9, and frozen in liquid nitrogen. For RNAi experiments, 10,000 embryos were placed onto NGM-lite plates containing 8 mM IPTG and 100 μg/ml carbenicillin seeded with control bacteria (HT115 transformed with empty L4440 vector) or nhr-49 RNAi bacteria (HT115 transformed with L4440-nhr-49). Worms were harvested at the L4 stage of development, washed twice with M9, and frozen in liquid nitrogen. For RNA preparation, worms were thawed at 65 °C for 10 min, and RNA was isolated using the Tri-Reagent Kit (Molecular Research Center, Cincinnati, Ohio, United States). Isolated total RNA was subjected to DNAase treatment and further purification using RNAeasy (Qiagen, Valencia, California, United States). ### QRT-PCR {#s4a6} cDNA was prepared from 5 μg of total RNA in a 100-μl reaction using the Protoscript cDNA preparation kit (New England Biolabs, Beverly, Massachusetts, United States). Primer pairs (primer sequences available upon request) were diluted into 96-well cell culture plates at a concentration of 3 μM. Next, 30-μl PCR reactions were prepared in 96-well plates. Each PCR reaction was carried out with TaqDNA Polymerase (Invitrogen, Carlsbad, California, United States) and consisted of the following reaction mixture: 0.3 μM primers, 1/500th of the cDNA reaction (corresponds to cDNA derived from 10 ng of total RNA), 125 μM dNTPs, 1.5 mM MgCl~2~, and 1X reaction buffer (20 mM Tris pH 8.4, 50 mM KCl). 0.15 μl (0.75 units) of TaqDNA Polymerase was used for each reaction. Formation of double-stranded DNA product was monitored using SYBR-Green (Molecular Probes, Eugene, Oregon, United States). All QRT-PCR reactions were carried out and analyzed on a DNA Engine-Opticon 2 (MJ Research, Waltham, Massachusetts, United States). Data were collected using RNA from at least three independent C. elegans growths. To determine the relationship between mRNA abundance and PCR cycle number, all primer sets were calibrated using serial dilutions of cDNA preparations. Primer sets were also calibrated by performing QRT-PCR reactions on serial dilutions of C. elegans genomic DNA. Relative abundance is reported as the mRNA abundance of each gene relative to the mRNA abundance of several control genes, which are expressed at constant levels throughout development. ### GC/MS {#s4a7} Fatty acids were isolated from 10,000 L4 animals grown on a single 15-cm NGM-Lite plate. For WT and nhr-49(nr2041) experiments, worms were grown at 23 °C on a lawn of OP50. For RNAi, 10,000 embryos were placed on control bacteria, or on fat-5, fat-7, ech-1, acs-2, or nhr-49 RNAi bacteria. Fatty acid extract was prepared as described previously \[[@pbio-0030053-b17]\]. GC/MS spectra were collected on an HP 6890 gas chromatograph outfitted with a J&W DB-XLB column. The mass spectrometer was an HP MSD 5973 (Agilent, Palo Alto, California, United States) and data were analyzed using Chemstation version A.03.00 software (Agilent). Peaks were assigned using fatty acid standards. ### GFP reporter analysis and ectopic expression of ACS-2::GFP {#s4a8} To make the P~nhr-49~GFP plasmid, a genomic fragment including 3.5 kb of the nhr-49 upstream sequence and 88 bp of the first exon of nhr-49 was ligated into the L3691 GFP expression vector (a gift of A. Fire). Germ-line transformation was performed following standard procedures \[[@pbio-0030053-b35]\]. P~nhr-49~GFP was injected at the following concentrations: 1 ng/μl, 5 ng/μl and 20 ng/μl along with the co-injection marker pRF4 rol-6(su1006) injected at 60 ng/μl. At all three P~nhr-49~GFP concentrations, we were unable to obtain stably transmitting lines. Animals used for GFP imaging were derived from the F1 generation and were mosaic, hosting the P~nhr-49~GFP transgene in only a subset of tissues. To construct acs-2::gfp, a 4-kb genomic fragment, including the full-length acs-2 gene plus 2 kb of upstream sequence, was cloned into the vector L3691. Germ-line transformation was performed using 50 ng/μl of acs:2::gfp and 50 ng/μl pRF4 rol-6(su1006). For N2/acs-2::gfp and nhr-49(nr2041)/acs-2::gfp, over ten independent transmitting lines were obtained and analyzed by GFP microscopy. GFP reporter expression was observed using a Zeiss Axiovert S100 fluorescence microscope (Carl Zeiss, Thornwood, New York, United States) under a 40X objective. For rescue experiments, 50--100 ng/μl of acs-2::gfp was injected into nhr-49(nr2041) and WT animals along with 50 ng/μl of pRF4 rol-6(su1006). Transgenic adults, selected for rolling behavior, were placed on Nile Red plates and assayed for fat content as described above. pRF4 rol-6(su1006) was injected into nhr-49(nr2041) in combination with unrelated plasmids to demonstrate that the lowered fat content was not a result of hosting a transgenic array or caused by expression of the rol-6(su1006) marker gene. Supporting Information {#s5} ====================== Table S1 ::: {.caption} ###### Raw Data for QRT-PCR Screen Data are shown as C~t~, the number of cycles required for amplification of a particular target gene from a cDNA preparation. Changes in gene expression in the nhr-49(nr2041) mutant were measured as ΔC~t~. ΔC~t~ are obtained by subtracting nhr-49(C~t~) from N2(C~t~) and adjusting for RNA concentration differences with a correction factor (CF). The CF was determined by aligning the data to a set of standard genes. Thus the equation for ΔC~t~ is as follows: Genes were designated as nhr-49 dependent if the C~t~ was \>1.0 in multiple developmental stages (and was greater than the standard error). nhr-49 targets are marked with an X. The QRT-PCR results of the major nhr-49 targets, acs-2, ech-1, fat-5, and fat-7, were confirmed with a second primer pair. (100 KB XLS). ::: ::: {.caption} ###### Click here for additional data file. ::: We thank Carl Johnson and Nemapharm Pharmaceuticals for the nhr-49(nr2041) strain and Trisha Brock and Jennifer Watts for the fat-7 RNAi vector. We also thank Kaveh Ashrafi, Barbara Meyer, Jennifer Watts, Neil Freedman, Hans Luecke, Marc Diamond, and Holly Ingraham for critical comments on the manuscript, and Jen-Chywan Wang, Ann Sluder, and members of the Yamamoto laboratory for useful discussions. MRVG was supported by a Helen Hay Whitney Fellowship and by a National Institutes of Health grant (NIH K01 DK61361--01). Research support was from NIH. Competing interests. The authors have declared that no competing interests exist. Author contributions. MRVG and KRY conceived and designed the experiments. MRVG, HH, and AJ performed the experiments. MRVG analyzed the data. MRVG contributed reagents/materials/analysis tools. MRVG and KRY wrote the paper. Citation: Van Gilst MR, Hadjivassiliou H, Jolly A, Yamamoto KR (2005) Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in C. elegans. PLoS Biol 3(2): e53. GC/MS : gas chromatography/mass spectrometry GFP : green fluorescent protein HNF4 : hepatocyte nuclear factor 4 LBD : ligand binding domain NGM : nematode growth media NHR : nuclear hormone receptor PCR : polymerase chain reaction. PPAR PUFA : polyunsaturated fatty acid QRT--PCR : quantitative RT-PCR RNAi : RNA interference SCD : stearoyl-CoA desaturase WT : wild-type	PubMed Central	2024-06-05T03:55:52.440163	2005-2-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547972/", "journal": "PLoS Biol. 2005 Feb 8; 3(2):e53", "authors": [ { "first": "Marc R. Van", "last": "Gilst" }, { "first": "Haralambos", "last": "Hadjivassiliou" }, { "first": "Amber", "last": "Jolly" }, { "first": "Keith R", "last": "Yamamoto" } ] }
PMC548128	Background ========== Recent technological advances have led to an explosive growth in high-throughput genomic and proteomic data such as DNA microarrays. The rapid growth in available data has led in turn to a need for novel quantitive methods for analysis. As a consequence of this need, the reconstruction of gene network architectures from DNA microarray expression data has become a major goal in the field of systems biology. An increased understanding of the network architectures and their respective dynamics will enable novel approaches to disease treatments by allowing us, for example, to identify drug targets in silicowhich manipulate the functional outputs of these networks. This process is expected to lead to novel classes of drug based on a network approach to cellular dynamics. Frequently, the gene expression data itself is derived from perturbation experiments such as stress conditions, temperature shifts, and chemical treatments; for example, the widely used yeast cell-cycle datasets of Cho \[[@B1]\] and Spellman \[[@B2]\]. Although these global perturbations are carried out in order to reveal causality between genes, it is not always clear how experiments should be designed so as to reveal as much causality as possible, while both minimising costly experimentation and remaining computationally tractable. A range of computational and mathematical techniques have been adopted in the effort to find a successful gene network reconstruction technique. Reconstruction methods often have to negotiate a tradeoff between intensive (often intractable) computations, and having to perform a large number of costly experiments. Certain progress can be achieved by making simplifications, such as imposing a limit on the number of inputs to each gene, or making steady state assumptions about the system \[[@B3],[@B4]\]. Some techniques described in the literature offer efficient algorithms, but require a large number of experiments, perhaps as many as there are genes \[[@B5]-[@B7]\]. On the other hand, theoretical work on Boolean models has shown \[[@B8]\] that perhaps as few as O(log(n)) experiments (input/output pairs) might be required for ngenes, but that to infer these relationships requires the use of computationally costly enumeration methods. In this paper, we propose to explore the issue of how perturbation microarray experiments might be designed, and to suggest how such experiments might be optimised so as to maximize inference capability. Logical gene network models have previously been used to investigate gene network robustness \[[@B9]\], perturbation dynamics \[[@B10]\] and evolutionary potential \[[@B11]\], and form the basis of the inference method used in this study. This inference method \[[@B11]\] is similar to others in which networks with a minimal number of connections are reconstructed through enumeration \[[@B12],[@B13]\]. Given the significant speed advantage of integer computation over floating point computation, and that most genes are expected to have few inputs (93% have between 1 and 4 \[[@B14]\]), the method is considered to be adequate for this investigation. In this study, exhaustive evaluation was performed up to a maximum of 4 inputs of both positive and negative sign (see Methods). Enumeration is computationally feasible on an ordinary desktop computer for medium-sized networks (n\~ 100), and still tractable for large networks (n\~ 1000), though this would require some parallelisation. The global perturbations themselves are simulated by changing the state of each gene at random. A perturbation intensity measure q, defines the probability that each gene will change state (see Methods). Results and discussion ====================== A limited number of perturbations significantly improve accuracy ---------------------------------------------------------------- A discrete dynamical model was used to generate time series data from random networks (see Methods). To measure the effect of adding perturbations on inference ability, inference sensitivity(defined as true positives/true positives + false negatives, see Methods) was measured against P, the number of additional perturbations. Figure [1](#F1){ref-type="fig"} shows the results for predicted solutions with one and two inputs, as well as overall sensitivity. The top graph in figure [1](#F1){ref-type="fig"} shows that overall sensitivity is clearly enhanced by including more perturbation experiments, with lower order solutions (one and two inputs) reaching higher levels of sensitivity. The bottom graph shows the corresponding inverse relationship for the standard deviation of the sensitivity (lower for higher P). It should be noted that the algorithm tends to underestimate the number of inputs a gene may have. This is to be expected in genes for which dynamics cannot be informative: for example, consider a gene iwhich has one or more negative inputs, as well as having default value OFF. Since the discrete dynamics for this gene will be the same as if it had no inputs at all (i.e. zero gene expression for t\> 0), the presence of the inputs is impossible to infer. This underestimation effect is clear in table [1](#T1){ref-type="table"}, which compares the distribution of inferred solution set sizes (\\|Y~i~\\|, see Methods) with the actual solution sizes (i.e. the indegree distribution), and shows that the method is only able to produce roughly half the number of one and two input solution sets that actually exist. The increase in sensitivity with Pcan be explained at least partially, in the following way. Since the time series are discrete, many of the genes may have identical behaviour over time despite having different inputs (i.e. s~i~(t) = s~j~(t) for two different genes iand j). If we define a \"concatenated\" time series vector ![](1471-2105-6-11-i1.gif) for gene i, and then map each gene ionto S~i~, we obtain a many-to-one mapping. As we increase the number of perturbations, we might expect the number of distinct time series also to increase. We define a simple measure to quantify this mapping, M= n\'/Nwhere n\' is the number of distinct vectors S~i~, and Nis the number of genes. The maximum value of M= 1 indicates that the mapping of genes to time series is one-to-one, whereas lower values indicate degenerate mappings. The manner in which Mincreases with the number of perturbations is shown in figure [2](#F2){ref-type="fig"}, and shows how the increase in M reflects the corresponding increase in sensitivity (figure [1](#F1){ref-type="fig"}). Network size and optimal perturbation intensity ----------------------------------------------- The experiments described above were repeated to consider variations in two other parameters: the network size N, and the perturbation intensity parameter q(roughly, the proportion of genes whose initial expression level is changed in each perturbation experiment -- see Methods). To consider the first case, the minimum number of perturbations P\* required to reach a given high accuracy criterion was measured for different values of the network size N. The high accuracy criterion was defined as average sensitivity = 0.95 for one-input solution sets (average sensitivity is found using a default value q = 0.5 and averaging for all the sensitivity measurements obtained from 250 random networks). To find P\, we first find the number of perturbations P^+^, such that average sensitivity P^+^≥ 0.95, and average sensitivity (P^+^- 1) \< 0.95. If average sensitivity P^+^\> 0.95, we use simple linear interpolation to find the (real) value of P\ between P^+^and (P^+^- 1) for which average sensitivity = 0.95. The resulting values for P\* are shown in figure [3](#F3){ref-type="fig"}. Since the relationship is expected to be logarithmic \[[@B8]\], the plot shows log(N) against P\* (logarithms used are base 10). A least squares best fit gives P\* ≃ 1.75 log(N) + 7.02, which, for N= 1000, gives P\* ≃ 12.26. In order to obtain a measure of variance for P\, we would need to calculate P\-equivalent values for many individual networks separately, then consolidate these values to obtain the relevant statistics. However, because it was only feasible to consider medium-sized networks (20 ≤ N≤ 70), and for any such network we often find only a small number of one-input solution sets, such statistics were found to be unreliable. The second case (varying perturbation intensity) suggests an optimal range for q. Figure [4a](#F4){ref-type="fig"} shows the inference sensitivity over a range of values for q, and figure [4b](#F4){ref-type="fig"} shows the corresponding standard deviation. Again, inference sensitivity for one-input solutions is higher than for two-input solutions, which in turn is higher than overall sensitivity. For one-input solutions, the results show a clear peak for sensitivity close to the range 0.5 \<q\< 0.6. Together with a corresponding minimisation of the standard deviation in this interval (though it still remains fairly high in absolute terms), these results suggest that perturbation intensity should be close to this range to optimise inference accuracy. Conclusions =========== A recent analysis of the yeast genetic network has shown that 93% of genes are regulated by between 1 and 4 genes \[[@B14]\]. This suggests that enumerative network reconstruction methods can be useful within computationally feasible limits. Experiments involving large-scale perturbations (such as temperature shifts, chemical stress) are a standard way of obtaining time-series of gene expression data \[[@B1],[@B2]\]. A key result of \[[@B14]\] is that indegree appears to follow an exponential distribution, whereas outdegree follows a scale-free distribution, which has enabled the generation of realistic artificial gene networks used here. A logical model \[[@B11]\] was used to simulate the perturbed expression data. Subsequently, experimental parameters were considered in relation to inference accuracy, namely: a) number of perturbations required, P, and b) perturbation intensity, q. The inference method itself is most useful for low order inputs, with inference accuracy maximized for predicted single input genes. More accurate methods have been proposed, though these generally require a much larger number of experiments \[[@B5],[@B15]\]. Methods such as the one proposed here, which infer relationships from expression data may well be more successful when used in conjunction with other methods such as promoter analysis \[[@B16],[@B17]\], or when used to drive experimental procedure \[[@B18]\]. Here, the results show that only a relatively small number of perturbations are necessary in order to achieve a substantial inference accuracy, even for large N. These relatively modest experimental requirements would presumably imply lower experimental costs. The results also suggest that the perturbations should be calibrated (by changing stress intensity, for example), so as to alter the expression levels of approximately half the genes in each experiment. Generating perturbations which alter the expression level of half the genes at random may be difficult to achieve in practice, though experiments can be designed to come as close to this goal as possible. Even in the absence of optimal perturbations, we hope the simulation approach described here will still serve as a useful tool for planning experiments. Methods ======= Discrete dynamical model ------------------------ For a system of Ngenes, the state of each gene s~i~(i= 1, .., N) is represented by the binary values 0(OFF) and 1(ON). Additionally, each gene is assigned a default ON/OFF state θ~i~∈ {0, 1}. The gene interactions are described by an (N× N) matrix C, composed of elements C~ij~∈ {-1, 0, +1}, representing the positive(+1), zero(0) or negative(-1) influence of gene jon gene i. State transitions are calculated as follows: ![](1471-2105-6-11-i2.gif) The state of the ith gene at the next timestep, s~i~(t+ 1), is therefore determined by the balance of positive versus negative inputs which are ON at the previous timestep t. If the balance is positive, then u~i~(t) \> 0 and the next state will be 1(ON). Similarly, if the balance is negative, then u~i~(t) \< 0 and the next state will be 0(OFF). If u~i~(t) = 0 (indicating either that there are no active input connections, or that they balance out), then the default value θ~i~determines the next state. This default value needs to be given a priori, and for the purpose of this study will be random. Network inference method ------------------------ Assuming we are given the state dynamics s(t) and the default vector θ, the problem is to find the necessary model parameters (C) which will reproduce these dynamics. Specifically, a system initialised at s(0) should reproduce the given dynamics s(t) for t\> 0. Note that multiple s(t) expression patterns may be defined, which will be denoted as s^r^(t) for r= 0, .., P, corresponding to time series with different initial states s^r^(0). Our problem is to find at least one interaction matrix that will reproduce all given dynamics s^r^(t). The problem of finding an appropriate matrix Cmay be broken up into Nsub-problems, since in this system, each gene imay be solved independently from the others. More precisely, the inputs to gene i(i.e. C~i~, the ith row of C), can be found independently of the other genes. This reduces the search space from ![](1471-2105-6-11-i3.gif) down to O(N3^N^). Each input z^i^to gene iis represented as an ordered pair (j, g), j∈ {1, .., N}, g∈ {± 1}, indicating an input from gene jof sign g. A solution y(i) for gene iis a set of Kinputs ![](1471-2105-6-11-i4.gif) (with y(i) = φif K= 0). For Kinputs there are ![](1471-2105-6-11-i5.gif) solutions to evaluate. Starting with K= 0 (no inputs), we progress up to a maximum of K= 4, exhaustively evaluating all possible solutions for each K. However, making a parsimony assumption, if solutions are found for some K~s~\< 4, the method no longer evaluates for K\>K~s~. Note that the method does not stop as soon as a solution is found, but evaluates all possible solutions for K~s~. The failure rate (percentage of genes for which no solution was found for K≤ 4) never exceeded 3% of the genes in any single network for which reconstruction was attempted. Global perturbations and the perturbation intensity measure ----------------------------------------------------------- The control time series s^0^(t) is generated by setting s^0^(0) = θ. The other time series s^r^(t), r\> 0 are obtained from initial conditions which are perturbations of θ, and correspond to standard experiments such as stress conditions, or chemical treatments. Since, experimental perturbations can usually be modulated in intensity (for example, a temperature shift), this was represented using modulated artificial perturbations. Perturbed initial states s^r^(0) were generated by randomly changing each state s^0^(0) with probability q. This means that, on average, there will be qNrandom state differences between each perturbed initial state s^r^(0), and θ. Measuring inference accuracy ---------------------------- Assuming one or more solutions y~1~(i), y~2~(i), \... are found for gene i, these are consolidated into a solution set, Y~i~= ∪~l~{y~l~(i)}. Note that some information about the solutions has been lost using this approach. For example, a solution set ![](1471-2105-6-11-i6.gif) obtained from a single two-input (K= 2) solution: ![](1471-2105-6-11-i7.gif), may be equal to another solution set ![](1471-2105-6-11-i8.gif) resulting from two single-input (K= 1) solutions: ![](1471-2105-6-11-i9.gif) with ![](1471-2105-6-11-i10.gif) and ![](1471-2105-6-11-i11.gif). However, this consolidation is convenient in that the solution set is easily compared with the known network structures using standard accuracy measures such as sensitivityand specificity. Here, accuracy was measured using sensitivity, defined as true positives / (true positives + false negatives). The relatively large number of true negatives, makes specificity, defined as true negatives / (true negatives + false positives), an uninformative statistic. Here, true positivesare members of the solution set Y~i~which are also true inputs (since the networks will be generated artificially, true inputs are known), and false negativesare those true inputs which are not members of the solution set Y~i~. Accuracy statistics were gathered from inferences performed on a large number of medium-sized random networks (20 ≤ N≤ 70). Inferences on Rrandom networks (each with Ngenes), will produce approximately RN sensitivitymeasurements (slightly fewer due to the nonzero failure rate). Artificial gene network generation ---------------------------------- It appears to be the case in gene networks that indegree follows an exponential distribution, whereas outdegree appears to follow a scale-free distribution. More specifically, for the yeast network, the probability distribution for indegree kfollows p~k~\~ C~in~e^-βk^with β\~ 0.45, whereas the distribution for outdegree follows p~k~\~ C~out~k^-τ^, with τ\~ 1 (C~in~, C~out~constants) \[[@B14]\]. Here, artificial gene networks \[[@B19]\] were created using the algorithm for generating directed graphs with arbitrary in/out degree distributions described in \[[@B20]\]. The exponential probability distribution for indegree kis given by: p~k~= (1 - e^-β^)e^-βk^, where β= 0.45 is a constant. Similarly, the power law distribution (including an exponential cutoff term which is both biologically realistic and necessary analytically when τ\< 2 \[[@B20]\]) for outdegree kis described by: p~k~= Ck^-τ^e^-γk^, where C, γ, and τ= 1 are constants. Since the algorithm begins by generating in/out-degree pairs for each node, we require equal means for both indegree (\<k~in~\>) and outdegree (\<k~out~\>). Following \[[@B20]\], we obtain expressions for the mean in/out degree: ![](1471-2105-6-11-i12.gif) Since βis given, we obtain a value \<k~in~\> = 1.76, and fit the free parameter γ= 0.436 to obtain \<k~out~\> = \<k~in~\> Since the resulting networks are unweighted, non-zero weights (C~ij~∈ {-1, +1}) are assigned at random with probability 0.5, as in \[[@B19]\]. It should be noted that autoregulatory interactions can be (and indeed were) generated, and that these present no particular problem for the inference method. An example of a network which was used in the analysis is shown in figure [5](#F5){ref-type="fig"}. Authors\' contributions ======================= TM devised and implemented the experiments and drafted the manuscript. RS and AP supervised the project. All authors read and approved the final manuscript. Acknowledgements ================ TM was supported by a grant from the UK Medical Research Council (MRC). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *Sensitivity vs. P.**(a) Sensitivity vs. number of additional perturbations used. (b) The corresponding standard deviation is shown here separately for clarity. The curves represent results for overall (i.e. all solutions) sensitivity, and specific sensitivity for (predicted) one and two-input solutions. Sensitivity is generally lower for higher order of inputs. Accuracy increases significantly with the number of additional perturbations used. The results shown are average values for 250 random networks at each data point. The remaining parameters are fixed: network size N= 50, perturbation intensity q= 0.5. ::: ![](1471-2105-6-11-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### *Mvs. P.*M (the number of distinct \"concatenated\" vectors S~i~divided by N, the number of genes) increases in value, as the number of perturbations (P) is increased. The graph shows curves for three values of perturbation intensity q. ::: ![](1471-2105-6-11-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Perturbations required for high accuracyThe minimum number of perturbations (P\) required to reach the high accuracy criterion (average sensitivity = 0.95) for different values of the network size N. Each point represents the average value for 250 random networks inferred. This is equivalent to finding the value of Pfor which sensitivity = 0.95 on the one-input curve of figure 1(a) for different values of N(figure 1(a) shows N= 50). A linear fit is also shown. ::: ![](1471-2105-6-11-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### *Sensitivity vs. q.**(a) Average inference sensitivity vs. perturbation intensity q. (b) The variance (one standard deviation) is shown here separately for clarity. The results show sensitivity for (predicted) one and two-input solutions being generally higher than the overall case. The results shown are average values for 250 random networks inferred. The remaining parameters are fixed: network size N= 50 and P= 12. ::: ![](1471-2105-6-11-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Example network.Example of an artificial gene network with N= 50. Positive interactions are shown in black, negative interactions in grey. Note the autoregulatory interaction on the upper right hand side. This diagram was generated using Pajek <http://vlado.fmf.uni-lj.si/pub/networks/pajek/>. ::: ![](1471-2105-6-11-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Solution set sizes Distribution for the inferred solution set sizes, compared to the distribution of indegree in the actual network for the simulations. These statistics were produced from 250 random networks run using the following parameter values: N= 50, P= 12, and q= 0.5. The table illustrates how the algorithm overestimates the number of solutions with zero inputs. ::: \\|Y~i*~\\| 0 1 2 3 ≥ 4 -------------- ------ ------ ------ ------ ------ inferred 0.57 0.12 0.07 0.05 0.19 actual 0.37 0.24 0.15 0.10 0.14 :::	PubMed Central	2024-06-05T03:55:52.444469	2005-1-19	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548128/", "journal": "BMC Bioinformatics. 2005 Jan 19; 6:11", "authors": [ { "first": "Thomas", "last": "MacCarthy" }, { "first": "Andrew", "last": "Pomiankowski" }, { "first": "Robert", "last": "Seymour" } ] }
PMC548129	Background ========== Systems biology is a data- and knowledge-driven discipline, which heavily relies on automated tools to support the generation and validation of hypotheses. Such tasks aim to provide novel and meaningful views of the functional relationships between biological components at different complexity levels. Over the past seven years hundreds of methods have been reported to analyse these data, with an emphasis on gene expression data classification \[[@B1],[@B2]\]. More recently, the analysis of gene regulatory and protein-protein networks has started to attract contributions from computer and physical sciences \[[@B3]-[@B5]\]. All of these tasks are linked by a need for comparing, classifying and visualising information. The ever-increasing number and sophistication of techniques may represent an obstacle to achieve more meaningful and rigorous data analysis and discovery tasks. One important problem is that users may not have the time and knowledge required to adequately understand the dynamics and operation of several tools. These deficiencies have been reflected, for example, in a lack of sound practices for assessing the statistical significance of results and for selecting the most suitable data sets and classification models \[[@B6],[@B7]\]. On the other hand, the emergence of multiple data sets and prediction models represents an opportunity for developing an integrative data mining paradigm, which is already significantly improving several predictive tasks in systems biology \[[@B5],[@B8],[@B9]\]. The problem domains mentioned above have mainly concentrated on the application of statistical and machine learning models for classification tasks. Emphasis has been placed on the development of supervised and unsupervised classification methods \[[@B2],[@B10],[@B11]\], as well as on the application of statistical tools for assessing the quality of classification results \[[@B12],[@B13]\]. Relatively fewer efforts have been reported on data visualisation techniquesto support exploratory analysis. It has been shown that information visualisation techniques may support predictive data mining applications, including data clustering \[[@B14]-[@B16]\]. These tasks should complement each other in order to achieve higher levels of knowledge integration and understanding. Furthermore, visualisation-based exploratory methods may support: a) the identification of key patterns in the data, and b) the selection of the most adequate models for data pre-processing and/or classification. The former task refers to the recognition of key groups of data, outliers and features based on computationally-inexpensive, user-friendly and robust analyses. Its outcomes may offer guidance to conduct the latter task by gaining a better insight into the high-level structure and relationships found in the data. Such an exploratory, visualisation-based approach may generate useful alternative views for supporting a more intelligent and meaningful application of classification models. One of the traditional approaches to functional genomics information visualisation has been the application of clustering-based visualisation techniques. Such an approach mainly consists of two steps: a) the implementation of a clustering algorithm, and b) the display of the obtained clusters. The resulting clusters may be visualised by generating, for example, dendrograms \[[@B16]\], other hierarchical structures \[[@B17]\] and maps \[[@B14],[@B18],[@B19]\], which highlight or summarise similarity relationships between groups of data. Clustering-based visualisation has become a fundamental tool for analysing gene and protein expression data. Different variations of hierarchical clustering, Kohonen Self Organising Maps(SOM) and Self-Adaptive Neural Networks(SANN) are relevant examples of techniques belonging to this approach. Their capabilities and applications have been widely reported \[[@B2],[@B15],[@B20]\]. They have been successfully tested on several classification and decision support problems. However, its application to visualisation-driven exploratory analysis is limited by several problems: Many of these techniques are not capable of explicitly and automatically detecting cluster boundaries; some of them are critically sensitive to several learning parameters that need to be selected by the user; some of these solutions were not originally designed to tackle cluster-based visualisation tasks of massive collections of data described by several thousands attributes; and they traditionally require assumptions about the inherent structure of the data, which may not be always possible in exploratory data analyses. Clustering visualisation has also become an important task for the analysis of protein-protein interaction networks. Clustering is a fundamental mathematical property of networks, which allows the identification of key connectivity patterns. Such patterns may be associated with significant functional behaviours and modularity \[[@B3]\]. Moreover, it allows researchers to identify relevant areas for further statistical or experimental analyses. For instance, hierarchical clustering of network nodes (proteins) has been applied to detect functional modules in S. cerevisiae\[[@B3]\]. Each node may be represented by a vector of connectivity values that reflects the node\'s interactions with other network members. Graph theoretic approaches have also been applied to detect significant clusters of interconnected proteins \[[@B21]\]. Another important data visualisation approach, which may be applied to clustering-based analysis, comprises the application of non-linear mapping techniques. They are based on the idea of transforming the original, n-dimensional input space into a reduced, m-dimensional one, where m\<n. These methods are also known as non-linear projection methodsor multidimensional scaling(MDS) methods \[[@B22]\]. They mainly aim to optimize a function, M, which reflects key aspects of the distance structure of the original n-dimensional space. Thus, these methods aim to preserve such global properties in the transformed, m-dimensional space. This principle has been followed by several models including those proposed by Kruskal \[[@B23],[@B24]\] and Sammon \[[@B25]\]. Principal component analysis(PCA) \[[@B26]\] is another relevant technique for reducing n-dimensional data. In this case the resulting transformation accounts for the greatest variation of the original space, but without preserving the distances observed between the points in the n-dimensional space. MDS, including Sammon\'s mapping, applications to gene expression analysis have been reported, which highlight their advantages for supporting the detection of clusters \[[@B27]\]. PCA may not be directly used to visualise clusters. But it may be applied as a pre-processing procedure, and its resulting components may be used as inputs to clustering and supervised classification models \[[@B28]\]. Although widely investigated techniques, such as SOM and Sammon\'s mapping, are usually useful to visualise clusters of high-dimensional data, they present several limitations. For example, the SOM may be highly sensitive to its training parameters, which have to be defined by the user. It also requires the user to define map topologies, and it does not provide automated mechanisms for cluster boundary detection. Its limitations for data exploratory analysis, particularly in relation to data topology preservation, have been stressed in \[[@B29]\]. These and other limitations, as well as adapted solutions, have been discussed in \[[@B14],[@B15]\]. Sammon\'s method may include several data overlaps when the n-dimensional input space contains noisy or weakly discriminatory information \[[@B30]\]. Depending of the size of the input data (number of points), the number of learning iterations and computational facilities available, Sammon\'s mapping might be computationally expensive. Empirical analyses have shown that Sammon\'s mapping may easily get stuck at local optima \[[@B30]\]. Moreover, it has been demonstrated that this method may be sensitive to the initialisation scheme applied \[[@B19],[@B31]\]. This paper assesses an alternative, non-linear mapping technique which aims to address key limitations exhibited by traditional methods. Its application to clustering-driven exploratory analysis of gene expression data is investigated. Furthermore, it provides the basis for an interactome network clustering visualisation system. The following section summarises relevant results. Results ======= A relaxation method for non-linear mapping was implemented to visualise relevant similarity relationships in data originating from gene expression and interactome data. Such a method was designed by Chang and Lee \[[@B32]\] to address key limitations observed in methods such as those proposed by Sammon \[[@B25]\] and Kruskal \[[@B23],[@B24]\]. These techniques are related because they aim to achieve a space reduction by preserving the structure of local distances in the data. However, unlike those traditional mapping techniques, the method assessed in this paper adapts a pair of points in the transformed m-dimensional space at every processing step, instead of adapting all points at every step. Thus, the term \"relaxation\" is taken from the relaxation method for linear equalities \[[@B32]\]. Chang\'s and Lee\'s method showed to outperform Sammon\'s mapping both in terms of cluster detection effectiveness and computational efficiency. A mapping iterationis defined as a complete sequence of adaptation steps involving pairs of points, p(i,j), for each i≠ j(see Methods for a more detailed description). In this study a point may encode a biological sample described by a gene expression profile (e.g. tumour sample), or a protein described by its interaction profile. Figure [1](#F1){ref-type="fig"} summarises the mapping mechanism of this approach. Analysis of gene expression data -------------------------------- Analyses were performed on three publicly available expression data sets. The first one includes 38 samples from a known leukaemia study \[[@B33]\], which are represented by 50 expression values. The samples are categorised into two classes: Acute myeloid leukemia(AML) and acute lymphoblastic leukemia(ALL). This data set has been previously validated by several experimental and in silicomethods. It may be considered as an adequate example for illustrating basic capabilities of clustering algorithms. The second data set includes samples originating from small, round blue-cell tumours(SRBCT) \[[@B28]\]. These data consisted of 63 samples categorised into four classes: Ewing family of tumors(EWS), rhabdomyosarcoma(RMS), Burkitt lymphomas(BL) and neuroblastomas(NB), which are represented by the expression values of 2308 genes with suspected roles in processes relevant to these tumours. The third data set offers another example of gene expression analysis complexity: A data set consisting of a relatively small number of samples described by thousands of gene expression values, without incorporating pre-processing by feature selection or transformation procedures. This application comprises 20 samples described by 9504 gene expression values for both normal brainand a pharmacological model of Parkinson\'s disease\[[@B34]\]. The reader is referred to the section of Methods for a more detailed description of the data and their prediction tasks. Several experiments were performed on each data set to assess possible, key advantages and limitations of the mapping approach introduced above. Each experiment requires an input file storing a matrix, A, where each entry, a~ij~, represents an expression value, j, for a sample, i. The user needs to define only one learning parameter, the number of mapping iterations, as defined above. The output of the algorithm is the projection of the Ksamples represented in Aon the reduced m-dimensional space. Mappings were analysed for m= 2 and m= 3, which allowed the generation of 2D and 3D visual displays. Experiments were also conducted for several numbers of mapping iterations. The results facilitated a high-level understanding of fundamental similarity relationships between the samples, which are also consistent with previous research. Three important exploratory data analysis tasks were accomplished: The automated, unsupervised detection of clusters relevant to the natural class structure of the data sets; the visualisation of a coherent preservation of local similarity (distance) relationships between samples; and the identification of potential outliers. Figure [2](#F2){ref-type="fig"} depicts mapping results for the leukaemia data. Panels (a) to (d) show results for 0, 1, 10 and 100 mapping iterations respectively. Circles are used to represent the samples. Labels \'0\' and \'1\' refer to ALL and AML samples respectively. In the initialisation of the mapping process (0 iterations) the samples are randomly assigned to positions in the 2D space. After 10 iterations there is a clear indication of separation of samples belonging to different classes. With 100 iterations the ALL samples are clustered on the upper left side of the map, and the AML samples are clustered at the bottom of this map. Such clusters were also distinguished in different experiments. As a consequence of the random initialisation process, clusters may occupy different areas on the resulting maps for different experiments using the same number of iterations. Nevertheless, the system correctly separated samples in different experiments with more than 20 iterations. Figure [3](#F3){ref-type="fig"} shows resulting maps for the SRBCT data with 100 mapping iterations. These data were pre-processed as explained in the section of Methods. EWS, RMS, BL and NB samples are represented by symbols \'1\', \'2\', \'3\' and \'4\' respectively. It suggests that the mapping process was able to identify key similarity relationships. Samples belonging to the same class tend to cluster together. For example, EWS samples are mainly located at the bottom of the map. RMS samples are clustered on its left side. The sample \'1\' (x: 0.54, y: -1.79) that was located on the left side of Figure [3](#F3){ref-type="fig"} far from the EWS cluster (its natural class) was consistently mapped in this fashion by different experiments. Moreover, this sample was also displayed closer to RMS samples for different experiments, which might suggest a significant relationship between such a sample and the RMS class. Furthermore, a previous study using more sophisticated, supervised learning models showed that this sample may be difficult to correctly classify \[[@B35]\]. This suggests that the map shown in Figure [3](#F3){ref-type="fig"} highlights a potential outlier in the EWS class data. Additional experiments using a smaller number of SRBCT classes (only EWS, RMS, BL) were performed. This was mainly done to explore the possibility of obtaining alternative graphical views of the data. Results are in general consistent with the results produced with 4 classes: Samples belonging to the same class were clustered together. However, these experiments allow a clearer graphical differentiation of classes on the resulting maps. Figure [4](#F4){ref-type="fig"} depicts an example obtained with 100 mapping iterations. For this and other experiments, it was also possible to detect the outlier that was observed in Figure [3](#F3){ref-type="fig"}. Such an EWS sample is located at the top of the map shown in Figure [4](#F4){ref-type="fig"}. SOM-based analyses using the SOM Toolbox\[[@B36],[@B37]\] were also implemented to establish comparisons. Figure [5](#F5){ref-type="fig"} shows the unified distance matrix(U-matrix) and the label map(panel on the right side) for a representative result. The section of Methods describes the construction of these maps. The SOM was able to correctly cluster the SRBCT samples. However, these standard SOM visualisation techniques do not provide clear information on sample-to-sample similarity relationships. Moreover, they do not adequately facilitate a direct visualisation of the distribution of samples assigned to each node and their associations. [Additional file 1](#S1){ref-type="supplementary-material"} includes the frequency map, which is another standard SOM visualisation technique, for these results. Figure [6](#F6){ref-type="fig"} shows results obtained by applying Sammon\'s mapping. Symbols \'1\', \'2\' and \'3\' represent classes EWS, RMS and BL respectively. This approach is able to detect class differences between samples. Sammon\'s mapping also isolated the EWS sample suggested above as a possible class outlier (right side of the map). Figure [3](#F3){ref-type="fig"} (near x: 1.2, y: -1.5) suggests another sample \'1\' as a potential outlier. However, the Sammon\'s mapping did not clearly depict it as a potential outlier because in this case, unlike Figure [3](#F3){ref-type="fig"}, this sample is located closer to class \'1\' samples (Figure [6](#F6){ref-type="fig"}, near x: 0, y: 0.4). 3D Mapping analyses were also implemented for this SRBCT application. Additional files [2](#S2){ref-type="supplementary-material"} and [3](#S3){ref-type="supplementary-material"} compare the results originating from the relaxation non-linear and Sammon\'s mapping methods. Both methods displayed coherent partitions of the data, which are consistent with the 2D mapping results presented above. Moreover, 3D mappings also suggest relatively strong similarities between EWS and RMS samples because of their proximity on the maps. Figure [7](#F7){ref-type="fig"} displays a relaxation non-linear map of the Parkinson\'s disease model data. Parkinson\'s disease and Normal samples are identified by symbols \'1\' and \'2\' respectively. After 100 mapping iterations, results suggest that the method is able to differentiate between these classes. Parkinson\'s disease samples mainly fall on the upper region of the map (y\> 0), and 7 (out of 10) Normal samples are located below that area. Figure [8](#F8){ref-type="fig"} shows SOM-based results, which adequately distinguish between classes. It also indicates that a few Normal samples may be closer to Parkinson\'s samples than to the main group of its own class. Figure [8](#F8){ref-type="fig"} may offer a clearer graphical discrimination between classes. However, the SOM-based results are more dependent on the user\'s selection of an optimum set of learning parameters (see Methods). [Additional file 4](#S4){ref-type="supplementary-material"} shows the frequency map for these results. Although the separation of clusters is less clear, Sammon\'s mapping (Figure [9](#F9){ref-type="fig"}) was in general capable of grouping same class samples. In this figure symbols \'1\' and \'2\' represent Parkinson\'s disease and Normal samples respectively. 3D maps originating from relaxation non-linear and Sammon\'s mapping methods are depicted in Additional files [5](#S5){ref-type="supplementary-material"} and [6](#S6){ref-type="supplementary-material"} respectively. Both techniques offer alternative, but consistent exploratory views of the cluster structure of this data set. Analysis of interactome networks -------------------------------- As a way of promoting alternative, potential applications of the methodology presented, this section illustrates an example of exploratory data analysis of interactome networks. The approach proposed encodes a network as a graph of interconnected nodes. For a network consisting of Nnodes, the mapping tool (from now on referred to as interClust) requires an N× Nmatrix, B, as the input data. In this symmetrical matrix each element, b~ij~, represents the connection strengthbetween nodes iand jin the graph. Such values may also be interpreted as weightsrepresenting the relevance of the interaction between a pair of proteins, e.g.: number of hits observed in interaction experiments or a path distance between two nodes on the graph (indirect interactions). Thus, each row in Bmay be seen as the connectivity profilefor a node, i. That is, the connectivity profile of a node becomes this node\'s coordinates in the n-dimensional, input space. An accompanying tool, inBuilder, automatically generates such a network representation from a list of pairwise protein-protein interactions and their respective connection strengths predefined by the user. The section of Methods provides more information about design and operation aspects of this approach. Before testing this approach on a real interactome data set, a simple example of a network (Figure [10](#F10){ref-type="fig"}) is used to illustrate its application. In this figure the length of the links does not reflect distances or connection strengths. All of the direct connections are considered equally. This network comprises 25 nodes forming 4 main clusters. In this case a cluster is defined as a compact group of interconnected nodes. Figure [11](#F11){ref-type="fig"} shows the results obtained by applying interClustto this network with 20 iterations. This map clearly distinguishes the clusters of the network. Moreover, it preserves local interaction relationships: i.e. if two nodes have similar connectivity profiles in the original space (Figure [10](#F10){ref-type="fig"}), then they are also close to each other in the resulting map. Adequate cluster visualisations were also obtained in experiments with more than 5 iterations. This algorithm was tested on the chromatin interactome in C. elegans. It includes 303 proteins and 349 interactions. This data set regroups interactions of proteins involved in transcriptional regulation at the chromatin level. This functional module is of major interest because it is at the crossroads of many biological processes such as development, sex determination, cellular differentiation and proliferation. Its misregulation may have strong consequences such as tumorigenesis or developmental defects \[[@B38]\]. This data set includes both retested \[[@B39]\] and non-retested protein interactions obtained by high-throughput two-hybrid screens (unpublished data). Figure [12](#F12){ref-type="fig"} displays resulting relaxation maps, at different regions and levels of detail, obtained with 100 iterations. Figure [12](#F12){ref-type="fig"} indicates a separation of proteins according to their connectivity patterns. The network encoding scheme and the non-linear mapping algorithm applied distinguished highly-connected proteins (hubs) from the other components of the network. Hubs are located in the outer regions of the map. The farther a node is from the centre, the more connections it has. Figure [13](#F13){ref-type="fig"} displays a partial view of a region enriched by hubs, which is well-separated from the main group of proteins. The hubs are also relatively well-separated between them. It reflects the fact that such proteins share very few direct, interacting proteins in common. [Additional file 7](#S7){ref-type="supplementary-material"} depicts some of the hubs automatically isolated by the mapping algorithm, as well as a few nodes located near the centre of the map (F15A2.6, C34E10.5, C14B9.6). These diagrams were drawn using the InterViewertool \[[@B40]\]. A closer examination of a group of proteins located in the outer regions of the map (Figures [12](#F12){ref-type="fig"} and [13](#F13){ref-type="fig"}), for example, reveals that they are involved in key processes such as mitosis and meiosis. This differentiation indicates that such hubs may act as connection components between different biological processes. This is not a surprising finding, but it highlights the capacity of the algorithm to automatically detect some of the most biologically-relevant proteins only on the basis of their connectivity profiles. Seven of these hubs (Y2H9A.1, F56C9.1, F11A10.2, T12D8.7, Y113G7B.23, Y37D8A.9, C53A5.3), for instance, show a significant enrichment of phenotypes obtained by depletion of the transcripts by RNA interference (5.6-fold enrichment compared to genome-wide levels \[[@B41]\]). Moreover, four of these hubs (F56C9.1, F11A10.2, T12D8.7, C53A5.3) are strongly linked to embryonic lethality (Emb) by exhibiting a 6-fold enrichment of this property in relation to genome-wide levels \[[@B41]\]. Further analyses on the map suggest that neighboring nodes may reflect functional similarity relationships between the corresponding proteins. One example is illustrated in Figure [14](#F14){ref-type="fig"} (upper area), which focuses on the region surrounding T20B12.2, also known as Tbp-1, a key component of the Polymerase II holoenzyme. In this region it is possible to identify several components of the core Polymerase II enzyme, as well as several related families (histones acetylases and deacetylases, nucleosomes positioning enzymes of the Swi/snf family) that share similar phenotypes. There is a strong enrichment of phenotypes such as embryonic lethality or problems in growth. These phenotypes (like embryonic lethality) are often attributed to proteins with high connectivity. Only eleven (out of twenty seven) proteins examined in this region exhibit wild type phenotype when they are depleted. This cluster shows a 5.4-fold enrichment of phenotypes, a 5.1-fold enrichment of Emb phenotype, and a 33.3-fold enrichment of sterile progeny phenotype in comparison to genome-wide levels \[[@B41]\]. This cluster also exhibits significant associations with sterility, growth defect and problems in larval development. [Additional file 8](#S8){ref-type="supplementary-material"} describes the composition of this representative cluster. Discussion ========== With regard to gene expression data, the relaxation non-linear mapping method was capable to support an automated, unsupervised detection of relevant clusters of samples. Results demonstrated that it may also be useful for the visualisation of local similarity relationships between samples and the identification of potential outliers. In general its performance was comparable to Sammon\'s mapping. One cannot of course expect that a single method would always be able to accurately map different types of high-dimensional data. However, the application of both techniques is recommended as a reliable approach to data exploratory analysis. In this study they offered consistent views of the problems under analysis. SOM, as well as many other cluster analysis techniques, may be more suitable for application after first gaining an adequate insight into the structure and organisation of the data. Such a global understanding may be facilitated through the application of different non-linear mapping methods. Even though a comparison with existing network clustering methods was not implemented, preliminary results suggest that the approach proposed might represent a useful tool for interactome network visualisation and clustering. It distinguished key hubs and facilitated the identification of functionally relevant clusters. It showed that, not surprisingly, most of the hubs detected are essential for the normal development, behaviour and reproduction of C. elegansby exhibiting an enrichment of phenotypes obtained from RNA interference. This can be explained by the fact that the partners connected to these hubs are involved in numerous fundamental processes such as mitosis or meiosis. Hence, the modification of the network caused by the absence of a hub may have strong consequences on the topology of the network and the organisation of the cellular processes. Isolation of functional clusters (e.g. chromosome condensation and segregation of the transcriptional core process) is essential to investigate relationships between groups of proteins or modules. Modules playing a role in the same process (e.g. chromosome condensation and segregation during the mitosis) also tended to be interrelated in the clustering analysis. This underlies the fact that these modules are also functionally interconnected and are interdependent to stringently regulate the cellular processes. Without these connections the process may be misregulated and generate aberrant behaviour (i.e. oncogenesis if the mitosis is not well regulated). In this way, interactions that connect these modules are likely to be at the intersection of several biological processes and to regulate the correct succession of events in a cellular process (e.g. condensation of chromosomes before segregation). We do not claim that the tool reported can be used as an interaction prediction technique. The example analysed aims to illustrate the application of exploratory data analysis for detecting regions, which may be biologically meaningful and relevant for assessing the outcomes from protein-protein interaction prediction techniques. Such patterns may be useful for guiding future computational or experimental analyses to validate interaction hypotheses. Meaningful patterns may be associated, for example, with functionally enriched regions, as shown in this paper. Moreover, because of the limitations regarding predictive accuracy and coverage exhibited by existing single-source techniques, it is important to offer user-friendly tools that may help scientists to detect possible, spurious associations. Future research should include a more exhaustive statistical description of all possible hub candidates detected by the mapping process for this and other network examples. Statistical and functional attributes represented by this method should be compared with previous findings from large-scale, comprehensive studies. Such an investigation was not implemented here because it is outside the main scope of this paper. The preservation of local distance structures is an important property to interpret the non-linear mapping techniques studied here. This is the main goal of their data transformation mechanisms. It basically means that the distance between two points, dm~ij~, in the transformed m-dimensional space should be very similar to dn~ij~(their distance in the original n-dimensional data) if dn~ij~is small. However, if dn~ij~is relatively large, dm~ij~is not required to be similar to dn~ij~. A fundamental difference between the relaxation non-linear and the original Sammon\'s mapping methods is that the former adapts a point-to-point distance at every processing step, instead of adapting all of the distances at every step. For relatively small data sets the computing times required by the Java-based implementation of the relaxation non-linear mapping method were comparable to those obtained from Matlab^®^implementations of SOM and Sammon\'s techniques, i.e. in the order of seconds. But for larger data sets, e.g. Parkinson\'s disease data, the non-linear mapping method may run in the order of minutes. This of course depends on the number of mapping iterations and memory resources available. This concern may be addressed by implementing an optimised version of the software, perhaps using another programming language. Another important solution is the implementation of a frame method\[[@B32]\], which has demonstrated to improve the computational efficiency of the algorithm without significantly compromising its data structure preservation capabilities. We also intend to expand this and related research within an open-source data analysis and visualisation platform, such as the TIGR Multiexperiment Viewer\[[@B42]\]. An important aspect of future research is the adaptation of the relaxation non-linear mapping method to perform tasks beyond exploratory data analysis. A desirable property would be its capacity to generalise solutions for new samples in an incremental fashion. That is, the system should be able to add new samples to a map without having to re-generate it. One possible solution is the application of an artificial neural network to interpolate and extrapolate the mapping as illustrated by Mao and Jain \[[@B43]\]. It is also important to develop hybrid systems to combine the strengths and advantages demonstrated by various mapping techniques \[[@B44]\]. A two- stage approach, for example, represents a feasible solution. In this approach a data set may be firstly partitioned into a set of Voronoispaces using clustering techniques such as k-means and SOM, and then independent mapping projections may be performed on each area. It has been suggested that such a hybrid model may be advantageous especially when dealing with massive data sets \[[@B45]\]. Other investigations will involve the assessment and comparison of related techniques \[[@B46],[@B47]\] The mapping algorithm successfully recognised key topological properties and functional relationships in an interactome network based on a graph encoding scheme, which only considers direct interactions. Moreover, it considered all graph connections as equals. Nevertheless, it would be important to implement applications in which the network encoding values, b~ij~, may also reflect non-direct, shared interactions. This may be done, for example, by defining a graph distance function between network nodes. Another input representation scheme may exploit information relevant to the significance or confidence assigned to the interactions based on experimental evidence. Since cellular networks are organized in a modular fashion, the identification of these modules is crucial to understand relationships between biological processes and offer a higher-order, more accessible representation of the interactomes. The clustering approach proposed in this paper provides a meaningful, simplified representation of complex interactomes. This representation may significantly facilitate exploratory analysis of networks for non-specialists in bioinformatics. This type of analysis is fundamental to detect key network components, such as hubs, which are implicated in many physiological disorders. Identifying these hubs and their associated clusters is also an important step toward the functional annotation of these proteins, as well as for obtaining possible explanations of their involvement in a specific disease. The cluster visualisation tool, interClust, may represent a useful technique to analyse other protein-protein interaction networks including a future human interactome. For instance, it may be applied to isolate proteins linked to a human pathology and to associate them with a cluster or functional modules (e.g. the transcriptional core complex of the Polymerase II enzyme). Another important component of future research is the adaptation of network clustering methods to take into account spatio-temporal aspects of interactions based on, for example, microarrays, in-situ hybridization or protein localization data. Non-linear mapping methods may also be applied to support the annotation of unknown proteins. This may be done by assigning a protein to a functional role that is significantly associated with the cluster under consideration. Furthermore, it is of course fundamental to compare this network clustering methodology with existing techniques. Thus, such approaches may aid researchers in the design of further experiments and the selection of more sophisticated bioinformatics analyses. Conclusions =========== This research studied a user-friendly cluster visualisation approach that is able to support the generation of biologically meaningful outcomes. It represents an effective and robust exploratory data analysis technique. Comparisons indicate that applying more than one mapping approach may improve the confidence of results. Moreover, this may facilitate the generation of alternative, meaningful views of the data. Relaxation non-linear and Sammon\'s mapping techniques may be more suitable for exploratory data analysis tasks than SOM. This study did not aim to add another algorithm to the existing collection of supervised and unsupervised classification tools. This methodology is not reported as a competing solution to clustering algorithms. Our study shows how an exploratory data analysis approach based on non-linear mapping can support the identification of relevant, biologically-meaningful patterns. We do not argue that the methodology proposed should necessarily offer more accurate results in relation to existing classification solutions. We recommend this methodology as a first step towards understanding complex data mining problems in functional genomics. Such an exploratory approach may also facilitate the selection of more sophisticated methods and highlight possible, critical features for successfully implementing clustering-based studies. This research indicates that the outcomes originating from an exploratory, pattern visualisation method may be as meaningful as those produced by more sophisticated classification approaches, i.e. SOM. Moreover, the methodology proposed does not require the user to define multiple learning parameters. Exploratory analysis frameworks may facilitate a better insight into a data set before applying more sophisticated, problem-specific classification or predictive models. Such an insight may be achieved by helping users to recognise key features of the underlying structure of the data, detect potential outliers or anomalies and test assumptions about the cluster composition of the data. An adaptation of the relaxation non-linear mapping technique, interClust, represents a promising solution to aid researchers to recognise key connectivity and functional patterns in interactome networks. Further research is underway to continue assessing its application to this area. Methods ======= Data ---- The leukaemia data set includes 38 samples originating from \[[@B33]\]. Each sample is represented by 50 expression values. The samples are categorised into two classes: Acute myeloid leukemia(AML) and acute lymphoblastic leukemia(ALL). The original data sets and experimental protocols can be found at the Broad Institute Web site \[[@B48]\]. For each feature standardisation was applied by subtracting each value from the mean and dividing it by the standard deviation. The SRBCT data consisted of 63 samples categorised into four classes: Ewing family of tumors(EWS), rhabdomyosarcoma(RMS), Burkitt lymphomas(BL) and neuroblastomas(NB), which were represented by the expression values of 2308 genes. The dimensionality of the SRBCT expression samples was reduced by applying PCA. It has been shown that the application of PCA is important to facilitate an adequate discrimination of samples in this data set. The 10 dominant PCA components for each case were used as the input to the analysis techniques as suggested by \[[@B28]\], who applied a supervised learning approach to classify the samples after reduction by PCA. Using the raw data (without PCA) the relaxation non-linear mapping was not able to adequately depict differences between the samples. The original data sets and experimental protocols can be found at the National Human Genome Research Institute Web site \[[@B49]\]. The Parkinson\'s disease data include 20 samples described by 9504 gene expression values for both normal brain(10 samples) and a pharmacological model of Parkinson\'s disease\[[@B34]\]. M. musculuswas the organism studied in this disease model. The data are available at The Gene Expression Omnibus \[[@B50]\] (accession number GDS22). Additional files [9](#S9){ref-type="supplementary-material"} to [11](#S11){ref-type="supplementary-material"} include, respectively, the leukaemia, SRBCT and Parkinson\'s disease data analysed in this paper. The network of interactions was derived from the chromatin interactome in C. elegans. It contains 303 proteins and 349 interactions. It comprises components of the Polymerase II holoenzyme, histones modifying enzymes, nucleosomes positioning proteins and several proteins containing domains known to be essential to this process such as the chromodomain, the bromodomain or the SET domain. This is an early version of the chromatin interactome, which includes a number of retested \[[@B39]\] as well as non-retested interactions determined by a stringent high-throughput two-hybrid screen. It also contains several interologs \[[@B38],[@B51]\]. A combination of experimental and bioinformatic factors (reporter genes used for the phenotypic tests, number of hits per interactions, a blast e-value less than 1E-10, a PHRED score \>20 for 15% of the ISTs (interaction sequence tags) and the frame verification method) were used to provide optimal accuracy. It is known that the two-hybrid approach has the tendency to generate more false positives than the pull-down/Mass spectrometry approach, for example. However, in the data set analysed the rate of false positives is reduced by using more reporter genes (up to 4 genes, unlike traditional large scale two-hybrid screens which commonly use 2 genes). Using 4 reporter genes can reduce the rate of false positives up to 50%. [Additional file 12](#S12){ref-type="supplementary-material"} contains this data set. Algorithms and tools -------------------- The relaxation non-linear mapping algorithm is summarised in Figure [1](#F1){ref-type="fig"} and details on its design are reported in \[[@B32]\]. The adaptation of a pair points, iand j, in the transformed, m-dimensional map is implemented as follows. Given two points, Pm~i~and Pm~j~, in the m-dimensional map, the adjusted new values, Pm~new,\ i~and Pm~new~, ~j~, are calculated using: ![](1471-2105-6-13-i1.gif) where dn~ij~and dm~ij~represent the Euclidean distances between the points, iand j, in the n- and m-dimensional spaces respectively. The SOM results were obtained using the SOM Toolbox, which is a Matlab^®^implementation \[[@B36],[@B37]\]. Each training process consists of two phases. The following parameters were used. Initial learning rates equal to 0.5 (first phase) and 0.05 (second phase). Learning rates were controlled by an inverse-of-time function. The SOM neighborhood radius starts covering one fourth of the map size. The number of training epochs was equal to 10 times the number of map nodes (first phase). For the second phase it was equal to 4 times the number of training epochs in the first phase. A U-matrix depicts the distances between neighbouring map units by displaying a grey scale. In a label map a SOM node represents a class based on a majority voting strategy for the samples associated with this node. In case of a draw, the first class encountered is used. Empty nodes are not labeled. The Sammon\'s mapping analyses were implemented using the SOM Toolbox with 100 mapping iterations, iteration step size equal to 0.2 and the Euclidian distance. For the interaction data set, the tool inBuilderwas used to transform it into interClustinput format. The cross-platform tools interClustand inBuilderare available for academic researchers on request from the authors. Graphical outputs for the relaxation maps were obtained with the proprietary software Statistica^©^. [Additional file 7](#S7){ref-type="supplementary-material"} was created using InterViewer\[[@B40]\], which is freely available at \[[@B52]\]. Analyses were performed on a PC with a Pentium^®^4CPU. Authors\' contributions ======================= FA designed the study, implemented the relaxation non-linear mapping algorithm, performed analyses and wrote the manuscript. HW did the SOM and Sammon\'s mapping experiments and helped with the preparation of the manuscript. AC selected the interactome data, interpreted results and helped with the preparation of the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 SOM frequency map for SRBCT dataIt shows the distribution of samples, X(Y), over each node in Figure [5](#F5){ref-type="fig"}, where Xrepresents the class label and Ystands for the number of Class Xsamples assigned to the corresponding node. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 3D visual display originating from relaxation non-linear mapping -- SRBCT dataEWS, RMS and BL samples are represented by symbols \'1\', \'2\', \'3\' respectively. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 3D Sammon\'s mapping results -- SRBCT dataSymbols \"1\", \"2\" and \"3\" represent classes EWS, RMS and BL respectively. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 4 SOM frequency map for Parkinson\'s disease dataIt shows the distribution of samples, X(Y), over each node in Figure [8](#F8){ref-type="fig"}, where Xrepresents the class label and Ystands for the number of Class Xsamples assigned to the corresponding node. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 5 3D Relaxation non-linear mapping of the Parkinson\'s disease model dataParkinson\'s disease and Normal samples are identified by symbols \'1\' and \'2\' respectively. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 6 3D Sammon\'s mapping of the Parkinson\'s disease model dataSymbols \"1\" and \"2\" represent Parkinson\'s disease and Normal samples respectively. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 7 Examples of key hubs in the interactomeIt depicts some of the hubs automatically isolated by the mapping algorithm, as well as a few nodes located near the centre of the map (F15A2.6, C34E10.5, C14B9.6). ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 8 Description of protein cluster obtained from Figure[14](#F14){ref-type="fig"} ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 9 Leukaemia data set analysed in this paperLog ratios are used to represent the expression levels. The last column shows the class labels. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 10 SRBCT data set analysed in this paperLog ratios are used to represent the expression levels. The last column shows the class labels. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 11 Parkinson\'s disease data set analysed in this paperLog ratios are used to represent the expression levels. The last column shows the class labels. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 12 *Chromatin interaction network in C. elegans*The last column shows the connection strength in the graph. All connections are considered equally. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We thank Claude Sardet at the CNRS-Montpellier for supporting AC\'s participation in this collaboration. AC is financially supported by a fellowship from La Ligue Contre le Cancer (ligue départementale de l\'Hérault) and by a grant from la Ligue Contre le Cancer (Equipe labélisée Ligue Nationale contre le Cancer). We thank the anonymous reviewers for their helpful comments and corrections. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Relaxation method for non-linear mapping.*The input to the algorithm is a collection of Kpoints described in an n-dimensional space. A mapping iteration refers to a complete sequence of adaptation steps involving pairs of points, p(i,j). A point may encode a biological sample described by a gene expression profile (e.g. tumour sample), or a protein described by an interaction profile. ::: ![](1471-2105-6-13-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Visualisation of clusters in leukaemia data for different numbers of mapping iterations.Panels (a) to (d) show results for 0, 1, 10 and 100 mapping iterations respectively. Circles are used to represent the samples on the maps. Labels \'0\' and \'1\' refer to ALL and AML samples respectively. ::: ![](1471-2105-6-13-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Resulting maps for the SRBCT data.EWS, RMS, BL and NB samples are represented by symbols \'1\', \'2\', \'3\' and \'4\' respectively. 100 mapping iterations. ::: ![](1471-2105-6-13-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Resulting maps for the SRBCT data considering only 3 classes.EWS, RMS and BL samples are represented by symbols \'1\', \'2\', \'3\' respectively. 100 mapping iterations. ::: ![](1471-2105-6-13-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### SOM-based analyses on the SRBCT data.U-matrix and label map (panel on the right side). U-matrix depicts the distances between neighbouring map units using a grey scale. In the label map a node represents a class based on a majority voting strategy for the samples associated with this node. EWS, RMS and BL samples are represented by symbols \'1\', \'2\', \'3\' respectively. [Additional file 1](#S1){ref-type="supplementary-material"} shows its frequency map. ::: ![](1471-2105-6-13-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Sammon\'s mapping analyses on the SRBCT data.Symbols \"1\", \"2\" and \"3\" represent classes EWS, RMS and BL respectively. ::: ![](1471-2105-6-13-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Relaxation non-linear mapping of the Parkinson\'s disease model data.Parkinson\'s disease and Normal samples are identified by symbols \'1\' and \'2\' respectively. 100 mapping iterations. ::: ![](1471-2105-6-13-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### SOM of the Parkinson\'s disease model data.U-matrix and label map (panel on the right side). U-matrix depicts the distances between neighbouring map units using a grey scale. In the label map a node represents a class based on a majority voting strategy for the samples associated with this node. [Additional file 4](#S4){ref-type="supplementary-material"} shows its frequency map. ::: ![](1471-2105-6-13-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Sammon\'s mapping of the Parkinson\'s disease model data.Symbols \"1\" and \"2\" represent Parkinson\'s disease and Normal samples respectively. ::: ![](1471-2105-6-13-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### Example of a network characterized by a number of clusters.The length of the links does not reflect distances or connection strengths. All of the direct connections are considered equally. ::: ![](1471-2105-6-13-10) ::: ::: {#F11 .fig} Figure 11 ::: {.caption} ###### A resulting map after applying interClust to the network shown in Figure 10.Clusters are identified and local interaction relationships tend to be preserved. 20 mapping iterations. ::: ![](1471-2105-6-13-11) ::: ::: {#F12 .fig} Figure 12 ::: {.caption} ###### Relaxation map for the chromatin interactome network.Each panel depicts different regions and levels of detail, obtained with 100 iterations. ::: ![](1471-2105-6-13-12) ::: ::: {#F13 .fig} Figure 13 ::: {.caption} ###### Partial view of chromatin interactome map.Its shows a region enriched by hubs, which is well-separated from the main group of proteins. ::: ![](1471-2105-6-13-13) ::: ::: {#F14 .fig} Figure 14 ::: {.caption} ###### Identification of key functional components based on cluster visualisation.*The region surrounding protein T20B12.2 includes several components of the core polymerase II enzyme, as well as several related families that share similar phenotypes. See [Additional file 8](#S8){ref-type="supplementary-material"} for further descriptions. ::: ![](1471-2105-6-13-14) :::	PubMed Central	2024-06-05T03:55:52.446292	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548129/", "journal": "BMC Bioinformatics. 2005 Jan 20; 6:13", "authors": [ { "first": "Francisco", "last": "Azuaje" }, { "first": "Haiying", "last": "Wang" }, { "first": "Alban", "last": "Chesneau" } ] }
PMC548130	Background ========== Detection of LOH is one of the most common molecular applications in the study of human diseases, in particular cancer. It is commonly used to examine whether a known tumour suppressor gene is inactivated or to map unknown tumour suppressor gene(s). Detection of LOH not only helps in understanding the molecular mechanisms underlying the development of cancer, but also provides important information useful for disease diagnosis and prognosis. LOH detection is commonly carried out by the analysis of microsatellite markers using an automated DNA sequencer. With the raw data from the sequencer being stored in one file per lane together with corresponding clinical information and patient follow up data, each LOH study \[[@B1],[@B2]\] generates hundred of files that need to be organised and related in a structured format. However, with the increasing number of samples analysed using different software, no tool is currently available to facilitate the extensive and efficient data retrieval and analyses, such as correlation of LOH data with various clinical data sets. We have developed a novel software package: LOH Data Management and Analysis Software (LDMAS) in order to satisfy these needs. LDMAS can retrieve LOH data from automated DNA sequencer platform and clinical data from any patient record system and correlate different data sets according to the user\'s choice. Here we present how LDMAS interfaces to Genotyper software (ABI, Foster City, CA) which is used to determine the presence of LOH, and the patient record system SunQuest (San Francisco, CA), facilitating the identification of LOH markers associated with the development of CIN \[[@B3]\]. CIN show variable clinical behaviour despite morphological homogeneity within each subgroup. Clinically, it is vital to distinguish CIN lesions with different behaviour and identify those likely to persist and progress despite treatment. Implementation ============== System architecture ------------------- LDMAS package is composed of three modules: \(1) MRES (Medical Report Extractor Software) which parses patient report files, extracts the information of interest and organises it into a structured format, applicable to LDAS \(2) LDAS (LOH Data Analysis Software) which obtains LOH data from Genotyper (Applied Biosystems, California) or GDAS (Affymetrix, California) software and correlates it to clinical data obtained from MRES \(3) LDMS (LOH Data Management Software) which is used to gather patients\' clinico-pathological data and extract significant relationship between the various data sets LDAS and LDMS work synergistically to manage and analyse LOH data. The MRES source code for automatic parsing of patient reports is written in C++ using C++ Builder 5.0 (Borland Software Corporation, Scotts Valley, CA), LDAS is written in Visual Basic for Application as an Excel 2000 add-in and LDMS is written as Visual Basic for Application modules embedded within Access 2000 as fully functional software. These modules can be run independently and used for applications other than LOH. LDMAS runs on Microsoft^®^Windows 2000 or Windows XP operating system. Figure [1](#F1){ref-type="fig"} shows LDMAS architecture. Type of input data for LDMAS modules ------------------------------------ The MRES module takes its input from any patient report file containing clinical details such as diagnosis, stage of the disease, treatment and follow up results, parses and formats patient\'s data into a structured format that can be saved as Excel spreadsheet. In this case, data were taken from SunQuest patient record system and MRES converted and produced the data as: \(1) Hospital Number \(2) Hospital ID \(3) Patient Name \(4) Date of Birth \(5) Pathological specimen Number \(6) Date of Diagnosis \(7) Histological diagnosis. The user can manually check the data and use it as template for analysis. Data analysis is carried out using LDAS which obtains LOH data from Genotyper and correlates it to the clinical data obtained from MRES. LDAS obtains data in plain text format and can thus be easily interfaced to any LOH platform generating software such as Genotyper and GDAS. Finally all data are entered into LDMS for storage and intelligent mining of data. Database query results can be exported back to LDAS allowing correlation between LOH and various clinico-pathological parameters such as age, histological grade, treatment modality and their responses, and HPV status as well as carry out multivariate analysis to determine the sensitivity and specificity of the markers involved in the LOH study. A more detailed description of all the modules is provided in LDMAS user\'s guide \[Additional file 1\] and the example below. Advantages of LDMAS ------------------- LDMAS offers several advantages to users. It is user friendly and its architecture is modular allowing versatility of use. It enforces the standardisation of procedures for studies involving large cohorts of individuals. The data is well organized since LDMAS systematically assigns LOH results of each case to its corresponding clinical information. Additionally, LDAS standardizes LOH data analysis implicitly and allows the user to edit the data manually if needed. Microsoft Excel has been chosen to implement LDAS because of its wide use, versatility and convenient statistical analysis features facilitating the implementation of multivariate analysis and correlation testing between LOH and clinico-pathological parameters. Results and discussion ====================== LDMAS application in identification of LOH markers associated with persistence / progression of cervical intraepithelial neoplasia ---------------------------------------------------------------------------------------------------------------------------------- We divided the CIN groups into disease free indicating cases that become CIN free after treatment, and disease persistence/progression indicating cases that develop show progression or persistence of CIN despite treatment. We used LDMAS to retrospectively examine the prognostic value of LOH at 12 microsatellite markers including 10 from 3p14, 3p22-21, 6p21 and 11q23 which are frequently deleted in cervical cancer \[[@B3],[@B4]\], in 164 cases of CIN lesions using archival cytological/histological specimens. LOH was further correlated with high risk HPV infection. Initially MRES was used to automatically parse 4300 patient records and extract clinico-pathological data including age, diagnosis, method of treatment and treatment response during follow up. Out of those, 164 cases with follow up of 3 or more years were chosen for the study and their clinico-pathological information was imported into LDAS. Initially, 71 out of the 164 selected cases were examined for LOH using 12 fluorescent microsatellite markers ran on ABI377 DNA Sequencer. LDAS was then used to identify the microsatellite markers for which LOH was significantly associated with disease persistence/progression of CIN using two tailed student t-test. Figure [2](#F2){ref-type="fig"} generated using LDAS shows that microsatellite markers D3S1300 (3p14.2), D3S1260 (3p22.2), D11S35 (11q22.1) and D11S528 (11q23.3) have the highest LOH in CIN lesions displaying persistence/progression than those who were disease free during follow up \[[@B5]\]. Validation of prognostic markers associated with persistence / progression of CIN --------------------------------------------------------------------------------- To validate this finding, LOH at these four markers was investigated in a further series of 93 cases. Compatible results were obtained from these additional cases. The two sets of data were combined and further compared using LDMS. Methodologies included : 1\) comparison using χ^2^(chi-squared) test of LOH at each of the four microsatellite markers with age, various methods of treatment, different subtypes of HPV infection and between CINs showing disease free or disease persistence/progression. 2\) correlation of LOH data with histological grade of CIN, treatment response and various HPV subtypes. Through such complex analysis, we showed that concurrent LOH at two of the four microsatellite markers could identify 47% of CINs that showed disease persistence/progression with 100% specificity \[[@B5]\]. Furthermore, LOH at D3S1300 was found to be significantly associated with HPV16 infection. Part of this data analysis is supplied in the LDMAS guide \[see Additional file 1\]. More detailed analysis of this study is described in \[[@B5]\]. Algorithm for identifying prognostic disease markers ---------------------------------------------------- Based on the above example, an algorithm can be developed to extract prognostic markers for other diseases. The algorithm can be summarised in the following pseudocode : \(1) Divide the disease in groups according to the pathology staging \(2) Parse patient data from clinical records and use the groups defined in part (1) \(3) FOR each microsatellite marker carry out a two tailed student t-test between the disease groups using LOH data IF t-test p ≤ 0.05 Marker is significant in prognosis of the disease ELSE Marker is not significant in prognosis of the disease \(4) Validate the prognostic markers using χ^2^(chi-squared) test of LOH with clinico-pathological data and correlation of LOH data with histological grade of CIN, treatment response and various HPV subtypes. LDMAS has been implemented using the above pseudocode. Conclusions =========== We have devised an effective algorithm to identify and extract useful markers that can be used to predict the outcome of disease and used the algorithm to successfully identify 4 novel prognostic markers that can be used to predict the outcome of CIN. The algorithm was implemented in a novel software called LDMAS which provides an essential platform for the extraction of useful information from large amount of data generated by LOH studies. Furthermore, LDMAS is used to efficiently store, manage and track the data. Its flexible nature allows the easy manipulation of data facilitating complex analysis as demonstrated in the current study. The various modules of LDMAS can be easily adapted and used with other applications such as high throughput LOH and genotyping using SNPs on Affymetrix^®^GeneChip Mapping arrays and fingerprinting studies. Modules such as MRES can be used independently to parse medical records facilitating extraction of specific clinical information of interest. Additionally, LDMAS can be used to extract clinically useful markers for other diseases. Availability and requirements ============================= The source code and executable files for LDMAS modules as well as user manual including examples from real study data are freely available and can downloaded from our website at : <http://molpath.his.path.cam.ac.uk/bioinformatics/LDMAS.shtml> Additionally examples of input files are provided from our website for users to test the software and assess its functionality. Authors\' contributions ======================= RH designed and developed and implemented LDMAS software and the web site. AE and RH did the experimental work to generate the data necessary to test and validate LDMAS. MD supervised the study and designed the experimental work using CIN biopsies and smears. All authors read and approved the final manuscript. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### LDMAS architecture. ::: ![](1471-2105-6-18-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Example of output from LDMAS. LDMAS generated histogram showing the best set of LOH markers in cervical cancer highlighted by \*. DF indicates Disease Free and DP indicates Disease Persistence/Progression CIN cases. ::: ![](1471-2105-6-18-2) :::	PubMed Central	2024-06-05T03:55:52.450434	2005-1-26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548130/", "journal": "BMC Bioinformatics. 2005 Jan 26; 6:18", "authors": [ { "first": "Rifat A", "last": "Hamoudi" }, { "first": "Amina", "last": "El-Hamidi" }, { "first": "Ming-Qing", "last": "Du" } ] }
PMC548131	Background ========== Paratuberculosis (Johne\'s disease) is caused by Mycobacterium aviumsubsp. paratuberculosis(referred to hereafter as M. aviumsubsp. paratuberculosis) and induces a chronic enteritis in ruminants. The disease signs include weight loss, diarrhea, and decreased milk production. In the United States alone the economic burden of Johne\'s disease is estimated at over \$200 million in lost annual revenue to the dairy industry \[[@B1]\]. Prevalence studies in the United States have estimated that between 20 to 30% of dairy herds are infected with M. aviumsubsp. paratuberculosis\[[@B2],[@B3]\]. Neonatal calves are most susceptible to infection and are likely to become infected after ingestion of contaminated milk or colostrum \[[@B4],[@B5]\]. During the subclinical stage of infection, the host cell-mediated immune response is robust and appears to control the infection. As the disease progresses from the subclinical to the clinical stage, the cell-mediated response diminishes, and a humoral immune response predominates \[[@B6]\]. Vaccines are not completely protective, but have been reported to reduce fecal shedding and delay the onset of clinical disease \[[@B7]-[@B9]\]. Lipoproteins have long been considered immunomodulators and mycobacteria are especially rich in these post-translationally modified proteins. There are approximately 100 open reading frames identified in the M. tuberculosisgenome that possess a characteristic amino-terminal acylation motif \[[@B10]\]. The 19-kDa lipoprotein from Mycobacterium tuberculosisis immunodominant in both mice \[[@B11]-[@B13]\] and humans \[[@B14],[@B15]\] and has been shown to stimulate CD4^+^T cell proliferation as well as the release of IL-2, IFN-γ, and IL-12 \[[@B16],[@B17]\]. Acylation near the N-terminal portion of the 19-kDa protein is believed to occur at amino acids 19--24 and contributes to its immunogenicity \[[@B12]\]. Furthermore, glycosylation of the M. tuberculosis19-kDa protein inhibits innate immune responses, such as the release of TNF-α, IL-6, and IL-10 from macrophages, but does not affect antibody binding \[[@B18]-[@B20]\]. The 19-kDa protein was also shown to induce CD8+ cells to secrete IFN-γ and specifically lyse M. tuberculosis-infected monocytes \[[@B21]\] as well as promote neutrophil priming and activation \[[@B22]\]. Finally, B cell epitopes have been described that localize to the linear sequences of amino acids 11--30, 29--47, 61--80, and 140--159, as well as a conformation-dependent epitope at the amino and carboxy-terminal ends because of intramolecular disulfide bonding of cysteine residues \[[@B21],[@B23]\]. Experimental infection and staining of macrophages has shown that the 19-kDa protein is secreted by live M. tuberculosisresiding within the phagolysosomal compartment \[[@B24]\]. Homologues of the 19-kDa lipoprotein exist in M. bovis, M. avium, and M. intracellularebut are absent from M. phlei, M. smegmatis, M. fortuitum, M. gordonae, and M. leprae\[[@B25]\]. Despite decades of research, little is known about the M. aviumsubsp. paratuberculosisproteins involved in metabolism, cell wall synthesis, macrophage entry and survival, disease pathogenesis, or host immune evasion. However, several antigens have recently been identified and their immunogenicity examined by serodiagnostic and/or lymphocyte stimulation assays \[[@B26]-[@B30]\]. With the genome sequence of M. aviumsubsp. paratuberculosisrecently defined \[[@B31]\], all proteins produced by this pathogen are now identified and can be characterized. Novel opportunities arising from the genome sequence of M. aviumsubsp. paratuberculosisenable us to select and characterize genes of interest. A major goal of this laboratory is to define a complete catalog of immunodominant antigens in M. aviumsubsp. paratuberculosis. With the immunostimulatory capabilities of the M. tuberculosis19-kDa antigen in mind, the objective of this study was to determine if the 19-kDa protein of M. aviumsubsp. paratuberculosispossessed a similar capacity. In this study, the 19-kDa lipoprotein from M. avium subspecies paratuberculosiswas cloned, expressed, and characterized. In addition, the purified recombinant protein was used to assess cellular immune responses in subclinically infected cattle as well as humoral immune responses in cattle with clinical Johne\'s disease. Results ======= Sequence analysis of the mycobacterial 19-kDa coding region ----------------------------------------------------------- The 19-kDa coding sequence was identified from the M. aviumsubsp. paratuberculosisgenome project as MAP0261c. Comparison of amino acid sequences from other species of mycobacteria show that this gene product is conserved. Sequence alignment shows the N-terminal half is more variable and the region between amino acids 99 and 123 is highly conserved (Figure [1](#F1){ref-type="fig"}). MAP0261c displays a 95% amino acid identity in M. avium, 84% in M. intracellulareand 76% in M. bovisand M. tuberculosis. MAP0261c has a G+C content of 66.2% and encodes for 161 amino acids with a predicted molecular mass of 15.2 kDa. The first 22 amino acids of the M. tuberculosis19-kDa protein are hydrophobic and were previously noted to represent a signal peptide that is post-translationally cleaved to expose an N-terminal cysteine \[[@B32]\]. Signal peptidase cleavage analysis of MAP0261c (SignalP3.0; <http://www.cbs.dtu.dk/services/SignalP/>) detected a signal peptide generated from a putative cleavage site between amino acids 34 and 35, to expose an N-terminal serine (Figure [1](#F1){ref-type="fig"}). The SignalP-NN (neural networks) model assigned the highest cleavage probability values to amino acid 35 (C score = 0.324; Y score = 0.439) with the predicted length of the signal peptide being 34 amino acids. This is slightly longer than most signal sequences which range from 18 to about 30 amino acid residues in length \[[@B33]\]. The predicted peptidase cleavage sight for M. tuberculosisis between amino acids 21 and 22 and therefore falls within this range (Figure [1](#F1){ref-type="fig"}). Despite a predicted signal peptidase cleavage site, PSORTb analysis software <http://www.psort.org/psortb/> could not predict if the protein was cytoplasmic or membrane located. Cloning and expression of the M. aviumsubsp. paratuberculosis19-kDa protein ------------------------------------------------------------------------------- In order to perform immunoassays with purified 19-kDa protein, MAP0261c was amplified from M. aviumsubsp. paratuberculosisgenomic DNA and cloned into the pMal-c2 expression vector and transformed in E. coli. Induced expression resulted in production of a maltose binding protein (MBP)-19 kDa fusion protein that was affinity-purified from E. colilysates. MBP-19 kDa was analyzed by SDS-PAGE (Figure [2](#F2){ref-type="fig"}) to assess yield, purity and size. The predicted mass of MBP alone is 42 kDa, while the predicted mass of the MBP-19 kDa fusion protein is 56 kDa. The purified MBP-19 kDa protein migrated to a position around 50 kDa in SDS-PAGE (Figure [2A](#F2){ref-type="fig"}). Approximately 5 mg of purified protein was easily obtained from a 500-ml broth culture at O.D.~600\ nm~= 0.9. The purified protein was further characterized by immunoblot analysis using a monoclonal antibody (mAb) that detects the MBP affinity tag (Figure [2B](#F2){ref-type="fig"}). Both the fusion protein and MBP alone are detected by the mAb. In addition, the fusion protein is expressed at higher levels under inducing conditions. Immunoblot analysis of the 19-kDa protein ----------------------------------------- The M. tuberculosis19-kDa protein is known to be immunodominant, therefore immunoblot analysis was performed to determine if cattle naturally infected with M. aviumsubsp. paratuberculosisproduce antibodies against the 19-kDa protein. Immunoblots were probed with sera from 9 non-infected and 14 clinically infected cattle. Sera from 8 of 9 non-infected cattle did not react with either MBP or MBP-19 kDa, while 1 non-infected cattle weakly recognized both MBP and MBP-19 kDa protein (data not shown). By comparison, sera from 9 of 14 infected cattle reacted specifically with the 19-kDa protein, but not MBP alone. Antibody reactivity to the 19-kDa protein from 3 clinical cows is shown in Figure [3](#F3){ref-type="fig"}. Sera from the remaining five infected cattle detected both MBP and MBP-19 kDa proteins. This result made it difficult to distinguish if sera from the animal recognized the MAP0261c gene product or if it simply recognized the MBP affinity tag. Collectively, these data suggest that the 19-kDa protein is detectable and immunogenic in cattle with Johne\'s disease. IFN-γ responses of infected cattle ---------------------------------- As an indicator of the cell-mediated responses of infected cattle to the 19-kDa protein, whole blood containing PBMCs from 9 control and 21 infected cows was stimulated with M. aviumsubsp. paratuberculosissonicated whole cell lysate (WCL), MBP, or MBP-19 kDa and IFN-γ production was assayed by ELISA. These cattle were selected from a larger group because they showed no IFN-γ stimulation in response to MBP alone. IFN-γ production in response to WCL stimulation allowed for the segregation of infected cattle into three groups: suspect, positive, and high positive. After subtracting the IFN-γ responses of non-stimulated cells, suspect animals had less than 0.1 absorbance units of IFN-γ production, positive animals had 0.1 -- 0.3 absorbance units of IFN-γ production, and high positive animals had more than 0.3 absorbance units of IFN-γ production. IFN-γ responses by blood mononuclear cells from infected cattle exceeded responses from control cattle for both the WCL and MBP-19 kDa (Table [1](#T1){ref-type="table"}. Significant differences (P \< 0.05) were found between control and high positive groups for both WCL and MBP-19 kDa protein stimulation. However, direct comparisons of the two antigen preps using mononuclear cells from the same animal clearly showed the WCL was a stronger stimulator of IFN-γ production (Table [1](#T1){ref-type="table"}). Discussion ========== A majority of the research on individual mycobacterial proteins has been performed in M. tuberculosis, whereas little is known about the M. aviumsubsp. paratuberculosisproteome. Indeed, all currently available antigen-based diagnostic tests for Johne\'s disease use an undefined mixture of proteins, such as purified protein derivative (PPD) or WCL, which may not be specific for M. aviumsubsp. paratuberculosis. Recent completion of the M. aviumsubsp. paratuberculosisgenome has already advanced efforts to identify novel antigens \[[@B26],[@B34]\]. Furthermore, the genome will be a critical resource in proteomic studies directed at defining the proteins present in mixtures such as johnin PPD. The present study was performed in order to characterize the M. aviumsubsp. paratuberculosis19-kDa protein, as well as assess its immunostimulatory capabilities in cattle. In this study, we show that the 19-kDa protein of M. aviumsubsp. paratuberculosiscan be readily overexpressed as a fusion protein in E. coli. This is not true for many other proteins encoded by M. aviumsubsp. paratuberculosis\[[@B34]\] and suggests the putative lipoprotein is not toxic to E. coli. Previous studies have suggested the M. tuberculosis19-kDa protein undergoes posttranslational modification by the addition of fatty acids to form a lipoprotein \[[@B35]\]. Although not demonstrated directly by our studies, it is possible that the 19-kDa was not posttranslationally modified by the heterologous E. colihost. It is unclear whether posttranslational modification of this protein would affect its immunological activity. The recombinant antigen was detected by sera from cattle with Johne\'s disease; however, it was not as strong a stimulator of proliferative T-cell responses as has been reported for its counterpart in M. tuberculosis\[[@B36]\]. Furthermore, the 19-kDa protein from M. tuberculosiswas shown to induce both cellular and humoral immune responses from mice and humans \[[@B11],[@B14]\]. These studies, combined with the present study, may suggest that acylation is more important in cell-mediated immune responses than in the humoral immune response. It is generally accepted that cellular and humoral immune responses of M. aviumsubsp. paratuberculosis-infected cattle are biphasic, with IFN-γ responses detected early and antibody responses detected late in infection. However, evidence suggests that an unknown M. aviumsubsp. paratuberculosisprotein can be detected by antibodies from cattle just 3 weeks after infection \[[@B37]\]. As a measure of cellular immune responses, blood mononuclear cells from infected cattle were stimulated with both the recombinant 19-kDa protein and a whole-cell sonicated lysate of M. aviumsubsp. paratuberculosis(WCL), and IFN-γ production was measured. Results from this study suggest that while the 19-kDa protein is a stimulator of IFN-γ production, it is not as potent when compared to WCL. Additionally, we found that a majority (9 of 14) of infected cattle produced antibodies to the 19-kDa protein, as determined by immunoblot analysis. By comparison, sera from the majority of control, non-infected cattle (8 of 9) did not react to the 19-kDa protein. The single non-infected cow that did show reactivity to the MAP0261c gene product may be attributed to exposure of environmental mycobacteria such as M. aviumsubsp. avium, which has a similar protein (Figure [1](#F1){ref-type="fig"}). Sera from four clinical cows reacted to the MBP protein, but this reactivity was extremely weak. MBP is found in environmental E. coliand likely accounts for reactivity seen in some cattle. In order to avoid potential cross-reactivity with MBP, we attempted to cleave the MBP portion from the MBP-19 kDa fusion protein, but cleavage was not 100% efficient (data not shown). The M. tuberculosis19-kDa protein was reported to contain a 21 amino acid N-terminal signal peptide \[[@B22]\], however our SignalP analysis for M. aviumsubsp. paratuberculosisidentified a putative 34 amino acid N-terminal signal sequence. Furthermore, the computer algorithm PSORTb predicts a putative signal sequence, but it cannot determine if the protein is actually secreted. Antibodies will be produced against the recombinant MBP-19 kDa protein to determine if the protein is secreted by M. aviumsubsp. paratuberculosis. Conclusions =========== The results from this study show that the recombinant 19-kDa protein stimulates a weak host immune response in infected cattle. The 19-kDa protein may be used in conjunction with other antigens from M. aviumsubsp. paratuberculosisto identify infected cattle but should not be used as a \"stand alone antigen\" in new diagnostic assays. Methods ======= Bacterial strains and culture conditions ---------------------------------------- M. aviumsubsp. paratuberculosisstrain 19698-1974 (originally isolated in 1974 from a clinical cow housed at the National Animal Disease Center, Ames, IA) was grown in Middlebrook 7H9 liquid media (pH 6.0) supplemented with 10% oleic acid albumin dextrose complex (Becton Dickinson Microbiology, Sparks, MD), 0.05% Tween 80 (Becton Dickinson Microbiology), and 2 mg/ml mycobactin J (Allied Monitor Inc., Fayette, MO). M. aviumsubsp. paratuberculosiscultures were grown to log phase at an optical density 540 nm (OD~540~) of 0.4, at 37°C without shaking. Escherichia coliDH5α cells were routinely grown in Luria-Bertani (LB) broth or LB agar plates at 37°C supplemented with ampicillin (100 μg/ml) for selection. Cattle ------ The Johne\'s Disease Research Project at the National Animal Disease Center has a repository of sera from cattle that were euthanized with clinical signs of Johne\'s disease, which included shedding, weight loss and diarrhea. Fecal samples from each of these clinical cattle were found to contain more than 100 CFU per gram of feces as determined by colony counts on Herrold\'s egg yolk media (HEYM) agar slants by standard culture methods \[[@B38]\]. Sera from 14 clinical cattle were selected for immunoblot analysis. The National Animal Disease Center also maintains a small herd of non-infected cattle as well as a herd of cattle naturally infected with M. aviumsubsp. paratuberculosis. The animals used in IFN-γ experiments were placed in four groups consisting of 9 non-infected healthy cows, 9 suspect subclinical cows (IFN-γ responses \< 0.1), 6 positive subclinical cows (IFN-γ responses 0.1 -- 0.3), and 6 high-positive cows (IFN-γ responses \> 0.3). The non-infected control cows were characterized by repeated negative fecal cultures performed quarterly over a 3- to 5-year period. In addition, these animals were negative on all immunological assays (i.e. ELISA and IFN-γ production) performed during that period. Subclinical cattle (suspect, positive, and high-positive) were characterized by shedding less than 10 CFU/g of feces and were intermittently positive by IFN-γ assays (response \> 0.1) performed quarterly over a 3- to 5-year period. The institutional Animal Care and Use Committee approved all animal procedures described in this study. Comparison of the mycobacterial 19 kDa coding region ---------------------------------------------------- The nucleotide sequences for the 19 kDa coding regions from M. tuberculosis, M. bovis, M. aviumand M. intracellularewere obtained from the NCBI nucleotide sequence database. The sequences were assembled and compared using MegAlign software (DNASTAR, Inc., Madison, WI). Cloning and expression of the M. aviumsubsp. paratuberculosis19-kDa gene ---------------------------------------------------------------------------- A maltose binding protein (MBP) fusion of the M. aviumsubsp. paratuberculosis19-kDa sequence (MBP-19 kDa) was constructed using the pMAL-c2 vector (New England Biolabs, Beverly, MA). To amplify the 19-kDa coding region from M. aviumsubsp. paratuberculosis, primers were designed directly from the MAP0261c sequence. MAP0261c was amplified with Expand High Fidelity PCR system using the primers 19-kDa-pMal5\' (5\'-GCGC[CAGCTG]{.underline}ACGATCGCGGTCGCGGGCGCGGC-3\') and 19-kDa-pMal3\' (5\'-GCGC[AAGCTT]{.underline}CAGGTCACATCGATCTCGAAC-3\'). The 19-kDa-pMal5\' and 19-kDa-pMal3\' primers contained PvuII and Hind III restriction sites (underlined) for cloning, respectively. This primer set amplified the 19-kDa coding sequence minus the N-terminal 4 amino acids and the C-terminal 3 amino acids. The pMal-c2 vector was digested with XmnI and Hind III and the PCR amplicon was digested with PvuII and Hind III. The two products were ligated overnight at 12°C with T4 DNA ligase (Life Technologies Inc., Rockville, MD), which resulted in an in-frame fusion between the vector-encoded malEgene and a majority of the 19-kDa gene. The resulting recombinant plasmid, designated pMal-19-kDa, was transformed into E. coliDH5α competent cells. Recombinant clones were selected by plating on LB-ampicillin plates overnight at 37°C. Individual clones were picked and inserts were confirmed by DNA sequencing. The resulting fusion protein was overexpressed and purified by maltose affinity chromatography using amylose resin (New England Biolabs). A detailed expression and purification protocol has been published previously \[[@B39]\]. Expression and purification of the MBP-19 kDa fusion protein was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels either stained with GelCode Blue (Pierce Biotechnology Inc., Rockford, IL) or checked by immunoblot analysis with a monoclonal antibody against MBP developed at the National Animal Disease Center. Expression and purification of the MBP alone (without 19-kDa) was previously described \[[@B26]\]. SDS-PAGE and immunoblotting --------------------------- E. colilysates expressing MBP-19 kDa were prepared as previously described \[[@B40]\]. SDS-PAGE was performed using 12% (wt/vol) polyacrylamide gels. Proteins were electrophoretically transferred onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH) using the Trans Blot Cell (Bio-Rad Laboratories, Hercules, CA) in sodium phosphate buffer (25 mM; pH7.8) at 0.9 amps for 90 minutes. After transfer, the blots were blocked overnight with PBS plus 2% bovine serum albumin (BSA) and 0.1% Tween 20 (PBS-BSA). For immunoblots, serum from cattle with Johne\'s disease was diluted 1:500 in PBS-BSA. Sera were incubated on the blots at room temperature for 2 hours. After 3 washes in PBS plus 0.1% Tween 20, blots were incubated for 1.5 hours in anti-goat peroxidase-conjugated secondary antibody diluted 1:20,000 in PBS-BSA (Pierce Biotechnology Inc.). After secondary antibody incubation, the blots were washed 3 times as described above and were developed for chemiluminescent detection using SuperSignal detection reagents (Pierce Biotechnology Inc.). IFN-γ assays ------------ Blood was collected from the jugular vein of subclinically-infected cattle into sodium heparin vacutainer blood collection tubes. One ml aliquots of whole blood from each animal were plated into 4 wells of 24-well culture plates and cultured alone (non-stimulated) or with 10 μg/ml of M. aviumsubsp. paratuberculosissonicate (WCL), 10 μg/ml of MBP, or 10 μg/ml of MBP-19 kDa. The WCL was prepared by sonication of bacilli and centrifugation exactly as described previously \[[@B37]\]. Blood-antigen mixtures were incubated for 18 hours at 39°C in a 5% CO~2~humidified atmosphere. Plates containing blood-antigen samples were centrifuged at 500 × g for 15 minutes and the plasma was harvested from each well. Plasma samples were frozen at -20°C until being analyzed for IFN-γ concentration by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Bovigam, BioCor, Omaha, NE) as recommended by the manufacturer. Samples were analyzed in duplicate and were determined to be positive for IFN-γ production if the absorbance of the stimulated sample (WCL, MBP, MBP-19 kDa) was 0.1 units greater than the absorbance of the nonstimulated well for that animal. This classification of IFN-γ positive samples has been previously reported by our laboratory as well as others \[[@B41],[@B42]\]. Statistical analysis -------------------- ANOVA and unpaired t tests were performed to analyze the IFN-γ stimulation data. Analyses were performed to compare average stimulation of control, non-infected cattle to the infected cattle groups (suspect, positive, high positive). Differences were considered significant when P \< 0.05. Authors\' contributions ======================= JFH carried out all the experiments and drafted the manuscript as part of his PhD dissertation. JRS provided advice, participated in its design and coordination and helped edit the manuscript. JPB conceived of the study, participated in its design and helped to draft the manuscript. Acknowledgements ================ The authors gratefully acknowledge the members of the NADC paratuberculosis research project for their assistance during this study. The lab members include: Trudy Tatum, Janis Hansen, Tonia McNunn, and Bart Olthoff. This work was funded by the USDA\'s Agricultural Research Service and CSREES NRI grant 2002-02228 to J. P. B. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Amino acid sequence comparison of the mycobacterial 19 kDa protein. Non-conserved residues are shaded in black and gaps in amino acid sequence are indicated by hyphens (-). The predicted signal peptidase cleavage site for M. tuberculosisis indicated by a single arrow (↓), while the site for M. aviumsubsp. paratuberculosisis indicated by a double arrow (⇓). The GenBank accession number and abbreviations are M. aviumsubsp. paratuberculosis(M. paratb; [AAS02578]{.underline}), M. aviumsubsp. avium(M. avium; [AAB25888]{.underline}), M. intracellulare(M. intracel; AAB25885), M. bovis(M. bovis; S11234) and M. tuberculosis(M. tuberc; NP\_218280). ::: ![](1471-2180-5-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### SDS-PAGE and immunoblot analysis of the MBP-19 kDa fusion protein. (A) An SDS-PAGE gel showing the noninduced and IPTG-induced E. coliprotein lysates. Lanes: 1- protein size markers; 2- noninduced E. coliMBP-19 kDa; 3- IPTG induced E. coliMBP-19 kDa; 4- Affinity purified MBP-19 kDa. (B) Immunoblot probed with a monoclonal antibody to MBP. Lane 1- purified MBP; Lane 2- noninduced E. coliMBP-19 kDa; Lane 3- IPTG induced E. coliMBP-19 kDa. ::: ![](1471-2180-5-3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The recombinant 19-kDa protein is recognized by serum from infected cattle. Affinity purified MBP (lane 1) and MBP-19 kDa (lane 2) were transferred onto membranes in equal amounts and probed with sera from three clinically infected cattle: (A) animal 107; (B) animal 116; (C) animal 167. Reactivity was observed for the 19-kDa protein but not the MBP protein. Size standards are indicated in kilodaltons in the left margin. ::: ![](1471-2180-5-3-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### IFN-γ production by PBMCs in response to WCL and MBP-19 kDa stimulation. ::: CONTROL WCL CONTROL 19 KDA SUSPECT WCL SUSPECT 19 KDA POSITIVE WCL POSITIVE 19 KDA HIGH POS WCL HIGH POS 19 KDA ---------- ----------------- -------------------- ----------------- -------------------- ------------------ --------------------- ------------------ --------------------- 0 0 0.0294 0.0017 0.1058 0.0056 1.2407 0.9212 0.0623 0.0513 0.0980 0 0.1013 0.7737 1.0966 1.125 0.0072 0.0280 0.0412 0.1036 0.1323 0 0.3719 0.0124 0 0 0.0051 0.0286 0.1909 0.0290 0.3348 0.0925 0 0.0429 0.0259 0.0735 0.1623 0.0245 1.1866 0.7230 0.0560 0.0076 0 0 0.1974 0.0299 2.5995 0.0536 0.0561 0.0183 0.0348 0 0.0066 0.0285 0.0203 0.0071 0.0293 0.0236 0.0268 0.0002 Mean 0.0242 0.0222 0.0313 0.0239 0.1483 0.1438 1.1384 0.4880 :::	PubMed Central	2024-06-05T03:55:52.451743	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548131/", "journal": "BMC Microbiol. 2005 Jan 21; 5:3", "authors": [ { "first": "Jason FJ", "last": "Huntley" }, { "first": "Judith R", "last": "Stabel" }, { "first": "John P", "last": "Bannantine" } ] }
PMC548132	Introduction ============ Apoptosis plays important roles in placentation and embryonic development \[[@B1]\]. The cells undergoing apoptosis have characteristic structural changes in the nucleus and cytoplasm. The nuclear disintegration involves DNA cleavage into oligonucleosomal length DNA fragments \[[@B2]-[@B4]\], and the DNA fragments can be detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labelling (TUNEL) technique. Expressions of apoptotic regulatory molecules, such as Fas, Fas ligand, P53, and the proteins of Bcl-2 family, have been reported in human placenta \[[@B5]-[@B8]\]. Bcl-2 and P53 are two of the key players in the apoptotic signaling cascades. Bcl-2, a proto-oncogene first discovered in human follicular lymphoma \[[@B9]\], is involved in the inhibition of apoptosis and the survival of a variety of cell types \[[@B10]\]. Bcl-2 protein is located in the membranes of endoplasmic reticulum, nuclear envelope, and mitochondria. Over-expression of Bcl-2 suppresses apoptosis by preventing the activation of caspases that carry out the process. P53 is well known as a tumor suppressor. It is a transcription factor that induces apoptosis mainly through inducing the expression of a batch of redox-related genes \[[@B11]\] and the down-regulating Bcl-2 \[[@B12]\]. The expression of Bcl-2 and P53 human placenta has been studied \[[@B1],[@B13]\]. However, their cellular distribution in the implantation site at early stage of pregnancy has not been reported. Because the monkey and the human share a very similar implantation process in terms of timing, morphological changes, and cell types involved \[[@B14]\], we aimed, in the present study, to investigate the expression, localization of Bcl-2 and P53 in the implantation site of the rhesus monkey, in order to gain some insights to the mechanism of time-dependent apoptosis occurring at the fetal-maternal interface. Materials and methods ===================== Animals ------- Healthy adult male and female rhesus monkeys (Macaca mulatta) were purchased from the monkey colony of the Primate Research Center (PRC), Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS). All experimental procedures were approved by the Animal Ethics Committees of both the Institute of Zoology and PRC. The animals were caged individually and were evaluated daily by visual examination of the perineum for menses, with the onset of menses defined as Day 1 of the menstrual cycle. Adult female monkeys with regular menstrual cycles of approximately 28 days were chosen for this study. Female monkeys on Day 11 of their menstrual cycle were caged with a male monkey of proven fertility from previous mating for 3 days. Vaginal smears were examined the next morning for the presence of sperm. The day when the smear was detected as positive for sperms was designated as Day 1 of pregnancy (D1). The presence of a conceptus was confirmed by ultrasound examination. The monkeys were anesthetized by pentobarbital sodium (3 animals each group), and the uteri were removed surgically from early villous to villous placenta stages: on D17, D19, D28 and D34 of pregnancy respectively and cut into pieces, the specimens were quickly washed in cold phosphate-buffered saline (PBS) to remove adherent blood, then placed in cold 4% paraformaldehyde fixative for 16 h at 4°C and further processed through graded dehydration, clearing and embedding in paraffin for immunohistochemistry and TUNEL assay. Part of the specimen was cryopreserved at -70°C for Western blot analysis. Reagents -------- Primary antibodies including rabbit anti-human P53 (SC-6243) and mouse anti-human Bcl-2 (SC-7382) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Their quality and specificity were confirmed by the result of the Western blot analysis of D34 placental proteins (Figure [1](#F1){ref-type="fig"}). Rabbit anti-human cytokeratin (ZA0070), mouse anti-human actin (ZA0001), and mouse anti-Ki67 (ZM0166) were purchased from Zymed laboratories (San Francisco, CA, USA). Biotin labeled secondary antibodies, alkaline phosphatase (AP) conjugated avidin, horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG, HRP-conjugated horse anti-mouse IgG, and AP substrates \"Vector-red\" were from Vector Laboratories (Burlingame, CA, USA). Digoxigenindideoxy (DIG)-11-dUTP, TdT, blocking reagent, AP-conjugated anti-DIG antibody and 5-bromo-4-chloro-3-indoxyl phosphate/nitro-blue tetrazolium chloride (BCIP/NBT) were purchased from Roche-Boehringer-Mannheim (Mannheim, Germany). Proteinase K was purchased from Merck-Schuchardt (Darmstadt, Germany). Levamisole were purchased from Sigma-Aldrich (St Louis, MO, USA). SuperSignal^®^West Pico substrate was from PIERCE Biotechnology (Rockford, IL, USA). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Western blot analysis of Bcl-2 and P53 for monkey tissue obtained on D34 of pregnancy. Specific signals of Bcl-2 and P53 proteins were detected. No band was found in the control when antibodies were replaced with normal IgG of the same concentration and origin. ::: ![](1477-7827-3-4-1) ::: Western blot ------------ Western blot was done as previously described \[[@B15]\] with slight modifications to verify the cross-reactive specificity of the antibodies with the monkey tissue. The tissue of implantation sites on D34 of pregnancy was homogenized and the supernatant (50 μg) from centrifugation was run on a 10% SDS-PAGE gel under reduced conditions. After being transferred to the polyvinylidene difluoride membrane, individual lanes were cut and blocked with 5% nonfat milk/PBS for 1 h, followed by incubation at 20°C for 1 h with the primary antibodies (IgG, 0.2 μg/ml) in 5% milk/PBS. The membranes were washed three times, 5 min for each, in 5% milk/PBS and incubated with HRP-conjugated horse anti-mouse IgG (0.2 μg/ml, for Bcl-2) or HRP-conjugated goat anti-rabbit IgG (0.04 μg/ml, for P53) in 5% milk/PBS for 1 h respectively. The membranes were washed in PBS three times 5 min for each, followed by 10 min of incubation with SuperSignal^®^West Pico substrate, then exposed on x-ray film. For negative controls, primary antibodies were replaced with normal IgG of the same concentration and origin. TUNEL ----- Apoptotic cells were identified by using the TUNEL technique \[[@B1],[@B16]\]. The procedure was slightly modified based on Gao et al. \[[@B17]\] as the following. Deparaffinized and hydrated 4 μm sections were first treated with 10 μg/ml proteinase K at 37°C for 20 min, and then subjected to 3\'-end-labelling of the DNA with 1 μM DIG-11-dUTP and 1 U/μl TdT at 37°C for 1 h. The sections were washes three times in Tris buffer, and incubated with blocking buffer (100 mM Tris, 150 mM NaCl, pH 7.5, and 1% blocking reagent) for 30 min at room temperature. Next, sections were incubated with the primary AP-conjugated anti-DIG antibody (1:500 in 1% blocking reagent, 100 mM Tris, and 150 mM NaCl, pH 7.5) at room temperature for 2 h, and then washed with Tris buffer. Staining was developed using the standard substrates NBT (337.5 μg/ml) and BCIP (175 μg/ml). Negative controls were similarly processed with the omission of TdT. Immunohistochemistry -------------------- Serial 4 μm sections of tissue were deparaffinized and rehydrated through degraded ethanol. Antigen retrieval was performed by incubating the sections in 0.01 M citrate buffer (pH 6.0) at 98°C for 20 min followed by cooling at room temperature for 20 min. Non-specific binding was blocked with 5% (v/v) normal goat serum in PBS for 1 h. The sections were incubated with primary antibodies specific for P53 (1 μg/ml), Bcl-2 (2 μg/ml) or Ki67 (2 μg/ml) respectively in 2% goat serum overnight at 4°C. Sections were then washed three times with PBS (10 min each) and incubated with biotinylated secondary antibody (2 μg/ml) at RT for 30 min. 3 × 10 min successive washes were followed by incubation with avidin-AP complex (1:200, RT, 20 min). Sections were developed with standard substrates (337.5 μg/ml NBT and 175 μg/ml BCIP) or Vector Red AP substrates according to the manufacturer\'s protocol after another three washes. Endogenous AP activity was inhibited by supplement of 1 mM levamisole into substrate. The sections stained with Vector Red substrates were counter-stained using haematoxylin. Sections incubated with normal IgG instead of primary antibody served as negative controls. A double immunostaining technique using the antibodies to cytokeratin and actin was performed to localize the extravillous endovascular trophoblast cells. De-paraffinized sections were incubated with 3% H~2~O~2~in methanol for 10 min at room temperature to quench endogenous peroxidase after antigen retrieval treatment as described above. To detect the cytokeratin signal, the sections were washed (3 × 10 min in PBS), blocked for nonspecific signals, incubated sequentially with primary anti-human cytokeratin anbibody (1 μg/ml, RT, 1 h), secondary biotinylated goat anti-rabbit IgG (2 μg/ml, RT, 30 min), and avidin-peroxidase complex (1:200, RT, 20 min), and developed with DAB substrate solution in a similar way as described above. To detect actin signal, the procedure was repeated one more time with anti-human actin antibody (1 μg/ml, RT, 2 h) as primary antibody, AP conjugated horse anti-mouse IgG (1 μg/ml, RT, 40 min) as secondary antibody, and Vector Red developing AP substrate. As a result, the trophoblast cells were labeled brown and the blood vessel wall red. Microscopic assessment ---------------------- Placental samples from three individual monkeys for each group were analyzed. Experiments were repeated at least three times, and one representative from at least three similar results was presented. The mounted sections were examined using a Nikon microscope. For Ki67, the percentages of immunoreactive cells were assessed on at least 2000 cells in each tissue section; For TUNEL, the percentages of positive nuclei were assessed out of at least 2000 nuclei in each tissue section; For assessment of Bcl-2 staining intensity in cells of different compartments, semi-quantitative subjective scoring was performed by three blinded investigators using a 4-scale system with \"-\"= nil; \"+/-\"= weak; \"+\" = moderate; and \"++\" = strong as described by Yue et al. \[[@B18]\]. Results ======= Apoptosis in implantation site of early pregnancy ------------------------------------------------- The TUNEL technique was used to identify cell types that underwent apoptosis in the implantation site of rhesus monkey on D17, D19, D28 and D34 of pregnancy. On D17 and D19, apoptotic nuclei were observed in the syncytiotrophoblast layer covering the basal feet of the anchoring villi (Figure [2 A, B](#F2){ref-type="fig"}, arrowhead) and in the villous stromal cells (Figure [2 A](#F2){ref-type="fig"}, arrow), but not in the cytotrophoblasts. The positive nuclei in the syncytiotrophoblast was only about 0.06%. On D28 and D34, the apoptotic nuclei were present in the syncytiotrophoblast covering the villi (Figure [2 C, D](#F2){ref-type="fig"}), in the villous stromal cells (Figure [2 C, D](#F2){ref-type="fig"}, arrow), in the syncytiotrophoblast layer covering the basal feet of the anchoring villi (Figure [2 E](#F2){ref-type="fig"}), and in the cytotrophoblasts within the cell columns (Figure [2 F](#F2){ref-type="fig"}). On D28, the percentage of TUNEL-positive nuclei in the syncytiotrophoblast was 0.21%. As pregnancy progresses, the percentage increased to 0.34% on D34. In maternal compartment, a lot of apoptotic nuclei were detected in the stromal cells (Figure [2 G](#F2){ref-type="fig"}) and glandular epithelium (Figure [2 H](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Apoptosis detected by TUNEL at the implantation sites of the rhesus monkey on D17 (A), D19 (B), D28 (C, G) and D34 (D, E, F, H) of gestation.Apoptotic nuclei were stained dark. Arrowhead and arrow in panel A -- D indicated the nuclei of syncytiotrophoblast and villous stromal cells respectively. The insets in C and D showed the positive nuclei under a higher magnification. Note the syncytiotrophoblast layer covering the basal feet of the anchoring villi in E and the cell columns in F. G and H represent the stromal cells and glandular epithelial cells respectively in the endometrium. I was the negative control. St, syncytiotrophoblast; CT, cytotrophoblast; Vi, placental villi. Scale bars represent 50 μm. ::: ![](1477-7827-3-4-2) ::: Proliferative activity in implantation site at early pregnancy -------------------------------------------------------------- Ki67 is a protein expressed in cycling cells from G1 to M phases and is widely used as a roliferative marker (19, 20). As shown in Figure [3](#F3){ref-type="fig"}, Ki67 was expressed in the cytotrophoblasts and the villous stromal cells, but not in the syncytiotrophoblasts. As pregnancy progresses, the percentage of Ki67-positive cytotrophoblast cells lining the villi decreased from more than 85% on D17 to less than 25% on D34 (Panel A-D, and E, F for a higher magnification). However, the cytotrophoblasts at the proximal tip of cell columns remained highly proliferative (more than 70%) at all stages (Panel G and H). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The proliferating activity revealed by Ki-67 immunostaining at implantation sites of the rhesus monkey on D17 (A, E, G), D19 (B), D28 (C) and D34 (D, F, H) of gestation.Panels A-D were under a lower magnification. Ki-67 protein was stained red, and nuclei blue. E and F were the placental villi under a higher magnification. G and H were the anchoring villi under a higher magnification. Vi, placental villi. ST, syncytiotrophoblast. CT, cytotrophoblast. Sc, stromal cell. En, endometrium. Scale bars represent 100 μm. ::: ![](1477-7827-3-4-3) ::: Bcl-2 expression in implantation site at early pregnancy -------------------------------------------------------- In order to study the mechanisms of the apoptosis observed at the fetal-maternal interface, the expression of Bcl-2 was investigated by using immunohistochemistry. At the early stages of placentation (D17, D19), Bcl-2 was only detected in the syncytiotrophoblast covering the cell columns (Figure [4, A](#F4){ref-type="fig"} and [4B](#F4){ref-type="fig"}) and the extravillous cytotrophoblast (Figure [4C](#F4){ref-type="fig"}, arrow). At the later stages (D28, D34) it was detected in all the syncytiotrophoblast (Figure [4,D](#F4){ref-type="fig"} and [4E](#F4){ref-type="fig"}), the villous stromal cells (Figure [4F](#F4){ref-type="fig"}, arrow), and the extravillous endovascular trophoblast cells (Figure [4G](#F4){ref-type="fig"}), the fetal origin of these cells were indicated by the anti-cytokeratin antibody staining (Figure [4 G](#F4){ref-type="fig"} inset, brown), and the vascular wall was stained by anti-actin antibody (red). The pattern of Bcl-2 expression in the syncytiotrophoblast was similar to that of the apoptotic nuclei distribution (Figure [2](#F2){ref-type="fig"}). In the maternal compartment, Bcl-2 could be detected in some stromal cells (Figure [4H](#F4){ref-type="fig"}). Notably, the cytotrophoblasts lining the villi, within the cell columns, and the glandular epithelia were negative for Bcl-2 staining. The semi-quantitative expression level of Bcl-2 in different cell types at the various stages was summarized in Table [1](#T1){ref-type="table"}. A gradual increase of Bcl-2 staining was observed in the syncytiotrophoblast as gestation advances. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Immunohistochemical staining for Bcl-2 at implantation sites of the rhesus monkey.Bcl-2 staining is red, and nuclear counterstain blue. A, villous plancenta on D17. B, villous plancenta on D19. C, extravillous trophoblast cells in the basal plate of D17. D, villous plancenta on D28. E, villous plancenta on D34. F, villous plancenta on D34 under a higher magnification. G, the extravillous endovascular trophoblast cells; in the inset, the fetal origin of these cells was confirmed by anti-cytokeratin antibody (brown), and their position within the vascular wall was confirmed by anti-actin antibody staining (red). H, decidua. I, negative control. Vi, placental villi. ST, syncytiotrophoblast. CT, cytotrophoblast. Sc, stromal cells. Ge, glandular epithelium. Evc, extravillous cytotrophoblast. Scale bars represent 50 μm. ::: ![](1477-7827-3-4-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Semi-quantitative assessment of the immunohistochemical staining of Bcl-2 in the placenta of rhesus monkey. ::: D17 D19 D28 D34 ---------------------------------------------- ----- ----- ----- ----- Syncytiotrophoblast lining the villi +/- +/- ++ ++ Syncytiotrophoblast covering the cell column \+ \+ ++ ++ cytotrophoblast lining the villi \- \- \- \- extravillous cytotrophoblast \+ \+ \+ \+ ::: P53 expression in implantation site at early pregnancy ------------------------------------------------------ The expression profile of P53 was also acquired by using the immunohistochemistry. On D17 and D19, the expression of P53 was only confined to a small number of nuclei in the syncytiotrophoblast (Figure [5,A,B](#F5){ref-type="fig"}). On D28 and D34, its expression was observed not only in the syncytiotrophoblast (Figure [5C,D](#F5){ref-type="fig"}) but also in the nuclei of cytotrophoblasts lining the villi (Figure [5E](#F5){ref-type="fig"}) and within proximal tip of cell columns (Figure [5F](#F5){ref-type="fig"}) where a proliferative activity was high as indicated by Ki67 staining (Figure [3](#F3){ref-type="fig"}). Clustered P53-positive nuclei were seen in the syncytiotrophoblast covering the basal feet of the anchoring villi (Figure [5G](#F5){ref-type="fig"}), coincident well with the strong apoptosis detected by TUNEL (Figure [2E](#F2){ref-type="fig"}). P53 was also expressed in some stromal cells (Figure [5H](#F5){ref-type="fig"}) of the uterine endometrium. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Immunohistochemical staining for P53 at implantation sites of the rhesus monkey on D17 (A), D19 (B), D28 (C, H), and D34 (D, E, F, G) of gestation. P53 was stained dark in nuclei.A-D were villous placenta under a lower magnification. The inset of panel A shows the staining in the syncytiotrophoblast covering the basal feet of the anchoring villi under a higher magnification. E, staining in villous placenta under a higher magnification. F, staining in cell columns. G, syncytiotrophoblast covering the basal feet of the anchoring villi under a higher magnification. H, the endometrium with arrows indicating stromal cells. ST, syncytiotrophoblast. CT, cytotrophoblast. Scale bars represent 50 μm. ::: ![](1477-7827-3-4-5) ::: Discussion ========== For the first time in present study, we investigated the expression of Bcl-2 and P53 in relation to apoptosis at the fetal-maternal interface of rhesus monkey at the very early stages (D17-D34) of gestation. Villous trophoblasts consist of cytotrophoblasts and syncytiotrophoblast. While cytotrophoblasts possess a brisk mitotic activity during the first trimester of gestation in human, the syncytiotrophoblast is incapable of cell division despite of a metabolic activity \[[@B1]\]. This fact implies that cell proliferation is differently regulated in these two cell types. The reports on the type of trophoblast cells undergoing apoptosis in the first trimester are controversial \[[@B1],[@B21],[@B22]\]. Our results further cleared that the apoptotic nuclei were distributed mainly in the syncytiotrophoblast at the early stages and in the cytotrophoblasts within the cell columns at later stages of pregnancy. In our previous study, Bax expression was found at the Fetal-Maternal Interface of Rhesus Monkey \[[@B17]\]. Bax is a Bcl-2 family member that promotes cell death susceptibility, possibly by countering the effect of Bcl-2 on cell survival through heterodimer interaction. Bax to Bcl-2 \"rheostat\" may be a critical factor in regulating apoptosis in multiplicate tissues. As shown in Figure [6](#F6){ref-type="fig"}, Bax was found expressed in the placenta and glandular epithelium of endometrium and all kinds of cells in placental villi, and no obvious change was observed between different time points from D17 to D34 in placental villi. Therefore, we speculated that Bcl-2 may play a more important role on controlling the apoptosis in placental villi. The diffusive expression of Bcl-2 in syncytiotrophoblast obtained from the first trimester human placenta has been reported recently \[[@B7],[@B23]-[@B25]\]. Our observation on the Bcl-2 expression in syncytiotrophoblast at later stages (D28-D34) agreed well with these data. As shown in this study, although Bcl-2 was expressed, apoptotic nuclei still exsisted in the same region. This phenomenon implies that the expression of Bcl-2 is not sufficient to completely inhibit the apoptosis in the syncytiotrophoblast. Therefore, the role of Bcl-2 here becomes an interesting question. Multiple nuclei sharing the same cytoplasm is a morphological characteristic of syncytiotrophoblast. In such cells, the apoptotic signal may be transmitted from one nuclear to another, and cause a spontaneous abortion. Therefore, the number of nuclei undergoing apoptosis in the syncytiotrophoblast should be limited by some mechanism in order to ensure normal embryo development in normal pregnancy \[[@B1]\]. We speculate that Bcl-2 may be included in this mechanism. The major role of apoptosis-associated Bcl-2 expression in the syncytiotrophoblast might be to limit the nuclear degradation to a special area and inhibit the spread of cell apoptosis signals to the other nuclei sharing the same cytoplasm, thus sustain cell survival in these multi-nucleated cells. Toki et al has also suggested that Bcl-2 might play a major role in avoiding the possible excessive nuclear degradation in syncytiotrophoblast \[[@B26]\]. Further studies, however, are needed to prove this speculation. The immunostaining for Bcl-2 was also detected in part of the extravillous and endovascular cytotrophoblast in our study. These subtypes of cytotrophoblast lost the capacity of proliferation (Ki-67-negative), but they did not undergo apoptosis (negative in TUNEL assay). Therefore, we hypothesize that Bcl-2 may also participate in regulation of the extravillous trophoblast apoptosis by stimulating the cellular survival. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Immunohistochemical staining for Bax at implantation sites of the rhesus monkey on D28.Bax staining is brown, and nuclear counterstain blue. A, villous plancenta, positive staining was found in all the cells. B, endometrium, glandular epithelium was positive for Bax staining. Vi, placental villi. Ge, glandular epithelium. ::: ![](1477-7827-3-4-6) ::: P53 was partly identified in some nuclei of the syncytiotrophoblast with the same position of apoptotic nuclei, in the basal feet of the anchoring villi in particular, but it is not clear whether the P53 was co-localized with the apoptotic signals. Activation of P53 in some cell types leads to either the cessation of cell growth or apoptosis \[[@B27]\]. Therefore, P53 protein might be related to cell cycle arrest or apoptosis in syncytiotrophoblast during early stage of placentation. Low level of P53 staining was detected in the cytotrophoblasts during the earlier stages of gestation (D17 and D19). However, at the later stages (D28 and D34), the expression was observed predominantly in the nuclei of cytotrophoblasts. The presence of P53 in cytotrophoblast in the primate was consistent with that observed in the human first trimester placenta \[[@B8]\]. Indeed, the TUNEL staining showed that the apoptosis seldom happened in the cytotrophoblast, with the exception of cytotrophoblast at proximal tip of cell columns during later stages of placentation (D28, D34) where a high proliferative activity and P53 expression were detected. This finding supports the hypothesis that a physiological upregulation of the P53 tumour suppressor gene might be a mechanism for controlling excessive trophoblastic proliferation in normal placentation \[[@B26],[@B28]\]. It is known that early pregnancy is unique in its methods of cell proliferation control, the existing data suggest that some growth factors and transcription factors from the embryo and endometrium, such as CSF-1, VEGF, and transcription factors of the helix-loop-helix family, provide at least part of this control \[[@B29]\]. In addition, other studies found maternal age and some diseases, such as diabetes can also influence the apoptotic and proliferative activities in trophoblast cells \[[@B30],[@B31]\]. Further investigations are required to uncover which endocrine event regulates the expression of Bcl-2 and P53. Acknowledgments =============== This study was supported by WHO/Rockefeller Fundation Project (RF96020\#78), the National Science Fundation of China (30270196), Chuang-Xin program of Chinese Academy of Sciences (KSCX-2-SW-201/IOZ-7), and the Chinese \"973\" Program (G1999055901).	PubMed Central	2024-06-05T03:55:52.453898	2005-1-14	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548132/", "journal": "Reprod Biol Endocrinol. 2005 Jan 14; 3:4", "authors": [ { "first": "Peng", "last": "Wei" }, { "first": "Xuan", "last": "Jin" }, { "first": "Xue-Sen", "last": "Zhang" }, { "first": "Zhao-Yuan", "last": "Hu" }, { "first": "Chun-Sheng", "last": "Han" }, { "first": "Yi-Xun", "last": "Liu" } ] }
PMC548133	Background ========== It is well known that cytotoxic factors, such as lipopolysaccharides, derange nitrogen metabolism in hepatocytes and nitric oxide (NO) is involved among the other factors regulating this metabolic pathway \[[@B1]\]. NO is a free radical that is involved in many cellular events. In the biological systems NO has an halflife long lasting few seconds. It is an oxidation intermediate, therefore is both an oxidant and a reducing agent of metabolic products. Its biosynthesis is mainly performed by converting L-arginine to L-citrulline. L-arginine analogues, such as N^g^-nitro-L-arginine methyl ester (L-NAME), act as false substrates and are selective inhibitors of NO synthesis. NO synthase (NOS) is either a constitutive or inducible enzyme. The endothelial isoform (e-NOS) and the neuronal isoform (n-NOS) are constitutive. The inducible form of the enzyme (i-NOS), has the main property to be not regulated by intracellular calcium concentration and Ca^2+^-calmodulin complex, unlike the constitutive form \[[@B2]\]. It is known that iNOS is expressed by many cell types including macrophages, smooth muscle cells and hepatocytes \[[@B3]\]. Hepatocytes have been shown to express large levels of NO following exposure to endotoxins, such as bacterial lipopolysaccharide and/or cytokines, such as tumour necrosis factor-α (TNFα), interleukin-1 \[[@B4],[@B5]\]. NO may posses both cytoprotective and cytotoxic properties, depending on the amount and the isoform of NOS by which it is produced \[[@B6]\]. NO generally mediates beneficial responses, but becomes deleterious when coexistence with enhanced superoxide formation leads to the synthesis of peroxynitrite, a potent oxidant and nitrating agent \[[@B7]\]. According to this hypothesis, authors studied the effect of bradykinin, a proinflammatory mediator kinin, on cell viability and on urea production in cultures of rat hepatocytes. Kinins exert numerous physiological and pathological actions; they partecipate in vascular and cellular events that accompany the inflammatory processes. In pathological states, kinins are thought to be implicated in inflammatory diseases and in haemorrhagic and endotoxic shock \[[@B8]\]. To demonstrate the decrease of cell viability and urea production by bradykinin, the authors studied its effects on NO production. The measurements of NO release from hepatocytes were investigated by using a NO-specific fluorescence indicator, 4,5 diaminofluorescein diacetate (DAF-2DA) \[[@B9]\]. Results ======= Effect of bradykinin treatment on NO production ----------------------------------------------- The amounts of released NO were measured using DAF-2DA, that specifically reacts with the oxidized form of NO, producing the fluorescent triazolofluorescein \[[@B9]\]. NO determination was performed after 2 hours of incubation in the presence of bradykinin (0.01 mM and 0.1 mM). As shown in figure [1](#F1){ref-type="fig"} the treatment with 0.01 mM bradykinin did not produce NO increase compared to control, but 0.1 mM bradykinin increased significantly the NO release. In contrast no appreciable NO release was observed during the same period in hepatocytes cultured with 0.1 mM bradykinin and 1.68 mM L-NAME. Effect of bradykinin treatment on urea production ------------------------------------------------- To evaluate urea synthesis after bradykinin treatment, the hepatocytes were treated with 1 mM NH~4~Cl for 2 h. Figure [2](#F2){ref-type="fig"} shows that only the treatment with 0.1 mM bradykinin significantly decreased urea production and that the treatment with 0.1 mM bradykinin and 1.68 mM L-NAME did not produce a significant urea level decrease in comparison to control. Effect of bradykinin treatment on cell viability ------------------------------------------------ To determine the effects of bradykinin on cell viability, the hepatocytes were exposed to bradykinin (either 0.01 mM or 0.1 mM) for an incubation time of 2 hours. In one experimental series, the cell viability was determined by MTT test after 2 hours of incubation. In a second one, culture medium containing bradykinin was removed and replaced with the same fresh medium at 2 hours after the addition of bradykinin, and then cell viability was measured 24 hours after the end of bradykinin treatment. The MTT test after 2 hours of incubation does not indicate any significant viability difference in treated hepatocyte cultures in comparison to control (figure [3A](#F3){ref-type="fig"}). By MTT test after 24 h (figure [3B](#F3){ref-type="fig"}), a significant lowering of viability is observed in bradykinin 0.1 mM treated hepatocytes in comparison to control. The decrease was significantly reduced by the simultaneous treatment with L-NAME 1.68 mM even if always significantly lower than in control. Cell viability was validated by Trypan blue exclusion test (Table [1](#T1){ref-type="table"}). Discussion ========== The role of NO as mediator of hepatic injury after endotoxic shock remains controversial \[[@B16]\]. Increased NO production in response to cytokines has been demonstrated in cultured hepatocytes \[[@B17]\]. Laskin et al. \[[@B18]\] demonstrated that the induction of acute endotoxemia, caused an increase in NO production in the liver. This was associated with expression of inducible nitric oxide synthase (iNOS) messenger m-RNA in hepatocytes. Also our data showed an increase of NO production after 2 hours treatment of culture with 0.1 mM bradykinin in an arginine supplemented medium, as substrate for the synthesis of NO. The simultaneous treatment with L-NAME, a known inhibitor of NOS, blocked the increase of NO production. In this work we analyzed the urea synthesis after bradykinin treatment. Urea synthesis was decreased after 2 hours treatment with bradykinin 0.1 mM and the simultaneous treatment with L-NAME leaves urea biosynthesis unaltered. These data can be attributed to the control role of arginine in both urea and NO biosynthesis. When NO is synthesized from arginine, by the NOS reaction, cytrulline, an intermediate of urea cycle, is produced. Thus, the urea cycle is bypassed by the NOS reaction \[[@B1]\]. Whether NO exerts cytotoxic or cytoprotective action remains unclear \[[@B6]\]. We also found a significant decrease of viability, at long term, in hepatocytes subjected to bradykinin treatment. The simultaneous treatment of hepatocytes with L-NAME improves cell viability even if control levels are not restored. The data show that the increased NO production plays a role in liver damage induction, that follows the proinflammatory mediator treatment. The hepatocellular injury attributed to NO may be due either to its direct cytotoxicity or its reaction with superoxide to produce the toxic nitrogen metabolite peroxynitrite \[[@B19]\]. Oldenburg et al. \[[@B20]\], demonstrated in other cell types, like cardiomyocytes, that bradykinin caused the increase in reactive oxygen species (ROS) generation. At last, our results show that the increased NO synthesis induces a reduced urea production, that is an index of cell damage. The simultaneous treatment of liver cell cultures with L-NAME decreases NO levels and sustains overall biosynthesis activities and cell viability. Conclusions =========== In summary, we conclude that 0.1 mM bradykinin treatment induces an increase of NO levels and reduction of urea synthesis in the hepatocytes. This increased NO production mediates, after 24 hours, cell toxicity as shown by MTT test. In contrast, the administration of the NOS inhibitor L-NAME protects against cell damage and increases urea levels, suggesting that NO plays a key role in the bradykinin-induced liver damage. Methods ======= Materials --------- Unless otherwise specified, all chemicals were obtained from Sigma (St. Louis, MO, USA). Isolation and culture of rat hepatocytes ---------------------------------------- Hepatocytes were isolated from male rats, Wistar strain, (180 to 200 gbw), by a modification of the method of Seglen \[[@B10]\]. All procedures on the animals were performed according to the CEE directive n. 86/609 on animal experimentation. Rats were anesthetized with diethylether, the pre-perfusion of the liver in situ was performed at a rate of 20--30 ml/min with Ca^2+^-free Hanks balanced salt solution. The liver was then excised and the digestion was carried out by adding 0.05% (w/v) collagenase (type IV) in Hanks balanced salt solution supplemented with CaCl~2~·H~2~O (0.0186 g/L) at a flux rate of 40 ml/min. At this point liver was transferred to a square plate containing 100 ml of RPMI 1640 medium supplemented with 200 mM L-glutamine, 20 ml/L essential amino acid solution and 10 ml/L non-essential amino acid solution, 1% antibiotic antimycotic stabilized solution and 100 μM L-arginine (incomplete medium). The cells were dispersed by gentle distruption with a stainless steel comb. After filtration through 200 μm Nytal mesh, parenchymal cells (hepatocytes) were separated from nonparenchymal cells (endothelial cells, Kupffer cells and stellate cells) by centrifugation at 50 g in Eppendorf Centrifuge 5810R at 4°C for 2 minutes and then washed twice in washing buffer \[[@B11]\]. Then the cells were resuspended in the same medium and filtered through 63 μm Nytal mesh. The viability of the cells was more than 80%, as estimated by trypan blue dye exclusion test \[[@B12]\]. After cell counting the cells were diluited at a concentration of 5 × 10^5^cells/ml with incomplete medium supplemented with 2% fetal calf serum, 0.1 U/ml insulin and 10^-6^M dexamethasone (complete medium). The hepatocytes were then plated in 24 well-plates coated with rat tail collagen at the final cell density of 2.5 × 10^5^cells per well and incubated at 37°C in an humidified atmosphere of 5% CO~2~and 95% air. After 6 hours incubation, the medium was changed and replaced with incomplete medium to remove dead cells. To verify the isolation method efficiency, the acid fosfatase activity per mg of proteins was evaluated. According to literature data, the specific activity of acid fosfatase in nonparenchimal cells is 1,7 folds the same activity in parenchimal cells \[[@B13]\]. Treatment --------- After 24 hours of culture the hepatocytes were exposed either to bradykinin (0.01 mM and 0.1 mM) or bradykinin 0.1 mM supplemented with L-NAME 1.68 mM \[[@B14]\]. Determination of NO from hepatocytes ------------------------------------ DAF-2DA (Alexis Biochemicals, Lausen, Switzerland) was dissolved in DMSO (1 mg/0.45 ml) and diluted to 10 μM in phosphate buffer (0.1 M, pH 7.4). Then the cells were either incubated in phosphate buffer containing 10 μM DAF-2DA, bradykinin (0.01 mM and 0.1 mM) and bradykinin 0.1 mM supplemented with L-NAME 1.68 mM. After 2 hours of incubation in this reaction mixture, the fluorescence from the reaction of DAF-2DA with NO released from hepatocytes was measured with Perkin-Elmer MPF-44B Spectrofluorimeter calibrated for excitation at 495 nm and emission at 515 nm. Results were expressed as a percentage of the fluorescence of the samples in comparison to control. Determination of urea synthesis ------------------------------- To determine the effects of bradykinin on urea production, cells were treated either with bradykinin (0.01 mM and 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM. At the same media 1 mM NH~4~Cl was added. After 2 hours urea levels in the media were measured by spectrophotometric method using Urea Color 2 Kit (Sclavo Diagnostics, Siena, Italia) measuring absorbance at 600 nm and blank sample with the same NH~4~Cl final concentration was used. Urea synthesis was calculated as ng urea per cell per hour. Determination of cell viability ------------------------------- Cell viability was determined by MTT test method \[[@B15]\] and confirmed by Trypan blue exclusion test \[[@B12]\]. MTT (5 mg/ml) was dissolved in RPMI-1640 without phenol red. The solution is filtered through a 0.2 μm filter and stored at 2--8°C for frequent use. To determine the effects of bradykinin on cell viability, cells were either treated with bradykinin (0.01 mM and 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM for a 2 h period. After that cells were used either immediately or after an additional 24 h incubation period in incomplete medium. For the determination of cell viability, the medium has been discarded and MTT solution was added and incubated for 3 hours. At the end of the incubation period the MTT solution was removed and the cells and dye cristals were dissolved by adding dimethylsulfoxide (DMSO). Absorbance was measured at 570 nm in a Shimadzu UV-2100 Spectrophotometer and the results were expressed as a percentage of the absorbance of the samples in comparison to control. Statistical analysis -------------------- At least four independent determinations of each parameter were compared to control using Student\'s T-test. Differences were considered significant when p \< 0.05 was obtained. Authors\' contributions ======================= SS: Fluorimetric analysis and overall statistical analysis of data MG: Director of research MS: Spectrophotometric analysis CR: Primary hepatocyte cultures and characterization All authors read and approved the final manuscript Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Determination of NO release after treatment of hepatocytes with bradykinin. Fluorescence intensity was measured after 2 h incubation: with 10 μM DAF-2DA in basal conditions (white column, reference), in presence of 0.01 mM bradykinin (hatched column), in presence of 0.1 mM bradykinin (crosshatched column) and in presence of 0.1 mM bradykinin with 1.68 mM L-NAME (black column). The excitation wavelength was 495 nm and the emission wavelength was 515 nm. Values, expressed as a percentage of control values, are the means ± S.E.M. (bars) of four independent experiments. ![](1472-6793-5-2-i1.gif) p \< 0,05 compared with control ::: ![](1472-6793-5-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Determination of urea production after treatment of hepatocytes with bradykinin. Urea production was spectrophotometrically determined at 600 nm after 2 h incubation with 1 mM NH~4~Cl in basal conditions (white column), in presence of 0.01 mM bradykinin (hatched column), in presence of 0.1 mM bradykinin (crosshatched column) and in presence of 0.1 mM bradykinin with 1.68 mM L-NAME (black column). Values, expressed as ng urea per cell per hour, are the means ± S.E.M. (bars) of four independent experiments. ![](1472-6793-5-2-i1.gif) p \< 0,05 compared with control ::: ![](1472-6793-5-2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Determination of cell viability in hepatocytes treated with bradykinin. The cell viability was spectrophotometrically determined at 570 nm by MTT assay in hepatocytes incubated in basal conditions (white column), in presence of 0.01 mM bradykinin (hatched column), in presence of 0.1 mM bradykinin (crosshatched column) and in presence of 0.1 mM bradykinin with 1.68 mM L-NAME (black column) for 2 h period. (A) Cell viability determined immediately after. (B) Cell viability determined after an additional 24 h incubation period in incomplete medium Results are expressed as a percentage of control. Values are the means ± S.E.M. (bars) of four independent experiments. ![](1472-6793-5-2-i1.gif) p \< 0,05 compared with control. ![](1472-6793-5-2-i2.gif) p \< 0,05 compared with control and 0.1 mM bradykinin treated cells ::: ![](1472-6793-5-2-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of bradykinin treatment on cell viability ::: Treatment: 2 h+Brad (Viability %) Treatment: 2 h+Brad + 24 h-Brad (Viability %) ------------------------------------ ----------------------------------- ----------------------------------------------- Control 100 ± 10 100 ± 6 0.01 mM Bradykinin 98 ± 12 104 ± 10 0.1 mM Bradykinin 104 ± 8 50 ± 11\* 0.1 mM Bradykinin + 1.68 mM L-NAME 102 ± 10 79 ± 7\\ Hepatocytes were isolated and cultured in presence and in absence of bradykinin (either 0.01 mM or 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM for a 2 h period. Cell viability was determined by Trypan blue exclusion test either immediately after 2 h or after additional 24 h incubation period in incomplete medium. Results are expressed as a percentage of control. Values are expressed as mean ± S.E.M., n = 4. \Statistically significant differences (p \< 0,05) from control levels as determined by Student\'s t-test. \\* Statistically significant differences (p \< 0,05) from control levels and from 0.1 mM bradykinin treated cells as determined by Student\'s t-test. :::	PubMed Central	2024-06-05T03:55:52.455819	2005-1-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548133/", "journal": "BMC Physiol. 2005 Jan 25; 5:2", "authors": [ { "first": "Settimio", "last": "Sesti" }, { "first": "Guglielmo", "last": "Martino" }, { "first": "Sergio", "last": "Mazzulla" }, { "first": "Rosa", "last": "Chimenti" } ] }
PMC548134	Background ========== Diabetes mellitus, the prevalence of which is reaching epidemic proportions in many parts of the world, is an increasingly important public health concern. In the United States, diabetes is present in 8% of the adult population, and is associated with a two-fold increase in age-adjusted mortality \[[@B1],[@B2]\]. In addition, there is a high prevalence of chronic medical conditions among subjects with diabetes \[[@B3]-[@B5]\]. For example, in the United States, the prevalence of cardiovascular diseases, stroke and depression in subjects with diabetes was at least twice as high as in subjects without diabetes \[[@B6]-[@B9]\]. Diabetes has detrimental effects on a range of health outcomes including health-related quality of life (HRQoL) \[[@B10]-[@B12]\]. For example, in the Medical Outcomes Study, diabetes was found to impair all dimensions of health except mental health and pain \[[@B13]\]. In a more recent multinational study, diabetes was found to have a notable impact on general health, measured using the Medical Outcomes Short-Form 36 (SF-36) \[[@B14]\]. The magnitude of impact of diabetes on HRQoL was reported to be equivalent to that of having cardiovascular conditions, cancer and chronic respiratory disease \[[@B15]\]. Subjects with diabetes and multiple co-existing chronic medical conditions have poorer HRQoL than those without these conditions \[[@B16],[@B17]\]. For example, subjects with diabetes and co-existing cardiovascular diseases reported significantly lower scores on RAND-36 social functioning, vitality and health-change scales \[[@B16]\]. In another study, subjects with diabetes and co-existing coronary artery disease, peripheral sensory neuropathy and peripheral vascular diseases reported significantly lower scores on several SF-36 scales \[[@B17]\]. In combination, the influence of multiple chronic medical conditions on HRQoL may exhibit an additive, synergistic or possibly subtractive relationship. Assuming that each chronic medical condition results in the lowering of HRQoL, in an additive relationship, the combined effect of two or more chronic medical conditions on HRQoL approximates the sum of the independent effect of each of these conditions, while in a synergistic relationship, the combined effect is greater than the sum of the independent effect of each of these conditions and in a subtractive relationship the combined effect is smaller than the sum of the independent effect of each of these conditions (Figure [1](#F1){ref-type="fig"}). For example, the SF-36 developers \[[@B18]\] reported an additive relationship between hypertension and other co-existing chronic medical conditions on HRQoL, while Gaynes et al. \[[@B19]\] reported synergistic relationships between depression and co-existing arthritis on physical functioning and between depression and co-existing diabetes on role functioning. Several mechanisms could account for these observed synergistic effects. For example, treatment for one medical condition might also adversely affect another pre-existing medical condition, leading to a greater lowering of HRQoL than would occur due to the pre-existing condition alone \[[@B20]\]. Additionally, a medical condition itself (e.g. depression) might adversely affect patient behavior and thus negatively affect treatment outcomes \[[@B21]\]. Although there have not been any reports in the literature, it is theoretically possible that subtractive relationships exist. For example, it was reported that patients undergo changes in their conception of poor levels of functioning, personal values (e.g. changes in life priority) and/or meaning of life in response to their chronic medical conditions, a concept known as response shift \[[@B22]\]. These changes in self-assessment and values may help to cushion the impact of the second medical condition, resulting in a smaller decrement in HRQoL than might otherwise be expected. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### An overview of the possible relationships among multiple chronic medical conditions on HRQoL. \These relationships (subtractive or synergistic) are discussed in relation to additive relationships. ^†^If net effect is zero, then discussion of subtractive and synergistic relationships do not apply. ::: ![](1477-7525-3-2-1) ::: Two published studies have evaluated the relationship between diabetes and other chronic medical conditions on HRQoL in the general population \[[@B23],[@B24]\]. In the first study \[[@B23]\], diabetes and stroke were found to increase the risks of disability (measured with the Activity of Daily Living and Instrumental Activity of Daily Living scales) among older Mexican Americans in an additive fashion. In the second study, the impact of co-existing obesity, hypertension or diabetes and heart disease on HRQoL among 17,195 U.S. middle- and older-age adults was evaluated \[[@B24]\]. A strong synergistic relationship between heart disease and diabetes on the odds of mobility difficulty (1.8 to 4.0 times), activity of daily living limitations (2.2 to 4.0 times) and poor perceived health (2.1 to 6.8 times) was found, after adjusting for gender, ethnicity, insurance status, history of cancer, and lung diseases \[[@B24]\]. However, there were two important limitations in both studies. First, these studies focused on the physical domains of HRQoL, without assessing the mental and social domains of HRQoL. Second, both studies were conducted among middle-aged and/or elderly adults. Thus, the burden of diabetes and other chronic medical conditions on HRQoL in the rest of the general population with diabetes are not known. Further studies to elucidate the relationship between diabetes and other chronic medical conditions on physical, mental and social domains of HRQoL in the general population are clearly needed. The primary purpose of this study was therefore to evaluate the impact of diabetes and co-existing chronic medical conditions on the physical, mental and social domains of HRQoL in the general population. We also sought to determine if the impact of diabetes and co-existing chronic medical conditions on HRQoL would be additive, synergistic or subtractive. For these purposes, we measured HRQoL using both the SF-36 and the SF-6D. The SF-36 is a comprehensive, generic HRQoL measure that has been extensively validated and allows comparison of HRQoL in subjects with various chronic medical conditions. The SF-6D is a single index utility score derived from the SF-36 which measures health preference, and which is therefore suitable for pharmacoeconomic analyses to assist healthcare resource allocation. We sought to determine the extent to which the SF-6D would reflect the multidimensional information provided by the SF-36. Methods ======= Study design ------------ We analyzed data from a cross-sectional, population-based disproportionately stratified sample of ethnic Chinese, Malays and Indians listed in the 1996 electoral register for the Bishan-Toa Payoh district (representative of the general population) in Singapore from April 1998 to January 1999, details of which have been reported previously \[[@B25]\] and are summarized here. Participants completed either the UK English or Chinese (Hong Kong) SF-36 and the Family Functioning Measure. Eligibility criteria were: age 21 to 65 years on 1^st^January 1998 and ability to read a newspaper in English or Chinese; Chinese, Malay or Indian ethnicity as reflected in the National registration identification card, which reflects parental ethnicity; and status as free-living adults in the community. Persons older than 65 years of age were excluded because of low literacy rates in this age group in Singapore \[[@B26]\]. Fieldworkers visited eligible subjects at their house within 7 days after an introductory letter was sent, invited them to self-administer the SF-36, checked returned questionnaires for completeness and obtained information on ethnicity, socioeconomic status and chronic medical conditions (including diabetes, hypertension, heart disease, lung diseases, musculoskeletal illnesses and mental illnesses) and other potential determinants of HRQoL through a structured interview. Participants were asked to indicate either \"yes\" or \"no\" on a list of chronic medical conditions including diabetes, high blood pressure, heart disease, etc. Instruments ----------- ### The Short-Form 36 Health Survey (SF-36) The SF-36 measures perceived health in the areas of physical functioning (PF), role-physical (RP), bodily pain (BP), general health (GH), vitality (VT), social functioning (SF), role-emotional (RE), and mental health (MH), with higher scores (range 0--100) reflecting better perceived health. The UK English \[[@B27]\] and Chinese (Hong Kong) \[[@B28]\] SF-36 with 4-week recall and metric units of measurement were used in this study (previously validated for use in Singapore) \[[@B25]\]. ### The SF-6D The SF-6D is a six-dimensional health classification system assessing physical functioning (PF), role limitations (RL), social functioning (SF), pain (PN), mental health (MH), and vitality (VT), with 4 to 6 levels per dimension \[[@B29],[@B30]\]. A SF-6D \"health state\" is defined by selecting one level from each dimension. For example, perfect health on the SF-6D would be represented by the health state 111111. A total of 18,000 health states are thus defined. All responders to the original SF-36 questionnaire can be assigned an SF-6D score provided the items used to construct the SF-6D are completed \[[@B29],[@B30]\]. The SF-6D preference-based measure can be regarded as a continuous variable scored on a 0.29 to 1.00 scale, with 0.29 corresponding to the worst health state (i.e. all dimension being at the worst level) and 1.00 corresponding to \"full health\" (i.e. all dimensions being at full functional level). ### Family Functioning Measure The Family Functioning Measure is a 3-item Likert scale assessing the quality of interactions among family members \[[@B31]\], with higher scores (range 0--100) reflecting better family functioning, and has been validated for use in Singapore \[[@B32]\]. Statistical analyses -------------------- Data from participants completing English and Chinese SF-36 versions were pooled to increase the power and representativeness of our study. This approach was supported by previous work demonstrating equivalence of these SF-36 \[[@B33]\] and SF-6D \[[@B34]\] versions in this same study sample. Data were entered into an Excel spreadsheet (Microsoft Corporation, Redmond, Washington) and analyzed using SPSS (SPSS Inc., Chicago, Illinois) software. SF-6D index scores were calculated using a utility function derived from the United Kingdom general population \[[@B29]\], which is currently the only published SF-6D scoring algorithm. Participants with missing scale scores were excluded listwise from analysis. To study the influence of diabetes and other chronic medical conditions on HRQoL, we developed 9 multiple linear regression models, with each SF-36 scale and the SF-6D index score being the dependent variable for a separate model. Each model was built in 3 stages. The first stage assessed the effect of diabetes on HRQoL while adjusting for the influence of sociodemographic factors. The second stage assessed the independent effects of diabetes and a second chronic medical condition on HRQoL while adjusting for the influence of sociodemographic factors. The third stage additionally assessed any interactions between diabetes and this second chronic medical condition on HRQoL. Thus, each model studied the effect of diabetes and one other chronic medical condition. The absence of a significant interaction term would suggest that the influence of two chronic medical conditions on HRQoL scores was additive. The presence of a significant interaction term would suggest that a synergistic or subtractive relationship existed, the former if the coefficient for the interaction term was negative, the latter if the coefficient was positive (since changes in HRQoL scores in the presence of chronic medical conditions were expected to be negative). Ethnicity was coded using dummy variables. Educational level was used as proxy for socioeconomic status. In anticipation that if the number of subjects with a given chronic medical condition was less than 50, the numbers of subjects with this medical condition and diabetes would be very small and not amenable to meaningful interpretation, two chronic medical conditions, stroke and cancer, were thus excluded from analysis because of small numbers of subjects reporting these conditions (n\<50). Two other chronic medical conditions were subsequently excluded from analysis because of small numbers of subjects with diabetes and these conditions (diabetes and lung diseases: n = 27 and diabetes and mental illnesses: n = 9). Results ======= Characteristics of study subjects --------------------------------- Complete data were available from 5,224 of 5,420 subjects in the study, with 196 (3.6%) subjects excluded from analysis (110 because of missing demographic information and 86 because of missing responses to the SF-36). Table [1](#T1){ref-type="table"} shows subjects\' characteristics and distribution of SF-36 scales and SF-6D index scores. Approximately 6% of subjects suffered from diabetes and two-fifths of subjects reported at least 1 chronic medical condition. Prevalence of co-existing conditions in subjects with diabetes ranged from 2.9% (mental illnesses) to 37.2% (hypertension). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of subjects and distribution of SF-36 scales and SF-6D index scores. ::: Subject characteristics (N = 5,224) N (%) unless specified otherwise --------------------------------------------------------------------------------------- ---------------------------------- Mean (SD) age in years 40.3 (11.52) Female gender 2,555 (48.9) Ethnicity Chinese 2,558 (49.0) Malay 1,189 (22.8) Indian 1,477 (28.3) Housing type Lower cost public housing 388 (7.4) Public housing 4,647 (89.0) Private housing 188 (3.6) Years of education ≤ 6 1,266 (24.2) 7--10 2,682 (51.3) \>10 1,276 (24.4) Marital status Married 3,686 (70.6) Single 1,272 (24.3) Divorced/Separated 136 (2.6) Widow 130 (2.5) Working 4,022 (75.0) Smoking 1,045 (20.0) Presence of acute medical conditions\ 3,493 (66.9) Presence of chronic medical conditions Diabetes mellitus 309 (5.9) Hypertension 557 (10.7) Heart disease 125 (2.4) Stroke 32 (0.6) Lung diseases^‡^ 278 (5.3) Cancer 35 (0.7) Musculoskeletal illnesses^‡^ 1,389 (26.6) Mental illness^‡^ 136 (2.6) Prevalence of co-existing chronic medical conditions in people with diabetes mellitus Hypertension 115 (37.2) Heart disease 46 (14.9) Lung diseases^‡^ 27 (8.7) Musculoskeletal illnesses^‡^ 107 (34.6) Mental illnesses^‡^ 9 (2.9) Mean (SD) Family Functioning Measure scores 10.3 (2.75) Mean (SD) SF-36 scores^†^ Physical functioning (PF) 80.3 (23.19) Role-physical (RP) 80.5 (32.94) Bodily pain (BP) 78.3 (21.73) General health (GH) 68.6 (17.21) Vitality (VT) 63.8 (16.98) Social functioning (SF) 80.6 (20.48) Role-emotional (RE) 79.2 (34.98) Mental health (MH) 72.4 (16.70) Mean (SD) SF-6D index scores^†^ 0.77 (0.130) \* Acute medical conditions were self-limiting illnesses and injuries in the preceding one month that might influence SF-36 scores and included acute illnesses (e.g. upper respiratory tract infections, gastroenteritis and headaches), injuries or poor sleep. ^†^Mean scores adjusted for the influence of diabetes mellitus, other chronic medical conditions (and their interactions), socioeconomic status and other factors. ^‡^Lung diseases included asthma and other lung diseases; musculoskeletal illnesses included rheumatism, back pain and other bone or muscle illness; mental illness included depression, anxiety disorder and schizophrenia. ::: The influence of chronic medical conditions on SF-36 scores ----------------------------------------------------------- The influence of chronic medical conditions on individual SF-36 scales is presented in Table [2](#T2){ref-type="table"}. Before adjusting for selected sociodemographic variables known to influence HRQoL (i.e. age, gender, ethnicity and years of education), subjects with self-reported chronic medical conditions (diabetes, hypertension, heart disease and musculoskeletal illnesses) generally reported lower scores for most SF-36 scales when compared to subjects without these conditions, indicating poorer HRQoL. After adjusting for the influence of these sociodemographic variables, chronic medical conditions continued to influence HRQoL, generally to a lesser degree than before adjustment. The influences of heart disease and musculoskeletal illnesses on SF-36 scores were of a similar magnitude and were larger than the influence of diabetes or hypertension. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Influence of diabetes mellitus and other chronic medical conditions on SF-36 scales. ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Unadjusted differences in mean scores ------------------------------------ ----------------------------------------------------------------- -------------- -------------- -------------- -------------- -------------- -------------- -------------- -------------- PF RP BP GH VT SF RE MH SF-6D No chronic medical condition^†^\ 82.0 (22.61) 81.9 (32.16) 80.4 (21.20) 70.0 (16.62) 64.8 (16.43) 81.5 (20.12) 77.5 (36.00) 73.1 (16.35) 0.79 (0.123) (n = 3155) Diabetes mellitus only (n = 309) -7.2 -7.3 -5.8 -3.5 -1.3 -1.8 0.7 0.2 -0.06 Hypertension only\ -4.3 -4.1 -3.4 -3.3 -0.5 0.5 1.0 0.3 -0.05 (n = 557) Heart disease only (n = 125) -9.5 -10.1 -7.3 -5.2 -3.6 -0.8 -4.7 -0.3 -0.09 Musculoskeletal illnesses only^‡^\ -4.7 -3.6 -6.9 -4.4 -3.1 -2.7 -0.8 -2.5 -0.08 (n = 1389) Adjusted differences in mean scores due to medical condition^§^ PF RP BP GH VT SF RE MH SF-6D No chronic medical condition^†^\ 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00 (n = 3155) Diabetes mellitus only (n = 309) -2.9 \* -3.7 -1.8 -2.36 \* -1.3 -0.1 0.3 -0.2 -0.03 \\ Hypertension only\ -2.6 \* -3.1 \* -1.5 -1.6 0.1 0.6 -1.0 0.1 -0.03 \\\* (n = 557) Heart disease only (n = 125) -5.0 \* -6.5 \* -3.5 -4.0 \* -3.9 \* 0.9 -5.9 -1.0 -0.06 \\\* Musculoskeletal illnesses only^‡^\ -3.8\ -3.1\ -6.4\ -3.9\ -2.9\ -3.1\ -3.4\ -2.6\ -0.08\ (n = 1389) \\\* \\ \\\* \\\* \\\* \\\* \\ \\\* \\\* ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ^†^Mean (SD) scores ^‡^Musculoskeletal illnesses included rheumatism, back pain and other bone or muscle illnesses. ^§^Multiple linear regression, adjusted for influence of ethnicity, age, gender and years of education. \p \< 0.05, \\p \< 0.01, \\\p \< 0.001 Abbreviations: PF = Physical Functioning, RP = Role-Physical, BP = Bodily Pain, GH = General Health, VT = Vitality, SF = Social Functioning, RE = Role -Emotional, MH = Mental Health ::: The influence of chronic medical conditions on SF-6D scores ----------------------------------------------------------- The influence of chronic medical conditions on SF-6D scores is also presented in Table [2](#T2){ref-type="table"}. Subjects with chronic medical conditions similarly reported lower unadjusted mean SF-6D scores. After adjusting for known determinants of HRQoL, lowering of SF-6D scores persisted for all chronic medical conditions, though again to a smaller magnitude. The influences of heart disease and musculoskeletal illnesses on SF-6D scores were of a similar magnitude and were again larger than the influence of diabetes or hypertension. The influence of diabetes mellitus and co-existing chronic medical conditions on SF-36 scores --------------------------------------------------------------------------------------------- Characteristics of subjects with diabetes, with and without other co-existing chronic medical conditions, are shown in Table [3](#T3){ref-type="table"}. Subjects with diabetes only (i.e. no co-existing chronic medical conditions) were generally younger and more likely to be male. There were more Indians with diabetes and co-existing heart disease or musculoskeletal illnesses and more Chinese with diabetes and co-existing hypertension. The distribution of years of education completed was fairly similar in the various ethnic groups. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Characteristics of subjects with diabetes, with and without other chronic medical conditions. ::: ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Co-existing chronic medical conditions among subjects with diabetes -------------------------------------- --------------------------------------------------------------------- --------------- ---------------- ------------------------------- Characteristics No co-existing chronic medical conditions\ Hypertension\ Heart disease\ Musculoskeletal illnesses^†^\ (n = 115) (n = 115) (n = 46) (n = 107) Mean age (years) 48.1 (10.52) 53.7 (8.46) 55.5 (7.95) 53.2 (8.68) Male (%) 64.3 56.5 71.7 54.2 Ethnicity (%) Chinese 23.5 40.0 21.7 36.4 Malays 27.8 22.6 21.7 17.8 Indians 48.7 37.4 56.5 45.8 Questionnaire language (English) (%) 86.1 73.9 91.3 76.6 Education (%) ≤ 6 years 36.5 44.3 30.4 41.1 7--10 years 50.4 42.6 56.5 48.6 \> 10 years 13.0 13.0 13.0 10.3 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: The impact of co-existing chronic medical conditions on adjusted SF-36 scores of subjects with diabetes is presented in Table [4](#T4){ref-type="table"}. In general, after adjusting for known determinants of HRQoL, the presence of concurrent hypertension, heart disease and musculoskeletal illnesses further reduced SF-36 scores in subjects with diabetes. For example, subjects with diabetes and co-existing hypertension or musculoskeletal illnesses experienced further lowering of physical functioning scores by 2.3 and 3.7 points, respectively. The influence of two chronic medical conditions on SF-36 scores was generally additive, in that statistically significant interaction terms were not present for most comparisons. There were some exceptions in which subtractive effects were observed, mainly clustered within subjects with diabetes and co-existing heart disease (physical functioning, role-physical, social functioning and role-emotional scales) or musculoskeletal illnesses (bodily pain and social functioning scales). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Multiple linear regression models of the influence of diabetes mellitus and a second chronic medical condition (i.e. hypertension, heart disease or musculoskeletal illnesses) adjusted for ethnicity, age, gender and years of education on SF-36 and SF-6D scores. ::: ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Co-existing chronic medical conditions among subjects with diabetes ------------------------------------ --------------------------------------------------------------------- --------------- ------------------------ ------------------------------- No co-existing chronic medical conditions\ Hypertension\ Heart disease (n = 46) Musculoskeletal illnesses^†^\ (n = 115) (n = 115) (n = 107) Physical Functioning Differences in scale scores due to Diabetes mellitus -2.9\* -2.4 -3.5\* -2.8\* 2nd chronic medical condition‡ na -2.3\* -7.9\\ -3.7\\\* Interaction termξ na ns 12.46\\ ns Role-Physical Differences in scale scores due to Diabetes mellitus -3.7 -3.1 -5.2\* -3.6 2nd chronic medical condition‡ na -2.8 -12.5\\ -3.1\\ Interaction termξ na ns 20.1\\ ns Bodily Pain Differences in scale scores due to Diabetes mellitus -1.8 -1.6 -1.5 -3.8\* 2nd chronic medical condition‡ na -1.3 -3.1 -6.9\\\* Interaction termξ na ns ns 7.0\\ General Health Differences in scale scores due to Diabetes mellitus -2.3\* -2.0 -1.9 -2.2\* 2nd chronic medical condition na -1.3 -3.5\* -3.9\\\* Interaction termξ na ns ns ns Vitality Differences in scale scores due to Diabetes mellitus -1.3 -1.4 -0.9 -1.3 2nd chronic medical condition‡ na 0.3 -3.7\* -2.9\\\* Interaction termξ na ns ns ns Social Functioning Differences in scale scores due to Diabetes mellitus -0.1 -0.2 -1.3 -1.9 2nd chronic medical condition^‡^ na 0.6 -2.5 -3.4\\\* Interaction term^ξ^ na ns 10.2\* 5.4\* Role-Emotional Differences in scale scores due to Diabetes mellitus 0.3 -2.8 -0.9 0.4 2nd chronic medical condition^‡^ na -2.7 -11.8\\ -3.4\\ Interaction term^ξ^ na 9.9\* 16.8\* ns Mental Health Differences in scale scores due to Diabetes mellitus -0.2 -1.7 -0.1 -0.1 2nd chronic medical condition^‡^ na -0.5 -0.9 -2.6\\\* Interaction term^ξ^ na 5.1\* ns ns SF-6D Differences in scale scores due to Diabetes mellitus -0.03\\ -0.02\* -0.02\* -0.02\\ 2nd chronic medical condition^‡^ na -0.03\\\* -0.06\\\* -0.08\\\* Interaction term^ξ^ na ns ns ns ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ^†^Musculoskeletal illnesses included rheumatism, back pain and other bone or muscle illnesses. ^‡^Second chronic medical condition refers to the chronic medical condition other than diabetes mellitus listed in the relevant column (e.g. hypertension, heart disease or musculoskeletal illnesses). ^ξ^Interaction term for diabetes mellitus and second chronic medical condition. \p \< 0.05, \\p \< 0.01, \\\p \< 0.001, ns denotes statistically insignificant. ::: The influence of diabetes mellitus and co-existing chronic medical conditions on SF-6D scores --------------------------------------------------------------------------------------------- The influence of diabetes and co-existing chronic medical conditions on SF-6D scores is presented in Table [4](#T4){ref-type="table"}. Subjects with diabetes and other co-existing chronic medical conditions reported lower unadjusted SF-6D scores than subjects with diabetes only. After adjustment for known determinants of HRQoL, the influence of co-existing chronic medical conditions on SF-6D scores persisted. As before, subjects with diabetes and co-existing heart disease or musculoskeletal illnesses reported the greatest impairment in HRQoL. Diabetes and other co-existing chronic medical conditions, including heart disease, reduced SF-6D scores in an additive fashion. Discussion ========== In this multiethnic, population-based study, we found that subjects with diabetes experienced lowering of HRQoL as compared to subjects without diabetes. The presence of other chronic medical conditions in subjects with diabetes led to further lowering of HRQoL, the effect of which was generally additive. Our findings further underscore the importance of preventing and treating complications of diabetes to prevent further deterioration in HRQoL among subjects with diabetes, and also highlight the need to identify factors that may be modulated to improve HRQoL in these subjects. Our findings are important for several reasons. First, this is the first study showing that the combination of diabetes and a second chronic medical condition may adversely affect the mental domains of HRQoL as measured by the vitality, social functioning and mental health scales of the SF-36 (previous studies having focused on the physical domains of HRQoL) \[[@B23],[@B24]\]. Second, it is reassuring that the effect of diabetes and a second chronic medical condition on HRQoL was in general additive rather than synergistic. Third, given that subjects were drawn from the general population, it is likely that our findings can be readily generalized to the population at large, especially given that this study was conducted in a multiethnic Asian population with one of the highest diabetes prevalence rates in the world \[[@B35]\]. We found that the effect of diabetes on HRQoL was generally mild, with greater impact on the SF-36 scales measuring physical (physical functioning, role-physical, bodily pain, general health, role-emotional (in this study sample)) relative to mental health components (vitality, social functioning and mental health). This was not surprising, given that our subjects were recruited from the general population and were therefore likely to have less severe illness than subjects in hospital or clinic-based studies. Further, other studies have shown that impact of diabetes on HRQoL is intermediate, relative to other chronic medical conditions \[[@B14]\]. For example, Egede \[[@B9]\] reported that the risk of functional disability was lower in subjects with diabetes than subjects with major depression (odds ratio (OR): 2.42 vs 3.00), while Otiniano et al. \[[@B23]\] found that the risk of disabilities in ADL were lower in subjects with diabetes than those with strokes (OR: 2.80 vs 5.55). We also found that with the exception of subjects with diabetes and heart disease, the presence of co-existing chronic medical conditions in subjects with diabetes generally resulted in further significant lowering of HRQoL. Our results are important because they demonstrate that the impact of these co-existing chronic medical conditions in diabetes is not only in increasing healthcare costs \[[@B36],[@B37]\] and mortality \[[@B38]\] but also in increasing the physical and psychosocial burden of diabetes. Given that complications of diabetes constitute the majority of chronic medical conditions commonly present in subjects with diabetes, our findings further underscore the importance of preventing and treating complications of diabetes, and also highlight the need to identify factors that may be modulated to improve HRQoL in these subjects. In our study, the relationships between diabetes and other chronic medical conditions on HRQoL were largely additive. This is in contrast with the study by Gaynes et al. \[[@B19]\], where synergistic effects between depression and diabetes on HRQoL were observed. Instead, we found in general an additive effect with several comparisons showing a subtractive effect on HRQoL (especially in subjects with diabetes and co-existing heart disease). There are several possible explanations for the presence of subtractive effects. One possible explanation is the presence of response shift related to the specific diseases involved. In this study, the subtractive effects were clustered among subjects with diabetes and co-existing heart disease. Diabetes (in particular Type 2 diabetes) is a well-recognized cardiovascular risk factor with subjects with diabetes commonly developing heart disease in the 5^th^or 6^th^decade of life \[[@B40]\], often many years after the diagnosis of diabetes \[[@B41]\] (although heart disease may on occasion be present at or before diagnosis) \[[@B42],[@B43]\]. In contrast, hypertension typically precedes or occurs soon after diagnosis in subjects with Type 2 diabetes, with up to 50% of subjects with diabetes presenting with hypertension at the time of diagnosis \[[@B44]\]. Hence, adaptation to the underlying diabetes may have led to response shift occurring in subjects with diabetes and co-existing heart disease but not with hypertension. A second plausible explanation for this observation is a healthy-responder effect. This would occur if subjects with diabetes and co-existing heart disease were more likely to have passed away or were too sick to participate in the study. Thus the subset of subjects with diabetes and co-existing heart disease who did participate in this study would reflect the healthier end of the spectrum, leading to the observed effect of higher scores for respondents with the two co-existing chronic medical conditions. A third possible explanation is that some conditions are so similar in their effects on HRQoL that having more than one such condition is not particularly problematic to the individual. For example, in our study, subjects with diabetes alone and subjects with heart disease alone experienced lower scores on the physical functioning scale of the SF-36; if these two conditions exerted a similar effect on HRQoL, then a subtractive effect would be expected, as was indeed observed among subjects with diabetes and co-existing heart disease. A secondary objective of this study was to understand the extent to which the single index SF-6D captured information from the multidimensional SF-36. We found that in general, the impact of co-existing chronic medical conditions on the SF-36 was well-reflected in the SF-6D. However, the subtractive effects of diabetes and co-existing heart disease on SF-36 scores were not reflected in SF-6D scores. This suggests that there is some inevitable loss of information associated with a reduction from a multi-dimensional scale to a unidimensional scale. Hence, more studies are needed to evaluate the adequacy of the SF-6D as a summary measure of the SF-36. We recognize several limitations of this study. First, identification of chronic medical conditions was based on self-report which may not be as accurate as physician diagnoses. However, reliability of self-report has been found to be acceptable for conditions that required medical or laboratory diagnostic procedures, including diabetes \[[@B45]-[@B47]\]. Second, although we captured information on subjects with diabetes and co-existing mental illnesses or lung diseases, we had to exclude these because of the small number of subjects. Finally, in this study we did not differentiate between subjects with Type 1 and Type 2 diabetes. However, given that more than 90% of subjects with diabetes are Type 2, this is not likely to affect our findings. Conclusions =========== In conclusion, in this large, multiethnic, population-based study, we found that subjects with diabetes experienced lowering of HRQoL as compared to subjects without diabetes. The co-existence of other chronic medical conditions in subjects with diabetes led to further lowering of HRQoL, the effect of which was generally additive. Finally, we found that the SF-6D is a reasonably good summary measure of the SF-36 although more studies are needed to confirm this observation. List of abbreviations ===================== ADL -- Activities of Daily Living, DM -- Diabetes mellitus, HD -- Heart disease, HRQoL -- Health-related quality of life, HTN -- Hypertension, MS -- Musculoskeletal illnesses, OR -- Odds ratio, SF-36 -- Medical Outcomes Short-Form 36. Authors\' contributions ======================= JT conceived of the study, and participated in its design and coordination. HL Wee and YB Cheung participated in the design of the study and performed the statistical analysis. SC Li and KY Fong participated in the design of the study and its coordination. All authors read and approved the final manuscript. Acknowledgements ================ We would like to thank Dr John Brazier for providing the scoring algorithm for the SF-6D, and Prof PH Feng, Drs ML Boey, KH Leong and Staff Nurse ST Thio for their contributions to the study on which this analysis is based. This study was funded by grants from the Biomedical Research Council of Singapore (03/1/27/18/226) and the National Medical Research Council of Singapore (0160/96).	PubMed Central	2024-06-05T03:55:52.457294	2005-1-12	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548134/", "journal": "Health Qual Life Outcomes. 2005 Jan 12; 3:2", "authors": [ { "first": "Hwee-Lin", "last": "Wee" }, { "first": "Yin-Bun", "last": "Cheung" }, { "first": "Shu-Chuen", "last": "Li" }, { "first": "Kok-Yong", "last": "Fong" }, { "first": "Julian", "last": "Thumboo" } ] }
PMC548135	Background ========== This and the following presentation are part of a project for the International Programme on Chemical Safety (IPCS) evaluating the possible use of transgenic animal mutagenicity assays in chemical toxicity testing and mechanistic research. It was decided to compare the results obtained from those transgenic mutagenicity test systems where considerable data was available, with other in vivo genotoxicity tests: in the first article (Part 1) with the mouse bone marrow micronucleus test and in the second (Part II) with the mouse spot test. The mouse bone marrow micronucleus test is one of several available in vivomammalian test system for the detection of chromosomal aberrations \[[@B1]-[@B5]\]. A documentation of the test procedure and evaluation of results is given in the OECD guideline 474 \[[@B6]\]. This test is routinely used with a widespread acceptance in industry and authorities. Mutagenicity assays using transgenic animals have been developed in particular the lacImodel \[[@B7]\] (commercially available as the Big Blue^®^mouse), and the lacZmodel \[[@B8]\] (commercially available as the Muta™Mouse). In this article, available data on the results of mouse bone marrow micronucleus test were compared with results from these two transgenic mouse assays for 39 substances. The advantages and disadvantages of the test systems are discussed. Recently, further transgenic rodent mutation assays have been developed, however, the data base is not sufficient for comparison with other test systems. The mouse bone marrow micronucleus test: principles and procedure ----------------------------------------------------------------- Micronuclei are chromatin-containing bodies in the cytoplasm arising from acentric chromosome fragments or from whole chromosomes that were not incorporated in the daughter nuclei during the last stages of mitosis. Chromosome fragments are associated with the clastogenic (chromosome breakage) activity of the test substance whereas the presence of a whole chromosome is indicative of an adverse effect of the test substance on the mitotic spindle apparatus (aneugenic effects). The difference in size of the micronucleus is therefore an indicator for clastogenicity (small micronucleus) or aneugenicity (large micronucleus). However, the size of the micronucleus is an inaccurate measure. Micronuclei can be distinguished by further criteria, for example by identification of the presence of a kinetochore or centromeric DNA, indicating aneugenic activity. Overall, an increase in micronuclei is an indirect measure of induced structural or numerical chromosome aberrations \[[@B1],[@B2]\]. In the micronucleus test according to the OECD guideline 474, erythroblasts in the bone marrow of mice (or rats) are used as target cells. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded. Any micronucleus that has been formed may remain in the otherwise anucleated cytoplasm and can easily been detected. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage \[[@B6]\]. In the last three decades, toxicologists have often used the mouse bone marrow micronucleus test because 1) it is part of the regulatory toxicology in the admission procedure for chemicals and drugs and 2) it has advantages in speed, simplicity, and cost effectiveness in comparison to other in vivosystems for testing chromosomal aberrations (e.g. the cytogenetic test). Transgenic mouse models ----------------------- Transgenic mutation test systems contain a foreign gene construct having two essential parts: the transgene containing the target genethat serves as target for mutations, and a shuttle vectorfor recovering the target gene DNA from the tissue of the transgenic animal. The transgene is constructed using recombinant DNA technologies. ### LacItransgenic mouse model (Big Blue^®^mouse) The proprietary mouse of the Big Blue^®^mutagenesis assay system contains about 30--40 copies of a lambda LIZα shuttle phage vector integrated into its genome at a single locus on chromosome 4. The target site for mutagenesis is the lacIgene \[[@B7]\]. The test compound is administered to the mouse either as a single or repeated dose. After the post treatment period for manifestation of the DNA lesions, the tissue of interest is isolated and the DNA extracted. A proprietary lambda DNA packaging extract automatically excises the lambda vector target and packages it into a lambda phage head and the phage is transfected to bacteria. These bacteria are plated on agar indicator plates containing the chromogenic substance (X-gal). The phage transfected bacteria with mutations in the lacIgene form blue plaques, whereas bacteria with a nonmutated lacIform colourless plaques. The ratio between blue and white plaques is a measure of the mutagenicity. \[[@B7],[@B9]\]. ### LacZtransgenic mouse model (Muta™Mouse) The lambda-gt10-lacZshuttle vector for the lacZmouse model contains the entire lacZ target gene \[[@B8]\]. The commercially available lacZmouse model contains about 80 copies of the shuttle vector at chromosome 3. As above, the test compound is administered to the mouse, the genomic DNA is isolated from the tissue of interest, and the lambda genomes are excised by and packed with a bacteriophage packaging extract. The resulting phage particles are then plated on a lacZ^-^E. colistrain (for transfection) in the presence of the chromogenic substance (X-gal) as indicator. The plaques containing an intact lacZare β-galactosidase active and are blue, whereas plaques containing mutated lacZwill be white/colourless. In this original model the ratio between colourless and blue plaques is a measure of the mutagenicity. Due to optical difficulties in the evaluation of plaques, this system has been improved using a selection assay in lieuof colour screening whereby only mutant particles form plaques \[[@B10]\]. The number of plaques under non-selective conditions is a measure for the total number of phage-transfected bacteria (intact ormutant lacZ gene). The ratio between the number of plaques produced under selective conditions versus the number of plaques under non-selective conditions is a measure of the mutagenicity \[[@B9],[@B10]\]. Methods ======= Data presented in this documentation are the results of an extensive literature research. Concerning data on transgenic mouse assays only primary literature was used. Data on the mouse bone marrow micronucleus assay were extracted from reliable reviews on this item or from primary literature. For all other data informations from secondary literature or data compilation bases were used. Results and Discussion ====================== Comparison of data from the mouse bone marrow micronucleus test and transgenic mouse test ----------------------------------------------------------------------------------------- The authors are aware that a comparison of the transgenic mouse assays with the mouse bone marrow micronucleus test is limited by the fact that different genotoxic endpoints are studied in these two systems. In transgenic mouse assays, point mutations and small insertions and deletions are detected whereas in the mouse bone marrow assay, chromosome breakage leading to light microscopically visible micronuclei resulting from chromosome fragment or micronuclei originated from whole chromosomes are investigated. However, both point mutations and micronuclei may be induced by a single agent, so some overlap of results is to be expected. From the literature, 39 substances were identified with data on the mouse bone marrow micronucleus test andthe Muta™mouse assay (n = 29) or the Big Blue^®^mouse assay (n = 21) or both transgenic mutation assays (n = 11); see [Additional file 1](#S1){ref-type="supplementary-material"} for references. Agreement between the Muta™mouse and the micronucleus test was seen with 18 out of 29 substances, no agreement with 8 substances and in 3 cases the comparison is inconclusive because of questionable results in the micronucleus assay. With the Big Blue^®^mouse assay, the results obtained with 14 out of 21 substances agreed with results in the mouse micronucleus test, but 6 showed no agreement and another was inconclusive. Most substances included in this comparison are also carcinogenic in long-term assays on mice. No data on carcinogenicity in mice are available for 4-acetylaminofluorene and negative results were obtained with bromomethane and 2,6-diaminotoluene. Carcinogenic substances with positive results in the bone marrow micronucleus and transgenic gene mutation assays ----------------------------------------------------------------------------------------------------------------- ### Studies with Muta™mouse The following 17 substances were gene mutagenic in at least one of the examined organs in the Muta™mouse assay, induced micronuclei in mouse bone marrow and showed positive results in carcinogenicity studies on mice: 2-acetylaminofluorene, 4-aminobiphenyl, benzo(a)pyrene, 1,3-butadiene, chlorambucil, cyclophosphamide, 7,12-dimethylbenz(a)anthracene, ethylmethanesulfonate, N-ethyl-N-nitrosourea, methylmethane-sulfonate, N-methyl-N\'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, 4-nitroquinoline-1-oxide, N-nitrosodimethylamine, procarbazine, quinoline, and urethane. Further studies on chromosome aberration in vitroand in vivo(see [Additional file 1](#S1){ref-type="supplementary-material"}) supported the results in the micronucleus test, except data on 4-aminobiphenyl (negative micronucleus test in rats) and quinoline (see below). Of the substances tested for mutagenic activity in bone marrow in Muta™mice, 14 gave positive results, only 1,3-butadiene, methylmethanesulfonate, and quinoline were negative, indicating that other organs are more sensitive. 1,3-Butadiene is clearly clastogenic in other studies on chromosome aberration in mice and induced gene mutation in the bone marrow of Big Blue^®^mice (see [Additional file 1](#S1){ref-type="supplementary-material"}). Quinoline, however, gave inconclusive results in other in vivostudies on clastogenic effects in bone marrow of rats and mice. ### Studies with Big Blue^®^mouse 12 substances showed gene mutagenic effects in at least one of the examined organs in the Big Blue^®^mouse assay, chromosome mutagenic effects in the mouse bone marrow micronucleus assay and induced carcinogenic effects in long-term assays on mice,: 2-acetylaminofluorene, aflatoxin B1, benzene, benzo(a)pyrene, 1,3-butadiene, cyclophosphamide, 7,12-dimethylbenz(a)anthracene, ethylene oxide, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, N-nitrosodimethylamine, urethane. In the Big Blue^®^mouse assay the target organ bone marrow revealed also increased mutation frequencies induced by benzene, 1,3-butadiene, and 7,12-dimethylbenz(a)anthracene. However, negative results in bone marrow were obtained with cyclophosphamide, ethylene oxide, and N-nitrosodimethylamine, indicating that other target organs are more sensitive in the Big Blue^®^mouse assay. All of these 12 substances induced also chromosome aberrations in majority of further in vitroand in vivostudies. Substances with carcinogenic effects in mice but no agreement between the transgenic mouse assay and the mouse bone marrow assay -------------------------------------------------------------------------------------------------------------------------------- ### Acrylamide The inconclusive result in the bone marrow micronucleus test \[[@B4]\] is contradictory to the Muta™mouse assay \[[@B21]-[@B23]\] which shows mutagenic activity in the target organ bone marrow but also contradictory to other in vivotests on the endpoint chromosome aberration including a cytogenetic test on mice \[[@B4],[@B19],[@B21]\]. Micronuclei were detected in spleen and testis of mice \[[@B4]\]. Overall, using other experimental design the mouse bone marrow micronucleus assay might give clearly positive results. ### 2-Amino-3-methylimidazo(4,5-f)quinoline (IQ) IQ is mutagenic in the liver of the Muta™mouse \[[@B34]\] but negative results were obtained in the mouse bone marrow micronucleus test \[[@B2],[@B35]\]. This negative result is supported by a negative cytogenetic assay on mice. Inconclusive results were obtained in in vitrostudies on chromosome aberrations but IQ induced micronuclei in rats (see [Additional file 1](#S1){ref-type="supplementary-material"}). This discrepancy might be due to the possibility that a) the liver but not the bone marrow of mice is target organ (the liver but not the blood is target organ in carcinogenicity \[[@B33]\]) or b) IQ is less clastogenic than gene mutagenic in mice. ### ortho-Anisidine The target organ of carcinogenesis in mice (and humans) is the bladder \[[@B36]\]. In the Big Blue^®^assay on mice mutagenic activity was detected in the bladder but not in the liver \[[@B37]\]. As expected, the mouse bone marrow micronucleus test \[[@B4],[@B36]\] gave negative results (also in rats; see [Additional file 1](#S1){ref-type="supplementary-material"}), because the bone marrow is presumably not a target organ of mutagenicity. ### Asbestos crocidolite Local carcinogenic effects were observed in carcinogenicity studies, the lung is the target organ after inhalation \[[@B38]-[@B40]\]. Mutagenic activity was detected in the lung of Big Blue^®^mice after inhalation \[[@B41]\]. As expected, clearly no systemic effects in the bone marrow could be observed in the mouse micronucleus test \[[@B4]\]. ### 2,4-Diaminotoluene The main target organ in carcinogenicity is the liver (also in rats) \[[@B81]\]. Positive results were reported in two Big Blue^®^mouse assays examining the liver \[[@B82]-[@B84]\]. The mouse micronucleus test gave negative results \[[@B4]\], however, systemic effects in the bone marrow are not expected from carcinogenicity studies. Results of other studies on chromosome aberration in vivoare inconclusive (see [Additional file 1](#S1){ref-type="supplementary-material"}). ### Hydrazine This substance induced no mutagenic effects in lung, liver, or bone marrow of the Muta™mouse which were target organs in mouse carcinogenicity studies \[[@B119],[@B120]\]. The mouse bone marrow micronucleus test gave positive results after repeated application \[[@B4],[@B119]\]. However, single exposure was used in the Muta™mouse assay \[[@B120]\]. Studies on other in vivogenotoxicity endpoints have shown almost negative results after single exposure but genotoxic activity after repeated application, for example in the mouse bone marrow micronucleus assay \[[@B4]\]. Positive results might be expected in the Muta™mouse assay using another experimental design since other in vivoas well as in vitrotest systems revealed gene mutagenic effects. ### Methyl methanesulfonate Only weak mutagenic effects in the liver but no effects in bone marrow were observed in the Muta™mouse \[[@B18],[@B124]\] and negative results in the Big Blue^®^mouse \[[@B111]-[@B113],[@B126]\]. In the mouse bone marrow micronucleus test this carcinogenic substance induced chromosome aberration \[[@B1],[@B127]\]; other in vitroand in vivoassays clearly supported the clastogenic activity \[[@B121],[@B122]\]. There is evidence that the chromosome mutagenic activity is detectable at much lower doses than the gene mutagenic activity. Tinwell et al. (1998) \[[@B18]\] have shown on Muta™mice a weak gene mutagenic effect in the liver but no effect in the bone marrow. The same dose induced in these animals a significant increase in bone marrow micronuclei indicating clear clastogenic activity. Overall, methyl methanesulfonate is more clastogenic than gene mutagenic. ### Mitomycin C A very similar situation is given with mitomycin C. No mutagenic activity was observed in the Muta™mouse assay in liver and bone marrow after single injection but the same dose induced chromosome aberrations in the bone marrow of the same mice \[[@B142]\]. The mouse bone marrow micronucleus test \[[@B1],[@B47]\] and all in vivoand in vitroassays on the endpoint chromosome aberration revealed clearly positive results \[[@B139]-[@B141]\]. ### N-Nitrosodiethylamine The main target organ in systemic carcinogenesis is the liver \[[@B146]-[@B148]\]. As expected, mutagenic effects were detected in the liver of the Muta™mouse, but none in the bone marrow \[[@B104],[@B106],[@B149],[@B150]\]. The same mice showed also no micronuclei at these dose levels \[[@B104]\]. Consequently, the mouse bone marrow micronucleus test gave negative results indicating that this is not a target organ of genotoxicity. However, there is also evidence that this substance shows more gene mutagenic activity than clastogenic activity because other in vivostudies gave no clear indication for chromosome aberration (see [Additional file 1](#S1){ref-type="supplementary-material"}). ### N-Nitrosodi-N-propylamine There is evidence that this substance shows more gene than chromosome mutagenic activity. Beside local carcinogenic effects in carcinogenicity studies on mice and rats systemic effects were located in the liver (mouse and rats) and in bone marrow (rat) \[[@B160]-[@B163]\]. Several target organs were detected in the Muta™mouse assay including liver and bone marrow \[[@B164]\]. But no chromosome mutagenic activity was recorded in the mouse bone marrow micronucleus test \[[@B4]\]. Further in vivodata on this endpoint are not available (see [Additional file 1](#S1){ref-type="supplementary-material"}). ### Phenobarbital Liver tumours were detected in carcinogenicity studies on mice and rats \[[@B165]-[@B167]\]. In the Muta™mouse assay no mutagenic activity was observed although the liver weight of the treated mice increased indicating systemic effects in this organ \[[@B149],[@B168]\]. Two studies are available on the Big Blue^®^mouse, one gave negative \[[@B16]\] and the other weak positive results \[[@B169]\]. Taken together the results in transgenic mice are inconclusive which is in accord with the mouse bone marrow nucleus test (inconclusive results). Inconclusive (in vitro) or negative results were obtained in other studies on clastogenicity or other endpoints of genotoxicity (see [Additional file 1](#S1){ref-type="supplementary-material"}). Overall, there is no clear indication of gene or chromosome mutagenic activity in transgenic mouse assays or other genotoxic tests systems. However, genotoxic mechanisms in carcinogenicity cannot be excluded. ### β-Propiolactone This is an alkylating substance with predominantly local carcinogenic effects\[[@B175],[@B176]\]. In the Muta™mouse assay \[[@B131]\] local effects in the stomach were observed in gavage studies plus systemic effects in the liver, but no mutagenic activity was seen in bone marrow of the Muta™mouse \[[@B131]\]. As expected, no increased incidence in micronuclei was detected in the bone marrow of treated mice \[[@B1],[@B4]\] although clastogenic effects were observed in in vitrostudies, in insects and plants (see [Additional file 1](#S1){ref-type="supplementary-material"}) indicating chromosomal aberration after direct contact with this alkylating substance. ### Trichloroethylene This substance gave clearly positive results in different carcinogenicity studies on mice \[[@B186]\]. Although the target organs of carcinogenicity (including bone marrow) were investigated in the Muta™mouse assay, no mutagenic activity was noted \[[@B187]\]. The positive results in the mouse bone marrow micronucleus test are contradictory to other in vivostudies on clastogenicity. However, a further (simple) reason for the negative results in the Muta™mouse assay might be that the MTD was not reached \[[@B187]\]. Overall, further discussion on the mechanisms of carcinogenicity is necessary. ### Tris(2,3-dibromopropyl)phosphate This might be a further example for a substance where genotoxic effects are not related to the target organ bone marrow but induce systemic effects in others. The target organ in oral carcinogenicity studies on mice and rats was the kidney, in mice systemic carcinogenic effects were also seen in lung and liver \[[@B188]-[@B190]\]. In the Big Blue^®^mouse assay, mutagenic activity was detected in the kidney in gavage studies\[[@B46],[@B191]\]. The mouse bone marrow micronucleus assay (i.p. injection)\[[@B4]\] as well as cytogenetic studies on rats and mice were negative \[[@B188]-[@B190]\]. Substance without data on carcinogenicity in mice and differing results in the micronucleus test and transgenic mouse assay --------------------------------------------------------------------------------------------------------------------------- ### 4-Acetylaminofluorene This substance showed mutagenic activity in the Muta™mouse assay \[[@B18]\] but inconclusive results in the mouse bone marrow micronucleus test \[[@B2]\]. No data on carcinogenicity are available on 4-acetylaminofluorene. However, data on two in vitromammalian test systems indicated gene mutagenic activity \[[@B17]\] supporting results in the transgenic assay. Substances without carcinogenic effects in mice and differing results in the micronucleus test and transgenic mouse assay ------------------------------------------------------------------------------------------------------------------------- ### Bromomethane No increased tumour incidences were observed in mice as well as in re-evaluated studies on rats \[[@B58],[@B59]\]. Negative results were obtained also in the examined organs including liver and bone marrow in the Muta™mouse assay. However, DNA methylation in the liver was observed in the same assay even at lower dose levels \[[@B60]\] indicating differences in the sensitivity of these two genotoxic endpoints. The mouse micronucleus test revealed chromosome mutagenic activity \[[@B61]\] although results of other in vivotests are equivocal (see [Additional file 1](#S1){ref-type="supplementary-material"}). ### 2,6-Diaminotoluene No carcinogenic effects were seen in a valid long-term study on mice and rats \[[@B85]\]. In the Big Blue^®^mouse no mutagenic activity was induced \[[@B82],[@B84]\]. However, only the liver was examined and the assays had some limitations (one dose tested, MTD possibly not reached). The mouse micronucleus test revealed chromosome aberrations \[[@B3],[@B47]\] as well as the available in vitrostudies but no clastogenic activity was detected in a cytogenetic study on rodents (see [Additional file 1](#S1){ref-type="supplementary-material"}). Substances with carcinogenic effects in mice but nongenotoxic mechanisms are presumed ------------------------------------------------------------------------------------- ### Chloroform (German MAK Classification 4 = substances with carcinogen effects where the genotoxic effects are absent or only play an insignificant role) Liver tumours were induced in B6C3F1 mice \[[@B72]\], however no mutagenic effects were detected in the liver of Big Blue^®^mice of the same strain in a valid assay \[[@B74]\]. Accordingly, no increased incidence in micronuclei were observed in the bone marrow of mice \[[@B1],[@B47]\] and no clastogenicity in in vitrostudies (see [Additional file 1](#S1){ref-type="supplementary-material"}). In contrast, rats showed clastogenic activity in the kidney, but only at toxic dose levels \[[@B73]\]. ### Di-(2-ethylhexyl)phthalate (German MAK Classification 4) This substance induced liver tumours in mice and rats \[[@B86]\]. No increased mutation frequency was seen in the liver of the Big Blue^®^mouse \[[@B16]\] (limited validity, MTD presumably not reached) and no chromosome aberration in the mouse micronucleus test \[[@B4]\]. The majority of in vitroand in vivotests revealed also negative results with di-(2-ethylhexyl)phthalate (see [Additional file 1](#S1){ref-type="supplementary-material"}). ### Tetrachloromethane (German MAK Classification 4) An increased incidence of liver tumours were induced in mice and in rats \[[@B185]\]. However, no mutagenic activity in the liver was reported in a Muta™mouse assay although the organ weight increased in the same mice indicating some hepatocellular regeneration \[[@B168]\]. No chromosome aberration was induced in the mouse bone marrow micronucleus test \[[@B4]\]. Also other studies on this endpoint and the majority studies on other endpoint of genotoxicity revealed negative results (see [Additional file 1](#S1){ref-type="supplementary-material"}). Discussion on the comparison of both assays ------------------------------------------- Most carcinogens in [Additional file 1](#S1){ref-type="supplementary-material"} are positive in transgenic mouse assays [and]{.underline} in the mouse bone marrow micronucleus test although different endpoints are studied. This indicates coincidence in both test systems and/or effects of the test substance on different genotoxic endpoints. However, there are several substances (see above for example ortho-anisidine) whose mutagenic (and carcinogenic) potential could not be demonstrated with the mouse bone marrow micronucleus test but with the transgenic mouse assay. This might be due to the fact that the micronucleus test is restricted to the bone marrow as target organ. In contrast, all organs can be examined in transgenic mouse assay for mutagenic activity without any restrictions. A special subgroup of substances should also be mentioned: substances with predominantly local mutagenic/carcinogenic effects and less systemic direction of effects. For these substances the mouse bone marrow micronucleus test is an unsuitable test system. Beside the restrictions on the target organ there is also given the possibility that a substance induces predominantly gene mutations and not or less chromosome aberrations at the same dose level. The example N-nitrosodi-N-propylamine has shown positive results in different target organs including the bone marrow using the transgenic mouse assay but negative results were shown in the mouse micronucleus test. On the other hand there are also substances for which the transgenic mouse assay is an unsuitable test system. The examples methylmethanesulfonate and mitomycin C have shown that chromosome aberration and not gene mutation is the predominant endpoint at the corresponding dose level in vivo. These genotoxic effects are not easily detectable with the transgenic mouse assay which is restricted to the detection of small deletion and insertions in the DNA. However, the negative Muta™mouse assay on mitomycin C has some limitations in the validity of the test system. With increased dose levels (MTD reached) and/or repeated application also gene mutagenic effects might be detected in the transgenic mouse assay. Interestingly, substances with carcinogenic effects induced by nongenotoxic mechanisms gave mainly correct negative results in both test systems although the protocols for the transgenic mouse assays were not optimised except for chloroform. Predictability of the transgenic animal assays and the mouse bone marrow micronucleus test for carcinogenicity -------------------------------------------------------------------------------------------------------------- The sensitivity, specificity and predictive values to cancer for the Muta™mouse assay and the Big Blue^®^mouse assay combined, and the mouse bone marrow micronucleus test are documented in Table [1](#T1){ref-type="table"}. In the present study data on 38 substances were available concerning carcinogenicity in mice [and]{.underline} mutagenic effects in transgenic mice as well as mutagenic effects in the mouse bone marrow micronucleus test ([Additional file 1](#S1){ref-type="supplementary-material"}). The two substances with inconclusive results in the mouse bone marrow micronucleus assay (phenobarbital & acrylamide) were not included in the final calculation data and in the comparison of the micronucleus test versus the transgenic mouse assay. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of the Muta™mouse assay and the Big Blue^®^mouse assay for predicting mouse carcinogenicity in comparison with the micronucleus test ::: Term\# Calculation for the mouse bone marrow micronucleus test Calculation for Muta™mouse and/or Big Blue^®^mouse combined \* ------------------------- --------------------------------------------------------- ---------------------------------------------------------------- Sensitivity 68% (23/34) 82% (28/34) Specificity 0% (0/2) 100% (2/2) Positive predictability 92% (23/25) 100% (28/28) Negative predictability 0% (0/11) 25% (2/8) Overall accuracy 64% (23/36) 83% (30/36) \#Sensitivity = % of carcinogens with a positive result in the specified test system (STS) Specificity = % of noncarcinogens with a negative result in STS Positive predictability = % of positive results in the STS that are carcinogen Negative predictability = % of negative results in the STS that are noncarcinogens Overall accuracy = % of chemicals tested where STS results agree with the carcinogenicity results Carcinogens with genetoxic and nongenotoxic mechanisms were considered but not noncarcinogenic substances; only data on mice were used Weak positive results in transgenic mouse assays judged as positive. \: judged as positive in transgenic assays if positive in one of the two test systems ::: Although the data pool in this document is not sufficient for a comprehensive comparison (low number of examples, especially for specificity and negative predictability; limitations of most transgenic mouse assays with negative results) some differences were apparent between the two test systems. The overall accuracy of the micronucleus test is lower than that of the transgenic mouse assays. This is mainly due to 11 negative results in the micronucleus test system (negative in the micronucleus test but positive in carcinogenicity studies) influencing the terms sensitivity and negative predictability. Three of these negative results in the micronucleus test are obtained with carcinogenic substances \[chloroform, di-(2-ethylhexyl)phthalate, and tetrachloromethane\] for which carcinogenic effects are considered to be of a nongenotoxic mechanism. However, chloroform, di-(2-ethylhexyl)phthalate and tetrachloromethane gave negative results in transgenic mice, so the comparison of both test system is not essentially affected and the evaluation „nongenotoxic" supported. For the other 8 substances out of these 11 with false negative results in the micronucleus test these results are explainable (see above): Ortho-anisidine (mutagenic/carcinogenic effects are restricted to the bladder), IQ (bone marrow presumably not target organ of genotoxicity in mice and more gene mutagenic than clastogenic), asbestos (local genotoxic/carcinogenic effects in the lung), 2,4-diaminotoluene (target organ liver, presumably not bone marrow), N-nitrosodiethylamine (target organ liver, more gene mutagenic than clastogenic), N-nitroso-N-propylamine (presumably more gene mutagenic than clastogenic), beta-propiolactone (mainly local effects and less systemic effects \[in bone marrow\]), tris(2,3-dibromopropyl)phosphate (systemic effects not related to the bone marrow). The term negative predictability is also low in the transgenic mouse assay due to false negative results on six carcinogenic substances; for three of them, hydrazine, mitomycin C, and tetrachloroethylene (detailed treatise in section „direct comparison", see above) genotoxic mechanisms are presumed. For hydrazine (no repeated application) and tetrachloroethylene (MTD not reached) limitations on the experimentel design might be the reason for the negative results. Mitomycin C is clearly more clastogenic than gene mutagenic, and the transgenic mouse with lacI and lacZ is possibly an unsuitable test system. For 3 out of the 6 substances the carcinogenic effects in mice were attributed to nongenotoxic mechanisms: chloroform, di-(2-ethylhexyl)phthalate and tetrachloromethane (see also above), all gave negative results in transgenic mice. Only two substances with negative results in long-term carcinogenicity studies are available in the data pool: bromomethane and 2,6-diaminotoluene. Both gave correct negative results in the transgenic mouse assay (although of limited validity) but false positive results in the micronucleus test (see term specificity). Generally, the differences between the two test systems might be due to the fact that 1) unequal genotoxic endpoints are investigated (chromosome aberration in the micronucleus test versus gene mutation in the transgenic mouse assay), 2) organotrophy of genotoxic effects (especially bone marrow not target organ) might play an essential role and 3) transgenic rodent assay conditions in the different systems may not be optimal for mutation detection. Advantages and disadvantages of both test systems ------------------------------------------------- A comparison of the transgenic mouse assays with the mouse bone marrow micronucleus test is limited by the fact that different genotoxic endpoints are studied in these two systems. In transgenic mouse assays, point mutations and small insertions and deletions are detected whereas in the mouse bone marrow assay, chromosome breakage leading to light microscopically visible micronuclei resulting from chromosome fragment or micronuclei originated from whole chromosomes are investigated. ### Sensitivity of the test system In comparison to other test systems in genotoxicity testing using endogenous target structures the spontaneous mutant frequency in the transgenic mouse assay is relatively high. This might be related to the fact that bacterial DNA is the target gene (high methylation rate) or the transgene is silent and no transcription related repair occurs like in endogenous genes which are more efficiently repaired \[[@B9]\]. In the mouse bone marrow micronucleus test the spontaneous rate of micronuclei is low ranging between 1--3 PCEs with micronuclei per 1000 PCEs. However, frequency of chromosome aberrations is not directly comparable with a gene mutantion frequency. Comparing the target organs and cells at risk at the time of exposure, the mouse micronucleus test is restricted to one target organ, the bone marrow, especially to the erythroblasts. This limitation is not given in transgenic mouse assays: target cells are cells in all organs \[[@B195]\]. ### Considerations of animal welfare Both test systems are similar in the number of animals used for a valid test. The minimal number of mice needed in the mouse bone marrow assay is 25 per gender (3 dose levels, vehicle control, positive control; 5 mice per group) using a treatment schedule with 2 or more applications at 24 h intervals and sampling 18--24 h following the final treatment. In the limit test (for a test substance of low toxicity) only one dose level of 2000 mg/kg bw is necessary (OECD guideline 474, \[[@B6]\]). In transgenic mutation assays ca. 20 animals (3 dose groups and 1 concurrent vehicle control group in laboratories which already established this test system) are recommended per species and gender \[[@B196],[@B197]\]. In terms of animal welfare, it is also desired to merge more than one in vivo genotoxicity assay such as transgenic mouse assay and micronucleus assay using the same animals for both assays. ### Cost effectiveness Due to the simplicity of the mouse bone marrow micronucleus assay and the use of systems for automated analysis, this test is less expensive than the transgenic mouse assay. A comparison both test systems is presented in Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Comparison of the mouse bone micronucleus assay with transgenic mouse models (Muta™mouse and the Big Blue^®^assay) ::: Mouse bone marrow micronucleus test\[1,2\] Transgenic mouse mutation assay\[9,195\] --------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------- Type of endpoint* Detects light microscopically visible micronuclei resulting from whole chromosomes or chromosome fragments following chromosome breakage Detects 1) gene mutation, 2) small deletions or insertions Regulatory use Widespread acceptance (OECD guideline established since 1983) Not widely used by the industry in toxicological screening; OECD guideline in preparation Background mutation rate Spontaneous incidence of micronuclei is low (ca. 0.3%) and almost uniform High spontaneous rate of mutations comparing with other mutation assays Negative predictivity low negative predictivity for cancer Low negative predictivity for cancer Implementation Simplicity of the test system; easily recognised end-point Higher Complexity of the test system (target cells in mice and expression of mutagenic effects in bacteria; vector system needed) Toxicokinetics and metabolism Restrictions in toxicokinetics: test substance or the toxic metabolites may not reach the bone marrow but other target organs No restrictions after absorption and distribution of the test substance Target tissue Restricted to erythroblasts in the bone marrow No tissue restriction; analysis of mutagenic potency in different organs; measurement of organotrophic effects Dependency of effects on application route Only systemic effects can be detected Systemic as well as local mutagenic effects can be detected Number of animals 5 animals per gender per dose recommended 5 animals per gender per dose recommended Restrictions on the used model Also some recommendations are given in OECD guideline 474, no limitation concerning species, strain, gender, age of animals, exposure duration Limitations: Muta™mouse assay only 1 species & 1 strain; Big Blue^®^2 species (mouse and rat) but 1 (rat) or 2 strains (mouse); no limitations on other parameters Costs Less expensive due to the simplicity of the test system More expensive test system Molecular mechanism Mechanisms of the induction of micronuclei originating from chromosome fragments could not be resolved Detection of the \"molecular signature\" of a particular mutagenic substance by DNA sequence analysis with standardised methods Parallel examination of different genetic endpoints Combination with other genotoxic endpoints is not recommended but possible if results of the micronucleus test are not influenced and vice versa The transgenic mouse assay can be combined with other in vivogenotoxic endpoints in the same animal: micronuclei, chromosomal aberration, UDS, SCE Type of mutational target In situ end point Target genes are integrated parts of foreign DNA and consequently no \"normal\" mutational target, no expression UDS: unscheduled DNA synthesis; SCE: sister chromatid exchange. ::: Conclusions =========== In a comparison of the tests available to genetic toxicologists, the results from studies on substances which had been tested in transgenic mutagenicity assays Big Blue^®^mouse and the Muta™mouse were compared with those from the more traditional mouse bone marrow micronucleus test. The transgenic animal mutation assay, which is not yet used in toxicological screening, is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation. However, from the 39 substances, the majority gave the same positive or negative result in both test systems. The substances where differences occurred were discussed in more detail. The advantages and disadvantages of the transgenic Big Blue^®^mouse and the Muta™Mouse transgenic model compared to the micronucleus test were discussed and both systems were found to have a place in mutagenicity testing and to supplement each other. The transgenic animal assay has, however, some distinct advantages over the micronucleus test in that it is not restricted to one target organ and detects systemic as well as local mutagenic effects. Authors\' contributions ======================= UW was the main author. The other authors were involved in the discussions, writing small parts of text and in final preparation of the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Table: Results in the transgenic mouse assay versus mouse bone marrow micronucleus test ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This paper is based on work performed by the authors in preparation of an Environmental Health Criteria document on Transgenic Animals in Mutagenicity Testing for the International Programme on Chemical Safety (IPCS). However, opinions expressed in this paper are the sole responsibility of the authors. We acknowledge the financial support of the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety.	PubMed Central	2024-06-05T03:55:52.461993	2005-1-17	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548135/", "journal": "J Carcinog. 2005 Jan 17; 4:3", "authors": [ { "first": "U", "last": "Wahnschaffe" }, { "first": "A", "last": "Bitsch" }, { "first": "J", "last": "Kielhorn" }, { "first": "I", "last": "Mangelsdorf" } ] }
PMC548136	Background ========== Lepidium meyenii, a traditional Peruvian cruciferous vegetable known as Maca, grows exclusively at altitudes over 4000 m. The hypocotyl, the edible part of the plant, is used as a nutritional supplement and for its enhancing sperm production properties \[[@B1]\]. Maca is presented in different ecotypes according to the colors of its hypocotyls, ranging from white to black \[[@B2]\]. Epidemiological studies have found that consumption of cruciferous vegetables is associated with a reduced risk of prostate cancer \[[@B3]-[@B5]\]. Cruciferous (Brassica) vegetables are broccoli, cabbage, mustard and collard greens, bok choy \[[@B3]\], and members of the genus Lepidium\[[@B1],[@B6]\], including Maca. In the many hundreds of cruciferous species investigated, all are able to synthesize glucosinolates \[[@B7]\]. Glucosinolate content in cruciferous vegetables is highly variable, depending of plant age and environmental factors which can cause the broad range of values reported to vegetables of the same variety \[[@B8]\]. This variability may explain differences in epidemiological studies related to protection of intake of cruciferous vegetables against prostate cancer \[[@B3]-[@B5],[@B9]-[@B11]\]. Upon ingestion by humans \[[@B12]\] or rats \[[@B13]\], glucosinolates are converted in isothiocyanates by gut microflora. As alternate mechanism, glucosinolates are catalyzed by dietary myrosinase \[[@B13]\]. Almost all of the mammalian chemoprotective activity from cruciferous is due to these isothiocyanates \[[@B7],[@B14]\]. Therefore, glucosinolates after conversion to isothiocyanates have both antiproliferative and proapoptotic properties in prostate cells \[[@B14]-[@B18]\]. The isothiocyanates formed from aromatic glucosinolates decompose spontaneously to indole-3-carbinol (I3C) \[[@B7]\]. I3C and metabolites induce apoptosis in human prostate cancer cells \[[@B16]-[@B18]\]. The absolute content of glucosinolates in fresh Maca hypocotyls is relatively higher than reported in other cruciferous crops \[[@B19]\]. The most abundant glucosinolates detected in Maca were the aromatic glucosinolates: benzyl glucosinolate (glucotropaeolin) \[[@B19]-[@B21]\]; as a result, it is possible that Lepidium meyenii(Maca) may have important effects on prostate. More recently, it has been demonstrated that an integral suspension of Lepidium latifoliumsignificantly reduced prostate size and volume in castrated rats where the hyperplasia was induced by steroid treatment \[[@B6]\]. For such reason, the present study has been designed to determine the effect of three ecotypes of Maca on ventral prostate of rats. Methods ======= Animals ------- Adult Holtzman rats were obtained from our Animal House at the Universidad Peruana Cayetano Heredia and used for the present study. The rats were maintained 4--6 per cage at environmental temperature (22°C) with a 12:12 h light/dark cycle. Also they were fed with Purina laboratory chow and tap water ad libitum. All animal experiments were conducted in compliance with \"Guide for the care and use of laboratory animals\" of the National Institutes of Health from the USA \[[@B22]\]. The Institutional Review Board of the Scientific Research Office from the Universidad Peruana Cayetano Heredia approved the study. Experimental protocol --------------------- ### Experiment 1: Effect of different ecotypes of Maca on prostate size in rats Rats were treated with vehicle, Yellow Maca, Red Maca or Black Maca for 7 days in dose of 2 g dried Maca hypocotyls/Kg BW. This dose was selected from a previous dose response study \[[@B23]\]. Each Maca treated group included 12 animals, and Control (vehicle) sample size included 35 animals. Maca or vehicle was orally administered using an intubation needle No 18 (Fisher Scientific, Pittsburgh, Pennsylvania). ### Experiment 2: Effect of Red Maca on prostate size and histology in rats treated with testosterone enanthate (TE) Rats were injected (i.m) with 0.1 ml (25 mg) of testosterone enanthate (TE) on day 1 and day 7. Control rats received 0.1 ml oil (im) at day 1 and at day 7. A group treated with TE received also Red Maca (2 g/Kg) for 14 days and another group treated with TE received Red Maca for 42 days. Control rats received vehicle by oral route for 14 or 42 days. Oral treatment (Maca or vehicle) and intramuscular treatment (TE or vehicle) both started on day 1. Preparation of aqueous extract of Lepidium meyenii (Maca) --------------------------------------------------------- The dried hypocotyls of Lepidium meyeniiwere obtained from Carhuamayo, Junin at 4000 m altitude. The ages of different Maca plants were similar. All hypocotyls were obtained at the same time. Irma Fernandez, a Botanist of the Department of Pharmaceutical Sciences at Universidad Peruana Cayetano Heredia, authenticated the identity of the plant by visual inspection. The biological activity of the plant is located in the hypocotyls, which are consumed by natives after natural drying. Traditionally, the dried hypocotyls of Maca are boiled and served as juice. For the present study, the aqueous extract of the hypocotyls was prepared according to the traditional method. First, 500 g of the dried hypocotyls were pulverized and placed in a container with 1500 ml of water, and boiled for 120 minutes. Next, the preparation was left standing to cool, and then it was filtered. Finally, the filtrate containing 333 mg of dry Maca hypocotyls in 1 ml was placed in small vials and kept in 4°C refrigerator. Sacrifice, Blood and sample of tissue ------------------------------------- One day after the last treatment, rats were sacrificed by decapitation. Blood sample was obtained from cervical trunk from rats treated for 7 days with different ecotypes of Maca. Blood samples were centrifuged at 1,000 g, and sera were separated, placed in vials and kept frozen until assayed for sex hormone levels. Ventral prostate was used for histological study. Organ Weights ------------- After animals were sacrificed several organs (testes, epididymis, ventral prostate, seminal vesicles, kidneys, liver, spleen, heart and lungs) were collected, dissected free of fat, and weighed. Measurement of serum estradiol and testosterone ----------------------------------------------- Serum estradiol and testosterone concentrations were measured by radioimmunoassay using commercial kits (Diagnostic Products Co, Los Angeles, USA). The hormone labeled with iodine-125 was used as radioactive marker. Samples were run in the same assay to avoid inter-assay variation. The intra-assay variation was 6.42% for estradiol, and 5.5% for testosterone. Sensitivity of testosterone assay was 4 ng/dl and for estradiol assay was 8 pg/ml. Histological study ------------------ Ventral prostate lobes obtained from experiment 2 were excised and dissected free of fat. Ventral prostates (VP) were immersion-fixed in Bouin\'s fixative. After their dehydration, VP were embedded in paraffin. The tissue blocks were sectioned into 5 um thickness and stained with hematoxylin and eosine (H&E), and then observed under a light microscope. Epithelial height (um) and duct lumen area (um^2^) were measured by sampling 10 random sections per slide in the peripheral region of the ventral prostate. In each duct, 20 cells were measured for epithelial height. All assessments were performed using an axiostar plus microscope (Carl Zeizz, Thornwood, New York, USA). The images were captured by a Moticam2000 (Richmond, B.C, Canada) coupled to a personal computer. Motic image plus 2.0 software (Motic Instruments Inc.) was used for measurements of prostatic epithelial height and duct luminal area and calculated by statistic ANOVA test. Pictures at 50× and 400× magnifications are included. Phytochemistry of Red Maca -------------------------- The phytochemical screening in the aqueous extract of Red Maca was performed using standard phytochemical procedures \[[@B24],[@B25]\]. Maca aqueous extract was lyophilized previously to extraction procedures. After extraction in methanol or ethanol, the presence of alkaloids (Dragendorff reagent; Mayer\'s test), flavonoids (Shinoda test), steroids (Liebermann-Burchard/Thin Layer Chromatography test), anthraquinones (Bornträger reaction), tannins (Gelatin/Ferric chloride test), saponins (Froth test), sesquiterpene lactones (Vainillin test and Ferric hydroxamate test), coumarins (Vainillin test and Ferric hydroxamate test), cardiotonic glycosides (Raymond reagent), and cardenolids (Kedde reagent) were assessed \[[@B24],[@B25]\]. Measurement of infrared (IR) spectra ------------------------------------ IR spectra of lyophilized aqueous extracts of Red, Yellow and Black Maca were measured from 3800 cm^-1^to 650 cm^-1^with an FT-IR spectrophotometer (SPECTRUM2000, Perkin Elmer Ltd., Beaconsfield, England). An overhead-attenuated total refraction (ATR) accessory was equipped as the sample stage for solid samples. All spectral measurements were done at 1 cm^-1^resolutions. Data are presented as absorbance units. Each peak represents the presence of a functional chemical group. Differences in the height of absorbance peaks reflect differences in amount of functional groups. Statistical analysis -------------------- Data were analyzed using the statistical package STATA (version 8.0) for personal computer (Stata Corporation, 702 University Drive East, College Station, TX, USA). Data are presented as mean ± standard error of the mean (SEM). Homogeneity of variances was assessed by the Bartlett test. If variances were homogeneous, differences between groups were assessed by analysis of variance (ANOVA). If F value in the ANOVA test was significant, the differences between pair of means were assessed by the Scheffeé test. If variances were non homogeneous, non parametric tests were used. A value of P\< 0.05 was considered statistically significant. Results ======= Red Maca but not Black or Yellow Maca reduced ventral prostate weight in rats ----------------------------------------------------------------------------- Ventral prostate weight was significantly reduced in rats treated for 7 days with Red Maca (P \< 0.05). Black Maca and Yellow Maca did not modify ventral prostate weight (Figure [1a](#F1){ref-type="fig"}). Seminal vesicles weights were not modified by treatment with any ecotype of Maca (Figure [1b](#F1){ref-type="fig"}). Body weight was similar in the control group (415.8 ± 3.3 g, mean ± SEM) and in rats treated with Red (407.5 ± 7.1 g), Yellow (421.5 ± 6.1 g) or Black Maca (426.5 ± 6.8 g). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Ventral prostate (A) and seminal vesicles (B) weights in adult rats treated for 7 days with different ecotypes of Maca (2 g/kg BW). Data are Mean ± SEM \P \< 0.05 with respect to control value. Number of animals was 35 for controls, 12 for Yellow Maca, 12 for Red Maca and 12 for Black Maca. None: control group receiving vehicle. ::: ![](1477-7827-3-5-1) ::: Maca and serum sexual hormone levels ------------------------------------ Means of serum testosterone and estradiol levels were similar between control group and groups treated for 7 days (Figure [2a,b](#F2){ref-type="fig"}) with Red, Yellow or Black Maca. The group treated with Yellow Maca showed higher serum testosterone levels than the group treated with Black Maca (P \< 0.05). Higher serum testosterone levels in the group treated with Yellow Maca were due to two rats with high serum testosterone levels. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effects of different Maca ecotypes administered for 7 days on serum testosterone (A) or estradiol (B) levels in rats. Data are mean ± SEM. Maca (2 gr/Kg BW) was administered for 7 days. Number of rats was 10 in the control group, 6 in the Red Maca treated group, 6 in the Yellow Maca, and 6 in the Black Maca treated group. P:NS between groups treated with Maca and control rats. ^a^P \< 0.05 with respect to the Yellow Maca treated group. ::: ![](1477-7827-3-5-2) ::: Red Maca reduced ventral prostate weight in rats treated with testosterone enanthate (TE) ----------------------------------------------------------------------------------------- Treatment with TE increased significantly ventral prostate weight almost to double value of the control group (P \< 0.05) (Figures [3a](#F3){ref-type="fig"} and [4a](#F4){ref-type="fig"}). The increase in ventral prostate weight was maintained in high levels up to 5 weeks after last TE injection. Seminal vesicles weight increased 2.5 times that control values (P \< 0.05) (Figures [3b](#F3){ref-type="fig"} and [4b](#F4){ref-type="fig"}). Treatment with only Red Maca for 14 days (P \< 0.05) (Figure [3a](#F3){ref-type="fig"}) or 42 days (P \< 0.05) (Figure [4a](#F4){ref-type="fig"}) resulted in low ventral prostate weight compared with control values. Seminal vesicles values were not affected after treatment with Red Maca for 14 or 42 days (Figures [3b](#F3){ref-type="fig"} and [4b](#F4){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Ventral prostate (A) and seminal vesicles (B) weights in adult rats treated for 14 days with Red Maca. Data are mean ± SEM.TE: rats treated on day 1 and 7 with testosterone enanthate (25 mg each) i.m, Red Maca (2 g/Kg BW) was given orally during 14 days. Rats were sacrificed on day 15. \ P \< 0.05 with respect to vehicle group; ^a^P \< 0.05 with respect to TE group. ^b^P \< 0.05 with respect to the group treated with TE+Red Maca. Number of rats was 13 for the control group, 6 for the Red Maca, 6 for TE, and 6 for the TE plus Red Maca groups. ::: ![](1477-7827-3-5-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Ventral prostate (A) and seminal vesicles (B) weights in adult rats treated for 42 days with Red Maca. Data are mean ± SEM.TE: rats treated on day 1 and 7 with testosterone enanthate (25 mg each) i.m. Red Maca (2 g/Kg BW) was given orally during 42 days. Rats were sacrificed on day 43. \P \< 0.05 with respect to vehicle group; ^a^P \< 0.05 with respect to TE group. ^b^P \< 0.05 with respect to the group treated with TE+Red Maca. Number of animals was 12 in the control group, 7 in the Red Maca group, 5 in the TE group and 5 in the TE plus Red Maca group. ::: ![](1477-7827-3-5-4) ::: Red Maca administered for 14 days also reduced ventral prostate weight in rats treated with TE (P \< 0.05). The effect was more noticeable after 42 days of treatment with Red Maca (P \< 0.05). After 42 days of treatment with Red Maca, the ventral prostate weight was reduced more than 50% (Figure [4a](#F4){ref-type="fig"}). After 14 or 42 days of treatment with Red Maca, the increased seminal vesicles weights induced by TE was not affected (Figures [3b](#F3){ref-type="fig"} and [4b](#F4){ref-type="fig"}). Ventral prostate weight related to body weight was lower in the group treated for 14 days with Red Maca (0.11 ± 0.01 g/100 g BW) than in controls (0.15 ± 0.006 g/100 g BW) (P \< 0.05). Rats treated with TE plus Red Maca had lower prostate weight related to BW (0.23 ± 0.02 g/100 g BW, P \< 0.05) than rats treated with TE (0.29 ± 0.01) but prostate weight relative to BW was still higher in rats treated with TE plus Red Maca than in controls (P \< 0.05). The same pattern was observed when rats were treated for 42 days with Red Maca (Data not shown). Rats treated with two TE injections showed lower body weight at day 42 after the first injection (P \< 0.05) compared to controls (290.63 ± 30.48 g and 383.60 ± 11.31 g in TE and vehicle treated groups). This low body weight was also observed in the group treated with TE plus Red Maca (319.30 ± 3.08 g) (P \< 0.05). Weights of testes, epididymis, kidneys, liver, spleen, lungs and heart were not affected by treatment with Red Maca for 7, 14 or 42 days (Data not shown). Histological study ------------------ Sections of ventral prostate in the peripheral region of the ductal system after 14 and 42 days of treatment with vehicle (control), Red Maca, ET, or ET plus Red Maca are shown in Figures [5](#F5){ref-type="fig"} and [6](#F6){ref-type="fig"}. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### The effects of Red Maca administered to rats for 14 days on ventral prostatic epithelial height and duct luminal area. A,B: Control group; C,D: Red Maca treated; E,F: TE treated; G,H: TE+Red Maca treated. HE stain. Left: ×50 magnification; Right: ×400 magnification. ::: ![](1477-7827-3-5-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### The effects of Red Maca administered to rats for 42 days on ventral prostatic epithelial height and duct luminal area. A,B: Control group; C,D: Red Maca treated; E,F: TE treated; G,H: TE+Red Maca treated. HE stain. Left: ×50 magnification; Right: ×400 magnification. ::: ![](1477-7827-3-5-6) ::: At 50× magnification, the number of ducts per field from rats treated with Red Maca for 14 days was slightly higher than in controls (Figures [5a](#F5){ref-type="fig"} and [5c](#F5){ref-type="fig"}). TE reduced the number of ducts per field as a consequence of increase in duct area (Figure [5e](#F5){ref-type="fig"}). Red Maca increased the number of ducts per field in rats treated with TE (Figure [5g](#F5){ref-type="fig"}). At 400× magnification, in Red Maca treated rats (alone or with TE), cell size was decreased and membrane blebbing and nuclear distortion are apparent (Figures [5d](#F5){ref-type="fig"} and [5h](#F5){ref-type="fig"} vs Figures [5b](#F5){ref-type="fig"} and [5F](#F5){ref-type="fig"}). At 50× magnification, compared to control (Figure [6a](#F6){ref-type="fig"}), the treatment with Red Maca for 42 days resulted in high number of ducts per field by Maca effect reducing the lumen area (Figure [6c](#F6){ref-type="fig"}). Treatment with TE resulted in lower number of ducts per field as a result of testosterone induced high luminal area (Figure [6e](#F6){ref-type="fig"}). Treatment with TE plus Red Maca (Figure [6g](#F6){ref-type="fig"}) compared to TE (Figure [6e](#F6){ref-type="fig"}) showed an increase in the number of ducts per field. At higher magnification (400×), it was observed secretory luminal cells lined with a single layer of columnar epithelium in the control group (Figure [6b](#F6){ref-type="fig"}). In specimens treated with only Red Maca, the epithelium of the ventral prostate showed a change from columnar to cuboidal shape (Figure [6d](#F6){ref-type="fig"}). TE caused an increase in proliferation of epithelial cells (Figure [6f](#F6){ref-type="fig"}). Red Maca reduced the epithelium in rats treated with TE (Figure [6h](#F6){ref-type="fig"}). Membrane blebbing and nuclear distortion are apparent in rats treated with Red Maca (Figures [6d](#F6){ref-type="fig"} and [6h](#F6){ref-type="fig"}). Quantitative analyses of epithelial height and luminal area in rats treated for 42 days are presented in Figure [7](#F7){ref-type="fig"}. Rats treated for 42 days with only Red Maca showed lower prostatic epithelial height (P \< 0.05) and duct luminal area (P \< 0.05) than control, TE and TE+Red Maca groups (Figure [7a--b](#F7){ref-type="fig"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Ventral prostatic epithelial height (A) and luminal area (B) in control rats, rats treated with Red Maca (RM) alone, testosterone enanthate (TE) alone or TE+Red Maca. Rats were treated for 42 days. \P \< 0.05 with respect to control, ^a^P \< 0.05 respect to TE group; ^b^P \< 0.05 respect to TE+ Red Maca. Differences in duct luminal areas were assessed with Mann-Whitney U test. ::: ![](1477-7827-3-5-7) ::: Rats treated during 2 weeks with injections of TE once a week showed higher prostatic epithelial height (P \< 0.05) and duct luminal area (P \< 0.05) at day 42 compared to controls (Figure [7a--b](#F7){ref-type="fig"}). Rats treated with TE plus Red Maca for 42 days showed that prostatic epithelial height and luminal area were similar to those observed in the control group (Figures [7a--b](#F7){ref-type="fig"}). Phytochemistry of Red Maca -------------------------- The phytochemical analysis of the ethanolic extract prepared from lyophilized aqueous extract of Red Maca hypocotyls revealed the presence of alkaloids, steroids, saponins and cardiotonic glycosides, and the absence of flavonoids, anthraquinones, tannins, sesquiterpene lactones and coumarins (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Result of phytochemical screening of extracts of Red Maca. ::: Tests Ethanolic extract Methanolic extract ------------------------------ ----------------------- ------------------------ Alkaloids Dragendorff test \+ \+ Mayer\'s test \+ \+ Flavonoids Shinoda test \- \- Steroids Liebermann-Burchard test \+ \+ Anthraquinones Bornträger test \- \- Tannins Gelatin/Ferric Chloride test \- \+ Saponins Froth test \+ \+ Sesquiterpene Lactones Ferric hydroxamate test \- \- Vainillin test \- \- Coumarins Ferric hydroxamate test \- \- Vainillin test \- \- Cardiotonic glycosides Raymond test \+ NA Cardenolids Kedde test NA \- -, test negative; +, test positive; NA, not available ::: The phytochemical analysis of the methanolic extract prepared from lyophilized aqueous extract of Red Maca hypocotyls revealed the presence of alkaloids, steroids, tannins and saponins, and the absence of flavonoids, coumarins, anthraquinones, sesquiterpene lactones and cardenolides (Table [1](#T1){ref-type="table"}). The positive tests were more intense for alkaloids than for the other compounds. The IR spectra of the three Maca\'s ecotypes extracts are shown in Fig. [8](#F8){ref-type="fig"}. The IR spectra of the three ecotypes of Maca in 3800-650 cm^-1^region had 7 peaks, which were at 3291 cm^-1^, 2927 cm^-1^, 1614 cm^-1^, 1406 cm^-1^, 1022 cm^-1^, 924 cm^-1^and 862 cm^-1^. These peaks are due to C-H, OH, amides, amines, carboxylic acids, aromatic, and alkyls groups, respectively. Highest peak values were observed for Red Maca, intermediate values for Yellow Maca and low values for Black Maca. These functional groups correspond among others to benzyl glucosinolate (Figure [9](#F9){ref-type="fig"}). ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Infrared (IR) spectra of lyophilized aqueous extract of three ecotypes of Lepidium meyenii(Maca). Data are expressed in absorbance units (A). Wave number is expressed in cm^-1^. IR spectra were measured from 4000 cm^-1^to 650 cm^-1^with a FT-IR spectrophotometer equipped with an ATR apparatus. Highest absorbance values correspond to Red Maca, intermediate values to Yellow Maca and lowest values to Black Maca. Peaks of absorbance are recorded at 3291 cm^-1^, 2927 cm^-1^, 1614 cm^-1^, 1406 cm^-1^, 1022 cm^-1^, 924 cm^-1^and 862 cm^-1^. ::: ![](1477-7827-3-5-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Structure of Glucotropaeolin (Benzyl glucosinolate) ::: ![](1477-7827-3-5-9) ::: Discussion ========== The present study was designed to determine if different ecotypes of Lepidium meyenii(Maca), a cruciferous plant that grows exclusively over 4000 m in Peruvian Andes, affect ventral prostate size. It was of great interest to demonstrate that Red Maca reduced significantly ventral prostate weight. This effect was not observed after treatment with Yellow or Black Maca. The effect of Red Maca was specific for prostate, since other organs as testes, epididymis, seminal vesicles, kidneys, spleen, liver, lungs and heart were not affected. It was also demonstrated an effect of Red Maca on rats in which ventral prostate size was enlarged by two injections of testosterone enanthate. In fact, Red Maca administered for 14 or 42 days reduced the effect of TE. At 42 days, the ventral prostate size of rats treated with TE plus Red Maca was similar to that of control rats treated only with vehicle. Epithelial height and luminal areas were proved to be sensitive parameters for the evaluation of androgen effects on prostates \[[@B26]\]. The present study shows that prostatic epithelial height increased after treatment with TE. The same effect has been observed when castrated rats were treated with testosterone \[[@B26]\] suggesting that prostatic epithelial height is androgen dependent. Red Maca was able to reduce the prostatic epithelial height of TE treated rats. This would means that Red Maca interferes the androgen action. Growth of the prostate is a hormone-mediated phenomenon regulated by both androgens and estrogens \[[@B27]\]. However, data showed that Red Maca affect ventral prostate size without affecting serum testosterone or estradiol levels. This is not surprising because previously, it has been published that dietary phytoestrogens may affect prostate size without modify circulating testosterone or estrogen level \[[@B28]\], but affecting the androgen action in the rat prostate \[[@B27]\]. Our data on effect of Red Maca on ventral prostate size in rats previously treated with testosterone enanthate suggest that this cruciferous is acting by interfering the androgen action. Maca is characterized by its higher content on aromatic glucosinolates \[[@B19]-[@B21]\]. Recently, it has been described a metabolite of the aromatic glucosinolates that specifically antagonizes androgen receptor \[[@B18]\]; therefore, it is possible that effect of Red Maca on ventral prostate size may be due in part to an action of glucosinolate metabolites on androgen receptor. However, further studies will be required to clarify mechanism of action of this cruciferous plant. Recently, increasing evidence has been presented suggesting that cruciferous (Brassicas) vegetables may reduce the risk of prostate cancer development \[[@B3],[@B4]\]. The genus Lepidiumcould be an important alternative for treatment of prostate diseases. Other Brassica from the genus Lepidium, as Lepidium latifoliumreduced prostate weight \[[@B6]\] suggesting that cruciferous from the genus Lepidiummay have important anti-proliferative and proapoptotic effects. In Red Maca treated rats, cell size has decreased and membrane blebbing and nuclear distortion are apparent suggesting a pro-apoptotic effect. It is still unknown the active principle for the effect of Red Maca on ventral prostate and why the action is specific since any other organ was affected. Moreover, the different effects among ecotypes seem to be due to different amount of active metabolites. This study used aqueous extract of dried hypocotyls of Lepidium meyenii. In the aqueous extract is possible to find glucosinolates \[[@B7]\] and anthocyanines \[[@B29]\]. Both compounds have antiproliferative and proapoptotic properties in prostate cancer cells \[[@B14]-[@B18],[@B30]\]. As effect was specific for Red Maca and not for Yellow or Black Maca, it is probably that Red Maca has more glucosinolate content than other ecotypes. Results from the infrared (IR) spectroscopy showed that peaks of absorbance were higher for Red Maca, intermediate for Yellow Maca and lower for Black Maca. Each peak reflects specific chemical functional groups. Several functional groups found in the different Maca ecotypes correspond among others to benzyl glucosinolate. In such sense, it is suggested that benzyl glucosinolate content is higher in Red Maca, intermediate in Yellow Maca and lower in Black Maca. In addition to the potential glucosinolates effects on prostate, it is possible that other active metabolites may be acting on prostate. Maca aqueous extracts were further extracted with ethanol or methanol and assessed for different compounds. The compounds found are potential candidates to affect prostate; however, it is difficult at this time to ascertain which specific compound has the prostate effect. In fact, the phyto-chemical screening data showed that aqueous extract of Red Maca has alkaloids, steroids, tannins, saponins and cardiotonic glycosides, all of them may have effects on prostate. Phytochemical study showed that Red Maca was more positive for alkaloids than from other compounds. The alkaloid, (1R,3S)-1-methyltetrahydro-β-carboline-3-carboxylic acid has been reported as a constituent of Maca \[[@B20]\]. This alkaloid acts as antioxidants and free radical scavengers \[[@B31]\]. Beta carbolines are also proapoptotic compounds \[[@B32]\] and they have antitumor activities \[[@B33]\]. Further studies will be required to determine the impact of tetrahydro-beta-carbolines from Maca on prostate. Conclusions =========== Indeed, the data presented here show that Red Maca reduced ventral prostate size in normal adult rats and also in rats treated with testosterone enanthate. Hence, it is proposed that Red Maca may have important implications under pathological conditions of the prostate. Authors\' contributions ======================= GFG conceived of the study participating in its design, coordination, and drafting the manuscript. SM participated in the design of the study and the study of different ecotypes of Maca. JN participated in the biological study with different ecotypes of Maca. GF participated in the phytochemical screening JR participated in the statistical analysis SY participated in the histological study PY participated in the histological study MG participated in the design and analysis of results, and its interpretation. All authors read and approved the final manuscript. Acknowledgements ================ This research was supported by a Grant from Vicerrectorate of Investigation at the Universidad Peruana Cayetano Heredia. We thank to Sharon Castillo for her technical support and Leon Villegas for his assistance on infrared measurement.	PubMed Central	2024-06-05T03:55:52.464882	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548136/", "journal": "Reprod Biol Endocrinol. 2005 Jan 20; 3:5", "authors": [ { "first": "Gustavo F", "last": "Gonzales" }, { "first": "Sara", "last": "Miranda" }, { "first": "Jessica", "last": "Nieto" }, { "first": "Gilma", "last": "Fernández" }, { "first": "Sandra", "last": "Yucra" }, { "first": "Julio", "last": "Rubio" }, { "first": "Pedro", "last": "Yi" }, { "first": "Manuel", "last": "Gasco" } ] }
PMC548137	Background ========== It is well established in mammals that gonadal steroid hormones are potent negative feedback regulators of the hypothalamic-pituitary-gonadal (HPG) axis. In ranid frogs, the first evidence supporting this notion came from a study on the bullfrog, Rana catesbeiana, in which gonadectomy elevated circulating gonadotropins, and estrogen and androgen replacement suppressed this elevation \[[@B1]\]. It was later shown that two gonadal steroids, 17β-estradiol (E~2~) and 5α-dihydrotestosterone (DHT), could directly target the pituitary to modulate the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in these frogs \[[@B2]-[@B4]\]. Despite the established role of DHT and E~2~as feedback regulators of gonadotropin secretion in ranid frogs, there is still some confusion regarding the exact nature of these feedback effects. For example, in female and juvenile R. catesbeiana, DHT suppressed the post-gonadectomy rise in circulating gonadotropins \[[@B1]\], yet it enhanced the responsiveness of the pituitary to gonadotropin-releasing hormone (GnRH) \[[@B1],[@B5]\], suggesting DHT is involved in both negative and positive feedback. On the other hand, in the leopard frog (R. pipiens), DHT had no effect on the post-castration rise in gonadotropin in males \[[@B2]\] but modestly stimulated pituitary responsiveness to GnRH, suggesting a role in only positive feedback. Although the effects of E~2~were less variable, some conflicting data also exist. In both R. catesbeianaand R. pipiens, E~2~consistently inhibited LH and FSH secretion both in vivoand in vitro, demonstrating a direct and powerful negative feedback effect of E~2~at the level of the pituitary \[[@B1]-[@B3]\]. However, recent studies reported E~2~treatment significantly stimulated the proliferation of primary spermatogonia (I SPG) in the green frogs, R. esculenta\[[@B6],[@B7]\], suggesting an additional role of E~2~in the positive feedback of the HPG axis. Results from the previous studies revealed the complex nature in which estrogen and androgen feed back on the reproductive axis, and suggest that the nature of the feedback effects might vary depending on the species, sex, reproductive stage of the frogs used, and the duration of steroid hormones administered. Further, there are still significant gaps in our knowledge regarding how these two steroid hormones feed back on the other two components of the HPG axis, the hypothalamus and the gonad. Therefore, the goal of the present study is to understand how E~2~and DHT impact the HPG axis in gonadally-intact male R. pipiens. We will do so by measuring parameters that reflect the function of the HPG axis, including hypothalamic GnRH, pituitary and circulating LH, and spermatogenic activity. Moreover, steroid treatments were administered over two periods, 10 days and 30 days, to examine if the nature of steroidal feedback effects remains consistent over time. These results should allow us to determine if DHT and E~2~act consistently as negative feedback regulators at different levels of the HPG axis. Methods ======= Animals ------- All experimental procedures were conducted in compliance with the animal protocol approved by the Institutional Animal Care and Use Committee at the University of Colorado. Mature male northern leopard frogs, Rana pipiens, were obtained from Carolina Biologicals (Burlington, NC) from April to July of 2004. Since the life histories of these animals were not entirely clear, all experiments were conducted within the three-month period to minimize the possible confounding effects of seasonality. As a control, we performed histological analysis on testes of representative frogs from every batch. When testicular histology was compared, little differences were observed among batches of frogs arriving at different times (data not shown). Frogs were kept under a 12L:12D photoperiod and fed live crickets every other day. All frogs were allowed to acclimate to the laboratory environment for at least one week before surgical implant. Surgical Implant ---------------- One-cm silastic capsules containing crystalline cholesterol (Ch: control), DHT, or E~2~were prepared as previously described \[[@B8]\], with some minor modifications. Briefly, silastic tubing (outer diameter = 1.96 mm; inner diameter = 1.47 mm) was filled with 1 cm length of crystalline Ch, DHT, or E~2~(Sigma, St Louis, MO) and sealed on both ends with silicon glue. Ch implant was widely used as a negative control for the experimental treatment of cholesterol-based compounds such as steroid hormones \[[@B8]-[@B11]\]. In a preliminary observation (data not shown), we noted no difference in the testicular morphology in animals that have lost Ch capsules compared to animals that have retained capsules throughout the 30-day period, indicating Ch had minimal effects on the reproduction of R. pipiens. Filled capsules were equilibrated in 0.6% saline for at least 48 hours before implant. For implant, frogs were anesthetized by immersion in 0.03% benzocaine. A small incision was made at the base of the left leg and a silastic capsule inserted subcutaneously. The incision was then closed with silk thread. Frogs were monitored for recovery from anesthesia and then returned to their respective holding tanks. Tissue Preparation ------------------ Ten or 30 days after implant, frogs were weighed and sacrificed by quick decapitation using a guillotine. Trunk blood was collected into heparinized tubes, centrifuged, and plasma stored at -70°C until the measurement of LH and steroid hormones by radioimmunoassays (RIAs). Testes were removed, their masses recorded, and immersion-fixed in Bouin\'s fixative overnight. Hypothalami were excised from the brain by four cuts: a coronal cut 1 mm rostral to the optic chiasm, a coronal cut on the caudal border of the optic tectum, and two sagittal cuts along the lateral margins of the median eminence. Hypothalami were flash-frozen on dry ice and stored at -70°C until extraction and the measurement of GnRH by RIA. Pituitary glands were removed, sonicated in 500 μl phosphate-buffered saline (PBS), and stored at -70°C until the measurement of LH by RIA. All carcasses were later inspected for the presence of the silastic capsule in the legs. Animals whose capsules were lost were excluded from data analysis. LH RIA ------ Plasma and pituitary LH levels were measured by a LH RIA developed for the bullfrog (R. catesbeiana) \[[@B12]\] and validated for R. pipiens\[[@B13]\]. The iodination stock, standard, and antiserum for the RIA were a generous gift of Dr. Paul Licht (University of California at Berkeley). The limit of detection was 0.1 ng/ml. The intra- and inter-assay coefficients of variation were 4.8% and 13.3%, respectively. Pituitary LH levels were normalized for protein content assessed by the Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Extraction of Hypothalami and GnRH RIA -------------------------------------- Hypothalamic GnRH was extracted with 1 N HCl from the frozen tissues as previously described \[[@B11]\]. The recovery for hypothalamic extractions, assessed by the post-extraction counting of a known amount of \[^125^I\]GnRH added to representative homogenates prior to extraction, was 86%. GnRH RIA was carried out with an antiserum specific for the mammalian form of GnRH (R1245, provided by Dr. Terry Nett at the Colorado State University) using a protocol described in detail elsewhere \[[@B11],[@B14],[@B15]\]. The intra- and inter-assay coefficients of variation were 7.3% and 5.0%, respectively. Hypothalamic GnRH levels were normalized for protein content determined by the Bradford protein assay. Steroid Hormone RIAs -------------------- E~2~and DHT RIAs were performed using the RIA kits from Diagnostic Systems Laboratories (Webster, TX). These RIA kits have been validated previously for the measurement E~2~and DHT in R. pipiens\[[@B11]\]. The limits of detection were 6.5 pg/ml for the E~2~RIA and 4 pg/ml for the DHT RIA. The intra- and inter-assay coefficients of variation were 5.3% and 4.9%, respectively, for the E~2~RIA, and 3.1% and 8.4%, respectively, for the DHT RIA. Both RIAs are highly specific and cross-react minimally with other steroid hormones. Histology --------- After fixation, testes were dehydrated through ascending concentrations of ethanol, defatted in Histoclear, and embedded in paraffin. Thirteen-μm sections were cut on a rotary microtome, mounted on poly-L-lysine-coated slides, and stained with hematoxylin and eosin. Immunocytochemistry (ICC) of Proliferating Cell Nuclear Antigen (PCNA) ---------------------------------------------------------------------- Testes were processed for ICC of PCNA, a cell cycle S phase marker, to identify I SPG and secondary spermatogonia (II SPG) undergoing cell proliferation \[[@B16]\]. Testicular sections, prepared as described above for histological staining, were deparaffinized in Histoclear, rehydrated through descending concentrations of ethanol, and immersed in Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) for 10 minutes at 90°C. After antigen retrieval, sections were washed with 1% hydrogen peroxide in 0.1 M PBS containing 0.4% Triton × 100 (PBST) for 10 minutes to quench the endogenous peroxidase activity, rinsed 5 times with PBST, and incubated for 48 hours at 4°C in PBST containing a monoclonal anti-PCNA antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:500) and 4% normal sheep serum. After incubation, sections were washed with PBST and incubated with a biotinylated sheep-anti-mouse IgG (Jackson Laboratory, West Grove, PA; 1:400), washed, and incubated with the Vectastain ABC reagent (Vector Laboratories) for 1 hour. Sections were washed and the immunoreactivity visualized using diaminobenzidine as the chromagen. After the color reaction, sections were washed, counterstained with hematoxylin, dehydrated through ascending concentrations of ethanol, cleared in Histoclear, and coverslipped. Controls for ICC included the preadsorption of the primary antiserum with 20 μg/ml of recombinant human PCNA (Spring Bioscience, Fremont, CA) and the omission of the primary antiserum. Histological Analysis --------------------- Five to eight testes (each from a different animal) per treatment group were assessed for the following histological parameters: seminiferous tubule diameter, the number of I SPG per tubule, and the number of cysts within each tubule containing II SPG, primary spermatocytes (I SPC), or secondary spermatocytes (II SPC). These germ cells were defined according to Rastogi et al. \[[@B17]\]. To score the number of cysts containing II SPG, I SPC, and II SPC, sections stained with hematoxylin and eosin were used. To score the number of I SPG, which were scattered and more difficult to locate, sections processed for PCNA ICC were used. Specifically, PCNA-positive cells that were large, isolated, and located at the periphery of the cysts were scored as proliferating I SPG. This method allowed us to identify more I SPG than if morphological criteria were used alone. Since most spermatids and mature spermatozoa were not confined within the cysts and were therefore difficult to measure, these two germ cell types were not quantified. For each testis, five randomly selected tubules were sampled on a slide that had been coded to conceal the identity of the animal. Histological parameters from five tubules were averaged to give a mean for a single animal. Tubular diameters were measured using a calibrated ocular micrometer. All histological parameters were assessed by one individual blind to the identity of the slides. Statistical Analysis -------------------- Differences among groups were analyzed by the one-way analysis of variance (ANOVA) on log~10~-transformed data followed by the Tukey\'s post-hoc test. Differences were considered significant when P\< 0.05. Results ======= Steroid hormone RIAs were performed to monitor circulating steroid hormone levels in Ch- and hormone-implanted animals. For animals implanted for 10 days, circulating E~2~levels were 540 ± 110.5 (Ch group; n = 5) and 1966 ± 13.2 pg/ml (E~2~group; n = 5), and circulating DHT levels were 0.9 ± 0.3 (Ch group; n = 4) and 37.5 ± 4 ng/ml (DHT group; n = 5). For animals implanted for 30 days, circulating E~2~levels were 488 ± 370 (Ch group; n = 5) and 2255 ± 650 pg/ml (E~2~group; n = 9), and circulating DHT levels were 2.1 ± 1.4 (Ch group; n = 4) and 24.6 ± 2.7 ng/ml (DHT group; n = 8). To assess the overall accumulation of GnRH in the hypothalami of control and steroid hormone-treated animals, hypothalami were removed, extracted, and measured for the concentration of GnRH. No significant differences in hypothalamic GnRH concentration were observed among Ch, DHT, and E~2~groups implanted for 10 or 30 days (Fig. [1](#F1){ref-type="fig"}). In animals implanted for 10 days, plasma LH levels were not different among the treatment groups (Fig. [2A](#F2){ref-type="fig"}). However, in animals implanted for 30 days, both DHT and E~2~significantly suppressed circulating LH (Fig. [2B](#F2){ref-type="fig"}). The suppressive effect of E~2~was significantly more potent than DHT, with all E~2~-treated animals having undetectable levels of circulating LH (Fig. [2B](#F2){ref-type="fig"}). In 10-day-implanted frogs, no differences in pituitary LH concentration were observed among the treatment groups (Fig. [3A](#F3){ref-type="fig"}). In 30-day-implanted frogs, E~2~, but not DHT, significantly decreased the accumulation of LH in the pituitary (Fig. [3B](#F3){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Hypothalamic GnRH concentrations in implanted frogs.Hypothalamic GnRH concentrations in frogs implanted for (A)10 days or (B)30 days with either Ch, DHT, or E~2~. No significant differences were observed among treatment groups in either 10- or 30-day-implanted animals. Each bar represents mean ± SEM. N = 6--11.+ ::: ![](1477-7827-3-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Plasma LH levels in implanted frogs.Plasma LH levels in frogs implanted for (A)10 days or (B)30 days with either Ch, DHT, or E~2~. Each bar represents mean ± SEM. Dissimilar letters indicate significant difference between groups. ND = not detectable. N = 7--10. ::: ![](1477-7827-3-2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Pituitary LH concentrations in implanted frogs.Pituitary LH concentrations in frogs implanted for (A)10 days or (B)30 days with either Ch, DHT, or E~2~. Each bar represents mean ± SEM. Dissimilar letters indicate significant difference between groups. N = 7--10. ::: ![](1477-7827-3-2-3) ::: Testicular function was assessed by six parameters: the gonadosomatic index (GSI; \[g testes mass/g body mass\] × 100), the diameter of the seminiferous tubules, and the presence of four germ cell types (I SPG, II SPG, I SPC, II SPC) in the testes. Overall, no differences were observed in any of the six parameters among treatment groups in the 10-day-implanted animals (Fig. [4](#F4){ref-type="fig"}). However, in the 30-day-implanted animals, significant steroid-induced changes in the testes were seen. E~2~significantly depleted the presence of I SPG, II SPG, I SPC, and reduced the GSI (Fig. [5](#F5){ref-type="fig"}), whereas DHT significantly reduced only the presence of II SPG and I SPC (Fig. [5](#F5){ref-type="fig"}). Neither steroid hormone affected the diameter of the seminiferous tubules or II SPC (Figs. [5B, F](#F5){ref-type="fig"}). Representative photomicrographs of testicular histology (Fig. [6](#F6){ref-type="fig"}) showed morphological changes parallel to the quantitative measurements in Figs. [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}. In animals implanted for 10 days, no visible differences in germ cell types were seen among treatment groups; I SPG, II SPG, and I SPC were present equally in the testes of all groups (Figs. [6A, B, C](#F6){ref-type="fig"}). In contrast, 30-day implant with DHT or E~2~resulted in highly pronounced and visible changes in the histology of the testes (Figs. [6D, E, F](#F6){ref-type="fig"}). Whereas Ch-treated tubules contained germ cells of all types (Fig. [6D](#F6){ref-type="fig"}), DHT- and E~2~-treated tubules showed a conspicuous absence of II SPG and I SPC (Figs. [6E, F](#F6){ref-type="fig"}). The loss of I SPG with DHT and E~2~treatments was less visible than the loss of other two germ cell types (Figs. [6E, F](#F6){ref-type="fig"}), a result consistent with the quantitative data (Fig. [5](#F5){ref-type="fig"}). Interestingly, the formation of spermatozoa appeared to be stimulated by DHT and E~2~; in fact, the most prominent germ cells in tubules of DHT and E~2~-treated were the large bundles of mature spermatozoa, which occupied most of the tubular lumen (Figs. [6E, F](#F6){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Testicular function in frogs implanted for 10 days.Measurements of testicular function in frogs implanted for 10 days with either Ch, DHT, or E~2~. (A)Average GSI, (B)average diameter of seminiferous tubules, (C)number of I SPG, and number of cysts containing (D)II SPG, (E)I SPC, and (F)II SPC were measured from 5--8 animals per treatment group. Each bar represents mean ± SEM. ND = not detectable. No significant differences were observed among treatment groups in any of the parameters measured. ::: ![](1477-7827-3-2-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Testicular function in frogs implanted for 30 days.Measurements of testicular function in frogs implanted for 30 days with either Ch, DHT, or E~2~. A)Average GSI, (B)average diameter of seminiferous tubules, (C)number of I SPG, and number of cysts containing (D)II SPG, (E)I SPC, and (F)II SPC were measured from 5--6 animals per treatment group. Each bar represents mean ± SEM. Dissimilar letters indicate significant difference between groups. ::: ![](1477-7827-3-2-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Testicular histology of implanted frogs.Representative testicular histology of frogs implanted with (A, D)Ch, (B, E)DHT, or (C, F)E~2~. (A, B, C)Testes from animals implanted for 10 days. (D, E, F)Testes from animals implanted for 30 days. Red arrow = I SPG; dark blue arrow = II SPG; light blue arrow = I SPC; yellow arrow = II SPC; green arrow = spermatozoa. Note DHT and E~2~had no visible effects on testes of animals implanted for 10 days (A, B, C). In contrast, DHT and E~2~visibly altered the germ cell composition in animals implanted for 30 days (D, E, F). Note the prominent spermatozoa and the absence of I SPC and II SPG in both DHT- and E~2~-treated testes (E, F). Scale bar = 100 μm. ::: ![](1477-7827-3-2-6) ::: PCNA ICC was conducted to determine the effects of gonadal steroids on the number of proliferating germ cells and to allow for the more consistent identification of I SPG. In animals implanted with Ch, intense PCNA immuoreactivity was observed in both I SPG and II SPG. In addition, the cytoplasm of I SPC was lightly stained (Figs. [7A, D](#F7){ref-type="fig"}), since PCNA was also shown to be expressed during the pre-meiotic S phase and meiotic prophase \[[@B18]\]. All spermatogonia (I SPG and II SPG) identifiable by the hematoxylin counterstain were positive for PCNA. Treatment with DHT (Fig. [7B](#F7){ref-type="fig"}) or E~2~(Fig. [7C](#F7){ref-type="fig"}) did not visibly alter the appearance of cells positive for PCNA in frogs implanted for 10 days compared to the Ch control (Fig. [7A](#F7){ref-type="fig"}). In contrast, in animals implanted for 30 days, a visible reduction in the number of PCNA-positive germ cells was observed in DHT- and E~2~-treated animals (Figs. [7E, F](#F7){ref-type="fig"}) compared to the Ch control (Fig. [7D](#F7){ref-type="fig"}). This reduction was primarily due to the decreased presence of I SPG, II SPG, and I SPC in the testes treated with steroid hormones. In control sections incubated with preadsorbed primary antiserum or no primary antiserum, only background staining was present (data not shown). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### PCNA ICC on testes of implanted frogs.Representative photomicrographs of PCNA ICC performed on testicular sections of frogs implanted with (A, D)Ch, (B, E)DHT, or (C, F)E~2~. (A, B, C)Testes from animals implanted for 10 days. (D, E, F)Testes from animals implanted for 30 days. Brown stain = PCNA immunoreactivity. Blue stain = hematoxylin counterstain. Red arrow = I SPG; dark blue arrow = II SPG; light blue arrow = I SPC. Note DHT and E~2~had no visible effects on testes of animals implanted for 10 days (A, B, C). In contrast, DHT and E~2~visibly reduced the presence of PCNA-positive germ cells in animals implanted for 30 days (D, E, F). Σχαλεβαρ = 100 μm. ::: ![](1477-7827-3-2-7) ::: Discussion ========== Under our experimental paradigm, both E~2~and DHT exerted negative feedback effects upon the reproductive axis of the sexually mature male leopard frogs. The manifestation of these inhibitory effects was time-dependent and required treatment duration for longer than 10 days. In frogs implanted for 30 days, E~2~decreased circulating LH to undetectable levels and significantly reduced the presence of I SPG, II SPG, and I SPC. The effects of DHT were less potent, reducing only circulating LH, II SPG, and I SPC. Unexpectedly, the presence of post-meiotic germ cells was either unaffected or stimulated with the DHT or E~2~treatment. These results suggest that along the reproductive axis, the most significant negative feedback effects were upon the pituitary and testes, although a stimulatory effect upon the testes might also exist. All hormone implants elevated circulating steroid hormones to levels above the Ch controls without exceeding the physiological range reported for male ranid frogs. For instance, depending on the reproductive status, the range of circulating E~2~reported for sexually mature male ranid frogs was approximately 100 to 3500 pg/ml, and the range of circulating DHT was approximately 1 to 30 ng/ml \[[@B6],[@B19]\]. Another study \[[@B20]\] reported circulating levels of approximately 10 ng/ml for DHT and 2 ng/ml for E~2~in captive male R. pipiens. Thus, circulating E~2~and DHT in the hormone-implanted animals were largely within the high end of the physiological range. DHT and E~2~did not significantly alter hypothalamic GnRH concentration in animals treated for 10 or 30 days. These results were consistent with our previous finding on frogs implanted for 20 days with DHT and E~2~, and speak to the highly stable nature of GnRH peptide accumulation. One should note that this study focused on the mammalian form (Type I) of GnRH because this is the predominant hypophysiotropic form of GnRH in the diencephalon of ranid frogs \[[@B21]\] and the form more sensitive to changes in reproductive status \[[@B22]\]. However, the chicken II (Type II) form of GnRH may also be hypophysiotropic since it binds to pituitary GnRH receptor with high affinity \[[@B23]\], and its presence is detected in the hypothalamic-pituitary portal blood \[[@B21]\]. The ability of steroid hormones to feed back upon the Type II GnRH system in R. pipiensis at present unclear and awaits further investigation. Only E~2~treatment for the longer duration (30 days) significantly reduced pituitary LH concentration, although a substantial amount of LH still remained in the pituitary glands of these E~2~-treated animals. Thus, the potent suppression of circulating LH in animals treated with E~2~for 30 days was most likely attributable to the low secretory activity of the pituitary gonadotropes rather than the depletion of LH stores. Somewhat surprising was the inability of E~2~treatment for 10 days to suppress circulating LH. The pituitaries of ranid frogs were shown to be extremely sensitive to the inhibitory actions of E~2~. In R. pipiens, in vitroexposure of the pituitary to E~2~concentrations as low as 100 pg/ml for only 48 hours significantly suppressed both basal and GnRH-stimulated gonadotropin secretion \[[@B2]\]. Extrapolating from this time course and from our current observation that E~2~implants could elevate circulating E~2~to about 2000 pg/ml, one might expect a substantial decline in circulating LH of these animals after only 10 days of implant, but this was not the case. It is possible that additional in vivomechanisms exist in these frogs to buffer against short-term estrogenic inhibition. Some of these might include changes in the clearance rate of circulating LH \[[@B24]\] and the levels of sex steroid binding proteins \[[@B25]\]. The former could prolong the half-life of LH in circulation; the latter could dampen the inhibitory effects of E~2~by binding to E~2~and decreasing the availability of the bioactive hormone. Another unexpected observation was that DHT treatment for 30 days also suppressed circulating LH. Our results differ from a previous study demonstrating the inability of DHT to suppress post-gonadectomy rise in LH \[[@B2]\] and showed, for the first time, that DHT participates in the negative feedback regulation of gonadotropin secretion in R. pipiens. It is at present unclear if reduced circulating LH is a direct consequence of suppressed secretory activity of the gonadotropes or reduced output from the GnRH system. Based on the previous report that DHT had no direct inhibitory effect on pituitary gonadotropin secretion \[[@B2]\], it seems likely that DHT may achieve negative feedback primarily by targeting the GnRH system to suppress GnRH release. This possibility does not conflict with our current observation that DHT lacks an effect on GnRH content. Since GnRH content is a measure of GnRH peptide accumulation which is a result of transcription, translation, mRNA stability, peptide turnover, or release, it is a poor indicator of GnRH release alone. One of the most pronounced negative feedback effects of DHT and E~2~was observed at the level of the testes. In animals implanted for 30 days with these two steroids, a marked loss of several germ cell types (I SPG, II SPG, and I SPC) was observed. Interestingly, although E~2~exerted a greater disruptive effect on spermatogenesis, similar trends of reduction with virtually no qualitative difference were also observed in DHT-treated testes, suggesting similar outcome might be attained with longer DHT exposure. That DHT- and E~2~-induced disruption of spermatogenesis differed only in the degree of severity suggests the disruption occurred primarily via a common pathway, possibly through the inhibition of gonadotropins. In this study, we could not measure circulating FSH because we lack a homologous FSH RIA. However, previous studies have reported that FSH secretion in R. pipienswas under the negative control of E~2~\[[@B2],[@B3]\] and possibly DHT \[[@B2]\], since gonadectomy significantly elevated the levels of circulating FSH. The observation that spermatogenic disruption occurred only when circulating gonadotropin was reduced (30 day-implants) further lends support to this hypothesis. Although the roles of FSH and LH in anuran spermatogenesis are not entirely clear, data from other amphibians suggest FSH is essential for supporting the proliferation and survival of spermatogonia \[[@B26],[@B27]\]. Importantly, FSH is required for the completion of the last spermatogonial mitosis, thus the entrance into meiosis and the generation of spermatocytes \[[@B27]\]. On the other hand, LH is specifically required for the stimulation of androgen production in ranid frogs \[[@B28]\] and could be responsible for maintaining high levels of intratesticular androgen required for androgen-dependent stimulation of germ cell formation \[[@B29]\]. Thus, low circulating levels of gonadotropins could be the common pathway leading to defective spermatogenesis in both DHT- and E~2~-treated animals. Although the significant reduction of I SPG in E~2~-treated testes could partially account for the loss of germ cell types that arose from I SPG, this cannot be the sole cause. For example, substantial PCNA-positive I SPG still remained in the testes of frogs implanted with E~2~for 30 days, yet virtually no II SPG remained in the testes of these animals. Similarly, in 30-day-DHT-treated testes, there was no significant decline in I SPG, but II SPG were markedly reduced. These observations indicate a disproportionate loss of II SPG that may have resulted from their failure to survive. These data were consistent with a previous report in the newt that the mitotic penultimate SPG failed to survive when circulating FSH was suppressed \[[@B26]\]. Taken together, we believe that II SPG was the germ cell type most severely affected by steroid treatments, and the loss of II SPG was the most important underlying cause for disrupted spermatogenesis. An interesting observation is that although pre-meiotic germ cells (I SPG, II SPG, and I SPC) were adversely affected by steroid hormone implants at 30 days, meiotic or post-meiotic germ cells (II SPC, spermatids and spermatozoa) appeared unaffected or stimulated. In fact, the most conspicuous germ cells in the seminiferous tubules of 30-day E~2~- or DHT-implanted frogs were mature spermatozoa, which occupied the largest bulk of the tubular lumen. It is possible that low circulating FSH had little influence on the germ cells once they entered meiotic division. Under low circulating LH, spermiation was inhibited, and mature spermatozoa continued to accumulate in the tubule. Another possibility is that E~2~and DHT, while suppressing the presence of pre-meiotic germ cells, actually stimulated the entrance of existing I SPC into meiosis and promoted the survival of post-meiotic germ cells. This possibility was partially supported by the previous observation that testes of frogs treated with DHT for 20 days had fewer II SPG, but significantly more I SPC in the midst of meiotic division \[[@B11]\]. Along the same line of reasoning, it is also possible that DHT and E~2~facilitated the progression of spermatogenesis to the extent that the intermediate germ cell types could no longer be adequately replenished, leaving tubules filled with post-meiotic cells (primarily spermatozoa) and very little else. Regardless, the trend towards reduced GSI in steroid hormone-treated animals, along with the reduced presence of pre-meiotic germ cells, indicate an overall negative effect of these hormones upon the testes. The abundance of spermatozoa in DHT- and E~2~-treated animals nevertheless raised an interesting possibility for the existence of a positive steroidal effect on spermatogenesis. Worth mentioning is the possibility that DHT and E~2~, in addition to affecting spermatogenesis by lowering circulating gonadotropins, have also been shown to act directly upon the amphibian testes. Both androgen and estrogen binding sites were found in the amphibian testes \[[@B30]-[@B33]\]. A number of physiological responses were presumably mediated through these testicular steroid hormone receptors. For instance, E~2~acted directly upon the amphibian testes to suppress androgen secretion \[[@B34]-[@B36]\], stimulate nuclear translocation of c-Fos \[[@B37],[@B38]\], and enhance proliferation of I SPG \[[@B6],[@B37]\]. Similarly, DHT has also been found to directly modulate androgen secretion \[[@B34]\]. Of interest to the present study is the demonstration that E~2~directly stimulated SPG I proliferation in R. esculenta\[[@B6],[@B7],[@B37]\]. Under our experimental paradigm, however, such a stimulatory effect was not seen in R. pipiens. Whether or not this discrepancy was due to the differences in experimental paradigms or species used is at present unclear. We previously showed that spermatogenesis in the ranid frogs was altered in mature male frogs implanted with DHT and E~2~for 20 days \[[@B11]\]. Specifically, E~2~reduced the presence of II SPG and I SPC, whereas DHT reduced only the presence of the former. However, the study represented only a snapshot in time, so no information was available regarding the progression of events that led to the altered formation of germ cells. Moreover, it was unclear if the treatment with these two steroid hormones for shorter or longer periods could impact the testes differently. Our current results showed that both steroids inhibited circulating gonadotropin and disrupted spermatogenesis progressively in a time-dependent manner, with the longer duration of treatment producing the more pronounced effects. Further, the changes in the testes were qualitatively similar between DHT and E~2~treatments, suggesting declining gonadotropin levels might be the common underlying cause for the disrupted spermatogenesis. These results reflect the highly sensitive nature of the anuran reproductive axis to estrogenic and androgenic modulation. We showed that the continuous exposure of mature frogs to high physiological levels of steroid hormones for a relatively short period could profoundly alter their pituitary and testicular function. In particular, the potency of estrogen hormones raises concerns regarding the potential reproductive disruption that can occur when mature frogs are exposed to short-term and low-level environmental estrogen mimics. Authors\' contributions ======================= PST designed the experiments, analyzed the data, and prepared the manuscript. AEK and JTJ prepared the silastic capsules, implanted the animals, removed the tissues, and performed all the hormone measurements. KBW implanted some animals, prepared all histological samples, counted the germ cells, and performed the PCNA ICC. All authors read and approved the final manuscript. Acknowledgments =============== We thank Dr. Paul Licht for the generous gift of frog LH RIA reagents. This work was supported by NSF IBN-9996398 and NIH HD-042634 to P.-S.T.	PubMed Central	2024-06-05T03:55:52.467464	2005-1-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548137/", "journal": "Reprod Biol Endocrinol. 2005 Jan 10; 3:2", "authors": [ { "first": "Pei-San", "last": "Tsai" }, { "first": "Ann E", "last": "Kessler" }, { "first": "Jeremy T", "last": "Jones" }, { "first": "Kathleen B", "last": "Wahr" } ] }
PMC548138	Background ========== Metastases to the umbilicus are universally referred to as Sister Joseph\'s (or Sister Mary Joseph\'s) nodule. Etiology is related to the presence of primary malignant disease in the abdominal cavity or occasionally in the chest and/or breast \[[@B1]-[@B5]\]. Historically, Sister Mary Joseph (1856--1939) was a surgical assistant under the guidance of Dr. William Mayo. She was the first one to note the connection between the umbilical nodule and intra-abdominal cancer. The first case reporting the presence of Sister Mary Joseph\'s nodule was in 1864 by Storer, however; Hamilton Bailey was the first one to use the term \"Sister Mary Joseph\'s nodule. Although skin metastasis is rare and range between 5% and 9%, it is estimated that 1% to 3% of abdomino-pelvic tumors metastasize to the umbilicus \[[@B2],[@B3],[@B5]\]. The most common primary neoplasm is adenocarcinoma (75%), more rarely squamous cell carcinoma followed by undifferentiated tumors or carcinoid can metastasize to umbilicus. In men, gastrointestinal tract (55%) is the most common location of the primary neoplasm that metastasizes to the umbilicus, followed by stomach, colon, rectum, small bowel, and pancreas in a decreasing order \[[@B1]-[@B5]\]. In women, on the other hand, gynecological neoplasms particularly ovarian cancer is the most common primary site, of which serous papillary cystadenocarcinoma (34%) is the most frequent. Further, endometrial carcinoma, cervix, vagina and vulva may also be responsible for the metastasis to the umbilicus. In addition to these the literature reports other but rare sites that could form potential grounds for metastasis to the umbilicus, namely gallbladder, liver, breast, lung, prostate, penis, peritoneum, lymphoma, bladder and kidney. In about 11% of the umbilical metastasis, the origin of the metastasis is unknown \[[@B4],[@B5]\]. Upon recognition of the positive nodules in the umbilicus, physician must consider other potential sources such as endometriosis, melanocytic nevi, fibroma, epithelial inclusion cysts, seborrheic keratosis, pilonidal sinus, keloid, foreign body granulomas, myxoma, omphalitis, abscesses, umbilical hernia and of course primary malignant umbilical tumor (melanoma, squamous and basal cell carcinoma, sarcoma and adenocarcinoma) \[[@B6]\]. In many instances, Sister Joseph\'s nodule is the first and often the only indication of an underlying or occult cancer and/or may indicate recurrence of previously treated malignancy. The presence of umbilical metastasis usually suggests already advanced metastatic process characterized by poor prognosis \[[@B4],[@B5]\]. Therefore, a biopsy of umbilical nodule is recommended which is safe and easy. Obtaining histological description can further direct the clinician to dictate a prompt treatment. In the case of recurrence of the neoplasm, fine needle aspiration cytology may be sufficient to provide definite diagnosis; however, sometimes clinical, cytological, histological, radiological and/or surgical investigations may not be sufficient to identify primary site of the metastasis \[[@B7]\]. Serous papillary cystadenocarcinoma has been documented previously as a cancer responsible for Sister Joseph\'s nodule because of the metastasis to the regional lymph nodes \[[@B6]-[@B9]\]. Herein, we report, to the best of our knowledge, the first case of Sister Joseph\'s nodule in a patient after liver transplantation complicated by serous papillary cystadenocarcinoma. In addition, we review the potential routes by which tumor spread may have occurred and the prognostic significance associated with umbilicus metastasis. Case presentation ================= A 59-year-old Caucasian woman was admitted to our hospital for progressively enlarging but painless mass in the region of the umbilicus. The umbilical mass of approximately 5 cm in diameter and has developed two months after liver transplantation. The nodule was firm with irregular borders, wine-red shade and fixed to the surrounding tissues. The overlying skin was not ulcerated. She never smoked, consumed alcohol or used oral contraceptive pills. The gynecological history was unremarkable. The patient underwent a liver transplantation for end stage liver disease (ESLD) secondary to hepatitis C in mid of 2003. Prior to the transplant, her serum alpha-fetoprotein (alpha-FP) and CEA concentration were measured and both were within normal limits. The transplant procedure and immediate post-transplant period were unremarkable. The post-transplant immunosuppressive regimen consisted of Tacrolimus (6 mg/day) and steroids (prednisone 10 mg/day). Two months after transplantation, during routine follow-up, physical examination revealed multiple subcutaneous satellite nodules in close proximity to the umbilical region and lower abdominal wall. There was bilateral inguinal lymphadenopathy. A plain anterior/posterior chest x-ray was normal. Ultrasound of the abdominal cavity showed a 4 × 4 cm mass below the umbilicus and additional smaller nodules confined to the lower abdominal wall. Paraaortic lymph nodes were not identified. The echogenicity of the mass consisted of alternative hyper- and hypoechoic with poorly defined edges. However, the general condition of the patient was good and the laboratory test showed: hemoglobin level 9.6 g/dl, total bilirubin 0.6 mg/dl, conjugated bilirubin 0.1 mg/dl, Alkaline Phosphates 273 U/l, GGT 40 U/L, AST 36 U/L, and ALT 56 U/L. There was serological evidence of past hepatitis C virus infection. The biopsy of the umbilical mass showed features characteristic for a poorly differentiated serous papillary cystadenocarcinoma. An extended resection of the umbilical and paraumbilical metastasis was performed. In addition, extensive investigation to detect the primary neoplastic origin was initiated. Tumor marker evaluation consisted of: CEA 0.22 \[normal 0--3.7 ng/ml\], alpha feto protein 1.49 (normal: 0.8--9.4 UI/ml), CA125 27.73 (normal: 0--30 UI/ml), CA 19.9 was 8.61 (normal: 0--36.2 UI/ml), CA 15.3 was 22.48 (normal: 0--35 UI/ml). Multiple tumor marker measurements were obtained and each time similar results were reported. A computed tomographic (CT) scan was unable to demonstrate any lesion that could be considered a primary source of the metastasis to the umbilicus. Trans-vaginal ultrasonography showed a normal uterus and ovaries; ultrasonographic examination of thyroid and breast did not show any pathological changes as well. In spite of laborious efforts, the tumor\'s primary site was not found at that time. However, considering the histological results, the umbilicus metastasis could be attributed almost entirely to the ovarian cancer stage 4, since the main primary site in females are the ovaries. In order to prevent further tumor extension and expansion, tacrolimus was reduced from 6 mg/day to 1.5 mg/day. In addition, chemotherapy with Taxol was initiated (50 mg/m^2^I.V) once a week for 16 weeks. To date, after 6 months of follow-up, the patient is doing well, the liver function is normal and tacrolimus serum level is 1 ng/ml. A recent whole body CT-scan showed regression of iliac and inguinal lymph nodes involvement. Discussion ========== Metastases may appear virtually anywhere in the body; however, certain sites are more common then others and umbilical metastasis are unusual sites \[[@B9],[@B10]\]. In majority of cases, the metastatic lesions accompany the symptoms and signs of the primary tumor; however, it can be the first and often the only sign of a carcinoma, although this happens rarely \[[@B10]\]. A variation in vascularity and embryological development makes the umbilicus easy target for metastasis from an intra-abdominal tumor. In fact, during fetal development, the ductus venous (ductus venous Arantii) connects the umbilical portion of the left branch of the portal vein to the inferior vena cava, thus shunting oxygenated umbilical cord blood away form the liver. After birth the duct obliterates and persists as ligamentum venosum or Arantius\' ligament. Because of its functional role, it is commonly believed that the ligament runs from the left branch of the portal vein to the vena cava itself. However, attention to anatomical detail demonstrates that the fibers of the ligament insert either on the left hepatic vein or at the junction between the left hepatic and the middle hepatic vein \[[@B1],[@B11]\]. We know that in cirrhotic patients, the umbilical vein remains open due to portal hypertension. There are numerous potential routes by which a carcinoma can metastasize to the umbilicus in a liver transplant recipient. Malignant carcinomatous cells in the portal venous system may reach the umbilicus by way of a patent umbilical vein \[[@B12]-[@B15]\]. This would be more likely to occur in the presence of portal hypertension and the resulting portal-systemic shunting of blood \[[@B16]\]. Since our patient received liver transplant for cirrhosis secondary to hepatitis C virus infection accompanied by portal hypertension, this could be a possible mode of spread of papillary serous cystadenocarcinoma in our patient. In addition, presence of severe esophageal varicies and large umbilical vein that was viewed by angiography examination before the liver transplantation could have contributed to the spread of tumor. Therefore, in this case, malignant cells could have reached the umbilicus via the umbilical vein. In addition, a pelvic carcinoma of unknown origin may spread via lymph nodes to the umbilical region \[[@B9],[@B17],[@B18]\]. Furthermore, dermal lymphatics are a potential route of spread to the umbilicus and should be considered, since the umbilicus can be reached by retrograde lymphatic flow \[[@B4]\]. Nevertheless, the spread of malignant cells could have occurred via embolization thru the arterial blood supply to the umbilicus. Furthermore, direct implantation of tumor cells may have occurred via direct extension of the neoplasm from the anterior peritoneal surface or via redistribution of the peritoneal fluid flow \[[@B19],[@B20]\]. Finally, cutaneous metastasis to the anterior abdominal wall, may perhaps cause malignant cells to spread in a retrograde direction, namely from the metastatic lesions along the subcutaneous lymphatic vessels to the umbilicus. It is known that the immunosuppression promotes tumor spreading and potentates its expansion, thus immunosuppressive regimen plays a crucial role in the tumor expansion and metastasis. Umbilical metastasis is one of many characteristic signs of extensive neoplastic disease. It suggests advanced distant metastasis and is associated with poor prognosis; mean survival is approximately 10--12 months, although long-term survival has been reported, but only in the presence of solitary metastatic umbilicus nodule \[[@B5]\]. Conclusions =========== Presence of umbilical nodule or nodules requires extensive, meticulous and laborious evaluations since the differential diagnosis for umbilical nodule are many. In our case although, the primary tumor was not found, we treated it as ovarian tumor considering the histological findings and the iliac-inguinal nodes involvement. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= FP: conception and design, interpretation of data, drafting the article and revising EA: interpretation of data, drafting the article and revising SDD: acquisition of data and revising NM: interpretation of data, design and coordination and helped to draft the manuscript GB: collection of data, design and coordination and helped to draft the manuscript RM: collection of data, design and coordination MM: acquisition of data and drafting the article TJ: drafting the article and interpretation FR: acquisition of data and design MC: interpretation of data, drafting the article UV: drafting the article, revising and supervision ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Preoperative Sister Mary Joseph\'s nodule ultrasonography: 4 × 4 cm mass confined below the umbilicus (arrows). The main lesion is partly hyperechoic and partly hypoechoic with a poorly defined edge. ::: ![](1477-7819-3-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Intraoperative specimen of the umbilical region: Sister Mary Joseph\'s nodule and umbilicus. ::: ![](1477-7819-3-4-2) ::: Acknowledgement =============== Patients consent was obtained for reporting her case.	PubMed Central	2024-06-05T03:55:52.470914	2005-1-14	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548138/", "journal": "World J Surg Oncol. 2005 Jan 14; 3:4", "authors": [ { "first": "Fabrizio", "last": "Panaro" }, { "first": "Enzo", "last": "Andorno" }, { "first": "Stefano", "last": "Di Domenico" }, { "first": "Nicola", "last": "Morelli" }, { "first": "Giuliano", "last": "Bottino" }, { "first": "Rosalia", "last": "Mondello" }, { "first": "Marco", "last": "Miggino" }, { "first": "Tomasz M", "last": "Jarzembowski" }, { "first": "Ferruccio", "last": "Ravazzoni" }, { "first": "Marco", "last": "Casaccia" }, { "first": "Umberto", "last": "Valente" } ] }
PMC548139	1 Introduction ============== The theme of this paper is that recent and proposed changes to the financing and delivery of health services in Australia have focused upon issues of relatively minor significance while failing to address adequately major inequities and system deficiencies. An intriguing question -- not discussed in the paper -- is how such drastic failures could continue year after year with little comment and no decisive policy commensurate with the magnitude of the problems. The political-sociological answer to this question is undoubtedly complex and contentious. The failure may (or may not) be attributed to understandable, even unavoidable obstacles arising from Australia\'s social history. Nevertheless it is important to be aware that these failures exist. The current state of our health services could justifiably be described as a \'silent crisis\'; service delivery is highly inequitable and inefficient; patients are dying unnecessarily and avoidable medical errors are imposing huge financial and human costs on the community. While this occurs, health policy at the political level has focussed upon cost shifting between the States and Commonwealth, between public and private sectors and between the well off and the poorer members of society. Reforms addressing the larger issue have progressed at glacial speed, relative to what is achievable, or they have stalled altogether. In Section 2 there is a brief overview of the economists\' analytical framework in order to introduce two preliminary issues, viz, the role of social values in health system reform and the constraints created by the limited availability of resources. Two commonly made but wrong inferences from this latter constraint are discussed. Section 3 is concerned with recent policy and, in particular, the changes to private health insurance (PHI) which have been introduced since July 1997. In contrast with the relativelyinconsequential (and possibly negative) impact of these policies, five major problems are outlined in Section 4, each of which has received insufficient or no attention. Policy implications and options for future policy are discussed and highlighted throughout the paper. In the final section I argue that the optimal health system -- entirely public, largely private or one of the myriad combinations between these polar options -- is the system which is most likely to address systemic failures. The most important changes to achieve the optimal system may have less to do with the public-private mix of services, or even funding, than with the extent to which these failures are addressed within any of the conceivable health systems of the future. This, in turn, may depend upon the willingness to create appropriate economic and other incentives. 2 Resources, values and the economic framework ============================================== The discipline of economics provides a framework for the analysis of options which is based upon a comparison of costs and benefits. If social welfare is to be maximised then the logic of the framework must be adopted explicitly or implicitly. The framework focuses attention upon the benefits which might be obtained when resources are used in a particular way, and the benefits which might have been obtained if they had been used somewhere else -- the (opportunity) cost of using those resources in the chosen way. Social wellbeing is maximised when the benefits exceed the (opportunity) costs in every setting and on every margin where choice is possible. While this statement is tautologically true, the focus upon choice highlights two important facts. First, choices generally do exist; the economy is flexible and choices are driven by individual and social preferences. Technical inevitabilities are rarely encountered. Second, and more fundamentally, it is necessary to define \'benefits\'. In principle, the abstract framework is consistent with an almost unlimited number of value systems. For example, in the context of an intensive care department with limited capacity, benefits and costs might be measured by lives saved and lives lost. In this simple example the cost benefit formula would translate into a policy of providing ICU beds to those most likely to live. Social objectives in the health sector are clearly more complex than in most other settings and the nexus between objectives, policies and the optimal health system is more problematical. Nevertheless, an important conclusion from this framework (which needs constant repetition) is that there is not a single \'best\' health system; rather, there are various options which are more or less consistent with different social goals. This conclusion is illustrated in Table [1](#T1){ref-type="table"} using two highly simplified but archetypal social objectives, viz, the egalitarian desire for equal access to health or health services and the social objective of maximising individual choice. As shown, the first of these objectives is more easily achieved through a compulsory public system with defined benefits and constrained choice. The second objective is most likely to be achieved in a less constrained and more competitive private system which responds to individual preferences, as described and generally prescribed by economic theory for less complex markets and social objectives. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The relationship between choice, values and the optimal health system ::: --------------------------------------------------------------------------------------------- Objectives/Social Values Option which maximises likelihood of success ------------------------------------------ -------------------------------------------------- Equalise access, outcome Universal (monopoly) Public Insurance/Financing Maximise choice; diversity + safety net\ Pure private (competitive) scheme Optimise the max of these 2 objectives Both of the above Mixed public-private scheme --------------------------------------------------------------------------------------------- ::: *Conclusion 1:The form of the optimal health scheme depends upon social objectives and disagreement about these translates into differences with respect to the funding and delivery of health services.* While the two objectives in Table [1](#T1){ref-type="table"} are archetypes, they broadly correspond with two important but conflicting \'world views\'; that is, with different ethical beliefs about the appropriate supply and financing of health services. The social values underpinning the competitive market model are well articulated and well labelled. Its \'liberal\' or \'libertarian\' value system emphasises the importance of individual responsibility and freedom of choice. It is the prevailing value system in many aspects of life in a democratic society and there is commonly a presumption that, in the absence of some compelling argument, liberty and choice should be maximised. In economic theory this objective is equivalent to the goal of maximising \'utility\' and the Welfarist theory of \'Social Welfare\'. Even those expounding liberal values, however, generally believe that some constraint upon choice, in the form of compulsory taxation, is justifiable to finance a limited number of public goods and that at least basic medical services should be provided for the medically and financially indigent. With this \'world view\' fairness generally equates with a vertical redistribution of income to help the most needy. In contrast, the value system underpinning the public model is less clearly articulated (at least in Anglo Saxon countries). The financing and provision of services to the entire population is often characterised as \'middle (and upper) class welfare\' and contrasted with the less intrusive \'safety net welfare\' which is all that is required to help those who cannot help themselves. This interpretation of egalitarianism does not, however, correctly represent the values which underpin the public health insurance system. These are nicely described in a report of a commission of enquiry into Canadian Medicare as follows: \'Canadian Medicare is far more than just an administrative mechanism for paying medical bills. It is widely regarded as an important symbol of community, a concrete representation of mutual support and concern\... it expresses the fundamental equality of Canadian citizens in the face of death and disease\... as the Premier of Ottawa pointed out\... \"There is no social program that we have that more defines Canadianism\"\' \[[@B1]\]. The social value or world view embodied in this quotation does not correspond with the simple notion of assistance for the indigent. Rather it corresponds with a desire to \'remove health and health care from the economic reward system\' in the same way as all citizens are, in principle, given equal protection by the law. A close analogy is the desire to have public parks which may be accessed by all members of the community without payment. The objective is not a redistribution of income or the provision of a safety net. Rather, with this view, access to public parks is one of the consequences of belonging to the community; it is a shared benefit and, as such, engenders a feeling of sharing, participation and belonging. The concept is closely linked to the notion of \'social capital\' which \'accumulates\' with an increase in communal sharing and participation. Pay parks are possible, but a fully informed community might reject this option. Its citizens may wish to live in a community where the Arts flourish, where its sportsmen and women are a source of national pride, where parks are free for all citizens, where an acceptable standard of living is guaranteed after retirement and where all citizens have access to the same range of medical services. In European countries the term \'solidarity\' is used to describe this value system. Unfortunately, in English, there is no commonly used and understood word for the concept. (\'Communitarianism\' is the closest translation.) The consequence of this is a degree of confusion in the expression of social values as both sides of the debate attempt to appropriate the word \'equity\' to support their world view. It is clearly desirable that the debate should not be derailed by linguistic ambiguities. *Conclusion 2:Health policy should be informed by a careful evaluation of the social values held by different groups of the community with respect to different elements of the health system.* While it is ultimately the responsibility of the government to decide which of these values should be embodied in policy, it is desirable for the government\'s decision to be informed by evidence concerning the community\'s values and the strength of preferences for different values systems by different groups in the community. In his influential book \'The Power of Public Ideas\' Robert Reich emphasises the importance of \'discovery\', or the process of getting the public to articulate these values \[[@B2]\]. To date, neither this nor research into health related social values has been carried out satisfactorily in Australia. *Conclusion 3:The choice between public and private funding of health services depends upon social values and, in particular, the strength of liberal-libertarian versus solidarity-communitarian values as they apply to the health sector.* Returning to the first defining characteristic of the economic framework, the emphasis upon choice conflicts with two commonly held technocratic beliefs about the inevitability of particular \'problems\'. First, the common perception that the country cannot, or shortly will not be able to afford health services is unambiguously false, at least in the foreseeable future. In the USA, per capita expenditures are about double the Australian level and the US Health Care Financing Agency (HCFA) has projected a doubling of these expenditures relative to GDP by 2030. This is technically possible. The relevant question is whether or not we obtain commensurate benefits from these expenditures and if, as a society, we chose these benefits in preference to the benefits foregone. Taking an extreme example, if Australians could spend 25 percent of GDP upon health services this option would probably be embraced enthusiastically if it resulted in an illness free life expectancy of 120. Optimal expenditures are entirely a function of the benefits we obtain and are not driven by technological imperatives. *Conclusion 4:There is no immediate limit to the optimal level of health expenditures. It is technically possible to increase present expenditures very significantly. The optimal level depends upon the costs and benefits of the various health services. Increased health expenditures should be enthusiastically embraced if they improve health and health related objectives sufficiently. Technocratic arguments asserting the economic impossibility of increased health spending or increased public funding are unambiguously wrong. At best they are based upon unstated political/ideological assumptions and not economic arguments.* A similar argument applies to the second non-problem -- the impossibility of funding health services through public taxation. Arguments of the form \'the Government can\'t afford to pay\' are also unambiguously false. The country which can afford to finance health expenditure from private health insurance can also afford to pay an equivalent amount through taxation and some have argued that PHI is, itself, a form of privatised tax. The government share of the health bill is smaller in Australia than in most developed countries. Likewise, taxation is relatively low. This implies that Australia could significantly raise its level of public funding without exceeding the tax burden which is presently experienced in most comparable countries. More fundamentally, however, the form of financing for health services is flexible and is again a matter of social choice. It is likely that this choice will be influenced by the relative costs and benefits arising from the choice, but in the health sector the known costs and benefits associated with public and private health care are not compelling. Privately funded health care is often a little more expensive, but countries with a strong preference for liberal-libertarian values might sensibly opt for a relatively larger private scheme even if it is more expensive. The principle of paying more for what is wanted should not be controversial! *Conclusion 5:The balance between public and private sources of revenue for a health service should be determined premanually by the social philosophy of the country. There are no compelling technical or economic constraints on the freedom of sound choice.* 3 Recent policy issues ====================== Public debate has recently focused upon three \'problems\', namely the high and rising cost of pharmaceuticals, the declining rate of bulk billing by General Practitioners (and particularly amongst health care card holders) and the declining number of people purchasing private health insurance. The comments below do not purport to be an exhaustive analysis of these subjects but are included to contrast the subject matter of the policy debate with the more substantive problems which will be discussed in the following section. Pharmaceuticals and PHI are discussed more fully in Richardson and Segal \[[@B3]\]. Pharmaceuticals --------------- Pharmaceuticals are included in the Pharmaceutical Benefits Scheme (PBS) after a detailed review of their effectiveness and cost effectiveness (see Salkeld et al 1998 for a description \[[@B4]\]). This process does not, by itself, reduce expenditures. Rather, it ensures that drugs whose effectiveness is low in relation to their cost will not be adopted. Expenditures will be lower if the manufacturers of relatively cost ineffective drugs reduce their prices to increase the likelihood of their inclusion in the PBS. However, this may be offset (more or less) if drug companies increase the price of new highly cost effective drugs as they know that the PBAC is aware of their cost effectiveness. Cost effective drugs may also be overused if doctors prescribe them for purposes or at thresholds not tested before their introduction. Partly for this reason the PBAC has sometimes negotiated a price-volume trade-off -- if drug use exceeds the initial expectation then the agreed price is lowered. These measures have not contained pharmaceutical costs. In view of rising drug prices in other countries it is likely that this is primarily or entirely attributable to the high cost of new generation drugs and is not attributable to policy. Nevertheless, expenditures have risen and in recent years copayments have been progressively increased in order to reduce government expenditures through the PBS. However it is not clear that the use of copayments, and particularly in a single sector, will have an overall beneficial effect. First, as demonstrated by the Rand Experiment copayments reduce demand somewhat (elasticities are low but not insignificant) \[[@B5]\]. There is a disproportionate effect upon low income households \[[@B6]\] which, in this case, implies low income non health care card holders. There is little evidence that lower income patients will discriminate between effective and less effective drugs and at least one study suggests that, perversely, the greatest impact will be upon life saving drugs which have a relatively small immediate impact upon symptoms \[[@B4],[@B7]\]. Secondly, relatively larger copayments in one sub-sector violates a fundamental principle for achieving allocative efficiency; viz, a \'level (financial) playing field\' between alternative products/interventions. Violation of this condition increases the likelihood that less cost effective services may be used because of the distorted price signals. Allocative efficiency depends upon relative prices rather than the existence of copayments per se. More specifically, high pharmaceutical prices at the point of service will encourage the use of potentially more expensive interventions including hospital services if a copayment results in the deterioration of a patient\'s health. Thirdly, the effects of copayments on national health expenditures will be relatively small. Wealthy individuals will simply pay the copayment and health care card holders will be largely shielded from them. The most important effect of the copayment is that it will shift costs from the taxpayer to the patient; that is, its major effect will be upon the distribution of income with the healthy-wealthy taxpayer gaining at the expense of the less healthy-less wealthy. In 1960, pharmaceutical costs represented an estimated 22.3 percent of health expenditure. By 2002 they were 13.5 percent of the total. The comparison indicates that the composition of expenditures may vary significantly through time and, taken out of context, focus upon a single sub-sector may be misleading. Cost control requires a full system perspective and there is no optimal level of pharmaceutical expenditures which is independent of the cost of alternative services. The problem is, in part, attributable to the program structure of delivery and financing which treats pharmaceuticals as an independent program as distinct from an input into an overall treatment regime. *Conclusion 6:Reliance on copayments for the control of pharmaceutical expenditures is probably an inappropriate policy as it violates an important principle for achieving allocative efficiency. A more global approach to health policy analysis and reform is needed.* Bulk billing ------------ Since the introduction of Medicare the percentage of GP attendances bulk billed rose from 52.5 percent in 1984/85 to a maximum of 80.6 percent in 1996/97, then fell to a low of 66.5 percent in December 2004. The measures discussed below appear to have arrested this trend and by March 2004 bulk billing had risen to 68.7 percent of GP attendances \[[@B8]\]. The chief problem the reforms were designed to overcome was the possibility that the falling level of bulk billing may have jeopardised access to health services by pensioner/health care card holders and other low income households. Importantly, data did not exist to demonstrate that this was, indeed, a problem and that bulk billing or service use by this group had declined by the average or near average percentage. That is, the existence and quantitative significance of the \'problem\' were not documented. The recent \'Medicare Plus\' package introduced a potentially important structural change. Rebates for bulk billed/health care card holders were separated from the rebate to the general public thereby allowing the separate treatment of the two groups. To encourage the bulk billing of the first group the benefit was increased and bulk billing doctors were permitted, for the first time, to direct-bill the Health Insurance Commission for general patients while simultaneously charging an \'over the counter\' extra payment, a measure widely perceived as encouraging an increase in fees as patients will commonly equate the relatively small copayment with the total fee. These measures have two important effects. First, the probable increase in general fees allows the benefit payable for GP bulk billed pensioners/health care card holders to be at a lower level than would occur than without the effective cross subsidy from increased general fees. Secondly, the government can preserve \'equity\' (ie bulk billing the needy) by adjusting only the pensioner/health care card holder benefit, while simultaneously allowing a deterioration of the general rebate. Cost shifting would have been further facilitated by an earlier agreement to allow private health insurers to reimburse medical expenses above the schedule fee as part of an agreement with doctors to remove out-of-pocket costs. But this proposal was rejected by the Senate. In sum, there is now a structure which facilitates cost shifting from the public to the private sector. There are two important caveats to this conclusion. First, a rebate structure which facilities additional cost shifting does not imply that government will use this option in the short or long run. Secondly, the effect of additional cost shifting will be mitigated by an additional element of the Medicare Plus package, namely, the introduction of a \'safety net\' which reimburses 80 percent of out-of-pocket, out-of-hospital costs including billing above the schedule fee once family expenses reach \$1,000 (or \$500 for a health care card holder\'s family). The long run effect of this latter cost off-set is difficult to assess. As reported in the announcement of Medicare Plus the expected cost of the safety net in its first three years would average \$89 million p.a. as compared with likely out of pocket medical expenses of about \$1,400 million. in 2004/05 (extrapolated AIHW) \[[@B9]\]. Even allowing for a probable cost overrun the amount is not large. Nevertheless, the \'safety net\' offers additional protection to those who would be most affected by additional cost shifting; that is, this policy also helps create a structure where equity, defined in terms of need, may be reconciled with increased cost shifting. However, the most significant feature of these changes to the reimbursement formula for medical expenses is that they deal with relatively\'small order\' issues. Under Medicare, pensioners/health care card holders with significant health problems have access to emergency and outpatient facilities at public hospitals. Poor access to these will increase inconvenience and queuing and, in an unknown number of cases, result in poorer health. But this more serious outcome is likely to be relativelyinfrequent as the majority of this group have always been bulk billed while others will have satisfactory access to hospital based care. *Conclusion 7:Proposed and actual policies towards pharmaceutical expenditures and bulk billing have had a common element. Both represent immediate or potential cost shifting from the government to the public and any net reduction in societal expenditure because of a reduction in patient initiated services will have potentially adverse effects upon the health of the \'near poor\'. However, the financial effects of the policies are relatively small and the indirect adverse effect upon health is likely to be correspondingly small.* Private health insurance (PHI) ------------------------------ The role and regulation of PHI after the introduction of compulsory national health insurance has been ambiguous and anomalous. Since Medicare was introduced as the vehicle for achieving fairness in the financing and delivery of health care it has never been clearly stated why PHI should be closely regulated to achieve fairness in he sub-group of the population which elects to purchase PHI and why it is regulated in a way which inhibits effective competition. But these are the effects of Australia\'s community rating and reinsurance requirements respectively. In the last one and a half decades there has been an ongoing concern that declining levels of private health insurance have had an adverse effect upon the public hospital system and that \'PHI reform\' has been needed to solve this problem. The argument is summarised in the following (constructed) quotation: \'Private health insurance was caught up in a downward spiral caused by the adverse selection identified in the Productivity Commission report \[[@B10]\]. \'Rising premiums triggered by increasing costs induced low risk members to withdraw. Without their cross subsidy of high cost members, premiums were forced to rise again, which further increased premiums, which induced further departures, etc. \"PHI is primarily purchased to cover the costs of private hospitalisation. Consequently, as PHI declined through time, fewer patients have been able to afford the cost of private hospitals, and this has created an excess demand for public hospital beds. The increasing length of queues is a confirmation of this problem\". While plausible, the account of history in the second half of this argument is wrong. Between 1985/86 and 1999/00 private hospitals increased their share of admissions by 32.4 percent from 25.9 to 34.3 percent of the total. The share of bed days rose from 21.9 to 28.1 percent of the total or by 28.3 percent \[[@B3]\]. These simple and readily available statistics unambiguously contradict the conventional wisdom propagated by the media and some politicians. It is true that queues in public hospitals have increased, but this is attributable to the increasingly draconian budget caps upon public hospitals throughout the 1990s. That is, queuing in the public sector has been primarily a result of supply side and not demand side factors. (Excess demand for beds cannot explain the closure of wards that occurred in many public hospitals!) The simplicity of the statistics contradicting the conventional wisdom calls into question the analytical capacity of many media commentators (and some independent commentators)! *Conclusion 8:Media analysis of the relationship between PHI and queuing in the public system highlights an important system failure; viz, the failure of the media to exercise rudimentary critical skills in their analysis of PHI and hospital queuing.* Even if PHI membership had fallen sufficiently to have reduced the share of private hospital admissions by almost a third, the balance of public and private hospital separations in 1999/00 would have been similar to the share in 1985/86. This suggests that it was the events summarised in the first half of the quotation which were of concern at the government and departmental level and that PHI membership and the public-private mix of health financing and delivery were explicitly or implicitly the real target of health policy. In response to the \'problem\' of falling PHI and the demand side problem for public hospitals (which did not exist!), the government introduced three enduring policy changes. In July 1997 individuals with an income above \$50,000 and families with a combined income above \$100,000 who did not purchase PHI became liable for a 1 percent tax surcharge upon their incomes. In December 1998 a 30 percent rebate on PHI premiums was introduced. (In August 2004 a selective increase in the rebate was foreshadowed); and in September 1999 lifetime community rating was enacted which has the effect of reducing future premiums for those who have held PHI from the age of 30. Discounts apply at any age between 30 and 64; 30 is the age of maximum discount. Failure to enrol by this age will result in a higher premium if PHI is subsequently purchased. The evidence demonstrates that these policies, and particularly the last policy, have succeeded in increasing PHI membership. However it has created an industry which, along with the platypus and echidna could be Australia\'s entrants into the world\'s strange but true contest. Australia would almost certainly win -- even without our egg laying marsupials. First, the surcharge results in a negative price for the wealthy. Individuals and families above the income threshold avoid an increasingly large tax payment as their income rises; that is, if they purchase PHI they will have a greater income at the end of the year than the individuals and families above the threshold who do not buy PHI. This is analogous to supporting the automobile industry by placing a surcharge on wealthy families who fail to buy an Australian car. It would be difficult to find any other support scheme which uses the income tax system to coerce the purchase of a particular product. It would be equally difficult to find a produce where the price is negative. Secondly, (and predating the recent legislation) the use of PHI to cover hospital bills generally results in a greater, not smaller, out of pocket expense. Public hospitalisation is free. Those with PHI commonly pay a copayment. (There is a perverse equity in these two anomalous outcomes. The wealthy are paid to take PHI but financially penalised if they use it!) Third, lifetime community rating also involves a leap of faith. The 30 year old must believe that public policy will remain stable for 50 years -- a belief that should have been challenged when both parties announced changes in the 2004 election campaign. However lifetime community rating also has a bizarre dimension. Insurance is generally purchased to reduce risk and uncertainty. In the health sector these arise because of the risk and uncertainty of ill health and the cost of medical care. Prior to lifetime community rating this uncertainty depended upon possible events in the following 1--3 years. After the change it depended upon the next 1--30 years. Events in 30 years are more uncertain than events in 3 years and, consequently, the legislation increased risk and uncertainty. The perceived risk was amplified by the publicly financed marketing of private insurance. Predictably, people responded to the increased uncertainty by buying more insurance. Thus, to encourage the uptake of insurance the government increased the very thing insurance is designed to reduce, viz, risk and uncertainty. A final anomaly which predates the recent PHI reforms is that private health insurance in Australia leaves the consumer with residual risk. Particularly for ancillary services, most benefits are capped and out of pocket expenditures rise with the price of the service. This pattern of benefits is exactly the opposite of the structure of benefits which will maximise the patient\'s \'expected utility\' \[[@B11]\]. Taken together, the changes introduced since July 1997 have created an extraordinarily complex and perverse set of incentives. The ethical basis of the free market and the liberal-libertarian model is that choices should be determined by individual\'s preferences in relation to real (opportunity) costs. The surcharge subverts this process and coerces choice and the strength of the coercion is based upon economic class. There is no justification for this in the economic theory of the efficient market. Despite this conclusion, it is a legitimate function of government to determine the balance between public and private delivery financing and the distribution of health care costs. As described earlier the preference for private sector funding and provision (albeitin the context of compulsory core insurance) is consistent with a legitimate and defensible world view, viz, the liberal-libertarian belief that in a free society individuals should be encouraged to take responsibility for their own lives. In particular, the 30 percent subsidy is the orthodox approach to encouraging an industry which has a special claim for protection and, private health insurance does not compete on a \'level playing field\': it competes with a public sector which is free at the point of service. There are, however, two important caveats. First, and as noted above, the measures taken have destroyed any nexus between potential benefits and the price paid by wealthy individuals. Secondly, the \'product\' is unlike usual insurance where the benefit -- a payment after an adverse event -- does not impinge upon other individuals. PHI is purchased to avoid queues and to select the best possible doctor. With fixed capacity, the avoidance of queues by one person imposes a longer queue on another person -- there is a \'negative externality\'. Selection of the best doctor reduces the access to these doctors by public patients. Consequently, the important debate should be about the \'right\' to purchase preferential care at the expense of those who do not have PHI. In a liberal democracy there is a presumption that individuals may spend their own income as they wish. For the individual there is probably no more important context for exercising this \'right\' than in the context of preserving life and its quality. There is, therefore, a head-on-head conflict between the liberal-libertarian \'right\' of the individual to spend his or her income on health care and the communitarian-solidarity based \'right\' of each individual in a community to have equal access to high quality medical care. The latter goal must necessarily be achieved by imposing some constraint upon income-based preferential care to a particular group in the community. In Australia the constraint has been a de facto financial penalty for seeking priority care. Until the advent of engineering of negative prices for the wealthy, the purchase and use of PHI resulted in \'double payment\' for some services, first via taxation and the Medicare Levy and secondly by premium paid for PHI. This \'penalty\' still exists for lower income individuals and households. *Conclusion 9:The public debate over Private Health Insurance has been misleading. The contentious issues do not only concern the most effective way of ensuring access to health care (with the erroneous presumption that public monies not spent on health services represents wasted resources). Rather, the contentious issues include the \'right\' or otherwise of the individual to spend their own income on whatever they wish without coercive financial penalties and the consequences for the remainder of the community when one group jumps the queue and has priority access to the most experienced doctors.* Distributional effects of recent policies ----------------------------------------- PHI policy has been consistent with the other policies discussed above in one important respect. The policies are likely to have a relatively small effect upon the deliveryof health services. Rather, they are about financing care and the public-private balance in the health system. The balance, in turn, affects the distribution of health care costs between different households. Copayments shift costs from the government to patients. As government payments are met by progressive taxation, copayments redistribute the cost of health care from wealthy-healthy taxpayers to the less healthy, less wealthy. Additionally, as copayments have a disproportionate effect upon the use of services by low income households, the proportion of the government subsidy returned to high income households rises as copayments rise. The redistributive effects of PHI are more complex. Low income households which purchase PHI unambiguously pay more for hospital and health services. Their taxes and the Medicare surcharge are unchanged and the purchase of PHI leaves them out of pocket. For higher income households, which are liable for the PHI surcharge, the effects are conceptually ambiguous as they depend upon the assumption made about the surcharge in a counterfactual world in which PHI did not result in a surcharge exemption. The surcharge was created specifically to permit an exemption for those who purchased PHI and the removal of the exemption might therefore be accompanied by the removal of the surcharge. Finally, and as noted earlier, the proposed changes to bulk billing represent a structural change which will facilitate the transfer of medical insurance costs from the public to the private sector. *Conclusion 10:Recent and foreshadowed legislative policy initiatives with respect to bulk billing, copayments and PHI, all concern the financing of health services. Recent policy has been introduced with measures to mitigate the impact upon the most needy and, in the case of medical rebates, in a way which accommodates the popularity of bulk billing. Nevertheless, a common feature is that each of the proposed or implemented policies assists with the creation of a structure which facilitates the transfer of expenditure from the public to the private sector. If this occurs, it will reduce the cross subsidy from healthy wealthy to unhealthy less wealthy households. This effect occurs immediately with copayments. The transfers are more complex in the case of PHI.* 4 Five major problem areas ========================== The five issues below have two common elements. First, they are directly concerned with health services and not the distribution of costs and incomes. Second, they have been almost ignored in the public policy debate despite being problem areas where there are quantitatively large inefficiencies and a corresponding potential to increase health outcomes and/or increase the overall cost effectiveness of the system. Efficacy, effectiveness and social objectives --------------------------------------------- In common with all other health systems many, and probably most, of the services provided in Australia have not been adequately evaluated and there is no ongoing process for the elimination of cost ineffective services. In 1987 Chassin et al \[[@B12]\] estimated that between one third and one half of coronary services in his study were \'inappropriate\' in the sense that they had no beneficial, or a detrimental affect upon health. An additional one third to one half of the procedures considered had equivocal benefits. Likewise, Brook \[[@B13]\] estimated that 51 percent of angiography and 42 percent of coronary artery bypass graft procedures were unnecessary. Other studies by the US Health Care Financing Agency and the OECD have likewise concluded that only a small number of services have been evaluated for efficacy \[[@B14]\]. In Australia Segal \[[@B15]\] demonstrated that in the context of diabetes the cost of obtaining a life year varied from \$70,000 (drug therapy) to \$2,400 for behavioural programs. Comprehensive diabetes care was estimated to have a negative cost per life year; ie the program saved life and saved cost. With respect to this issue, the Australian record is comparatively good. It has led the world with the introduction of mandatory economic evaluations for the drugs and services to be subsidised through the PBS and the Medical Benefits Scheme respectively. However the failure of other countries does not indicate that Australian procedures are satisfactory. The overwhelming majority of the services which were accepted before the introduction of mandatory economic evaluations have not been assessed and, with the passage of time, there is a need for the reassessment of services and drugs. A related problem is that drugs or services which are efficacious when used appropriately in a random control trial may be used inefficiently with inappropriate patients who do not have the clinical indications of the patients in the trial. Gold standard medical care requires that cost effective therapy, drawing upon evidence-based medicine, should be the norm and not the exception in medical practice. Cost effectiveness should be determined by a broad ranging comparison of options form across the full range of potential interventions; that is, comparator interventions should take account of substitute services from across the entire health sector. This does not presently occur. Guidelines for the PBS require a comparison between drugs and exclude comparison with lifestyle/behaviour change programs. *Conclusion 11:The scale of present evaluation activities is inadequate. In an industry absorbing 9 percent of the GDP -- the country\'s largest industry -- there should be ongoing and large scale evaluation and re-evaluation of the cost effectiveness of the services provided. Evaluation should be based upon a comparison with the full spectrum of substitute services. A failure to do this almost certainly ensures that there will be widespread and significant allocative inefficiency in the level and mix of services.* Implementation of evidence based practice will inevitably be resisted by service providers whose practice and income may be adversely affected. Consequently, provider education for a \'culture change\' is likely to be a slow and ineffective vehicle for change. In contrast, once the desired practice pattern has been determined, financial incentives to encourage this form of practice may be implemented relatively quickly and do not involve direct coercion. The economics literature is replete with examples where such incentives have significantly altered provider behaviour \[[@B16]-[@B19]\]. Australian examples include the use of GP incentives to bulk bill; to undertake rural practice and to increase child immunisation. DRG based financial incentives were spectacularly successful in increasing hospital throughput in Victoria in the early 1990s \[[@B20]\]. *Conclusion 12:Reimbursement formula for service provision should include financial incentives at all levels of service delivery for the use of the most cost effective therapies.* The more general principle which should, but does not, permeate the market for health services is that the willingness to pay for services should vary with social objectives, a principle which is successfully incorporated in simple competitive markets when social and individual objectives are identical. As one example where this principle has recently and successfully been used, if society wishes to encourage bulk billing (which clearly benefits patients but lessens provider control over their incomes) then the benefit for services that are bulk billed should be increased relative to the rebate for other services and the differential increased until the target of bulk billing is achieved. Likewise, if society wishes providers to adopt evidence based medicine, hospitals to install clinical pathways, some procedures to be encouraged and others discouraged, then payment for the desired service or process should be increased relative to the reward when these services or activities do not occur. At the level of the individual service, recent research has demonstrated health-related social objectives which are often broader than the minimisation of morbidity and mortality, for example, preferential treatment for particular age groups, particular classes of patients and health benefits \[[@B21],[@B22]\]. Presently these additional \'social preferences\' are ignored. This is unsurprising as the research to identify and measure them is still underway. However, where these preferences are significant and consistent, payments should eventually be adjusted to ensure that they are achieved. *Conclusion 13:The payment of service providers should incorporate the principle that society should pay for what it wishes to have in accordance with its social objectives, rather than what it is given. This implies the need for ongoing enquiry into health related social objectives (the numerous objectives loosely grouped under the heading of \'equity\').* Practice variations ------------------- In 1982 John Deeble and I used data from the first full year of compulsory health insurance, (Medibank), to determine the level of service use in each of Australia\'s statistical divisions \[[@B23]\]. An example of the results is shown in Table [2](#T2){ref-type="table"}. Huge variations in service use were detected even between the relatively large geographical units used in the study. Within these units small area variation would have further increased the discrepancies. These differences do not appear to have diminished with time. Thus, for example, Robertson and Richardson \[[@B24]\] found startling variation in the use of well-defined hospital procedures even after standardisation for age, sex and population. In this study data were collected for a two year period for each of Victoria\'s statistical sub-divisions. Procedural rates were expressed as a percentage of the rates which would be expected from the State average service use per age-sex cohort and from the demographic characteristic of the Statistical sub-division (SSD). Results are summarised in Figure [1](#F1){ref-type="fig"}. The bar, lines and circles give an indication of the frequency distribution of the procedure utilisation rates (ie the 25 and 75th percentiles (bars), two standard deviations (lines) and outlying SSDs). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Practice Variations 1976 ::: Consultations per capita ------------------------------ ------------- ------------- ----------- Statistical Division GP/(GP) Q(Spec) Total Sydney 5.1 2.3 7.4 Tasmania 3.1 1.3 4.4 Darwin 1.1 0.5 1.6 Sydney/Darwin 4.6 4.6 4.6 ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Standardised Rate Ratios for Various Operations in the Statistical Local Areas in Victoria, Compared to the Rate Ratios for All Victoria, Source: Richardson 1999 p198. ::: ![](1743-8462-2-1-1) ::: The results reveal an 8 to 10 fold variation in service use. Part of this is attributable to the random variation that would be expected because of the uncertainty of the episodes of ill health. Using state-wide data the ratio of actual to expected variance was calculated. This is reported in the column of numbers to the left of the bar diagram. For the first procedure, coronary angiography, the observed variance was 13.4 times greater than expected; ie actual variance was 1,440 percent of the expectation. This extraordinary result is reproduced for all of the procedures examined. A second example of uneven service delivery was also published by the same authors. This related to the likelihood of obtaining a high tech procedure -- angiography or revascularisation -- after a heart attack. Results shown in Table [3](#T3){ref-type="table"} indicate that in the 14 days following the heart attack, men and women admitted to a private hospital were 2.2 and 2.27 times more likely to receive angiography than their counterparts at a public hospital. They were 3.43 and 3.86 times more likely, respectively, to undergo revascularisation (coronary artery bypass surgery angioplasty, stent). These discrepancies did not diminish significantly in the following 12 months. The same study identified statistically significant differences in the likelihood of a procedure between men and women, young and old, urban and rural populations. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Ratio: (Likelihood of procedure after admission to a private hospital) ÷ (Likelihood of procedure as a public patient) ::: Angiography Revascularisation ------------------ ----------------- ----------------------- Within 14 days Men 2.20 3.43 Women 2.27 3.86 Within 12 months Men 2.16 2.89 Women 2.22 2.84 ::: Taken together these studies suggest that there is a highly erratic pattern of service delivery across Australia and between social groups. One of three conclusions is inevitable. Some groups are under-serviced; some groups are over-serviced or both of these problems occur to different sub-groups of the population. This indicates both allocative inefficiency (more health could be obtained with a redistribution of existing resources) and significant inequity for those with poor access to health services or a different form of inequity for those persuaded to undergo procedures where risks exceed likely medical benefits. Government has shown almost no interest in this type of result. There has been recognition of an \'urban-rural\' discrepancy but, 20 years after the demonstration of a more complex pattern than a simple urban-rural dichotomy, remarkably little has been achieved. *Conclusion 14:Significant variation in the use of services has been allowed to continue more than two decades after it was identified. Small area variation across Australia almost certainly reflects significant allocative inefficiency and an inequitable access to health services. There appears to have been relatively little interest in this problem.* Lack of coordination -------------------- The health system at present consists of a series of financially independent programs (\'silos\'), which are poorly linked to other health services or programs. There appears to be near universal agreement that a sensible health scheme cannot be built upon the current Federal-State division of financing, responsibilities and powers or upon the existing Commonwealth program structure. In the context of a recent review of hospital care, the Tasmanian Government accepted a recommendation for the pooling of all health and aged care resources \[[@B25]\]. However implementation of the recommendation requires Commonwealth support which may or may not be forthcoming. The fact that a simple solution exists for this most elementary problem but, to date, has not been seriously addressed, represents a fundamental failure of government in Australia. Two case studies are used below to illustrate what gold standard allocative efficiency would imply for the use and coordination of services. They indicate the distance that health services reform must travel in Australia before we have gold standard delivery. The first case study is real. A Seattle-based \'pure\' Managed Care company, Ethix, was asked to establish a health scheme for a small town close to Seattle. Routine surveillance of the medical claims over the first two years of the new scheme highlighted an anomaly. There were excessively large numbers of youths receiving surgery for spinal injuries. Further investigation found that the problem was attributable to a toboggan run on the outskirts of the town which had a tree stump half way down the slope. Youths were crashing into the stump and damaging their spine. The health scheme paid for a bulldozer to remove the stump \[[@B26]\]. Medicare does not pay for bulldozer services. However, in the circumstances described here it should do so. More generally, the vignette illustrates two of the characteristics of gold standard delivery, namely, routine data surveillance to locate problems with any aspect of social or medical behaviour which might be modified to improve health and, secondly, the flexibility of funding which is needed to adopt the most cost effective solution to the identified problems. In contrast, in the Australian health scheme a problem of this sort would not be detected by the health system. It is possible that a similar type of accident could fall under the jurisdiction of an occupational health and safety or traffic accident authority. Otherwise there would be neither the will nor the means to respond. If such a problem was eventually identified the typical response would be accusation, blame shifting and, possibly, litigation. The cause of this problem is related to the practice of Management by Objectives, an aspect of managerialism, which encourages local rather than system-wide thinking. Interestingly, Peter Drucker, one of the early advocates of MBO, in recent years has publishes warnings about its over-use and limitations. *Conclusion 15:A key challenge is to establish a single payer (for each person) with flexibility and incentives to purchase the most cost effective services. Services should not be determined by historical program boundaries and rigid budgets. There has been a serious failure by government to address this fundamental issue. The achievement of this relatively simple reform is necessary (but not sufficient) for the achievement of a range of system reforms.* The second case study is a hypothetical scenario constructed by Duckett to illustrate gold standard system coordination and processes \[[@B27]\]. \'A woman with dizziness is concerned about her health. She rings the State call centrewhich advises her to visit her local health team. She is able to see the GP quickly who asks her a series of questions from the relevant research based protocoland undertakes a clinical examination. The GP emails the results to a local specialist\... who orders some further investigations consistent with the state research based care path\... Adviceof (an) impending admission is automatically conveyed electronically to the GP and the social worker in the referring health team. The social worker contacts the hospital to discuss discharge planning\... The specialist\... suggest(s) a number of sources for information about the patient\'s condition. The patient contacts the call centre for further information\... The case is randomly selected by the hospital audit committeefor quality review. The committee suggests some slight changes to the state-wide protocol committee.\' p204. The key elements of this scenario relate to information access and transferral. It illustrates the role of evidence based medicine, routine service review, the adaptation of protocols, universal electronic transfer of all information and the absence of incentives to depart from best practice. Parts of this scenario correspond with Australian practice. But the events in italics would be unusual. It appears to be serendipitous whether a particular problem of a particular person and in a particular part of Australia results in a response which even partially mirrors the gold standard response in this scenario. *Conclusion 16:Commonwealth and State authorities should mandate practices which improve the coordination of services. To facilitate this there should be universal use of electronic data systems for patient notes and information transfer. Evidence based protocols and clinical paths should be adopted when available and relevant. Feedback and error learning should be a routine part of the system. All of these desirable features are presently difficult because of the fragmentation of the system which is, in part, attributable to the failure to establish a single funder for all health services received by an individual.* Quality of care --------------- Results from the 1995 \'Quality in Australian Health Care Study\' (QAHCS) suggest that the quality of health care in Australia is a problem which overshadows all others. In the initial study, reported by Wilson et al \[[@B28]\] medical records for more than 40,000 admissions to 28 hospitals in NSW and SA were individually examined to determine whether or not an adverse event (AE) was associated with the admission (prior to or during the episode of hospitalisation). A judgement was made concerning the consequences of the AE and whether or not it might have been avoided. By extrapolating results the authors estimated that about 470,000 admissions were associated annually with an AE and that these would have resulted in 18,000 deaths and 50,000 cases of permanent disability. In a subsequent report Runciman et al \[[@B29]\] estimated that in 50 percent of the AEs in the QAHCS had a high preventability score. Sixty percent of deaths could have been avoided. In this latter study, incidence and not prevalence scores were reported as part of the effort to standardise the methodology with an earlier Harvard Medical Practice Study (HMPS) reported by Brennen et al \[[@B30]\]. This reduced the annual rate of AEs to 10.6 percent of admissions. From the response to these events (discussed below) it appears likely that many have been unable to appreciate the scale of the carnage implied by the report. If the results from the original QAHCS are not discounted then medical errors have been responsible for the death of more Australians per annum than the average annual death rate of Australian soldiers in World War 1 (15,800). Permanent disabilities per annum approximate the annual rate of casualties in The Great War (62,500). Unlike The Great War, the problem of AEs has probably been ongoing for 50 years or more. In Table [4](#T4){ref-type="table"} below the death rate from AEs is compared with mortality rates from other causes which are of particular social concern. To be conservative and to take account of undetected bias, the AE rate reported in a 1995 study is reduced by 50 percent. Preventable deaths are assumed to be 50, not 60, percent of deaths associated with an AE. The resulting, conservative number of deaths from AEs in 1999 were about 40 percent higher than the number of deaths from AIDS, suicide, motor vehicle accident, accidental falls, homicide, drowning and poisoning combined. In Table [5](#T5){ref-type="table"} some equivalent events are listed. The conservative estimate of the unnecessary death rate is about the same as would occur if the Bali bombing occurred every week of the year, year after year. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Perspective: Selected Causes of death, 1999 ::: Cause No of deaths ------------------------------------------------- ------------------ AIDS 122 Suicide (intentional self harm) 2,492 Motor vehicle accidents 1,741 Accidental falls 520 Homicide 300 Accidental drowning/submersion 278 Poisoning by drugs/medications 1,015 Subtotal 6,468 All deaths from adverse events^(1)^ 9,000 All preventable adverse events^(2)^ 4,500 \(1) 50 percent of reported estimate \(2) assuming 50 not 60 percent are preventable ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Events equivalent to avoidable AE deaths\* ::: Cause --------------------------------------------------------------------------- 1 in 10 customers in restaurants poisoned each year: annual deaths 4,500 13 Jumbo jets crash each year, each with 350 Australian passengers killed 45 Bali bombing type attacks, each with 100 Australians killed each year \'September 11\' every 8 months: only Australians die \assuming preventable deaths = 25% QAHCS (1995) ::: While those affected by adverse events will, on average, be older and frailer than those who died in Bali or during World War 1, the magnitude of the problem is still staggering. Considering the reaction to the Bali bombing it might have been expected that the publication of the QAHCS would have caused a seismic shock, with the public demanding immediate, comprehensive reform and government passing urgent legislation to mandate any achievable system reform which ameliorated the problem. If the system failures which preceded the Bali bombing warrant a Parliamentary inquiry, equivalent interest might have been expected with respect to a problem responsible for an equivalent number of deaths every week. An effectively unlimited budget might have been approved for the upgrading of quality and safety. In contrast, the actual reaction to the report must constitute one of the more puzzling episodes of Australia\'s social history. At all levels the response was sedate, cautious and incremental and there appears to have been greater concern that our health system might be perceived as unsafe than with the fact that it actually is unsafe. The level of activity in the 5 years following publication of QAHCS suggests that the results may not have been truly believed. But no subsequent study was funded to validate or refute the results. The QAHCS results were not ignored. As summarised in Table [6](#T6){ref-type="table"} an advisory body was immediately established which evolved into the Australian Council for Safety and Quality in Health Care (ACSQHC). Its activities are summarised in five successive annual reports. In addition, the Australian Health Care agreement between the Commonwealth and State governments allocated budgets of \$680 million and \$785 million for quality assurance activities for the periods 1998--2003 and 2003--2008. In each of the States sub-committees and working groups were created which, along with the ACSQHC have resulted in a very large number of reports, publications, some legislation and local initiatives. The ACSQHC alone lists 35 publications \[[@B31]\]. State activities are summarised in ACSQHC, \[[@B32]\]. These initiatives have resulted, inter alia, in moves to tighten up hospital accreditation processes; to monitor adverse events more closely; to improve consumer participation in the evaluation of health care; to encourage health professionals to report adverse events; to improve health information technology; to establish practice guidelines, etc. The large number of funded projects are described and listed in ACSQHC, \[[@B33]\]. ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Response to QAHCS ::: ------ ------------------------------------------------------------------------------------------------------------ 1995 QAHCS published 1996 Taskforce established on QAHC 1998 Health ministers ask Advisory Group for report 1998 Interim Report 1999 Report of the National Expert Group \'Actions identified by the taskforce\... need to be implemented at all levels of the health system \...\' 1999 Australian Council for Safety and Quality in Health Care Established ------ ------------------------------------------------------------------------------------------------------------ ::: Despite the high level of activity, the importance, priority and sense of urgency reflected appear more appropriate for an (important) ongoing reform process in an already well-operating system. There is a yawning gulf between this and what the public would undoubtedly demand if our TV and news services were reporting deaths on the scale of the September 11 New York disaster very two months accompanied by about three times this number of injuries. The philosophy of the reform process appears to be summarised by the ACSQHC when it approvingly quotes Berwick as saying that \'there are no quick fixes. We must re-examine all that we do\', p14 \[[@B31]\]. As a description of history the \'no quick fixes\' statement is correct. The 1999 report -- 4 years after the publication of the QAHCS recommended that \'actions identified by the taskforce\... need to be implemented at all levels of the health system\'. By 2001 NSW had passed legislation which, inter alia, created the Institute of Clinical Excellence. By 2002 -- 6 years after publication -- the Victorian Quality Council Plan had been established. In 2003 the updated version of this plan set as its goals, inter alia, the establishment of a framework, the involvement of consumers and education. Almost 10 years after the report, the ACSQHC called for mandatory participation of all hospitals in a process of assessment \[[@B31]\] and (with possible irony or ill concealed frustration) the Chairman of the ACSQHC argues in the 2004 Annual Report that \'Action must be taken without untimely delay where culpability is clear\', p5 \[[@B34]\]. Between publication of the QAHCS and the Health Ministers\' request for a report in 1995, 13,500 Australian would have died and 37,500 become permanently disabled. By the time the 1999 report was recommending the implementation of various policies at least 18,000 would have died. By the time of the NSW legislation cumulative national deaths would have reached 27,000. The Victorian Strategic Plan to develop a framework was published after the death of at least 31,500. It is scarcely surprising that in 1999 an editorial in the Medical Journal of Australia commented that: \'Welcome though (various initiatives) are, the pace of change nevertheless seems slow given the stark message of the original QAHCS study four years ago\... 50,000 Australians suffer permanent disability and 18,000 die at least in part as a result of their health care\', p404--50 \[[@B35]\]. By 2002 Siddons could still comment that: \'On the 10^th^anniversary of the study year, the most striking outcome has been the paucity of reform currently exhibited at the coalface of tertiary health care\', p823 \[[@B36]\]. The inadequacy of the response is surprising as the evidence suggests that a reduction in AEs would be spectacularly cost effective. The interim report from the national expert group estimated the potential savings from preventable adverse events in 1995/96 would be \$4.17 billion per annum. Consequently, expenditures of this amount could be justified if they eliminated the unnecessary AEs or if the cost of the achievable 50 percent reduction in AEs was less than \$2 billion per annum. In contrast with the view that little could be done quickly there are a number of examples where, prima facie, very significant and effective change could have been/can be rapidly implemented. The chief distinguishing feature of each of the suggestions below is that they involve legislation and regulatory enforcement which appears to be inconsistent with the apparent emphasis upon persuasion and voluntary culture change in many of the present activities. Mandated options include the suggestions below. AccreditationFor decades health professionals have believed that a significant number of small hospitals are dangerous. However there has been no decisive action. With full knowledge of the QAHCS results, hospital accreditation remains voluntary in all States except Victoria. There will clearly be self-selection. Low quality hospitals will opt not to seek accreditation and poorly qualified doctors will seek out these hospitals. Universal accreditation could be mandated. Multiple accreditation teams could have the power to randomly inspect hospitals or units within hospitals and close those judged to be dangerous -- as occurs with restaurants with sub-standard hygiene. It is unclear whether or not present accreditation is sufficiently rigorous to reduce avoidable adverse events significantly. There appears little reason why the accreditation process should not itself be reviewed to ensure that credentialed hospitals satisfy rigorous safety standards in their facilities and procedures. Doctor AccreditationPatterns of private practice patterns are already subject to scrutiny in Australia. But the chief purpose is to detect medical fraud. Legislation could require the examination of practices to detect those which deviate significantly from evidence based guidelines constructed by the relevant Royal Colleges. When there is a known relationship between the small number of procedures carried out by a doctor and negative outcomes, as occurs with surgery, critical annual procedure rates may be established which trigger the provision of information to the doctor, the mandatory review of the practice and finally the dis-accreditation of the doctor for the conduct of these procedures. While it is true that some doctors take on the hard cases partial standardisation for case complexity is possible and this problem would obviously be taken into account by those conducting the review. The appropriate systems could be established in 1 to 2 years. Mandatory Disclosure and Error LearningIt is not compulsory for hospitals or doctors to register AE and routinely provide feedback to facilitate error learning. This means that the most important vehicle for improving quality and reducing patient risk is not compulsory. The opportunity for error leaning is almost certainly under-utilised in a large number of hospitals and probably ignored by the doctors whose performance most needs monitoring. Legislation might ensure the universality of this critical system reform. The adverse events register could and should be linked to doctors and appropriate threshold levels installed which sequentially trigger information feedback, review, and finally dis-accreditation of the doctor. It is likely that the first of these steps will be sufficient to effect satisfactory change. Despite this, 9 years after publication of the QAHCS the chairman of the ASCQHC notes that \'we have insufficient accurate data for fully appreciating the current size of the multiple causes of this problem \... we need the data from multiple sources, including incident monitoring systems, routine administration data sources and the use of screening tools to practically identify areas that may cause harm.\' (p5) \[[@B34]\]. Protection from litigationThe published research on \'high reliability organizations\' suggests that it is wise to separate inquisitorial from punishment processes, such as dis-accreditation. Adverse events are unlikely to be reported if there is a financial incentive to hide the AE. For this reason legislative protection of doctors from the financial outcome of litigation is probably a prerequisite for a successful and comprehensive system of error learning. The consequences for a doctor associated with AE should be based upon medical criteria and uncoupled from the social mechanism for compensating patients. Legal protection alone is unlikely to improve the quality of the information used for error learning. Rather, it should be part of a package of requirements which includes penalties for the failure to report an AE. Information transferPatient notes are still transferred within hospitals using 19th Century clipboards. It is known that this commonly causes potentially lethal errors. The mandated use of (long available) electronic forms of transmission could alert staff to the risk of inappropriate procedures, the administration of conflicting drugs or the failure to administer a drug. Likewise X-ray films are often misplaced or lost. The consequences may be lethal. Legislation could ensure the use of digital technology to ensure immediate access of results. New wireless technologies make it possible for roving staff -- doctors and other professionals -- to have constant access to text and basic technical data. As with electronic note taking this might take 1 to 2 years to fully install in all Australian hospitals, clinics and nursing homes. Hospital systemsHospital systems in Australia are commonly ramshackle or antique. There are no required pathways or mandatory discharge criteria. There are no internal or external financial incentives for the optimal treatment of patients. These changes are more complex but could be installed comfortably in 4--6 years. Conclusion 17:There is no reason why much of the health system should have missed the IT revolution which has transformed other parts of the community. In relation to the size of the AE problem, the cost of implementing late 20^th^Century information technology throughout the health system is likely to be small relative to the human and financial cost of AEs averted.* *Minimum staffing requirementsThere is no regulation which links on site expertise and the complexity or riskiness of the procedures which may be undertaken in a hospital. For example, it is possible for a hospital to permit significant surgery but have no on-site medical practitioner post-operatively. This potentially lethal practice could be proscribed and minimum staffing ratios implemented within the 1--2 weeks needed to reschedule staff or the location of procedures. It was not until 2003 that the ACSQHC released a paper considering issues of staff rostering, skill mix, staff numbers, staff supervision and team functioning \[[@B33]\]. While endorsing the AMA (voluntary) code of practice, \[[@B37]\] it comments -- almost 8 years after the QAHCS -- that \'responsibility for improving the management of staffing variables cannot (ie should not but still is being) left to individuals. It is a governance responsibility\...\' pii (words in brackets added)\] \[[@B38]\]. QueuingThe airline industry operates a highly efficient computerised system of booking and queuing which may be accessed by travel agents throughout the world. By this standard most hospital queuing systems -- if they exist -- are rudimentary. It is possible and desirable for different hospitals in the public hospital system to be interconnected to provide patients or their doctors with available times for treatment city, state or nation-wide. Queuing and scheduling can and should be operated using publicly known criteria. In the USA there is a nation-wide system for matching patients with available organs. (There is, interestingly a prioritisation criterion which, for reasons of equity, assigns compatible organs on the basis of need, not prognosis.) Australia has no such system. Conclusion 18:Queuing should be regulated by a nation-wide prioritising system based upon explicit criteria. This would increase efficiency and, for the patient, increase choice and certainty.* The private sector would have greater difficulty in operating such a system because of the patient\'s attachment to a particular doctor. Private health insurance organisations could, however, offer a similar service to patients who are willing to accept treatment from contracted providers. Australia\'s technologically conservative PHI organisations appear uninterested in these initiatives. *Information and system auditThe suggestions above and, to date, the majority of the reforms contemplated in reports, represent process measures of success. However, their objective is to reduce adverse errors and for this reason, record analysis of the form conducted by the QAHCS should be an ongoing feature of the system. The QAHCS research was relatively expensive, but these costs are infinitesimal in relation to the importance of the surveillance, the costs, the morbidity and deaths averted. Conclusion 19:A policy of persuasion and culture change is an insufficient response to a problem of the magnitude of the AE epidemic. As a matter of highest priority, legislation should be passed requiring extensive system regulation and reform to reduce the incidence of adverse events. The required changes should be fully funded (or they will not occur) and should include the use of state of the art IT, minimum staffing requirements and system feedback on both individual hospital and individual doctor performance. Evaluation and random audit of all hospitals and health care providers and the reporting of all AEs should be mandatory. Doctors should be given comprehensive protection against the results of successful litigation. The review of doctor performance should be uncoupled from compensation for the patient.* *Public informationThere is no legitimate reason for information relating to hospitals and individual doctors to be withheld from the public. There is a persuasive argument that the public has a right to such information. Additionally public awareness and the likely response from the public to such information are likely to accelerate the pace of reform. There are persuasive reasons for providing information more generally concerning the performance of all parts of the system, including individual care providers. Patient choice in the absence of information is a charade. Failure to forewarn patients that the hospital of their (doctors\') choice has a substandard safety record and that capacity exists in hospitals with a better record could, and arguably should, be regarded as professional negligence. There can be little doubt that, if consulted, the public would overwhelmingly endorse the need for this information. More generally, the provision of information is an effective method for effecting change and it is unlikely that the pace of reform would have been as sedate if the public were properly aware of the safety record of various health care providers. One argument against this option is that the provision of information might result in a loss of confidence in hospitals and doctors. However, the argument that the public should be kept in ignorance to engender unjustified confidence is, at best, dubious and if this ignorance allows an inadequate policy response then it is additionally harmful. In some states of the USA, and most notably New York, severity adjusted mortality rates are available for every hospital and for every doctor. This has not resulted in a significant change in the pattern for public demand but it has galvanised doctors and hospital staff to successfully review and upgrade their procedures \[[@B12]\]. League tables have recently been introduced in England to allow doctors and patients to evaluate the performance of particular hospitals \[[@B39]\]. From late 2004 the performance of individual surgeons will probably be available \[[@B40]\]. Conclusion 20:Information is a key element in achieving system reform and for the efficient operation of all markets, private or public. All institutions and individuals should receive rapid feedback on their performance. Information should be routinely collected and also obtained from random audit of hospitals. In particular, information should be publicly available with respect to the safety record of hospitals and providers of medical care.* *Financial incentivesAs discussed earlier, financial incentives are one some of the most effective, non-coercive ways of achieving desired outcomes. There has been very limited use of this powerful instrument and the financing of medical services has generally been perceived as a reward for providers doing what they select to do rather than as an opportunity for influencing what is done. This is an important missed opportunity. Importantly, financial incentives are non-coercive and avoid the head-to-head conflict between \'clinical autonomy\' and the \'patient\'s right to evidence based medicine\' which may accompany direct regulation. Conclusion 21:Financial incentives should be used flexibly throughout the health system to induce behaviour which minimise AEs.* *Governance and ownership of the problemA key theme of this paper has been that government policy has focused almost exclusively upon financial issues and that there has been little concern from within the legislature with health services and health. This is possibly the root cause of the government failure. Activities from within the various bureaucracies have largely been bureaucratic. In its 2004 review of State initiatives the ACSQHC reports that Queensland now has \'a program to develop a workforce culture that values a multi-disciplinary evidence based approach to improvement\'; in 2003 the NT implemented an \'overarching quality committee\'; NSW had \'involved the appointment of patient safety managers\'. The ACT now has a framework which \'provides explicit lines of accountably\'; Victoria now \'has a policy of an open and transparent approach to the provision of information\' etc (p8) \[[@B34]\]. There is no reference to national legislative action to enforce safety. The ACSQHC itself appears frustrated with the scale of the national effort when it comments that \'over the last year with public failures reported in some Australian hospitals it is clear that a small program of national investment and development needs to be matched by the will, skill and capacity of stakeholders\....\' (p5). On the same page it comments that \'Council is actively working to further develop data sources, but cannot do so without continued support by all governments\' (p5). Almost 10 years after the 1995 report the ACSQHC comments that \'future work \... needs to consider what governance and responsibilities are required at all levels of the health care system to ensure the provision of safe health care\' (p84). Conclusion 22:Despite the unnecessary death of between 40,000 and 80,000 Australians since the publication of the QAHCS, Australian government has not taken ownership of the problem of adverse events. As a result, those attempting to effect change have been forced to \'coax and cajole\' Options to \'mandate and enforce\' have been largely ignored.* Disregard of evidence --------------------- The last conclusion relates to the need to generate and disseminate information relating to system safety. The arguments for this apply more generally to health system data. Australia has a wealth of administrative databases which could be employed to investigate and improve system performance. As noted earlier, service use is very uneven across Australia. To the extent that this violates the usual notion of \'equity\' this information should be an important input into policy formulation. It does not appear to be used for this purpose outside NSW. However the quality of the data also presents other opportunities. The effect of different treatments, such as the varying rates of angiography and revascularisation observed between public and private hospitals could be traced through time if record linkage were possible. This would allow an assessment of downstream costs, mortality and morbidity associated with the two patient groups. There are clearly many opportunities for longitudinal research of this sort. A further option which may be piggy-backed on administrative data is the routine provision of information to different groups of patients who have been identified by their service mix. Information of this sort is probably a highly effective way of \'empowering patients\'; that is, enabling patients, and particularly those with a chronic disease, to take greater control of their disease management. These developments have not occurred for a variety of reasons. First, for an \$80,000 million industry, research funding for \'product development and marketing\' -- health services research -- is astonishingly small. In the USA six Federal agencies alone spent \$US 1,658 million in 2002 upon HSR. The agencies and their expenditures are detailed in Table [7](#T7){ref-type="table"}. Significant US funding is also obtained form the US network of Foundations which does not exist in Australia. Benchmarking against the Federal agencies alone, at an exchange rate of \$US 0.70 = \$AUS 1.0 (0.65) and scaling these expenditures down in relation to the size of the US and Australian economies, Australia should be spending about \$AUS 120 million on HSR. Australia does not currently spend a fraction of this amount. As a major initiative, the NHMRC is to provide \$10 million per annum for HSR -- or about 7.7 percent of the US Federal benchmark. ::: {#T7 .table-wrap} Table 7 ::: {.caption} ###### US expenditure upon Health Services Research ::: Federal Agency Expenditure (\$US millions) --------------------------------------------- --------------------------------- Agency for Health Care Research and Quality 300 National Centers for Health Statistics 127 Extra Mural Prevention Research CDC 18 Centers for Medicare and Medicaid Services 55 Veteran\'s Health Administration 371 National Institute of Health 787 Source: Annual reports ::: A second and possibly related reason is that there is no dedicated instrumentality, similar to the AIHW, which has taken \'ownership\' of the need to provide and periodically to review the need for information generated by HSR. Funding is currently inadequate but also ad hoc. Third, concern over the confidentiality of records has been elevated to such a level that easy and routine data linkage to observe the outcome of different service patterns does not seem to be a possibility. For example, access to Australia-wide, de-identified public hospital records requires the separate consent of all States and Territories as well as the cooperation of the Commonwealth Department of Health or AIHW. Data linkage to determine the consequences of different treatment patterns -- who lives and who dies -- is so difficult that the research is effectively proscribed. It is extremely doubtful that this concern in the bureaucracy over privacy would reflect the preferences of a well-informed population. Patients almost certainly suffer and die because of the interpretation and implementation of our confidentiality laws in a way which seriously inhibits the capacity of the public, researchers and private and public agencies to investigate the outcome of system performance and differences in individual treatments. The bureaucratic fetish with confidentiality does not occur in the USA where the risk of litigation is significantly greater than in Australia. In some states data relating to hospital and doctor mortality rates are regularly published and in California unit record hospital data is available on CD in university libraries. *Conclusion 23:Routinely collected administrative data should be fully used to monitor system performance. In particular it should be employed to monitor equity of access to services regularly and to provide disease-related information to population groups identified as having particular needs and interests. A statutorily independent national institute for health services research should be established whose terms of reference require the achievement of these objectives.* 5 Discussion and conclusion =========================== There are various options for the macro reform of the health system and the corresponding reform of financial incentives and the roles and responsibilities of the various players. In particular, Dick Scotton has cogently argued for the adoption of Managed Competition \[[@B41]-[@B43]\]. A partial movement in this direction could be achieved by transferring responsibility for the purchase of health services to the various health regions \[[@B44]\]. There has been no attempt to review this large topic here. Rather, the paper has reviewed the \'micro\' elements of such reform. The most appropriate \'macro\' model for the health system is the model which maximises the likelihood of implementing satisfactory solutions to the numerous problems facing the system including those that have been discussed above. The chief conclusion from this paper is that the \'health care debate\' and recent policy \'reform\' has focussed upon issues which are best described as \'very small order\' as judged by their likely effect upon either health or the cost effectiveness of the health system. They have been primarily concerned with dollars, not health and, more specifically, with the distribution of the cost between the public and private sectors. None of the policies discussed in Section 3 of the paper is likely to increase cost effectiveness. Cost shifting from the PBS to the public creates differential copayments which encourages allocative inefficiency. PHI reforms have created an industry with bizarre financial incentives and is a spectacular example of negative micro economic reform. The common feature of the three policies is that each moves the health system in a direction which is more consistent with the liberal/libertarian world view in which responsibility is transferred to the individual and away from the community. While PHI helps the individual to avoid financial decisions at the point of service, the individual is responsible for the purchase of the insurance and for the payment of (net) premiums. Copayments are the most direct method for shifting responsibility to the individual users of health services. Bulk billing for pensioners/health care card holders preserves the safety net which is needed for the achievement of equity as commonly defined by this world view. Over the longer term the pursuit of these values would redistribute income to the healthy, wealthy and away from the unhealthy unwealthy which is the antithesis of the communitarian/solidarity value system. The vehicle for the transfer is both a decline in community financed expenditures and a corresponding decline in taxation. As discussed in Section 2, decision making with respect to social objectives is the legitimate role of government. However, good economic and social policy seeks to achieve these objectives in a way that is cost effective. The reforms discussed here have not achieved this. In contrast with these policies, the five neglected areas discussed in Section 4 deal with problems which are \'large order issues\' as judged by their likely impact upon health and the cost effectiveness of the health system. Also contrasting with the first group, these issues are relatively \'value neutral\' as judged by either the communitarian or libertarian perspectives. The failure to address satisfactorily these issues is attributable to neglect, not the dominance of a particular ideology. This failure over a very long period itself refects a failure in the governance structure and a failure to identify and act upon opportunities for quantitatively large system improvements. The failure is probably replicated in most other developed countries, reflecting the complexity of the health sector. But benchmarking against similarly impaired systems does not alter the fact that there are opportunities for significantly improving the community\'s health which have been largely ignored. The reasons why this has occurred has not been discussed here and, to a greater or lesser extent, these failures have probably been replicated in most other developed countries, reflecting similar social and technical histories. Competing interests =================== The author(s) declare that they have no competing interests. Acknowledgements ================ The author would like to acknowledge the very helpful suggestions and corrections made by two anonymous reviewers.	PubMed Central	2024-06-05T03:55:52.472431	2005-1-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548139/", "journal": "Aust New Zealand Health Policy. 2005 Jan 11; 2:1", "authors": [ { "first": "Jeff RJ", "last": "Richardson" } ] }
PMC548140	Background ========== Oxidative stress is an important factor of acute lung injury. Prolonged exposure to high concentrations of oxygen (hyperoxia) during mechanical ventilation represents a life-saving intervention for critically ill patients. However, it also induces oxidative stress to the lung. The development of therapeutic strategies, aiming to prevent lung injury depends on a better understanding of the underlying pathways of hyperoxia-induced pulmonary damage. Severe, long lasting hyperoxia causes an inflammatory reaction with an influx of inflammatory cells, cell proliferation and hypertrophy, an increase of cytokines, apoptotic activity and subsequent morphologic evidence of lung injury \[[@B1]\]. The first 24 to 48 hrs of oxygen exposure constitute the initiation phase of the pulmonary oxygen toxicity \[[@B1]\]. Even though no morphologic injury has been described during this phase, several changes occur due to the hyperoxic exposure. Highly reactive oxygen species are likely to cause lipid peroxidation, protein and DNA modification that will further cell injury \[[@B2],[@B3]\]. On the other hand, antioxidant enzymes are also induced and may counteract the oxidative stress \[[@B4]-[@B6]\]. Perkowski et al. analysed more then 8700 genes during the early response (0 up to 48 hrs) to hyperoxia in total lung of mice. Out of 385 genes in the lung, 175 showed an increased and 210 a decreased expression \[[@B6]\]. These results indicate that the initiation phase of hyperoxic-induced lung injury already marks a very complex process that is still poorly understood. From previous investigations it may be concluded that in response to oxidative stress, the number of endothelial cells strongly decreases in the post-initiation phase, whereas epithelial cells seem to be relative resistant to oxidative stress \[[@B1],[@B7]\]. In contrast, it has also been shown that in response to hyperoxic ventilation \[[@B8]\], emphysema \[[@B9]\], activation of the Fas/FasL system \[[@B10]\], exposure to donors of nitric oxide or hydrogen peroxide \[[@B11]\], hyperoxia and nitric oxide \[[@B12]\], respiratory distress syndrome \[[@B13]\], and hyperoxia-mediated increase of total lung p53 protein expression \[[@B14]\] alveolar type II cells (TIIcells) are severely damaged, culminating in apoptotic death. TIIcells are functionally highly important epithelial lung cells. They are responsible for the metabolism of alveolar surfactant, serve as progenitor cells of type I pneumocytes, and take part in the inflammatory response of the lung \[[@B15]-[@B17]\]. Thus, damage and apoptotic elimination of TIIcells will severely alter pulmonary function. Following the concept that hyperoxic lung injury is a continuous process, we assumed that appropriate metabolic changes of TIIcells start during the initiation phase. This would then, in response to longer lasting severe hyperoxia or an additional stress, merge in apoptosis in the post-initiation phase \[[@B18]\]. Factors which induce such pro-apoptotic sensitisation of TIIcells in the initiation phase are yet unknown. Elevation of tumour necrosis factor α (TNFα) represents one of the first pulmonary responses to hyperoxia. Pre-treatment of animals with antibodies directed against TNFα reduces hyperoxia-induced lung injury, strongly suggesting a causal relationship between TNFα and hyperoxic lung \[[@B19]\]. TNFα is a classic regulator of cell death by apoptosis or necrosis \[[@B20],[@B21]\]. Cellular response to TNFα is mediated by TNF receptor type I and type II (TNFRI and TNFRII; \[[@B22]\]). Pryhuber et al. \[[@B23]\] studied the contribution of both, TNFRI and TNFRII to hyperoxia-induced lung injury and found that the average length of early survival under hyperoxic conditions is significantly improved in mice that lack the TNFRI (-/-), when compared with wild type or TNFRII (-/-) mice, respectively. However, the blockade of the TNFα receptor function does not protect against pulmonary inflammation and toxicity induced by prolonged hyperoxia \[[@B23]\]. In fact, during prolonged hyperoxia severe lung injury is most likely initiated by additional factors beside TNFα, as the inhibition of TNF receptors does not further affect oxygen-induced mortality \[[@B23]\]. Thus, TNFα and signal transduction via TNFRI seems to be responsible for metabolic changes that regulate the length of survival under short term hyperoxia. In this paper, we tested the hypothesis that TNFα activates caspases in the initiation phase of pulmonary oxygen toxicity in TIIcells without a significant increase in apoptosis. Since apoptosis is modulated by cell-matrix and cell-cell interactions in cultured TIIcells isolated from animals with acute lung injury \[[@B24],[@B25]\]. we used freshly isolated TIIcells for our study. Materials and methods ===================== Hyperoxia --------- Wistar rats (body wt 120 g), each in an individual plastic chamber were continuously gassed with 100 % oxygen for 48 hours. Water and food was available ad libitum. Preparation of the bronchoalveolar lavage, alveolar macrophages and TIIcells was carried out as previously described \[[@B26]\]. Immunohistochemistry -------------------- Immunohistochemistry and microscopy were carried out as previously described \[[@B26]\]. The following antibodies were used: Rabbit polyclonal anti-rat TNFα antibody from Biosource Europe (Nivelles, Belgium), rabbit polyclonal anti TNFRI antibody raised against a recombinant peptide (amino acids 30--301) including the extracellular domain of TNFRI (Santa Cruz Biotechnology, Heidelberg, Germany), and anti-active caspase 3 polyclonal antibody was from Promega (Mannheim, Germany). Secondary antibodies conjugated with Alexa 499 and Alexa 594 were from Molecular Probes Europe BV (Leiden, Netherlands). Lung tissue sections were labelled with specific antibodies directed against TNFRI, TNFα, caspase 3, and p180 \[[@B27]\], an integral lamellar body-limiting membrane protein (clone 3C9, Covance/Berkeley Antibody, Richmond, CA, USA). For double staining, the labelled preparations were analysed using a confocal laser scanning microscope (CLSM, Leica Microsystems AG, Wetzlar, Germany), equipped with an argon/krypton laser. Images were taken using a 40 × NA 1.3 oil objective to fluorescent excitation and emission spectra for Alexa 488 (excitation 490 nm, emission 520 nm) and for Alexa 594 (excitation 541 nm, emission 572 nm). With the dual-channel system of the confocal microscope, dual-emission (535/590 nm) images were recorded simultaneously with a scanning speed at 16 s/frame (512 lines). Images were obtained and processed using TCS NT Version 1.5.451 (Leica Microsystems AG, Wetzlar, Germany). As controls, the tissue slides were incubated with the Alexa-labelled second antibodies only. No unspecific binding of the second antibodies occurred (results not shown). For threefold staining (Figure [6](#F6){ref-type="fig"}), fixed lung tissue and freshly isolated TIIcells were incubated in 0.01 M phosphate buffered saline containing 1% (w/v) BSA and 0.3% (w/v) Triton X-100 for 1 hr at room temperature (RT). Detection of lamellar bodies and active Caspase 3 was achieved by incubation with mab 3C9 (20 hrs at 4°C) followed by Alexa 488-labelled goat anti-mouse IgG (2 hrs at RT) and with anti-active caspase 3 followed by Alexa 594-labelled goat anti-rabbit IgG (2 hrs at RT), respectively. Nuclear DNA was stained with 4\',6-diamidino-2-phenylindole (DAPI; Molecular Probes Europe BV, Leiden, Netherlands) for 20 minutes at RT. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Hyperoxia activates caspase 3 in TIIcells. Rats were kept normoxic (control) or subjected to hyperoxia. Lung sections (A) and freshly isolated TIIcells (B) were immunohistochemically threefold stained as described in Materials and Methods. Cell nuclei stained light blue (DAPI). TIIcells are distinguishable by the close proximity of their nuclei to green stained lamellar bodies (A; arrows). Active caspase 3 appears red labelled and is predominantly found in the cytosol of TIIcells upon hyperoxic treatment of rats (A and B). Bar 10 μm; ::: ![](1465-9921-6-10-6) ::: Laser scanning confocal microscopy was performed using a ZEISS LSM 510 system with Axiovert microscope (Carl Zeiss Jena GmbH, Jena, Germany) with 40×/1.3 Oil Dic or 63×/1.4 Oil Dic objective, equipped with an argon, helium/neon and violet laser set to 488, 543 and 405 nm, respectively. The multitrack standard FITC/Rhodamine/DAPI configuration was selected. Determination of apoptosis in lung tissue ----------------------------------------- ### TUNEL reaction Sections of rat lung were prepared as described \[[@B26]\]. After deparaffinization and proteinase K-treatment, apoptotic cuts of chromatin DNA were specifically detected by nick end labelling of 3\'-OH DNA ends with fluorescein-dUTP using terminal deoxynucleotidyl transferase. (MEBSTAIN Apoptosis Kit Direct, MBL, Naka-ku Nagoya, Japan). Sections were analyzed using a confocal laser scanning microscope as described above for double staining. Determination of apoptosis in freshly isolated TIIcells ------------------------------------------------------- ### Cell Death Detection ELISA Cytoplasmic histone-DNA fragments were quantified using the Cell Death Detection ELISA (Roche, Mannheim, Germany). ### Flow cytometry We used the TACS™ Annexin V-FITC Detection kit (R&D Systems, Wiesbaden, Germany) to quantify the population of early and late apoptotic cells in percent of total cells. The tests were performed according to the protocols of the manufactures. Determination of caspase activities ----------------------------------- Activities of caspases 3, 8 and 9 were determined in the lysates of 8 × 10^6^TIIcells for each group and for each caspase with the Colorimetric assays from R&D Systems Inc. (Wiesbaden, Germany). When the pro-caspases had to be determined, an aliquot of the lysate (corresponding to 2 × 106 cells) was preincubated with 0.1 μg granzyme (Calbiochem, Bad Soden, Germany; dissolved in 5 μl 0.9% NaCl) for 30 min at 37°C. Determination of NF-κB activation --------------------------------- ### Immunocytochemistry Activation of NF-κB was measured by immunocytochemistry using a monoclonal anti-NF-κB-antibody (MAB3026, Chemicon International, Temecula, USA), that recognises an epitope which includes the nuclear location signal of p65, the DNA binding subunit mainly responsible for the strong gene-inductory potential of NF-κB. Thus, only the activated form of NF-κB was measured. The semiquantitative estimation of NF-κB subunit by confocal microscopy was carried out as recently described in detail \[[@B28]\]. ### Immunoblotting Translocation of NF-κB to the nucleus was assessed as described by Li et al. by immunoblotting of nuclear extracts using a rabbit polyclonal antibody (biomol GmbH, Hamburg, Germany) directed against the p65-subunit \[[@B29]\]. ### Electrophoretic mobility shift assay (EMSA) was employed to detect the activated transcription factor NF-κB. Because this method is based on the binding of the transcription factors to their specific DNA recognition sequences, it is highly specific. Labelling of the NF-κB consensus oligonucleotide and handling of the assay were as described by the manufacturer (Gel Shift Assay Systems, Promega GmbH, Mannheim, Germany). Briefly, 50 micrograms of TIIcell nuclear extract were preincubated in reaction buffer for 10 minutes at RT. A \[32P\]-labelled oligonucleotide (Promega GmbH, Mannheim, Germany) which contains DNA binding sites for NF-κB transcription factors was then added to the reaction mixture and incubated for 20 minutes at RT. The complexes were separated on a 4% polyacrylamide gel that was dried and exposed to autoradiography. The specificity of the DNA-binding protein for the putative binding site was established by competition experiments using unlabelled NF-κB consensus oligonucleotide. Intratracheal application of anti-TNFα antibodies ------------------------------------------------- Wistar rats were lightly anaesthetised by inhalation of ether. The rats obtained intratracheally 50 μg goat IgG (PERBIO SCIENCE, Bonn, Germany) or 50 μg goat polyclonal anti-rat TNFα antibody (Santa Cruz Biotechnology, Heidelberg, Germany) per animal, respectively. Thereafter the rats were kept at hyperoxic conditions as described in hyperoxia. After 48 hrs, the TIIcells were isolated and TNFRI expression and caspase-3 activity were determined as described above. Other methods ------------- For the determination of the TNFα concentration in TIIcells, macrophages, plasma or cell-free bronchoalveolar lavage, we used a commercially available ELISA kit from Biosource (Ratingen, Germany). The determination of different apoptotic markers \[[@B18]\], Western blot analysis \[[@B17]\], mRNA isolation and Real-time quantitative PCR reaction for the determination of the expression of mRNAs in TIIcells was described in detail previously \[[@B26]\]. GSH, GSSG and GSH-reductase were determined by HPLC with subsequent fluorescence detection as previously described \[[@B17]\]. Statistical analysis -------------------- Differences between two groups were assessed using the Student\'s t-test. Probability values \< 0.05 (two-tailed) were considered significant (see legends of Tables and Figures). Results ======= Hyperoxia-induced changes in lung tissue ---------------------------------------- To test the general usefulness of our hypothesis, we first characterised the oxygen-induced changes in lung-tissue-sections. Immunohistochemically, we observed a clear increase in TNFα, TNFRI, and caspase 3 activities in lung tissue of hyperoxic rats in relation to normoxic animals in the initiation phase (Figure [1](#F1){ref-type="fig"}). Albeit the increase of these pro-apoptotic parameters in lung tissue corroborated our hypothesis, this approach does not allow to identify the participating cell types. Furthermore, we tested lung sections for DNA-degradation products using the TUNEL reaction (Figure [2](#F2){ref-type="fig"}). Here, TIIcells are distinguishable from other cells of the lung by their content of lamellar bodies. Lamellar bodies were immunohistochemically labelled in lung sections with an antibody directed against the 180-kDa lamellar body-limiting membrane protein (red, Figure [2](#F2){ref-type="fig"}). Upon hyperoxia, we found sporadically a fragmentation of DNA (green, Figure [2](#F2){ref-type="fig"}), but no co-localisation of the 180-kDa lamellar body-limiting membrane protein and DNA-fragments. The intensity of red lamellar body stain increased in lung sections from hyperoxic rats. This is in good accordance with previously published electron micrographs showing swollen and deformed lamellar bodies after hyperoxia \[[@B30]\]. From these results, we conclude in agreement with the literature \[[@B1],[@B7],[@B25]\]. that sublethal hyperoxia of rats did not induce apoptosis in TIIcells in vivo; at least not in the initiation phase. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Hyperoxia increases the expression of TNFRI, TNFα and caspase 3 in vivo. Lung sections of normoxic (control) and hyperoxic rats were prepared as described in Materials and Methodsand were labelled by single immunofluorescence with antibodies directed against TNFRI, TNFα or active caspase 3 ::: ![](1465-9921-6-10-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### No significant apoptosis was found in TIIcells upon hyperoxia in vivo for 48 hrs. Lung tissue of normoxic rats (left) and of hyperoxic rats (right) were tested for DNA fragmentation by TUNEL reaction as described in Materials and Methods. The positive TUNEL reaction is represented by green fluorescence. The presence of lamellar bodies is indicated by red fluorescence. Pseudo-colour blue was used to highlight the contours of lung tissue. Bar: 25 μm. ::: ![](1465-9921-6-10-2) ::: The results indicate that apoptotic parameters as are TNFα content, TNFRI expression and caspase-3 activity increase in lung tissue during the initiation phase but do not induce TIIcell apoptosis in vivo. Following our hypothesis, we examined whether TIIcells undergo oxidative stress at our conditions, and whether an increase of TNFα, TNFRI expression, and caspase-3 activity appears in isolated TIIcells. Hyperoxia-induced oxidative stress of freshly isolated TIIcells --------------------------------------------------------------- The determination of cellular GSH, oxidised GSH (GSSG), and the activity of the GSH-reductase showed the oxidative burdening of TIIcells. In response to hyperoxia, the GSSG content significantly increased and the GSH reductase activity significantly decreased (Table [1](#T1){ref-type="table"}). The GSH content in freshly isolated TIIcells has rarely been determined. In our TIIcell population, the GSH content differs in relation to previously published data of freshly isolated TIIcells from rat \[[@B31]\] and rabbit \[[@B32]\] by the factor of about 2 and 4, respectively. These differences might be explained by different methods of TIIcell isolation and by species specificity. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of hyperoxia on GSH reductase activity, and on GSH and GSSG content in TIIcells ::: Control Hyperoxia ------------------------------ ---------------- ----------------- GSH reductase (% of control) 100 76 ± 10\* GSH (μmol/mg protein) 0.022 ± 0.015 0.020 ± 0.008 GSSG (μmol/mg protein) 0.005 ± 0.0015 0.013 ± 0.001\* GSH/(GSH+GSSG) (ratio) 0.81 0.61 Activity of glutathione (GSH) reductase and concentration of GSH and GSSG were determined in freshly prepared TIIcells of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) as described in Materials and Methods. Values are means ± standard deviation of n = 3 independent experiments. Asterisk indicates a significant difference to control (p\< 0.05). ::: The ratio GSH/(GSH+GSSG) is one of the most sensitive parameters to describe oxidative burdening. Our values are comparable to the values found by van Klaveren et al. in freshly isolated type II cells (0.816 versus 0.815) \[[@B30]\]. This ratio decreased in response to hyperoxia in vitro to 0.74 \[[@B31]\] and in our in vivo approach to 0.61 (Table [1](#T1){ref-type="table"}). With respect to the published data, we conclude that our treatment induced oxidative burdening in freshly isolated type II cells. Hyperoxia activates NF-κB ------------------------- NF-κB activation has been described as an indicator of oxidative stress \[[@B33],[@B34]\]. In response to sublethal hyperoxia, the content of activated NF-κB in TIIcells clearly increased (Figure [3A](#F3){ref-type="fig"}). The picture shows that the larger portion of activated NF-κB seems to be localised in cytosol. The semiquantitative determination of activated NF-κB showed a significant increase (control: 12.7 ± 9.0; hyperoxia: 59.5 ± 11.6; n = 12; p \< 0.01) in the nucleus of TIIcells in response to hyperoxia. In the cytosol, there was also an increase of activated NF-κB (1.6-fold), however, this difference curtly fails the level of significance. Additionally, we detected a translocation of the NF-κB-subunit p65 into the nuclear protein fraction as estimated by Western blot analysis (Figure [3B](#F3){ref-type="fig"}). Hyperoxia increased the content of the p65-subunit in the nuclear protein fraction of TIIcells 1.68-fold (SD 0.58, n = 4) compared to normoxic control, but the difference did not reach significance. Whether the immunoreactive band above the p65-subunit in the Western blot of the hyperoxic group is non-specific or a possible post-translational modification can not be decided. In Figure [3C](#F3){ref-type="fig"} we confirm the hyperoxia induced activation of NF-κB by EMSA of the nuclear protein fractions of TIIcells freshly isolated from control (lanes 1, 2) and hyperoxic (lanes 3, 4) rats. Addition of unlabelled specific oligonucleotide clearly competes with the \[32P\]-labelled probe confirming the specificity of the bands. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Activation of NF-κB in TIIcells and its enrichment in the nuclear protein fraction after hyperoxic treatment of rats. Freshly isolated TIIcells from normoxic (control) and hyperoxic rats were prepared for immunocytochemistry, SDS-PAGE and immunoblotting as described in Materials and Methods. A, activation of NF-κB was measured by immunocytochemistry using a monoclonal anti-NF-κB-antibody overlapping the nuclear localisation signal of the p65 subunit in the NF-κB heterodimer. Activated NF-κB in the nucleus and cytosol was quantified as described recently in detail \[28\]. The signal of activated NF-κB in the nucleus increased 4.7 fold in respose to hyperoxia (n = 12; p \< 0.05). Bar: 10 μm. B, the nuclear protein fraction \[52\] of TIIcells was subjected to SDS-PAGE and immunoblotting. The p65 subunit of NF-κB was visualised using a rabbit polyclonal antibody, and its expression was densitometrically estimated. Values of n = 4 independent experiments are given as mean ± SD in arbitrary units (control = 1). C, Electrophoretic mobility shift assay for NF-κB in freshly isolated TIIcells from normoxic (lanes 1, 2) and hyperoxic (lanes 3, 4) rats. Signal competition upon addition of unlabelled oligonucleotide (lanes 2, 4). ::: ![](1465-9921-6-10-3) ::: Hyperoxia increases synthesis and secretion of TNFα by TIIcells --------------------------------------------------------------- The concentration of TNFα significantly increased in response to hyperoxia of rats in plasma, alveolar fluid, lung macrophages and TIIcells (Table [2](#T2){ref-type="table"}) as determined by ELISA. Flow cytometric analysis of the TNFα content in freshly isolated TIIcell preparations from control and hyperoxic rats confirmed the increase of the TNFα concentration in macrophages and TIIcells. In response to hyperoxia, TNFα increased in TIIcells 1.45-fold and in macrophages 1.87-fold compared to control. In TIIcells, TNFα seems to be localised in lamellar bodies (Figure [4](#F4){ref-type="fig"}), whereas cytoplasmic caspase 3 showed no co-localisation with lamellar bodies as expected. The latter result attaches value to the histochemically detected localisation of TNFα in lamellar bodies, because it is unlikely an artefact. The spontaneous secretion of TNFα by TIIcells significantly increased in response to hyperoxia (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Effect of hyperoxia on the TNFα content in different specimen from rat ::: Control Hyperoxia ------------------------------------------------------------------------------ ------------ -------------- Plasma (pg/ml) 106 ± 31 149 ± 11 Macrophages (ng/mg protein) 11.4 ± 3.7 19.4 ± 2.8\* TIIcells (ng/mg protein; n = 6) 18.5 ± 2.1 27.2 ± 6.7\* Bronchoalveolar lavage (pg/ml) 109 ± 1 172 ± 38\* Spontaneous secretion of TNFα by TIIcells (ng × mg cell protein^-1^× hr^-1^) 6.3 ± 1.3 21.2 ± 7.5\* Concentration of TNFα was determined in plasma, macrophages, TIIcells and bronchoalveolar lavage of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) by ELISA as described in Materials and Methods. Spontaneous secretion of TNFα was measured in cell-free supernatant upon incubation of freshly isolated TIIcells in DMEM for 30 min at 37°C. TNFα concentrations are given as means with standard deviation of n = 3 independent experiments unless stated otherwise. Asterisk indicates a significant difference to control (p\< 0.05). ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### TNFα is localised in lamellar bodies and caspase 3 in the cytosol of TIIcells. After hyperoxia, rat lungs were fixed and the sections were immunohistochemically double labelled as described in Materials and Methods. A: bar 50 μm; B: higher magnification of the indicated area of A (arrow); bar 10 μm. By confocal microscopy, TIIcells were identified by the green labelling of lamellar bodies (see Methods). The red labelled TNFα appears yellow (arrow in B) when co-localised in lamellar bodies as shown in two TIIcells in B. ::: ![](1465-9921-6-10-4) ::: Hyperoxia induces the expression of TNFRI and activates caspases in TIIcells ---------------------------------------------------------------------------- Hyperoxia induced an significant increase of TNFRI on TIIcells (4.9-fold ± 2.7, n = 6), whereas the expression of Fas, a member of the same receptor family, did not change (Figure [5](#F5){ref-type="fig"}). TNFRI-mediated action of TNFα depends on the so called \"death domain\" representing a part of the intracellular segment of the receptor protein responsible for the activation of pro-caspase 8. Caspase 8 in turn can activate pro-caspase 3, a feature previously reviewed \[[@B35]-[@B37]\]. We show in situthat the activation of caspase 3 actually occurs in TIIcells and that caspase activation as a response to an unspecific stress, e.g. isolation, can be excluded (Figure [6](#F6){ref-type="fig"}). This staining technique excepts cytoplasm and membranes, thus, cells can hardly be delimited. However, TIIcells are identifiable by the immediate proximity of their nuclei to lamellar bodies (green). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Hyperoxia increases the expression of TNFRI but not of Fas in TIIcells. For the expression analysis of TNFRI and Fas the membrane fraction of freshly isolated TIIcells was prepared. TNFRI (A) and Fas (B) were visualised by Western blot technique, and their expression was densitometrically determined. Values of n = 6 independent experiments are given as means ± SD in arbitrary units (control = 1). Asterisk indicates a significant difference to control (p\< 0.05). ::: ![](1465-9921-6-10-5) ::: As shown in Table [3](#T3){ref-type="table"}, in TIIcells the activity of caspase 8 and 3 increased in response to hyperoxia, whereas the activity of caspase 9 did not change. Pre-incubation with granzyme activated pro-caspases and resulted in an increase of caspase 8 and -3 activities in control and hyperoxic TIIcells (Table [4](#T4){ref-type="table"}). However, the activation in control cells clearly exceed that in hyperoxic TIIcells, indicating that pro-caspases were already, at least in part, activated in response to hyperoxia. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Effect of hyperoxia on the activity of caspases in TIIcells ::: Control Hyperoxia ------------------- --------- ------------ Caspase 8 (n = 6) 100 143 ± 14\* Caspase 3 (n = 6) 100 168 ± 23\* Caspase 9 (n = 3) 100 108 ± 11 Caspase activity in freshly prepared TIIcells of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) was determined colorimetrically as described in Materials and Methods. Values are means ± S.D. given in arbitrary units (control = 100). Asterisk indicates a significant difference to control (p\< 0.05). ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Effect of granzyme-treatment on the activity of caspases in TIIcells from normoxic and hyperoxic rats ::: Control Hyperoxia ----------- --------- ------------ ----- ------------ Caspase 8 100 155 ± 7\* 100 110 ± 4\* Caspase 3 100 216 ± 14\* 100 155 ± 10\* Caspase activity in freshly prepared TIIcells of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) was determined colorimetrically as described in Materials and Methods. In some experiments, cell lysates were incubated in the presence of granzyme prior to determination of caspase activity (see Methods). Values are means ± s. d. given in arbitrary units (values without granzyme = 100) of n = 3 independent experiments. Asterisk indicates a significant difference between granzyme-treated and untreated samples (p\< 0.05). ::: Anti-TNFα in vivo prevents hyperoxia-driven increase in TNFRI and in active caspase 3 ------------------------------------------------------------------------------------- In order to demonstrate the causality between hyperoxia and TNFRI-mediated activation of caspases, we attempted to bind TNFα by anti-TNFα antibodies. Table [5](#T5){ref-type="table"} shows that a single intratracheal application of anti-TNFα antibodies immediately preceding hyperoxic treatment prevents the hyperoxic-induced increase of TNFRI expression and caspase-3 activity in freshly isolated TIIcells. These results indicate that both TNFRI expression and caspase-3 activation were induced by TNFα. This corroborates the concept that in a cascade starting by the TNFα/TNFRI-interaction caspase 8 is activated which in turn activates caspase 3. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Effect of intratracheal application of anti-TNFα antibodies on hyperoxic induced parameters of TIIcells ::: Hyperoxia -------------------- ----------- ----------- TNFRI 100 26 ± 19\* Caspase 3 activity 100 64 ± 5\* Rats obtained intratracheal 50 μg goat-IgG (without) or 50 μg goat polyclonal anti-TNFα antibodies (with anti-TNFα antibody) preceding hyperoxic treatment. Hyperoxia of rats was carried out as described in Material and Methods. Values are means ± SD of n = 3 independent experiments given in percent (hyperoxia without anti-TNFα antibody = 100). Determination of TNFRI (Western blot) and of caspase 3 activity (colorimetrically) was as described in Material and Methods.). Asterisk indicates a significant difference compared to control (p\< 0.05). ::: Hyperoxia upregulates genes of TNFRI, TNFα and caspases 3 and 8 --------------------------------------------------------------- The content of individual mRNAs in TIIcells was determined by Real-time PCR. In agreement with microarray analysis in total lung of mice \[[@B6]\] we found that the amount of GAPDH mRNA did not change in the first 48 hrs of hyperoxia (results not shown). Therefore, GAPDH was used as a house keeping gene. In contrast, Ho et al. found a small but significant increase of GAPDH mRNA in lung tissue \[[@B5]\]. This difference may be caused by different base material (TIIcells versus lung tissue) and methodical differences (Taqman versus densitometry of autoradiographs). In response to sublethal hyperoxia the mRNA content of TNFα, TNFRI and caspases increased (Table [6](#T6){ref-type="table"}). The increment of caspase-8 mRNA was not significant, but even a ΔΔc~t~of -1.15 indicates a 2.2-fold increase of mRNA content. ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Effect of hyperoxia on the mRNA content of TNFα, TNFRI, and caspases in TIIcells ::: number of cycles (Δc~t~) increase of mRNA ----------- -------------------------- ------------------ ------- ----- TNFα 3.66 ± 0.66 0.93 ± 0.30\* -2.73 6.6 TNFRI 5.02 ± 0.19 4.44 ± 0.11\* -0.58 1.5 Caspase 8 10.3 ± 0.89 9.15 ± 0.35 -1.15 2.2 Caspase 3 5.33 ± 0.25 4.29 ± 0.48\* -1.04 2.1 The mRNA of three animals per group was isolated and combined. The content of specific mRNA was determined using Real time-PCR. In parallel, GAPDH was determined in each run as internal standard. The values are given as number of GAPDH-corrected cycle (Δc~t~) ± SD of n = 3 determinations. ΔΔc~t~is the difference of the number of cycles between control and hyperoxia; a decrease of Δc~t~indicates an increase of the mRNA, also given as a factor of increase. Asterisk indicates a significant difference to control (p\< 0.05). ::: Hyperoxia of rats does not induce apoptosis in freshly isolated TIIcells ------------------------------------------------------------------------ As mentioned above in this section, no increase of apoptosis was detected in TIIcells in response to sublethal hyperoxia by immunohistochemistry (Figure [2](#F2){ref-type="fig"}). To confirm this result, we analyzed biochemical parameters of apoptosis in freshly isolated TIIcells. In agreement with our immunohistochemical results, sublethal hyperoxia of rats did not increase apoptosis in freshly isolated TIIcells (Table [7](#T7){ref-type="table"}). Hyperoxia was without effect on anti-apoptotic Bcl-2, and cytosolic cytochrome C, although the pro-apoptotic Bax increased and the mitochondrial transmembrane potential slightly decreased (Table [7](#T7){ref-type="table"}). In accordance with these results, the activity of caspase 9 did not change. Therefore, activation of caspase 3 seems to be catalysed by caspase 8. ::: {#T7 .table-wrap} Table 7 ::: {.caption} ###### Effect of hyperoxia on apoptotic parameters in TIIcells ::: Control Hyperoxia ---------------------------------- --------- ------------ Bcl-2 (n = 5) 100 96 ± 8 Bax (n = 5) 100 134 ± 28\* Cytochrome c (n = 5) 100 102 ± 15 Mitochondrial membrane potential 100 81 ± 8\* Early apoptotic TIIcells 100 101 ± 26 Late apoptotic TIIcells 100 94 ± 22 Cell death detection ELISA 100 104 ± 13 Several apoptotic parameters were determined in freshly prepared TIIcells of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) as described in Materials and Methods. Values are means ± S.D. given in arbitrary units (control = 100) of n = 3 independent experiments unless stated otherwise. Asterisk indicates a significant difference to control (p\< 0.05). ::: Hyperoxia of rats followed by normoxia reduced caspase 3 activity but did not increase apoptosis in TIIcells ------------------------------------------------------------------------------------------------------------ After hyperoxia for 48 hrs, we detected an activation of caspase 8 and caspase 3. Much to our surprise, we found no increase in apoptosis of TIIcells although the activity of these caspases increased. However, apoptosis might occur later than 48 hrs and will not necessarily arise concomitantly with caspase activation. Thus, following hyperoxia the animals were kept for 24 and 48 hrs under normoxic conditions to test TIIcells for appearance of apoptosis at a later time point. During normoxia, caspase 3 activity gradually decreases compared to control, whereas apoptosis did not change significantly as detected by cell death detection ELISA and by the number of early and late apoptotic cells (Table [8](#T8){ref-type="table"}). ::: {#T8 .table-wrap} Table 8 ::: {.caption} ###### Apoptotic parameters upon sublethal hyperoxia of rats followed by normoxia ::: Hyperoxia followed by normoxia for ---------------------------- ------------------------------------ ---------- ---------- Caspase 3 activity 171 ± 18\* 97 ± 16 73 ± 29 Early apoptotic cells 102 ± 13 95 ± 14 109 ± 26 Late apoptotic cells 106 ± 29 125 ± 25 85 ± 17 Cell death detection ELISA 97 ± 23 104 ± 6 95 ± 14 Activity of caspase 3 and parameters of apoptosis detected in freshly prepared TIIcells of rats exposed to oxygen for 48 hrs (hyperoxia) followed by normoxia for 24 or 48 hrs as described in Materials and Methods. Values are means ± S.D. given in arbitrary units (normoxic control = 100) of n = 3 independent experiments. Asterisk indicates a significant difference compared to control (p\< 0.05) ::: Discussion ========== Short-time hyperoxia, as used in this experiments represents the initiation phase of lung injury \[[@B1]\]. We started our investigations with the aim to characterise metabolic changes in TIIcells taking place in this phase. On the one hand, these changes should be less complex than in post-initiation phases. On the other hand, therapeutic interventions to avoid or minimise hyperoxia-induced lung injury should focus in particular on this phase, because morphologic injury of lung tissue, inflammation, and death of lung cells originate from here. Furthermore, the metabolic changes in the initiation phase may, at least in part, be still reversible. For the first time, we show that sublethal hyperoxia causes not only an increase in TNFα concentration in lung tissue (as previously published \[[@B38],[@B39]\]), but also in freshly isolated TIIcells and that this increase is combined with an enhanced expression of TNFRI and an activation of caspase 3. It has been widely assumed that macrophages are the source of TNFα in alveolar fluid \[[@B40]\]. Our findings provide evidence that hyperoxic treatment of rats provokes a rise in cellular TNFα not only in alveolar macrophages \[[@B41]\]. We show that the TNFα-gene is up-regulated in TIIcells; in parallel, the TNFα-protein content increased. To the best of our knowledge, our data suggests for the first time that TNFα is localised in lamellar bodies. In the light of this assumption, the secretion of TNFα as a constituent of the lamellar bodies by TIIcells might represent a significant contribution to the increased TNFα-content in alveolar fluid. Whether the small increase of the TNFα concentration in plasma observed by us indicates a beginning systemic inflammation or rather reflects a transfer of TNFα from the alveolar space to plasma, remains open (Table [2](#T2){ref-type="table"}). Cellular effects of TNFα are mediated mainly by its specific receptor, TNFRI. In agreement with our hypothesis, the expression of TNFRI in TIIcells is up-regulated on mRNA and protein level in response to sublethal hyperoxia, while the Fas-expression did not change, although both receptors belong to the same family (Figure [5](#F5){ref-type="fig"}). It may be speculated that a parallel increase of effector and specific receptor always occurs when the cellular metabolism in TIIcells can be affected by an autocrine mechanism. The increase of Fas/Fas-Ligand might be characteristic in post-initiation phases. The intracellular part of the transmembrane TNFRI protein contains the \"death domain\" which is responsible for the activation of caspase 8. This can trigger the path to the final part of apoptosis via activation of caspase 3 \[[@B36]\]. In line with this, we found enhanced levels of caspases 8 and 3, yet caspase 9 was not activated. The latter result is corroborated by the absence of an increased mitochondrial cytochrome C release. The data presented here demonstrate that sublethal hyperoxia of rats did not induce apoptosis in TIIcells despite of the increase in TNFα content, TNFRI expression, and activation of caspase 3. To check whether this pro-apoptotic state in TIIcells is indeed triggered by TNFα and whether it may be reversible, anti-TNFα antibodies were administered intratracheally prior to hyperoxic exposure. We show that this treatment completely prevented hyperoxic-induced increase of TNFRI expression and caspase 3 activation. It is a widely accepted concept that activation of caspase 3 marks the \"point of no return\" in the pathway of apoptotic death of mammalian cells. However, we found that neither in lung tissue nor in freshly isolated TIIcells apoptosis occurs in response to sublethal hyperoxia despite the significant activation of caspases 8 and 3. In other words, the increase of caspase 8 and 3 in response to sublethal hyperoxia did not mark the \"point of no return\" in TIIcells. This interpretation of our results is strongly corroborated by Perfettini and Kroemer \[[@B37]\]. They summarised that caspase inhibition does not avoid but actually encourage death in TNF induced shock, indicating that caspase activation is not basically synonymous with apoptotic cell death. It could be argued that no apoptosis was found in freshly isolated TIIcells because the increase of caspase 3 activity and the increase of apoptotic parameters does not occur concomitantly. However, we found no increase in apoptosis of TIIcells in a normoxic period of 24 and 48 hrs directly succeeding the hyperoxic treatment of animals, whereas the caspase 3 activity gradually decreased. These results indicate that caspase 3 activation in response to sublethal hyperoxia is reversible and does not kill TIIcells essentially. The reason why the activation of caspases did not induce apoptosis of TIIcells is not clear. On the one hand, activation of NF-κB has often been implicated as an anti-apoptotic event \[[@B42]-[@B45]\], and TNFα induces also the expression of anti-apoptotic TNFα-receptor-associated-factors in lung cells and might inhibit by this way TNFα-induced cell death or apoptosis \[[@B46]\]. Therefore, it may be assumed that both NF-κB activation and expression of anti-apoptotic TNFα-receptor-associated-factors arrest the hyperoxia-induced metabolic changes of TIIcells in the pro-apoptotic state. On the other hand, it can not be excluded that the extend of caspase 3 activation (1.68-fold compared to control) is not high enough to induce apoptosis of TIIcells although a 1.8-fold increase of caspase 3 is combined with apoptosis in total lung of a hyperoxia/pneumonia model \[[@B47]\]. We hypothesise that caspase 3 activation is then followed by apoptosis, when its activation occurs viastrong mitochondrial damage resulting in cytochrome c release and caspase 9 activation in post-initiation phases of pulmonary oxygen toxicity. This concept is supported by recent findings that mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury \[[@B48]\]. In fact, the mitochondrial membrane potential and Bax significantly changed in TIIcells in the initiation phase of hyperoxic lung injury, but without cytochrome c release or activation of caspase 9. Recently, it has been shown that in response to severe hyperoxia of mice apoptosis and necrosis contribute to an extensive cell death; p53, bax, bcl-x, and Fas increased in mRNA and protein level, but the activity of caspase 3 and caspase 1 did not change \[[@B49]\]. This independence of apoptosis from caspase activities in the lung has also been shown in freshly isolated TIIcells. Previously, we showed that an increase of TIIcell-apoptosis in response to vitamin E deficiency of rats is independent of caspase activation \[[@B18]\]. Furthermore, using p53-deficient and Fas-null mice, Barazzone et al. \[[@B49]\] showed that Fas and p53 activation exhibits no linkage to lung injury in response to severe hyperoxia of mice. Contrariwise, it has also been shown that Fas activation in vitro \[[@B50]\] and in vivo results in TIIcell apoptosis and lung inflammation \[[@B10],[@B51]\]. These results confirm that hyperoxic-induced lung injury is multifactorial, and that the elimination of one factor in the network of factors activated in the post-initiation phases often can not avoid lung injury in response to severe hyperoxia. In summary, TIIcells were not killed in the initiation phase of pulmonary oxygen toxicity. More precisely, its pro-apoptotic sensitisation is the background that, together with an additional stress factor, hyperoxia causes lung injury probably by apoptotic elimination of TIIcells. In agreement with this idea, we showed that the combination of the stress factors hyperoxia and vitamin E deficiency increases TIIcell apoptosis \[[@B18]\]. Taking into account that premature neonates exhibit vitamin E deficiency, it is consequential that ventilation of premature neonates suffering from respiratory distress syndrome with high levels of inspired oxygen amplifies lung injury, which is associated with TIIcell apoptosis \[[@B13]\]. Conclusions =========== In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFα effect in vivo by anti-TNFα antibodies prevents the pro-apoptotic sensitisation of TIIcells. List of abbreviations ===================== DAPI -- 4\',6-diamidino-2-phenylindole; EMSA -- electrophoretic mobility shift assay; GSH -- gluthatione; GSSG -- oxidised gluthatione; NF-κB -- nuclear factor-κB; TIIcell -- type II pneumocyte; TNFα -- tumour necrosis factor α; TNFR -- TNFα receptor; TUNEL -- terminal transferase dUTP nick end labelling Authors\' contributions ======================= HW carried out the immunohistochemistry, CS performed the mRNA analysis, AT carried out the protein measurements and participated in data analysis, MR applicated anti TNFα antibodies intratracheally, FS participated in the mRNA analysis and the design of the study, FG and BR conceived of the study, participated in its design and co-ordination, analysed the data and wrote the manuscript. All authors read and approved the final manuscript.	PubMed Central	2024-06-05T03:55:52.482954	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548140/", "journal": "Respir Res. 2005 Jan 21; 6(1):10", "authors": [ { "first": "Florian", "last": "Guthmann" }, { "first": "Heide", "last": "Wissel" }, { "first": "Christian", "last": "Schachtrup" }, { "first": "Angelika", "last": "Tölle" }, { "first": "Mario", "last": "Rüdiger" }, { "first": "Friedrich", "last": "Spener" }, { "first": "Bernd", "last": "Rüstow" } ] }
PMC548141	Background ========== Most, if not all, forms of chronic obstruction pulmonary disease (COPD) as well as asthma begin in the small airways. While the pathogenesis of small airway diseases is poorly understood \[[@B1],[@B2]\], it is generally accepted that the fluid and electrolyte transport properties of the epithelia lining these peripheral bronchioles play a crucial role in maintaining normal airway hygiene and patency. Some argue that these fluids are the primary defense because coupled with the ciliated escalator they form the first mechanism for clearing the airway of foreign debris and noxious agents. At the same time, almost nothing is known with certainty about the transport properties of distal airway epithelia or how fluid movements help maintain hygiene. No doubt, the paucity of understanding is due to the inaccessibility and the fragility of the tissue. Most concepts of the mechanisms and functions at this level have been taken from findings in the upper respiratory tract or from the larger cartilaginous ringed structures of the trachea and bronchi \[[@B3]-[@B7]\]. More extrapolations have been made from primary cultures of the same sources \[[@B8],[@B9]\]. Two previously published attempts were made to measure electrolyte transport parameters in isolated segments of small airways dissected from the peripheral airways of sheep \[[@B10]-[@B12]\] and pigs \[[@B13],[@B14]\]. However, in these studies the electrical signals, reflecting underlying transport properties may have been severely muted by tissue trauma during dissection and preparation. For standard electrophysiological studies of epithelia, dissection of the bronchiole would seem mandatory in order to maintain control of solutions on both sides of the epithelium. In order to minimize trauma, however, we attempted to microperfuse small bronchioles (i.d. 0.5--0.8 mm) in the periphery of pig lung without dissection. Unfortunately, since the bronchioles are embedded in a parenchyma of bronchioli and alveoli, this approach sacrifices control of the contra-luminal solution. Nonetheless, under this condition, we now find striking improvements in electrophysiological responses and strong evidence of a highly Cl^-^selective conductance that dominates the electroconductive properties of this epithelium, that is most probably duo to CFTR. Methods ======= Tissue ------ Lungs were excised intact immediately after sacrifice of young pigs (30--60 kg). Lungs were maintained inflated through a ligated plastic tube connected to an aquarium air pump (\~1 L/min) to maintain a positive airway pressure of 10--14 cm-H~2~O. The assembly was wrapped in a plastic bag and transported from the abattoir to the laboratory (\<60 min) in an insulated box chilled with ice. In the laboratory, small pieces of about 0.5 cm^3^were cut from the peripheral lung parenchyma, usually from along the costal diaphragmatic ridge of the lower lobes. In general, the freshest tissue gave the best responses although some tissue responded well after 6--8 hours of storage in a cooled environment. Microperfusion -------------- Under a dissecting microscope, the opening to a small airway was visualized on the proximal cut surface of a small block of lung tissue. The airway opening was then cannulated with a system of two concentric micropipettes \[[@B15]-[@B17]\] with tips fabricated so that the identified open end of the bronchiole could be aspirated into the outer pipette. (Fig. [1](#F1){ref-type="fig"}). Simultaneously, a double barreled inner pipette was inserted into the lumen of the bronchiole for delivering experimental solutions and monitoring electrical potentials through one barrel while constant current pulses were delivered through the other barrel \[[@B18]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Pipette assembly for microperfusing segments of undissected bronchiole. The bronchiole is held in outer large pipette (A) by suction. An inner, septated cannulating pipette provides current passing capacity through one barrel (B~1~) and perfusing fluid to the duct lumen through the opposite barrel (B~2~), which also contains a small cannula pipette (C) that allows changes of perfusing solutions. ::: ![](1465-9921-6-7-1) ::: Solutions --------- The perfused airways were intactand therefore remained embedded in the mass of connective tissue and air filled alveoli that normally surround the bronchi in vivo. The surrounding parenchymal tissue effectively prevented changing the solution in contact with the serosal surfaces of the airway epithelium, which in vivois the extracellular fluid and in theintact preparation could not be readily removed. NaCl Ringer solution is designed to mimic mammalian extracellular fluid. Therefore, we used NaCl Ringer in the bath to establish electrical continuity with the serosal surface of the bronchiolar epithelium during the entire experimental period. The Ringer solution contained in (mM): Na^+^(\~155), K^+^(4.5), Mg^2+^(1.2), Ca^2+^(1.0), PO~4~^3-^(3.5), Cl^-^(152), SO~4~^2-^(1.2), Glucose (5) buffered to pH 7.4 with NaOH. For ion diffusion studies, 150 mM of Cl^-^was replaced with an equivalent amount of gluconate (taken as impermeant), HCO~3~^-^, NO~3~^-^, I^-^, or Br^-^. Luminal solutions perfusing the bronchiolar airway were rapidly changed as needed via a manifold distributing stores of the above solutions through a needle tube to the tip of the perfusing pipette (Fig. [1](#F1){ref-type="fig"}). Agonists were added to solutions (in μM) as needed as forskolin (1), IBMX (100), ionomycin (1), ATP (100), and adenosine (100). Inhibitors were added (in μM) as needed as amiloride (10), NPPB (100), CFTR~Inh~-172 (5) \[[@B19],[@B20]\], GlyH-101 (50) \[[@B21]\] (CFTR~Inh~-172 and GlyH-101 were generous gifts from Dr. A. Verkman, University of California, San Francisco, CA.). Electrical Measurements ----------------------- The basic electrical circuit for recording potentials and conductance during microperfusion has been described previously \[[@B18]\]. The lumen of the bronchiole can be considered as a conductive core of fluid (perfusate) surrounded by an insulating epithelium.Unfortunately, the complex arborizing geometry of the bronchiole make it impossible to calculate the specific conductance of the epithelium from cable analysis as is possible with straight, unbranching tubes like sweat ducts and renal tubules. Thus, in the present protocol, the current pulse induced voltage deflections reflect the total resistance of the preparation and can only be used to compare changes in the epithelial resistance within the same preparation when identical solutions are present in the lumen and bath; e.g., pre- and post-drug application. Although we recorded the total resistance (R~t~) of the system, which includes the summed resistances of the epithelium (including parallel shunts through it) plus the core resistance of the lumen plus the extracellular fluid resistance, the resistance of the epithelium relative to R~t~was small (even after floating the current passing circuit), and therefore changes in the epithelial resistance were obscured. Consequently, the response of TEP\'s was taken as the primary indication of the permeability properties of the epithelium. Temperature ----------- The bathing solution was maintained at 35 ± 2°C. mRNA expression --------------- Total RNA was isolated from the dissected bronchioles of 4 pigs by using RNeasy Mini Kit (QIAGEN Inc. CA). RNA was reversely transcribed using Sensiscript RT Kit (QIAGEN Inc. CA). The resulting first-strand cDNA was directly used for PCR amplification (TaqPCR Core Kit, QIAGEN Inc. CA). The conditions for PCR reactions were as follows: 3 min at 94°C (initial melt); 35 cycles of 1 min at 94°C, 1 min at 55--60°C, 1 min at 72°C and then 72°C 10 min (final extension). For the negative control, RT-PCR was performed in the absence of RT. The PCR products were analyzed by agarose gel electrophoresis stained with ethidium bromide. The primers were constructed on the basis of the published cDNA sequence of CFTR, ENaC, NKCC1 and β-Actin from GenBank. Since the pig gene sequence was not complete, primers were obtained from the human accordant gene, in which highly conserved regions were selected. The pairs of primers for CFTR (accession no. NM\_000492) were sense 5\'-TCCTAAGCCATGGCCACAA-3\' and antisense 5\'-GCATTCCAGCATTGCTTCTA-3\'; sense 5\'-GCCTGGCACCATTAAAGAAA-3\' and antisense 5\'-CTTGCTCGTTGACCTCCACT-3\', which generated a 197-bp and 171-bp CFTR PCR product respectively; for α-ENaC (Z92978) were sense 5\'-CAACAACACCACCATCCAC-3\' and antisense 5\'-TAGGGATTGAGGGTGCAGA-3\', which generated a 225-bp PCR product; for β-ENaC (NM\_000336) were sense 5\'-TGCTGTGCCTCATCGAGTTTG-3\' and antisense 5\'-TGCAGACGCAGGGAGTCATAGTTG-3\', which generated a 277-bp PCR product; for γ-ENaC (X87160) were sense 5\'-TCAAGAAGAATCTGCCCGTGA-3\' and antisense 5\'-GGAAGTGGACTTTGATGGAAACTG-3\', which generated a 237-bp PCR product; for NKCC1 (U30246) were sense 5\'-TCCAGGTAATGAGTATGGTGTCAG-3\' and antisense, 5\'-GTTAAGATGTAGCCACGAAGAGGT-3\', which generated a 205-bp PCR product; and for β-Actin (BC004251) were sense, 5\'-TTCAACTCCATCATGAAGAAGTGTGACGTG-3\' and antisense, 5\'-CTAAGTCATAGTCCGCCTAGAAGCATT-3\', which generated a 312-bp PCR product. All primers showed products closely corresponding to the predicted size for expression of RNA transcripts for these genes. Immunocytochemistry ------------------- The bronchioles were dissected and then fixed in ice-cold 4% formaldehyde buffered in phosphate at 4°C for 3 hours, infiltrated with cryoprotectant (30% sucrose in PBS) overnight, and frozen in OTC medium (Triangle Biomedical Sciences) at -35°C. Sections of 5 μm thickness were cut on a cryostat microtome (Thermo Electron) and mounted on glass slides (Fisher Superfrost Plus). Antigen retrieval was performed using a pressure cooker (10 min in 10 mM citrate buffer, pH 6). To reduce autofluorescence, sections were treated for 20 min with 1.5% sodium borohydride in PBS. Sections were incubated sequentially with blocking solution (30 min), primary antibody (overnight at 4°C), and secondary antibodies conjugated to Alexa Fluor-488 and/or -546 (Molecular Probes). Confocal images were acquired with a Zeiss LSM-510 microscope and assembled using Adobe Photoshop. CFTR was labeled with rabbit antibody R3194 (courtesy of C. Marino), and ENaC with a rabbit antibody against the β-subunit of human ENaC (kindly courtesy of C. Fuller and D. Benos). Tight junctions were labeled with a mouse antibody against the junction-associated protein zonula occludens-1 (Zymed). Nuclei were stained with TO-PRO-3 (Molecular Probes). Statistical treatment --------------------- Differences in mean measurement were assayed by applying the Student T test to paired or unpaired means as appropriate. A probability (P value) of ≤ 0.05 was taken as significantly different. Results ======= Basic electrical properties --------------------------- When the bronchiolar lumen was perfused with NaCl Ringers, which we assumed represented insignificant ion gradients except for a small lumen (145 mM) to serosa (110 mm) Cl^-^gradient. Despite the fact that this small Cl^-^gradient should render the lumen positive, there was a small spontaneous lumen negative potential of about -3 mV (Fig. [2](#F2){ref-type="fig"}; Table [1](#T1){ref-type="table"}). When we applied amiloride (10 μM) to the lumen to block Na^+^conductance (gNa^+^), the TEP decreased slightly, but without statistical significance (Fig. [2](#F2){ref-type="fig"}; Table [1](#T1){ref-type="table"}). We were unable to detect a change in total conductance with amiloride applied to the lumen. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effect of amiloride and Forskolin (Fsk, 5 μM) + IBMX (100 μM) on transepithelial potential (TEP) of bronchiole. In the presence of luminal Cl^-^, the effects of both amiloride and Fsk+IBMX on TEP were almost imperceptible (left side). However, when Cl^-^was substituted with Gluconate to more effectively reveal the Cl^-^conductance, addition of amiloride depolarized TEP and Fsk+IBMX hyperpolarized the TEP (right side), suggesting that the large Cl^-^conductance present in the epithelium mutes (shunts) the smaller changes in conductance occasioned by amiloride and Fsk when isotonic Cl^-^is present bilaterally. NaGlu: Na-gluconate ::: ![](1465-9921-6-7-2) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Amiloride Inhibition ::: NaCl NaCl+Amil NaGlu NaGlu+Amil ------------ ------------ ------------ ------------- ------------- TEP (mV) -3.1 ± 0.6 -2.6 ± 0.7 -57.3 ± 2.7 -43.6 ± 2.7 Δ TEP (mV) \-- +0.5 \-- +13.7 n 25 10 25 16 P value \-- 0.676 \-- \<0.001 ::: Gluconate Substitution ---------------------- However, when we replaced luminal Cl^-^with the impermeant anion, gluconate, the TEP hyperpolarized to as much as -90 mV (Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}). Addition of amiloride (10 μM) to the lumen depolarized the mean TEP of these tissues by an average of 14 mV (Fig. [2](#F2){ref-type="fig"}; Table [1](#T1){ref-type="table"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effect of luminal Cl^-^substitution in a perfused small bronchiole on TEP. The substitution of Gluconate, an impermeant anion, for permeable Cl^-^in the lumen markedly hyperpolarized the TEP. This response indicates a predominant Cl^-^conductance that appears to be constitutively active. ::: ![](1465-9921-6-7-3) ::: Anion Conductance Inhibitors ---------------------------- Under conditions of symmetrical \[Cl^-^\] concentrations, luminal applications of anion conductance inhibitors had virtually no detectable effect on the spontaneous TEP. However, under hyperpolarizing conditions created by Cl^-^substitution in the lumen, NPPB (100 μM), GlyH-101 (50 μM) and CFTR~Inh~-172 (5 μM) significantly depolarized the TEP by 36.7, 20.0 and 8.7 mV (Fig. [4](#F4){ref-type="fig"}; Table [2](#T2){ref-type="table"}). Bumetanide (1 mM) had no effect on the TEP in either the presence or absence of a Cl^-^gradient (not shown). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effect of Cl^-^conductance inhibitors on TEP. In the absence of luminal Cl^-^(Gluconate substitution), two new inhibitors GlyH-101 (50 μM) and CFTR~Inh~-172 (5 μM, re: A. Verkman) showed at least partial inhibition of the Cl^-^:Gluconate anion diffusion potential. NPPB (100 μM) seemed to be the most effective inhibitor in terms of depolarizing the TEP. ::: ![](1465-9921-6-7-4) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Cl^-^Conductance Inhibition ::: CFTR~inh~-172 NPPB GlyH-101 ------------ --------------- ------------- ------------- TEP (mV) -48.6 ± 4.7 -16.5 ± 2.1 -26.6 ± 2.5 Δ TEP (mV) +8.7 +36.7 +20.0 n 3 5 4 P value 0.03 0.001 0.03 ::: Anion selectivity ----------------- We assayed for the relative permeability of several monovalent anions by substituting them for Cl^-^in NaCl Ringer. Amiloride (10 μM) was added in order to block Na^+^transport. For luminal Cl^-^, Br^-^, I^-^, NO~3~^-^, HCO~3~^-^and gluconate, the mean estimated P~x~/P~Cl~were, respectively, 1.0, 0.92, 0.79, 0.65, 0.33 and 0.28 (Table.[3](#T3){ref-type="table"}). When the NaCl Ringer perfusion solution was diluted by 1:2, the potential depolarized by 12 ± 1 mV, indicating Cl^-^permeability exceeded the Na^+^permeability by about 5.4 fold (Fig. [5](#F5){ref-type="fig"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Anion selectivity sequence of the perfused bronchiole. The sequence of the bronchiole (upper data) roughly fits the known sequence of CTFR in other tissues (lower data taken from literature; cf. text) ::: Airway Anion selectivity: Cl^-^ ≈ Br^-^ \> I^-^ \> NO~3~^-^ \> HCO~3~^-^ \>\> Gluconate --------------------------- -------------- ------- ----------- -------- ----------- -------- -------------- -------- --------------- ---------- --------------- Transepithelial TEP : \~0 -1.9 -5.1 -9.2 -19.5 -44.8 Estimated P~x~/P~Cl~: 1.0 0.92 0.79 0.65 0.33 0.28 CFTR anion selectivity: NO~3~^-^ ≈ Br^-^ ≈ Cl^-^ \> I^-^ \> HCO~3~^-^ \>\> Gluconate Estimated P~x~/P~Cl~: 1.1 1.1 1.0 0.39 0.014 \~0 ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### NaCl dilution diffusion across the bronchiolar epithelium on TEP. The hyperpolarization of the TEP with diluted NaCl (75 mM) indicates that Cl^-^must be significantly more permeable through the epithelium than Na^+^. ::: ![](1465-9921-6-7-5) ::: Agonists -------- When we added forskolin (5 μM) plus IBMX (100 μM), adenosine (100 μM), ATP (100 μM) or ATP (100 μM)+adenosine (100 μM) to the perfusate to activate CFTR gCl^-^in the presence of isotonic Cl^-^concentrations and in the absence of a hyperpolarizing gradient, the TEP did not change perceptibly (not shown), but in the presence of the Cl^-^gradient, the TEP hyperpolarized significantly to all agonists; the response appeared to be increased when both ATP and adenosine were added together (Fig. [6](#F6){ref-type="fig"}; Table [4](#T4){ref-type="table"}). In order to observe the maximum effect on the activation of Cl^-^conductance, amiloride (10 μM) was present in all luminal perfusates to block ENaC Na^+^conductance. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Effect of Cl^-^conductance agonists on TEP: Applying Fsk (5 μM)+IBMX (100 μM), adenosine (100 μM), ATP (100 μM) and ATP (100 μM)+adenosine (100 μM) to the perfusate to activate CFTR gCl^-^in the presence of a hyperpolarizing Cl^-^gradient, the TEP hyperpolarized significantly; however, the response appeared to be increased when both ATP and adenosine were added together. In order to observe maximum effect on activation of Cl^-^conductance, amiloride (10 μM) is present in all luminal perfusate above to block Na^+^conductance. ::: ![](1465-9921-6-7-6) ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Cl^-^Conductance Agonists ::: Fsk+IBMX ATP Adenosine ATP+Adenosine Ionomycin ------------ ------------ ------------- ------------- --------------- ------------- TEP (mV) 53.0 ± 2.8 -51.0 ± 0.8 -55.8 ± 6.7 -52.0 ± 6.5 -40.1 ± 7.5 Δ TEP (mV) -26.0 -5.5 -5.5 -6.3 0.4 n 13 4 5 6 5 P value \<0.001 0.007 0.003 0.011 0.836 ::: RT-PCR ------ Two sets of different primers for CFTR as well as primers for α-, β-and γ-ENaC, NKCC1, and β-Actin all produced products of predicted sizes for each of the corresponding mRNAs (Fig. [7](#F7){ref-type="fig"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### RT-PCR bands for CFTR (lanes 2 &3), α,β,γ subunits of ENaC (lanes 5,6,7 respectively), and NKCC1 (lane 9) and β-Actin (lane 10). Two independent sets of primers were used to detect CFTR. The presence of CFTR, ENaC, and NKCC1 may indicate that the epithelium has both absorptive and secretory functions. β-Actin was used as a housekeeper marker for control. Size ladders in 100 bp increments with lowest band equal to 100 bp (lanes 1,4,8). ::: ![](1465-9921-6-7-7) ::: Immunocytochemistry of CFTR --------------------------- Immunoreactive CFTR antibody (gift of C. Marino) was detected in the apical domains of the bronchiolar epithelia in a continuous border of the bronchiole. The tight junctions were labeled simultaneously and appeared as punctate areas within the border staining for CFTR consistent with the expected location for these structures (Fig. [8A](#F8){ref-type="fig"} and inset). Negative controls consisted of omission of primary antibodies and showed no labeling of the antibody in any tissue (Fig. [8B](#F8){ref-type="fig"}). ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Immunocytochemical localization of CFTR in bronchiole epithelium. A.). Antibody R3194 against CFTR prominently labels (green) the apical margin of epithelial cells lining the bronchiole cut in cross section. Inset: high magnification view of epithelial cells showing demarcation of apical margin by tight junction-associated protein ZO-1 (red). B.) Control serial section stained without primary antibody. ::: ![](1465-9921-6-7-8) ::: Discussion ========== The airways are extraordinarily specialized conduits for air to and from the alveoli for gas exchange. They must remain moist in order to remain flexible and to effectively filter air before it enters the delicate tissues of the alveoli. Hence, a principal function of the airway epithelia is to provide and service a continuous layer of aqueous fluid on the airway surfaces. For decades, the upper airways, trachea, and large bronchi have served as models for the entire tracheobronchial epithelial function \[[@B14],[@B22],[@B23]\], and yet it is known that there are distinct regional differences progressing from nares to alveoli. For example, anatomically, the upper airways and bronchi are characterized by submucosal glands that secrete fluid directly into the airway, but are absent in the peripheral small airways \[[@B24]\]; \[[@B25]\]. Likewise, the trachea and larger airways are kept patent by cartilaginous rings whereas the peripheral airways are generally held open (but may collapse) by internal retractile forces of the lung parenchyma \[[@B26]\]. Similarly, the cell populations change. The epithelium of the upper respiratory tract changes from ciliated, pseudostratified columnar to simple cuboidal cells in the smaller airways where the proportion of Clara cells increase and that of ciliated cells decrease \[[@B27]\]. Functionally, there are differences as well. For example, in humans and in dogs, the spontaneous transmural electrical potential appears to become less negative (lumen: blood) from upper to lower airways \[[@B13],[@B28],[@B29]\]. Differences in airway surface fluid composition may also exist \[[@B30]\]. The functions of the lower peripheral airway epithelium is poorly defined and understood because this tissue is inaccessible and relatively unamenable to standard physiological techniques for study. Nonetheless, a few courageous attempts to unravel these mysteries have been made. About ten years ago, Ballard \[[@B13],[@B14]\] and Al-Bazzaz \[[@B10]\] isolated and perfused small airways and bronchioles from pig and sheep, respectively. Ballard perfused porcine airways of about 1 mm diameter and reported a mean spontaneous TEP potential difference in bilateral NaCl Ringer solution of about -3.4 mV and in lumen Cl^-^free solution of about -16 mV \[[@B14]\]. Al-Bazzaz perfused ovine bronchioles of about 250 μm diameter and reported a mean spontaneous TEP of -2.5 mV in bilateral NaCl Ringer and of about -4.2 mV mean TEP in lumens perfused with Cl^-^free Ringers \[[@B11]\]. In all of these studies, it has been difficult to ascertain to what degree the electrical responses were muted or altered by trauma to the tissue during dissection because similar measurements are not possible in vivo. We, too, found similar, relatively small TEP voltages in microdissected porcine airways. Undissected bronchioles ----------------------- Therefore, in order to maximally preserve, and minimally traumatize the airway epithelium, we avoided dissecting the bronchial structure and microperfused the airways imbedded in lung parenchyma. We found that the spontaneous TEP in bilateral Ringer solution was about -3 mV, slightly more negative than reported earlier. But in striking contrast to the previous studies (including our own dissected preparations), we found that the bi-ionic TEP with Cl^-^free solutions in the lumen was as much as -90 mV (mean: -57 mV; Table [1](#T1){ref-type="table"}; Fig. [2](#F2){ref-type="fig"}). These differences almost certainly reflect differences in trauma to the airway. Because of the extremely complicated morphology that arises from arborization of the airway in route to hundreds of alveolar acini, it is impossible to physically remove the surrounding tissues (very small bronchioles, respiratory bronchioles, blood vessels, and alveolar sacs) without breaking or tearing smaller \"branches\" from the \"tree\" of airways. Both Ballard and Al-Bazzaz attempted to patch these breaks by micro-suturing obviously dangling limbs; unfortunately, only larger branches are amenable to such heroic attempts and many smaller, even microscopic transepithelial openings, must remain. In order for an epithelium to reflect its in vivoelectrical properties in vitro, it is fundamental that the integrity of the epithelial sheet be conserved because TEP measurements depend on separation of charge. Breaks, holes, or tears in the barrier inescapably create electrical shunts, which, by allowing simultaneous back leak of epithelial current, prevent separation of charge. Even though the individual cells or groups of cells of the epithelia function and respond physiologically, the transepithelial voltage signals will be erroneously muted or lost through such shunts. In the present case, it appears that perfusion of the bronchiole without dissection from surrounding parenchyma, minimizes trauma induced shunts and allows detection of a much more complete and robust electrical signal. Unfortunately, the price for this preservation of signal is the inability to control the contra luminal solution. Keeping in mind that the undissected bronchiole is embedded in a mass of air filled alveoli together with numerous other elements of the tissue at this level, it is easy to see that changing the bath solution can have no acute effects on the composition of the contra luminal solution at the basilateral membrane of the epithelium. Consequently, we did not change the bath solution and assumed that normal Ringer solution is sufficiently similar to extracellular fluid in vivoto avoid introducing significant free solution diffusion potentials or altering the physiological composition of the native fluid present in the extracellular compartment of the in tact preparation. Cl^-^Conductance ---------------- The simple fact that substituting Cl^-^in the lumen spontaneously created a large lumen negative transmural potential (Figs. [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}; Table [1](#T1){ref-type="table"}) immediately indicates that the small airway is characterized by a dominant constitutively active anion selective conductance. The fact that imposing a putative 1:2 NaCl diffusion gradient across the epithelium resulted in -12 mV hyperpolarization indicates that the intact bronchiole epithelium is at least 5 times more permeable to Cl^-^than to Na^+^(Fig. [5](#F5){ref-type="fig"}). The facts that this Cl^-^conductance is immediately evident upon initial perfusion without any prior agonist stimulation and that additions of agonists did not significantly hyperpolarize the bronchiole perfused with 150 mM NaCl and hyperpolarized bronchioles perfused with Na Gluconate only by about 20--25% demonstrates that the Cl^-^conductance is constitutively open under these conditions (Fig. [6](#F6){ref-type="fig"}). That is, from the first moment of measurement after cannulation, the large Cl^-^conductance is present (Figs. [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}). There was no need to activate PKA or wait for the transmural potential to develop even though the addition of forskolin, adenosine, and ATP appeared to increase Cl^-^conductance (Figs. [2](#F2){ref-type="fig"}, [6](#F6){ref-type="fig"}; Table [4](#T4){ref-type="table"}). In secretory cells where secretory activity is usually an acute, temporal event, CFTR is assumed to remain closed until activated by PKA and ATP. However, in the human sweat duct, also a rich source of CFTR, where the transport function is exclusively absorptive and where CFTR is thought to be the only anion conductance through the tissue, CFTR appears with a large Cl^-^conductance at the first moment of perfusion and measurement, indicating a constitutively open state for the CFTR channel in this tissue as well. It is well established that CFTR can be regulated in the classic sense by PKA phosphorylation and ATP in the sweat duct, but the cytoplasm must first be \"rinsed\" of small solutes by permeabilizing the basilateral membrane \[[@B31]\]. CFTR ---- Since obstructive airway disease in Cystic Fibrosis arises in the small airways \[[@B32]-[@B34]\] and CF is known to be due to mutations in the CFTR gene that expresses a Cl^-^channel, we asked if this conductance could be due to CFTR. We found several lines of evidence that are consistent with CFTR being responsible for the Cl^-^conductance. First, the anion selectivity sequence, excepting NO~3~^-^, is grossly the same as that for CFTR (Table [3](#T3){ref-type="table"}); i.e., Cl^-^≈ Br^-^\> I^-^\> NO~3~^-^\> HCO~3~^-^\> Gluconate, and compares favorably with that of CFTR: SCN \> NO~3~^-^\> Br^-^\> Cl^-^\> I^-^\> HCO~3~^-^\> F^-^\> ClO~4~^-^\> gluconate \[[@B35]-[@B37]\]. Second, it is well known that CFTR is activated characteristically by cAMP mediated protein kinase A, which is driven pharmacologically by forskolin and IBMX. Here, we see (Figs. [2](#F2){ref-type="fig"}, [6](#F6){ref-type="fig"}; Table [4](#T4){ref-type="table"}) that these agonists routinely elevate the bionic Cl: gluconate diffusion potential by an average of -22 mV (n = 13). Similarly, ATP and adenosine may activate CFTR in airway epithelia since these agonists can elevate cAMP via purinergic receptors. \[[@B38]-[@B40]\]. In contrast, when we applied ionomycin to elevate intracellular Ca^2+^, there was no effect (not shown), suggesting that since CFTR is not sensitive to Ca^2+^mediated activation either, a.) the conductance is due to CFTR, b.) other Ca^2+^activated Cl^-^conductances must be fully constitutively activated or not present, or c.) luminal application of the ionophore drug does not increase intracellular Ca^2+^effectively. Third, CFTR is known to be inhibited by NPPB, CFTR~Inh~-172, and GlyH-101\[[@B19],[@B21]\]. These inhibitors had varying, but consistently inhibitory effects on the TEP (Fig. [4](#F4){ref-type="fig"}; Table [2](#T2){ref-type="table"}) indicating inhibition of Cl^-^conductance in the airway epithelium. However, the fact that none of the inhibitors appeared to completely inhibit the transepithelial potential might argue that another anion channel conductance is present. Two points diminish this argument. First, if the tissue is actively transporting, anion channel inhibitors will not completely ablate the TEP because that component of potential generated by the basilateral K^+^emf and the apical Na^+^emf should not be blocked and should be reflected across the epithelium in the TEP. Secondly, even when the CFTR Cl^-^channel has been isolated from active transport components, anion channel blockers do not usually completely block its conductance in native tissue \[[@B41]\]. Moreover, recently, in contrast, to our results, GlyH-101 was reported to be ineffective in blocking the Cl^-^conductance of pig nasal airways \[[@B42]\]. It is not known whether CFTR is present in the nasal epithelium of pigs, but biochemically, we found markers for conserved regions of expressed CFTR RNA from reverse transcriptase polymerase chain reactions (Fig. [7](#F7){ref-type="fig"}) in bronchioles dissected free of parenchyma post perfusion. Appropriate bands for CFTR protein in lysates of frozen peripheral lung tissue were also observed with affinity purified antibodies (J. Riordan, personal communication), and as shown in (Fig. [8](#F8){ref-type="fig"}) CFTR localized immunocytochemically, specifically to the apical surface of the bronchiolar epithelia. These data strongly suggest that CFTR is a part of, or probably is, the primary source of the anion conductance in small airways. Transport Function ------------------ Not only does the bronchiole resemble the sweat duct with respect to exhibiting a constitutively active Cl^-^channel that responds to activation of PKA, but both are also apparently insensitive to ionomycin. They both show equally large bionic diffusion potentials for Cl^-^and are several fold more permeable to Cl^-^than to Na^+^, and yet both have very low transmembrane potentials in bilateral isotonic Ringer solutions \[[@B43]\]. Both are incompletely inhibited by anion channel blockers \[[@B20],[@B41],[@B44]\], and both are sensitive to amiloride. On the basis of these observations and by analogy with the sweat duct, it is tempting to propose that the bronchiole at least in its basal state is a constitutively absorptive epithelium. Conclusions =========== We have found that the epithelium of terminal airways of the pig appears to express an anion permeability that constitutively dominates the electroconductive properties of this zone of the airway. Its anion selectivity sequence is similar to that expected for CFTR, and its activity can be enhanced by forskolin/IBMX or decreased by anion channel blockers known to inhibit CFTR. RT-PCR amplification products and specific antibodies identify CFTR in this tissue. The small airway appears to share a number of properties with the human sweat duct and may, by analogy, belong to a class of highly absorptive epithelia. Abbreviations ============= CFTR: Cystic Fibrosis Transmembrane Conductance Regulator ENaC: Epithelial Na^+^Channel NKCC: Na^+^-K^+^-2Cl^-^Cotransporter TEP: Transepithelial Potential PKA: Protein Kinase A Acknowledgments =============== This work was supported by CFRI, the Nancy Olmsted Trust, and the USPHS- NIH 5R01 DK51899-04 and DE 14352. We gratefully acknowledge the assistance of Ms. Joanne Albrecht, Ms. Suchi Madireddi, and Mr. Kirk Taylor. We sincerely thank Dr. Chris Marino (University of Tennessee, Memphis) for the CFTR antibody and Dr. Allen Verkman (UCSF) for supplying the Cl- conductance inhibitors.	PubMed Central	2024-06-05T03:55:52.486980	2005-1-17	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548141/", "journal": "Respir Res. 2005 Jan 17; 6(1):7", "authors": [ { "first": "Xiaofei", "last": "Wang" }, { "first": "Christian", "last": "Lytle" }, { "first": "Paul M", "last": "Quinton" } ] }
PMC548142	Background ========== Sickle cell disease is a common debilitating hematologic disease occurring in 1 in 650 African Americans. Lung disease is a major cause of cardiopulmonary disability and mortality \[[@B1],[@B2]\]. Progressive restrictive lung disease related to recurrent episodes of acute chest syndrome may develop with advancing age \[[@B3]\]. Acute chest syndrome is a clinical manifestation triggered by pathological processes including infections, fat embolism, infarction and bronchospasm \[[@B4]\]. Nearly 70% of patients with acute chest syndrome are hypoxemic (as measured by pulse oximetry \< 90% or PO~2~\< 80 mmHg by blood gas analysis) \[[@B5]\]. Hypoxemia causes sickle hemoglobin to gel, inducing red blood cell sickling and vaso-occlusion within pulmonary blood vessels. Asthma is the most common chronic disease of childhood occurring in 6% of the general population. Moreover, the African American population has the highest prevalence (8%), emergency department (ED) visits, hospitalizations and risk of mortality of any ethnic population in the United States \[[@B6]\]. Reversible airway obstruction, airway inflammation and nonspecific bronchial hyperresponsiveness are the hallmarks of asthma. Exacerbations of asthma result in mucous plugging, bronchoconstriction with decreased air exchange, ventilation-perfusion mismatch and subsequently hypoxemia. Aggressive treatment with oxygen, bronchodilators and oral corticosteroids are recommended for symptomatic relief of acute episodes. The African American population is at significant risk for the occurrence of both diseases simultaneously but little is known of the affect of asthma on individuals with sickle cell anemia. In this study, we examined hospitalized children with sickle cell disease with concomitant asthma establishing whether there was an increased rate of acute chest syndrome or other complications compared to patients with sickle cell disease without asthma in those presenting with vaso-occlusive pain crises. Methods ======= We performed a 5-year retrospective chart review of 48 children with asthma and sickle cell disease and compared them to a control group of 48 children with sickle cell disease alone. The 48 children with sickle cell and asthma represented all patients with complete medical records with both diseases that were cared for at our center. These children had no hospitalizations for sickle cell crises at other facilities. Children in the control group were matched for age, hemoglobinopathy and gender. Sickle cell disease was determined by high performance liquid chromatography and isoelectric focusing analysis and grouped into phenotypes of hemoglobin SS or hemoglobin SC. Children were included in the asthma group if they had medical record documentation of a discharge diagnosis of asthma (ICD-493) and had been prescribed asthma medications. Vaso-occlusive pain was defined as pain that could not be explained by injury or infection requiring hospital admission and treatment with intravenous pain medication. The criteria for a diagnosis of acute chest syndrome included respiratory distress, hypoxemia, and a new infiltrate on chest x-ray that required hospitalization and a transfusion of packed red blood cells. A cerebral vascular accident diagnosis was based on the new onset of an acute neurological syndrome with a focal neurological finding on examination associated with ischemic changes (images compatible with stroke) on a brain MRI or computed tomography scan. Inpatient admissions were recorded from March 1997 to March 2002 for episodes of acute chest syndrome, vasoocclusive pain crises, cerebral vascular accidents, total blood transfusions, exchange transfusions and chronic transfusions (monthly blood transfusions). Exclusion criteria included any child who had incomplete documentation of their hospital records or those who had moved into or out of the Milwaukee area during the 5-year study period. Six children were excluded because they moved from Milwaukee. The study was approved by the Investigational Review Board. Statistical Methods ------------------- The Mann-Whitney test was used to evaluate differences between the incidents of vasoocclusive pain crises, acute chest syndrome, total blood transfusions and cerebral vascular accidents. Chi-squared analysis was used to evaluate the number of exchange and chronic transfusions. Statistical significance was given to p values 0.05 or less. Results ======= All subjects in the asthma group had asthma recorded in their medical record with symptoms consistent with asthma. All had been prescribed albuterol; while 28 (58%) had been prescribed controller medications (24 inhaled corticosteroids, 12 inhaled cromolyn sodium, and 5 leukotriene modifier). Most patients had been prescribed combination therapy with more than 1 controller medication or had been switched from 1 controller to another. There was no reliable means to confirm adherence to the prescribed asthma drug regimen. Seventeen (35%) subjects had not had prior ED [or]{.underline} hospital admissions for asthma. Of these children, 13 (76%) had been prescribed only albuterol. Twelve (25%) had at least 1 ED visit [and]{.underline} at least 1 hospital admission for asthma. Twenty-six (54%) subjects had only hospital admissions for asthma, while 15 children had only been treated in the ED for acute asthma. Of 14 children with a severity category documented, 9 had mild intermittent asthma, 1 each with mild persistent and moderate persistent asthma, and 3 with severe persistent asthma. No patient had documented bronchoprovocation with methacholine and only 3 had documented spirometry with 1 consistent with airway obstruction, 1 with reversibility of airway obstruction, and one with poor technique and unreliable results. Two patients (age 3) were too young for spirometry. Four subjects had been seen in the Asthma and Allergy clinic for consultation and the diagnosis of asthma reaffirmed. Twenty-one (44%) subjects had a primary family member with asthma. Only 5 children were born preterm (27, 33, 34, 36, and 37 weeks gestation) and none were diagnosed with bronchopulmonary dysplasia. The cases and controls were well-matched for age, gender and type of hemoglobinopathy (Table [I](#T1){ref-type="table"}). The cases (21 males and 27 females) consisted of 42 children with HgbSS and 6 with HgbSC. The control group (17 males and 31 females) included 41 children with HgbSS and 7 with HgbSC. The age of the children ranged from 3 to 18 years old with a mean age of 10.1 years (median 10 years) for the case patients and 10.3 years (median 10 years) for the control children. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Subject Demographics ::: Patients with Sickle Cell & Asthma Patients with Sickle Cell Disease -------------- ---------------------------------------- --------------------------------------- Males 21 17 Females 27 31 Mean Age 10.1 years 10.3 years HgbSS 42 41 HgbSC 6 7 ::: Patients with sickle cell disease and asthma had significantly more episodes of acute chest syndrome, cerebral vascular accidents, blood transfusions and chronic transfusions as compared to the control group (Table [2](#T2){ref-type="table"}). Admissions for vaso-occlusive pain crisis and exchange transfusions were not statistically significant between groups. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Results in children with sickle cell disease with and without asthma ::: Patients with Sickle Cell & Asthma (n = 48) Patients with Sickle Cell Disease (n = 48) Statistical Significance ------------------------------------------- ------------------------------------------------- ------------------------------------------------ ------------------------------ Admissions for Acute Chest Syndrome 90 58 P = 0.03 Number of Cerebral Vascular Accidents 10 2 P = 0.05 Total Blood Transfusions 432 226 P = 0.01 Chronic Blood Transfusions 13 4 P = 0.04 Vaso-occlusive Pain Crises 248 223 P = 0.52 Exchange Blood Transfusions 9 4 P = 0.21 ::: No patient with SC disease in either group had a history of a cerebral vascular accident. Only 1 patient with SC disease had acute chest syndrome, while 3 children in the asthma group had acute chest syndrome with one child experiencing 2 episodes. Discussion ========== Despite the high prevalence of these two diseases that affect the African American population, there is paucity of research investigating patients with concomitant asthma and sickle cell disease. In this study, we discovered that children with both asthma and sickle cell disease are significantly more likely to develop severe complications of sickle cell disease including acute chest syndrome and cerebral vascular accidents compared to children with sickle cell disease alone. The significance of these findings relates to the hypothesis that appropriately aggressive treatment of asthma in children with sickle cell disease may diminish the frequency of pulmonary complications. Patients with reactive airway disease and sickle cell disease have a lower transcutaneous oxyhemoglobin saturation \[[@B7]\]. The lower transcutaneous oxyhemoglobin saturation increases sickling of red blood cells, causing subsequent gelling and vaso-occlusion in multiple organs leading to numerous complications. A high prevalence (73%) of airway hyperreactivity to cold-air challenge occurs in children with sickle cell anemia even in the absence of clinical symptoms of asthma \[[@B8]\]. Cold air or other provocative challenge tests had not been performed in our group of patients. Our results are in agreement with an abstract that showed 18 children with both asthma and sickle cell disease had increased hospital admissions for pain crises and acute chest episodes compared with patients with only sickle cell disease \[[@B9]\]. Our results are unique because we assessed for an increased frequency of transfusions and cerebral vascular accidents in a larger group of patients. Cerebral vascular accidents affect 10% of the children with sickle cell disease often with devastating long term implications. The Cooperative Study of Sickle Cell Disease reported that recent and recurrent episodes of acute chest syndrome are risk factors for cerebral vascular accidents \[[@B10]\]. Our findings of increased cerebral vascular accidents in patients with sickle cell disease and asthma may be due to their increased and recurrent episodes of acute chest syndrome. The mechanism linking stroke and acute chest syndrome is unknown but may be related to hypoxemia due to pulmonary disease. Hypoxemia has been reported to increase adhesion of red blood cells to endothelium \[[@B11]\]. Likewise, increased red blood cell adhesion to pulmonary endothelium has been associated with bone marrow vaso-occlusive crises due to hypoxia, cytokine expression and fat embolization \[[@B12]\]. Whether hypoxemia secondary to asthma affects red blood cell and pulmonary/cerebral endothelium adhesion characteristics is unknown. Our study demonstrates that pediatric patients with sickle cell disease and asthma are more likely to require acute and chronic blood transfusions. It is not surprising that these patients needed more frequent transfusions since treatment for cerebral vascular accidents and severe acute chest syndrome is blood transfusions. Although SS type of sickle cell anemia is more severe than SC type, the small numbers of patients with SC prevent us from making broad conclusions regarding the degree of risk based on hemaglobinopathy. Two studies have demonstrated increased airway hyperreactivity with reversibility in patients with sickle cell disease without known asthma \[[@B8],[@B13]\]. Thirty-five percent of sickle cell patients had evidence of lower airway obstruction and 78% of these reversed with bronchodilator. Even in 30% of those with normal lung function, 30% had a positive response to bronchodilator. Therefore, even methods to determine the presence bronchial hyperresponsiveness are not sufficient to discriminate asthma from acute chest syndrome. Whether this hyperreactivity is due to asthma or is secondary to the pathophysiology of sickle cell disease is still unclear. Additional studies are needed to confirm that sickle cell disease is associated with the development of reversible airway obstruction. If the association is valid, the effects of routine use of anti-inflammatory controller agents prophylactically or therapeutically would deserve investigation. A randomized, placebo-controlled trial of 43 episodes of acute chest syndrome in 38 children revealed that intravenous dexamethasone prevented clinical deterioration in mild to moderately severe episodes of acute chest syndrome \[[@B14]\]. Mild and moderately severe acute chest syndrome was defined as respiratory distress, and normal mental status without pulmonary infiltrates or arterial hypoxemia. The study excluded children with an exacerbation of reactive airways disease. If patients with sickle cell disease are at increased risk of airway inflammation and obstruction, successful treatment with intravenous steroids may have been due to aggressive treatment of underlying asthma. Limitations of our study include those inherent in a retrospective analysis including selection bias, measurement bias and confounding factors. The children who received transfusions for acute chest syndrome likely represent the more severe form of disease. In contrast, those patients deemed to have asthma were primarily being treated as if they had mild to moderate asthma. Only 3 had been diagnosed with severe persistent asthma and none were on daily or every-other-day oral steroids. Importantly, even individuals with mild intermittent asthma can experience severe asthma exacerbations. Other sources of potential error that deserve recognition include the diagnosis of asthma (whether over-diagnosed or under-diagnosed), medication compliance and unknown admission to other hospitals. Although a strict definition of asthma was lacking, the history gleaned from the medical records supported an asthma diagnosis. Most patients with asthma are not cared for by asthma specialists and frequently the diagnosis is made on clinical grounds without formal pulmonary function testing. Additionally, spirometry was not routinely performed during ED and hospital admissions. Although other EDs and hospitals in the metropolitan area evaluate, treat, and admit pediatric patients with asthma exacerbations, the concomitant diagnosis of sickle cell disease likely prompted evaluation at the children\'s hospital. Conclusions =========== In this small series, children with a history of asthma and sickle cell disease developed acute chest syndrome and cerebral vascular accidents more frequently than children with sickle cell disease without asthma. The implications of this retrospective study are wide ranging and should lead to further prospective investigations. Whether aggressive asthma therapy in patients with sickle cell disease and asthma reduces the incidence of serious complications is unknown. The potential gains are far reaching and could make enormous impacts on the morbidity, quality of life and mortality of many patients. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= MEN conceived the study, participated in its design and coordination and drafted the manuscript. JL participated in data collection. MCZ participated in coordination and manuscript preparation. JPS participated in design, coordination and manuscript preparation. KJK participated in design and coordination.	PubMed Central	2024-06-05T03:55:52.490770	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548142/", "journal": "Clin Mol Allergy. 2005 Jan 21; 3:2", "authors": [ { "first": "Mark E", "last": "Nordness" }, { "first": "John", "last": "Lynn" }, { "first": "Michael C", "last": "Zacharisen" }, { "first": "Paul J", "last": "Scott" }, { "first": "Kevin J", "last": "Kelly" } ] }
PMC548143	Introduction ============ Estrogen is considered to elicit different growth responses in various tissues through binding to the estrogen receptor (ER) α and ERβ \[[@B1]-[@B3]\]. The modulation of estrogen responsive gene transcription by the ER is termed a \"genomic\" action of estrogen. In contrast, \"non-genomic\" effects of estrogen are characterized by a rapid onset of action within seconds to minutes after hormone exposure through post-translation modification of signaling proteins. ER-mediated signaling pathways are considered to support the growth of normal, preneoplastic and neoplastic cells \[[@B1]-[@B3]\]. In contrast to the classical genomic pathways of estrogen action that occur over the course of several hours or days, recent studies have shown evidence of a rapid signaling pathway mediated by cell surface ERs and non-genomic estrogen-induced signal transduction pathways which contribute to cell proliferation \[[@B4]\]. Recently, we have identified that estrogen stimulated the growth of HEK 293 cells in an ER-independent manner given that this cell line does not contain ER (unpublished Singh KP, Venkat S, Roy D). These findings suggest that in addition to ER mediated actions, other factor(s) must be involved in the stimulation of cell growth by estrogen. More recently, mitochondria have been implicated in the control of cell proliferation \[[@B5]\]. For instance, the mitochondrial peripheral benzodiazepine receptor (PBR) has been implicated in the regulation of human breast cancer cell proliferation \[[@B6]\]. Similarly, we have demonstrated that mitochondria can modulate the expression of nuclear cell cycle genes and human breast tumor growth \[[@B7],[@B8]\]. For instance, the growth of estrogen-dependent and estrogen-independent cells, is inhibited by controlling mitochondrial biogenesis \[[@B8]\]. In this paper, we critically review the role of mitochondria in the growth of estrogen-dependent cancer and non-cancer cells. Mitochondria are important targets of estrogen action. The cross-talk between the cell nucleus and the mitochondria appears to control estrogen-induced signaling involved in the apoptosis, proliferation, and differentiation of both normal and malignant cells. Mitochondria through its interaction with the cytoskeleton, export of cleaved signaling peptides, or generation of ROS appears to transduce signals to the nucleus for the activation of transcription factors involved in the cell cycle progression of estrogen-dependent cells. The understanding of the regulation of mitochondrial biogenesis by estrogenic compounds would open a new way to better understand steroidal and non-steroidal estrogen action at the cellular level. Estrogen actions at the mitochondria ------------------------------------ Besides apoptosis, respiration, and oxidative phosphorylation; mitochondria also control ion homeostasis and the synthesis of heme, lipids, amino acids, and nucleotides. Steroidogenesis is also controlled by mitochondria. Estrogen biosynthesis-related enzymes, 3 β-hydroxysteroid dehydrogenase and aromatase, have been demonstrated in the mitochondria of ovarian tumor epithelial cells \[[@B9]\]. ### Estrogen transport to mitochondria Although estrogen synthesis occurs in the mitochondria, exogenously added estrogen is also transported to this organelle. For instance, in vivoexposure of ovariectomized rats to tritiated 17-β-estradiol (E2) showed with increasing time, the translocation of this hormone from the plasmalemma mainly to the mitochondria (75%) rather than the nuclei in liver, adrenal gland, and spleen tissues \[[@B10]\]. The lipophilic property of E2 allows this molecule to easily diffuse into lipid bilayers. Since mitochondria are enriched with lipids, the organelle has the ability to act as an estrogen-sink within the cell. Although passive diffusion of estrogen into the mitochondria exists, a rapid delivery of E2 via receptor-mediated endocytosis from the plasma membrane to the mitochondria has been reported as a potential new pathway in HepG2 cells \[[@B11]\]. The uptake of E2-BSA from the medium by HepG2 cells occurred as early as 30 min. post-exposure and the ligand could be viewed in organelles that resembled vesiculated mitochondria found in steroid producing cells of the adrenal cortex and testes \[[@B11]-[@B13]\]. ### Estrogen effects on mitochondria morphology The impact of estrogen on mitochondrial morphology has previously been reported in the human breast cancer cell line MCF7. Transmission electron microscopy (TEM) revealed that an 8 day treatment of MCF7 cells with E2 (10 nM) resulted in large, clear mitochondria \[[@B14]\]. These alterations in mitochondria structure were observed as early as 2 days after treatment with a physiologically relevant dose of estrogen. The delamellated cristae of E2 treated mitochondria resemble an early anaerobic state of mitochondria development seen in embryonic rats and primates in which the cell depends on glycolysis \[[@B15]\]. However, it is not known whether the reported estrogen induced morphological changes had an affect on mitochondrial function. ### Estrogen receptor localization in mitochondria The function of estrogen at the mitochondria is not clear, however, recent studies have identified ERα and ERβ within the mitochondria implicating its role in the regulation of mitochondrial genome transcription. Subcellular fractionation of rabbit ovarian and uterine tissue revealed isoforms of ERα and ERβ in the mitochondrial enriched fraction as detected by western Blot analysis using ER specific antibodies \[[@B16]\]. More recently, ERβ was shown to be localized in the mitochondria of human lens epithelial cells (HLE-B3), human heart, rat primary neuron and primary cardiomyocyte, and in a murine hippocampal cell line HT-22 \[[@B17],[@B18]\]. Western blot analysis using polyclonal antibodies against human ERα and -β showed both ERs present in purified mitochondria isolated from the human breast cancer cell line MCF7 \[[@B19]\]. Using immunohistochemistry with confocal microscopy and immunogold electron microscopy, ERα and ERβ were identified in the MCF7 mitochondrial matrix. Mitochondrial ERα and ERβ were reported to account for 10% and 18%, respectively, of total cellular ER-α and -β when MCF7 cells were treated with E2. Treatment of MCF7 with E2 (10^-8^M and 10^-9^M) significantly increased the mitochondrial level of ERα and ERβ by 2.5-fold. ### Estrogen influence on mitochondrial gene expression An increasing body of evidence has shown that mitochondrial transcription is enhanced by estrogen treatment. For instance, a 16-fold increase in cytochrome oxidase II (CO II) mRNA is reported in the GH4C1 rat pituitary tumor cell line when treated for 6 days with E2 (0.5 nM) \[[@B20]\]. The mitochondrial gene for subunit III of cytochrome oxidase (CO III) is induced as early as 3 h following a single dose of E2 in the hippocampus of ovariectomized female rats \[[@B21]\]. Other mitochondrial transcripts have also been reported to increase in the human hepatoma cell line, HepG2, and rat hepatocytes when exposed to ethinyl estradiol (EE). A 40 h exposure to an EE concentration ranging from 0.5 to 10 μM resulted in a 2- to 3-fold induction of CO I, CO II, and NADPH dehydrogenase subunit 1 (NADPH-DH1) mRNA \[[@B22]\]. E2 (20 μM), although less potent than EE, showed a similar effect of induction from 1.5- to 1.8-fold in mitochondrial transcripts CO I, CO II, and NADPH-DH1 when treated for 12 h. The E2 catechol metabolite 4-OH-E2 caused a greater response in CO I and CO II transcript levels as compared to E2 after 24 h of treatement with a dose of 10 μM. The mitochondrial gene for ATP synthase subunit 6 (ATPase 6) was also elevated in female rat liver tissue exposed to EE (5 μg/day) for 42 days. An increase in the transcript level of COX7RP (cytochrome c oxidase subunit IV-related protein) was reported after a 6 h E2 (100 nM) treatment in MCF7 cells \[[@B23]\]. It is not known whether the COX7RP transcript is translated to a functional protein in the mitochondria, but the study proposed that COX7RP may represent a regulatory subunit of cytochrome c oxidase that modulates a high state of energy production in estrogen sensitive target tissues. More recently, a 12 h E2 (0.3 μM) treatment of MCF7 cells was demonstrated to enhance the mitochondrial transcript levels of CO I approximately fourfold and CO II\~2.5-fold \[[@B19]\]. The mechanism of estrogen-induced mitochondrial gene transcription is not clearly understood. The involvement of estrogen responsive elements (EREs) and/or the ER may be a possible mechanism in the increase of these mitochondrial transcripts. Sequences with partial similarity to the ERE consensus sequence, (AGGTCANNNTGACCT), have been reported in the mouse mitochondrial genome \[[@B24]\]. These partial EREs were detected in genes CO I and CO II which may account for the observed increases in these two transcripts in rat GH4C1 pituitary cells and rat hepatocytes \[[@B20],[@B22]\]. Other genes in which the various EREs were detected include 12S rRNA, 16S rRNA, tRNA-gln, cytochrome oxidase b, unidentified reading frame (URF) 4, URF5, and the D-loop region \[[@B24]\]. In the human mitochondrial genome, we identified partial or ERE 1/2 sites in the D-loop region, CO II, tRNA-met, 12S rRNA, 7S rRNA, URF1, and URF5 (unpublished Felty Q and Roy D). The presence of these partial EREs in the mitochondrial genome may lend support to a novel ER signal transduction pathway. A mechanism of ER translocation into the mitochondria and ER binding to mitochondrial EREs remains unclear. Using electrophoresis mobility shift assay (EMSA) and plasmon resonance analysis, the recombinant human ERα- and ERβ-containing mitochondrial proteins were demonstrated to specifically bind putative EREs in the mtDNA D-Loop, and this ER binding was enhanced by E2 treatment and inhibited by ICI 182780 \[[@B19]\]. Based on this evidence, it is biologically plausible that ER mediates mitochondrial transcription in the same manner as the glucocorticoid receptor (GR) which is translocated into the mitochondria and binds glucocorticoid response elements (GRE) after treatment with glucocorticoid \[[@B25],[@B26]\]. Whether estrogen-induced mitochondrial transcription participates in the development and growth of estrogen dependent breast cancer is not known. Long-term stilbene estrogen (diethylstilbestrol = DES) treatment of Syrian hamsters produced tumors in the kidney with a 5- to 10-fold higher transcript level of CO III than age-matched control kidneys \[[@B27]\]. Estrogen and the electron transport chain ----------------------------------------- Besides transcription of mitochondrial genes, estrogen has also been demonstrated to effect mitochondria at the protein level. Estrogen has been demonstrated in several studies to inhibit mitochondrial respiratory complex I, II, III, IV, and mitochondrial ATP synthase (F~0~F~1~-ATPase) \[[@B28]-[@B31]\]. Several studies have reported estrogen specific inhibition of mitochondrial respiratory proteins, but it is not clear whether estrogen can modify mitochondrial proteins at the post-translational level. However, there is a report that E2 increased the phosphorylation of a 76 kDa protein in the mitochondrial fraction of the rat corpus luteum \[[@B32]\]. The presence of protein kinases within the mitochondria together with evidence for estrogen-induced phosphorylation of mitochondrial proteins suggest that estrogen may regulate mitochondrial respiratory physiology at the post-translational level \[[@B33]\]. Besides a direct interaction with mitochondrial proteins, estrogen may indirectly effect the electron transport chain (ETC) through an increase of membrane fluidity. Given that thyroid hormone increases mitochondrial membrane fluidity \[[@B34]\] and that physiologic concentrations of estrogen can alter the fluidity of human red blood cell membranes \[[@B35]\], it is likely that a low dose of estrogen may facilitate electron transfer by increasing respiratory protein interactions through more membrane fluidity. Thus, the rate of electron transfer may increase as a consequence of more frequent collisions/interactions between respiratory chain complexes and electron carriers. The three proton pumps of the ETC depend on electron flow to generate the mitochondrial membrane potential (ΔΨ~m~). In human neuroblastoma cells, the following ER ligands: tamoxifen (30.2 μM), clomiphene (10.6 μM), and nafoxidine (2.8 μM); were reported to modulate ΔΨ~m~while E2 did not change ΔΨ~m~at concentrations up to 100 μM \[[@B36]\]. Assuming that estrogen can effect the fluidity of the inner mitochondrial membrane, a rise in ΔΨ~m~could result from an increased rate of electron transfer. The formation of ROS in mitochondria is reported to occur at high ΔΨ~m~\[[@B37],[@B38]\] and therefore suggests that estrogen modulation of ΔΨ~m~may be a possible mechanism for the generation of ROS. Whether physiologic concentrations of estrogen can increase ΔΨ~m~in target tissues is not clear, but these lines of evidence suggest that estrogen may modulate ΔΨ~m~in a dose- and tissue-specific manner. This effect is significant because estrogen-induced mitochondrial reactive oxygen species (ROS) may participate in cell signaling. In the following section, we provide a detailed review of the effects of E2 on mitochondrial respiratory complexes. ### NADH dehydrogenase (complex I) The effect of natural estrogens and synthetic estrogens on the mitochondrial ETC has been demonstrated in several studies. Human NADH dehydrogenase (complex I) is the largest respiratory chain complex consisting of 7 mitochondrial genome encoded subunits and more than 41 subunits encoded from the nuclear genome. This integral membrane protein is located within the mitochondrial inner membrane and the matrix. Two electrons enter the ETC from the oxidation of NADH by ubiquinone (CoQ) at complex I which is coupled with proton movement across the inner membrane from the matrix to the intermembrane space. Natural estrogens, 17-α-estradiol, E2, and estrone, at concentrations of approximately 10 μM were demonstrated to inhibit mitochondrial electron transport in homogenates of rat uterus, liver, and skeletal muscle \[[@B39]\]. The synthetic estrogen DES was also reported to inhibit electron transfer from complex I to CoQ at a half-maximal inhibitory concentration range of 0.2--2.6 μM \[[@B28]\]. Although DES at a dose of 20--30 μM could inhibit electron transfer by 90% it was much less effective than rotenone and piericidin A which could inhibit electron tranfer by 90--95% at a dose of 30--50 nM due to a tighter binding affinity to complex I. The researchers postulated that at relatively low doses, DES reversibly inhibits electron transfer at complex I. Additionally, DES displayed specific binding to a site in which rotenone and piercidin A bind to complex I. Since photoreactive analogues of rotenone have been reported to label complex I \[[@B40]\], these results implicate estrogen specific binding to respiratory complex I. Another class of compounds, phytoestrogens, which are found in our diet can also inhibit the activity of complex I. Phytoestrogens, genistein (found in soy beans) and resveratrol (found in red wine), can inhibit the activity of complex I, but are considered less active than rotenoid compounds \[[@B41]\]. There are very few reports that have investigated co-treatment of estrogen with mitochondrial inhibitors of oxidative phosphorylation (OXPHOS). Complex I inhibitors rotenone, piericidin A, and amytal have been used in co-treatment with DES and/or estrogens to elucidate the site of estrogen action on electron transfer \[[@B28],[@B39]\]. In MCF7 cells, it was demonstrated that co-treatment with rotenone (10 nM) and E2 (10 nM) strongly inhibited ornithine decarboxylase activity by 86% \[[@B42]\]. More recently, a study reported that treatment with 10 μM of 2-methoxyestradiol (2-Me) induced apoptosis in Ewing sarcoma cells through hydrogen peroxide (H~2~O~2~) production \[[@B43]\]. Since a 2 h prior treatment of rotenone (6 μM) inhibited 2-Me induced apoptosis and H~2~O~2~production, it was suggested that the H~2~O~2~source was the mitochondria. Although the biological significance of estrogen interaction with complex I is unknown, evidence of estrogen inhibition of electron transfer support a novel role of estrogen in the formation of mitochondrial ROS. ### Succinate dehydrogenase (complex II) Human succinate dehydrogenase (complex II) is a membrane bound protein located on the matrix side of the inner mitochondrial membrane. Complex II consists of 4 subunits all encoded from the nuclear genome. Two electrons enter the mitochondrial ETC from the oxidation of FADH~2~by CoQ at complex II. Although previous studies have reported complex I and cytochrome bc~1~reductase (complex III) as the major sites of ROS generation, ubiquinone radicals have been reported to contribute to basal levels of ROS at complex II \[[@B44]\]. Physiologically relevant ROS generation supported by the complex II substrate succinate occurs at complex I through reversed electron transfer \[[@B45]\]. Research studies of the protective effect of estrogen on the brain use the complex II inhibitor, 3-nitroproprionic acid (3-NPA) to model the condition of ischemia. Although the mechanism of estrogen neuroprotection is not clearly understood, estrogen has been proposed to modulate cerebral energy/glucose metabolism. Both 17-β-E2 and 17-α-E2 have been demonstrated in vivo to reduce ischemic brain damage induced by middle cerebral artery occlusion in ovariectomized rats \[[@B46],[@B47]\]. To model an in vitro state of interrupted energy metabolism as seen in cerebral ischemia and chronic neurodegenerative disease, the human neuroblastoma cell line SK-N-SH was treated with 3-NPA (10 mM) \[[@B48]\]. This study demonstrated that pretreatment of SK-N-SH with E2 (2 μM) restored ATP levels to 80% at 12 h as compared to the control cells treated with 3-NPA alone. Whether mitochondrial function can be preserved with a physiological concentration of estrogen is not clear as this study used a high dose (2 μM) which can be cytotoxic in certain tissues. The maintenance of ΔΨ~m~and ATP levels by estrogen when faced with 3-NPA toxicity was proposed to be due to the antioxidant effect and/or ATP increasing effects. At the 2 μM E2 dose an antioxidant effect is likely because estrogen is reported to be an effective neuroprotective antioxidant in the micromolar dose range \[[@B49]\]. However, another possibility may be due to an estrogen-induced increase in complex II activity which would overcome inhibition by 3-NPA as succinate dehydrogenase activity has been reported to increase in the brain of E2 treated rats \[[@B29]\]. Estrogen has been shown to cause multiple effects at the level of complex II that include the inhibition of electron transfer, maintenance of ΔΨ~m~and ATP levels, and the enhancement of succinate dehydrogenase activity. ### Cytochrome bc~1~reductase (complex III) The human cytochrome bc~1~reductase (complex III) is an integral membrane protein located in the inner mitochondrial membrane consisting of 10 subunits encoded from the nuclear genome and 1 subunit encoded from the mitochondria. Complex III transfers electrons from ubiquinol (CoQH~2~) to cytochrome c which is coupled to proton movement across the inner membrane. The synthetic stilbene estrogen, DES (10 μM--50 μM), is reported to inhibit complex III at the site of electron flow from ubiquinone to cytochrome c~1~\[[@B50]\]. The ER ligand tamoxifen is also reported to inhibit electron transfer at the site of complex III in isolated rat liver mitochondria \[[@B30]\]. The phytoestrogen resveratrol has been reported to bind both ER α/β, increase expression of estrogen responsive genes, and stimulate cell proliferation of MCF7 and T47D breast cancer cells \[[@B51],[@B52]\]. Interestingly, resveratrol has been shown to inhibit complex III activity (20%) by competition with ubiquinol (CoQH~2~) and preserve mitochondrial function by its action on complex III in isolated rat brain mitochondria \[[@B53],[@B54]\]. Besides the inhibition of electron transfer at complex III, the phytoestrogen genistein (50 μM) induced mitochondrial permeability transition (MPT) in isolated rat liver mitochondria. Since estrogen can inhibit electron transfer at complex III, the potential of estrogen to modulate the formation of mitochondrial ROS exists at multiple respiratory complexes. ### Cytochrome c oxidase (complex IV) In humans, the mitochondrial cytochrome c oxidase (Complex IV) is located in the inner membrane and catalyzes the transfer of electrons to oxygen with water being the final product of the reduction reaction coupled with proton movement across the inner membrane. The mitochondria genome encodes 3 subunits of complex IV while the other 10 subunits are encoded from the nuclear genome. The effect of estrogen and ER ligands on complex IV has been reported to increase and decrease enzymatic activity. During the oestrus cycle in rats, E2 decreased complex IV activity in brown adipose tissue, which suggests a role for E2 in the modulation of oxidative capacity \[[@B55]\]. The ER ligand tamoxifen was reported to restore complex IV activity to normal levels in disrupted rat liver mitochondria \[[@B56]\]. In rat liver mitochondria, tamoxifen was demonstrated to have a biphasic effect on complex IV in which a low concentration of 10--15 μM increased enzyme activity while a higher concentration of 50 μM inhibited activity by 50% \[[@B30]\]. Although the biological effect of estrogen actions on complex IV remains to be elucidated, it has been suggested that estrogen modulation of complex IV activity may increase energy production in estrogen sensitive tissues \[[@B23]\]. Furthermore, it has been proposed that ΔΨ~m~and ROS formation may be controlled by a hormone mediated reversible phosphorylation of complex IV \[[@B57]\]. Thus, the possibility for estrogen control of ROS formation by modulating complex IV activity may provide a mechanism of estrogen-induced redox signaling by the mitochondria. ### Mitochondrial ATP synthase (F~0~F~1~-ATPase/Complex V) Mitochondrial ATP synthase (F~0~F~1~-ATPase/Complex V) is composed of two distinct parts: 1) the F~1~-ATPase portion which protrudes into the matrix and synthesizes ATP when protons pass through it down their electrochemical gradient. 2) the F~0~-ATPase which forms a transmembrane proton channel through the inner membrane. F~0~F~1~-ATPase is encoded by 2 mitochondrial genome encoded subunits and 14 nuclear encoded subunits. F~0~F~1~-ATPase like respiratory chain complexes I-IV represents another enzyme in the mitochondrial inner membrane that is sensitive to estrogen. Inhibition of rat liver F~0~F~1~-ATPase by DES (10 μM) has been demonstrated and the F~0~portion was reported to contain a distinct binding site for DES \[[@B58]\]. This specific binding site for the F~0~portion has made DES a unique probe for the rapid isolation of functional F~0~from rat liver mitochondria \[[@B59]\]. Using E2-BSA conjugates, a 23 kDa estrogen binding protein was identified in rat brain mitochondria \[[@B60]\]. The 23 kDa protein was identified as the oligomycin-sensitivity conferring protein (OSCP) which forms the stalk region between F~0~and F~1~subunits of F~0~F~1~-ATPase in mitochondria. The OSCP was proposed to be the specific site on F~0~F~1~-ATPase where estrogen modulates ATPase activity. Differential effects of estrogen on F~0~F~1~-ATPase activity in isolated rat heart, liver, and brain mitochondria were observed when treated with E2, DES, and resveratrol \[[@B31]\]. E2 (13 nM) stimulated F~0~F~1~-ATPase activity in the heart by 10%, but not in the liver and brain. The phytoestrogen resveratrol (13--15 μM) inhibited F~0~F~1~-ATPase activity in the heart and liver while lower doses (133 pM--1.3 nM) stimulated F~0~F~1~-ATPase activity in the liver by 10%. Both phytoestrogens resveratrol (19 μM) and genistein (55 μM) can inhibit F~0~F~1~-ATPase activity in rat brain mitochondria \[[@B61]\]. In rat heart, liver, and brain the mitochondrial F~0~F~1~-ATPase activity was inhibited by DES (6.7 μM) 61%--67% \[[@B31]\]. In rat brain mitochondria, 17α-estradiol and E2 partly inhibited F~0~F~1~-ATPase activity at low concentrations of 15 nM and 3.4 nM, respectively, while 17α-E2 preserved mitochondrial function altered by the stress of anoxia-reoxygenation \[[@B62]\]. Although these studies of estrogen effects on F~0~F~1~-ATPase have been shown in isolated mitochondrial preparations, F~0~F~1~-ATPase inhibition by E2 was demonstrated to occur with intact human osteolclastic FLG 29.1 cells \[[@B63]\]. These studies of F~0~F~1~-ATPase activity demonstrate that estrogens and estrogen-like compounds possess cell-type and dose-specific effects on mitochondrial function. Modulation of mitochondrial ROS by estrogens -------------------------------------------- Within the cell, mitochondria are considered to be a major source of ROS, which include superoxide anion (O~2~^•-^), H~2~O~2~, and the hydroxyl free radical (^•^OH) \[[@B64]-[@B66]\]. Since mitochondria consume 85% of the oxygen used by the cell, the mitochondrial ETC generates a substantial amount of intracellular ROS \[[@B67]\]. As electrons pass through the mitochondrial ETC, some electrons leak out to molecular oxygen (O~2~) to form O~2~^•-^which is dismutated by manganese superoxide dismutase (MnSOD) to form H~2~O~2~\[[@B64],[@B68]\]. During mitochondrial respiration, 2% of the electron flow is reported to result in the formation of H~2~O~2~\[[@B66]\]. However, lower values of free radical leak were reported in the range of 0.4%--0.8% for heart mitochondria respiring on physiological concentrations of succinate (\<0.5 mM) \[[@B37]\]. In support of these findings, intact rat skeletal muscle, heart, and liver mitochondria were reported not to produce measurable amounts of ROS when respiring on complex I and complex II substrates \[[@B69]\]. In addition, this study reported a lower estimate of electron flow (0.15%) that contributed to H~2~O~2~production under resting conditions. These results suggest that mitochondria produce low levels of ROS that can be effectively scavenged by the cell\'s antioxidant defenses at resting conditions. It is this point, the low basal level of ROS produced by the mitochondria at rest, which makes mitochondrial ROS ideal signaling molecules since its contribution to the intracellular level of ROS is not at so high a level to induce oxidative stress; instead, a low oxidant level provides a physiologically safe window for redox signaling which allows the cell to regulate mild to moderate oxidative changes and critically respond to them by activating cellular processes such as proliferation and differentiation rather than triggering cell death. ### Characteristics of mitochondrial ROS Mitochondria are a predominant source of ROS in most cell types with unique characteristics that may allow it to participate in growth signal transduction. First, mitochondria are unique because they are a regulatable source of ROS in response to external stimuli. For example, cortical neurons exposed to N-methyl-D-aspartate (NMDA) were reported to couple a rise in intracellular calcium with mitochondrial O~2~^•-^production \[[@B70]\]. Tumor necrosis factor alpha (TNF-α) is another example of stimulated mitochondrial generation of O~2~^•-^in L929 cells and this ROS generation is coupled to the cytokine by the TNF-α receptor \[[@B71],[@B72]\]. Few other examples exist of mitochondria producing ROS in response to external stimuli, but more recently integrins (cell surface receptors that interact with the extracellular matrix) were reported to modulate mitochondrial ROS production for signal transduction \[[@B73]\]. Although signal pathways involved in triggering mitochondrial ROS remain largely unknown, it has been proposed that mitochondria participate in integrin signaling in a nonapoptotic manner, which leads to gene expression and cell differentiation. Mitochondrial ROS can enter the cytosol as either H~2~O~2~or O~2~^•-^where it can participate in redox signaling. Within the mitochondria, MnSOD can dismutate O~2~^•-^to H~2~O~2~which is a highly diffusible signaling molecule that can exit the mitochondria. In addition to H~2~O~2~, O~2~^•-^was demonstrated to be released by mitochondria to the cytosol via the voltage-dependent anion channels (VDACs) \[[@B74]\]. In regard to turning the mitochondria ROS signal off, cellular antioxidant defenses such as SOD, catalase, and glutathione peroxidase easily degrade ROS, which terminates the signal. Therefore, mitochondrial ROS fulfill the prerequisites of a 2^nd^messenger since they are short-lived (rapidly generated and degraded), produced in response to a stimulus, highly diffusible, and ubiquitously present in most cell types. Mitochondria are highly dynamic structures capable of changing their shape (by elongation, branching, swelling) and their location inside a living cell \[[@B75]\]. It is becoming clear that the morphological, functional, and genetic differences (heteroplasmy) that exist within the mitochondria population may reflect a division of labor within the cell. Mitochondria have been reported to be morphologically heterogeneous and unconnected within individual cells \[[@B76]\]. Pancreatic acinar cells were reported to contain distinct groups of mitochondria classified by their cellular location that included perinuclear, subplasmalemmal, and perigranular mitochondria \[[@B77]\]. In light of this finding, the highly diffusible H~2~O~2~generated by mitochondria may become a specific signaling molecule as a function of location. For example, perinuclear mitochondria may generate H~2~O~2~that only transduces signals to the nucleus or the subplasamlemmal mitochondria may only activate signal cascades of plasma membrane origin. Additionally, perinuclear, subplasmalemmal, and perigranular mitochondria were independently activated by intracellular calcium signals in their immediate environment, which supports distinct calcium functions for each type of mitochondria. It is significant that mitochondria can create subcompartments or \'microzones\' within the cytoplasm because signal transduction depends on the close proximity of substrates and effector molecules to be an efficient process. In addition, given the presence of other endogenous ROS sources besides the mitochondria such as NADPH oxidase, peroxisomes, cytochrome p450, xanthine oxidase, cyclooxygenase, lipooxygenase, and γ-glutamyl transpeptidase \[[@B78]\]; and because ROS is involved in a variety of signal cascades, understanding how mitochondrial ROS is activated at the right place and at the right time is vital in understanding the organelle\'s role in signal transduction. Compartmentalization has already been reported to play a key role in redox signaling and we consider this attribute when describing the mitochondria as a signal transducer \[[@B79]\]. In adult cells, mitochondrial clustering functions to create steep gradients of low molecular weight species such as O~2~, ATP, and pH resulting in specialized microzones that may facilitate signal specificity \[[@B80]\]. In the cytosol, the volume occupied by mitochondria in cells is highly variable and ranges from 15% to 50%. Based on volume, mitochondria compose a significant compartment within the cytosol that harbors signaling molecules. H~2~O~2~produced within the mitochondria is highly diffusible in contrast to O~2~^•-^, which cannot diffuse through membranes making it easily compartmentalized. Thus, mitochondrial generated O~2~^•-^may be kept separated from the cytosol until an appropriate stimulus releases it through VDACs. Another route for O~2~^•-^release may be through the mitochondrial permeability transition pore (MPTP) as low molecular weight compounds up to molecular weight 1500, can be exchanged between the mitochondrial matrix and the cytosol via this pore \[[@B81]\]. Since the MPTP is reported to reversibly open/close naturally in intact cells without resulting in apoptosis, mitochondrial signaling molecules could be exchanged with the cytosol by the transient \'flickering\' (open/closing) of the MPTP in response to certain stimuli \[[@B82]\]. In addition to location and compartmentalization, protein scaffolds mediate the selective activation of the mitogen activated protein kinase (MAPK) signaling pathway which raises the question of whether mitochondria may also act as a protein scaffold for signaling complexes \[[@B83]\]. A-kinase anchoring protein (AKAP) is reported to tether protein kinase A (PKA) to the mouse mitochondrial outer membrane \[[@B84],[@B85]\]. What makes AKAPs unique is their ability to simultaneously bind multiple signaling enzymes such as other kinases and phosphatases \[[@B86]\]. This multivalent scaffold has been described as a \'transduceosome\' capable of integrating signals from multiple pathways \[[@B87]\]. Whether these multivalent scaffolds exist on mitochondria is not clear at this time, but these signaling complexes could be a mediator of signals between the mitochondria and the nucleus during cell division. For example, AKAP84/121 has been demonstrated to concentrate on mitochondria in interphase and on mitotic spindles during metaphase transition alluding to its role in the cell cycle \[[@B88]\]. In addition to AKAP, a mitochondrial signaling complex has been reported to activate MAPKs. The PKCε can form signaling complexes with ERKs, JNKs, and p38 MAPKs in the murine heart \[[@B89]\]. Activated PKCε was shown to increase phosphorylation of mitochondrial ERK and p38 MAPKs. Whether the anchoring of PKC to mitochondria depends on AKAP is not known at this time. ### Mechanisms of estrogen-induced mitochondrial ROS Studies of the mitochondrial ETC have reported only two ROS forming sites, the FMN group of complex I and the ubiquinone site in complex III \[[@B45],[@B90]\]. The topology of ROS production, on which side of the mitochondrial inner membrane O~2~^•-^is produced, was reported to occur on the cytosolic side by complex III and on the martix side of the inner membrane by complex I \[[@B69]\]. Estrogen is known to act as either an antioxidant or pro-oxidant depending on the concentration \[[@B91]\]. Whether physiological concentrations of estrogen can stimulate mitochondrial ROS at complex I and complex III is not clear since most studies have been performed with cytotoxic doses. In the following section, we provide evidence for potential mechanisms of estrogen induced mitochondrial ROS. Electrons feed into the mitochondrial ETC at complex I and complex II. At complex I and complex II, ubiquinone or co-enzyme Q~10~(CoQ) oxidizes NADH and FADH~2~, respectively. CoQ functions as a mobile electron carrier within the mitochondrial inner membrane and transfers 2 electrons from both NADH and FADH~2~to complex III \[[@B92]\]. CoQ is known to offer protection from heart disease by increased ATP production and antioxidant actions \[[@B93]\]. It is a highly lipophillic compound due to its structure which includes an isoprenoid tail of usually 10 isoprene units in length, hence the designation Q~10~. Other than its role as an electron carrier and antioxidant, CoQ is also reported to act as a pro-oxidant. Although the pro-oxidative action of CoQ within the mitochondria is a matter of debate, O~2~^•-^formation occurs from a single electron transfer from ubisemiquinone to molecular oxygen. Exogenously added CoQ has been demonstrated to enhance O~2~^•-^generation in isolated respiratory complex I and III \[[@B94]\]. Further evidence in support of CoQ redox-cycling come from a study that demonstrated H~2~O~2~derived from decomposing O~2~^•-^was inhibited after the removal of CoQ \[[@B90]\]. Upon the re-addition of CoQ, H~2~O~2~was detected again which indirectly demonstrated that the O~2~^•-^may originate from CoQ. Although some reports make a case for O~2~^•-^formation by CoQ redox cycling in mitochondria, arguments against its role as a source of O~2~^•-^come from a study which demonstrated that O~2~^•-^formation did not occur in a water-free nonpolar reaction system that mimics the lipophilic nature of the inner mitochondrial membrane \[[@B95]\]. However, pretreatment of the membrane with toluene which increased its permeability to protons provided conditions in favor of O~2~^•-^formation by CoQ. Thus, it was proposed that under certain pathological conditions in which the inner mitochondrial membrane is protonated, CoQ becomes a significant source of O~2~^•-^\[[@B96]\]. In line with this result, a study demonstrated that CoQ (100 μM) enhanced the release of H~2~O~2~from mitochondria in the presence of antimycin A (2 μM) and to a lesser extent with Ca^2+^(10 μM) \[[@B97]\]. The antimycin A and Ca^2+^pretreatment was thought to induce a leaky inner mitochondrial membrane thereby allowing protons to interact with CoQ and enhance ROS production by redox cycling. Interestingly, CoQ shares similar characteristics to catechol metabolites of estrogen. The catechol metabolites 2- and 4-OH-E2 contain hydroxyl groups that can be oxidized to semiquinones, which in the presence of molecular oxygen can be further oxidized to quinones with the formation of O~2~^•-^\[[@B98]\]. Since CoQ undergoes reduction/oxidation (redox) reactions which result in the radical semiquinone intermediate (semiubiquinone) and quinone, it is biologically possible for catechol estrogens to participate in shuttling electrons and to act as a pro-oxidant like CoQ. Given that MCF7 cells treated with the o-quinone form of estrogen and NADPH produce significant amounts of H~2~O~2~in the mitochondrial subfraction \[[@B99]\]; and that mitochondrial enzymes catalyze redox reactons of stilbene estrogen in the mitochondria \[[@B100]\], the capacity of catechol estrogens to redox cycle within mitochondria suggests that these metabolites could facilitate mitochondrial ROS formation. Assuming that estrogen is a weak electron carrier compared to CoQ, it may have a tendency to leak electrons to molecular oxygen instead of transferring its electrons to complex III. The phytoestrogen and dietary flavinoid quercetin is reported to act as a pro-oxidant. Foods of plant origin contain flavinoids known to act as antioxidants or pro-oxidants depending on the concentration and metal chelates which catalyze the oxidative process \[[@B101]\]. Estrogen is similar to some flavinoids with respect to inhibition of the mitochondrial respiratory chain at complex I and complex II \[[@B102],[@B103]\]. Based on these reports, the formation of O~2~^•-^by estrogen at the level of electron transport could be one mechanism of increased mitochondrial ROS. Mitochondrial Ca^2+^, \[Ca^2+^\]~m~, accumulation is reported to promote the generation of ROS \[[@B104]\]. For example, an increase in \[Ca^2+^\]~m~was reported to stimulate H~2~O~2~production by rat brain mitochondria in the presence of rotenone \[[@B105]\]. Using confocal microscopy, we have shown a time-dependent increase in \[Ca^2+^\]~m~with E2 (100 pg/ml) treatment of MCF7 cells \[[@B106]\]. Another study reported an increase in \[Ca^2+^\]~m~with E2 (10 ng/ml) treatment of hippocampal neurons \[[@B107]\]. The mechanism for these increases in \[Ca^2+^\]~m~is not clear, however, the inhibition of Na-dependent Ca^2+^efflux from mitochondria was reported to increase calcium retention in E2 (1 nM) treated synaptosomal mitochondria \[[@B108]\]. There is some evidence which suggests allosteric inhibition of a respiratory complex may be a mechanism for hormone induced ROS formation. Allosteric inhibition of the respiratory complex IV (COIV) or cytochrome c oxidase is reported to occur by cAMP-dependent phosphorylation; and this inhibition is turned-off by Ca^2+^-activated dephosphorylation \[[@B109]\]. A study proposed that a hormone stimulated increase of cellular Ca^2+^may activate a mitochondrial protein phosphatase which dephosphorylates cytochrome c oxidase. In turn, cytochrome c oxidase is activated which results in a rise of ΔΨ~m~and ROS \[[@B57]\]. Interestingly, the ER ligand tamoxifen (15 μM) showed a slight stimulatory effect on cytochrome c oxidase \[[@B30]\]. Since estrogen is capable of increasing \[Ca^2+^\]~m~, it is possible for estrogen to signal the formation of mitochondrial ROS through a similar mechanism. The inhibition of respiratory complex I is known to favor ROS generation. Rat brain mitochondria that respired on complex I substrates produced a substantial level of ROS when inhibited with rotenone concentrations as low as 20 nM \[[@B110]\]. Since estrogen is known to inhibit respiratory complex I, we speculate that complex I interactions with the hormone could favor ROS production in a manner similar to rotenone. The phytoestrogen genistein is another flavinoid besides quercetin that acts like a pro-oxidant at the level of mitochondria. Genistein (50 μM) treatment of rat liver mitochondria was shown to increase ROS formation through interaction with respiratory complex III which resulted in the opening of the membrane transition pore \[[@B111]\]. Besides hormone interactions with respiratory enzymes, post-translational modifications such as phosphorylation-/-dephosphorylation that affect the activity of mitochondrial proteins should also be considered in ROS generation. The cAMP-dependent protein kinase is reported to phosphorylate 6-, 18-, 29-, 42-kDa mitochondrial proteins in bovine heart and phosphorylate the human 18-kDa subunit which promotes the activity of complex I \[[@B112]-[@B115]\]. Since estrogen is reported to stimulate cAMP-dependent protein kinase activity in hippocampal neurons, it raises the possibility for estrogen to induce cAMP accumulation in mitochondria \[[@B116],[@B117]\]. If estrogen increased cAMP levels within mitochondria, then cAMP-dependent phosphorylation of mitochondrial respiratory complexes may modulate ΔΨ~m~and/or \[Ca^2+^\]~m~in favor of ROS generation. Several isoforms of the enzyme nitric oxide synthase (NOS) are reported to exist which include inducible-(i), endothelial-(e), neuronal-(n), and mitochondrial-(mt) NOS. Estrogen has been reported to induce various isoforms of NOS. The activity of eNOS is modulated by estrogen in human aortic endothelial cells, uterine artery, heart, and skeletal muscle \[[@B118],[@B119]\]. Estrogen has also been shown to stimulate protein expression of nNOS in human neutrophils and the transcription of iNOS in rat macrophages \[[@B120],[@B121]\]. These effects are not limited to E2 because the estrogen-like chemical bisphenol A and the phytoestrogen resveratrol are also reported to stimulate NO synthesis \[[@B122],[@B123]\]. These lines of evidence demonstrate a significant role of estrogen compounds in the modulation of NOS and NO. In regards to redox signaling, a NO-dependent inhibition of cytochrome c oxidase has been proposed to generate O~2~^•-^which is dismutated into the membrane permeable second messenger H~2~O~2~\[[@B124]\]. Since estrogen is capable of inducing NOS activity and expression, we postulate that an estrogen induced rise in NO could participate in a similar manner whereby generating mitochondrial H~2~O~2~. Interestingly, NO induced mitochondrial biogenesis has been demonstrated in several cell lines, which include brown adipocytes, 3T3-L1, U937, and HeLa cells \[[@B125]\]. We have shown that estrogen can influence mitochondrial biogenesis (data unpublished) and postulate that estrogen-induced NO could be one possible mechanism. More specifically, since mtNOS activity is dependent on Ca^2+^, we propose that an estrogen-induced rise in \[Ca^2+^\]~m~could stimulate mtNOS activity ultimately leading to the generation of ROS via NO-dependent inhibition of cytochrome c oxidase \[[@B126]\]. Estrogen-induced growth of cells in relation to mitochondria ------------------------------------------------------------ The rapid stimulation of intracellular ROS by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and nerve growth factor (NGF) suggests that this underlying mechanism of cell growth may be shared with other growth factors including estrogen \[[@B127]\]. Exogenous addition of low concentrations of H~2~O~2~and/or O~2~^•-^has been demonstrated to stimulate cell growth in a variety of cell types including muscle cells, fibroblasts, amnion cells, prostate cancer cells, and aortic endothelial cells \[[@B78]\]. The molecular signaling mechanism that initiates ROS production by mitochondria is not clear, however, other cell processes besides apoptosis may be coupled to this signaling event. Tumor necrosis factor alpha (TNF-α) induces gene expression via mitochondrial respiratory chain dependent activation of NF-κB, AP-1, JNK, and MAPKK \[[@B73]\]. The proliferative response of endothelial cells to hypoxia was demonstrated to be initiated upstream by mitochondrial ROS which activated the MEK/ERK pathway \[[@B128]\]. Although other endogenous ROS sources besides mitochondria such as NAD(P)H oxidase exist, mitochondria will be the focus of this paper for the following reasons: (i) mitochondria are the principal source of intracellular ROS in epithelial cells. (ii) the growth of adenocarcinomas occur in tissue of epithelial cell origin. A characteristic of rapidly dividing cancer cells is their capacity to produce significant amounts of intracellular ROS, which has been implicated in the promotion of accelerated cell cycle activity in neoplastic cells. Mitochondria have long been suspected to play a role in the development and progression of cancers. The ROS molecules H~2~O~2~and NO have been demonstrated to stimulate mitochondrial biogenesis, a process that depends on the flow of molecules into and out of the organelle \[[@B125],[@B129]\]. Since mitochondrial proteins are encoded in two separate genomes (mitochondria and nuclear genome), biogenesis is a coordinated effort in which mitochondria transmit signals to the nucleus and vice versa. The question of how mitochondria transmit these signals in the process of cell proliferation has risen from reports of its involvement in cell growth. Cerebral granular cells isolated from newborn rats with high mtNOS activity were reported to exhibit maximal proliferation rates which depended on NO and H~2~O~2~levels. In addition, MnSOD displayed an increased pattern of activity similar to mtNOS \[[@B130]\]. NO has been proposed to inhibit cytochrome c oxidase in favor of O~2~^•-^production and therefore MnSOD may dismutate O~2~^•-^generated by NO-dependent inhibition into the signaling molecule H~2~O~2~. Ethinyl estradiol, E2, and estrogen catechol metabolites at a dose of 0.25 to 5 μM are reported to increase mitochondrial O~2~^•-^in cultured rat hepatocytes and HepG2 cells \[[@B131]\]. Although the biological significance of the estrogen-induced mitochondrial O~2~^•-^is not known at this time, ROS has been demonstrated to modulate ER protein expression in various cell lines. Treatment of human breast cancer cells MCF7 and T-47D with H~2~O~2~(2.5 μM) increased the protein level of ERβ \[[@B132]\]. In addition, PMA (100 ng/ml) treatment increased the expression of ERβ in the macrophage cell line J774A.1. Evidence for the involvement of redox signaling with estrogen-induced cell proliferation has been demonstrated in several studies. Liposomes containing SOD or catalase inhibited in vitroestrogen-induced proliferation of Syrian hamster renal proximal tubular cells \[[@B133]\]. The cytokines IL-1β and TNF-α are known to cause the release of O~2~^•-^from human fibroblast cells. Co-treatment with an inhibitor of IL-1β and TNF-α synthesis, pentoxifylline, inhibited stilbene estrogen-induced increase in myeloperoxidase activities, 8-hydroxydeoxyguanosine (8-OHdG) formation, mutations in the testicular genome, and prevented estrogen-induced testicular preneoplastic lesions \[[@B3]\]. Recently, we have shown that estrogen-induced stimulation of macrophage cells and MCF7 cells in part occurs through ROS \[[@B134],[@B135]\]. We have also observed inhibition of estrogen-induced MCF7 cell growth by ROS scavengers such as N-acetylcysteine, ebselen, and catalase (unpublished Singh M, Felty Q and Roy D). ROS can modulate effector molecules such as PKC, p53, extracellular regulated kinase (ERK), nuclear factor-κB (NF-κB), and the c-fos/c-jun heterodimer (AP-1); and these effector molecules are known to participate in growth signal transduction \[[@B136]\]. Therefore, estrogen-induced production of mitochondrial ROS may activate cell growth in estrogen-sensitive tissues. Oxidative stress has been shown to affect mitochondrial proteins of chronically estrogenized Syrian hamster kidney. A decrease in thiol/sulfhydryl groups was reported in the mitochondrial fraction at a preneoplastic stage of carcinogenesis \[[@B137]\]. Estrogen-induced oxidative stress may be responsible for these post-translational modifications in mitochondrial proteins. This finding is significant in the context of cell signaling because redox reactions involving cysteine thiol groups transduce signals by breaking or forming protein dithiol/disulfide bridges \[[@B138]\]. Since estrogen can induce mitochondrial ROS, we infer that the oxidation of thiols in response to estrogen converts the oxidative stress to a change in protein function involved in cell growth. Oxidative stress modifies mitochondrial matrix protein thiols \[[@B139]\]. Similarly, thiols on protein subunits 51-kDa and 75-kDa of NADH dehydrogenase (complex I) have been reported to form mixed disulfides with glutathione (glutathionylation) in response to mitochondrial oxidative stress. This post-translational modification was reversible and correlated with an increase in mitochondrial O~2~^•-^production \[[@B140]\]. Evidence in support of a ROS signal transduction pathway originating from complex I comes from a study which reported that the mitochondrial complex I inhibitor, rotenone, blocked ROS mediated signaling. Interestingly, this study demonstrated that a co-treatment of rotenone (10 nM) and E2 (10 nM) inhibited ornithine decarboxylase activity by 86% in MCF7 cells \[[@B42]\]. Since ornithine decarboxylase activity is a marker for cell growth, it appears that a signal transduction pathway for estrogen-induced cell growth may originate from the mitochondria assuming that rotenone inhibition is specific to complex I. The antitumor arotinoid, mofarotene (Ro 40-8757), has been demonstrated to down-regulate mitochondrial encoded NADH dehydrogenase subunit 1 (MtND1) expression in breast cancer cell lines MDA-MB-231, ZR-75-I, and MCF7 \[[@B141]\]. Since MtND1 has been reported to form part of the rotenone-binding site in complex I \[[@B40]\], the absence of MtND1 may remove an important site of estrogen action in mofarotene treated cells and may account for the anti-proliferative effects of this compound. Whether these protein interactions and/or modifications can occur as a result of estrogen exposure remains to be investigated. From these investigations, we infer that estrogen mediated cell growth via mitochondrial generated ROS signaling molecules may exist and merits future exploration to address this novel pathway. The mitochondrial thioredoxin system has been demonstrated to play a role in cell cycle progression. In general, the two antioxidant oxidoreductase enzymes thioredoxin (Trx) and thioredoxin reductase (TrxR) that compose the system modulate signal transduction properties of ROS by the reduction of intracellular disulfides. Trx acts as a protein disulfide reductant for ribonucleotide reductase and several transcription factors including p53, NF-κB, and AP-1 \[[@B142]\]. Once oxidized the active disulfide site is reduced by TrxR re-generating the reductant form of Trx. Enzyme isoforms Trx-2 and TrxR2 are reported to exist in the mitochondria. A biological role for TrxR2 in cell growth was demonstrated in HeLa cells using a dominant negative form of TrxR2 (TrxR2DN) \[[@B143]\]. An increase of G~1~to S phase transition, cell growth, and transcription of cell cycle genes was induced by TrxR2N expression. TrxR2DN expression was suggested to increase intracellular H~2~O~2~, which in turn signaled cell proliferation. Although it is not clear whether estrogen can modulate the H~2~O~2~levels of mitochondria, estrogen (10 nM--100 nM) treatment of primary human endometrial stromal cells in vitroshow an increase in Trx protein and mRNA which implicate Trx involvement in cell growth and differentiation of estrogen responsive tissue \[[@B144]\]. Alterations in cellular redox status by increased expression of TrxR2 have been suggested to play a role in the growth of hepatocellular carcinomas \[[@B145]\]. Whether estrogen can signal cell growth through Trx2 and/or TrxR2 is not known, but these findings suggest that estrogen may modulate signal transduction of mitochondrial derived ROS via the thioredoxin system. Transduction of estrogn-induced mitochondrial signals to nucleus ---------------------------------------------------------------- Mitochondrial ROS fulfill the characteristics of a 2^nd^messenger since they are short-lived (rapidly generated and degraded), produced in response to a stimulus, highly diffusible (H~2~O~2~), and ubiquitously present in most cell types. It is not known whether mitochondrial ROS like H~2~O~2~are involved in signaling pathways that control estrogen-induced cell proliferation. In this section, we provide evidence in support of redox signaling pathways of mitochondrial origin, which may be involved in cell cycle progression of estrogen-dependent cells. ### Redox sensor proteins and transcription Protein kinases are known to participate in phosphorylation signal cascades, however, zinc finger domains contained in some proteins may allow them to participate in redox signaling networks. Zinc finger structures within a protein consist of at least two zinc-coordinated thiolates. Upon oxidation zinc is released from the protein, which converts cysteine thiol groups to disulfide. A conformational change in the protein may result in either its activation or inhibition. There are several protein kinases such as a-raf and PKC that contain zinc finger domains. In addition, zinc finger domains are also found in hormone receptors such as the GR and ER \[[@B146]\]. Both PKC and c-raf have been demonstrated to be redox activated at the zinc finger domain \[[@B147],[@B148]\]. For instance, ROS can trigger the release of zinc ions from PKC which results in its activation. Another protein kinase, c-raf, known to participate in the MAPK signal cascade was also demonstrated to be redox activated at the zinc finger domain. The mitochondrial localization of protein kinases src, Akt, a-raf, and PKC is evidence that this subcellular compartment harbors oxidant sensitive proteins that may facilitate cross-communication between redox and phosphorylation networks \[[@B33],[@B89]\]. Although the role of the protein kinase a-raf in the mitochondria is not clear, a-raf mRNA is highly expressed in normal murine tissues such as the epididymis, ovary, kidney, and urinary bladder \[[@B149]\]. In Hela cells, epidermal growth factor rapidly (2 min.) and transiently activated a-raf, which in turn phosphorylated the MAP kinase activator MEK1 \[[@B150]\]. Therefore, mitochondrial ROS may activate MAPK signaling via a-raf. It is interesting to note that E2 can stimulate the phosphorylation of a-raf and cell cycle progression in MCF7 cells \[[@B151]\]. Whether the estrogen induced phosphorylation of a-raf depends on ROS is not known. Mitochondrial PKCδ and PKCε could also activate the raf/MEK/ERK pathway or directly activate MAPKs, respectively \[[@B152],[@B153]\]. Rapid effects of estrogen have been demonstrated to mediate the DNA binding activity and phosphorylation of transcription factors. E2 treatment of rat adipocytes doubled AP-1 DNA binding and phosphorylated CREB protein within 15 min \[[@B154]\]. The redox sensitive protein Akt is known to phosphorylate an upstream kinase, IKKα, which stimulates the degradation of Iκ-B \[[@B155]\]. Estrogen-induced mitochondrial ROS may stimulate Akt leading to the degradation of Iκ-B and activation of the transcription factor NF-κB. Whether estrogen treatment can activate Akt via mitochondrial derived ROS is not clear, however, phosphorylation and translocation of Akt to the mitochondria was demonstrated when cells are treated with estrogen \[[@B156]\]. Given that E2 can stimulate mitochondrial ROS generation; ER, src, a-raf, Akt, and PKC are targets of oxidative stimuli localized at the mitochondria; and the transcription factors AP-1, NF-κB, and CREB are stimulated by oxidants \[[@B127],[@B131],[@B157]\]; it is possible that estrogen specific effects at the level of mitochondria can activate these transcription factors. Based on these studies we postulate that estrogen-induced mitochondrial ROS stimulates oxidant sensors a-raf, Akt, or PKC, which in turn activate transcription factors such as NF-κB, CREB, or AP-1 via the MEK/ERK pathway resulting in the transcription of cell cycle genes containing DNA responsive elements for NF-κB, CREB, or AP-1 and ultimately estrogen-induced cell proliferation (Fig. [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Hypothetical scheme outlining three E2-induced signaling pathways from mitochondria. (i) E2 binding to a plasma membrane receptor and/or mitochondrial respiratory complexes generates ROS which leads to kinase activation. (ii) E2-induced rise in mitochondrial calcium leads to the activation of calcium-dependent proteases which process signal peptides, in turn responsible for kinase activation. (iii) E2-induced cytoskeleton modifications by mitochondria leads to kinase activation. Increased kinase activity results in the activation of transcription factors responsible for cell cycle progression. ::: ![](1477-3163-4-1-1) ::: ### Estrogen and mitochondrial-cytoskeleton interactions Mechanical signals associated with cytoskeletal tension generation and cytoskeleton restructuring are a requirement for anchorage dependent cells to pass through the late G~1~restriction point \[[@B158]\]. Since these cytoskeleton dependent effects on the G~1~checkpoint are independent from the MAPK signaling pathway, a new question rises of whether mitochondria can modulate cell growth by interacting with the cytoskeleton. Mitochondria are reported to be associated with three major cytoskeletal structures, which include microtubules, microfilaments of actin, and intermediate filaments \[[@B159]\]. Mitochondrial tubulin and microtubule associated proteins (MAPS) are reported to bind to porin or VDAC a component of the permeability transition pore \[[@B160]\]. The association of the cytoskeleton with VDAC could be biologically significant because the actin filament severing and capping protein gelsolin has been reported to modulate ΔΨ~m~by its interactions with VDAC \[[@B161]\]. Epithelial cell spreading results from the binding of integrins to the extracellular matrix which depends on the actin cytoskeleton \[[@B162],[@B163]\]. The survival of epithelial cells depends on this interaction with the extracellular matrix, which if disrupted leads to a specific form of apoptosis called anoikis \[[@B164]\]. Actin filaments are necessary to cluster integrin receptors and proteins linked to their cytoplasmic domain into focal adhesion complexes \[[@B165]\]. These focal adhesion complexes provide a direct link between the extracellular matrix and the actin cytoskeleton. Anchorage-independent growth is a property of cancer cells, which may depend on the mitochondria based on evidence from the following studies. Long-term exposure of cells to ethidium bromide, an intercalation agent which inhibits mtDNA replication, results in the depletion of mitochondria. Mitochondria-depleted (ρ°) brain and breast tumor cells have been shown to lose their ability for anchorage-independent growth \[[@B166]\]. In addition, human ρ° cell lines derived from ovarian carcinoma, cervical carcinoma, and osteogenic sarcoma were demonstrated to be non-tumorigenic or poorly tumorigenic when administered subcutaneously to nude mice \[[@B167]\]. Taken from these reports is the interesting possibility that cancer cells maintain tension on the cytoskeleton via the contraction and expansion of mitochondria instead of binding to the extracellular matrix. Actin assembly and disassembly is regulated by the protein gelsolin. Since gelsolin is reported to prevent apoptotic mitochondrial changes by binding and closing VDAC, perhaps other diverse functions are modulated by interactions between the mitochondria and cytoskeleton. More recently, it was demonstrated that mitochondrial ROS production is stimulated by integrin induced changes \[[@B73]\]. Although integrin receptors are linked to the actin cytoskeleton, it is not clear whether the signal that is transduced to the mitochondria occurs via the cytoskeleton. Furthermore microtubules have also been implicated in the biogenesis of mitochondria based on the inhibition of mitochondrial mass increase and mtDNA replication caused by the microtubule-destabilizing drug colchicine and the estrogen metabolite 2-methoxyestradiol in mammalian cells \[[@B168]\]. Mitochondria biogenesis is reported to occur in the G~1~phase of the cell cycle but also starts in the late S phase \[[@B169]\]. Although mitochondria were not reported to be an upstream signaling event for generating cytoskeleton tension, physical effects of mitochondria such as shape changes and stretching or contraction could generate tension based on its association with the cytoskeleton. Changes in cytoskeleton tension may be mediated by mitochondria in response to estrogen. For instance, transmission electron microscopy (TEM) showed an increase in mitochondria size of MCF7 cells treated with E2 \[[@B14]\]. This change in mitochondrial size may generate mechanical forces that, in turn, may transduce a signal to the nucleus. Another possibility is that estrogen stimulated mitochondrial ROS may affect the elasticity of the actin network. Actin filaments are a prevalent feature of the cytoskeleton, which partially determine the overall mechanical strength of a cell. Thiol oxidation forms disulfide-bonded actin dimers resulting in interfilament cross-links \[[@B170]\]. Estrogen-induced oxidative stress has been recently reported to oxidatively modify cysteine residues of proteins \[[@B137]\]. Thus, estrogen-induced mitochondrial ROS could stimulate the formation of actin dimers that modulate cytoskeleton tension which in turn transduces a signal to the nucleus (Fig. [1](#F1){ref-type="fig"}). ### Mitochondrial proteolysis and peptides as signals The mitochondrial protein cytochrome c is known to be released to the cytosol where it initiates a signal for apoptosis. Given the role of cytochrome c the existence of other mitochondrial protein signaling molecules is a likely possibility that could mediate a diverse number of cellular processes including cell growth. It has been shown that mitochondria have the capability to export mitochondrial-matrix proteins to other cellular compartments such as the nucleus, peroxisome, endoplasmic reticulum, and secretory vesicles \[[@B171]\]. For example, mitochondrially transmitted factors (MTFs) are peptides derived from mitochondrial encoded proteins that are presented on the cell surface as minor histocompatability antigens. MTFs are derived from the mitochondrial encoded NADH dehydrogenase subunit 1 gene in murine and humans while rat MTFs are derived from the mitochondrial encoded ATPase 6 gene \[[@B172],[@B173]\]. Although the synthesis and cell surface expression of MTFs was inhibited by the mitochondria specific protein synthesis inhibitor chloramphenicol, it is not clear whether post-translational modifications of mitochondria proteins are also responsible for MTFs; given that chloramphenicol is also an inhibitor of proteolysis in rat liver mitochondria \[[@B174]\]. Thus, mitochondria may serve as a subcellular compartment of proteolysis that generates signaling peptides that are exported to the cytosol. It is possible that proteolysis plays a significant role in mitochondrial protein processing because the chemical rhodamine 6G (R6G) inhibits matrix catalyzed processing of rat-liver mitochondrial precursors which include iron-sulfur protein, cytochrome c~1~, and core protein I of the cytochrome bc~1~complex; the α and β subunits of F1 ATPase and subunit IV of cytorochrome oxidase \[[@B175]\]. The molecular mechanism of R6G inhibition of protein processing was not identified, but it was proposed to be due to an interaction between R6G and a matrix protease. The import of proteins into mitochondria has been investigated in great detail while the process of export is minimally explored. A novel ATP-binding cassette (ABC) transporter, Mdl1, located in the inner mitochondrial membrane of yeast is required for the export of mitochondrial peptides with a molecular mass of 2100 to 600 daltons generated by proteolysis \[[@B176]\]. It was suggested that the export of peptides from the mitochondria may allow the mitochondria to communicate with its environment. This novel mode of communication may exist based on studies that demonstrate mitochondrial specific cleavage and export of cytokines. The cytokine IL-1β is localized in the mitochondria of LPS stimulated human peripheral blood monocytes and the 31 kDa form of IL-1β is reported to be cleaved into the 17 kDa mature active form within the mitochondria upon exposure to the HIV coat glycoprotein 120 \[[@B177],[@B178]\]. Since macrophages secrete IL-1β by the ABC transporter, it is possible that proteins up to 17 kDa may be exported from the mitochondria \[[@B179]\]. In addition to IL-1β, mitochondrial Trx protein may also participate in a similar protein export mechanism. A truncated form of Trx, Trx80, is reported to be a potent mitogenic cytokine, however, it is not known whether Trx80 is derived from mitochondrial Trx \[[@B180]\]. Based on these reports it appears that cleaved proteins may be released as growth factors in response to an estrogen-induced rise in mitochondrial calcium that activates proteolysis. Thus, cleaved signaling peptides from the mitochondria may stimulate cell growth in an autocrine manner (Fig. [1](#F1){ref-type="fig"}). Several proteases are known to exist in the mitochondrial matrix such as ATP-dependent human Lon protease, ATP-dependent human Clp proteinase chain P (hClpP), and Ca^2+^dependent neutral protease \[[@B181]-[@B183]\]. Since endogenous proteolysis is a mechanism that regulates cell cycle progression, we postulate that E2-induced rise of mitochondrial calcium can activate calcium dependent proteases such as calpeptin and possibly hClpP. Once activated mitochondrial matrix proteins could be cleaved into bioactive forms that are exported to the cytosol. The heterogeneous association between ERα cleavage products and regulatory proteins has been suggested to play a role in physiological or pathological processes \[[@B184]\]. Low molecular weight ERα isoforms (\~35--28 kDa) have been identified in mitochondria \[[@B16]\]. The 44-kDa protein related to the nuclear RXRα receptor is reported to be enzymatically cleaved and imported into the mitochondrial matrix \[[@B185]\]. It may interact with mitochondrial proteins or bind the organelle genome. Once in the cytosol mitochondrial clevage products may also regulate the function of various enzymes. For example, a truncated ERα 46-kDa protein in human endothelial cells mediated an acute activation of eNOS in response to a 15 min E2 (30 nM) treatment \[[@B186]\]. A significant finding from this report is that the truncated ERα 46-kDa mediates acute responses of estrogen rather than transcriptional responses in endothelial cells. Recently, it was reported that physiologic concentrations of E2 (\<10 nM) induced NOS1 and activates the cGMP signal transduction pathway leading to sustained expression of Trx and MnSOD in human SH-SY5Y cells \[[@B187]\]. Thus, the acute activation of mtNOS by ERα cleavage products is yet another interesting possibility for redox signaling. Conclusion ========== In summary, mitochondria are a major target of estrogen. It appears that mitochondria through its interaction with the cytoskeleton, export of cleaved signaling peptides, and/or generation of ROS may transduce signals to the nucleus for the activation of transcription factors, such as, AP-1, NF-κB, and CREB involved in the cell cycle progression of estrogen-dependent cells. These interactions between estrogen and mitochondria merit furture investigations, which may shed new light on the role of mitochondria in cell growth. Acknowledgements ================ This work was partly supported by the NIEHS grant ES10851 to DR; financial support to QF through NCI Cancer Prevention and Control Training Grant (CA 4788).	PubMed Central	2024-06-05T03:55:52.492207	2005-1-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548143/", "journal": "J Carcinog. 2005 Jan 15; 4:1", "authors": [ { "first": "Quentin", "last": "Felty" }, { "first": "Deodutta", "last": "Roy" } ] }
PMC548144	Background ========== The establishment of successful pregnancy requires a profound reorganization of uterine tissues. Rapid growth and differentiation of the endometrial stroma is the earliest and most striking event in pregnancy. Differentiation of stromal cells leads to the formation of unique cells, termed decidual cells, which differ greatly from the original stromal cells \[[@B1]\]. The decidua is an important component in the maternal recognition of pregnancy and can be induced by either the implantation of the blastocyst or by artificial stimuli. An interesting feature is that -- the growth and differentiation of the endometrial cells -- occurs differently in different regions of the uterus \[[@B1]\]. Mesometrial decidual cells are formed after the antimesometrial decidua and their death takes place after antimesometrial cell degeneration. Regression of the decidual cells appears to be controlled by an intrinsic cell death program of apoptosis, which takes place after day 10 of pregnancy in the antimesometrial region first, followed by the mesometrial region \[[@B2]\]. Prostaglandin F~2α~can induce different biological actions at the beginning and at the end of pregnancy. PGF~2α~, Prostaglandin E~2~(PGE~2~), and 6-keto-PGF~1α~are produced by the pregnant uterus \[[@B3]\]. An increase of uterine PGE~2~and PGF~2α~is observed on day 5 of pregnancy, allowing the decidualization process to take place. When embryo access to the uterus is impaired, production of prostaglandins (PGE~2~and PGF~2α~) is suppressed \[[@B4]\]. During postimplantation, PGE2 returns to the original preimplantation levels, but PGF~2α~decreases \[[@B4]\]. Whereas PGF2α contributes to the process of decidualization, implantation and recognition of pregnancy. An increase in PGF~2α~over certain values can terminate pregnancy \[[@B6]\]. A high level of PGF~2α~is known to induce inhibition of implantation, alteration of embryo development, and induction of luteal regression \[[@B5]\]. During infection, an inflammatory response mediated by cytokines can be generated \[[@B7]\]. This release of PGF~2α~can induce premature uterine contraction and premature labor \[[@B8]\]. Depending on which stage of pregnancy an increase of PGF~2α~secretion occurs, it can alter implantation, induce abortion or even embriolethality \[[@B7],[@B8]\]. Because an increase in PGF~2α~levels after implantation can be detrimental for the progression of pregnancy, the aim of this investigation was to determine whether PGF~2α~affects directly decidual cells and leads to disturbance in the expression of genes crucial for decidual survival. Methods ======= Animal model ------------ Pseudopregnancy was induced by mating Holtzman Sprague Dawley female rats with vasectomized male rats. The day a vaginal plug was found was designated day 1 of pseudopregnancy. Decidualization of the uterine endometrium was induced by scratching the antimesometrial surface with a hooked needle on day 5 of pseudopregnancy under ether anaesthesia. Animals were housed in a controlled environment (22--24°C) and kept under controlled conditions (lights on; 0500--1900 hrs) with free access to standard rat chow and water. The University of Illinois at Chicago animal care and use committee approved the animal care and handling. Primary decidual cell culture and hormone treatment --------------------------------------------------- For each experiment 15 pseudopregnant rats were used. Decidual cells were isolated as previously described \[[@B9]\]. Cells were pooled and seeded into six-well plates (1.4 × 10^6^cells/ well). They were allowed to attach 3--4 hrs before washing in PBS to remove erythrocytes. Cells were incubated in RPMI 1640 without phenol red (Mediatech, Whashington DC), containing 1% CD-FBS (HyClone Laboratories Inc, Logan, UT), and treated with or without high levels (5 μM \[[@B10],[@B11]\]) of PFG~2α~(Sigma, St. Louis, MO) for 12 hr). After incubation, the cells were harvested in cold PBS, quick frozen in liquid nitrogen and kept at -80 C until RNA isolation. Gene identification by cDNA array --------------------------------- Total RNA was isolated from primary decidual cells (PDC) by using a RNAII isolation kit (Clontech, Palo Alto, CA), following the manufacture\'s instructions. RNA isolated from wells treated with either PGF~2α~or vehicle were pooled independently and subjected to cDNA array. cDNA probes were generated from 4 μg of total RNA in a reverse transcriptase reaction using a mix of dATP, dTTP and dGTP (Takara Biomedicals, Shiga, Japan) plus \[α^32^P\]-dCTP (Amersham, MO), and a mixture of primers specific to each gene present in the array. All probes used had 5 to 10 × 10^6^cpm and the difference between control and experimental probes in each assay was less than 10%. cDNA were hybridized to an Atlas Rat 1.2 Array (\#7854-1) nylon membrane (Clontech, CA). Hybridization and post-hybridization washes were performed according to the manufacturer\'s protocol. The signal was scanned with a phosphorImager (Molecular Dynamics, CA) after 48 h of exposure. Control and experimental RNA were always processed in parallel. Data analysis ------------- Spot intensities from scanned membranes were analyzed using the AtlasImage 1.5 software (Clontech, CA). Grids were orientated manually and adjusted to ensure optimal spot recognition using AtlasImage\'s fine tuning tools, discarding spots with dust or locally high background. The software makes the analysis for background correction and normalization versus housekeeping genes. It also calculate the ratio and indicates which genes has a ratio \> 2 (up regulated), or ratio \< 0.5 (down regulated) or 0.5 \< ratio \< 2 (equal expression). Gene expression data were normalized using the Sum method included in the Atlas Image software. Data points where the expression was not greater than two standard deviations were discarded. For the final analysis, data points were the averages from triplicates and any non-reproducible data were discarded. The relative RNA expression with differences between control and treatment being higher or equal to 2 and lower than 0.5 were considered significantly different \[[@B12]\]. Results ======= Effect of PGF~2α~on gene expression in primary decidual cells ------------------------------------------------------------- From the total genes available in the rat 1.2 membrane arrays, only 20% of the genes were detected in rat primary decidual cells. Sixty genes were significantly down regulated, whereas only six genes were up regulated by PGF~2α~(Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}). PGF~2α~-receptor gene expression was similar in both control and treated groups. On the other hand, no differences in the expression of housekeeping genes such as ribosomal proteins and β-actin could be detectedin control and PGF~2α~treated cells (data not shown). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Representative cDNA expression array using mRNA obtained from rat primary decidual cells treated with either PGF~2α~or vehicle. ::: ![](1477-7827-3-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### cDNA expression array of rat primary decidual cells treated with PGF~2α~or vehicle. ::: ![](1477-7827-3-3-2) ::: Effect of PGF~2α~on the expression of genes related to the extracellular matrix (ECM) ------------------------------------------------------------------------------------- The majority of genes whose expression was affected by PGF~2α~in primary decidual cells are genes involved in the regulation of the ECM. Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (uPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase -- all of which were significantly up regulated by PGF~2α~(Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of PGF~2α~on the expression of genes related to the extracellular matrix (ECM). ::: Gene Bank number Swiss protein number Name Function Ratio % decrease Fold increase ---------------------- -------------------------- ---------------------------------------------- -------------------- -------------- ---------------- ------------------- U65656 P97581 Gelatinase A Metalloproteinase 0.04 ± 0.02 87.53 Y00697 P07154 Cathepsin L Cysteine protease 0.09 ± 0.05 91.09 L31884 P30121 Tissue inhibitor metalloproteinase 2 (TIMP2) Protease inhibitor 0.05 ± 0.01 95.54 U27201 P48032 Tissue inhibitor metalloproteinase 3 (TIMP3) Protease inhibitor 0.04 ± 0.01 91.95 X63434 P29598 Urokinase type Plasminogen activator (uPA) Serine protease 0.14 ± 0.09 94.86 M24067 P20961 Plasminogen activator inhibitor (PAI) Protease inhibitor 0.09 ± 0.05 91.68 M2367 P19367 Tissue type plasminogen activator (tPA) Serine protease 0.17 ± 0.02 89.90 X71071 Q08290 Calponin Cytoskeleton 0.08 ± 0.004 92.50 U06755 P37397 Calponin, acidic Cytoskeleton 0.24 ± 0.13 76.76 U62897 O35850 Carboxypeptidase D Carboxypeptidase 0.24 ± 0.14 76.48 M64711 P22388 Endothelin 1 Hormone 0.34 ± 0.2 77.48 X02601 P03957 Polypeptide, 53 kDa, growth factor induced Metalloproteinase 2.54 ± 1.04 3 J03552 P14046 Plasma proteinase inhibitor α1-inhibitor III Protease inhibitor 3.64 ± 0.64 4 M32247 P17475 α1 antiproteinase Protease inhibitor 2.45 ± 0.44 3 Ratio = net intensity of PGF~2α~normalized/net intensity of control normalized, and represent the mean ± SEM of each ratio from separate experiments. Ratio \> 2 = up regulation; ratio \< 0.5 = down regulation; 0.5 \< Ratio \< 2 = equal expression. ::: PGF~2α~regulation of genes related to the proteasome protein component system ----------------------------------------------------------------------------- Messenger RNA encoding proteasome Iota, proteasome component C2, proteasome subunit RC7-I, 26-S proteasome regulator and proteasome component C3 were down regulated by PGF~2α~. Only proteasome component C9 was up regulated after PGF~2α~treatment (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### PGF~2α~regulation of genes related to the proteasome component system. ::: Gene Bank number Swiss Protein number Name Function Ratio % decrease Fold increase ---------------------- -------------------------- ---------------------------- --------------------- ------------- ---------------- ------------------- D10755 P34062 Proteasome Iota Proteosomal protein 0.07 ± 0.04 93.64 M29859 P18420 Proteasome component C2 Proteosomal protein 0.31 ± 0.15 78.28 U77918 Q63569 26-proteasome component C3 Proteosomal protein 0.39 ± 0.08 61.94 J02897 P17220 Proteasome component C3 Proteosomal protein 0.31 ± 0.02 59.02 D21799 P40307 Proteasome subunit RC7-I Proteosomal protein 0.18 ± 0.01 82.62 X55986 P21670 Proteasome subunit C9 Proteosomal protein 2.32 ± 0.72 3 ::: PGF~2α~regulation of genes involved in trafficking and signal transduction -------------------------------------------------------------------------- The mRNAs encoding for 14-3-3ε and z/δ proteins, and Annexin IV were down regulated in PDC after PGF~2α~treatment (Table [3](#T3){ref-type="table"}). PGF~2α~down regulated the expression of serum glucocorticoid kinase (SGK), casein kinase I, extracellular signal-regulated kinase (ERK-1), male germ cell-associated kinase (MAK), and Wee tyrosine kinase. PGF~2α~also significantly inhibited Crk-associated kinase (CAS), calcium calmodulin kinase II (CAMKII) and IV (CAMKIV), phospholipase C1, and protein phosphatases such as phosphatase 2A and protein tyrosine phosphatase 1B (Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### PGF~2α~regulation of genes encoding proteins involved in trafficking and signal transduction. ::: Gene Bank number Swiss protein number Name Function Ratio % decrease Fold increase ---------------------- -------------------------- ------------------------------------------------- ------------------------- ------------- ---------------- ------------------- D17615 P97286 14-3-3 z/δ protein Traficking protein 0.14 ± 0.07 86.07 M84416 P29360 14-3-3 Epsilon protein Traficking protein 0.29 ± 0.15 71.63 L01624 Q06226 Serum Glucocorticoid kinase (SGK) Kinase 0.25 ± 0.12 93.49 L07578 Q06486 Casein kinase I δ Kinase 0.29 ± 0.15 72.18 M61177 P21708 Extracellular signal-regulated kinase 1 (ERK1) Kinase 0.29 ± 0.13 66.67 D29766 Q63766 Crk-associated kinase Kinase 0.09 ± 0.06 87.16 M35862 P20793 Male germ cell-associated kinase (Mak) Kinase 0.20 ± 0.11 78.6 D31838 Q63802 Wee tyrosine kinase Cell cycle kinase 0.23 ± 0.12 69.47 M20637 P10688 Phospholipase C δ1 Phospholipase PI kinase 031 ± 0.15 68.24 J04503 P20650 Protein phosphatase 2C α Phosphatase 0.18 ± 0.03 58.87 L12385 P26438 Protein Phosphatase 2A Phosphatase 0.38 ± 0.13 65.91 L13408 P15791 Ca^+2^/Calmodulin dependent kinase II δ subunit Kinase 0.34 ± 0.12 72.12 M63334 P13234 Ca^+2^/Calmodulin dependent kinase IV Kinase 0.29 ± 0.15 70.90 M33962 P20417 Protein Tyrosine phosphatase 1B Phosphatase 0.21 ± 0.08 79.77 D38224 P55260 Annexin IV (ANX4) Exocytosis protein 0.14 ± 0.08 86.34 L24810 Q64572 Ca^+2^/Calmodulin Phosphorylase B Kinase 5.06 ± 2.06 5 ::: PGF2α effect on the expression of genes associated with G-proteins, growth factors, chemokines and cytokines ------------------------------------------------------------------------------------------------------------ The 5-hydroxytryptamine receptor 2A (HTR2A), adenosine A2A and A2B receptors (ADORA2A and ADORA2B respectively), guanine nucleotide-binding protein G α3 subunit (GN-BPG α~3~), guanine nucleotide-binding protein α stimulating (GN-BP α stimul.), Rab 11A and guanine nucleotide-binding protein α12 subunit (GN-BP α~12~) gene expression were down regulated by PGF~2α~(Table [4](#T4){ref-type="table"}). The mRNA expression of growth factors such as bone morphogenetic protein 4 (BMP4), ADP ribosylation factor 5 and 6 (ADPRF5 and 6 respectively), glia maturation factor β (GMFB), and transforming growth factor β I (TGF-βI) decreased after PGF~2α~treatment as well (Table [1](#T1){ref-type="table"}). Interferon inducible protein (Table [4](#T4){ref-type="table"}) and leukocyte common antigen (data not shown) were the only cytokine and chemokine related genes found significantly down regulated in response to PGF~2α~. Conversely, PGF~2α~stimulated the expression of basic fibroblast growth receptor factor 1 (bFGRF1) (Table [4](#T4){ref-type="table"}). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Effect of PGF~2α~on the expression of genes associated with G-proteins, growth factors, chemokines and cytokines. ::: Gene Bank number Swiss protein number Name Function Ratio % decrease Fold change ---------------------- -------------------------- ------------------------------------------------------ -------------------------- -------------- ---------------- ----------------- X52498 P17246 Transforming Growth Factor β1 Growth factor 0.33 ± 0.2 72 M60921 P27049 Antiproliferative B-cell translocation gene 2 (BTG2) Growth factor 0.28 ± 0.15 51 X61381 P26376 Interferon-induced protein Chemokine 0.25 ± 0.14 56 L11586 Q64604 Leukocyte common antigen Intracell. phosphatase 0.07 ± 0.02 83 Z22607 Q06826 Bone Morphogenetic protein 4 (BMP4) Growth factor 0.34 ± 0.17 78 L12384 P26437 ADP Ribosylation factor 5 Intracellular transducer 0.02 ± 0.008 67 L12385 P26438 ADP Ribosylation factor 6 Intracellular transducer 0.18 ± 0.07 83 Z11558 Q63228 Glia maturation factor β (GMFB) Growth factor 0.17 ± 0.07 83 M30705 P14842 5-hydroxytryptamine receptor 2A (HTR2A) G proteins 0.06 ± 0.03 93 S47609 P30543 Adenosine A2A receptor (ADORA2A) G proteins 0.13 ± 0.08 87 M91466 P29276 Adenosine A2B receptor (ADORA2B) G proteins 0.17 ± 0.07 83 M20713 P08753 Guanine nucleotide-binding protein G α3 subunit G proteins 0.22 ± 0.14 78 M17525 P04894 Guanine nucleotide-binding protein α stimulating G proteins 0.24 ± 0.13 75 M75153 P24410 Rab-11A G proteins 0.18 ± 0.11 82. D85760 Q63210 Guanine nucleotide-binding protein α12 subunit G proteins 0.15 ± 0.09 85 D12498 Q04589 Basic Fibroblast Growth Receptor factor 1 Growth factor 2.32 ± 1.07 3 ::: Discussion ========== Levels of PGF~2α~in the uterus need to be under tight control to avoid interference with the establishment and progression of pregnancy. Pathophysiological elevations in PGF~2α~lead to excessive uterine contractions. Therefore, PGF~2α~production must be avoided to maintain a quiescent uterus \[[@B13]\]. In this paper we present initial data demonstrating that elevated PGF~2α~can target the decidua, an endocrine tissue whose integrity is fundamental for the success of implantation and for the progression of pregnancy. Our results demonstrate that elevated PGF~2α~down regulate the expression of decidual genes related to the proteasome protein component system, those involved in trafficking and signal transduction and genes associated with G-proteins, growth factors, chemokines and cytokines. However, interestingly, the main effect of PGF~2α~is related to the turnover and degradation of the ECM which provides mechanical strength to the tissue. Thus, in addition to inducing uterine contractions, PGF~2α~can have a noxious impact on the success of implantation and progression of pregnancy, at least in part by deregulating the turnover of the ECM in the decidua. The largest group of genes down regulated by PGF~2α~is directly or indirectly related with the turnover and degradation of the ECM. Genes such as gelatinase A (MMP-2), TIMP2 and 3 \[[@B14],[@B15]\], cathepsin L \[[@B16]\], carboxypeptidase D are included in different groups of proteases, and can directly affect the degradation of the ECM. Carboxypeptidase D (CPD) or metallocarboxypeptidase D is a 180-kDa protein that contains almost three carboxypeptidase-like domains, a transmembrane domain, and a cytosolic tail. This gene participates in the processing of proteins transiting the secretory pathway \[[@B17]\]. TGF-β is a key factor that favours accumulation of collagen, laminin and fibronectin in the ECM \[[@B17]\]. TGF-β can stimulate PAI-1, inhibiting the protease degradation of the ECM. Other factors included in the plasminogen/plasmin and fibrinolytic system, such as tPA, uPA and PAI-1, can participate in the tissue remodelling of the ECM directly through the binding to specific receptors, and through the transformation of the plasminogen precursor bound to the cell surface to plasmin, which is an active serine protease. Plasmin is able to degrade most of the components of the ECM either directly or indirectly by the activation of MMPs. All of the plasminogen/plasmin factors mentioned previously participate in the process of decidualization \[[@B19]-[@B21]\]. Endothelin-1 is a peptide characterized as a potent endothelial cell-derived vasoconstrictor. It is synthesized as an inactive precursor (preproendothelin) and processed to a mature active form (endothelin) by zinc metalloproteinases. The active form, endothelin-1, promotes synthesis of collagen types I and II by fibroblasts, affects the ECM remodelling and promotes the proliferation of mesangial cells \[[@B21]\]. Endothelin-1 is also associated with neovascularization and regulation of blood flow \[[@B22]\]. This vasoactive factor is also involved in the genesis of endothelial cells behaviour \[[@B22],[@B23]\]. Annexin IV (ANX4, also called Lipocortin) was differentially down regulated by PGF~2α~. This protein belongs to a family of intracellular proteins that binds membrane phospholipids in a calcium-dependent manner. Thus, it can also inhibit phospholipase A2 (PLA2) \[[@B24]\]. ANX4 can also directly bind glycosaminoglycan (GAG) and can be localized not only to the cytoplasm but also the cell surface or the extracellular compartment \[[@B25]\]. It has been proposed that ANX4 could affect the mobilization of different substrates involved in the regulation of the ECM. Moreover, Annexin IV and other annexins can act as ligands for proteoglycans localized on the cell surface, in the ECM, or in secretory granules \[[@B24],[@B25]\]. Calponin (CaP) has three genetic isoforms, h~1~, h~2~and acidic calponin, and can be identified by the individual C-terminal tail sequences. The c-terminal sequences regulate actin association and the cytoskeleton \[[@B26],[@B27]\]. It is known that Cap or basic Cap inhibit the actomyosin ATPase in a calmodulin dependent manner \[[@B27]\]. Basic Cap, by affecting microtubules assembly, can modify the cytoskeleton of the cells, and indirectly, the associated ECM \[[@B27],[@B28]\]. On the other hand, acidic Cap belongs to the family of actin-associated proteins. It can interact with F-actin but not with microtubules, desmin filaments, and tropomyosin as basic Cap does. These properties suggest that acidic Cap is functionally distinct from basic Cap and could affect the ECM in a different manner \[[@B29]\]. Pro-stromelysin, α~1~antiproteinase, plasma proteinase-Iα inhibitor, basic fibroblast growth receptor factor 1, phosphorylase B and proteasome component C9 were the only genes up regulated by PGF~2α~in PDC. Pro-stromelysin is a 53 kDa peptide and a pro-MMP3 precursor and is directly related with the ECM regulation \[[@B30]\]. Plasma proteinase 1α inhibitor belongs to a major group of proteins that includes α2-macroglobulin \[[@B31]\], an important protein secreted by the decidual cells which has a critical role in the control of implantation \[[@B31]\]. Growth factor receptors, such as bFGRF, are part of a multigene family of structurally related factors (FGFs 1 to 9), some of which bind heparin sulphate proteoglycans that are components of the ECM \[[@B32]\]. bFGRFs as well as bFGF are also temporally and spatially present in the pregnant rat uterus \[[@B33]-[@B35]\]. Another member of the TGFβ superfamily expressed in the uterus during early implantation and down-regulated by PGF~2α~was BMP4, a gene which appears to be involved in specific stages of embryo development and is \[[@B36]\]. Proteasome component system proteins, such as proteasome Iota, proteasome component C2, proteasome subunit RC-7 I, 26 S-proteasome and proteasome component C3 were down regulated by PGF~2α~. Proteosomal component systems are the main non-lysosomal proteolytic structures of the cells that participate in the elimination of abnormal proteins, short half-life proteins, and proteins controlling cell cycle \[[@B37]\]. During the process of cell differentiation, the level of proteasome expression and its localization varies. The proteasomal proteins can be intermediaries of ECM by contributing to the modulation of the cell cycle through the induction of proteasomal degradation of cyclin dependent kinase 2. Cell attachment to ECM components, such as fibronectin (FN), does not affect p21 mRNA levels, but the stability of the p21 protein decreased \[[@B36]\]. Kinase activities such as ERKs, calcium/calmodulin kinase II and IV, SGK, and casein kinase can affect the ECM downstream or upstream \[[@B38]\]. On the other hand, ECM can regulate the availability of substrates, as well as factors or effectors downstream or upstream related with different cascades of signal transduction pathways. For example, proteins such as decorin are components of the ECM in many tissues and appear to be involved in matrix assembly \[[@B39]\]. Decorin can cause the rapid phosphorylation of EGF with the concurrent activation of mitogen-activated kinase protein kinase signal pathway. Via TGFβ, decorin can interact with the MAP kinases signal transduction pathways and cross talk with calcium /calmodulin-dependent kinase II \[[@B40]\], which in the cDNA array was down-regulated by PGF~2α~. Crk-associated substrate (CAS) was down-regulated by PGF~2α~and is another gene related to the ECM. Active CAS can modulate changes in cell motility and gene expression by the various MAP kinase cascades, and modify the organization of the actin cytoskeleton \[[@B39]\]. Another gene down-regulated by PGF~2α~was SGK, which is a transcriptionally-regulated serine/threonine protein kinase with 45--55% homology to the catalytic domain of Akt/PKB protein kinase A \[[@B40]\]. Skg is expressed in decidual tissue and can be activated by the phosphoinositide 3-kinase pathway (PI3-Kinase) through PDK1-mediated phosphorylation \[[@B41]\]. Traficking proteins such as 14-3-3 z/δ and ε are involved in the regulation of genes related to the ECM because it can block the activation cascade of signal transduction pathways \[[@B42]\]. Protein phosphatase type 2A (PP2A) was down regulated by PGF~2α~. The first and most important point of control of PP2A is at the transcriptional level. The increase of phosphatase activity corresponded with a decrease in the phosphorylation of cellular proteins in anchorage-dependent cells, but much lesser regulated in anchorage-independent cells \[[@B43]\]. It is well known that members of the proinflammatory cytokine family can induce MMP expression in numerous tissues \[[@B44]\]. Also, cytokines and chemokines, such as interferon inducible protein and leukocyte common antigen, are associated with the local induction of MMP expression in response to proinflammatory cytokines. The indirect action of these molecules through MMPs may aid to generate different changes in the endometrial stroma during maternal recognition of pregnancy \[[@B44]\] We have also compared the pattern of gene expression in rat decidual tissue in vivoon day 12 of pseudopregnancy (when the decidua undergoes regression) with that of the PGF~2α~treatment in rat PDC in vitro(data not shown). Interestingly, we found a 49% coincidence in the genes that were down regulated in both experimental situations. This coincidence suggests a relationship between the physiological regression and reorganization of the decidual tissue that occurs on day 12 of pseudopregnancy, with the pattern of gene expression in rat PDC after PGF~2α~treatment. On the other hand, many of the genes whose expression was affected by PGF~2α~in the rat PDC, are expressed in the decidua during decidualization and implantation. This suggests a possible connection between the action of PGF~2α~and the physiology of the decidual tissue. Moreover, the ECM is an important component of the decidualization and implantation process. Any modification in its turnover or degradation could affect the formation of the decidua, blastocyst invasion, and the timing of decidual regression and reorganization. It is known that the expression of MMPs/TIMPs plays an important role in the control of implantation. If PGF~2α~silences or decreases the expression of genes related with the systems aforementioned, it also could affect tissue remodelling by directly modifying the proteases involved in this process, or indirectly by affecting the ECM turnover and degradation, important in the accumulation of a spongy mass of tissue around each embryo during decidualization \[[@B45]\]. Most probably, many of PGF~2α~\'s effects on the expression of genes involved in ECM turnover are indirect. Alterations of genes involved in different signalling pathways such as ERK-1, CaMCKII, PLCδ1 and G-protein subunits may impact the expression of a great number of transcripts. In summary, our results show, for the first time, that pathophysiological concentrations of PGF~2α~have a severe impact on the expression of numerous genes associated with the turnover of the ECM in the rat decidua. Future investigation should corroborate the differentially regulated genes, at the level of the message, protein, and in some cases, such as for the metalloproteinases, at the level of activity of the proteins. These data will contribute to the design of future studies on a cluster of gene candidates as targets of PGF~2α~action in this endocrine tissue. Abbreviations ============= TIMP2 = tissue inhibitor metalloproteinases 2; TIMP3 = tissue inhibitor metalloproteinases 3; PAI 1 = plasminogen activator inhibitor 1; uPA = urokinase type; tPA = tissue type palsminogen activator; TGFβ = transforming growth factor β; SGK = serum glucocorticoid kinase; ERK-1 = extracellular signal-regulated kinase-1; MAK = male germ cell-associated kinase; CAS = crk-associated kinase kinase; CamKII = calcium calmodulin kinase II; CamKIV = calcium calmodulin kinase IV; PLC δ~1~= phospholipase C delta 1; BMP 4 = bone morphogenetic factor 4; ADPRF 5 = ADP ribosylation factor 5; bFGFRF1 = basic fibroblast growth factor receptor F1; HTR2A = 5-hydroxytryptamine receptor 2A; ADORA2A = adenosine receptor A2A; ADORA2B = adenosine receptor A2B; GN-BPG α~3~= guanine nucleotide-binding protein G α~3~; GN-BP α stimul. = guanine nucleotide-binding protein α stimulating; GN-BPG α~12~sub. = guanine nucleotide-binding protein α~12~subunit Authors\' contributions ======================= EC and SFG carried out the decidualization, and primary decidual cell culture. EC isolated the RNA isolation, performed the cDNA array assay, the analysis of the results, and drafted the manuscript. GG conceived the study and edited the manuscript. All authors read and approved the final manuscript. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### PGF~2α~has major effects on extracellular matrix (ECM) regulation ::: ![](1477-7827-3-3-3) ::: Acknowledgements ================ This research was supported by NIH grant number NIH HD 12356 and NIH, U54 HD 40093. We thank Dr. CM Telleria and Gil Gibori for constructive reading of the manuscript.	PubMed Central	2024-06-05T03:55:52.497858	2005-1-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548144/", "journal": "Reprod Biol Endocrinol. 2005 Jan 11; 3:3", "authors": [ { "first": "Eduardo A", "last": "Callegari" }, { "first": "Susan", "last": "Ferguson-Gottschall" }, { "first": "Geula", "last": "Gibori" } ] }
PMC548145	Introduction ============ Severe acute respiratory syndrome (SARS) is the first new infectious disease of this millennium. SARS has originated from Southern China at the end of 2002 and has a high mortality and morbidity. Within a period of six months beginning at the end of 2002, the disease has affected more than 8,000 people and killed nearly 800 \[[@B1]\]. The disease poses a new threat for respiratory medicine and represents a challenge for antiviral drug development and administration \[[@B2],[@B3]\]. SARS is caused by a novel, SARS-associated coronavirus (SARS-CoV) \[[@B4]-[@B6]\] which has been identified by a World Health Organization (WHO)-led global laboratory network. The first cases of SARS were reported from a hospital in Hanoi, Vietnam, by Carlo Urbani, a WHO scientist who himself died from the disease \[[@B7]\]. After reports from health authorities in Hong Kong on the outbreak of a new form of epidemical atypical pneumonia in public hospitals, the WHO issued a global alert on the disease. During this period, cases of SARS were also reported from China, other Asian countries and even other continents including America (Canada, U.S.A.) and Europe (Germany). Shortly after the initial global alert, the WHO initiated a collaborative multi-center research project on SARS diagnosis, led by eleven principal laboratories in nine countries \[[@B8]\]. Using modern communication technologies to optimize the analysis of SARS tissue samples, it was soon shown that a novel coronavirus is the causative agent of SARS (SARS-CoV) \[[@B4]-[@B6]\]. Due to the death of Carlo Urbani who first identified the new disease, the first isolate of the virus was proposed to be named Urbani strain of SARS-associated coronavirus, but a final terminology has not been proposed so far \[[@B9]\]. Since Koch\'s principles have been shown to be fulfilled by the new pathogen \[[@B10],[@B11]\], it is not necessary to call the virus SARS-associated and the general agreement is now to call it SARS coronavirus (SARS-CoV). Parallel to the progress made in the epidemiology and clinical diagnosis which has recently been demonstrated by numerous case reports, clinical studies and definitions \[[@B1]\], scientists have also revealed basic mechanisms of the underlying causative agent, the SARS coronavirus. As it is crucial for future strategies that SARS is detected in its earliest stages and that therapeutic options are optimized, insights into the molecular mechanism of SARS have to be used to develop new therapeutic strategies and vaccines. While other reviews have focused on the epidemiology, clinical presentation and potential treatment of SARS, the present overview aims to analyze and present the currently available data on molecular mechanisms of SARS. In this respect, it is important to underline that in the present state of no specific drug or vaccine being available, research on molecular mechanism is crucial to identify potential treatment targets. Etiology -------- Prior to the development of therapeutic regimes based on molecular mechanisms of the disease, the causative agent had to be isolated and analysed. Soon after the fast establishment of the international WHO laboratory network, rapid progress was made in the identification process of the causative agent, and it was reported that SARS is most probably caused by a novel strain of the family of coronaviruses \[[@B4]-[@B6]\]. These viruses are commonly known to cause respiratory and gastrointestinal diseases of humans and domestic animals \[[@B12],[@B13]\]. The group of coronaviruses is classified as a member of the order nidovirales, which represents a group of enveloped positive-sense RNA viruses consisting of coronaviridae and arteriviridae \[[@B14]\]. Viruses of this group are known to synthesize a 3\' co-terminal set of subgenomic mRNAs in the infected cells \[[@B15]\]. Origin of the SARS virus ------------------------ Soon after the identification of a new coronavirus as the causative agent of SARS and of a southern Chinese province as the first area of occurrence, animal species of this area have been speculated to be the origin of the SARS-CoV. As analysis of the SARS-CoV genetic sequence revealed large differences to any other currently known coronaviruses in humans or domestic animals \[[@B16],[@B17]\], it was hypothesized that the new virus might originate from wild animals. This hypothesis was supported by a search for coronaviruses in wild animals sold on markets in southern China, which identified the presence of a coronavirus in civet cats. This animal coronavirus was shown to have a sequence identity of more than 99% to the SARS coronavirus \[[@B18]\] with only a limited number of deletions and mutations between both viruses. SARS-CoV has a deletion of 29 nucleotides relative to the civet cat virus, indicating that if there was direct transmission, it went from the animal to man, because deletions occur probably more easily than insertions. Recent reports indicate that SARS-CoV is distinct from the civet cat virus and it has not been answered so far if the civet cat virus is the origin of the SARS-CoV or if civet cats were also infected from other species \[[@B19]\]. Therefore, there are no data available on the possibility of horizontal transmission between animals, and the question whether the jump of the virus from an animal to humans was a single accident or may frequently occur in future with the animals as dangerous reservoirs for future SARS epidemics remains unanswered. So far, the SARS-CoV has been reported to be able to infect not only humans but also macaque monkeys \[[@B11]\], domestic cats, and ferrets \[[@B20]\]. However, transmission of the virus from the domestic cat to man has not been shown. The ability of the SARS-CoV to infect other animal species could point to potential natural reservoirs of the virus. In this respect, coronaviruses are known to relatively easily jump to other species. I.e., the human coronavirus OC43 shares a high degree of genetic sequence homology to bovine coronavirus (BCoV) and it is commonly assumed that it has jumped from one species to the other \[[@B21],[@B22]\]. In the same way, BCoV has been reported to be able to infect humans and cause diarrhea \[[@B23]\]. Whereas the precise mechanisms of these species jumps remain unclear, it is most likely that they represent the results of mutations and epidemiological studies of coronavirus infections in wild animals will therefore be crucial for future understanding and control of new SARS outbreaks. SARS virus taxonomy ------------------- Until the identification of the new SARS-CoV, the coronaviruses have been divided into three subgroups, which differ with respect to their genome \[[@B24]\]. The first group consists of viruses such as the human coronavirus 229E (HCoV-229E), porcine respiratory coronavirus (PRCV), porcine transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus (FIPV) and feline enteritis virus (FEV) or the canine coronavirus (CCoV). The second group comprises human coronavirus OC43 (HCoV-OC43), bovine coronavirus (BCoV), and mouse hepatitis virus (MHV), and the third group mainly consists of avian species such as the chicken infectious bronchitis virus (IBV). Whereas the SARS-CoV has been shown to cross-react with some group I coronavirus antibodies \[[@B6]\], its genetic sequence does not belong to this group. Within the nucleic acid or protein sequence phylogenetic trees of the coronavirus family, the SARS-CoV has first been located at an equal distance from the second and third group, irrespective of which SARS-CoV RNA region is used for analysis \[[@B6],[@B16],[@B17]\]. Therefore, the SARS-CoV may represent the first member of a new group of coronaviruses (Figure [1](#F1){ref-type="fig"}). However, the taxonomy is still no clear \[[@B19],[@B25]\], and recent studies that focused on the N-terminal domain of the spike protein and on poorly conserved proteins such as Nsp1, matrix protein, or nucleocapsid, have suggested a relation to group II viruses \[[@B26]\]. A similar conclusion can be drawn if the polymerase gene is examined, pointing to an early split-off from the coronavirus group 2 lineage \[[@B27]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Coronavirus classification. The family of coronaviruses belongs to the order of nidovirales and consists of three groups so far. It is still debatable whether the new SARS-CoV should be assigned to group II or to a new fourth group. Group I includes human coronavirus 229E (HCoV-229E), transmissible gastroenteritis virus (TEGV), porcine epidemic diarrhea virus (PEDV), canine coronavirus (CCoV), and feline coronavirus (FIPV). Group II viruses include human coronavirus OC43 (HCoV-OC43), murine hepatitis virus (MHV), and bovine coronavirus (BCoV), and group III species are turkey coronavirus (TCoV), and avian infectious bronchitis virus (IBV). ::: ![](1465-9921-6-8-1) ::: Despite the fact that this new virus most likely jumped to humans from wild animal species, it has remarkably well adapted to the human organism as shown by its high person-to-person transmissibility. SARS virus genome structure --------------------------- The structure of the SARS viral RNA is organized in 13--15 open reading frames (ORF) and contains a total of approximately 30,000 nucleotides \[[@B6],[@B16],[@B17]\]. Recently, 61 SARS-CoV sequences derived from the early, middle, and late phases of the SARS epidemic together with two viral sequences from palm civets were analyzed \[[@B28]\]. Genotypes characteristic of each phase were discovered, and it was found that the neutral mutation rate of the viral genome was constant but the amino acid substitution rate of the coding sequences slowed during the course of the epidemic. The spike protein showed the strongest initial responses to positive selection pressures \[[@B28]\]. Only ORFs exceeding fifty amino acids in translational capacity are considered relevant as they contain the sequences for the structural and functional properties of the virus and are therefore of potential interest for the development for future therapeutic strategies. The comparison of the different SARS-CoV ORFs with those of other coronaviruses reveals a familiar pattern of structural gene arrangement with replicase and protease genes (gene 1a-1b) and the spike (S), envelope (E), membrane (M) and nucleocapsid (N) genes in a typical 5\'- to 3\' order of appearance \[[@B29]\]. The proteins encoded by these genes may be targets for novel treatments. Between these well-known genes, a series of ORFs of unknown function was found: There are two ORFs situated between the spike and the envelope genes and three to five ORFs between the membrane and nucleocapsid genes. Comparison of this gene organization with other known coronaviruses does not indicate a closest proximity to group II coronaviruses. Also, the SARS-CoV genomic sequence does not contain a gene for hemagglutinin-esterase (HE) protein, which is present in the majority of group II coronaviruses. Two-thirds of the SARS RNA is organized in the gene 1a-1b. The sequence of this gene is highly conserved among all coronaviruses \[[@B17]\]. ORFs 1a and 1b encode two polyproteins, pp1a and pp1ab, the latter through a ribosomal frameshifting mechanism. These polyproteins are processed by virus-encoded proteinases, to yield 16 individual proteins. Most potential gene 1a-1b products are fairly well conserved between SARS-CoV and other coronaviruses \[[@B17],[@B29]\]. Many of their functions are unknown but it is suggested that they participate in viral RNA replication, making them potential targets for the development of antiviral compounds. Therefore, research efforts will focus on these proteins. One exception from the overall conservation of SARS-CoV gene 1a-1b is the lack of a sequence coding for PL1^pro^, one of the two papain-like proteinases operating on cleavage sites at the N-terminus of the polyproteins (Figure [2](#F2){ref-type="fig"}). The main proteinase (M^pro^), also called 3C-like protease (3CL^pro^), is responsible for the cleavage of all the remaining proteins encoded by gene 1a-1b \[[@B29],[@B30]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### SARS-CoV genome organization. The structure of the SARS viral RNA is organized into 13--15 open reading frames (ORFs) and contains an overall amount of approximately 30,000 nucleotides. The sequence can be separated into different elements and genomic and subgenomic mRNAs. ::: ![](1465-9921-6-8-2) ::: SARS virus gene expression -------------------------- Apart from gene 1, coronavirus genes are known to be usually expressed from subgenomic mRNAs. They share a common leader sequence at the 5\'-end and initiate at different places in the genome extending toward the 3\'-end of the virus genome \[[@B31]\]. Some ORFs may also be unconventionally translated from a single mRNA. As these uncommon translation mechanisms are not very efficient and the gene products are not very abundant, these ORFs typically encode nonstructural proteins. Whereas the ORFs between the structural protein genes are very heterogeneous among the different coronaviruses and not essential for viral replication, recent studies suggested that deletion of non-essential ORFs may result in a reduced virulence \[[@B32]\]. In agreement with this, some of these non-essential ORFs of the new SARS-CoV genome may be responsible for the high SARS-CoV virulence. So far, five to eight subgenomic mRNAs were found in SARS-CoV-infected cells \[[@B17],[@B27]\]. Thiel and colleagues performed the first detailed study on mechanisms and enzymes involved in SARS-CoV genome expression (Figure [2](#F2){ref-type="fig"}) \[[@B29]\]. They determined the sequence of the SARS-CoV isolate Frankfurt 1 and characterized the major RNA elements and protein functions involved in the genome expression by characterizing regulatory mechanisms such as the discontinuous synthesis of eight subgenomic mRNAs, ribosomal frameshifting and post-translational proteolytic processing. Also, the activities of SARS-CoV enzymes such as the helicase or the two cysteine proteinases (PL2^pro^and M^pro^) were addressed as they are involved in replication, transcription or post-translational polyprotein processing \[[@B29]\]. In conclusion, research in the area of coronavirus gene expression is important to delineate components which directly affect SARS-CoV virulence. SARS virus structural proteins ------------------------------ The structural proteins of the new SARS-CoV are potential targets for new treatment options. The new SARS-CoV only contains the three envelope proteins, spike (S), envelope (E), and membrane (M) but not the hemagglutinin-esterase (HE) protein, which is present in some coronaviruses of the second group. The spike glycoprotein is responsible for the characteristic spikes of the SARS-CoV (Figure [3](#F3){ref-type="fig"}). Intra- and extracellular proteases often cleave the S protein into S1 and S2 domains, with the cleavage process often increasing infectivity of the virus. Molecular modelling has been performed for the S1 and S2 units of the SARS-CoV spike protein \[[@B33],[@B34]\]. The spike proteins of coronaviruses are reported to bind to receptors on their target cells and the domains responsible for receptor-binding are commonly situated in the N-terminal region of S1 \[[@B35]-[@B40]\]. The spikes consist of oligomeric structures, that are formed by heptad repeats of the S2 domain which also represent a fusion peptide sequence. This peptide is responsible for the coronavirus fusion activity. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### SARS-CoV transmission electron microscopy. In the supernatant of SARS-CoV infected cytopathic Vero E6 cells, characteristic virus particles can be found. The diameter of the viruses ranges between 60 nm and 120 nm and the virus shapes are round or oval. There are many protrusions from the envelope which are arranged in order with wide gaps between them. There are also many virus particles in the infected cells present. They often form a virus vesicle with an encircling membrane. A: Higher magnification B: Lower magnification. Scale bars represent 100 nm. Reproduced with permission from Acta Biochimica et Biophysica Sinica 2003, 35(6):587--591 \[126\]. ::: ![](1465-9921-6-8-3) ::: The SARS-CoV has also been reported to cause the formation of syncytia in vivo, but so far only under the condition of cultured Vero cells \[[@B6]\]. The SARS-CoV S protein seems to have most of its characteristics in common with the S proteins of other coronaviruses, but it will be important for the understanding of the SARS-CoV pathogenic properties to identify the exact conditions of membrane fusion, i.e. pH dependency and protease sensitivity, which can increase the infectivity. The envelope and membrane proteins are integral membrane proteins and required for virus assembly \[[@B41]\]. In the case of the murine coronavirus MHV-A59 the coexpression of the E and M proteins but not the S or N proteins is needed for the release of virus-like particles (VLP) \[[@B42]\]. The nucleocapsid and viral core of the SARS-CoV are likely to be formed by the N protein. An interesting feature of the SARS-CoV and other coronaviruses is the resistance against the gastrointestinal fluids despite the lipid composition of their envelope. It has been reported that the SARS-CoV can survive in diarrheal stool for four days and also, patients with SARS often suffer from gastrointestinal symptoms with the virus to be detected in the stool \[[@B4]\]. As the molecular basis for the envelope\'s resistance against acidic environments and gastrointestinal enzymes is unclear, further research has to be carried out in this area which is important for the control of future SARS outbreaks. Evolution of the SARS virus --------------------------- It is unclear when and how novel pathogens such as the SARS-CoV cross the barriers between their natural reservoirs and human populations, leading to the epidemic spread of novel infectious diseases \[[@B43]\]. As with the SARS-CoV, new pathogens are believed to emerge from animal reservoirs and a variety of molecular mechanisms may contribute to the evolution of the viruses or bacteria. Due to the estimated error frequency of 1 × 10 ^-4^for RNA-dependent RNA polymerases \[[@B44]\], RNA viruses such as the SARS-CoV can undergo mutation at a high frequency. The SARS-CoV seems to be relatively genetically stable as the RNA sequences from different SARS patients were quite homogeneous. Even the entire genomic sequences of virus isolates from different continental areas did not differ by more than ten amino acids and it seems that two lineages of the virus can be traced \[[@B45]\]. This obvious contradiction to the high potential error rate of the RNA-dependent RNA polymerase suggests the presence of some proofreading mechanism connected with this enzyme. In fact, a detailed analysis of the SARS-CoV genome by bioinformatics indicates the presence of an exonuclease activity \[[@B27]\]. Next to mutations, a further threat of the SARS-CoV is based on the ability of coronaviruses to undergo RNA recombination at a high frequency \[[@B15]\]. For a variety of other coronaviruses, both recombination and mutation in natural infections have been shown to contribute to the diversification of the coronaviruses. Because of the demonstrated ability of coronaviruses to recombinate, the question whether the SARS-CoV will show a higher frequency of mutations within possible future seasonal changes or in respond to drug treatment is an issue of major concern. It was reported that in the initial phases of the SARS epidemic, the mutation rate was high in the gene for the spike protein, but this stabilized during the middle and final stages of the 2003 epidemy \[[@B28]\]. Thus, the virus had experienced great pressure to adapt to the new host after crossing the species barrier, but has then been optimized \[[@B28]\]. Duration of infection --------------------- Although human coronaviruses are characteristically causing self-limiting short diseases, the question of potential chronic SARS infections is of major importance for a future disease control. If the SARS-CoV is able to cause a chronic persistent infection, chronic carriers may serve as sources for new SARS outbreaks. However, the detection of SARS-CoV in stool of patients for longer periods than 6 weeks after hospital discharge has not been reported so far. Therefore, the danger of chronic carriers may not be relevant. In contrast to common human coronavirus infections with short durations, most animal coronaviruses cause persistent infections. As an example, the feline coronavirus FIPV infects animals which then continue to shed virus for periods reaching up to seven months after infection without carrying disease symptoms \[[@B46]\]. Also, TGEV and MHV tend to cause chronic infections as these viruses may be found in the airways and small intestine (TGEV) or the nervous system (MHV) several months after infection \[[@B47],[@B48]\]. Although the SARS-CoV has jumped to humans it may still have this property of inducing chronic infections. Thus, SARS-CoV RNA was found in patients\' stool specimen more than 30 days after the infections. Clinical picture of SARS ------------------------ The mean incubation period of SARS was estimated to be 6.4 days (95% confidence interval, 5.2 to 7.7). The mean reported time from the onset of clinical symptoms to the hospital admission varied between three and five days \[[@B49]\]. Main clinical features of the disease are in the initial period common symptoms such as persistent fever, myalgia, chills, dry cough, dizziness, and headache. Further, although less common symptoms are sore throat, sputum production, coryza, vomiting or nausea, and diarrhea \[[@B50],[@B51]\]. Special attention has been paid to the symptom of diarrhea: Watery diarrhea has also been reported in a subgroup of patients one week after the initial symptoms \[[@B52]\]. The clinical course of the disease seems to follow a bi- or triphasic pattern. In the first phase viral replication and an increasing viral load, fever, myalgia, and other systemic symptoms can be found. These symptoms generally improve after a few days. In the second phase representing an immunopathologic imbalance, major clinical findings are oxygen desaturation, a recurrence of fever, and clinical and radiological progression of acute pneumonia. This second phase is concomitant with a fall in the viral load. The majority of patients is known to respond in the second phase to treatment. However, about 20% of patients may progress to the third and critical phase. This phase is characterized by the development of an acute respiratory distress syndrome (ARDS) commonly necessitating mechanical ventilation. SARS in adults and children --------------------------- Rapid progress has been made in understanding the clinical presentation of SARS in adults and children \[[@B53]-[@B56]\]. In comparison to adults, SARS seems to be less aggressive in younger children, with no children in one case series requiring supplementary oxygen \[[@B57]\] while in adults, systemic infection as well as respiratory infection may be the rule. SARS is much milder with non-specific cold-like symptoms in children younger than 12 years than it is in adolescents and adults \[[@B58]\]. The reason for the milder clinical presentation of SARS in children is most likely due to differences in developmental stage of the immune system. The course of the disease in teenagers more likely resembles adults in concerning clinical presentation and disease progression \[[@B58]\]. SARS may also develop severe illness requiring intensive care and assisted ventilation in these adolescent patients. The common presenting features are fever, malaise, coryza, cough, chills or rigor, headache, myalgia, leucopaenia, thrombocytopaenia, lymphopaenia, elevated lactate dehydrogenase levels and mildly prolonged activated partial thromboplastin times \[[@B59]\]. The radiographic findings are non-specific: However, high-resolution computed chest tomography in clinically suspected cases may prove to be an early diagnostic aid when initial chest radiographs appeared normal. While rapid diagnosis with the first-generation RT-PCR assay was not satisfactory, improved RT-PCR assays may help to diagnose SARS in early stages. In this respect, a sensitivity approaching 80% in the first 3 days of illness when performed on nasopharyngeal aspirates may be achieved. The best treatment strategy for SARS among children still has to be determined while no case fatality has been reported in children. In comparison to the prognosis in adults, there is a relatively good short- to medium-term outcome. However, it is crucial to emphasize that continued monitoring for long-term complications due to the disease or its treatment is of major importance \[[@B60]\]. Molecular mechanisms of SARS virus pathogenesis ----------------------------------------------- ### Cytocidal mechanisms Coronaviruses are known to exert their effects by cytocidal and immune-mediated mechanisms. In vitro studies using cell culture assays have shown that coronavirus infection commonly results in cytopathic effects such as cellular lysis or apoptosis \[[@B61]\]. Also, the virus can cause cellular fusion leading to the formation of syncytia. These cytopathic effects are caused by steps of the viral replication such as the mobilisation of vesicles to form the viral replication complex \[[@B18]\], leading to the disruption of Golgi complexes \[[@B62]\]. Parallel to results on other coronaviruses, SARS-CoV has been shown to cause cytopathic effects in Vero cells and the formation of syncytia in lung tissues. A further similarity with other coronaviruses seems to be the potential of the SARS-CoV to cause tissue fibrosis \[[@B63]\]. As molecular mechanism for this fibrosis which has been reported for infections with the coronavirus MHV, the N protein has been demonstrated to induce promoter activity of the prothrombinase gene that correlates with fibrin deposition \[[@B64]\]. Immune-mediated mechanisms -------------------------- Next to cytocidal effects, also immune-mediated mechanisms of both the innate and adaptive immune system seem to contribute to the pathogenesis of SARS-CoV infections. In this respect, it has been shown that in MHV infection, T cells and cytokines play an important role in development of the disease \[[@B65]\]. Also, humoral antibodies have been reported to be crucial in infections caused by coronaviruses such as FIPV. Herein, antibodies against the spike protein were shown to be related to the induction of peritonitis \[[@B66]\]. For SARS-CoV infections, it has been reported that there seems to be an inflammatory cell influx consisting in particular of macrophages in the airways, and a massive release of cytokines during the peak of the infection \[[@B67],[@B68]\]. It is therefore crucial that these immune mechanisms are further analysed on the molecular level as it seems appropriate that not only antiviral but also anti-inflammatory strategies are evaluated for a use in the clinical management of future SARS cases. The pharmacotherapy for SARS with anti-inflammatory steroids is controversial and largely anecdotal \[[@B69]\]. It was reported that the initial use of pulse methylprednisolone therapy appears to be more efficacious and equally safe when compared with regimens with lower dosage and should therefore be considered as the preferred steroid regimen in the treatment of SARS, pending data from future randomized controlled trials \[[@B70]\]. A further preliminary, uncontrolled study of patients with SARS, reported that the use of interferon alfacon-1 plus steroids was associated with reduced disease-associated impaired oxygen saturation and more rapid resolution of radiographic lung abnormalities \[[@B71]\]. Mechanisms of target cell specificity ------------------------------------- The most obvious gene which is likely to be a key modifier of SARS pathomechanisms is the spike (S) protein gene. As known for other coronaviruses, it does not only affect viral pathogenesis by determining the target cell specificity but also by other mechanisms. In this respect, a single mutation in the S gene of MHV has significant effects on the viral virulence and tissue tropism \[[@B72]\]. Also, mutations in the S gene led to the emergence of the weakly virulent PRCV from the virulent enteric TGEV \[[@B73]\]. Further potentially important genes are the \'non-essential\' ORFs which show a significant divergence between SARS-CoV and other coronaviruses. In this respect, it was reported that the civet cat coronavirus has a 29-nucleotide deletion leading to a fusion of two non-essential ORFs into one new ORF in the SARS-CoV \[[@B18]\]. It was shown that deletion mutants of \'non-essential\' ORFs of the group 2 mouse hepatitis virus (MHV) leads to a lower virulence without an impact on viral replication \[[@B74]\]. It has to be established if this also applies to \'non-essential\' ORFs of SARS-CoV. Also, other viral gene products such as the M or E proteins may have an impact on the pathogenesis of the disease as they may induce interferon production or apoptosis \[[@B75],[@B76]\]. Molecular targets for antiviral treatment ----------------------------------------- The primary target cells of SARS-CoV infection are respiratory epithelial cells. As the virus can also be detected in stool specimen and patients with SARS often also have gastrointestinal symptoms, epithelial cells of the gastrointestinal tract also seem to be major target cells. Next to these epithelial cells, the SARS-CoV has also been found in macrophages and many other cells as it has been detected in not only in the respiratory tract and stool specimen but also in the blood, liver, kidney and urine \[[@B6]\]. In this respect, pathological examination did not only show changes in the respiratory tract, but also in splenic lymphoid tissues and lymph nodes. Furthermore, signs of a systemic vasculitis were found which included edema, localized fibrinoid necrosis, and infiltration of monocytes, lymphocytes, and plasma cells into vessel walls in the heart, lung, liver, kidney, adrenal gland, and the stroma of striated muscles. There was also thrombosis present in veins. Systemic toxic changes included necrosis and degeneration of parenchymal cells of the lung, heart, liver, kidney, and adrenal gland \[[@B77]\]. It may therefore be concluded that SARS can induce a systemic disease and thereby injuring many other organs apart from the respiratory tract. Target cell receptors --------------------- The SARS-CoV target cell specificity is determined by the spike protein affinity to cellular receptors. In contrast to the all group III coronaviruses and the SARS-CoV for which the receptors have not been finally analyzed, it is known that group I coronaviruses bind to aminopeptidase N (CD13) as receptors \[[@B78]\], while group II coronavirus such as MHV use carcinoembryonic antigen (CEA) as receptor \[[@B79]\]. Recently, it was shown that a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), efficiently binds the S1 domain of the SARS-CoV S protein. SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Also, anti-ACE2 but not anti-ACE1 antibodies blocked viral replication on Vero E6 cells, indicating that ACE2 is a functional receptor for SARS-CoV \[[@B80]\] which was also identified by a further study \[[@B81]\]. Recently, the C-type lectin CD209L (also called L-SIGN) was discovered to be a further human cellular glycoprotein that can serve as an alternative receptor for SARS-CoV \[[@B82]\]. The interruption of virus-receptor interactions could be a potential target for future therapeutic strategies (Figure [4](#F4){ref-type="fig"}). In this respect, the receptor-binding S1 domain of the SARS-CoV S protein represents a possible target for new SARS antiviral drugs. Also, antibodies against ACE2, but not inhibitors binding to the active site of ACE2 may be useful for the development of therapeutic strategies. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Potential target sites for therapeutic strategies. In view of the viral life cycle, there are several potential targets for the development of antiviral drugs. Starting from the binding of the virus to the target cell, the spike protein or receptors such as angiotension-converting enzyme 2 (ACE2), cell entry or the different replication steps may be targeted. After replication, virus assembly and exit mechanisms may also be used for antiviral strategies. VLP, virus-like particles. ::: ![](1465-9921-6-8-4) ::: Virus entry ----------- After binding to the receptor, the next molecular step of potential use for the development of anti-SARS drugs is the virus entry into the cells. While most coronaviruses enter their target cells via plasma membrane fusion, a further entry mechanism may be acidic pH-dependent endocytosis \[[@B83]\]. Focusing on these mechanisms, it will be crucial to gain further knowledge about SARS-CoV fusion activity. As a drug development candidate, a putative fusion peptide has good potential (Figure [4](#F4){ref-type="fig"}). Intracellular replication ------------------------- After the binding to a host cell receptor and entry into the cells, the molecular steps of transcription, translation and protein processing display further potential targets for new therapeutic strategies. In this respect, the RNA-dependent RNA polymerases (SARS-CoV RdRp) may be a potential target for a future anti-SARS therapy. A recent study located its conserved motifs and built a three-dimensional model of the catalytic domain \[[@B84]\]. The authors suggested that potential anti-SARS-CoV RdRp nucleotide-analog inhibitors should feature a hydrogen-bonding capability for the 2\' and 3\' groups of the sugar ring and C3\' endo sugar puckering. Also, the absence of a hydrophobic binding pocket for non-nucleoside analog inhibitors similar to those observed in hepatitis C virus RdRp and human immunodeficiency virus type 1 reverse transcriptase seems to be crucial \[[@B84]\]. Also, protease activity is crucial for SARS-CoV RNA replication and protein processing \[[@B29],[@B85]\], and the inhibition of protease function leads to an immediate stop of viral RNA synthesis. Most of the coronaviruses express one major cysteine proteinase, called the main proteinase (M^pro^) or the 3C-like proteinase (3CL^pro^), and two auxiliary, papain-like proteinases (PL1^pro^and PL2^pro^). The latter two are responsible for the cleavage of the viral polyproteins, pp1a and pp1ab, at three sites near the amino-terminus, while the M^pro^processes these proteins at as many as 11 additional sites. Interestingly, SARS-CoV lacks the PL1^pro^\[[@B16],[@B17]\], but it can be assumed that its action is taken over by the PL2^pro^\[[@B29]\]. This is conceivable since operation of the PL2^pro^on PL1^pro^cleavage sites has been shown in IBV and HCoV \[[@B86]\]. Roughly at the position of the PL1^pro^gene in other coronavirus genomes, SARS-CoV displays a domain within ORF1a that lacks any detectable sequence homology and has therefore been named the SARS-unique domain (SUD) \[[@B27]\]. It is not known whether the SUD protein is ever expressed in the life cycle of SARS-CoV but if it is, it may be connected to the high pathogenicity of SARS-CoV compared to other human coronaviruses and, therefore, it may constitute an attractive target for therapeutic intervention. Crystal structures have been determined for the M^pro^s of TGEV \[[@B87]\], HCoV 229E \[[@B85]\], and, more recently, SARS-CoV \[[@B88]\]. They all show a similar overall architecture for the 34 kD enzyme which forms a dimer in the crystals and also at intermediate and high concentrations in solution. The monomer consists of three domains of which the first two are β-barrels with an overall similarity to the 3C proteinases of picornaviruses and to the serine proteinase, chymotrypsin. The third domain is α-helical and was shown to be essential for dimerization \[[@B85],[@B87],[@B88]\]. The active site of the enzyme is located in a cleft between domains I and II and comprises a catalytic dyad of Cys\...His, rather than the catalytic triad common for cysteine and serine proteinases. Anand et al. \[[@B85]\] have synthesized a substrate-analogous hexapeptidyl chloromethylketone inhibitor and bound it to TGEV M^pro^in the crystalline state. The X-ray structure of the complex revealed binding of the P1 glutamine, P2 leucine, and P4 threonine side chains of this compound to the respective subsites in the substrate-binding cleft, in agreement with the pronounced specificity for cleavage by the M^pro^after the substrate sequence (Thr, Val, Ser)-Xaa-Leu-Gln. The structure also showed the expected covalent attachment of the methyl ketone group at P1 of the inhibitor to the catalytic cysteine of the enzyme. In spite of 40% and 44% sequence identity, respectively, to the M^pro^s of HCoV 229E and TGEV, the crystal structure of the SARS-CoV M^pro^revealed some surprises \[[@B88]\]. Within the dimer, one molecule was in the active conformation seen in the other structures, whereas the other one adopted a catalytically incompetent conformation. This enzyme had been crystallized at a pH value of \<6, which in one of the monomers apparently led to the protonation of a histidine residue at the bottom of the S1 specificity pocket. This resulted in major conformational rearrangements leading to the collapse of this binding site for the P1 glutamine residue of the substrate and to a catalytically incompetent conformation of the oxyanion-binding loop. However, when the crystals were equilibrated at higher pH values, their X-ray structures revealed the active conformation for both monomers in the dimer. This pH-dependent activation mechanism allows interesting conclusions to be made for the self-activation of the M^pro^from the viral polyprotein, which probably involves a pH-dependent step. The same hexapeptidyl chloromethylketone inhibitor used by Anand et al. \[[@B85]\] in their crystallographic study of the TGEV M^pro^was employed by Yang et al. \[[@B88]\] to characterize the interaction of the SARS-CoV enzyme with substrate. This was performed by soaking the inhibitor into crystals grown at the low pH. In spite of the inactive conformation of one of the two monomers in the dimer being preserved, the compound was found to bind to it, but with its P1 glutamine side chain pointing towards bulk solvent rather than into the S1 binding site, because of the collapse of the latter. The binding mode of the inhibitor to the active monomer was also somewhat unusual and is not fully understood at present. On the basis of their crystallographic work, Anand et al. \[[@B85]\] found that the binding mode of their hexapeptidyl chloromethylketone inhibitor to the TGEV M^pro^resembled that of AG7088 in complex with its target, the 3C proteinase of human rhinovirus \[[@B89]\], even though the respective target enzymes displayed large structural differences except in the immediate neighbourhood of the active site. AG7088 is in phase II/III clinical studies as an inhalation treatment for the common cold as caused by human rhinovirus. Anand et al. \[[@B85]\] therefore proposed that AG7088 should be a good starting point for the design of anti-SARS drugs, and indeed, the manufacturer of AG7088 confirmed only a few days after their proposal had appeared on-line that the compound was effective against SARS coronavirus in cell culture. AG7088 is now the subject of intensive optimization efforts \[[@B90]\]. Other studies used molecular dynamics simulations of the M^pro^and screened 29 approved and experimental drugs against a model of the SARS CoV proteinase as well as the experimental structure of the transmissible gastroenteritis virus (TGEV) proteinase \[[@B91]\]. It was suggested that existing HIV-1 protease inhibitors, L-700,417 for instance, may have high binding affinities and may therefore provide another good starting point for the future design of SARS-CoV proteinase inhibitors \[[@B92]\]. However, this has to be proved experimentally. Further potential targets are the E and M proteins (Figure [4](#F4){ref-type="fig"}) as they represent the minimum essential components for the assembly of coronaviruses which form the virus-like particles \[[@B41],[@B42]\]. Ultrastructurally, the process of SARS-CoV assembly is most likely localised to the ER-Golgi intermediate compartment \[[@B93]\]. Together with strategies that may focus on the inhibition of virus assembly, the virus exit through secretory pathways is also of interest for the development of new antiviral compounds. With regard to the multitude of potential epithelial target cells, specific endogenous drug delivery systems may also be of relevance. In this respect, the family of peptide transporters consisting of PEPT1 and PEPT2 which are differentially expressed in potentially infected cells of the respiratory tract \[[@B94],[@B95]\], small intestine \[[@B96]\], kidneys \[[@B97],[@B98]\], nervous system \[[@B99]\] and other organs \[[@B100]\], may serve a target for the rational drug design of antiviral drugs. So far, a variety of antiviral drugs or prodrugs such as valacyclovir \[[@B101]\], valganciclovir \[[@B102]\] or the valyl ester of zidovudine \[[@B103]\] have been shown to be transported via these systems and minimal structure requirements for substrate transport have been determined \[[@B104]\]. A further tool which may be used to approach antiviral therapies is the technique of small interfering RNAs (siRNAs). SiRNAs are double-stranded RNAs which lead to a sequence-specific degradation of mRNAs \[[@B105]\]. Recent in vitro studies used six 21-mer siRNAs that were targeted to different sites of the replicase 1A region of SARS-CoV \[[@B106]\]. Monkey kidney cells (FRhk-4) were infected with the SARS-CoV GZ50 strain and transfected eight hours later with the siRNAs. Three of the six siRNAs led to a marked inhibition of virus cytopathic effects and a reduction of virus copies between 85 and 92 %, indicating that siRNAs may have a potency as antiviral treatment options and that the 1A region displays a promising region to suppress virus replication \[[@B106]\]. Vaccines against the SARS virus ------------------------------- As most patients develop an immunity against the SARS-CoV and survive the infection, the possibility of creating an effective and safe vaccine seems to exist \[[@B107]\]. There are several options to develop vaccines against the SARS-CoV \[[@B108]\]. Live-attenuated vaccines ------------------------ Live-attenuated coronavirus vaccines can be generated by deletions in \"group-specific genes\". The deletions of these genes do not change replication properties but attenuate the virus \[[@B109]\]. Examples for the use of live-attenuated vaccines to prevent coronavirus infections are live attenuated IBV vaccines which are used in broiler chickens \[[@B110]\]. For the animal coronavirus infections, live attenuated vaccines have been proven to be significantly more effective than whole killed vaccines, indicating that cell-mediated immunity is a crucial defence mechanism. However, the great threat remains that a vaccine strain can recombine with a circulating wild type strain \[[@B111]\] and without evidence that recombination and reversion of a live-attenuated SARS-CoV to virulence can not occur, it is unlikely that a live attenuated SARS-CoV vaccines will be developed and used. Whole killed vaccines --------------------- Whole killed vaccines are generally safe and easy to generate. In fact, this technique has been applied in veterinary medicine to generate vaccines for BoCV and IBV \[[@B112]\]. Also, an inactivated canine coronavirus vaccine has been produced \[[@B113]\]. A SARS inactivated vaccine was recently developed using the SARS coronavirus (SARS-CoV) strain F69 treated with formaldehyde and mixed with Al(OH)(3) \[[@B114]\]. However, killed vaccines may not protect against different strains of coronaviruses, and live attenuated vaccines have been shown to be more effective than whole killed vaccines in preventing coronavirus animal infections \[[@B115]\]. Recombinant subunit vaccines ---------------------------- Using molecular biology techniques to generate large quantities of recombinant viral proteins, recombinant subunit vaccines, e.g. against the spike protein, are expected to be created relatively easy as shown by two recent studies \[[@B116],[@B117]\]. Eight recombinant human single-chain variable region fragments (scFvs) against the S1 domain of spike (S) protein of the SARS-CoV from two nonimmune human antibody libraries were screened and one scFv 80R efficiently neutralized SARS-CoV and inhibited syncytia formation between cells expressing the S protein and those expressing the SARS-CoV receptor angiotensin-converting enzyme 2 (ACE2) \[[@B117]\]. A recent study used the SARS-CoV spike protein receptor binding domain (aa 318--510) for immunization, which resulted in the induction of effective neutralizing antibodies \[[@B118]\]. However, recombinant subunit vaccines may have a limited ability to protect against SARS-CoV infections in view of the variations which may arise in the viral genome in future outbreaks. Therefore, the approach of recombinant subunit vaccines may have to be supplemented by further vaccine strategies which focus on cell-mediated immunity. Recombinant vectored vaccines ----------------------------- An approach using recombinant vectored vaccines with DNA or a viral vector could be a promising target. The DNA prime and adenovirus or MVA boost approach which is currently analysed for a potential use in the development of HIV vaccines, may also offer a strategy to prevent SARS infections. In this respect, a multi-valent approach which induces both humoral and T cell-mediated host responses seems to be the most attractive strategy. From the field of veterinary medicine, data on this approach are already available: A recombinant fowlpox with the S1 gene of IBV was demonstrated to be relatively protective against IBV \[[@B119]\]. Also, a DNA vaccine was developed which contains the nucleocapsid protein gene of porcine transmissible gastroenteritis virus (PTGV). This vaccine was shown to initiate both humoral and cell-mediated immune host responses \[[@B120]\]. Recently, three murine studies demonstrated that DNA vaccines encoding different SARS-CoV antigens are capable of generating humoral and cellular immunity and may potentially be useful for control of infection with SARS-CoV \[[@B121]-[@B123]\]. However, it was also shown that immunization with modified vaccinia virus Ankara-based recombinant vaccine against SARS is associated with enhanced hepatitis in ferrets \[[@B124]\]. Epitope-based vaccines ---------------------- A further strategy is based on the use of epitopes which can be delivered using viral or DNA vectors. Such an epitope-based strategy for coronavirus vaccination has already been reported \[[@B125]\] and the major advantages is the prevention of a possible vaccine reversion to virulence. A further benefit of this technique is the possibility to eliminate any regions of the viral genomic sequence which be associated with a potential autoimmune effects. The limitation of this approach is mainly based on potential variations. In this respect, epitopes which frequently undergo mutations will not protect against the SARS-CoV infections if used in epitope-based vaccines. If the SARS-CoV evolves as a highly variable virus, it will be crucial to identify highly conserved epitopes of the virus. In summary, the important development of SARS vaccines can be approached using several techniques which should ideally encompass the induction of both humoral and cell-mediated mechanisms. As coronavirus vaccines in animals have partly been reported to cause an enhancement of viral infections \[[@B66]\], a cautious approach has to be followed. A first study has investigated the ability of adenoviral delivery of a codon-optimised SARS-CoV spike protein S1 fragment, membrane protein, and nucleocapsid protein to induce immunity in rhesus macaques. The immunization with a combination of these three Ad5-SARS-CoV vectors and a booster vaccination on day 28 demonstrated antibody responses against the spike protein S1 fragment. Also T-cell responses against the nucleocapsid protein were found and all vaccinated animals displayed strong neutralising antibody responses in vitro. These results indicated that an adenoviral-based vaccine can induce SARS-CoV-specific immune responses in monkeys. Conclusion ========== In summary, the onset of the SARS epidemic in different continents has led to the formation of a successful laboratory network to identify the molecular mechanisms underlying the SARS infection. Next to the development of early diagnostic tests and effective treatment strategies, it is most important to orchestrate research activities which lead to the development of vaccines and antiviral agents, as there is no established therapy to date. Even now in a situation of only a handful of new cases, SARS remains a major global health hazard which may reappear. Acknowledgements ================ Part of this work was supported by grants from the European Commission and the DFG to RH (Hi 611/4-1) and to DAG (Gr 2014/2-1). Support from the Fonds der Chemischen Industrie (RH) and the Deutsche Atemwegsliga (DAG) is also gratefully acknowledged.	PubMed Central	2024-06-05T03:55:52.501415	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548145/", "journal": "Respir Res. 2005 Jan 20; 6(1):8", "authors": [ { "first": "David A", "last": "Groneberg" }, { "first": "Rolf", "last": "Hilgenfeld" }, { "first": "Peter", "last": "Zabel" } ] }
PMC548146	Background ========== Articular cartilage is composed of extracellular matrix (ECM), the matrix-secreting chondrocyte and water, which all account for the tissue\'s characteristic rigidity as well as its flexibility. These features are necessary in order to warrant life-long survival of the cartilage tissue, especially in the joint where it has to endure pressure forces caused by movement. Chondrocytes arise from a mesenchymal progenitor during development, the same progenitor that gives rise to other mesenchymal cell types including osteoblasts, adipocytes and myocytes. Bone formation can either be endochondral, when chondrocytes mature and calcify to provide a matrix for the invading osteoprogenitors, or intramembraneous involving ossification directly from a mesenchymal ancestor. All these diverse cell types may arise from the same precursor, but are distinguished by specific morphological features and with that, a certain set of characteristic proteins including transcription factors that control their differentiation. Two chondrocyte-specific transcription factors have been identified, Sox9, a member of the SOX-family of transcription factors, and Scleraxis, a member of the basic helix-loop-helix transcription factors \[[@B1],[@B2]\]. However, most of the exclusive markers for cartilage tissue reside in the ECM. The predominant form of collagen in mature cartilage is collagen type IIB, whereas the alternatively spliced collagen IIA is found primarily during development \[[@B3]\]. Aggrecan is the major proteoglycan species in cartilage \[[@B4]\]. The transition of chondrocytes into hypertrophy is distinguished by a change in expression of Cbfa1, the osteoblast-specific transcription factor, which is also switched on during intramembraneous ossification. Distinctive to cartilage, the major collagen molecule in osseus matrix is collagen type I. In contrast, the transcription factors controlling adipogenesis are C/EBPα and PPARγ, which transactivate subsets of genes as a function of either trans-acting factor alone or requiring the co-operative effort of both \[[@B5]\]. C/EBPα is known to bind to and transactivate particularly the promotors of the SCD1, aP2 and the Glut4 genes \[[@B6],[@B7]\], all highly characteristic of the adipocyte phenotype. For decades, the treatment of degenerative cartilage and bone diseases has been a challenge for orthopaedic surgeons due to the apparent inability of cartilage and bone to repair itself. Arthritis, a degenerative joint condition, is one of the most prevalent chronic health conditions in North America. Arthritis can devastate people, but to date there is no effective therapy available and patients can only be helped by surgical joint replacement. An inherent major concern is the limited availability of autografts, which significantly reduces the choice of treatable defects. However, new approaches to cell grafting are being developed in this field: increased yields of cells are achieved by the usage of bioreactors and growth factor administration, such as TGFβ~1~and BMPs \[[@B8],[@B9]\]. Additionally, stem cells are being discovered as a new source of transplantable material. Embryonic stem cells represent a valuable source for cell transplantation since their characteristic features include an unlimited self-renewing capacity and a multilineage differentiation potential \[[@B10],[@B11]\]. In fact, ES-derived glial precursors and cardiomyocytes have been successfully transplanted, integrated and shown to be functionally active in the transplantation site \[[@B12],[@B13]\]. The yield of differentiation of ES cells into an intended lineage can be greatly enhanced by the addition of growth factors or induction substances. Whereas protocols for the differentiation of cardiomyocytes, neuronal cell types, insulin-producing cells or adipocytes from ES cells have been available for many years \[[@B14]-[@B17]\], only recently their differentiation into elements of the skeleton has been reported \[[@B18]-[@B20]\]. Our group has previously shown that vitamin D~3~forces ES cells to undergo osteogenesis \[[@B18]\]. Kramer et al. have reported in 2000 that BMP-2 pushes ES cells to the chondrogenic fate when added during days 3--5 of EB differentiation \[[@B21]\]. Those ES-derived chondrocytes possess a certain plasticity to undergo hypertrophy and calcify \[[@B22]\]. We show here, that prolonged treatment of differentiating ES cell cultures with BMP-2 in synergy with TGFβ~1~, insulin and ascorbic acid leads to improved chondrogenesis in vitro. Compared to the brief supplementation described by Kramer et al. \[[@B21]\], the expression of chondrocyte-specific marker genes was highly up-regulated while proteoglycan content revealed an increased chondrocytic yield from 7.26% to 57.03%. As described by other groups \[[@B22]\], ES-derived chondrocytes become hypertrophic and calcify. However, spontaneous calcification did not reach mineralization levels that are found in vitamin D~3~induced ES-derived osteoblasts \[[@B18]\]. Yet, supplementation of chondrocyte-cultures with β-glycerophosphate, ascorbic acid and vitamin D~3~starting at day 20 rescued the osteoblast phenotype. In many differentiations, we also observed an accumulation of lipid droplets and an up-regulation of adipocyte-specific genes. This direction towards adipocyte differentiation varied with the use of specific co-factors, suggesting that in the future, such spurious differentiation may be controlled, once the pathways involved in adipogenesis are better understood. Results ======= Characterization of chondrocyte-like cells derived from ES cells ---------------------------------------------------------------- Embryonic stem cell cultures supplemented with BMP-2, TGFβ~1~, insulin and ascorbic acid show typical morphological changes compared to the untreated cultures. Starting with the fourth week of culture, aggregates consisting of small round cells formed in the supplemented cultures, which stained positive with alcian blue (fig. [1A](#F1){ref-type="fig"}). Little alcian blue staining was seen in control cultures (fig. [1B](#F1){ref-type="fig"}). Polygonal cells, which could also be found in treated cultures, did not stain with alcian blue. Significant immunostaining for the collagen type II (COL II) protein was observed at day 32 in treated cultures corresponding to the active secretion and formation of an extracellular matrix found with chondrocytes. The COL II antibody identified the fibrillary organization of the collagen molecules in the extracellular matrix (fig. [1C](#F1){ref-type="fig"}). Chondrogenic differentiation was confirmed by positive immunostaining for adult proteoglycans (fig. [1D](#F1){ref-type="fig"}), which is detectable in the aggregates identified by alcian blue staining. The distribution was associated with the extracellular matrix similar to that found with the COL II antibody. Staining appeared to be diffuse as extracellular matrix and not individual cells are stained. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Morphology and characteristics of ES-derived chondrocytes after 32 days of culture. (A, B) Determination of proteoglycans in EBs with alcian blue in chondrocyte cultures induced with TGFβ~1~\[2 ng/ml\] and BMP-2 \[10 ng/ml\] from d3--5 of culture and with BMP-2 \[10 ng/ml\], ascorbic acid \[50 μg/ml\] and insulin \[1 μg/ml\] from day 5 onwards (A) compared to control cultures (B). Bar = 8.3 μm. (C, D) Analysis of cartilage-specific matrix proteins in 32 day old EBs induced with the same supplements as in (A, B) by means of immunohistochemistry. Staining with anti-collagen type II (C) and anti-cartilage proteoglycan (D), respectively, both visualized by a secondary AlexaFluor 488 conjugated antibody. Bar = 106 μm. (E) Concentration-dependent effect of BMP-2 on chondrocyte-specific gene expression in 32-day old EBs. Values of cultures supplemented with 10 ng/ml BMP-2 are shown compared to 2 ng/ml as used by Kramer et al. \[2000\], which was set as 1. Values represent means of three independent experiments ± standard deviation, obtained by quantitative RT-PCR analysis. \\P \< 0.01; \\\P \< 0.001. (F) Proteoglycan content of EB extracts on day 32. BMP-2 directs increased proteoglycan synthesis in both 2 ng/ml and 10 ng/ml. \P \< 0.1; \\P \< 0.01. Mean ± standard deviation, n = 3. ::: ![](1471-213X-5-1-1) ::: Dependence of gene expression patterns and proteoglycan synthesis on BMP-2 -------------------------------------------------------------------------- Quantitative RT-PCR was used to examine the variability of RNA expression of various cartilage-specific genes in response to BMP-2. Previously, chondrogenesis was induced in ES cells using 2 ng/ml BMP-2 on days three to five of EB formation, when early mesodermal markers such as Brachyuryand BMP-4are expressed \[[@B21],[@B23],[@B24]\]. To improve chondrogenesis, we investigated whether a higher concentration of BMP-2 (10 ng/ml) could increase chondrocyte-specific gene expression. Total RNA was extracted on day 32 and quantitative real-time PCR was carried out. Expression of genes of interest in cultures supplemented with 10 ng/ml BMP-2 was normalized to GAPDH expression and compared to cultures supplemented with 2 ng/ml (fig. [1E](#F1){ref-type="fig"}), which were set as 1. Expression of the small leucine-rich proteoglycans biglycan and decorin was increased 1.6 fold upon supplementation with 10 ng/ml BMP-2 (P \< 0.01). Neither link protein expression nor Sox9 or scleraxis expression were affected by the higher dosage. However, expression of aggrecan and the collagen type II isoforms A and B, which are specific for mature chondrocytes, were significantly increased compared to the low BMP-2 concentration described by Kramer et al. (P \< 0.001). The degree of chondrogenic differentiation under influence of BMP-2 was further quantified by metachromatic detection of proteoglycans (fig. [1F](#F1){ref-type="fig"}). Secreted proteoglycans were extracted on day 32 of culture with guanidine/HCl and combined with dimethylmethylenblue. Based on our measurement of aggrecan, the synthesis of proteoglycan proteins is also increased under the influence of BMP-2. BMP-2 at 2 ng/ml initiated an induction in proteoglycan synthesis of about 2 fold (P \< 0.01), whereas 10 ng/ml BMP-2 increased the proteoglycan content of EBs 2.3 fold (P \< 0.1) compared to non-treated controls. This data shows, that BMP-2 causes the induced synthesis of negatively charged extracellular matrix, characterized by proteoglycans. Additive effect of TGFβ~1~, insulin and ascorbic acid on BMP-2 induced chondrogenesis ------------------------------------------------------------------------------------- Since BMP-2 is believed to play a role in late chondrogenesis \[[@B25]\], we studied the effect of prolonged BMP-2 supplementation beyond day 5 of culture. Additionally, the anabolic effect of insulin and ascorbic acid and the influence of the growth factor TGFβ~1~on BMP-2 induced differentiation was determined quantitatively by PCR analysis at various stages throughout EB differentiation. Table [1](#T1){ref-type="table"} shows the particular combinations of medium supplements used at different culture stages of the \'hanging drop\' protocol used for differentiation, in which day 1--3 represent the hanging drop stage. During days 3--5 the resulting embryoid bodies are cultured in suspension and then plated onto tissue culture treated plastic ware on day 5. Applied concentrations were 10 ng/ml BMP-2, 2 ng/ml TGFβ~1~, 1 μg/ml insulin and 50 μg/ml ascorbic acid. Dexamethasone, which is also known to be a chondro-inducing agent \[[@B26]\], did not evoke a mentionable chondrogenic response in the D3 ES cell line (data not shown). Figure [2](#F2){ref-type="fig"} shows changes in cartilage-specific gene expression under the influence of various differentiation co-factors throughout the 32 days of culture. TGFβ~1~(combination B) evoked a 2-fold increase in collagen II expression for both splice forms. The synergistic effect of both growth factors, TGFβ~1~and BMP-2 (combination C) began to show an increased expressions of aggrecan, link protein and COL IIA compared to cultures that were treated with one supplement alone. Additional supplementation with insulin and ascorbic acid between culture days 3 and 5 (combination D) barely increased aggrecan, link protein and COL II expression, but when given from day 5 onwards enhanced both the BMP-2 and TGFβ~1~effects (supplement combinations E and H). Addition of insulin and ascorbic acid to BMP-2 induced cultures increased collagen type IIA and aggrecan expression minimally to 1.2-fold, link protein and collagen type IIB were induced 2.7- to 2.8-fold compared to BMP-2 alone. The TGFβ~1~response was increased 3- to 4-fold. When cultures were induced with BMP-2 and TGFβ~1~in suspension (d3--5) and supplemented with insulin and ascorbic acid starting on day 5 onwards (combination F), aggrecan, link protein and COL II were up-regulated 10-fold compared to controls. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Combinations of medium supplements used at different culture stages ::: -------------------------------------------------------------------------------------------------- Supplement combination Day 3--5 (hanging drops) Day 5 onwards (attached culture) ---------------------------- ------------------------------ -------------------------------------- A BMP-2 B TGFβ~1~ C BMP-2\ TGFβ~1~ D BMP-2\ TGFβ~1~\ insulin\ ascorbic acid E BMP-2 insulin\ ascorbic acid F BMP-2\ insulin\ TGFβ~1~ ascorbic acid G BMP-2\ insulin\ TGFβ~1~ ascorbic acid\ BMP-2 H TGFβ~1~ insulin\ ascorbic acid -------------------------------------------------------------------------------------------------- Applied concentrations were 10 ng/ml BMP-2, 2 ng/v ml TGFβ~1~, 1 μg/ml insulin and 50 μg/ml ascorbic acid. The end of the culture period varied with every experiment carried out. ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Changes on cartilage markers in response to various combinations of BMP-2, TGFβ~1~, insulin and ascorbic acid as outlined in table 1. (A) Diagram of expression niveaus of cartilage-specific genes of 32 day old EBs obtained by quantitative RT-PCR and standardized to GAPDH. Untreated control values were set as 1. Mean ± standard deviation, n = 3. \P \< 0.1; \\P \< 0.01; \\\P = 0.001. (B) Proteoglycan content of extracts of 32 day old EBs treated with different combinations of BMP-2, TGFβ~1~, insulin and ascorbic acid (see table 1). Ctrl = control. Means ± standard deviation, n = 3. \P \< 0.1; \\P \< 0.01; \\\P = 0.001. n.d. = not determined. ::: ![](1471-213X-5-1-2) ::: Surprisingly, when BMP-2 supplementation was maintained throughout the culture period (combination G), a dramatic increase of marker gene expression was seen. Aggrecan and COL IIB were up-regulated over 80-fold above controls and link protein and COL IIA expression was increased to 35- and 16-fold, respectively. Here, scleraxis and Sox9 expression behaved conversely. Supplement combination G showed significant decreases of scleraxis and Sox9 expression to 50 and 87% of the control, respectively (P = 0.001). Deferral of the expression of these transcription factors in favour of chondrogenic differentiation characterizes BMP-2 induced differentiation, whereupon chondrogenic processes are furthermore enhanced by TGFβ~1~, insulin and ascorbic acid. The determination of proteoglycan content was used to confirm the results obtained by quantitative RT-PCR for all supplement combinations (fig. [2B](#F2){ref-type="fig"}). TGFβ~1~alone (combination B) did not alter proteoglycan levels compared to controls, and in combination with BMP-2 decreased the proteoglycan content of EBs compared to BMP-2 alone (combination C). Insulin and ascorbic acid did not enhance the proteoglycan synthesis in combination with TGFβ~1~, but caused a 4.2-fold increase in combination with BMP-2 (combination E, P = 0.01). In agreement with aggrecan gene expression, combinations F and G generated a massive 7-fold induction of proteoglycans in EBs (P = 0.001). Genetically manipulated ES cells that express GFP under the control of chondrocyte-specific aggrecan promotor were then used to quantify chondrocyte yield using fluorescence-activated cell sorting. ES cells were differentiated along the chondrocytic lineage using BMP-2 at 2 ng/ml or supplement combination G \[d3--5: TGFβ~1~10 ng/ml, BMP-2 10 ng/ml; d3--32: BMP-2 10 ng/ml, ascorbic acid 50 μg/ml and insulin 1 μg/ml\]. Green fluorescing chondrocytes in both cultures appeared either organized in clusters or scattered as seen in figure [3A](#F3){ref-type="fig"}. The Kramer protocol gave a 7.26 percent yield of chondrocytes, which was increased to 57.03% using our modified protocol (fig. [3B](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Quantification of chondrogenic yield by flow cytometry. (A, B) Genetically modified ES cells expressing GFP from the chondrocyte-specific aggrecan promotor. Green fluorescing chondrocytes appear as clusters of cells within the remaining cell population (A), but are also scattered in the entire population starting at day 28 of differentiation (B). (C) FACS analysis of ES-derived chondrocytes sorted by their GFP expression. Differentiation with BMP-2 only \[2 ng/ml, d3--5\] produced 7.26% GFP expressing cells compared to spontaneously differentiated controls, which contained very low levels of fluorescing cells (1.54%). Prolonged BMP-2 administration together with TGFβ~1~, ascorbic acid and insulin (supplement combination G, see table 1) raised chondrocyte outcome to 57.03%. ::: ![](1471-213X-5-1-3) ::: Kinetic analysis of cartilage-specific gene expression during EB differentiation -------------------------------------------------------------------------------- The degree of chondrogenic differentiation using BMP-2, TGFβ~1~, insulin and ascorbic acid supplementation was then examined over the 35-day culture period using quantitative RT-PCR of various cartilage matrix genes (fig. [4](#F4){ref-type="fig"}). Since the extent of changes in cartilage-specific genes seemed to be dependent on acute (d 3--5) or chronic (d 3--32) application of BMP-2 (supplement combination F versus supplement combination G), the stronger induction by chronic supplementation of BMP-2 was monitored throughout the culture duration. During the first three weeks of the BMP-2 induced differentiation, only minor changes in aggrecan, link protein or collagen type II expression could be detected. Starting with day 26 however, aggrecan expression was up-regulated significantly to 14-fold over control niveaus (P \< 0.1). Reaching day 32, aggrecan, link protein and collagen type II A and B approached peak levels. Consistent with the aggrecan expression profile, link protein expression was found to be significantly up-regulated on days 24--27 (factor 6.3; P = 0.001). Increased values were detectable as early as day 16. On day 26, the quantification of the collagen type IIA and B showed 11- and 24-fold increases respectively. On day 14, splice form B had already reached an 8-fold increase over control values. In contrast, during the early phases of differentiation, between day 5 and 20, collagen type IIA expression was continuously increased 2-fold. Transcripts for biglycan and decorin were detectable throughout all stages of chondrogenic differentiation. With the beginning of the maturation phase in the fourth week of culture (day 21--28), both genes were up-regulated 1.5--2 fold. Transcription factors scleraxis and Sox9 (fig. [4B](#F4){ref-type="fig"}) showed a similar expression profile constantly over the entire culture period. This level did not change significantly during chondrogenic differentiation, but rather decreased compared to controls. Both were already transcribed in day 5 EBs, hallmarking their participation in early differentiation events, where lineage specificity is determined. Additionally, scleraxis transcripts were engaged in later phases of development between days 16--19. Sox9 expression was also increased between days 9--11 and between days 25--27. In conclusion, cultures supplemented with BMP-2, TGFβ~1~, insulin and ascorbic acid express mRNAs of a chondrogenic phenotype, whose expression was time-dependent. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Expression of cartilage-specific genes in the course of EB differentiation induced by BMP-2, TGFβ~1~, insulin and ascorbic acid (supplement combination G, table 1). (A) Aggrecan, collagen type II A and B and link protein, biglycan and decorin. (B) Scleraxis and Sox9. Results show induction factors of expression obtained by quantitative RT-PCR and normalized to GAPDH expression in comparison to corresponding spontaneously differentiated controls, which were set as 1 (mean ± standard deviation, n = 3 independent experiments, 20 EBs each). ::: ![](1471-213X-5-1-4) ::: ES-derived chondrocytes undergo hypertrophy and mineralize ---------------------------------------------------------- During embryo development endochondral ossification occurs in two steps: chondrocytes arise after mesenchymal condensation and become hypertrophic, characterized by expression of collagen type X, and calcification. To test whether this was true for the ES-derived chondrocytes generated with our protocol, we assayed for the Ca^2+^content of the cultures, a measure for mineralization (fig. [5A](#F5){ref-type="fig"}). Calcium was increased 1.2-fold in cells that were supplemented with BMP-2, TGFβ~1~, insulin and ascorbic acid compared to controls (P \< 0.01). Previously, we have described the induction of mineralization in osteoblasts derived from ES cells with vitamin D~3~, which reach maturity at day 32 of culture \[[@B18],[@B27]\]. Here, we observed that the level of mineralization was considerably higher in direct osteoblast differentiation \[11.57 mg/dl\] compared to indirect differentiation using our improved chondrocyte protocol \[3.22 mg/dl\]. We observed, however, that the level of calcium found in VD~3~induced ES-derived osteoblasts could be rescued in the chondrocytes by adding VD~3~on day 20 of differentiation, a time when chondrocytes could be morphologically identified in the cultures (11.18 mg/dl, P \< 0.001). As shown by quantitative RT-PCR, VD~3~treated ES cell derived chondrocytes could alter their expression profile to that of d32 ES cell derived osteoblasts (fig. [5B](#F5){ref-type="fig"}). However, expression of osteocalcin and bone sialoprotein was less than in VD~3~rescued chondrocyte cultures than in VD~3~osteoblast cultures. Interestingly, chondrocyte-specific genes could not be detected in VD~3~osteoblasts and waned in VD~3~rescued chondrocytes. As we have shown previously, ES-derived mineralized osteocalcin expressing osteoblasts can be identified as black appearing cells in phase contrast microscopy \[[@B18]\]. Figure [5C](#F5){ref-type="fig"} shows these black cells in the VD~3~treated cultures. No such cells are visible in control cultures or in ES-derived chondrocyte differentiations. However, by adding VD~3~back in at day 20 to the chondrocytes, mineralization can be detected, although the localization pattern is slightly different. These observations support the hypothesis that VD~3~induces intramembraneous bone formation directly from mesenchymal progenitors while BMP-2 controls endochondral bone formation. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Degree of osteogenesis in cultures treated with chondroinducing medium (BMP, supplement combination G), osteoinducing medium (VD~3~as described in ref. 18) and control ES medium (ctrl). Osteoblast phenotype and mineralization could be increased in chondrocyte cultures by adding VD~3~to chondroinducing supplements at day 20 (BMP+VD~3~). (A) Extent of mineralization as measured by Ca^2+^content. \\P \< 0.01; \\\P = 0.001. (B) RT-PCR for chondrocyte-specific and osteoblast-specific marker genes as well as Collagen X as a marker for the hypertrophic state of chondrocytes. (C) Morphology as seen in phase contrast. Black appearing cells were identified as being mineralized osteocalcin expressing osteoblasts previously \[18\]. No such cells can be seen in control (ctrl) and chondrocyte (BMP) but in osteoblast (VD~3~) and rescued osteoblast cultures (BMP+VD~3~). ::: ![](1471-213X-5-1-5) ::: It has been shown by others that the BMP-2 induced alteration in cell fate is both concentration- and time-dependent \[[@B28]\]. Lower concentrations of BMP-2 support chondrogenesis whereas higher concentrations promote osteogenesis. In ES cells, BMP-2 at concentrations of 2 ng/ml, 10 ng/ml and 100 ng/ml did not increase the expression of bone markers with the exception of osteocalcin and osteopontin, which were significantly increased as shown by quantitative RT-PCR (fig. [6](#F6){ref-type="fig"}). In combination with VD~3~(given on days 5--30) however, alkaline phosphatase and Cbfa1 were also significantly up-regulated above controls (5.4- and 4-2-fold, respectively, P \< 0.001). As we noted earlier, BMP-2 induced osteogenesis with or without VD~3~supplementation did not meet the levels that were attained by VD~3~alone, but the late addition of VD~3~on day 20 rescued bone-specific gene expression arguing for an involvement for BMP-2 in endochondral bone formation. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Dependence of genes expressed in cartilage tissue on BMP-2 with and without vitamin D~3~(VD~3~). All cultures contained β-glycerophosphate \[10 mM\] and ascorbic acid \[50 μg/ml\]. Mean ± SD of independent triplicates was quantified by qPCR and is normalized to GAPDH expression. Controls were set as 1. \\P \< 0,01; \\\P = 0,001. OCN = osteocalcin, BSP = bone sialoprotein, Cbfa1 = Core binding factor alpha, ALP = alkaline phosphatase, OPN = osteopontin, ONT = osteonectin, Col1 = Collagen type I. Concentrations of BMP-2 up to 100 ng/ml do not lead to osteoblast-specific expression levels that are reached with VD~3~only. 100 ng/ml BMP-2 plus VD~3~given together early during differentiation can significantly up-regulate osteoblast genes. However, when VD~3~is administered later (d20) in combination with BMP-2, osteoblast gene expression is almost restored. ::: ![](1471-213X-5-1-6) ::: Induction of adipocyte differentiation -------------------------------------- During treatment of EBs with various chondrocyte differentiation inducing factors, we noticed accumulation of lipid droplets, which could not be found in untreated controls. Those droplets could indeed be characterized as lipid-containing by means of Oil-Red-O staining (fig. [7A](#F7){ref-type="fig"}). Quantitative real-time PCR analysis revealed a slight up-regulation of adipocyte-specific genes (ADD1, aP2, C/EBPα, GLUT-4, LPL, PPARγ and SCD1) on day 30 in most of the supplement combinations used for induction of chondrocyte differentiation (fig. [7B,C](#F7){ref-type="fig"}). Supplement combination G, which was the most successful for inducing chondrocyte differentiation suppressed adipocyte differentiation. GLUT-4 was most up-regulated in combinations B and C, whereas combinations E and B induced a increase in LPL expression compared to our chondrocyte differentiation protocol. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Characterization of ES-derived adipocytes. (A) Oil-Red-O staining of lipid droplets in ES-derived adipocytes. Bar = 42 μm. (B, C) Influence of BMP-2 alone and in combination with TGFβ~1~, insulin and ascorbic acid (see table 1) on expression of adipocyte-specific genes. Quantitative RT-PCR was performed on 30 day old EBs. Expression of genes of interest was normalized to GAPDH and compared to untreated controls. Values show means ± standard deviations on n = 3 independent experiments. \P \< 0.1; \\P \< 0.01; \\\P = 0.001. ::: ![](1471-213X-5-1-7) ::: Discussion ========== In this study, we demonstrate an improved method for driving ES cells to a cartilaginous fate when stimulated with BMP-2 and TGFβ~1~. In the early phase of differentiation, BMP-2 operates by directing differentiation towards the cartilage lineage and acting on chondroprogenitors. During later differentiation adding mineralizing agents can trigger hypertrophy and mineralization of the ES-derived chondrocytes. In the embryo, maturation of chondrocytes in the process of endochondral ossification follows a timely regulated developmental program, whereby cellular stages can be delimitated molecularly. During ES cell differentiation into chondrocytes, developmental processes follow the same pattern, as judged by the gene expression patterns observed. Chondrocytes, osteoblasts and adipocytes are thought to arise from the same mesenchymal progenitor. Based on our previous observations around VD~3~-induced osteogenesis, we have already hypothesized that during the first 5 days of differentiation mesodermal progenitors develop, which then are susceptible to the VD~3~treatment \[[@B18]\]. Treatment of the cultures with TGFβ~1~at days 3--5 may augment the number of mesenchymal progenitors, as TGFβ~1~is thought to inhibit the proliferation of most cells, but to stimulate some mesenchymal cells such as osteoblasts and chondrocytes \[[@B8]\]. It was not surprising to see adipocytes develop in many of our cultures, as they represent another member of the TGFβ~1~promoted mesodermal lineage. The gain of adipose characteristics in culture is hallmarked by: a) the appearance of cytoplasmic lipid droplets, b) the acquisition of insulin sensitivity with regard to glucose uptake (GLUT4) and c) the expression and secretion of numerous bioactive molecules \[[@B5]\]. All of these characteristics of in vivoadipogenesis were met in the EBs treated with BMP-2, TGFβ~1~, insulin and ascorbic acid. Indeed, a future challenge for improving adipogenic cultures will be the discovery of regulatory pathways in adipogenesis. Once identified, such pathways may be antagonized in order to enhance ES differentiation into chondrocytes. During the revision of this paper, a study was published describing that the overexpression of the sox triad, namely Sox9, Sox5 and Sox6, markedly increased chondrocyte marker gene expression (collagen type 2, aggrecan) in ES cells within 3 days \[[@B29]\]. Here, TGFβ~1~, BMP-2, IGF-1 and FGF-2 had no affect on the immediate regulation of these genes. In our differentiation system, Sox9 is elevated very early during differentiation at day 5 (data not shown) underlining its role as an early controller of chondrogenesis. We have shown here that BMP-2 regulates later processes in cartilage development. Marker gene expression levels reached upon overexpression of the Sox trio do not meet the levels we have observed in this study, suggesting that Sox9 may be necessary but not sufficient do direct all progenitors to the chondrocytic lineage. Earlier this year, another study portrayed chondrogenesis in ES cells using encapsulation in alginate \[[@B26]\]. The usage of a 3D culture system led to an increase in Col II and aggrecan expression of about 1.5- to 2-fold above the regular 2D system of plating EBs. Comparing those values to the 80- to 90-fold up-regulation described here, it is clear that three-dimensional signals also need to be incorporated into chondrocyte differentiations to increase differentiation efficiency. Articular cartilage has been refractory to repair following degeneration. Despite its limited capacity to self-repair, cartilage is replete with cells capable of undergoing mitotic division \[[@B30]\]. Current hypotheses suggest that cells may be constrained by their ECM, thus preventing expansion and differentiation or that there is a limit in the bioactive molecules, which support chondrogenesis \[[@B31],[@B32]\]. Engineering bone or cartilage usually requires the handling of autologous cells. Cells are released from the ECM using collagenase and hyaluronidase. However, the small number of available progenitors, within the tissue can be problematic \[[@B33]\]. Moreover, the ability of these harvested cells to proliferate is limited in the elderly, where most degenerative joint disorders occur. The outcome of conventional surgical treatment including joint resurfacing or biological autografts has been unsatisfactory following long-term evaluation \[[@B34]\]. This failure is caused by insufficient repair resulting in the formation of mechanically inadequate resident fibrocartilage. These disappointing results and the limited therapeutic opportunities have led investigators to focus on more appropriate bioregenerative tissue engineering approaches, which could be specifically tailored for a patient\'s needs. Pluripotent ES cells are now being contemplated as a new cell source for tissue engineering since they are the most multifaceted cells amongst all stem cells. While their pluripotency offers a huge potential for cell therapies directed against a wide spectrum of degenerative diseases that are ineffectively treated by traditional approaches, it also poses challenge for controlling developmental fate in the recipient. Understanding the developmental pathways regulating ES cell differentiation, will enable the creation of a cell source that can be manipulated to correct for a particular defect. This study was designed to improve upon the number of differentiated cells needed for the in vitrodevelopment of functional cartilage as this represents a first critical step in applying ES cells clinically. Additionally, the presented in vitromodel of chondrogenesis may be useful for in vitroembryotoxicity tests \[[@B27]\] as also serve as a new tool in identifying molecular pathways that underlie chondrogenesis. Compared to other in vitromodels of chondrogenesis, this model incorporates all steps of development beginning with a pluripotent, uncommitted cell and thus might further our understanding of normally unamendable stages of development. Conclusions =========== This study was particularly designed to improve chondrocyte yields from ES cells by investigating the effect of higher BMP-2 concentrations and the complementary effects of TGFβ~1~, insulin and ascorbic acid. By examining gene expression responses and cell sorting for GFP-expressing chondrocytes, we demonstrate that using a higher concentration of BMP-2 in combination with appropriate co-factors, we can significantly enhance ES cell derived chondrogenesis compared to known protocols. In addition, we document that ES-derived chondrocytes behave naturally as they undergo hypertrophy. We show that EBs supplemented with BMP-2 also result in a small amount of adipogenesis in vitro. This observation is consistent with knowledge that adipocytes, chondrocytes and osteoblasts arise from the same mesenchymal ancestor. Accordingly, in the future, it will be necessary to understand how to discriminate these populations for cytotherapeutic applications. At this time, the presented in vitromodel allows the study of mechanisms involved in BMP-2 induced chondrogenesis, osteogenesis and adipogenesis. Methods ======= Cell culture and differentiation of embryonic stem cells -------------------------------------------------------- Cells of the mouse ES cell line D3 (American Type Culture Collection, Rockville, Maryland, USA) were kept in permanent culture as described \[[@B35],[@B18]\] with the additive Leukemia Inhibitory Factor (1000 U/ml, Gibco Life Technologies, Karlsruhe, Germany). Differentiation was initiated in hanging drops. Cells condensed to form EBs, which were transferred on day 3 into suspension culture. At day 5, EBs were plated into 24-well tissue culture primaria plates (Falcon, Heidelberg, Germany). The effects of BMP-2 \[2 or 10 ng/ml\], TGFβ~1~\[2 ng/ml\], insulin \[1 μg/ml\] and ascorbic acid \[50 μg/ml\] in various combinations on the differentiation of chondrocytes were examined. Medium was changed every second day. Alcian blue staining -------------------- Proteoglycans secreted by ES-cell derived chondrocytes were stained with Alcian blue. Cultures were fixed in 2.5% glutaraldehyde, 25 mM sodium acetate, 0.4 M MgCl~2~containing 0.05% Alcian blue for 48 h. Wash steps in 3% acetic acid, 3% acetic acid/25% ethanol and 3% acetic acid/50% ethanol reduced unspecific binding of the dye. Metachromatic test with 1.9-dimethylmethylenblue ------------------------------------------------ Proteoglycan content of differentiated cultures was determined with the DMMB-assay \[[@B36]\]. Proteoglycans were extracted in 4 M guanidin-HCl/0.05 M sodium acetate (pH 5.8) containing 100 mM 6-amino-caproic acid, 10 mM EDTA, 5 mM benzamidine/HCl, 10 mM N-ethylmaleimide, 0.4 mM pepstatin, 1 mM PMSF and 1 μg/ml soy bean trypsin inhibitor for 48 h at 4°C. Non-completely digested cells were separated from the lysate by centrifugation. Lysate was mixed with DMMB reagent (0.16% w/v DMMB in 0.2% formic acid containing 2 mg/ml sodium formate, pH 3.5) and changes in absorption were detected at 535 nm in a spectrophotometer. Concentration of proteoglycans in samples was read against a standard curve of chondroitin sulfate C. Immunofluorescence ------------------ Embryoid bodies were differentiated to the chondrocyte lineage with BMP-2, TGFβ~1~, insulin and ascorbic acid and fixed on day 32 with ice-cold methanol:aceton (7:3) at -20°C for 10 min. For staining with anti-collagen type II (Chemicon, MAB 8887) cultures were digested with pepsin for 15 min at 37°C. Staining with anti-adult proteoglycan (Chemicon MAB 2015) was performed after a 1 h digest with chondroitinase ABC at room temperature. Cells were overlaid with the appropriate dilution of the first antibodies in PBS, 10% FCS at 4°C over night. The corresponding secondary antibody, an AlexaFluor 488 goat anti-mouse IgG (H+L), F(ab\')~2~fragment (A-11006, Molecular Probes, Leiden, The Netherlands) was incubated with the cells for 2 h at room temperature. Cultures were observed in a Leica Fluovert FU fluorescent microscope (Leitz, Wetzlar) with an excitation wavelength of 495 and an emission wavelength of 519 nm. RNA isolation, RT-PCR and real-time quantitative RT-PCR ------------------------------------------------------- Total RNA was isolated from 20 EBs per probe using the RNeasy Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer\'s instructions with on-column DNase I digestion. The amount of RNA was determined using the RiboGreen™ RNA quantitation reagent and kit (Molecular Probes). 275 ng total EB RNA was used as a template for cDNA synthesis with Superscript II (Invitrogen, Paisley, Scotland) as described \[[@B27],[@B35]\] in a total reaction volume of 25 μl. 5 μl aliquots of the first strand reaction were used for amplification performed with specific primers (see table [2](#T2){ref-type="table"}). Primer sequences for osteoblast-specific genes and PCR conditions have been described previously \[[@B18]\]. PCR products were visualized on 3% agarose gels containing 0.1 μg/ml ethidium bromide. Quantitative real-time PCR analysis was performed in an ABI Prism^®^7700 Sequence Detector. The accumulation of reaction products during PCR was monitored by measuring the increase in fluorescence caused by the binding of SYBR^®^Green (Applied Biosystems, Perkin Elmer, Weiterstadt, Germany) to double-stranded DNA. Expression analysis of collagen type II A and B was done in the same PCR run with two differently labelled probes specific for either the A isoform (5\'-CGAGATCCCCTTCGGAGAGTGCTGT-3\'/VIC) or the B isoform (5\'-CCAGGATGCCCGAAAATTAGGGCCAA-3\'/FAM) \[[@B27]\]. Reaction mixtures were set up as suggested by the manufacturer containing either SYBR Green or probes for collagen type II. Following a 10 min Taq Polymerase activating step at 95°C, the reactions were cycled by denaturing at 94°C for 30 s and annealing and elongation for 30 s at the corresponding temperatures (table [2](#T2){ref-type="table"}). Target gene C~T~-values were standardized against GAPDH expression and induction of expression in treated EBs was normalized to control EBs. Primer sequences for murine GAPDH were 5\'-GCACAGTCAAGGCCGAGAAT-3\' and 5\'-GCCTTCTCCATGGTGGTGAA-3\' (T~a~= 60°C). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Sequences and annealing temperatures for the primer sets used in RT-PCR ::: --------------------------------------------------------------------------- Gene Primer sequence T~m~in °C -------------------------- -------------------------------- --------------- Chondrocyte-specific Aggrecan 5\'-GATCTGGCATGAGAGAGGCG-3\'\ 61 5\'-GCCACGGTGCCCTTTTTAC-3\' Collagen type II 5\'-GCTGCTGACGCTGCTCATC-3\'\ 60 5\'-GGTTCTCCTTTCTGCCCCTT-3\' Collagen type X 5\'-CAAGCCAGGCTATGGAAGTC-3\'\ 60 5\'-AGCTGGGCCAATATCTCCTT-3\' Link protein 5\'-TTCTGGGCTATGACCGCTG-3\'\ 60 5\'-AGCGCCTTCTTGGTCGAGA-3\' Biglycan 5\'-CATGACAACCGTATCCGCAA-3\'\ 60 5\'-ATTCCCGCCCATCTCAATG-3\' Decorin 5\'-ATGACCCTGACAATCCCCTG-3\'\ 60 5\'-CCCAGATCAGAACACTGCACC-3\' Scleraxis 5\'-GGACCGCAAGCTCTCCAAG-3\'\ 62 5\'-ACCCACCAGCAGCACATTG-3\' Sox9 5\'-GCAGACCAGTACCCGCATCT-3\'\ 62 5\'-CTCGCTCTCGTTCAGCAGC-3\' Adipocyte-specific ADD1 5\'-CAGTGACTCTGAGCCCGACA-3\'\ 61 5\'-ATGCCTCGGCTATGTGAAGG-3\' PPARγ 5\'-ATCATCTACACGATGCTGGCC-3\'\ 59 5\'-CTCCCTGGTCATGAATCCTTG-3\' SCD1 5\'-ACACCATGGCGTTCCAAAAT-3\'\ 61 5\'-CGGCGTGTGTTTCTGAGAACT-3\' C/EBPα 5\'-CGCAAGAGCCGAGATAAAGC-3\'\ 60 5\'-GCGGTCATTGTCACTGGTCA-3\' GLUT-4 5\'-ATGGCTGTCGCTGGTTTCTC-3\'\ 59 5\'-ACCCATAGCATCCGCAACAT-3\' AP2 5\'-TGATGCCTTTGTGGGAACCT-3\'\ 58 5\'-GCAAAGCCCACTCCCACTT-3\' acrp30 5\'-AAGAAGGACAAGGCCGTTCTC-3\'\ 60 5\'-GAGGAGCACAGAGCCAGAGG-3\' LPL 5\'-CCAATGGAGGCACTTTCCAG-3\'\ 60 5\'-CCACGTCTCCGAGTCCTCTC-3\' --------------------------------------------------------------------------- Forward primer sequence is depicted from the 5\' to the 3\' end followed by the reverse primer. The appropriate annealing temperature for each set is given as T~m~in °C. ::: Quantification of chondrocyte yield by FACS ------------------------------------------- The GFP expressing plasmid pEGFP-1 (Clontech) under the control of the Aggrecan promotor (kind gift of John R. Matyas) was stably transfected into D3 ES cells using Invitrogen\'s Effectene system followed by neomycin selection. Genomic integration of the reporter was analyzed by RT-PCR with specific GFP primers (data not shown). ES cells were differentiated along the chondrocyte lineage, trypsinized into a single cell suspension and subjected to fluorescence-activated cell sorting on day 32 using a FACS Calibur instrument and the CellQuest software from Becton Dickinson (Germany). Ten thousand events were registered per sample and analysis of whole cells was performed using appropriate scatter gates to avoid cellular debris and aggregates. Oil-Red-O staining ------------------ Cells were washed with PBS and without any fixation directly overlaid with Oil-red-O working solution (0.18% Oil-Red-O dye/60% propanol) After a staining period of 15 minutes, ES cell cultures were rinsed in distilled water until desired colour was achieved. Statistical analysis -------------------- Data analysis was performed with ONE-WAY ANOVA on n = 3 experiments using SigmaStat version 2.03 (SPSS Inc., San Rafael, CA, USA). List of abbreviations ===================== ADD1 Adipocyte determination- and differentiation-dependent factor 1 aP2 Fatty acid-binding protein BMP Bone Morphogenetic Protein Cbfa1 Core binding factor alpha 1 C/EBP CCAAT/enhancer-binding protein Col Collagen DMMB Dimethylmethylenblue ECM extracellular matrix ES embryonic stem EB embryoid body FCS Fetal calf serum FGF Fibroblast growth factor GAPDH Glyceraldehyde-3-phosphate dehydrogenase GLUT4 Glucose transporter 4 IGF Insulin-like growth factor PPAR Peroxisome proliferator-activated receptor SCD Steroyl CoA desaturase Sox Sry-related high mobility group box TGFβ Transforming Growth Factor beta VD~3~Vitamin D~3~ Authors\' contributions ======================= NZN carried out cell culture, biochemical and molecular studies and drafted the manuscript. GK, DER and HJA participated in its design and coordination. All authors read and approved the final manuscript. Acknowledgements ================ This study was funded in part by the German Ministry of Education, Science, Research and Technology (BMBF) grant no. 0312314. NZN is supported by a post-doctoral fellowship from the Alberta Heritage Foundation of Medical Research (AHFMR). DER is an AHFMR Senior Scholar. The authors are thankful to the FACS core facility of the Faculty of Medicine for performing flow cytometry and to Dr. John R. Matyas for providing the Aggrecan-GFP construct. We furthermore appreciate the technical assistance of Lesley Davis.	PubMed Central	2024-06-05T03:55:52.505934	2005-1-26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548146/", "journal": "BMC Dev Biol. 2005 Jan 26; 5:1", "authors": [ { "first": "Nicole I", "last": "zur Nieden" }, { "first": "Grazyna", "last": "Kempka" }, { "first": "Derrick E", "last": "Rancourt" }, { "first": "Hans-Jürgen", "last": "Ahr" } ] }
PMC548147	Background ========== In recent years, numerous publications -- over 50 pages of them in current listings of the bibliographic search engine Medline -- have detailed changes in biological systems and organisms that appear associated with the climatic changes commonly referred to as global warming (e.g., \[[@B1]-[@B5]\], dealing with observed or impending shifts in the habitats of various organisms). Few studies, however, have documented long-term changes in the genetic structureof species populations on a regional scale, necessary for understanding the microevolutionary consequences of global change \[[@B6]\]. In the genus Drosophila, comprised of over 1500 species \[[@B7]\], only four species have received such attention. While some long term increases have been documented in the frequencies of chromosomal gene arrangements Standard (ST), Tree Line (TL), and Pike\'s Peak (PP) in Drosophila pseudoobscurain the western United States and Canada over ca 40 years \[[@B8]-[@B10]\] and of several arrangements in Drosophila melanogasterin Japan \[[@B11]\], little is known of the causes for these changes. Orengo and Prevosti \[[@B12]\] first suggested that climatic warming was responsible for long-term temporal changes in the frequencies of certain gene arrangements in European populations of D. subobscura. Later, Rodriguez-Trelles and co-workers \[[@B13]-[@B15]\] and Sole\', [et al]{.underline}. \[[@B16]\] convincingly demonstrated that climatic changes, including global warming, were likely the driving force of microevolutionary changes in these populations. Recently, significant changes have also been documented in the chromosomal variation of D. robustaSturtevant, some of them possibly attributable to global warming \[[@B17]-[@B19]\]. Here we describe additional data that underscore the variety of historical changes being experienced by populations of this species. Clearly, the chromosomal polymorphisms in D. robustaare also dynamic, and when compared with the considerable geographical and experimental data available for this species collected over the last 60 years, strongly implicate regional climatic changes as a cause for these temporal frequency shifts. Results ======= Regional patterns of climate change were revealed from ANCOVA analysis of temperature and precipitation data from 1945 -- 2003. No long term tends were detected for precipitation except for a significant increase in Central Park, NY (data available from the authors). Significant long-term temperature changes were apparent at all six 2003 study sites (Fig. [1](#F1){ref-type="fig"}; plus Philadelphia, last studied in 2002) for the three available temperature indicators: average monthly minimum temperature (MINTMP), average monthly temperature (AVETMP), and average maximum monthly temperature (MAXTMP; all 3 ANCOVA models, P \< 0.0001). AVTEMPand MAXTMPvaried significantly from site to site in different years, but MINTMPsignificantly increased over the 58-year period (P \< 0.0001) with no heterogeneity among sites. Thus, the most consistent change in climate across sites in this study was due to significant increases in minimum monthly temperatures. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Map of the eastern United States showing the locations of collecting sites in this study. This modified map is used by permission of the University of Texas Libraries, the University of Texas at Austin. ::: ![](1471-2148-5-4-1) ::: Evidence for site-specific temperature increases from 1945 -- 2003 was evident for these three temperature indicators (Fig. [2](#F2){ref-type="fig"}). Five of the seven sites showed linear or curvilinear increases in MINTMP, usually starting in the 1970\'s and extending until 2000. In contrast, both Fayetteville and Englewood, NJ have experienced significant temporal decreases in MAXTMP(with a concurrent significant increase in MINTMPin Fayetteville). Causes for these decreases are obscure, but in New Jersey may be due to the exaggerated temperature fluctuations in the early 1990\'s (as well as missing data for 1992 and 1993, Fig. [2](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Temperature data for each site in this study plotted from 1945 to 2003. Regression lines and equations are shown only for statistically significant trends in mean maximum monthly temperature (MAXTMP), mean monthly temperature (AVTMP), and mean minimum monthly temperature (MINTMP) at each site. Significance and sign of the regression coefficients are indicated (\P \< 0.05, \\* P \< 0.01, \\\* P \< 0.001, \\\\ P \< 0.0001). Y axes are in degrees Centigrade. Replicate data plotted for Fayetteville and New Jersey show incomplete secondary weather station data at sites closer to the locations where flies were collected. See text for details. ::: ![](1471-2148-5-4-2) ::: Concurrent with these temperature shifts, chromosome elements in populations of D. robustashow systematic temporal frequency changes (all frequency data are available from the authors). X chromosome combinations 1S and S1 show substantial frequency changes with time, with S1 doing so in a site-specific manner (Fig. [3](#F3){ref-type="fig"}). Frequencies of combination 1S have decreased over time, and are negatively correlated with increasing temperatures (Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}). Increases in S1 are only marginally correlated with temperature, but this may be due to other factors, including its low frequency in eastern populations (Fig. [3](#F3){ref-type="fig"}). Gene arrangements 2L-1 and 3R-1 have also increased in frequency since 1945 (Fig. [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}; Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}), and the changes are significantly site-specific as indicated by the significant year × site interactions. While temporal changes in 2L-1 and 3R-1 are correlated (r = 0.419, P = 0.0016, n = 54), only those of 3R-1 are significantly correlated with increasing temperatures (Fig. [5](#F5){ref-type="fig"}; Table [2](#T2){ref-type="table"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Frequencies (in percent) of D. robusta X-chromosome arrangement combination S1in the seven study localities, including Philadelphia \[18\]. When frequencies across years were statistically homogeneous, they were combined within each locality. Y axes for eastern populations reflect the much lower frequencies for S1 in that part of the species range. ::: ![](1471-2148-5-4-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### ANCOVA results for temporal changes in the frequencies of several X chromosome arrangement combinations and autosomal inversions across the seven collecting sites in this study. r^2^is an estimate of the proportion of the total variance explained by the model used. ::: X chromosome combination 1S ------------ ----------------------------- ------------- --------- ----------- ------- Source df Type III SS F Value Pr \> F r^2^ Model 13 1.151 50.31 \< 0.0001 0.942 Year 1 0.014 5.97 0.019 Site 6 0.207 14.93 \< 0.0001 Year\Site 6 0.199 14.35 \< 0.0001 X chromosome combination S1 Source df Type III SS F Value Pr \> F r^2^ Model 13 2.589 50.94 \< 0.0001 0.943 Year 1 0.000 0.02 0.894 Site 6 0.206 8.79 \< 0.0001 Year\Site 6 0.197 8.39 \< 0.0001 Gene arrangement 2L-1 Source df Type III SS F Value Pr \> F r^2^ Model 13 1.317 20.55 \< 0.0001 0.870 Year 1 0.038 7.66 0.0085 Site 6 0.215 7.25 \< 0.0001 Year\Site 6 0.216 7.29 \< 0.0001 Gene arrangement 3R-1 Source df Type III SS F Value Pr \> F r^2^ Model 13 2.054 54.09 \< 0.0001 0.946 Year 1 0.043 14.76 0.0004 Site 6 0.057 3.28 0.0102 Year\Site 6 0.060 3.40 0.0084 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Pearson product-moment correlations between average monthly temperature and frequencies of several X chromosome arrangement combinations and autosomal arrangements for all seven localities studied. n = 54 for all tests. ::: 1S S1 2L-1 3R-1 --------------------------------------------- ----------- ----------- ----------- ----------- Average temperature for month of collection \- 0.341 0.250 0.195 0.336 P = 0.012 P = 0.071 P = 0.163 P = 0.014 ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Frequencies (in percent) of D. robustagene arrangement 2L-1 in the seven study localities, including Philadelphia \[18\]. See Fig. 3 for details. ::: ![](1471-2148-5-4-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Frequencies (in percent) of D. robustagene arrangement 3R-1 in the seven study localities, including Philadelphia \[18\]. See Fig. 3 for details. ::: ![](1471-2148-5-4-5) ::: The most consistent historical changes have been gains in the frequency of arrangement 3R-1 with concomitant decreases of its allelic form, 3R (Figure [5](#F5){ref-type="fig"}). Coupled with the clinal tendency of 3R-1, varying between 100% in the southernmost latitudes and zero in the northernmost \[[@B20]\], its similar directional increases of recent years in at least four states, some far apart, and concordance with long term temperature increases indicates that 3R-1 is responding to regional climatic shifts. The frequency of arrangement 2L-1 increased directionally in most of the same localities as 3R-1 (Figure [4](#F4){ref-type="fig"}). The frequency of 2L-2, which is also more common in the south than in the north, has been decreasing steadily, almost to the point of extinction, in the four northern 2003 populations. This is probably not a temperature effect, however, as in the past 2L-2 has been irregularly distributed across the southern states, rarely rising above frequencies of 20 percent, without evidence of a latitudinal cline \[[@B20]-[@B22]\]. Like 2L-1 and 3R-1, the frequency of gene arrangement XL (the sum of data for SS, S1, and S2), tends to be higher in southern populations than in northern ones \[[@B20]\]. Except for a few deviations in small samples, it has increased steadily -- with concomitant decreases in northern arrangement XL-1 -- in all the localities studied except Olivette (where it is already about 95%) and Iowa. At Englewood, for example, it rose from about 40% in 1948 to an average of 78% in 2000--2003, that of northern arrangement XL-1 falling from about 60% to about 22% in the same period. Frequencies of X chromosome combination 1S (XL-1.XR) are clearly consistent with this pattern (Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}). Discussion ========== Studies of Drosophilainversion polymorphisms have now provided historical insights into environmental change. On different continents, temporal genetic changes correlated with increasing temperatures in populations of D. subobscura\[[@B12]-[@B16]\] and D. robusta\[\[[@B17],[@B18]\], this study\] bear the imprint of large scale climate change. For D. robusta, not all temporal changes were consistent with this hypothesis. In the course of comparing plant growth in rural and urban areas in relation to ozone exposure, rural areas tended to have lower average temperatures than urban ones \[[@B23]\]; this may explain the smaller (or lack of) change in 3R-1 frequencies in relatively rural Fayetteville and Iowa City as compared to the more urban locales. Also, there are fewer data available for these sites collected over time spans long enough to document temporal trends. Another possibility is that the climatic changes have not been of equal intensity in all localities, as indicated by regional increases and decreases in local temperatures (Fig. [2](#F2){ref-type="fig"}) suggested by the site by year interactions for AVTMPand MAXTMPin the ANCOVA analyses. Carson \[[@B24]\] noted that the chromosomal variation in populations of Drosophila robustaat Olivette, Missouri over a 10-year period was characterized by \"extraordinary stability,\" wherein \"certain frequencies may shift significantly as compared with the previous year, but in every case the observed frequencies approximate some previously observed level.\" This pattern continued until at least 1967 (Fig. [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}). Temperature records from this period seem rather stable before significantly increasing through 2000 (AVTMPand MINTMPsecond order regression slopes are both positive; Fig [2](#F2){ref-type="fig"}). Similar stability, frequency changes of less than 10 percent, has also been recorded near Blacksburg, Virginia from 1950 to 1962 (M. Levitan, unpublished data) and in northeastern New Jersey from 1948 until at least 1975 (Fig. [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}). By contrast, every population sampled in 2003 evidenced at least one significant change of chromosomal polymorphism frequency compared to the numbers in the same area ten or more years previously, with indication that the changes in at least three are part of similar, if not identical, directional historical processes. Therefore, documentation of temporal genetic changes in these populations requires at least 10 to 20 years of comparative data. Influences of natural selection due to ambient temperature variation on frequencies of these gene arrangements and X chromosome associations have been demonstrated in laboratory experiments \[[@B24],[@B25]\], and inferences from latitudinal and multiple elevational clines \[[@B26]-[@B31]\]. Frequencies of arrangements XL-1 and 2L-3 are clearly associated with cooler temperatures at higher elevations and latitudes, and 2L-1 increases in frequency in the laboratory under warmer temperatures. Carriers of 2L-3 have shorter egg to adult development times expressed in cooler temperatures, explaining increases in this gene arrangement with elevation and latitude \[[@B26]\]. Evidence of strong natural selection maintaining earlier observed genetic stability has also come from perturbation experiments in the wild \[[@B20]\]. At four time periods, large numbers of flies carrying X-chromosome combinations S2and 22, autosomal combination 2L-1.2R-1 (11), and 3R-1 from South Carolina, Alabama, and Mississippi were released in midsummer in the Englewood, NJ woods. In other years the released flies carried XL-1, 2L-3, and 3R from Minnesota and Michigan. Despite evidence of hybridization of the introduced flies with the local population, in each case the following spring saw return to frequencies of the previous year. According to Wright \[[@B32]\], \"The alternative to natural selection in a changing environment is, as noted by Dobzhansky \[[@B33]\], the emergence of superior genetic systems\" in the gene arrangements that have been increasing in frequency. He envisioned this happening in one population by recombination in inversion homozygotes, with the new adaptive gene complex in one or a few flies spreading to other localities by \"occasional very long dispersion by the wind.\" He conceded that such a hypothesis would be most satisfactory for cases where a very rare inversion suddenly started increasing, such as the rises of TLand PPof D. pseudoobscuramentioned above, since high frequencies of inversion homozygotes would undermine the structural stability of the new adaptive complex. The sudden rises of D. robustaX-chromosome combination S1in New York and New Jersey and of S2in Missouri could be cases in point, but it would not explain the near doubling of S1in Arkansas from a 35% base between 2001 and 2003 nor the three most consistent directional changes, those of 3R-1, 2L-1, and XL (Figures [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}). Indeed, the nearly simultaneous changes in places as far apart as New York and Allentown, Pennsylvania, let alone New York and Missouri, would be very unlikely to depend on chance wind-blown dispersions. Here, causes for systematic frequency changes in widespread populations can be inferred to be a result of common environmental causes given the large amount of background data available for D. robusta. When compared to the inversion frequency data collected over 60 years from more than 150 natural populations \[[@B20],[@B21]\] and results from numerous laboratory experiments, temporal frequency shifts across the range of D. robustastrongly suggest temperature variation as a likely mechanism driving microevolutionary change \[[@B17],[@B18]\]. Such regional frequency shifts suggest a common response to temperature fluctuations or causes correlated with them, whether due to regional climatic changes or global warming. Conclusions =========== Although direct evidence for climatic change has been accumulating for many years, its consequences for causing evolutionary changes have only recently been observed. Chromosome polymorphisms in Drosophilaspecies have been historically important genetic systems for understanding mechanisms of evolutionary change, and have now been studied long enough to begin revealing widespread, systematic temporal frequency shifts in response to environmental change. These polymorphisms thus represent excellent indicators of future climatic shifts. Methods ======= There are 14 commonly encountered gene arrangements segregating in natural populations of D. robustalocated on five of the six arms of the 3 major chromosomes. The \"Standard\" arrangements were labeled for the respective chromosome arms: XL, XR, 2L, etc. Others were named in order of their discovery, e.g., XL-1, XL-2, XR-1, 2L-1 \[described and configured in \[[@B20],[@B24]\]\]. The Standard arrangement of each arm was dubbed \"S,\" and the other arrangements are referred to by the Arabic numerals in their names \[[@B34]\]. A fly with karyotype XL/XL-1, XR/XR-2, for example, would be S/1, S/2 in this notation. Depending on the linkage combination of the arrangements, it is also either SS/12or S2/1S. Linkage relationships are inferred from karyotypic analyses of adult males and females \[[@B35]\]. Female D. robusta, unlike many other drosophilids, quickly deplete stored sperm in the absence of remating so that wild-caught females can be despermed by repeated transfers to fresh food vials and then crossed to homokaryotypic males in a controlled fashion. Karyotypes of at least 6 larvae from these test crosses were prepared in order to infer the linkage combination of X chromosome gene arrangements. Salivary gland smears from larvae derived from matings in the wild, so-called \"egg sample\" data, were included when collected females did not survive the desperming transfers. This report compares data obtained in 2003 to earlier summer collections at a number of geographically isolated populations going back in some cases to 1946: Olivette, a suburb of St. Louis, Missouri; woods alongside Route 4 in Englewood, New Jersey; Trexler Memorial Park on the western outskirts of Allentown, Pennsylvania; the North Woods of Central Park in New York City; Fayetteville, Arkansas; and woods along the Iowa River at Iowa City, Iowa. The significance of year-to-year differences in frequency of each chromosome or chromosome arm obtained prior to 2003 was determined by G-tests \[[@B36]\]. Chronologically contiguous results that proved statistically homogeneous are combined in the figures. Individual X- and second-chromosome arrangements could not be tested in this way due to problems of independence. Analysis of meteorological data =============================== Temperature and precipitation data were obtained online from the National Climatic Data Center <http://www.ncdc.noaa.gov/oa/ncdc.html> for stations with complete records extending from 1945--2003 nearest to each site. Continuous data for Fayetteville and Englewood were not available for the closest weather stations, so records from those stations with complete records were used. Correlations between temperatures from these stations were highly significant: Drake Field and the Agricultural Experiment Station for Fayetteville (r = 0.48 -- 0.90, P \< 0.01), Little Falls and Ridgefield for Englewood (r = 0.84 -- 0.98, P \< 0.0001); see Fig. [2](#F2){ref-type="fig"}. Analysis of covariance in PROC GLM \[[@B36]\] was used to test for overall significance of temperature trends and chromosome frequency changes across sites, and to evaluate regional patterns of temperature and precipitation change. Polynomial regression analyses of temperature and precipitation data with time were performed with PROC REG \[[@B37]\]. In all cases, linear or second order polynomial regression explained the most variation for a particular model. Pearson product-moment correlations between arcsin transformed chromosome frequencies and the average temperature of the month for each collection were calculated with PROC CORR \[[@B37]\]. Non-parametric correlation analyses produced equivalent results. We also assessed correlations with temperatures for the month prior to collection, and the average temperature of the 3 months prior to collection: none were significant. Authors\' contributions ======================= ML collected the Allentown, New York, and New Jersey flies and carried out the chromosomal analyses. WJE contributed the Fayetteville flies. Both authors contributed to planning the study, analyzing the data, and writing and revising the manuscript. Acknowledgments =============== The authors thank the 1996 M. M. Kaplan Family Foundation for grant GCO 00-1223 to ML and NSF grant DEB-0211125 to WJE; A. R. Templeton for the 2002 and 2003 collections in Missouri; B. McAllister for the 2003 collections in Iowa; M. M. Kaplan for advice and comments on the manuscript; S. Beaupré for statistical advice; N. Maj for computer and graphics assistance; and S. Hanada, N. Khanna, S. Gmuca, and K. Chan, sponsored by the New York Academy of Sciences, for assistance in the analysis of the 2003 collections.	PubMed Central	2024-06-05T03:55:52.509995	2005-1-6	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548147/", "journal": "BMC Evol Biol. 2005 Jan 6; 5:4", "authors": [ { "first": "Max", "last": "Levitan" }, { "first": "William J", "last": "Etges" } ] }
PMC548148	Background ========== Positional clustering of co-expressed genes is mainly due to operons in prokaryotes. Genes located within the same operon are transcribed together and thus co-regulated. In general, operons are missing from eukaryotes. Eukaryotic genes appear to be transcribed individually and are thought to be scattered on chromosomes without apparent organization by function or positional expression except for tandem duplication. However, positional clustering has recently been reported in several eukaryotes such as Saccharomyces cerevisiae\[[@B1],[@B23]\], Drosophila melanogaster\[[@B4],[@B5]\], Caenorhabditis elegans\[[@B2],[@B3],[@B7]\], and Homo sapiens\[[@B6],[@B8]\]. Cho et al. first showed clustering of co-expressed yeast genes on a genome-scale. They found that about 25% of genes with cell-cycle-dependent expression pattern were adjacent to those induced in the same phase of the cell cycle \[[@B23]\]. This fact was also reported by Cohen et al. \[[@B1]\]. Cohen et al. computationally analyzed the whole-genome gene expression using the same dataset. They consider two ORFs to be adjacent if they occur on the same chromosome and if there are no other ORFs or functional elements such as Ty element, long terminal repeats, tRNAs, snRNAs between them. They observed that adjacent or nearby non-adjacent pairs of genes showed correlated expression independent of their orientation in the yeast genome using chromosome correlation maps that display correlations between the expression patterns of genes on the same chromosome. In addition, they also showed that genes with similar functions tend to occur in adjacent positions along the chromosomes. Spellman et al. \[[@B4]\] found that numerous clusters that span 10 to 30 physically adjacent genes share strikingly similar expression profile in Drosophila melanogasterand these clustered genes account for over 20% of the total assayed genes. By mapping Expressed Sequence Tags (EST) back to the Drosophilagenome, Boutanaev et al. \[[@B5]\] observed almost one thirds of 1661 testis-specific genes are clustered on chromosomes. Similarly, positional clustering of co-expressed genes was also reported in Caenorhabditis elegans. Although operon and tandem duplication are major mechanisms for the observed co-expression gene clusters in the worm, co-expression gene clustering is still evident after exclusion of these two causes \[[@B3],[@B7]\]. Clusters of highly co-expressed genes were also revealed in the human and other mammalian genomes \[[@B6],[@B8],[@B5],[@B12]\]. Most of these studies focused on a set of tissue-specific genes that are highly expressed. For example, by analyzing the expression profiles of genes in carious tissues, Caron et al. \[[@B8]\] showed that highly expressed carious genes tend to cluster in large domains, called RIDGEs (Region of IncreaseD Gene Expression). However, these RIDGES seem to consist mainly of housekeeping genes \[[@B6]\]. Gabrielsson et al. \[[@B9]\] performed a microarray analysis of genes expressed in the human adipose tissue. By mapping these genes back to the human genome, they observed clusters of adipose tissue specific genes. Dempsey et al. \[[@B10]\] investigated genes from chromosomes 21 and 22 expressed in the human cardio-vascular system. Using EST (Expressed Sequence Tag), they showed positional clustering of these genes. These studies suggest that clusters of tissue-specific genes do exist, and might be more frequent than initially thought. Here we performed a genome-wide analysis of testis-specific genes in the mouse genome using a method similar to one proposed in \[[@B5]\]. We used a publicly available EST (Expressed Sequence Tag) database to identify differentially expressed genes. After mapping testis-expressed ESTs back to the genome, we obtained the testis-expressed gene expression profile by assembling overlapped ESTs. In the same way, we got embryo-expressed gene expression profile. Testis-specific genes were generated by subtracting embryo-expressed gene expression profile from testes-expressed one. From the testis-specific gene expression profile, clustered genes were observed. To further investigate testis-specific gene clusters in the mouse genome, we used the full-length cDNA sequences publicly available in the Fantom 2.1.1 database to identify differently expressed genes. After mapping 60,770 cDNA sequences back to the genome, we obtained the gene expression profile by assembling overlapping cDNA sequences into a single gene model. Testis-specific genes were selected by using only sequences from libraries where the tissue type included the keyword \"testis\" and not the keyword \"embryo\". Again, our statistical analysis shows the existence of testis-specific gene clusters along the mouse chromosomes even after removal of tandem duplicated genes. This suggests that considerable clusters of tissue-specific genes do exist in higher eukaryotes. Our results provide further support for the hypothesis that chromatin domain model may be involved in the regulation of gene expression in higher eukaryotes Results ======= The testis-expressed profile is a global view of gene expression and provides a foundation for understanding how the tissue functions in molecular level. We assume that most of the testis-specific genes are expressed after embryo phases in mouse. Therefore, we selected only testis genes models that exclude any expression in embryo for the construction of the testis-specific gene profile, which reveals positional clustering along chromosomes. Here, we analyzed the cluster distribution on all the chromosomes except for chromosome Y. (In Y chromosome, we only identified 12 testis-specific genes and 7 other genes.) Based on our study, we obtain the following observations. a. Density of testis-specific genes ----------------------------------- The testis-specific gene expression profile along the mouse genome shows that testis-specific genes are distributed on all the chromosomes with different gene density. The ratio (R) of the number of the observed testis-specific genes (O) to the expected number (E) is used to measure the gene density, where E is calculated according to the chromosome\'s size on assumption that the testis-specific genes are uniformly distributed throughout the genome. The lowest gene density appears on chromosomes 1 (R = 0.834), 15 (R = 0.893), 16 (0.855), and 18 (R = 0.767) except for sex chromosomes. But, chromosomes 9 (R = 1.145), 11 (R = 1.374), 14 (R = 1.207) and 19 (R = 1.48) have rather high gene density. Within a chromosome, the distribution of testes-expressed genes also shows diverse density. b. Genomic location is highly correlated with testis-specific gene expression ----------------------------------------------------------------------------- We observed numerous groups of physically neighboring testis-specific genes that shared strikingly similar expression levels. In eukaryotes, it is clear that the activity of a promoter can be modified by transcription factors binding to DNA sequence, which are located from hundreds to thousands of base pairs from the promoter. Recently, increasing evidences show that genes may also be regulated in a group. For example, when transgenes are removed from their local environment and reinserted elsewhere in the same genome, they tend to show variant levels of expression \[[@B13]\]. More commonly, most eukaryotic chromosomes exhibit transcriptionally active (euchromatin) or inactive (heterochromatin) regions. In animals, heterochromatin is typically found near the centromere and other regions of low sequence complexity. Since RIDGEs do exist and neighboring genes show similar expression level, we conclude that genomic location has impact on gene expression. c. A large proportion of testis-specific genes are in adjacent pairs -------------------------------------------------------------------- We consider two genes are adjacent if there are no other genes between them on the same chromosome. We observed that 1170 testis-specific genes appear in adjacent pairs, which account for 31.8% of the testis-specific genes after removal of tandemly duplicated genes. There are 350 adjacent pairs, 86 adjacent triplets, 23 adjacent quadruplets, and 4 quintuplets (see Table [1](#T1){ref-type="table"} for details), significantly more than would be expected by chance. In fact, the number of observed adjacent gene clusters (including singletons) is smaller than the expected number minus the standard deviation for every chromosome other than chromosomes 15 and 19. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Chromosomal distribution of adjacent testis-specific genes after removal of tandemly duplicated genes. ::: Size Cluster distributions of adjacent testis-specific genes on chromosomes ------- ------------------------------------------------------------------------ ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- --- ---- ---- --- --- ---- ----- 2 26 22 21 26 29 22 17 20 14 24 19 18 18 20 6 15 13 9 9 12 350 3 2 5 3 6 6 4 4 4 12 4 8 1 2 5 5 4 3 3 1 4 86 4 2 1 1 2 2 3 1 2 1 2 3 2 1 23 5 1 1 1 1 4 ::: The genes in the quintuplets are probably worthy to be investigated biologically. There is a quintuplet on each of chromosomes 6, 12, 14 and X. The quintuplet on chromosome 6 is located in a 400 kb segment starting at 144691608 and ending at 145141155; the one on chromosome 12 is located in a 600 K segment starting at 101700908 and ending at 102297618; the one on chromosome 14 is located in a 700 K segment starting at 62802300 and ending at 63500814. However, the one on chromosome X seems less significant, which is located in a 3719 K segment starting at 113253694. d. Testis-specific genes show obvious clustering ------------------------------------------------ We evaluated the positional clustering of testis-specific genes using a model called the neighborhood model. We define clusters using the distance between two neighboring testis-specific genes along the chromosome. Two testis-specific genes are in a cluster if there is a series of testis-specific genes between them such that the distance between two neighboring genes in the series is less than a specified distance (D). We obtained 3679 testis-specific genes on all the chromosomes except for Y after removal of tandemly duplicated genes. We conducted statistical analysis with 4 different values of D: 25 K, 50 K, 75 K, and 100 K (see Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Distribution of testis-specific gene clusters after removal of tandemly duplicated genes. Here only the numbers of clusters having at least 2 genes on all the chromosomes except for chromosome Y are given. ::: Size Cluster distributions on chromosomes (distance = 25 K) ------- --------------------------------------------------------- ------- ------- ------- ------- ------- ------- ------- ------- -------- -------- -------- -------- -------- -------- -------- -------- -------- -------- ------- ------- 2 18 22 17 22 23 13 17 17 18 22 16 12 15 15 6 8 7 7 7 7 288 3 1 1 2 1 2 3 1 2 1 3 1 21 4 1 1 2 5 6 Size Cluster distributions on chromosomes (distance = 50 K) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X Total 2 26 30 22 32 34 20 21 22 19 31 22 16 16 20 15 14 12 8 6 6 392 3 2 5 1 2 6 1 8 3 6 3 3 3 4 3 2 1 2 1 56 4 1 5 2 1 2 2 1 1 1 1 2 19 5 1 1 6 Size Cluster distributions on chromosomes (distance = 75 K) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X Total 2 27 28 23 37 47 29 24 29 17 34 29 17 18 24 16 16 19 11 9 10 454 3 1 4 4 5 4 5 9 2 9 2 7 3 3 6 5 4 1 1 3 1 79 4 2 6 3 1 3 1 2 4 1 1 1 1 1 1 28 5 1 2 1 4 6 1 1 Size Cluster distributions on chromosomes (distance = 100 K) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 X Total 2 32 33 24 31 42 33 28 27 19 39 23 21 21 25 16 15 24 9 9 12 463 3 2 6 4 7 8 6 12 8 12 3 11 2 4 7 4 6 2 3 6 1 114 4 3 6 5 3 1 1 3 1 3 7 2 2 2 2 1 1 2 1 46 5 1 1 1 2 1 1 7 6 1 1 2 ::: When D = 25 K, we obtained 288 two-gene, 21 three-gene, and 2 four-gene clusters. In total, 18% of the testis-specific genes are contained in these clusters. By incorporating the variance of gene density in different regions on a chromosome, we conclude that 45 two-gene, 7 three-gene and 2 four-gene clusters are significant with p-value less than 4.9·10^-2^, 3.1·10^-2^and 1.2·10^-2^respectively. The most significant cluster is the four-gene cluster on chromosome 2 (p-value = 1.3·10^-3^). When D = 50 K, we obtained 392 two-gene, 56 three-gene, 19 four-gene and 1 five-gene clusters. In total, 28% of the testis-specific genes are in these clusters. By incorporating the variance of gene density in different regions on a same chromosome, we conclude that 14 two-gene, 6 three-gene and 2 four-gene clusters are significant with p-value less than 4.9·10^-2^, 3.1·10^-2^and 1.2·10^-2^respectively. The most significant one is a two-gene cluster on chromosome 18 (p-value = 5.3·10^-3^). When D = 75 K, we obtained 454 two-gene, 79 three-gene, 28 four-gene, 4 five-gene and 1 six-gene clusters. In total, 35% of the testis-specific genes are in these clusters. By incorporating the variance of gene density in different regions on a chromosome, we conclude that 10 two-gene, 2 three-gene and 3 four-gene clusters are significant with p-value less than 4.1·10^-2^, 3.1·10^-2^and 4.9·10^-2^respectively. The most significant one is a two-gene cluster on chromosome 16 (p-value = 5.2·10^-3^). When D = 100 K, we obtained 463 two-gene, 114 three-gene, 46 four-gene, 7 five-gene and 2 six-gene clusters. In total, 41% of the testis-specific genes are in these clusters. By incorporating the variance of gene density in different regions even on the same chromosome, we conclude that 12 two-gene, 4 three-gene and 1 four-gene clusters are significant with p-value less than 4.9·10^-2^, 4.8·10^-2^and 3.2·10^-2^respectively. The most significant one is a two-gene cluster on chromosome 16 (p-value = 9.2·10^-3^). We also observed that there are more significant gene clusters on chromosomes 5, 7, 12 X than any other chromosomes. Discussion ========== It seems that in most eukaryotes, the transcription factor machinery is sufficient for ensuring co-regulation so that co-localization of genes in a genome is not necessary. However, there could be some degree of selection for keeping co-regulated genes in clusters on a chromosome, for instance, to make them available to transcription more efficient as a group. Gene clusters due to tandem duplication --------------------------------------- Clustering of co-expressed genes could be due to tandem duplication. Tandem duplication and subsequent divergence of amplified copies might result in an array of paralogous genes that may retain common regulatory element. Such a type of clustering is exemplified by the Hox gene family \[[@B14]\]. From the testis-specific gene representation profile, we can observe that besides co-expressed adjacent gene pairs, there are large-scale clusters on chromosomes. To determine to what degree tandem duplication contributes to the clustering of testis-specific co-expressed genes, we analyzed cluster distribution after removal of duplicated genes. To identify duplicated testis-specific genes, we performed an all-against-all BLAST homology search using default BLASTN settings. (See the method part for details.) In total, 240 duplicated genes were identified and removed from the list of the testis-specific genes (see Table [3](#T3){ref-type="table"} for details). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Testis-specific gene distribution before and after removal of tandem duplications. ::: Chromosome Number of genes Difference ---------------- --------------------- ---------------- ----- 1 249 234 15 2 298 280 18 3 223 209 14 4 230 220 10 5 253 232 21 6 210 201 9 7 229 211 18 8 220 205 15 9 217 207 10 10 223 207 16 11 250 239 11 12 171 162 9 13 179 168 11 14 213 199 14 15 142 136 6 16 128 119 9 17 155 145 10 18 104 94 10 19 109 106 3 X 113 105 8 Y 12 9 3 Total 3928 3688 240 ::: In the most of chromosomes except for chromosomes 2, 5, 7, 10, no more than 15 duplicated genes were identified. On chromosomes 2, 5, 7, 10, we identified 18, 21, 19, 16 duplicated genes respectively. Since, for each of distance threshold value (25 K, 50 K, 75 K and 100 K), we obtained more than 300 gene clusters, we conclude that duplication has no obvious effect on the positional clustering of testis-specific genes in the mouse genome. Non-duplicated clusters of genes -------------------------------- Eukaryotic genes have not only promoters, but also one or more enhancers. An enhancer is a DNA sequence that influences transcription of neighboring genes. Enhancers are different from promoters in their ability to act over thousands of base pairs upstream or downstream of a gene. While a promoter is responsible for initiating low levels of transcription and determining the transcription start site, enhancers are responsible for increasing or \"enhancing\" transcription levels, as well as for regulating cell- or tissue-specific transcription. There are presently two models for non-duplicated gene clustering. One is the incidental expression mode. In this model, co-regulation within an expression neighborhood is due to incidental interactions between transcriptional enhancers and promoters \[[@B16]\]. When transcription factor binds at the appropriate sites and activates nearby genes together with the target gene, a group of nearby genes tend to express together. In this case sites that bind strong transcriptional activators should create new neighboring co-expressed genes. An alternative model of clustering is that gene clusters may correspond to regions of active chromatin. This explanation is called the chromatin domain model. In mammalian genomes, gene clusters are on a much larger scale (Mb rather than 5--20 kb) and may involve long-range chromatin interactions \[[@B18],[@B19]\]. The structure of the chromatin domain model assumes that opening of the chromatin of an entire cluster depends on activation of a target gene within the cluster. Our findings can most be explained as regulation at the level of chromatin structure for the following reasons. First, the regions showing similarities in expression are quite large; each gene presumably has its own core promoter. Second, one or two genes in a group often displays a high level of differential expression. If the chromatin in a region that contains many genes was \'opened\' so that a single target gene could be expressed, it might increase the accessibility of the promoters and enhancers of other genes to transcriptional machinery, leading to corresponding increases in their expression. Such an effect could account for the observations we have made. Although the organization of neighborhoods along a chromosome indicates that there must be cis-acting structures, no known structure correlates with the blocks. The structure basis of positional gene clustering is still under investigation. After removal of tandemly duplicated genes, significant clusters of co-expressed testis-specific genes were still found on all the chromosomes. However, the mechanism responsible for this observation is still unknown. Conclusion ========== We revealed the positional clustering of testis-specific genes in the mouse genome by using EST profiling. Our analysis implies that the mouse genome is enriched with clusters of testis-specific genes. A significant trend for large clusters (at least 3 genes) was discernible. Positional clustering of co-expressed, non-homologous genes has also been reported in nematode \[[@B3]\] and in mammals \[[@B6]\]. This indicates that positional clustering is common in higher eukaryotes. Clustering of tissue-specific genes suggests that there exists some higher order in regulation of gene expression, most probably due to the chromatin domain. From this fascinating starting point we expect further insight into the significance of gene clustering and the mechanisms that generate them as more genomes are sequenced and more expression patterns are studied in the coming years. Methods ======= Testis expressed gene models ---------------------------- A total of 60,770 full-length cDNA sequences are available in the Fantom (Funtional Annotation of Mouse) 2.1.1 database. These sequences were aligned back to the mouse genome using BLAT \[[@B21]\] (a program that specializes in the alignment of EST/cDNA sequences to genomic sequences). Overlapping alignments were assembled to produce a complete gene model. This resulted in 34,402 genes. The 3,928 testis-specific genes were identified on the basis of having the keyword \"testis\" but not the keyword \"embryo\" in the tissue type information associated with the library. Removal of tandem repeats ------------------------- Clusters of co-expressed genes could be due to tandem duplication. To remove effect of local duplication, we did an all-against-all BLAST search on all 3,928 testis-specific genes. As a result, 240 duplicated genes were identified on the basis of an expectancy value less than 10^-50^as proposed by Friedman and Hughes \[[@B20]\]. This results in the final 3,688 testis-specific genes used for our statistical analysis. Statistic analysis ------------------ ### The neighborhood model Recall that we define clusters using the distance between two neighboring testis-specific genes along the chromosome. Two testis-specific genes are in a cluster if and only if there is a series of testis-specific genes between them such that the distance between two consecutive genes in the series is less than a specified threshold (D). To incorporate the variance of gene density in different regions on a chromosome, we divide a chromosome into segments of length L(2000 Kbp here) so that genes on such a segment are roughly uniformly distributed and analyze the significance of a cluster within the segment according to the number Nof all the genes (not just testis-specific genes) in the segment and the values of parameters Land D. Since we assume the (start) position of a gene is uniformly distributed in a segment of length L, it falls in an interval (x, x+ D) in the segment with probability D/L. Thus, the number of genes that fall in this interval has a binomial distribution with mean ND/L. In our case, D/Lis smaller than 0.1. This distribution is approximately Poisson with mean ND/L. Here we use this approximation in our analysis. The Poisson approximation implies that the number of the genes that falls in a randomly chosen interval of length Dhas a Poisson distribution with mean m= ND/L, and so the probability that at least one gene falls in this interval is 1 - e^-m^. Suppose g~1~, g~2~, ..., g~n~form a cluster in the segment. Then, all the distances of two successive genes are less than D. Thus, the number of genes in a cluster minus one has a geometric distribution with p = 1 - e^-m^(see page 10, \[[@B22]\]). This implies that the probability that a cluster has n genes is (1 - p) p^n-1^, n= 0, 1, 2, .... Hence, the p-value of a cluster with ngenes is the probability that a cluster has more than n genes, which is p^n^. In our analysis, D= 25 K, 50, 75 K, 100 K. Actually, the distance between two successive testis-specific genes in the most of observed clusters is much smaller than D in each case. Hence, the real p-value for each cluster could be much smaller than one reported in the result section. ### Adjacent gene clustering To remove the effect of non-uniform distribution of genes on analysis, we adopt an idea from order statistics. Here, we focus on the position rank of a gene rather than its specific position by ordering all the genes according to their positions on a chromosome. We also treat the set of the testis-specific genes and the set of all the other genes as two types of identical objects. In this way, a specific gene distribution on a chromosome is modeled as a 0--1 string in which 0 represents a test-specific gene and 1 a non-testis-specific gene. Assume there are Mgenes in a chromosome and Tof them are testis-specific genes, where T≤ M. Then, there are ![](1471-2164-6-7-i1.gif) possible gene arrangements on the chromosome and the number of arrangements with radjacent testis-specific gene clusters is ![](1471-2164-6-7-i2.gif). Thus, the probability that a random arrangement has radjacent testis-specific gene clusters is ![](1471-2164-6-7-i3.gif) Using above formula, we can obtain that the mean number of adjacent testis-specific gene clusters in a random arrangement is ![](1471-2164-6-7-i4.gif) and the standard deviation is ![](1471-2164-6-7-i5.gif). Take chromosome 4 for example. We have M= 2060, T= 220, we obtain that the mean number is 196.6 and the standard deviation is 4.4. However, we only observed 182 adjacent gene clusters (including singletons) on the chromosome. Authors\' contributions ======================= BL and QL carried out the software development, sequence analysis, and the design of the study. LXZ conceived of the study, performed the statistical analysis, and participated in its coordination. All authors read and approved the final manuscript. Acknowledgement =============== The authors would like to thank the anonymous reviewers for their extremely helpful comments and suggestions particularly on using Fantom database and incorporating gene density variance into statistic analysis. The work was partially supported by Singapore BMRC research grant BMRC01/1/21/19/140.	PubMed Central	2024-06-05T03:55:52.512581	2005-1-19	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548148/", "journal": "BMC Genomics. 2005 Jan 19; 6:7", "authors": [ { "first": "Quan", "last": "Li" }, { "first": "Bernett TK", "last": "Lee" }, { "first": "Louxin", "last": "Zhang" } ] }
PMC548149	Background ========== The nested case-control design employs a case-control approach within an established cohort \[[@B1],[@B2]\] to obtain estimates from a sample of the cohort that are similar to estimates obtained from analysis of the entire cohort \[[@B3],[@B4]\]. The nested case-control design is being increasingly used in large cohorts of patients from prospective studies and randomized clinical trials. This design has become popular because it allows for statistically efficient analysis of data from a cohort with substantial savings in cost and time \[[@B5],[@B6]\]. When studying exposures that vary with time, an additional level of complexity is introduced by the need to account for time-dependent exposure in both the design and analysis \[[@B7],[@B8]\]. This can be accomplished by including time-dependent covariates in a Cox proportional-hazards regression model \[[@B9]\]. Alternatively, a nested case-control approach can be used provided that the exposure and covariate information for controls reflects values corresponding to the time of selection of their respective case. This study compares nested case-control and survival analysis methodologies for evaluating time-dependent exposure. The risk of pacemaker insertion associated with dosage of amiodarone (an anti-arrhythmic medication used for the treatment of atrial fibrillation (AF)), represented by a time-dependent covariate, is evaluated in cohort of patients with AF using both methods for illustrative purposes. The comparability of results is evaluated and differences in computational efficiency are quantified for increasing cohort sizes. Advantages and limitations of the respective methodologies are discussed. Methods ======= Study cohort ------------ A cohort of 11395 elderly (\>65 years of age) Quebec residents with AF and a myocardial infarction (MI) between 1991 and 2000 was created by linking the provincial hospital discharge summary database with the provincial physician and drug claims database, using methods described previously \[[@B10]\]. Approval for the study was obtained from the McGill University Faculty of Medicine Institutional Review Board. In order to evaluate the effect of amiodarone dose on the previously demonstrated association between amiodarone therapy for AF and an increased risk of permanent pacemaker requirement \[[@B10]\], only patients newly started on amiodarone after their diagnosis of AF were included in the study cohort. Amiodarone dose was represented as a binary time-dependent variable comparing daily doses \>200 mg to ≤200 mg. Covariate information included age, sex, calendar year of cohort entry, baseline sinus node or conduction disorder, ventricular arrhythmia, and time-dependent exposure to five categories of medications. The final study cohort included 1340 subjects followed from the date of their first prescription of amiodarone until the first of pacemaker implantation, death, or March 31, 2001. Statistical analysis -------------------- The data for the entire cohort of 1340 subjects including all fixed and time-dependent variables was represented in counting process notation suitable for Cox regression of time-dependent exposure \[[@B11]\]. Multiple records (with consecutive start and end times) were created for each subject to account for every change in exposure to any of the time-dependent variables over the study period. The hazard ratio (HR) of pacemaker insertion associated with amiodarone doses \>200 mg per day was estimated using a Cox proportional-hazards model including all fixed and time-dependent covariates. The timescale used in the model was time since first prescription of amiodarone. Non-significant variables (other than age and sex) were sequentially removed if the resultant model had no significant increase in Akaike Information Criteria (AIC) and no significant change in the HR for amiodarone dose. The nested case-control approach was also used to estimate the HR of pacemaker insertion associated with amiodarone doses \>200 mg per day. Cases of pacemaker insertion were identified and controls were randomly selected from the risk-set of each case (i.e. subjects present in the cohort at the time the case is defined). After selecting all controls and recording their index dates (i.e. the time, in cohort time, at which the respective case is defined) the relevant time-dependent covariate information was retrieved by merging with the database configured in counting process notation. The relevant subject record was selected by requiring that the index date fall within the start and end time of the subject record for each control. The nested case-control approach was repeated using 4, 8, 16, 32, and 64 controls per case. For each number of controls per case, random sampling of controls for all cases and conditional logistic regression analysis was repeated 100 times using the OUTEST option in the PROC PHREG statement to create an output SAS data containing all the parameter estimates \[[@B11]\]. The mean and standard deviation (SD) of the parameter estimates for each number of controls per case was calculated. Computational times for regression models of time-dependent exposures using nested case-control and survival analysis methodologies were compared. The nested case-control samples with 4 and 32 controls per case were analyzed using conditional logistic regression with the PHREG procedure in SAS Release 8.2 \[[@B12]\]. The full cohort was analyzed using Cox regression adapted for analysis of time-dependent covariates with the PHREG procedure in SAS Release 8.2. Ties were handled using the TIES = EFRON option in the PHREG procedure \[[@B11]\]. All analyses were performed using an Intel Pentium 4 computer with a 1.80 GHz central processing unit (CPU) and 256 MB of random access memory (RAM). Relative computational efficiencies were evaluated by comparing the CPU times of the three regression models used to analyze the cohort. Relative increases in computational time as a function of sample size were quantified by repeating the analyses on progressively larger cohorts. This was done by progressive doubling of the original cohort to 2, 4, 8, and 32 times its original size. Given that the objective was to compare computational times, all fixed and time-dependent exposures were included in all models regardless of statistical significance. The computational efficiency of the Cox regression model was also compared to the conditional logistic regression model where the nested case-control sample included all possible controls for each case. This comparison was performed for the original cohort of 1340 subjects with 53 cases (including 2 ties). All analyses were performed using the PHREG procedure. Ties were handled using the TIES = EFRON option in the PHREG procedure, and subsequently using the TIES = DISCRETE option in the PHREG procedure for comparison. Results ======= Comparative risk estimates -------------------------- Pacemaker implantation occurred in 53 of the 1340 subjects during the study period. In the final Cox regression model, amiodarone daily dose (\>200 mg vs. ≤200 mg) was associated with an increased risk of pacemaker insertion (HR: 2.03; 95 percent confidence interval: 1.00, 4.14; p= 0.05; Parameter Estimate: 0.71; Standard Error of Parameter Estimate: 0.36), after adjusting for age, sex, as well as baseline sinus node or conduction disorder (the only covariate that was an independent predictor of outcome). The results of 500 nested case-control analyses (i.e. 100 for each of 4, 8, 16, 32, and 64 controls per case) are summarized in Table [1](#T1){ref-type="table"}. When using any number from 4 to 64 controls per case, the mean point estimate (of 100 analyses repeating the random sampling of controls) of the parameter estimate (and HR) was very similar to that obtained using Cox regression on the full cohort (i.e. HR: 2.03; Parameter Estimate: 0.71). The SDs of the parameter estimates decreased with increasing numbers of controls per case (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Nested case-control analyses with repeated sampling for increasing numbers of controls per case: Hazard ratio of pacemaker insertion associated with amiodarone dose\* in 1340 elderly Quebec residents with atrial fibrillation ::: Controls per Case (n) Repeated Sampling (n) Mean HR† SD† of HR Min HR Max HR Mean Parameter Estimate SD of Parameter Estimates ----------------------- ----------------------- ---------- ----------- -------- -------- ------------------------- --------------------------- 4 100 2.14 0.51 1.27 3.59 0.73 0.23 8 100 2.19 0.41 1.39 3.86 0.77 0.18 16 100 2.02 0.22 1.55 2.51 0.70 0.11 32 100 2.07 0.15 1.78 2.55 0.73 0.07 64 100 2.02 0.11 1.82 2.33 0.70 0.05 \* The effect of amiodarone dose, represented by a binary time-dependent covariate (\>200 mg vs. ≤200 mg), was adjusted for age, sex, and baseline sinus node or conduction disorder in all models. † HR, hazard ratio; SD, standard deviation. ::: Comparative computational efficiencies -------------------------------------- The computational time required to analyze the cohort of 1340 subjects with 53 events (cases) in models including four fixed variables and seven time-dependent variables is presented in Table [2](#T2){ref-type="table"}. CPU times are displayed for the Cox regression models and the nested case-control regression models (with 4 or 32 controls per case). For the cohort in its initial size, using 32 rather than 4 controls per case increased CPU time by a factor of 3, whereas using Cox regression increased CPU time by a factor of 42. As the cohort size was increased to 32 times the original (i.e. 42880 subjects), the increase in CPU time was greater for the Cox regression model than the nested case-control models. Figure [1](#F1){ref-type="fig"} displays graphically the increase in CPU time with increasing sample size for nested case-control and Cox regression models. The relative computational efficiency was magnified as the sample size increased, such that the CPU time for Cox regression with a cohort of 42880 subjects was 125 times greater than the nested case-control model with 4 controls per case (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Computational times for nested case-control and survival analyses of time-dependent data for cohorts of increasing sizes: Models\* of the risk of pacemaker insertion in elderly Quebec residents with atrial fibrillation ::: Cohort Size†: \#Subjects (\#Cases) 1340 (53) 2680 (106) 5360 (212) 10720 (424) 21440 (848) 42880 (1696) ------------------------------------ ----------- ------------ ------------ ------------- ------------- -------------- Cohort Size: multiple of original 1 2 4 8 16 32 CPU‡ Time (seconds): Nested: 4 Controls per Case 0.03 0.05 0.07 0.11 0.18 0.4 Nested: 32 Controls per Case 0.08 0.1 0.2 0.41 0.7 1.49 Survival Analysis 1.26 2.51 5.06 9.54 19.53 49.91 CPU Time (multiple of Nested 4): Nested: 4 Controls per Case 1 1 1 1 1 1 Nested: 32 Controls per Case 3 2 3 4 4 4 Survival Analysis 42 50 72 87 109 125 \* All models included four fixed variables (age, sex, calendar year of cohort entry, and baseline sinus node or conduction disorder) and seven time-dependent variables (amiodarone dose, ventricular arrhythmia, and exposure to sotalol, class I antiarrhythmic agents, beta-blockers, calcium channel blockers, and digoxin). † The original cohort of 1340 subjects was increased by progressive duplication to 2, 4, 8, 16, and 32 times its original size. ‡ CPU, central processing unit. ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Increase in computational time with increasing sample size for nested case-control and survival analysis of cohort data with time-dependent covariates ::: ![](1471-2288-5-5-1) ::: The computational time of the Cox regression model was also compared to the nested case-control model including all possible controls for each case. When ties were handled using the TIES = EFRON option, the CPU time for Cox regression was 1.06 times greater than the CPU time for nested case-control model (1.26 vs. 1.19 seconds). When ties were handled using the TIES = DISCRETE option, the CPU time for Cox regression was 3.91 times greater than the CPU time for nested case-control model (14.65 vs. 3.75 seconds). Discussion ========== In this study we illustrate empirically that a nested case-control approach can be used to analyze a cohort with time-dependent covariates, with results that are similar to those obtained by Cox regression. Additionally, given that the nested case-control approach obviates the computationally intensive calculations involved in Cox regression when time-dependent covariates are used, the example also illustrates quantitatively the large reduction in CPU time required for analysis. The similarity between the two methodologies is expected given that conditional logistic regression used to analyze nested case-control studies (as well as other matched case-control studies) is based on inference procedures adapted from Cox regression; i.e. the conditional likelihood used in conditional logistic regression is exactly the same form as the partial likelihood used in Cox regression except that the denominator includes only a selected number of sampled controls as opposed to all subjects available in the risk set \[[@B13]\]. The inclusion of time-dependent covariates adds an additional level of complexity to the analysis but remains based on the same inference procedures. The statistical efficiency of the nested case-control approach for cohort analysis depends on the number of controls per case selected. Our example demonstrates the expected decrease in the SD of the parameter estimates as the number of controls per case increases. This decrease in variance is explained by the fact that as the number of controls per case increases (towards the total number of controls in a case\'s risk-set), the probability of choosing the same controls increases, as does the proportion of available controls selected (i.e. approximating the situation in Cox regression where every case is compared to all controls in its risk-set). In general, the use of 4 controls per case provides a relative statistical efficiency of 0.8 compared to the use of an infinite number of controls \[[@B14]\]. However, the relative efficiency also depends on the probability of exposure among the controls and on the magnitude of the estimated relative risk. Gains in statistical efficiency are possible by using greater than 4 controls per case particularly when the probability of exposure among the controls is \<0.1 \[[@B4],[@B15]\]. In addition to situations where exposure prevalence in controls is low, increasing the number of controls per case is beneficial when the number of case-control sets is small \[[@B16]\]. The major reason for the superior computational efficiency of the conditional logistic regression method for nested-case control analysis of time-dependent covariates is that only a sample of all possible controls are included in the risk set of each case (whereas all are included in Cox regression). As illustrated in Figure [1](#F1){ref-type="fig"} and Table [2](#T2){ref-type="table"}, the impact on computational efficiency of sampling a fixed number of controls per case is greater for larger cohorts because the sample of controls represents a smaller proportion of the all the possible controls for each case. While this effect of sampling controls may be the main reason for the computational efficiency of the nested case-control approach is not the only reason. As demonstrated, even when all possible controls are included in the risk set of each case, the computational time of the conditional logistic regression increases significantly but remains faster than Cox regression. This is because the two analyses process time-dependent covariates differently. In Cox regression, risk sets and time-dependent covariates are calculated at the time of each case failure. In conditional logistic regression, the risk sets and time-dependent covariates are calculated in advance. The relative efficiency also depends on how ties are handled, with Cox regression relatively less efficient when ties are handled using the TIES = DISCRETE option compared to the TIES = EFRON option. The nested case-control approach for cohort analysis offers some advantages over analysis of an entire cohort that may be important regardless of the type of cohort used. A potential advantage with respect to design is the option to match controls to cases on the basis of possible confounding covariates for which estimation of effect is not of interest. Another advantage is that substantial savings in cost and time can be achieved by analyzing the cases and only a sample of the controls (as opposed to the entire cohort), particularly when the collection and/or processing of exposure information is very expensive and/or time consuming \[[@B6]\]. While cost is often a major factor in preferring a nested-case control approach over analyzing an entire cohort, there may be advantages even when differences in cost are not significant. In recent years, large administrative healthcare databases, such as the one from which the example cohort for this study was selected, have become particularly useful in studying outcomes that are very rare because they allow for adequate sample sizes \[[@B17]-[@B20]\]. Once a database with all exposure and outcome information is available, analyzing a sample of rather than the entire cohort does not necessarily decrease costs. However, depending on the size of the cohort (as well as the speed of the processor and amount of memory in the computer), it may not be possible to analyze an entire cohort when complex modeling of time-dependent covariates is needed. Such was the case in another study based on a cohort derived from the Quebec provincial healthcare database, where in one analysis the number of covariates was restricted to four and in another analysis only a sub-cohort (i.e. 15529 of 31062 subjects) could be included because the substantial computational resources required were prohibitive \[[@B21]\]. Depending on the rarity of the outcome under study, it may not be possible to analyze the required sample size when performing Cox regression on the whole cohort, whereas it may be possible to do so using a nested case-control approach. While it is recognized that issues of computational resources are overcome with time as computers and software become more efficient, limitations are likely to remain as the size of databases and complexity of time-dependent analyses will also increase. Both the nested case-control approach described (using conditional logistic regression) and the Cox proportional-hazards model with time-dependent covariates similarly account for the time-dependence of exposure when levels of exposure in subjects vary over time. A different and more complex issue is the possibility that the effect of a given exposure varies over time. This can be addressed by analyzing latency-weighted exposures using either Cox regression or a nested case-control approach, the latter being computationally faster \[[@B22]\]. Alternatively, Cox regression can accommodate changes in the hazard ratio over time with a flexible generalization of the Cox proportional hazards model using a regression spline technique \[[@B23]-[@B25]\]. Conclusions =========== A nested case-control approach is a useful alternative for analysis of a cohort when time-dependent covariates are used. The expectedly similar risk estimates are obtainable with superior computational efficiency. Particularly when studying the effects of time-dependent exposures on rare outcomes in very large databases, study power can be improved by being able to run complex regression models on a larger number of affected subjects. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= All authors participated in the conception and design of the study. VE performed the statistical analysis and drafted the manuscript. All authors contributed to the interpretation of the study and revision of the manuscript. The final manuscript was read and approved by all authors. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2288/5/5/prepub> Acknowledgements ================ This study was funded in part by grant \#53181 from the Canadian Institutes of Health Research (CIHR) and grant \#014100 from the Fonds de la Recherche en Santé du Québec (FRSQ). Dr. Vidal Essebag is the recipient of a Clinician Scientist Award from CIHR. Dr. Platt is a recipient of an Investigator Award from CIHR. Dr. Abrahamowicz is a James McGill Professor at McGill University, Montreal, Canada. Dr. Pilote is a recipient of an Investigator Award from CIHR and the Dawson chair at McGill University, Montreal, Canada.	PubMed Central	2024-06-05T03:55:52.517398	2005-1-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548149/", "journal": "BMC Med Res Methodol. 2005 Jan 25; 5:5", "authors": [ { "first": "Vidal", "last": "Essebag" }, { "first": "Robert W", "last": "Platt" }, { "first": "Michal", "last": "Abrahamowicz" }, { "first": "Louise", "last": "Pilote" } ] }
PMC548150	Background ========== Huntington\'s disease (HD) is a severe, dominantly inherited neurodegenerative disorder that typically has its onset in mid-life, though it may occur in the juvenile years or in the elderly, and that produces an inexorable decline to death 10--20 years later \[[@B1]\]. Its cardinal clinical feature is a characteristic motor disturbance involving progressive choreoathetosis, but the disorder also involves psychological changes and cognitive decline. The neuropathological hallmark of HD is the loss of medium spiny striatal projection neurons in a dorso-ventral/medio-lateral gradient that eventually decimates the caudate nucleus, but considerable neuronal loss also occurs in other parts of the basal ganglia and in the cortex \[[@B2]\]. The pathogenic process of HD is initially triggered by an expanded polyglutamine segment near the amino terminus of huntingtin, an \~350 kDa protein whose precise physiological function is uncertain \[[@B3]\]. Huntingtin is required for normal embryonic development and neurogenesis, based on the lethal consequences of mutational inactivation in the mouse \[[@B4]-[@B6]\]. By contrast, the HD mutation itself does not impair this developmental activity but rather produces a \"gain-of-function\" that acts to cause the disorder \[[@B7]\]. Genotype-phenotype studies of HD patients, in comparison with other polyglutamine neurodegenerative disorders, have delineated a number of genetic criteria for the mechanism that triggers HD pathogenesis: 1) a threshold polyglutamine length (within a normal human lifespan); 2) progressive severity with increasing polyglutamine length above the threshold; 3) complete dominance over the wild-type protein; 4) greater dependence on polyglutamine length than on huntingtin concentration (within a physiological range) and 5) striatal selectivity, due to the huntingtin protein context in which the polyglutamine tract is presented \[[@B8],[@B9]\]. The \"gain-of-function\" due to the HD mutation is thought to lie in a novel conformational property conferred on mutant huntingtin by the expanded polyglutamine tract \[[@B10]\]. This has been supported by in vitrostudies of a small amino-terminal huntingtin fragment, where an expanded polyglutamine tract promotes self-aggregation in a manner that conforms to the first four genetic criteria \[[@B10]-[@B12]\]. The in vitroaggregation involves a conformational change of the polyglutamine segment from a random coil to an amyloid structure and is paralleled in cell culture in some ways by the formation of cytoplasmic and nuclear inclusions that also incorporate other proteins \[[@B13]\]. Neuronal inclusions containing amino-terminal fragment have also been detected in HD brain, though their role in pathogenesis remains a matter of debate, as they may occur late in the pathogenic process as a consequence of huntingtin degradation \[[@B14]\]. Precise genetic modeling of HD in the mouse supports the view that in vivo, the \"gain-of-function\" property conferred by the expanded polyglutamine acts within full-length huntingtin to cause abnormalities that do not initially involve formation of an insoluble aggregate \[[@B15],[@B16]\]. Knock-in mice in which the HD mutation has been introduced into Hdh, the mouse orthologue, display early biochemical and histological phenotypes that are associated with expression of full-length mutant huntingtin at normal physiological levels and in a normal developmental pattern \[[@B7],[@B15]-[@B20]\]. Indeed, the phenotypes associated with expression of full-length mutant huntingtin in these mice, and in neuronal progenitor cells derived from them, also fulfill the genetic criteria for the mechanism triggering HD pathogenesis \[[@B15],[@B20]-[@B22]\] One of the earliest phenotypes is the nuclear localization of full-length mutant huntingtin in the nucleus of striatal neurons \[[@B16]\]. Together, the knock-in mouse data suggest that the process of pathogenesis is triggered by the presence of expanded polyglutamine in full-length huntingtin and leads only after many months to the formation of amino-terminal huntingtin fragment and inclusion formation \[[@B15]\]. We have postulated that the same conformational property that promotes aggregation in the context of a small fragment may also act with the context of full-length huntingtin to trigger pathogenesis, possibly by altering huntingtin\'s interaction with another cellular element. Consequently, we have identified small molecules from the NINDS Custom Collection of bioactive compounds that inhibit in vitroaggregation of amino-terminal mutant huntingtin \[[@B23]\]. These have been tested for their ability to reverse the huntingtin localization phenotype associated with full-length mutant huntingtin in cultured striatal progenitor cells from Hdhknock-in mice. Our findings indicate that some of these compounds reverse the effects of the expanded polyglutamine in both assays and support the view that some inhibitors of polyglutamine aggregation may lead to viable therapeutics targeted at full-length mutant huntingtin, early in the disease process. Results ======= Screening for inhibitors of aggregation --------------------------------------- We have previously demonstrated that, when released from the protection of a GST fusion protein, the amino terminal fragment 1--171 of mutant huntingtin, forms aggregates in a manner consistent with the genetic criteria for the mechanism of HD pathogenesis \[[@B10]\]. We used a modified version of this assay, implemented using a 96-well format ELIFA dot blot apparatus, to screen the NINDS Custom Collection (NCC) which consists of 1040 small bioactive compounds, both FDA-approved drugs and natural products (Figure [1A](#F1){ref-type="fig"}). The screening was carried out in a blinded fashion as part of the NINDS Neurodegeneration Drug Screening Consortium, with the identities of compounds in the NCC only being made available after completion of the screens \[[@B23]\]. In our primary screen (Figure [1A](#F1){ref-type="fig"}), GST-Q58-Htn (20 μg/ml) was mixed with thrombin (0.5 unit/μg GST-Q58-Htn) and immediately dispensed into a 96-well PCR plate containing compounds diluted to a final concentration of 100 μM. Incubation was continued for 24 hours at room temperature to allow aggregate formation. The aggregation was stopped by 2% SDS/10 mM 2-mercaptoethanol followed by boiling for 5 minutes. The mixture was filtered through a cellulose acetate membrane by using a 96-well ELIFA dot blot apparatus. The aggregates retained on the membrane were detected and quantified by immunoblotting and subsequent image analysis. A typical immunoblot result is shown in Figure [1B](#F1){ref-type="fig"}. Congo Red, a known huntingtin aggregation inhibitor, was used as the positive control \[[@B24]\]. 10 μM Congo Red can completely inhibit the Q58-Htn aggregation. DMSO, used for the negative control, had no impact on Q58-Htn aggregation. Potential inhibitors were distributed evenly cross the whole NCC library (Figure [2](#F2){ref-type="fig"}). Sixty compounds that showed more than 50% inhibitory effect were selected to be retested in a second screen at a lower concentration of 10 μM. The 8 compounds in column 5 of plate 9, were missed in the primary screening at 100 μM, and were therefore also tested in the second screening at 10 μM. In the primary screening, a \"hit\" could have resulted either from direct inhibition of polyglutamine-induced aggregation or indirectly, by inhibition of the thrombin and consequent failure to cleave GST-Q58-Htn, which does not by itself aggregate. Consequently, the second screening at 10 μM was carried out after thrombin digestion, to eliminate thrombin inhibitors. Western blotting showed that more than 95% of GST-Q58-Htn is cleaved by thrombin (at ratio of 0.5 unit/1 μg protein) within 30 minutes (data not shown). Consequently, the mixture of GST-Q58-Htn and thrombin was preincubated for 45 minutes, followed by centrifugation to remove any aggregates already formed, before adding the test compounds. Nineteen of the compounds tested at 10 μM, showed significant direct inhibitory effects on aggregation. The 10 most potent compounds, corresponding to a \'hit\' rate of 1%, are shown in Figure [3](#F3){ref-type="fig"}. Characteristics of aggregation inhibitors ----------------------------------------- To determine the potency of each inhibitor, we performed dose response assays at concentrations ranging from 0.01 μM to 500 μM. Representative curves for the 6 most potent compounds are shown in Figure [4](#F4){ref-type="fig"}. Gambogic acid and celastrol showed strong but incomplete inhibition even at the maximum concentration, permitting approximately 20% residual aggregate to form. The average IC~50~(half-maximal inhibition) values for the most potent 10 compounds, which range from 0.7 to 15 μM, were obtained from at least two independent experiments each (Figure [3](#F3){ref-type="fig"}). The most effective aggregation inhibitor was gossypol-acetic acid complex, followed closely by gambogic acid, and then juglone, celastrol, and sanguinarine nitrate, which all had IC~50~values less than 6 μM. Effect on striatal cells expressing endogenous full-length mutant huntingtin ---------------------------------------------------------------------------- To test the hypothesis that compounds, which inhibit the aggregation-promoting property of amino-terminal mutant huntingtin will also rescue effects of full-length mutant huntingtin, we tested the top six inhibitors in a striatal cell-based assay. Mutant Hdh^Q111/Q111^and wild-type Hdh^Q7/Q7^striatal cell lines, ST7/7 and ST111/111, respectively, which have been prepared by transformation with a tsSV40 vector, can be propagated in culture and used for cytological and biochemical comparisons \[[@B25]\]. These cells express full-length mutant or wild-type huntingtin, respectively, with no evidence of truncated amino-terminal fragments, no formation of polyglutamine aggregates and no cell death-producing toxicity. However, like the striatal neurons of Hdh^Q111/Q111^knock-in mice, the ST111/111 cells show nuclear staining of huntingtin when tested with an amino-terminal huntingtin antibody that is sensitive to the conformation of the full-length protein (Figure [5A](#F5){ref-type="fig"}). By contrast, ST7/7 cells expressing wild type huntingtin show both nuclear and cytoplasmic immunostaining with the same huntingtin antibody (Figure [5A](#F5){ref-type="fig"}). This differential localization phenotype occurs early in the cascade of events detected during the lifespan of Hdhknock-in mice, months before the appearance of huntingtin amino-terminal fragment, and fulfills the genetic criteria from genotype-phenotype studies in HD patients, including polyglutamine length progressiveness and striatal specificity, suggesting that it follows from the same property that triggers HD pathogenesis. This huntingtin localization phenotype was used to monitor the effect of inhibitors in the mutant cells (Figure [5B](#F5){ref-type="fig"}), and the results are shown in Table [1](#T1){ref-type="table"}. About 89% of ST7/7 striatal cells showed both nuclear and cytoplasmic immunostaining signal, while ST111/111 striatal cells showed only nuclear signal (99%). Of the six compounds tested, celastrol and juglone both reversed the mutant phenotype in a dose-dependent manner. Juglone showed no evident cell toxicity up to 10 μM, where 68% of mutant cells had reverted to wild-type phenotype. Celastrol reverted up to 81% of the cells but showed toxicity, killing \~4% of cells at 10 μM. Gossypol acetic acid complex was less effective, but showed no toxicity. At 50 μM, only 15% of the mutant cells displayed the wild-type phenotype. Gambogic acid showed a comparable small effect at 10 μM but was very toxic at high concentration, as all cells were killed at 50 μM. Sanguinarine nitrate and anthralin showed little effect on the huntingtin localization phenotype in this assay. Discussion ========== A dramatic marker of pathology in many neurodegenerative disorders is the appearance of intracellular inclusions in some surviving neurons \[[@B13]\]. In HD, these inclusions stain positively for huntingtin, ubiquitin and a number of other proteins, but are thought to be initiated by the aggregation of an amino-terminal fragment of mutant huntingtin, due to its expanded polyglutamine tract \[[@B14],[@B26]-[@B28]\]. A number of model systems have been developed to investigate the polyglutamine-driven aggregation process and its consequences both in vitroand in vivo, but it remains unclear in HD whether the formation of aggregates plays an essential role in the pathway of pathogenesis or is a downstream by-product of neuronal dysfunction induced by full-length mutant huntingtin \[[@B29]\]. In either event, the search for drugs that alter in vitroaggregation of amino-terminal huntingtin fragment is attractive, since the aggregation-promoting physical property exhibits characteristics comparable to the disease-producing property of corresponding human alleles, as defined from genotype-phenotype studies of HD patients. For example, if accumulation of huntingtin inclusions is the proximate cause of neuronal death, compounds that inhibit aggregation would have therapeutic potential. Conversely, if the inclusions are only a downstream marker of the pathogenic process, the same drugs may still have therapeutic potential if they act on the property of full-length mutant huntingtin that triggers pathogenesis. It was with a view to testing whether the in vitroaggregation assay could act as a proxy for monitoring the disease-producing property of mutant huntingtin that we undertook this study. A comparable screening assay to the one used here has been employed to screen a large chemical library and has demonstrated the feasibility of identifying small molecule inhibitors of polyglutamine aggregation, including a family of benzothiazole-related compounds \[[@B30]\]. However, the long, arduous and expensive process of developing compounds for use as drugs in humans prompted us to screen a smaller chemical library biased toward drugs already approved by the U.S. Food and Drug Administration. The aggregation-inhibiting compounds that we identified came from a collection of mostly FDA-approved compounds and bioactive natural products that were specifically assembled for a neurodegenerative disease drug screening consortium supported by the National Institute for Neurological Disorders and Stroke and several disease foundations, including the Huntington\'s Disease Society of America \[[@B23]\]. The drugs were distributed to 27 different labs for blinded testing in assays of potential relevance to neurodegenerative disease. Unfortunately, despite the preponderance of FDA approved drugs in the collection, none of our top hits (IC~50~\< 10 μM) is a compound approved for internal use in humans. The most effective inhibitor of aggregation was gossypol-acetic acid complex. Gossypol, a polyphenol found in cottonseed, has been studied extensively as a male contraceptive in China but the World Health Organization has argued against its use because of induced hypokalemia, high toxicity and the risk of permanent sterility \[[@B31]\]. Almost as potent, gambogic acid is a complex ring-structured natural product that is the main active ingredient of gamboge resin from the Garcinia hanburyitree. It has long been used as a pigment for painting and in traditional medicine as a potent purgative. Recently, it was identified in a high-throughput screen as an apoptosis inducer with potential for development as an anti-cancer agent \[[@B32]\]. Juglone is a napthoquinone found in the bark of the black walnut (Juglans nigra), which has been used as an herbal medicine for its antihaemorrhagic and antifungal properties \[[@B33]\]. Celastrol is a triterpenoid from the vine Tripterygium wilfordiiused as an alternative medicine for rheumatoid arthritis that has anti-oxidant, anti-inflammatory activity, immunosuppressive and anti-angiogenic activities. It has been proposed as worthy of exploration as a therapeutic in Alzheimer disease \[[@B34]\]. Sanguinarine, a benzophenanthridine alkaloid from bloodroot (Sanguinaria canadensis) has broad antimicrobial and anti-inflammatory, as well as potential anti-angiogenic activity \[[@B35]\]. It has been used as an oral rinse and as a potential antigingivitis/antiplaque agent in toothpaste \[[@B36]\]. Anthralin is a synthetic derivative of chrysarobin, a traditional remedy for various skin ailments from Andira araroba, that has been widely used as topical treatment for psoriasis and alopecia areata \[[@B37],[@B38]\]. Although none of these bioactive compounds is a candidate for immediate human trials, they provided a means to test whether compounds that inhibit polyglutamine aggregation might also block the neuronal phenotype caused by an elongated polyglutamine tract in full-length huntingtin. While we do not have a direct physical measure of huntingtin conformation and it remains possible that compounds could reverse the cellular phenotype by different pathways, our finding that juglone and celastrol, two drugs of different structure selected as hits in our primary aggregation assay, are both effective at restoring cytoplasmic huntingtin staining in the striatal cell assay suggests that they both act via a conformational property of mutant huntingtin. That gossypol, gambogic acid, sanguinarine and anthralin were not effective could be due to any of a number of reasons, including cellular uptake, toxicity, interaction with other cellular components, etc. However, it may also indicate that these compounds do not directly modify the conformational property of mutant huntingtin, but instead block a different step in the in vitroaggregation process or that they do so by a different physical effect than juglone and celastrol. A detailed analysis of structure-activity relationships using structurally-related compounds and testing in vivoin knock-in mice for their ability to reverse the cascade of mutant huntingtin-associated phenotypes will be needed to adequately assess the potential of any of these different types of compounds for testing in HD clinical trials. The remaining compounds identified as weaker aggregation blockers in our primary screen include selamectin, a veterinary anti-parasitic \[[@B39]\], pararosaniline pamoate, a treatment for schistosomiasis \[[@B40]\], tyrothricin, a cyclic peptide antibiotic, and meclocycline, a tetracycline-related antibiotic. The latter is of particular interest since it was the most potent of several tetracycline-related antibiotics present among the NCC compounds, including tetracycline, chlortetracycline, demeclocycline, doxycycline, methacycline, oxytetracycline and notably, minocycline, which has been proposed as a therapeutic in HD and other neurodegenerative disorders. Minocycline is an FDA approved antibiotic used for a variety of infections that has variably been reported to improve symptoms in the R6/2 exon 1 overexpression HD model \[[@B41]-[@B44]\]. It has anti-inflammatory and anti-apoptotic activity that has been proposed to involve several potential mechanisms of action. In our hands, minocycline is a weak inhibitor of polyglutamine aggregation with an IC~50~of 43 μM (unpublished data). This is consistent with the inhibitory effect on huntingtin exon 1 aggregation reported previously at 30 μM in long-term hippocampal slice cultures from the R6/2 mouse \[[@B44]\]. Two safety and tolerability studies of minocycline in human HD are completed \[[@B45],[@B46]\] and can be expected to lead to efficacy trials earlier than any trials for strong aggregation inhibitors. However, it is conceivable that long-term, low level inhibition of mutant huntingtin\'s aggregation-promoting conformational property, independent of minocycline\'s anti-apoptotic activity, may be sufficient to alter detectably the timing of disease onset or early progression. If the hoped for positive results are obtained in minocycline HD trials, this alternative mechanism should be considered since it would have implications for testing of meclocycline and for assessing the potential trade-off between potency and toxicity in choosing other aggregation inhibitors as potential long-term therapeutics. Interestingly, the same set of 1040 NCC compounds were screened for their ability to block toxicity in a PC12 cellular assay where induced expression of huntingtin exon 1 encoding 103 glutamines leads to the accumulation of aggregates and rapid cell death \[[@B47]\]. Although eighteen compounds were found to be completely protective, none was among our hits, suggesting that the mechanism of polyglutamine toxicity in the PC12 cells is fundamentally different than the mechanism(s) involved in the in vitroaggregation assay. Among a secondary class of partially protective compounds in the PC12 assay, only celastrol overlapped with our hits. The NCC compounds were also screened in a cellular assay in HEK 293T cells expressing androgen receptor with 112 glutamines \[[@B48]\]. In this model for spinal bulbar and muscular atrophy, accumulation of intracellular inclusions, accompanied by caspase 3 activation, is followed by cell death within 72 hours. Twenty compounds that blocked caspase 3 activation included celastrol, gambogic acid, sanguinarine and tyrothricin, though all but sanguinarine showed toxicity. The major finding from this assay was that several cardiac glycosides were protective, presumably by a different mechanism than our hits. Indeed, although most of the assays in the NINDS consortium involved disorders associated with protein aggregation, including various polyglutamine disorders, amyotrophic lateral sclerosis and Parkinson disease, there was a remarkable lack of overlap in hits suggesting that the individual assays targeted fundamentally different mechanisms. A possible exception was celastrol, which was found as a hit in our aggregation assay, the two assays noted above, and other assays which will be discussed in a summary article describing the consortium. Conclusions =========== The identification and further characterization of chemical inhibitors of in vitroaggregation of an amino-terminal fragment of mutant huntingtin offer promise for the development of potentially therapeutic compounds that also target the deleterious conformational property of full-length mutant huntingtin. Methods ======= Chemical library, enzymes and antibodies ---------------------------------------- A library containing 1040 small chemical compounds consisting of FDA-approved drugs and bioactive natural products, the National Institute of Neurodegenerative Diseases and Stroke Custom Collection (NCC), was provided in thirteen 96-well plates by MicroSource Discovery Systems, Inc (Gaylordsville CT). The complete list of NCC compounds is available \[[@B49]\]. All compounds were dissolved in 100% DMSO at a concentration of 10 mM. Thrombin was purchased from Amersham Pharmacia Biotech (Piscataway NJ). Anti-huntingtin antibody HP1, used in the screening assay was described by Persichetti et al. \[[@B50]\]. AP229 used in the cell-based assay was previously described and was a gift of Dr. A.H. Sharp \[[@B25]\]. GST-huntingtin construct and expression --------------------------------------- A recombinant pGEX-2TK expression vector with cDNA fragment encoding amino terminal 171 amino acids of human huntingtin with a polyglutamine tract of 58 was used to prepare the GST-Q58-htn fusion protein. The GST-Q58-htn was overexpressed in BL21 cells and purified by affinity chromatography over glutathione-sepharose 4B beads (Amersham Pharmacia Biotech). The purified proteins were stored at concentration of 2.0 mg/ml at -80°C. Aggregation assay ----------------- For primary screening of the chemical library, 1 μl of 4.0 mM small compound stock, diluted from the original plates, was placed in wells of 96-well plates. The fusion protein, GST-Q58-Htn was mixed with thrombin (0.5 unit/1 μg protein) at a concentration of 20 μg/ml in a buffer of 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2.5 mM CaCl~2~, 1.0 mM EDTA. The mixture was immediately distributed into the 96-well plates containing diluted compounds at 40 μl/well and mixed well. The final concentration of the small compounds was 100 μM. After 24 hours incubation at room temperature, the reaction was stopped by adding 10 μl of 10% SDS/50 mM 2-mercaptoethanol to each well followed by boiling in a PCR machine for 5 minutes. The mixture from each well was filtered through a cellulose acetate membrane ((0.2 μm, GE Osmonics labstore, Minnetonka MN) by using a 96-well ELIFA (Pierce Biotech). The aggregates retained on the membrane were detected by a specific anti-huntingtin antibody, HP1 (diluted 1:1000), followed by incubation with peroxidase conjugated anti-rabbit antibody (diluted 1:10,000, Sigma). Signals from SDS insoluble aggregates were scanned and quantified by using ImageMaster Totalab image analysis software (Amersham Pharmacia Biotech). In the secondary screening, all steps were the same except the final concentration of compound in each well was reduced to 10 μM and the GST-Q58-Htn/thrombin mixture was preincubated for 45-minutes at room temperature and clarified by centrifugation at 28,000 × g for 30 minutes before being added to the test wells. For IC~50~determinations, the in vitroaggregation assay and signal quantification were performed as in the second screening but varying the final concentration of input drug. The data for each inhibitor were obtained from at least two independent experiments in which every sample was analyzed in triplicate with Prism 3.0 software (Graphpad Software, Inc., San Diego, CA). Striatal cell line assay ------------------------ Conditionally immortalized wild-type Hdh^Q7/Q7^striatal neuronal progenitor cells (ST7/7) expressing endogenous normal huntingtin, and homozygous Hdh^Q111/Q111^striatal neuronal progenitor cells (ST111/111), expressing endogenous mutant huntingtin with 111-glutamines, have been described previously \[[@B25]\]. The striatal cell lines were grown at 33°C in Dulbecco\'s modified Eagle\'s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 1% nonessential aminoacids, 2 mM L-glutamine and 400 μg/ml G418 (Geneticin (GIBCO-BRL, Life Technologies, Gaithersburg, MD). In the immunofluorescence and confocal analysis experiment, wild-type ST7/7 and homozygous mutant ST111/111 cells, grown to confluence on glass coverslips, were treated with 6 different drugs at four different concentrations (0.5, 5, 10 or 50 μM) for 30 min at 33°C. After the treatment the drugs were removed and the cells washed twice in PBS. The cells were then fixed by incubation in 4% formaldehyde for 15 min, permeabilized by 0.1% Triton X-100 for 5 min and incubated for 30 min with blocking solution (1% bovine albumin in PBS). Coverslips were then incubated in primary antibody (AP229 1:500 dilution) for 2 h at room temperature and washed three times in PBS before a further 1 h in blocking solution containing the secondary antibody (goat anti-rabbit Cy3, Jackson ImmunoResearch, West Grove, PA. USA). After three washes in PBS coverslips were mounted onto glass slides with Vectashield (Burlingame, CA. USA) and the images were analyzed with a laser confocal microscope (Bio-Rad, Hercules, CA. USA) using 20 × objective. Cell death was quantified by scoring the percentage of cells with apoptotic nuclear morphology, i.e. condensed or fragmented nuclei, under the confocal microscope. In each case five to ten randomly selected fields were counted, comprising at least 200 cells, and each experiment was repeated 3 times. Authors\' contributions ======================= JW carried out the library screen and aggregation assays. SG carried out the cell-based assays. MEM and JFG contributed to the conception of these studies. JW and JFG drafted the manuscript and MEM and SG contributed to its final version. All authors read and approved the final manuscript. Acknowledgements ================ This work was supported by grants from NINDS (NS16367) to JFG and MEM, (NS32765) to MEM and the Huntington\'s Disease Society of America\'s Coalition for the Cure. JW received a fellowship from the Harvard Center for Neurodegeneration and Repair. We thank Jill Heemskerk for organizing the NINDS neurodegenerative disease drug screening consortium and to consortium members for sharing data prior to publication and for helpful discussions. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Schematic diagram of the aggregation screening assay (A) and typical results (B)A: Scheme of an in vitromutant huntingtin aggregation assay modified for drug screening. In the primary screening, the mixture of fusion protein, GST-Q58-Htn (20 μg/ml) and Thrombin (0.5 unit/μg protein) was immediately distributed into the 96-well plates containing diluted compounds at 40 μl/well. The final concentration of the small compounds is 100 μM. 10 μl of 10% SDS/50 mM 2-mercaptoethanol was added into each well to stop the reaction after 24 hours incubation at room temperature. The aggregates were separated by filtering through a cellulose acetate membrane (0.2 μm). Immunoblotting was done with a specific anti-huntingtin antibody, HP1, followed by incubation with peroxidase conjugated anti-rabbit antibody. The signals of the retained aggregates were scanned and quantified. In the secondary screening, compounds tested positive in were tested at 10 μM and a 45-minute incubation at room temperature was followed after mixing the protein and enzyme. B: A typical immunoblot of the huntingtin aggregation inhibitor screening. The aggregates retained on the membrane were visualized by ECL. The intensity of dot reflects the amount of aggregates. The blot shows the positions of each drug in a 96-well plate. Congo Red: positive control (rowE, column12 and rowF, column12). DMSO: negative control (rowG, column12 and rowH, column12). Drugs in wells F2, E4, E9 and D3 are huntingtin aggregation inhibitors. ::: ![](1471-2202-6-1-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Distribution of potential huntingtin aggregation inhibitors in the NCC library. Most compounds show no or little inhibitory effect on huntingtin aggregation. The potential inhibitors are evenly distributed across the whole chemical library. Due to very high background on particular regions of some dot blotting membranes, some compounds (compounds around 200) show a high apparent promotional effect that is artefactual (as shown in \~-70% inhibition). The 8 compounds located in column 5 of plate 9 were missed and were tested in the second screening at 10 μM. ::: ![](1471-2202-6-1-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Ten most potent aggregation inhibitors from the NCC library ::: ![](1471-2202-6-1-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Dose-response curve of mutant huntingtin aggregation inhibitors. Compounds were added in 1 μl DMSO to 40 μl 20 μg/ml (0.25 μM) GST-Q58-Htn pre-incubated with thrombin. The reaction was carried out at room temperature for 24 hours. The intensity of each dot of the dot blot was normalized to the DMSO control. Assays were performed twice, in triplicate at each concentration each time (n = 6). ::: ![](1471-2202-6-1-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Localization of huntingtin in wild type and mutant striatal cell from knock-in mice. Cells were cultured for 48 hours and immunostaining was done with a specific antibody against huntingtin, AP229, followed by Cy3 labeled secondary antibody. The signal was visualized by confocal microscopy (magnification: ×20). A. The localization of full-length huntingtin of striatal cell lines: ST7/7 and ST111/111 with 2.5% DMSO. B. The localization of huntingtin of mutant striatal cell, ST111/111, after treated with drugs at 10 μM. ::: ![](1471-2202-6-1-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Percentage of ST111/111 cells showing cytoplasmic/nuclear huntingtin localization comparable to wild-type ST7/7 cells in response to chemical treatment\* ::: Compounds Concentration (μM) ---------------------- ------------------------ ------------------- ------------------ -------------- Gossypol-acetic acid 0 4.5 ± 4.3% 3.5 ± 3.5% 15.9 ± 11.1% Gambogic acid 1 ± 2.6% 0 2.1 ± 4.6% Toxic (100%) Juglone 10.4 ± 12.4% 37.9 ± 19.9% 68.8 ± 18.4% N.A. Celastrol 8.7 ± 8.7% 69.2 ± 3.8% (1%) 78.1 ± 3.5% (4%) N.A. Sanguinarine 0 (4.3%) 3.7 ± 5.2% (5.5%) 0 (22.5%) N.A. Anthralin 4.6 ± 7.6% 6.6 ± 9.7% 9.6 ± 12.1% 11.8 ± 14.4% ST111/111 cells with both nuclear and cytoplasmic staining were counted, as well as total cells and dead cells. The effectiveness of the drugs was measured by percentage of cells with nuclear and cytoplasmic staining versus total cells. In untreated cultures, 89% of ST7/7 striatal cells show both nuclear and cytoplasmic immunostaining, while only 1% of ST111/111 striatal cells show this pattern. Entries in the table represent the average values from 5--10 different microcope fields ± standard deviation. Numbers in brackets indicate percentage of dead cells in cases where toxicity was observed. :::	PubMed Central	2024-06-05T03:55:52.520423	2005-1-13	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548150/", "journal": "BMC Neurosci. 2005 Jan 13; 6:1", "authors": [ { "first": "Jin", "last": "Wang" }, { "first": "Silvia", "last": "Gines" }, { "first": "Marcy E", "last": "MacDonald" }, { "first": "James F", "last": "Gusella" } ] }
PMC548151	Background ========== Lung cancer is the leading cause of cancer related deaths all around the world. About 80% of all lung cancers are non-small cell lung cancer (NSCLC) and more than 50% of these patients present with locally advanced or metastatic disease. Meta-analysis of several randomized trials have demonstrated a modest survival advantage for treatment with cisplatin-based regimens in patients with advanced stages of NSCLC \[[@B1],[@B2]\]. Furthermore, chemotherapy (CT) also has been shown to ameliorate symptoms and increase quality of life \[[@B3]\]. Addition of second generation CT regimens with cisplatin or carboplatin plus newer agents, such as taxanes (paclitaxel and docetaxel), gemcitabine, vinorelbine have shown increased response rates and 1-year survival ratios, but overall survivals have not been altered \[[@B4]-[@B6]\]. Being the first of the taxane antimicrotubule agents, paclitaxel (P) demonstrated overall response rates of 21--24% and 1-year survival rates of 37--42% in the phase II trials where it was used as a single agent \[[@B7],[@B8]\]. Antiangiogenic effect of P has also been reported (9). Carboplatin (C) has also demonstrated comparable activity but better toxicity profile than cisplatin in the treatment of advanced NSCLC \[[@B10],[@B11]\]. P and C used in combined chemotherapy regimens have significant activity in NSCLC. PC given every three weeks is considered to be one of the standard regimens being used worldwide \[[@B10]\]. The weekly administration of P is active, dose intense, and has a favorable toxicity profile. To evaluate the efficacy and toxicity of C and day 1 and 8 P in advanced stage NSCLC, we retrospectively reviewed 51 consecutive patients receiving this CT regimen. Methods ======= All patients with stage III or IV NSCLC treated at Medical Oncology Units of Marmara University Hospital, Dr. Lutfi Kirdar Research and Training Hospital, SSK Sureyyapasa Chest and Cardiovascular Diseases Hospital and Gulhane Military Medical Academy Hospital within July 2002 and August 2003 were considered for this protocol. Eligible patients were required to have pathologically proven NSCLC, stage III or IV disease at presentation or progressed after surgery, performance status (PS) ECOG 0--2, objective measurable disease, adequate bone marrow functions (white blood cell count ≥ 3500/mm^3^, hemoglobin ≥ 9 g/dl, and platelet count ≥ 100000/mm^3^), and adequate liver functions (bilirubin ≤ 1.5 mg/dl and alanine aminotransferase ≤ 2 times upper limit of normal and ≤ 5 times upper limit of normal for the patients with liver metastases) and kidney functions (creatinine ≤ 1.5 mg/dl). No prior chemotherapy or radiotherapy (except to bone metastases for palliation) was allowed. Patients presenting with known central nervous system (CNS) disease and uncontrolled cardiac arrhythmia were excluded from this study and they were treated with other chemotherapy regimens (single agent or combination of platinum and vinorelbine, docetaxel or gemcitabine). Patients were treated with P (112.5 mg/m^2^/day) on days 1 and 8, followed by C (AUC 5/6) on day 1, repeated in every three weeks. Both drugs were diluted in 250 ml of normal saline and given intravenously (IV) over 1 hour. No growth factors were administered. Anti-allergic premedication included IV diphenhydramine 50 mg, IV ranitidine 50 mg, and IV dexamethasone 16 mg 1 hour prior to P administration. Toxicity evaluation and routine physical examination were performed in every 3 weeks. Complete blood count (CBC) was done on days 1 and 8 of each cycle, liver and kidney function tests on every 2 cycles. Cranial computed tomography scans (CT), magnetic resonance imaging (MRI) and bone scans were performed as clinically indicated. Side effects of the treatment were graded according to the National Cancer Institute Common Toxicity Criteria (CTC), version 2.0 \[[@B12]\]. Colony stimulating factors were not used. Response was evaluated with CT of chest and/or abdomen on every 3^rd^cycle and standard World Health Organization (WHO) criteria were used to determine response \[[@B13]\]. Independent of the stage at presentation, patients having partial response (PR), stable disease (SD) or progressive disease (PD) during CT were consulted for radiotherapy (RT) for either primary treatment or palliation. The treatment was stopped for patients with PD. Patients with CR, PR or SD after 3 cycles continued their treatment. PC was given for maximum of 6 courses to the patients having PR or SD. Patients were irradiated with CT based treatment planning and multiple fields arrangements with custom blocking to all fields and involved hilar and mediastinal lymph nodes up to 40--41.4 Gy. Boost was given to the primary tumor. Total dose of 60--61.2 Gy was administered in 1.8--2 Gy daily fractions for 5 days a week and completed in 6 weeks. Statistical analysis -------------------- Overall survival (OS) and time to progression (TTP) were assessed from the date of diagnosis to the date of death (any cause) and the date of objective disease progression (death was considered a progression event in patients who died before disease progression), respectively. Survival rates were calculated by using the Kaplan-Meier method \[[@B14]\]. The pre-specified prognostic value of age (\< 60 years vs. ≥ 60 years), gender, PS (0 vs. 1--2), histology (adenocarcinoma vs. squamous cell vs. NSCLC), stage (III vs. IV), smoking history (present vs. absent), and response after third cycle of CT (PR vs. other) were evaluated in univariate and multivariate analyses. Log rank test was used for univariate survival analysis \[[@B15]\]. The multivariate Cox proportional hazard model was applied to identify the variables that can independently influence survival. Results ======= The data of 51 patients receiving PC treatment were collected retrospectively between July 2002 and November 2003. Median follow-up time was 7 months (range, 1--20). Median age was 58 years (range 39--77) and 45% of patients were 60 year-old or above. Eighty percent were male. PS was 0 in 57% of patients and 67% had presented with stage IV disease. Most frequent metastatic sites were the other lung (17), adrenal (10), liver (7) and bone (7). Eighty-two percent of the patients had smoking history, median of which was 40 pack-years (range, 0--135). Patients\' baseline characteristics are presented in Table [1](#T1){ref-type="table"}. The median number of cycles administered was 3 (range, 1--6). Seven patients (14%) did not complete the first 3 cycles either due to death (2), progression (3), grade 3 hypersensitivity reaction to P (1) or lost to follow up (1). Best evaluable response was PR in 45% and SD in 18%. Only 22 (43%) patients continued the treatment after the 3rd course. At the end of treatment of these 22 patients 10 (46%) had PR and 6 (27%) had SD, but the other 6 patients (27%) had PD. No complete remission was seen. Twelve patients (24%) received local RT and 4 of these patients were given low dose gemcitabine (75 mg/m2/week × 5--6 weeks) as radiosensitizing agent. Of these 12 patients 3 presented with stage IIIA and all had PR to PC therapy. But of the 5 patients with IIIB disease who were irradiated only one patient had PR, 3 had SD and another one had PD after the 3 cycles of CT. Four patients with stage IV were offered RT for palliation. Thirteen patients (25%) received 2nd line CT at progression and of those only one patient had PR and another SD to this treatment. For 2nd line CT gemcitabine ± cisplatin or C was used in 10 patients (78%) and the rest received other agents like vinorelbine, C or docetaxel. Details of this data can be seen in Table [2](#T2){ref-type="table"}. At a median follow-up of 7 months 25 (49%) patients died and 35 patients (69%) progressed. Median OS time was 11 ± 2 months (95% CI; 6 to16), 1-year OS ratio was 44% (Figure [1](#F1){ref-type="fig"}). Median TTP was 6 ± 1 months (95% CI; 4 to 8), 1-year progression free survival (PFS) ratio was 20% (Figure [2](#F2){ref-type="fig"}). The most frequent toxicity related symptoms were asthenia (61%), neuropathy (42%) and anorexia (35%). We observed the following grade 3 toxicities: asthenia (10%), neuropathy (4%), anorexia (4%), anemia (4%), hypersensitivity to P (2%), nausea/vomiting (2%), diarrhea (2%) and neutropenia (2%). Two patients (4%) died of febrile neutropenia due to a three day delay in referral to hospital after the onset of fever \> 38°, although they were warned about the side effects of the therapy. Doses of CT was reduced or delayed in 12 patients (24%) (Table [3](#T3){ref-type="table"}). Univariate analysis showed that patients presenting with PS of 0, stage III disease and having PR after the 3rd cycle of PC have statistically higher OS (p = 0.015, p = 0.018 and p = 0.047, respectively)(Table [4](#T4){ref-type="table"}). PS and stage of the disease at presentation and response to the CT after the 3rd cycle were also statistically significant independent prognostic factors influencing the OS in multivariate Cox regression analysis (p = 0.034, p = 0.049 and 0.021, respectively). Discussion ========== Paclitaxel and carboplatin have been shown to be an effective and well tolerated CT regimen in advanced stage NSCLC \[[@B10]\]. PC given once in every three weeks is one of the most widely used standard schedules worldwide based on the spectrum of activity and the ease of administration. This regimen results in an objective response rate of 17--25% with a median survival time of 8 months in stage IIIB and IV NSCLC patients. The major toxicities of this regimen are neuropathy and neutropenia \[[@B10],[@B16]\]. Weekly P is a relatively new strategy for lowering toxicity and increasing dose-intensity and possibly efficacy. Alvarez et al. have used weekly P on patients who progressed or remained stable on P administered in every three weeks and reported that it can induce response in 62.5% of patients with low toxicity \[[@B17]\]. Akerley has also studied weekly P administration on phase I and phase II settings \[[@B18]-[@B20]\]. They started with a P dose of 175 mg/m^2^/week × 6 every 8 weeks in the phase II trial, but they had to reduce the dose up to 50% due to primarily neutropenia and neuropathy with extended therapy. Therefore, they recommended 150 mg/m^2^as the weekly dose of P \[[@B20]\]. Weekly dose of P in combination with cisplatin or C had been administered in NSCLC patients by Belani et al. \[[@B21],[@B22]\]. They used this combination in a multicenter three arm trial in 401 patients with stage IIIB and IV disease \[[@B21]\]. In that trial P was given 100 mg/m^2^/week for 3 weeks out of 4 week cycles in arms I and II, with C either AUC of 6 on day 1 or AUC of 2 on days 1, 8 and 15 of each of four 4-week cycles. Arm III of this trial consisted of P (150 mg/m^2^) and C (AUC = 2) given weekly for 6 out of 8 weeks for a total of two cycles. Greater percentage of the patients on arm I received intended CT (30% of P and 55% of C) compared with the other arms (28--29% of P and 21--22% of C). Patients on arm I received more than half of the planned C dose. The main reasons for discontinuation of therapy were progression of disease (31%) and adverse events (15%). Median time to progression and median survival time were significantly higher for arm I than arm II for patients with stage IIIB disease. Performance status of the patients was also statistically related to the survival times. Patients with PS-0/1 had longer median PFS with treatment arm I than arm II and patients with PS-2 had higher median OS with arm I than arm II. Although arm I was the most easily tolerable schedule between the three arms, grade 3 or 4 neutropenia was observed in 22% of the patients included. In this trial treatment arm I had a response rate of 32%, median TTP of 6.9 months, median OS time of 11.3 months and 1-year survival rate of 47%. In our study, response rate was 45%, median TTP was 6 months, median OS time was 11 months and 1-year survival rate was 44%. The majority of our patients comprised of stage IIIB and IV disease, similar to the patient group in Belani\'s study resulting in similar response rates and survival data \[[@B21]\]. These results also seem more effective than the regimen given once in every three weeks of the same drugs \[[@B10],[@B16]\]. We used the standard dose of P (225 mg/m^2^) given in every three weeks and divided into two consecutive weeks. C dose was calculated according to Calvert formulation with an AUC of 5. This is a lower dose than the dose of C being used in other phase III trials in the literature. In our study only 4 patients (8%) had dose reduction of 10% and 16% of patients had treatment delays of 1 week because of side effects. According to this data, 76% of patients have received the total planned doses of the drugs on scheduled date. Two patients (4%) died of febrile neutropenia due to a three day delay in referral to hospital after the onset of fever \> 38°, although they were warned about the side effects of the therapy. It is worth mentioning that none of our patients received any colony stimulating factors. We have shown that the response to treatment after the third cycles of CT was one of the independent prognostic factors influencing OS. It has already been reported by Socinski et al. that 4 cycles of CT give the maximum benefit which could be obtained from CT in patients with stage IIIB and IV NSCLC \[[@B23]\]. Smith et al. also studied 3 cycles versus 6 cycles of CT in the same group of patients and failed to show any survival advantage for longer treatment durations \[[@B24]\]. In addition, there was an increase in the side effects such as fatigue, nausea and vomiting in the patients receiving six courses. It has been shown that PC combination has relatively mild toxicity profile. Belani et al. observed in their phase I trial that patients who received the PC combination in every three weeks experienced less severe thrombocytopenia than would be expected from C alone. In the view of this finding they suggested that there was a platelet-sparing effect of P on the dose-limiting thrombocytopenia side effect of C \[[@B25]\]. This phenomenon was also shown by Akerley \[[@B18]\] and Kearns \[[@B26]\]. Akerley reported that platelet counts rose by 17000/mL/week with weekly P administration \[[@B18]\]. Belani also speculated on the mechanism for this platelet protective effect and said that it may involve some alteration of megakaryocytopoiesis or thrombocytopoiesis, which could result in increased levels of endogenous thrombopoietin or other cytokines \[[@B27]\]. Kearns et al. suggested that prior exposure to P may suppress the inhibition of platelet formation, which is associated with C \[[@B26]\]. None of our patients experienced thrombocytopenia during our CT treatment with day 1 and 8 P with day 1 C on every three weeks. One of the most frequent side effects during our treatment was neuropathy, but it was usually mild (Grade 1 or 2), only 4% of our patients experiencing grade 3 sensory neuropathy. Grade 3 or 4 neuropathy has been reported to be 10--20% in schedules given every three weeks \[[@B10],[@B16]\]. Belani reported 3--13% of grade 3 or 4 neuropathy, but the incidence was lower for arms 1 (P given weekly and C every four weeks) and 2 (P and C both given weekly), at only 5% and 3%, respectively \[[@B21]\]. This result for arm 1 is similar to the neuropathy rate in our study. Besides the reduced toxicity, weekly administration of P also increases the drugs\' anti-angiogenic and apoptotic effects. The metronomic schedule of P has been studied widely during the last few years. P had been shown to inhibit endothelial cell proliferation, motility, invasiveness, and cord formation both in vitro and in vivo Matrigel assays in a dose dependent manner \[[@B9]\]. Belani et al. randomized the patients having objective response to weekly P and C regimens into two arms (maintenance and observation arms). Patients were either treated with weekly P (70 mg/m^2^/week × 3 weeks out of four weeks cycles) in maintenance arm or observed until disease progression has occurred. They reported that the maintenance arm was compared to the observation arm and had a median PFS of 38 weeks vs. 29 weeks, median OS of 75 weeks vs. 60 weeks, respectively \[[@B21]\]. Although there was not a statistically significant difference between the two arms, authors concluded that this might be a result of low number of patients enrolled in the study (only 65 patients in each arm). It is not yet known whether these responses have an anti-angiogenic basis, or whether such responses will translate into a significant prolongation of survival. Although our study is a retrospective analysis, it is one of the few manuscripts on this PC scheduling in NSCLC in the literature. Conclusions =========== Paclitaxel on day 1 and 8 and carboplatin every three weeks is a practical and fairly well tolerated outpatient regimen. This regimen seems to be comparably active to regimens given every three weeks. This schedule needs to be further evaluated by well planned randomized phase III trials where it could be compared to the standard regimens in patients with advanced stage NSCLC. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= PFY designed the study, followed the patients, collected the data, performed the statistical analysis and drafted the manuscript. NST followed the patients and helped with the manuscript. MG followed the patients and helped with statistical analysis. NFH, OT, AO, TS, MA followed the patients. RA confirmed the diagnosis. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/10/prepub> Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Overall survival Kaplan-Meier curve ::: ![](1471-2407-5-10-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Progression free survival curve ::: ![](1471-2407-5-10-2) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Patient characteristics ::: Characteristics Number of patients Percentage (%) ----------------------------- -------------------- ---------------- Age, years Median 58 Range (39--77) Gender Male 41 80 Female 10 20 Performance Status 0 29 57 1 17 33 2 5 10 Stage IIIA 3 6 IIIB 14 27 IV 34 67 Histology Adenocarcinoma 16 31 Squamous cell carcinoma 18 36 NSCLC 17 33 Sites of metastases Lung 17 50 Adrenal 10 29 Bone 7 21 Liver 7 21 Distant LAP 3 9 Number of metastatic organs 1 24 71 2 9 26 3 1 3 Smoking history Yes 42 82 No 9 18 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Response rates and second line treatments with CT and RT ::: Number of patients Percentage (%) ----------------------- -------------------- ---------------- Number of PC received 1 2 4 2 5 10 3 20 39 4 3 6 5 3 6 6 18 35 Response to PC PR 23 45 SD 9 18 PD 16 31 Not assessed 3 6 Second line CT 13 G ± Cis / C 10 78 Other 3 23 Local RT 12 Stage IIIA 3 25 Stage IIIB 5 42 Stage IV 4 33 CT: Chemotherapy; RT: Radiotherapy; P: Paclitaxel; C: Carboplatin; G: Gemcitabine; Cis: Cisplatin ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Toxicities seen during PC treatment ::: Toxicity Overall (%) Grade 1--2 (%) Grade 3 (%) Grade 4 (%) ----------------------- ------------- ---------------- ------------- ------------- Neutropenia 12 10 2 Febrile neutropenia 4 4 Anemia 22 18 4 Asthenia/Fatigue 61 51 10 Neuropathy 42 38 4 Anorexia 35 31 4 Arthralgia 31 31 Nausea/Vomiting 22 20 2 Diarrhea 8 6 2 Mucositis 6 6 Hypersensitivity to P 2 2 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Prognostic factors in the univariate analyses for overall survival ::: Variables Median OS (months) p ------------------------------- -------------------- --------- Age 60 years and over 11 ± 2 0.87 Less than 60 years 12 ± 3 Gender Male 9 ± 3 0.22 Female 15 ± 6 PS 0 15 ± 3 0.015\* 1--2 6 ± 2 Histology Adenocarcinoma 15 ± 5 0.73 Squamous cell 13 ± 7 NSCLC 11 ± 3 Stage at presentation III NR^†^ 0.018\* IV 9 ± 2 Smoking history Yes 9 ± 2 0.20 No NR^†^ Response after 3 cycles of CT PR 13 ± 2 0.047\* Other 6 ± 1 \* Statistically significant † Mean OS is 15 ± 2 months PS: ECOG Performance status; NR: Not reached; CT: Chemotherapy :::	PubMed Central	2024-06-05T03:55:52.523340	2005-1-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548151/", "journal": "BMC Cancer. 2005 Jan 25; 5:10", "authors": [ { "first": "Perran F", "last": "Yumuk" }, { "first": "Nazim S", "last": "Turhal" }, { "first": "Mahmut", "last": "Gumus" }, { "first": "Nilgun F", "last": "Hatabay" }, { "first": "Orhan", "last": "Turken" }, { "first": "Alper", "last": "Ozkan" }, { "first": "Taflan", "last": "Salepci" }, { "first": "Mehmet", "last": "Aliustaoglu" }, { "first": "Rengin", "last": "Ahiskali" } ] }
PMC548152	Background ========== The diagnostic stage of colon cancer determines survival. Patients diagnosed with localized tumours have a 75% probability of 5 year survival, whereas patients diagnosed with distant metastases only have a 5--10 % probability of 5 year survival (reviewed in \[[@B1]\]). Recently a number of studies have been published in which Surface Enhanced Laser Desorption/Ionisation-Time Of Flight/Mass Spectrometry (SELDI-TOF/MS) has been applied to biological samples from patients with various forms of cancer \[[@B2]-[@B4]\] leading to the identification of protein markers with clinical potential. Here we present a SELDI-TOF/MS study of colon cancer serum and tumours. We show that the expression of Human Neutrophil Peptides -1, -2 and -3 (HNP 1 -3), also known as alfa-defensin-1, -2 and -3, is upregulated in the tumour microenvironment, as compared to normal colon tissue. This finding is reflected in serum. We find that HNP 1-3 is present in elevated concentrations in serum from patients diagnosed with tumours in the colon, as compared to serum from a healthy control group matched by age and gender. By size-exclusion analysis we add to the existing evidence that HNP 1-3 bind to high mass plasma proteins, explaining the presence of HNP 1-3 in serum. By microflow analysis, we show that HNP 1-3 purified from colon tumours are lethal to mammalian cells. The HNP 1-3 peptides are part of the defensin family of peptides (reviewed in \[[@B5]-[@B7]\]), which are a fundamental component of the immune system and have the capacity to kill / inactivate a broad range of pathogens. Defensins are also known to function as regulators of both the innate and the adaptive immune system. We discuss the possible effects of HNP 1-3 in the tumour microenvironment. Methods ======= Biological samples ------------------ All biological samples were obtained by trained staff at Glostrup Hospital, Denmark. Written consent was obtained from all donators. Permission was obtained from the Danish Scientific Ethical Committee and the Danish Data Protection Agency. Tissue screening ---------------- Normal colon tissue samples and colon tumour samples were obtained from the removed fragment of the patient\'s colon after surgical treatment for colon cancer. Tissue samples were stored at -80°C until use. The protein content was extracted from the tissue: 100 mg tissue sample was thawed on ice and homogenised on a Wheaton Overhead Stirrer for 2 minutes at speed step 2, in 500 ul Lysis buffer (100 mM TRIS-HCl, pH 8.0, 9.5 M UREA, 2% CHAPS). The samples were centrifuged at 14,000 rpm for 10 minutes and the pellet was discarded (repeated twice). The tissue protein extracts were stored at -80°C until use. Pilot studies were performed on different chips (data not shown) and the NP20 (Normal Phase) (Ciphergen) chip was chosen for the tissue screening. NP20 chips were placed in Bioprocessor (Ciphergen) and pre-treated with 50 ul tissue binding buffer (50 mM TRIS-HCl, pH 8.0) for 5 minutes on shaker (250 rpm) (repeated twice). 5 ul tissue protein extract was diluted in 50 ul tissue binding buffer and incubated in Bioprocessor on NP20 chips for 40 minutes at room temperature on shaker (250 rpm). Spots were washed twice in 250 ul tissue washing buffer (50 mM TRIS-HCl, pH 8.0) for 5 minutes. The chips were air dried for 10 minutes, followed by treatment with two times 0.6 ul 100% sinapinic (SPA) matrix solution. SPA was obtained from Ciphergen in 5 mg aliquots and dissolved (150 ul MQ water, 150 ul acetonitrile, 1.5 ul Tri-Fluoro-acetic-Acid (TFA)) immediately before the screenings. Serum screening --------------- Colon cancer serum samples were obtained from patients before surgical treatment. Normal serum was obtained from a group of healthy individuals matched by age and gender to the cancer patients. All serum samples were stored at -80°C until use. Serum pilot studies were performed on different chips to monitor the presence of HNP 1-3 in serum (data not shown). The immobilised metal affinity capture (IMAC30) chip was chosen for the actual screening and was pre-treated with nickel before analysis: 5 ul 100 mM NiSO4 were added to each spot and left on shaker (250 rpm) for 5 minutes (repeated twice). The chips were placed in Bioprocessor and incubated with 100 ul MQ for 5 minutes on shaker (250 rpm). Each spot was treated with 50 ul serum binding buffer (100 mM TRIS-HCl, pH 7.5, 500 mM NaCl, 0,1% Triton X-100) and left on shaker for 5 minutes (250 rpm). Serum samples were thawed on ice and 1 ul serum was diluted in 50 ul serum binding buffer and applied to spots and left on shaker (250 rpm) at room temperature for 40 minutes. The sample solution was removed and the spots were washed twice in 200 ul serum washing buffer (100 mM PBS, pH 7.4, 700 mM NaCl), followed by one wash in 200 ul MQ water. The chips were removed from the Bioprocessor and left to air dry for 20 minutes followed by treatment with two times 0.6 ul SPA. Only freshly made matrix solutions were used and the instrument was calibrated daily. Cancer and normal samples were run side by side. The chips were analysed on a PBS II instrument (Ciphergen). All spectra in each screening were normalised based on total ion current. Purification and identification of HNP 1-3 ------------------------------------------ 100 ul protein extract from colon tumour tissue in tissue lysis buffer was loaded unto a RP-HPLC column (uRPC C2/C18 ST 4.6/100, Pharmacia Biotech, Flow rate: 0.5 ml/min, Fraction size: 0.5 ml) in buffer A (0.065% TFA in MQ-water) and proteins were eluted in a gradient of 0--100% buffer B (0.05% TFA in acetonitrile (ACN)). Elution of peptides was monitored by absorbance at 280 nm. All protein-containing fractions were analysed by Matrix Assisted Laser Desorption/Ionisation-Time of flight (MALDI-TOF) on the PBS II instrument: 1.5 ul fraction was incubated with 0.6 ul SPA on a Gold array (Ciphergen) and left to crystallise, followed by an additional treatment with 0.6 ul SPA and analysed in the PBSII instrument. The HNP 1-3 containing fraction (32% buffer B) was further purified on a peptide gelfiltration column (Superdex Peptide HR 10/30, Pharmacia Biotech, Flow rate 0.9 ml/min, Fraction size: 0.5 ml, Buffer: 50% ACN, 0.1 % TFA). Elution of peptides was monitored by absorbance at 280 nm and protein-containing fractions were again analysed by MALDI-TOF. Purified HNP 1-3 were identified by on-chip trypsin digestion: 10 ul of HNP 1-3 fraction was applied to an NP20 chip and left on shaker (250 rpm) at room temperature for 40 minutes. The solution was removed and the spot was washed twice with 10 ul water. In order to denature peptides prior to digestion, the chip was left on heating block (80°C) for 5 minutes. The chip was cooled on ice for 2 minutes. 10 ul trypsin digestion solution (0.01 ug/ul trypsin in 50 mM NH~4~HCO~3~, pH 8,0) was added, and the chip was left for 10 hours at 40°C in humidity chamber after which the chip was left to air dry for 20 minutes. 1 ul CHCA matrix (prepared as the SPA matrix solution) was added and the peptide map was analysed on the PBS II instrument. Peptide maps of trypsin autodiggest were used as controls. Identification was done with the PepIdent software on the Expasy server. For the reduction experiment, HNP 1-3 were first denaturation by heating (10 minutes at 80°C) followed by treatment with DTT (200 mM, 30 minutes at room temperature) and the peptides were incubated on an NP20 chip and analysed on the PBS II instrument. Size exclusion chromatography of HNP 1-3 ---------------------------------------- 50 ul colon cancer serum was loaded unto a peptide gelfiltration column (Superdex Peptide HR 10/30, Pharmacia Biotech, optimal separation range: 1 to 7 kDa, flow rate: 0.5 ml/min, fraction size: 0.5 ml, buffer: 10 mM Ammonium carbonate, pH: 8.0). Elution of peptides was followed by absorbance at 280 nm. All protein containing fractions were analysed by MALDI-TOF on PBS II (Ciphergen) as described above. Maximum signal intensity of 40 individual peaks was plotted as a function of elution volume and an approximate elution curve was calculated. Study of HNP 1-3 by microflow ----------------------------- For micro flow experiments, canine kidney cells (MDCK cells) were plated onto poly-d-lysine coated cover slips at a concentration 3000 cells/well, grown in Dilbeccoo\'s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) for five days with the result of confluent islands. Microflow was performed in an Eppendorf micromanipulator 5171 and transjector 5246 system mounted on a Leica DMIRBE inverted research microscope. Micro capillaries (borosilicate with filament, Sutter Instruments Company, Novato, California, USA) were pulled to an outer diameter of 0.85 nm on a Sutter P-97 Micropipette Puller. The dye-loaded cells were visualized by excitation at 470 nm and recorded at 509-nm emission using Haupage version 3.3.18038 software and Kappa CF 15/4 MC-S camera (Leica). The MDCK cells were recorded (in CO2 independent media) on the inverted DMIRBE inverted research microscope. The capillary was placed 20 nmover the confluent cells with a constant flow (1300 hPa). The MDCK cells were exposed to peptide and calcein (20 mM) fractions for 60 minutes. Results ======= HNP 1-3 expression in tissue and serum -------------------------------------- We performed pilot studies of colon tumour and normal colon tissue on a variety of chips with different chemical properties and with different binding and washing conditions. Based on these preliminary studies, we found that the expression of three peptides with mass/charge ratio (m/z) values of 3372, 3443 and 3486 (subsequently identified as HNP 2, 1 and 3, respectively), were upregulated in the tumour samples. The three peptides were visible on different chips and under different binding conditions (data not shown). The strongest signal of HNP 1-3 in tissue extract was obtained on the NP20 (Normal Phase) chip, whereas the strongest signal of HNP 1-3 in serum was observed on the IMAC30 (Ni) (immobilised metal affinity capture chip, activated with nickel), and these conditions were chosen for the actual screenings. We emphasize that in general the protein profiles of serum and tissue were very different when using the same protein chip for serum and tissue extract. However, individual peaks were present on several types of chips, and observed in both serum and in tissue extract, for example the characteristic triplet with m/z 3372, 3443 and 3486. Protein extract from 40 colon tumour and 40 normal colon tissue samples were analysed on NP20 chips and 125 colon cancer serum samples and 100 normal serum samples were analysed on IMAC30 (Ni) chips. All spectres in each screening were pooled and normalised based on overall ion current. Each spectrum produced approximately 40 to 90 protein peaks in the range from 2 to 80 kDa (FIG. [1A--C](#F1){ref-type="fig"}). Statistical analysis of the intensity values of HNP 1-3 in the tissue screening (FIG. [2A](#F2){ref-type="fig"}.) showed that HNP 1-3 were significantly upregulated in tumours (p \< 0.0005) and statistical analysis of HNP 1-3 expression in the serum screening (FIG. [2B](#F2){ref-type="fig"}.) showed that HNP 1-3 were significantly upregulated in cancer serum (p \< 2.2e^-16^). Compared to other peptides in the same range, HNP 1-3 showed average signal intensity in most normal colon tissue extract, whereas the HNP 1-3 signal was extremely high in most tumour samples (in some tumour spectres the HNP 1-3 peaks were the strongest of all peaks). In the normal serum samples the HNP 1-3 signals were weak and only slightly stronger in the cancer serum. Identification of HNP 1-3 ------------------------- The markers were purified by RP-HPLC, peptide gelfiltration and on-chip purification, after which they were identified by peptide mapping as HNP-2 (3372 Da), HNP-1 (3442 Da) and HNP-3 (3486 Da) (Table [1A](#T1){ref-type="table"}). The masses correspond to the peptides in their oxidised states with three disulfide bridges. After reduction with DTT, HNP-1 and HNP-2 increased 6 Daltons in mass, due to reduction of the six cysteines (Table [1B](#T1){ref-type="table"}). We were not able to reduce HNP-3. Size exclusion chromatography of HNP 1-3 ---------------------------------------- 50 ul colon tumour extract in Lysis buffer was applied to a peptide gelfiltration column. Elution of peptides was followed by absorbance at 280 nm. All fractions were analysed by MALDI-TOF on the PBS II instrument (Ciphergen). Maximum signal intensity of 40 individual peaks was plotted as a function of the elution volume and an approximate elution curve was calculated (FIG. [3](#F3){ref-type="fig"}). HNP 1-3 peptides were primarily eluted in early fraction together with high mass proteins above 20 kDa and also, but to a lesser degree, in fractions together with other peptides of similar mass interval (2 to 4 kDa) (FIG. [3](#F3){ref-type="fig"}). Cytoxic assay ------------- The cytotoxicity of HNP 1-3 purified from colon tumours was tested by exposing MDCK cells to different fractions purified from colon tumours. Calcein were added to the fractions and the solutions were left to flow over the cells for one hour. By fluorescence microscopy calcein was observed to accumulate in cells exposed to HNP 1-3/calcein fractions, whereas cells treated with fractions containing other (unidentified) tissue peptides did not uptake calcein (FIG. [4C&D](#F4){ref-type="fig"}). Further, by microscopy we observed that cells exposed to HNP 1-3 appeared more diffuse and had enlarged nuclei, indicating apoptosis (FIG. [4A&B](#F4){ref-type="fig"}). Discussion ========== Elevated concentrations of HNP 1-3 in colon cancer serum -------------------------------------------------------- Abnormal concentration of HNP 1-3 in blood has previously been demonstrated in connection with benign conditions. Elevated concentrations of HNP 1-3 following infection (bacterial- / non-bacterial- infection and pulmonary tuberculosis) has been found in plasma, blood and other body fluids \[[@B8]\], and plasma HNP 1-3 concentrations have been shown to be elevated in patients with septicemia or bacterial meningitis \[[@B9]\]. HNP 1-3 have been found in urine from patients with transitional cell carcinoma of the bladder \[[@B10]\] and HNP-1 has been found in salvia of patients with oral carcinomas \[[@B11]\]. Finally, HNP 1-3 are found in excess amounts in tears after ocular surface surgery \[[@B12]\]. Our study is the first that demonstrate elevated concentrations of HNP 1-3 in blood following tumour growth. Elevated concentrations of HNP 1-3 in colon tumours --------------------------------------------------- HNP expression has previously been linked to different types of tumours and cell lines. HNP-1 has been detected in lung tumours \[[@B13]\] and in the submandibular glands of patients with oral carcinomas \[[@B14]\]. By RT-PCR, mass spectrometry and flow cytometric analysis, HNP 1-3 have been shown to be expressed by cell lines deriving from renal cell carcinomas \[[@B15]\] and the expression of a specific HNP precursor peptide has been shown to be upregulated in human leukemic cells \[[@B16]\]. Our results suggest that HNP 1-3 are extremely abundant in colon tumours. This is in agreement with a study of HNP-1 in lung tumours, where the maximum observed level was 26 nanomoles per gram wet tissue \[[@B13]\]. In order for these excessive amounts of peptide to be detectable in serum, the peptides must be present extracellular in the tumour environment, such as observed in studies of HNP 1-3 expression in kidney \[[@B14]\] and brain \[[@B17]\]. Usually tumour expressed peptides are not easily detected in serum or plasma by SELDI-TOF mass spectrometry techniques; the highly concentrated plasma proteins out compete the signal of low abundance peptides. We suggest that HNP 1-3 are detectable in serum only because they are expressed in exceptionally high amounts in the tumour microenvironment. Previous studies indicate that HNP 1-3 expression in tumours primarily originate from tumour invading eosinophils \[[@B13]\] and neutrophils \[[@B14],[@B18]\]. However, it has been shown that the excess amounts of HNP 1-3 observed in urine from bladder cancer patients were often produced by the actual bladder cancer cells \[[@B10]\], and that highly invasive bladder cancer cells produced more HNP 1-3 than less invasive ones (Holterman DA, in press, personal communication). Since our tissue screening is based on comparison of tissue samples, we cannot say whether the peptides are produced by the colon cancer cells or by tumour infiltrating neutrophils. In the case of active inflammatory bowel disease, it is not clear whether epithelial expression of HNP1-3 is induced by the inflammatory state or the whether the peptides are released by adjacent neutrophils and taken up by the epithelial cells \[[@B6]\]. Here it is believed that the peptides provide protection against microbial invasion when the mucosal barrier is damaged (such as is the case in inflammatory bowel diseases). HNP 1-3 are known to stimulate bronchial epithelial cells to upregulate interleukin-8 production \[[@B19]\], a potent neutrophil chemotactic factor. Thus, the upregulated expression of HNP 1-3 in tumours may primarily originate from invading immune cells, but could be initiated by HNP 1-3 producing cancer cells. Size exclusion chromatography of HNP 1-3 ---------------------------------------- We explain the elevated concentrations of HNP 1-3 in colon cancer serum by unspecific binding between HNP 1-3 and high mass plasma proteins. We suggest that the peptides attach to plasma proteins in the tumour area and are carried into the bloodstream. The HNP 1-3 we observe in high mass fractions from size exclusion, could also be explained by multimerisation: In one study it was demonstrated that defensins form voltage dependent channels in lipid bilayer membranes and further conductance investigations suggested that the channels were formed by multimers containing 2--4 molecules \[[@B20]\] and a crystal structure study \[[@B21]\] of HNP-3 revealed an amphiphilic dimmer. We interpret the size exclusion results as evidence for interaction between HNP 1-3 and unidentified high mass proteins through unspecific interactions; the peptides are eluted in very early fractions which would probably require the presence of multimers of more than five peptides and our study does not reveal the presence of multimers containing four, three or two peptides. Further, our interpretation is in agreement with a number of previous studies that show that HNPs are bound to plasma protein in vitroand that high concentrations of HNPs causes precipitation of plasma proteins; specifically 2-macroglubulin and C1 complement \[[@B22],[@B23]\] has been shown to bind defensin. Another study \[[@B24]\] showed that HNP-1 bind to various plasma proteins, notably serum albumin, and it was found that serum, or serum albumin, was able to inhibit the anti-viral activity of HNP-1. This ability to bind to plasma proteins could also explain why HNP 1-3 lysis of mammalian cells is hindered in the presence of serum \[[@B25]\]. Together these observations add to the growing realisation that common plasma proteins may carry disease specific peptides and therefore should not be ignored in biomarker research. Common to beta-defensin 2 (another member of the defensin family) and HNP 1-3 is an uneven distribution of surface charges. Beta-defensin 2 has been shown to bind to a chemokine receptor \[[@B26]\]. It has been suggested that the positively charged cluster found in defensin peptides and chemokines, may play a common role in binding to receptors, but is not important for determining receptor specificity \[[@B27]\]. This surface charge may also explain the binding of HNP 1-3 to plasma proteins. The observation that defensins are localised to lymphocyte nuclei \[[@B28]\], could similarly be explained by unspecific binding to shuttle proteins. The function of HNP 1-3 ----------------------- The exact concentration of HNPs in the tumour microenvironment may decide the in vivofunction of HNP 1-3. One study showed that HNP 1-3 mediates lysis of tumours in a concentration dependent manner \[[@B25]\]. This is in agreement with another study that show that only relatively high concentrations of HNP-1 (10^-4^M) are cytotoxic for human monocytes, whereas lower concentration of HNP-1 (10^-8^to 10^-9^M) increases TNF-alpha production by monocytes \[[@B29]\]. In a study of renal cell carcinoma lines \[[@B14]\] it was shown that HNP 1-3 were cytotoxic to all tested cell lines when present in high concentrations (above 25 ug/ml), but at lower concentration HNP 1-3 stimulated growth of a subset of tumour cell lines. We add to these results by demonstrating that in vivoHNP 1-3 purified from colon tumours are capable of lysing MDCK cells. Our study was based on a 60 minutes microflow study. This screening set-up did not allow us to investigate the minimum concentration of HNP 1-3 necessary for lysis. Conclusions =========== The high concentration of HNP 1-3 observed in tumours and the observation that HNP 1-3 are capable of lysing mammalian cells may lead to the conclusion that the peptides serve to the benefit of the host by killing tumour cells. However, in one study HNP 1-3 were found to bind to HLA-Class II molecules and were capable of reducing the proliferation of a HLA-DR-restricted T-cell line after stimulation \[[@B30]\] and could in this way help the cancer cells avoid local immune recognition. Defensins also regulate the systemic immune response. Through interaction with the chemokine receptor CCR6, beta-defensins recruits dendritic and T cells\[[@B26]\] (discussed in \[[@B31]\]) and HNP 1-3 are capable of recruiting leukocytes to sites of infection in mice \[[@B32]\]. Upregulated immune responses are known to stimulate tumour proliferation; immune cells are actively recruited by tumours to exploit their pro-angiogenic and pro-metastatic effects (reviewed in \[[@B33],[@B34]\]). Whether the high concentrations of HNP 1-3 in the tumour limits the tumour growth or on the contrary stimulate tumour proliferation is not clarified; we emphasise that HNP 1-3 are expressed in inflammatory bowel disease and could be involved in the early stages of carcinogenesis. We suggest that the prominent surface charge on defensins, their unspecific binding to other proteins (such as high mass plasma proteins) and the observed excess amounts of peptides found in tumours, could provide the peptides with broad antagonising effects on numerous receptors in the tumour microenvironment. In this way HNP 1-3 peptides may serve to the benefit of the tumour. Our results add to the evidence that HNP 1-3 bind to high mass plasma proteins. We suggest that the peptides are released in the inflammatory site and passively diffuse into the blood stream. HNP 1-3 have been observed in primary tumours of different tissues and the peptides are known to play a fundamental role in the innate immune system. We suggest that the peptides may serve as markers for colon cancer in combination with established diagnostic tools and as prognostic markers following therapy. Competing interests =================== This study was partly financed by Colotech ltd. and partly by Glostrup Hospital. Jakob Albrethsen, Rikke Bøgebo and Hans Raskov receive salary from Colotech A/S. Steen Gammeltoft, Jesper Olsen and Benny Winther receive salary from Glostrup Hospital. Authors\' contributions ======================= JA performed the SELDI-TOF/MS screenings, the protein identification experiments and the size exclusion study. RB did the statistical analysis, BW did the microflow study, JO obtained the biological samples. HR and SG planned the project. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/8/prepub> Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Protein profiles of serum and tissue.Representative SELDI-TOF/MS spectra of normal colon tissue (FIG. 1A) on the NP20 chip and normal serum (FIG. 1B) on the IMAC30 (Ni) chip. The two spectra differ significantly and each produce a total of 40 to 60 peaks. The majority of the peaks are present in the specified range from 2 to 10 kDa. FIG. 1C Comparison of a typical colon tumor spectrum (above) and a normal colon spectrum (below) in the range from 3 to 4 kDa. The arrows point to the three differentially expressed peptides, subsequently identified as HNP 1-3. The three peptides are expressed in normal colon tissue and the expression is upregulated in the tumor samples. The three peptides are present in normal serum, but are present in higher concentrations in colon cancer serum. The average peak intensity for the three peptides were significantly lower in serum than in tissue. ::: ![](1471-2407-5-8-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Expression of HNP 1-3 in tumors and serum.FIG.2A HNP 1-3 profiles of normal and colon tumor tissue. 40 colon tumor and 40 normal colon tissue samples were analysed on NP20 chips. Differences in mean intensities of HNP 1-3 in normal and colon tumor tissue were statistical significant at 5% level (p \< 0.0005). FIG. 2B HNP profiles of normal and colon cancer serum. Serum samples (125 colon cancer and 100 normal) were analyzed on IMAC30 chips. The mean intensities were significantly different at 5% level (p \< 2.2e-16). The reproducibility was significant for all three peptides in both tissue and serum. The standard deviation of HNP 1, 2 and 3 was 70%, 136% and 57% in normal tissue and 11%, 15% and 8% in tumor tissue. The standard deviation of HNP 1, 2 and 3 was 96%, 154% and 81% in normal serum and 234%, 365% and 282% in cancer serum. The boxplot show the 25th quantile, median, 75th quantile, and whiskers extent to min. and max. values. ::: ![](1471-2407-5-8-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Size exclusion study of HNP 1-3.Protein extract from tumor tissue was separated on a peptide gelfiltration column. The elution volumes of forty (unidentified) peptides was plotted against their respective mass values and an approximate elution curve was calculated. The arrows point to HNP 1-3, which were eluted in two fractions: primarily in the void volume (8 ml) together with high mass proteins (above 20 kDa) and after 14 ml together with peptides of similar mass range (2--4 kDa). We interpret this as evidence for binding between HNP 1-3 and high mass proteins. ::: ![](1471-2407-5-8-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Functional study of HNP 1-3.Normal microscopy (A&B) and flourescence microscopy (C&D) of MDCK cells. MDCK cells were exposed to calcein with (A&C) and without HNP 1-3 (B&D). By fluorescence microscopy (C&D) the cells were observed to uptake calcein only when treated with fractions containing HNP 1-3/calcein (C). Fractions containing unidentified peptides purified from colon tumors were used as negative controls together with calcein and did not stimulate the cells to uptake calcein (D). Cell islands treated with HNP 1-3 appeared diffuse and showed enlarged nuclei, indicating apoptosis (A). ::: ![](1471-2407-5-8-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Identification of HNP 1-3. Table 1A HNP 1-3 peptides were identified by peptide mapping (on-chip trypsin digest). Three sequence specific fragments were produced (615.8, 930.6, 1061.4 Da.). The peptides were identified as HNP 1-3 using the PepIdent server on the ExPASy homepage. Table 1B The measured masses of the HNP 1-3 peptides were approximately 6 Daltons lower than the expected theoretical masses. This deviation was caused by the in vivoformation of three disulfide bridges. By heat denaturation and treatment with DTT we were able to protonate the six cysteines in HNP 1 and 2. (HNP-3 was consistently degraded during the reduction procedure). ::: A -------------------------------------------------- ----------------------------------------------------- ------ ---------------------------------------------------------- 615.8 615.7 +0.1 ACYCR 930.6 930.1 +0.5 IPACIAGE 1061.4 1061.2 +0.2 YGTCIYQ B Measured masses of HNP 1-3 peptides (Dalton) Theoretical masses of HNP 1-3 peptides (Dalton) Measured masses of reduced HNP 1-3 peptides (Dalton) 3371.2 3377.0 (HNP-2) 3376.2 3442.3 3448.1 (HNP-1) 3448.6 3486.3 3292.1 (HNP-3) \- :::	PubMed Central	2024-06-05T03:55:52.526429	2005-1-19	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548152/", "journal": "BMC Cancer. 2005 Jan 19; 5:8", "authors": [ { "first": "Jakob", "last": "Albrethsen" }, { "first": "Rikke", "last": "Bøgebo" }, { "first": "Steen", "last": "Gammeltoft" }, { "first": "Jesper", "last": "Olsen" }, { "first": "Benny", "last": "Winther" }, { "first": "Hans", "last": "Raskov" } ] }
PMC548262	Background ========== First described in 1997, Parachlamydia acanthamoebaeis an obligate intracellular bacterium naturally infecting free-living amoebae \[[@B1],[@B2]\]. It was isolated from Acanthamoebaspp. recovered from the nasal mucosa of healthy volunteers \[[@B1]\]. Later, additional strains of Parachlamydiaceaehave been found within about 5% of Acanthamoebaspp. and once within Hartmanella vermiformis\[[@B2],[@B3]\]. The 16S rRNA sequences of these Parachlamydiaceaeare about 14% different from those of both genera Chlamydophilaand Chlamydia\[[@B2],[@B3]\]. Since the 16S rRNA sequence difference between Chlamydophilasp. and Chlamydiasp. is 6% only, it clearly appears that the speciation between the two latter occurred after the divergence between Parachlamydiaceaeand Chlamydiaceae. Like other Chlamydiales, Parachlamydiaceaecan present two developmental stages: the reticulate body, a metabolically active dividing form, and the elementary body, an infective stage; the crescent body is another infective form, not observed in Chlamydiaceae\[[@B4]\]. Differentiation of the infective stages in reticulate bodies and multiplication of the latter were recently shown to occur within amoebal vacuoles, that may contain hundreds of bacteria \[[@B4]\]. Depending on the symbiotic/pathogenic relationships prevailing between both organisms, the escape of the bacteria from the amoeba may occur either by the release of secreted vesicles or by the lysis of the host \[[@B4]\]. There is a growing evidence of the human pathogenicity of Parachlamydiaceae\[[@B2]\]. For instance, positive Parachlamydiaserologies were shown to be associated with a febrile epidemic \[[@B5]\], community-acquired pneumonia \[[@B6]\], and inhalation pneumonia \[[@B7]\]. The role of Parachlamydia-related bacteria as agents of inhalation pneumonia is further suggested by the temperature-dependent release of the bacteria from their amoebal reservoir \[[@B8]\]. PCR amplification of parachlamydial DNA from monocytes, sputa and bronchoalveolar lavages collected from patients suffering of bronchitis or pneumonia also supports the pathogenic potential of Parachlamydia\[[@B9]-[@B12]\]. The survival of these Chlamydia-like organisms within human macrophages \[[@B13]\] is an additional hint of parachlamydial pathogenicity. Horn et al. \[[@B14]\], by sequencing and annotating the whole genome of the Parachlamydia-related UWE25 contributed much to the understanding of the evolution of chlamydiae. Indeed, they demonstrated that major virulence mechanisms of Chlamydiaceaesuch as the Type Three Secretion System (TTSS) and the Chlamydial Protease-like Activity Factor (CPAF) are also encoded by the chromosome of the evolutionary early-branching ParachlamydiaceaeUWE25. Genome analysis of the parachlamydial endosymbiont also identified Open Reading Frames (ORFs) homologous to Type Four Secretion Systems (TFSS) and characterized by a high G+C content, suggesting that they result from an horizontal transfer. Based on their annotation revealing the apparent absence of genes necessary for DNA transfer, Horn et al. \[[@B14]\] proposed that this TFSS was involved in protein export but not in DNA transfer. To date, numerous genomic islands (GIs) were already identified along whole chromosomal sequences of various bacterial species. For instance, 140 GIs are described in the Islander database, including GIs of proteobacteria, firmicutes, actinobacteria and cyanobacteria \[[@B15]\]. Thus, we wondered whether any GIs were located along the bacterial chromosome of the amoebal endosymbiont UWE25. GIs are genetic elements which length vary from 10 to 200 kb and are inserted in a chromosome after a lateral transfer occurring, in some instances, between phylogenetically-distant microorganisms. Their heterologous origins are generally evidenced by a G+C content different from that of the remaining bacterial chromosome and by the presence of various mobility genes (i.e. involved in transposition, transduction or conjugative transfer), that are occasionally source of GI instability \[[@B16],[@B17]\]. They are often flanked by particular DNA sequences, such as direct repeats or insertion sequences. Moreover, tRNA lociare generally used as insertion sites by GIs for their chromosomal integration \[[@B16]-[@B18]\]. Since no genetic tools are available for the study of this obligate intracellular bacteria, a bioinformatic approach was chosen to locate putative GIs. Results ======= A genomic island is present in the genome of UWE25 -------------------------------------------------- Using standard G+C content analyses of Parachlamydia-related UWE25 chromosome, we observed a G+C-rich region (Figure [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}), similar to that shown by Horn et al. \[[@B14]\]. Using the residual cumulative G+C content analysis adapted from the GC profile of Zhang and Zhang \[[@B19]\], we were able to precisely define a 19-kb region (Figure [1C](#F1){ref-type="fig"}). The presence of 17-bp direct repeats flanking a 100-kb chromosome region (1648 to 1748 kb, Table [1](#T1){ref-type="table"}) that encompasses the 19-kb DNA sequence enabled us to define a new region composed of 100 ORFs (See [additional data file 1](#S1){ref-type="supplementary-material"} for the description of these genes and their location on the chromosome of UWE25). Interestingly, this 100-kb region is characterized by a higher level of local gene coorientation (75/100) than that characterizing the remaining of the genome (1015/1931, 52.6%, p \< 0.001) and by a particular signature in the cumulative GC skew analysis. Two identical gly-tRNAgenes in tandem are located at the proximal end of this 100-kb genetic element (Figure [1A,1C](#F1){ref-type="fig"} and Table [1](#T1){ref-type="table"}). Several mobility genes (eight putative transposases, one recombinase and seven bacteriophage related-proteins) are encoded within the 100-kb region (Figure [1C](#F1){ref-type="fig"}, Table [1](#T1){ref-type="table"}). Thus, this region largely fulfills the accepted criteria of GIs \[[@B16]-[@B18]\]. We termed this newly described GI \"Pam100G\" (Protochlamydia amoebophila, 100-kb, Gly-tRNA) according to the nomenclature used in the Islander database \[[@B15]\]. Mosaicism of the 100-kb genomic island -------------------------------------- Interestingly, this GI can be divided into clearly distinct regions, according to their G+C content (Figure [1B](#F1){ref-type="fig"}, Table [2](#T2){ref-type="table"}). The residual cumulative G+C content analysis highlights a modular structure with different slopes, each linear segment indicates that genes of this unit present a rather constant local G+C content (Figure [1C](#F1){ref-type="fig"}). A positive or a negative slope would indicate that each block of genes presents a G+C content higher or lower that of the UWE25 chromosome, respectively. The first module begins with a direct repeat and two identical gly-tRNAsin tandem. Composed of 28 ORFs, this unit exhibits a G+C content (36.4%) similar to that of the remaining of the genome of UWE25 (1931 ORFs, 36.1%). Sixteen homologs to these 28 genes (57%) were found in databases, 12 of them (75%) exhibiting a best score in BLAST analyses with a ChlamydiaceaeORF (See [additional data file 1](#S1){ref-type="supplementary-material"}). Interestingly, no gene of the other modules of the 100-kb GI exhibited a best hit in similarity analyses with any Chlamydiaceaecounterpart. Some of the genes present in the first module, such as sctNand sctQ, are part of a TTSS also present in Chlamydiaceae. The other TTSS genes are disseminated along the chromosome of UWE25. The presence of some TTSS genes in the first module and of a gene encoding a putative transposase at the distal end of the first module of this 100-kb GI suggests that this first unit was acquired by chromosomal rearrangements. (See [additional data file 1](#S1){ref-type="supplementary-material"} for the results of BLAST analyses). Characterized by a low G+C content (34.1%), the 2^nd^module encodes 18 ORFs. Only five are similar to known protein sequences (28%), four of them being identified as mobility genes (three bacteriophage-related genes and one putative transposase encoding gene). The 3^rd^module (19 kb), exhibiting the second highest G+C content of the UWE25 genome (40.9%), comprises 21 ORFs. Some of these genes were identified as tragenes by Horn et al. \[[@B14]\]. Using BLAST analyses and alignment tools, we re-annotated the whole module (see below) and, if we except two transposase genes and one ORF of unknown function, we unveiled that all ORFs of this module belong to a genetic unit similar to the traoperons encoding the TFSS previously described in proteobacterial genomes (Figure [2](#F2){ref-type="fig"}, See [additional data file 1](#S1){ref-type="supplementary-material"} for the re-annotations of this module). Presenting a low G+C content (33.4%), the 4^th^module (10 kb) is composed of 13 ORFs. All these ORFs were previously annotated by Horn et al. \[[@B14]\] as encoding hypothetical proteins or without homolog. Our BLAST analysis identified one ORF homologous to genes encoding bacteriophage-related proteins and two genes of proteins involved in DNA metabolism (Table [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}, see also the table in the [additional data file 1](#S1){ref-type="supplementary-material"}). Interestingly, a direct repeat is located between the 9th and 10th genes of the module. This 17-bp direct repeat, that presents 3 mismatches is similar to those present at the proximal and distal ends of the GI, exhibiting the same 14 conserved nucleotides. It may reflect a complex evolutionary history of the GI, possibly enabling it to be mobile as 25-kb, 75-kb or 100-kb DNA segments. A single large protein is encoded along the 5^th^module (6 kb). Its G+C content is one of the highest of the UWE25 chromosome (41.8%). By BLAST analysis, this protein exhibits the strongest similarity with the human Nod3 protein. The 6^th^module (12 kb) is characterized by a low G+C content (33.3%). This unit is composed of 13 ORFs, the first ORF encoding a product similar to the Death on cure (Doc) protein of P1 bacteriophage. Two ORFs code for proteins involved in DNA metabolism and an additional ORF encodes a putative transposase. The 7^th^module is short (2 kb) and present a G+C-rich unit (38.7%). Five of the six ORFs of this unit encode a probable resolvase, three putative transposases and a phage-related Doc protein. The final direct repeat is located at the end of this module. With the only exception of the phage-related protein, all other ORFs of the 7^th^module appear to be similar to gamma-proteobacterial proteins, possibly explaining the observed different signal in the G+C content analysis. Role of the type IV secretion system encoded by the 100-kb genomic island ------------------------------------------------------------------------- The functions of genes encoded by GIs may be related, among others, to pathogenicity such as the ability to exploit the host intracellular environment. Since no genetic system has been described for any obligate intracellular chlamydiae, we investigated the putative functions of this GI by bioinformatics. We focused our analyses on the TFSS, for which a previous annotation of the tragenes showed a genetic unit unable to transfer DNA \[[@B14]\]. Using different protein comparison methods described in the [additional data file 1](#S1){ref-type="supplementary-material"}, we identified supplementary tragenes, and compared the general organization of this traunit with other genetic elements encoding TFSS genes \[[@B20]\]. The UWE25 traunit displays a striking colinearity with traoperons encoding F-like conjugative DNA transfer system, especially to those of the F and pNL1 plasmids of Escherichia coliand Novosphingobium aromaticivorans, respectively (Figure [2](#F2){ref-type="fig"}). All homologous genes essential for DNA transfer in plasmid F (traF, traG, traH, traN, traU, traW, and trbC) and involved in sex pilus retraction and mating pair stabilization \[[@B20]\] are present, strongly suggesting that, similarly to the other F-like TFSSs, the gene products encoded by the UWE25 traunit are devoted to DNA transfer. With the only exception of traG, these genes are not present on P-like and I-like plasmids, reinforcing the close relationship prevailing between the UWE25 traunit and their F-like plasmids counterparts. Figure [3](#F3){ref-type="fig"} shows that the UWE25 traunit clusters within F-like TFSSs, confirming that it may function as a F-like conjugative system. Drawn as an UPGMA tree (Figure [3A](#F3){ref-type="fig"}), the comparison of the genetic organization of all traunits was performed as a gene order breakpoint analysis developed for the study of the mitochondrial genome evolution \[[@B21]\]. This analysis clearly shows that the closest relatives of the UWE25 traunits are the traoperons of the F-like conjugative plasmids. The Fitch-Margoliash- and the minimum evolution comparisons performed on the same dataset presented the same tree topologies, confirming the former UPGMA results (data not shown). An omittest performed on this tree confirms that the results are robust: with one exception (involving the deep branching of one cluster on one tree), all 11 trees were congruent in all their nodes. Figure [3B](#F3){ref-type="fig"} shows an UPGMA tree comparing the Kimura corrected p-distances (the proportion pof nucleotide sites at which two sequences are different, taking into account the proportion of transversion- and transition-substitution rates) of nucleotide sequences of the concatenated traA, traK, traB, traV, and traCgenes. A similar topology is observed with (i) neighbor-joining- and minimum evolution trees inferred using the Kimura-corrected p-distances and (ii) UPGMA, neighbor-joining- and minimum evolution trees performed on p-distance of the whole coding sequences of the concatenated tragenes (See [additional data file 2](#S2){ref-type="supplementary-material"} for these trees). Neighbor-joining- and minimum evolution methods comparing Kimura-corrected p-distances of the complete coding sequences confirmed that the traunit of UWE25 is phylogenetically closely related to the traoperons of the F-like plasmids: the bootstrap values of 94% and 91% respectively, support the node separating the concatenated tragenes of the chromosomal UWE25 and the R27 plasmid, a gamma-proteobacterial F-like conjugative plasmid, from those of all other plasmids (See the [additional data file 2](#S2){ref-type="supplementary-material"} for these trees). In neighbor-joining and minimum evolution analyses of p-distances, the traunit of UWE25 also clusters with the traoperons of gamma-proteobacterial F-like plasmids: the bootstrap of 96% and 92%, respectively, support the node separating the concatenated tragenes of UWE25 and RTS1, SXT, R391, three gamma-proteobacterial F-like conjugative plasmid, from their closest relative, R27 plasmid (See [additional data file 2](#S2){ref-type="supplementary-material"} for these trees). Taken together, all these data strongly suggest that the UWE25 traunit is closely related to F-like conjugative traoperons. Origin of the genomic island and of its type four secretion system ------------------------------------------------------------------ Our BLAST analyses \[[@B22]\] reveal that a majority (24/43) of genes not presenting a best hit for chlamydial genes but having homologs in other taxa are more related to proteobacterial genes (see Table [2](#T2){ref-type="table"} and the [additional data file 1](#S1){ref-type="supplementary-material"} for similarity analyses indicating for each parachlamydial tragene the most similar gene and its taxonomical background). Moreover, the BLAST analyses of the 21 ORFs of the third module, encoding the tragenes, show that most ORFs of this unit (15/21) are of proteobacterial origin. However, since six of them present the highest similarity to alpha-proteobacterial genes and six others to gamma-proteobacterial genes, a more precise origin of the parachlamydial traunit could not be precisely defined by this first approach. The presence of gly-tRNAat the proximal end of the GI of UWE25 is consistent with a close relatedness between this GI and proteobacteria: out of 14 GIs described in the Islanderdatabase of Mantri et al. \[[@B15],[@B22]\] inserted along a chromosome by a gly-tRNA(14/140), 12 of them (86%) were sequenced in a proteobacterial genome. No GI of Gram-positives described in the Islander database are inserted in a chromosome within a gly-tRNAgene. Again, a precise proteobacterial origin could not be proposed, because the distribution of gly-tRNAgenes in alpha- (4/22) and gamma-proteobacterial (8/72) GIs is not significantly different: by including only the non-redundant GIs, the distribution of gly-tRNAgenes in alpha and gamma-proteobacterial GIs is 2/20 and 7/71, respectively. Comparison of gene order between all traunits also failed in assigning a precise origin to the UWE25 traunit since it branched near the alpha- and gamma-proteobacterial traoperons (Figure [3A](#F3){ref-type="fig"}). The only first hint of a possible gamma-proteobacterial origin for the UWE25 traunit was brought by the phylogenetic analyses (Figure [3B](#F3){ref-type="fig"} and additional files [1](#S1){ref-type="supplementary-material"} &[2](#S2){ref-type="supplementary-material"}). Thus, bootstraps values of 94, 91, 96 and 92% supported the node separating the concatenated tragenes of UWE25 and several traoperons of gamma-proteobacterial F-like plasmids from the F-plasmids of an alpha-proteobacteria and of other gamma-proteobacteria. (See above, and [additional data file 2](#S2){ref-type="supplementary-material"} for these trees). Discussion ========== We showed that the Parachlamydia-related endosymbiont UWE25 presents a 100-kb region largely fullfilling the criteria of GIs \[[@B16]-[@B18]\]. Indeed, this DNA region characterized by a high level of gene co-orientation presents a G+C content different from that of the remainder of the genome. The presence of direct repeats flanking this chromosome region enabled us to focus on 100 ORFs. Two identical gly-tRNAgenes in tandem are present at the proximal end of this genetic element. Moreover, several mobility genes encoding transposases and bacteriophage related-proteins are located within this chromosome region. The cumulative residual G+C content analysis shows that this GI is composed of seven modules. Such a chimeric organization was already described in other GIs \[[@B23],[@B24]\]. The first module contains chlamydiae genes probably brought by chromosome rearrangements. Some of these genes, homologous to TTSS genes of Chlamydiaceae, might provide selective advantages to strains that retained the GI. The 2^nd^, 4^th^and 6^th^modules are mainly composed of bacteriophage-related protein genes, that could reflect a putative phage implication in GI formation. The 3^rd^module codes for a TFSS similar to traoperons. We propose that this traunit is devoted to DNA transfer, based (i) on similarity analyses demonstrating the presence of all genes encoding proteins used during a DNA transfer, (ii) on phylogenetic analyses of traunit genes and, (iii) on comparison of gene order. These analyses clearly demonstrate that the UWE25 traunit is strongly more related to F-like conjugative system than to P-like and I-like secretion systems. The significant bootstraps of all trees obtained by standard gene phylogeny and their congruent topologies with others obtained by the gene order breakpoint analysis not biased by codon usage homing, strongly support the validity of these analyses confirming the F-like conjugative nature of the parachlamydial traunit. Thus, our model significantly differs from the other proposed by Horn et al. \[[@B14]\], who did not identify traA, traL, traK, traV, and concluded that the UWE25 traunit is involved in protein export, and not in DNA transfer. The 5^th^module presents a nucleotide composition similar to the traunit and is composed of a single high G+C 6-kb gene, whose product is similar to the human Nod3 protein. The Nod (Nucleotide-binding oligomerization domain) proteins are members of a family that also includes the apoptosis regulator Apaf1 (Apoptotic protease activating factor 1) and plant disease-resistance gene products \[[@B25]\]. The function of the human Nod3 is still unknown. Like Nod1 and Nod2, Nod3 might be involved in the recognition of conserved motifs present at the surface of bacteria, such as peptidoglycan. The nucleotide G+C composition of the 2^nd^, 4^th^, and 6^th^modules are similar, explaining the observed similar negative slope of the residual G+C curves. Moreover, these three modules encode phage-related proteins and proteins involved in DNA metabolism. These modules probably involved in mobility might have a common origin, the ancestral single phage module being currently separated in three pieces by the presence of the traunit and of the Nod3-like protein encoding gene. The positive slope in the G+C analysis of the 7^th^module echoes those of the traunit (3^rd^module) and of the Nod3-like protein (5^th^module). The 7^th^module encodes a transposition resolvase and three transposases similar to gamma-proteobacterial homologs. With the only exception of the phage-related Doc protein, that has an homolog at the beginning of the sixth module, and that might be located there after transposition, the 7^th^module appears thus to have a different origin than the 2^nd^, 4^th^and 6^th^modules, though also encoding mobility genes. The presence of a F-like traunit along the sequences of UWE25 is the first evidence of a putative conjugative system in chlamydiae. If conjugation occurs, it most probably takes place within free-living amoebae, that may contain several hundreds of Parachlamydiabacteria tightly packed in their vacuoles \[[@B4]\]. Such a conjugation system would be a mechanism to transfer DNA between internalized bacteria sharing an amoebal vacuole. Moreover, it may provide molecular genetic tools for obligate intracellular bacteria. The presence of traunits/operons in the parachlamydial UWE25 and in proteobacteria could be explained by an emergence of this unit in a common ancestor of both clades, and by its subsequent loss in Chlamydiaceae. Another evolutionary scenario is that the traunit was acquired from a proteobacteria by a Parachlamydiaceaein a common amoebal vacuole. Since the traunit is absent from all sequenced Chlamydiaceaegenomes, this transfer would have occurred after the divergence of Parachlamydiaceaeand Chlamydiaceae, at a time when Parachlamydiawas already an intracellular bacteria. An intra-amoebal transfer of this GI is supported by the permissivity of free-living amoebae to proteobacteria \[[@B26]\], and by several hints suggesting its proteobacterial origin. Though phylogenetic analyses suggested a gamma-proteobacterial origin of the F-like parachlamydial tra, further analyses have to confirm whether this GI module was acquired from an alpha-, beta-, or gamma-proteobacteria unit. We hypothesize that the F-like parachlamydial traunit has been brought by a lateral transfer from a proteobacterial genome. This hypothesis is strongly supported by the cumulative GC skew analysis \[[@B27]-[@B30]\] producing a signal of the GI differing from that of the remaining of the genome (Figure [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}). The value of nucleotide skew analyses as good taxonomical markers is supported by (i) routine analyses on prokaryotic genome by cumulative TA-skews \[[@B30]\] and (ii) comparison of intragenic nucleotide skews of small subunit ribosomal RNA of the whole living world \[[@B31]\]. The genometric approach appeared to be able to identify GIs of Chlamydiales. Sequencing additional genomes of environmental chlamydiae, that present a large biodiversity \[[@B3]\], will provide major insights on bacterial evolution and hopefully a better comprehension of the emergence of this parachlamydial GI. Conclusions =========== We showed that a GI present on the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of Parachlamydiaceaebacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of Chlamydiaceae, we hypothesize that it was acquired after the divergence between Parachlamydiaceaeand Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired by a Parachlamydiaceaefrom phylogenetically-distant bacteria sharing an amoebal vacuole. Since Parachlamydiaceaeare emerging agents of pneumonia \[[@B2]\] and since many GIs are also considered as pathogenicity islands \[[@B17]\], the Pam100G GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for studying Chlamydiales. Methods ======= Sequence -------- The genome sequence of UWE25 \[[@B14]\] (Accession number: NC\_005861) is available at the NCBI website \[[@B32],[@B33]\]. In this contribution, the acronym UWE25 refers only to the Parachlamydia-related endosymbiont UWE25, and thus not to the Acanthamoebasp. strain UWE25 from which the parachlamydial endosymbiont UWE25 was recovered \[[@B3]\]. Horn et al. recently proposed UWE25 as the type strain of a new bacterial species: Protochlamydia amoebophila\[[@B34]\]. BLAST analyses -------------- BLAST analyses were performed with BLASTP 2.2.9 \[[@B35]\] available on the NCBI website \[[@B36]\] using the BLOSUM62 matrix, and gap penalties of 11 and 1. Each ORF was compared against all genes of non-redundant databases available at the NCBI website. An e-value of 0.001 was selected as a standard cut-off. To further identify possible homologous ORFs, we also BLASTed each tragene of F plasmid versus all genes of the full genome of Parachlamydiaand conversely, each ORF of the putative parachlamydial traunit versus counterparts of the different F-like plasmids. CLUSTALW was used to detect the best relatedness of a given parachlamydial Tra protein with its possible homologs encoded by the F and pNL1 plasmids. Residual cumulative GC content ------------------------------ The residual cumulative G+C content, a slightly modified version of the cumulative GC profile defined by Zhang and Zhang \[[@B19]\], was used to reveal local variations of G+C content of a genome, without using sliding windows of arbitrary size. First, a G+C content analysis was performed on 100-bp windows of the selected chromosome sequence, as for a cumulative GC skew analysis. The cumulative G+C content GC~n~of the n^th^window is obtained by cumulating the G+C contents from the first to the n^th^window: ![](1471-2180-4-48-i1.gif) where, in the window i, G~i~and C~i~are the numbers of Gs and Cs, respectively, and N~i~is the total number of nucleotides. To visualize genomic regions differing from the average G+C content, a linear regression ydefined by a slope kis performed on the cumulative curve using the least square methods: y(n) = kn where nis the position of the center of the n^th^window. The residual cumulative G+C content curve GC\' can then be drawn as a function of the position of each window center: GC\'~n~= GC~n~- kn Zhang and Zhang \[[@B19]\] recently demonstrated that, in some instances, abrupt changes in the residual cumulative G+C content curve correspond to genomic islands. Repeats identification ---------------------- The perfect tandem repeats identification was first performed using the EQUICKTANDEM software (Richard Durbin, Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK) \[[@B37]\] on a 200-kb DNA sequence (UWE25 genome position: 1.6 to 1.8 Mb) encompassing the tragenes previously identified by Horn et al. \[[@B14]\]. The duplicated genes and the ORFs containing internal repeats were removed. For each pair of direct repeats, potential unperfect matches of flanking nucleotides were scanned using DNA strider 1.2.1 \[[@B38]\], with the following settings: a minimal size of 11 bp and 3 mismatches. Furthermore, sequences similar to direct repeats were searched along the whole chromosome, and sequences also found outside the selected 200-kb region were discarded from our analysis. Finally, the direct repeats positions were compared to the G+C content analysis, the cumulative GC skew curve, and to tRNA genes locations. Phylogenetic analyses --------------------- Since Horn et al. \[[@B14]\] did not identified traA, traL, traK, traV, re-annotation of the UWE25 traunit was necessary for phylogenetic analyses. We used i) the genes of F-like plasmids encoding the following tragenes, i.e. traA, traK, traB, traV, traC, and ii) the corresponding ORFs of P- and I-like plasmids \[[@B20]\], i.e. trbC/VirB2, trbG/VirB9, trbI/VirB10, trbH/VirB7, trbE/VirB4of P-plasmids and traX, traN, traO, traI, traUof I-plasmids, respectively. The genes were concatenated to obtain a single nucleotide sequence and aligned with CLUSTALW (\[[@B39]\] as it was already performed for genes of ribosomal proteins \[[@B40]\]. Using this alignment and the MEGA 2.1 software \[[@B41]\], we inferred phylogenetic relationships by drawing trees using p-distances (the proportion pof nucleotide sites at which two sequences compared are different) and Kimura corrected p-distance (correction for the rates of transition and transversion) with Unweighted Pair Group Method with Arithmetic Mean (UPGMA), neighbor-joining, and minimum evolution methods. To prevent alignment biases, trees were drawn using the complete deletion option implemented on MEGA 2.1. Gene order breakpoint analyses ------------------------------ To quantify the inversion and transposition events leading to the current organization of traoperons, the gene order breakpoint analysis developed for small genomes (mitochondria) by Blanchette et al. \[[@B21]\] was used to estimate the similarity of gene order existing between the traunit of UWE25 and the traoperons reviewed by Lawley et al. \[[@B20]\]. The distance calculated for two given operons O~i~and O~j~containing homologous genes proposed by Blanchette et al. \[[@B21]\] was slightly modified to take into account the variation of gene numbers of traoperons: instead of counting the number of minimal breakpoints existing between two traoperons, a distance was estimated by measuring the proportion of conserved gene pairs between both genomic entities. Next, a comparison matrix is established by calculating the distance for each pairwise comparison. Finally, a dissimilarity matrix is obtained by subtracting each distance from 1. For instance, if the operon O~i~encodes sequentially four genes (a, b, c, and d) and the operon O~j~, six genes (a, b, e, -d, -c, and -f; genes labeled by a minus sign are encoded by the complementary strand), the gene order breakpoint analysis reveals that two gene pairs are conserved: aband cd. The dissimilarity distances existing between the operons i) O~i~and O~j~, and ii) O~j~and O~i~would be: 1-(2/3) = 1/3 and 1-(2/5) = 3/5, respectively. From the square dissimilarity matrix, phylogenetic trees were drawn. Three different distance-matrix analyses were used: the UPGMA, the Fitch-Margoliash- and the minimum evolution methods. To assess the robustness of the tree, an omittest \[[@B42]\] was performed on 11 UPGMA trees, in each one organism is missing. Authors\' contributions ======================= GG and CAR initiated the project. GG and FC reannotated the parachlamydial traunit and performed all BLAST analyses. FC and LG delimited the GI by direct repeat analyses. GG drew the phylogenetic trees of the concatenated tragenes. After developing the residual cumulative G+C content analysis used for a software development, LG performed the G+C content analyses and the gene order comparison of traunits by the gene order breakpoint analysis. CAR established the correlation existing between the tRNA genes and the GIs according to taxonomy and coordinated the team work. GG wrote the first draft of the paper. All authors improved the manuscript and approved its final version. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Supplementary table. Results of BLAST \[[@B35],[@B36]\] analyses of 100 ORFs present in the 100-kb region. BLAST analyses were performed using BLOSUM62 matrix and gap penalties of 11 and 1. Chromosome location of each ORF, its G+C content, coding strand, and the presence of at least one homolog in Chlamydiaceaeare presented. Direct repeats (DR), gly-tRNAgenes and limits of each modules of the GI are highlighted. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Supplementary figure. Phylogenetic analyses suggest that the UWE25 traunit is phylogenetically closely related to F-like DNA conjugative traoperons: (A) UPGMA-, (B) Neighbor-joining-, and (C) minimum evolution-trees comparing p-distances and Kimura corrected p-distances of nucleotide sequences of the concatenated traA, traK, traB, traV, and traCgenes (the UPGMA tree comparing the Kimura corrected p-distances of tragenes, shown in Figure [3B](#F3){ref-type="fig"}, is presented here to facilitate comparison with the other trees). Interestingly, in neighbor-joining and minimum evolution analyses of the p-distances, the traunit of UWE25 is clustered with traoperons of gamma-proteobacterial F-like plasmids: bootstrap values of 96% and 92%, respectively, support the node separating the concatenated tragenes of UWE25 and RTS1, SXT, R391, three gamma-proteobacterial F-like conjugative plasmids, from their closest relative R27 plasmid. Similarly, in neighbor-joining and minimum evolution analyses of the Kimura corrected p-distances, bootstrap values of 94% and 91%, respectively, support the node separating the concatenated tragenes of the chromosomal UWE25 and the R27 plasmid, another gamma-proteobacterial F-like conjugative plasmid, from those of all other plasmids. ::: ::: {.caption} ###### Click here for file ::: Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The genomic island (GI) present in the chromosome of the endosymbiont UWE25. (A)Position of the GI on the UWE25 genome, a 100-kb region (grey area) delimited by two direct repeats (Table 1) at both ends and by two gly-tRNAs genes in tandem (all tRNAs genes are represented by \'+\') at its proximal end. A third copy of the direct repeat (Table 1) is indicated by a white line disrupting the grey area. The region is characterized by a different slope in the cumulative GC skew analysis (black curve) and by a higher G+C content (grey curve, windows of 20 kb, 0.1-kb step). The horizontal line indicates the genomic G+C content average. (B)Closer view of the 100-kb region (black curve, cumulative GC skew; grey curve, G+C content windows of 5 kb, 0.1-kb step; horizontal line, average genomic G+C content). (C)Residual cumulative G+C content (GC\') and genomic features of the 100-kb GI. This region encompasses the region with the highest G+C content in the 20-kb windows analysis of the UWE25 genome. The position of genes is represented by an \'X\' on the upper line if encoded on the positive strand, otherwise by an \'X\' on the bottom line (For details, see Table in Supplementary Material 1). A large majority of genes are co-oriented in the genome region flanked by the direct repeats. The traoperon (thick line), present on this GI, exhibits a G+C content (40.0%) clearly higher than that of the whole genome (34.7%). The positions of transposases (open circles) and of phage-related genes (full circles) are indicated. ::: ![](1471-2180-4-48-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Comparison of the traunit of the endosymbiont UWE25 with similar operons of pNL1 (Novosphingobium aromaticivorans), F (Escherichia coli), R391 (Providencia rettgeri) and R27 (SalmonellaTyphi) plasmids. In the upper part of the figure: the G+C content of the UWE25 chromosome, around the 1.71 Mb location (1-kb sliding window average, 0.1-kb step). The horizontal line represents the genomic G+C content average. Only the ORFs composed of more than hundred amino-acids are presented on genetic maps of traunits/operons by arrows according to their transcription direction (adapted from Lawley et al. \[20\]). Colors or patterns are used to indicate tragene homologs. White genes represent non-conserved transfer genes. Upper case letters refer to the corresponding tragenes, whereas lower case letters f and c stand for trsFand trbC, respectively. Double slashes indicate non-contiguous regions. Interestingly, the G+C-rich genes encoded by the UWE25 chromosome correspond to the ORFs presenting trahomologs. ::: ![](1471-2180-4-48-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Similarity and phylogenetic analyses of traunits showing the close relatedness of the UWE25 traunit with the operons involved in the F-like conjugative systems: (A)UPGMA tree of gene order analysis and (B)UPGMA tree comparing the Kimura corrected p-distances of the concatenated traA, traK, traB, traV, and traCgene present along the UWE25 traunit and the F-like, I-like and P-like plasmids \[20\]. The bar represents estimated evolutionary distance scale. The numbers at each node are the results of a bootstrap analysis; each value is derived from 100 samples. ::: ![](1471-2180-4-48-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Description of main features of the parachlamydial 100-kb genomic island. Chromosome location of direct repeats, tRNA genes, traoperon, transposases, bacteriophage-related proteins and proteins involved in DNA metabolism is listed below. ::: Protein number^a^ Position^a^ ----------------------------------------------------------- ------------------- ------------------ Direct repeat \- 1648147--1648157 gly-tRNA^a,b^ \- 1648172--1648243 gly-tRNA^a,b^ \- 1648332--1648403 Transposase^c^ pc1402 1679924--1680400 Phage-related protein^c^ pc1404 1681569--1682441 Putative transcriptional regulator^c,d^ pc1405 1679924--1680400 Phage-related protein^c^ pc1410 1685329--1686447 Putative transposase^c^ pc1419 1695418--1696245 traoperon^e^ pc1420-1441 1696410--1716241 Transposases^e^ pc1426-1427 1700887--1701896 Putative DNA-binding protein^c^ pc1443 1716648--1717004 Phage-related protein^c^ pc1444 1717137--1717400 Direct repeat \- 1723093--1723103 Putative ATPase involved in DNA repair^c^ pc1451 1723169--1723504 Probable Doc (death on cure) protein, bacteriophage P1^a^ pc1456 1732622--1732999 Putative DNA-binding protein pc1461 1735745--1736065 Putative transposase^c^ pc1465 1740371--1741198 Probable DNA double-strand break repair ATPase pc1467 1742079--1744181 Putative transposase pc1468 1744634--1745023 Probable resolvase^a^ pc1469 1745398--1745955 Probable transposases, partial length^a^ pc1470-1471 1745807--1746692 Probable Doc (death on cure) protein, bacteriophage P1^a^ pc1473 1747135--1747512 Phage-related protein^c^ pc1474 1747618--1747809 Direct repeat \- 1747915--1747925 ^a^, according to Horn et al. \[14\]; ^b^, positive strand (cooriented as the majority of the genes of the GI); gly-tRNAs are separated by 88 nt; ^c^, identified by BLAST \[35; 36\] and CLUSTALW \[39\] by ourselves; ^d^, phage-related protein based on additional BLAST hit; ^e^, partially annotated by Horn et al. \[14\] and further characterized by ourselves by BLAST \[35, 36\]. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The genomic island of the UWE25 endosymbiont presents seven different modules. The limit of each module was determined by residual cumulative G+C content analysis. ::: Modules 1^st^mod. 2^nd^mod. 3^rd^mod. 4^th^mod. 5^th^mod. 6^th^mod. 7^th^mod. --------------------------------------- ----------- ----------- ----------- ----------- ----------- ----------- ----------- Length 32 kb 16 kb 19 kb 10 kb 6 kb 12 kb 2 kb Mean G+C content (%) 36.4%^a^ 34.1% 40.9% 33.4% 41.8% 33.3% 38.7% Number of ORFs 28 18 21 13 1 13 6 Number of genes having an homolog 16 (57%) 5 (28%) 16 (85%) 7 (54%) 1 (100%) 5 (38%) 5 (83%) Number of genes having no homolog 12 13 5 6 0 8 1 Best hits with homologs in: \- chlamydiae 12 0 0 0 0 0 0 \- cyanobacteria 2 2 0 3 0 2 0 \- plants^b^ 0 0 0 0 0 0 0 \- α-proteobacteria 0 0 6 0 0 0 0 \- β-proteobacteria 1 0 3 1 0 0 0 \- γ-proteobacteria 0 1 6 2 0 0 4^c^ \- Bacteroidetes group 0 0 1 0 0 2 1 \- others 1 2 0 1 1 1 0 Homologous^d^to: \- phage-related protein 0 3 0 1 0 1 2 \- putative transposase 1 1 2 0 0 2^e^ 2 \- resolvase 0 0 0 0 0 0 1 \- protein involved in DNA metabolism 0 1 0 2 0 2 0 ^a^the G+C content is similar to that of 36.1% of the remaining genome (calculated on 1931 ORFs); ^b^5% of the 2031 ORFs of the genome of UWE25 have products homologous to plant proteins, but no ORFs of the GI were homologous to plant counterparts; ^c^two ORFs which presented best homologs encoded by a plasmid of an uncultured bacteria present in activated sludge have a second best BLAST hit encoded by a gamma-proteobacterial ORFs (Pseudomonassp.); ^d^not only the best BLAST hit is taking into consideration to determine the putative function encoded by the ORF; ^e^one of them has an e-value above 0.001. :::	PubMed Central	2024-06-05T03:55:52.528953	2004-12-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548262/", "journal": "BMC Microbiol. 2004 Dec 22; 4:48", "authors": [ { "first": "Gilbert", "last": "Greub" }, { "first": "François", "last": "Collyn" }, { "first": "Lionel", "last": "Guy" }, { "first": "Claude-Alain", "last": "Roten" } ] }
PMC548263	Background ========== Alpha1-adrenergic receptors (α~1~-ARs) are members of the G-protein-coupled receptor superfamily. Both pharmacological and molecular cloning studies have indicated the existence of multiple subtypes of α~1~-ARs including α~1A/C~-AR, α~1B~-AR, and α~1D~-AR \[[@B1]-[@B4]\]. α~1~-ARs play a key role in a variety of physiological processes, such as contraction of vascular and cardiac muscle, contraction of the spleen, liver glycogenesis, or melatonin secretion in the pineal gland \[[@B3],[@B4]\]. Activation of α~1A~-AR promotes hypertrophy of cardiac myocytes \[[@B5],[@B6]\]. Recently it has been shown that all three subtypes of α~1~-AR are also expressed in rat aortic adventitial fibroblasts and vascular smooth muscle cells (SMC) \[[@B7]\] and their activation with norepinephrine stimulates migration, proliferation and protein synthesis \[[@B8],[@B9]\]. However, norepinephrine increased SMC hypertrophy, but not DNA synthesis, through α~1A~-AR stimulation in uninjured aorta whereas norepinephrine stimulated proliferation of adventitial fibroblasts through the α~1B~-AR subtype \[[@B8]\]. Nonvascular fibroblasts, including cardiac fibroblasts \[[@B7],[@B10]\], generally do not express α~1~-AR and have been used for stable transfection of different subtypes of α~1~-AR to study their respective functions. However, a recent study showed the expression of a functional α~1A~-AR in primary tendon fibroblasts \[[@B11]\]. In rat-1 cells, a transformed cell line from embryonic fibroblast, expressing different subtypes of α~1~-AR, phenylephrine (PE), an α~1~-AR agonist, activates phospholipase D and releases arachidonic acid \[[@B12]\]. However, unlike SMC, activation of α~1~-ARs in rat-1 cells also increases cAMP levels and PKA activity \[[@B12]\]. α~1A~-AR is more efficiently coupled to phospholipase D activation, arachidonic acid release and cAMP than α~1B~-AR or α~1D~-AR in these cells \[[@B12]\]. Activation of α~1A~-AR expressed in COS-7 and HeLa cells \[[@B13]\] and α~1B~-AR or α~1D~-AR in COS and CHO cells \[[@B14]\] also increase cAMP levels. In HepG2 and M12 cells expressing α~1B~-AR, PE causes cell scattering and inhibits proliferation through activation of MAP kinases \[[@B15]\]. The family of connective tissue cells includes fibroblasts, cartilage cells, bone cells, fat cells and smooth muscle cells. Fibroblasts seem to be able to transform into any of other members of the family -- in some cases reversibly -- although it is not clear whether this is a property of a single type of fibroblast that is pluripotent or of a mixture of distinct types of fibroblasts with more restricted potentials. These transformations of connective tissue cell type are regulated by the composition of the surrounding extracellular matrix, by cell shape, and by hormones and growth factors \[[@B16]\]. Heterologous expression of α~1A~-ARs in CHO cells inhibits basal and growth factor-stimulated DNA synthesis, in contrast to the α~1D~-AR \[[@B17]\]. A recent study in the same model has reported cAMP as the mediator of the antiproliferative effect of α~1A~-AR stimulation \[[@B18]\]. Therefore, it is possible that activation of α~1A~-AR with PE in rat-1 cells affects their growth and/or differentiation status. To test this hypothesis, we have investigated the effect of PE and cAMP modulators on proliferation, growth and morphology in rat-1 cells expressing α~1A~-ARs. Moreover, we have examined the effect of PE and cAMP modulators on the expression of cell cycle regulators and muscle cell markers, because of the ability of fibroblasts to differentiate into myofibroblasts. Our results show that activation of α~1A~-ARs in rat-1 cells exerts profound effects promoting hypertrophy and expression of specific smooth muscle cell markers. We also show here that α~1A~-AR-induced cessation of DNA synthesis is independent of cAMP and involves the expression of cyclin-dependent kinase (Cdk) inhibitor, p27^kip1^. Results ======= Stimulation of α~1A~-AR inhibits DNA synthesis at the G~1~/S checkpoint of the cell cycle in rat-1 fibroblasts -------------------------------------------------------------------------------------------------------------- Rat-1 fibroblasts stably transfected with α~1A~-AR expressed 288 ± 2 fmol/mg protein of receptors \[[@B12]\]. Cells at 80% confluency were serum-deprived for 48 h in DMEM and then incubated with PE (2, 5 and 10 μM) for different periods prior to the addition of \[^3^H\]thymidine. PE decreased basal \[^3^H\]thymidine incorporation in a concentration-dependent manner with a maximum effect at 10 μM PE (Fig [1A](#F1){ref-type="fig"}). Reduction of basal DNA synthesis was significant after 12 h of treatment with a maximum effect at 18 h of incubation with PE (\> 65% decrease) (Fig [1B](#F1){ref-type="fig"}). Rat-1 fibroblasts stably transfected with α~1A~-AR incorporate \[^3^H\]thymidine in DNA in a time-dependent manner (from 6 to 24 h), even after 48 h of serum-deprivation (Fig [1B](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Effect of PE on \[^3^H\]thymidine incorporation in rat-1 cells. Cells were grown in 24-well plates in DMEM containing 10% FBS at a density of 150,000 cells/well until they reached 80--90% confluency, and then serum-deprived for 48 h in DMEM. A, Cells were treated with different concentrations of PE (2, 5, and 10 μM) for 18 h prior to the addition of \[^3^H\]thymidine (0.50 μCi/ml) for the last 4 h. B, Cells were treated with PE for different incubation periods prior to the addition of \[^3^H\]thymidine (0.50 μCi/ml). Cells were incubated with \[^3^H\]thymidine for the last 4 h and thymidine incorporation was determined as described in Methods. Data are expressed as dpm of \[^3^H\]thymidine incorporated in DNA per well. Values are the mean ± S.E. of six independent experiments performed in quadruplicates on different batches of cells. \* Values significantly different from vehicle, p \< .05. ::: ![](1471-2121-5-47-1) ::: To determine whether PE at different concentrations delays the re-entry of rat-1 cells into the cell cycle, we examined the kinetics of cell cycle re-entry. Flow cytometry studies showed that incubation of the rat-1 cells with 2, 5, and 10 μM PE resulted in inhibition of the re-entry of 83, 81, or 85%, respectively, of the cell population into the S phase of the cell cycle (Table [1](#T1){ref-type="table"}). Therefore, stimulation of α~1A~-AR inhibits DNA synthesis at the G~1~/S checkpoint in rat-1 fibroblasts. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of PE on cell cycle phases. ::: Treatment G~0~/G~1~phase S phase G~2~/M phase % inhibition (S) --------------- -------------------- -------------- ------------------ ---------------------- vehicle 9269 ± 0.5 319.5 ± 13.5 238.5 ± 23.5 0 % 2 μM PE 9770 ± 9.0 54.0 ± 2.0 125 ± 21.0 83% 5 μM PE 9699 ± 29.0 59.0 ± 4.0 170 ± 11.0 81% 10 μM PE 9761.5 ± 89.5 47.5 ± 13.5 110.5 ± 39.5 85% Cells were subjected to flow cytometric DNA analysis as described in Methods. Different concentrations of PE inhibited the re-entry of the cell population into S phase of the cell cycle. Data show the number of cells in each phase of the cell cycle. Values are the mean ± S.E. of three independent experiments performed in duplicate on different batches of cells (Total events per cycle is 10,000 cells). ::: Stimulation of α~1A~-AR increases protein synthesis and promotes hypertrophy in rat-1 fibroblasts ------------------------------------------------------------------------------------------------- Stimulation of α~1~-ARs in vascular smooth muscle cells \[[@B19]\] and in adult rat ventricular myocytes \[[@B8]\] increases hypertrophy and protein synthesis. However, the α~1~-AR subtype mediating these effects is not known. Exposure to 5 μM PE for 18 h increased \[^3^H\]leucine incorporation by 64% (Fig [2B](#F2){ref-type="fig"}) and decreased basal \[^3^H\]thymidine uptake (Fig [2A](#F2){ref-type="fig"}), as shown earlier in Fig [1](#F1){ref-type="fig"}. These effects were blocked by prazosin (PRZ, 100 nM), an α~1~-AR antagonist. Propranolol (2 μM), a β-AR antagonist (Fig [2A,B](#F2){ref-type="fig"}), or yohimbine (1 μM), an α~2~-AR antagonist (data not shown), had no effect on the decrease in basal \[^3^H\]thymidine or the increase in \[^3^H\]leucine incorporation in rat-1 cells caused by exposure to PE. These results show that PE stimulates protein synthesis and inhibits DNA synthesis in rat-1 cells through activation of α~1A~-ARs. The hypertrophy index, an indicator of cellular hypertrophy, is defined as the ratio of protein synthesis (\[^3^H\]leucine incorporation) to DNA synthesis (\[^3^H\]thymidine incorporation) \[[@B20]\]. At 18 h, this ratio was 4.4 fold as high in PE-treated cells (index = 0.22) than in control cells (index = 0.05, n = 6), indicating that PE promotes rat-1 cell hypertrophy. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Stimulation of α~1A~-AR mediates the effect of PE on DNA and protein synthesis.A, Effect of prazosin (α~1~-AR antagonist) and propranolol (β-AR antagonist) on PE-induced inhibition of DNA synthesis. Cells were treated with prazosin and/or propranolol for 30 min before the addition of 5 μM PE for 18 h. \[^3^H\]thymidine incorporation was measured as described in Methods. Data are expressed as dpm of \[^3^H\]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in quadruplicates on different batches of cells. \* Value significantly different from the corresponding value obtained without PE treatment, p \< .05. B, Effect of prazosin and propranolol on PE-induced increase in protein synthesis. Experimental conditions were similar as described in A. \[^3^H\]leucine incorporation was measured as described in Methods. Data are expressed as dpm of \[^3^H\]leucine incorporated per well. Values are the mean ± S.E of three independent experiments performed in quadruplicates on different batches of cells. \* Value significantly different from the corresponding value obtained without PE treatment, p \< .05. ::: ![](1471-2121-5-47-2) ::: Protein synthesis elicited by α~1~-AR stimulation was time-dependent and remained significant from 6 to 72 h of exposure with PE (Fig [3A](#F3){ref-type="fig"}). EGF and cAMP have opposite effects on rat-1 fibroblasts proliferation \[[@B21],[@B22]\]. Therefore, we tested the effect of EGF and forskolin (FN), an activator of adenylyl cyclase \[[@B12]\], on protein and DNA synthesis. EGF (30 ng/ml) slightly increased, whereas FN decreased, \[^3^H\]leucine incorporation at 6, 18, 48 and 72 h (Fig [3A](#F3){ref-type="fig"}). Surprisingly, PE was a better activator of protein synthesis than EGF. In contrast, EGF increased \[^3^H\]thymidine incorporation by 272% (Fig [3B](#F3){ref-type="fig"}), an effect blocked by FN and the cAMP analog, 8-cpt-cAMP, as well as by PE. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effect of PE, cAMP-elevating agents and EGF on protein and DNA synthesis. A, Cells were treated with 5 μM PE, 1 μM forskolin (FN) and 30 ng/ml EGF for different periods. \[^3^H\]leucine incorporation was measured as described in Methods. Data are expressed as dpm of \[^3^H\]leucine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in sextuplicates. \* Values significantly different from vehicle, p \< .05. B, Effect of FN, 8-cpt-cAMP and PE on EGF-induced \[^3^H\]thymidine incorporation. Cells were preincubated with FN (1 μM), 8-cpt-cAMP (5 μM) and PE (5 μM) for 1 h. Cells were then treated with EGF (30 ng/ml) for 18 h. Incorporation of \[^3^H\]thymidine into rat-1 fibroblasts was determined as described in Methods. Data are expressed as dpm of \[^3^H\]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in duplicate in different batches of cells. \* Value significantly different from vehicle, p \< .05. § Value significantly different from the corresponding value obtained without treatment by FN, cpt-cAMP or PE, p \< .05. ::: ![](1471-2121-5-47-3) ::: PE inhibits DNA synthesis and promotes hypertrophy by a mechanism independent of cAMP ------------------------------------------------------------------------------------- Previously, we have shown that PE induces cAMP accumulation in rat-1 cells \[[@B12]\]. It is also known that cAMP-dependent PKA-mediated inhibition of cell division is due to blockade of growth factor-stimulated cell cycle progression from G~1~to S phase \[[@B23]\]. Therefore, to determine whether cAMP mediates the antiproliferative effect of PE, we examined the effect of FN (0.1--10 μM) and 8-cpt-cAMP (5--20 μM) and of FN and PE on DNA synthesis in the presence and absence of adenylyl cyclase and PKA inhibitors. FN and 8-cpt-cAMP inhibited \[^3^H\]thymidine incorporation to the same extent as PE (Fig [4A](#F4){ref-type="fig"}). The FN but not the PE-induced decrease in \[^3^H\]thymidine incorporation was blunted by the selective inhibitor of adenylyl cyclase SQ 22536 (Fig [4B](#F4){ref-type="fig"}) and by the P-site inhibitors of adenylyl cyclase 2\',5\'dideoxyadenosine, and 2\'-deoxyadenosine 3\'-monophosphate (5 μM each-data not shown). The selectivity of cAMP modulators, FN, 8-cpt-cAMP and SQ 22536 has been tested previously in rat-1 fibroblasts \[[@B12]\]. To further determine the contribution of cAMP pathway, rat-1 cells were transfected with πLXX-PKI \[[@B1]-[@B31]\], a vector that expresses the PKA inhibitor, PKI \[[@B23],[@B24]\]. Transfection with PKI reversed the inhibitory effect of FN, but not that of PE, on \[^3^H\]thymidine incorporation in rat-1 cells (Fig [4C](#F4){ref-type="fig"}). These observations indicate that cAMP and PKA do not mediate the PE-induced inhibition of DNA synthesis. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effect of PE and cAMP-elevating agents, FN and 8-cpt-cAMP, on DNA synthesis. Cells were serum-deprived for 2 days. A, Cells were treated with PE (5 μM), FN (0.1, 1, and 10 μM), and 8-cpt-cAMP (5, 10, and 20 μM) for 18 h. Incorporation of \[^3^H\]thymidine into rat-1 fibroblasts was determined as described in Methods. Data are expressed as dpm of \[^3^H\]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in sextuplicates in different batches of cells. \* Value significantly different from vehicle, p \< .05. B, Effect of inhibitors of adenylyl cyclase inhibition on PE and cAMP-elevating agents. Cells were pre-incubated with SQ22536 (25 μM), an specific adenylyl cyclase inhibitor for 1 h. Cells were then treated with 5 μM PE and/or 1 μM FN for 18 h. \[^3^H\]thymidine incorporation was determined as described in Methods. Values are the mean ± S.E. of three independent experiments performed in quadruplicates on different batches of cells. Data are expressed as dpm of \[^3^H\]thymidine incorporated per well. \* Value significantly different from the corresponding vehicle, p \< .05. § value significantly different from that obtained in the presence of agonist alone, p \< .05. C, Effect of transfection of πLXX-PKI \[1-31\] on PE-induced inhibition of DNA synthesis. Preconfluent cells were transiently transfected with πLXX-PKI \[1-31\], a plasmid encoding for the protein kinase inhibitor, (PKI), using Lipofectamine Plus transfection reagent according to the manufacturer\'s protocol (Life Technologies). Cells were cultured in serum-free DMEM for 24 h and then treated with 5 μM PE and/or 1 μM FN. Cells were assayed for \[^3^H\]thymidine incorporation 48 h post transfection as described in Methods. Data are expressed as dpm of \[^3^H\]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed on different batches of cells. \* Value significantly different from the corresponding vehicle, p \< .05. § value significantly different from that obtained in the presence of agonist alone, p \< .05. ::: ![](1471-2121-5-47-4) ::: The antiproliferative effect of PE, FN and 8-cpt-cAMP could be the result of cell loss due to a cytotoxicity. However, the trypan blue exclusion test and LDH assay showed that more than 90% of cells were viable (Table [2](#T2){ref-type="table"}) and less than 1% of the cells were found in the supernatant, indicating very little cell loss. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Effect of PE and cAMP elevating agents on viability of rat-1 fibroblasts using the hemocytometer trypan blue method. ::: Treatment Total number of cells Blue cells % of viability ------------------ --------------------------- ---------------- -------------------- Vehicle 78 8 90% 5 μM PE 142 10 92% 10 μM PE 87 12 86% 10 μM FN 78 8 90% 20 μM 8-cpt-cAMP 84 4 96% 20 μM 8-Br-cAMP 66 2 97% Cells were incubated for 18 h with PE (5 and 10 μM), forskolin (FN), 8-cpt-cAMP and 8-Br-cAMP. The percentage of viable cells was calculated. Values are representative of five different experiments. ::: Morphological change elicited by α~1A~-AR stimulation and by cAMP in rat-1 cells -------------------------------------------------------------------------------- Although both PE and FN inhibited DNA synthesis in rat-1 cells, they produced different effects on cell morphology. PE increased, whereas FN decreased, the apparent size of rat-1 cells as shown in Fig [5](#F5){ref-type="fig"}. Prazosin (100 nM), an α~1~-AR antagonist, reversed the shape change produced by PE. To determine if PE-induced cell cycle arrest and hypertrophy of rat-1 fibroblasts is also independent of cAMP, we preincubated the cells with SQ22536 (25 μM) and then treated them with PE (5 μM) or FN (1 μM) for different time periods. SQ22536 reversed the morphological changes produced by FN and restored the normal fibroblast shape, whereas it enhanced the effect of PE, i.e. the cells became more hypertrophic. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Effect of prazosin and SQ22536 on PE-and FN-induced morphological change in rat-1 cells. Preconfluent, serum-deprived cells were either stimulated with 5 μM PE and/or 1 μM FN for different periods of time (Fig 5B) and/or pretreated for 30 min with 100 nM prazosin (Fig 5A) or 25 μM SQ22536 (Fig 5C), and then further incubated with 5 μM PE and/or 1 μM FN for 24 h. Morphological studies were performed as described in Methods. The same morphological patterns were obtained in three different experiments. ::: ![](1471-2121-5-47-5) ::: p27^kip1^mediates PE-induced inhibition of DNA synthesis -------------------------------------------------------- Cyclin-dependent kinase (cdk) inhibitors p27^kip1^and p21^cip1/waf1^play an important role in cell-cycle regulation \[[@B25],[@B26]\]. High levels of p27^kip1^and p21^cip1/waf1^in quiescent (G~0~) cells decline upon induction of mitogenesis \[[@B27]\]. This decrease in Cdk inhibitors appears to be critical in enabling cell cycle entry. Western blot analysis showed that α~1A~-AR stimulation increased the expression levels of p27^kip1^(642% of control at 48 h) and p21^cip1/waf1^(935% of control at 48 h) (Fig. [6A](#F6){ref-type="fig"}). The protein levels remained elevated for 48 h, with levels of p27^kip1^reaching 389% of control at 18 h of incubation and remaining elevated for up to 48 h. This increase was concomitant with the inhibitory effect of PE on DNA synthesis, which was also maximal at 18 h of incubation. PE increased the expression of p27^kip1^(552% of control at 24 h) more efficiently than p21^cip1/waf1^(87% of control at 24 h) suggesting a greater contribution of p27^kip1^to PE-induced cell cycle arrest and a delayed induction of p21^cip1/waf1^. Retinoblastoma protein (pRb) hyperphosphorylation and subsequent release of E2F is required for cell cycle progression \[[@B28]\]. However, α-AR stimulation of neonatal myocytes has been shown to induce pRb hyperphosphorylation without induction of cell proliferation \[[@B29]\]. Our results demonstrate that PE promotes an early increase in pRb levels (607% of control at 6 h) in rat-1 cells (Fig [6C](#F6){ref-type="fig"}), associated with a decrease in DNA synthesis (Fig [1B](#F1){ref-type="fig"}). The nuclear envelope proteins lamin A and C, used as control, were not altered by PE treatment (Fig [6D](#F6){ref-type="fig"}). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Effect of PE on the expression of cell cycle regulators, p27^kip1^, p21^ip1/waf1^and pRB. Preconfluent, serum-deprived cells were incubated with PE (5 μM) or vehicle for up to 48 h. Expression of cell cycle regulators was determined by Western blot analysis of whole cell lysates as described in Methods. The panels are representative Western blots showing the effect of PE on the expression of p27^kip1^(A), p21^cip1/waf1^(B), pRb (C), and lamin A/C (D) protein levels respectively. Percentages representing the quantitation of protein bands by densitometric analysis of blots obtained from three different experiments are indicated on top of each immunoblot. ::: ![](1471-2121-5-47-6) ::: In VSMC, p27^kip1^level is a critical determinant of the cell response, hyperplasia vs. hypertrophy, to angiotensin II and other growth factors \[[@B20]\]. We postulated that the response of rat-1 cells to PE was mediated by p27^kip1^. PE upregulates the expression of p27^kip1^at 18 h of treatment in rat-1 cells at all concentrations (Fig [7A](#F7){ref-type="fig"}). We therefore explored further the role of p27^kip1^in modulating the antiproliferative response of rat-1 cells to PE by transfecting these cells with p27^kip1^antisense and sense ODN. p27^kip1^antisense (102% of control), but not sense ODN (347% of vehicle vs. 338% for PE alone), diminished p27^kip1^level (Fig [7B](#F7){ref-type="fig"}) and blocked the antiproliferative effect of PE in rat-1 cells (Fig [7C](#F7){ref-type="fig"}). These observations suggest that PE-induced inhibition of DNA synthesis is mediated by p27^kip1^. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Effect of PE on p27^kip1^level and effect of p27^kip1^antisense oligonucleotides on PE-induced inhibition of DNA synthesis. A, Preconfluent rat-1 cells were stimulated with 2, 5, and 10 μM PE for 18 h. Total cell lysates were subjected to Western blot analysis as described in Methods. Percentages representing the quantitation of protein bands by densitometric analysis of blots obtained from three different experiments are indicated on top of each immunoblot. B, Representative Western blot showing the effect of p27^kip1^antisense and sense oligonucleotides treatment on PE-induced increase in p27^kip1^protein level. Cells were transiently transfected with p27^kip^antisense or sense oligonucleotide for 48 h using Oligofectamine™ Reagent according to the manufacturer\'s instruction (Invitrogen). C, Transfected cells as in B were serum-starved for 24 h and then treated with 5 μM PE. Thymidine uptake was measured as described in Methods 48 h post-transfection. Data are expressed as the percent decrease in \[^3^H\]thymidine uptake above the basal value obtained in unstimulated cells. Values are the mean ± S.E of three independent experiments performed in different batches of cells. \* Value significantly different from the corresponding vehicle, p \< .05. ::: ![](1471-2121-5-47-7) ::: PE increases the expression of cyclin D1, proliferating cell nuclear antigen (PCNA), Cdk2 and Cdk4 in rat-1 cells ----------------------------------------------------------------------------------------------------------------- We next investigated the effect of PE on several cell cycle proteins important for G~1~/S phase progression. Fig. [8](#F8){ref-type="fig"} shows that treatment of serum-deprived rat-1 cells with PE increased the levels of cyclin D1 (548% of control at 24 h), PCNA (158% of control at 24 h), and Cdk2 (236% of control at 24 h) in a time-dependent manner. The levels of nuclear envelope proteins lamin A and C, were not altered. These results are unexpected since PE promotes cell cycle arrest at G~1~/S checkpoint (Table [1](#T1){ref-type="table"}). However, the up-regulation of p27^kip1^seems to be sufficient to block cell cycle entry into S phase and DNA synthesis. Surprisingly, we also observed an increase in the protein levels of Cdk4 (280% of control at 24 h), which normally do not change even during mitogenic stimulation. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Effect of PE on the expression of cyclin D1, PCNA Cdk 2, and Cdk 4. Preconfluent, serum-deprived cells were stimulated with PE (5 μM) for 6, 12, 24 and 48 h. Cell-cycle regulators expression was determined by Western blot analysis of whole cell lysates as described in Methods. The bar graph represents quantitation of protein bands by densitometric analysis of blots obtained from three different experiments. \* Value significantly different from the corresponding vehicle, p \< .05. ::: ![](1471-2121-5-47-8) ::: PE increases expression of specific smooth muscle differentiation markers as well as MyoD ----------------------------------------------------------------------------------------- Cell cycle arrest is closely coupled to muscle cell differentiation and is required for activation of muscle cell specific gene expression \[[@B16]\]. Since stimulation of α~1A~ARs promoted a change in morphology associated with hypertrophy, we examined the effect of PE on expression of the specific smooth muscle cell markers, α-smooth muscle actin (SMα actin), caldesmon and myosin heavy chain (SM-MHC) \[[@B30]\]. The smooth muscle markers were not expressed in serum-deprived rat-1 cells (Fig [9A,C](#F9){ref-type="fig"}). Stimulation of α~1A~ARs with PE (5 μM) promoted a time-dependent increase in the levels of SMα actin (695% of control at 24 h) (Fig [9A](#F9){ref-type="fig"}), caldesmon (284% of control at 24 h) (Fig [9B](#F9){ref-type="fig"}) and SM-MHC (445% of control at 24 h) (Fig [9C](#F9){ref-type="fig"}) as shown by Western blot analysis We also investigated the expression of MyoD, a helix-loop-helix protein that plays an important role in the regulation of muscle development \[[@B31]\]. Western blot analysis showed that PE also increased the expression of MyoD in a time-dependent manner (457% of control at 24 h), further supporting the evidence that stimulation of α~1A~AR induces differentiation of rat-1 fibroblasts into muscle-like cells, which is closely coordinated with cell cycle arrest (Fig [9D](#F9){ref-type="fig"}). The expression of vimentin, an intermediate filament protein used as a control, was not altered by treatment with PE (Fig [9E](#F9){ref-type="fig"}). The α~1A~AR stimulation of smooth muscle differentiation markers was independent of cAMP because FN did not increase the expression of SMα actin, caldesmon or SM-MHC (Fig [10A,C](#F10){ref-type="fig"}). MyoD expression was not altered by FN (Fig [10D](#F10){ref-type="fig"}). The level of vimentin expression was not altered by PE or FN (Bar Graph). Together, these data demonstrate that stimulation of α~1A~ARs increases expression of smooth muscle markers and MyoD in rat-1 cells, independently of cAMP. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Time course of the effect of PE on the expression of the smooth muscle differentiation markers and MyoD. Preconfluent, serum-deprived fibroblasts were stimulated with PE (5 μM) for up to 48 h. The protein expression of smooth muscle differentiation markers and MyoD was determined by Western blot analysis of whole cell lysates as described in Methods. A, smooth muscle α actin (SMα actin). B, caldesmon. C, smooth muscle myosin heavy chain (SM-MHC). D, MyoD, E, vimentin. VSMC lysate is shown as a positive control. Percentages representing the quantitation of protein bands by densitometric analysis of blots obtained from three different experiments are indicated on top of each immunoblot. ::: ![](1471-2121-5-47-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### Effect of PE and FN on the expression of smooth muscle differentiation markers and MyoD.Preconfluent, serum-deprived fibroblasts were stimulated with either PE (5 μM) or FN (1 μM) for up to 48 h. Expression of markers was determined by Western blot analysis of whole cell lysates as described in Methods. A, smooth muscle α actin (SMα actin). B, caldesmon. C, smooth muscle myosin heavy chain (SM-MHC). D, MyoD. VSMC lysate is shown as a positive control in panel A, B, C and D. The bar graph represents quantitation of protein bands by densitometric analysis of blots obtained from three different experiments. \* Value significantly higher than the corresponding vehicle, p \< .05. ::: ![](1471-2121-5-47-10) ::: Discussion ========== This study is the first demonstration of the ability of an α~1A~-AR subtype to promote differentiation of a fibroblast into a SMC/myocyte-like cell. Further, it demonstrates that α~1A~-AR-induced cell cycle arrest and hypertrophy as well as differentiation is mediated through a mechanism dependent upon the increased expression of the critical cell cycle protein p27^kip1^and selective smooth muscle markers. Stimulation of α~1A~-ARs promotes hypertrophy of cardiac myocytes \[[@B6]\]. Norepinephrine increases SMC hypertrophy, but not cell proliferation, through α~1A~-AR stimulation in uninjured aorta \[[@B8]\]. In HepG2 and M12 cells transfected with α~1A~-AR, PE stimulates cell scattering and inhibition of proliferation \[[@B15]\]. Similar observations have been made in CHO cells expressing α~1A~-ARs \[[@B17],[@B18]\]. In the present study, PE inhibited cell proliferation, promoted hypertrophy and differentiation through stimulation of α~1A~-ARs in rat-1 cells expressing this subtype. Analysis of the effect of α~1A~-AR stimulation on cell cycle progression revealed that it inhibits the re-entry of cells from G~0~/G~1~into the S phase of the cell cycle. Next we attempted to define the mechanism mediating α~1A~-AR-induced cell cycle arrest, hypertrophy and differentiation of rat-1 cells. Previous studies have shown that in rat 1-cells expressing α~1A~-ARs, PE promotes increase in cAMP accumulation and PKA activation \[[@B12]\]. Since cAMP has been reported to inhibit growth in various cell types, including cells of fibroblastic origin \[[@B32]\], it seemed possible that cAMP mediates the inhibitory effect of PE on rat-1 cells proliferation. Stable cAMP analogs or adenylyl cyclase activation promoted cell cycle arrest similar to α~1A~-AR stimulation. However, activation of the cAMP/PKA pathway did not promote hypertrophy or differentiation of rat-1 cells. Moreover, inhibition of cAMP/PKA did not reverse the α~1A~-AR-induced inhibition of DNA synthesis. Therefore, α~1A~-AR-induced cell cycle arrest, hypertrophy and differentiation, is independent of the cAMP/PKA pathway in rat-1 cells. Recently, the cAMP pathway has been implicated in α~1A~-AR-induced cell cycle arrest in CHO cells expressing α~1A~-ARs \[[@B18]\]. The difference between these cell lines may be due to the inhibitory effect of PE on ERK in rat-1 cells \[[@B33]\] vs. PE-induced ERK activation in CHO cells \[[@B18]\]. In addition, the potential effect of α~1A~-AR stimulation on CHO cells hypertrophy and differentiation is not known. During our investigation of the possible mechanism by which PE induces cell cycle arrest, we observed that although both PE and FN inhibited DNA synthesis, these two agents produced remarkably different morphological changes in rat-1 cells. FN promoted a change in rat-1 cells to a small, shrinked, round shaped morphology whereas PE shifted the cells to a hypertrophied, enlarged and spindle shaped smooth muscle-like phenotype. This α~1A~-AR-induced morphological change was again independent of cAMP. Differences between PE and cAMP on different cell functions are reported in Table [3](#T3){ref-type="table"}. EGF increases DNA synthesis in rat-1 fibroblasts \[[@B22]\] and slightly increased protein synthesis (our data). However, EGF was a far less potent hypertrophic agent than PE and did not change the morphology of rat-1 cells. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Comparison of the effects of PE and FN on different cell parameters. ::: Parameter PE FN ------------------------- -------------------------- ----------------- cAMP production ↑ ↑ Protein synthesis ↑ ↓ DNA synthesis ↓ ↓ Morphology enlarged; spindle-shaped shrunk; rounded Differentiation markers ↑ ↓ Cdk inhibitors ↑ ↑ The arrows indicate a significant increase or decrease elicited by PE or FN in the corresponding cell parameter studied. These agents\' stimulated cAMP production, decreased basal DNA synthesis, and up-regulated cyclin-dependent kinase inhibitors. PE and cAMP have opposite effects on protein synthesis, cell morphology and smooth muscle differentiation markers. ::: The different effects of α~1A~-AR stimulation and cAMP on rat-1 cell hypertrophy and morphology led us to explore the effects of PE and FN on different cell parameters associated with morphological changes, i.e. cell cycle regulators and smooth muscle cell differentiation markers. Recently, it has been reported that the Cdk inhibitor p27^kip1^mediates angiotensin II-induced cell cycle arrest and hypertrophy in cultured renal tubular cells \[[@B34]\], and vascular smooth muscle cells \[[@B20]\] and induces intestinal epithelial cell differentiation \[[@B35]\]. In the present study, stimulation of α~1A~-ARs increased the expression of p27^kip1^to a much greater extent than p21^cip1/waf1^, suggesting an important role for the former Cdk inhibitor in the action of α~1A~-AR stimulation. Interestingly, the time of upregulation of p27^kip1^by PE correlated closely with its effect on inhibition of DNA synthesis, which was maximal at 18 h. Depletion of p27^kip1^prevented both PE-induced upregulation of p27^kip1^and reversed PE-induced decrease in DNA synthesis. Therefore, p27^kip1^plays a central role as a mediator of α~1A~-AR-induced inhibition of DNA synthesis and probably hypertrophy and differentiation of rat-1 fibroblasts. An important finding in the present study was that PE produced a change in the morphology of rat-1 cells characterized by an increase in cell size. Supporting this phenotypic and hypertrophic change was our demonstration that PE increased global protein synthesis and the expression of markers specific to smooth muscle cells such as smooth muscle actin, caldesmon, and myosin heavy chain. The protein levels of these smooth muscle cell markers were found to remain elevated for up to 48 h, consistent with the PE-induced morphological change that persisted for the same time period. Although stimulation of α~1A~-ARs shifted rat-1 fibroblasts to SMC/myocyte-like phenotype, it also increased the expression of the helix loop helix protein MyoD, a skeletal muscle-specific regulatory transcription factor \[[@B31]\]. Surprisingly, PE also caused an increase in pRb expression to a level similar to p27^kip1^. MyoD has been shown to interact with pRb and to promote muscle gene activation and cell cycle arrest \[[@B28],[@B36]\]. Indeed, pRb has been found to contain a differentiation-promoting activity that is distinct from its cell-cycle progression functions \[[@B37]\]. More recent evidence has indicated an essential role for pRb in promoting functional synergism between MyoD and MEF2 proteins \[[@B38]\]. Although PE increased the expression of MyoD in our study, the rat-1 cells had a smooth muscle/myocyte-like phenotype. It has been reported that vascular smooth muscle cells can spontaneously adopt the skeletal muscle phenotype \[[@B31]\]. Therefore, it is possible that PE increases expression of MyoD in rat-1 cells during differentiation into myocyte-like cells. However, expression of MyoD is not sufficient for the coordinated program of skeletal myogenesis in smooth muscle cells \[[@B31]\]. PE-induced hypertrophy and differentiation of rat-1 cells was also associated with increased levels of cell cycle proteins pRb, cyclin D1, PCNA and Cdk2 that are important for G~1~/S phase progression \[[@B39]\]. These data indicate that stimulation of α~1A~-ARs promotes the transcriptional/translational activation of the machinery required for G~1~/S cell cycle progression. However, a simultaneous increase in Cdk inhibitors such as p27^kip1^prevented DNA synthesis. Elevation of p27^kip1^protein level alone is sufficient for induction of cell cycle arrest, independent of cyclins or Cdk level \[[@B40]\]. Surprisingly, PE also increased the protein level of Cdk4, which is normally not affected by mitogenic stimuli. Although the Cdk inhibitors bind to cyclin/Cdk complexes and reduce their activity, their interaction is probably much more complex \[[@B25],[@B26]\]. Cdk inhibitors may paradoxically activate these kinases, particularly cyclin D/Cdk4, 6 complexes \[[@B26]\]. The mechanism by which stimulation of the α~1A~-AR promotes the up-regulation of Cdk inhibitors (p27^kip1^, p21^cip1/waf1^), smooth muscle cell markers, MyoD, and G~1~/S transition cell cycle proteins (pRb, cyclin D1, PCNA, Cdk2/4) leading to inhibition of proliferation and stimulation of hypertrophy and differentiation is not known. The cAMP/PKA pathway was excluded by our results as well as the ERK pathway \[[@B33]\]. The phosphatidylinositol 3-kinase and Akt/PKB pathway, a pro-survival/mitogenic and hypertrophic pathway is also unlikely to be involved, because PE does not stimulate this pathway in rat-1 cells \[[@B42]\]. Recently, a study on the genetic profiling of rat-1 fibroblasts expressing different subtypes of α-AR has shown that in cells expressing the α~1A~-AR, epinephrine (one hour stimulation) increased the gene expression of IL-6, gp-130 (an IL-6 high affinity receptor and signal transducer) and STAT-3 (an IL-6 activated transcription factor) \[[@B42]\]. Moreover, in cells expressing α~1A~-adrenergic receptor, epinephrine also increased IL-6 secretion and STAT-3 Ser^727^phosphorylation \[[@B42]\]. Therefore, it is possible that IL-6, gp130, and/or STAT-3 contributes to the upregulation of one or more of the cell cycle associated proteins and smooth muscle cell markers responsible for the cells arrest, hypertrophy and/or differentiation caused by α~1A~-AR activation in rat-1 cells. With regard to functional significance in vivo, our model uses a transformed embryonic fibroblast cell line that expresses high levels of α~1A~-AR \[[@B12]\]. Therefore, the relevance of these results to fibroblasts in tissues remains to be determined. These features in our model may underlie the ability of PE to cause expression of SMC markers and MyoD through α~1A~-AR. Conclusions =========== This study demonstrates that stimulation of α~1A~-ARs in rat-1 cells promotes cell cycle arrest by increasing levels of Cdk inhibitors and promotes hypertrophy and differentiation into a phenotype having the characteristics of smooth muscle cells by a mechanism independent of cAMP or EGF. Moreover, cell cycle progression was blocked at G~1~/S transition without causing apoptosis, and this cycle arrest was critical for rat-1 cell hypertrophy and differentiation. Reducing p27^kip1^levels reversed α~1A~-AR-promoted inhibition of DNA synthesis. Furthermore, it shows that cell cycle arrest and differentiation are closely coordinated processes but temporally separable. Further studies are underway in our laboratory to characterize the signaling pathway(s) involved in α~1A~-AR-induced differentiation of fibroblasts to smooth muscle cells. Methods ======= Materials --------- \[Methyl-^3^H\]thymidine (20 Ci/mmol) was purchased from NEN Life Science Products, Inc. (Boston, MA). L-\[4,5-^3^H\] leucine from Amercham Pharmacia Biotech. (Piscataway, NJ.). L-phenylephrine hydrochloride, penicillin, streptomycin, prazosin, propranolol, epidermal growth factor (EGF), and propidium iodide were obtained from Sigma (St. Louis, MO). Forskolin (FN), 8-cpt-cAMP and SQ 22536 were purchased from Calbiochem (La Jolla, CA). 2\'-5\'dideoxyadenosine and 2\'-deoxyadenosine 3\'-monophosphates were purchased from Biomol (Playmouth Meeting, PA). G418 sulfate from Invitrogen (Carlsbad, CA). Hanks\' balanced salt solution and fetal bovine serum were from Mediatech, Inc. (Herndon, VA). Dulbecco\'s Modified Eagle\'s Medium (DMEM) and trypsin/EDTA were obtained from Life Technologies Inc. (Grand Island, N.Y). Rat-1 cells and culture conditions ---------------------------------- Rat-1 cells transfected with bovine α~1A~-AR kindly provided to us by Drs. L. Allen, R. J. Lefkowitz, and M. G. Caron (Duke University), were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum, 400 μg/ml G418 sulfate, 50 μg/ml streptomycin and 50 units/ml penicillin at 37°C in an humid atmosphere (5% CO2, 95% air). Cells were serially passaged upon reaching confluence, and all experiments were performed on subculture passages 5--15. Prior to all experiments, preconfluent cell cultures were serum-starved for 48 h. Measurement of DNA synthesis ---------------------------- Cells at a density of 150,000 cells/well were seeded in DMEM containing 10% FBS on 24-well plates until they reached 80--90% confluency and were serum-deprived for 48 h prior to the addition of agonists. Rat-1 cells, even after serum-deprivation for 2 days, exhibit a measurable level of \[^3^H\]thymidine incorporation. Inhibition of \[^3^H\]thymidine incorporation was used as a quantitative measure of reduction in DNA synthesis. Cells were serum-starved in the presence or absence of agonists for the indicated time period, and \[^3^H\]thymidine (0.5 μCi/ml) was added for the last 4 h of the incubation. This time period was chosen because it gave the maximum incorporation of \[^3^H\]thymidine into DNA after addition of agonists. The medium was then removed, and the cells were washed twice with phosphate-buffered saline and three times with 10% ice-cold trichloroacetic acid. Cells were kept in contact with trichloroacetic acid for 10 min during each wash. The precipitated material was dissolved in 1 M NaOH containing 0.1% SDS, and radioactivity was determined by liquid scintillation spectrometry. Measurement of protein synthesis -------------------------------- Rat-1 cells were seeded at a density of 150,000 cells/well in 24-well plates and just prior to confluency were serum-deprived for 48 h. Cells were rinsed with DMEM and 1 μCi/ml \[^3^H\]leucine was added for the last 4 h of each incubation with or without the indicated concentrations of the agonists. Cells were fixed and washed three times with ice-cold trichloroacetic acid (10%). The precipitated material was solubilized with 0.1 M NaOH and the incorporated radioactivity was counted by liquid spectrometry. Flow cytometry -------------- Flow cytometry studies were performed to determine the effect of different concentrations of PE on cell cycle phases. Cells were subjected to flow cytometric DNA analysis as described \[[@B43]\] with some modifications. Briefly, rat-1 cells plated on 100 mm dishes were grown in DMEM containing 10% FBS until they reached 80--90% confluency. Preconfluent cell cultures were serum-starved for 48 h to stop the mitogenic effect of growth factors. The medium was aspirated and cells were incubated for 18 h with different concentrations of PE. Cells were trypsinized in 1 ml trypsin/EDTA for at least 5 min, and then the reaction was stopped by the addition of 1 ml of serum-containing DMEM. The samples were centrifuged at 1000 rpm for 5 min, washed three times in ice-cold PBS containing 1% bovine serum albumin by centrifugation at 1000 rpm for 5 min each, resuspended in 0.5 ml of the same solution, and then fixed with 1 ml of 70% ethanol (-20°C) added dropwise. The fixed cells were stored at 4°C until analyzed. Cells were then washed three times by centrifuging at 1000 rpm for 10 min and resuspended in 3 ml of the BSA buffer. The pellet was then finally resuspended in 1 ml of the BSA buffer to which 100 μg/ml of RNAse A was added to remove interfering RNA, and 5 μg/ml propidium iodide was added to stain DNA. Cells were incubated at 37°C for 10--15 min in the dark to facilitate staining. The cells were analyzed for DNA content using an Epics Profile Analyzer (Coulter Electronics) with an Argon laser emitting at 448 nm. Percentages of cells in various stages of the cell cycles were determined using a multi-cycle program (P. Rabinovitch, Phoenix Flow Systems). At least 10,000 cells per cycle were counted for each treatment. Cell viability -------------- Preconfluent cultures were serum-starved for 48 h. Cells were incubated with different concentrations of PE, FN and 8-cpt-cAMP for 18 h, trypsinized with 1 ml trypsin/EDTA for 5 min and centrifuged for 10 min at room temperature. The pellet was resuspended in 1 ml plain DMEM. 10 μL of the suspension was mixed with 10 μL of 0.4% trypan blue in a 0.5 ml microtube. The total number of cells and the number of blue cells and the percentage of the viable cells in 10 μL of the trypan blue mixture was calculated as follows: % viable cells = \[1-(blue cells/total cells)\] × 100. Morphological study ------------------- Rat-1 cells resuspended in culture medium were seeded on six-well plates (Corning, N.Y.). Preconfluent cells were serum-deprived for 2 days, pre-incubated with inhibitor (s) for 30 min, and then stimulated with 5 μM PE and /or 1 μM FN for the indicated time. Cells were rinsed twice in PBS to remove non-adherent cells and then fixed for 10 min in \[1:1\] methanol-acetone mixture at room temperature. Cells were washed once in distilled water and stained in hematoxylin solution (Sigma, St. Louis, MO) for 15 min at room temperature. Cells were again washed three times in water, air dried and were finally observed under a phase contrast microscope. Images were captured and saved as TIFF files. For each condition, data were collected by random observation. Hypertrophic phenotype was defined as both enlarged, elongated and spindle-shaped whereas rat-1 fibroblasts are round and/or polygonal-shaped cells. Transfection procedures ----------------------- To determine whether protein kinase A (PKA) mediates the antiproliferative effect of PE, rat-1 cells were transiently transfected with πLXX-PKI \[[@B1]-[@B31],[@B24]\], a plasmid encoding for the PKA inhibitor, PKI (A generous gift from Dr. J. Avruch, Massachusetts General Hospital, Boston, MA), using Lipofectamine Plus (Life Technologies, Inc., Grand Island, N.Y.) according to the manufacturer\'s instructions. For p27^kip1^experiments, phosphorothionate oligonucleotides (ODN) (Life Technologies, Grand Island, N.Y.) were used. The antisense ODN sequence used in the experiments was 5\'-CACTCTCACGTTTGACAT-3\' (nuc 1--18 of rat p27 kip1); the sense ODN sequence was 5\'-ATGTCAAACGTGAGAGTG. ODN transfection was performed with oligofectamine in Opti-MEM according to the manufacturer\'s protocol (Invitrogen, Carlsbad, CA). Cells were left in the transfection mixture for 48 h and then incubated for 24 h with or without agonists and harvested for Western blot, or labeled with \[^3^H\]thymidine to determine DNA synthesis. Preparation of cell lysates and western blot analysis ----------------------------------------------------- Cells were rinsed twice with PBS and lysed in ice-cold lysis buffer (1% Igepal CA-630, 25 mM HEPES pH 7.5, 50 mM NaCl, 50 mM NaF, 5 mM EDTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 5 mg/ml pepstatin A, 100 mM PMSF, 100 mM sodium orthovanadate, and 10 μM okadaic acid). Lysates were centrifuged at 4°C for 15 min in a microfuge at maximum speed, and the supernatant was collected for Western blot analysis. Equal amounts of protein were separated on denaturing SDS/polyacrylamide gel and transferred on nitrocellulose blots (Hybond-ECL; Amersham Life Sciences Inc., Arlington Heights, IL) by electrophoresis. Blots were blocked for 1 h in 5% nonfat dry milk in TBST, washed three times 5 min each in TBST and then incubated with the indicated specific primary antibody overnight at 4°C: p27^kip1^, p21^waf1/cip1^, pRb (Biosource International); caldesmon (smooth muscle), myosin (smooth muscle), α-smooth muscle actin, β-actin (Sigma Biosciences); MyoD, cyclin D1, Cdk 2, Cdk 4, PCNA, lamin A/C or vimentin (Santa Cruz Biotechnology). Following incubation, the membranes were washed three times 10 min each in TBST and incubated with a secondary antibody coupled to peroxidase for 1 h at room temperature. Then the membranes were washed three times 10 min each in TBST. Specific proteins were detected by enhanced chemiluminescence (ECL; Amersham Life Sciences Inc.) according to the manufacturer\'s instructions and analyzed with an Alpha Innotech Fluorochem imaging system (Packard Canberra). A lysate from cultured aortic vascular smooth muscle cell (VSMC) was used as a control for the expression of smooth muscle markers. Statistical analysis -------------------- The basal values of incorporation of \[^3^H\]thymidine and/or leucine were variable in different batches of cells. However, the effect of various agents on the incorporation of \[^3^H\]thymidine and \[^3^H\]leucine in rat-1 cells was consistent within each batch of cells. The results are expressed as mean ± SEM. The data were analyzed by one-way analysis of variance; the Newman-Keuls multiple range test was applied to determine the differences among multiple groups, the unpaired Student\'s t-test was applied to determine the difference between two groups. The null hypothesis was rejected at p \< 0.05. The protein level were estimated by densitometric analysis of the Western blots and performed on the indicated number of blots using NIH Image software, and expressed as a percentage of the control, arbitrarily chosen as 100%. Abbreviations ============= AR, adrenergic receptor; Cdk, cyclin-dependent kinase; FN, forskolin; MHC, myosin heavy chain; PCNA, proliferating cell nuclear antigen; PE, phenylephrine; pRb, retinoblastoma protein; SMC, smooth muscle cell Authors contributions ===================== AES performed thymidine/leucine incorporation, morphological studies, cell viability, flow cytometry and Western blot analysis. JHP carried out the antisense design, transfection experiments, statistical analysis and some thymidine/leucine incorporation and Western blot analysis. KUM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Acknowledgements ================ We would like to dedicate this article to the memory of Dr. Abdelwahab El Saeed, who unexpectedly died in September 2004. He performed this work with great dedication, high enthusiasm and passion and we will miss him. This work was supported by NIH-NHLBI grant 19134. A.E.S was supported by a NIH Minority Postdoctoral Fellowship, supplement to NIH-NHLBI grant 19134-26. J.H.P is the recipient of a Beginning Grant-In-Aid from American Heart Association Southeast Affiliate. We thank Anne Estes for her technical assistance, Felicia Walker (Molecular Resource Center, UTHSC) for performing flow cytometry analysis and Dr. Lauren Cagen for editorial comments. We gratefully acknowledge Dr. J. Avruch (Massachusetts General Hospital, Boston, MA) for generously supplying us with π LXX-PKI \[1-31\].	PubMed Central	2024-06-05T03:55:52.532898	2004-12-16	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548263/", "journal": "BMC Cell Biol. 2004 Dec 16; 5:47", "authors": [ { "first": "Abdelwahab E", "last": "Saeed" }, { "first": "Jean-Hugues", "last": "Parmentier" }, { "first": "Kafait U", "last": "Malik" } ] }
PMC548264	Background ========== An earthquake measuring 6.3 on the Richter scale struck the city of Bam in Iran on the 26th of December 2003 at 5.26 A.M. It was devastating, the city was destroyed, left over 40,000 dead and around 30,000 injured, as well as destroying approximately 20,000 homes, leaving more than 45,000 people homeless \[[@B1]\]. The profound tragedy of thousands killed has caused emotional and psychological trauma for tens of thousands of people who have survived. In general earthquakes are known to be related to increased psychological symptoms among survivors and in particular it has been shown that earthquakes might cause post-traumatic stress disorder (PTSD). Several studies on psychological morbidity have been reported following earthquakes in different parts of the world \[[@B2]-[@B5]\], but there is no report from Iran. Iran is known to be a country of frequent severe earthquakes causing thousands of deaths in recent years. Therefore for the first time a study was conducted to measure psychological distress among the survivors of Bam earthquake in Iran. The objective was to indicate prevalence of psychological distress among survivors and to specify factors associated with psychological symptoms and thus provide information for interventions needed in Bam and in similar conditions in the country. In addition it was thought this might add to a small body of existing literature on the topic from a different population. Methods ======= This was a population-based study of psychological distress among Bam earthquake survivors aged 15 years and over at five months after the event. The pre-earthquake population of Bam was reported to be 97,000 and surprisingly it was increased to around 120,000 at the post-earthquake period (listing from the authorities). This was due to the fact that because of worry many people living in rural areas were moved to the city of Bam after earthquake. At the time of screening about 80% of people stayed inside their homes and the remaining 20% were moved to four specially designated sites with around 4000 tents. However, to select a representative sample of individuals living in Bam a stratified multi-stage area sampling was applied. The stratification was used because people living in the southern region of the city were less affluent and exposed to more damage as compared to those living in the northern part of the city. Every household or tent within the city had the same probability to be sampled. For the purposes of the first stage, units (census sections) were randomly selected after stratifying by region and size of residence. Then, within each census section the homes and tents to be sampled were selected using the procedure of random routes. Finally the last stage sampling units (the individuals) were selected randomly from all persons living in the same home or tent. Random sampling was based on a list of all households (N = 23000) derived from the electricity bills. A sample of 1150 households (5%) was selected as explained. However, we were actually able to screen 1000 households, giving a success rate of 87%. The main reason for the failed attempts for contact was due to the fact that the targeted house was uninhabited because the residents had moved out and there was no possibility to trace the survivors. In addition considering logistic difficulties we did not replace them with other households. A team of trained interviewers collected data in a week and all participants were interviewed in their homes or designated camps. However, the data were collected in a disorganized environment and thus data gathering strategy was based on offering help to survivors. Since there was no validated measure of PTSD in the Iranian language, only psychological distress was measured using the Iranian version of the 12-item General Health Questionnaire, GHQ-12 \[[@B6]\]. The scale examines whether the respondent has experienced a particular symptom or behavior recently. Each item is rated on a four-point scale (less than usual, no more than usual, rather more than usual, or much more than usual). The GHQ-12 is brief, simple, easy to complete, and its application in research settings as a screening tool is well documented, and there is evidence that the GHQ-12 is a consistent and reliable instrument when used in general population samples \[[@B7]\]. The study used the original scoring method. In this method response categories score 0, 0, 1, and 1 respectively. This gives scores ranging from 0 to 12 and higher values indicate more psychological symptoms \[[@B8]\]. In addition, we collected information about demographic and the number of family members that died due to the earthquake. To analyze data Student\'s t-test and one-way analysis of variance were used to find out differences in psychological distress among different sub-groups of the study sample and the logistic regression analysis was performed to investigate factors associated with distress. For the purpose of the logistic regression analysis we used the population mean score on the GHQ-12 as cut-off point as recommended \[[@B9]\]. Results ======= Data were collected from 1000 randomly selected households (600 homes and 400 tents). In total 999 survivors were interviewed, and data for 916 respondents were complete, giving a response rate of 91.7%. The mean age of the respondents was 32.9 years (SD = 12.4), and mostly were male (53%), married (66%) and had secondary school education (50%). The mean score of the survivors on the GHQ-12 was 8.7 (SD = 3.2). The findings showed that 58% of the respondents suffered from severe psychological distress as measured by the GHQ-12. Forty-one percent of the respondents reported that they lost three to five members of their family in the earthquake. The characteristics of the respondents are shown in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The characteristics of the study sample (n = 916) ::: No. % --------------------------- -------------------- ------------- ------- Age ≤ 20 126 14 21--30 352 38 31--40 205 23 41--50 148 16 \> 50 85 9 Mean (SD) 32.9 (12.4) Gender Male 486 53 Female 432 47 Marital status Single 257 28 Married 600 66 Divorced/widowed 59 6 Educational level Illiterate 158 17 Primary 181 20 Secondary 459 50 College/university 118 13 Employment status Employed 233 25 Housewife 277 30 Student 97 11 Unemployed 275 30 Retired 34 4 Number of family loss None 185 20 \< 3 261 29 3--5 377 41 \> 5 93 10 GHQ score Mean (SD) 8.7 (3.2) \< mean score 385 42 \> mean score 531 58 ::: To examine differences in psychological distress among sub-groups of the study sample Student\'s t-test and one-way analysis of variance were performed. The findings showed that there were significant differences between people with different characteristics. People with old age, females, less educated individuals, divorced or widowed and unemployed respondents, and those with loss of more family members in the earthquake reported more severe psychological distress. The results are shown in Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The GHQ-12 score by demographic characteristics (n = 916) ::: Mean (SD) P --------------------------- -------------------- --------------- ----------- Age groups 0.006 ≤ 20 7.8 (3.2) 21--30 8.6 (3.3) 31--40 8.9 (3.0) 41--50 8.9 (3.1) \> 50 9.3 (3.0) Gender 0.04 Male 8.5 (3.2) Female 8.9 (3.1) Marital status \< 0.0001 Single 8.1 (3.3) Married 8.9 (3.1) Divorced/widowed 9.9 (2.5) Educational level \< 0.0001 Illiterate 9.7 (2.7) Primary 8.7 (3.2) Secondary 8.5 (3.2) College/university 8.3 (3.1) Employment status \< 0.0001 Employed 8.4 (3.2) Housewife 8.8 (3.4) Student 7.0 (3.2) Unemployed 9.3 (2.9) Retired 9.0 (3.2) Number of family loss \< 0.0001 None 7.7 (3.5) \< 3 8.7 (3.2) 3--5 9.1 (2.8) \> 5 9.3 (2.7) ::: To investigate factors associated with severe psychological distress all the significant findings from the univariate analysis were entered into the logistic regression analysis. The analysis indicated that being female (OR = 2.73, 95% CI 1.19--6.26, P = 0.02), illiterate (OR = 3.36, 95% CI 1.11--10.2, P = 0.03), unemployed (OR = 4.39, 95% CI 1.56--12.4, P = 0.005), and the loss of more family members (OR for loss of more than five family members = 2.11, 95% CI 1.22--3.61, P = 0.007) were associated with more severe psychological distress. The results are shown in Table [3](#T3){ref-type="table"}. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### The results of logistic regression analysis ::: OR (95% CI) P --------------------------- ------------------- ------- Age 0.98 (0.95--1.01) 0.25 Gender Male 1.0 (ref.) Female 2.73 (1.19--6.26) 0.02 Marital status Single 1.0 (ref.) Married 1.56 (0.71--3.41) 0.26 Divorced/widowed 2.17 (0.50--9.37) 0.30 Educational level College/university 1.0 (ref.) Secondary 1.09 (0.51--2.31) 0.82 Primary 1.02 (0.43--2.43) 0.96 Illiterate 3.36 (1.11--10.2) 0.03 Employment status Housewife 1.0 (ref.) Employed 2.29 (0.85--6.18) 0.10 Student 1.90 (0.57--6.32) 0.29 Unemployed 4.39 (1.56--12.4) 0.005 Retired 1.43 (0.33--6.17) 0.63 Number of family loss None 1.0 (ref.) \< 3 1.76 (1.21--2.55) 0.003 3--5 1.98 (1.32--2.98) 0.001 \> 5 2.11 (1.22--3.61) 0.007 ::: Discussion ========== This was a population-based study measuring psychological distress among survivors of the Bam earthquake in Iran. Psychological morbidity was higher among females, less educated respondents and unemployed individuals. Perhaps such observation might relate to the fact that these groups of people were exposed to a relatively homogenous set of psychological stressors, leading to relatively homogenous mental health outcomes \[[@B10],[@B11]\]. In addition although in the univariate analysis there were significant differences among sub-groups of the study sample with regard to their GHQ-12 mean scores, in the logistic regression analysis age and marital status no longer remained significant. This is consistent with other research findings where female gender, lower education, and lower socio-economic status were found to be related to higher PTSD and depression among earthquake survivors \[[@B12],[@B13]\]. However, recent findings on the topic showed a differential predictor pattern for PTSD and depression among earthquake survivors indicating that although certain factors (e.g. grater fear during the earthquake and female gender) relate to PTSD, lower education and loss of family members tend to relate to depression and not to PTSD \[[@B13],[@B14]\]. This suggests that when interpreting the study results one might relate female gender to possible PTSD which we did not measure, and the other variables to depression which was measured using the GHQ-12. Most notably it was found that people who lost family members reported significant severe psychological distress compared to those who did not. Also there was a distinct pattern of psychological distress among those who lost their family members in the earthquake showing a dose-response relationship between loss and the risk of more psychological distress. One might argue that the relationship between loss of relatives and psychological distress may be a reflection of the relationship between severity of trauma exposure and psychopathology. Since no other variables reflecting trauma-exposure (e.g. fear during the earthquake, level of damage to home, injury, etc.) were entered in the regression equation, \'loss of relatives\' was the only variable tapping that, as there is high correlation between loss of first degree relatives (with whom survivors often share the same roof) and exposure to serious damage or collapse of the house. In addition studies have indicated that loss is a strong determinant of PTSD among earthquake survivors and thus it is argued the observation that the risk of PTSD is linked to the amount of loss is an important issue that needs to be incorporated in the development of any effective preventive strategy \[[@B15]\]. As discussed earlier such finding has been challenged and studies have shown that PTSD and depression are distinct issues and loss only relates to depression whereas PTSD is linked to factors relating to exposure to threat or fear for life \[[@B14]\]. However, since in the present study only the GHQ-12 was used, there is no way of knowing which factors related to PTSD and which to depression. The GHQ-12 has items tapping depression but little is known as to how sensitive is to picking up PTSD. Psychological distress among earthquake survivors alongside experience of other problems could be considered a serious issue for people\'s health status living in such difficult conditions. Evidence suggests that severe earthquakes even can cause long-standing morbidity \[[@B12]\]. However, past psychiatric illness also might contribute to this situation \[[@B15],[@B16]\]. Unfortunately one of the shortcomings of the present study was that we did not measure previous psychiatric conditions among survivors and thus it was not possible to comment on this further. It is recommended that the mean GHQ score for the whole population of respondents provide a rough guide to the best cut-off threshold \[[@B9]\]. Thus considering people who scored above the mean, the findings from the present study indicated that 58% of the respondents showed an indication of severe mental health problems. Comparing the figure with the national data this was found to be three times higher than reported mental disorders in the general population \[[@B17]\]. A study using the GHQ-28 measuring prevalence of psychiatric disorder following the China earthquake showed relatively a similar result where the rate of psychological morbidity for earthquake survivors was found to be 51% \[[@B18]\]. Thus it is argued that there is an urgent need to deliver mental health care to disaster victims in local medical settings and health care professionals who work with the earthquake victims need to be promptly and efficiently trained in mental health crisis interventions \[[@B19]\]. The results reported here is an estimate for the overall population experiencing the earthquake. Studies have shown that there is variation in psychological morbidity among earthquake survivors by epicenter proximity and rate of property damage \[[@B12],[@B18]\]. However one should be aware that these variables are not the only information that is needed for studying the relationship between psychological distress and the impact of the earthquake. There is also need to obtain information from earthquake survivors on the extent of damage to their homes, experience of being buried under rubble, participation in rescue operations, and witnessing grotesque sites \[e.g. see \[[@B13],[@B14]\]\]. The present study had certain methodological limitations. Firstly, the results were based on the GHQ-12 scores and we did not measure PTSD symptoms. In this respect also it is important to acknowledge that the GHQ is a general measure of mental health and it is not a measure of diagnostic depression. Thus, since depression is also common in earthquake survivors, our study is limited in not including a validated diagnostic measure of depression. Secondly, no information was obtained on important trauma-exposure variables such as extent of fear or perceived life-threat during the earthquake, rubble experience, disability or injury, et cetera. Thirdly, data on important demographic variables such as past psychiatric illness, and family psychiatric illness were not collected. However in spite of these limitations, the results from this study are useful. This study is the only paper addressing reports on the psychological consequences of the most disastrous earthquakes of the last 50 years. In addition, to our knowledge the international literature does not contain any study on the psychological status of Iranian earthquake survivors, despite the fact that Iran is an earthquake-prone country. It seems that the future research should carefully assess the psychological distress and disruption experiences of the survivors in order to implement necessary interventions. Given that Iran is a country that suffers catastrophic earthquakes relatively frequently, there is need to develop and validate standard instruments such as PTSD measures to include in the future research. Conclusions =========== In conclusion the findings from this study indicated that the survivors of Bam earthquake suffer from psychological distress three times higher than the normal population. In addition female gender, lower education, unemployment, and loss of family members were found to be associated with more severe psychological morbidity among survivors. This suggests that to reduce negative health impacts of the earthquake adequate psychological counseling is needed for those who survived the tragedy. Abbreviations ============= GHQ-12: The 12-item General Health Questionnaire; PTSD: Post-traumatic stress disorder; SD: Standard deviation; OR: Odds ratio, CI: Confidence interval. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= AM was the principle investigator and contributed to the study design, the data analysis and wrote the paper. All other investigators contributed to the study design, the data collection and analysis. All authors reviewed and contributed to the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/4/prepub> Acknowledgements ================ The authors wish to thanks all those who helped to carry this study especially colleagues in the Iranian Academic Centre for Education, Culture and Research-Kerman Province Branch (ACECR). Thanks also go to the Iranian Students\' Polling Agency (ISPA) for helping the authors and to Maria Livanou (Section of Trauma Studies, Division of Psychological Medicine, Institute of Psychiatry, University of London, United Kingdom) for her thoughtful comments to improve the paper.	PubMed Central	2024-06-05T03:55:52.537303	2005-1-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548264/", "journal": "BMC Public Health. 2005 Jan 11; 5:4", "authors": [ { "first": "Ali", "last": "Montazeri" }, { "first": "Hamid", "last": "Baradaran" }, { "first": "Sepideh", "last": "Omidvari" }, { "first": "Seyed Ali", "last": "Azin" }, { "first": "Mehdi", "last": "Ebadi" }, { "first": "Gholamreza", "last": "Garmaroudi" }, { "first": "Amir Mahmood", "last": "Harirchi" }, { "first": "Mohammad", "last": "Shariati" } ] }
PMC548265	Background ========== Invasive squamous cell carcinoma(SC) is the most common malignancy of the larynx\[[@B1]\]. Distinguishing between preinvasive squamous neoplasia (high grade squamous cell dysplaisa/ squamous cell carcinoma in-situ, SCIS) and SC may be difficult in small biopsy specimens, particularly when the tissue is superficial and fragmented, a prominent inflammatory infiltrate obscures the epithelial-stromal interface, and/or there is tangential sectioning of the acanthotic neoplastic squamous epithelium. Even in larger resection specimens, the presence of invasion may sometimes be elusive if the invasive element lacks paradoxical maturation characterized by prominent eosinophilic cytoplasm that may undergo either central or individual cell keratinzation, well developed cell borders, and large vesicular nuclei with prominent nucleoli. The existence of an adjunctive feature associated with invasion would be helpful in assessing whether there is any degree of invasion in these challenging cases, or whether such a feature should raise the suspicion that the lesion may harbor an invasive component when it is absent by conventional diagnostic criteria. A moderate to marked stromal eosinophils that may infiltrate into the neoplastic epithelium has occasionally been reported in invasive carcinoma \[[@B2]-[@B4]\]. Spiegel et alrecently reported that the presence of eosinophils is associated with invasion in the neoplastic squamous lesions in the female genital tract, and proposed that eosinophilia provided as adjunctive morphologic feature in identifying SC in the cervix and vulva\[[@B5],[@B6]\]. One of us (DT) has observed moderate to marked stromal eosinophilia in cases of SC of the larynx, whereas stromal eosinophils were usually either absent or rare in cases of laryngeal SCIS. We speculated that the degree of stromal eosinophilia is a pathologic feature that would provide an adjunctive criterion for distinguishing SC from SCIS in the larynx, and undertook a systematic study to test this hypothesis. In this study, we focused on a single head and neck region, the larynx, to avoid any potential selection bias, since squamous neoplasia and the associated host response and changes in the head and neck are heterogeneous and varied in different anatomic locations\[[@B7]\]. Methods ======= The biopsy and resection specimens with available H&E stained slides of laryngeal SC and SCIS diagnosed at Roswell Park Cancer Institute from 1993 through 2000 were reviewed by two of the authors simultaneously (MZ and DT). Cases with prior radiation and/ or chemotherapy were excluded. All histology specimens at Roswell Park Cancer Institute were fixed in 10% formalin. Paraffin blocks of 5 μm thickness were cut and the sections were stained with conventional hematoxylin and eosin. For each biopsy and resection specimen, the original diagnosis was recorded and compared with the review diagnosis. Cases with any degree of invasion including \"minimal invasion\" or \"microscopic invasion\" in either a biopsy or resection specimen or both were classified as SC. Cases with SCIS only in a biopsy specimen that subsequently had invasion in the resection specimen were classified as SC. On the other hand, a resection specimen lacking invasion was required for a case to be classified as SCIS. For each of specimen, the high-power field(hpf) (Olympus BH2 ×10 ocular and ×40 objective lens) with a maximum number of eosinophils was identified and recorded as eos/hpf. Then, the eosinophils in the adjacent nine contiguous hpf were counted, added to those in the first, and recorded as eos/10hpf. Only nucleated cells with intensely red cytoplasmic granules were accepted as eosinophils, and care was taken to exclude red blood cells with superimposed mononuclear and polymorphonuclear inflammatory cells. And those that were confined to lymphovascular spaces were excluded. During the course of the study, it was noted that frozen section preparation results in the degranulation of eosinophils and leads to difficulty in their being recognized; however, under these conditions, collections extracytoplasmic typical red granules approximately the expected size of an eosinophil allows for their identification. As an internal control, non-neoplastic portions of the specimens, whenever available, were also evaluated. Statistical methods ------------------- Frequency was computed for each eosinophil category in invasive and non-invasive squamous neoplasia specimens obtained from biopsy and excision. Chi-square test was utilized to examine the difference of frequency distribution between each elevated eosinophil category and the referent eosinophil category (0--4 eos/10hpf). This analysis was conducted independently for specimens obtained from biopsy and excision. Sensitivity, specificity, positive predictive value and negative predictive value were computed for eosinophil counts that were exceeding 10 eos/hpf or 20 eos/10hpf to evaluate if these two classifications of eosinophil count would suggest any significance of clinical implication from statistical point of view. P value less than 0.05 was used to determine statistical significance. Elevated eosinophils were defined and categorized as: focally and moderately elevated (5--9 eos/hpf), focally and markedly increased(\>10/hpf), diffusely and moderately elevated(5--19 eos/10hpf), and diffusely and markedly increased (\>20/10hpf), while 0--4 eosinophils/10hpf was used as a baseline. The recorded eosinophil counts were analyzed to determine whether there were thresholds of stromal versus neoplastic squamous eosinophils per 1 hpf and 10 hpf that were significantly associated with invasive tumor in both biopsy and follow-up excisional/resectional specimens. Results ======= A total of 87 cases were evaluated and sixty-eight percent of the cases (n = 59) displayed a chronic inflammation in the stroma. Fifty-seven were biopsy specimens and 30 were ablative resection specimens. The diagnoses of 57 biopsy specimens were 35 invasive carcinoma, 4 minimal invasive carcinoma, 2 suspicious for invasion, and 16 were preinvasive squamous cell neoplasia. 27 of the biopsy specimens (18 invasive carcinoma, 1 minimal invasive carcinoma, 1 suspicious for invasion and six preinvasive squamous cell neoplasm) were followed by ablative resections (7 wide excisional resection, and 20 total laryngectomy). The follow-up specimens confirmed 19 invasive carcinoma and six preinvasive carcinoma, and revealed an invasive carcinoma in the suspicious case. In this case, there were stromal nests of neoplastic squamous cells that were associated with 15 and 34 eosinophils per 1 and 10 hpf, respectively, and clinical image studied revealed an advanced disease (stage IIIa). Additionally, Other 3 ablative resection specimens (all excisional biopsies) were initial operations. There were 2 invasive carcinoma and 1 preinvasive lesion. The distribution of eosinophil counts is summarized in Table [1](#T1){ref-type="table"}. Both diffuse and focal elevated eosinophilic infiltration were noted in invasive tumor and, to much less extend, in the non-invasive counterparts. Typical examples of eosinophilic infiltration in the stroma tissue were illustrated in Figures [1A--1C](#F1){ref-type="fig"}. As shown in Table [2](#T2){ref-type="table"}, thirty-six (57%) of patients with invasive squamous cell carcinoma were found to have diffuse eosinophilic infiltration (\>20/10hpf), whereas elevated eosinophils were not diffusely observed in all non-invasive lesions (p \< 0.05). The same held true for focally marked eosinophilia (\>10/hpf) when compared invasive group with non-invasive group. One exception was observed in a non-invasive neoplasia (Case 37, high grade dysplasia/SCIS) which showed a marked increased eosinophils (10 eosinophils/hpf). Noticeably, this high grade dysplasia with elevated eosinophil infiltrate uncovered an invasive carcinoma in the follow-up resection specimen. These results indicated that a close association exists between the stromal invasion and the presence of elevated tissue eosinophils. Stromal eosinophilia was statistically significantly associated with invasion in squamous cell carcinoma (Table [2](#T2){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Distribution of eosinophils in invasive and non-invasive squamous neoplasia ::: ------------------------ ----------------- ------------------- -------- --------- --------- ----------- ----------- ------------ --------------- Specimen and Diagnosis Eosinophils/hpf Eosinophils/10hpf 0 1--4 5--9 10--20 \>20 0 1--4 5--19 20 or greater Biopsy Invasive CA(41) 1(2%) 5(12%) 8(20%) 15(36%) 12(29%) 1(3%) 5(12%)^1^ 10(24%)^1^ 25(61%)^1^ Non-invasive(16) 13(81%) 1(6%) 1(6%) 1(6%) none 7(44%)^2^ 6(38%)^2^ 3(18%)^2^ none Excision/Resection Invasive CA(22) 1(5%) 4(18%) 8(36%) 6(23%) 3(14%) 1(5%) 3(14%) 7(31%) 11(50%) Non-invasive(8) 6(76) 1(12%) 1(12%) none none 4(50%) 3(38%) 1(12%) none ------------------------ ----------------- ------------------- -------- --------- --------- ----------- ----------- ------------ --------------- ^1^Two invasive carcinoma case less than 10hpf in size ^2^One non-invasive carcinoma case less than 10hpf in size. ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### a\. Absence of eosinophils in normal squamous epithelium. Note that a moderately inflamed submucosal tissue. Arrows point to the inflammatory cells. 200× b. A squamous cell carcinoma in-situ (non-invasive tumor) with no elevated eosinophils in a chronic inflammatory background. Arrows point to the inflammatory cells. 200× c. Markedly increased eosinophils in an invasive squamous cell carcinoma. Note that the eosinophils (arrows) were a major component of the infiltrating nucleated cells. 200× ::: ![](1472-6890-5-1-1) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Significance of eosinophils in invasive and non-invasive squamous neoplasia ::: --------------------------------- -------------------- -------- ------------------- -------- ---------------------- -------- --------------------- --------- Lesion Eosinophils Counts 5--9 eos/hpf ^1^ p \>or = 10 eos/hpf p 5--19 eosin/10hpf^2^ p \>or = 20 eos10/hpf p Invasive CA in biopsy 8/41(20%) \>0.05 27/41(66%) \<0.01 10/41(24%) \>0.05 25(61%) \<0.005 Non invasive lesion in biopsy 1/16(6%) 1/16(6%) 3/16(18%)) 0/16(\--) Invasive CA in Excision 8/22(36%) \>0.05 9/22(41%) \<0.05 7(31%) \>0.05 11((50%) \<0.05 Non-invasive lesion in excision 1/8(12%) 0/8(\--) 1(12%) 0/8(\-\-\--) --------------------------------- -------------------- -------- ------------------- -------- ---------------------- -------- --------------------- --------- ^1^eos/hpf:for each specimen, a high power field (hpf) (Olympus BH2 ×10 ocular and ×40 objective lens) with a maximum number of eosinophils in the lesion area ^2^eos/10hpf: for each specimen, one high power field (hpf) (Olympus BH2 ×10 ocular and ×40 objective lens) with a maximum number of eosinophils in the lesion area and additional nine hpfs in the contiguous areas ::: There is no association of elevated tissue eosinophils with overall inflammatory response of the stroma in the specimens studied (p \< 0.05). Specifically, among the 37 cases with \> = 10 eosinophils/hpf, 24 cases displayed a non-specific inflammation, while 50 cases with \<10 eosinophils/hpf, 35 displayed a non-specific inflammation. Among the 36 cases with \> = 20 eosinophils/10hpf, 24 cases revealed a non-specific inflammation, while 51 cases with \<20 eosinophils/10hpf, 34 revealed a non-specific inflammation. In fact, some invasive carcinomas (n = 6) virtually contained no chronic inflammatory background, but showed a marked elevated tissue eosinophila (Fig. [2](#F2){ref-type="fig"}). In addition, a number of cases (n = 21) with elevated eosinophila showed a distinct polarization of the infiltrating cells, namely eosinophilic cells accumulating in the tumor invading front (Fig. [2C](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### a\. A low power view of an invasive carcinoma. Note that there was no significant inflammatory background. 40× b. A higher power view of the square area labeled as A in Figure 2a. No eosinophils were present in the stromal tissue between the tumor nests. 200× c. A higher power view of the square area labeled as B in Figure 2a. Elevated eosinophils were present at the invading front of the carcinoma. 200× ::: ![](1472-6890-5-1-2) ::: The predictive values of tissue eosinophils in assessing stromal invasion in squamous neoplastic lesions of larynx are presented in Table [3](#T3){ref-type="table"}. In biopsy specimens, diffusely elevation of eosinophils (\>20/10 hpf) had a sensitivity, specificity and positive predictive value of invasion of 80%, 100% and 100%, respectively. In these specimen, the presence of \>10 eosinophils/hpf predicted invasion in all cases with a sensitivity of 81% and positive predictive value 96%, respectively, while values below this threshold had a predictive value of an absence of invasion 68%. Similarly, the presence of \> 20 eosinophils/10hpf in the excisional specimens had a sensitivity, specificity, and positive predictive value of invasion of 69%, 100% and 100 %, respectively. In these excisional specimens, the presence of \>10 eosinophils/hpf had sensitivity, specificity, and positive predictive values for invasion of 64%, 100% and 100%, respectively. Values below the thresholds of \>10 eosinophils/hpf or 20 eosinophils/10hpf had a predictive value of an absence of invasion of 40% and 42%, respectively. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Predictive value of eosinophils in assessing stromal invasive in squamous neoplasia of larynx ::: Eosinophils in lesion Sensitivity(%) Specificity(%) Positive predictive value(%) Negative predictive value(%) ------------------------ ---------------- ---------------- ------------------------------ ------------------------------ Biopsy specimens \>or = 10 eos/hpf 66% 94% 96% 52% \>or = 20/10hpf 80% 100% 100% 68% Excisonal specimens \>or = 10 eos/hpf 64% 98% 100% 58% \>or = 20 eos/10 hpf 68% 100% 100% 58% Sensitivity, specificity, positive predictive value and negative predictive value were computed for eosinophil counts that were exceeding 10 eos/hpf or 20 eos/10hpf to evaluate if these two classifications of eosinophil count would suggest any significance of clinical implication from statistical point of view ::: Sections with adequate non neoplastic epithelium were available in twelve cases. Eight of them were absent for eosinophils. The highest counts for non-neoplastic epithelium were 4 eosinophils/hpf and 8 eosinophils/10hpf. Although it seems that non-neoplastic epithelium contains less eosinophils than squamous neoplasia, no statistical analyses were performed since the number of available non-neoplastic regions in this series was too small. Discussion ========== For decades, pathologists have used a variety of histologic features, including desmoplastic stromal reaction, intrastromal foreign body reaction to keratin, and the presence of separate minute clusters of intrastromal neoplastic cells, to assess and identify invasion\[[@B8],[@B9]\]. However, when evaluating a small, poorly-oriented, tangentially-cut specimens, one sometimes enters an area replete with uncertainties. The presence of a morphologic feature associated with invasion would be helpful in determining whether any degree of invasion has occurred in the equivocal cases. In practice, we have noticed a frequent presence of eosinophilic infiltration in invasive squamous cell carcinoma of the larynx, which is usually absent in non-invasive neoplastic counterparts. Such a consistent observation has prompted us to carry out the current study. In this series, a systematic study of eosinophils in tissues of squamous neoplasia of larynx suggests that elevated eosinophils are a morphologic marker for assessing tumor invasiveness. We observed that in the invasive squamous carcinomas eosinophils were significantly elevated focally and /or diffusely, statistically more frequent than in non-invasive neoplasia. The increased eosinophil counts (\>10 hpf, and \>20/10 hpf) in laryngeal biopsy and excisional specimens were all statistically significantly associated with stromal invasion. In contrast, values below both of these thresholds had a significant predictive value for the absence of invasion. The slight decrease in the correlation of \>10 eosinophils/hpf with invasion in excisional specimens, relative to that in biopsy counterparts, may be attributed to the increased chance of observing microscopic clusters of eosinophils unrelated to invasion in the larger specimens. It is not surprising to observe inflammation in the specimens examined, likely due to several factors including the specific anatomic location and an overall inflammatory response of the stroma to the tumor, among others\[[@B7],[@B9]\]. However, there is no association of elevated eosinophils with overall inflammatory response of the stroma in the specimens studied. Furthermore, a number of cases with elevated eosinophila showed a distinct polarization of the infiltrating cells, specifically eosinophilic cells accumulating in the tumor invading front (Fig. [2C](#F2){ref-type="fig"}). Cumulatively, our findings strongly indicate that elevated tissue eosinophila is a specific cell response independent of a non-specific inflammatory reaction. Although elevated eosinophil counts are statistically significantlly associated with stromal invasion in squamous cell carcinoma of larynx, occasionally the presence of high number of eosinophils were observed in the non-invasive counterpart tissues (Table [1](#T1){ref-type="table"}). In other words, the presence of eosinophils in squamous neoplasia of larynx is not pathognomonic for stromal invasion and caution must be exerted when evaluating the number of infiltrating eosinophils. However, the quantitation method and thresholds identified in the current experiment may represent an adjunctive feature in assessment of stromal invasion in squamous neoplasia. Specifically, the presence of eosinophils at these thresholds should raise the suspicion that invasive or microinvasive carcinoma is present within the specimen, particularly when \>10 eosinophils/hpf and or 20 eosinophils/10hpf are observed. Since the first observation of malignancy with marked blood eosinophilia described by Rheinbach in 1893, eosinophilia has been described in human cancers from a variety of organs \[[@B10]-[@B12]\]. In head and neck squamous cell carcinoma, it has been reported that the presence of tissue eosinophils ranges between 22 and 89% \[[@B13]-[@B16]\]. Most of these series have focused on whether the presence of a prominent eosinophilic infiltrate has a prognostic value, or is an indicator of response to treatment. Some authors have claimed that the presence of a marked or moderate eosinophilic is associated with a poor prognosis\[[@B12],[@B17]\], while others have found that eosinophilia is a favorable prognostic feature\[[@B13],[@B14]\]. No study has addressed the value of eosinophils in distinguishing invasive from non-invasive squamous neoplasia in the head and neck. The mechanism of eosinophilic accumulation in cases of invasive carcinoma remains largely unknown. It has been suggested that such eosinophilic infiltration may be induced by a tumor-derived eosinophil chemotactic factor\[[@B18],[@B19]\]. A recent study further indicated that stromal eosinophils in squamous cell carcinoma may play a key role in tumor invasion through activation of gelatinase\[[@B20],[@B21]\]. It was found that 92-kd gelatinase, a key member of the matrix metalloproteineaes which are involved in tumor invasion by breaking down the basement membrane and extracellular matrix, is actively expressed by eosinophils. In conclusion, although the etiology of tissue eosinophils in invasive carcinoma is unknown, our study is the first to suggest that an elevated eosinophil count in the squamous neoplasia of larynx may serve as a morphologic feature associated with tumor invasion. The presence of more than individual eosinophils, specifically when the number of infiltrating eosinophils exceeds 10/hpf and or \>20/10 hpf in a biopsy of larynx with squamous neoplasia, represents a histologic marker for the presence of tumor invasion. Similarly, the presence of eosinophils reaching these thresholds in an excisional specimen should prompt a thorough search for invasiveness when evidence of invasion is absent, or when invasion is suspected by conventional criteria in the initial sections. Although the present study assesses a quantitative parameter of tumor invasion, in our daily practice we find it useful that a readily appreciable elevation of tissue eosinophilia alerts us to search for possible invasiveness in tissue biopsy of laryngeal lesions. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= Drs. Said, Speiegel, Tan for study design; Drs. Said, Tan, for pathology evaluation; Drs. Alrwi, Douglas, Hicks, Loree, Riguel, Wiseman for surgical evaluation and clinical follow-up as well as card review. Dr. Yang for statistical analyses. Dr. Cheney for administrative and financial support. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6890/5/1/prepub>	PubMed Central	2024-06-05T03:55:52.539900	2005-1-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548265/", "journal": "BMC Clin Pathol. 2005 Jan 7; 5:1", "authors": [ { "first": "Mahmoud", "last": "Said" }, { "first": "Sam", "last": "Wiseman" }, { "first": "Jun", "last": "Yang" }, { "first": "Sadir", "last": "Alrawi" }, { "first": "Wade", "last": "Douglas" }, { "first": "Richard", "last": "Cheney" }, { "first": "Wesley", "last": "Hicks" }, { "first": "Nestor", "last": "Rigual" }, { "first": "Thom", "last": "Loree" }, { "first": "Gregory", "last": "Spiegel" }, { "first": "Dongfeng", "last": "Tan" } ] }
PMC548266	Background ========== In both Canada and the United States efforts are underway to develop systems to assess the quality of health care as a first step to improving services. In the US nursing home sector, the implementation of the Minimum Data Set has been linked to improvements in quality of care \[[@B1]-[@B4]\]. Recently, the Centers for Medicare and Medicaid Services (CMS) has developed web pages to give consumers, and the public at large, further information about the quality of nursing homes and home care services across the country. The \"Nursing Home Compare\" and more recent \"Home Health Compare\" web sites allow individuals to view several quality indicators (QIs) for individual providers\[[@B5],[@B6]\]. When comparing health care providers across a set of QIs there is a concern that they may differentially admit clients with a greater likelihood of triggering on quality issues. Since the indicators are defined as events that are preferable to avoid (e.g., skin ulcers, untreated pain, weight loss), in the absence of risk adjustment, these providers would appear to be delivering poorer quality of care. Risk adjustment attempts to adjust for different populations of clients who may be at greater risk for experiencing quality issues that is a function of their clinical status rather than the quality of care. In the US nursing home sector, the CMS recently funded a large-scale project to review all potential long-term care QIs and the risk adjustment process. The evaluation of risk adjustment included a review of both client-level and agency-level covariates. The research team revised the original set of client covariates to create a set of new models and assessed the effects of using an agency-level covariate, the Facility Admission Profile (FAP). The FAP represents the prevalence of the quality issue for residents newly admitted to the facility and was intended to adjust for potential selection bias (i.e., facilities preferentially admitting clients with a greater likelihood of triggering on the indicator) and ascertainment bias (i.e., ability of providers to detect a quality issue that might increase the QI rate). However, after extensive analyses, the research team did not recommend the use of the FAP, given that the FAP did not appear to be a particularly useful measure of ascertainment bias \[[@B7]\]. Nevertheless, in October 2002, the CMS included the FAP in three quality measures reported on the Nursing Home Compare web page\[[@B8]\]. As part of this same initiative, Kidder et al.\[[@B9]\] examined the Nursing Case Mix Index from the RUG-III grouping system as well as seven of the RUG scales as potential risk adjusters. In their final list of quality measures, 12 indicators for chronic care residents and four indicators for post-acute care residents, included a covariate related to the RUG-III system. Home Care Quality Indicators (HCQIs) ------------------------------------ The current research was part of a project to develop a set of 22 Home Care Quality Indicators (HCQIs) based on items in the Minimum Data Set for Home Care (MDS-HC). Implementation of the MDS-HC is complete or underway in 15 states and in 7 Canadian provinces and territories, where it is being used mainly as a clinical assessment instrument. In Michigan, the MDS-HC is being used as part of the MI-Choice waiver program to reduce nursing home admissions. In Ontario, Community Care Access Centre (CCAC) case managers use the instrument to determine needs, allocate services and make placement decisions. The HCQI derivation was accomplished using data from Ontario and Michigan since these were the regions with the largest scale implementation in Canada and the US, respectively. In their paper describing the HCQI derivation, Hirdes et al.\[[@B10]\] recommended the use of client-level risk adjustment for all but four indicators, and suggested the use of the Agency Intake Profile (AIP) as another method for risk adjustment. These various approaches to risk adjustment are the focus of this study. The set of HCQIs used in this study were developed by members of interRAI, a non-profit multinational organization dedicated to the development and refinement of assessment instruments for older adults and persons with disabilities, and their related applications. The HCQIs represent outcome measures that document an agency\'s rate for triggering on a quality issue. For example, one indicator measures the prevalence of weight loss among individuals who are not considered to be palliative clients. The HCQIs include a mix of prevalence measures (i.e., measured on a cross-section of clients at one point in time) and incidence measures (i.e., failure to improve or incidence of an event measured across two points in time). All HCQIs are defined as events to be avoided such that a higher rate on the indicator is indicative of poorer performance. This paper uses a dataset from two Canadian provinces to further explore these three types of risk adjustment for the interRAIHCQIs. It explores the effects of risk adjustment at both the level of the region and at the level of the individual home care provider. Methods ======= Data ---- The RAI Health Informatics Project was a two and a half year research study begun in 1999 in which fourteen of Ontario\'s 43 CCACs used the MDS-HC as part of usual practice for all adult (18 years and older) home care clients. CCACs provide several different services: information and referral, case management and placement in long-term care facilities. CCACs purchase in-home services from external contracted agencies through a \"request for proposal\" process. Services provided include in-home physiotherapy, occupational therapy, personal support and homemaking, medical supplies and equipment, and care by other professional such as social workers, dietitians and speech-language pathologists. Case managers oversee the assessment process, make referrals to service providers and then monitor the care provided in the home. Funding for CCACs is based on an annual budget provided by the Ontario Ministry of Health and Long-term Care. CCACs are governed by independent, non-profit boards of directors, accountable to the Ministry of Health and Long-term Care. In Ontario, it is now mandatory for all long stay home care clients to be assessed with the MDS-HC. However, at the time of the RAI Health Informatics project, the MDS-HC was not mandated for use. As such, some clients (e.g., those expected to be on service less than two weeks) did not receive an MDS-HC assessment and were not included in the current project. At this same time, Manitoba conducted a pilot implementation of the MDS-HC in the fourteen offices of the Winnipeg Regional Health Authority (WRHA). The WRHA is one of twelve health authorities in the province, and is responsible for providing health services to the approximately 646,000 residents of Winnipeg and the surrounding suburban area. The WRHA receives annual funding from the government of Manitoba. The home care program of the WRHA was established in 1974 with a mandate to provide effective and responsive health care services in the community to support independent living and facilitate admission into LTC facilities when independent community living is no longer an option. Home care services include personal care, nursing, counselling, occupational therapy and physiotherapy assessment, referral to other agencies and coordination of services. In the WRHA region, an MDS-HC assessment was completed by care coordinators only if the client was anticipated to be on service for at least 90 days. As a result, the study sample was focused on a long stay population of home care clients. All participating sites in the WRHA and in Ontario identified a set of case managers who received a two-day training session led by a member of the research team. They then used the MDS-HC instrument as part of their usual in-home assessment. The data used for this study represent a cross-sectional cohort of home care clients assessed between November, 1999 and December, 2002. Two CCACs in Ontario and four WRHA sites, were removed from the database because they submitted fewer than 20 assessments, resulting in a total of ten WHRA and twelve Ontario sites. Of these, three Ontario CCACs and eight offices of the WRHA also submitted client reassessments at approximately 90 days, which allowed for the calculation of failure to improve/incidence HCQIs. The protocol for data collection was reviewed and received ethics clearance through the Office of Research Ethics at the University of Waterloo, Canada. Risk adjustment --------------- Risk adjustment attempts to adjust for differences in client populations that may bias the HCQI rates. Organizations that provide care to more impaired clients will tend to have higher unadjusted rates, regardless of the quality of care they provide. As such, risk adjustment methods are used to maximize the ability to make fair comparisons across providers. With any type of risk adjustment, caution must be exercised to prevent over adjustment (i.e., adjusting away poor practice). The choice of risk adjustment covariates is therefore highly important and should not include variables that would be considered to reflect suboptimal clinical care. There are two generic types of risk adjustment that have been recommended for the HCQIs. The first adjusts for differences in the population at the client-level\[[@B10]\]. Potential adjusters can include both individual assessment items and summary scales embedded within the MDS-HC. The HCQI developers evaluated a large range of potential covariates, considering their distributional properties, strengths of association with the outcomes of interest, consistency of findings across jurisdictions and potential for clinically inappropriate adjustment (e.g., benzodiazepine use was not considered a reasonable adjuster for falls). As a result, from zero to five risk adjusters were recommended for the 22 HCQIs and these client covariates were used in the current project\[[@B11]\]. The second type of risk adjustment was intended to control for two types of bias at the agency level: a) the agency\'s ability to identify differences in clients\' clinical characteristics and b) differences in who an agency selects for admission. These risk adjustments are performed at the agency level after the individual-level risk adjustments are applied. The Agency Intake Profile (AIP) was used following the methodology outlined by Morris et al., for the MDS 2.0 quality indicators\[[@B7]\]. The AIP was calculated for each agency based on clients for whom the MDS-HC assessment was their intake assessment or for clients who had been on service for no more than 30 days. This group of clients was considered to be the intake cohort. More recently, an alternative form of agency-level risk adjustment has been proposed, to control for potential selection and ascertainment bias. This adjustment employs the Case Mix Index (CMI) associated with the RUG-III/HC methodology\[[@B9]\]. In this instance, the CMI for the intake cohort was calculated, and adjustments to the HCQIs were performed similar to those used for the AIP. The process followed for adjusting HCQI rates was the same as that used by Morris et al. in adjusting the US nursing home QI rates\[[@B7]\]. Three sources of information are required: the agency-level observed rate, the agency-level expected rate and the grand mean across all agencies. The first data element was simply the raw observed HCQI rate for a given home care agency. The next data element involved the creation of an expected rate for each client within a given agency, based on output from a logistic regression model. In this model, each HCQI acts as a dichotomous dependent variable (i.e., triggering on the HCQI or not) and the client-level and/or agency-level covariates are entered simultaneously as independent variables. The expected value for each client is then pooled to create an expected value for the agency. Three separate risk adjusted HCQI rates are thus computed, based on which method is used for adjustment: the expected rate based on the client covariates only (CC), on client covariates plus AIP (+AIP) and on client covariates plus CMI (+CMI). The final adjusted value can be thought of as an estimate of an agency\'s HCQI rate if the agency had clients with an average level of risk\[[@B7]\]. This risk adjustment method is similar to the concept of indirect standardization, in which the ratio of the observed to expected events is calculated then multiplied by the crude rate in the standard population\[[@B12]\]. In the current project, the standard population used was the combined set of agencies from both the Winnipeg Regional Health Authority (WRHA) and from Ontario. HCQI rates ---------- For each participating agency, the unadjusted HCQI rates were calculated for all 22 indicators. These rates represent the average rate across all eligible clients for a given agency. For prevalence indicators, the rates were calculated only for clients who had been on service for at least 30 days to avoid penalizing an organization for quality issues that were newly recognized. Several methods were utilized to assess the impact of risk adjustment, both at the regional and at the agency-level. The unadjusted and three adjusted rates were compared between Ontario and the WRHA, to assess the effects at the regional level. At the level of the individual agency, ranks were calculated for each HCQI and a count was created to examine how often a given agency was among those with the four highest rates. Since higher rates on each HCQI were indicative of higher prevalence or incidence of undesirable outcomes, agencies ranked within the four highest rates were considered to be among the \"worst performers.\" The range between agencies with the highest and fourth highest rates was also examined for both unadjusted and adjusted rates to assess the degree of variation among the group of worst performers. Results ======= The two regions were very similar on average age and sex. The WRHA clients were significantly less likely to have some level of cognitive impairment, as measured by the Cognitive Performance Scale (CPS)\[[@B13]\], compared to Ontario clients, although the actual difference was only 2.6% (WRHA: 37.0% vs. Ontario: 39.6%; p \< 0.0001). They were also significantly less likely than clients in Ontario to require some assistance with ADLs (22.0% vs. 27.1%, respectively; p \< 0.0001), as measured by the ADL Self-performance Hierarchy Scale\[[@B14]\]. Severe daily pain was experienced by 17.2% of the Ontario clients compared to 14.2% of the WRHA clients (p \< 0.0001). Although the differences were statistically significant, with Ontario having significantly lower rates of both arthritis and hypertension, the absolute difference between the regions on the most common diagnoses was less than 5% (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of home care clients in the WRHA and Ontario\* ::: WRHA (n = 6704) ON (n = 5063) p value ------------------------------------------------- --------------------- ------------------- ------------- % % Age Mean (95% CI\\) 76.0 (74.9, 77.0) 75.6 (75.2, 76.0) 0.49 18--64 13.4 16.5 \<0.0001 65--74 16.5 19.2 75--84 39.9 40.7 85 and older 30.0 23.6 Sex Female 69.2 70.5 0.14 Marital status Never married 11.5 8.7 \<0.0001 Married/widowed 79.4 82.7 Separated/divorced 8.2 7.9 Other 0.8 0.7 Primary language English 85.6 85.1 0.63 French 3.5 3.8 Other 10.9 11.1 Aboriginal status Origin is Inuit, Metis or North American Indian 3.2 1.6 \<0.0001 Cognitive Performance Scale Mean (95% CI) 0.8 (0.8, 0.8) 0.8 (0.8, 0.9) 0.07 0 -- intact 63.0 60.4 \<0.0001 1 -- borderline intact 15.0 18.4 1 -- borderline intact 15.0 18.4 2 -- mild impairment 8.4 7.3 3 -- moderate impairment 10.9 10.1 4 -- moderate to severe impairment 0.8 1.0 5 -- severe impairment 1.5 2.5 6 -- very severe impairment 0.5 0.4 ADL Self-Performance Hierarchy Scale Mean (95% CI) 0.5 (0.5, 0.6) 0.7 (0.6, 0.7) \<0.0001 0 -- independent 78.0 72.9 \<0.0001 1 -- supervision required 5.2 7.9 2 -- limited impairment 8.9 8.5 3 -- extensive assistance required (level I) 4.7 5.4 4 -- extensive assistance required (level II) 1.6 2.6 5 -- dependent 1.1 2.0 6 -- total dependence 0.5 0.7 Pain Scale Mean (95% CI) 1.2 (1.2, 1.2) 1.3 (1.3, 1.4) \<0.0001 0 -- no pain 39.8 34.9 \<0.0001 1 -- less than daily pain 14.4 14.1 2 -- daily pain but not severe 31.6 33.8 3 -- severe daily pain 14.2 17.2 *Top 3 medical diagnoses\\\** Arthritis 49.4 44.8 \<0.0001 Hypertension 42.2 37.4 \<0.0001 Diabetes 19.1 20.1 0.24 \* client characteristics and scale values based on first submitted MDS-HC assessment \\ CI = confidence interval \\\* disease was present and was or was not being treated or monitored by a home care professional ::: Unadjusted rates ---------------- When comparing the two regions, there were statistically significant differences for five of the 22 unadjusted HCQIs (Table [2](#T2){ref-type="table"}). In only one of these five cases (ADL rehabilitation potential and no therapies) was the rate in the WRHA significantly higher than the rate in Ontario. However, the actual size of the absolute difference between regions was small (0.3% on average). Among the prevalence HCQIs, the largest unadjusted difference was for disruptive/intense daily pain, which was 9.1% higher in Ontario. There were no statistically significant differences between the regions for any of the incidence HCQIs. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Unadjusted HCQI rates comparing the WRHA and Ontario\* ::: WRHA Offices Ontario CCACs *p value\\* ----------------------------------------------------------------- ------------------ ------------------- ----------------- ------------ -------- n = 10 n = 12 Prevalence HCQIs Mean (sd) 95% CI Mean (sd) 95% CI Inadequate meals 2.7 (1.2) 1.9, 3.6 4.4 (1.9) 2.1, 7.1 0.02 Weight loss 5.7 (3.4) 3.2, 8.1 7.6 (2.2) 5.1, 10.3 0.13 Dehydration 0.6(0.7) 0.08, 1.1 2.4 (1.6) 1.0, 5.0 0.003 Not receiving a medication review by a physician 3.8 (1.8) 2.5, 5.0 10.3 (4.3) 6.6, 17.1 0.0002 No assistive device among clients with difficulty in locomotion 13.8 (16.4) 2.1, 25.5 10.2 (5.2) 4.2, 15.6 0.52 ADL/rehabilitation potential and no therapies 84.0 (7.4) 78.7, 89.3 73.7 (5.5) 69.6, 78.5 0.001 Falls 20.8 (7.8) 15.2, 26.3 24.4 (4.5) 19.7, 30.3 0.18 Social isolation 21.6 (4.5) 18.4, 24.8 24.3 (5.9) 17.6, 32.4 0.24 Delirium 5.1 (2.3) 3.4, 6.7 5.9 (2.6) 3.4, 9.0 0.45 Negative mood 7.9 (3.2) 5.6, 10.1 11.6 (6.2) 5.2, 18.4 0.10 Disruptive or intense daily pain 30.5 (4.0) 27.6, 33.3 39.6 (6.3) 33.6, 44.3 0.0008 Inadequate pain control among those with pain 26.6 (5.3) 22.8, 30.4 29.7 (7.4) 22.3, 35.8 0.27 Neglect/abuse 3.0 (2.6) 1.2, 4.9 2.5 (2.1) 0.8, 5.2 0.58 Injuries 13.6 (6.4) 9.0, 18.2 11.0 (3.7) 8.6, 14.3 0.26 No influenza vaccine 28.4 (7.1) 23.2, 33.5 30.3 (9.8) 21.1, 44.0 0.61 Hospitalization 32.0 (7.2) 26.8, 37.2 36.4 (4.9) 31.1, 40.4 0.11 Incidence HCQIs n = 8 n = 3** Failure to improve/incidence of bladder incontinence 29.5 (9.7) 21.4, 37.5 23.1 (7.4) 4.8, 41.4 0.33 Failure to improve/incidence of skin ulcers 3.4 (4.54) 0.0, 7.1 3.4 (3.2) -5.0, 11.3 0.96 Failure to improve/incidence of decline on ADL long form 41.5 (12.5) 31.1, 51.9 30.3 (4.4) 19.3, 41.3 0.17 Failure to improve/incidence of impaired locomotion in the home 11.0 (9.0) 3.5, 18.5 16.5 (9.2) -6.5, 38.4 0.44 Failure to improve/incidence of cognitive decline 44.6 (13.7) 33.1, 56.0 36.1 (3.9) 26.3, 45.9 0.33 Failure to improve/incidence of difficulty in communication 13.7 (8.1) 6.9, 20.4 14.4 (3.4) 6.0, 22.9 0.88 \* in the event that the lower value of the 95% confidence interval was less than zero, the value was set to zero. \\ based on a t-test of independent means. ::: Agency-level covariates ----------------------- The AIP was calculated for each home care agency for each of the 19 HCQIs for which client-level risk adjustment was recommended. The AIP values shown represent the HCQI rate among an admission cohort within each region and were used in the risk adjustment models that included client-level covariates together with the AIP covariate. Ontario CCACs had a significantly higher AIP value for eight indicators, demonstrating that they admit or at least assess individuals as more likely to have these conditions on admission (Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### AIP values for the WRHA and Ontario for each of the 19 risk-adjusted HCQIs\* ::: WRHA n = 10 Ontario n = 12 p value ----------------------------------------------------------------- ------------------- -------------------- ------------- *Prevalence HCQIs* mean (95% CI) Inadequate meals 2.9 (1.7, 4.1) 6.8 (5.3, 8.2) 0.0003 Weight loss 8.2 (4.7, 11.7) 12.8 (10.7, 14.8) 0.02 Dehydration 0.6 (0.2, 1.1) 4.0 (2.5, 5.5) 0.0005 No assistive device among clients with difficulty in locomotion 13.4 (10.8, 16.0) 19.1 (12.7, 25.4) 0.09 Falls 24.4 (20.6, 28.2) 31.4 (27.5, 35.3) 0.01 Social isolation 22.4 (19.4, 25.3) 25.3 (21.2, 29.4) 0.23 Delirium 5.2 (4.0, 6.4) 10.2 (7.8, 12.6) 0.0008 Negative mood 7.7 (5.5, 9.9) 12.8 (8.7, 17.0) 0.03 Disruptive or intense daily pain 31.5 (28.1, 34.9) 42.3 (37.5, 47.0) 0.0008 Inadequate pain control among those with pain 30.6 (26.6, 34.6) 31.4 (26.5, 36.4) 0.79 Neglect/abuse 2.5 (1.5, 3.5) 2.9 (1.3, 4.6) 0.65 Injuries 11.5 (8.5, 14.5) 12.8 (9.1, 16.5) 0.55 Hospitalization 35.3 (31.6, 39.1) 52.0 (47.4, 56.6) \<0.0001 *Incidence HCQIs* n = 8 n = 3 Failure to improve/incidence of bladder incontinence 0.9 (0.8, 1.0) 0.9 (0.5, 1.2) 0.81 Failure to improve/incidence of skin ulcers 6.3 (5.6, 7.0) 7.6 (4.8, 10.3) 0.07 Failure to improve/incidence of decline on ADL long form 1.9 (1.6, 2.3) 2.8 (0.0, 6.9) 0.45 Failure to improve/incidence of impaired locomotion in the home 0.3 (0.2, 0.4) 0.6 (0.0, 1.6) 0.36 Failure to improve/incidence of cognitive decline 0.8 (0.7, 0.9) 1.0 (0.2, 1.7) 0.21 Failure to improve/incidence of difficulty in communication 0.5 (0.4, 0.6) 0.6 (0.0, 1.6) 0.65 \* for prevalence HCQIs and failure to improve/incidence of skin ulcers, the AIP value represents the mean rate (expressed as a percent) of the HCQI on an intake cohort of clients and for the remaining incidence HCQIs, the AIP value represents the mean value for the MDS item(s) involved in calculating the HCQI. ::: Among new home care clients in Ontario, 52% of clients triggered on the HCQI for the prevalence of hospitalization versus 35% in the WRHA (difference of 17%). Ontario also had a significantly (p \< 0.0001) higher Case Mix Index (CMI) on intake (i.e., the CMI covariate), at 0.84 (95% CI: 0.81, 0.86), compared with the WRHA at 0.70 (CI: 0.68, 0.71). Risk adjustment at the regional level ------------------------------------- In general, the risk adjustment process minimized the differences between the two regions compared with the unadjusted rates. For example, the unadjusted difference between regions for the prevalence of disruptive/intense daily pain was 9.1%, however, the difference was reduced to 8.0% with the CC adjustment, 5.7% with the +CMI adjustment and 2.8% with the +AIP adjustment (Table [4](#T4){ref-type="table"}). The +AIP adjusted rates showed the most variability, so that for five HCQIs, the direction of the difference was reversed. For example, the unadjusted rate of falls was 3.6% *higherin Ontario than in the WRHA. Following the +AIP adjustment, Ontario had a rate that was 1.4% lowerthan in the WRHA. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Difference between Ontario and WRHA HCQI unadjusted and adjusted rates\ ::: Unadjusted Client covariates only AIP + client covariates CMI+ client Covariates ----------------------------------------------------------------- ------------------------------------------- ---------------------------- ----------------------------- ---------------------------- Difference (ON-WRHA) expressed as a % *Prevalence HCQIs* Inadequate meals 1.7 1.7 0.2 1.5 Weight loss 1.9 1.9 0.2 1.5 Dehydration 1.8 1.9 1.1 1.7 No assistive device among clients with difficulty in locomotion -3.6 -1.9 -2.8 -3.3 Falls 3.6 1.7 -1.4 0.7 Social isolation 2.7 0.8 1.1 0.6 Delirium 0.8 1.1 -0.4 0.8 Negative mood 3.7 3.1 -1.0 1.8 Disruptive or intense daily pain 9.1 8.0 2.8 5.7 Inadequate pain control among those with pain 3.1 2.8 1.7 3.3 Neglect/abuse -0.5 -0.4 -0.5 -1.0 Injuries -2.6 -3.0 -3.3 -1.8 Hospitalization 4.3 2.8 -1.4 2.1 *Incidence HCQIs* Failure to improve/incidence of bladder incontinence -6.4 -5.7 -5.6 -2.3 Failure to improve/incidence of skin ulcers 0.0 0.0 -1.2 -0.8 Failure to improve/incidence of decline on ADL long form -11.2 -11.0 -10.7 -6.6 Failure to improve/incidence of impaired locomotion in the home 5.0 5.3 -0.1 4.2 Failure to improve/incidence of cognitive decline -8.5 -6.1 -7.7 -4.1 Failure to improve/incidence of difficulty in communication 0.7 1.3 0.0 1.7 *Mean* *0.3* *0.2* *-1.5* *0.3* \* differences were always calculated as Ontario rate minus WRHA rate ::: Agencies with the highest rates ------------------------------- A summary measure was created to count the frequency with which an agency was ranked among the worst performers. Overall, only three of the ten offices within the WRHA did not change their ranking when rates were adjusted (Table [5](#T5){ref-type="table"}). For example, Office 6 was ranked within the four highest (i.e., worst) agencies for three out of the 19 HCQIs both for unadjusted and for the three adjusted rates. In another six offices, the effect of risk adjustment was a decline in their standing (i.e., poorer quality) so that after at least one type of risk adjustment they were ranked among the worst performers. For example, in Offices 2, 3 and 7, the number of times they were ranked within the four highest rates increased as a result of the +AIP adjustment. The most dramatic effect was evident in Office 7 such that the unadjusted rates ranked this group among the worst performers only once, but the +AIP adjustment increased this frequency to five. There was no instance of an office in the WRHA consistently benefiting from risk adjustment. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Number of times a given agency was ranked within the four highest rates ::: Number of times agency ranked within the four highest rates ------------- ----------------------------------------------------------------- ----------------------- ----------------------------- ----------------------------- Unadjusted Client covariates AIP + client covariates CMI + client covariates WRHA 1 8 9 10 9 2 1 1 4 3 3 2 2 5 3 4\* 0 0 0 0 5 3 5 4 5 6 3 3 3 3 7 1 2 5 2 8 0 0 2 0 9 7 6 8 6 10\* 4 4 4 4 Ontario 100\* 3 3 2 4 200 5 5 4 4 300\* 1 1 2 2 400\* 5 5 6 4 500\* 1 2 0 2 600\* 4 3 3 1 700\* 1 1 2 1 800\* 5 4 2 4 900\* 6 6 6 6 1000\* 1 1 0 1 1100 10 9 3 8 1200 6 4 1 4 \* indicates agencies for which the count was based on 13 HCQIs since incidence HCQIs could not be calculated ::: In Ontario, only CCAC 900 experienced no change in their ranking following risk adjustment (Table [5](#T5){ref-type="table"}). In another nine cases, at least one type of adjustment resulted in an improved standing, with the agency ranking among the four highest rates less often. For example, CCAC 1100 ranked among the worst performers ten times across the 19 HCQIs prior to risk adjustment, but the +AIP adjustment reduced this value to three. A similar effect was observed for CCAC 1200 which was ranked in the worst four performers six times across the unadjusted rates, but only once following +AIP adjustment. Examining only the ranking of agencies may be misleading if very small changes in the actual rate resulted in an increase or decrease in the rank for a given agency. For example, the change in the rate for a given agency could be very small, say 5%, but could result in the organization moving from the fifth highest rate (i.e., fifth worst performer) to the third highest rate. Examining only the ranking tells one little about the actual magnitude of change among the worst performers. Therefore, it is also important to explore the range in the rates across the four highest agencies. The unadjusted and CC adjusted rates had the largest degree of variation between the highest and fourth highest agency, with a mean of 11% across the 19 HCQIs. The +CMI adjustment had the next largest amount of variation at 10% and the +AIP adjustment at 8% (Table [6](#T6){ref-type="table"}). These results further reinforce the finding that the +AIP method of adjustment consistently had the largest impact compared with the other types of risk adjustment. ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Range in the HCQI rates comparing agencies with the highest rates ::: Unadjusted Client covariates AIP + client covariates CMI + client covariates ----------------------------------------------------------------- ------------------------------------------------ --------------------------------------------- ------------------------------------------------ --------------------------------------------- ------------------------------------------------ --------------------------------------------- ------------------------------------------------ --------------------------------------------- Range between 1^st^and 4^th^highest agency Difference between 1^st^and 4^th^agency Range between 1^st^and 4^th^highest agency Difference between 1^st^and 4^th^agency Range between 1^st^and 4^th^highest agency Difference between 1^st^and 4^th^agency Range between 1^st^and 4^th^highest agency Difference between 1^st^and 4^th^agency % % % % *Prevalence HCQIs* Inadequate meals 7.4 -- 4.9 2.5 8.3 -- 5.4 2.9 6.4 -- 4.6 1.8 8.2 -- 5.4 2.9 Weight loss 13.3 -- 9.3 4.0 13.3 -- 9.2 4.1 10.7 -- 9.2 1.5 13.6 -- 9.8 3.8 Dehydration 6.1 -- 2.6 3.5 6.6 -- 3.0 3.6 3.8 -- 2.3 1.5 6.5 -- 2.8 3.7 No assistive device among clients with difficulty in locomotion 60.0 -- 15.3 44.7 50.4 -- 14.8 35.6 53.8 -- 15.6 38.2 52.0 -- 14.3 37.7 Falls 38.4 -- 28.8 9.6 38.4 -- 28.8 9.6 36.5 -- 28.4 8.1 39.1 -- 29.1 10.0 Social isolation 33.1 -- 28.6 4.5 31.8 -- 30.2 1.6 31.5 -- 30.3 1.2 31.9 -- 30.2 1.7 Delirium 11.0 -- 8.1 2.9 14.3 -- 12.4 1.9 12.8 -- 11.9 0.9 14.2 -- 12.9 1.3 Negative mood 26.2 -- 13.4 12.8 30.4 -- 16.1 14.3 18.0 -- 15.0 3.0 23.4 -- 15.9 7.5 Disruptive or intense daily pain 54.1 -- 42.5 11.6 51.6 -- 40.8 10.8 43.7 -- 39.4 4.3 44.5 -- 41.2 3.3 Inadequate pain control among those with pain 46.5 -- 32.6 13.9 47.6 -- 33.8 13.8 36.6 -- 33.0 3.6 47.4 -- 34.2 13.2 Neglect/abuse 9.7 -- 3.9 5.8 9.5 -- 4.0 5.5 6.7 -- 3.7 3.0 9.8 -- 4.4 5.4 Injuries 22.4 -- 18.9 3.5 22.8 -- 18.3 4.5 21.9 -- 16.1 5.8 22.0 -- 19.0 3.0 Hospitalization 46.8 -- 40.1 6.7 44.6 -- 40.8 3.8 47.7 -- 38.4 9.3 46.2 -- 41.2 5.0 *Incidence HCQIs* Failure to improve/incidence of bladder incontinence 48.0 -- 29.0 19.0 48.4 -- 28.4 20.0 44.5 -- 31.9 12.6 47.3 -- 27.9 19.4 Failure to improve/incidence of skin ulcers 12.9 -- 4.2 8.7 13.2 -- 4.3 8.9 11.1 -- 5.5 5.6 14.1 -- 4.9 9.2 Failure to improve/incidence of decline on ADL long form 56.0 -- 42.9 13.1 55.1 -- 43.4 11.7 54.9 -- 43.3 11.6 53.9 -- 41.2 12.7 Failure to improve/incidence of impaired locomotion in the home 29.0 -- 14.1 14.9 33.4 -- 19.1 14.3 36.6 -- 22.2 14.4 34.1 -- 18.4 15.7 Failure to improve/incidence of cognitive decline 72.0 -- 48.4 23.6 70.1 -- 46.7 23.4 69.5 -- 46.3 23.2 69.6 -- 46.0 23.6 Failure to improve/incidence of difficulty in communication 23.8 -- 18.3 5.5 30.7 -- 22.3 8.4 29.3 -- 22.4 6.9 30.6 -- 22.4 8.2 Mean difference 11.1 10.5 8.2 9.9 ::: Discussion ========== Ontario CCACs exhibited several key differences when compared to the WRHA. Ontario sites had significantly higher unadjusted rates across four HCQIs. However, the degree of variation between the sites was small, with a mean difference across indicators of less than 1%. Ontario as a region had significantly higher AIP values for eight HCQIs, indicating a greater propensity in Ontario to admit clients exhibiting quality issues, and they also had a higher mean Case Mix Index. Overall, the change in the actual rates at the regional level was small following risk adjustment. When risk adjustment was applied, for the client covariates alone or the client covariates with the CMI covariate, there was little effect on the mean difference between the two regions. In each case, the mean difference was positive (i.e., Ontario had a higher rate on average) and less than 1%. The +AIP adjustment, however, resulted in a negative mean difference (i.e., WRHA higher than Ontario on average) across the set of risk-adjusted HCQIs. At the agency level, there was a greater influence of risk adjustment, as assessed by agency rankings across the set of indicators. In general, sites in Ontario benefited from risk adjustment and were less likely to be ranked among the worst performers. The opposite was true within the WRHA. Again, the +AIP adjustment had the largest influence when compared to the other two types of risk adjustment. It is possible that the level of variation between agencies on the AIP covariate was greater than the level of variation between clients on the individual-level covariates. Although a detailed analysis is beyond the scope of this paper, use of a single HCQI example may prove beneficial. When examining the prevalence of falls (an HCQI that showed the largest changes in the agency rankings following risk adjustment), the coefficient of variation (CV) for the AIP value was 23.0. The CV is a relative measure of dispersion about the mean and is calculated by dividing the standard deviation by the mean and multiplying by 100. This CV value was much lower than any of the corresponding CV values for the various MDS outcome scales that reflect differences at the level of the individual client. For example, the CV for the Cognitive Performance Scale was 158.5 and for the ADL Self-performance Hierarchy Scale, the CV was 195.6 (data not shown). Clearly, the +AIP adjustment had the effect of minimizing differences between the individual providers and it also resulted in rates that had less variability, as demonstrated by the drop in the CV value when comparing the unadjusted and +AIP adjusted rates for the prevalence of falls indicator (CV of 27.7 and 19.1, respectively; data not shown). However, this effect cannot be explained by differences in the variances of the adjusters since the AIP had a lower level of variance than the individual covariates. It is also possible that the AIP covariate serves as a proxy for regional differences in practice patterns. If the Ontario agencies are grouped into four main geographic regions, the AIP value for the falls HCQI ranged from 24.1% to 34.0%. The degree of variation appears modest, but cannot be discounted as one factor in the ability of the AIP adjustment to minimize differences between providers and between regions. Although there continues to be support for the conceptual notion of risk adjustment, the potential to over adjust remains a concern. In a recent publication, Mor et al\[[@B15]\] recognize the possibility for over adjustment and conclude that in the absence of a simple solution to this problem, researchers must carefully consider each performance measure on an individual basis in an attempt to minimize this issue. In the current project, the AIP covariate represents a continuous, numeric value that can range from zero to one. Thus, it does not simply serve as an agency identifier, but represents the rate among an intake cohort. Furthermore, in the US, Morris et al. determined that the Facility Admission Profile, the conceptual cousin to the AIP, did not substantially improve the risk adjustment models and they did not recommend its use. This decision was not based on a fear of over adjustment, but rather a concern that the FAP added increased complexity without significantly improving the risk adjustment process\[[@B7]\]. It is also important that to develop a clearer understanding of what is meant by \"over adjustment\". For example, it would seem reasonable to differentiate between instances of truly inappropriaterisk adjustment where variance is due to poor practices (e.g., adjusted for benzodiazepine use for a falls indicator) and \"over adjustment\" due to the excessive use of spurious risk adjusters resulting in suppression of variance. Another possible example of over adjustment is the use of individual level covariates that are too closely related to the quality indicator. For example, one might argue that using dressing of the upper body as a risk adjuster for a QI on dressing the lower body is a form of over adjustment because the dependent variable is represented on both sides of the regression equation. This area of research continues to present many challenges. Given the current results and those from the long-term care sector, it appears advantageous to undertake some form of risk adjustment, even though the definitive method may not yet be best understood. Given that these HCQIs are new and so little is known about the risk adjustment process, it seems appropriate that all three type of risk adjustment be considered by researchers and policy makers. The +AIP adjustment represents the most conservative approach and may therefore be most appropriate for public reporting of HCQI results. The assessment of quality of care, and ongoing refinement of risk adjustment methods, is ultimately intended to provide information to different audiences to assist in continuously improving the quality of care. To date, many initiatives in the US have taken place to provide additional information on the quality of LTC and home health services\[[@B6]\]. Similar efforts have not begun in the Canadian home care sector. Several important issues should be addressed prior to moving towards more public reporting of these types of quality data. For example, there needs to be a discussion of the relative importance of the HCQIs. The current project made no attempt to prioritize the indicators, although clearly some would have higher clinical priority than others. To some degree, individual agencies must determine their own priorities. However, a simple system based on prevalence, severity and modifiability may be useful in determining relative importance. For example, pain is a prevalent condition in this population, can be severely debilitating for clients and is clearly modifiable. One might argue that it is of higher importance to address than declining cognitive performance, which is less prevalent and in many cases not modifiable. On the other hand, cognitive decline can have extremely serious consequences for the individual In addition, a decision is needed regarding the calculation of the HCQIs, in particular, which pairs of assessment are to be used in the calculation of failure to improve/incidence HCQIs and what time period in between assessments is acceptable (e.g., a minimum of 90 or 120 days). There will need to be further analysis to explore the relationship between the length of stay and triggering on the HCQIs. It is anticipated that clients who have been on service longer will show important clinical differences from short-stay clients and would therefore be expected to have differential HCQI rates. Tracking of the HCQI rates over time will be essential to determine the level of stability in the indicators. It would be useful to measure the amount of variation and to assess methods to summarize the rates over time in order to maximize their stability. For example, it may be important to report annual HCQI rates for a given organization or province if in fact the rates are found to be highly variable over shorter periods of observation. A shorter time frame may also lead to unstable rates if the number of observations is small. In this paper, a minimum of 20 observations was required and a decision would also be needed for the minimum sample size for public reporting of HCQIs. Finally, it will be essential to assess possible methods to create summary measures across the set of HCQIs. This is not a simple task and one for which little research has been conducted to date. Previous research has pointed to the lack of correlation between measures of quality, but has also suggested that summary measures would be useful for consumers who would likely prefer less complicated information\[[@B15]\]. Knowledge about the home care sector in Canada remains rudimentary, and our knowledge and understanding of quality assessment in this sector is still in its infancy. The results from the current project serve to enhance the overall understanding of the issues involved in quality assessment and our ability, through the risk adjustment process, to create fair comparisons across providers. Conclusions =========== Risk adjustment of quality indicators is important to enable fair comparisons across geographic regions or across home care providers. To date, little research has examined the quality of home care services. This project, using a set of HCQIs developed by interRAI, provides an important first step in assessing quality and the variable effects of different types of risk adjustment. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= DD participated in the coordination of the study, assisted in the conceptual design of the study, carried out the data analysis and took the lead in developing the manuscript. JH designed the original study, oversaw the development of the indicators, gave input into the data analysis and development of the draft manuscript. BF was the Co-Principal Investigator in the development of the indicators and provided feedback in terms of data analysis and choice of statistical methods. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/5/7/prepub> Acknowledgements ================ The authors would like to acknowledge John Morris, Naoki Ikegami, David Zimmerman and Richard Jones as well as Pablo Aliaga and Suzanne Hammer who were instrumental in the development of the home care quality indicators. The authors are grateful for contributions to the development effort by Mary James, Nancy Curtin-Telegdi, Jeff Poss, Colleen Maxwell, Lori Mitchell, Trevor Smith, Paula Fletcher, Gary Teare, Michael Stones, Kim Voelker, and Judy Bowyer. The authors also wish to thank Roy Cameron, John Goyder and Margaret Denton for helpful comments on an earlier report on which this manuscript is based. The authors wish to thank the following agencies for financial support of this research: Agency for Health Care Policy Research (Grant 5 U18 HS09455), the Health Transition Fund -- Health Canada (ON 421), interRAI, the State of Michigan and the Robert Wood Johnson Foundation.	PubMed Central	2024-06-05T03:55:52.542305	2005-1-18	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548266/", "journal": "BMC Health Serv Res. 2005 Jan 18; 5:7", "authors": [ { "first": "Dawn M", "last": "Dalby" }, { "first": "John P", "last": "Hirdes" }, { "first": "Brant E", "last": "Fries" } ] }
PMC548267	Background ========== Alzheimer\'s disease (AD) is the most common form of dementia affecting elderly people in the United States. Prevalence is 1% to 2% at age 65 years, but increases markedly to 35% or greater by age 85. Because of a demographic shift toward a more aged population, the percentage of affected individuals is rapidly increasing. This trend is expected to continue for the foreseeable future. Therefore, accurate and timely diagnosis and effective treatments are critical to optimal outcomes over the 8- to 10-year course of the illness \[[@B1]\]. Traditionally, a probable diagnosis of AD was accomplished by history, clinical examination, neuroimaging, and neuropsychological and laboratory testing to rule out treatable causes for the patient\'s symptoms and to differentiate AD from other possible causes of dementia \[[@B2],[@B3]\]. Much effort has gone into defining risk factors for the development and progression of Alzheimer\'s dementia, as well as to identify biological markers for the disease. Clinical-demographic variables that are consistently associated with AD in prior studies include family history of AD, age, and Down\'s syndrome \[[@B1],[@B3]\]. None of these variables has been demonstrated to affect the rate of disease progression or show any utility in defining subgroups that may be more amenable to therapy. Currently, predominant symptoms of dementia are treated primarily with second-generation cholinesterase (ChE) inhibitors. These drugs have demonstrated efficacy, as measured by cognitive, behavioral, and functional outcomes, in randomized, placebo-controlled clinical trials, the majority of which have been of 6 months\' duration \[[@B4]-[@B6]\]. In an open-label extension study of the cholinesterase inhibitor donepezil, Doody et al \[[@B7]\] concluded that donepezil was safe and effective for treating the symptoms of mild to moderate AD for up to 2 1/2 years. Cognitive, behavioral, and functional outcomes in patients treated with ChE inhibitors over the longer term are of great interest given the substantial social and economic implications of AD, which has a course that averages 8 to 10 years. Due to their relatively recent approval, however, longer-term data on the clinical benefits and/or limitations of ChE inhibitor therapy in AD patients is virtually nonexistent \[[@B8]\]. Rivastigmine\'s approval by the FDA in 2000 was supported by several pivotal trials, including a randomized US trial (ENA 713 B352)\[[@B5]\]. In this pivotal trial, 699 patients with mild to moderately severe AD were randomized to high dose rivastigmine (6--12 mg/day), low dose (1--4 mg/day) or placebo with a 7 week fixed dose-titration phase followed by a flexible dosing phase during weeks 8--26. Results of the 26-week open-label extension of this study found that at 52 weeks, patients originally treated with 6--12 mg/day rivastigmine had significantly better cognitive function than patients originally treated with placebo \[[@B9]\]. In this paper the authors present descriptive findings for a cohort of 37 patients who participated in the long-term open-label extension of the ENA713B352 rivastigmine trial. Much work remains to be done to more definitively answer questions about when to start therapy, which patients are most likely to benefit, what constitutes clinically relevant beneficial effects over the longer term, and when these drugs are no longer clinically effective. Consideration should also be given to withdrawal of therapy. Findings presented in this article will add to the current limited dataset for long-term efficacy and outcomes with cholinesterase inhibitor therapy for persons with probable AD. This report describes our experience in following the cohort of patients at our center with AD treated with the ChE inhibitor rivastigmine (a medication that inhibits both butyl- and acetylcholinesterase) as part of the ENA 713 B352 pivotal trial for a period up to 5 years. Methods ======= Data in this analysis came from a subgroup of 37 patients with originally mild- to moderate-stage (defined by a Mini-Mental State Examination \[MMSE\] score of 10 to 26) AD followed at a large Mid-Western university site in a 26-week, prospective, randomized, double-blind, placebo-controlled, parallel-group study of rivastigmine as therapy for AD conducted at 22 research sites across the United States (ENA713, B352 Study Group, 1998) \[[@B5]\]. Patients were enrolled according to previously described inclusion criteria \[[@B5]\]. Of note, this study allowed rather broad inclusion of AD patients with other comorbid illnesses; presumably this would allow this cohort to more closely mirror real-world populations. The study was conducted in accordance with ethical standards of the institutional committee on human experimentation and with the Helsinki Declaration of 1975, revised 1983. The initial study protocol was therefore reviewed in our center by the institutional review board and all patients or caregivers consented to inclusion based on appropriate informed consent. Additional consent was obtained for the open-label extension of the study. Cholinesterase inhibitor treatment ---------------------------------- Immediately following the double-blind phase of the study, open-label rivastigmine was flexibly titrated over a 12-week time period to a maximum tolerated dosage of up to 6 mg BID. By the end of the 12 week titration 25 participants were on 4--6 mg. of rivastigmine and 11 participants had dropped from the study. One participant remained on a 2 mg. dose and dropped from the study between weeks 52 and 78. Assessment of treatment response -------------------------------- Outcomes measures included the Alzheimer\'s Disease Assessment Scale-cognitive subscale (ADAS-cog) and the Clinician\'s Interview-Based Impression of Change, with caregiver input (CIBIC-Plus). Ability to carry out activities of daily living (ADL) was assessed by the Progressive Deterioration Scale (PDS). Disease-staging measures included the Geriatric Deterioration Scale (GDS) and the MMSE. In the open-label phase of the study, efficacy evaluations were performed every 6 to 8 weeks for titration and early maintenance, and every 26 weeks for the next 5 to 6 years. Statistical analysis -------------------- These data are predominantly descriptive, with analyses including Kaplan- Meier survival plots when appropriate. All statistical analyses on our single center extension were performed at our center using SPSS. Subgroup analyses by initial treatment randomization were also performed. Results ======= Demographics and population --------------------------- Twenty-one Caucasian women and 11 Caucasian men participated in the open label extension of this pivotal study. Twenty-two participants reported a family history of AD. Selected demographic and baseline characteristics of the subsample are presented in Table [I](#T1){ref-type="table"}. Of the 32 patients, 11 were originally randomized to the high dose (6--12 mg/day) group, 10 to the low dose group, and 11 to placebo. Five of the patients in the cohort chose not to participate in the extension. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Selected baseline and demographic characteristics (N = 32). ::: Characteristic Mean (SD) --------------------------- ------------- Age (y) 71.8 (7.5) Length of symptoms (mo)\* 29.6 (18.3) Baseline MMSE 21.3 (4.1) Baseline GDS 3.7 (0.78) MMSE = Mini-Mental Status Examination; GDS = Geriatric Deterioration Scale. \Length of symptoms prior to enrollment in the double-blind study. ::: A total of 25 patients during the 5-year term eventually withdrew from the study. Reasons for termination, stratified by original group during the double-blind phase, are summarized in Figure [1](#F1){ref-type="fig"}. The most frequent reason for termination of participants initially randomized to the high-dose group was the lack of availability of free rivastigmine following FDA approval, which had been provided at pre-launch at no charge as part of the clinical study. It is of interest that no deaths occurred in the group initially randomized to high-dose rivastigmine during the initial double-blind, placebo-controlled trial. Furthermore, disease severity, as measured by the GDS, was greater in the original high-dose group (mean = 4.0; median = 4.0) as compared with the low-dose (mean = 3.7; median = 3.5) and placebo groups (mean = 3.4, median = 3.0). A total of 8 terminations were due to withdrawal of consent (n = 3) or caregiver discontinuation (n = 5). Five of the 8 were in the original placebo group. Seven patients withdrew because of adverse events; 2 each in the original low-dose and placebo groups (57%) and 3 in the original high-dose group (43%). Treatment failure was cited as the reason for termination of one 69-year-old man in the original placebo group. His baseline MMSE score was 21, and his final MMSE score at week 26 was 17. No other patients withdrew as a result of treatment failure. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Reason for termination stratified by original group. ::: ![](1471-2318-5-3-1) ::: Of the 4 patients who withdrew from the study due to disease progression, 3 were men between the ages of 57 and 64 years. The 57-year-old man was in the original high-dose group, with a baseline MMSE score of 16 and an MMSE score of 0 at the 234-week data collection. The other two male participants were aged 62 and 64 years with baseline MMSE scores of 22 and 20, respectively. The 62-year-old man in the original low-dose group withdrew at week 104 with an MMSE score of 9; the 64-year old man in the original high-dose group withdrew at week 156 with an MMSE score of 11. Interestingly, the 76-year-old woman in the original low-dose group, with a baseline MMSE score of 22, withdrew after the 26-week data collection (MMSE = 21). Quantitative (objective) analysis --------------------------------- The Kaplan-Meier survival analysis, shown in Figure [2](#F2){ref-type="fig"}, illustrates time to dropout from week 26 to week 234. The survival curve reveals a relatively steep decline in participation in the first 9 months of open-label extension, mostly related to adverse events (AEs), since almost all AEs causing discontinuation occurred during this phase, followed by a \"flattening\" of the curve. Fourteen participants were still taking high-dose rivastigmine at 2 years, 12 participants at 3 years, and 10 participants at 4 years. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Kaplan-Meier analysis of time to dropout from week 26 to week 234 ::: ![](1471-2318-5-3-2) ::: Of the subjects starting open-label rivastigmine, 25% were still participating at week 234. Of the 8 subjects still participating, 5 were female and only 1 reported a family history of AD. Interestingly, this group was characterized by a broad age range (57--85 years), a broad range of baseline MMSE scores (15--26), and by the fact that 5 of the 8 remaining participants at week 234 were in the original high-dose rivastigmine group. For the end point defined as a 5-point drop from the baseline MMSE score, the group mean was 94 weeks. However, an age-related observation was noted such that the mean time to a 5-point drop from baseline MMSE for the ≤ 70 age group was 67 weeks as compared with 122 weeks for the \>70 years age group. A similar trend was observed for scores on the ADAS-cog. For the group as a whole, mean time to a 4-point deterioration on the ADAS-cog was 84 weeks; with a mean of 70 weeks for the younger group and 104 weeks for the older group. \[In the original study, mean age across the 22 research sites was 74.5 years; mean age for participants in this report was 71.8 years\] Figures [4](#F4){ref-type="fig"} through 8 summarize mean scores for the ADAS-cog, CIBIC, GDS, PDS, and MMSE from week 26 through week 234. As expected, mean values for cognitive and functional status decline over time; however, significant within subject variability is present. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Mean scores on the Alzheimer\'s Disease Assessment Scale-cognitive subscale (ADAS-cog) for weeks 26--234. ::: ![](1471-2318-5-3-3) ::: Qualitative analysis -------------------- When evaluating qualitative data, it is important to examine particular patients in terms of the data collected and treatment response at end of study. Data for the youngest man and the elderly woman, considered side-by-side, seem counterintuitive. Part of the explanation, however, may rest with the caregivers\' experiences with and beliefs about the patients. This information can be accessed by reviewing the \"symptoms most troubling\" item from the CIBIC-Plus. This item asks, \"With respect to the above symptoms, which are the biggest problems for you and/or other caregivers?\" The spouse of the 57-year-old man reported that the \"symptoms most troubling\" for her were the patient\'s \"selective difficulties and his attitude problem\" (week 104, MMSE = 11) and that the patient \"doesn\'t try\" (week 130, MMSE = 7). These comments suggest a lack of acceptance of the patient\'s diagnosis and a lack of belief regarding the patient\'s documented decline that could explain continued clinic visits until, at week 234, the patient\'s MMSE score was 0. In contrast, the caregiver for the 76-year-old woman cited the fact that the patient \"doesn\'t want to leave the house\" (week 12) and the patient\'s anxiety about coming to clinic appointments (week 26) as the \"most troubling\" symptoms. These comments suggest that the caregiver was encountering patient resistance and observing patient distress, which were exacerbated by efforts to participate in the study. Therefore, further clinic visits were declined. Retrospectively, it is impossible to determine how the caregiver\'s beliefs and experience with the patient affect perceptions about disease status, treatment response, and decisions to continue or terminate treatment. These are interesting hypothesis-generating observations that should be explored in future investigations. Discussion ========== Limited data are available on the tolerability and effectiveness of cholinesterase inhibitor therapy for periods up to 3 years \[[@B4]-[@B7]\], but nothing has been reported in the literature concerning the percentages of patients remaining on therapy and its effect beyond this time point. This study adds to the limited long term data on patients treated with high-dose rivastigmine therapy by reporting descriptive data for up to 5 years. Findings presented in this report indicate that, in this sample of patients, high-dose rivastigmine therapy was well tolerated over a 5-year period. Of note, approximately two thirds of the participants still enrolled at week 234 were in the original high-dose rivastigmine group during the double-blind phase; this finding suggests that early therapy with rivastigmine may confer some benefit in delaying long-term progression of symptoms, as has been previously suggested by analysis of the combined 26 weeks of double-blind and first 26 weeks of open-label data from the B352 US trial \[[@B9]\]. Throughout the initial 26-week double-blind portion, patients receiving placebo steadily deteriorated, while those treated with high-dose rivastigmine were able to maintain their baseline level of performance on the ADAS-Cog \[[@B5]\]. This approximated a delayed-start design for the open-label portion, which demonstrated that patients who started rivastigmine late never \"caught up\" with patients who had been on high-dose rivastigmine from the beginning of the trial. This suggests a disease-progression-delaying effect of the drug, which may allow this population to maintain their autonomy for a longer period of time. However, it is important to emphasize the limitations of this data; the analysis was retrospective, the sample was small, there were significant numbers of drop-outs, and the availability of free rivastigmine ceased with FDA approval which occurred near to the end of the study Conclusions =========== In summary, long-term therapy with the ChE inhibitor rivastigmine was well tolerated with no dropouts due to adverse effects past the first 9 months of the open-label extension. In contrast to the generally reported community experience of relatively brief duration of therapy with ChE inhibitors in AD, 25% of patients in this study were still taking rivastigmine by the end of 5 years. Of interest is the multifactorial nature of reasons for treatment discontinuation. Only 1 of these patients withdrew from the study because of perceived treatment failure over the 5-year period. Disease progression accounted for the withdrawal of 4 patients. These results are informative both for the duration of treatment, and because the majority of the patients who continued treatment during this 5 year period were from the group originally titrated up to a high dose during the initial double-blind phase. Given that the sample size prohibits significance testing, these data suggest that rivastigmine therapy may be sustained over long periods of time in a significant percentage of patients with AD, and that high-dose therapy, particularly when begun early, may have an advantage in delaying the progression of the illness. Competing interests =================== MRF has received grant support and has served as consultant and received honoraria from Novartis Pharmaceuticals Corporation. MLL has no financial interest to declare. Authors\' contributions ======================= MF carried out the original clinical trial, conceived of the design for the observational study, and participated in the drafting and revisions of the manuscript. ML participated in the design of the observational study, performed statistical analysis, and participated in the drafting and revisions of the manuscript. Both authors read and approved the final manuscript. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Change in Clinician\'s Interview-Based Impression of Change for weeks 26--56. ::: ![](1471-2318-5-3-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Mean scores on Geriatric Deterioration Scale for weeks 26--234. ::: ![](1471-2318-5-3-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Mean scores on Progressive Deterioration Scale for weeks 26--156. ::: ![](1471-2318-5-3-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Mean scores on Mini-Mental Status Examination for weeks 26--234. ::: ![](1471-2318-5-3-7) ::: Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2318/5/3/prepub> Acknowledgments =============== \The principal investigators for the ENA713 B352 Study Group are listed in the Acknowledgments. This study was supported by Novartis Pharmaceuticals Corporation, East Hanover, NJ. The participants in the ENA 713 B352 Study Group were as follows: Marc Bauder, MD, and Troy Willaims, MD, Clinical Studies, Peoria, AZ; Louis Cohen, MD, Clinical Studies, Sarasota, FL; Martin Farlow, MD, Indiana University Alzheimer\'s Clinic, University Hospital, Indianapolis; Michael Gold, MD, Behavioral Neurology Memory Disorders Clinic, University of South Florida, Tampa; John Feighner, MD, Feighner Research Institute, San Diego, CA; Donald Hay, MD, St Louis University Medical Center, St Louis, MO; Ranga Krishnan, MD, Duke University Medical Center, Durham, NC; Vinod Bhatnagar, MD, Center for Clinical Trials & Research Inc, Venice, FL; David Margolin, MD, Fresno, CA; Peter Ripley, MD, Clinical Studies, Cape Cod, S Yarmouth, MA; Nunzio Pomara, MD, Nathan S Kline Institute for Psychiatric Research, Orangeburg, New York and Brookdale Hospital Medical Center Neurology Department, Brooklyn, NY; Benjamin Seltzer, MD, Tulane University Medical Center, New Orleans, LA; James Webster, Jr, MD, McGaw Medical Center, Northwestern University, Chicago, IL; Lorna Charles, MD, Southern New Jersey Medical Institute, Stratford, NJ; George Hanna, MD, Neurology Suite, Fontaine Research Park, Charlottesville, VA; Walter Brown, MD, Clinical Studies, Ltd, Providence, RI; Robert Linden, MD, Pharmacology Research Institute, Long Beach, CA; Barry Gordon, MD, Cognitive Neurology, Johns Hopkins University School of Medicine, Baltimore, MD; Stuart Stark, MD, The Neurology Center, Alexandria, VA; Patricia Walicke, MD, and Michael Jann, PharmD, Buckhead Neurology, PC, Atlanta, GA and Center for Clinical Research, Mercer University Southern School of Pharmacy, Atlanta, GA; Jody Corey-Bloom, MD, PhD, Department of Neuroscience, UCSD, La Jolla, CA; Charles Echols, Jr, MD, Phoenix, AZ. Also, Richard D Hartman, PhD, Stephen M Graham, PhD, Peggy E Hayes, PharmD, John C Messina, Jr, PharmD, Peter Irwin, Novartis Pharmaceuticals Corporation, East Hanover, NJ. This study was monitored by Quintiles Pacific, Inc, San Diego, CA. The authors would also like to thank Amy Rothman Schonfeld, PhD, Robert Gilbert, STL, Mary Ellen S Congleton, and Pietro Mastrangeli for their editorial and artistic assistance.	PubMed Central	2024-06-05T03:55:52.548307	2005-1-19	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548267/", "journal": "BMC Geriatr. 2005 Jan 19; 5:3", "authors": [ { "first": "Martin R", "last": "Farlow" }, { "first": "Mary L", "last": "Lilly" } ] }
PMC548268	Background ========== Suction drainage in the management of mastectomy patients was used for the first time in 1947 \[[@B1]\] and has been found in various studies superior to other methods of fluid evacuation to minimize the dead space. The mechanism proposed is that the suction helps skin flaps to adhere to the chest wall and axilla sealing off all the leaking lymphatics\[[@B2],[@B3]\]. This reduces the incidence of post-operative seromas, hematoma formation and flap necrosis, which are, recognized complications of modified radical mastectomy \[[@B2],[@B3]\]. When no postoperative suction drains were used the incidence of seromas was found to be unacceptably high in various studies \[[@B4]\]. Prolonged drainage on the other hand, may increase the hospital stay and increase the risk of infection by allowing retrograde migration of bacteria \[[@B4]\]. Indiscriminate or premature withdrawl of postoperative drains irrespective of the amount of fluid drained may be accompanied by an increase in the incidence of axillary seromas \[[@B4]-[@B6]\]. If kept for longer periods it has been observed that drain itself might contribute to increased drainage and the risk of infection in addition to the increased hospital stay resulting in to wasteful utilization of the hospital resources. The amount of postoperative drainage is influenced by various factors like the clinical profile of the patient including the body mass index, extent of axillary lymph node dissection, number of lymph nodes dissected, use of elctrocautery, co morbid conditions and also the negative pressureon the suction drain \[[@B4]-[@B10]\]. The amount of postoperative fluid drained has been found to be significantly influenced by the negative pressure on the suction drainage. While the negative suction drain is logically expected to drain the fluid, a high negative suction drain may prevent the leaking lymphatics from sealing off thus leading to prolonged drainage leading to increased hospital stay \[[@B4]\]. The present prospective randomized clinical trial compared the postoperative wound drainage in patients with full suction drain (high) and those with half vacuum drainage system (low). The study also compared the drain volume, average hospital stay and postoperative morbidity between full vacuum and half vacuum suction groups. Methods ======= The study was conducted in one surgical unit of a tertiary care center over a period of two years.85, FNAC (fine needle aspiration cytology) proven cases of locally advanced breast cancer were randomized (using randomly ordered sealed envelops, which were opened immediately before the closure of the wound) into full vacuum suction (pressure = 700 g/m2) group -- (A) and 35 cases into half vacuum suction (pressure = 350 g/m2) group -- (B). The two groups were comparable in respect of age, weight and type of operation i.e. modified radical mastectomy (MRM). Following complete routine and metastatic work up, all patients received three cycles of Neoadjuvant chemotherapy (NACT) using CAF regime (Cyclophosphamide, Adriamycin, 5-Fluorouracil) and underwent Patey\'s modified radical mastectomy after three weeks of the last cycle. Surgery was performed by the same surgical team comprising of five surgeons (two senior and three resident surgeons) using a standardized technique with electro cautery. Axillary dissection was done up to level- III in all the cases. The boundaries of axillary dissection were defined by superior limit as the posterolateral border of the Pectoralis major muscle and axillary vein, medial limit being clavipectoral fascia or Hallstead\'s ligament, lateral limit as the anterior border of lattismus dorsi and the inferior limit being the angular vein joining the thoracodorsal vein. The long thoracic and thoracodorsal nerves were identified, dissected and preserved. Two silicone tube drains (12Fr) (one axillary and pectoral) were inserted in all the patients. All resected specimens were examined and the lymph nodes dissected, counted and assessed histo-pathologically for metastases. No patient received intra-operative blood transfusion. Both the drains were connected to a single 600 ml suction bottle (Romovac -Romson). In-group A (n = 50), drainage was performed using complete vacuum negative suction (700 g/m2) and in-group B (n = 35) with half vacuum suction drainage (350 g/m2). The pressure was also measured by attaching a manometer to the exit opening of the drainage bottle. The two groups were comparable with respect to age, weight (body mass index), type of operation indicating the success of randomization (Table. [1](#T1){ref-type="table"}). The drain was emptied every 24 hours to reset suction at the respective pressures and to measure the daily drain out put. External compression dressing was provided over the axilla for first 48 hrs and following that the patients were encouraged to do active and passive shoulder exercises. The outcomes measured were morbidity and the length of hospital stay. The total drain output was measured and recorded daily in both the groups, the drains were removed once the output was less than 30 ml in 24 hrs and the patients were discharged on the same day. The mean total drain output was measured in each group and compared. The mean hospital stay in both the groups was calculated and compared. The associated morbidity in the form of seroma formation, flap necrosis and wound infection during the postoperative period was recorded and compared in both the groups Statistical methods used ------------------------ Descriptive studies were performed with SPSS version 10 and group characteristics were compared using student t-test (Tables. [2](#T2){ref-type="table"}&[3](#T3){ref-type="table"}). Results ======= 1\. Half vaccum suction drains were removed earlier than full vacuum suction drains without any significant addition to the postoperative morbidity. 2\. The use of half vacuum drains after modified radical mastectomy reduced the hospital stay significantly without any increase in the postoperative morbidity. 3\. The high negative suction is an important contributory factor to the amount of drainage following breast surgery along with axillary dissection. While negative suction helps in prevention of postoperative seroma formation, a high negative suction may affect adversely by increasing the amount and duration of drainage. This probably is on account of persistent drainage from the lymphatics, which do not close due to high negative suction. Discussion ========== Seroma formation is the most frequently observed early complication after breast and axillary surgery. The use of closed suction drainage is a common practice that has been shown to reduce the incidence of seroma formation \[[@B1]-[@B6]\]. These drains are generally removed once the lymph production falls to less than 35--50 ml/24 hours, a level generally reached between 3--17 days after surgery \[[@B1]\]. The length of postoperative axillary drainage is a major cause of morbidity after axillary dissection as the patients are usually discharged once the drains are removed. The patients with suction drains in situ are normally managed in the hospital (although some authors advocate discharge with the drains in situ)\[[@B13]\]. Migration of bacteria along these drains has also observed to increase the risk of infection if the drains stay in situ for a long time \[[@B7]\]. Early or premature removal however has been found to be associated with an unacceptably high incidence of seroma formation and its continuation until fluid discharge is acceptably low leads to a prolonged stay in the hospital, which has a bearing on the cost of surgical management of breast cancer \[[@B1],[@B11]-[@B13]\]. Shortening the hospital stay has been shown to be an effective way of reducing the costs in the case of surgery for breast cancer and axillary drains are the main obstacles in achieving it \[[@B1],[@B8]-[@B10]\]. To reduce the hospital stay after MRM, early discharge with the drains in situ has been reported but discharging patients with drains in situ has an inherent difficulty faced by the patients in management of drains besides higher incidence of wound infection \[[@B13],[@B14]\]. The other disadvantages are discomfort for the patients, with difficulties undressing or using the toilet. It may be feasible with patients of higher cultural and social standing, but not all the patients have the required background. In a third world country where the patients are poor, uneducated coming from far and remote areas with limited medical facilities, there is an added difficulty in management of the drains away from the hospital. As most of our patients come from far flung rural areas with limited education, poor medical and communication facilities they were managed indoors until the drains were removed. There are other solutions proposed for prevention or reduction of fluid accumulation and early discharge after axillary dissection e.g by Patrek et al \[[@B15],[@B16]\] where several parallel drains were used. Suture obliteration of axillary space under skin flaps with sutures to the chest wall, approximation of the pectoralis major and the latissimus dorsi muscle in the form of axillary padding has been suggested by some authors \[[@B9],[@B17]\]. The incidence of seroma formation had reduced but the length of drainage was not specified in these studies. Further more suture approximation of the muscles may limit movement of the arm leading to shoulder dysfunction. Harada et al \[[@B14]\] used fibrin glue in rats to occlude transected lymph channels and obliterate the subcutaneous cavity. The association of seroma formation with large amounts of drainage before removal of the drain has already been established \[[@B18]-[@B20]\]. In one study it was observed that when the amount of fluid drained before removal of the catheter was less than 250 ml in three days no seromas developed and they concluded that it is safe to remove drains if the total amount of fluid drained during the first postoperative days is low. Yii et al \[[@B20]\] reported that removal of drains after 48 hours did not result result in seroma formation if the total amount of fluid drained before removal was less than 150 ml Proposed factors contributing to the increased drainage and seroma formation \[[@B2],[@B3],[@B8]-[@B15]\] --------------------------------------------------------------------------------------------------------- Patrek et al \[[@B15]\] examined 13 factors influencing fluid drainage. Only two (a large number of positive lymph nodes and previous biopsy) predicted greater drainage. 1.Body mass index. A significant linear relation exists between BMI and increased seroma formation was reported by (Boonman et al) 2\. Technique: Use of elctrocautery has been reported to be associated with increased incidence of seroma formation as compared to cold knife. It has also been reported that tissue ligation around the axillary vein rather than mere transection with knife or diathermy may reduce the amount of postoperative discharge, the technique was followed in the presented study \[[@B8]\]. 3 Drains themselvesencourage drainage by stimulating tissue reactions or by suction \[[@B20]\] 4.Early shoulder exerciseshave been implicated but were not observed to be a factor in the present study \[[@B10],[@B11]\] Although early mobilization of the shoulder did not increase fluid discharge in various studies but it was reported to be an additional factor leading to increased drainage after axillary dissection \[[@B10],[@B11]\]. 5 The negative suctionapplied may prevent the lymphatics from closing leading to continuous leakage and discharge \[[@B18]\]. 6.Extent of axillary dissection. More seromas were seen when more lymph nodes were dissected from the axilla. The higher lymph node yield may well be an indirect measure of more extensive dissection performed. \[[@B3]\]. The drainage may also reflect the damage to the lymph vessels and therefore the number of lymph nodes dissected may have a bearing on the amount of drainage. In our study also, it was observed that patients with higher lymph node yield had a higher volume and duration of drainage although it was not found to be significantly different in both the groups because they were matched in all respects except the negative suction pressure of the drainage. Negative suction and the drainage --------------------------------- It is an accepted fact that negative suction prevents seroma collection and helps in the adherence of the walls of the axilla thus reducing the dead space and allowing the lymphatics to close. High negative suction pressure generated by the drain can maintain lymph drainage by a negative pressure gradient \[[@B18]\]. It is also reported that the high negative suction pressure does not allow the lymphatic channels to close leading to continuous drainage and a higher incidence of seroma formation \[[@B18]-[@B20]\]. There are studies to suggest that high negative suction may be beneficial in the sense that the amount of drainage would be more thus allowing an early adherence of walls of the axilla to the chest wall and reduction in the seroma formation \[[@B18]\]. However in the present study it was observed that high suction caused prolonged drainage, which can possibly be explained by the hypothesis that high negative suction may not allow, leaking lymphatics to close. Therefore no suction or high suction drainage both may contribute to the same result that is higher incidence of seroma formation and longer hospital stay. To strike a balance between not having suction at all and having a very high or full negative suction, half negative suction drainage was used in the present study to achieve a shorter hospital stay without any increase in the rate of post operative seroma formation. The external compression dressings in the first forty-eight hours perhaps helped in the adherence of the flaps and reduction of dead space without compromising on the shoulder mobility. This was found to effectively reduce the Hospital stay and also did not increase the postoperative morbidity as compared to high (full) negative suction group. Conclusions =========== Reducing the negative suction pressure applied to the drain (making it half suction) along with external compression dressings applied for first 48 hours can significantly reduce drainage from the axilla following modified radical mastectomy without increasing the incidence of seroma formation as was observed in this randomized prospective clinical study. The hospital stay was reduced considerably compared to a matched group with full suction drain (p \< 0.001). Half suction drain following axillary dissection in patients with carcinoma breast may thus be recommended as an effective approach to reducing the hospital stay and the cost of treatment without adding to the morbidity. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= CM was the surgeon in charge of the project and prepared the study design. VS was the first surgical assistant and did the data processing and statistical analysis. JP, the second surgical assistant assisted in the preparation of the manuscript. SS the Director of ICMR and AB the research officer, Tumor Biology Lab ICMR were responsible for the histopathological analysis and contributed to the preparation of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/11/prepub> Figures and Tables ================== ::: {#T1 .table-wrap} Table 1 Full vaccum group n = 50 Half vaccum group n = 35 p- value ------------------------------------ -------------------------- -------------------------- ---------- Mean Age (years) 49.54 (S.D = 9.821) 46 (S.D = 7.580) \- Mean Weight (kg) 54 (S.D = 6.543) 55 (S.D = 5.285) \- No. Of lymph nodes resected (mean) 12.5 (S.D = 1.619) 12 (S.D = 1.328) \- \% With positive lymph nodes 28% 30% \- NACT regime CAF CAF \- Surgery MRM MRM \- Mean volume drained (ml) 525 (S.D = 66.282) 325 (S.D = 39.612) \<0.001 Mean hospital stay (days) 10.8 (S.D = 1.603) 6 (S.D = 1.414) \<0.001 BMI 21.6 (S.D = 2.347) 21.7 (S.D = 1.800) \- Morbidity Flap necrosis 1 1 NS Wound infection 4 3 NS Seroma formation 2 1 NS Seroma aspirations required None None \- ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Non parametric test ::: Group n Mean Rank ----------- ------- ---- ----------- Drain vol 1.00 50 60.49 2.00 35 18.01 Total 85 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Mann Whitney U test ::: Drain vol ------------- ----------- Chi square 61.044 df 1 Asymp. Sig. \<0.05 :::	PubMed Central	2024-06-05T03:55:52.550300	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548268/", "journal": "BMC Cancer. 2005 Jan 27; 5:11", "authors": [ { "first": "Vinay", "last": "Singhal" }, { "first": "JP", "last": "Singh" }, { "first": "Anju", "last": "Bansal" }, { "first": "Sunita", "last": "Saxena" } ] }
PMC548269	Background ========== Insulin-like growth factor binding protein (IGFBP)-3 is a multifunctional protein ferrying insulin-like growth factors (IGFs) in circulation and mediating growth suppression signals in cells. Serum IGFBP-3 protein (\< 5000 ng/ml) complexes with IGFs and an acid labile subunit (ALS), to extend the half lives and modulate the bio-availability of IGFs \[[@B1]\]. While a precise mechanism of action is not clear, the growth suppressive activity of IGFBP-3 depends on its nuclear translocation \[[@B2]\]. Other growth suppressors such as p53, retinoic acids, transforming growth factor (TGF)-β, and tumor necrosis factor (TNF)-α induce IGFBP-3 as a mediator of growth suppression \[[@B3]-[@B6]\]. The growth suppression by IGFBP-3 is independent from the modulation of IGFs action \[[@B7]-[@B9]\]. The functional importance of the IGFBP-3 in the growth suppression is noteworthy. IGFBP-3 is produced in most tissues, but the main site of production is liver. It is produced by non-parenchymal cells (endothelial and Kupffer cells) while parenchymal cells (hepatocytes) do not produce it under normal condition \[[@B10]\]. We postulate that IGFBP-3 is a gene induced by growth suppression signals such as p53 in hepatocytes. While the growth suppression imported by IGFBP-3 suggests the potential for tumor suppression, polymorphisms, but no significant mutations were observed in a survey of several tumors \[[@B11]\]. As gene silencing may occur without mutations, we recently investigated IGFBP-3 promoter hypermethylation in human hepatocellular carcinoma \[[@B12]\]. These promoter hypermethylations were subsequently reported in other tumors systems \[[@B13],[@B14]\]. Promoter analysis of IGFBP-3 indicated that the NaB-RE sequence is essential for the sodium butyrate (NaB) induced IGFBP-3 expression \[[@B15]\], but the importance of the eleven upstream p53 binding sites reported by Bourdon et al. were not confirmed until now \[[@B16]\]. The methylation hot spot we identified exactly matched the putative p53 binding sites that Bourdon et al. indicated. Thus, we postulated that these sites are important for the expression of IGFBP-3 induced by p53. Moreover, we hypothesized that the suppression of apoptosis mediated by IGFBP-3 due to the promoter hypermethylation will be a possible pathway of hepatocarcinogenesis. To explore this possibility, the functions of the promoter upstream binding sites of p53 were examined precisely in this study. Methods ======= Cell culture ------------ HepG2 cells were obtained from Japanese Cancer Research Resources Bank (Tokyo, Japan), and maintained in D-MEM supplemented with 10 % FCS (Life Technologies, Tokyo, Japan), antibiotic-antimycotics at 37°C in a humidified atmosphere of 95 % air and 5 % CO~2~. Plasmids -------- pGL2-IGFBP-3, kindly provided by Dr. Youngman Oh (Oregon Health Sciences University, Portland, OR), carries a 1.9 kb IGFBP-3 promoter (-1805/+69) in pGL2-Basic (Promega Corp., Madison, WI). A series of deletion mutant constructs, pGL2-270, pGL2-240, pGL2-210, pGL2-180, pGL2-150, pGL2-120, pGL2-90, pGL2-60, pGL2-30, and pGL2-1 containing the indicated fragments upstream of the transcription start site and 60 bp of fragments downstream of the transcription start site, were generated by PCR amplification of the promoter fragment and subsequent subcloning of the MluI-BglII fragment to pGL2-Basic (Table [1](#T1){ref-type="table"}, Fig. [2](#F2){ref-type="fig"}). The transcription start site of the IGFBP-3 promoter, +1, is based on the sequence determined by Cubbage et al. \[[@B17]\]. The plasmid containing site-directed mutations in putative p53 binding sites was generated by replacing the wild type MluI-XhoI fragment of pGL2-210 with a mutant fragment synthesized artificially (pGL2-210B, Table [1](#T1){ref-type="table"}, Fig. [3](#F3){ref-type="fig"}). A fusion gene with site-directed methylation was constructed by methylating the MluI-XhoI fragment (-210/-174) of pGL2-210 in vitrowith SssI methylase (New England Biolabs, Inc., Beverly, MA), and reconstituting the fragment and unmethylated vector (Fig. [4](#F4){ref-type="fig"}). pCMV-p53 and pCMV-p53mt135 were obtained from BD-Biosciences (East Meadow Circle, Palo Alto, CA). Transient transfection ---------------------- HepG2 cells were transiently transfected using the FuGENE 6 transfection reagent according to the manufacturer\'s instructions (Roche Molecular Biochemicals, Indianapolis IN). Cells were seeded at a density of 5 × 10^4^cells/well in 24-well plates. After 24 hours, cells were transfected with 0.25 μg/well of reporter plasmid DNA in serum-containing medium. Forty-eight hours post transfection, cells were washed twice with PBS and collected for luciferase assays. Transfections were performed in quadruplicate and experiments were performed at least two times. Luciferase assay ---------------- Luciferase activities of cell lysates were measured according to the manufacturer\'s instructions (Promega Corp. Madison, WI) using a liquid scintillation counter (Aloka, LSC-700, Tokyo, Japan). Luciferase activities were normalized for total protein determined using the Bradford Assay (Bio-Rad Laboratories, In., Hercules, CA). EMSA (electrophoresis mobility shift assay) ------------------------------------------- 278 bp of the MluI-BglII fragment of pGL-210 were labelled using \[α-^32^P\]dCTP by end filling with Klenow fragment, and used for EMSA. Oligonucleotide DNAs (BP3WPSF: 5\'-GGCTGCAGCG GGCGTGCGCA CGAGGAGCAG GTGCCCGGGC GAGTCTCGAG CTGCACGCCC CCGAGCTCGG-3\', BP3WPSR: 5\'-CCGAGCTCGG GGGCGTGCAG CTCGAGACTC GCCCGGGCAC CTGCTCCTCG TGCGCACGCC CGCTGCAGCC-3\'), comprising the promoter sequence of -210/-149, were custom-made (Sigma-genosys Japan, Ishikari, Japan), annealed one hour at room temperature, and used as cold competitor in the assay. EMSA was performed in a 20 μl reaction containing 10 mM Tris (pH 7.5), 2.5% Glycerol, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, and in the presence of 50 ng/μl of double-stranded poly \[d(I-C)\], 5000 cpm (2 ng) of ^32^P-labeled probe DNA, and 2.5 μg of H~2~O~2~treated MCF7 nuclear extract (Active motif LLC, Palomar, CA). The reaction mixture was incubated at 14°C for 20 min. 20 μl of each reaction mixture was then loaded onto a native 4 % polyacrylamide gel containing 0.5 × Tris-Glycine buffer (25 mM Tris, 190 mM Glycine, 1 mM EDTA pH 8.3), and electrophoresed at 14°C, 100 V for 1 hr. For the supershift assay, a p53 antibody (Ab-2, Oncogene Research Products, San Diego, CA) was used. Results ======= Deletion analysis ----------------- We identified a hot spot of promoter hypermethylation in human hepatocellular carcinoma and its cell lines in the promoter upstream region of IGFBP-3 (Fig. [1](#F1){ref-type="fig"}). As methylation sites were identified to the cluster of putative p53 binding sites, we examined the role of these sites in IGFBP-3 expression. First, we constructed promoter deletion mutants of IGFBP-3 and examined the expression of the reporter gene in the absence (Fig. [2A](#F2){ref-type="fig"}) and presence (Fig. [2B](#F2){ref-type="fig"}) of p53 expression by the co-transfection of a p53 expression plasmid (pCMV-p53). As the transfection efficiency was not fully controlled, and p53 overexpression may to cause massive changes in cellular conditions, we did not compare results between the presence and absence of p53. Even though, we can obtain clear results about the effect of p53 to IGFBP-3 expression. The pattern of expression of deletion mutants was clearly different in the absence and presence of p53. In the absence of p53 overexpression, we identified the NaB-RE sequence as an essential site for expression, and deletion of p53 binding site enhanced the gene expression (Fig. [2A](#F2){ref-type="fig"}). These observations were similar to those of Walker et al. \[[@B15]\], but differed as HepG2 cell does not require NaB or Tricostatin A (TSA) for its activation. In contrast, in the presence of p53 over-expression, we identified that four p53 binding sites between -210 to -150 (relative transcription start site as +1) were essential for IGFBP-3 expression (Fig. [2B](#F2){ref-type="fig"}). When the deletion constructs were co-transfected with pCMV-p53mt135, the expression of the IGFBP-3 promoter was suppressed to a background level (almost same as blank constructs) in all constructs (data not shown). This indicates that IGFBP-3 expression we observe is tightly regulated by p53. Site-directed mutagenesis ------------------------- To confirm the importance of p53 binding sites between -210 to -150, we abrogated one of p53 binding sites by site-directed mutagenesis (C to T at -179 and G to C at -176, Fig. [3A](#F3){ref-type="fig"}). In the absence of p53 overexpression, there exist little differences in expression between the wild type and mutant construct (74 % relative to the wild type) (Fig. [3B](#F3){ref-type="fig"}). However, in the presence of p53 overexpression, IGFBP-3 expression was strongly decreased in the mutant construct (3.4 %) relative to wild type (Fig. [3B](#F3){ref-type="fig"}). For reasons already mentioned, we did not compare the result between in presence and absence of p53. Site-directed methylation ------------------------- Next, we constructed in vitromethylated promoter constructs to evaluate the effect of methylation (Fig. [4A](#F4){ref-type="fig"}). For methylation, the MluI-XhoI fragment of pGL2-210 was methylated with SssI methylase and reconstituted with unmethylated reporter vector fragment. As we used linear constructs for the transfection, the expression of luciferase was strongly suppressed compared to circular plasmids (0.7 %). But similar patterns compared to site directed mutation were observed. Although the expression of IGFBP-3 was slightly enhanced in the absence of p53 overexpression in the methylated construct, it was decreased in the presence of p53 overexpression in the methylated construct (Fig. [4B](#F4){ref-type="fig"}). In this experiment, at a glance, we observed induction of IGFBP-3 expression by p53, but the transfection efficiency of linear plasmids is low, while the relative levels of p53 and the availability of putative negative regulators are extremely different from other experiments. Thus, we cannot conclude in this case, whether or not there is induction by p53. EMSA ---- EMSA was performed to determine whether p53 protein binding was disturbed by methylation. In this assay, the wild type and methylated promoter fragments (278 bp, -210/+60) were incubated with H~2~O~2~-treated MCF7 nuclear extract that express high level of p53. In the wild type promoter probe, we observed supershifted complex (Fig. [5](#F5){ref-type="fig"}, lane 4, indicated by arrows) when p53 antibody (Ab-2) is added to the reaction mixture. A 100-fold molar excess of a cold 70 bp sequence containing a p53 binding site (-210/-149), competed the probe (Fig. [5](#F5){ref-type="fig"}, lane 5). In methylated probe, we observed no supershifted complex (Fig. [5](#F5){ref-type="fig"}, lane 9). As we used probes that contain a Sp1/GC box and TATA box, we observed the shift and supershift bands in the methylated probe as well as unmethylated probe. These bands were postulated to be due to p53 binding to the nuclear factor such as p300. These observations indicate that p53 binding to the IGFBP3promoter sequence is blocked or at least attenuated by hypermethylation. Discussion ========== We have indicated that the p53 binding sites upstream of IGFBP-3 promoter are essential for its induction by p53, and that the induction can be suppressed by promoter hypermethylation in the human hepatoblastoma cell line HepG2. A working model of the p53 action in IGFBP-3 promoter upstream binding sites is summarized in Fig. [6](#F6){ref-type="fig"}. In this model, in normal cells, IGFBP-3 is induced by factors such as growth hormone (GH) and IGFs, and steady levels of expression are observed in some cells. That the deletion of p53 binding sites enhances the expression of IGFBP-3, suggests existence of negative regulators (Fig. [6A](#F6){ref-type="fig"}). In apoptotic cells, p53 tetramers at high levels of expression bind to upstream binding sites. These tetramers recruit the p300 complex to its binding site, thereby a p53-dependent high level of expression (Fig. [6B](#F6){ref-type="fig"}). In tumor cells, deletions, mutations, methylations of p53 binding sites, or mutations of p53 such as p53mt135, disturb the binding of p53 tetramers to their binding sites, and this prevents the binding of the p300 complex to IGFBP-3 promoter. As a result, the expression of IGFBP-3 is suppressed in tumor cells (Fig.[6C](#F6){ref-type="fig"}). In this model, promoter hypermethylation has the same effect as promoter mutation in determining IGFBP-3 expression. Our observations of promoter hypermethylation in human hepatocellular carcinomas and derivative cell lines (12), and the observations in this report strongly support the notion that IGFBP3is a true tumor suppressor gene. IGFBP3is a gene that is silenced by biallelic hypermethylation or hypermethylation and loss of heterogeneity (LOH) in human hepatocellular carcinoma. As reported recently \[[@B14]\], we have also observed the reduced expression of IGFBP-3 in several tumors such as, breast (9/41), uterus (11/42), ovary (6/16), kidney (6/20), and prostate (1/4) using the cancer profiling array (BD bioscience, data not shown). We therefore postulate that the tumor suppressor role of IGFBP-3 will not be limited to HCCs. In addition, there are also many reports of IGFBP-3 overexpression in tumors from breast \[[@B18]\], prostate \[[@B19]\], kidneys \[[@B20]\], and lung squamous cells \[[@B21]\], so on. We thus anticipate the existence of additional defects, such as papilloma virus infections that inactivate IGFBP-3 \[[@B22]\], TGF-β / Rb signalling abnormalities that often coincide with IGFBP-3 overexpression \[[@B23]-[@B25]\], or as yet unknown defects in IGFBP-3 receptor function leading to IGFBP-3, for these overexpression in tumors. IGFBP-3 is a ubiquitous, multifunctional protein, whose importance as a carrier of IGFs is evident. The absence of gross loss-of-function mutations of IGFBP-3 observed to date likely underscores its functional importance. We hypothesize that IGFBP-3 is a gene whose basal level of expression is essential for cell survival, but upon induction by p53, high levels of expression of IGFBP-3 induces apoptosis. Alternatively, IGFBP-3 may be a gene that is essential for cell survival when induced by growth hormones or IGFs, but functions as an apoptotic mediator when induced by p53. We observed slight base changes within p53 binding sites strongly influenced the induction of IGFBP-3 by p53. As SNPs that change the expression level of IGFBP-3 were within p53 binding sites \[[@B26]\], and it was reported that the IGFBP-3 is differentially activated by p53 mutants \[[@B27],[@B28]\], we postulate that the expression and functions of IGFBP-3 is controlled in some way by p53 binding sites in the promoter of IGFBP-3. This may include the intronic p53 binding sites as well as the upstream sites explored here. IGFBP-3 may, therefore, have an important function in tumor development through p53 control. We identified the MyoD (-195/-186) and WT1 (-164/-156) binding sites as well as p53 binding sites at the hypermethylation hot spot in HCC by promoter analysis using TRANSFAC (v 4.0). As MyoD is a transcription factor that can induce apoptosis, and WT1 is a tumor suppressor gene, the existence of a binding site for these putative regulator genes in the hot spot of the promoter of IGFBP-3 suggest the possibility that these genes also use IGFBP-3 as a mediator of their actions. Conclusions =========== We conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells. As IGFBP-3 functions downstream of many growth suppressors and its growth suppression effects are drastic, and as it is a small-sized secreted protein, the use of IGFBP-3 in tumor therapy will be a promising option. Abbreviations ============= ALS; acid labile subunit, D-MEM; Dulbecco\'s modification of Eagle\'s medium DTT; dithiothreitol, EDTA; ethylenediaminetetra-acetic acid, EMSA; electrophoresis mobility shift assay, FCS; fetal calf serum, HCC; hepatocellular carcinoma, IGF; Insulin-like growth factor, IGFBP-3; Insulin-like growth factor binding protein-3, LOH; loss of heterogeneity, NaB; sodium butyrate, NaB-RE (sodium butyrate-responsive region), PBS; phosphate buffered saline, PCR; polymerase chain reaction, TGF-β; transforming growth factor-β, TNF-α; tumor necrosis factor-α, SEM; standard error of mean, SNP; single nucleotide polymorphism. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= TH carried out these studies and manuscript preparation, KN and YI participated plasmid construction and reporter assay, TS and HS participated the EMSA, EY helped the array study, TO participated in the design of the study and performed the statistical analysis. NK conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/9/prepub> Acknowledgements ================ We Thank Dr. Y. Oh (Oregon Health Sciences University, Portland, OR) for providing pGL2-IGFBP-3 plasmid and Dr. R. Chaparro (Albert Einstein College of Medicine, New York, NY) for critical reading of this manuscript. This work was supported in part by grants-in aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (\#11770273). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *Schematic genomic structure of the IGFBP3gene. There exist 11 p53 consensus sequences upstream of the promoter \[16\] and two binding sites in introns \[3\]. Polymorphisms and hypermethylations are identified in the promoter upstream p53 binding sites \[26, 12\]. ::: ![](1471-2407-5-9-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Deletion analysis of IGFBP-3 promoter for the induction by p53. HepG2 cells were transiently transfected with the panel of IGFBP-3 promoter-luciferase reporter constructs with (B) or without (A) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown. ::: ![](1471-2407-5-9-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Mutant analysis of IGFBP-3 promoter for the induction by p53. A, Schematic of four wild type p53 consensus sequence of IGFBP-3 (-159/-209) of pGL2-210 and mutant sequence carrying point mutations in the core consensus sequence (-179 C to T, -176 G to C) of pGL2-210B. B, HepG2 cells were transiently transfected with wild type IGFBP-3 promoter-luciferase reporter constructs pGL2-210 or mutant IGFBP-3 promoter-luciferase reporter constructs pGL2-210B with (filled box) or without (open box) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown. ::: ![](1471-2407-5-9-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Methylation analysis of IGFBP-3 promoter for induction by p53. A, Schematic of four wild type p53 consensus sequence of IGFBP-3 (-159/-209) of pGL2-210 and a sequence of the methylated construct carrying the CpG methylation in the core consensus sequence (underlined) of pGL2-210. B, HepG2 cells were transiently transfected with wild type IGFBP-3 promoter-luciferase reporter construct or methylated IGFBP-3 promoter-luciferase reporter construct with (filled box) or without (open box) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown. ::: ![](1471-2407-5-9-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Specific binding of p53 to IGFBP-3 promoter and its inhibition by methylation. 2 ng of end-labelled 278 bp of MluI-BglII fragment of pGL-210 were used for EMSA. To construct the methylated probe, a labelled fragment was methylated in vitrowith SssI methylase. Each reaction contained the components listed in Materials and Methods. 2.5 μg of H~2~O~2~-treated MCF7 with an induced p53 expression was used as nuclear extract. Supershift was obtained using 0.1 μg of anti-p53 (Ab-2). As competitor, 200 ng of the fragment that carries the core p53 binding site (-210/-149) were used. The mixture was incubated at room temperature for 20 min and analyzed on 4 % nondenaturating polyacrylamide gel in 0.5 × Tris-Glycine buffer. Supershift bands observed in lane 4 were indicated by arrows. ::: ![](1471-2407-5-9-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Working model of p53 action to IGFBP-3 promoter. In normal cells, IGFBP-3 is induced by such as growth hormone and IGFs, and steady level of expression is observed in some cells. As the deletion of p53 binding sites enhances the expression of IGFBP-3, the binding of negative regulators is expected (Fig. 6A). In apoptotic cells, p53 tetramers at high level of expression bind to upstream binding sites. These tetramers recruit the p300 complex to its binding site, and a p53 dependent high level of expression is observed (Fig. 6B). In tumor cells, deletions, mutations, methylations of p53 binding sites, or mutations of p53 such as p53mt135, disturb the binding of p53 tetramers to its binding sites, and this prevents the binding of p300. As a result, the expression of IGFBP-3 is suppressed in tumor cells. ::: ![](1471-2407-5-9-6) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Oligonucleotide DNAs used for construction of IGFBP-3 promoter mutants. ::: ------------------------------------------------------------------------------------------------------------------- Plasmid* Cloned fragment\* *sense primer\\*(5\' to 3\') antisense primer\\*(5\' to 3\') ----------------- ----------------------- ---------------------------------- -------------------------------------- pGL2-270 -270/+60 ggacgcgtctggagtgccggggtggc ggagatctgctgtggaatccaggcag pGL2-240 -240/+60 ggacgcgtggttcttgtagacgacaa ggagatctgctgtggaatccaggcag pGL2-210 -210/+60 ggacgcgtcgggcgtgagcacgagga ggagatctgctgtggaatccaggcag pGL2-180 -180/+60 ggacgcgtgcgagtctcgagctgcac ggagatctgctgtggaatccaggcag pGL2-150 -150/+60 ggacgcgtggccccggctgctcaggg ggagatctgctgtggaatccaggcag pGL2-120 -120/+60 ggacgcgtcccgcagccgtgcctgcg ggagatctgctgtggaatccaggcag pGL2-90 -90/+60 ggacgcgtcctcccaacccccactcc ggagatctgctgtggaatccaggcag pGL2-60 -60/+60 ggacgcgtccggggcgtgtcctgggc ggagatctgctgtggaatccaggcag pGL2-30 -30/+60 ggacgcgttatatacgggccggcgcg ggagatctgctgtggaatccaggcag pGL2-1 +1/+60 ggacgcgtagatgcgagcactgcggc ggagatctgctgtggaatccaggcag pGL2-210B\\\ -210/+60 ggacgcgtctgcagcgggcgtgagcacga\ agctcgagagtcacccgggcacctgctcc\ ggagcaggtgcccgggtgactctcgagct tcgtgctcacgcccgctgcagacgcgtcc ------------------------------------------------------------------------------------------------------------------- \; The number is indicated by transcription start site as +1. \\; MluI restriction site is included in sense primers and BglII restriction site is included in antisense primers. \\\; pGL-210B was made by replacing MluI-XhoI fragment of pGL2-210 with the oligonucleotide DNA indicated. :::	PubMed Central	2024-06-05T03:55:52.552213	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548269/", "journal": "BMC Cancer. 2005 Jan 20; 5:9", "authors": [ { "first": "Tadashi", "last": "Hanafusa" }, { "first": "Toshiyuki", "last": "Shinji" }, { "first": "Hidenori", "last": "Shiraha" }, { "first": "Kazuhiro", "last": "Nouso" }, { "first": "Yoshiaki", "last": "Iwasaki" }, { "first": "Eichiro", "last": "Yumoto" }, { "first": "Toshiro", "last": "Ono" }, { "first": "Norio", "last": "Koide" } ] }
PMC548270	Background ========== Type II restriction endonucleases (REases) comprise one of the major families of endonucleases and one of the largest groups of experimentally characterized enzymes (comprehensively reviewed in: \[[@B1]\]). The \"orthodox\" Type IIP REases are dimeric, they recognize a short (4--8 bp) palindromic sequence of double-stranded DNA and in the presence of Mg^2+^, catalyze the hydrolysis of phosphodiester bonds at precise positions within or close to this sequence, leaving \"blunt\" or \"sticky\" ends (with a 5\' or 3\' overhangs). The enzymes that do not fit this definition or exhibit certain structural and functional peculiarities, have been classified into several subtypes (review: \[[@B2]\]). REases coupled with DNA methyltransferases (MTases) of similar specificity form restriction-modification (RM) systems, which are ubiquitous among Bacteria and Archaea \[[@B3]\]. While cleavage at specific sequences provides efficient means of destroying foreign DNA, methylation of these sequences in the prokaryotic chromosome renders them resistant to REase and thereby protects the own DNA from cleavage. Because cleavage of the chromosomal DNA in unmodified sequences would be deletorious for the cell, the REases must maintain extremely high specificities, tightly coupled with that of the methyltransferase. A change in just one base pair of the \"cognate\" site can reduce the ratio k~cat~/K~m~for DNA cleavage by a factor ≥10^6^\[[@B4]\]. To date, only crystal structures of 15 REases have been solved, compared to over 3000 biochemically characterized enzymes (review: \[[@B1]\]; see also \[[@B3]\] for updates). It was found that they share a characteristic structural core and a very weakly conserved catalytic motif (P)[D]{.underline}-X~n~-([D/E]{.underline})-X-[K]{.underline} (where X is any amino acid), together with a number of non-specific and structure-specific nucleases, suggesting that these proteins are evolutionarily related despite the absence of overall sequence similarity (review: \[[@B5]\]). The comparison of crystal structures of members of this so-called \"PD-DxK\" superfamily suggested that the catalytic and DNA-binding regions are major determinants of structural stability of these proteins \[[@B6]\]. Structural comparisons revealed also two major branches or classes, α and β, whose archetypal members were the enzymes that cleave DNA to generate 4 nt long 5\' \"sticky ends\", for instance EcoRI (α-class), and those that generate \"blunt\" ends after the cleavage, for instance EcoRV (β-class) \[[@B7]-[@B9]\]. Using the experimentally solved structures as templates, bioinformatics methods such as iterative sequence database searches and protein fold-recognition have been used to predict the active site in some REases \[[@B10]-[@B13]\]. From these analyses it was learned that certain functional peculiarities, like the requirement of a binding of an uncleaved effector site for cleavage of another site characteristic for Type IIE enzymes, evolved independently in the α and β branches of the PD-DxK superfamily \[[@B11],[@B13]-[@B15]\]. It was also found that other REases belong to completely unrelated superfamilies, with different three-dimensional folds and catalytic sites (review: \[[@B9]\]): BfiI is a member of the phospholipase D (PLD) superfamily \[[@B16]\], Eco29kI belongs to the GIY-YIG superfamily \[[@B17]\], and KpnI and a few other REases belong to the HNH superfamily \[[@B17]-[@B19]\]. While the structural information is essential to infer the molecular basis of sequence specificity in REases, the lack of overall sequence conservation in these enzymes, the absence of invariable residues even in the active site and the presence of several alternative folds make structure prediction and classification extremely difficult. SfiI is a REase isolated from Streptomyces fimbriatus. It recognizes the interrupted palindromic sequence 5\'GGCCNNNN\^NGGCC3\', where N denotes any base, and cleaves it as indicated by \"\^\", leaving 3\' extensions 3 nt long \[[@B20]\]. SfiI is a prototype of Type IIF enzymes, which function as tetramers that bind simultaneously to two recognition sites and cleave both sites concertedly \[[@B21]\]. However, a structural model of SfiI, which could be used as a platform to study its sequence-function relationships, is not yet available. Thus, despite the availability of a large body of biochemical data on how SfiI interacts with the DNA substrate (mainly on the kinetics of protein-DNA interactions with different substrates and the geometry of DNA looping \[[@B22]-[@B25]\], but not on the \"residue-level\" details thereof), the identity of amino acid residues important for dimerization, tetramerization, DNA binding and cleavage remains completely unknown. We have therefore carried out bioinformatics analyses of SfiI that allowed to identify its closest relative amongst REases with known structure and use this information to construct a tertiary model of SfiI in complex with its target DNA. Results and discussion ====================== In the absence of experimentally determined protein structures, homology-based models may serve as working models for the investigation of sequence-structure-function relationships between diverged enzymes \[[@B26]\]. Homology-modeled structures may be of too low resolution to characterize the protein-protein or protein-DNA contacts at the atomic level, but they can suggest which sequence regions or individual amino-acids are essential components of the binding surfaces. In particular, identification of amino acids potentially involved in protein-DNA contacts may guide mutagenesis experiments aimed at the engineering protein variants with novel specificities. However, homology modeling requires a homologous template structure to be identified and the sequence of the protein of interest (a target) to be correctly aligned to the template. Identification of the three-dimensional fold of SfiI ---------------------------------------------------- The sequence of SfiI showed no significant similarity to any other protein sequences. Also among the proteins reported by BLAST with sub-optimal scores, there were no proteins of known structure and no nucleases (data not shown) that could hint at potential relationships of SfiI to any previously characterized protein superfamily. Thus, in order to identify a template structure for modeling of SfI we used the threading approach, which allows to assess the compatibility of the target sequence with the available protein folds based not only on the sequence similarity but also on the structural considerations (match of secondary structure elements, compatibility of residue-residue contacts, etc.) (reviews: \[[@B27],[@B28]\]). The SfiI sequence was therefore submitted to the GeneSilico protein fold-recognition metaserver \[[@B29]\]. As expected, fold-recognition methods that rely only on sequence similarity (PDB-BLAST, and FFAS) failed to identify any significant matches between SfiI and proteins with known structrues. However, several threading methods that explicitly use the structural information from the templates reported a match between SfiI and the structure of a Type II REase BglI \[[@B30]\], a member of the PD-DxK superfamily of nucleases (FUGUE \[[@B31]\]: 4.25, INBGU \[[@B32]\]: 3.8, SAM-T02 \[[@B33]\]: 0.13, 3DPSSM \[[@B34]\]: 4.4; note that these scores are not normalized as each server uses a different evaluation system; see the individual references for details). Additionally, FUGUE reported a match (low score 3.17) between SfiI and the structure of another REase, EcoRV \[[@B35]\]. Despite the scores reported by the individual threading methods (except FUGUE for BglI) were hardly significant, the consensus server Pcons5 \[[@B36]\] assigned a significant score (1.35) to the BglI structure as a potential modeling template. Homology modeling of the SfiI monomer ------------------------------------- A homology model of SfiI was constructed based on the alignments reported by threading methods, using the \"FRankenstein\'s Monster\" approach \[[@B37]\] (see Methods). Since the PD-DxK nuclease fold was selected by Pcons as the only reasonable template and no other nuclease folds were identified by the FR methods, only alignments between SfiI and the PD-DxK superfamily members BglI and EcoRV were used. The final model was constructed by iterating the homology modeling procedure (initially based on the raw FR alignments), evaluation of the sequence-structure fit by VERIFY3D, merging of fragments with best scores, and local realignment in poorly scored regions. Local realignments were constrained to maintain the overlap between the secondary structure elements found in the bglI structure used as the modeling template, and predicted for SfiI. This procedure was stopped when all regions in the protein core obtained acceptable VERIFY3D score (\>0.3) or their score could not be improved by any manipulations, while the average VERIFY3D score for the whole model could not be improved. The final model, comprising residues 13--240 obtained the average VERIFY3D score of 2.6. The alignment between SfiI and BglI is shown in Figure [1](#F1){ref-type="fig"}, the corresponding final model of the monomer is shown in Figure [2](#F2){ref-type="fig"}. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Alignment between SfiI and structurally characterized REases.A) Fold-recognition alignment between full-length sequences of SfiI and BglI. Amino acids are colored according to the physico-chemical properties of their side-chains (negatively charged: red, positively charged: blue, polar: magenta, hydrophobic: green. Pairs of residues conserved between SfiI and BglI are highlighted. Putative catalytic residues are indicated by \"\#\", putative DNA-binding residues are indicated by \"\\". Secondary structure elements of BglI are shown below the alignment. Numbers of amino acid residues at the N-terminus of each panel are shown. B) Structure-based sequence alignment of the conserved core, corresponding to the PD-(D/E)XK motif, including SfiI, BglI, and other selected REases from the β-class. Conserved residues of the active site are highlighted. ::: ![](1472-6807-5-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Homology model of the SfiI monomer.A) Superposition of the BglI template structure (red) and the SfiI model (blue). The CCGG half-site of the DNA target is shown in green. The Ca^2+^ions from the BglI structure are shown as white dots. B) SfiI model colored according to the sequence conservation: residues identical between SfiI and BglI are shown in blue, residues with physico-chemically similar side chains are in green, dissimilar residues are in yellow and red. The putative conserved active site is shown in the wireframe representation. ::: ![](1472-6807-5-2-2) ::: Modeling of the SfiI dimer in complex with the DNA -------------------------------------------------- BglI belongs to the \"EcoRV-like\" β-class of PD-DxK nucleases. The most typical features of REases from this class are: antiparallel orientation of the 5^th^strand of the common β-sheet and recognition of the DNA by an additional β-sheet formed by extended loops between the common secondary structure elements \[[@B5],[@B7],[@B9]\]. Most of β-class PD-DxK REases (including EcoRV) exhibit a similar mode of dimerization, which results in positioning of the two active sites as to cut the pair of the opposite phosphodiester bonds in the middle of the recognition sequence and thereby produce the \"blunt\" ends. BglI is exceptional in that its mode of dimerization is completely different, which leads to a different arrangement of the active sites and the sequence-recognition loops, resulting in the recognition of an interrupted sequence 5\'GCCNNNN\^NGGC3\' and cleavage in the position indicated by \"\^\" that yields 3\' ends 3 nt long. SfiI also recognizes an interrupted sequence 5\'GGCCNNNN\^NGGCC3\' and cleaves it in the same manner and therefore can be regarded as a more specific variant of BglI. These striking functional similarities, together with the results of the threading analysis, suggest that SfiI is indeed closely related to BglI and that both enzymes interact with their substrate DNA in a similar manner. Thus, we modeled the structure of the SfiI dimer in complex with the DNA based on the available crystal structure of BglI \[[@B30]\]. Briefly, the SfiI monomer model was duplicated and each of the copies was superimposed onto the corresponding monomer in the BglI dimer. A few minor steric clashes between the side-chains of residues at the protein-protein interface were removed by choosing alternative rotamers for the respective amino acids. The DNA duplex (sequence 5\'ATCGCCTAATAGGCGAT3\') was copied from the BglI co-crystal structure (1 dmu) \[[@B30]\] and \"mutated\" to 5\'AT[G]{.underline}GCCTAATAGGC[C]{.underline}AT3\' using HyperChem 7.1 (Hypercube, Inc.), followed by local geometry optimization. One of the adenine residues in the mismatched A/A base pair in the middle of the DNA molecule was \"mutated\" to T/A. The curvature of the DNA remained unchanged. Essentially, the global structure of the protein-DNA complex for SfiI remains exactly as in the BglI structure, as reliable modeling of macromolecular interactions remains beyond the capabilities of the existing methods. The model of SfiI dimer is shown in Figure [3](#F3){ref-type="fig"} and is available for download from <ftp://genesilico.pl/iamb/models/R.SfiI/>. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Model of the SfiI dimer.Individual subunits are shown in yellow and blue. The modeled DNA sequence is shown in green, the specifically recognized CCGG half-sites are in red. ::: ![](1472-6807-5-2-3) ::: Model-based identification of amino acid residues important for catalysis, DNA-binding and dimerization of SfiI --------------------------------------------------------------------------------------------------------------- In the proposed model of SfiI, the spatial configuration of the catalytic residues is typical for an active site architecture conserved among PD-DxK nucleases. We predict that the active site of SfiI comprises residues: E55, D79, D100 and K102, which superimpose well on the catalytic residues of BglI: E87, D116, D142 and K144, respectively (Figure [4](#F4){ref-type="fig"}). We predict that the DNA-binding mode of SfiI will be very similar to that of BglI, with the side chains of residues S210 and R218 (homologs of D268 and R277 in BglI) involved in the recognition of the inner C/G base pair (Figure [5a](#F5){ref-type="fig"}), backbone oxygen and the side chain of K208 (a homolog of K266 in BglI) recognizing the middle C/G base pair (Figure [5b](#F5){ref-type="fig"}), and the side chain of R220 (a homolog of R279) recognizing the G of the middle G/C base pair (Figure [5c](#F5){ref-type="fig"}). The specificity of SfiI towards the outer G/C base pair, not discriminated by BglI, can be explained by the development of new contacts made by residues from a divergent loop adjacent to the REase active site and comprising residues 104--110 of SfiI and 146--155 of BglI. In BglI, D150 makes specific contacts to the middle C/G base pair and the G/C base pair \[[@B30]\], however this residue is not conserved in SfiI (Figure [1](#F1){ref-type="fig"}). Instead, we predict that the changes of the loop length and the amino acid substitutions lead to a different conformation of the corresponding loop in SfiI, which allows R109 (not present in BglI) to make a specific contact to the G of the outer G/C base pair (Figure [5d](#F5){ref-type="fig"}). Other residues from the same loop, such as K107 may also contribute to the specific sequence recognition by SfiI by making contacts to either of the two G/C base pairs. It is noteworthy that according to our model of SfiI, the majority of specific contacts are achieved by three Arg residues (R109, R218, and R220). These predictions can be tested by site-directed mutagenesis of the respective residues to Ala and testing whether the mutant proteins are proficient in DNA cleavage and/or binding. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Superposition of SfiI and BglI structures.The predicted active site of SfiI (in blue) superimposed onto the BglI structure (red) Individual subunits are shown in yellow and blue. The Ca^2+^ions from the BglI structure are shown as cyan spheres. Only the two nucleotides adjacent to the scissile phosphodiester bond are shown. ::: ![](1472-6807-5-2-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Predicted specific protein-DNA contacts.A) Recognition of the inner C/G pair B) Recognition of the middle C/G pair. C) Recognition of the middle G/C pair. D) Recognition of the outer G/C pair. ::: ![](1472-6807-5-2-5) ::: Interestingly, our model suggests that SfiI lacks the counterpart of a loop corresponding to aa 63--80 in BglI used by this enzyme to interact with the target site from the minor groove side. Thus, SfiI appears to recognize its target solely from the major groove side and to use fewer specific contacts than BglI to recognize its cognate site. This suggests that SfiI may be an easier target for the engineering of REases with new sequence specificities. The dimerization interface of SfiI is comparable to that of BglI. We predict that the following residues may be important for monomer-monomer interactions: Q59, Y60, E63, E66, R73, F74, G76 and that mutating them to change the volume of the side chain (for instance G76R) or introducing (or reversing) the charge (E63R, E66R, R73D, F74R) could disrupt the formation of the SfiI dimer and destroy the REase activity. Prediction of the dimer-dimer interaction surface in the SfiI tetramer ---------------------------------------------------------------------- SfiI is a Type IIF enzyme, i.e. a tetramer that binds simultaneously to two recognition sites and cleaves both sites concertedly \[[@B21]\], while BglI is an orthodox IIP enzyme, i.e. a dimer that acts on single sites \[[@B38]\]. Therefore, the crystal structure of BglI cannot be used to model the tetrameric structure of SfiI. To date, the only Type IIF enzymes, for which crystal structures have been solved, are NgoMIV \[[@B39]\], Cfr10I \[[@B40]\] and Bse634I \[[@B41]\], which are all relatively closely related to each other and exhibit similar mode of interactions between two dimers within the tetramer. These enzymes, however, even at the level of a dimer exhibit a completely different arrangement of monomers, compatible with the generation of 5\' overhangs 4 nt long (compared to 3\' overhangs 3 nt long in the case of SfiI and BglI). Therefore, it is impossible to obtain a meaningful superposition of the BglI or SfiI dimer onto any pair of subunits in the NgoMIV, Cfr10I or Bse634I tetramer. However, it is tempting to speculate that SfiI may tetramerize in a similar manner to these enzymes, i.e. to use surface regions on the opposite sites of the molecule to the protein-DNA and protein-protein binding. A highly speculative model of SfiI tetramer obtained by manual docking of two dimers is shown in Figure [6](#F6){ref-type="fig"}. Based on this model, we predict that the dimer-dimer interface will be composed mostly of hydrophobic and polar residues (and very few charged ones), involve the following segments of the amino-acid sequence, corresponding to loops on the surface of the dimer: 26--34, 67--69, and 86--93. The putative dimer-dimer interactions involve contacts between hydrophobic regions (aa 86--93 from different subunits) as well as hydrophilic ones (aa 26--34). It is possible that the C-terminal region of the SfiI sequence, which could not be modeled (aa 241--269) may also participates in tetramerization. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Putative structure of the SfiI tetramer.*Individual subunits are shown in yellow, green, red, and magenta. The two DNA substrates are shown in white. ::: ![](1472-6807-5-2-6) ::: Conclusions =========== Implications for the evolutionary history of different (sub) Types of REases ---------------------------------------------------------------------------- Comparative analysis of nucleases from the PD-DxK superfamily suggests that they can be classified into two remotely related lineages: α (EcoRI-like) and β (EcoRV-like) \[[@B7]-[@B9]\]. It was proposed that extant REases evolved independently from non-specific or structure-specific nucleases from both lineages (review: \[[@B5]\]). Interestingly, the phylogenetic tree of the PD-DxK superfamily revealed intriguing cases of convergent evolution. So far, it was found that Type IIE enzymes that bind two copies of the recognition site (the actual target of cleavage and the non-cleaved allosteric effector), evolved independently at least three times: EcoRII is an α-lineage member that apparently evolved from IIP enzymes similar to SsoII or PspGI by acquisition of an N-terminal effector-binding domain \[[@B12],[@B42]\]. NaeI is a β-lineage member remotely related to IIP enzymes EcoRV and HincII that acquired a C-terminal effector-binding domain unrelated to that of EcoRII \[[@B7]\]. Finally, Sau3AI is a β-lineage member that apparently evolved by a duplication of a catalytic domain closely related to a DNA repair enzyme MutH, followed by the loss of catalytic residues in the C-terminal domain, thereby adapted to function as an effector-binding domain \[[@B14],[@B15]\]. Another type of evidence for convergent evolution is provided by the finding that the specificity for the GATC sequence appeared independently in the α-lineage (MboI and its close homologs \[[@B5],[@B13]\]) and in the β-lineage (Sau3AI and its close homologs \[[@B14],[@B15]\]). Our results strongly suggest that the archetypal Type IIF enzyme SfiI is closely related to a β-lineage member, an \"orthodox\" Type IIP REase BglI. Another well-characterized group of Type IIF REases comprises α-lineage members for which crystal structures were solved (NgoMIV, Cfr10I, Bse643I) \[[@B39]-[@B41]\]. Thus, our analysis provides evidence that the ability of Type IIF REases to tetramerize and cut two target sites in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily. Different Type IIF REases appear to have evolved independently from the simplest, \"orthodox\" Type IIP enzymes, like previously found for Type IIE REases. It was previously demonstrated that deletion of the effector-binding domain converts the Type IIE REase EcoRII to function as a Type IIP enzyme \[[@B43]\]. However, tetramerization seems to be important for the catalytic activity of the Type IIF enzyme Cfr10I, since the DNA cleavage activity of the dimeric W220A mutant of this REase is \<0.1% of that of the wild-type enzyme \[[@B44]\]. In the absence of a high resolution co-crystal structure of SfiI in complex with DNA, our model will serve as a convenient platform to study sequence-structure-function relationships in this enzyme. In particular, it will facilitate the mutagenesis of residues potentially involved in tetramerization, dimerization, DNA-binding and catalysis. Methods ======= Structure prediction -------------------- Sequence searches of the non-redundant (nr) database and of the putative translations from finished and unfinished microbial genomes were carried out at the NCBI using PSI-BLAST \[[@B45]\]. Secondary structure prediction and tertiary fold-recognition was carried out via the GeneSilico meta-server gateway \[[@B29]\]. Secondary structure prediction was predicted using PSIPRED \[[@B46]\], PROFsec \[[@B47]\], PROF \[[@B48]\], SABLE \[[@B49]\], JNET \[[@B50]\], JUFO \[[@B51]\], and SAM-T02 \[[@B33]\]. Solvent accessibility for the individual residues was predicted with SABLE \[[@B49]\] and JPRED \[[@B52]\]. The fold-recognition analysis (attempt to match the query sequence to known protein structures) was carried out using FFAS03 \[[@B53]\], SAM-T02 \[[@B33]\], 3DPSSM \[[@B34]\], INBGU \[[@B32]\], FUGUE \[[@B31]\], mGENTHREADER \[[@B54]\], and SPARKS \[[@B55]\]. Fold-recognition alignments reported by these methods were compared, evaluated, and ranked by the Pcons server \[[@B36]\]. Homology modeling ----------------- The alignments between the sequence of SfiI and the structures of selected templates (members of the fold identified by Pcons) were used as a starting point for modeling of the SfiI tertiary structure using the \"FRankenstein\'s Monster\" approach \[[@B37]\], comprising cycles of model building by MODELLER \[[@B56]\], evaluation by VERIFY3D \[[@B57]\] via the COLORADO3D server \[[@B58]\], realignment in poorly scored regions and merging of best scoring fragments. The positions of predicted catalytic residues and secondary structure elements were used as spatial restraints. This strategy has previously helped us to build accurate, experimentally validated models of other REases, such as SsoII \[[@B11]\], PspGI \[[@B12]\], MboI \[[@B13]\], and KpnI \[[@B19]\]. List of abbreviations ===================== aa, amino acid(s); bp, base pair(s); nt, nucleotide; e, expectation; REase, restriction endonuclease; MTase, methyltransferase; ORF, product of an open reading frame, RM, restriction-modification; Authors\' contributions ======================= JMB carried out the fold-recognition analysis for SfiI, built the preliminary models, drafted the manuscript and coordinated the whole study. AC built the final models and identified functionally important residues. KS participated in interpretation of the data and writing the manuscript. All authors have read and accepted the final version of the manuscript. Acknowledgements ================ This analysis was funded by KBN (grant 3P04A01124 to JMB). The work of KJS and AAC was supported by the NIH (Fogarty International Center grant R03 TW007163-01). JMB was supported by the EMBO/HHMI Young Investigator Award and by the Fellowship for Young Scientists from the Foundation for Polish Science. We would like to thank Alfred Pingoud and Virgis Siksnys for stimulating discussions concerning the evolution of sequence-structure-function relationships in REases of various subTypes.	PubMed Central	2024-06-05T03:55:52.554423	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548270/", "journal": "BMC Struct Biol. 2005 Jan 24; 5:2", "authors": [ { "first": "Agnieszka A", "last": "Chmiel" }, { "first": "Janusz M", "last": "Bujnicki" }, { "first": "Krzysztof J", "last": "Skowronek" } ] }
PMC548271	Background ========== There is a long tradition of studying at population level the quality of medical care provided to patients who died from conditions amenable to medical intervention. This type of study (so called \'in-depth\' or \'audit\' study), aims to identify deficiencies in medical care that may have contributed to death. It was first systematically carried out on maternal death, and later on other causes of avoidable death \[[@B1]-[@B4]\]. This method can be applied to other potentially avoidable conditions, e.g. those that could be avoided by appropriate preventive care. The general approach is to document in detail the process of care provided to a single patient preceding the occurrence of an adverse event, followed by an assessment of the quality of care by an expert panel, either with or without the use of explicit criteria \[[@B5]\]. An important limitation of this type of study, without control subjects, is its inability to fully establish a causal relationship between identified deficiencies in care and the adverse outcome, and to determine to what extent identified deficiencies are associated to the occurrence of such an event. Identified deficiencies in care are expected to indicate only to a certain extent an increase in risk of an adverse health outcome, while the probability of having an adverse outcome can be calculated only if we compare the care provided to patients who suffered an adverse outcome with that of patients who did not suffer such an event. For this reason it has been proposed to perform a case-control study with patients with an adverse event as \'cases\' and a comparable group of patients without an adverse outcome as \'controls\' \[[@B6]\]. We performed a case-control study with the aim to assess the effect of guideline adherence for stroke prevention on the occurrence of stroke in general practice. Unfortunately, we encountered various obstacles in the design and conduct of this study, in particular related to the recruitment of cases and controls, in availability of information on the care delivery process in the GP\'s data registration system, and in controlling for differences other than differences in the quality of care. The aim of this paper is to highlight the problems related to a variant of confounding by indication, that may be common in quality of care studies. Observational studies of intended treatment effects are particularly prone to \'confounding by indication\', and can produce misleading estimates on either, or both, the size and direction of treatment effects \[[@B7],[@B8]\]. Confounding by indication refers to an extraneous determinant of the outcome parameter that is present if a perceived high risk or poor prognosis is an indication for intervention. This means that differences in care, for example, between cases and controls may partly originate from differences in indication for medical intervention such as the presence of risk factors for particular health problems. The latter has frequently been reported in studies evaluating the efficacy of pharmaceutical interventions \[[@B9],[@B10]\], screening tests \[[@B11]\], and vaccines \[[@B12]\]. We hypothesise that this may not only apply to indications for medical intervention but also for guideline adherence and quality of care. In comparing retrospectively the quality of care between patients with and without a stroke, stroke patients may have received more preventive care because more indications for preventive interventions were present. Because differences in indications for preventive intervention correspond with the probability of an adverse outcome (more indications will be associated with a higher risk of an adverse outcome), when comparing care between cases and controls it is necessary to control for these differences. If one omits to control for confounding by indication, it is expected that more and probably better care, correlates with a higher risk of stroke. In quality of care research, there is, as yet, little information regarding the role of confounding by indication in studies that investigate the effect of quality of care on disease outcomes. Methods ======= Sample ------ From the Dutch national GP register, a random sample was taken of 58 GPs working in Rotterdam and the surrounding region. The study was restricted to patients with a first-ever stroke meeting the following criteria for inclusion: (a) diagnosis of intracerebral haemorrhage or infarction according to the World Health Organization (WHO) definition of stroke \[[@B13]\], (b) age between 39--80 year, (c) occurrence of stroke in the period 1996--1997, (d) stroke caused by cardiovascular disease (CVD) and not by trauma, infection or malignancy, (e) presence of hypertension, (f) GP of the patient practising in the southern part of Rotterdam or surrounding region, (g) patient registered with local GP for not less than two years, and (h) patient not living in a nursing home during the two years period prior to stroke. Cases and controls were selected from the GPs\' patient register, using health outcome (stroke) and risk factor (e.g. hypertension) entries. For each case, two controls were randomly selected and matched with the cases in terms of overall distribution on sex, age, and hypertension (most important risk factor for stroke). Cases and controls were not matched on the same GP. Data collection --------------- In a pilot study among 32 GPs, the quality of care measurement instruments (audit procedure and questionnaire) were tested. GPs participating in the pilot study did not participate in this study. Data on the process of care, two years prior to the occurrence of stroke (for controls from January 1995 to January 1997), were collected by means of structured face-to-face interviews with the GP, using separate questionnaires for each stroke patient. GPs were interviewed between March and October 1999. At the time of interview, GPs used either hand-written or electronic patient records to retrieve patient information. In case information was not available in the patient\'s record, information was drawn from the GP\'s memory. For each question, the type of data source was registered. The questionnaire comprised questions related to patient characteristics and family and medical history of CVD and risk factors, and the detection and treatment of cardiovascular risk factors such as hypertension, diabetes mellitus, transient ischemic attack (TIA) and cardiac failure. Similarly, data were collected on lifestyle-related risk factors such as smoking status, overweight, and excessive alcohol intake. Expert panel and assessment method ---------------------------------- The quality of preventive care and its potential to prevent stroke was assessed and valued by a six-member panel of experts. The panellists (three neurologists and three GPs) were selected on the basis of their clinical expertise with respect to stroke prevention, experience in quality of care evaluation, academic or non-academic background and professional discipline. Six practice guidelines relevant to stroke prevention (hypertension, diabetes mellitus, TIA, peripheral vascular disease, cardiac failure and angina pectoris) were selected by the panel \[[@B14]\]. These guidelines, based on scientific evidence, broad consensus, and clinical evidence, are developed and implemented by the Dutch College of General Practitioners as part of a national guideline program operational since 1987 \[[@B15]\]. From each guideline, the panellists identified specific elements of care and systematically converted these into review criteria (n = 65), allowing detailed measurement of GP\'s adherence \[[@B16]\]. All these criteria were all used to construct the patient questionnaire. In a two-round evaluation, with a final plenary round, cases were assessed by the panellists (panellists were divided in sub-panels). Each sub-panel assessed a specific number of cases. Based on identified elements of sub-optimal care and seriousness of shortcoming in terms of \'minor\' and \'major\', the panellists allocated grades on a scale of 0 to 3 (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Grades of (sub)optimal care given by the expert panel (in both groups allpatients are hypertensive) ::: Cases Controls ------------------ --- ------------------------------------------------------------------------------------------------------------------------ --------- ------------ ---- ----- Grading: 0 No sub-optimal factors have been identified 12 43 18 31 1 Sub-optimal factor(s) have been identified, but are unlikely to be related to the occurrence of stroke in this patient 8 29 18 31 2 Sub-optimal factor(s) have been identified, and possibly have failed to prevent the stroke in this patient 4 14 18 31 3 Sub-optimal factor(s) have been identified, and are likely to have failed to prevent the stroke in this patient 4 14 4 7 Sub-optimal care Grading 1, 2, 3 16 57 40 69 Total Grading 0, 1, 2, 3 28 100 58 100 ::: The two-round process was focussed on detecting consensus among the panellists (providing the same grade), and no attempt was made to force the panellists to consensus. The intersubpanel agreement was k = 0.63 (overall agreement on assigned grades between sub-panels was 74%). A detailed description of the assessment method is provided elsewhere \[[@B17]\]. Analysis -------- Analysis of the data was done by using simple cross-tabulations, and by using logistic regression analysis to model the chance of getting a stroke as a function of the presence of sub-optimal care (as ascertained by the panel), age and sex, and risk factors for stroke. Results ======= GP Participation and recruitment of cases / controls ---------------------------------------------------- The rate of participation was 62% (36 GPs). The main reason for GPs not to participate in the study was lack of time and interest (68%). Participating and non-participating GPs did not differ significantly in age, practice type, and date of qualification. Ninety-two percent of the GPs used electronic GP information systems. Among cases and controls there was a nonsignificant difference in mean age, however, cases were slightly older than controls (67 versus 65 years). Initially, before we excluded patients \'without\' hypertension, GPs identified and selected 50 cases and 58 controls (1.4 case and 1.6 control per GP). Expected number of cases was 2.5 stroke patients per GP per year \[[@B18]\]. After excluding patients without hypertension, 28 cases and 58 controls with hypertension entered the study. Availability of data -------------------- Overall, data for verification of the initial diagnosis of stroke, assessment of GPs\' guideline adherence, and judgement of the causality of the relationship between non-adherence and the occurrence of stroke could be collected from the patient records. However, information on risk factors such as family history of CVD, body weight (overweight), excessive alcohol intake, and smoking was less easily obtained. Depending on the type of risk factor, in 8--56% of all subjects, information on risk factors was unknown to the GP (8% in patients with overweight, 11% in patients smoking cigarettes, 17% in patients with excessive alcohol consumption, and 56% in patients with a family history of CVD). In 41--58% information was taken from the GP\'s memory, instead of the patient register. Indications for confounding by indication ----------------------------------------- In 43% of the cases and 31% of the controls, no sub-optimal care could be identified (grade 0), whereas in 57% and 69%, respectively, sub-optimal care was identified (grade 1, 2 or 3). Thus the Odds Ratio for a case to receive sub-optimal care was 0.60 (95%CI 0.24 -- 1.53) compared to a control (Table [1](#T1){ref-type="table"}). Compared with controls receiving sub-optimal care, the number of shortcomings in care per case receiving sub-optimal care was higher (28/16 = 1.7 versus 41/40 = 1.0) (Table [2](#T2){ref-type="table"}). The percentage of shortcomings in hypertensive care, however, was considerably higher among controls (90% versus 57%, respectively). The latter, apparently, correlates with the fact that controls less often have risk factors other than hypertension (next paragraph). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Guideline-derived elements of care used to indicate shortcomings in care among stroke patients and controls ::: Practice guideline Elements of care Cases Controls -------------------------------------------------------------- ------------------------------------------------ --------- ------------ Arguments derived from practice guideline: Hypertension \- Detection of hypertension 1 2 \- Confirmation diagnosis hypertension 2 1 \- Pharmacologic therapy (anti-hypert. med) 2 1 \- Follow-up (quarterly) 8 17 \- Follow-up (annually) 3 16 Arguments derived from practice guideline: Diabetes mellitus \- Follow-up (quarterly) 4 3 \- Laboratory evaluation 1 0 \- Referral to eye specialist 1 0 Arguments derived from practice guideline: TIA \- Treatment (therapy and follow-up after TIA) 1 1 Arguments derived from more than one practice Guideline \- Advice to quit smoking 2 0 \- Dietary advice (overweight) 1 0 \- Evaluation of cardiovascular risk profile 2 0 Total number of shortcomings 28 41 Total number of patients with shortcomings 16 40 TIA, Transient Ischemic Attack Note: each patient could have more than one element of sub-optimal care ::: The mean number of risk factors among cases (6.1 per patient) was higher than among controls (4.4 per patient) (Figure [1](#F1){ref-type="fig"}). Multivariate logistic regression indicates that cases receiving sub-optimal care (grade 1, 2, or 3) have a lower risk of stroke (crude OR 0.60) (Table [3](#T3){ref-type="table"}). If adjusted for sex and age distribution, the odds ratio does not change significantly (adjusted OR 0.64). Subsequently, in an attempt to investigate the possible role of confounding by indication we adjusted for risk factor prevalence. Indeed, with an adjusted OR of 0.82 (95% CI 0.29--2.30), it seems that risk factor prevalence to some extent explains why patients receiving sub-optimal care have a lower risk of stroke. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Risk factor distribution. Prevalence (%) of risk factors for stroke among stroke patients (n = 28) and controls (n = 58). Total number of risk factors among stroke patients is 172, and among controls 277. Mean number of risk factors per case is 6.1, and for controls 4.4. This relationship is statistically borderline significant (p = 0.096), and could be an explanation for the somehow surprising result found earlier, that is, that cases receive sub-optimal care less often than controls. ::: ![](1472-6963-5-10-1) ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Relationship between quality of care and the occurrence of stroke(Odds Ratio and 95% CI) ::: MODEL 1 MODEL 2 MODEL 3 --------------- ------------------- ------------------- ------------------- Care: Optimal 1.00 (ref.) 1.00 (ref.) 1.00 (ref.) Sub-optimal 0.60 (0.24--1.53) 0.64 (0.25--1.65) 0.82 (0.29--2.30) Sex: Male 1.00 (ref.) 1.00 (ref.) Female 0.90 (0.36--2.30) 0.61 (0.22--1.72) Age: 1.03 (0.98--1.08) 1.03 (0.98--1.08) Risk factors: 0.76 (0.61--0.94) Note: to control for risk factors, the number of risk factors per patient were included in the regression model. ::: Patients with a higher number of risk factors for stroke, indeed have a lower risk of sub-optimal care (OR for the number of risk factors present 0.76; 95% CI 0.61--0.94). As expected, higher numbers of risk factors per patient also increases the risk of stroke (OR for the number of risk factors present 1.34; 95% CI 1.10--1.62). Discussion ========== This study demonstrated confounding by indication in a case-control study analysing the association between guideline adherence and the occurrence of stroke in general practice. It also provided insight into the possibilities for controlling for this confounding bias. We learned that, at present, difficulties in patient recruitment and data retrieval seriously limit the potential use of a case-control method to assess the relationship between guideline adherence for stroke prevention and stroke in general practice. We found that in specific domains data were incomplete and not readily available in the patient records. As a consequence, in many cases GPs were unable to identify stroke patients from their patient register, which most likely introduced under-reporting of stroke patients. As compared to national frequencies (2.5 stroke patients per GP per year) \[[@B18]\], GPs participating in our study identified less stroke patients, 1.7 stroke patient per GP. The same applies to information on patients\' family history of CVD and lifestyle-related risk factors, which was inaccurate and in many cases not available in the patient\'s register. The latter finding is consistent with previous work on the accuracy of information on CVD risk factors in GPs\' patient records \[[@B19],[@B20]\], indicating that data from GP\'s record on lifestyle-related risk factors of CVD are frequently incomplete or absent. Incomplete information on risk factors for stroke is a serious threat to the validity of the results of case-control studies investigating the relationship between process of care and health care outcome. It complicates evaluation of GP\'s adherence to recommended guidelines, and makes it difficult, if not impossible, to control for confounding by indication. Apart from that, information on risk factors that was available in the patient records is presumably not 100% valid. Strong indications for the existence of confounding by indication were found, albeit different from how it is usually described in literature. Confounding by indication, which is conceived as a substantial problem in observational studies of treatment efficacy, usually refers to a situation in which patients who are more in need both receive more care have a higher risk of adverse health outcome \[[@B8]\]. In our study, we show that confounding by indication can also cause patients with an adverse health outcome (stroke) to appear to receive better quality of care. A more detailed analysis showed, similar to results found in previous studies, that this result partly emanates from a higher prevalence of risk factors for stroke among patients suffering stroke at a later stage in life, which not only increases the risk of stroke but also GPs\' compliance to guidelines. We hypothesize that, on average, patients with more risk factors for stroke receive more attention or visit their GP more frequently, which in turn facilitates guideline adherence (e.g. compliance to quarterly follow-up of treated hypertensive patients) and at the same time results in better quality of care. Controlling for (recorded) risk factors reduced the counter-intuitive result by approximately one half, and we hypothesise that incomplete registration of risk factors for stroke explains why the risk of stroke in stroke-prone or high-risk patients associated with sub-optimal care remained below 1.00, even after controlling for risk factors. We hope that our paper draws the attention of quality of care researchers to this variant of confounding by indication, that may lead to biased associations between process measures of quality of care and care outcomes. Conclusions =========== This study shows that, at present, difficulties in patient recruitment and data retrieval seriously limit the potential use of a case-control method to assess the relationship between guideline adherence for stroke prevention and stroke in general practice. It demonstrates the role of confounding by indication, causing patients with an adverse health outcome to appear to receive better quality of care. Competing interests =================== The author(s) declare that they have no competing interests Authors\' contributions ======================= JK, NK, PK, AP and JM conceived and designed the study. Analyses were performed by JK and GB. All authors contributed to this article and earlier drafts of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/5/10/prepub> Acknowledgements ================ The audit was carried out by a panel of experts. The members were: Chairman: Jan W. Van Ree, MD. Members: Diederik W.J. Dippel, MD (neurologist); Gabriël J.E. Rinkel, MD (neurologist); Jan Heeringa, MD (general practitioner); R.P. Kleyweg, MD (neurologist); Arie Knuistingh-Neven, MD (general practitioner); Jan W. Van Ree, MD (general practitioner).	PubMed Central	2024-06-05T03:55:52.557201	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548271/", "journal": "BMC Health Serv Res. 2005 Jan 27; 5:10", "authors": [ { "first": "Johan S", "last": "de Koning" }, { "first": "Niek S", "last": "Klazinga" }, { "first": "Peter J", "last": "Koudstaal" }, { "first": "Ad", "last": "Prins" }, { "first": "Gerard JJM", "last": "Borsboom" }, { "first": "Johan P", "last": "Mackenbach" } ] }
PMC548272	Background ========== Priority setting, also known as rationing or resource allocation, occurs at all levels of every health care system, including governments, funded provincial/territorial agencies, pharmaceutical benefit-management organizations, hospitals and clinical programs \[[@B1]\]. Countries with very different health care systems and levels of health care are all grappling with the problem of how to reconcile growing demands and constrained resources \[[@B2]\]. Hospitals, in particular, are struggling to meet growing demands, affordably, without compromising delivery of services \[[@B3],[@B4]\]. Daniels and Sabin have proposed a framework for priority setting in health care institutions called \'accountability for reasonableness\' \[[@B5]-[@B7]\], which links priority setting to theories of democratic deliberation, operationalizing the ethical concept of fairness. Fairness is a key goal of priority setting. According to \'accountability for reasonableness\', health care institutions engaged in priority setting have a claim to fairness if they satisfy four conditions of relevance, publicity, appeals/revision, and enforcement (described in Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The four conditions of \'accountability for reasonableness\' ::: ------------------ -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Relevance Rationales rest on evidence, reasons, and principles that fair-minded parties can agree are relevant to deciding how to meet the diverse needs of a covered population under necessary resource constraints. Publicity Limit-setting decisions and their rationales must be publicly accessible. Appeals/Revision There is a mechanism for challenge and dispute resolution regarding limit-setting decisions, including the opportunity for revising decisions in light of further evidence or arguments. Enforcement There is either a voluntary or public regulation of the process to ensure that the first three conditions are met. ------------------ -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: \'Accountability for reasonableness\' may be an effective framework for describing the components of priority setting, evaluating the fairness of priority setting in hospitals and identifying \'good\' practices and opportunities for improvement. It can help hospitals improve their priority setting practices and can be an effective driver of health care reforms \[[@B8]-[@B10]\]. \'Accountability for reasonableness\' has been used to evaluate priority setting in health systems \[[@B11]\]. However, only a limited number of studies have addressed how priority setting occurs in hospitals \[[@B12]-[@B17]\], and no study has attempted to survey the views of hospital decision makers throughout an entire health system about the fairness of priority setting in their institutions. The purpose of this study is to elicit hospital decision-makers\' self-report of the fairness of priority setting in their hospitals using an explicit conceptual framework, \'accountability for reasonableness\'. Methods ======= Design ------ We conducted a survey of Chief Executive Officers, or their designates, of Ontario hospitals. The survey questionnaire covered 102 items, including hospital profile information (e.g. hospital name, number of beds, operating budgets). In this paper we focus on the results of 9 questions concerning priority setting, fairness, and the four conditions of \'accountability for reasonableness\' (refer to Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Survey Questions -- Rating\* and Open Scale ::: --------------------------------------------------------------------------------------------------------------------------- Overall, how fair is priority setting at your hospital? (Rating Scale). Please explain your response (Open-ended). How well does your hospital meet its priority setting goal(s)? (Rating Scale). Please explain your response (Open-ended). What do you see as the goal(s) of priority setting in your hospital? (Open-ended) How well is the relevance condition met at your hospital? (Rating Scale) How well is the publicity condition met at your hospital? (Rating Scale) How well is the appeals condition met at your hospital? (Rating Scale) How well is the enforcement condition met at your hospital? (Rating Scale) --------------------------------------------------------------------------------------------------------------------------- \* Ratings were on a five-point scale, from 1 (not well) to 5 (very well) or from 1 (not fair) to 5 (very fair). ::: Setting and sample scope ------------------------ With 12 million people, Ontario is the largest province in Canada. Like the rest of the country, it is a single payor, predominately publicly-funded health care system with some privately funded services and products (e.g. dental services, drugs). There are 160 hospitals in Ontario and all were invited to participate in this study. Participants ------------ 160 Ontario hospital Chief Executive Officers, or their designates, were asked to participate. 86 Ontario hospitals completed this survey, for a response rate of 54%. The average bed count of the responding hospitals was 250.4, with a range of 18 to 1265 beds. The average operating budget of the hospital sample was \$75.8 million, ranging from \$3.3 million to \$733 million. The sampled hospitals represented a blend of teaching, small, community and specialized service facilities across the province. Data collection --------------- The survey was pre-tested and mailed to Chief Executive Officers of all Ontario hospitals in January 2001. Data from returned surveys were entered into an electronic database for further analysis. Of the 9 questions analyzed in this study, 6 asked for a rating on a 5-point scale ranging from \'Not Well\' or \'1\' to \'Very Well\' or \'5\', and 3 were open-ended. Data analysis ------------- Close-ended ratings were analyzed statistically \[[@B18]\], with ratings equal to and below the mid-point combined to identify proportions of respondents suggesting improvement, using a conservative bivariate cut-off point to discriminate among reported responses. P values for all hypothesis tests were two tailed. Open-ended responses were analyzed using a modified thematic analysis involving open and axial coding techniques \[[@B19],[@B20]\]. In open coding, data was segmented and coded with a label identifying parts of text relating to a concept or idea. For axial coding, concepts were organized into overarching themes, and compared, both within and between questions, in search of patterns in responses and to ensure consistency. Results ======= Overall, 60.7% of respondents indicated their hospitals\' priority setting was fair, while 79% stated their hospitals met their priority setting goals. With respect to the \'accountability for reasonableness\' conditions, respondents indicated their hospitals performed best for the relevance (75.0%) condition, followed by appeals/revision (56.6%), publicity (56.0%), and enforcement (39.5%). (Refer to Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Summary of Decision Maker Responses ^a,b^ ::: How well does your hospital meet its priority setting goal(s)? Overall, how fair is priority setting at your hospital? How well is the relevance condition met at your hospital? How well is the publicity condition met at your hospital? How well is the appeals condition met at your hospital? How well is the enforcement condition met at your hospital? ----------------- ---------------------------------------------------------------- --------------------------------------------------------- ----------------------------------------------------------- ----------------------------------------------------------- --------------------------------------------------------- ------------------------------------------------------------- \# Respondents 81 84 84 84 83 81 5 \'Very Well\' 15 (18.5%) 15 (17.9%) 21 (25.0%) 14 (16.7%) 12 (14.4%) 9 (11.1%) 4 49 (60.5%) 36 (42.8%) 42 (50.0%) 33 (39.3%) 35 (42.2%) 23 (28.4%) 3 12 (14.8%) 28 (33.3%) 17 (20.2%) 26 (30.9%) 23 (27.7%) 34 (41.9%) 2 3 (3.7%) 4 (4.8%) 3 (3.6%) 9 (10.7%) 10 (12.0%) 9 (11.1%) 1 \'Not Well\' 2 (2.5%) 1 (1.2%) 1 (1.2%) 2 (2.4%) 3 (3.6%) 6 (7.4%) (SD) 3.89 (0.84) 3.71 (0.86) 3.94 (0.84) 3.57 (0.97) 3.52 (1.00) 3.25 (1.04) a Due to rounding, frequencies may not add up to 100% b Based on survey scale from 1 to 5 ::: Respondents rated the relevance (![](1472-6963-5-8-i1.gif) = 3.94) condition significantly higher than the publicity (![](1472-6963-5-8-i1.gif) = 3.57), appeals (![](1472-6963-5-8-i1.gif) = 3.52), and enforcement (![](1472-6963-5-8-i1.gif) = 3.25) conditions (p \< .05) (paired-samples t test). While publicity and appeals conditions were not significantly different, respondents rated these conditions significantly higher than the enforcement condition (p \< .05). The distribution of ratings suggested that there was ample opportunity for improvement, with most room for improvement in meeting the enforcement condition. Priority setting and fairness ratings were positively correlated (r = .51, p \< .01). Fairness ratings were also positively correlated with each of the 4 \'accountability for reasonableness\' conditions (p \< .01), as were meeting priority setting goals (p \< .05). The bivariate correlation between each of the accountability variables of relevance, publicity, appeals/revision and enforcement was strongly positive (p \< .01). The Pearson correlation coefficients among the 4 conditions ranged from 0.41 to 0.57. The internal consistency (Cronbach\'s α) of all of the \'accountability for reasonableness\' conditions was very good (α = .78). Bed size (r = -.24, p \<.05) and operating budget (r = -.32, p \< .01) were negatively correlated with the rating of fairness in priority setting. Analysis of respondent comments ------------------------------- In this section we describe participants\' responses to the open-ended questions organized according to themes. We have included verbatim responses to help illustrate the themes. What do you see as the goals of priority setting in your hospital? ------------------------------------------------------------------ Decision makers emphasized four priority setting goals which were complex, interdependent, and required balancing. Some respondents said that (1) meeting needs existed in relation to delivering (2) quality services, or was constrained by resource availability. Examples of these goals include \"quality care within available resources\" and \"access to high quality service in areas of greatest need\". While (3) meeting budgets was identified as a goal by some decision makers, it did not exist in isolation from other goals such as meeting strategic directions or needs. One decision maker said his goal was \"ensuring a balanced situation at year end while meeting the direction set out in our strategic plan\". Achieving (4) organizational goals was expressed as a limiting, or organizational focusing process, which involved alignment or balancing of multiple goals and values. How well does your hospital meet its priority setting goals? Please explain your response ----------------------------------------------------------------------------------------- Decision makers described a deliberative process by which their priority setting goals were operationalized, providing examples of good practices and challenges or barriers for achievement. Overall, decision makers appeared confident that their priority setting goals were the correct ones, in no case did they comment that expressed goals were unrealistic, so contributing to lack of achievement. Decision makers pointed to five factors of importance in meeting their priority setting goals. Review processes (1) were described in which different review processes are brought to bear pointing to areas requiring additional resources. For example, a decision maker said \"annually we review and ensure that resources are being allocated to priority programs and services\". Leadership (2) was implicated as an important factor in meeting priority-setting goals. For example, a decision maker said \"We do not set goals that are pie in the sky, they must be achievable\". With respect to (3) stakeholder consultation, some respondents pointed to the need to involve the wider community in decision making. Decision-makers felt that improvements in priority setting were contingent upon (4) access to relevant information. Finally, some decision-makers emphasized the importance of (5) decision making tools, or benchmarking, to improve decision making. Overall, how fair is priority setting at your hospital? Please explain your response ------------------------------------------------------------------------------------ Respondents gave information to support their self-evaluation, and provided examples to demonstrate the fairness of priority setting processes from their perspective. In evaluating the fairness of priority setting, decision makers emphasized five themes. Stakeholder consultation raises the level of (1) inclusivity, bringing different points of views and interests to bear in solving common problems. For example, a decision maker said \"we have a service providers network in our community that has direct input into resource allocation and program planning\". Respondents described review processes (2) involving the deliberation of concerned parties, using relevant data, based on need, cost and other values (e.g. evidence). Reporting systems (3) provide an institutional feedback loop, pointing to areas where limits may be fairly set. With respect to (4) revision/appeals, revising arguments or values based on iterative processes involving various stakeholders is a feature of fairness, and helps to improve buy-in, but may increase organizational tensions. Finally, (5) governance may also be involved in developing decision-making models, and in ensuring the conditions of relevancy, appeals/revision, and publicity are met. For example, a decision maker said \"The Board and senior management have developed decision-making models for key program/service changes\...our appeals process is informal, but open to access including the Board\". Discussion ========== To our knowledge, this is the first published survey of hospital decision makers covering an entire health system, using Daniels and Sabin\'s framework of \'accountability for reasonableness\'. It provides data to understand how fair priority setting processes are, what decision makers\' priority setting goals are, and how well these goals are met. Several studies, using either survey or qualitative methodologies sometimes in combination with hypothetical \"tradeoffs\", have examined priority setting from theoretical perspectives aimed at capturing the public or professional\'s preferences or values in priority setting \[[@B21]-[@B24]\]. Relatively few studies have described what is happening in real-world contexts with a view of evaluating and improving priority setting activities \[[@B25]-[@B31]\]. These have described actual priority setting in individual hospitals, focusing on strategic planning \[[@B12]\], surgery \[[@B13]\], critical care \[[@B14]\], new technologies \[[@B15]\], and the rationing of new drugs \[[@B17]\], with no study reviewing priority setting in hospitals across the health system. This study makes five contributions to knowledge on priority setting. First, there is ample room for improvement in fair practices within Ontario hospitals as described by their Chief Executive Officers. Although many hospital CEOs felt that their priority setting was fair, ample room for improvement was noted, especially for the enforcement condition, followed by publicity, appeals/revision and relevance. While 79% of decision makers felt their hospitals met their priority setting goals, only 60.7% felt their processes were fair. According to decision makers, the perceived \'gap\' was greater in meeting fairness than in priority setting goals. Second, it is feasible for decision makers to assess the fairness of priority setting in their institutions on a quantitative basis according to the \'accountability for reasonableness\' framework, with greater than half of respondents saying their processes are fair. There is a high degree of association between \'fairness\' and the internal components of \'accountability for reasonableness\', lending support that fairness can be operationalized through \'accountability for reasonableness\'. As well, the internal conditions of \'accountability for reasonableness\' are highly related, and positively correlated, suggesting relevance, publicity, appeals/revision, and enforcement are measuring comparable aspects of fairness. Consistent with this, the high Cronbach\'s α finding is promising for the development of an \'accountability for reasonableness\' scale of perceived fairness of priority setting in health care institutions. Measuring decision makers\' self-report of their perception in meeting the various conditions of \'accountability for reasonableness\', the scale would provide a practical tool for decision makers to assess self-reported views over time, noting progress in meeting conditions, while providing a reference point for needed improvement (i.e. enhance meeting \'publicity\' condition). Finally, the finding of a negative relationship between number of hospital beds or budgets and self-report on fairness is consistent with the view that smaller hospitals may be perceived to have fairer processes, with greater involvement of their local communities in decision making and emphasis on transparency and \"trust\". Third, decision makers\' views were surveyed at a health system level to determine what their actual priority setting practices were. Four complex, interrelated priority setting goals were described, suggesting a balance of need, quality, budget and organizational goals in priority determination. In addition, the study points to a close alignment of factors required in the meeting of priority setting goals and in the elements of fairness. In providing decision maker perspectives on characteristics of fairness, this study also adds to previous empirical work suggesting fair priority setting depends on a fair priority setting process \[[@B31]\]. Additional research is required to understand the capacity of organizations to improve such practices. Four, this study has shown, from a blend of qualitative and quantitative approaches, that it is feasible to operationalize the \'accountability for reasonableness\' framework, through the design of survey instruments to facilitate self-evaluation, and identification of good practices. This data can be shared with decisions makers to improve the fairness of their priority setting processes in meeting the four conditions of \'accountability for reasonableness\'. Finally, this study expands on the likely relationship between leadership and priority setting in which leadership was found to contribute to perceptions of fairness in two committees engaged in priority setting for new health care technologies. Study results indicate that greatest room for improvement exists in meeting the \'enforcement\' condition, with decision-makers describing critical leadership success factors, including: the role of governance in establishing policy and in meeting this condition, the need to set achievable corporate goals, and the significance of a lobbying funding function. Further study is needed to clarify the nature of this leadership contribution. Limitations ----------- The study is limited, first, in the response rate from hospitals. Only 54% of Ontario\'s 160 hospital CEO\'s responded to the survey. It is possible that hospitals responding to this survey were those doing, or perceived doing, better on self-report than others. However, there is no evidence to suggest that this is the case, and the sample did include small, medium and larger teaching hospitals, as well as specialized facilities to mitigate against representative selection bias. Second, social desirability bias was possible in that the views of decision makers expressed in the survey may not have corresponded to what they actually believed, or did. However, unpublished data \[[@B32]\] based on in-depth interviews with Ontario hospital decision makers suggest self-reported views in relation to fairness were similar in both survey respondents and non-respondents, and that reasons for non-response were related to convenience, workload and priority, and not social desirability. Third, corroborative evidence of \'fairness\', obtained through in-depth interviews with staff or community stakeholders, or through review of other relevant information (e.g. meeting minutes, publication of rationales on web sites) was not done. The study design was a survey, focusing on the self-report of fairness from the perspective of Chief Executive Officers, or delegates. In-depth interviews with Chief Executive Officers, or additional individual hospital case studies would be helpful in understanding actual priority setting practices, building on these survey results. Conclusions =========== In this first survey of Chief Executive Officers within a health region reporting on their assessment of the fairness of priority setting practices in their institutions according to \'accountability for reasonableness\', ample room for improvement was observed, especially for the enforcement condition. Additional study is required to understand how application of \'accountability for reasonableness\' will vary within and across institutions, and is shaped or influenced by various parameters, including the nature of corporate leadership. These evaluations can help to identify good practice opportunities for improvement that can be shared between local institutions or used within government to drive health care reforms. Competing interests =================== The author(s) declare they have no competing interests. Authors\' contributions ======================= DR was the primary analyst and principal author of the manuscript. DKM, CK and PAS were involved in study conception, analysis and drafting the manuscript. CK and DKM were involved in data collection. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/5/8/prepub> Acknowledgements ================ Funding for study provided by a grant from The Change Foundation and by an Interdisciplinary Capacity Enhancement grant from the Canadian Institutes of Health Research. DKM is supported by an Ontario Ministry of Health and Long-Term Care Career Scientist Award. PAS is supported by a Canadian Institutes of Health Research Distinguished Scientist Award.	PubMed Central	2024-06-05T03:55:52.560423	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548272/", "journal": "BMC Health Serv Res. 2005 Jan 21; 5:8", "authors": [ { "first": "David", "last": "Reeleder" }, { "first": "Douglas K", "last": "Martin" }, { "first": "Christian", "last": "Keresztes" }, { "first": "Peter A", "last": "Singer" } ] }
PMC548273	Background ========== The glucose-6-phosphatase system consists of the glucose-6-phosphate catalytic subunit (EC 3.1.3.9), embedded in the membrane of the endoplasmic reticulum (ER) via nine transmembrane domains, and the membrane spanning translocases, responsible in carrying either the substrate into the ER or the product from the ER \[[@B1]\]. Transport of substrate and product is necessary due to the orientation of the active site of the glucose-6-phosphatase enzyme towards the luminal side of the ER. Glucose-6-phosphate, the end product of both gluconeogenesis and glycogenolysis in the liver, is hydrolyzed by the glucose-6-phosphatase system allowing the liberation of glucose into the circulation. Thus, glucose-6-phosphatase plays a key role in the homeostasis of blood glucose. Mutations in the gene encoding the catalytic subunit of glucose-6-phosphatase are responsible for the development of glycogen storage disease type 1, also known as Gierke disease \[[@B2]\]. In animal models of diabetes mellitus, glucose-6-phosphatase activity is increased along with mRNA and protein levels \[[@B3]\]. Accordingly, inhibitors of glucose-6-phosphatase or the G6PT transporter are used for the treatment of type 2 diabetes. The glucose-6-phosphatase encoding gene is regulated by a variety of extracellular signaling molecules, including glucose, insulin, glucocorticoids, and cAMP. Insulin decreases the level of glucose-6-phosphatase mRNA in the liver and in hepatoma cells and three functionally distinct insulin response elements have been identified \[[@B4]\]. Glucocorticoids elevate glucose-6-phosphatase promoter activity mediated by a glucocorticoid response element and hepatocyte nuclear factor 1 \[[@B5]\]. Conflicting results were published concerning the stimulation of glucose-6-phosphatase promoter activity by elevated intracellular cAMP concentrations. While dibutyryl cAMP alone did not significantly stimulate transcription of a luciferase reporter gene under control of 1.2 kb of the human glucose-6-phosphatase promoter in H4IIIE hepatoma cells \[[@B6]\], a later report by the same group described a ≈ 2-fold stimulation of glucose-6-phosphatase promoter activity with dibutyryl cAMP \[[@B7]\]. The sequence from -161 to -152 of the human glucose-6-phosphatase promoter, including the motif 5\'-TTTACGTAA-3\', was proposed to mediate the effect of dibutyryl cAMP on reporter gene transcription \[[@B7]\]. In HepG2 cells a glucose-6-phosphatase promoter/chloramphenicol acetyltransferase reporter gene showed a 2 to 3-fold enhancement in transcription following stimulation of the cells with dibutyryl cAMP \[[@B8]\]. Here, the sequence from -136 to -129 of the human glucose-6-phosphatase gene, including the motif 5\'-TTGCATCAA-3\', was proposed to be essential to couple dibutyryl cAMP stimulation with enhanced reporter gene transcription \[[@B8]\]. The most important and best characterized protein that connects an elevation of intracellular cAMP concentrations with enhanced transcription of selected genes is the CRE binding protein CREB, a basic region leucine zipper protein. CREB plays an essential role in the regulation of glucose-6-phosphatase gene transcription in the liver, as exemplified by the fact that transgenic mice expressing a dominant-negative CREB mutant in the liver show reduced mRNA levels of glucose-6-phosphatase \[[@B9]\]. CREB not only mediates stimulus-transcription coupling of the cAMP signaling pathway but functions as a point of convergence of many other signaling molecules involving calcium, neurotrophins, tumor promoters, and growth factors \[[@B10]\]. CREB is inactive in the dephosphorylated state and turns into an activator upon phosphorylation. The key enzyme leading to CREB activation is the cAMP-dependent protein kinase (PKA), but CREB serves also as a substrate for calcium/calmodulin-dependent protein kinase IV and the mitogen-and stress-activated kinases MSK1 and 2 \[[@B11],[@B12]\]. To study CREB regulated gene transcription, a signaling cascade needs to be activated that leads to phosphorylation and activation of CREB. This can be accomplished by adding cAMP analogues such as dibutyryl cAMP that passes the plasma membrane or via a direct activation of adenylate cyclase by forskolin. This experimental setting is problematic in that high levels of phosphodiesterase enzymes in the cells may immediately hydrolyze cAMP, with the result that no cAMP-mediated gene transcription can be monitored \[[@B13]\]. Moreover, elevated levels of cAMP may additionally activate the EPAC/Rap1 pathway, making it somewhat difficult to attribute the effect of cAMP solely to the cAMP/PKA signaling pathway. Many functions of cAMP previously attributed to PKA may be in fact the result of EPAC activation \[[@B14]\]. Instead of increasing the intracellular levels of cAMP some investigators employed an overexpression strategy using the catalytic subunit of cAMP-dependent protein kinase to analyze cAMP regulated genes. This approach has the advantage that by excluding a parallel signaling cascade via the cAMP-inducible nucleotide exchange factor EPAC, only the biological outcome of cAMP-dependent protein kinase activation is studied. However, the translocation of the catalytic subunit of cAMP-dependent protein kinase into the nucleus is not very efficient and may rely solely on diffusion \[[@B15]\]. Thus high amounts of expression vector in the range of 1 to 5 μg/plate are often transfected \[[@B16]-[@B18]\] in order to observe an effect on gene transcription. Naturally, these high amounts of expressed catalytic subunit are far away from the physiological concentrations within the cells. Here, we report on the regulation of glucose-6-phosphatase gene transcription by CREB and PKA. Using a constitutively active CREB2/CREB fusion protein, that does not require phosphorylation for activation, and a nuclear-targeted mutant of the catalytic subunit of PKA, we show that CREB activates transcription of glucose-6-phosphatase promoter/luciferase reporter genes via two distinct genetic elements. In contrast, the bZIP protein ATF2, known to compete with CREB for binding to the canonical CRE, did not transactivate these reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Results ======= The proximal region of the human glucose-6-phosphatase gene contains two cyclic AMP response element like motifs ---------------------------------------------------------------------------------------------------------------- Fig. [1A](#F1){ref-type="fig"} shows part of the proximal region of the human glucose-6-phosphatase gene. As indicated, two CRE-like sequences are present, resembling the canonical CRE sequence 5\'-TGACGTCA-3\'. The distal CRE-like site (CRE1) and the proximal CRE-like site (CRE2) differ on two or three positions, respectively, in comparison to the canonical CRE. To investigate the biological function of these elements we constructed a battery of reporter plasmids containing both CRE-like elements or only one of them (Fig. [1B,C](#F1){ref-type="fig"}). Mutations were introduced into CRE1 and CRE2 sites to prevent CREB binding \[[@B19]\]. As a control, we generated a reporter plasmid containing two copies of a CRE/AP1-like element derived from the human tumor necrosis factor (TNF) α gene promoter (Fig. [1D](#F1){ref-type="fig"}). The TNFα gene belongs to the target genes of CREB \[[@B10]\]. Additionally, the reporter genes contained a TATA box derived from the HIV long terminal repeat, an initiator element from the adenovirus major late promoter and the luciferase open reading frame. A constitutively active CREB2/CREB fusion protein transactivates reporter genes with either CRE1 or CRE2 of the glucose-6-phosphatase gene in their regulatory regions ---------------------------------------------------------------------------------------------------------------------------------------------------------------------- The fact that unphosphorylated CREB is transcriptionally silent excludes a simple overexpression strategy to analyze the biological impact of the CRE-like sequences within the glucose-6-phosphatase gene. The use of natural inducers of CREB such as glucagon or epinephrine, that all elevate the cAMP concentration in the cells, also triggers pleiotropic responses by activating the cAMP-dependent protein kinase and the EPAC/Rap1 pathway, so that the biological outcome cannot be attributed solely to CREB activation. We therefore designed a constitutively active CREB2/CREB fusion protein to uncouple the investigation of glucose-6-phosphatase gene transcription from signaling pathways in the cell. Fig. [2A](#F2){ref-type="fig"} shows the structural domains of the basic region leucine zipper (bZIP) transcription factors CREB and CREB2. Both proteins contain a bZIP domain on their C-termini responsible for dimerization and DNA-binding. The N-termini of CREB and CREB2 contain activation domains. While the activation domain of CREB2 is constitutively active and transferable to heterologous DNA-binding domains \[[@B20]\], the major activation domain of CREB is controlled by phosphorylation. We expressed the bZIP domain of CREB, which is responsible for DNA binding and dimerization, as a fusion protein with the constitutively active transcriptional activation domain of CREB2, generating the chimeric transcription factor C2/CREB (Fig. [2A](#F2){ref-type="fig"}). The C2/CREB fusion protein contains additionally an immunological tag used for the detection of the protein. Proteins derived from nuclear extracts of HepG2 human hepatoma cells (mock) or HepG2 cells transfected with an expression vector encoding C2/CREB were fractionated by SDS-PAGE. The fusion protein was identified by Western Blot analysis using antibodies targeting the FLAG epitope. Fig. [2B](#F2){ref-type="fig"} shows that the CREB2/CREB fusion protein was synthesized as expected. To study the regulation of the glucose-6-phosphatase promoter/luciferase reporter genes by C2/CREB, we decided to use the human hepatoma cell line HepG2, as it has been reported that activation of the cAMP signaling pathway stimulates glucose-6-phosphatase gene transcription in these cells \[[@B17],[@B21],[@B22]\]. HepG2 cells were transfected with one of the luciferase reporter plasmids pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc, pG6PCRE1/CRE2mutluc, pG6PCRE1luc or pG6PCRE2luc together with either the \"empty\" expression vector pCMV5 (denoted \"-\") or an expression vector encoding C2/CREB (plasmid pCMV-FLAG-C2/CREB) (denoted \"+\"). As a control, we transfected the pTNFα(CRE/AP1)^2^luc reporter plasmid that contains two copies of the composite CRE/AP1 element of the TNFα promoter. We transfected additionally plasmid pRSVβ, encoding β-galactosidase under the control of the Rous sarcoma virus long-terminal repeat, to correct for variations in transfection efficiencies. Luciferase activities were normalized for transfection efficiency by dividing luciferase light units by β-galactosidase activities. The results of the transfection experiments are depicted in Fig. [3](#F3){ref-type="fig"}. Transcription of the pG6PCRE1/CRE2luc reporter gene, that contains both CRE-like sequences in its regulatory region, was strongly induced following expression of C2/CREB (Fig. [3A](#F3){ref-type="fig"}, upper panel). Mutation of CRE1 or CRE2 did not abolish the transactivation potential of C2/CREB (Fig. [3A](#F3){ref-type="fig"}, middle, bottom panel), indicating that both CRE-like sequences function independently as bona fideCREs. This conclusion was confirmed by transfection experiments of reporter plasmids having only one of the CRE-like sequences in the regulatory regions. Fig. [3B](#F3){ref-type="fig"} shows that both reporter genes pG6PCRE1luc and pG6PCRE2luc were transactivated by C2/CREB. Finally, C2/CREB also transactivated the pTNFα(CRE/AP1)^2^luc reporter gene via the CRE/AP1 element (Fig. [3C](#F3){ref-type="fig"}). We conclude that both CRE1 and CRE2 motifs of the glucose-6-phosphatase gene promoter function as bona fideCREs. Expression of a nuclear-targeted catalytic subunit of cAMP-dependent protein kinase ----------------------------------------------------------------------------------- The structural domains of the catalytic subunit of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) are depicted in Fig. [4A](#F4){ref-type="fig"}. The protein is myristylated on the N-terminus, but myristylation is nonessential for enzyme activation \[[@B23]\]. We modified the N- terminal region of the protein by adding a nuclear targeting signal derived from the SV40 large T antigen (NLS) and a FLAG epitope to facilitate immunological detection. This mutant termed NLSCα should be sorted to the nuclear compartment, due to the presence of the NLS. HepG2 cells were transfected with an expression vector encoding NLSCα. Nuclear extracts of these cells were fractionated by SDS-PAGE and analyzed by Western blotting. Fig. [4B](#F4){ref-type="fig"} shows that the modified catalytic subunit of cAMP-dependent protein kinase was synthesized as expected. To test the biological activity of NLSCα, in comparison to the wild-type form of the catalytic subunit (Cα), we measured the activity of the phosphorylation-regulated transcription factor CREB using a fusion protein consisting of the GAL4 DNA-binding domain fused to the kinase inducible activation domain of CREB. The modular structure of the GAL4-CREB fusion protein is depicted in Fig. [4C](#F4){ref-type="fig"}. Transcriptional activation was monitored by co-transfection of the reporter plasmid pUAS^5^luc that contains five copies of the GAL4 binding site termed \"upstream activating sequence\" (UAS) upstream of a luciferase reporter gene (Fig. [4C](#F4){ref-type="fig"}). Since mammalian cells do not express transcription factors that bind to the UAS, this system directly measures the effect of NLSCα or Cα on the transcriptional activation potential of CREB. The reporter plasmid pUAS^5^luc and the expression plasmid that encodes GAL4-CREB were transfected into HepG2 cells together with either an \"empty\" expression vector (denoted \"-\") or expression vectors encoding Cα or NLSCα. Transfection efficiency was monitored by co-transfecting pRSVβ. As a control, plasmid pM1 encoding only the DNA binding domain of GAL4 (GAL4~DBD~) was transfected. Cell extracts were prepared forty-eight hours later and β-galactosidase and luciferase activities were determined. Fig. [4C](#F4){ref-type="fig"} shows that the modified form of PKA catalytic subunit (NLSCα) was highly potent in stimulating the transcriptional activation potential of CREB. The activation was on the order of 60-fold. In contrast, no transcriptional activation was observed following overexpression of the wild-type form of the catalytic subunit. Next, we tested the biological activity of NLSCα with regard to transcription of the glucose-6-phosphatase promoter/luciferase reporter genes. In the experiments, we additionally overexpressed wild-type CREB, the CREBS133A containing a serine to alanine mutation at position 133, and K-CREB, a CREB mutant containing the point mutation R286L within the basic DNA-binding domain, impairing DNA-binding \[[@B24]\]. Transfection of expression vectors encoding the wild-type form of CREB, CREBS133A, or K-CREB did not significantly change basal transcription of the reporter genes. However, transfection of an NLSCα expression vector strongly stimulated transcription of the glucose-6-phosphatase promoter/luciferase reporter genes, containing both or one of the CRE-like sequences of the glucose-6-phosphatase promoter (Fig. [5A,B](#F5){ref-type="fig"}). These results confirm the previous observations, obtained via overexpression of C2/CREB, that both CRE motifs of the glucose-6-phosphatase gene function as independent genetic elements responsive to couple elevated cAMP and PKA levels with enhanced glucose-6-phosphatase gene transcription. Similarly, coexpression of wild-type CREB and NLSCα stimulated reporter gene transcription mediated by the CRE/AP1 element derived from the TNFα gene (Fig. [5C](#F5){ref-type="fig"}). Surprisingly, we still detected reporter gene activation following coexpression of NLSCα with CREBS133A, lacking the major PKA phosphorylation site. Compared to the coexpression experiments of NLSCα with the wild-type form of CREB, the reduced activation of reporter gene transcription in coexpression experiments of NLSCα with CREBS133A indicates that NLSCα catalyzed phosphorylation of serine residue 133 of CREB is important for reporter gene transcription. The fact that CREBS133A is still able to transactivate the reporter genes following expression of NLSCα suggests that NLSCα triggers further phosphorylation reactions leading to enhanced transcription via the CRE-like sequences within the glucose-6-phosphatase gene. In contrast, expression of K-CREB is transcriptionally inactive, in the presence or absence of NLSCα, indicating that DNA-binding is a prerequisite for CREB and CREBS133A to transactivate the reporter genes. Biological activity of a constitutively active CREB2/ATF2 fusion protein on glucose-6-phosphatase promoter/luciferase reporter genes ------------------------------------------------------------------------------------------------------------------------------------ The transcription factor ATF2, a substrate of stress-activated protein kinases, has been reported to bind to the classical cAMP responsive element (CRE) 5\'-TGACGTCA-3\' \[[@B25]\]. Recently, we confirmed this observation showing that ATF2 and CREB compete for binding to the CRE of the secretogranin II gene \[[@B26]\]. ATF2 was proposed to also bind with high affinity to the related DNA target sequence 5\'-TTACGTAA-3\' \[[@B19]\], which is identical to the CRE1 of the glucose-6-phosphatase gene promoter. We tested the biological activity of ATF2 on reporter genes containing sequences of the glucose-6-phosphatase promoter region. As a control, we used a reporter gene containing two copies of the CRE/AP-1 element of the TNFα gene. ATF2 has been shown to stimulate TNFα promoter activity \[[@B27]\]. The unphosphorylated form of ATF2 is transcriptionally inactive, due to an inhibitory intramolecular interaction between the activation domain of ATF2 and the bZIP domain of ATF2 \[[@B28]\]. We therefore expressed a constitutively active CREB2/ATF2 fusion protein to investigate whether ATF2 functions as a transactivator for reporter genes containing the CRE1 and/or CRE2 sequences of the glucose-6-phosphatase gene in the regulatory region. The domain structure of this C2/ATF2 fusion protein is depicted in Fig. [6A](#F6){ref-type="fig"}. HepG2 cells were transfected with one of the reporter plasmids (pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc, pG6PCRE1/CRE2mutluc, pG6PCRE1luc, and pG6PCRE2luc), the internal standard plasmid pRSVβ, and an expression vector encoding C2/ATF2. As a control, we analyzed the reporter plasmid pTNFα(CRE/AP1)^2^luc. The results show that C2/ATF2 was unable to transactivate the reporter genes containing CRE1, CRE2, or both CRE-like sequences derived from the glucose-6-phosphatase gene in the regulatory region, indicating that glucose-6-phosphatase gene transcription is not regulated by ATF2 via the two CRE-like sequences. In contrast, C2/ATF2 strongly activated transcription of the pTNFα(CRE/AP1)^2^luc reporter gene, confirming that TNFα gene transcription is regulated by ATF2. Discussion ========== The gene encoding glucose-6-phosphatase is regulated by increased levels of intracellular cAMP, but the regulatory sites responsible for cAMP-induced gene transcription are still a matter of controversy. The objective of this study was to characterize the genetic elements that function as cis-acting sites for transactivation by CREB and that also respond to activated PKA in the nucleus. Two distinct genetic elements had been suggested to couple enhanced levels of cAMP and elevated PKA activity with increased glucose-6-phosphatase gene transcription \[[@B8],[@B17]\] and we intended to clarify which of these elements is required for transactivation of the glucose-6-phosphatase gene by CREB. The reason for the differences in the published reports concerning cAMP-regulated glucose-6-phosphatase gene transcription can be explained by the technical problems that occurs following elevation of the intracellular cAMP concentration, as outlined in the introduction section. To solve these problems, we prefered to measure \"transcriptional activation\" instead of \"transcription factor/DNA-binding\" because although DNA-binding is required for a subsequent transcriptional activation by CREB, an enhanced binding activity of a transcription factor to DNA, monitored by an in vitrobinding assay, does not necessarily prove an enhanced transcriptional activation potential of this protein \[[@B29]\]. In addition, we expressed a constitutively active CREB2/CREB fusion protein that was highly active in transactivating CREB-responsive target genes. Using this strategy we avoided the use of dibutyryl cAMP or forskolin, that may trigger other biological responses. Furthermore, only nanomolar levels of the expression vector encoding C2/CREB were required to show that both CRE-like sequences (CRE1 and CRE2) within the glucose-6-phosphatase gene function as target sites for an active CREB. Thus, both the reported genetic elements, the sequence from -161 to -152 \[[@B7]\] and the sequence from -136 to -129 \[[@B8]\] of the human glucose-6-phosphatase promoter, mediated reporter gene transactivation by CREB. The fact that the glucose-6-phosphatase gene contains two distinct genetic elements for the regulation by CREB is not surprising as multiple CREs have been found in other genes encoding for instance ICER, MKP-1, Nur77 or Egr-4 \[[@B30]\]. Using an overexpression strategy for the catalytic subunit of PKA, it has been reported that PKA directly stimulates reporter gene transcription under control of CRE1, whereas CRE2 was unable to connect elevated PKA activity with enhanced CRE2-controlled reporter gene transcription \[[@B17]\]. The authors concluded that only the CRE1, but not the CRE2 functions as bona fideCRE \[[@B17]\]. In this study a concentration of 5 μg of expression vector encoding the catalytic subunit of PKA was used. In the past, we also employed this overexpression strategy for the catalytic subunit of PKA in the laboratory \[[@B29],[@B31]\] and observed that high levels of expression vectors are required to monitor an effect on gene transcription. We also observed that transcription of the reference gene encoding β-galactosidase under control of the Rous sarcoma virus long terminal repeat was changed following overexpression of the catalytic subunit, although no CRE has been mapped within this promoter/enhancer region. This fact indicates that transfection of micromolar levels of expression vectors encoding the catalytic subunit of PKA disturbs the machinery of transcription and the data obtained by this approach may not always depict the real picture. Likewise, it has been known for many years that expression of the strong transcriptional activator VP16 induces transcriptional repression within the cells, due to squelching. In the study reported here, expression of a modified catalytic subunit that had a nuclear targeting signal, activated reporter gene transcription very efficiently following transfection of nanomolar amounts of the expression vector. In fact, we used a 50-fold lower amount of expression vector compared to the study by Streeper et al. \[[@B17]\]. Transfection of nanomolar concentrations of an expression plasmid encoding the wild-type catalytic subunit of PKA did not show any effect on reporter gene transcription. These experiments, involving an overexpression of NLSCα, showed an enhanced transcription of reporter genes having either an intact CRE1 or CRE2 in its regulatory region, indicating that both CRE1 and CRE2 functioned as bona fideCRE. We also observed an activation of glucose-6-phosphatase promoter/luciferase reporter gene transcription following coexpression of CREBS133A with NLSCα. Phosphorylation of the serine 133 residue of CREB is the predominant mechanism to enhance the transcriptional activation potential of CREB via recruitment of the coactivator CREB binding protein (CBP) and its paralogue p300 to the promoter. Accordingly, we observed a clear reduction of reporter gene transcription in coexpression studies of NLSCα together with CREBS133A instead of wild-type CREB. However, expression of CREBS133A still contributed to reporter gene transcription in the presence of NLSCα, suggesting that either other residues of CREBS133A or other promoter-bound proteins involved in the regulation of CREB-mediated transcription are phosphorylated. PKA can phosphorylate CBP directly or other components of the transcriptional machinery downstream from CREB \[[@B32],[@B33]\]. Nonetheless, the experiments confirmed that both CRE-like sequences within the glucose-6-phosphatase gene function as genetic elements to mediate transactivation via CREB. In contrast to the expression experiments involving C2/CREB, we did not detect an effect of C2/ATF2, a constitutively active CREB2/ATF2 fusion protein, on glucose-6-phosphatase promoter/luciferase reporter gene transcription, despite the fact that the sequence 5\'-TTACGTAA-3\', which is identical to the CRE1 site of the glucose-6-phosphatase promoter, has been shown to function as a high affinity binding site for ATF2 in vitro \[[@B19]\]. This discrepancy supports the view that transcriptional bioassays and not in vitro DNA-protein binding experiments describe the biological activities of transcription factors. The CRE-like sequences CRE1 and CRE2 of the glucose-6-phosphatase gene are therefore strikingly different from the CRE/AP1 site of the TNFα gene, that is strongly activated by C2/ATF2. Recently, we showed that ATF2 is able to specifically transactivate CRE-containing genes and we identified the secretogranin II gene as a target gene for ATF2 \[[@B26]\]. We also showed that C2/ATF2 only marginally enhanced transcription of a reporter gene carrying four copies of the c-Fos CRE in its regulatory region, while transcription regulated by the tyrosine hydroxylase promoter was not upregulated at all. Thus, ATF2 distinguishs between different CRE-containing genes. The biological activity of ATF2 is regulated by stress-activated protein kinases and ATF2 is thought to play an important role in the cellular stress response. Our data sheds light on the fact that expression of glucose-6-phosphatase is not connected -- via ATF2 -- to the cellular stress response, in contrast to the TNFα gene. Conclusions =========== We have shown that there are two distinct CREB-responsive sites in the glucose-6-phosphatase gene promoter that are responding to either a constitutively active CREB or elevated concentrations of the catalytic subunit of cAMP-dependent protein kinase in the nucleus. This study further shows that the expression vectors encoding C2/CREB and NLSCα are valuable tools for the investigation of CREB-mediated gene transcription and the biological functions of CREB. CREB is a key molecule in neuronal survival, as demonstrated by the fact that a dominant-negative A-CREB induced apoptosis in sympathetic neurons grown in NGF \[[@B34]\]. The C2/CREB fusion protein, that directs CREB-mediated gene transcription in the absence of PKA activation, can be used in gain-of-function experiments to investigate the cytoprotective activity of CREB on the molecular level. Most importantly, the C2/CREB protein can be used to identify anti-apoptotic genes that are controlled by CREB. Likewise, expression of NLSCα will permit the analysis of PKA-regulated gene transcription, without influencing other functions of PKA in the cytosol. Methods ======= Reporter constructs ------------------- The glucose-6-phosphatase promoter/luciferase reporter genes pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc, and pG6PCRE1/CRE2mutluc were constructed by the insertion of the annealed oligonucleotides depicted in Fig. [1B](#F1){ref-type="fig"} with KpnI/XhoI cohesive ends into the KpnI/XhoI sites of plasmid pHIVTATAluc \[[@B35]\]. The glucose-6-phosphatase promoter/luciferase reporter genes pG6PCRE1luc and pG6PCRE2luc that contain either the CRE1 or the CRE2 sequence derived from the human glucose-6-phosphatase gene were constructed by the insertion of the annealed oligonucleotides depicted in Fig. [1C](#F1){ref-type="fig"} with XhoI/SalI cohesive ends into the XhoI/SalI sites of plasmid pHIVTATA-CAT \[[@B36]\]. These plasmids termed pG6PCRE1CAT and pG6PCRE2CAT were digested with XbaI, filled in with the Klenow fragment of DNA polymerase I, recut with XhoI and cloned into plasmid pGL3-Basic (Promega). The reporter gene pTNFα(CRE/AP-1)^2^luc, that contains two copies of the CRE/AP1 element of the human tumor necrosis factor α gene, was generated in a similar way with annealed oligonucleotides encompassing the sequence depicted in Fig. [1D](#F1){ref-type="fig"}. Plasmid pUAS^5^luc containing the luciferase reporter gene, a TATA box derived from the HIV long terminal repeat, an initiator element from the adenovirus major late promoter, and five binding sites for GAL4 (termed \'upstream activating sequence\', UAS) has been described \[[@B35]\]. Expression vectors ------------------ The expression vector pCMV-FLAG-C2/CREB, encoding a constitutively active CREB2/CREB chimera, has been described \[[@B26]\]. The CREB2/CREB fusion protein consists of the amino-terminal 187 amino acids from CREB2, encompassing the phosphorylation-independent transcriptional activation domain, and amino acids 182 to 326 of CREB, including the bZIP domain. Expression vectors encoding CREB, CREBS133A, and K-CREB were kind gifts of Wilhart Knepel and Elke Oetjen, Department of Molecular Pharmacology, University of Göttingen, Germany. The expression vector encoding a constitutively active CREB2/ATF2 fusion protein termed C2/ATF2 has been described \[[@B27]\]. The GAL4 expression plasmid pFA2CREB was purchased from Stratagene. The fusion protein encodes the transcriptional activation sequence of CREB (amino acids 1--281) fused to the DNA-binding domain of GAL4. An expression vector encoding the catalytic subunit of PKA (pCMVCα) was a kind gift of Michael Uhler from the University of Michigan, Ann Arbor \[[@B37]\]. The construction of the NLSCα encoding expression vector has been described \[[@B26]\]. NLSCα has a triple FLAG-tag and a nuclear localization signal on the N-terminus, followed by amino acids 19 to 351 of Cα. The expression vector pRSVβ, encoding β-galactosidase under the control of the Rous sarcoma virus long terminal repeat, has been described \[[@B29]\]. Cell culture, transfections, and reporter gene assays ----------------------------------------------------- Human HepG2 hepatoma cells were cultured and transfected as described \[[@B38]\]. The amounts of expression vectors transfected are indicated in the figure legends. The luciferase reporter plasmids (1 μg) and the internal reference plasmid pRSVβ were transfected into cells grown on 60 mm plates. Lysates were prepared forty-eight hours later using cell culture lysis buffer (Promega) and β-galactosidase and luciferase activities were measured as described \[[@B35]\]. Western Blots ------------- Nuclear extracts were prepared as described \[[@B39]\]. 20 μg of nuclear proteins were separated by SDS-PAGE and the blot was incubated with the M2 monoclonal antibody directed against the FLAG epitope (Sigma, \# F3165). Blots were developed with a horseradish peroxidase conjugated anti-mouse secondary antibody and ECL (Amersham, Freiburg, Germany). Abbreviations ============= ATF activating transcription factor bZIP basic region leucine zipper CRE cAMP response element CREB cAMP response element binding protein G6P glucose-6-phosphatase PKA protein kinase A TNF tumor necrosis factor Authors\' contributions ======================= GT designed the study, generated the reporter plasmids and the expression vectors encoding C2/CREB, C2/ATF2, and NLSCα, performed part of the transfection experiments and drafted the manuscript. JAS performed the transfection experiments depicted in Figs. [3](#F3){ref-type="fig"}, [4C](#F4){ref-type="fig"}, and [5](#F5){ref-type="fig"}. LS performed the C2/ATF2 transfection experiments depicted in Figs. [6](#F6){ref-type="fig"}. All authors read and approved the manuscript. Acknowledgement =============== We thank Karl Bach for excellent technical help, Wilhart Knepel, Elke Oetjen and Michael Uhler for plasmids, and Libby Guethlein and Oliver Rössler for critical reading of the manuscript. This work was supported by the Medical Faculty of the University of Saarland via HOMFOR and by fellowships from the \"Deutscher Akademischer Austauschdienst\" (DAAD) to Jude Al Sarraj and from the European Graduate School of Neuroscience (EURON) to Luisa Stefano. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The human glucose-6-phosphatase gene promoter. (A) Sequence of a portion of the human glucose-6-phosphatase gene promoter including the CRE-like sequences CRE1 and CRE2. (B) The reporter plasmid pG6PCRE1/CRE2luc contains a minimal promoter consisting of the human immunodeficiency virus TATA box, the adenovirus major late promoter initiator element, the luciferase open reading frame, and glucose-6-phosphatase promoter sequences encompassing both CRE-like sequences CRE1 and CRE2. The reporter plasmids pG6PCRE1mut/CRE2luc and pG6PCRE1/CRE2mutluc carry mutations in either the CRE1 or the CRE2, to inactivate these sites. (C) Reporter plasmids pG6PCRE1luc and pG6PCRE2luc contain glucose-6-phosphatase promoter sequences encompassing either the CRE1 or the CRE2. (D) The reporter plasmid pTNFα(CRE/AP1)^2^luc contains two identical copies of the composite CRE/AP1 sequence derived from the human TNFα gene, upstream of a minimal promoter. The sequence of one of these motifs is depicted. ::: ![](1471-2199-6-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Schematic representation and expression of the constitutively active CREB2/CREB fusion protein C2/CREB. (A) Schematic representation of the modular structure of CREB, CREB2, and C2/CREB. The basic region leucine zipper domain (bZIP) is indicated. The chimeric bZIP protein C2/CREB consists of the constitutively active transcriptional activation domain of CREB2 and the bZIP domain of CREB, responsible for dimerization and DNA-binding. (B) Western Blot analysis of HepG2 cells transfected with an expression vector encoding C2/CREB. As a control, extracts from mock transfected HepG2 cells were analyzed. Western blots were probed with an antibody against the FLAG-tag. ::: ![](1471-2199-6-2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Biological activity of C2/CREB, a constitutively active CREB2/CREB fusion protein. One of the reporter plasmids pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc pG6PCRE1/CRE2mutluc (A), pG6PCRE1luc, pG6PCRE2luc (B), or pTNFα(CRE/AP1)^2^luc (C) (1 μg/plate) was transfected into HepG2 cells together with the pRSVβ internal standard plasmid (2 μg/plate), encoding β-galactosidase under the control of the Rous sarcoma virus long terminal repeat, and either the \"empty\" expression vector pCMV5 or an expression vector encoding C2/CREB (20 ng plasmid/plate). The data are presented as the ratio of luciferase activity (light units) to β-galactosidase units (OD units) measured in the cell extracts. The mean +/- SD is depicted. ::: ![](1471-2199-6-2-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Schematic representation and biological activity of the nuclear targeted mutant of the catalytic subunit of cAMP-dependent protein kinase. (A) Schematic representation of the modular structure of the catalytic subunit of cAMP-dependent protein kinase (Cα) and the mutant NLSCα. The wild-type enzyme is myristylated as indicated (Myr). The sequence of the nuclear localization signal derived from the SV40 large T antigen and the triple FLAG epitope in NLSCα are shown. (B) Western Blot analysis of HepG2 cells transfected with an expression vector encoding NLSCα. As a control, extracts from mock transfected HepG2 cells were analyzed. Western blots were probed with an antibody against the FLAG-tag. (C) Modular structure of the GAL4-CREB fusion protein. The protein consists of the DNA-binding domain of GAL4 (amino acids 1--147) and the activation domain of CREB (amino acids 1--281). The reporter plasmid pUAS^5^luc contains a transcription unit encompassing the luciferase open reading frame and a minimal promoter that consists of five copies of the upstream activating sequence (UAS), a TATA box derived from the HIV long terminal repeat and the initiator element from the adenovirus major late promoter. The reporter plasmid pUAS^5^luc (1 μg/plate) was transfected into HepG2 cells together with the pRSVβ internal standard plasmid (2 μg/plate) and either an expression vector encoding the DNA binding domain of GAL4 (plasmid pM1) or the GAL4-CREB fusion protein (1 μg plasmid/plate). In addition, cells were transfected with an expression vector encoding either the wild-type (Cα) or mutated (NLSCα) form of cAMP-dependent protein kinase (100 ng/plate). Forty-eight hours post-transfection cell extracts were prepared and the β-galactosidase and luciferase activities of these extracts were determined. ::: ![](1471-2199-6-2-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Transcriptional activity of CREB and CREB mutants in the presence of a nuclear targeted mutant of the catalytic subunit of cAMP-dependent protein kinase. One of the reporter plasmids pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc pG6PCRE1/CRE2mutluc (A), pG6PCRE1luc, pG6PCRE2luc (B), or pTNFα(CRE/AP1)^2^luc (C) (1 μg/plate) was transfected into HepG2 cells together with the pRSVβ internal standard plasmid (2 μg/plate) and the \"empty\" expression vector pCMV5 or an expression vector encoding either CREB, CREBS133A, or K-CREB (20 ng plasmid/plate). In addition, an expression vector encoding NLSCα (100 ng/plate) was transfected. Forty-eight hours post-transfection cell extracts were prepared and the β-galactosidase and luciferase activities of these extracts were determined. The data are presented as the ratio of luciferase activity (light units) to β-galactosidase units (OD units) measured in the cell extracts. The mean +/- SD is depicted. ::: ![](1471-2199-6-2-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Biological activity of constitutively active ATF2 fusion protein towards glucose-6-phosphatase promoter-containing reporter genes. (A) Schematic representation of the modular structure of C2/ATF2. This chimeric bZIP protein consists of the constitutively active transcriptional activation domain of CREB2 and the bZIP domain of ATF2, responsible for dimerization and DNA-binding. (B, C, D) HepG2 cells were transfected with one of the reporter plasmids pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc pG6PCRE1/CRE2mutluc (B), pG6PCRE1luc, pG6PCRE2luc (C), or pTNFα(CRE/AP1)^2^luc (D), the pRSVβ internal reference plasmid, and either the \"empty\" expression vector pCMV5 or an expression vector encoding C2/ATF2 (100 ng expression plasmid/plate). Lysates were prepared forty-eight hours post-transfection and β-galactosidase and luciferase activities were measured. The mean +/- SD is depicted. ::: ![](1471-2199-6-2-6) :::	PubMed Central	2024-06-05T03:55:52.563018	2005-1-19	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548273/", "journal": "BMC Mol Biol. 2005 Jan 19; 6:2", "authors": [ { "first": "Gerald", "last": "Thiel" }, { "first": "Jude", "last": "Al Sarraj" }, { "first": "Luisa", "last": "Stefano" } ] }
PMC548274	Background ========== Among many trophic factors that act on sensory neurons, three have been studied extensively: nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). During development, NGF, GDNF and BDNF, along with neurotrophin-3 (NT3), support the survival of subpopulations of sensory neurons through their cognate trk receptors \[[@B1]-[@B3]\]. In the adult, uninjured rat, receptors for NGF, GDNF and BDNF are found in partially distinct subpopulations of sensory neurons. NGF-responsive, trkA-containing neurons are mostly small diameter, contain the neuropeptide calcitonin gene-related peptide (CGRP), and are generally thought to have nociceptive properties \[[@B4],[@B5]\]. The BDNF-responsive, trkB-containing population of sensory neurons is predominantly larger diameter and mechanosensitive \[[@B6]\], although there is considerable overlap with the trkA population \[[@B7]\]. GDNF receptors are more widespread, with as many as 60% of dorsal root ganglion (DRG) neurons containing c-ret, while GFR components are found throughout the c-ret population, as well as in c-ret negative neurons \[[@B8],[@B9]\]. Additionally, sensory neurons that are postnatally dependent on GDNF for survival bind the isolectin B~4~(IB~4~, \[[@B2]\]). NGF is thought to play a primary role in the development and maintenance of several pro-algesic states. NGF produces sensitization of nociceptive responses in vivo and increases responsiveness to chemical stimuli associated with nociceptive neurotransmission in vitro \[[@B10]-[@B12]\]. NGF increases the expression of the pro-inflammatory neuropeptide CGRP \[[@B13]\] and increases substance P (SP) and CGRP content in sensory neurons \[[@B14]-[@B17]\]. NGF also promotes the development and maintenance of hyperalgesia following chronic constriction injury \[[@B18],[@B19]\]. Additionally, NGF increases capsaicin (CAP) sensitivity both in vivo and in vitro \[[@B20]-[@B24]\]. In accordance with this increased CAP sensitivity, NGF increases the expression of the CAP- and noxious heat-sensitive ion channel vanilloid receptor type 1 (TRPV1) through the ras/p38 MAP kinase signaling pathway, thereby promoting thermal hyperalgesia \[[@B25],[@B26]\]. Additionally, NGF has recently been shown to modulate TRPV1 by releasing the receptor from phosphotidylinositol-4,5-bisphosphate-mediated inhibition \[[@B27]\]. GDNF also plays a role in modulating nociceptive processing, but with contrasting in vitro and in vivo effects. In cultured DRG neurons, GDNF increases CAP sensitivity and TRPV1 expression \[[@B16],[@B26]\] and increases neuropeptide content \[[@B16]\]. On the other hand, intrathecal injection of GDNF has no effect on C-fiber evoked outflow of SP and does not induce thermal hyperalgesia, while NGF does both \[[@B15]\]. This, coupled with the observation that GDNF-overexpressing mice do not develop thermal or mechanical hypersensitivity \[[@B28]\], suggests that GDNF might not lead to nociceptor sensitization in vivo. In fact, in nerve injured rats, GDNF has antihyperalgesic effects \[[@B29]\] and promotes the functional regeneration of DRG-spinal cord connections \[[@B30]\]. It is not understood how this dissociation of the in vitro and in vivo effects of GDNF is manifested. Inflammation and nerve injury both increase BDNF content in DRG neurons and in the spinal cord, and this increase in BDNF is associated with the maintenance of a hyperalgesic state \[[@B31]-[@B36]\]. Furthermore, BDNF is released in the spinal cord upon noxious afferent stimulation \[[@B37],[@B38]\], and peripheral CAP application increases BDNF release at central terminals of sensory neurons \[[@B39]\]. Hence, BDNF might act as a neurotransmitter in the pain pathway in adult animals \[[@B40]\]. The trophic properties of BDNF on adult sensory neurons, particularly nociceptors, are poorly understood. In addition to CAP, a number of other TRPV1 agonists have been described. Among these are compounds that are also cannabinoid receptor agonists, including the endogenous cannabinoid anandamide (AEA, \[[@B41],[@B42]\] and the synthetic AEA analogue arachidonyl-2-chloroethylamide (ACEA, \[[@B43]\]). AEA causes antinociception in vivo through central \[[@B44]\] and peripheral mechanisms \[[@B45],[@B46]\]. It is not known, though, whether the endogenous production of AEA can reach sufficient concentrations under pathophysiological states to act as an endogenous activator of TRPV1. One potential mechanism whereby growth factors could contribute to the development of nociceptor sensitization is through the modulation of the efficacy or potency of AEA at TRPV1.Thus, growth factors might unmask conditions under which AEA could be capable of functioning as an endogenously produced TRPV1 agonist, potentially leading to neurogenic inflammation and thermal hyperalgesia. The aim of the present work was to gain a more precise understanding of NGF-, GDNF- and BDNF-dependent alterations of cultured TG sensory neuron survival and phenotype, with attention to markers and functions that are related to nociception. Furthermore, by relating these findings to neurosecretion associated with pharmacological stimulation of TRPV1, we hope to present a rationale for differences in how these neurotrophins might contribute to altered nociception at the level of the TG sensory neuron. Moreover, there is a gap in knowledge concerning neurotrophic factor influence over TG neurons, as the vast majority of our understanding of how neurotrophins alter sensory neurons stems from studies of the DRG. The recent advances of CGRP receptor antagonists for the treatment of migraine \[[@B47],[@B48]\] raises the possibility that manipulations which influence CGRP expression and/or secretion might be beneficial in the treatment of migraine or conditions related to the cerebral vasculature. A fuller understanding of the impact of NGF, GDNF and BDNF on TG neurochemical properties has the potential to lead to novel therapeutic strategies concerning disease states involving neuropeptide secretion in the trigeminal system, such as migraine. Results ======= Effects of NGF, GDNF or BDNF treatment on neuronal survival in vitro -------------------------------------------------------------------- TG neuronal cultures were plated at equal densities (\~5000 neurons / well) on 8-well culture slides (experimental design shown in Figure [1](#F1){ref-type="fig"}). After five days of growth factor treatment, immunocytochemistry (ICC) for 200 kD neurofilament (NF-H) present in all sensory neurons \[[@B49]\] and not other ganglion cells (i.e., glia), was performed to determine the number of neurons in each well. Representative photomicrographs are shown in Figure [2](#F2){ref-type="fig"}. NGF did not significantly influence the number of neurons in culture following five days of treatment (Fig [3](#F3){ref-type="fig"}). On the other hand, GDNF significantly enhanced the number of neurons per well versus no growth factor treated cultures at both 1 and 100 ng/ml (Fig. [3](#F3){ref-type="fig"}). The enhanced neuronal survival seen with GDNF was biphasic, as 10 ng/ml GDNF did not significantly enhance neuronal survival. It should be noted that in GDNF-treated cultures we observed a robust sprouting of TG neurons, appearing far more robust than in NGF- or BDNF-treated cultures (although sprouting was still evident in these cultures). Effects of NGF, GDNF or BDNF treatment on TRPV1 mRNA and protein levels ----------------------------------------------------------------------- To directly assess changes in TRPV1 mRNA levels with growth factor treatment, quantitative, realtime PCR was conducted. NGF, GDNF or BDNF (100 ng/ml) did not significantly alter TRPV1 mRNA levels after 5 days of treatment (Fig. [4](#F4){ref-type="fig"}). To evaluate whether growth factor treatment might exert post-transcriptional regulation of TRPV1 gene expression, we next examined their effects on TRPV1 protein levels by Western blot. As shown in Fig. [5](#F5){ref-type="fig"}, a major band was detected with the TRPV1 antibody at \~130 kD, consistent with the glycosylated form of TRPV1 \[[@B25]\]. NGF and GDNF (100 ng/ml) each was demonstrated to have increased TRPV1 protein levels by approximately 50% compared with no growth factor-treated cultures (Fig [5](#F5){ref-type="fig"}). BDNF effects on TRPV1 protein levels were not assessed, because preliminary studies indicated that BDNF did not influence CAP-evoked CGRP release. Effects of NGF, GDNF or BDNF treatment on K^+^-evoked CGRP release and CGRP content ----------------------------------------------------------------------------------- We next examined the effect of growth factor treatment on K^+^-evoked CGRP release and total CGRP content in TG neuronal cultures. NGF treatment significantly and concentration-dependently increased 50 mM K^+^-evoked CGRP release at every concentration, peaking (10.5-fold increase vs. control) at 100 ng/ml (Fig. [6A](#F6){ref-type="fig"}). Similarly, GDNF significantly and concentration-dependently increased K^+^- evoked release at every concentration, again peaking (9.7-fold increase vs. control) at 100 ng/ml. On the other hand, BDNF did not alter K -evoked CGRP release. Because there were differences in neuron numbers between growth factor conditions, we examined under each condition the proportion of neurons that were positive for CGRP, TRPV1 or IB~4~. Representative photomicrographs for each of these conditions are shown in Figure [7](#F7){ref-type="fig"}. The proportion of neurons positive for CGRP, TRPV1 or IB~4~are shown in Table [1](#T1){ref-type="table"}. In no cases were CGRP- or TRPV1-immunoreactive or IB~4~-binding neurons not likewise immunoreactive for NF-H. We then utilized the proportion of CGRP-expressing neurons under NGF, GDNF or BDNF treatment conditions to normalize K^+^- evoked CGRP release to the number of CGRP-immunoreactive neurons under each condition, respectively (see Methodsfor normalization calculation). Following this transformation, NGF treatment still augmented K^+^-evoked CGRP release at every concentration tested, with the magnitude of the effect reaching 18 times that of control (Fig [6B](#F6){ref-type="fig"}). While GDNF treatment again enhanced K^+^-evoked CGRP release at all concentrations following normalization, its peak effect was relatively lower. BDNF treatment continued not to have an effect following normalization to the number of CGRP-immunoreactive neurons. Much like K^+^-evoked CGRP release, total CGRP content was increased significantly by NGF and GDNF treatment, while BDNF had no effect (Fig. [6C](#F6){ref-type="fig"}). Both NGF and GDNF increased CGRP content concentration-dependently at every concentration (p \< 0.001), with peaks at 10 ng/ml (3.2-fold increase) for NGF and 100 ng/ml (3.0-fold increase) for GDNF (Fig. [6C](#F6){ref-type="fig"}). Again, we normalized these data to CGRP-immunoreactive neurons to assess changes in relation to alterations in CGRP-immunoreactive neurons between the growth factor conditions. Interestingly, the GDNF augmentation of CGRP release appeared to be due primarily to the large increases in neuron survival, because only the 10 ng/ml GDNF condition was significantly greater than control (p \< 0.001), but still substantial lower compared with the untransformed lysis figures (Fig. [6D](#F6){ref-type="fig"}). On the other hand, normalization led to a further augmention in the effect of NGF on total CGRP content, up to a 5.8-fold increase. BDNF treatment did not alter the normalized CGRP content. Effects of NGF, GDNF or BDNF treatment on CAP-evoked CGRP release ----------------------------------------------------------------- To assess the effect of growth factor supplementation on the neurosecretory function of nociceptors in culture, we evaluated CAP-evoked CGRP release. Treatment with 100 nM CAP for 10 min led to a significant enhancement of CGRP release over baseline under all conditions. Treatment of cultures with NGF or GDNF resulted in a concentration-dependent enhancement of this effect, with significant increases in CAP-evoked CGRP release at 1, 10 and 100 ng/ml. BDNF also enhanced the ability of CAP to evoke CGRP release; however, the effect was only seen at the 100 ng/ml concentration. In every case, GDNF and NGF (except 1 ng/ml NGF) supplementation significantly enhanced CAP-evoked CGRP release over the same concentration of BDNF (Fig [8A](#F8){ref-type="fig"}). To determine to what extent the enhancement of CAP-evoked CGRP release in response to growth factor treatment might derive from increased neuronal survival and/or up-regulation in the proportion of neurons expressing TRPV1, release data were normalized to the number of TRPV1-immunoreactive neurons (Fig. [8B](#F8){ref-type="fig"}; TRPV1 neuron proportions shown in Table [1](#T1){ref-type="table"}). In this case, NGF treatment still significantly increased CAP-evoked CGRP release over no growth factor treatment at 1, 10 and 100 ng/ml. However, this normalization procedure led to a relative reduction in the GDNF effect on CAP-evoked CGRP release, such that only the 10 ng/ml condition reached a significant increase in CGRP outflow. After normalization, BDNF treatment had no significant effect on CAP-evoked CGRP release at 1 or 100 ng/ml and actually reduced the amount of release at 10 ng/ml. Moreover, following normalization, NGF-treated neurons displayed a significantly higher CGRP release than either GDNF- or BDNF-treated neurons at every concentration of growth factor tested (Fig [8B](#F8){ref-type="fig"}). Because treatment with either GDNF or NGF induced a large increase in 100 nM CAP-evoked CGRP release, we assessed the effect of supplementation with these growth factors on the receptor pharmacodynamics of this response compared with no growth factor supplementation. GDNF or NGF treatment significantly increased the Hill slope of the concentration-response function from 1.12 ± 0.47 in the no growth factor condition to 3.760 ± 0.057 for 100 ng/ml NGF and to 4.48 ± 0.90 for 100 ng/ml GDNF (Fig [9A](#F9){ref-type="fig"}) but had no effect on the EC~50~(41 nM for GDNF or NGF and 47 nM for no growth factor). GDNF (100 ng/ml) or NGF (100 ng/ml) treatment caused a significant, five-fold increase in the E~max~of the CAP concentration-response curve (Fig. [9B](#F9){ref-type="fig"}). A common feature of CAP concentration-response functions is that they frequently exhibit an inverted U-shape, likely attributable to TRPV1 desensitization at higher concentrations. Thus, whereas the concentration-response function in the absence of growth factor displayed desensitization only at 1 uM, that for either GDNF- and NGF-treated TG cultures showed desensitization of the CAP-evoked CGRP response at concentrations above 100 nM (Fig. [9B](#F9){ref-type="fig"}). Effects of NGF, GDNF or BDNF treatment on AEA- and ACEA-evoked CGRP release --------------------------------------------------------------------------- To test whether growth factor-mediated enhancement of neuropeptide release might be generalizable to other TRPV1 secretagogues, we evaluated the dual cannabinoid-vanilloid agonists AEA and ACEA. As in the case with CAP, the effects of AEA and ACEA on evoked CGRP release were augmented by supplementation of TG cultures with NGF (Fig. [10A](#F10){ref-type="fig"}) or GDNF (Fig. [10B](#F10){ref-type="fig"}). While TG neurons not treated with growth factors were largely unresponsive to AEA or ACEA, in terms of CGRP release, treatment of cultures with either NGF or GDNF for 5 days at 1, 10 or 100 ng/ml caused a concentration-dependent increase in CGRP release evoked by 30 μM AEA or ACEA. Furthermore, the estimated EC~50~values for each of the individual growth factors to increase evoked CGRP release were equivalent for CAP, AEA and ACEA (NGF = 3.5 ng/ml; GDNF = 1.3 ng/ml). In contrast, BDNF supplementation did not augment AEA- or ACEA-evoked CGRP release, while a small, yet significant, increase in CAP-evoked CGRP release was observed with increasing BDNF concentration (Fig. [10C](#F10){ref-type="fig"}). When TG neurons were supplemented with either 100 ng/ml NGF or GDNF, the E~max~and EC~50~values for AEA did not change (F = 2.082~(4,85)~) ; however, when compared with non-growth factor-treated TG neurons, the E~max~was significantly augmented (Fig. [10D](#F10){ref-type="fig"}). Discussion ========== This study demonstrates that NGF, GDNF and BDNF differentially influence neuronal survival, neuropeptide content and stimulated secretion of TG neurons in culture. GDNF significantly augments TG neuronal survival, while both NGF and GDNF modulate CAP sensitivity, and alter the pharmacodynamics of the concentration-response function for CAP-evoked CGRP release. Furthermore, NGF and GDNF increased the releasable pool and total content of CGRP while increasing TRPV1 protein, without increasing its mRNA. These data support the hypothesis that GDNF and NGF, but not BDNF, alter CAP sensitivity in cultured TG neurons, and taken together, suggest that the differential effects of NGF and GDNF in vitro may reflect their differential effects in vivo, particularly with regard to TRPV1-mediated nociception. GDNF, but not NGF or BDNF significantly increased the number of neurons present in TG culture 5 days post-plating. The finding that this effect of GDNF was biphasic, as the 10 ng/ml concentration did not enhance the number of neurons in TG culture, suggests the possible involvement of different receptors with different concentrations of GDNF. Multiple receptors exist in the GDNF receptor family, and they function in concert with the protein c-ret. GDNF binds most readily to GFR alpha-1, but also binds to GFR alpha-2 / c-ret heterodimers \[[@B50]\]. GFR alpha-1 and -2 are expressed in sensory ganglia and are found mostly in IB~4~-binding neurons \[[@B51]\] that also contain c-ret \[[@B8]\]. Notably, it has been suggested that GFR alpha-1 and GFR alpha-2-containing neurons in the adult rat make up two distinct populations in the DRG within the IB~4~-binding class \[[@B8]\]; hence, the effects observed here may be due to GDNF acting through its high affinity GFR alpha-1 site at the 1 ng/ml concentration and through either or both GFR alpha-1 and GFR alpha-2 / c-ret, for which it has a lower affinity, at the 100 ng/ml concentration. While the observation here that GDNF promotes survival is a novel finding for rat TG sensory neurons in primary culture, the neuroprotective effects of GDNF are well documented. GDNF is protective against the loss of dopaminergic neurons in animal models of Parkinson\'s disease \[[@B52],[@B53]\], and GDNF reduces the number of apoptotic bodies in DRG explants from adult mice \[[@B54]\]. GDNF also rescues the reduction of P2X3 expression that occurs in the DRG following axotomy \[[@B55]\]. Although BDNF and NGF did not significantly increase the number of TG neurons in culture, we did observe a significant, linear trend for increased neuron survival as a function of increasing BDNF concentrations, an observation not present in NGF-treated TG cultures. Hence, BDNF and GDNF both promoted the survival of TG neurons in vitro. Primary cultures generated here were grown in the presence of mitotic inhibitors, which greatly reduce the supportive glial cells and the trophic factors they normally provide TG neurons in the native ganglia (although some of these cells are still present). Removal of astroglial support promotes apoptosis in cerebral neuronal cultures, an effect which is reversed by addition of GDNF or BDNF, but not NGF, to the culture medium \[[@B56]\]. The addition of GDNF or BDNF to the culture medium here may have re-supplied, at least partially, the withdrawn glial-supplied trophic factors that maintain sensory neurons in vivo, thereby increasing the number of neurons in culture at 5 days. It should be noted, however, that despite the significant effects of the growth factors observed in this study on neuronal survival, from an original density of nearly 5,000 neurons / well in the original culture homogenate, only about 10% of neurons could be counted at 5 days post-platting, in the control condition. A maximum of just over 20% were counted in the 100 ng/ml GDNF treated TG cultures. Therefore, it stands to reason that GDNF and BDNF are supporting the survival of certain classes of sensory neurons that are not supported either without growth factors or with NGF alone. The proportions of TG neurons in culture that expressed CGRP- or TRPV1-immunoreactivity or IB~4~-binding sites were assessed with attention to how these proportions changed in the presence of NGF, GDNF and BDNF. Notably, the proportion of sensory neurons in vitro that expressed CGRP (\~65%) was much larger than the known proportion of CGRP-containing neurons in native TG (\~35%, \[[@B57]\]). This likely indicates that the culturing process conditions are either selectively preserving peptidergic neurons or that normally non-peptidergic neurons novelly express CGRP in culture. On the other hand, discrepancies on reports of the percentage of IB~4~-binding neurons in native TG, ranging from \~35--60% \[[@B58],[@B59]\] make it difficult to assess whether there is an increased proportion of IB~4~-binding neurons in culture; however, our data indicate that this proportion is at least in the upper range, if not greater than native TG. Furthermore, a probable significant overlap exists between CGRP-immunoreactive and IB~4~-binding neuronal populations observed here, as both were found in the majority of TG neurons in culture, in agreement with the demonstration that these populations overlap significantly in native, adult rat TG and DRG (Price and Flores, unpublished observations). We observed that a greater proportion of TRPV1-immunoreactive neurons were present with GDNF supplementation (nearly 30% increase), again indicating either that GDNF preferentially supports the survival of TRPV1-expressing sensory neurons or that neurons that do not normally express TRPV1 begin expressing TRPV1 when GDNF is included in the culture medium. The latter proposition is supported by the finding that peripheral treatment with anti-GDNF antibodies suppresses the novel expression of TRPV1 in IB~4~-binding neurons following peripheral inflammation \[[@B60]\]. The percentage of TRPV1-expressing neurons in TG cultures not treated with growth factors was essentially equivalent to the percentage in native TG \[[@B57]\]. This finding indicates that, not only does GDNF promote survival of TG neurons in culture, but also enriches for TRPV1-expressing neurons, suggesting that GDNF might be preferentially neuroprotective for sensory neurons in adult animals that express TRPV1. We have also illustrated that chronic application of NGF or GDNF, but not BDNF (except at high concentrations, and to a much lesser degree), increases CAP-evoked CGRP release from TG neurons in vitro. NGF and GDNF each increased TRPV1 protein, and both upregulated the CGRP content of TG neurons. Both the NGF- and GDNF-induced upregulation of TRPV1 appears to be translationally regulated, as neither of these growth factors altered TRPV1 mRNA levels. NGF, in the setting of inflammation, is known to increase TRPV1 protein, but not mRNA, through activation of the p38/MAP kinase pathway \[[@B25],[@B26]\], and NGF-mediated upregulation of TRPV1 is blocked by over-expression of dominant-negative ras \[[@B26]\]. Interestingly, the study by Bron et al. (2003) indicated that GDNF is also able to upregulate TRPV1 expression; although this conclusion was based on immunofluorescence and cobalt uptake assays, it is consistent with our direct demonstration of GDNF-induced upregulation of TRPV1 protein by Western blot. After normalization to the number of neurons that express TRPV1 and CGRP, we found that NGF had a much greater effect on CAP-evoked CGRP release and neuropeptide content, on a per cell basis, than did GDNF. On the other hand, GDNF increased the proportion of neurons in culture that express TRPV1, such that GDNF-maintained cultures contained a higher number of TRPV1-expressing neurons compared with NGF-maintained cultures. Hence, our findings suggest that NGF increases CAP responsiveness in individual cultured TG neurons. On the other hand, GDNF appears to increase the responsiveness of the in vitro population by altering the proportion of TRPV1 neurons and upregulating the releasable pool of CGRP, as evidenced by the persistent increase in 50 mM K^+^-evoked CGRP release after normalization. This difference in the ability of GDNF and NGF to enhance individual neuronal neuropeptide content and CAP responsiveness could partially explain why NGF induces hyperalgesia \[[@B10],[@B12],[@B19],[@B61]\] while GDNF does not \[[@B15],[@B28],[@B29]\]. Our findings suggest that while GDNF might play a role in maintaining CAP sensitivity in vitro, its effects in vivo might be of a preservative nature that prevents the development of pain exacerbation following experimental manipulation. Furthermore, NGF and GDNF might differentially/predominantly subserve two of the main physiologic processes following injury: hyperalgesia to protect the organism from further injury (NGF) and regeneration/repair to restore function (GDNF). While it has been shown, using a variety of dependent measures, that both NGF and GDNF increase CAP responsiveness \[[@B16],[@B17]\], this is the first demonstration that these growth factors qualitatively alter the pharmacodynamics of the neuronal response to CAP in TG neurons. The increase in the Hill slope of the CAP response following exposure to NGF or GDNF indicates that these growth factors induce positive cooperativity, possible at the level of TRPV1. While the mechanism underlying this effect is not known, it could involve an alteration in post-translational modifications and/or protein interactions of TRPV1 in response to NGF or GDNF. NGF or GDNF also decreased the concentration of CAP necessary to induce tachyphylaxsis compared with control cultures. These findings indicate that TRPV1-mediated sensory neuron desensitization might be a more efficacious therapeutic strategy in pathologies known to be associated with increased levels of NGF and/or GDNF. While we are unaware of any data linking GDNF with migraine, increased cerebrospinal fluid levels of NGF are associated with chronic headache \[[@B62]\]. Interestingly, a TRPV1 targeted approach has been utilized in a clinical trial for migraine treatment in which intranasal civamide (a TRPV1 agonist) was shown to be effective for acute treatment of migraine \[63\], indicating that TRPV1 agonists might be employed in this condition, possibly to desensitize CGRP-containing TG nerve endings. Similarly to CAP-evoked CGRP release, the ability of AEA and ACEA to evoke release was concentration-dependently augmented by NGF or GDNF supplementation but was unaltered by BDNF. Moreover, the respective potencies of NGF and GDNF to enhance the CGRP release in response to CAP, AEA and ACEA were equivalent. This suggests that the pharmacology of CAP, AEA and ACEA at TRPV1, at least with respect to neuropeptide secretion, is similarly regulated in the presence of either of these growth factors. Insofar as NGF and GDNF have been implicated in the development of nociceptor sensitization in a number of pathological states, it should be considered that endocanninoids are apt to have enhanced TRPV1-mediated peripheral neuromodulatory effects under these conditions. Conclusions =========== Taken together, our results illustrate that NGF, GDNF and BDNF differentially alter sensory neuron survival, neurochemical properties and TRPV1-mediated neuropeptide release of TG neurons in culture. GDNF and, to a lesser extent, BDNF promote survival of TG neurons and GDNF enhances the proportion of neurons that exhibit TRPV1-immunoreactivity. GDNF or NGF enhanced CAP-, AEA- and ACEA-evoked CGRP release from TG neurons in vitro and increased TRPV1 protein, likely through translational regulation, and overall CGRP content. On the other hand, our findings suggest that GDNF and NGF differentially modulate TRPV1-mediated neuropeptide secretion sensitivity, with NGF having a much greater effect on a per neuron basis, providing a possible explanation for why NGF promotes thermal hypersensitivity in vivo while GDNF apparently does not. Although the present studies were conducted on cultured neurons under artificially controlled conditions, these findings contribute to a growing body of work concerning neurotrophin modulation of the properties of nociceptors. Thus the results described here have implications for advancing our understanding of how this system might be advantageously manipulated in a therapeutic setting especially as it concerns the TG system. Methods ======= Experimental chemicals ---------------------- Capsaicin (CAP, 8-methyl-N-vanillyl-trans-6-nonenamide) was from Fluka-Aldrich (St Louis, MO). AEA (N-(2-hydroxyethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide, in water soluble emulsion), and ACEA (N-(2-chloroethyl)-5Z,11Z,14Z)-eicosatetraenamide were from Tocris (Ellisville, MO). Recombinant rat GDNF and recombinant human (100% homologous to rat) BDNF were from Sigma (St Louis, MO). Rat NGF was from Harlan (Indianapolis, IN). All other chemicals were from Sigma, unless otherwise stated. TG culture ---------- Adult, male Sprague-Dawley rats weighing 250--300 g were used in this study. All procedures utilizing animals were approved by the Institutional Animal Care and Use Committee of The University of Texas Health Science Center at San Antonio and were conducted in accordance with policies for the ethical treatment of animals established by the National Institutes of Health. Animals were euthanized by decapitation and their TGs were rapidly dissected (\~30 s) and placed in ice-cold Ca^++^- and Mg^++^- free Hank\'s balanced salt solution (HBSS, Gibco, Carlsbad, CA). TGs were enzymaticaly digested for 30 min with 5 mg/ml collagenase followed by 25 min with 0.1% trypsin type IX supplemented for the last 10 min with 10 units of DNase I (Roche, Indianapolis, IN). TG cell suspensions were then centrifuged at 2000 RPM for 2 min, vortexed briefly and centrifuged again. They were then resuspended in basal culture medium containing high glucose Dulbeco\'s Modified Eagle\'s Medium (DMEM, Gibco), 1X pen-strep (Gibco), 1X glutamine (Gibco) and 3 μg/mL 5-FDU and 7 μg/mL uridine as mitotic inhibitors. TG cell suspensions were gently triturated with a Pasteur pipette followed by successive triturations through 19- and 23-gauge needles. TG cell suspensions were then transferred to a separate container and adjusted to the total volume needed for plating at a density of \~5000 neurons / well. The appropriate growth factors were added to this suspension prior to plating. Immunocytochemistry (ICC) and Image Aquisition ---------------------------------------------- TG cultures were first washed in PBS and fixed for 1 hr in 3.7% formaldehyde in PBS. Next, TG cultures were washed 3 times in PBS and permeabilized in PBS containing 10% normal goat serum (NGS, Gibco) and 0.2% Triton X-100 (Sigma) for 1 hr. Finally, TG cultures were blocked 3 × 10 min in PBS containing 10% NGS and then exposed to NF-H mouse-monoclonal antibody (1:300; Sigma) overnight at 4°C. Primary antisera were then washed off and goat anti-mouse Alexa-Fluor-488 (1:300, Molecular Probes, Eugene, OR), or goat anti-mouse Alexa-Fluor-594 (1:300) was applied for 1 hr at room temperature. For double-labeling either CGRP rabbit polyclonal (1:750, Peninsula Labs) or TRPV1 guinea pig polyclonal (1:3000, Neuromics) or IB~4~-conjugated to Alexa-Fluor-488 (1:1000, Molecular Probes) was then added overnight at 4°C. CGRP or TRPV1 antisera were followed by goat anti-rabbit Alexa-Fluor-594 (1:300) for 1 hr at room temperature. All images were acquired using a Nikon E600 microscope (Melville, NY) equipped with a Photometrics SenSys digital CCD camera (Roper Scientific, Tucson, AZ) connected to a computer equipped with Metamorph V4.1 image analysis software (Universal Image Corporation, Downingtown, PA). Twenty 20X images were taken of each well to capture the complete cellular area for neuron counts. For these experiments, all neurons displaying fluorescent signal above background were counted as positive for the specific marker. This was defined by scaling the image, using Metamorph\'s built-in scaling feature, to an average pixel value for negative neurons in the case of TRPV1, CGRP and IB~4~(no scaling was performed for NF-H ICC) and establishing all neurons as positive that were above that threshold. For double labeling experiments, the thresholded images were then overlaid and analyzed for the presence of both signals to assess colocalization. Neuronal Counting (Survival) ---------------------------- TG cell suspensions (\~5000 neurons/well) were added to 8-well poly-D-lysine Lab-Tek II chamber slides (Nalge/Nunc, Naperville, IL). Neuron density for plating was determined by counting neurons with a hemacytometer with Neubauer rulings and the cultures for each slide were generated independently. Neurons were easily distinguished from other cell types for plating density measurements due to their large size and opaqueness. Medium was changed after 24 hr and 72 hr, and on day 5, ICC was performed (as described above). In total, 12 chamber slides were utilized. The experimental design is shown in figure [1](#F1){ref-type="fig"} with 4 slides each for NGF, GDNF and BDNF. Neurons on the NF-H alone slides were not counted as these slides were used only for images shown in figure [2](#F2){ref-type="fig"}. To assess neuronal survival, all NF-H- immunoreactive neurons were counted for each growth factor concentration and are presented as mean ± SEM. The 2 matching wells for each slide were averaged and this average was used for 1 observation (as described in Fig [1](#F1){ref-type="fig"}). For colocalization studies, the previously generated NF-H-immunoreactive neuron counts for each well were followed by counting of CGRP- or TRPV1-immunoreactive or IB~4~-binding neurons in the same wells to generate the proportion of neurons expressing these markers. The total for the 2 matching wells were summed to yield the final proportion. At least 500 NF-H-immunoreactive neurons were counted under every growth factor condition to calculate proportions of neurons expressing the three markers examined. In no cases were CGRP-, TRPV1- or IB~4~-binding neurons not likewise positive for NF-H. Colocalization is presented only as an overall percentage. Realtime PCR ------------ TG cultures were prepared on 48-well poly-D-lysine pre-coated plates (Becton Dickinson, Franklin Lakes, NJ) at an initial density of \~5000 neurons/well. For each 48 well plate 12 wells received no growth factor, 12 received 100 ng/ml NGF, 12 received 100 ng/ml GDNF and the remaining 12 received 100 ng/ml BDNF. A total of 3 plates were utilized and each culture plate was generated independently. Following 5 days of culture, RNA was extracted from TG cultures using a ToTALLY RNA (Ambion, Austin, TX) total RNA extraction kit and each of the 12 wells per condition were pooled together to yield sufficient RNA. RNA samples were subsequently treated with DNA-free DNAse (Ambion) for removal of trace amounts of DNA. RNA concentrations were determined by UV absorbance, and RNA samples were then diluted to equal concentrations in TE buffer. For realtime PCR assessment of TRPV1 mRNA levels, samples were loaded in triplicate in 96-well reaction plates with each sample containing 150 ng RNA, 2X RT-PCR Taqman Master Mix, 40X Multiscribe and RNase Inhibitor solution, forward primer (tcc agt caa gcc cca cat c), reverse primer (tcc gag tca ccc ttc cca) and Taqman probe (6FAM tca cta cca gga gtc gta ccc ggc ttt TAMRA) (all 300 nM, all reagents Applied Biosystems, Foster City, CA) in a final reaction volume of 50 μL. Controls were run concomitantly using the same reaction recipe with a rodent GAPDH control kit (Applied Biosystems), primers and probe at 50 nM. Reactions were run on an ABI Prism 7700 (Applied Biosystems) with an initial RT step of 48°C for 30 min followed by a 95°C 10 min denaturation step and then a repeating denaturation, extension cycle of 95°C for 15 s and 60°C for 1 min for 55 cycles in order to reach a full plateau for all samples. All data were normalized to GAPDH mRNA levels to account for any variation in RNA concentrations between samples. Western blotting ---------------- TG cultures were prepared on 48-well poly-D-lysine pre-coated plates at an initial density of \~5000 neurons/well. For each 48 well plate 12 wells received no growth factor, 12 received 100 ng/ml NGF, 12 received 100 ng/ml GDNF and the remaining 12 received 100 ng/ml BDNF. A total of 3 plates were utilized and each culture was generated independently. After five days, total protein was extracted by first lysing cells with lysis buffer (1 mM Na pyrophosphate, 50 mM HEPES, 1% Triton X-100, 50 mM NaCl, 50 mM NaF, 5 mM EDTA and 1 mM Na orthovanadate) supplemented with 1% protease inhibitor cocktail and then homogenizing the combined lysate from the 12 wells per condition by pumping it through a 25 gauge needle 20 times. Extracted proteins were cleared of nuclei and cellular debris by spinning the homogenate at 1000 × g for 5 min at 4°C. Protein levels were then measured by the Bradford method. Proteins were run at a concentration of 20 μg/lane on a 12.5% SDS-PAGE gel and transferred to Immobilon -- P membranes (Millipore). Membranes were blocked in 5% dry milk for 1 hr and then exposed to rabbit anti-TRPV1 antibody (Neuromics, Minneapolis MN), at a concentration of 1:1000, overnight at 4°C. Membranes were then incubated with donkey anti-rabbit horseradish peroxidase-linked secondary antibody (Amersham, 1:5000) for 1 hr followed by ECL Western blotting detection for 1 min (Amersham). Blots were next exposed to film and subsequently scanned on a flatbed scanner. To control for protein loading, membranes were then stripped and reblotted for β-actin. Images were assessed for changes in TRPV1 protein using NIH image and normalized to β-actin protein levels. CGRP release and CGRP content ----------------------------- All experiments were performed in 48-well poly-D-lysine pre-coated plates. Data shown are representative of at least 3 independently conducted CGRP release experiments with consistent results and neurons were plated at the same density as indicated for survival, ICC, realtime PCR and Western blotting experiments (\~5000 neurons/well). Culture medium was changed at 24 and 72 hr, and all CGRP assays were performed on day 5. TG cultures were washed free of culture medium by 2 successive washes with release buffer (Hank\'s balanced salt solution (Gibco) supplemented with 10.9 mM HEPES, 4.2 mM sodium bicarbonate, 10 mM dextrose and 0.1% bovine serum albumin (BSA), pH 7.4). Growth factors were not included in the release buffer. Following washing, TG cultures were exposed for 10 min to the indicated concentrations of CAP, AEA or ACEA or to 50 mM K^+^buffer (containing 2.5 mM CaCl~2~, 50 mM KCl, 1.2 mM MgCl~2~, 90 mM NaCl, 25 mM NaHCO~3~, 1 mM NaH~2~PO~4~, 10 mM dextrose, 15 mM Hepes, 16 uM thiophan and 0.1% BSA at pH = 7.4), after which the CGRP-containing supernatant was removed and transferred to glass culture tubes (Fisher). Content was assessed by hypotonic lysis with deionized H~2~O supplemented with 1% protease inhibitor cocktail (Sigma) for 30 min. CGRP release or content for each well was subsequently measured by radioimmunoassay. CGRP radioimmunoassay --------------------- Following culture release assays, individual aliquots of the superfusate (0.5 ml) were incubated with a C-terminally directed anti-CGRP antiserum (kindly donated by Dr Michael Iadarola, NIDCR, NIH, Bethesda, MD, USA). After 24 h, 100 μL of \[125^I^\]-CGRP~28--37~(approximately 20000--25000 cpm) and 50 μL of goat anti-rabbit antibody conjugated to ferric beads were added. Following another 24 h, bound peptide was separated from free peptide via immunomagnetic separation (PerSeptive Biosystems, Framingham, MA, USA). All incubations were carried out at 4°C. The minimum detection limit for this assay is approximately 1--2 fmol per tube, with 50% displacement occurring at 20--40 fmol per tube. To account for the possibility of any nonspecific effects on the RIA, all drugs used in the release experiments were included in separate standard curves for the purposes of data analysis. We did not observe any alterations in the standard curve for any of the compounds utilized in these studies Data analysis and statistics ---------------------------- All data are presented as mean ± SEM unless otherwise stated. When normalizations for CGRP release or content to neuron numbers for either CGRP or TRPV1-immunoreactive neurons were made the equation shown in figure [11](#F11){ref-type="fig"} was used. All data were analyzed using GraphPad Prism for Mac OSX (GraphPad, San Diego, CA). To assess statistical differences, data were analyzed by one-way ANOVA followed by Tukey\'s post-test, for multiple comparisons, or Dunnett\'s post-test to compare all groups to the control group. For differences between growth factors at the same concentrations differences were assessed by two-way ANOVA with Bonferroni post-test for between group comparisons. All nonlinear regressions were fit to a sigmoidal curve with variable slope. Abbreviations ============= ACEA: arachidonyl-2-chloroethylamide, AEA: anandamide, BDNF: brain-dervied neurotrphic factor, CAP: capsaicin, CGRP: calcitonin gene-related peptide, DRG: dorsal root ganglion, GDNF: glial cell-line derived neurotrphic factor, IB~4~: isolectin B~4~, ICC: immunocytochemistry, NGF: nerve growth factor, SP: substance P, TG: trigeminal ganglion, TRPV1, transient receptor potential receptor vanilloid type 1 Author\'s Contributions ======================= TJP performed and conceived (or participated in their conception) experiments in each section, analyzed all data and authored the manuscript. MDL performed immunocytochemistry and neuron counts. DCS conducted the intitial CAP-evoked CGRP release study. GOD assisted in conceiving the experiments and performing the pilot studies. NAJ conducted the Western blots. AP assisted in conducting the CGRP release experiments. AD assisted in real-time PCR experiments. AAT assisted in generating cultures and in conducting pilot studies for CAP-evoked CGRP release. KMH assisted in conceiving the experiments and gave critical readings of the manuscript. CMF supervised all studies and conceived the original experimental designs as well as critically editing the manuscript. Acknowledgments =============== This work was supported by National Institute of Drug Abuse grants DA06085 and DA11959. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Experimental design for neuronal counting. 12 slides were utilized in these experiments, with 4 slides for each growth factor. The antibodies used on each slide are shown to the right of the slide along with the wavelength for the corresponding secondary antibody. For the 2 matching wells for each slide (growth factor concentration), the neuron numbers were averaged to give one observation (since the wells were not derived from independent cultures). Each slide contained a no growth factor control (blue), hence n = 9 for this condition. All other growth factors at 1 (i.e. NGF, red), 10 (i.e. GDNF, green) or 100 ng/ml (i.e. BDNF, yellow) are n = 3. The same slides were then utilized to ascertain the proportions of neurons expressing CGRP- or TRPV1-immunoreactivity or IB~4~-binding, as described in Methods. This figure refers to Figures 2 and 3 and Table 1. ::: ![](1471-2202-6-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Representative 20X photomicrographs of growth factor-treated TG neurons. TG cultures were fixed and labeled for NF-H-immunoreactivity (green), and the 20X photomicrographs depicted here are representative of the 20 images taken per slide for each condition for neuron counting. The upper frame shows the no growth factor treated control with corresponding growth factor treatments shown for BDNF, GDNF and NGF at 1, 10 and 100 ng/ml concentrations. ::: ![](1471-2202-6-4-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### NGF, GDNF and BDNF and TG neuronal surival. All NF-H-immunoreactive neurons were counted for each growth factor-treated TG culture (n = 9 no growth factor; n = 3 all other conditions) and compared with the control (no growth factor-treated) TG cultures to assess neuronal survival at day 5 post-plating (\* p \< 0.05, \\\* p \< 0.001). ::: ![](1471-2202-6-4-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Assessment of TRPV1 mRNA levels by Realtime PCR. Levels of TRPV1 mRNA are depicted as fold change compared with no growth factor-treated cultures for each growth factor condition normalized to its corresponding GAPDH mRNA levels (n = 3). ::: ![](1471-2202-6-4-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Assessment of TRPV1 protein by Western Blot. Panel A, 20 μg total protein was electrophoretically seperated per lane and transferred to membranes that were subsequently probed with an anti-TRPV1 antibody and then reprobed for standardization with β-actin (X = no growth factor; N 100 = NGF 100 ng/ml; G 100 = GDNF 100 ng/ml). Image is of a representative Western blot. Immunoreactivity to a protein corresponding to the size of the glycosyated form of TRPV1 was detected at \~130 kD. Panel B depicts alterations in TRPV1 protein levels standardized to β-actin (\* p \< 0.05, n = 3) ::: ![](1471-2202-6-4-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### 50 mM K+ -evoked CGRP release and total CGRP content in TG cultures. Panel A illustrates the effect of growth factor treatment on 50 mM K+ -evoked CGRP release, while panel B shows the data standardized to the number of CGRP-positive neurons per condition as a proportion of the no growth factor-treated cultures. Panel C shows total CGRP content by growth factor treatment, and again, panel D shows this data standardized to CGRP-positive neurons, as stated above (\\\* p \< 0.001, n = 6). ::: ![](1471-2202-6-4-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Representative 20X photomicropgraphs of colocalization of sensory neurons markers with NF-H in growth factor-treated TG neurons. Immunoreactivity for CGRP and TRPV1 and staining for IB~4~-binding sites (red) was assessed following immunocytochemistry for NF-H (green) to assess the proportion of neurons expressing these population markers. Representative 20X photomicrographs of each growth factor at 100 ng/ml for CGRP (left), TRPV1 (middle) and IB~4~(right) are shown as well as control (no growth factor-treated) TG cultures (top panels). All cell bodies containing both NF-H-immunoreactivity and CGRP- or TRPV1-immunoreactivity or IB~4~-binding appear yellow from the overlay of the red with green. In no cases were neurons observed that contained CGRP- or TRPV1-immunoreactivity or IB~4~-binding without the co-presence of NF-H-immunoreactivity. ::: ![](1471-2202-6-4-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Growth factor treatment increases CAP (100 nM)-evoked CGRP release in TG cultures. Panel A illustrates the total amount of CGRP released by 10 min treatment with capsaicin for each growth factor. Panel B illustrates the same data standardized to the number of TRPV1-positive neurons under each growth factor condition (\\ p \< 0.01, \\\* p \< 0.01 for comparisons to no growth factor treated controls; \#\# p \< 0.01, \#\#\# p \< 0.001 for comparisons to NGF at the equivalent growth factor concentration by two-way ANOVA with Bonferroni post-test; n = 6). ::: ![](1471-2202-6-4-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### NGF and GDNF alter capsaicin-evoked CGRP release. Concentration-response functions (molar, log units) are shown for capsaicin-evoked CGRP release in TG cultures maintained in 100 ng/ml NGF, 100 ng/ml GNDF or no growth factor conditions (n = 6). Panel A illustrates the maximum relative effect of capsaicin under each condition matched to the maximum evoked-CGRP release for that given condition. Panel B depicts the CAP-evoked CGRP release data in terms of CGRP released in fmoles in the presence or absence of NGF or GDNF to illustrate the magnitude of increases in E~max~and the biphasic nature of the capsaicin-evoked CGRP concentration-response function. ::: ![](1471-2202-6-4-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### The effect of growth factor treatment on AEA- and ACEA-evoked CGRP release. TG neurons were grown in the presence of the indicated concentrations of NGF (panel A), GDNF (panel B) or BDNF (panel C) for five days and then exposed to 100 nM CAP, 30 μM AEA or 30 μM ACEA for 10 min, and the released CGRP was quantified (n = 12). Panel D, TG neurons were grown in the absence of growth factors or in the presence of 100 ng/ml NGF or GDNF for 5 days and exposed to the indicated concentrations of AEA (molar, log units) to assess CGRP release (\#\#\# p \< 0.001; GDNF and NGF vs. no growth factor; two-way ANOVA, Bonferroni post-test, n = 6). ::: ![](1471-2202-6-4-10) ::: ::: {#F11 .fig} Figure 11 ::: {.caption} ###### Equation used to normalize CGRP release. \"No GF\" refers to no growth factor controls and \"GF condition\" refers to any given growth factor treated condition ::: ![](1471-2202-6-4-11) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Percentage of neurons expressing markers. ::: X N1 N10 N100 G1 G10 G100 B1 B10 B100 ----------- ------- -------- --------- ---------- -------- --------- ---------- -------- --------- ---------- TRPV1 37.9 37.5 37.4 40.0 48.4 57.5 66.6 50.1 50.9 47.8 CGRP 67.1 61.4 61.0 54.3 64.6 74.4 76.0 65.0 68.7 63.5 IB~4~ 56.5 61.3 73.1 62.0 63.3 65.0 63.9 59.6 61.8 47.2 The percentage of total NF-H-positive neurons expressing TRPV1, CGRP or IB~4~is shown for each growth factor condition (X: no growth factor, N: NGF, G: GDNF, B: BDNF). :::	PubMed Central	2024-06-05T03:55:52.566587	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548274/", "journal": "BMC Neurosci. 2005 Jan 24; 6:4", "authors": [ { "first": "Theodore J", "last": "Price" }, { "first": "Michael D", "last": "Louria" }, { "first": "Damaries", "last": "Candelario-Soto" }, { "first": "Gregory O", "last": "Dussor" }, { "first": "Nathanial A", "last": "Jeske" }, { "first": "Amol M", "last": "Patwardhan" }, { "first": "Anibal", "last": "Diogenes" }, { "first": "Amanda A", "last": "Trott" }, { "first": "Kenneth M", "last": "Hargreaves" }, { "first": "Christopher M", "last": "Flores" } ] }
PMC548275	Background ========== Over recent years increased emphasis has been given to performance monitoring of NHS hospitals and various quality indicators based on analysis of routine administrative data have been devised\[[@B1]\]. The 2003 dataset of the Commission of Health Improvement indicators includes 28-day hospital readmission rates following any hospital admission, and also readmission rates following admission for stroke and hip fracture\[[@B2]\]. Although indicators are currently standardised for age and sex^1^, they are not adjusted for potential case-mix differentials in disease severity, co-morbidity and patient deprivation status. Unplanned hospital re-admissions may represent adverse events and could therefore indicate poor quality of care\[[@B3],[@B4]\]. The interpretation of variation in readmission rates between healthcare organisations is nevertheless complicated\[[@B5],[@B6]\]. Broadly a readmission could be due to healthcare factors (e.g. sub-optimal health and social care, either at hospital or within primary/social care structures), patient factors (e.g. poor treatment adherence), disease factors (e.g. natural disease progression), or a combination of all the above. Readmissions due to healthcare and patient factors could be assumed to be potentially avoidable. There is insufficient evidence about the proportion of hospital readmissions that could be judged to be due to healthcare factors, as estimates of the proportion of medical readmissions that are due to healthcare factors vary widely between 9 and 48%\[[@B4]\]. Although current NHS performance indicators use readmission rates at 28-days\[[@B1],[@B2]\], there is lack of consensus in the literature about the choice of optimal time interval, with different studies choosing different intervals, ranging from one week to one year\[[@B4]\]. Intuitively shorter time intervals are more likely to represent \"avoidable\" readmissions due to poor quality care, nevertheless this may be more applicable in the case of elective surgical admissions rather than emergency medical ones. Moreover, for chronic medical conditions (such as diabetes, chronic obstructive pulmonary disease and heart failure), even a \"delayed\" readmission may represent a failure of disease management due to deficiencies in healthcare quality\[[@B4]\]. It can be hypothesised that factors such as patient sex, length of stay, number of coded co-morbidities, method of admission (e.g. presentation to the Accidents and Emergency department or GP referral), as well as patient deprivation, independently influence the probability of hospital readmission. Lower socioeconomic status in particular is independently associated with increased risk for many adverse health outcomes\[[@B7]\], including hospital readmission due to conditions such as heart failure\[[@B8]\]. The increasing demand for emergency medical admissions\[[@B9]\] makes epidemiological studies of medical readmissions a priority. Additionally, emergency medical admissions constitute a sizeable proportion of all hospital admissions, and can therefore play an important role in the overall performance of NHS hospitals under the current set of indicators\[[@B1],[@B2]\]. We therefore conducted a study to examine the effect of several patient and disease factors on the risk of emergency medical readmission at various time intervals following an index emergency medical admission. Methods ======= Context ------- This work was carried out as part of the routine function of Stockport NHS Trust Clinical Effectiveness Unit, and in relation to work commissioned by the then \"Emergency Demand Management Group\" of the Stockport Primary Care Trust. The objective was to accurately describe the epidemiology of emergency medical readmissions so that demand management strategies (e.g. appropriate targeting of resources to patients with certain conditions, or certain types of presentation) could be informed. Setting ------- Stockport NHS Trust is a district general hospital in Greater Manchester, serving a reference population of about 300,000. About 85% of all patients emergency medical admissions are from Stockport, a population with a slightly lower, compared to the England and Wales average, Standardised Mortality Ratio from all causes (all ages) of 96 (95% CI 94--98)\[[@B10]\]. Data source, population and follow-up period -------------------------------------------- Hospital Episodes Statistics Data from April 1997 to September 2001 for Stockport NHS Trust were analysed and all emergency medical admissions in Stockport residents leading to live discharge were identified. An emergency medical admission was defined as an emergency hospital admission to any medical specialty in person over 18 years of age. Some persons had more than one emergency medical admission during the study period, and emergency medical admissions other than the index admission (defined as the chronologically first admission during the 4.5-year study period) were excluded. This was because not restricting analysis to index admissions would have meant that any deprivation gradients in readmission rates would have been confounded by deprivation gradients in index admissions, as previously described\[[@B11]\]. This is an important difference of the methodology used in this study in relation to the way the relevant performance indicators are presently calculated, including \"all\" (as opposed to index/first only) admissions in the denominator\[[@B12]\]. An emergency medical re-admission was defined as the first subsequent emergency medical admission during a follow-up period of either 28-days, or 3, 6 and 12 months respectively, following a first (index) emergency medical admission that led to a live discharge, and through the use of a single patient identifier. Observations were censored at the end of the chosen follow-up periods (as above) or at the time of intervening death unrelated to readmission to the study hospital. The latter was ascertained by data-linking to the Stockport Health Authority Public Health Mortality File produced by the Office for National Statistics. Measurement and definitions --------------------------- Index admission data were originally available on: sex; age; length of stay of index admission; International Classification of Diseases (ICD)-10 coded primary diagnosis; number (up to four) of coded co-morbidities; patient post code and admission method (referral by Accidents and Emergency Department, General Practitioner or other). Information on primary diagnosis was aggregated into five categorical groups comprising chronic obstructive pulmonary disease / asthma (ICD codes J44.0--45.9 respectively), heart failure (I50.0--50.9), acute coronary syndrome (I20.0 \[unstable angina\] and I21.0--9 \[acute MI\]), stroke (I60.0--I67.0) and all other conditions (all other codes). Length of stay was divided into quartiles (\<2, 2--5, 6--11 and \>11 days). Deprivation status was subsequently ascribed with an area-based measure, using the 1991 Census enumeration district (ED) of patient\'s post-code, and by the use of Townsend multiple deprivation index score. Four deprivation groups were defined, using quartiles of the range of the Townsend scores between Stockport EDs. Analysis -------- Kaplan-Meier readmission-free curves at 28 days, and 3, 6 and 12 months were constructed for each of the following variables: sex, age group, diagnostic group(defined as above), admission method, number of coded co-morbidities(0--4), length of stay group(quartile) and deprivation group(quartile). Statistical significance for each of the above variables was assessed by the log rank test. A series of proportional hazards models with follow-up at 28 days, 3, 6 and 12 months were subsequently constructed to examine the adjusted hazard (risk) ratio of emergency medical readmission. Each model included all variables found to be significant in the uni-variable analysis at the 0.05 probability level. The proportional hazards assumption was tested using the Schoenfeld residuals as per the stphtestcommand in STATA. This showed that a time varying co-variate should be included in all four models, in relation to the length of stayvariable (i.e. that an interaction term between length of stayand time of follow-up should be included), and this was hence included in the models. Additionally, for the number of coded co-morbidities and deprivation group variables, a test for trend was performed, entering the actual values as continuous variables. In this context, the test for trend value indicates the proportional change in the risk of readmission associated with one unit change in the exposure variable (i.e. number of coded co-morbidities, Townsend deprivation score). Results ======= There were 21,118 index emergency medical admissions corresponding to an equal number of patients leading to a live discharge during the study period, but primary diagnosis information was only available for 20,209 index emergency medical admissions (Table [1](#T1){ref-type="table"}). Cases without diagnostic information were excluded from further analysis. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Basic characteristics of index admissions in study participants (n = 20,209) ::: Variable Category n % -------------------------- ------------------------- ------- ------- Sex Male 9397 46.5 Female 10812 53.5 Age group \<60 8094 40.1 60--74 5526 27.3 75+ 6589 32.6 Diagnostic group Acute coronary syndrome 4283 21.2 COPD/asthma 1594 7.9 Heart failure 587 2.9 Stroke 575 2.8 All other diagnoses 13170 65.2 Length of Stay \<2 5666 28.0 2--5 5184 25.7 6--11 5174 25.6 \>11 4185 20.7 Deprivation Group Affluent 5057 25.0 2 5003 24.8 3 5113 25.3 Deprived 5015 24.8 Unknown 21 0.1 Admission method A&E 12604 62.4 GP referral 7113 35.2 Other 492 2.4 No of co-morbidities 0 2963 14.7 1 3796 18.8 2 3790 18.8 3 8801 43.5 4 859 4.3 ::: Uni-variable analysis --------------------- The proportion of patients readmitted at 28 days and 3, 6, 12 months progressively increased from 7.2% at 28-days to 23.3% at 12 months respectively (Table [2](#T2){ref-type="table"}). Male sex, older age group, length of stay, higher number of coded co-morbidities and any primary diagnosis other than the \"all other diagnoses\" category were significantly associated with higher readmission rates independently of duration of follow-up (Table [2](#T2){ref-type="table"}). Higher deprivation status was significantly associated with increased readmission rates in follow-up periods longer than three months, but not at 28 days (Table [2](#T2){ref-type="table"} and Figure [1](#F1){ref-type="fig"}). Admission method was not significantly associated with deprivation risk. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Proportion of patients readmitted (%) by patient subgroup and different periods of follow-up (log rank test p values from relevant Kaplan-Meier readmission-free curves) ::: 28 days p\* 3 months p\* 6 months p\* 12 months p\* ------------------------------------ --------------- ------------- --------- -------------- --------- -------------- --------- --------------- --------- Sex Men 7.7 0.017 13.5 0.009 18.3 0.003 24.1 0.01 Women 6.8 12.4 16.8 22.7 Age group \<60 5.4 \<0.001 8.8 \<0.001 11.8 \<0.001 15.8 \<0.001 60--74 8.2 15.1 19.7 26.4 \>75 8.5 15.8 22.3 29.6 Admission method A&E 6.9 0.07 12.7 0.2 17.4 0.79 23.3 0.93 GP referral 7.7 13.4 17.7 23.4 Other 7.8 11.6 17.6 23.9 Length of Stay \<2 5.9 \<0.001 8.6 \<0.001 11.2 \<0.001 14.7 \<0.001 2--5 6.5 10.9 15.5 21.4 6--11 8.0 15.6 21.2 28.0 \>11 8.8 17.4 22.3 31.0 Number of coded co-morbidities None 6.1 \<0.001 9.6 \<0.001 13.2 \<0.001 17.3 \<0.001 1 6.0 9.8 13.3 17.5 2 7.0 12.3 16.4 22.2 3 8.3 15.8 21.6 28.9 4 7.3 14.3 18.7 24.4 Diagnosis ACS 7.2 \<0.001 13.0 \<0.001 17.2 \<0.001 22.3 \<0.001 COPD/Asthma 8.7 15.7 22.1 30.1 Heart Failure 11.1 22.7 31.3 37.5 Stroke 4.5 9.6 14.6 23.0 All other 7.0 12.3 16.6 22.3 Deprivation Affluent 7.1 0.44 11.8 0.002 15.8 \<0.001 21.0 \<0.001 2 7.2 12.5 17.0 22.2 3 6.9 13.0 18.1 24.2 Deprived 7.7 14.3 19.2 25.9 *Total* *7.2* *12.9* *17.5* *23.3* \Log Rank test A&E: Accident and Emergency, GP: General Practitioner, ACS: Acute Coronary Syndrome, COPD: Chronic Obstructive Pulmonary Disease ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Kaplan-Meier readmission-free curves by deprivation group. ::: ![](1471-227X-5-1-1) ::: Multi-variable analysis ----------------------- Male sex, older age group, and primary diagnosis of heart failure and chronic obstructive pulmonary disease/asthma were significantly associated with increased readmission risk, independently of length of follow-up (Table [3](#T3){ref-type="table"}). With the \"all other diagnoses\" as the reference category, primary diagnosis of acute coronary syndrome was associated with a significantly increased risk of readmission at 3 and 6 months, but not at 28 days or 12 months. Independently of length of follow-up and all other variables, primary diagnosis of stroke was significantly associated with reduced readmissions risk compared with the \"all other diagnoses\" category as reference. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Hazard ratios (HR) by variable and follow-up length, with associated 95% confidence intervals ::: Variables in the Equation* 28 days HR (95% CI) 3 months HR (95% CI) 6 months HR (95% CI) 12 months HR (95% CI) ---------------------------------------- ------------------------- -------------------------- -------------------------- --------------------------- ------------ ------------------ ------------- ------------------ Female \- \- \- \- Male 1.17\\ (1.06 -- 1.30) 1.14\\\* (1.06 -- 1.23) 1.15\\\* (1.08 -- 1.23) 1.13\\\* (1.07 -- 1.20) Age \<60 \- \- \- \- Age 60--74 1.41\\\* (1.23 -- 1.62) 1.47\\\* (1.33 -- 1.64) 1.44\\\* (1.32 -- 1.58) 1.46\\\* (1.35 -- 1.58) Age \>75 1.45\\\* (1.26 -- 1.67) 1.45\\\* (1.30 -- 1.62) 1.56\\\* (1.42 -- 1.71) 1.57\\\* (1.45 -- 1.70) Affluent \- \- \- \- 2 1.01 (0.87 -- 1.17) 1.05 (0.94 -- 1.17) 1.07 (0.97 -- 1.18) 1.07 (0.98 -- 1.16) 3 0.98 (0.84 -- 1.13) 1.09 (0.98 -- 1.22) 1.15\\ (1.05 -- 1.27) 1.17\\\* (1.08 -- 1.27) Deprived 1.09 (0.94 -- 1.26) 1.21\\ (1.08 -- 1.35) 1.21\\\* (1.10 -- 1.33) 1.25\\\* (1.16 -- 1.36) Test for trend (Deprivation Index)\^ 1.02 (0.98 -- 1.07) 1.06\\\* (1.03 -- 1.10) 1.07\\\* (1.04 -- 1.10) 1.08\\\* (1.05 -- 1.11) All other diagnoses \- \- \- \- Heart Failure 1.32\* (1.02 -- 1.70) 1.43\\\* (1.19 -- 1.71) 1.47\\\* (1.26 -- 1.71) 1.33\\\* (1.16 -- 1.53) COPD/asthma 1.25\* (1.04 -- 1.50) 1.23\\ (1.08 -- 1.41) 1.29\\\* (1.15 -- 1.45) 1.34\\\* (1.21 -- 1.48) ACS 1.12 (0.97 -- 1.28) 1.15\\ (1.04 -- 1.27) 1.12\* (1.02 -- 1.22) 1.07 (0.99 -- 1.15) Stroke 0.54\\ (0.37 -- 0.81) 0.57\\\* (0.44 -- 0.75) 0.65\\\* (0.52 -- 0.81) 0.76\\ (0.63 -- 0.90) Without co-morbidity \- \- \- \- 1 co-morbidity 1.02 (0.83 -- 1.25) 1.04 (0.89 -- 1.23) 1.05 (0.91 -- 1.20) 1.10 (0.97 -- 1.24) 2 co-morbidities 1.13 (0.93 -- 1.38) 1.21\* (1.03 -- 1.41) 1.19\* (1.04 -- 1.36) 1.29\\\* (1.15 -- 1.45) 3 co-morbidities 1.25\* (1.04 -- 1.49) 1.39\\\* (1.21 -- 1.60) 1.41\\\* (1.25 -- 1.59) 1.54\\\* (1.38 -- 1.71) 4 co-morbidities 1.26 (0.93 -- 1.69) 1.46\\ (1.17 -- 1.82) 1.42\\\* (1.18 -- 1.73) 1.49\\\* (1.26 -- 1.76) Test for trend (number of com.)\^ 1.08\\ (1.03 -- 1.14) 1.11\\\* (1.08 -- 1.17) 1.13\\\* (1.09 -- 1.17) 1.15\\\* (1.12 -- 1.18) A&E referral \- \- \- \- GP referral 1.09 (0.98 -- 1.22) 1.00 (0.92 -- 1.09) 0.96 (0.89 -- 1.03) 0.93\* (0.88 -- 0.99) Other referral 1.17 (0.85 -- 1.60) 0.84 (0.64 -- 1.09) 0.91 (0.74 -- 1.13) 0.94 (0.78 -- 1.13) \<2 days LoS \- \- \- \- 2--5 days LoS 0.54\\\* (0.42 -- 0.69) 0.82\* (0.69 -- 0.99) 0.92 (0.79 -- 1.08) 1.06 (0.93 -- 1.21) 6--11 days LoS 0.52\\\* (0.41 -- 0.66) 0.89 (0.74 -- 1.05) 1.14 (0.98 -- 1.32) 1.31\\\* (1.15 -- 1.49) \>11 days LoS 0.54\\\* (0.41 -- 0.70) 0.95 (0.79 -- 1.14) 1.23\\ (1.06 -- 1.44) 1.42\\\* (1.25 -- 1.62) *\<2 days LoS \ Time\^\^** \- \- \- \- 2--5 days LoS \* Time\^\^ 1.07\\\* (1.05 -- 1.09 1.01\\\* (1.01 -- 1.02) 1.01\\\* (1.00 -- 1.01) 1.002\\\* (1.001 -- 1.003) 6--11 days LoS \* Time\^\^ 1.08\\\* (1.06 -- 1.11 1.02\\\* (1.01 -- 1.02) 1.01\\\* (1.00 -- 1.01) 1.002\\\* (1.001 -- 1.003) \>11 days LoS \* Time\^\^ 1.09\\\* (1.06 -- 1.11 1.02\\\* (1.02 -- 1.02) 1.01\\\* (1.00 -- 1.01) 1.002\\ (1.001 -- 1.002) \: p \< 0.05, \\: p \< 0.01, \\\: p \< 0.001 \^ : Denotes the proportion change in probability of outcome associated with one unit change in continuous variable (e.g. Townsend deprivation score index, number of co-morbidities) \^\^ In days HR: Hazard Ratio, COPD: Chronic obstructive airways disease, ACS: Acute coronary syndrome, LoS: Length of stay, A&E: Accident and Emergency, GP: General Practitioner. ::: More than two coded co-morbidities were associated with higher readmission risk only in follow-up periods of more than three months duration. However test for trend indicated a strong and significant positive effect of the number of coded co-morbidities independently of follow-up length. Higher deprivation status was independently associated with higher readmission risk at 3, 6 and 12 months, but did not significantly influence readmission risk short term (at 28 days). Test for trend confirmed the strong and statistically significant effect of deprivation at 3--12 months but there was no effect at 28 days. Admission method via a GP referral was significantly associated with a lower readmission risk at one year, but not at any other time intervals. Length of stay of the index admission influences readmission risk differently, depending on the length of follow-up as there was a highly significant interaction between length of stay group and time of follow-up (see [Additional file 1](#S1){ref-type="supplementary-material"}). Taking into account the relevant time varying co-variate, shorter length of stay is associated with higher readmission risk at discharge and immediately afterwards, but with lower readmission risk thereafter. Discussion ========== The findings indicate that in the study hospital about a quarter of patients with an index emergency medical admission will be readmitted in the same hospital during the subsequent year. Male sex and older age were strongly and independently associated with higher readmission risk, along with diagnosis of heart failure and chronic obstructive pulmonary disease or asthma. Effective measures to reduce readmission rates for patients suffering from these two conditions in particular are available\[[@B13],[@B14]\] but not always used\[[@B13],[@B15],[@B16]\]. Improving the availability of effective treatments for these two conditions could contribute greatly to the management of demand for emergency care in general. The present study, at the local health economy level, has helped support the decision making process that allocated increased resources to the management of these two conditions by the expansion or creation of relevant specialist services. As originally hypothesised, patient deprivation status exerted a significant independent effect on the risk of emergency medical readmission at 3--12 months of follow-up, with more deprived patients having had a higher readmission risk. There are several theoretical reasons why deprived patients may be at higher readmission risk, including: disease factors, such as greater disease severity in deprived patients\[[@B17]\]; patient factors, such as poor adherence to treatment and advice because of educational or behavioural reasons; and health and social care factors, such as differentials in the type and quality of primary care in particular, in a way analogous to the \"inverse care law\", originally describing differentials in access, rather than quality, of care\[[@B18]\]. A clearer understanding of the exact mechanisms responsible for deprivation group gradients through further research is necessary for future policy measures aiming at reducing such gradients. Although this is a single-centre study, the results may also have implications for the way current and future NHS performance indicators relating to readmission rates are both constructed and interpreted. NHS hospitals serving pre-dominantly deprived populations might in principle be disadvantaged if indicators are not adjusted for the impact of deprivation on case-mix. Although this study showed no significant effect of deprivation status on readmission risk at 28-days, which is the follow-up period currently used by the performance indicators\[[@B1],[@B2]\], care should be taken when interpreting this \"negative\" finding. Firstly, this analysis included in the denominator only index (as opposed to \"all\") admissions, in contrast to the technical specification of the performance indicators\[[@B12]\]. Because more deprived patients have higher rates of index emergency medical admissions\[[@B11]\], including all index admission in the calculation will accentuate any deprivation differences in readmission risk, and the performance indicators as they are currently calculated may for this reason be misleading. Previous analysis of the same dataset including \"all\" admissions provides empirical evidence that this is true\[[@B19]\]. Secondly, it is possible that a true effect of deprivation status on index readmission rates at 28-days also exists but it was not detected by our study due to its single-centre nature, or insufficient sample size. A larger study, ideally using data from more than one hospital may be warranted. The Department of Health includes performance indicators in the calculations of award of \"three-star\" status, which in turn is the \"gateway\" to \"Foundation\" status\"\[[@B20]\]. Standardising, or otherwise adjusting, for patient deprivation is feasible using the HES data, as this study indicates. Standardisation of readmission indicators for patient deprivation status would be prudent. This would ensure that NHS organisations serving deprived communities would not be unfairly \"punished\" for poor performance because of factors outside their control. It will also increase the perception of validity of the indicators. Unlike information on disease severity, which is difficult to measure accurately for most medical conditions, information about patient socioeconomic status using area-based (ecological) deprivation measures is relatively easy to obtain, using patient postcodes, routinely included in the Hospital Episodes Statistics dataset. All studies using administrative data are sensitive to the quality of routine data collection. The validity of HES data in relation to age, sex, length of stay, admission method, and area of residence is generally good, but misclassification errors may occur in relation to diagnostic codes and the extent to which co-morbidities are recorded and coded\[[@B21]\]. Currently the degree of miscoding in our data is uncertain, but an audit of 200 cases in the study hospital has shown diagnostic inaccuracy to be in the order of 7.5%, comparable with levels quoted in the published literature\[[@B22]\]. Misclassification of primary diagnosis might be assumed to have occurred non-differentially between patients of different deprivation groups, and is so it would have diminished rather than exaggerated any association observed in this study, including the observed effect of deprivation. Misclassification error may have also resulted by the use of ecological measures of socioeconomic status (ecological fallacy). Again, this would reduce the effect size, if one exists. Therefore the effect of deprivation status on readmissions risk reported in our study may be an under-estimate of a true association. A limitation of the study is that, besides the very large sample size, the findings are based on one single hospital in an urban English setting, and in principle the results are not generalisable. Similarly, the study was not population-based, so readmissions that may have occurred to other hospitals (either because of where patients happened to be taken if fallen acutely ill, or due to migration) were not ascertained. In theory such readmissions may have occurred at a differential rate between different deprivation groups. However this factor is unlikely to have biased the results in any considerable way for two reasons. First, the emergency (as opposed to elective) nature of the studied condition (emergency medical admission) makes it unlikely that either patients or doctors exercise an important degree of choice on which hospital a patient is admitted or readmitted, independently of patient deprivation status. Second, due to local geography and service configuration, 85% of the total medical admissions in Stockport residents occur at Stockport NHS (unpublished data). Similarly, by the nature of the hospital-centred nature of the study, admissions to private hospitals could not have been accounted. However, most admissions to private hospitals are for elective surgical procedures (rather than emergency medical reasons) for which we believe this is unlikely to have introduced considerable degree of bias. Lastly it is worth remembering that current NHS performance indicators for hospital Trusts are not population-based. Conclusions =========== Our study suggests that there is an important potential for both managing emergency demand and improving individual patient experience by focusing on the effective management of heart failure and chronic obstructive pulmonary disease. Although there is a similar potential by reducing differentials in readmission risk between deprivation groups, more research is required in order to understand reasons for such differentials in order to inform relevant policy measures. In the mean time, standardisation or other adjustment of hospital readmission indicators for patient socio-economic status in the future would be prudent. Failure to do so may disadvantage hospitals serving primarily deprived communities. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= The study was conceived and designed by GL and GC. GL, DH and IG analysed data. All authors contributed in the interpretation of findings and in the writing of the paper. GL and GC are guarantors. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-227X/5/1/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Length of stay group and readmission free Kaplan-Meier curves (0--28 days). This file demonstrates that during follow-up of 0--28 days, length of stay is not proportional to readmission risk, reason for which a time varying co-variate was included in the Cox regression model (see main article Text). ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We are grateful to Professor RF Heller for useful comments on the manuscript.	PubMed Central	2024-06-05T03:55:52.571760	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548275/", "journal": "BMC Emerg Med. 2005 Jan 21; 5:1", "authors": [ { "first": "Georgios", "last": "Lyratzopoulos" }, { "first": "Daniel", "last": "Havely" }, { "first": "Islay", "last": "Gemmell" }, { "first": "Gary A", "last": "Cook" } ] }
PMC548276	Background ========== Case-control research on the association between surgery and sporadic Creutzfeldt-Jakob Disease (CJD) encompasses six studies and one meta-analysis \[[@B1]-[@B8]\], which frequently yielded diverging, partly inconsistent, positive \[[@B5],[@B6]\] or negative \[[@B7],[@B8]\] results, potentially attributable to methodological difficulties. This paper constitutes the first part of methods development for a case-control study summarily described in the Methods/Design section. \"Development\" in this sense refers to theoretical development built on scientific evidence and will focus on classification of surgical procedures, using technical and biological criteria appropriate for identifying characteristics of the intervention reflecting the putative risk level of sporadic CJD transmission. Methods ======= In this section, we summarily describe the on-going study, review reports that possibly shed light on the biological plausibility of surgery-related CJD transmission, and propose principles for re-classification of surgical procedures. Study design ------------ The on-going case-control study, entitled \"Surgery and risk of Creutzfeldt-Jakob Disease\" (EUROSURGYCJD), constitutes a Concerted Action funded by the EU Research Commission, contract QLG3-CT-2002-81223. The main objective of this study is to quantify a putative excess risk of CJD associated with surgery. A secondary objective is to establish a basis for the design of preventive strategies. The most relevant methodological characteristics are: case-control design; exposure measurement prior to disease onset, registered as codes for surgical procedures; matched 5:1, randomly chosen population controls and random sample of population controls. The population base is the resident population in Denmark, Finland and Sweden, covered by the respective hospital in-patient registers. Cases are individuals with diagnoses corresponding to ICD-9 codes 046.1 and 331.5 and ICD-10 code A81.0 at death or at hospital discharge for the period 1987--2002, identified from the respective national hospital in-patient registers and corresponding national surveillance units. A questionnaire will be mailed to the heads of the registered hospital department or surveillance unit, and a copy of the medical record will be obtained for diagnosis validation. Approximately 300 patients will fulfil criteria for definite or probable sporadic CJD, and constitute the study cases. Population controls are: 1) 5 × 1 controls (approximately 1500), matched to the corresponding case by age, sex, county of residence of the case (at first discharge from hospital with CJD diagnosis or death if never hospitalised with CJD diagnosis); and 2) a 20/million sample of the 1987--2002 resident population aged \>40 years, randomly selected from the corresponding national population registers. Individual person-numbers will be used for each resident case or control. Diagnoses and surgical procedures at hospital discharge of the corresponding CJD case at any registered time before date of death will be obtained from the three national hospital discharge registers in Sweden, Denmark and Finland. Perusal of surgical records might be undertaken for selected associations in order to understand transmission mechanisms and interpret results. Open-care surgery or dentistry will not be studied. Surgical procedures coded in registers as per national or NOMESCO classifications will be re-classified in accordance with putative levels of transmission risk based on scientific evidence/plausibility. The quality of CJD diagnoses will be assessed by a review of medical records. The accuracy of surgical history given by the registers will be assessed by comparison with that of a sample of controls and surrogate respondents obtained by interview. Centralised data analyses will be conducted by the Spanish team. In specific instances, risk due to blood transfusion, whether or not performed during surgical procedures, might also be studied. Review of reports and proposal ------------------------------ Surgery may be a pathway for patient-to-patient transmission of sporadic Creutzfeldt-Jakob Disease (CJD). In many invasive surgical procedures, non-disposable surgical instruments come in contact with tissues that are known to be infective in CJD patients. These same instruments may retain a considerable level of infectivity after routine sterilisation, and in successive patients can come into contact with tissues that may act as entry sites for CJD transmission. Among the almost 300 recorded cases of iatrogenic transmission of CJD, 5 cases have been attributed to surgical instruments employed in neurosurgical procedures, whilst 2 additional cases were caused by the use of a contaminated intracerebral EEG electrode \[[@B9]\]. To date, proven surgical transmission of CJD has only been shown to have taken place through instruments contaminated with high-infectivity tissues (brain). However, stainless steel instruments exposed to infective tissue can acquire a maximum load of infectivity in a considerably short period of time (5 minutes) and are highly efficient in transmitting disease even after thorough washing \[[@B10]\]. The possibility of prion transmission through surgical interventions involving nervous or peripheral tissue has raised concern about decontaminating procedures, particularly after the emergence of variant CJD in the United Kingdom and several other countries \[[@B11]\]. In the above-mentioned studies \[[@B1]-[@B7]\], surgical procedures have been grouped and analysed according to gross anatomical regions (e.g., thyroid, gallbladder, prostate, etc.), which limits an interpretation of results based on biological inference. For a surgical instrument to act as a vehicle of prion transmission, it should come into contact with infective tissue during surgery of the \"donor\" (contaminating procedure), should maintain any adhered infectivity after being washed and sterilised, and, finally, should make contact with receptive tissues in the \"recipient\" patient (transmitting procedure). Different surgical interventions on the same organ may result in direct exposure of different tissues to surgical instruments, and may consequently involve a different risk of prion transmission. Within the context of a case-control study designed to address surgical risk in sporadic CJD in Nordic European countries (EUROSURGYCJD Project), we adopted the strategy of categorising all reported surgical procedures (putative transmitting procedures) in terms of potential risk of CJD acquisition. For this purpose, a classification of exposed tissues and anatomic structures has been drawn up on the basis of their specific putative role as entry site for prion transmission through surgical instruments. This classification can serve, both in our study and in further epidemiological studies, as a reference for a categorisation of surgical procedures in terms of risk of CJD acquisition. According to the \"protein only\" hypothesis \[[@B12]\], pathogenic prion protein (PrP^Sc^) is a conformational isoform of PrP^C^, a normal host protein present in neurons and other cell types. In sporadic and familial transmissible spongiform encephalopathies (TSEs), \"spontaneous\" conversion of PrP^C^into PrP^Sc^is the key pathogenic event, followed by the accumulation, deposition and further conversion of PrP^Sc^in tissues, together with its propagation along specific neural pathways. In the case of transmitted TSEs -when these are not due to direct inoculation into the CNS- a peripheral phase of neuroinvasion by PrP^Sc^is followed by a subsequent phase of prion replication and propagation along the peripheral nervous system, with final access to the central nervous system \[[@B13]\]. In scrapie, bovine spongiform encephalopathy (BSE) and variant CJD, neuroinvasion follows widespread deposition of PrP^Sc^in mucosae-associated lymphoid tissue. For the purpose of classifying tissues susceptible to prion inoculation, the following considerations can be derived from this pathogenic model: i) a tissue can act as entry site for prion transmission if it normally expresses PrP^C^; ii) the level of PrP^Sc^expression of an infected tissue correlates positively with the risk of prion acquisition by that tissue; and, iii) all tissues involved in the propagation chain of infection from peripheral tissues to the central nervous system can act as entry sites for prion transmission. The recently published WHO classification of tissue infectivity in TSEs \[[@B14]\], though aimed at public health issues radically different from those addressed in our study, may nonetheless serve as a conceptual framework for a tissue classification in terms of risk level of prion acquisition. This approach is based on our above-mentioned assumption (ii). The WHO classification groups tissues in three levels (high, lower and no detected infectivity) on the basis of bioassay infectivity data and/or detection of PrP^Sc^by Western blot. This three-level classification correlates quite closely with the distribution and levels of PrP^C^expression in normal nervous and non-nervous tissues in mammals.\[[@B15]\] The WHO tissue classification presents data on vCJD, other human TSEs, BSE and scrapie. Since no vCJD cases have been registered in Nordic countries, our epidemiological study must be limited to sporadic CJD. Consequently, our working classification excludes all tissues where positive data on infectivity or PrP^Sc^detection have been obtained exclusively in animal TSEs and/or vCJD. This is the case of the small bowel, large bowel (including enteric nerve plexuses), adrenal tissue, pancreas and bone marrow. Further relevant data for tissue classification derive from iatrogenic CJD cases and from experimental transmission of prion diseases to animals. In roughly half of iCJD cases the entry site has been the CNS or the eye (dura mater transplants, neurosurgical instruments or devices, corneal transplants), whilst in the other half, injection of pituitary hormones means that a peripheral route of entry has to be assumed \[[@B9]\]. Experimental efficiency of prion disease transmission to animals depends on various factors, such as the inoculum dose, the species barrier between the species of origin of the inoculum and the host, and the route of administration, among others. Under the same experimental conditions, different routes of administration show different efficacy of disease transmission, in terms of length of incubation period and percentage of infected animals \[[@B16],[@B17]\]. While the most efficient route of transmission is intracerebral administration, other routes, such as intraperitoneal, intraneural, intraocular, intravenous, subcutaneous and intramuscular administration, have been used successfully in bioassays and other experimental models \[[@B18]\]. Still other routes, such as oral administration and conjunctival instillation \[[@B19]\], have shown a lower efficiency of transmission. Accordingly, clinical and experimental evidence includes several routes of prion transmission that cannot be easily reduced to a simple tissue classification involving tissues of known infectivity in CJD and/or expression of PrP^C^under normal conditions. This is the case of anterior ophthalmic tissues, skeletal muscle, peritoneum, and subcutaneous tissue rich in sensitive nerve fibres. These anatomical structures have therefore been independently added to our classification as putative routes of entry, with a lower level of risk compared to the high level represented by the central nervous system, sensitive ganglia and posterior eye tissues. The fact that PrP^Sc^has been recently found in 1/4 skeletal muscle samples of sCJD cases\[[@B20]\] prompted us to classify it as a tissue for potential entry rather than a route. A final classification of entry sites for putative surgical transmission of CJD contains tissues, including all those showing positive results for sporadic and familial CJD in the WHO classification \[[@B14]\], with minor additions (tonsil and thymus), and maintains the three risk levels of the original classification along with several putative routes of entry, based on clinical and experimental evidence (see Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Proposed classification of entry sites for putative surgical transmission of CJD by risk level. ::: ------------------------------------------------------------------------------------------------------ Risk level Tissues Anatomical structures / routes ---------------- ----------------------- ------------------------------------------------------------- High Brain\ Spinal cord\ Retina, optic nerve\ Spinal ganglia\ Trigeminal ganglia\ Pituitary gland\ Dura mater^a^ Lower Peripheral nerves^b^\ Anterior ophthalmic\ Spleen\ Peritoneum\ Lymph nodes^c^\ Subcutaneous (high density of sensitive nerve terminals)^f^ Tonsil^d^\ Thymus^d^\ Placenta\ Lung\ Liver\ Kidney\ Blood vessels^e^\ Olfactory mucosa\ CSF\ Skeletal muscle Lowest Other Other ------------------------------------------------------------------------------------------------------ ^a^Dura mater does not contain pathological PrP in CJD patients and its infectivity has not been tested. It is included among high-infectivity tissues in the WHO classification because of evidence of iatrogenic transmission through dura mater grafts.^11^The same rationale has been applied to its inclusion in the present table. ^b^Only surgical procedures on peripheral nerves (e.g., amputation, vagotomy, etc.) will be classified according to this tissue. ^c^Surgical procedures that include this tissue as putative risk are those involving direct manipulation of lymph node chains, e.g., lymph node excision, oncological lymphadenectomy, and intra-abdominal procedures with extensive section of lymph node chains, such as cholecystectomy, gastrectomy and diverse types of bowel resection. ^d^Although tonsil and thymic tissue have yielded negative results for infectivity and presence of pathological PrP in sporadic CJD tissue (tonsillar tissue has not yet been tested for infectivity), they are included in the table in the lower level group, together with the spleen and lymph nodes for biological reasons, in order to assess the role of peripheral lymphoid tissue in surgical transmission. ^e^Only procedures involving direct surgery on blood vessels will be classified according to this tissue. ^f^Hand and facial subcutaneous tissue will be included under this heading. ::: Discussion ========== We propose a list intended to be used to generate attributes of single, well-defined surgical procedures and criteria for their classification in terms of putative risk level of transmission. The two main attributes will be: 1) use of non-disposable surgical instruments; and, 2) exposure during surgery of tissues included in the preceding list. High- and lower-risk surgical procedures will be defined by exposure of tissue corresponding to the respective risk level during surgery. In addition, the lowest risk level is represented by those surgical procedures where disposable surgical instruments are not employed or where no listed tissue or anatomical structure is exposed to surgical instruments. A categorisation of surgical procedures based on the above attributes is inevitably prone to some degree of subjectivity, something that should be minimised by adequate methodological assessment. However, this drawback is more than offset by the possible benefits of identifying specific surgical procedures that pose a significant risk of CJD transmission, in terms of increased study power and control of misclassification of exposure by the removal of surgical procedures, which are probably irrelevant for CJD transmission, from gross anatomical classifications of surgery. We are also aware of the fact that whereas the same surgical instruments are commonly employed in an homogeneous group of procedures in some countries, as is the case of Nordic countries, the same instruments may circulate through a much wider range of procedures and putative risk levels in other countries. Final risk for disease transmission in each surgical procedure combines postulated risk related to tissues exposed in that procedure with the highest risk level of tissues to which instruments have been previously exposed. Accordingly, results should always be interpreted in the light of a known or assumed pattern of instrument circulation between surgical procedure groups. Finally, it is worth stressing that the aim of the approach presented here is exclusively to produce a useful tool for epidemiological research in CJD transmission. Under the present state of knowledge, no consequences for the possible adoption of any practical recommendation concerning surgery or further preventive measures are to be drawn from this approach. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= AR assumed the basic task of reviewing literature, proposing tissues and structures, and drafting the first manuscript version. JPC indicated the subject domain, suggested differences between disease transmission and disease acquisition as seen from experimental and observational epidemiological research, generated first paragraphs relating to epidemiological aspects. KM suggested changes in epidemiological aspects. ÅS provided some criticisms. MC gave diverse comments, particularly on biological plausibility. HL clarified the need for methodological refinement in future work. All authors read and accepted the final version. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/9/prepub> Acknowledgements ================ The EUROSURGYCJD group members are grateful to Prof. Maurizio Pocchiari, Italy, for the substantial contribution to this proposal as regards his stance on the biological plausibility of surgical transmission of prion disorders, and to Maria José Bleda and Margarita Ramírez, Spain, for editorial help. EUROSURGYCJD group members.Spanish team: Javier ALMAZÁN, María J. BLEDA, Miguel CALERO, Pablo MARTÍNEZ-MARTÍN, Jesús de PEDRO-CUESTA (P. Leader), Alberto RÁBANO. Danish team: Kåre M∅LBACK, Henning LAURSEN. Finnish team: Jussi KOVANEN. Swedish team: Åke SIDEN, Inger NENNESMO. Consultant experts: Dr. Annick ALPEROVITCH, Prof. Paul BROWN, Prof. James IRONSIDE, Prof. Maurizio POCCHIARI, Prof. Robert G. WILL	PubMed Central	2024-06-05T03:55:52.576382	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548276/", "journal": "BMC Public Health. 2005 Jan 24; 5:9", "authors": [ { "first": "Alberto", "last": "Rábano" }, { "first": "Jesús", "last": "de Pedro-Cuesta" }, { "first": "Kåre", "last": "Mølbak" }, { "first": "Åke", "last": "Siden" }, { "first": "Miguel", "last": "Calero" }, { "first": "Henning", "last": "Laursen" } ] }
PMC548277	Background ========== Reflection is considered by many \[[@B1]-[@B3]\] in the Western world to be an important part of clinical practice and clinical decision-making. In order to learn from experience, health care professionals must reflect on the events and determine where the information \'fits\' in relation to their assumptions, previous experience and knowledge. Reflection is the process where individuals think about and evaluate their experience in order to come to new understandings and appreciations \[[@B2]-[@B4]\]. All promoters of reflection refer to it as a process of thinking about experiences. The process leads to a change in perspective or understanding. Schön \[[@B2],[@B3]\] suggested that reflective practitioners are better able to manage the uncertainty and complexity of clinical practice, and therefore asserted that a major part of preparation of professionals should be centred on enhancing their ability to reflect. Journal writing is a method that has been used to promote reflective thinking in health science students and professionals \[[@B5],[@B6]\], to facilitate the link between academic learning and clinical practice \[[@B7]\], and to assist students to explore and change their attitudes toward patients \[[@B7],[@B8]\]. When writing a reflective journal, students are asked to describe a learning event or situation, explain how the experience led to new understandings and appreciations, and consider how they might act differently in the future. Students have indicated that the writing of the journal provides a framework for the reflective process).)\[[@B9],[@B10]\]. Shepard and Jensen \[[@B11]\] specifically referred to the need for physiotherapists to have \'reflective\' knowledge in order to deal with uncertainties and value conflicts. Two recent Canadian studies \[[@B12],[@B13]\]. have described the reflections of physiotherapy students during clinical placement. The students wrote weekly in journals, and reflected on a number of topics that were classified into six major themes \[[@B12]\]. These included: 1) the process of making clinical decisions; 2) the complexity and richness of interactions with clients; 3) the influence of the practice environment on learning and patient care; 4) acquisition of clinical and administrative skills; 5) the value of clinical experiences in validating and integrating previous learning; and 6) acknowledgment and evaluation of different learning methods. Students frequently commented on their own values and beliefs and whether these were in harmony or conflict with the beliefs and actions of other health professionals or clients \[[@B13]\]. They discussed professional behavior, professional collegiality, respect for others, advocacy and informed consent. In considering the student reflections, the investigators wondered about the potential impact of culture and working environment on the content of the reflective journals. Would students from a non-Western culture and a different health care system be concerned about the same issues? Would they feel comfortable with the reflective process, particularly documenting their thoughts in a journal? How might the hospital/institutional infrastructure and norms affect their learning? We had a unique opportunity to answer some of these questions by comparing the themes from reflective journals of physiotherapy students in the United Arab Emirates (UAE) with those taken from the above mentioned studies \[[@B12],[@B13]\]. conducted at McMaster University in Canada. The physiotherapy program at the University of Sharjah in the UAE was developed with a philosophy similar to that of the program at McMaster, and the Chair of the program was a McMaster faculty member. Students in both programs have worked in small groups, studied similar content, and considered the same professional and attitudinal issues such as moral reasoning, client-centred care, professional behavior and professional code of ethics. Although both the UAE and Canada have many of the conveniences of modern society, there are major societal and cultural differences. The students in both countries have similar standards of living and have ready access to education and health services. Many of the health problems of modern society -- diabetes, heart disease, arthritis, cancer, and trauma from traffic accidents -- are common in Canada and the UAE. However, Canada is a democracy with a predominantly Christian and European population, whereas the UAE is a system of emirates with a population comprising mainly Arabic and Eastern cultures and where the Muslim religion predominates. The students in the two countries might be expected to have different values and beliefs, and different societal norms that could affect their reflections. The purpose of this study was to describe the reflections of physiotherapy students from the University of Sharjah during their clinical placements. A second objective was to compare the themes generated from the Sharjah data to the themes from the previous two studies on reflections of McMaster students during their clinical placements. Because the students in the two programs have similar socioeconomic status and similar curricula, it was anticipated that differences in reflections would be due to differences in culture and life experiences related to the cultures. Methods ======= This qualitative study was conducted during the students\'clinical placements in a summer semester of the Physiotherapy Program at the University of Sharjah, United Arab Emirates, a program developed in affiliation with McMaster University, Hamilton, Ontario, Canada. Curriculum and semester description ----------------------------------- The baccalaureate physiotherapy program in the UAE comprises one pre-professional year (year 1) and three professional years (years 2--4) of study. To be admitted to the program, students must have completed the UAE Secondary School Scientific Certificate (equivalent to grade 12), or equivalent program accredited by the Ministry of Education. They must have also scored at least 500 on the Test of English as a Foreign Language (TOEFL). While in the physiotherapy program, students need to complete university required and elective courses. Basic science courses are offered in years 1 and 2. Physiotherapy courses in years 2--4 include small-group, problem-based learning tutorials and clinical skills laboratories based on problems/ case scenarios enhanced with resource sessions. The male and female students are taught separately in discrete areas of the campus. Students -------- Six senior physiotherapy students (3 females and 3 males) with a mean age of 22.6 years (range 20.5 to 23.1) and 15 junior, female students with a mean age of 20.1 years (range 19.9 to 22.8) formed the subject group of this study. These students were respectively from the first and second cohorts of students admitted to the new physiotherapy program at the University of Sharjah. All students spoke Arabic and English. The senior students had completed the academic component of year 3 and were in their third clinical placement, which provided them with six weeks experience primarily with clients with cardio-respiratory conditions. The junior students had just completed two academic semesters of year 2, and were in their first clinical placement -- four weeks with clients with peripheral musculoskeletal conditions. Students practiced in five different clinical sites in the UAE, under the supervision of clinical preceptors and university instructors/coordinators. The study was approved by the Ethics Committee of the College of Health Sciences, and all participating students signed an informed consent. Evaluators ---------- Two instructors were involved in reading, coding and identifying the major themes that emerged from the students\' reflective journals. One instructor (HL) from the University of Sharjah, Department of Physiotherapy, had taught one course to the junior group and two courses to the senior group. The second instructor (JW) from McMaster University Physiotherapy Program, had taught one course to the senior group as a consultant to the program. The two instructors were not involved with the students during their summer clinical placements. A third evaluator (AAS) independently read all the journals. She was an instructor in the Sharjah program, a UAE national, and fluent in both Arabic and English. She had taught two courses to the junior students. Her analysis was used to confirm the interpretation of the entries made by the other two evaluators who were both from Western cultures. Procedure --------- The students were asked to write a reflective journal, a new exercise for all students. This assignment was one of the requirements for a Pass mark in their summer clinical placement. The students were required to make one entry per week for the duration of their clinical placement (four weeks for junior students and six weeks for senior students), and to submit the journal weekly to the university instructor involved in their clinical supervision. However, the instructor did not provide any feedback to the student at that time. The students were instructed to include the following in their reflective journals: Reflective journals should include observations, impressions, and reactions to what you have learned in the academic portion of the semester and how you are applying it to clinical practice. How does the clinical experience change what you thought, felt, or did in the past, and how you may respond in the future? You are expected to write at least one journal entry per week during your clinical placement. 1\. Describe the learning event, issue or situation. Describe prior knowledge, feelings or attitudes with new knowledge, feelings or attitudes. What happened? 2\. Analyse the learning event, issue or situation in relation to prior knowledge, feelings or attitudes. What was your reaction to the learning event, issue or situation? Your response may include cognitive and emotional reactions. Why did it happen? 3\. Verify the learning event, issue or situation in relation to prior knowledge, feelings or attitudes. What is the value of the learning event, issue or situation that has occurred? Is the new knowledge, feeling or attitude about the learning event, issue or situation correct? 4\. Gain a new understanding of the learning event, issue or situation. What is your new understanding of the learning event, issue or situation? 5\. Indicate how the new learning event, issue or situation will affect future behavior. Determine the clarification of an issue, the development of a skill or the resolution of a problem. How will you approach the same or similar event, issue or situation in the future? Data analysis ------------- The method described by Coffey and Atkinson \[[@B14]\] was used to analyse and interpret the data in the students\' journals. The analysis was conducted in two phases after the students had completed their clinical placements and submitted their entire journal. In Phase 1, one evaluator (HL) selected five journal entries based on their readability level and diverse content and forwarded them to the second evaluator (JW). The two evaluators read and coded the five journals independently. Then they met to discuss and establish agreement on the coding. In Phase 2, the two evaluators independently read and coded all remaining journals. They met again to 1) determine agreement on the content of each entry; 2) group the codes into categories; and 3) determine major themes. When coding discrepancy occurred, the evaluators discussed the discrepancy and came to a consensus. The following two methods were used to further validate the analysis and interpretation of the data. 1) The third evaluator independently read and labelled all journal entries to confirm the interpretation of the content. 2) The themes established by the evaluators were presented to two students who had completed the journals, and who volunteered to provide feedback on the authors\' interpretation of their reflections. Themes for the present study were developed without direct reference to the themes found in the Canadian studies \[[@B12],[@B13]\]. The Emirati and Canadian themes were then compared by examining their descriptions and content. Results ======= The two initial evaluators (HL, JW) came to a consensus on four themes concerning the clinical learning experience of the students. These themes did not change as a result of the coding of the third reader (AAS) or the feedback from the students. However, these additional data occasionally resulted in a change in emphasis of some of the content within a theme. Whenever this happened, the differences are described. All students outlined the positive impact and value of their learning from the clinical placement. They discussed events and issues that fit into the following four themes: 1\. Professional behaviors: skills and attitudes, scope of practice, time management, professional boundaries, and respect for clients and colleagues. 2\. Awareness of learning: clinical versus academic learning, gaining of important new knowledge, self-directed and life-long learning, and client as a source of learning. 3\. Self-development and shift to a more client-oriented focus over the time of the clinical placement. 4\. Identification and analysis of ethical issues. Each of these themes is discussed below with quotes provided for illustrative purposes. Professional behaviors ---------------------- The students appeared very aware of their own behavior and that of other health professionals and students. They reflected on how they should behave as professionals, but this also included how they should behave in general. The events that precipitated discussion on this theme included actions of themselves and other health professionals and included what they regarded as both positive and negative behaviors. They referred to physiotherapists and other health professionals who helped the students and who modelled good professional behavior. They also described some situations where health professionals did not show sufficient respect for their clients or their colleagues, or did not provide \'best practice\'. They commented on professional boundaries, being concerned about the client-therapist relationship becoming too personal and limits to the scope of physiotherapy practice. In addition, the students noted that self-directed learning and life-long learning were professional responsibilities. This topic is discussed under the next theme, Awareness of Learning. The students providing feedback emphasized that professional behavior (both negative and positive) was also learned from patients who might show their appreciation, be patient or uncooperative, or provide specific feedback regarding the student\'s communication or skills. The students also provided examples of \"crossing boundaries\" in terms of professional competencies and relationships with their patients and supervising therapists. One patient asked me angrily where \[were the physiotherapists\]? I responded politely to wait until they finish their work with the patients\... I calmed her down and asked her to be patient\...I really felt that the \[physiotherapist\] and I should maintain and manage our time\...It is really a big responsibility we have. \[student 11, week 3\] The child came crying and refusing to look at her forearm. We should respect the emotional status of the child and try to understand it because the child has been through a lot. The accident\..., surgery,\...and functional limitations\...The stress should be controlled before giving physiotherapy\...I should be patient and try to give the most enjoyable set of exercises to gain the child\'s trust. \[student 10, week 3\] Sometimes patients are shy to tell that there is no benefit from the treatment programme. So I asked the patient if he feels any improvements and if not to inform me in order to change the plan of treatment. The patient responded very well to that. \[student 5, week 3\] Awareness of learning --------------------- Although the entire reflective journal assignment was designed to have students reflect on their learning, this theme was included to capture the many components of learning discussed by the students. They gained knowledge, but also commented on the unique aspects of clinical learning. Clinical experiences provided an opportunity to apply and enhance their academic learning. Junior students particularly noted how observing surgery helped them understand their anatomy, or how working with \"real patients\' was not as straight forward as their academic learning. The senior students realized the need to be life-long and self-directed learners because they were always encountering new conditions/situations and they wanted to provide their clients with \'best practice\'. Students acknowledged the clients as a source of knowledge, and noted that clinical situations provided a stimulus to learn. In the feedback session, students particularly emphasized how the clinical experience had enhanced their learning \"a lot!\" They also indicated that clinicians reinforced the need for life-long learning because of the constant changes in science. Some therapists welcomed new knowledge from students, for example, learning about outcome measures, while others indicated they did not have the time. It was really good to have a mix of people \[clinicians\] and to learn from their experience in the work field\...Each one of them has given us a new way to deal with patients and\... also a new experience to add to our future knowledge\... The learning is not finished\...We have to keep our hands on the best information available and the best experience. \[student 4, week 6\] \[I\] try to apply the theoretical bases learned at the university. \[I\] interact with patients and take their histories, try to know their goals and what they need from physiotherapy. I try to invent new materials for the exercises. In my opinion, when we apply what we learned in the theory, the information sticks in our minds and is hardly forgotten. \[student 21, week 2\] Self-development and orientation shift -------------------------------------- The theme of self-development was derived from the content of individual student entries, but was also seen in the change in the reflections as students proceeded through their placements. Often at the beginning of the clinical encounter, the students were unsure of themselves, but they gained confidence as they worked with the client and/or learned from their colleagues. The change across the journal entries was even more noticeable. In many of the early entries, students described their own emotional response to a client or a situation. They were concerned about their own performance, and about what they were learning from the placement. They indicated how an event had an impact on them. The later entries were more client-centred. The focus was more on how the client responded to an event, or how helpful a physiotherapist was. Students also referred to an increased confidence, the more experiences they had. It became evident from the third reader\'s summary that students were aware of how their professional behavior, outside direct patient care, could ultimately have an impact on the patient. They referred specifically to the maintenance of knowledge and skills and to relationships with other health professionals. Students in the feedback session referred to their initial lack of confidence which improved during the clinical placement. They added, however, that it was difficult to demonstrate the \"correct confidence level\" to their instructor/evaluator, particularly when students were occasionally told they were \"over-confident\". They felt that self-development was affected by behavior of other physiotherapy students, method of feedback from instructors and whether their clients improved or not. The quote immediately below illustrates how a student confronted his/her uncertainties during one client encounter. The next quote concerns a student\'s acknowledgement of the change in his/her confidence with increasing experience. First patient with tetraplegia\...What if I do something wrong?\...First, worried\... and unable to do anything, I observed other colleagues\... I could do it too!\[student 6, week 2\] A one-month old infant was in the ICU. I thought I would not be able to touch her as usual, but things are now different. I did all the treatment\... with my hands with no hesitation and I was not scared\... My reaction was totally changed and it is becoming more regular\... I am happy\... I trust myself better and it is increasing with time\...and will increase the more cases I face. \[student 3, week 6\] In the quotes below, the student initially talks about his/her own emotional response to a patient, but later in the placement discusses the need to adapt to patients\' situations and provide them with the necessary treatment. In both journal entries, the student discusses a client with a novel and somewhat \"unpleasant\" condition. However, the student reacts differently in week 5 than week 1. At that moment I was shocked because I was not familiar with the neurology cases. When the physiotherapist asked me to help\...I hesitated in the beginning. \...This situation had a bad effect on me \... I was depressed and I couldn\'t do any single movement\[student 4, week 1\] I have seen a patient who was always under sedation due to his pain and his anger which was there almost all the time. \... I was always confused \...not knowing what to do for him and what is the best for him. \... Such a case made me think \[very\] much before any and each simple movement. \... So this made me prevent \[other\] problems such as ulcers, and invent any simple movement just to change his lying position\[student 4, week 5\] As the quotes illustrate, the student was concerned about what to do in both cases, but by week 5 was able to overcome the emotional response, problem solve about the management of the client, and take action. Ethics ------ Students commented on what they believed to be ethical or unethical behavior of the health professionals they were in contact with. They noted when informed consent was not obtained from clients and they discussed ways to get consent from uncooperative clients. They talked about having respect for their clients, and maintaining confidentiality. They noted \"ethical\" and \"unethical\" behavior in the same professionals under different circumstances. The staff is cooperative and helpful, respect each other, but there is no confidentiality or respect of time with patients. \[student 20, week 1\] They were concerned when individuals (clients or professionals) were being treated unfairly or differently because of their health condition or socio-economic status. In some cases they thought that the client might be harmed by the practice. One student was concerned about how to \'tell the truth\' to a client who had a poor surgical result possibly due to the questionable practice of the surgeon. One student described an event where a therapist refused to see a patient when she changed her appointment time, but treated another patient who did not have an appointment. The student felt that the latter patient was treated because she was a \"VIP (very important person)\". I felt sad for the \[person who did not receive treatment\], and I said to myself, where are the ethics? I learned before \... that all patients should be at the same level without any bias. \...all persons who work in health care fields specially the professional therapists must \[follow\] the code of ethics because they have humans in their hands. \... I will delete the word VIP from my life work. \[student 12, week 2\] In the feedback session, the students also indicated how clients could be discriminated against because of their health condition and the therapist\'s level of confidence. Therapists might provide less treatment to a child if they felt uncomfortable treating children. They might leave a student to treat an older person if they felt the management of older persons was less important or less effective. Feedback from students ---------------------- The students agreed with the four themes and did not think that any major concept was omitted in this analysis. However, they added some insight to the content and interpretation of each theme. The students emphasized that many people affect their learning -- university instructors, therapists, other health professionals, students and patients/clients. They felt that the information derived from the analysis of the reflective journals was very important. They strongly recommended that the results be disseminated to clinicians, instructors and future students to increase their awareness of the learning process and help the students to improve. Comparisons of themes of students from McMaster University and the University of Sharjah ---------------------------------------------------------------------------------------- The content of the journals indicated that students from both universities were comfortable with the reflective process. They frequently shared quite personal information, including their positive and negative emotions, concerns about their own behavior, and a discussion of their own beliefs. Some students in both countries, simply described an event and provided minimal personal reflection, but these students were in the minority. Table [1](#T1){ref-type="table"} summarizes the comparable themes of the Emirati and Canadian students. Themes from both countries related to professional behaviors, learning and ethical issues. Self-development was not identified per se as a theme in the Canadian studies, but change and growth were mentioned under the other themes. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Themes from Emirati and Canadian students during clinical placements ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------ UAE Canada ----------------------------------------------- ------------------------------------------------------------------------------------------------------------------ Professional behaviors † Process of making clinical decisions † Complexity and richness of interactions with patients ‡ Respect: for uniqueness of individual ‡ Professionalism: responsibility and behavior as a member of a profession ‡ Professional collegiality: interaction with and respect for health professionals including physical therapists Awareness of learning † Effects of practice environment on learning and patient care † Acknowledgment and evaluation of different learning methods † Value of clinical experiences in validating and integrating previous learning † Acquisition of knowledge and clinical and administrative skills Identification and analysis of ethical issues ‡ Allocation of resources\ ‡ Advocacy: for clients, society or health policy\ ‡ Informed consent: right of person to decide what will happen to him/her ------------------------------------------------------------------------------------------------------------------------------------------------------------------ † Themes from Williams et al \[12\] ‡ Themes from Geddes et al \[13\] ::: In the area of professional behavior, students in both countries commented on role models (both positive and negative) in the clinical environment, as well as on their own behavior or response to clients. Both groups demonstrated a respect for their clients and saw their role as going beyond physical treatment. Canadian students mentioned advocacy in the community while students in the UAE discussed supporting their clients within the hospital milieu. Both groups talked about the uniqueness of learning in clinical placements. The clinical experience enhanced and confirmed their academic learning, and stimulated them to learn more. They enjoyed learning new information and using their knowledge to help others. They were anxious when they \"didn\'t know what to do\", but expressed increased confidence with each experience. They appreciated constructive feedback on their performance from clinical supervisors, health professionals and clients. Both groups acquired new skills related to direct client care (therapeutic techniques and communication) and administrative activities (time management, organizational skills and charting). Only Canadian students referred to writing reports and billing, tasks that students in the UAE were not expected to do in their clinical placements. Both groups, however, broadened their view of the scope of practice of physiotherapy. Students from both countries noted ethical issues pertaining to inequalities in their respective health care systems. Canadian students were concerned about the shortfall of resources (time, personnel, funding), and about differences in health care provided to private clients or those funded through provincial health insurance. Emirati students noted that the health or societal status of a client could affect their treatment within the health system. Discussion ========== Physiotherapy students from Sharjah demonstrated the ability to reflect on their clinical practice at the highest level (discussion of how experience will impact on future behavior). They described not only the learning event, but also their emotional and cognitive reactions to it. They analysed the situations to arrive at new understandings and considerations of how they would behave differently in the future. Like the students in the Canadian studies, the subjects in the present study described three types of learning that are considered essential for clinical decision-making. These are propositional (academic learning -- facts, concepts), professional craft (learning from experience), and personal knowledge (knowledge of self and unique frame of reference) \[[@B15]\]. The students commented on the knowledge that they gained (e.g., anatomy, treatment techniques, medications), and the unique learning opportunities of fieldwork (e.g., application and enhancement of academic learning, learning new skills, response to novel situations). They acknowledged that their personal frame of reference affected their clinical interactions, but was also modified by their clinical experience. Although the students did not categorize their learning in the three areas, they were able to describe the acquisition of knowledge and skills required for making clinical decisions. The reflection on one\'s own development is a common theme in the reflections of health professionals. Jensen and Denton\[[@B7]\] referred to themes of student self-adequacy and inadequacy in their study of 23 physiotherapy students completing journals in their first clinical placement. Tryssenaar and Perkins\[[@B16]\] demonstrated that reflection on personal growth was still evident at the end of the educational programs and the beginning of careers for physical and occupational therapists. Subjects in their study described initial anxiety and then increasing confidence about their ability as graduate therapists. They became more patient-centred with experience. Jensen and Paschal\[[@B17]\] suggest that the shift to a client-centred approach is part of the transition that students go through from their first to later clinical placements. Because we did not follow one group of students over several placements, we cannot comment on the change in the focus of students with each additional clinical experience. However, both junior and senior students described their personal reactions more frequently at the beginning of the placement, and the patient\'s unique situation more frequently in the later weeks. Perhaps with each new experience (e.g., start of a clinical placement) students will be temporarily more self-centred as they deal with their anxieties and uncertainties. As they become more comfortable with their own abilities, they can focus more on the client. Such a pattern is in agreement with the finding mentioned above, that therapists become more client-centred with experience. \[[@B16]\] One other study \[[@B18]\] examined the reflections of non-Western physiotherapy students during their clinical placements. Although the students were practicing in an English, Western environment, rather than in their own culture and language, the students and their supervisors did raise some issues that may have relevance to the present study. The Chinese students and their Australian supervisors noted that the students were reluctant to self-evaluate, to express their opinions, to admit they did not understand or to take active steps to acquire needed information. In the present study, the students certainly voiced their opinions, admitted their perceived inadequacies, and expressed pride in their achievements in the reflective journals. However, some may have been less forthcoming with their clinical preceptors or university instructors/coordinators, particularly when they did not agree with them. In the sample quotes in the Results section, the students did not mention discussing their concerns and feelings with their supervisors. On the other hand, the students in the present study were working in their own language and culture, and would be better able to express their views, and to do it in a \"politically correct\" manner. Unlike the Chinese students, the students in the UAE discussed the need for self-direction in their learning. This difference could be cultural and/or due to the self-directed nature of the problem-based learning method used in the physiotherapy program at the University of Sharjah. Language might have been a factor in the reflections of the students from the UAE, however. Although the students worked in Arabic in the clinical environment, they conducted their physiotherapy education and wrote their journals in English. It is possible that some students had difficulty accurately expressing their reflections in English, and that there was some misinterpretation by the evaluators. However, two of the evaluators (HL, AAS) knew the students well and were used to their writing. One of them (AAS) was a UAE national and fluent in both English and Arabic. When the two initial evaluators (HL, JW) worked together in Phase 1 to determine coding methods, part of the discussion was on how to interpret the \"second-language writing\" of the journals. In spite of these precautions, it is still possible that the themes may have varied somewhat if the journals had been written in Arabic and interpreted by Arabic-speaking evaluators. Conclusions =========== Physiotherapy students from a Middle East culture consider many of the same issues as students from a Western culture when asked to reflect on their clinical experience. They reflect on their personal growth, on how they learn in a clinical setting, and on the ethical and professional behaviors of themselves and others. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= HL and JW were involved in the conception and design of the study, in the qualitative analysis, and in the writing of the manuscript. HL coordinated the data collection. AAS assisted with data analysis. All authors were involved in the preparation of the manuscript, and read and approved the final version. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6920/5/3/prepub> Acknowledgements ================ We thank all the students who participated in the study and provided feedback on the analysis. This study was supported by an Education Grant from the School of Rehabilitation Science, McMaster University.	PubMed Central	2024-06-05T03:55:52.578117	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548277/", "journal": "BMC Med Educ. 2005 Jan 20; 5:3", "authors": [ { "first": "Hélène", "last": "Larin" }, { "first": "Jean", "last": "Wessel" }, { "first": "Amal", "last": "Al-Shamlan" } ] }
PMC548278	Background ========== Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation within species. As a result, SNPs are now becoming the most popular type of marker in genetic association and mapping studies. SNPs are also most likely to be the molecular basis for the majority of phenotypic variation in (outbred) populations. In particular, SNPs in regulatory and protein-coding regions can have an effect on gene expression levels and protein activity, respectively. The phenotypic differences observed between selected (sub) strains in model organisms may be the result of specific (combinations of) natural occurring polymorphisms. Hence, a comprehensive inventory of SNPs, including extensive annotation will be extremely valuable in the search for functional polymorphisms. There is often a vast unexplored potential in large sequence datasets that have been collected for other purposes, for example, EST and whole genome sequencing (WGS) projects. In an effort to address these two issues, we have developed an in silicocandidate SNP mining pipeline that uses all publicly available sequence data for a specific organism, and designed a database, CASCAD (CAscad SNP CAndidates Database), that allows storage of a wide variety of primary source data, cross-annotation to other databases, and analysis parameters for SNPs associated with expressed sequences. Construction and content ======================== We applied the SNP discovery pipeline to both rat \[[@B1]\] and zebrafish (unpublished results) and identified about 33,000 and 52,000 high-quality candidates, respectively, that were extensively annotated and stored in the CASCAD database (Table [1](#T1){ref-type="table"}). The database includes detailed primary information on which the discovery of the candidate SNPs was based. This information, including sequence quality information (Phred score), the number of supporting reads for every nucleotide observed at the SNP position, and expected alignment lengths, was found to be very valuable for filtering for predicted variants that have the highest likelihood to be experimentally confirmed. Two verification experiments for the rat resulted in confirmation rate estimates of 59% (68 candidate SNPs in 10 different laboratory rat strains) and 50.3 % (340 candidates in 5 laboratory and 2 wild rat isolates) \[[@B1]\]. A set of 139 CASCAD entries tested in 7 zebrafish isolates confirmed 67.6% of them as true polymorphisms (unpublished data). The success rate values obtained are likely to be underestimates since only limited number of isolates/samples were used and we were unable to include exactly the same isolates that were used for generating the primary data (e.g. EST sequencing). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Input data (number of sequence reads) for the CASCAD pipeline and number of predicted candidate SNPs. ::: *Rattus norvegicus* *Danio rerio* -------------------------- ------------------------- ------------------- Input data mRNA 25, 634 3, 366 EST 244, 518 283, 572 WGS 19, 813, 313 11, 588, 394 Candidate SNPs predicted 33, 305 51, 769 synonymous 3, 842 9, 111 non-synonymous 3, 708 6, 217 nonsense 162 158 ::: We designed a web-based interface with Perl scripts communicating to a MySQL database, and displaying HTML pages through Apache server running on SuSE Linux. The interface provides simple, advanced (Figure [1](#F1){ref-type="fig"}), and sequence-based search forms. Parameters that can be used in a search include strain information, different formats of sequence identifiers (e.g. GenBank, UniGene, LocusLink accessions or gene symbol), map positions (genetic and physical), and a wide variety of SNP characteristics, such as experimental evidence, a likelihood score for verification as deduced from extensive verification experiments \[[@B1]\], and information regarding restriction sites that have been affected, facilitating the design of RFLP based assays. To this end CASCAD is tightly linked to primer design \[[@B2]\] and local genome assembly \[[@B3]\] interfaces, enabling a fast, reliable, and universal primer design for a chosen SNP candidate even when there is no assembled genome sequence available. To facilitate the retrieval of candidate SNPs with higher verification rates, represented by more reliable and common SNP candidates, we implemented the possibility to restrict searches to entries characterized by location at hypervariable CpG site, by minimal basecalling quality (Phred score), sequence match size, and/or minimal number of supporting reads for either allele. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### CASCAD advanced search form. ::: ![](1471-2164-6-10-1) ::: In addition to primary sequence data analysis, the effect of all SNPs on protein coding capacity was evaluated and non-synonymous SNPs were categorized in classes reflecting the severity of the polymorphism using a BLOSUM-based score. The predicted missense SNPs were analyzed by SIFT \[[@B4]\] and Polyphen \[[@B5]\] programs that utilize not only substitution information but also phylogenetic conservation and structural protein information to predict a potential effect of the polymorphism on protein function. Query results are summarized on the SNP details page (Figure [2](#F2){ref-type="fig"}), listing the SNP characteristics and including active links to other databases and resources, such as dbSNP, Ensembl, UniGene, and LocusLink. More detailed information regarding raw data underlying the candidate SNP (links to the original sequence files and a full nucleotide and protein alignment) can be obtained by clicking on the observed nearly exact hits between nucleotide sequences. Moreover, statistics on the data that support the SNP (number of occurrences for every nucleotide at SNP position, range of Phred basecalling quality scores) are provided. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### SNP details page ::: ![](1471-2164-6-10-2) ::: Utility and discussion ====================== For many applications, it is important to be able to distinguish between SNP candidates by their characteristics, as they may be predictive for verification success rate or carry biologically relevant information. Non-confirmed candidate polymorphisms may represent variants uncommon for a given population, but also sequencing errors (all types of sequences), RNA editing events and reverse transcriptase errors (EST reads). In order to minimize the contribution of false positives, one can exclude polymorphisms based on a single read for either allele, as is common for many in silicodiscovery pipelines \[[@B6]\]. Although this is a valid approach when selecting SNPs for population or association genetics, one could inadvertently discard many rare variants that may be associated with phenotypic variation, for example by affecting protein structure or function. Information on such polymorphisms can be very useful when mapping disease or QTL alleles. We have developed our database to fulfill the needs of any particular SNP application by providing control over every parameter we used in the polymorphism discovery step. Applications of the CASCAD database include queries for potentially deleterious SNPs in a specific genomic region of interest, for example a QTL interval, design of SNP-based mapping panels using either RFLP or any other technology, and identification of informative SNPs for fine-mapping. Custom sequences can be provided to search for known SNPs in any sequence of interest. In addition, the CASCAD pipeline \[[@B1]\] can be used to build a candidate SNP database for any model organism of interest for which sufficient sequencing data is available. Conclusions =========== The main purpose of CASCAD database is to provide flexible access to candidate single nucleotide polymorphisms, which were predicted using a computational approach from publicly available sequence data of the rat and zebrafish. The resulting database is crosslinked to most common public databases and can be queried for SNPs using accession numbers, sequence context, SNP characteristics, but also using parameters specific to the SNP discovery process, allowing stringent or relaxed conditions suitable for different types of applications. Availability and requirements ============================= The database is freely accessible through the website <http://cascad.niob.knaw.nl>. Programs, scripts, MySQL database dumps, and instructions for setting up a species-specific SNP database can be obtained from the authors upon request. Authors\' contributions ======================= VG designed and implemented the CASCAD database. EB tested database and interface. EC provided supervision and guidance for the project. Acknowledgements ================ This work was supported by the Dutch Ministry of Economic Affairs through the Innovation Oriented Research Program on Genomics, grant \#IGE010017.	PubMed Central	2024-06-05T03:55:52.582399	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548278/", "journal": "BMC Genomics. 2005 Jan 27; 6:10", "authors": [ { "first": "Victor", "last": "Guryev" }, { "first": "Eugene", "last": "Berezikov" }, { "first": "Edwin", "last": "Cuppen" } ] }
PMC548279	Background ========== In many developing countries, social marketing program have become an essential component, if not the main component, of the national family planning and HIV prevention strategy. Social marketing programs in reproductive health all aim to improve reproductive health of the target population by using commercial approaches to promote healthy behaviors and/or to expand access to essential products and services. While improving reproductive health of the target population is the ultimate aim, donors recognize the potential of social marketing programs to build the sustainable delivery of reproductive health and family planning products and services in developing countries. Different types of social marketing models have been used to achieve these objectives. Social marketing programs have often been classified as either using the non-governmental organization (NGO) model (sometimes also referred to as the social marketing organization model) or the manufacturer\'s model. \[[@B1]-[@B3]\]. The NGO model typically focuses on achieving the largest possible health impact among the target population. Hence, the NGO model typically heavily subsidizes products. By contrast, the manufacturer\'s model was specifically developed in response to the need not only to improve reproductive health, but also to do so in a financially sustainable manner. Programs under the manufacturer\'s model are treated as temporary interventions with a realistic exit strategy. Such programs typically use experts to provide temporary technical assistance to existing private sector companies, which eventually are expected to continue the program without subsidies. Although the two models are defined largely by the type of management and financial structure, they often also differ in terms of branding, pricing, distribution, and other factors. Moreover, context-specific variations on these two models are common, and some programs use hybrid approaches that integrate features from each of the two broader models \[[@B4]\]. While many social marketing variants have proven successful, it is generally assumed that the manufacturer\'s model is more feasible in middle-income countries with a fairly well developed commercial infrastructure, while NGO models are more appropriate in lower-income countries with less less-developed commercial infrastructure \[[@B4]\]. Although scattered case studies support these assumptions, as yet there has not been any comprehensive empirical assessment of the socio-economic context in which each model works best. This paper analyzes a comprehensive database of annual social marketing statistics. The database is unique in that it includes annual data on social marketing programs in reproductive health in over 70 countries, which cumulatively represent 555 years of experience with social marketing programs. We analyze these data to provide empirical information on the extent to which different social marketing models have been used in different socio-economic contexts, and to identify the socio-economic context in which each model is most effective. Our analysis is restricted to programs that social market condoms or oral contraceptives. As only limited data on the features of social marketing programs are routinely collected, we focus on differences in management structure. In many countries, social marketing programs are an essential component of the national family planning and HIV prevention programs. At present, large-scale social marketing programs for reproductive health are in operation in over 60 countries worldwide. A growing body of evidence shows that social marketing programs can make an important contribution to the reproductive health of the target population \[[@B5]-[@B19]\]. In 2002, social marketing programs in 69 countries sold almost 1.6 billion condoms \[[@B20]\] accounting for more than half of all condoms available from public sources. Total sales of condoms, pills, vaginal foaming tablets and IUDs amounted to over 28 million couple-years of protection (a measure of the number of couples that could be protected from pregnancy for one year). In these countries (excluding China and Russia) social marketing sales of temporary methods of contraception excluding condoms accounted for almost 10 percent of all use \[[@B21]\]. Social marketing tends to be a cost-effective approach to achieving widespread access to health products and services \[[@B22],[@B23]\]. It uses private sector incentives and organizations to achieve efficiency and revenues from sales offset some of the program costs. The total cost of \$6 per couple-year of protection compares favorably with costs to implement public sector family planning programs, which are around \$15--20 per couple-year of protection \[[@B24]-[@B27]\]. Some programs may eventually become self-sustaining and graduate from donor assistance altogether. Some social marketing programs in low-income countries are now operated by independent local social marketing foundations (e.g., the Ghana Social Marketing Foundation (GSMF) and ADEMAS in Senegal) and in some middle-income countries social marketing sales are continuing well after donor assistance has ended (e.g., condoms in Turkey, oral contraceptives in Morocco, and injectables in Brazil) \[[@B28]-[@B30]\]. Three different management structures are common in social marketing programs: 1) management by an affiliate of an international NGO, 2) management by local clinic-based or non-clinic-based organizations, and 3) partnerships with a commercial organization. The most commonly used management structure in health-oriented social marketing programs is management by an NGO. Typically this implies management by an affiliate of an international social marketing organization that provides an expatriate resident advisor. Several social marketing programs are managed by local organizations. For example, some social marketing programs are managed by a specially developed new organization that is independent of international affiliation. These are often called social marketing foundations. This organization may receive technical assistance from an international social marketing organization but it operates as an independent entity. Implementation may also be by an existing organization, such as a family planning association (FPA) or another type of clinic-based organization. In such case an international NGO provides technical assistance to the local organization to develop and operate the social marketing program. Social marketing programs may also be implemented through a partnership between an international NGO and an existing commercial organization. In this case, the international NGO often helps to develop the market for the product through research and promotion, while the commercial partner handles all aspects of packaging, sales and distribution. Not all management approaches are feasible in every context. For example, in countries with relatively high-income levels and a strong commercial sector there may be many opportunities for commercial partnerships, while in other countries there may be few such opportunities, if any \[[@B4]\]. Similarly, opportunities for management through existing local organizations do not exist in all countries. Furthermore, the context in which social marketing programs are implemented has a crucial impact on the types of programs that can be successful. For example, in a poor country with a rudimentary commercial sector, it is unlikely that commercial partnerships will be successful. On the other hand, in a middle-income country with a well-developed commercial sector and a rapidly developing middle class, it may be counterproductive to use an NGO affiliate model with a highly subsidized product that competes with existing brands. Methods ======= This section analyses empirical data to study differences in social marketing models and their impact. Since data about program characteristics are scarce, we focus on differences in management structure. Specifically, our objectives are to 1) describe to what extent the type of social marketing management structure actually varies according to the socio-economic context in which the programs are implemented, and 2) to examine the effectiveness of various management structures in increasing socially marketed product sales in different contexts. Data ---- For the purpose of this study, we compiled a database that comprises data on most of the major social marketing programs that were in operation at any time during the period between 1988 and 2001. Many, but not all, of these programs were still in operation in 2001. The database contains annual data on several program characteristics and on indicators of the local socio-economic context. The main sources of information for data on program characteristics were DKT International\'s \"Contraceptive Social Marketing Statistics,\" Population Services International\'s (PSI) MIS database, and published data on Futures Group International (TFGI) programs \[[@B26]\]. These three programs are the dominant implementing organization in contraceptive social marketing \[[@B1]\]. Data on the local context were obtained from the \"World Development Indicators 2003 CD-ROM\" \[[@B31]\]. For each social marketing program, the database contains one case for each year of operation within the 1988--2001 study period. In other words, our unit of analysis is the \"program-year.\" Using \"program-years\" as our unit of analysis has the advantage that all indicators, including program characteristics, can change over time. In addition, it implies that in the analyses programs that have been in operation for a longer period of time are given more weight than newer programs. For example, a social marketing program that has been operating since 1988 will contribute 14 program-years to the database (one case for each year of operation); a program that started in 1995 and was terminated in 1997 will contribute three program-years, and a program that started in 2001 will contribute only one program-year. After restricting the database to those social marketing programs that have a reproductive health component, the database contains information on social marketing programs in over 70 countries, which represent a total of 555 years of program experience. This includes 508 years of experience with condom social marketing and 208 years of experience with OC social marketing. ### Indicators Our measures of market potential are based on the indicators used by Michigan State University\'s Center for International Business Education and Research \[[@B32]\]. Specifically, we use the following indicators of market size, market intensity, and commercial infrastructure: ### Market size • Population size • Percentage of population living in urban areas ### Market intensity • Per capita GNI in current international dollars. This indicator measures the GNI converted in international dollars using purchasing power parity. An international dollar has the same purchasing power as a dollar in the U.S. \[World Bank 2003\] ### Commercial infrastructure • Telephone mainlines per 1,000 inhabitants • Television sets per 1,000 inhabitants The database also includes selected indicators of the characteristics of the social marketing programs, including: • Management structure (NGO affiliate, local clinic-based and non-clinic-based organizations, and commercial partnerships) • Program maturity/years of operation (less than 3 years, 4--6 years, and 7+ years) • Number of reproductive health products social marketed (one vs. two or more). Only condoms, oral contraceptives, injectables and IUDs are considered. In addition to these indicators of the socio-economic context and of the characteristics of the programs, information is available on the following outcome measures: • Per capita condom sales • Per capita OC sales While some social marketing programs distribute and/or promote injectables or IUDs, their number is not sufficiently large to allow multivariate analyses. Hence, data on sales of injectables and IUDs are not examined in this study. Other indicators of program effectiveness, such as user-prevalence rates are not collected on an annual basis. Ideally, one would also want to examine the cost-efficiency of implementing different social marketing models. A commonly used approach to estimate cost-efficiency is to conduct a cost-effectiveness analysis. By definition, any cost-effectiveness analysis requires obtaining data on both the effectiveness of the program, i.e. the impact of the program, and on the cost of implementing the program \[[@B33],[@B34]\]. Unfortunately, only limited cost information on social marketing programs is available, and the cost data that are routinely collected by the main social marketing organizations are not comparable. Hence, we are unable to assess the cost-efficiency of the programs in our database. Methods ------- In the next sections, we use cross-tabulations to examine to what extent management structure varies by socio-economic context. Since our unit of analysis consists of program-years of operation, these tabulations show the percentage of program-years that had each management type. We also test the extent to which different indicators of socio-economic context affect effectiveness of social marketing programs with different management structures. For this purpose, we use multiple regression analyses to assess the effect of indicators of socio-economic context on per capita condom sales for different management structures. Because each social marketing program has multiple entries in the data set (one for each year of operation), the standard errors in ordinary linear regression are incorrect. We use random-effects regression to solve the problem that observations for a given country-program are not independent \[[@B35]\]. For each type of management structure, we assess the net effect of socio-economic context on per capita condom sales, after controlling for the number of reproductive health products marketed, program maturity, time period (1995--2001 vs. earlier). Similar analyses are conducted for per capita OC sales. Results ======= Variations in management structure by socio-economic context ------------------------------------------------------------ We now analyze our database to illustrate to what extent different management structures have been used in social marketing programs (see Table [1](#T1){ref-type="table"}). Overall, data on the management structure of the social marketing programs were available for 555 program years. Of these 55 program years, 72% were managed through an NGO affiliate, 16% by local organizations, and 12% through a commercial partnership. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Distribution of Years of Social Marketing Program Experience by Management Structure and by Socio-Economic Context ::: Distribution of Program Years --------------------------- ------------------------------- ------- ------- ----- Population Size \<10 Million 76.3% 17.1% 6.6% 211 10--25 Million 62.7% 24.0% 13.3% 150 25+ Million 72.5% 9.0% 18.5% 189 Urban Population \<25% 86.1% 13.9% 0.0% 101 25--49% 80.8% 7.7% 11.5% 260 50+% 47.0% 31.0% 22.0% 168 GNI per capita \<\$1000 97.0% 3.0% 0.0% 132 \$1000--3000 68.2% 18.6% 13.2% 258 \$3000+ 52.3% 24.5% 23.2% 151 Phone mainlines per 1,000 \<5 87.1% 11.8% 1.1% 178 5--30 73.4% 9.2% 17.4% 184 30+ 53.8% 27.4% 18.8% 186 Television sets per 1,000 \<20 88.2% 10.1% 1.8% 169 20--100 70.9% 18.7% 10.4% 182 100+ 48.5% 23.3% 28.2% 163 Time Period 1986--93 56.4% 25.4% 18.3% 126 1994--97 66.2% 18.5% 15.3% 216 1998--01 85.9% 8.0% 6.1% 213 Region Eastern Europe 100.0% 0.0% 0.0% 16 Africa 89.7% 7.7% 2.6% 273 Asia 64.2% 17.5% 18.3% 137 Latin America 42.9% 41.9% 15.2% 105 Mid. East/N. Afr. 13.6% 0.0% 86.4% 22 Total 71.5% 16.0% 12.4% 555 ::: Social marketing programs in countries with populations smaller than 10 million have predominantly been managed by NGO affiliates (76% of program-years). In larger countries, other management structures have been somewhat more common. Breakdown by level of urbanization confirms that the range of management strategies used increased with socio-economic status. The percentage of social marketing program-years managed by NGO affiliates ranged from 86% when urbanization was low to 47% when urbanization was high. In highly urbanized populations, 31% of program-years were managed by existing local organizations and 22% by commercial partnerships. Social marketing programs in countries with a low per capita GNI (\<\$1,000) have been managed predominantly by NGO affiliates (97% of program-years), with local organizations being a distant second (3%). In countries with higher GNI levels, a wider range of management strategies has been used. When the per capita GNI levels are medium-low, management through NGO affiliates was still the most common (68% of all program-years). However, in medium-low GNI countries local organizations accounted for 18% of program years and commercial partnerships for 13%. Finally, when the GNI exceeded \$3,000 per capita, NGO affiliates accounted for only 52% of program-years, followed by local organizations (25%) and commercial partnerships (23%). Our indicators of the level of development of the commercial infrastructure show that the NGO model has dominated in settings with a relatively poorly developed commercial infrastructure. For example, NGO affiliates accounted for roughly 87% of program years in countries with fewer than 5 phone mainlines per 1,000 inhabitants, and for 88% of program years in countries with fewer than 20 television sets per 1,000 in habitants. Management through existing local organizations and commercial partnerships is most common in settings with over 30 phone mainlines and over 100 television sets per 1,000 inhabitants. Management structures have also changed over time. For example, the percentage of program-years managed through NGO affiliates has steadily increased from 56% in 1986--93 to 86% in 1998--2001. Simultaneously, management through existing local organizations and commercial partnerships has gradually declined. The last panel of Table [1](#T1){ref-type="table"} shows that management through commercial partnerships has been most common in the Middle East and Northern Africa (87% of program-years), while management through existing organizations has been most common in Latin America (86% of program-years). In Eastern Europe and Africa management through NGO affiliates has dominated (90% and over). In Latin America, management through NGO affiliates and through local organizations have both been common (43% and 42%, respectively). The effect of socio-economic context on the effectiveness of different social marketing management structures ------------------------------------------------------------------------------------------------------------- We now use random-effect GLS regression analyses to examine to what extent market size, market intensity, commercial infrastructure, and program features (number of products social marketed and program maturity) affect per capita condom sales and per capita OC sales. The variable \"number of television sets per 1,000 inhabitants\" was removed from the multivariate analyses as it correlated too much with some of the other predictor variables. We conduct separate analyses for social marketing programs managed by NGO affiliates, commercial partnerships, and local organizations. Due to the small number of cases, we are unable to differentiate between existing organizations and new organizations. ### Management by NGO affiliates The second column in Table [2](#T2){ref-type="table"} shows the predictors of per capita condoms sales in social marketing programs managed by NGO affiliates. The results indicate that among social marketing programs managed through NGO affiliates per capita condoms sales increase significantly with the number of years of program maturity (β = .181 for programs with 4--6 years of maturity; β = .348 for programs with 7 or more years of maturity). Programs that social marketed two or more products have higher per capita condom sales than programs that just market condoms (β = .159). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Effect of Socio-Economic Context on Condom Sales and CYP Among Social Marketing Programs Managed Through an NGO Affiliate (Random-Effects Regression GLS Coefficients) ::: Per Capita Condom Sales OC Sales ---------------------------------- ------------------------- ----------- Markets Two or More Products .159\\\* .017 Program Maturity 1--3 (ref) 4--6 .181\\\* .001 7+ .348\\\* .003 Time Period \<1994 (ref) 1995--2001 .143\\ .004 Population (in 10 millions) -.006\\\* -.000\* Percent Urban -.002 -.000 Per Capita Gross National Income -.003 .007 Phone mainlines per 1,000 -.001 -.000 Constant .260\\\* (dropped) R Square .236 .082 Number of Program-Years 374 90 Number of Countries Included 58 26 \\\* p \< .01; \\ p \< .05; \* p \< .10 ::: As expected, social marketing programs managed through an NGO affiliate tend to be more effective, as measured by per capita condom sales, in countries with smaller populations (β = -.006). Per capita condom sales do not vary with the level of urbanization, per capita Gross National Income (an indicator of market intensity), nor with the number of phone mainlines (an indicator of the strength of commercial infrastructure). The third column in Table [2](#T2){ref-type="table"} shows the predictors of per capita OC sales. The results indicate that there is no evidence that our indicators of market size, market intensity, and commercial infrastructure have any significant impact on per capita OC sales. ### Management by commercial partnerships Table [3](#T3){ref-type="table"} shows the predictors of per capita condom sales in social marketing programs managed using the manufacturer\'s model. The results shown in Table [3](#T3){ref-type="table"} indicate that among social marketing programs managed through commercial partnerships, per capita condom sales tend to be higher in countries with smaller populations (β = -.012), and with a lower per capita gross national income (β = -.111). The level of commercial infrastructure, as measured by the number of telephone mainlines per 1,000 inhabitants, has a small but significant positive effect on per capita condom sales (β = .001). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Effect of Socio-Economic Context on Condom Sales and CYP Among Social Marketing Programs Managed Through the Manufacturer\'s Model (Random-Effects Regression GLS Coefficients) ::: Per Capita Condom Sales OC Sales ---------------------------------- ------------------------- ---------- Markets Two or More Products .078 -.015\* Program Maturity 1--3 (ref) 4--6 -.018 .003 7+ -.069 .007 Time Period \<1994 (ref) 1995--2001 .099 .007 Population (in 10 millions) -.012\\\* -.002 Percent Urban .001 -.000 Per Capita Gross National Income -.111\\ .015\\ Phone mainlines per 1,000 .001\\ -.000 Constant .278\\ .030 R Square .384 .185 Number of Program-Years 54 55 Number of Countries Included 9 12 \\\* p \< .01; \\ p \< .05; \* p \< .10 ::: The third column in Table [3](#T3){ref-type="table"} reveals a different pattern for factors affecting per capita OC sales. Among social marketing programs managed through commercial partnerships, per capita OC sales appear to be lower for those programs that also market other products (β = -.015; p \< .010). Our indicator of market intensity, per capita gross national income, has a significant positive effect on per capita OC sales (β = .015). However, population size and urbanization do not have any significant effect. ### Management by local clinic-based and non-clinic-based organizations Table [4](#T4){ref-type="table"} shows similar analyses for programs managed by local organizations (including both clinic-based and non-clinic-based organizations). The results show that programs in countries with larger populations have significantly higher per capita condom sales (β = .036). However, program in countries with higher levels of urbanization have lower per capita condom sales (β = -.012). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Effect of Socio-Economic Context on Condom Sales and CYP Among Social Marketing Programs Managed Through Local Organizations (Random-Effects Regression GLS Coefficients) ::: Per Capita Condom Sales OC Sales ---------------------------------- ------------------------- ------------- Markets Two or More Products .075 -.012 Program Maturity 1--3 (ref) 4--6 .158 .022\* 7+ .181 .028\\ Time Period \<1994 (ref) 1995--2002 .138 -.010 Population (in millions) .036\\ .008\\ Percent Urban -.012\\\* .001 Per Capita Gross National Income .006 .028\\\* Phone mainlines per 1,000 .001 -.001\\\* Constant .436\\ -.021 R Square .309 .401 Number of Program-Years 80 63 Number of Countries Included 17 12 \\\* p \< .01; \\ p \< .05; \* p \< .10 ::: The third column in Table [4](#T4){ref-type="table"} shows that among social marketing programs implemented by local organization, OC sales are significantly higher for those programs that have been in operation for seven or more years (β = .031). For these types of social marketing programs per capita OC sales increase with population size (β = .008) and with level of urbanization (β = .028). The level of commercial infrastructure, as measure through the number of telephone mainlines per 1,000 inhabitants, has a small negative effect on per capita OC sales. Discussion ========== Several studies have commented on the differences between social marketing programs that use the non-governmental organization model and those that use the manufacturer\'s model \[[@B1]-[@B3]\]. Although it is increasingly recognized that context-specific variations on these models are common, and that hybrid models exist \[[@B4]\], it is generally assumed that social marketing programs that use the manufacturer\'s model are most feasible in middle-income countries with a fairly well-developed commercial infrastructure, while NGO managed models work best in lower-income countries with less-developed commercial infrastructure. As yet, however, there has been little empirical evidence to verify these assumptions. This paper has used a database of annual data on social marketing programs in over 70 countries to verify the extent to which the different social marketing models that have been used vary across socio-economic contexts, and to test in which socio-economic context each of the models is most successful at increasing use of socially marketed products. A rigorous analysis of the relative effectiveness of different social marketing models would require detailed data about the characteristics of the program, including data about financial structures, branding, pricing (and level of subsidies), distribution, and promotion. Because data on the features of social marketing programs are limited, we focused on differences in the programs\' management structure. Ideally, we would also like to examine are range of outcome measures, including per capita sales, the user-prevalence, and the unit-cost for each method. Since such prevalence and unit-cost data are unavailable, we have focused on per capita condom and OC sales. We have used empirical analyses to obtain a better understanding of the variations that exist in social marketing programs. Specifically, we have analyzed a database of annual statistics on social marketing programs in over 70 countries, accounting for a total of 555 years of program experience. The database contains annual data on the management structure, program maturity, the number of reproductive health products marketed, and on product sales. The database also contains indicators of the local socio-economic context (market size, market intensity, and commercial infrastructure), including the population size, level of urbanization, per capita GNI, number of telephone mainlines and television sets per 1,000 inhabitants. Our analyses have shown that of the 555 years of social marketing program experience covered by the database, over 70% has used the NGO management structure. Nevertheless, there are considerable variations in management structure. The NGO affiliate management structure has been the dominant model when the per capita GNI is below \$3,000 and the level of urbanization below 50%. The data also show that use of the NGO management structure has increase dramatically over the course of our study period. The findings support the theory that there may be more opportunities for successful partnerships with the private sector in countries with a high per capita GNI. The highest percentage of such partnerships (23%) was found in countries with a GNI exceeding \$3,000 per capita. This percentage decreases to 13% in countries with a per capita GNI between \$1,000 and \$3,000. None of the commercial partnerships in our database were in countries with a per capita GNI below \$1,000. One of the reasons for this pattern may be that there is more commercial activity and a wider range of possible partners in countries with a higher per capita income. Users in those countries are also more likely to be able to afford the commercially sustainable brands that are marketed under this approach. Although the program characteristics usually appear to be appropriate for the local socio-economic context, it is questionable whether this has always been the case. For example, over 50% of program-years in countries with a medium-high GNI (over \$3,000) used the NGO model, even though countries with such income levels typically provide opportunities for commercial partnerships. Our multivariate analyses identified the factors that affect per capita condom and OC sales for each of the four social marketing models. The reader is cautioned, however, that condom and OC sales may also be influenced by other factors that were not measured in our data base, such as HIV prevalence and overall levels of contraceptive prevalence. Bearing these cautions in mind, the analyses yielded the following key findings: Programs managed by NGO affiliates ---------------------------------- • Per capita condom sales are higher in countries with smaller populations. This may be influenced by the difficulty of achieving high distribution coverage in large, mostly rural countries (e.g., India, Indonesia, Vietnam, Nigeria) and/or the time it takes to large such large populations with social marketing campaigns. Also, social marketing programs in countries with a larger population may have more competition (e.g., India, South Africa). By contrast, several small countries have a high HIV/AIDS prevalence and/or limited method choice (e.g., sub-Saharan Africa); • Per capita condom sales are higher for more mature programs and those that market more than one product. This probably reflects improvements in distribution capability over time, as well as the cumulative effect of behavior change programs that stimulate demand. More developed programs with stronger distribution are also more likely to market several products; • Per capita condom sales do not vary with the level of urbanization, per capita income, or commercial infrastructure. These variables are more likely to affect access-related factors, such as product access and ability to pay. Other factors to look for that may affect sales are related to demand: level of effort of the social marketing program (which reflects funding levels), health context (HIV/AIDS and contraceptive prevalence) and competition from the public sector and other NGOs that may cancel out the higher market potential in the more developed countries; • None of the factors examined had any significant influence on the per capita OC sales of social marketing programs managed through an NGO affiliate. Programs managed by commercial partnerships ------------------------------------------- • Per capita condom sales tend to be higher in countries with smaller populations and with a lower per capita growth. At first glance, this finding is counter-intuitive. That is, initially one would expect sales for commercial partnerships to increase with population and per capita GNI, because commercial program need both a high demand and ability to pay. However, as population size and income increase sales may decline due to increased competition from commercial and other suppliers. Several countries with middle-high incomes (Turkey, the Philippines, and Jordan) or large populations (Turkey, the Philippines, and Indonesia) have strong public sector programs that offer free contraceptives, as well as many commercial suppliers. Also, demand for condoms in these countries has been very low, given that HIV prevalence is low that other contraceptive methods are available. By contrast, smaller countries such as Haiti and Zimbabwe have high HIV rates, less choice of methods, and probably less competition from other suppliers. Programs managed by local clinic-based and non clinic-based organizations ------------------------------------------------------------------------- • Programs in countries with larger populations have significantly higher per capita condom sales. One likely explanation for this pattern is that local organizations tend to have good distribution networks, which pays off in countries with a larger population. Some of the larger countries (e.g., Bangladesh and Colombia) have well-established organizations with a big market share. For example, in Bangladesh SMC provides almost all condoms and the demand is very high; • OC sales are significantly higher for programs that have been in operation for seven or more years. The longer a program operates, the more developed its distribution is likely to be. Social marketing communication efforts are also more likely to pay off over the long run; • Programs in countries with a higher level of urbanization have lower per capita condom sales. This was anticipated, given that NGOs and family planning associations have a strong track record of serving users in peri-urban and rural areas. In addition, when urbanization increases, access to commercial products sold in pharmacies and other private sector outlets increases as well, which allows commercial suppliers to grow their market share and compete with social marketing and public sector products; • The level of commercial infrastructure, as measured through the number of telephone mainlines per 1,000 inhabitants has a small negative effect on per capita OC sales. This finding may reflect that a better commercial infrastructure implies more competition from commercial suppliers. Conclusions =========== Our analyses of records on 555 years of experience with social marketing programs have illustrated the tendency to design social marketing programs with a management structure suitable for the local context. However, the evidence also shows examples where this has not been the case. While socio-economic context clearly influences the effectiveness of some of the social marketing models, program maturity and the size of the target population appear equally important. Consequently, it is crucial that social marketing programs are designed using the model or approach that is most suitable for the local context. Competing interests =================== Dr. Meekers is the former Research Director of PSI (1996--2001), and has been a paid consultant for other PSI research projects. Author\'s contributions ======================= Dr. Meekers developed the study design, assisted with the analysis, and contributed to the writing of the final manuscript. Mr. Rahaim collected data, conducted literature reviews, and contributed to the editing of the paper. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/10/prepub> Acknowledgements ================ This study was funded by the United States Agency for International Development (USAID) through the Commercial Market Strategies (CMS) Project. The authors are grateful to Ruth Berg and Françoise Armand for comments and suggestions on earlier drafts of this report.	PubMed Central	2024-06-05T03:55:52.583437	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548279/", "journal": "BMC Public Health. 2005 Jan 27; 5:10", "authors": [ { "first": "Dominique", "last": "Meekers" }, { "first": "Stephen", "last": "Rahaim" } ] }
PMC548280	Background ========== Phenotypic robustness refers to the invariance of the specified target phenotype given the genetic makeup and environmental conditions. Whereas the presence of naturally occurring phenotypic variation is at the core of evolutionary biology, developmental geneticists have traditionally considered it as a nuisance. Instead, they have relied on the study of single or multiple mutant combinations to reveal the generation of phenotypic patterns (e.g. \[[@B1]\]). A resurgence of interest in the issue of phenotypic robustness has emerged in recent years, partly due to experimental results showing that many knock-out mutations have little effect on phenotype (\[[@B2]\]; although Papp\'s et al. \[[@B3]\] metabolic network analysis found that the majority of genes that looked dispensable turn out to be such only under laboratory conditions), and that developmental systems show a high degree of stability with respect to perturbations \[[@B4],[@B5]\]. Three major processes are involved in the control of phenotypic variability (the potential or propensity to vary, in the terminology of Wagner and Altenberg \[[@B6]\]): canalization, developmental stability (DS), and plasticity \[[@B7]\]. As first defined by Waddington \[[@B8]\] the term canalization could be understood as a morphogenetic constrain \[[@B9]\], where development appears to be buffered so that slight abnormalities of genotype or slight perturbations in the environment do not lead to the production of abnormal phenotypes. However, evolutionary geneticists define canalization as the tendency of traits to evolve a reduction in variability \[[@B4],[@B10]\]. DS can be defined as the ability of organisms to buffer against the random noise that arises spontaneously as a consequence of stochastic variation in the cellular processes that are involved in the development of morphological structures \[[@B11]\]. Therefore, canalization and DS are subcategories of developmental buffering: the first can be appraised by estimating interindividual variance whereas the most commonly used estimate of DS in bilaterally symmetrical organisms is fluctuating asymmetry (FA); i.e. the intraindividual variation due to random differences between left and right sides. The question of whether or not canalization and DS are different buffering mechanisms has been a constant source of debate. Two recent reviews implicitly \[[@B4]\] or explicitly \[[@B10]\] assume that DS is a special case of canalization, a viewpoint also embraced by several authors (e.g. \[[@B12]-[@B14]\]). Thus, by using geometric morphometrics Klingenberg and McIntyre \[[@B13]\] found that the vectors describing inter- and intraindividual variation of landmark position for fly vein traits were highly concordant. On the other hand, Debat et al. \[[@B15]\] came to the opposite conclusion applying the same methods to cranial landmarks in the house mouse -- although Klingenberg\'s et al. \[[@B16]\] work with mouse mandibles found patterns of intra- and interindividual variation that were only partly consistent --. At first glance, the different results may suggest that the mechanisms that affect canalization and DS are related in some developmental contexts but not in others. The problem is, however, that according to the causes of phenotypic variation a distinction between genetic and environmental canalization is necessary \[[@B17],[@B18]\]. Selection for environmental canalization may produce genetic canalization as a by-product \[[@B4],[@B10]\], but this may not always be the case. The better way to address these contentious issues is to rely on quantitative genetic analyses devised to partition phenotypic variation into genetic and environmental components \[[@B19]\]. Environmental variation can be further partitioned into general (![](1471-2148-5-7-i1.gif)) and special (micro) environmental effects (![](1471-2148-5-7-i2.gif)): the first refer to influential factors (e.g. temperature) that are shared by groups of individuals, whereas the latter are residual deviations from the phenotype that would be specified on the basis of genotype and general environmental effects. Such deviations are unique to individuals and are largely unpredictable. The variance associated with special environmental effects can be estimated when experiments are performed on completely inbred lines (i.e., there is no genetic variance). In bilaterally symmetrical organisms it is also feasible to estimate the two sources that contribute to those special environmental effects: among-individual (![](1471-2148-5-7-i3.gif)) and within-individual variance (![](1471-2148-5-7-i4.gif)). If the only real cause of asymmetry is variation due to stochasticity in development, then FA can be taken as an estimated of ![](1471-2148-5-7-i4.gif). Therefore, FA is only one source of the phenotypic variation within environments (excluding environmentally induced asymmetry), contrarily to the arguments in Nijhout and Davidovitz \[[@B20]\]. The other source is ![](1471-2148-5-7-i3.gif). The third process involved in the control of phenotypic variability is plasticity, which can be defined as the ability of an individual to express one phenotype under one set of environmental circumstances and another phenotype under another set. The expressed phenotypes can be discontinuous thus eliciting discrete morphs (i.e., polyphenism), or there can be a continuous range of potential phenotypes (i.e., reaction norm). The reaction norm is thus a property of the genome: genetic canalization and phenotypic plasticity are not mutually exclusive and can combine to form canalized reaction norms \[[@B7],[@B17]\]. Plasticity is thus an alternative to genetic change allowing populations to adapt to changing environmental conditions. To summarize, phenotypic plasticity increases the variance among groups of individuals that produce different phenotypes in different environments, canalization decreases the within-group interindividual variance around the target phenotype by reducing the sensitivity to genetic and environmental conditions, and DS buffers against random perturbations in development (i.e., decreases FA). Because the left and right body sides share the same genome (barring unusual somatic mutation or somatic recombination) and in most organisms also very nearly the same environment, FA provides an intrinsic control for genetic and environmental effects and the important question is to what extent these two sources of variation share underlying regulatory mechanisms. Within the framework of recently developed geometrically based methods for the statistical analysis of size and shape variation (collectively referred to as geometric morphometrics \[[@B21],[@B22]\]), the wing vein network of Drosophilais regarded as an excellent model system to investigate those problems \[[@B23],[@B24]\]. Wing development in Drosophilais well understood \[[@B25]\], and the vein pattern is highly conserved across species (e.g. \[[@B26]\]). When flies are reared at low temperatures it is well known that the final wing size increases because of an increase in adult cell size \[[@B27]\]. This plastic response is parallel to what has been commonly observed in laboratory experiments on thermal evolution, where adaptation to lower temperature resulted in increased wing size (a proxy for body size) entirely as a consequence of cell size divergence \[[@B28]\]. However, there is circumstantial evidence suggesting that developmental and evolutionary temperature-related cell size divergence have contrasting effects on wing shape. Thus, Birdsall et al. \[[@B29]\] concluded that wing shape in Drosophila melanogasteris quite resistant to developmental temperature. Conversely, in D. subobscurathere are changes in wing proportions along a latitudinal size cline mediated by cell area \[[@B30],[@B31]\]. These populations exhibit, in addition, prominent latitudinal clines for chromosomal inversion polymorphisms, and there is compelling evidence showing that the inversion clines underlie the latitudinal changes in wing proportions \[[@B32],[@B33]\]. Here we report on the effects of clinal genetic variation (inversion polymorphism) on wing form (size and shape) and bilateral asymmetry using isochromosomal lines of D. subobscura. We consider the consequences of inbreeding and temperature on the two components of developmental homeostasis (canalization and DS), and the relationship between them. The remainder of the paper is planned as follows. First, we provide a short account of the inversion polymorphism in D. subobscuraand the experimental settings. Then, based on the well balanced data set rendered by the experimental design we used the standard least-squares (ANOVA) method to decompose sources of variation for wing size and shape into causal components at the core of further analyses. Furthermore, because the underlying assumption to use FA as a measure of DS is that left -- right-side variation has not heritable basis, the genetic and environmental components of bilateral asymmetry were partitioned. As a result, our approach is unusual in studies of DS in providing estimates of the two components of special environmental effects (co-) variance under different genetic backgrounds and general environmental settings. We also present some evidence for the presence of genetic variation in directional asymmetry (DA) but not in FA. Next, we test whether or not the vectors describing variation of landmark position for fly vein traits are concordant, and finally we discuss the main findings in relation with the evolution of buffering mechanisms and the putative adaptive value of natural wing shape variation in D subobscura. Experimental settings ===================== D. subobscurais a particularly inversion-rich species, with up to 38 natural chromosomal arrangements already reported for the largest chromosome O (homologous to arm 3R in D. melanogaster\[[@B34]\]) for which a balancer stock is available. In colonizing populations of the New World only six gene arrangements are segregating for that chromosome: O~st~, O~3+4~, O~3+4+2~, O~3+4+7~, O~3+4+8~and O~5~(arrangement O~7~is also present at very low frequency but it is probably the result of a recombination event in the O~st~/O~3+4+7~heterokaryotype \[[@B35]\]). In native Palearctic populations arrangements O~3+4+2~and O~3+4+8~are restricted to the Mediterranean region (the likely area from which the original American colonists derived \[[@B36]\]) and are not involved in latitudinal clines \[[@B35]\]. On the other hand, arrangement O~st~shows a world-wide positive correlation with latitude, while arrangements O~3+4~and O~3+4+7~show a contrasting pattern \[[@B35]\]. Therefore, six independent isochromosomal lines for each of these three chromosome arrangements (i.e., ![](1471-2148-5-7-i5.gif), \..., ![](1471-2148-5-7-i6.gif); j= st, 3+4, 3+4+7) were used in the present experiments. The experimental flies were obtained from 54 crosses, which will be referred to as inbred (isogenic; i.e., ![](1471-2148-5-7-i7.gif)) with 18 crosses in total, or outbred (including both structural homo- and heterokaryotypes) with 36 (18 + 18) crosses in total. The six lines with a given gene arrangement were crossed to produce the three different outbred homokaryotypes (i.e., ![](1471-2148-5-7-i8.gif)). The three kinds of heterokaryotypic flies were similarly obtained but using lines with different gene arrangements (i.e., ![](1471-2148-5-7-i9.gif)). Since all isochromosomal lines were homogeneous for the same genetic background (except for the male sex chromosome), maternal effects were not considered to be critically important. Anyhow, experimental flies were randomly derived form reciprocal crosses for all outbred combinations. Two developmental temperatures were used in the experiment: optimal (18°C) and warm (23°C). Results and discussion ====================== Variation and asymmetry in size ------------------------------- ### a) Basic statistics Signed left-right (![](1471-2148-5-7-i10.gif)) differences of centroid size did not significantly departure from normality in any case (D~max~ranging from 0.032 for inbred females at 18°C to 0.073 for inbred males at 23°C; P\> 0.05). In addition, none of the regressions of centroid size FA on average wing size was statistically significant (ranging from β= -0.045 (95% C.I.: -0.091, 0.001) for inbred females at 18°C to β= 0.030 (-0.005, 0.064) for inbred females at 23°C), thus suggesting independence between size and size FA. ### b) Causal components of variation For each sex two-way mixed ANOVAs were separately performed for inbred and outbred crosses at each experimental temperature (Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}). Size variation (CS: centroid size) among individuals comprised the largest part (\> 90%) of the variation. The fraction of the total phenotypic variance in wing size associated to genetic differences among karyotypes and/or lines (i.e., ![](1471-2148-5-7-i11.gif)) ranged from 0.235 (inbred males at 18°C) to 0.602 (inbred females at 23°C). (Bear in mind that there is nothing in the ANOVA method of estimation that will prevent a negative variance estimate \[[@B37]\].) ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Asymmetry of overall wing size for females raised at 18°C Drosophila subobscuraflies raised from inbred (isogenic) and outbred crosses reared at 18°C. Centroid size (CS, estimated in a normalized form \[22\]) is the dependent variable (values in pixels^2^: 1 mm = 144 pixels). The ANOVAs assess measurement error, directional asymmetry (Sides effect), fluctuating asymmetry (Individuals × Sides interaction effect), and genetic components of the trait () and DA of the trait ((DA)). (CS) and (DA~CS~) provide here unbiased estimates of the among-fly (i.e. ) and within-fly ( or FA) special environmental effects. (⊂ means \'nested in\'.) ::: Inbred Outbred --------------------- -------------------- -------- --------------- -------------------- ------ --------------- -------------------- Source of variation Variance component d.f. Mean Square Estimated variance d.f. Mean Square Estimated variance Individuals (I) 107 39.747\\\* 9.6175 215 45.773\\\* 11.2830 Karyotypes (K) (CS) 2 114.593^n.s.^ 0.1389 5 214.204^n.s.^ 0.7014 Cross ⊂ K (CS) 15 94.589\\\* 2.7352 30 113.199\\\* 3.4726 Among flies (CS) 90 28.944\\\* 6.9166 180 29.857\\\* 7.3040 Sides (S) 1 15.982\\\* 1 18.549\\\* I × S (CS) 107 1.278\\\* 0.5467 215 0.641\\\* 0.2225 Karyotypes (K) (DA~CS~) 2 0.067^n.s.^ -0.0520 5 0.457^n.s.^ -0.0123 Cross ⊂ K (DA~CS~) 15 1.938^¶^ 0.1239 30 0.899^¶^ 0.0492 Within flies (DA~CS~) 90 1.194\\\* 0.5051 180 0.603\\\* 0.2036 Measurement error (CS) 216 0.184 0.1841 432 0.196 0.1962 Average CS for left (L) and right (R) wings: inbred females = 0.9918 mm, = 0.9891 ; outbred females = 1.0022, = 1.0002. ^n.s.^P\> 0.10; ^¶^0.10 \>P\> 0.05; \\\* P\< 0.001. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Asymmetry of overall wing size for males raised at 18°C Same as in Table 1. ::: Inbred Outbred --------------------- -------------------- -------- -------------- -------------------- ------ --------------- -------------------- Source of variation Variance component d.f. Mean Square Estimated variance d.f. Mean Square Estimated variance Individuals (I) 107 43.303\\\* 10.4842 215 38.600\\\* 9.4200 Karyotypes (K) (CS) 2 232.359^¶^ 1.1331 5 115.718^n.s.^ 0.1908 Cross ⊂ K (CS) 15 69.186\* 1.4332 30 88.246\\\* 2.5026 Among flies (CS) 90 34.788\\\* 8.3554 180 28.184\\\* 6.8159 Sides (S) 1 1.140^n.s.^ 1 22.492\\\* I × S (CS) 107 1.366\\\* 0.5045 215 0.920\\\* 0.3297 Karyotypes (K) (DA~CS~) 2 0.385^n.s.^ -0.0176 5 1.156^n.s.^ 0.0035 Cross ⊂ K (DA~CS~) 15 1.017^n.s.^ -0.0715 30 1.031^n.s.^ 0.0226 Within flies (DA~CS~) 90 1.446\\\* 0.5444 180 0.895\\\* 0.3172 Measurement error (CS) 216 0.357 0.3574 432 0.261 0.2609 Average CS for left (L) and right (R) wings: inbred males = 0.8942, = 0.8935; outbred males = 0.9003, = 0.8980. ^n.s.^P\> 0.10; ^¶^0.10 \>P\> 0.05; \* P\< 0.05; \\\* P\< 0.001. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Asymmetry of overall wing size for females raised at 23°C Same as in Table 1 for Drosophila subobscuraflies reared at 23°C ::: Inbred Outbred --------------------- -------------------- -------- --------------- -------------------- ------ --------------- -------------------- Source of variation Variance component d.f. Mean Square Estimated variance d.f. Mean Square Estimated variance Individuals (I) 107 64.857\\\* 15.8796 215 49.100\\\* 11.9347 Karyotypes (K) (CS) 2 27.808^n.s.^ -1.8893 5 457.293\\\* 2.6178 Cross ⊂ K (CS) 15 299.873\\\* 11.3901 30 80.332\\\* 1.9907 Among flies (CS) 90 26.511\\\* 6.2931 180 32.556\\\* 7.7987 Sides (S) 1 33.413\\\* 1 16.825\\\* I × S (CS) 107 1.339\\\* 0.5446 215 1.361\\\* 0.5916 Karyotypes (K) (DA~CS~) 2 0.681^n.s.^ -0.0125 5 4.333\* 0.0781 Cross ⊂ K (DA~CS~) 15 1.132^n.s.^ -0.0427 30 1.520^n.s.^ 0.0447 Within flies (DA~CS~) 90 1.388\\\* 0.5692 180 1.252\\\* 0.5371 Measurement error (CS) 216 0.250 0.2496 432 0.178 0.1778 Average CS for left (L) and right (R) wings: inbred females = 0.8999 mm, = 0.8960; outbred females = 0.9203, = 0.9184. ^n.s.^P\> 0.10; \* P\< 0.05; \\\* P\< 0.001. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Asymmetry of overall wing size for males raised at 23°C Same as in Table 1 for Drosophila subobscuraflies reared at 23°C ::: Inbred Outbred --------------------- -------------------- -------- --------------- -------------------- ------ --------------- -------------------- Source of variation Variance component d.f Mean Square Estimated variance d.f. Mean Square Estimated variance Individuals (I) 107 44.690\\\* 10.9045 215 28.772\\\* 6.8138 Karyotypes (K) (CS) 2 41.926^n.s.^ -0.6021 5 112.284^n.s.^ 0.3480 Cross ⊂ K (CS) 15 128.628\\\* 4.0778 30 62.165\\\* 1.7199 Among flies (CS) 90 30.762\\\* 7.4224 180 20.887\\\* 4.8425 Sides (S) 1 36.691\\\* 1 13.586\\ I × S (CS) 107 1.072\\\* 0.3553 215 1.517\\\* 0.6465 Karyotypes (K) (CS) 2 2.596^n.s.^ 0.0366 5 0.862^n.s.^ -0.0110 Cross ⊂ K (DA~CS~) 15 1.277^n.s.^ 0.0454 30 1.259^n.s.^ -0.0532 Within flies (DA~CS~) 90 1.004\\\* 0.3213 180 1.578\\\* 0.6771 Measurement error (CS) 216 0.361 0.3615 432 0.224 0.2240 Average CS for left (L) and right (R) wings: inbred males = 0.8112, = 0.8072 ; outbred males = 0.8277, = 0.8260. ^n.s.^P\> 0.10; \\ P\< 0.01; \\\* P\< 0.001. ::: No significant size differences were generally detected among karyotypes for average CS, although O~3+4~flies were always the biggest within inbred lines (Fig. [1](#F1){ref-type="fig"}). On the other hand, in outbred crosses heterokaryotypes were bigger than homokaryotypes (females: 18°C F~(1,195)~= 9.78, P= 0.002; 23°C F~(1,195)~= 9.19, P= 0.003; males: 18°C F~(1,195)~= 1.84, P= 0.176; 23°C F~(1,195)~= 4.23, P= 0.041), but interactions of dominance effects were observed in all samples with discernible heterosis in O~st~/O~3+4~lines when compared to their homokaryotypic counterparts. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Inbreeding and temperature effects on sizeHomokaryotipic averages for centroid size and centroid size FA (index FA1 in \[39\]) in inbred (black symbols) and outbred (open symbols) crosses. Small symbols give the average values for each of the three different homokaryotypes to appreciate the dispersion from the corresponding grand average (large symbols connected by lines). Squares give the values at 23°C and circles at 18°C. ::: ![](1471-2148-5-7-1) ::: In concert with some independent preliminary results using a set of O~st~isochromosomal lines \[[@B38]\] a quite remarkable finding here was that left wings were consistently bigger than the right ones, thus causing a generally highly significant DA (i.e., \"sides\" effect in Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}) of overall wing size even though DA was fairly subtle (see bottom statistics in Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}). Each Drosophilawing vein has dorsal and ventral components that come together after the apposition of the dorsal and ventral surfaces, but each vein protrudes only in one wing surface (\"corrugation\") \[[@B25]\]. When wings were mounted no attempt was made to standardize the surface position: in females 394 (60.8%) left and 387 (59.7%) right wings were mounted on the slides with the dorsal side up (χ^2^= 0.16, 1 df, P= 0.691); in males the corresponding figures were 383 (59.1%) and 401 (61.9%), respectively (χ^2^= 1.05, 1 df, P= 0.306). Potential biasing effects when measuring wings; namely, dorsal or ventral Bitmap images or possible differences between left and right wings when Bitmap images are captured from the top or bottom of the microscope slide, were checked from a subset of 75 females and 75 males. An additional set of two images for each wing were taken in the same session from the top and bottom of the slide and digitized once. The centroid size differences between the averages of both measurements was apparently random with respect to digitizing procedure and always lower than 0.07%, whereas left wings were 0.26% bigger than the right ones in females and 0.34% in males. We are, therefore, quite confident that the fairly subtle DA for wing CS is not an experimental artifact but a real phenomenon. In addition to DA, there was subtle but significant FA in all crosses (i.e., \"individuals × sides\" interaction effect in Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}) together with a small amount of genetic variation for DA in some of them. This last finding could hardly be attributable to a type I error because similar results had been previously obtained \[\[[@B38]\]; see below\]. Conversely, two-level nested ANOVAs to test for genetic components of overall size FA (using index FA1 in Palmer \[[@B39]\]) failed to show any statistically significant effects whatsoever (variance components ranging from -0.0047 to 0.0071 for karyotypes, and from -0.0343 to 0.0406 for crosses within karyotypes; values in pixels^2^). ### c) Consanguinity and temperature effects Inbreeding and environmental effects were simultaneously analyzed by contrasting isogenic vs. outbred homokaryotypic flies reared at both experimental temperatures (Fig. [1](#F1){ref-type="fig"}). Flies were obviously bigger when raised at the lowest temperature, and three-way factorial ANOVAs performed separately for each sex using CS (as log~e~(pixels), but results were qualitatively identical without a log-transformation) as the dependent variable, with karyotype, temperature and inbreeding as fixed effects, and crosses nested within karyotypes, clearly indicated inbreeding depression together with temperature by inbreeding interaction (i.e., inbreeding was most noticeable at the sub-optimal temperature of 23°C), but no karyotype by temperature interaction was detected. These results confirm that wing size is not a purely additive trait in D. subobscura, in agreement with the previous observation that heterokaryotypes were bigger than homokaryotypes in outbred crosses (see also \[[@B40]\]). Both inbreeding and (sub-optimal) temperature effects were also apparent in females when overall size FA (index FA1) was used as the dependent variable in three-way factorial ANOVAs, with no differences among karyotypes. On the other hand, no statistically significant effects were detected for males, basically because inbred crosses performed approximately equal at both temperatures (Fig. [1](#F1){ref-type="fig"}). However, overall asymmetry augmented in inbred crosses because DA largely increased (mainly in males) at the highest temperature (\"temperature × inbreeding\" interaction: F~(1,856)~= 9.46, P= 0.002). It is worth mentioning here that in outbred crosses overall size FA was about the same for homokaryotypes and heterokaryotypes: the only significant effect was again an increase in FA at the sub-optimal temperature (more than two-fold; c.f. ![](1471-2148-5-7-i13.gif)(DA~CS~) values in Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}). Finally, inbreeding appears to have affected among-fly variation only in males as suggested by the consistently lower ![](1471-2148-5-7-i13.gif)(CS) estimates in outbred crosses within rearing temperature. In conclusion, overall size DS was positively correlated with levels of heterozygosity (i.e., inbred vs. outbred homokaryotypes) and development at the optimal temperature of 18°C. However, no positive association was found between DS and chromosomal heterozygosity in outbred crosses. Variation and asymmetry in shape -------------------------------- ### a) Sources of variation Two-way MANOVA analyses to quantify inter- and intra-individual variation in wing shape are shown in Tables [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}, [7](#T7){ref-type="table"}, [8](#T8){ref-type="table"}. For the present study of 13 landmarks, with 2 coordinates each, the shape dimension is 22. Sums of squares and cross-products (SSCP) matrices are therefore not full-ranked, and we retained 22 PC (principal components \[[@B41]\]) scores for outbred crosses and only 15 PC scores -- which accounted for more than 98% of the total shape variance -- for inbred crosses to be capable of testing for genetic components. The degrees of freedom in Tables [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}, [7](#T7){ref-type="table"}, [8](#T8){ref-type="table"} (columns \"df 1\") are simply the corresponding degrees of freedom in the ANOVAs for centroid size (Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}) times the number of PC scores retained in each sample. Likewise, the overall covariation in wing shape (\"individuals\" effect) was decomposed into causal components (karyotypes, crosses in karyotypes, and among flies); and the overall covariation in wing shape FA (\"individuals × sides\" interaction effect) was decomposed into causal components attributable to wing shape DA (karyotypes, crosses in karyotypes, and within flies). ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Asymmetry of overall wing shape for females raised at 18°C Flies raised from inbred (isogenic) and outbred crosses of Drosophila subobscurareared at 18°C. For the inbred crosses 15 PC scores were retained for analyses (proportion of total shape variance accounted is given in parenthesis). For the outbred crosses 22 PC scores were retained. (⊂ means \'nested in\'.) ::: Inbred (98.6%) Outbred --------------------- ---------------- --------- ------ --------- ---------------- ------ ------ --------- Source of variation Wilks\' lambda df 1 df 2 P Wilks\' lambda df 1 df 2 P Individuals (I) 1.13 × 10^-11^ 1605 1464 \<0.001 5.14 × 10^-15^ 4730 4442 \<0.001 Karyotypes (K) 0.002 30 2 0.505 7.44 × 10^-5^ 110 48 \<0.001 Cross ⊂ K 1.51 × 10^-4^ 225 843 \<0.001 3.68 × 10^-4^ 660 2932 \<0.001 Among flies 2.23 × 10^-8^ 1350 1457 \<0.001 7.55 × 10^-12^ 3960 4430 \<0.001 Sides (S) 0.597 15 93 \<0.001 0.563 22 194 \<0.001 I × S 1.58 × 10^-9^ 1605 3083 \<0.001 6.46 × 10^-11^ 4730 9192 \<0.001 Karyotypes (K) 0.003 30 2 0.546 0.002 110 48 0.301 Cross ⊂ K 0.074 225 843 0.362 0.018 660 2932 0.023 Within flies 9.69 × 10^-9^ 1350 3070 \<0.001 7.91 × 10^-10^ 3960 9169 \<0.001 ::: ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Asymmetry of overall wing shape for males raised at 18°C Same as in Table 5. ::: Inbred (98.5%) Outbred --------------------- ---------------- --------- ------ --------- ---------------- ------ ------ --------- Source of variation Wilks\' lambda df 1 df 2 P Wilks\' lambda df 1 df 2 P Individuals (I) 7.18 × 10^-12^ 1605 1464 \<0.001 3.51 × 10^-14^ 4730 4442 \<0.001 Karyotypes (K) 0.004 30 2 0.633 2.49 × 10^-4^ 110 48 0.006 Cross ⊂ K 1.61 × 10^-4^ 225 843 \<0.001 2.98 × 10^-4^ 660 2932 \<0.001 Among flies 1.47 × 10^-8^ 1350 1457 \<0.001 3.62 × 10^-11^ 3960 4430 \<0.001 Sides (S) 0.658 15 93 \<0.001 0.569 22 194 \<0.001 I × S 5.58 × 10^-8^ 1605 3083 \<0.001 1.28 × 10^-10^ 4730 9192 \<0.001 Karyotypes (K) 0.004 30 2 0.605 0.003 110 48 0.449 Cross ⊂ K 0.068 225 843 0.236 0.019 660 2932 0.036 Within flies 3.05 × 10^-7^ 1350 3070 \<0.001 1.73 × 10^-9^ 3960 9169 \<0.001 ::: ::: {#T7 .table-wrap} Table 7 ::: {.caption} ###### Asymmetry of overall wing shape for females raised at 23°C Same as in Table 5 for Drosophila subobscuraflies reared at 23°C. ::: Inbred (98.3%) Outbred --------------------- ---------------- --------- ------ --------- ---------------- ------ ------ --------- Source of variation Wilks\' lambda df 1 df 2 P Wilks\' lambda df 1 df 2 P Individuals (I) 1.07 × 10^-12^ 1605 1464 \<0.001 1.08 × 10^-13^ 4730 4442 \<0.001 Karyotypes (K) 2.18 × 10^-4^ 30 2 0.200 0.001 110 48 0.146 Cross ⊂ K 3.31 × 10^-4^ 225 843 \<0.001 1.81 × 10^-4^ 660 2932 \<0.001 Among flies 2.32 × 10^-9^ 1350 1457 \<0.001 1.54 × 10^-10^ 3960 4430 \<0.001 Sides (S) 0.450 15 93 \<0.001 0.585 22 194 \<0.001 I × S 2.57 × 10^-9^ 1605 3083 \<0.001 3.21 × 10^-13^ 4730 9192 \<0.001 Karyotypes (K) 0.007 30 2 0.725 0.006 110 48 0.842 Cross ⊂ K 0.055 225 843 0.062 0.034 660 2932 0.889 Within flies 1.95 × 10^-8^ 1350 3070 \<0.001 3.54 × 10^-12^ 3960 9169 \<0.001 ::: ::: {#T8 .table-wrap} Table 8 ::: {.caption} ###### Asymmetry of overall wing shape for males raised at 23°C Same as in Table 5 for Drosophila subobscuraflies reared at 23°C. ::: Inbred (98.3%) Outbred --------------------- ---------------- --------- ------ --------- ---------------- ------ ------ --------- Source of variation Wilks\' lambda df 1 df 2 P Wilks\' lambda df 1 df 2 P Individuals (I) 5.39 × 10^-12^ 1605 1464 \<0.001 6.41 × 10^-14^ 4730 4442 \<0.001 Karyotypes (K) 8.81 × 10^-4^ 30 2 0.364 1.18 × 10^-4^ 110 48 \<0.001 Cross ⊂ K 2.75 × 10^-4^ 225 843 \<0.001 1.96 × 10^-4^ 660 2932 \<0.001 Among flies 8.92 × 10^-9^ 1350 1457 \<0.001 7.94 × 10^-11^ 3960 4430 \<0.001 Sides (S) 0.642 15 93 \<0.001 0.540 22 194 \<0.001 I × S 8.58 × 10^-9^ 1605 3083 \<0.001 9.84 × 10^-12^ 4730 9192 \<0.001 Karyotypes (K) 5.13 × 10^-5^ 30 2 0.102 6.40 × 10^-4^ 110 48 0.052 Cross ⊂ K 0.060 225 843 0.111 0.024 660 2932 0.250 Within flies 5.79 × 10^-8^ 1350 3070 \<0.001 1.26 × 10^-10^ 3960 9169 \<0.001 ::: Similarly to what had been found for CS, differences between left and right wings were also highly significant (\"sides\" effect), thus indicating that DA was present for overall wing shape. This finding is contrary to our previous claim from a subset of O~st~isochromosomal lines, where DA for some landmarks (e.g. those defining the position of the anterior crossvein) but not for overall wing shape was detected \[[@B38]\]. After plotting the Procrustes grand mean shapes of both wings it also became apparent here that the location of the anterior crossvein was indeed slightly more distal in the right wings. Furthermore, the individuals × sides interaction effects were highly significant in all cases and, hence, wing shape FA greatly exceeded measurement error. ### b) Causal components of variation As has been forcefully stressed \[[@B42]\] shape is an inherently multidimensional concept and cannot be easily reduced to a scalar index without severe loss of information. Therefore, for a quantitative genetic analysis of shape data a multivariate approach is required \[[@B43]\]. For overall wing shape, genetic differences among karyotypes were mostly detected for outbred crosses (Tables [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}, [7](#T7){ref-type="table"}, [8](#T8){ref-type="table"}), and we have estimated the covariance matrices P= K+ C+ Eas a simple multivariate extension of the two-level nested ANOVAs, where Pis the phenotypic covariance matrix and K, C, and Eare, respectively, the covariance matrices for karyotypes, crosses within karyotypes, and the residuals. Fig. [2](#F2){ref-type="fig"} shows the amount of variation associated with the different dimensions in shape space. Much of the variation was concentrated in the first few PCs, but the Kmatrices showed the clearest trend to quickly decrease after the first PC. Permutation tests indicated that matrix correlations (MCs) between Kand Cmatrices were generally higher at 18°C (females MC = 0.258, P= 0.1908 ; males MC = 0.305, P= 0.1963) than at 23°C (females MC = 0.157, P= 0.3356 ; males MC = 0.250, P= 0.2665), but none of the MCs was statistically significant. On the other hand, VCV matrices were correlated across rearing temperatures (females: MC K= 0.716, P= 0.0163; MC C= 0.818, P= 0.0001; males: MC K= 0.706, P= 0.0160 ; MC C= 0.587, P= 0.0399 ; this last correlation was no longer significant after the Bonferroni procedure \[[@B44]\]). A close inspection to Fig. [2](#F2){ref-type="fig"} reveals an increase in the genetic components of overall wing shape at 23°C, which agrees with our preliminary findings \[[@B38]\]. Thus, the ratio between the total variance of genetic (G= K+ C) covariance matrix onto the total variance of the phenotypic covariance matrix was lower at 18°C in both sexes (females: 0.1312 vs. 0.5450; males: 0.2365 vs. 0.2522). A caveat: these ratios cannot be interpreted as estimates of shape heritability \[[@B43]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Eigenvalues of causal covariance matrices for wing shapeFirst 15 eigenvalues of the phenotypic (black bars), karyotype (hatched) and crosses (open) covariance matrices from outbred crosses. ::: ![](1471-2148-5-7-2) ::: MANOVA results in Tables [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}, [7](#T7){ref-type="table"}, [8](#T8){ref-type="table"} also point to the presence of genetic variation for overall shape DA, mainly at 18°C (i.e., the \"crosses in karyotypes\" component from the decomposition of the I × S interaction effect). As far as we are aware, these are the first experiments that found detectable genetic variation in DA for wing traits. The uncovering of DA (i.e., \"side\" effect) for fly wings is quite general when quantitative analyses of form are carried out using the powerful methods of geometric morphometrics to reveal even small morphological variation that otherwise would remain hidden with less effective techniques \[[@B13],[@B45]\]. This has raised concerns against the conventional wisdom that left and right are not distinguished in Drosophiladevelopment \[[@B46]\] because it provides compelling evidence that DA in fly wings may signal the presence of genetic variation in a phylogenetic conserved left-right developmental axis (i.e., an imaginary plane between the two lateral sides of the body), as discussed by Klingenberg et al. \[[@B45]\]. Actually, modern treatises in developmental biology (e.g. \[[@B9]\]) distinguish the left-right axis besides the customary anterior-posterior and dorsal-ventral axes, and several asymmetrically expressed genes (e.g. sonic hedgehog) have recently been discovered. In Drosophila, Ligoxygakis et al. \[[@B47]\] were the first (and to our knowledge the only ones) who showed a developmental mechanism for the developmental asymmetry. It seems, therefore, that the detection of genetic variation for DA in this genus appears to be basically a methodological problem, including statistical power and the environmental conditions where the experiments are performed. The mechanisms that constitute the genetic basis of morphological asymmetry in Drosophilaobviously require further study. ### c) Genetic components of wing shape FA Following \[[@B13]\] a multivariate equivalent of FA1 (i.e., the \"unsigned\" left-right differences) was defined by changing the signs of all coordinate differences (from left-right to right-left) whenever the inner product (also referred to as the dot product) of a left-right difference vector with the vector of mean left-right difference was negative. For the univariate case (CS) this procedure would render here the absolute (![](1471-2148-5-7-i10.gif)) differences, but notice that for the multivariate case it is not equivalent to calculate the absolute (![](1471-2148-5-7-i10.gif)) differences of all Procrustes coordinates. MANOVA analyses of these \"unsigned\" shape asymmetries in outbred crosses did not detect any genetic variation for shape FA at 18°C or 23°C (Tables [9](#T9){ref-type="table"}, [10](#T10){ref-type="table"}). However, the approach used to define the multivariate equivalent of FA1 might be influenced by the arbitrary choice of the plane (i.e., the mean left-right differences) to subdivide the shape space into \"positive\" and \"negative\" halves (Christian P Klingenberg, pers. comm. 2004). A modified Procrustes shape distance for non-isotropic variation (i.e., landmarks usually differ in their amounts of variation) has been recently developed by Klingenberg and Monteiro \[[@B48]\], and can be used here as a scalar measure of the amount of shape asymmetry because FA is random in origin (i.e., only the magnitude and not the direction may usually be the interesting component of FA shape variation). When this scalar was used in our data set the same conclusion was obtained; namely, there was no detectable genetic variation for wing shape FA in any case (results not shown). ::: {#T9 .table-wrap} Table 9 ::: {.caption} ###### MANOVAs for female wing shape fluctuating asymmetry A multivariate equivalent of FA1 (i.e., the \"unsigned\" left-right differences) was defined as explained in the text. Flies raised from outbred crosses of Drosophila subobscura(⊂ means \'nested in\'). ::: 18°C 23°C --------------------- ---------------- ------ ------ ------- ---------------- ------ ------ ------- Source of variation Wilks\' lambda df 1 df 2 P Wilks\' lambda df 1 df 2 P Karyotypes (K) 0.008 110 48 0.908 0.007 110 48 0.856 Cross ⊂ K 0.022 660 2932 0.169 0.029 660 2932 0.604 ::: ::: {#T10 .table-wrap} Table 10 ::: {.caption} ###### MANOVAs for male wing shape fluctuating asymmetry Same as in Table 9. ::: 18°C 23°C --------------------- ---------------- ------ ------ ------- ---------------- ------ ------ ------- Source of variation Wilks\' lambda df 1 df 2 P Wilks\' lambda df 1 df 2 P Karyotypes (K) 0.004 110 48 0.627 0.009 110 48 0.938 Cross ⊂ K 0.024 660 2932 0.243 0.042 660 2932 0.988 ::: ### d) Consanguinity and temperature effects on wing shape To investigate allometric and nonallometric temperature effects on overall wing shape we performed a multivariate analyses of covariance (MANCOVA) of the Procrustes coordinates (after averaging both sides and the two replicated measurements per side) considering temperature and inbreeding (i.e., isogenic vs. outbred homokaryotypic flies) as the categorical predictors and CS (as log~e~(pixels)) as the covariate. Temperature effects were only significant in males, but inbreeding and temperature × inbreeding interaction effects were highly significant in both sexes (results not shown), which suggests a strong effect of the categorical predictors on the nonallometric component of shape. Size effects were also found to be significant (females: Wilks\' λ= 0.881, F~(22,405)~= 2.496, P\< 0.001; males: Wilks\' λ= 0.915, F~(22,405)~= 1.715, P= 0.024), but the allometric effect on shape remained relatively consistent at both temperatures in females (size × temperature interaction: Wilks\' λ= 0.930, F~(22,405)~= 1.395, P= 0.111) but not in males (Wilks\' λ= 0.853, F~(22,405)~= 3.165, P\<0.001). The association between size and temperature (Fig. [1](#F1){ref-type="fig"}), measured by the variance inflaction factor (VIF\< 5; \[[@B49]\]), was found to be lower than the suggested guideline for serious collinearity (i.e. VIF≥ 10), which indicates that the effects of temperature and size on wing shape could be effectively separated. The conclusion is, therefore, that Drosophilawing shape does not seem to be as resistant to environmental temperature as previously claimed from the analysis of 12 highly inbred D. melanogasterlines \[[@B29]\]. Inbreeding effects (isogenic vs. outbred homokaryotypic flies) on wing shape FA were tested from the ratio between the traces of the corresponding \"individual × side\" VCV matrices. Notice that the traces of these interaction matrices are equal to the respective mean squares of the Procrustes ANOVA as implemented by Klingenberg and McIntyre \[[@B13]\], and are simply the sum of ![](1471-2148-5-7-i20.gif) (index FA4 in \[[@B39]\]) for each xand ycoordinates of the corresponding aligned configurations divided by the shape dimension. We performed 10,000 randomization runs for each test. Inbreeding effects were detected at 18°C but only in females (18°C: female F= 1.694, P= 0.0003 ; male F= 0.963, P= 0.6037 ; 23°C: female F= 0.834, P= 0.9231; male F= 0.984, P= 0.5541). Patterns of wing shape variation -------------------------------- ### a) Fluctuating asymmetry Principal component analyses were only implemented for the outbred crosses since they are more representative of the natural situation. The percentages of total shape variation, together with the features of variation associated with the dominant PCs, are graphically plotted in Figs. [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}, [6](#F6){ref-type="fig"}. For the individual variation several PCs accounted for relatively large amounts of variability. On the contrary, for FA and measurement error PC1 explained almost all total variance (\>80%). For all levels in the analysis (i.e. individuals, FA and measurement error) the dominant PCs were connected to the relatively large variability of landmarks 3, 6, 7 and, to a lesser extent, landmark 2. However, the disproportionate amount of variation associated with these landmarks did not spread to all sources of causal variation because their coefficients were relatively small for the PC1 of karyotype variation (which explained \~60% of the total variance; see below). Furthermore, for the individual variation the first two PCs were also linked to the shift of the anterior (landmarks 11 and 12) and posterior (landmarks 7 and 13) crossveins along the adjoining longitudinal veins. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Vectors of the landmarks displacementsFirst two axes of wing shape variation for each effect in the two-way mixed MANOVA (individuals, individuals × sides interaction, and measurement error) for females from outbred crosses reared at 18°C. Also plotted are the percentages of total wing shape variation explained by the principal components for the corresponding covariance matrices. ::: ![](1471-2148-5-7-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Vectors of the landmarks displacementsSame as Fig. 3 for males from outbred crosses reared at 18°C. ::: ![](1471-2148-5-7-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Vectors of the landmarks displacementsSame as Fig. 3 for females from outbred crosses reared at 23°C. ::: ![](1471-2148-5-7-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Vectors of the landmarks displacementsSame as Fig. 3 for males from outbred crosses reared at 23°C. ::: ![](1471-2148-5-7-6) ::: Permutation tests indicated that VCV matrices were mostly correlated for FA and measurement error effects within samples (MCs \> 0.95, P\< 0.01; Table [11](#T11){ref-type="table"}). The individual VCV matrix was significantly correlated with the FA and measurement error matrices only for females at 18°C. Between temperatures the VCV matrices were highly correlated for FA and measurement error (results not shown), but loosely correlated for the individual variation (females MC = 0.668, P= 0.0355 ; males MC = 0.494, P= 0.1066 ; statistical significance vanishes after the Bonferroni procedure). ::: {#T11 .table-wrap} Table 11 ::: {.caption} ###### Correlations between VCV matrices of landmarks displacements within groups Results of the permutation tests used for the analyses within sexes and temperatures. ::: Group Effects Correlation P(permutation) P(Bonferroni) -------------- -------------------- ------------- ------------------ ----------------- Females 18°C Individual / FA 0.7699 0.0001 \\ Karyotype / FA -0.1691 0.7583 n.s. Cross / FA 0.5773 0.0871 n.s. Between-fly / FA 0.7517 0.0001 \\ Individual / error 0.7550 0.0001 \\ FA / error 0.9953 0.0001 \\ Males 18°C Individual / FA -0.3998 0.8694 n.s. Karyotype / FA 0.0067 0.4393 n.s. Cross / FA -0.0706 0.6202 n.s. Between-fly / FA 0.2060 0.3296 n.s. Individual / error -0.4280 0.9436 n.s. FA / error 0.9964 0.0001 \\ Females 23°C Individual / FA 0.1233 0.2881 n.s. Karyotype / FA -0.0151 0.4771 n.s. Cross / FA 0.6516 0.0264 n.s. Between-fly / FA 0.5764 0.0744 n.s. Individual / error 0.1093 0.3141 n.s. FA / error 0.9959 0.0001 \\ Males 23°C Individual / FA 0.5278 0.0523 n.s. Karyotype / FA -0.1469 0.7545 n.s. Cross / FA 0.2752 0.1817 n.s. Between-fly / FA 0.4033 0.1241 n.s. Individual / error 0.5165 0.0519 n.s. FA / error 0.9922 0.0001 \\ n.s. = P\> 0.05; \\ = P\< 0.01. ::: The angles between the PC1s for FA and measurement error were very much alike (ranging from angle α= 2.1° to α= 3.4° ; recall that the 0.1% quantile of the resulting distribution between pairs of random vectors in 22-dimensional space was 50.3°), which reflects the similarity due to landmarks 3, 6 and 7. However, the first three PCs for interindividual variation were generally distinct to those of FA: the only clear correspondences were between the PC1s for females at 18°C (α= 21.5°), and the PC2 of interindividual variation with the PC1 of FA for males at 18°C (α= 11.8°). (The correspondences were qualitatively the same for interindividual variation and measurement error; results not shown.) Overall, these results seem to suggest that canalization and DS do not generally share the same underlying regulatory mechanisms (but see below). A potentially important problem with the foregoing approach to compare the patterns of intra- and interindividual variation is to rely on the interaction VCV matrix as the source of variation due to FA. As has been previously argued the uncovering of DA is almost ubiquitous for shape data when using the methods of geometry morphometrics, and there was evidence here for statistically significant genetic variation of overall shape DA at 18°C (Tables [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}). Therefore, the VCV matrix from the \"individuals × sides\" interaction effect gives a biased estimate of developmental stability and cannot be taken as the covariance matrix for FA. In other words, this VCV matrix also includes all causal components due to genetic variation for DA, and the corresponding unbiased VCV matrix for FA is that for the within-fly component of the interaction effect (i.e., after removing the genetic variation for DA \[[@B50],[@B51]\]). In any case, all results were qualitatively similar and, hence, the conclusion that canalization and DS seem to be different mechanisms remains unchanged. However, it is difficult to appraise how this potential problem could have affected the previously published conclusions when comparing interindividual variation and \"FA\" in fly wings and mouse skulls (see Background section). Between rearing temperatures the congruence of PC1 eigenvectors was also very high for FA (females α= 4.0°; males α= 3.5°) and measurement error (females α= 3.1°; males α= 4.1°). For the interindividual variation the correlations between PC1s were significant only in males (females α= 74.3°; males α= 19.3°); however, the PC1 vector describing the joint interindividual variation of landmark position in females at 18°C matched the PC2 of the interindividual covariance matrix at 23°C (α= 49.6°; recall that the direction of PCs is arbitrary and all the movements in Figs. [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}, [6](#F6){ref-type="fig"} can be simultaneously reversed by 180°) and vice versa(i.e., PC1 at 23°C vsPC2 at 18°C: α= 26.4°). ### b) Causal components Besides the interindividual variation in the two-way MANOVAs (which comprises genetic plus environmental covariances due to special environmental effects) it is important here to asses the patterns of joint displacements of landmarks for each of the causal components of wing shape variation (Figs. [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}, [9](#F9){ref-type="fig"}, [10](#F10){ref-type="fig"}). For karyotype variation PC1 accounted for \~60% of the total variance and was linked to a great extent with equivalent movements of those landmarks defining the location of the crossveins, which shifted in the same direction. Landmarks 4 and 5 tended to move away each other, stretching the wing margin between longitudinal veins III and IV. Landmark 9 budged in the opposite direction to crossveins shifts, thus shaping the relationship between L1 to the total length of longitudinal vein IV (i.e. shape index L1 WL ; Fig. [12](#F12){ref-type="fig"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Vectors of the landmarks displacementsFirst two axes of wing shape variation in the two-level nested MANOVA (karyotypes, crosses nested in karyotypes, and within crosses) for each causal component effect pertaining to the inter-individual variation in females from outbred crosses reared at 18°C. Also plotted are the percentages of total wing shape variation explained by the principal components for the corresponding covariance matrices. ::: ![](1471-2148-5-7-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Vectors of the landmarks displacementsSame as Fig. 7 for males from outbred crosses reared at 18°C. ::: ![](1471-2148-5-7-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Vectors of the landmarks displacementsSame as Fig. 7 for females from outbred crosses reared at 23°C. ::: ![](1471-2148-5-7-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### Vectors of the landmarks displacementsSame as Fig. 7 for males from outbred crosses reared at 23°C. ::: ![](1471-2148-5-7-10) ::: ::: {#F12 .fig} Figure 12 ::: {.caption} ###### *Left wing of Drosophila subobscura**The image shows the thirteen landmarks (1 -- 13) used in this work. I -- VI longitudinal veins; cv-a and cv-p anterior and posterior crossveins; Co costal or marginal veins; L1 and L2 lengths of the proximal (Euclidian distance between landmarks 9 and 13) and distal (Euclidian distance between landmarks 13 and 5) segments of longitudinal vein IV, respectively. Wing shape index ![](1471-2148-5-7-i21.gif) has been previously used to study shape clines in this species \[30\]. ::: ![](1471-2148-5-7-12) ::: A relative shortening of the basal length of longitudinal vein IV relative to the total wing length with an increasing dose of standard gene arrangements in all five major chromosomes of D. subobscurahad been previously identified in an outbred stock \[[@B32],[@B33]\]. A similar pattern regarding O~st~dose is also clear here when considering the six karyotypes (Fig. [11](#F11){ref-type="fig"}), but rearing temperature quantitatively modified the shape index (L1/WL was lower at the highest temperature). However, there was no statistically significant karyotype × temperature interaction. The wing shape index appears to be a purely additive trait since heterokaryotypes were always intermediate to their corresponding homokaryotypes (Fig. [11](#F11){ref-type="fig"}). Actually, none of 12 within- group (i.e., sex and temperature) possible contrasts comparing all three heterokaryotypes with the average of the corresponding homokaryotypes was statistically significant (the mean square for \"crosses\" was used as the error term; see legend in Fig. [11](#F11){ref-type="fig"}). ::: {#F11 .fig} Figure 11 ::: {.caption} ###### Wing shape indexAverages of the relative length (with 95% confidence intervals) of the basal portion of longitudinal vein IV (L1) to the total wing length (WL = L1 + L2) versus karyotype for outbred crosses at the two rearing temperatures. A two-way factorial ANOVA using the shape index as ![](1471-2148-5-7-i22.gif), with karyotype and temperature as fixed effects, and crosses nested within karyotypes, detected statistically significant differences for the main effects (karyotype: female F~5,30~= 12.625, P\< 0.001; male F~5,30~= 9.785, P\< 0.001. Temperature: female F~1,390~= 30.219, P\< 0.001; male F~1,390~= 61.835, P\< 0.001) but no karyotype × temperature interaction (females: F~5,390~= 1.570, P= 0.168; males: F~5,390~= 1.111, P= 0.354). ::: ![](1471-2148-5-7-11) ::: PC2 for karyotypes was also connected to the variability of landmarks 3, 6 and 7. For the crosses component, several PCs explained relatively large amounts of variation, and shifts of crossveins now seem to be independent of each other at 18°C but not at 23°C. Finally, for the within-fly variation several PCs accounted for relatively large amounts of variability. PC1s were again connected to the variability of landmarks 3, 6 and 7; and PC2s to shifts in the anterior crossvein. The large amount of variation of the anterior and posterior crossveins for karyotypes and crosses can be interpreted in terms of developmental processes. The crossveins are determined after the longitudinal veins, and mutations that eliminate crossveins (e.g. crossveinless) do not affect the longitudinal veins; however, some mutants that affect the longitudinal veins also influence the crossveins (e.g. the vngroup in \[[@B1]\]). Intra- and interespecific studies in several Drosophilaspecies have found displacements of one or both crossveins along their longitudinal veins, and such shifts also occur in a number of mutants (see \[[@B23]\]). However, these shifts do not occur in isolation an also include other landmarks as well (e.g., landmarks 9 and 5 on L4; landmarks 1 and 2 on L1; Figs. [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}, [9](#F9){ref-type="fig"}, [10](#F10){ref-type="fig"}). The matrix permutation tests (Table [11](#T11){ref-type="table"}) indicated that the VCV matrices of karyotypes and crosses were never significantly correlated with the VCV matrices of FA and measurement error. The high correlation between the VCV matrices of the interindividual and FA effects for females at 18°C was basically due to the (micro-) environmental component. Also notice that all correlations between the VCV matrices of karyotype and FA effects were close to zero or even negative, which clearly suggests that this genetic component of canalization is unrelated to DS. In addition, the PC1s of karyotypes and FA were nearly at right angles (18°C: females α= 85.8°, males α= 77.3°; 23°C: females α= 75.6°, males α= 78.5°). The only matches were between PC2 of karyotypes and PC1 of FA for females at 18°C (α= 13.0°) and males at 23°C (α= 31.2°). The PC1s of crosses and FA were also poorly correlated; the only exception being females a 23°C (α= 43.3°). These results clearly support the hypothesis that genetic canalization and DS are not functionally the same mechanism. On the other hand, all observed angles involving PC1s between \"replicated genotypes\" (i.e. the between-fly component) and FA were relatively small and highly significant (18°C: females α= 20.1°, males α= 15.9° ; 23°C: females α= 22.7°, males α= 36.7°). (Results were qualitatively the same for all observed angles involving PC1s of the between-fly and measurement error covariance matrices; results not shown.) Together with the overall comparisons of the covariance matrices (Table [11](#T11){ref-type="table"}), these results indicate that (micro-) environmental canalization and DS share underlying regulatory mechanisms but are not identical. There was not a complete congruence as PC1 of FA accounted for most part of the variation, while PC1 of between-fly variation usually explained less than 50% of the total variance (Figs. [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}, [9](#F9){ref-type="fig"}, [10](#F10){ref-type="fig"}). To conclude, the theoretical lower limit for (micro-) environmental canalization (i.e., the environmental variance among genetically identical individuals) would be FA because the two sides share the same genome (barring unusual somatic mutation or somatic recombination) and nearly the same environment, so differences between sides are likely to be small. Under stabilizing selection this lower limit is obviously associated with higher fitness. However, this \"canalization limit\" would hardly ever be observed because of unavoidable additional sources of environmental variance (e.g. variation between vials, the position of the pupae in a vial, etc.). A similar logic than the one used in this work has been applied to distinguish between intrinsic and extrinsic stochastic variation in gene expression: intrinsic noise can be separated by contrasting the levels of gene expression in a construct with two identically regulated but fluorescently distinguishable gpfgenes in the Escherichia colichromosome, whereas extrinsic noise is inferred by the correlated variation between the two copies in the same environment \[[@B52],[@B53]\]. Conclusions =========== This study applied the methods of geometric morphometrics in the context of quantitative genetics of wing form variation using isochromosomal lines of D. subobscura. The main findings can be summarized as follows: (i) for the analysis of overall size, DS was positively correlated with levels of heterozygosity (i.e., inbred vs. outbred homokaryotypes) and development at the optimal temperature; however, no positive association was found between DS and chromosomal heterozygosity in outbred crosses; (ii) there was detectable genetic variation (mainly for overall shape) for the directional component of morphological asymmetry (i.e., DA) but not for FA, which likely reflects variation due to stochasticity in development; (iii) for analyses of shape, the patterns of covariation for FA and measurement error were highly concordant in all samples, which also provides strong reasons to conclude that FA is generated by random perturbations of developmental processes (obviously, this does not imply that DS is independent of the genetic background: wing shape FA was found to be higher in inbred females at 18°C when compared to their outbred homokaryotypic counterparts); (iv) the inter- and intraindividual variation patterns were generally poorly correlated, which supports the hypothesis that canalization and DS are distinct mechanisms; however, (v) the patterns of variation due to the (micro-) environmental component of canalization (i.e., the among-fly special environmental effects covariances) were quite similar to those observed for FA; (vi) the lack of a significant within-group correlation between the VCV matrices associated with the interindividual genetic components of canalization and FA, as well as the low similarity between the corresponding vectors describing variation of landmark position, strongly suggest that genetic and environmental canalization are not similar mechanisms. In addition, (vii) a discrepancy between sexes was observed in some situations; e.g. overall size FA increased with inbreeding and (sub-optimal) temperature effects mainly in females, and the allometric effect on wing shape at both experimental temperatures was similar in females but not in males. It is also interesting to note here that wing size (measured as WL; Fig. [12](#F12){ref-type="fig"}) clines for D. subobscuradeveloped in North America after \~20 years since colonization, but males were clearly lagging behind females \[[@B54]\]. What is not obvious, however, is why there is a difference between the sexes. It has been suggested that a relationship between canalization and DS could only reflect a common underlying association between character and fitness \[[@B55],[@B56]\]. Those traits under strong stabilizing selection may not be genetically canalized and the major source of selective pressure for canalization can result from the benefits gained by buffering the effects of environmental perturbations \[[@B4],[@B10]\]. The strongest evidence in favor of this hypothesis comes from the well-known genotype-phenotype mapping of RNA folding. Conservation of RNA secondary structure is under strong selection, and low structural plasticity is achieved through increasing the thermodynamic independence of any one structural component from the remaining structure \[[@B57]\]. Likewise, the flux summation theorem developed in the field of metabolic control analysis implies, if true, that phenotypic robustness is an inevitable outcome of the underlying metabolism and not a result of evolution (see \[[@B58]\]). However, it is still an open question whether or not natural wing shape changes in Drosophilaare adaptive. There are no consistent patterns between latitude and wing shape (e.g. \[[@B30]\]), contrarily to what happens for size-related traits where world-wide latitudinal clines are found with genetically larger individuals derived from higher latitudes (e.g. \[[@B30],[@B59]\]). Many genes with small additive effects on features of wing shape are dispersed along the Drosophilagenome (e.g. \[[@B60],[@B61]\]), and we have shown here that the wing shape index L1/WL appears to be a purely additive trait since heterokaryotypes were always intermediate to their homokaryotypic counterparts. The wing shape cline in North America colonizing populations of D. subobscura\[[@B30]\] can be largely accounted for parallel latitudinal clines in chromosomal gene arrangements \[[@B32],[@B33]\], and the small shifts of (e.g.) the anterior and posterior crossveins in relation to karyotype variation (Figs. [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}, [9](#F9){ref-type="fig"}, [10](#F10){ref-type="fig"}; notice that the plotted joint variation in landmark positions is an exaggeration of the actual variation in the data set) are difficult to link with any adaptive response to a better flight capacity. Actually, we lack even hypothetical functional explanations for subtle shape variation: Gilchrist et al. \[[@B54]\] speculated that wing shape variation in D. subobscuramay simply represent drift around an optimum. Our present results (points (v) and (vi) above) give some credence to that conjecture. Genetic canalization on wing shape does not seem to arise as a by-product of environmental canalization and, therefore, canalization is not a single mechanism to buffer any source of variation as has been suggested \[[@B10]\]. According to Graham et al. \[[@B62]\] the classical linear theory of DS can successfully account for both normally distributed error distributions and leptokurtic distributions caused by the admixture of individuals having different levels of DS, but cannot account for transitions between FA and DA. We have previously suggested, however, that a transition from \"ideal\" FA (i.e., a normal distribution of left -- right-side scores whose mean is zero) to a distribution showing DA could be made entirely compatible with what it is already known from classical quantitative genetics \[[@B38]\]. Shifts between asymmetry types (FA, DA and antisymmetry) have been reported to happen along a species distribution range \[[@B63]\], but unless the genetic component can be partitioned out the variation in left-right differences cannot be assumed to describe DS. From the results of outbred crosses reared at 18°C (Table [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}) it is possible to test here for the congruence between patterns of morphological variation with respect to the variation attributable to FA (i.e. the within-fly environmental component of the interaction term) and that attributable to genetic variation for DA (the within-fly genetic component due to crosses in karyotypes of the interaction term). The corresponding VCV matrices were highly correlated for females (MC = 0.914, P~(permutation)~= 0.0001) but not for males (MC = 0.064, P~(permutation)~= 0.485). The angles between the PC1s also reflect this discrepancy between samples (females α= 8.6° ; males α= 45.9°). When considered together, these results clearly suggest that FA and genetic variation for DA may or may not be functionally linked. Methods ======= Extraction of O chromosomes and fly handling -------------------------------------------- A large number of D. subobscuraisochromosomal lines for the O chromosome in an otherwise homogeneous genetic background were derived from an outbred stock collected at Puerto Montt (Chile; 41° 28\' S) in November 1999 as previously indicated \[[@B38]\]. Briefly, wild-type males were individually crossed to three or four virgin females from the highly homogeneous ch-cumarker strain, which is homozygous for the morphological recessive markers on the O chromosome cherryeyes (ch) and curledwings (cu) and fixed for the gene arrangement O~3+4~. A single ![](1471-2148-5-7-i23.gif) / O~+\ cu+\ ch~male from the offspring was backcrossed to ch-cufemales, and its arrangement on the wild-type chromosome was identified after four generations of backcrosses. Followed by at least another backcross to ch-cufemales, a single male from each line carrying the wild chromosome was crossed to two virgin females from the Va/Babalanced marker stock. This strain was derived from the ch-custock and carries the dominant lethal genes Varicose(Va) and Bare(Ba) on the O chromosome. The isochromosomal lines were established from the final crosses ♀♀ O~Va\ ch+\ cu~/ ![](1471-2148-5-7-i23.gif) × ♂♂ O~Va\ ch+\ cu~/ ![](1471-2148-5-7-i23.gif). All lines used here had a quasi-normal viability according to the recorded proportions of wild-type flies raised in the final crosses to obtain the isochromosomal lines \[[@B38]\]. The lines were kept at 18°C (12:12 light/dark cycle) in 130-mL bottles with low adult density to standardize the rearing conditions before egg collections. As previously indicated the experimental flies were obtained from 54 crosses. Reciprocal crosses were made for all outbred combinations by mating one-week virgin females and males. After three days the males were discarded and equal numbers of females from each reciprocal cross were placed together in a plastic chamber with a spoon containing non-nutritive agar with a generous smear of live yeast for egg collections. To standardize the experimental conditions, eggs from the inbred (isogenic) crosses were also obtained in a similar way; namely, after mating the flies in bottles and transferring the females to plastic chambers. Eggs were placed in six 2 × 8 cm vials with 6 mL of food (26 eggs/vial); three vials were kept at 18°C (optimal temperature) and the other three at 23°C (sub-optimal temperature). Within each experimental temperature the vials were randomly placed on the same incubator shelf. As a result, the total experiment consisted of 324 vials (162 vials at each experimental temperature), and all eggs were sampled on the same day. Emerging flies (not less than 2 or 3 days old) were stored in Eppendorf tubes with a 3:1 mixture of alcohol and glycerol at 4°C before wing measurements. All fly handling was done at room temperature using CO~2~anesthesia on flies not less than 6 h after eclosion. Wing measurements ----------------- Two randomly sampled females and males emerged from each vial were used for morphometric analyses. Both wings were removed from each fly and fixed in DPX under coverslips on microscope slides. Bitmap images were captured with a video camera (Sony CCD-Iris, Tokyo, Japan) connected to a PC computer with MGI VideoWave software and mounted on a compound microscope (Zeiss Axioskop, Jena, Germany), using a 2.5 × objective. Calibration of the optical system was checked at each session. The images were stored on a Dell Workstation PWS350. To quantify and minimize measurement error all wings were digitized two times at different sessions as follows: images of both the left and right wings were captured during a given session and after an entire round on all individuals the same process was repeated again. A similar procedure was also used to record the xand ycoordinates of 13 morphological landmarks (i.e., labeled geometric points located at the intersections of wing veins or at sites where veins reach the wing margin; Fig. [12](#F12){ref-type="fig"}) by using the Scion Image for Windows software \[[@B64]\]. Therefore, the process we used guaranteed that the observer was blind with respect to the results from previous measurements. Analysis of wing size and shape ------------------------------- Geometric morphometrics precisely separates morphological variation (i.e., variation in form) into size and shape components \[[@B21],[@B22]\]. Size is a one-dimensional trait and the measure most widely used in geometric morphometrics is centroid size (CS), computed here in a normalized form as the square root of the sum of squared Euclidian distances between each landmark to the centroid (center of gravity) of all landmarks divided by the square root of the number of landmarks. Individual size is therefore represented by four scalars, one for each side and session. The shape of an original configuration of landmarks is the geometrical information that is invariant to uniform scaling (variation in size), translation (differences in position), and rotation (differences in orientation). In contrast to size, shape is an inherently multidimensional space and we used Procrustes superimposition to characterize shape variation. This method allows comparing configurations of landmarks by optimally superimposing (according to a least-squares criterion) homologous landmarks in two or more specimens to achieve an overall best fit \[[@B65]\].Because the data set included both left and right wings (i.e., we are dealing with \"matching symmetry\" \[[@B66],[@B67]\]) our analyses also removed differences due to reflection by changing the sign of the xcoordinate of every landmark for configurations from the right side. The reflection, scaling, and superposition steps were performed for all wings within each cross and temperature simultaneously, which allows contrasting wing shapes between different lines or crosses. The final iteration to minimize the sum of the squared distances between the landmarks of all wings in the sample was done without additional scaling and, consequently, we performed a partial Procrustes fit according to Dryden and Mardia \[[@B22]\]. Given the small amounts of shape variation in this analysis rescaling the coordinates of each configuration by the scaling option 1/cos(ρ) \[[@B65]\] would have negligible effects on the results. The landmark coordinates after Procrustes superimposition are amenable to standard multivariate analyses. However, it is important to remember that the removal of size, position (in two dimensions), and orientation reduces the dimensional space to 2p-- 4, where pis the number of landmarks \[[@B22]\]. Thus, for the present study of 13 landmarks, with 2 coordinates each, the shape dimension is 22. Sums of squares and cross-products (SSCP) matrices are therefore not full-ranked, and the degrees of freedom need to be adjusted. There are three alternative ways of avoiding these difficulties \[[@B22],[@B67]\]: (i) to omit, after Procrustes superimposition of the complete configurations, the coordinates of any two landmarks; (ii) to retain 22 PC scores from the covariance matrix of the data set; (iii) to slightly modify the multivariate statistics (see below) by using the Moore-Penrose generalized inverse of the SSCP matrices so they can tolerate singular matrices, and compute the product of nonzero eigenvalues instead of the determinant of SSCP matrices. We have used here the second scheme. Experimental design and asymmetry analysis ------------------------------------------ Quantitative genetic studies of directional and fluctuating asymmetry obviously require measures from individuals that can be grouped into families or independent lines. Our final data set was a fully balanced design, comprising 54 crosses × 3 vials per cross × 2 females per vial × 2 males per vial × 2 sides per fly × 2 measurements per wing × 2 temperatures = 5,184 wing landmark configurations in total. Within each sex and temperature, least-squares (ANOVA) estimates of variance components (i.e. CS) can be easily obtained from the linear model: Y~ijkl~= μ+ κ~i~+ l~j(i)~+ ν~k(ji)~+ ε~l(kji)~, where μis the overall grand mean, κ~i~is the effect of the ith karyotype, l~j(i)~is the random effect of the jth cross within karyotype i, ν~k(ji)~is the random effect of the kth vial within cross jand karyotype i, and ε~l(kji)~is the residual error associated with the trait (i.e. the individual means computed from both sides and the two replicated measurements per side) of the lth individual within vial k, cross j, and karyotype i. Since there was no genetic variation within crosses, the residual error provides an estimate of the total special environmental effects variance (i.e. ![](1471-2148-5-7-i2.gif)). Variation among the three replicated vials was generally negligible (results not shown) and, therefore, we have conveniently reduced the previous model to a two-level nested ANOVA after grouping flies across vials. To first partition the total phenotypic variation into interindividual, intraindividual and measurement error components, we used the conventional mixed model, two-way ANOVA (or its MANOVA generalization; see below) for the study of left-right asymmetries \[[@B39]\]. In this ANOVA the main random effect of individuals stands for phenotypic variation in the trait (i.e. CS), the main fixed effect of body side is for directional asymmetry (DA) and tests whether or no the signed differences between the left and right wings \[designated as (![](1471-2148-5-7-i10.gif))\] have a mean of zero, the interaction term is a measure of fluctuating asymmetry (the variation in left-right differences among individuals) provided that there is no genetic variation for DA \[[@B51]\], and the error term gives an estimate of the measurement error. The two-level nested ANOVA can be straightforwardly subsumed within the two-way ANOVA. We now digress slightly to point out some inconsistencies in the literature on what is the appropriate error term to test for the \"interindividual\" effect in the mixed model, two-way ANOVA (either the individual × side interaction effect or the measurement error \[[@B13],[@B15],[@B68],[@B69]\]). Interindividual variation, even if of no general interest in most studies of asymmetry, comprises here genetic components (\"karyotype\" plus \"crosses within karyotypes\") and special environmental effects variance (![](1471-2148-5-7-i2.gif); there is no genetic variance within crosses). An estimated of the among-fly special environmental effects variance (i.e. ![](1471-2148-5-7-i3.gif)) is therefore obtained by subtracting the individual × side interaction effect (which includes ![](1471-2148-5-7-i4.gif) plus measurement error) as the appropriate error term. However, when genetic variation for DA is present the unbiased within-fly special environmental effects variance (i.e. FA) is estimated after partitioning the individual × side interaction effect into its causal components \[[@B51]\]. As pointed out by Klingenberg et al. \[[@B67]\] it is fairly straightforward to extent the preceding ANOVA approach to a full two-factor MANOVA to analyze wing shape asymmetry since all effects are computed from averages or contrasts in the same shape space. Recall that the traces of the corresponding SSCP matrices are just the sum of squares in the Procrustes ANOVA as implemented by Klingenberg and McIntyre \[[@B13]\], but this ANOVA is based on an isotropic model (i.e., it assumes that there is an equal amount of non-directional variation at each landmark \[[@B70]\]) that is not generally correct for any real data. Covariance (VCV) matrices for each effect in the MANOVA were calculated as a simple multivariate extension of the two-way ANOVA. Thus, the SSCP matrices were divided by the appropriate degrees of freedom, and effects were separated according to the expected mean squares in the ANOVA by subtracting the interaction covariance (VCV) matrix from the interindividual VCV matrix, and the error VCV matrix from the interaction one. Therefore, for (e.g.) outbred crosses the interindividual covariance components were calculated as ![](1471-2148-5-7-i24.gif) and the covariance components of FA as ![](1471-2148-5-7-i25.gif), were SSCP~I~is the interindividual SSCP matrix, SSCP~IS~is the interaction SSCP matrix, and SSCP~ME~is the measurement error SSCP matrix. The SSCP~I~matrix was further partitioned into among-karyotype SSCP~K~matrix, among-cross within karyotype SSCP~C⊂K~matrix, and the residual SSCP~e~matrix corresponding to the special environmental effects. As a result, genetic effects for overall wing shape were separated from special environmental effects according to the expected means squares in the two-level nested ANOVA. Therefore, for (e.g.) outbred crosses the karyotype covariance components were calculated as ![](1471-2148-5-7-i26.gif) (remind that the entries in the SSCP~I~matrix are equal to those computed from individual means times twice the number of independent measurements per wing), the cross covariance components as ![](1471-2148-5-7-i27.gif), and the among-fly special environmental effects covariance components as ![](1471-2148-5-7-i28.gif). Similarly, genetic effects for DA can be investigated after partitioning the SSCP~IS~matrix into their causal components \[[@B51]\]. Morphological patterns of variation ----------------------------------- Within each sex and temperature, principal component analyses \[[@B41]\] of the VCV matrices were performed for each source of variation with the purpose of describing the landmark displacements corresponding to each emerging principal component (PC), and also to test for the congruence of these displacements between effects. This technique extracts new shape variables (PCs) which successively account for the maximal amount of shape variation and contain information on how the variables relate to each other. The PCs form an orthonormal set of vectors (i.e., the inner product ![](1471-2148-5-7-i29.gif) for i ≠ j, ![](1471-2148-5-7-i30.gif) for i = j; superscript \'denotes transposition) in an n-dimensional space. Correlations between corresponding VCV matrices were computed from the upper triangular part (diagonal entries were included) since covariance matrices are symmetrical, and statistical significance was assessed using permutation tests designed to maintain the associations between pairs of x- and y-coordinates (i.e., by permuting pairs of rows and columns \[[@B13],[@B15]\]); otherwise the null hypothesis would imply the complete absence of all geometric structure. The permutation procedure was carried out 10,000 times. Correlative patterns of whole shape variation are difficult to interpret: a significant correlation would suggest a real congruence, but a weak congruence does not imply a significant correlation. A second test examined the congruence of the landmark displacements corresponding to each emergent PC for the different effects within groups. Because the PCs correspond to directions in the multivariate shape space, correlations can be obtained by angular comparisons of component vectors. Statistical significance of these correlations was then assessed by comparing those observed values to a null distribution of absolute angles between 100,000 pairs of 22-dimensional random vectors \[[@B71]\]. The 0.1% and 0.001% quantiles of the resulting distribution were 50.3° and 41.6°, respectively. Antisymmetry and allometric effects ----------------------------------- The occurrence of antisymmetry (AS: a bimodal distribution of signed (![](1471-2148-5-7-i10.gif)) \[[@B39]\]) for centroid size was investigated within each sample using the Lilliefors (Kolmogorov-Smirnov) test for the composite hypothesis of normality \[[@B69]\]. The independence between size and size FA within each sample was assessed by a linear regression of unsigned (![](1471-2148-5-7-i31.gif)) against mean centroid size ![](1471-2148-5-7-i32.gif). Scatter plots of left-right differences for each landmark after Procrustes superimposition were visually checked to see whether or not there was evidence for clustering of these vectors that would have argue for the occurrence of AS \[[@B13],[@B15]\]. No indication of AS was detected. Finally, to test for size effects on shape asymmetry within each sample we used multivariate regression of vectors of both signed and \"unsigned\" shape asymmetries onto mean centroid size \[[@B13]\]. Shape asymmetries were not related to size (P*-values \> 0.10) and, therefore, no size corrections were necessary. Computer software for statistical analysis ------------------------------------------ The computer programs used for statistical data analyses were MATLAB (V.6. \[[@B72]\]) together with the collection of tools supplied by the Statistics Toolbox (V.3. \[[@B73]\]). Some helpful functions in morphometrics from the MATLAB toolboxes Res5 and Res6 developed by R. E. Strauss \[[@B74]\] were also used. Results (e.g., derivation of SSCP matrices) were checked with the statistical software packages STATISTICA V.6 \[[@B75]\] and SPSS V.11 \[[@B76]\]. Authors\' contributions ======================= MS conceived the study, carried out extraction of O chromosomes, experimental crosses, egg collections, statistical analyses, and drafted the final manuscript. PFI carried out extraction of O chromosomes, experimental crosses, egg collections, wing measurements, and preliminary statistical analyses and drafts of results. WC read all salivary gland squashes for gene arrangement identification and mounted the wings on microscope slides. All authors read and approved the final manuscript. Acknowledgments =============== We thank Chris Klingenberg for illuminating discussions during the preparation of the manuscript, for providing insightful comments on an earlier draft, and for sharing with us unpublished manuscripts. We are also grateful to Andrew Pomiankowski and two anonymous reviewers for their thoughtful and highly constructive criticisms that substantially improved the manuscript. PFI was supported by a postdoctoral fellowship (SB2000-0370) from the Secretaría de Estado de Educación y Universidades del Ministerio de Educación, Cultura y Deporte (Spain). WC is supported by a postgraduate fellowship (FP2000-7001) from the Ministerio de Ciencia y Tecnología (Spain). This work was supported by grants BOS2000-0295-C02 and BOS2003-05904-C02 from the Ministerio de Ciencia y Tecnología (Spain), 2001SGR-00207 from the Direcció General de Recerca (Generalitat de Catalunya) to the GBE, and by Fundación Ramón Areces (Spain).	PubMed Central	2024-06-05T03:55:52.587143	2005-1-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548280/", "journal": "BMC Evol Biol. 2005 Jan 22; 5:7", "authors": [ { "first": "Mauro", "last": "Santos" }, { "first": "Pedro Fernández", "last": "Iriarte" }, { "first": "Walkiria", "last": "Céspedes" } ] }
PMC548281	Background ========== Ribosomal protein (RP) S6 is a phosphorylated protein that resides on the small subunit of eukaryotic ribosomes. Phosphorylation occurs on a cluster of five serine residues near the C-terminal end of the protein. Although details remain unclear, the phosphorylation state of RPS6 is believed to influence translational efficiency of some mRNAs \[[@B1]\], possibly mediated by direct contact between RPS6 and the 28S rRNA in the large subunit. RPS6 has also been implicated in ribosome biogenesis, and is thought to play a conserved role in the initiation of protein synthesis \[[@B2]\]. In Aedes aegyptiand Aedes albopictusmosquitoes, the RPS6 protein is \~17 kDa larger than its Drosophilahomolog, and on polyacrylamide gels, it migrates as the largest protein from the small ribosomal subunit. Ae. aegyptiand Ae. albopictusRPS6 cDNAs encode an approximately 100 amino acid extension at the C-terminal end of the protein. The extension is particularly rich in lysine, alanine and glutamic acid, and most closely resembles the sequence of histone H1 proteins from diverse sources \[[@B3]\]. Because RPS6 is thought to have regulatory function(s) in a variety of cell signaling pathways \[[@B2]\], we were surprised to uncover this difference between mosquito and DrosophilaRPS6 proteins. We have recently shown that RPS6 protein isolated from ribosomal subunits retains its histone H1-like tail \[[@B4]\]. Thus, unlike the case with the ubiquitinated ribosomal protein S27a in the rat \[[@B5]\], the histone tail is not removed from the mosquito ribosomal protein prior to ribosome assembly. RpS6cDNA from an Anopheles stephensicell line encodes an approximately 170 amino acid histone H1-like C-terminal extension, and in silico analysis reveals a similar modification encoded by the rpS6gene in Anopheles gambiae. In both Aedesand Anophelesmosquitoes, the C-terminal extension was completely encoded in Exon 3, directly contiguous with upstream open reading frame encoding the series of serines that may be phosphorylated \[[@B4]\]. Anopheline mosquitoes are believed to be ancestral to the Culicidae, which includes the genera Aedesand Culex\[[@B6]\]. Thus, to a first approximation, we infer that the longer tail in Anophelesmosquitoes represents the ancestral state, and that the RPS6 tail has been lost in the higher Diptera, which include D. melanogaster. Although mosquito RPS6 tails in general resemble histone H1 proteins, their divergence between Aedesand Anophelesmosquitoes was high, relative to the conventional portion of the RPS6 coding sequence. Because histone H1 is the most variable of the histone proteins, and functions as a linker, rather than as a component of the histone octamer, we set out to clone a cDNA encoding a bona fide histone H1 protein from an An. stephensicell line. In a phylogenetic comparison, the An. stephensihistone H1 protein clusters with homologs from Drosophilaand Chironomus, rather than with RPS6 histone H1-like tails from mosquitoes. These results indicate that the histone H1-like tails on mosquito RPS6 proteins are evolving independently of conspecific histone H1 proteins. Results ======= Design of PCR primers --------------------- The gene encoding Drosophila melanogasterhistone H1 spans 1204 nucleotides, and encodes a 256 amino acid protein in a single exon \[[@B7]\]. There is a single recorded His1allele in Drosophila\[[@B8]\], while multiple histone H1 variants have been described in Chironomid flies \[[@B9]-[@B11]\]. When the deduced sequence of the Drosophilahistone H1 protein (Accession NM\_058232) was compared to the Anopheles gambiaegenome using the program BLAST \[[@B12]\] on the NCBI website (National Center for Biotechnology Information; <http://www.ncbi.nlm.nih.gov/>), we obtained 5 accessions with E values ranging from 3e-35 to 8e-43, distributed on mosquito chromosomes 2 and 3. Upon further examination, we noted that XP\_314184 and XP\_314186 (chromosome 2) corresponded to the same protein. Two additional histone H1 candidates (XP\_311486 and XP\_309451) were encoded on chromosome 3. These three conceptual Anophelesproteins shared 70--80% identity to one another, and about 50% identity to the DrosophilaH1 protein sequence. In the EST-other database, we found a single uninformative match to an unidentified An. gambiaeentry (dbEST id = 11236311), with the relatively modest E value of 0.055. Histone H1 sequences from Aedesmosquitoes are not yet in existing databases. The 50% identity between Drosophilaand Anopheleshistone H1 proteins was relatively low, compared to approximately 80% amino acid identity between Drosophilaand AnophelesRPS6, exclusive of the histone-H1-like tail in the mosquito protein. The DrosophilaH1 histone was also \~50% identical to that from Chironomus thummi, a fly closely related to mosquitoes in the infraorder/superfamily Culicomorpha \[[@B13]\]. To design primers that would amplify a histone H1 gene, and not the histone H1-like tail in mosquito rpS6, we aligned one of the An. gambiaeH1 candidate proteins (XP\_311486) to a histone H1 protein from C. thummi, and examined the alignment for precise matches (Fig. [1A](#F1){ref-type="fig"}) that did not match well in a separate alignment of the An. gambiaehistone H1 protein with the An. gambiaeRPS6 tail (Fig. [1B](#F1){ref-type="fig"}). The forward primer (F1) corresponded to amino acids PKKPKKP in An. gambiae, and a reverse primer (R1) corresponded to residues AAKKPKA (Fig. [2](#F2){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Primer design. To design primers, we aligned an An. gambiaeputative histone H1 candidate XP\_311486 (Panel A, top) with a histone H1 protein (Q07134; Panel A, bottom) from C. thummi. Boxed residues were chosen for design of primers, according to the An. gambiaenucleotide sequence. Panel B shows these primer residues aligned between the An. stephensiRPS6 tail (top), and the putative Anopheles gambiaehistone H1 (bottom). Vertical bars designate identities. ::: ![](1471-2164-6-8-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Sequence of An. stephensihistone H1 gene. The positions of internal primers F1 and R1, and primers F2 and R2 are designated by arrows. The ATG start codon and TAA stop codon are boxed. ::: ![](1471-2164-6-8-2) ::: Recovery of An. stephensihistone H1 gene ------------------------------------------ We used F1 and R1 primers with HindIII-digested genomic DNA from An. stephensicells to obtain an approximately 450 bp PCR product, which was sequenced and verified to encode a histone H1 protein. The 5-end of the gene, which extended 81 nucleotides upstream of the ATG start codon, was obtained using primer R1 with the GeneRacer kit (Invitrogen, Carlsbad, CA), with total RNA as the template. The absence of a poly(A) tail on histone mRNAs required an unconventional strategy to obtain the 3\'-end of the coding sequence. First, we used HindIII-digested genomic DNA template, with a primer based entirely on the 3\'-UTR of An. gambiaeXP\_314184, without success. When we designed a second primer (R2, in Fig. [2](#F2){ref-type="fig"}) extending from the 3\'-UTR through the TAA stop codon and into the coding region, we obtained the 3\'-end of the coding sequence. Finally, primers F2 and R2 (Fig. [2](#F2){ref-type="fig"}) were used to verify the complete nucleotide sequence. Southern blots with An. stephensigenomic DNA ---------------------------------------------- The likelihood that the mosquito genome contains multiple histone H1 gene variants is consistent with the multiple H1 variants that have been described in Chironomus\[[@B9]-[@B11]\] and eight histone H1 subtypes that have been described in mammals \[[@B14],[@B15]\]. When we used the An. stephensicDNA to probe Southern blots of genomic DNA digested with various restriction enzymes with 6 bp recognition sites, most enzymes gave multiple bands, with the notable exception of BamHI, which hybridized to a single band longer than 10 kb (Fig. [3](#F3){ref-type="fig"}). Based on the observation that D. melanogasterH1, H2A, H2B, H3 and H4 histone genes are organized in approximately one hundred 5 kb repeats per haploid genome \[[@B16]\], the large BamHI fragment from An. stephensimay be a starting point for recovery of a complete cluster of the An. stephensihistone gene family. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Southern blot of An. stephensigenomic DNA hybridized to the An. stephensihistone H1 probe. DNA was digested with BamHI (B), EcoRI (E), HindIII (H) and PvuI (P). Positions of size markers are shown at right. ::: ![](1471-2164-6-8-3) ::: The An. stephensinucleotide sequence (GenBank accession \# AY672907) matched An. gambiaehistone H1 candidates on chromosomes 2 and 3 with an E value of 0.0. In addition, 6 unmapped sites also had E values of 0.0. A final two sites had E values of 4e-170 and 3e-127. The deduced An. stephensiprotein sequence was 92% identical to An. gambiaeprotein XP\_314184 on chromosome 2 (Fig. [4A](#F4){ref-type="fig"}). A similar level of identity was obtained with An. gambiaeXP\_309451 on chromosome 3, but the alignment required introduction of a 58 amino acid gap in the shorter (190 residue) deduced Anopheles gambiaeprotein (not shown). Identity with An. gambiaeXP-311486 was 79%. Based on these criteria, we have cloned the An. stephensihomolog of An. gambiaeXP\_314184. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Comparison of mosquito histone H1 proteins and RPS6 histone H1-like tails. Panel A shows the alignment of the experimentally-determined An. stephensihistone H1 amino acid sequence, compared to An. gambiaeconceptual protein XP\_314184. Panel B shows a phylogram produced in PAUP\* by neighbor joining, with the nematode C. eleganshistone H1-like protein 2 (AAM44399) designated as the outgroup. The alignment includes histone H1 proteins from various Diptera, and the known histone H1-like tails on mosquito RPS6. Values on the horizontal lines indicate branch lengths, defined as the fraction of substitutions between the nodes that define the branch. Bootstrap values based on 1000 replicates are shown within circles. A single tree with identical topology was obtained with the optimality criterion set to parsimony. ::: ![](1471-2164-6-8-4) ::: Comparisons of histone H1 proteins with mosquito RPS6 C-terminal extensions --------------------------------------------------------------------------- The identity between Drosophilaand Anopheles(or Drosophilaand Chironomus) histone H1 proteins was only 50%. This divergence undoubtedly reflects the \~250 million years \[[@B6]\] separating Nematoceran from Cyclorrhaphan diptera. In this study, we were interested in comparing mosquito histone H1 proteins to the histone H1-like tails of mosquito RPS6. Fig. [4B](#F4){ref-type="fig"} shows a neighbor-joining analysis in which we compared protein sequences from Aedesand AnophelesRPS6 histone H1-like tails, exclusive of the conventional RPS6 protein sequence, with histone H1 proteins from the nematode Caenorhabditis elegans(AAM44399), the closely-related flies Chironomus thummi(Q07134) and Chironomus tentans(AAB62239), Drosophila, and the Anopheles gambiaeand Anopheles stephensihomologs (Fig. [4A](#F4){ref-type="fig"}). With the C. eleganssequence designated as the outgroup, the phylogram shows that the RPS6 tails cluster into a distinct group relative to the Dipteran histone H1 proteins. Circled values indicate bootstrap values based on 1000 replicates. When the analysis was repeated with the optimality criterion set to parsimony, we obtained a tree with the same topology, with the 77% value shown in Fig. [4B](#F4){ref-type="fig"} reduced to 59%, and the 97% value reduced to 94%. The 100% values remained unchanged. In an alignment of mosquito RPS6 tails with the AnophelesH1 histones (Fig. [5](#F5){ref-type="fig"}), we note that while some degree of identity covers the entire histone H1 protein, the C-terminal half of the H1 histone has a higher proportion of identities to the RPS6 tail, as indicated by the distribution of consensus residues. Within the RPS6 tails, however, the boxed motifs:VAKK(D/E)A, KKEVKK, AAPA, KKEAPKRKPE, KG(D/E)ASAAK(E/D) are shared by all four mosquitoes. In contrast, the additional amino acids in the AnophelesRPS6 tails, which are represented by gaps in the Aedessequences (Fig. [5](#F5){ref-type="fig"}), did not show regions of homology with Anopheleshistone H1. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Alignment of mosquito RPS6 tails with mosquito histone H1 proteins. Angam (CAD89874), An. gambiae; Anstep (AY237124), An. stephensi; Aealbo (Q9U762), Ae. albopictus; Aeaegy (Q9U761), Ae. aegypti. The alignment was produced with ClustalX (version 1.83), using default settings. Indicators of consensus residues are shown below the alignment. Boxes in the top four entries indicate identities (aside from D, E substitutions) shared by the mosquito RPS6 tails. ::: ![](1471-2164-6-8-5) ::: Discussion ========== An important rationale for cloning an An. stephensihistone H1 was to compare its sequence to the histone H1-like tails on mosquito RPS6 ribosomal proteins. Our choice of an Anopheleshistone H1 was based on the existing database for An. gambiae, the observation that the tail in AnophelesRPS6 is nearly twice as long as that in AedesRPS6 proteins \[[@B4]\], and evidence that the genus Anophelesis ancestral to Aedes\[[@B6]\]. Because putative homologies to Drosophilahistone H1 protein could be recovered as conceptual translation products from the An. gambiaedatabase, we used these sequences to design primers that would discriminate between an An. stephensihistone H1 gene, and the histone H1-like extension in An. stephensiRPS6. Because the Drosophilagene was encoded in a single exon, and the histone message was unlikely to be polyadenylated \[[@B14]\], we used genomic DNA from An. stephensias a template for our PCR reaction. The gene we recovered had more than 90% identity to XP\_314184 in An. gambiae. The proteins differed in length by a single amino acid residue, and showed 92 % identity. When we analyzed RPS6 tails and histone H1 genes, we found that the Dipteran histone H1 proteins and the RPS6 tails each fell into distinct groups, suggesting that in present-day mosquitoes, these proteins are evolving independently. Although these data are consistent with the possibility that present-day histone H1 proteins and the histone H1-like tails on mosquito RPS6 protein share a common ancestral gene, the histone tails seem to be evolving independently in the two mosquito genera, and have changed more rapidly than the conventional portion of mosquito RPS6 proteins. Because RPS6 is considered an important functional component of the ribosome, it seems surprising that a histone H1-like tail occurs at the C-terminal end of this particular protein. However, histone H1-like tails have been reported at the N-terminus of Drosophila melanogasterribosomal proteins L22 and L23a \[[@B17]\]. The An. gambiaehomolog of D. melanogasterL23a also contains an N-terminal histone-like extension. The N-terminal tails of DrosophilaL22 and L23a were found in an effort to identify proteins that interact with poly (ADP-ribose) polymerase (PARP). In future studies, we plan to explore whether the histone H1-like tail undergoes posttranslational modification, and whether it plays a functional role in ribosome biogenesis, perhaps through the activity of PARP. Experimental procedures ----------------------- ### Mosquito cells and culture conditions We used the ASE-IV Anopheles stephensimosquito cell line \[[@B18]\], which was adapted to Eagle\'s minimal medium, supplemented with non-essential amino acids, glutamine and 5% heat-inactivated fetal bovine serum \[[@B19]\]. This formulation is called E-5 medium. ### Genomic DNA preparation Cells grown as suspended vesicles for 4 to 5 days in twenty 60 mm plates were collected by centrifugation, and the cell pellet was washed twice with phosphate-buffered saline (PBS; \[[@B20]\]). The cell pellet was resuspended in 20 ml lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 200 μg/ml proteinase K), and SDS was added to a final concentration of 0.5%. The lysate was incubated at 37°C overnight. NaCl was added to a final concentration of 0.4 M, and the DNA was extracted once with 20 ml phenol, twice with an equal volume of phenol:chloroform (1:1), and twice with an equal volume of chloroform. Two volumes of ethanol were added, and DNA was spooled onto a clean glass rod. The DNA was dried, and dissolved in 10 ml of TE (10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA) at 37°C. RNase A was added to a final concentration of 200 μg/ml and incubated at 37°C for 4 hours. DNA was phenol extracted, ethanol precipitated and dissolved in TE as described above. ### DNA amplification by PCR Genomic DNA (0.4 mg) was digested with HindIII (Promega) at 37°C overnight. Enzyme was removed by phenol:choloroform extraction, and the DNA was recovered by precipitation with ethanol and dissolved in TE. Digested DNA (100 ng) was used as template for the PCR reaction, which contained 1X PCR buffer, 1.5 mM MgCl~2~, 0.2 mM of each of the four dNTPs, 0.4 μM of primer F1 (5\'CCG AAG AAG CCG AAG AAG CCC) and R1 (5\'TGC TTT CGG CTT CTT GGC AGC) and 2.5 units of Taq DNA polymerase (Promega, Madison, WI). PCR was performed with an initial denaturation at 94°C for 2 minutes. The next 35 cycles included 94°C denaturation for 45 sec, 55°C annealing for 1 minute, and 72°C extension for 1 minute. The reaction was terminated by a final elongation cycle at 72°C for 2 minutes. The PCR product was recovered from a 0.9% agarose gel, purified using Ultra-Clean 15 (MO Bio Laboratories Inc., Solana Beach, CA) and cloned into PGEM T-Easy vector (Promega). The 3\'-end of the gene was obtained in a similar manner, using primers R2 (Fig. [2](#F2){ref-type="fig"}) and F1. ### Amplifying the 5\'-end of the cDNA Total RNA was recovered from ASE-IV cells by guanidine isothiocyanate extraction and cesium chloride centrifugation as described by Davis et al. \[[@B21]\]. The final RNA pellet was dissolved in DEPC-treated water and stored at -70°C. RNA (1 μg) was used with the GeneRacer kit (Invitrogen) to obtain the 5\' end of the mRNA, using primer R1 as the reverse primer. ### Programs and accession numbers The analysis in Fig. [4A](#F4){ref-type="fig"} was produced using the Genetics Computer Group (GCG; Madison, WI) program \"gap\". The tree in Fig. [4B](#F4){ref-type="fig"} and the alignment in Fig. [5](#F5){ref-type="fig"} were produced by an alignment of amino acid residues using default parameters of Clustal X (version 1.83) \[[@B22]\]. The tree was created in PAUP\* \[[@B23]\], with the C. elegansH1 protein designated as an outgroup. The An. stephensihistone H1 sequence has GenBank accession \# AY672907. Authors\' contributions ======================= YZ did the experimental work, AMF helped with experimental design and manuscript preparation. Both authors read and approved the final manuscript. Acknowledgments =============== We acknowledge support from the National Institutes of Health (AI 20385) and from the University of Minnesota Agricultural Experiment Station, St. Paul, MN.	PubMed Central	2024-06-05T03:55:52.595713	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548281/", "journal": "BMC Genomics. 2005 Jan 24; 6:8", "authors": [ { "first": "Yongjiao", "last": "Zhai" }, { "first": "Ann M", "last": "Fallon" } ] }
PMC548282	Background ========== Relatively little documentation supports a scientific basis for alternative healing therapies involving the manipulation of a purported healing energy associated with the body. Despite this paucity of evidence, such energy healingmodalities are becoming increasingly popular. Recent surveys indicate the majority of the United States public has used alternative medical therapies, and that energy healing therapies are among the fastest growing complementary and alternative medicine treatments \[[@B1],[@B2]\]. Johrei is one such energy healing practice with origins in Japan. Johrei was founded by Mokichi Okada in the 1920\'s and is now practiced worldwide with significant numbers of adherents in Asia, Europe and the Americas. Practitioners of Johrei believe it possible to improve the health of others by directing a universal healing energy toward them. The Johrei philosophy, as it relates to healing energy, emphasizes what can be described as \"spiritual purification.\" Other aspects of the Johrei philosophy include fostering an appreciation of beauty, and a form of organic farming. Two scientific studies evaluating Johrei healing practices have found provocative results. In one, Johrei was shown to have a beneficial affect on the mood of practitioners \[[@B3]\]. Results from a second study suggested that those practicing Johrei may display an immune profile consistent with stress reduction \[[@B4]\]. Clinical studies evaluating potential efficacy of Johrei are ongoing \[[@B5],[@B6]\]. An in vitro study revealed an apparent influence of Johrei treatment on the germination rate of irradiated seeds but the study did not follow standard scientific methods \[[@B7]\]. To investigate whether Johrei can have a direct effect on human cells, we exposed cultured cancer cells to Johrei treatment from a short distance. Johrei treatments were maintained continuously for four hours by teams of Johrei practitioners. Experiments were conducted using strict blinding procedures, exceeding standards commonly used to evaluate conventional therapeutics. Methods ======= Overall study design -------------------- For each session Johrei practitioners were asked to direct healing intention toward cell cultures in a temperature, CO~2~, and humidity controlled time-lapse microscope incubator chamber. Johrei practitioners participated in teams of two, alternating every half hour such that a total of 4 hours of Johrei treatment was delivered. A total of eight control and eight Johrei intervention experiments were accomplished. Johrei treatments were initiated after 4 hours of baseline data had been collected and the observation period extended for a total of 22 hours. We quantified tumor cell death and proliferation throughout the observation period. We also quantified the rate of cellular emigration (i.e. how many tracked cells left the microscopic field per hour) in order to accurately assess cell population dynamics (i.e. cell death and proliferation rates). Blinding procedures ------------------- Experiments were conducted with blinding applied to each of the scientists based on previously reported methods \[[@B8]\]. The experimental protocol was divided among scientists such that those responsible for preparation of the cell cultures, data acquisition, and data analysis were blind to each other\'s activities and results until data analysis was complete. Cell culture ------------ As the target of Johrei healing intentionality in these studies we used a human cell line derived from a brain tumor biopsy specimen from a patient with glioblastoma multiforme (SF188 GBM). This cell culture model is used widely and can show responsiveness to conventional therapeutic agents including ionizing radiation and chemotherapeutics. SF188 GBM cells were grown in RPMI media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate, and 0.25 μg/ml amphotericin. A fresh aliquot of cryogenically preserved cells was thawed at the start of each experiment to ensure uniformity in the genetic profile of the target cells throughout the experiments. Cells were plated at a density of 5,000 cells per well in six-well culture plates and allowed to grow uninterrupted for 20 hours in a humidified incubator maintained at 37°C and 5% CO~2~before beginning each time-lapse experiment. Time-lapse microscopy --------------------- For each experiment cell cultures were transferred from the incubator to a time-lapse microscope in our laboratory equipped with a heated stage, CO~2~chamber, and a plexi-glass environmental chamber (Axiovert 200; Zeiss, Gottingen, Germany). Cell cultures were maintained at routine incubation settings (37°C, 5% CO~2~) and optimum humidity. Temperature and CO~2~concentration were independently maintained using digital controlling units (Zeiss, Gottingen, Germany). Two sets of phase contrast images (100 X magnification) from each well were taken in 300 second intervals using a Cohu 2600 Series compact monochrome interline transfer CCD camera. Images were acquired during a four-hour baseline period and then continuously for another 18 hours. An Openlab software automation (Improvision, Lexington, MA) drove the camera and stage movements, and compiled the acquired phase images. Images were subsequently processed as Quicktime movies using Openlab. Johrei intervention ------------------- Johrei practitioners were selected based on experience and willingness to participate by the Center for the Science of Life, a Johrei organizational body in the United States. Five Johrei practitioners with a minimum of 17 years experience participated in the experiments in teams of two. Johrei treatment was administered by the practitioner teams for a total of four hours, beginning after the four-hour baseline period. This exceptionally long treatment period (4 hours) was chosen as the highest \"dose\" that was practical within the experimental model. Each practitioner treated the cells for a total of 2 hours per experiment, switching every half hour with the team member. Treatment began with one of the two practitioners being seated in front of the time-lapse microscope and raising one hand toward the cellular target. One hand remained raised toward the cell cultures for the duration of any given Johrei treatment. Johrei treatments were delivered from a distance of 6 -- 9 cm, from outside the plexiglass environmental chamber. In collaboration with The Center for the Science of Life, we developed a set of standard mental procedures for practitioners to follow to minimize variability in the mental states among practitioners. These mental cues were used by all practitioners in all experiment. The mental cues can be summarized briefly as: 1) establishing a connection to the divine, 2) consciously relaxing body and mind, 3) visualizing healing energy penetrating the cellular target, 4) taking enjoyment in participating in the experiment, 5) maintaining a feeling of gratitude. Time-lapse microscopy data analysis ----------------------------------- Every cell in the initial microscopic field was identified and numbered. For each experiment, we counted the initial number of cells in each of 12 microscopic fields. All numbered cells and their progeny were then tracked for the duration of their onscreen viability using compiled Quicktime movies. Each cell\'s life events were identified using a modified version of a previously described cell pedigree system \[[@B9]\]. Cells identified as dead at the start of the video or that entered the microscopic field after the initial frame were not included in this analysis. Cataloged data was entered into a Microsoft Excel spreadsheet (using blinding codes) for further analysis. Cell deaths and divisions occurring per half hour were recorded. Statistical analysis -------------------- Statistical analysis was based on a model which categorizes a cell as engaged in any one of four activities at any time: 1) division, resulting in an additional cell (division), 2) death, resulting in the loss of a cell (death), 3) movement out of the microscopic field (emigration), or 4) the cell can remain unchanged \[[@B10]\]. Under this model three transition probabilities plus the total number of cells at a previous time determine the number of cells at a future time. The expected number of cells at time t, N(t), is given by the equation N(t) = N(t-1) exp(λ(t) - μ(t) - ν(t))\], where λ(t), μ(t) and ν(t) are the transition probabilities for division, death and emigration, respectively, at time t. We estimated the transition probabilities in one-hour time blocks. For example, the estimate for λ(t) is λ(t) = \[ln(N(t) + div(t)) - ln(N(t-1))\], where div(t) is the number of divisions during (t-1, t). A similar equation was used for estimating death and emigration transition probabilities at each hour. Statistical analyses focused on two types of comparisons: comparisons within the Johrei treatment experiments, where division and death rates (transition probabilities) pre-treatment were compared with those during and post-treatment; and comparisons between Johrei and control experiments at similar times. Data from each experiment was pooled at 30 minute time intervals. Each of the cellular events (division, death or emigration) occurs infrequently to each cell, so it is necessary to pool results from many cells in order to have nonzero data. Additionally, in instances where few events were recorded due to relatively few cells being observed it was necessary to pool over several time intervals in order to have enough data to perform statistical tests. Results ======= Overall we documented the behavior of 336 cells in the eight control experiments and 351 cells in the eight Johrei intervention experiments. Initial numbers of cells per microscopic field ranged from 5 to 10; the average cell count per field was 7.0 for controls and 7.3 for Johrei. The numbers of cells per experiment ranged from 40 to 48 with averages of 42.0 and 43.9 for controls and Johrei. Observation over 22 hours showed 312 divisions, 15 deaths and 17 emigrations in the control experiments and 316 divisions, 21 deaths and 7 emigrations in the Johrei experiments. Differences in number of divisions or deaths is not significant (p = 0.19 for divisions and p = 0.37 for deaths based on chi-square test comparing frequencies). While the difference in emigrations is statistically significant (p = 0.03, chi-square test comparing frequencies), this was largely affected by a difference present in the 0--4 hour baseline period making it unlikely that the observed effect is due to Johrei treatment. Experimental treatments (control or Johrei) were confined to a four-hour period starting after collection of four hours of baseline data. During the four-hour treatment period the numbers of divisions were 84 (control), 59 (Johrei); deaths were 4 (control), 5 (Johrei); and emigrations were 0 (control) and 2 (Johrei). Taking into account the numbers of cells at the start of the treatment period, only the difference in divisions is statistically significant (p = 0.018 based on comparing the division rates during the treatment period). This result is based on pooling observations from all cells over the four-hour period and the p-value is based on an assumption that counts observed follow a Poisson distribution. It is possible that counts do not follow a Poisson distribution and that their variability is greater than that expected for Poisson counts. To examine this we calculated division rates separately for each of the eight control experiments and eight Johrei experiments and then compared these rates using the nonparametric Mann-Whitney rank sum test. This analysis confirmed the result based on the total counts (p = 0.016 for Mann-Whitney test). We also tried fitting parametric models to the data in an effort to gain degrees of freedom for performing statistical tests. For example, we tried fitting low order polynomials to division rates over time, but the fits were unsatisfactory, as judged by assuming counts were Poisson distributed with rates predicted from the polynomial fit. The fits were particularly deficient in following the rapid initial rise in division rates over the first four hours of baseline data (Figure [1](#F1){ref-type="fig"}). We obtained similar results attempting to fit a sum of exponentials model (Figure [2](#F2){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Rates of cell division are shown as plotted against time in hours. The plot depicts a decrease in divisions in Johrei treated cells (pink line) during the 4.0--8.0 treatment period which is statistically offset by a similar dip in the control period. Control and pooled data are depicted by green and blue lines respectively. The solid black line depicts the fit of a 4^th^degree polynomial to half-hourly cell division rates for all experiments. ::: ![](1472-6882-5-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The fit of the sum of two exponentials to pooled division rate data for all experiments is depicted. The maroon line is pooled data and the blue line is fit. ::: ![](1472-6882-5-2-2) ::: The division rates for control and Johrei experiments plotted in Figure [1](#F1){ref-type="fig"} suggest another method of comparison. The experimental conditions should be identical during the 0--4 hour baseline period, yet we see wide variation in division rates for the two conditions. We can measure the variance between control and Johrei experiments at each half-hour time point and use the pooled value, over the first eight time points, as a baseline standard. We computed the pooled variances for the 4--8 hour time period (treatment) and compared the two using an F-test for equality of variances. The pooled variance for the 0--4 hours period was 5.8 × 10^-5^compared to 6.8 × 10^-5^for the 4--8 hours period. The ratio of these is 1.17 which is not statistically significant (p = 0.41 based on F distribution with numerator and denominator df = 8). This suggests that the discrepancies between the rates during the 4--8 hour period are statistically no larger than those observed during the 0--4 hour period when there were no treatment differences. This comparison requires no assumption of Poisson variability of counts and takes advantage of a priori knowledge that treatment conditions were identical in the first four hours of the experiments. The only assumption is that the variances arise from normally distributed data, so this requirement was tested. A Mann-Whitney comparison of the ranks of the absolute differences in control and Johrei rate during 0--4 hours vs. 4--8 hours give a p-value of 0.92. Thus, there appears to be no evidence that differences in rates of division during 4--8 hours for control and Johrei were any different from those during 0--4 ours. A plot of these absolute differences is shown in Figure [3](#F3){ref-type="fig"}. The original data are provided as supplementary material \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Absolute differences in half-hourly rates of cell division for all experiments are depicted. The plot suggests that differences between control and Johrei experiments were largest during the first 8 hours and that the differences during Johrei treatment (4--8 hrs) were not statistically larger than those during the previous baseline time period, when the treatment conditions were identical. ::: ![](1472-6882-5-2-3) ::: Discussion ========== Our initial examination of the data suggested that Johrei may have affected the rate of division of cultured human brain tumor cells. This analysis, however, was based on the assumption that the baseline period in both the control and Johrei treated cultures were statistically identical with respect to cell divisions and cell deaths. Subsequent analysis revealed that a significant difference existed in the baseline period between control and Johrei treated samples, with Johrei cultures exhibiting fewer divisions. Taking this into account it appears that there was no observable effect of Johrei on these cell behaviors. The failure to observe evidence of a reproducible cellular response to Johrei treatment is consistent with prior studies in our laboratory evaluating another popular energy medicine modality, external Qigong. Like Johrei, practitioners of external Qigong generally claim the ability to emit or direct healing energy to treat patients. Qigong practices originate from China and are based on the manipulation of a purported healing energy called \"Qi.\" Our prior study investigated the ability of experienced Qigong practitioners to enhance the growth of normal human brain cells in culture as measured by a colony-forming efficiency assay. Following a rigorously designed protocol with randomization, blinding and controls for variability, we did not observe reproducible effects of external Qigong treatment on the growth of these cells \[[@B11]\]. Such \"negative\" data do not negate the possible therapeutic effects of such practices. Cultured cells offer an incomplete system that may not be sensitive to treatments of this kind. More complete models (e.g., a clinical research model) may be more useful to evaluate Johrei treatment for a variety of reasons. Conclusion ========== Cell death and proliferation rates of cultured human cancer cells do not appear responsive to Johrei treatment from a short distance. Competing interests =================== The Center for the Science of Life, a Johrei organizational body in the United States, has provided funding for this work. Authors\' contributions ======================= RT and GY conceived of the study and participated in its design, coordination and implementation. DM performed statistical analysis. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6882/5/2/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Johrei Experimental Data. The supplementary file includes the raw data for all experiments, including number of events for cell death, division, and emigration by half-hourly intervals and cumulative totals. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This work was supported by grants from the Samueli Institute for Information Biology, the Rockefeller-Samueli Center for Research in Mind-Body Energy and the Center for the Science of Life. The authors thank Tri Luu and Aly Pennucci for preparation of samples and performing data analysis.	PubMed Central	2024-06-05T03:55:52.597687	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548282/", "journal": "BMC Complement Altern Med. 2005 Jan 24; 5:2", "authors": [ { "first": "Ryan", "last": "Taft" }, { "first": "Dan", "last": "Moore" }, { "first": "Garret", "last": "Yount" } ] }
PMC548283	Background ========== In the Netherlands, little information is available about prevalence, incidence, etiology and prognosis of pregnancy-related pelvic girdle pain (PPGP). It is hypothesized that during pregnancy many women (about 80%) experience some degree of pain in the pelvic region and/or the low back and that in some of these patients pain becomes chronic or recurrent. Often, symptoms impact on activities of daily life, hobbies, participation in society, planning of next pregnancies and sometimes lead to a chronic disabling condition with considerable work absenteeism in the future \[[@B1]\]. Treatment and indirect costs of these chronic or recurrent patients constitute a considerable burden on health care services, health care insurers and other parties. The total health care expenditures incurred by patients with back pain in the United States in 1998 were approximately \$91 billion, accounting for about 1 % of the Gross National Product \[[@B2]\]. Van Tulder et al. estimated the costs of back pain for Dutch society in 1991 at 1.7% of the Gross National Product \[[@B3]\]. These costs consist almost totally of indirect costs (97%) such as absenteeism and disablement; for that reason chronic back pain can be considered a major economical problem. It is therefore important that PPGP can be diagnosed and treated before PPGP becomes chronic. Consequently, tracking risk factors and characteristics influencing etiology and prognosis of pregnancy-related pelvic girdle pain is important. Absence of knowledge of risk and prognostic factors and the absence of evidence-based treatment strategies about PPGP has prompted the start of a large cohort study in the Netherlands. In January 2000, the Maastricht PPGP cohort study started. It was established (I) to examine the prevalence and incidence of pregnancy-related pelvic girdle pain (PPGP) during and after pregnancy, (II) to identify risk factors involved in the onset of PPGP and to identify which factors can play an important role in the early detection of PPGP and finally (III) to determine the prognosis of PPGP and to identify prognostic factors. Furthermore, a clinical trial is embedded in this cohort, aimed at studying the effectiveness of a tailor-made treatment program in PPGP after delivery \[Bastiaenen et al., treatment, submitted\]. Methods/design ============== Design and study population --------------------------- In an observational prospective cohort study, etiology and prognosis of PPGP will be studied in 7526 pregnant women. The source population for the study comprises of pregnant women from the southeastern area of the Netherlands. Both midwives and gynecologists recruit women for the study when pregnant between 10 and 14 weeks. Women are considered eligible if they meet the following inclusion criteria: women are well versed in the Dutch language and at least 18 years. At inclusion, women receive a leaflet containing information about the research project, an informed consent form and a baseline questionnaire. After providing informed consent and filling out the first questionnaire, the women receive a second questionnaire at 30 weeks of pregnancy, a third one at 2 weeks after delivery and a fourth and fifth at 6 months and 12 months postpartum, respectively. In this study, extensive questionnaires are used with questions ranging from physical complaints (past and present), limitations in activities, restriction in participation, work situation, demographics, lifestyle, pregnancy-related factors and psychosocial factors. With the help of these questionnaires the prevalence and incidence of PPGP in the Netherlands will be described. In addition, possible risk factors and prognostic factors of PPGP will be examined. Exposure variables ------------------ In the Maastricht PPGP cohort study several domains of exposure were measured, including individual characteristics, lifestyle, work situation, pregnancy-related factors and psychosocial factors. The majority of factors were assessed with existing, validated questionnaires. The Dutch translation of the Quebec Back Pain Disability Scale (QBPDS) \[[@B4]\] will measure low back functional status \[[@B5]\]. The QBPDS is a 20-item 6-point scale describing activities commonly affected by back pain. This questionnaire is not developed to study a pregnant population and some activities were unsuitable for women who were pregnant or just gave birth. We therefore added a 7^th^option to the questionnaire, namely \"not applicable\". We also changed the phrase \"because of my back \" into \"because of my back and/or pelvic pain\" in questionnaires. Fear of movement is measured by means of the Dutch translation of the Tampa Scale for Kinesiophobia (TSK) \[[@B6],[@B7]\]. The TSK consists of 17 items; each rated on a 4-point Likert scale. Pain catastrophizing is measured by the Pain Catastrophizing Scale (PCS) \[[@B8]\]. The PCS is a 13-item 5-point scale. A woman is said to catastrophize pain, when she views pain as extremely threatening. To measure the experience of negative affect and positive affect we used the 14-item Negative Emotionality Scale (NEM) and the 11-item Positive Emotionality Scale (PEM) \[[@B9]\]. Current mental health was measured by the General Health questionnaire (GHQ). The questionnaire was originally designed as a 60-item instrument, but we used the shortened version GHQ-12 \[[@B10]\]. The perceived stress scale (PSS) was used to measure to assess stress. The PSS is a 14-item instrument with a 5-point scale \[[@B11]\]. Outcome measurements -------------------- Pregnancy-related pelvic girdle pain is currently not an entity that can be clearly diagnosed and described. Therefore, Bastiaenen et al. studied separate diagnostic strategies of four international authors in the field of PPGP \[Bastiaenen et al., submitted\]. They concluded that there was no similarity in the selection of patients with PPGP between the authors. Most of these classification-strategies of PPGP are based on expert-opinions. Therefore, a possible reason for the lack of similarity in the selection of patients can be that they all select different small parts of the same large patient-group. Because of the relatively unknown etiology of pregnancy-related pelvic girdle pain and the lack of an all-embracing definition, we will use an extensive description of PPGP. We expect that during pregnancy almost all women experience some form of pain in the lower back, the buttocks, the symphyses, the groins and/or radiation into the legs. This pain is probably caused by hormonal and physiological changes which are considered normal during pregnancy. However, some women experience pain in a very early stage of pregnancy while others only experience pain in the final stages of pregnancy. In addition, some women are more limited in their activities (due to pain) than others. This suggests that other factors might influence the hormonal or physiological changes during pregnancy \[[@B12]\]. Most women who had developed PPGP during pregnancy quickly recover after delivery \[[@B13]\]. In this study, pain during or after pregnancy is measured by using patients\' self-reports. Women with pain can be identified by the question whether they experienced pain in the lower back, the buttocks, the symphyses, the groins or radiation into the legs during or after this pregnancy. To study etiology of PPGP, women who gave a positive answer to this question during this pregnancy were selected. For the prognosis of PPGP it is important that women have pain that started during pregnancy and persisted after delivery. At several moments during and after pregnancy, experienced pain in the lower back, the buttocks, symphyses, groins or radiation into the legs was measured. The answers to this question were assimilated into a flowchart (Fig. [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Flowchart of pain in the lower back, the buttocks, the symphyses, groins and/or radiation into the legs: description of cases. ::: ![](1471-2458-5-1-1) ::: Based on their self-reports, the women are stratified into a case group without a history of LBP/PPGP or a case group with a history of LBP/PPGP. Some women (N = 246) were not classified into groups for the following reasons; specific disorders of the spine, rheumatism, neurological disorders and cancer. The first group consists of women without a history of LBP/PPGP, but they experience pain during pregnancy and this pain does not disappear until (at least) 2 weeks after delivery. The second group experiences pain during and after pregnancy, but they also have a history of LBP/PPGP. Both groups of cases will be analysed to study the prognosis of PPGP. Women who experience recurrent pain episodes during pregnancy, that resolve within 2 weeks after delivery, will not be considered cases in the analyses for the prognosis of PPGP. They form a miscellaneous group. However, this miscellaneous group and women who experience no pain after delivery were not excluded from follow-up. Data analyses ------------- Participation rates and descriptions of baseline characteristics of the Maastricht PPGP cohort study will be presented. We calculated prevalence rates for PPGP by dividing the numbers of prevalent cases at several moments during pregnancy by the total number of subjects. Characteristics of the study population at baseline --------------------------------------------------- Recruitment of women into the study began in November 2000 and ended in November 2002. Approximately 10,850 women were asked to participate in the study by midwives and gynecologist in the southeast of the Netherlands. The locations of the participating midwives and gynecologists are shown in Fig. [2](#F2){ref-type="fig"}. At the end of the recruitment period 7,526 pregnant women (73.4%) were willing to participate and were included in the cohort. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The location of participating midwives and gynaecologists in the Netherlands. ::: ![](1471-2458-5-1-2) ::: In Table [1](#T1){ref-type="table"} a number of selected characteristics of the study population at baseline are presented, including details of their age, education, BMI, smoking, work and reproductive history. Mean age of the study population is 31.5 years. The educational level of the participants is very high. Approximately 38 % of the participants have had higher vocational or academic education. The use of alcohol during pregnancy is limited. While 44.8% of the study population did not use alcohol before pregnancy, 91.2% did not use alcohol during pregnancy. With exception of the country of birth, the study population is heterogeneous with respect to demographics, work status and pregnancy-related factors. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of the study population (N = 7526) at baseline (14 wks pregnancy) ::: Aspect \% ----------------------------------------------- --------------------------------------------------------------------------------------- -------------------- ------- ------ Age (yrs) Mean 31.54 N = 7523 \< = 20 0.4 21--25 5.3 26--30 32.4 31--35 47.8 36--40 12.8 \>40 1.2 Country of origin Netherlands 96.5 N = 7516 Belgium, Germany, France, Austria, Switzerland, Luxemburg, Ireland and United Kingdom 1.9 Other countries 1.6 Education Primary school 0.6 N = 7498 Preparatory vocational education 7.2 Lower general secondary education 6.6 Vocational education 31.9 Higher general secondary education 8.5 Pre-university education 2.3 Higher vocational education 27.7 Academic education 10.3 Different 4.9 BMI before pregnancy \< 18.5 Underweight 3,1 N = 7437 18.5--24.9 Normal weight 68,1 25.0--29.9 Overweight 20,5 \> = 30 Extreme overweight 8,3 Smoking habits at 14 wks pregnancy Never 59.7 N = 7488 Ex 29.8 Current during pregnancy 10.5 Alcohol-usage (glasses/week) before pregnancy 0 44.8 N = 7482 1--10 53.5 11--20 1.6 \>20 0.2 Alcohol-usage (glasses/week) during pregnancy 0 91.2 N = 7210 1--10 8.8 11--20 0.0 \>20 0.0 Work (hours/week) No job 13.9 N = 7428 1--10 3.8 11--20 22.9 21--30 21.1 31--40 36.9 \>40 1.5 Number of pregnancies 1 42.3 N = 7519 2 36.6 3 14.2 4 4.6 \>4 2.3 ::: To examine whether the response in our study affected the determinant distributions (e.g. did primarily women in their first pregnancy respond?), a comparison of response rates was carried out. We performed a pilot-study in which data was recorded from April 2001 until November 2001 of every pregnant woman who attended one of the cooperating practices. Information about parity, PPGP during pregnancy, delivery and PPGP after delivery (until 6 weeks) was collected of 283 women (170 participants and 113 non-participants). Results of this pilot-study showed that primipara compared to multiparous women were more willing to participate in the cohort study. Furthermore, data from the responders in the cohort was compared to available data on pregnant Women in The Netherlands from the \"Centraal Bureau voor Statistiek\". The results of this comparison are shown in table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### A comparison between the total Dutch female population and the study population ::: ------------------------------------------------------------------------------------------------------------------------------------------------ Total Dutch population Study population --------------------------------- ---------------------------- ---------------------- ------------- -------------- -------------- -------------- Period 2001 Period 2002 Period 2003 Period 2001\ Period 2002\ Period 2003\ (N = 2892) (N = 3567) (N = 1061) BMI \%\* \%\* \%\* \% \% \% - Underweight (\<18.5) 5.3 4.9 3.0 2,9 3,2 3,5 - Normal weight (18.5--24.9) 70.3 71.9 71.2 66,8 68,5 70,4 - Overweight (25.0--29.9) 18.5 17.8 18.0 21,3 20,3 18,9 - Extreme overweight (\> = 30) 5.9 5.4 7.8 9,1 8,1 7,2 Smoking \%\* \%\* \%\* \% \% \% - Current/before pregnancy 35.7 34.0 33.3 29.7 29.3 31.7 Pregnancy-related factors Mean age mother - Total pregnancies 30.8 30.9 31.0 31.1 31.7 32.4 - First pregnancies 29.2 29.2 29.3 29.6 30.2 30.9 Child \% \% \% \% \% \% - Boy 51.2 51.3 51.4 51.0 52.2 50.5 - Girl 48.8 48.7 48.6 49.0 47.8 49.5 Number of pregnancies: \% \% \% \% \% \% - First 46.3 45.8 45.5 44,6 41,0 41.0 - Second 36.3 36.7 36.9 35,6 37,2 37.3 - Third 12.3 12.5 12.6 13,6 14,7 13.9 - Fourth or more 5.1 5.0 5.0 6,2 7,1 7.8 Multiple pregnancy \% \% \% \% \% \% - Twin 1.9 1.9 1.8 1.0 0.9 1.1 ------------------------------------------------------------------------------------------------------------------------------------------------ \* Age-group comparable to study-population ::: Data are presented in 3 separate years to show possible fluctuations. It is noticeable that the mean age of pregnant women is increasing during the 3-year period in the study population and in the total Dutch population. In the study population, pregnant women are slightly older and heavier compared to the total Dutch population. Approximately 34% of all Dutch women (age 18--45 years), compared to 30% in the study population, are smoking (see table [2](#T2){ref-type="table"}). During pregnancy, 10.5% of the study population will continue smoking (see table [1](#T1){ref-type="table"}). In general, data of the study population correspond with data of the total Dutch population. Prevalence of pregnancy-related pelvic girdle pain during pregnancy ------------------------------------------------------------------- To determine the prevalence of PPGP during pregnancy, data at baseline (14 wks), 30 weeks of pregnancy and 2 weeks after delivery (information about 34--40 weeks of pregnancy) were used. Almost every woman develops pain in the lower back, the buttocks, the symphyses, the groins or radiation into the legs at some time in their pregnancy. Of the 7527 women, 84% reported pain in any or all of these areas during pregnancy. Women with a history of LBP/PPGP are more likely to develop PPGP during pregnancy then women without a history of LBP/PPGP (see fig. [3](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The point prevalence of PPGP during pregnancy for women with (■) and without (▲) a history of LBP/PPGP. ::: ![](1471-2458-5-1-3) ::: Discussion ========== In this article, we describe the main characteristics of the Maastricht PPGP cohort study in terms of study design, study population, exposure variables and outcome measures. This design, in which both risk factors and outcomes are frequently measured between 14 weeks of pregnancy and 1 year after delivery, enables us to examine the etiology and prognosis of PPGP. Advantages and disadvantages of the study ----------------------------------------- Although a cohort design (even incorporating a randomized controlled trial) has advantages over other epidemiological designs, it poses also a burden on the project in terms of logistics and recruitment. First of all, we are totally dependent on the cooperation and recruiting power of midwives and gynecologists. The number of pregnant women is about evenly divided between the two professions. Although professional workload for midwives in the Netherlands is extremely high, 62 % of the midwife practices took part in this study, recruiting about 90% of the patients. Whereas 58% of the hospitals in the recruitment area participate, they only recruit about 10% of the women. Coordination of the recruitment and frequent staff changes in the hospitals themselves seems to be a key problem. In etiological research there should be sufficient contrast in exposure. In this study, the study population at baseline is heterogeneous with respect to demographic variables, lifestyle characteristics and work-related factors. However, due to logistic constraints we have restricted the recruiting area to the southeast of the Netherlands, posing questions about the representation of our sample for the whole Dutch population. For instance the number of immigrants is significantly less in the southeast of the Netherlands. However, for future genetically oriented studies, this could be a major advantage. We have shown previously that our sample is slightly different from the national population of pregnant women with regards to several determinants (e.g. age, BMI). At baseline, 7526 pregnant women participated in our study. This is a response rate of 73.4%. Non-response and loss to follow-up might introduce selection bias in prospective studies. Loss to follow-up, especially in time series designs with repeated measurements, pose threats to representation of the sample. Especially when losses-to-follow-up are connected with negative pregnancy outcomes (miscarriage, birth defects), co-morbidity of the mother or factors predicting for PPGP. To evaluate whether differential loss to follow-up occurs, we will compare the profile of those lost to follow-up with other participants. In this study we were able to evaluate if there are significant differences between participants and non-participants. This evaluations shows that primiparous women are more willing to participate in the study compared to multiparous women. We expect that multiparity plays an important role in the etiology and prognosis of PPGP. Therefore, an underestimation of the prevalence of PPGP reported in this study cannot be ruled out. However, the collected sample should be able to provide reliable preliminary insights in incidence and prevalence of PPGP and factors related to etiology and prognosis of PPGP in the Netherlands. PPGP is a complex syndrome and for a greater understanding of pregnancy-related pelvic girdle pain, future studies should further disentangle the multifactorial etiology and prognosis of PPGP. Future research within the framework of the Maastricht PPGP cohort study will focus on disentanglement of the complex syndrome. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= All authors participated in the design of the study. JMB drafted the manuscript with input from the other authors. All authors read, revised and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/1/prepub> Acknowledgements ================ The Maastricht PPGP cohort study was funded by the Dutch board of health insurance companies. The authors would like to thank the participating midwives and gynecologists as well as all the women who completed the questionnaires. We also would like to thank Conny de Zwart for the logistic assistance.	PubMed Central	2024-06-05T03:55:52.599351	2005-1-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548283/", "journal": "BMC Public Health. 2005 Jan 3; 5:1", "authors": [ { "first": "Janneke M", "last": "Bastiaanssen" }, { "first": "Rob A", "last": "de Bie" }, { "first": "Caroline HG", "last": "Bastiaenen" }, { "first": "Annie", "last": "Heuts" }, { "first": "Mariëlle EAL", "last": "Kroese" }, { "first": "Gerard GM", "last": "Essed" }, { "first": "Piet A", "last": "van den Brandt" } ] }
PMC548284	Introduction ============ Fetal development and morphogenesis of various tissues and organs such as hair follicles, teeth, salivary glands, mammary glands, kidneys, liver, pancreas, and lungs depend on epithelium-mesenchyme interactions \[[@B1],[@B2]\]. Such interactions are believed to be important for the tissue regeneration necessary for wound healing in adults \[[@B3]\]. Pulmonary fibrosis is thought to be a result of wound healing or regeneration after lung injury \[[@B4]-[@B11]\]. Lung injury causes the epithelial basement membrane to be destroyed, which enables migration of interstitial cells into intra-alveolar spaces where they produce and deposit extracellular matrix (ECM). Regenerated epithelial cells then cover the surface of the intra-alveolar fibrotic area. If the injury is mild and focal and re-epithelialization has occurred, these epithelial cells appear widespread over early intraluminal fibrotic lesions and form intra-alveolar buds, which later become small collagen globules and do not contribute to alveolar structural remodeling \[[@B5]-[@B7],[@B9]-[@B13]\]. For example, intra-alveolar buds, in a so-called organizing pneumonia pattern, are frequently observed in nonspecific interstitial pneumonia (NSIP); NSIP usually has a better prognosis than does usual interstitial pneumonia (UIP), which has fibroblastic foci without good re-epithelialization \[[@B8]-[@B10],[@B13],[@B14]\] as early fibrotic lesions. The re-epithelialization around early fibrotic lesions is similar to the process of fetal epithelial development \[[@B4]-[@B7]\], which suggests that epithelium-mesenchyme interactions may play a key role in repair of pulmonary fibrosis. Epimorphin is a mesenchymal cell surface-associated protein that modulates epithelial morphogenesis in embryonic mouse organs including lungs and skin \[[@B15]\]. Epimorphin expression was found in fetal lungs and skin rudimentary cells, at the mesenchyme-epithelium interface. Epimorphin also directs epithelial morphogenetic processes in other gastrointestinal organs and mammary glands \[[@B15]-[@B21]\]. For example, primary cultured rat hepatocytes (an epithelial cell) were used to show that epimorphin induces formation of hepatocyte spheroids with a bile canaliculi-like structure, which maintained albumin production even without growth factors \[[@B16]\]. Epimorphin mediated mammary luminal morphogenesis by controlling expression of CCAAT/enhancer binding protein Î² \[[@B22]\], which is essential for proper mammary morphogenesis and for determination of the fate of mammary epithelial cells \[[@B23]\]. In cultured mammary epithelial cells, epimorphin augmented expression of matrix metalloproteinase 2 (MMP-2), an important proteinase in matrix degradation, and both epimorphin and MMP-2 were required for mammary gland morphogenesis \[[@B24]\]. During fetal rabbit lung development, MMP-2 and MT1-MMP (an activator of MMP-2) were detected in epithelial cells, and expression of active MMP-2 and MT1-MMP increased dramatically; MMP-2 and MT1-MMP were especially predominant during late development, in which there was an extremely wide alveolar surface, which indicated an important role for MMP-2 in alveoli formation \[[@B25]\]. MMP-2 was also up-regulated and activated in regenerated alveolar epithelial cells, which may lead to elongation and migration of these cells for repair of pulmonary fibrosis \[[@B26],[@B27]\]. The epimorphin gene is highly conserved among mouse, rat, and human \[[@B15],[@B28]\]: the 289 amino acid sequence of rat epimorphin/syntaxin 2 exhibits 86% homology to human epimorphin \[[@B29]\]. However, the distribution and function of epimorphin in human lungs, including during fibrosis, are unknown. To clarify the role of epimorphin in human lungs, especially in fibrosis, we evaluated epimorphin expression and localization in normal control lungs and in lungs affected by NSIP or UIP, especially in early intra-alveolar fibrotic lesions. We also used cultured human alveolar epithelial cells to assess the effect of recombinant epimorphin on MMP-2 expression. Materials and Methods ===================== Patients -------- All histologic slides from open or thoracoscopic lung biopsies in the files of the Department of Pathology, Kumamoto University Hospital, from 2000 to 2003, were evaluated. From among these samples, 17 patients were selected, with 8 fulfilling the histologic and clinical criteria for NSIP and 9 showing UIP. To confirm the histologic diagnosis, two pulmonary pathologists reviewed the slides; they were informed only of the age and sex of each patient and the presence of bilateral pulmonary disease determined by chest posteroanterior radiography and high-resolution computed tomography. All patients had had no steroid therapy before the biopsy. Normal control lung tissues were obtained from the normal areas of lungs that had been surgically removed from eight patients because of cancer. Table 1 shows the characteristics of the nine patients with UIP, eight patients with NSIP, and eight normal control subjects. In addition to the histologic diagnosis, we confirmed that the clinical data, pulmonary function test results, bronchoalveolar lavage findings, chest radiographic findings, and follow-up information obtained from the patients were consistent with the diagnoses. Diagnostic criteria of the American Thoracic Society/European Respiratory Society consensus classification system were applied \[[@B30]\]. We also verified that none of the patients with UIP had a connective tissue disease. In the group of patients with NSIP, two with SjÃ¶gren\'s syndrome and one with rheumatoid arthritis were included. In all other cases, no etiologic agent was found. These tissue samples were used for microscopic immunohistochemistry, Western blotting, and Northern blotting assays. The procedures used in this study were in accordance with those recommended by the regional ethical committee on human experimentation. Northern Blotting Studies ------------------------- Human epimorphin DNA fragments were isolated by reverse transcriptase-polymerase chain reaction. Strand cDNA was synthesized with random primers from human lung total RNA. Polymerase chain reaction assays were carried out at 95Â°C for 1 minute, 64Â°C for 1 minute, and 72Â°C for 1 minute for 35 cycles. The following primer pairs were used: human epimorphin forward 5\'-GGA ACC GGA CTT CAG TGG ATC-3\' and reverse 5\'-CAGC CAA TGA TTA GAG CCA GGA-3\'. Polymerase chain reaction products were subcloned into the NcoI and SpeI sites of the pGEM-T Vector (Promega, Madison, WI), and authenticity was confirmed by sequencing. Total cellular RNA was isolated from lung tissues from each case by using the acid guanidinium-isothiocyanate-phenol-chloroform method. The RNA samples (10 Î¼g/lane) were fractionated by electrophoresis on 1% agarose-formaldehyde gels under denaturing conditions and were transferred to Nytran by capillary action. The blots were then probed with the 339-bp NcoI/SpeI fragment of the human epimorphin cDNA labeled with \[^32^P\]dCTP (3000 Ci/mmol) by means of the Nick Translation System kit (GIBCO/BRL, Grand Island, NY). After hybridization, blots were washed and were processed by autoradiography. Washed blots were analyzed by using a Fujix BAS2000 Bio-Imaging analyzer system (BASTM, Fuji Photo Film Co., Ltd., Tokyo, Japan) and were visualized on Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY). Signal intensity was quantified in digital images via BASTM analysis (FUJIX BAStation, Fuji Photo Film Co., Ltd.). To control for differences in gel loading, for each sample the RNA hybridized with the epimorphin probe was normalized to the expression of Î²-actin mRNA in the same sample. Western Blotting Studies ------------------------ Although the monoclonal antibody to mouse epimorphin (MC-1) has been reported to cross-react with human epimorphin \[[@B31],[@B32]\], we performed Western blotting to confirm whether the anti-epimorphin antibody (MC-1, a gift from Dr. Hirai, Osaka R&D Laboratory (Yokohama-lab), Sumitomo Electric Industries, Yokohama, Japan) cross-reacted with the human epimorphin in our lung samples and to make comparisons of epimorphin peptide expression between normal and fibrotic groups. For immunoblotting, lung tissues from each case were dissolved in sample buffer containing 2% sodium dodecyl sulfate in 8 M urea and were kept at 4Â°C overnight; the same protein concentration (30 Î¼g) in samples from each lung extract was ensured by using Protein Assay (Bio-Rad Laboratories, Hercules, CA). Lung tissues from normal adult mice were also dissolved in sample buffer as above and served as positive controls. Those samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to the method of Laemmli (1970), and proteins in the gels were transferred onto nitrocellulose filters and were incubated sequentially with 5% skim milk in 0.5% Tween 20 overnight. Blots were then incubated at 4Â°C overnight with 10 Î¼g/ml rat anti-mouse epimorphin primary monoclonal antibody MC-1 used at a dilution of 1:1000 in 1% bovine serum albumin. Samples were then stained by incubation with horseradish peroxidase-conjugated anti-rat goat IgG F(ab\')~2~antibody (Biosource International Inc., Camarillo, CA) used at a dilution of 1:1000 in Tris-buffered saline with 0.05% Tween 20 for 2 hours at room temperature. The bound antibody was detected by using the enhanced chemiluminescence method (Amersham Pharmacia Biotech UK Limited, Little Chalfont, Buckinghamshire, England) according to the manufacturer\'s protocol. Light Microscopic Immunohistochemistry -------------------------------------- A portion of each lung specimen was fixed immediately in a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), after which samples were sequentially washed for 4 hours in 10% sucrose in 0.01 M phosphate-buffered saline (pH 7.4, 4Â°C), 4 hours in 20% sucrose in phosphate-buffered saline, and overnight in 30% sucrose in phosphate-buffered saline. They were then snap-frozen in OCT embedding medium and stored at -80Â°C. Frozen tissues were cut into 4-Î¼m sections, which were incubated for 30 minutes with a biotin blocking system (Dako Corporation, Carpinteria, CA) and for 30 minutes with 0.3% hydrogen peroxide in methanol to eliminate endogenous peroxidase activity. After treatment with normal goat serum, tissues were incubated overnight at 4Â°C with 10 Î¼g/ml rat anti-mouse monoclonal epimorphin antibody and were then incubated for 1.5 hours at 37Â°C with 1 Î¼g/ml biotinylated goat anti-rat IgG (ZYMED, San Francisco, CA) and streptavidin-biotin-horseradish peroxidase complex (Dakopatts, Glostrup, Denmark). Bound antibody was visualized after incubation for 10 minutes in a Coplin jar with 100 ml of Tris-HCl buffer (pH 7.6) containing 20 mg of diaminobenzidine and 17 ml of 30% H~2~O~2~. Counterstaining was with Mayer\'s hematoxylin. Lung tissue of normal adult mice was stained as a positive control. Nonspecific labeling of primary antibody was evaluated with normal rat serum. Each tissue sample was also stained for keratin (rabbit anti-bovine antibody raised against the 58-, 56-, and 52-kd subunits of muzzle epidermal keratin; Dakopatts, Santa Barbara, CA), Î±-smooth muscle actin (mouse anti-human Î±-smooth muscle actin monoclonal antibody; Dakopatts, Santa Barbara, CA), and MMP-2 (mouse anti-human MMP-2 monoclonal antibody; clone 42-5D11, IgG1 isotype, purified antibody; Daiichi Fine Chemical Co., Ltd., Takaoka, Japan). Serial sections were also stained with hematoxylin-eosin (H&E) and Alcian blue-periodic acid-Schiff (AB-PAS). Confocal Microscopy ------------------- To localize epimorphin as precisely as possible, epithelial and stromal cells were double labeled by means of immunofluorescent probes, and the distribution of epimorphin was compared with that of keratin and vimentin (mouse monoclonal anti-vimentin \[V9\] antibody; Dakopatts, Glostrup, Denmark). Briefly, after sections were exposed to primary antibodies, they were exposed to secondary antibodies: for the first analysis: fluorescein isothiocyanate-conjugated goat anti-rat IgG (American Qualex, San Clemente, CA) or Texas Red-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR); for the second analysis: fluorescein isothiocyanate-conjugated goat anti-rat IgG (American Qualex) or Texas Red-conjugated goat anti-mouse IgG (Molecular Probes). The nuclei were counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (Vector Laboratories, Inc., Burlingame, CA). Specimens were examined under a confocal laser scanning microscope (TCS-SP; Leica Lasertechnik, Heidelberg, Germany), based on an upright microscope (DMRB, Leica Lasertechnik) equipped with a krypton/argon laser \[[@B33]\]. The excitation wavelengths for fluorescein isothiocyanate and Texas Red were 498 nm and 568 nm, respectively. Green fluorescein isothiocyanate emission was selected and recorded by using a 500- to 550-nm bandpass filter; red Texas Red emission was selected and recorded by using a 581- to 631-nm bandpass filter. In addition, 4,6-diamidino-2-phenylindole dihydrochloride was excited at 350-nm by using a UV laser, and its blue emission was recorded via a 401- to 551-nm bandpass filter. Localization of MMP-2 and Epimorphin in Lungs with NSIP ------------------------------------------------------- To compare the precise localization of MMP-2 and epimorphin in NSIP, a confocal microscope was used for immunohistochemical study of lung tissues from patients with NSIP, by means of the same technical immunohistochemical staining and analysis method as that described previously for epimorphin and keratin detection \[[@B34]\]. Mouse anti-human MMP-2 monoclonal antibody and rat anti-mouse monoclonal epimorphin antibody served as primary antibodies. Secondary antibodies for double-labeling immunohistochemistry studies via confocal microscopy were goat anti-mouse IgG antibody (Alexa Fluor 546; Molecular Probes) and goat anti-rat IgG antibody (Alexa Fluor 488; Molecular Probes). Cell Culture and Treatment Conditions ------------------------------------- Human lung-derived alveolar epithelial cells (A549), an epithelial cell line derived from lung adenocarcinoma (Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University), were cultured in RPMI (GIBCO/BRL) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 50 U/ml penicillin, and 50 Î¼g/ml streptomycin. All cells were maintained at 37Â°C in a humidified incubator containing 5% CO~2~and 95% air. Detection of MMP-2 Expression in A549 Cells ------------------------------------------- Four-well chamber slides (Lab-Tek II, Nalge Nunc International, Naperville, IL) were coated with human recombinant epimorphin fragment (H12; 20 Î¼g/well, dissolved in 1.5 mM HCl; a gift from Dr. Hirai), negative control solution prepared from non-transfected Escherichia colistrain BL21 (BL; 20 Î¼g/well, dissolved in 1.5 mM HCl; a gift from Dr. Hirai), or Type IV collagen from human placenta collagen (20 Î¼g/well, dissolved in 0.4% glacial acetic acid; Sigma Chemical Co., St. Louis, MO), after which samples were dried at room temperature. After A549 cells were washed two times with phosphate-buffered saline, the cells (5 Ã--- 10^5^) were suspended in RPMI medium with 2% fetal calf serum and were incubated at 37Â°C on the chamber slides coated with recombinant epimorphin, BL, or Type IV collagen. After incubation for 9 h, culture dishes were placed on ice, medium from each dish was collected, and cells were washed twice with phosphate-buffered saline. Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline for 5 minutes at -20Â°C and then were washed in phosphate-buffered saline and permeabilized in acetone for 5 minutes at -20Â°C. Cells were again washed in phosphate-buffered saline and incubated with a blocking solution containing 1% bovine serum albumin/phosphate-buffered saline for 1 hour at room temperature. Immunohistochemistry was performed via the same technical method described above for epimorphin detection. The primary antibody was 5 Î¼g/ml mouse anti-human MMP-2 monoclonal antibody, and the secondary antibody was horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Amersham Pharmacia Biotech UK Limited). The A549 culture supernatant collected after incubation for 9 hours on the chamber slides coated with recombinant epimorphin, BL, and Type IV collagen was used to determine the amount of MMP-2 activity by means of the MMP-2 Biotrack Activity Assay System (Amersham Pharmacia Biotech UK Limited), according to the manufacturer\'s instructions. All samples were assayed in triplicate, and assays were repeated three times. Statistical Analysis -------------------- Densitometric analysis of the bands of Northern and Western blotting was performed with Macintosh G4 computer (Apple Japan, Inc., Tokyo, Japan) using high-resolution scanner and NIH image software (version 1.62, National Institutes of Health, Bethesda, Maryland). The data are expressed as a ratio of a standard normal control subject to each case\'s band density units (arbitrary units) and reported as mean Â± standard error of the mean (SEM). Statistical significance was established using the one-way analysis of variance test followed by Tukey-Kramer multiple intergroup comparison test. Probabilities less than 0.05 were considered significant. Results ======= Expression of Epimorphin mRNA in Normal Human Lungs and Lungs of Patients with NSIP or UIP ------------------------------------------------------------------------------------------ Expression of 3.2-kb epimorphin mRNA was revealed in all lung samples from patients with NSIP, UIP and normal control subjects by Northern blotting (Figure [1A](#F1){ref-type="fig"}). To confirm that the blots reflected samples of equal size, they were reprobed for expression of Î²-actin mRNA. Compared with corresponding densities of Î²-actin, the densities of epimorphin expression in NSIP samples were significantly higher than those in control lungs and lungs with UIP, as determined by NIH Image analysis (P\< 0.05) (Figure [1B](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### \(A) Representative Northern blots of epimorphin mRNA and Î²-actin mRNA. Epimorphin mRNA (3.2-kb) was expressed in samples of normal lung (Control: lanes 1â€"3) and lungs from patients with UIP (lanes 4â€"6) and patients with NSIP (lanes 7â€"9) (upper blot). To confirm that the blots reflected samples of equal size, they were reprobed for expression of Î²-actin mRNA (lower blot). (B) The data are expressed as a ratio of epimorphin to Î²-actin band density units (arbitrary units) and reported as mean Â± SEM measured by means of NIH Image analysis. In NSIP samples, the density of epimorphin expression was significantly higher than that in control and UIP samples (\*P\< 0.05). ::: ![](1465-9921-6-6-1) ::: Tissue Localization of Epimorphin in Normal Human Lungs ------------------------------------------------------- Western blotting showed that rat anti-mouse epimorphin monoclonal antibody (MC-1) recognized the 150-kd bands in the extracted samples from both mouse lung and normal human lung (Figure [2A](#F2){ref-type="fig"}), similar to results reported elsewhere \[[@B31],[@B32]\]. We thus confirmed that anti-mouse epimorphin antibody demonstrated specificity and cross-reactivity with the human epimorphin of our lung samples. Normal lung tissues of the human samples had normally opened alveoli with infiltration of a few inflammatory cells. In immunohistochemical studies of the human samples, as with normal mouse lung specimens \[[@B34]\], epimorphin was weakly stained in vascular and alveolar walls (Figure [2B](#F2){ref-type="fig"}). Negative control staining with normal rat serum showed no positive findings in the serial section (data not shown). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Representative Western blots for epimorphin in normal mouse and human lung samples (A), and representative epimorphin immunohistochemistry (B) in normal mouse lung. (A) Recognition of the 150-kd bands by rat anti-mouse epimorphin monoclonal antibody (MC-1) in extracted lung samples. (B) Weak epimorphin staining in vascular walls (arrowheads) and alveolar walls (inset). Scale bar = 50 Î¼m. ::: ![](1465-9921-6-6-2) ::: Amount of Epimorphin in Normal Human Lungs and Lungs of Patients with NSIP or UIP --------------------------------------------------------------------------------- In the lung tissue homogenates of patients with NSIP, UIP and of control subjects, Western blotting with antibody for epimorphin also recognized the 150-kd bands (Figure [3A](#F3){ref-type="fig"}) and the densities of the bands of patients with NSIP are relatively higher than those of UIP and control subjects by methods of NIH image analysis (p \< 0.05) (Figure [3B](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### \(A) Representative Western blots for epimorphin in normal control subjects and fibrotic groups. Epimorphin protein (150-kd) was expressed in samples of normal lung (Control: lanes 1â€"3) and lungs from patients with UIP (lanes 4â€"7) and patients with NSIP (lanes 8â€"10). (B) The data are expressed as a ratio of a standard normal control subject to each case\'s band density units (arbitrary units) and reported as mean Â± SEM measured by means of NIH Image analysis. In NSIP samples, the density of epimorphin protein expression was significantly higher than that in control and UIP samples (\*P\< 0.05) ::: ![](1465-9921-6-6-3) ::: Tissue Localization of Epimorphin in NSIP and UIP by Means of Immunohistochemical Analyses ------------------------------------------------------------------------------------------ In lungs of all patients with NSIP, alveolar walls were thickened with edema, fibrosis, and inflammatory cell infiltration; the appearance was typically uniform (Figure [4A](#F4){ref-type="fig"}). There was slight or no dense fibrosis; intra-alveolar organizing fibrosis was seen as pale eosinophilic fibrosis with H&E staining and as Alcian blue positive area with AB-PAS staining as early stage of fibrosis (Figure [4A](#F4){ref-type="fig"} and [4B](#F4){ref-type="fig"}). Epimorphin immunostaining was strong in the early intra-alveolar fibrotic areas with a covering of regenerated alveolar epithelial cells (Figure [4C](#F4){ref-type="fig"} and [4E](#F4){ref-type="fig"}), which contained a few Î±-smooth muscle actin-positive cells (Figure [4F](#F4){ref-type="fig"}), in addition to immunostaining seen in vascular and alveolar walls. No positive findings was shown using normal rat serum as fist antibody for negative control (Figure [4D](#F4){ref-type="fig"}). The epimorphin immunohistochemical staining patterns for all eight patients with NSIP were quite similar. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Representative histology stained with hematoxylin-eosin (H&E)(A) and Alcian blue-periodic acid-Schiff (AB-PAS)(B) and immunohistochemistry for epimorphin (C), rat normal serum as negative control for epimorphin (D), keratin (E), and Î±-smooth muscle actin (F) for all patients with NSIP. (A) Thickened alveolar walls showed edema, fibrosis, and inflammatory cell infiltration. Dense fibrosis was inconspicuous or absent. (A and B) Intra-alveolar organizing fibrotic areas were seen as pale eosinophilic fibrosis with H&E staining and as Alcian blue positive area with AB-PAS staining as early stage of fibrosis (asterisks). (C) Positive epimorphin immunostaining appeared in areas of early fibrosis (insetin C shows a close-up view; asterisks indicates early intra-alveolar fibrotic area) covered with regenerated alveolar epithelial cells (arrowheadin E), in addition to the alveolar walls. A few Î±-smooth muscle actin-positive cells were noted in the fibrotic areas (arrowheadin F). Scale bars = 60 Î¼m. ::: ![](1465-9921-6-6-4) ::: In lungs of all patients with UIP, the fibrotic zones showed temporal heterogeneity, with dense acellular collagen and scattered fibroblastic foci with intervening nearly normal alveoli. Most fibrotic zones had honeycombing with complete destruction of the architecture (Figure [5A](#F5){ref-type="fig"}). Though clear immunostaining in the vascular walls, only weak epimorphin immunostaining occurred in dense fibrotic lesions and scattered fibroblastic foci, as in areas of early fibrosis (Figure [5B](#F5){ref-type="fig"} and [5F](#F5){ref-type="fig"}) with Alcian blue positive (Figure [5E](#F5){ref-type="fig"}). The scattered fibroblastic foci contained many Î±-smooth muscle actin-positive cells (Figure [5D](#F5){ref-type="fig"}) with the desquamative regenerated alveolar epithelial cells overlying these fibroblastic foci (Figure [5C](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Representative histology (A, C \[close-up view\]; H&E staining) and immunohistochemistry for epimorphin (B, F \[close-up view\]) and Î±-smooth muscle actin (D \[close-up view\]) of scattered fibroblastic foci in early fibrotic areas as Alcian blue positive area (E; AB-PAS staining) for patients with UIP. (A and C) Honeycombing with complete architectural destruction occurred in fibrotic zones. (C and E) Arrowheads indicate scattered fibroblastic foci. In addition to clear immunostaining in the vascular and alveolar walls (arrows), only weak epimorphin immunostaining was seen in scattered fibroblastic foci (arrowheads) (B and F); many Î±-smooth muscle actin-positive cells (D) and desquamative regenerated alveolar epithelial cells (C) were found. Scale bars = A, B: 200 Î¼m; Câ€"E: 80 Î¼m. ::: ![](1465-9921-6-6-5) ::: Tissue Localization of Epimorphin in NSIP by Means of Double-labeled Immunohistochemical Analyses ------------------------------------------------------------------------------------------------- Double-labeled confocal fluorescent images confirmed the presence of epimorphin in early fibrotic lesions and keratin-positive epithelial cells overlying the lesions (Figure [6A](#F6){ref-type="fig"} and [6B](#F6){ref-type="fig"}). Double-labeled confocal fluorescent images also verified the localization of epimorphin in vimentin-positive stromal cells and in surrounding ECM (Figure [6C, D](#F6){ref-type="fig"}, and [6E](#F6){ref-type="fig"}) in early fibrotic lesions. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Representative double-labeled confocal fluorescent images of staining for epimorphin plus keratin (A), epimorphin (C), vimentin (D), and epimorphin plus vimentin (E) in an early fibrotic lesion from the lung of a patient with NSIP. (A) The presence of epimorphin (green) in areas of early fibrosis and the presence of keratin (red) in epithelial cells overlying the lesions were confirmed. (B) The same image shown in part A but with Nomarski optics used. (Câ€"E) Epimorphin (C, green) localized in vimentin-positive (D, red) stromal cells (yellow-orange) and in surrounding ECM in early fibrotic areas (E). Nuclei were 4,6-diamidino-2-phenylindole dihydrochloride-positive (blue). Asterisks indicate early fibrotic areas. Scale bars = B: 20 Î¼m, E: 5 Î¼m. ::: ![](1465-9921-6-6-6) ::: Localization of MMP-2 and Its Relation to Epimorphin in NSIP ------------------------------------------------------------ Areas of early fibrosis in lung tissues from patients with NSIP showed epimorphin immunoreactivity (Figure [7A](#F7){ref-type="fig"}), and regenerating epithelial cells overlying these lesions demonstrated MMP-2 labeling (Figure [7B](#F7){ref-type="fig"}). Furthermore, in tissues from patients with NSIP, double-labeled confocal fluorescent images confirmed the expression of MMP-2 (Figure [7C](#F7){ref-type="fig"}) in re-epithelialized cells overlying fibrotic lesions in which epimorphin was also clearly expressed. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Representative images of immunostaining for epimorphin (A) and MMP-2 (B) in NSIP; the double-labeled confocal fluorescent image of in an early fibrotic lesion from the lung of a patient with NSIP was stained for epimorphin plus MMP-2 (C). (A) Epimorphin immunoreactivity was observed in early fibrotic areas, with (B) MMP-2 labeled in regenerated epithelial cells overlying the fibrotic lesion. Asterisks indicate early fibrotic areas. (C) MMP-2 (red) was strongly expressed in re-epithelialized cells overlying early fibrotic lesions with clear epimorphin expression (green). Nuclei were 4,6-diamidino-2-phenylindole dihydrochloride-positive (blue). Asterisks indicate early fibrotic areas. Scale bars = 20 Î¼m. ::: ![](1465-9921-6-6-7) ::: Increased Expression of MMP-2 Induced in A549 Cells by Epimorphin ----------------------------------------------------------------- Nine hours after plating of BL or Type IV collagen-coated chamber slides with cells of the human lung-derived alveolar epithelial cell line A549, the cells had spread and formed a loose sheet (Figure [8A](#F8){ref-type="fig"}, left panel). However, during the same time period, in recombinant epimorphin-coated chamber slides, the A549 cells formed some monolayer cell islands in addition to the loose cellular sheet, in about 50% of the chamber slide area (Figure [8A](#F8){ref-type="fig"}, right panel). Moreover, the immunoperoxidase method revealed strong MMP-2 immunostaining in cells of these cell islands in epimorphin-coated slides after 9 hours of incubation (Figure [8A](#F8){ref-type="fig"}, right panel). The MMP-2 Biotrack Activity Assay System revealed significantly higher MMP-2 activity levels in the supernatants of cultures of A549 cells with recombinant epimorphin compared with those for cultures with BL or Type IV collagen after 9 hours of incubation (Figure [8B](#F8){ref-type="fig"}). ::: {#F8 .fig} Figure 8 ::: {.caption} ###### \(A) Representative images of MMP-2 immunostaining in A549 cells cultured with recombinant epimorphin (right panel) or with BL (left panel) after 9 hours of incubation. In the epimorphin-coated slides, plated cells formed some monolayer cell islands in addition to loose sheets of cells, and cells in these cell islands showed strong MMP-2 immunostaining (by the immunoperoxidase method). Scale bars = 30 Î¼m. (B) Via the MMP-2 Biotrack Activity Assay System, MMP-2 activity in the supernatants of A549 cells cultured with recombinant epimorphin was higher than in supernatants of cells cultured with BL or Type IV collagen (P= 0.01). ::: ![](1465-9921-6-6-8) ::: Discussion ========== This is the first study to examine in detail the distribution and magnitude of epimorphin expression in normal human lungs and lungs from patients with NSIP or UIP. We found clear expression of epimorphin mRNA and protein in lung samples from normal control subjects and patients with NSIP or UIP. Expression of Epimorphin in Normal Human Lungs ---------------------------------------------- Consistent with previous reports \[[@B32]\], epimorphin was expressed in connective tissue components of walls of the alveoli and blood vessels in normal lungs, as determined by immunohistochemistry. Epimorphin expression was also confirmed at the mRNA and protein levels by Northern and Western blotting assays, similar to results found previously for mice \[[@B15],[@B32]\]. Although the specific function of epimorphin in normal lungs is not yet known, it may serve as an epithelial and endothelial cell morphogen to maintain the cell turnover necessary for normal structure and function. Alveolar epithelial cells are replaced at regular intervals under physiologic conditions. For example, the estimated turnover time for normal mouse alveolar epithelial cells ranges from 28 to 35 days \[[@B35]\], and daily endothelial cell turnover is reported to be about 1% \[[@B36]\]. Also, in the normal adult human kidney, similar to results for normal adult mouse kidney, low levels of epimorphin mRNA and protein were detected in the glomerular mesangium and peritubular interstitium (interstitial fibroblast-like cells), as revealed by reverse transcriptase -polymerase chain reaction and immunohistochemistry \[[@B37]\]. These findings are consistent with our result that epimorphin is expressed in the interstitium in human control lungs, as in normal adult mouse lungs \[[@B34]\]. Expression of Epimorphin in Lungs of Patients with NSIP or UIP -------------------------------------------------------------- We found, by means of Northern blotting assays, distinct expression of epimorphin mRNA and protein in normal lungs and lung samples of patients with NSIP or UIP, with significantly higher epimorphin expression in lungs of patients with NSIP than in other lung samples. Also, serial sections and double-labeled immunohistochemistry analyses of NSIP samples demonstrated strong expression of epimorphin in mesenchymal cells situated within active intra-alveolar fibrotic lesions and the presence of epimorphin in the ECM in the vicinity of these cells. These findings are consistent with our previous report that epimorphin was synthesized by mesenchymal cells and localized to these cells and the ECM of early intra-alveolar fibrotic lesions in a murine bleomycin-induced pulmonary fibrosis model \[[@B34]\]. They are also consistent with a report that epimorphin was synthesized by normal human skin fibroblasts \[[@B38]\]. It is well known that regeneration of alveolar and bronchiolar epithelial cells is crucial for repair of pulmonary fibrosis, and the ECM in early fibrotic lesions provides fibronectin and other adhesion molecules as ligands for regenerating epithelial cells to ensure successful repair \[[@B3],[@B4],[@B39]-[@B41]\]. In the murine bleomycin-induced pulmonary fibrosis model, we suggested that epimorphin in the early fibrotic lesions participated in adhesion of the regenerating alveolar epithelial cells. Involvement of epimorphin in the repair process was also reported for other organs including the gut during tissue repair in an isograft in rats \[[@B20]\] and the liver during regeneration after partial hepatectomy in mice \[[@B17]\]. Increased expression of epimorphin during epithelial cell regeneration was also reported in human skin ulcers and active ulcerative colitis \[[@B32],[@B42]\]. We observed strong epimorphin immunostaining in early fibrotic areas with a few Î±-smooth muscle actin-positive cells in NSIP, as well as weak epimorphin immunostaining in fibroblastic foci with many Î±-smooth muscle actin-positive cells and desquamative regenerated alveolar epithelial cells in UIP. It is believed that in UIP, a failure of re-epithelialization of fibroblastic foci maintains fibroblast/myofibroblast activity and ECM synthesis \[[@B43],[@B44]\]. We suggest that highly expressed epimorphin in the lungs of patients with NSIP may be necessary during wound healing, especially for regeneration of alveolar epithelial cells in early fibrotic lesions, and we also suggest that poorly expressed epimorphin in the lungs of patients with UIP may lead to failure of re-epithelialization of fibroblastic foci and the contraction of fibrotic lungs during the end stage of UIP. Role of Epimorphin Associated with MMP-2 in Lung Repair ------------------------------------------------------- To understand the role of epimorphin in the repair process in the lungs of patients with NSIP, we evaluated whether epimorphin enhanced expression of MMP-2 (gelatinase A). Recombinant epimorphin induced formation of some monolayer cell islands in addition to a loose cellular sheet and augmented expression of MMP-2 in cultured human alveolar epithelial cells, as demonstrated by immunohistochemistry and the MMP-2 Activity Assay System. We suggest that epimorphin-induced monolayer cell islands are a type of formation of differentiated alveolar epithelial cells, like the epimorphin-induced spheroid formation of rat hepatocytes, and suggest that the augmented expression of MMP-2 may be similar to the increased albumin production of rat hepatocytes. We also demonstrated expression of MMP-2 in re-epithelialized cells overlying epimorphin-positive early fibrotic areas by means of double-labeled confocal fluorescent images. MMP-2, which is an important proteinase needed for matrix degradation, is secreted by type II alveolar epithelial cells in vitro\[[@B45]\]. Fukuda et al. \[[@B25]\] observed the localization of MMP-2 in fetal rabbit alveolar epithelial cells and gelatinolytic activities of MMP-2 in fetal rabbit lung, which indicated an important role for MMP-2 in formation of alveoli. These authors also observed the localization of MMP-2 in regenerating alveolar epithelial cells covering intra-alveolar fibrotic areas in a study of bronchiolitis obliterans organizing pneumonia, which is a reversible fibrotic human lung disease \[[@B13]\]. Yaguchi et al. \[[@B26]\] reported that MMP-2 was found in regenerating alveolar epithelial cells and that gelatinolytic activities of the active forms of MMP-2 increased in later repair stages in bleomycin-induced pulmonary fibrosis, during reconstruction of alveoli. Moreover, Fukuda et al. \[[@B46]\] reported that in bleomycin-induced pulmonary fibrosis, re-epithelialization on the surface of early intra-alveolar fibrotic lesions in gelatinase A^-/-^mice was markedly reduced compared with that of gelatinase A^+/+^controls. Thus, MMP-2 was up-regulated and activated in regenerating alveolar epithelial cells, which may allow elongation and migration of these cells for successful repair of pulmonary fibrotic lesions \[\[[@B26],[@B27]\], 47\]. We therefore suggest that epimorphin expression in early fibrotic lesions in the lungs of patients with NSIP may contribute to the repair process in pulmonary fibrosis, in part by inducing MMP-2 expression. What may follow this MMP-2 expression may be elongation and migration of regenerating epithelial cells and degradation of ECM in alveolar spaces. Thus, epimorphin, as a component of proteolytic systems, may contribute to cell migration and tissue remodeling during repair of human lung fibrosis. Although epimorphin receptors have not yet been identified, binding ECM molecules has been suggested as involved in establishing epithelial polarity, thus helping form an organized basement membrane and cell-ECM junctional complexes \[[@B21]\]. Our results are consistent with the idea \[[@B22],[@B34]\] that epimorphin has a high affinity for ECM molecules and could modify functions of adhesion and migration of regenerating alveolar epithelial cells by altering signaling through MMP-2. In conclusion, epimorphin expressed in lungs may have important roles as a morphogen not only in mice but also in humans, and epimorphin may contribute to the repair process in human pulmonary fibrosis via epithelium-mesenchyme interactions. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Table 1. Characteristics of subjects, including pulmonary function test results ::: ::: {.caption} ###### Click here for file ::: Acknowledgments =============== The authors thank Dr. Yohei Hirai, Osaka R&D Laboratory (Yokohama-lab), Sumitomo Electric Industries, Yokohama, for generous gifts of anti-epimorphin antibody and human recombinant epimorphin; Mr. Osamu Nakamura, Mr. Takenobu Nakagawa, Mrs. Emi Miyata, and Ms. Makiko Tanaka for their skillful technical assistance; and Ms. Judith B. Gandy for editing the manuscript. This work is supported by a Grant-in-Aid for Young Scientists from the Ministry of Education, Science, Sports, Science and Technology of Japan.	PubMed Central	2024-06-05T03:55:52.602942	2005-1-16	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548284/", "journal": "Respir Res. 2005 Jan 16; 6(1):6", "authors": [ { "first": "Yasuhiro", "last": "Terasaki" }, { "first": "Yuh", "last": "Fukuda" }, { "first": "Moritaka", "last": "Suga" }, { "first": "Naoki", "last": "Ikeguchi" }, { "first": "Motohiro", "last": "Takeya" } ] }
PMC548285	Background ========== The current generation of adolescents -- over one billion -- is the largest in history \[[@B1]\] and, far from representing a picture of health \[[@B2]\], many will suffer untimely disease and death. HIV and malaria are responsible for much of the disease burden affecting female adolescents, who suffer disproportionately from these combined infections relative to other age groups. This is due to a high HIV incidence during the period when many adolescents become pregnant for the first time -- an event which greatly increases their susceptibility to Plasmodium falciparummalaria \[[@B3]\]. Biological interactions between malaria and HIV in pregnancy complicate therapy, which can also be compromised by inappropriate adolescent health-seeking behaviour. The public health importance of HIV and malaria synergism is only now emerging. It has led to a recommendation by the World Health Organization (WHO) that the two disease programs should collaborate to ensure integrated service delivery, especially within the framework of reproductive health services \[[@B4]\]. A case has also been made for linking disease control programs as a more efficient and effective way for lowering the malaria and HIV disease burden \[[@B5]\]. Nevertheless, it is difficult to seen how these goals will be attained unless adolescents are accorded a much higher priority by disease control programs. In this paper we consider the justification, as well as the challenges, of using common approaches to improve delivery of HIV and malaria interventions to female adolescents. HIV and malaria burden in pregnant and non-pregnant adolescents --------------------------------------------------------------- Prevalence estimates for HIV infection \[[@B1]\] for 35 sub-Saharan African countries, for young males and females (15--24 years), are shown in Figure [1](#F1){ref-type="fig"}. Estimations are often derived from sentinel surveillance of women attending antenatal care (ANC) and tend to overestimate prevalence in adolescents due to the selection of sexually active \[[@B6]\] and less-educated groups \[[@B7]\]. Accepting this bias, in every country, listed HIV prevalence is two to three times higher among females than males. Having an older male partner is associated with a modest increased HIV risk \[[@B8]\] but probably does not explain a sex difference that occurs uniformly across all countries and cultures and at all levels of HIV endemicity. In Rakai, Uganda, only 12.4% of HIV cases in 15--19 year olds was attributed to relationships with men 10 years older \[[@B9]\]. Other sexually transmitted infections (STIs) increase susceptibility to HIV but do not account for the sex difference \[[@B10]\]. Host biological factors may be critical \[[@B11]\]. Sexual maturation takes several years to complete, but menarche is delayed amongst socio-disadvantaged girls, and many will be sexually active before biological maturation is complete. Interventions that delay the onset of sexual activity could reduce exposure at a time of peak biological susceptibility to HIV and highest risk of malaria in pregnancy. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Female and male HIV prevalence among young people 15--24 years of age in sub-Saharan Africa. Compiled from UNFPA^1^ ::: ![](1475-2875-4-2-1) ::: Malaria is an important cause of adolescent hospital admissions in many sub-Saharan African countries with stable malaria transmission \[[@B12]\]. Malaria mortality in African adolescents (both sexes) aged 10--14 years has been estimated at over 45,000 deaths per annum \[[@B13]\]. The highest prevalence of P. falciparuminfection after childhood is also in young, mostly adolescent, women. Data from Malawi (Table [1](#T1){ref-type="table"}) shows that non-pregnant and pregnant adolescents had significantly higher parasite rates than women above 19 years. Primigravidae also have a markedly increased susceptibility to malaria \[[@B3]\]. Young age and nulliparity in the same individual lead to greatly increased malaria risk. This partly relates to reduced acquisition of acquired malaria immunity in young individuals \[[@B14]\] as well as the lack of parity-specific malaria immunity acquired during the first pregnancy \[[@B15]\]. Risk factors for symptomatic malaria in adolescents have not been studied, but both symptomatic and asymptomatic malaria infection will contribute to the development of adolescent and newborn anaemia \[[@B16]\]. HIV infection increases P. falciparumprevalence during pregnancy and this is marked during the first pregnancy \[[@B17],[@B18]\], which in Africa, is nearly always in adolescence. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Malaria prevalence in non-pregnant and pregnant adolescents and adults in the Shire Valley, Malawi ::: Adolescent Adult ------------------ ----------------- ------------------ ---------------- Non-Pregnant Pregnant \* Non-Pregnant Pregnant\* % (n) % (n) % (n) % (n) 41.4(29)† 45.9 (122)‡ 20.3 (74) 35.4 (345) \* At delivery. Includes women who had received anti-malarials during pregnancy † Difference with non-pregnant adults χ^2^= 4.8; p = 0.028 ‡ Difference with pregnant adults χ^2^= 4.3; p = 0.04 ::: HIV and malaria interventions during adolescence ------------------------------------------------ For both HIV and malaria there are effective interventions for disease control. HIV and malaria frequently occur in the same African populations and the same individuals, yet control activities are directed to different target groups, using different channels. Adolescent HIV prevention, with its focus on behavioural change, has taken place largely outside conventional health settings. Few HIV strategies have been directed at pregnant adolescents and/or their partners -- an omission noted by a WHO Technical Consultation on married adolescents \[[@B19]\]. In some African countries, by the age of 19 years, half of all female adolescents were in marital or permanent consensual unions \[[@B9]\] that will almost certainly lead to pregnancy. In contrast, malaria control efforts for women have focused on pregnancy, but not on adolescents and certainly not on adolescents before they become pregnant. As a result, in many sub-Saharan African settings, adolescents are often parasitaemic and anaemic when they first become pregnant. The problem is that currently no overall strategy for adolescent health has been approved for most African countries. The burden of infection and disease among adolescents is of sufficient magnitude that a way must be found to provide services to this population. The existence of an appropriate strategy would allow both HIV and malaria interventions to be delivered within a common framework. In traditional cultures, menarche often marks the transition from childhood to adulthood because it signifies reproductive potential. Nevertheless, adolescence is a physical process that takes a number of years and is usually completed by the late teens. This developmental process should be encompassed within health care provision. Pregnancy may punctuate that process, but during it, the adolescent continues to grow physically and mentally \[[@B20]\]. Strategies to reduce adolescent malaria or HIV should, therefore, provide a continuum of care -- before conception, during and after pregnancy -- encompassing appropriate information, preventive and curative services. For prevention, adolescents need access to condoms and bednets, information on how to use them and, when and where to go to deal with infection. Services must be available for infection monitoring and treatment, including HIV testing and drug therapy for malaria, anaemia and HIV-related opportunistic infections. System improvements may not be strictly about adolescent health services, but about support for ANC. Adolescents need good access to pregnancy care and this includes attendance for early pregnancy assessment and screening for HIV and other sexually transmitted infections (STIs). ANC presents an opportunity for HIV prevention, support for discordant couples and care and referral for HIV-infected adolescents. Early assessment is important as the HIV positive individual needs to plan for anti-retroviral treatment (ART) at delivery to prevent mother-to-child transmission. It is also important for malaria control in pregnancy. Intermittent preventive antimalarial treatment (IPT) is the key malaria prevention policy that improves birthweight outcomes and reduces pregnancy anaemia \[[@B21]\]. Its effectiveness depends on the prevalence of HIV infection \[[@B22]\]. It also depends on adherence to the treatment regimen. Coverage with the standard two dose intermittent sulphadoxine-pyrimethamine preventive malaria treatment was lower in younger adolescents in the Shire Valley, Malawi ≤ 17 years, 35.1% versus 18--19 years, 53.7%; p = 0.028, Verhoeff, personal communication), and more than half of all pregnant adolescents received inadequate antimalarial treatment. This demonstrates the importance of pre-conceptual malaria health education for girls. Adolescent mothers need instruction on how to recognize and respond to infant malaria infection and encouragement for using bednets for themselves and their babies. A major challenge for malaria control is to improve adolescent health prior to the first pregnancy. For non-pregnant adolescents there are several conundrums. On account of drug resistance, malaria combination therapy with artemisinin derivatives is being introduced into an increasing number of sub-Saharan African countries. Artemisinin may be foeto-toxic and should be avoided, except for symptomatic infections with multi-drug resistant malaria, in the first trimester of pregnancy \[[@B23]\]. Inadvertent use or self-treatment by adolescent girls could frequently occur in early pregnancy, before they recognise their pregnancy. The issue of pregnancy testing adolescent girls before treatment with potentially foeto-toxic drugs will need to be considered. A further issue is that the benefits of IPT extend to pregnant but not nulliparous adolescents who may be anaemic or parasitaemic at the start of their pregnancy. The use of permethrin-treated bednets would protect against anaemia and parasitaemia during the first trimester, but adolescent pregnant girls and their newborns are the least likely to be net users \[[@B24]\]. Reaching the target population ------------------------------ The growing literature on adolescent-friendly health services shows that adolescents have substantial concerns about the delivery of interventions through health services \[[@B25]\]. Almost universally, adolescents complain of lack of information, confidentiality issues and judgmental attitudes of service providers. There may be barriers of physical inaccessibility, service fees and non-supportive community attitudes. A WHO consultation in Africa concluded that adolescents had \"a right to access health services that can protect them from HIV/AIDS and from other threats to their health and well-being, and that these services should be made adolescent friendly\" \[[@B26]\]. A WHO Global Consultation on Adolescent Friendly Health Services stated that government ministries should take appropriate action to ensure service provision for adolescents, taking into account cost, epidemiological factors and national health priorities \[[@B27]\]. The role of non-governmental organizations and the private sector in spearheading adolescent projects was acknowledged, but alone, these organizations cannot obtain sufficient coverage of the adolescent population to substantially reduce the HIV and malaria burden. Governments and disease control programs must also consider training health care providers and modifying service delivery, so as to reduce barriers to adolescent uptake. The only country in Africa that has scaled up adolescent health services is South Africa \[[@B28]\]. The South African National Adolescent-Friendly Clinic Initiative has strengthened the public health sector\'s ability to respond to adolescent health needs by building capacity and establishing good clinical services which, eventually, will be maintained by district and provincial health authorities. It has demonstrated that an adolescent strategy is achievable if there is political will. The South African approach may not be suitable or affordable for lower income countries. Well-focused services, training of key staff at selected sites, outreach preventive services and good quality, adolescent-oriented ANC are potential service reconfigurations that could be used to extend coverage and increase uptake of both HIV and malaria interventions without creating a separate adolescent service. What about young men? Available evidence suggests that young men are not attracted to clinic services which are perceived as \"female \"spaces \[[@B29]\]. In Ghana, for example, 76--89% of all adolescent clinic users were female \[[@B30]\]. Malaria is a reproductive health issue for females, but not for males, and the disease burden of both malaria and HIV infections is much lower in young males. Adolescent females require more health-service based care than males, and this should be reflected in service configuration. Creating an enabling environment to promote adolescent access to HIV and malaria interventions ---------------------------------------------------------------------------------------------- Behavioural change, including appropriate health-seeking practices, is essential for disease reduction. A decline in HIV prevalence amongst adolescents has been reported from Zambia \[[@B7]\], Uganda \[[@B31],[@B32]\] and Tanzania \[[@B33]\] and is attributed to changes in sexual behaviour. In Zambia, between 1996 and 1999, urban adolescent males reported less frequent sexual activity, fewer partners and were more likely to have used a condom at the last sexual encounter. Changes in these indices were less marked, or even deteriorated, among rural female adolescents \[[@B7]\]. Whether behaviour change is the result of HIV programme interventions or other factors is unclear. Recent surveys in sub-Saharan Africa reported that higher female education was associated with lower HIV prevalence and lack of schooling with higher HIV incidence \[[@B6]\] and lower condom use \[[@B34]\]. In western Uganda the proportion of illiterate women fell from 41.6% to 24.6% over six years and may have facilitated behavioural changes, but this link has been little investigated. Illiterate females are likely to be young and married, and to be in a situation where they cannot reduce frequency of intercourse, negotiate condom use or delay having children \[[@B19]\]. In a large pregnancy study in southern Malawi, 73% of 469 nulliparous adolescents were illiterate \[[@B35]\]. They made significantly fewer ANC visits, were less likely to have a supervised delivery and had a higher risk of low birthweight. Reducing illiteracy and increasing educational and vocational opportunities for female adolescents may be critical enabling actions to promote risk reduction, to enhance their social status and to decrease female vulnerability. \[[@B36]\] Developing policies to support HIV and malaria control for female adolescents ----------------------------------------------------------------------------- Few developing countries have well-defined policies for delivering care to the non-pregnant or pregnant adolescent, especially for sexual health. Existing malaria guidelines also do not mention adolescents specifically \[[@B12]\]. An important aspect is the issue of the adolescent\'s right to sexual and reproductive information and services vis à vis the legitimate rights of parents to act in the best interests of a child, and the health care provider\'s right to work within the law. These are issues of consent and confidentiality for adolescents considered \"minors\" and under parental control, or wives under their husband\'s or husband\'s family\'s jurisdiction. When these issues are ignored, adolescents cannot be assured of confidentiality, and health workers are neither bound, nor protected by, the legal system. Rights established in law, or by signed Conventions (eg Convention of the Rights of the Child), may not be implemented. Health policy falls down because it is not explicit about the vulnerability of female adolescents and does not challenge cultural and social norms that can perpetuate the disease burden. Similarly, encouragement for improving female literacy and education may be lacking, reducing the supportive environment for promoting uptake of adolescent health services and safer sex. Advocacy is essential to co-ordinate law, custom and health practice. At another level, it is important to acknowledge that interventions for female adolescents raise ethical issues that are avoided when they are excluded from care. The policy of avoiding treatment of adolescent girls (and adult women) because they might be in the first trimester of pregnancy, raises issues of gender discrimination and has been a serious issue for parasitic disease control programs \[[@B37]\]. Reducing the necessity for first trimester treatments for malaria can be achieved by increasing efforts to redress the malaria burden prior to pregnancy, but this requires reconfiguring service delivery. HIV-positive adolescents who are at the start of their sexual and reproductive lives might be prioritized to receive anti-retroviral therapy, but their relative poverty and lack of social status may lead to their exclusion from access to this intervention. Conclusions =========== For both HIV and malaria, although interventions are available to reduce the disease burden, adolescents are not prioritized. Effective policies and services are needed to reduce the substantial disease burden they experience from these infections. To secure this goal, concerted action across HIV and malaria disease control programs is required. This will reinforce support for adolescent programs rather than weakening them by multiple separate activities, which can confuse implementation. A schedule of activities is required that is reinforced by other systems (e.g. education) to facilitate embedding it into a way of life. These issues need to be addressed head-on by the adolescent health community to achieve the focus needed, and to establish the realistic steps forward. In the short term, developing services to deliver HIV and malaria interventions can act as a catalyst for addressing broader adolescent health service needs. In the medium-term adolescent-friendly health services provide the required platform for the evaluation and delivery of future vaccines and drug therapies for HIV, malaria \[[@B38]\] and other diseases. In the longer term an inter-sectoral adolescent strategy provides a basis for breaking the cycle of maternal-child ill health. Authors\' Contributions ======================= Both authors made substantial contributions to the analysis, interpretation, drafting and revision of this article. Both have agreed to authorship. Acknowledgements ================ L. Brabin is funded by the Max Elstein Trust. Dr. F. Verhoeff provided unpublished data. B. Brabin has contributed to this review as a member of the PREMA-EU Concerted Action on Malaria and Anaemia Control in Pregnancy (European Commission Research Directorate General, Fifth Framework. Contract: PREMA-EU-ICAL-CT-111012).	PubMed Central	2024-06-05T03:55:52.605506	2005-1-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548285/", "journal": "Malar J. 2005 Jan 10; 4:2", "authors": [ { "first": "Loretta", "last": "Brabin" }, { "first": "Bernard John", "last": "Brabin" } ] }
PMC548286	Background ========== Leishmaniais an intracellular pathogen that causes Leishmaniasis, which remain an important medical problem in several countries. Protozoan parasites of the genus Leishmaniaresult in a spectrum of human disease that range from self-healing cutaneous ulcers to potentially fatal visceral infection, depending primarily upon the species of parasite involved \[[@B1],[@B2]\]. Leishmanialive as either extracellular flagellated promastigotes in the digestive tracts of their sand fly vectors or as nonflagellated amastigotes within macrophages where they survive and replicate within phagolysosome. Leishmaniaare known to export large range of proteins and glycoconjugates including lipophosphoglycan \[[@B3]\]. In a previous paper we have characterized a Leishmania majorgene encoding a protein carrying extensive homology to SIR2 of yeast that we consequently termed LmSIR2rp \[[@B4]\]. It belongs to a large family of closely related NAD-dependent deacetylase named Hst proteins (Homologous of Sir two) or sirTuins, present in both prokaryotic and eukaryotic species \[[@B5]\]. Post-translational modifications of histones, like acetylation by histone acetylase (HAT) and deacetylation by histone deacetylase (HDAC), within the context of chromatin has been shown to regulate gene expression \[[@B6],[@B7]\]. To date, three classes of HDACs are characterized in eukaryotes, the class I and II HDACs, and the class III defined by the sir2 family. In eukaryotic species some members of the Sir2 family are much more closely related to the core domain of the yHst2p protein than to the core domain of ySir2p itself \[[@B8]\]. These are: the SIR2 family members from Schizosaccharomyces pombe, C albicans, Leishmania, chicken, human (hSirT2p, hSirT3p) and the closely related mouse protein (MmSirT2p and MmSirT3p) \[[@B8]\]. This group has been designated Hst2-like, as it forms an independent branch within the Sir2 family tree. Five members of this group (LmSIR2rp), like it yeast (yHst2) human (SIR2L) and mouse (MmSIR2L2 and MmSIR2L3), are cytoplasmic \[[@B4],[@B9],[@B8],[@B11]\]. The implication of SIR2 proteins bearing nuclear localization, in aging process has been extensively studied. In yeast, the deletion of SIR2 shortens lifespan and over expression extends lifespan \[[@B12]\]. This phenotype of life extension has been also observed in Caenorhabditis eleganscarrying extra copies of the SIR2 orthologue, Sir-2.1 \[review in \[[@B13]\]\]. In mammalian cell, SIRT1 promote cell survival \[[@B14]\]. SIRT1 is able to deacetylate FOXO3 and/or FOXO4, thus attenuating FOXO-induced apoptosis and potentiating FOXO-induced cell-cycle arrest. Thus SIRT1 might increase longevity by shifting FOXO dependent responses away from cell death and toward cell survival \[[@B15]\]. Also, the unusual NAD-requirement for the Sir2 deacetylase may link metabolic rate to silencing and life span \[[@B16]\]. We have shown that the cytosolic LmSIR2rp protein, when over expressed in Leishmaniaamastigotes, was able to promote parasite survival in in vitroculture systems \[[@B9]\]. Further, we also observed that LmSIR2rp can be detected in the excreted/secreted material of Leishmaniaparasites when using radiolabelled immunoprecipitation technique, suggesting therefore that fraction of LmSIR2 is actively excreted by parasite (personal observations). We then initially surmised that this protein could also be involved in some aspect of host cell-parasite interplay, particularly in pathways that could contribute to cell survival. To test this hypothesis, the leishmaniaLmSIR2 gene was cloned in a mammalian shuttle vector and the expression of the protein was performed in fibroblasts. Unexpectedly, expression of the LeishmaniaSir2 protein leads to a cell growth arrest phenotypes, which correlates, with the expression by the cells of an acidic (pH 6.0) β-galactosidase activity, known to be a senescence biomarker. Furthermore, fibroblasts expressing LmSIR2 were more permissive towards Leishmaniainfection than control ones. Altogether our results may suggest that the excreted forms of LmSIR2 could participate in the perturbation observed during chronic disease. Materials and Methods ===================== Molecular construct and transfection procedure ---------------------------------------------- LmSIR2was amplified using sense GAA TTC GAT ATG ACA GGG TCT CCG and antisense CTC GAG CAG TCA CCA TGT TGG CAG primers. The 1119 bp PCR amplified genomic fragment subcloned into pCR2.1 was digested with EcoRI and inserted into the EcoRI digested and dephosphorylated pcDNA3.1 shuttle vector (Amersham). Restriction analysis of the recombinant plasmids using EcoRI and XhoI allowed the identification of recombinant vector carrying the LmSIR2gene in sense orientation. Large-scale preparations of pcDNA-LmSIR2and pcDNA empty vectors were performed using the QIAGEN plasmid maxi kit. Transfected cells were selected in RPMI 1640 medium containing 10% FBS and 400 μg/ml G418. Each G418-resistant clone, isolated by limiting dilution, were propagated and screened by immunofluorescence. Four clones were isolated and named Cl2, Cl9, Cl10 and Cl11. Production of monoclonal antibodies (mAb) against the fusion protein -------------------------------------------------------------------- LmSIR2 recombinant protein containing 6 X histidine residues at its N-terminal (hisLmSIR2) was previously obtained after having subcloned the encoding gene in the pQE-expression vector \[[@B17]\]. Mice of BALB/c strain were injected intraperitoneally with 50 mg of hisLmSIR2 in saline solution emulsified with 0.1 ml of Freud\'s Complete Adjuvant. After 2 weeks, a booster injection of the same preparation was given. The antibody response of the animals was tested 1 month later. The same dose of antigen was injected intravenously 3 days before the hybridization experiment. The fusion procedure and the screening of mAb were carried out as previously described \[[@B18]\]. The class of mAb was determined by agarose double diffusion of supernatants against antisera specific for immunoglobulin subclasses obtained from cliniscience (Montrouge, France). Several hybridoma clones producing mAb reacting in an ELISA test with LmSIR2 protein were obtained; only one mAb of the IgG1 isotype (IIIG4) reacted in Western blot against the fusion as well as the native LmSIR2 protein. Growth kinetic and ^3^H-thymidine incorporation ----------------------------------------------- L929 cells were inoculated at 400 cells/cm^2^, after various period of incubation they were collected by using Trypsin-EDTA (0.025%). The mean number of viable cells was determined at 100-× magnification after staining with trypan blue. In order to evaluate the rate of ^3^H-thymidine incorporation, cells were seeded in 96 wells plates at 1000 cells/cm2, after various periods of incubation 1 μCi of ^3^H-thymidine was added to each well. Cells were further incubated for 4 hours before reading the level of incorporated radioactivity. Giemsa staining and Immunofluorescence analysis ----------------------------------------------- L929 cells were seeded in 16-well Labteck chambers for 48 hrs. Cells were then washed with PBS and fixed for 30 minutes with 4% paraformaldehyde at 4°C. After two washes with PBS, cells were permeabilized with 0.01 M PBS containing 0.5% TritonX-100 and 2% BSA for 30 min at 4°C. Slides were then washed and incubated for 1 hour with mAb IIIG4 in 0.01 M PBS containing 2% BSA. After two washes, slides were incubated for 45 min with fluoresceine-conjugated rabbit anti-mouse IgG (Diagnostic Pasteur, Marnes la Coquette, France) diluted 1:100 in PBS containing Evan\'s blue (final dilution in PBS: 1: 10.000) and mounted in glycerol PBS (1:1). Giemsa staining of the cell culture was performed as follows: cells were inoculated at 400 cells/cm^2^in 25 cm^2^flasks, after 7 days of growth they were fixed with methanol and stained with Giemsa and examined observed on inverted microscope (40× magnification) after adding a solution of PBS 0,01 M-Glycerol (1/1). RT-PCR analysis --------------- Total RNA was isolated from wild type and transformed cells using the Rneasy Mini Kit following the manufacturer\'s instructions. One microgram of RNA was reverse transcribed to cDNA with an oligonucleotide \[poly (dT)~12--18~\] using the Superscript II RNase H-reverse transcriptase. PCR amplifications were performed using 1--2 μl of reverse-transcription of each product and 20 pmol of each primer, using Taq DNA polymerase. Each PCR cycle consisted of a denaturation step (94°C, 1 min), an annealing step (55°C, 1 min) and an elongation step (72°C 1 min). For the last cycle, the elongation step was extended to 10 min at 72°C. Reactions were carried out for 25--35 cycles in a thermocycler (PTC-100™, MJ Research, Inc.). PCR products were analyzed on 1.5% agarose gel and visualized with ethidium bromide. As an internal control, a housekeeping gene, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript, was amplified. LmSIR2primers were: Sense GAA TTC GAT ATG ACA GGG TCT CCG and antisense CTC GAG CAG TCA CCA TGT TGG CAG. GAPDH primers were: Sense GTC TTC ACC ACC ATG GAG and antisense CCA AAG TTG TCA TGG ATG ACC. β-galactosidase pH6 analysis ---------------------------- Acidic (pH 6.0) β-galactosidase was assayed according to Dimri et al. \[[@B19]\]. Briefly, stationary-phase cells were washed three times in phosphate buffered saline (0.1 M PBS, pH 7.4), fixed in 2% formaldehyde/0,2% glutaraldehyde for 5 min at room temperature, washed three times with PBS, then incubated at 37°C without CO~2~for 12 h with freshly prepared β-Gal staining solution pH 6.0, containing the 5-bromo-4-chloro-3-indoyl-b-D-galactosidase (X-Gal) chromogenic substrate which produces blue 5-bromo-4-chloro-3-indole upon hydrolysis. Cell preparation was stored at 4°C in 70% glycerol upon analysis. Results and Discussion ====================== Expression of LmSIR2 by L929 fibroblasts ---------------------------------------- The Level of the LmSIR2 gene transcription was evaluated by RT-PCR analysis. As shown in Figure [1](#F1){ref-type="fig"}, an amplification product of around 1.2 Kb was observed only in recombinant L929 cells transformed with the pcDNA carrying LmSIR2 gene (Fig [1](#F1){ref-type="fig"} lane 2), No amplification could be detected in Wild-type fibroblasts or in fibroblasts carrying the empty pcDNA vector (Lane 1 and 3 respectively). Accordingly, Western blot analysis of cellular extracts derived from the clones 2 and 11 reacted with with the mAb IIIG4 showed a polypeptide of 50 kDa (Fig [2](#F2){ref-type="fig"} lanes 3 and 5), which is slightly higher than the molecular size of the pcDNA encoded protein (≈ 43 kDa). However, it is noteworthy that this molecular size is similar to that of the parasite native protein shown in previous studies \[[@B9]\]. It is therefore likely that the transfected gene product is submitted to posttranslational modifications that occur during its processing. No cross reactivity was observed on cell extract derived from Wild-type cell or fibroblast carrying the empty vector (Fig [2](#F2){ref-type="fig"} Lane 1 and 2). Thus, these results confirm that mouse L929 fibroblast cell line properly expressed the LmSIR2gene. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Expression of LmSIR2 gene in L-929 cells.RT-PCR analysis of total RNA from various L-929 clones was carried out using LmSIR2 or GAPDH primers. The lanes correspond to the following samples: standard size marker (PM); WT L929 cells (1); L929 clone 2 (Cl2) (2); L929 carrying an empty pcDNA plasmid (3). ::: ![](1475-9292-4-1-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Detection of the LmSIR2 protein in L929 cell extracts.The lanes correspond to the following samples: WT L929 cells (1); L929 carrying an empty pcDNA plasmid (2), L929 Cl2 (3); Molecular weight markers (4); L-929 Clone 9 (5). ::: ![](1475-9292-4-1-2) ::: The LmSIR2gene was originally isolated via immunoscreening of a cDNA library, using polyclonal antibodies raised against excreted factors which could bind to glutathione \[[@B4]\]. The presence of such protein in the leishmania\'s excreted material as previously demonstrated raised the question of its function during the infection process. Thus, heterologous expression in eukaryotic cells could represent a useful approach to give some insight into the biological activity of secreted parasite material. Immunolocalization of LmSIR2 shows that cells expressing LmSIR2 (Figure [3B](#F3){ref-type="fig"}) react with the monoclonal antibody as compared to cells carrying the empty pcDNA vector (Figure [3A](#F3){ref-type="fig"}). Interestingly, high number of cells positive for LmSIR2 bears abnormal morphology i.e. giant multinucleated cells (Figure [3B](#F3){ref-type="fig"}). In these cells, the protein is localized both in the cytosol and the nucleus. Functional studies of several mammalian SIR2 related proteins have been carried out including those who are mainly localized in the cytosol. Nevertheless, similar cellular localization pattern has never been reported nor such modification of cell physiology. Our observations suggest that LmSIR2 possesses unrelated specificity that can strongly affect the biological properties of the host cell. Interestingly, LmSIR2 protein presents a NES (the nuclear export signal or NES)-like motif LX~3~L~X~LX~3~L at its carboxy-terminal region, where L represents Leucine and X any amino acid \[[@B20]\]. The intracellular localization of key regulatory proteins tagged with a short Leucine-rich motif (nuclear export signal) is controlled by CRM1/exportin1, which is involved in the export of these proteins from the nucleus \[reviewed in \[[@B21]\]\]. However, the ability of the LeishmaniaSIR2 to shuttle between the cytoplasm and nucleus of the host cell through the NES putative sequence, await further investigations. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Indirect immunofluorescence analysis and morphology of transgenic cells. Localisation of LmSIR2 using immunofluorescence analysis (A) L929 cells carrying an empty pcDNA vector (B) L929 (Cl2) expressing LmSIR2 protein. Morphology of cells in culture: (C) L929 cells carrying an empty pcDNA vector and (D) L929 Cl2. Note the reduced cell density and the presence of cells bearing abnormal morphology (arrow). ::: ![](1475-9292-4-1-3) ::: When L929 cell culture, of either clone 1, clone 2, clone 9 or clone 10, were stained with Giemsa, a high rate of cells with unusual shape and size associated with the presence of multiple nuclei was revealed (Figure [3 D](#F3){ref-type="fig"}) as compared to cells carrying the empty pcDNA vector (Figure [3C](#F3){ref-type="fig"}). The rate of multinucleated cells was determined by counting the number of cells bearing more than 2 nucleus per 10 champs at 10-× magnification. In fact all the clone cultures present a 10 to 15-fold increase in giant multinucleated cells as compared to cultures of L-929 fibroblast transformed with the empty vector (Figure [3 C](#F3){ref-type="fig"}). Moreover, majority of the cells if not all, present an enlarged and flattened morphology (Figure [3 D](#F3){ref-type="fig"}). Growth kinetic parameters of L929 cell expressing LmSIR2 -------------------------------------------------------- In order to investigate the potential functions of LmSIR2 protein, when delivered intracellularly in L929 fibroblasts, we have determined the growth kinetic parameters of cells via two different methodologies. Using a counting method, we observed a high reduction in the saturation density of cells expressing LmSIR2 (Figure [4A](#F4){ref-type="fig"}). The reduction observed in this experiment was as high as 40% as compared to cells carrying the empty pcDNA vector. These preliminary observations were further confirmed using ^3^H thymidine. Indeed, as shown in figure ([4B](#F4){ref-type="fig"}), expression of LmSIR2 strongly reduced the thymidine incorporation over the time course of the experiment. The most dramatic reduction of 3H-thymidine incorporation was observed a day 7 which correspond to the maximal cell density observed for both Wild type control and the four clones studied (reduction in ^3^H-thymidine incorporation of about 61% for clone 2, 60% for clone 9, 52% for clone 10 and 42% for clone 11). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Growth kinetics analysis.Cell proliferation was determined using two methods: cell counting (A), the results are given as a mean value of a triplicate experiment ; and ^3^H-thymidine incorporation (B), the results are given as mean value of a sextuplate experiment ::: ![](1475-9292-4-1-4) ::: Expression of senescence biomarker ---------------------------------- Parameters of senescence include increased cell size, reduced saturation density as well as altered expression of some gene products that have been proposed to serve as biomarkers of aging. Reduction in the final cell yield or saturation density is known to occur in human diploid fibroblast senescence and is considered as a criterion of the onset of senescence \[[@B22]\]. With respect to biomarkers, pH 6 β-galactosidase is particularly useful, since this activity has been found to distinguish senescent from quiescent cells both in vivoand in vitro\[[@B19]\]. We found that L929 cells expressing LmSIR2 exhibit reduced saturation densities (40% to 60%) and altered cell morphology. We have thus evaluated the occurrence of the pH 6 β-glactosidase activity. As shown on Figure [5](#F5){ref-type="fig"} (B and C), a strong pH-6 β-glactosidase was detected in L929 cells expressing the LmSIR2gene but was not detected in wild-type cells or cells transformed with the empty pcDNA vector (Figure [5A](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Expression of a senescence biomarker.(pH6.0) β-galactosidase activity detected in L929 cells expressing LmSIR2 (Cl2) (B and C) but not in L929 carrying an empty pcDNA plasmid (A). Cells were seeded at 40 cells/mm^2^in 25 cm^2^flasks and after 13 days of growth, the presence of pH6-galactosidase activity (Arrow) was determined as described in the Materials and Methods section. N nucleus, note the perinuclear galactosidase activity. ::: ![](1475-9292-4-1-5) ::: Permissivity of fibroblast expressing LmSIR2 towards Leishmania amazoensis ---------------------------------------------------------------------------- Fibroblasts at the lymph are used as safe target for long lasting residual parasite population \[[@B23]\]. However, only few reports deal with in vitroinfection of fibroblast. In 1978, Chang \[[@B24]\] demonstrated that promastigotes of L. amazonsensisbut not L. donovaniwere able to infect human skin fibroblasts. However, once differentiated into amastigotes the parasites were unable to multiply inside the cell. Dedet and co-workers further confirmed these observations \[[@B25]\]. Thus, we decided to test the capacity of L. amazonensisto infect L929 cell lines. When L. amazonensispromastigotes were incubated with Wild-type fibroblasts the proportion of infected cells reached a peak of about 50% after 24 hours of contact, after what the proportion of infected fibroblasts decreased quickly. On day 2, only few parasites could be seen and after 3 days of incubation less that 1% of fibroblasts remain infected (data not shown). As shown in Figure [6](#F6){ref-type="fig"}, fibroblasts expressing LmSIR2 were more permissive toward Leishmaniainfection since the mean percentage of infected fibroblast reach 70% after 24 hours of contact. However, once inside fibroblasts no differences in the capacity of parasites to survive and/or multiply was observed and the intracellular amastigote population was eliminated after three days of contact (data not shown). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### *Permissivity of L929 cells toward Leishmania amazonensisinfection.L929 cells were seeded in 25 cm2 flasks and treated with mitomycin. They were then infected, at different parasite/fibroblast ratio, with freshly differentiated stationary phase L. amazonensispromastigotes. After 24 hours of contact cells were washed and the percentage of fibroblats bearing attached or internalized parasites was determined. Results are expressed as a mean value of a triplicate experiment. ::: ![](1475-9292-4-1-6) ::: Conclusions =========== Our study demonstrates that LmSIR2 is able to down regulate the proliferation of fibroblasts and to induce a senescence-like state in L929 fibroblast cell line. The most interesting finding is that a parasitic protein is able to modify the physiology of the host cell making them more permissive toward Leishmaniainfection. These results suggest that LmSIR2 has dual function according to the cell type in which it acts: in Leishmaniaparasites, LmSIR2 is able to promote cell survival while in the host fibroblast it induces a senescence phenotype. This effect could be beneficial for Leishmaniasince senescent cells are no longer capable of dividing yet remaining metabolically active. They could thus represent safe target for parasite. It is documented that senescent fibroblasts present an inherent higher capacity to resist to apoptotic death \[[@B26]\]. In this view, it will be of interest to determine if fibroblasts that express LmSIR2 were more resistant to apoptotic stimuli. It is known that infected macrophages are usually more resistant to apoptosis, however it is not know if such mechanism operate in infected fibroblast. Thus, the implication of LmSIR2 in a general mechanism leading to apoptosis resistance of both macrophages and fibroblasts await further investigations. The in vivorelevance of our observation is rather difficult to appreciate because; (1) the level of the protein produced in fibroblasts transformed with the pcDNA is certainly far higher than the level achievable in vivo, and (2) the presence of excreted parasite material inside the fibroblast cytosol has yet not been reported. However, an action of the excreted form of LmSIR2 on he host cell could no be ruled out. Two series of observations support this possibility: (1) it has been shown that the LeishmaniaEF1-a could be translocated from the parasitophorous vesicle into the macrophage cytosol \[[@B27]\]; (2) the ultrastructural examinations of skin biopsies obtained from patients with cutaneous leishmaniasis showed that intracellular amastigotes could be frequently identified in vacuoles and in the cytosol of the host cell \[[@B28]\]. In conclusion, although the effect of the protein, when delivered extracellularly, on the host cells have not being examined in the present study, our data support the notion that the excreted form of LmSIR2 is able to interfere with the host cell physiology, when delivered intracellularly. Unexpectedly the expression of the protein is associated with the appearance of a senescence biomarker. Relevance of such finding during the in vivo*infection process awaits further investigations Acknowledgement =============== This work was supported in part by a grant from IRD and INSERM. AMA was supported by a DSF IRD (France) grant. BV was supported by an ANRS (France) grant.	PubMed Central	2024-06-05T03:55:52.607218	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548286/", "journal": "Kinetoplastid Biol Dis. 2005 Jan 24; 4:1", "authors": [ { "first": "Denis", "last": "Sereno" }, { "first": "Laurent", "last": "Vanhille" }, { "first": "Baptiste", "last": "Vergnes" }, { "first": "Adriano", "last": "Monte-Allegre" }, { "first": "Ali", "last": "Ouaissi" } ] }
PMC548287	Review ====== Assessments of patient-focused health status and health-related quality of life (HRQL) are being recognized increasingly by clinicians, patient advocates, regulatory authorities, administrators and policy makers as primary measures of the need, efficacy, effectiveness and efficiency associated with health care services. These types of measures are commonly reported in the published literature of many health care disciplines although there are few published reports concerning orthopedic surgery. However, the orthopedic community is becoming interested in using evidence reported by patients and patients\' family members to determine what is best for patients. Patient-focused evidence (including patient-reported outcomes, PROs) refers to results from functional health status (FHS) and HRQL studies of orthopedic outcomes with measurements obtained from the perspective of patients or their non-clinician caregivers (e.g., parents of patients too young to self-report). The field of orthopedics is concerned with all musculo-skeletal problems from the top to the bottom of the human body. This broad anatomical range is associated with a wide range of functional problems. Some orthopedic problems are highly focused in regards to anatomy and associated with highly effective therapies. However, other orthopedic problems are of a diverse nature, the etiology is often poorly understood, and effective treatment strategies are sometimes elusive. The breadth and depth of some particularly challenging orthopedic problems has stimulated an interest in patient-based perspectives of their own FHS and HRQL. These types of measures will be referred to as \"health measures\" for the purposes of this paper. Health measures can be used to assess the burden of morbidity associated with orthopedic diagnoses, and to assess the effectiveness and efficiency of therapies. The diversity of issues also led the American Academy of Orthopedic Surgeons and the Pediatric Orthopedic Society of North America to develop the Pediatric Outcomes Data Collection Instrument (PODCI), and led other researchers to develop function-specific instruments such as the Activities Scale for Kids (ASK). However, there are a large variety of potentially appropriate health measures and there are limits to the number of these measures that can be used in any given study. The most appropriate set of health measures should be selected on the basis of underlying study objectives and design criteria \[[@B1]\]. Objectives and designs vary greatly across orthopedic studies. In general, orthopedic procedures and programs are undertaken to improve the health of patients. The health of patients can be measured in different ways. Conventional clinical or physiological measures are generally very useful for diagnostic and therapeutic purposes. Conventional clinical measures may, however, provide an incomplete assessment of health. For example, clinical examination and gait measures have been used to describe the quality and quantity of limitations in walking ability associated with hip flexion contracture but these types of assessments provide little information about the importance of this contracture relative to that of other serious health problems. In addition, various measures may provide conflicting results: some indicating improvement while others suggest decline in patients\' health. Therefore, in the interest of scientific rigor, protocols to assess the overall effectiveness of therapy should include a priorispecification of the comprehensive measure of health and a credible assessment viewpoint for the purposes of primary analysis of study results. Secondary study objectives may involve use of data collected from other health measures or from other assessment perspectives. FHS and HRQL measures are important for a variety of reasons that complement conventional clinical measures \[[@B2]\]. FHS measures provide descriptive information and HRQL measures add some type of valuation about the desirability of overall health status. Valuation may be based on preference-based scores or by including only items identified as important to patients. HRQL is a more comprehensive concept than FHS as noted in this leading definition. \"Health-related quality of life is the value assigned to duration of life as modified by the impairments, functional states, perceptions, and social opportunities that are influenced by disease, injury, treatment or policy\" \[[@B3]\]. It is generally accepted that a goal of therapy is to make patients feel better \[[@B4]\] and this is supported by statements of leading policy analysts such as \"The goal of health care is to protect, promote, and maintain the health status of people\" \[[@B5]\] and \"Since the ultimate goal of all health care is to improve, restore, and preserve HRQL\" \[[@B6]\]. However, physiological measures may change without people feeling better and people may feel better without measurable change in physiological function. Furthermore there is often a need for trade-offs between treatment-related benefits and adverse side effects. There is an increasing awareness also that various stakeholders in clinical decisions often have differing opinions about disability and that the opinions of patients should count. Numerous valid and reliable questionnaires are now available to collect FHS and HRQL measurements from patients and their representatives. The measures may be used for discriminative (comparing groups at a point in time such as assessing the burden of illness), evaluative (assessing within-person change over time as in clinical trials), and predictive purposes (providing prognostic information) \[[@B7]\]. A series of studies from a randomized controlled clinical trial (RCT) and prospective cohort study of elective total hip arthroplasty are illustrative of these purposes. These examples, while not from a pediatric setting, are orthopedic and demonstrate how data can be used for a variety of purposes. In the RCT, Rorabeck and colleagues \[[@B8]\] undertook a discriminative analysis of a set of FHS and HRQL measures, and reported there were no differences in outcomes between cemented and non-cemented protheses. Evaluative analyses of data from the same trial have documented improvements according to six HRQL measures and the six-minute-walk test \[[@B9]\], and have shown important differences among results from four major generic HRQL measures \[[@B10]\]. In the prospective cohort study by Mahon et al \[[@B11]\], waiting time for surgery was inversely related to baseline WOMAC (Western Ontario and McMaster University Osteoarthritis Index) scores for patients at time of referral to an orthopedic surgeon. There are many factors to consider when designing studies using patient-focused health measures for orthopedic patients. Among the most important factors are the study objectives, types of patients, the dimensions and FHS constructs associated with the health problem under study, the appropriateness of the questionnaire for the age range of the patients, the viewpoint of the assessors answering the questionnaire, the method of collecting questionnaire information (self-completion or interviewer-administration), the period of time respondents are asked to consider when answering the questionnaire (i.e., assessment recall period), and measurement properties of assessment tools. Measurement properties include content and construct validity, test-retest and inter-rater reliability, responsiveness, and practical limitations of data collection. It takes many years, and often decades, for credible and relevant evidence to be accumulated about the measurement properties of instruments. Instruments with well-established measurement properties should be used whenever possible. Two prime issues for assessments of children are the developmental stage of the subjects and the most appropriate respondent for questionnaires. The methods section of the paper presents a framework of important factors for consideration in designing studies to assess FHS and HRQL for orthopedic patients. The results were generated by applying the framework in the context of developing a proposal for a comprehensive international study of patients with neurofibromatosis type 1 (NF1) and congenital tibial dysplasia (CTD). The NF1-CTD protocol provides a rich illustration because it encompasses great variability in the instrument selection factors, and these patients with their supportive caregivers face numerous complex health status challenges with long-term implications for HRQL. Furthermore, there is little information in the published literature about the comprehensive health status and HRQL of patients with NF1-CTD. The rationale and approach to studying FHS and HRQL from a patient perspective have relevance to many other areas of orthopedics. Methods: a framework for orthopedic applications ================================================ This section identifies specific study design and measurement instrument factors that should be considered in selecting measures for use in studies, and presents some background information for illustrating the application of these factors. Study design factors -------------------- Study design factors are specified in detailed protocols that clearly define the study objectives and conditions. Well-defined study objectives identify the study subjects, and type and number of measurements required. Study conditions describe practical issues including data collection sites, and budgets for time and resources. Variability in study objectives ------------------------------- A highly focused, single objective study may require relatively few instruments while a broad-based, multi-faceted study will require numerous measures. For example, a RCT of efficacy for an experimental technique to achieve union of bone compared to a conventional technique might specify patients\' reports from a single well-established walking ability scale as the primary outcome measure. However, an economic evaluation of the cost-effectiveness and cost-utility of the experimental technique would require at least two instruments: the single well-established walking ability scale to estimate effectiveness for the numerator in the cost-effectiveness ratio; and a utility-based scale of overall HRQL to estimate quality-adjusted life years for the numerator in the cost-utility ratio. Age range of subjects --------------------- The age of study subjects affects the choice of measurement instruments and the method of collecting data. Each instrument is valid for a specific age range and the age range is quite small for many pediatric instruments. For example, developmental changes during childhood make it difficult for single instruments to be valid from infancy through adolescence. Many instruments are valid for one or more of the following age categories: infants, pre-school children, primary-school children, adolescents, young / middle-age adults, seniors and elderly. Inability to read well (e.g., young children or populations with high illiteracy rates), or see well (e.g., many elderly populations), or concentrate well (e.g., people on certain strong medications) inhibits use of self-complete questions. Well-designed interviewer-administered questionnaires pose clear, short, well-focused questions with readily understood and easily remembered response options (e.g., yes or no response options). Assessment perspective ---------------------- It is becoming widely accepted in many disciplines that studies of health care programs and technologies should include measures of patients\' perceptions of their FHS and HRQL \[[@B12]\]. Measurements of these types have been shown to vary by assessment viewpoint and, although many different viewpoints are valid, the patient perspective is considered to be one of the most important. It has been shown that children as young as 7 years can reliably complete interviewer-administered disease-specific and generic questionnaires about their own health \[[@B13]\]. Type of data collection sites ----------------------------- The locations and cultural characteristics of the study population will determine the language and sometimes the choice of assessors. Relatively few instruments have been carefully translated and culturally-adapted to facilitate use in a large variety of communities. Some cultures accept a variety of assessment viewpoints (e.g., patient, family, nurse, physician, and other allied health professionals) while other cultures recognize only a physician viewpoint for health assessments. Mode of data collection ----------------------- The literature suggests that there may be important effects due to mode of data collection \[[@B14]\] and, therefore, mode of administration should be standardized across subjects, assessors, and assessment points. In general, self-complete questionnaires tend to elicit reports of greater morbidity than interviewer-administered questionnaires. It is hypothesized that subjects may be less inhibited to report disabilities on self-complete questionnaires than to report disabilities to interviewers. The mode of data collection for a study may be determined by numerous factors other than the characteristics of study subjects mentioned above. A low budget study may rely on a mail survey, or distribution of questionnaires in a busy clinic setting, and use self-complete questionnaires. Alternatively, a study involving serial assessments may require follow-up data to be collected by telephone and therefore require use of questionnaires designed for interviewer-administration. Assessment recall period ------------------------ FHS questionnaires should have well-specified assessment periods to help ensure that the subjects and researchers know the period of time covered by the responses. Assessment recall period refers to the period of time the assessor is asked to consider when answering the questionnaire. The period should match the objectives of the study. For instance, if a new surgical technique is thought to reduce the peri-operative burden of morbidity then frequent assessments with a brief recall period (24 hours) might be suitable. Standard assessment periods include the past 24 hours, the past 1 week, the past 2 weeks and the past 4 weeks. Short assessment periods should be used in studies of patients whose FHS varies over time and in studies involving serial assessments to ensure that there is no overlap in assessment times. Relatively long recall assessment durations may be used when it can be assumed patients\' health status is fairly stable. For example, a four-week recall assessment period was used to measure the HRQL of patients in a randomized clinical trial and economic evaluation of two alternative treatment strategies for patients with knee osteoarthritis \[[@B15],[@B16]\]. Other factors ------------- There are limits to the number and type of measures that respondents can be expected to complete without getting tired or frustrated, and/or that the study budget can afford in regards to both data collection and analysis. The evidence about the limits of respondent burden is sparse. All else being equal, studies involving serial assessments should expect to collect fewer measurements per assessment than single-assessment cross-sectional surveys. To maximize efficiency, instruments should be selected to provide complementary rather than overlapping information. Instrument factors ------------------ ### Types of measures There are numerous definitions of FHS and HRQL. For the purposes of the paper, a FHS measure is defined as being descriptive in terms of functional ability and HRQL is defined as involving some form of valuation of that health status. One published taxonomy \[[@B7]\] suggests that measures may be classified as specific or generic. Specific measures are focused on a specified health problem, disease, or age group of subjects. An example is the Western Ontario McMaster Osteoarthritis Index \[[@B17]\]. Specific measures designed for evaluative purposes are often able to detect small but clinically important differences among subjects and be responsive to small but clinically important intra-subject changes over time. Generic measures are applicable to a broad range of subjects, including a wide variety of clinical groups and general populations. There are 2 types of generic measures: generic health profile instruments such as the Rand-36 \[[@B18]\] and SF-36 \[[@B19]\]; and generic preference-based instruments. There are 2 types of generic preference-based instruments: direct measurement instruments, such as standard gamble \[[@B20]\]; and multi-attribute classification systems with preference-based scoring functions \[[@B21]\], such as the Health Utilities Index \[[@B22],[@B23]\] and the Quality of Well-Being Scale \[[@B24],[@B25]\]. A detailed description of direct measurement techniques is beyond the scope of this paper but provided in a recent review paper by Torrance and colleagues \[[@B20]\]. Direct preference measurement is generally not practical for application in most clinical studies, especially those involving very young children. Customized instrumentation, usually relying on administration by highly skilled interviewers, must be developed for each study. Further, measurement questions are cognitively demanding. Direct preference measurement will, therefore, not be considered further in this paper. Each multi-attribute system includes a descriptive classification scheme to describe and assess health status, and a preference-based valuation system. HRQL scores for health states defined by multi-attribute systems are calculated from models fitted from directly measured preference measurements (see below). Multi-attribute systems provide descriptive information about comprehensive health status, and interval-scale preference scores of overall HRQL from a community perspective on a scale where 0.00 is the score for being dead and 1.00 is the score for being in perfect health. Several multi-attribute systems define negative scores of overall HRQL to represent preferences for states considered worse than being dead. A few systems include single-attribute preference-based scales of morbidity. Single-attribute morbidity scales are defined such that the least desirable level within an attribute (dimension of health status such as vision) has a score of 0.00 (blind) and the most desirable level has a score of 1.00. The community perspective is most widely recommended for technology assessment and reference case economic evaluation analyses \[[@B26]-[@B29]\]. Interval-scale properties, and a score of 0.00 for being dead, are important features of HRQL scales for integrating the effects of morbidity and mortality in descriptive studies and in cost-utility economic evaluations. Interval-scale preference scores of HRQL may be either utilities (e.g., Health Utilities Index Mark 3) or values (e.g., EQ-5D). Utility preference measures are based on von Neumann-Morgenstern utility theory, include an element of risk attitude, and are therefore appropriate for decision problems with uncertainty. Value scores are preferences measured under certainty. Details about differences between utilities and values, and about direct preference measurement, appear in recent papers by Torrance et al \[[@B20],[@B30]\]. Uncertainty is an important factor in many orthopedic procedures and therefore utility scores are more appropriate than value scores in this context. Evidence of measurement properties: validity, reliability, and responsiveness ----------------------------------------------------------------------------- A valid measure is \"sound and sufficient\" \[[@B31]\]. There are many ways to assess validity of measures. Assessments of FHS and HRQL measures should consider at least six types of validity: face validity, content validity, construct validity, convergent validity, discriminative validity, and predictive validity \[[@B32]\]. Face validity requires that a measure appear on the surface to make sense in regards to being relevant and useful. Content validity requires that the measure include all important and relevant domains or dimensions of health status. Construct validity describes the extent to which a measure corresponds to theoretical concepts and convergent validity describes the association between related variables. Discriminant validity is a lack of correlation between dissimilar variables or groups. Predictive validity, one type of criterion validity, describes the relationship between current and future measurements. A measure is reliable if it is sound and dependable \[[@B31]\]. Reliability is assessed by tests of repeatability or reproducibility. Reliability is often assessed in terms of agreement between intra-subject test-retest measurements and inter-assessor measurements \[[@B33]\]. Responsiveness is also referred to as sensitivity to change. It is an important feature for determining a measure\'s ability to detect effects of treatments or natural changes over time (e.g., due to the aging process). Husted and colleagues reviewed the literature and defined two major types of responsiveness: internal and external responsiveness \[[@B34]\]. Internal responsiveness describes the ability of a measure (instrument) to change and has been assessed using a variety of techniques including the magnitude of statistical significance tests (e.g., p \< 0.05 versus p \< 0.001), the mean change score divided by the standard deviation of scores at baseline (effect size), and a sensitivity coefficient calculated as the proportion of the variance in change scores due to treatment \[[@B32]\]. It has also been assessed as the ratio of the mean change in patients\' scores and the pooled standard deviation of the mean change scores \[[@B35]\], and as the mean change score among those who changed divided by the standard deviation of change scores among stable patients \[[@B33]\]. External responsiveness is concerned with the relationship between change in a measurement and change in a reference measurement of health status. External responsiveness has been assessed using the receiver operating characteristic method, correlations (e.g., Pearson product moment correlation), and regression models. The minimum important difference (MID) is the smallest size of difference that is important from patients\' or clinicians\' perspectives. The MID between two measurements is a concept closely related to responsiveness when assessing change over time \[[@B36]\]. Ceiling and floor effects are undesirable properties that reduce the validity, reliability, and responsiveness of measures. A ceiling effect may occur when a large proportion of measurement observations are close to the upper bound of the measurement scale. A ceiling effect results in a positively skewed distribution of measurements, limited ability of the measure to discriminate among subjects at the upper end of the scale, and attenuated responsiveness to improvements in health in longitudinal studies. A floor effect may occur when a large proportion of measurement observations are close to the lower bound of the measurement scale. Floor effects create a negatively skewed distribution of measurements, limited ability of the measure to discriminate among subjects at the lower end of the scale, and decreased responsiveness to decrements in health in longitudinal studies. Many generic and specific measures of HRQL may be subject to ceiling effect problems in that they may not be able to describe patients or subjects with above average (supra-normal) health. Some measures are subject to floor effect problems. Some are subject to both. Typically floor effect problems are more serious in clinical studies (which often involve patients with disabilities) and ceiling effect problems may be more problematic in general population studies. Limits of respondent burden --------------------------- The limits of respondent burden depend upon many factors including the number of questions presented, how the questions are presented, the complexity of questions, the sophistication of respondents, and the respondents\' interest in the questions. In general, the allowable length of questionnaire is shorter for mail and phone administration than for face-to-face interviewer administration \[[@B37]\]. One set of guidelines specifies the following maximum lengths: 20 questions for phone surveys; 60 questions for mailed surveys; and 80 questions for face-to-face interview surveys \[[@B38]\]. Another guideline recommends that telephone interviews not exceed 5 to 10 minutes \[[@B39]\]. These guidelines are in general agreement with maximum recommended number of pages for self-administered questionnaires: 2 to 4 page upper limit for topics not especially salient \[[@B40]\]; 12 page upper limit for self-administered questionnaires \[[@B41]\]; and 4 to 6 page upper limit for mailed surveys \[[@B42]\]. For mail-out surveys, the evidence suggests no effect of length on response rates for questionnaires varying from 3 to 9 pages \[[@B43],[@B44]\] but reduced response rates with questionnaires greater than 12 pages \[[@B41]\]. Availability of support services -------------------------------- Applications of FHS and HRQL measures are greatly facilitated by expert advice, detailed instructions and other services designed to support users of a measure. Supporting documentation is usually protected by copyright and should not be used without written permission of the original developers. Documentation obtained from third-party sources should be considered suspect because it is frequently invalid. Licensing fees are used to fund high quality, readily accessible service centers. Permission to use copyright materials is typically granted one study at a time. Support services may also include consultation about the most appropriate versions of questionnaires for use in a specific study. Application packages may include data collection instruments such as questionnaires, procedure manuals, coding algorithms and scoring systems, as well as background information about the conceptual and measurement properties of the instrument. NF1-CTD: A case study --------------------- Recently there has been interest in using measures of FHS and HRQL to evaluate treatment alternatives for NF1-CTD. NF1 is one of the most common genetic disorders in childhood \[[@B45]\]. It is estimated that at least 1 million people throughout the world have NF1 \[[@B46]\]. NF1 has a wide range of clinical manifestations including abnormalities of the skin, nervous system, bones and soft tissues \[[@B46]\]. Other conditions experienced by children with NF1 include short stature and neurologic problems such as learning disabilities or unspecified school performance problems (36%), frequent headaches (28%), mental retardation (6%), and reduced reproductive potential \[[@B46]-[@B49]\]. CTD is rare in the general population, approximately 1 per 140,000 \[[@B46]\]. It has been estimated that approximately 1% of people with NF1 have CTD \[[@B46]\]. CTD is diagnosed usually during the first year of life and fractures often occur before 3 years of age. Frequently, initial presentation is tibial bowing followed by subsequent fracture and pseudoarthrosis \[[@B45]\]. There is no generally accepted standard for management of CTD although most surgeons would suggest initial treatment of either intramedullary fixation with bone grafting or resection and bone transplant. Surgical procedures for the treatment of CTD are fraught with complications and failure of union. For the treatment of CTD, pre-fracture bracing until skeletal maturity may be a better alternative than surgery. CTD is associated with severe complications due to nonunion or pseudoarthrosis after osteotomy and amputation may be required. Conventional clinical measures of CTD include the Crawford classification system \[[@B46]\]. These measures provide clinicians with important information used in diagnosis and management of well-established symptoms. A list of important concerns could be prepared by interviewing patients and members of their families. Standardized comprehensive tools that integrate multi-dimensional effects would also be useful in quantifying the number and extent of problems experienced by NF1-CTD patients, and other pediatric orthopedic patients with complex issues. The published literature on NF1 and NF1-CTD contains virtually no information based on FHS or HRQL measurements. The exception is a recent paper by Wolkenstein and colleagues \[[@B50]\] who reported results from 128 adult patients in France using the generic health profile SF-36 and a skin-disease-specific measure, Skindex-France. Surveys of the published literature, experts in the fields, web sites and other sources of information were conducted to determine the dimensions of health that are affected by NF1-CTD, the types of FHS and HRQL measures that have been used, which measures should be considered as potentially useful for studies of NF1-CTD, the measurement properties of potentially useful measures, and the relative merits of various measures. A review of the on-line Quality of Life Instruments Database (QOLID) developed by Dr. Marcello Tamburini and the MAPI Research Institute \[[@B51]\], and correspondence with instrument developers, identified a short list of potentially useful measurement tools in each of the following categories: generic preference-based HRQL systems, major pediatric and other generic health profiles, and disease or function specific measures. Selected measures should have demonstrated properties in accordance with currently accepted criteria \[[@B12],[@B52],[@B53]\] and should provide commensurate measurements for patients across a wide age range. Problems with mobility, cognition, pain, emotion (including impacts of problems with self-image), self-care, vision, and fertility are aspects of health reported in the published literature to be compromised in NF1 patients. Illustrative study design criteria ---------------------------------- There are five important research objectives of an NF1-CTD study that provide a context for applying the framework described in the Methods section: 1\) to document long-term health outcomes associated with the disease and its treatment; 2\) to measure the burden of disease and treatment during active therapy; 3\) to investigate the hypothesis that improved HRQL is associated with initial amputation compared with multiple limb-saving procedures; 4\) to determine relationships of FHS and HRQL with conventional clinical variables used in diagnosis and management; and 5\) to assess the measurement properties (e.g., construct validity, patient versus parent inter-rater reliability, and responsiveness to change) of selected FHS and HRQL measures in NF1-CTD patients. These detailed objectives require the identification and assessment of leading FHS and HRQL measures for use in both cross-sectional and prospective longitudinal surveys. The prevalence of NF1-CTD is relatively low. Patients will need to be recruited from numerous clinical centers in North America to generate precise estimates of FHS and HRQL. Questionnaires should be available in at least 3 major languages: English, French and Spanish. The survey population ranges in age from newborn into adulthood and linking results across the study objectives requires that at least some of the assessment tools be in common across the age range of study patients. To avoid potential confounding effects, data collection techniques should be consistent across subjects and measures. The patient-focus will be represented by collecting measurements from all patients old enough to provide self-assessments, and from parents acting as proxy assessors for all children and adolescents. Self-complete questionnaires requiring minimal supervision should be used to eliminate the need for interviewers at each clinical center, to facilitate use of mail-out surveys, and to avoid potential \"interviewer\" effects. The number and type of measures per assessment, and the number of serial assessments per patient, should be sufficient to address all the study objectives within the limits of study resources and assessor burden. Measures of morbidity associated with NF1-CTD should be comparable with data on norms from surveys of general populations and other patient groups, and be useful for assessing the effectiveness and efficiency of health care services. Existing patient-focused health measures ---------------------------------------- The HRQL measure should be comprehensive and preference-based, to facilitate a broad variety of comparisons. A pediatric health profile measure and other specific measures will be selected to complement the selected preference-based HRQL measure. FHS measures may be focused on one or more of the following: the population of interest (e.g., pediatrics); the major underlying disease (e.g., NF1); the major human function of most interest (e.g., walking ability); the medically-defined health problem of most interest (e.g., tibial dysplasia); the medical speciality most involved with treatment of the health problem (e.g., pediatric orthopedics). There are six major generic preference-based HRQL utility systems \[[@B21]\], presented here in chronological order of development: QWB \[[@B25]\], 15D \[[@B54]\], HUI \[[@B23]\], EQ-5D \[[@B55]\], AQOL \[[@B56]\] and SF-6D \[[@B57],[@B58]\]. HRQL scores from these systems represent mean community preference scores. The 15D and AQOL have not been widely used outside of Finland and Australia respectively and, therefore, will not be described further in this paper. The SF-6D has been developed only recently so there is as yet little evidence to report. The major features of QWB, HUI, EQ-5D, and SF-6D systems are summarized in Table [1](#T1){ref-type="table"}\[[@B21],[@B25],[@B57],[@B59],[@B60]\]. The major characteristics vary greatly among the systems. For example, linear additive scoring models do not include effects of preference interactions among attributes or domains but multiplicative scoring functions include these effects. The QWB is available in both self-complete and interviewer-administered formats \[[@B61]\]. The symptoms attribute is a dominant feature of the QWB health status classification system. This emphasis is reflected in the population-derived preference weights. HUI health status classification systems cover more than 10 attributes. There is evidence that HUI scores agree well with mean directly measured standard gamble utility scores from a representative sample of the general population \[[@B59],[@B60],[@B62],[@B63]\]. Numerous versions of HUI questionnaires are available and HUI has a service center \[[@B64]-[@B66]\]. It is available in numerous languages. A closely-related comprehensive health status classification system for pre-school children (CHSCS-PS) has been developed recently \[[@B67]-[@B69]\] for children age 2 through 5 years of age. EQ-5D is very simple and concise. It consists of 5 attributes with 3 levels per attribute, assesses \"current\" health status, has been used in a large number of studies, and is available in numerous languages. Information, including a long list of references, about EQ-5D is available on the EuroQol Group web site \[[@B70]\]. SF-6D is a multi-attribute health status classification system based on the SF-36 \[[@B19],[@B71],[@B72]\]. The SF-36 was not designed to be commensurate with the fitting of a multi-attribute utility function. The SF-6D health status classification system is a sub-set of the attributes defined in the SF-36 health status classification system \[[@B57]\]. SF-6D utility scores may be useful in retrospective studies analyzing previously collected SF-36 data. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Major Characteristics of Five Generic Preference-Based Multi-Attribute Systems ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Instrument QWB HUI EQ-5D SF-6D --------------------------------------------- -------------------------------------------------------------------- ------------------------------------------- --------------------------------------------------------------------------- --------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------- Developed 1970s 1980s 1990s 1990s 1990s \# Attributes (\# levels per attribute) 4 (3 to 27) 7 (3 to 5) 8 (5 or 6) 5 (3) 6\* (4 to 6) \# of unique health states 1215 24000 972000 243 18000 Attributes Mobility, Physical activity, Social activity, Symptoms / problems. Sensation, Mobility, Emotion, Cognition,\ Vision, Hearing, Speech, Ambulation, Dexterity, Emotion, Cognition, Pain. Mobility, Self-care, Usual activities, Pain / discomfort, Anxiety / depression. Physical functioning, Role limitations, Social functioning, Pain, Mental health, Vitality. Self-care,\ Pain, Fertility. Types of HRQL scores and measurement Values; VAS Utilities; VAS and SG combination Utilities; VAS and SG combination Values; TTO Utilities; SG HRQL scoring model form Linear additive Multiplicative Multiplicative Modified linear additive Modified linear additive HRQL scale interval 0.00 to 1.00 -0.03 to 1.00 -0.36 to 1.00 -0.59 to 1.00 0.00 to 1.00 Questionnaire formats (languages) 2 (numerous) 16 (numerous) 1 (numerous) 2 SF-36 versions (numerous) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- SG -- standard gamble; TTO -- time trade-off; VAS -- visual analog scale. \* -- 6 attributes but role limitations attribute above includes emotional and physical so encompasses 7 of 8 SF-36 domains. ::: The major population-specific health profiles include the Child Health Questionnaire (CHQ), Pediatric Inventory of Quality of Life (PedsQL), Pediatric Evaluation of Disability Inventory (PEI) and TNO-AZL Pre-School Children Quality of Life questionnaire (TAPQOL). The PEI is limited to children age 0.5 -- 7 years of age and requires a structured parent interview or clinician observation \[[@B73]\]. TAPQOL \[[@B74],[@B51]\] is limited to children 0.5 to 5 years of age. Therefore, PEI and TAPQOL will not be discussed further. The major pediatric disease-/function-/specialty-specific instruments include the PODCI (also referred to as the POSNA or Pediatric Orthopedic Society of North America instrument), ASK, Gillette Functional Assessment Questionnaire Walking Scale (FAQ walking scale), and Wee-FIM. Wee-FIM \[[@B75]\], a popular measure of functional independence, is not being considered because it involves clinician assessments rather than assessments from a patient or parent perspective. In general, disease-specific scales in orthopedics focus on pain and physical function because these factors are major areas of concern for orthopedic patients and no generic health measures have been developed specifically for orthopedic application \[[@B73]\]. No relevant disease-specific measures or disease-specific preference-based tools were identified. A summary of the major pediatric generic health profiles appears in Tables [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}. The CHQ \[[@B76]\] covers relevant physical domains and provides detail on emotion/psychological health. The PedsQL \[[@B77]\] assesses physical, emotional, social and school functioning. It has demonstrated a return to health 3 months after acute limb fractures \[[@B78]\] and has been used in large general population surveys \[[@B79]\]. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Major Characteristics of Two Pediatric Health Profile Systems ::: ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Instrument CHQ PedsQL 4.0 ------------------------------- -------------------------------------------------------- ----------------------------------------------- ------------------------------ ------------------------------------------------------ ---------------------- Full Name Child Health Questionnaire Pediatric Inventory of Quality of Life Origin 1996 1998 Version Parent Forms (PF) Parent Form for Infants & Toddlers Youth- Completion Form Child Self-Report Forms\ Parent Report Forms\ 1) ages 5--7 years\ 1) ages 2--4 years\ 2) ages 8--12 years\ 2) ages 5--7 years\ 3) ages 13--18 years 3) ages 8--12 years\ 4) ages 13--18 years Measurement Constructs Measures of physical and psycho-social health concepts Measures of core dimensions delineated by WHO Age Bounds (years) 5 to 17 2 months to 5 years 11 to 17 5 to 18 2 to 18 \# Items 98 (PF98) or 50 (PF50) or 28 (PF28) short form 102 87 (working on shorter form) 23 \# Domains 10 child & 4 parent concepts 8 infant & 5 parent concepts 10 child concepts 4 \"Generic Core\" Overall Score 2 Global Physical & Global Psycho-Social 1 overall, for primary analyses Sub-scale Scores 14 13 10 4 sub-scales, for secondary and descriptive analyses Completion Time (minutes) 10 to 20 for PF50;\ 20 to 30 15 to 30 n/a \< 4 7 to 15 for PF28 Spanish Language Yes Yes Assessment Perspective Parents\' Parents\' Patients\' Patients\' Parents\' ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Legend: WHO -- World Health Organization. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Domains and Constructs of Forms for Two Pediatric Health Profile Systems ::: ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Instrument CHQ: Child Health Questionnaire PedsQL 4.0 ------------------ ----------------------------------------------------------------------------------------------------------------------- ------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- --------------------------------------------------- ---------------------------------------------------------- Version Parent Forms (PF) Youth-Completion Form Parent Form for Infants & Toddlers Child Forms (3 versions: 5--7, 8--12, 13--18 yrs) Parent Forms (4 versions: 2--4, 5--7, 8--12, 13--18 yrs) Domain Names 1\. Physical functioning -- 6 items.\ 1\. Physical Functioning.\ 1.. Physical Functioning -- 8 items.\ - e.g., Child is greatly limited in performing all physical activities, including self-care, due to physical health.\ 2. Behavior (perception).\ - hard to walk more than one block, hard to run, hard to do sports or exercises, hard to lift something heavy, hard to take a bath, hard to do chores around the house, hurt or ache, low energy.\ 2. Role (school and activities) limitations due to emotional.\\ 3. Growth & Development.\ 2. Emotional Functioning -- 5 items.\ 3. Role limitations due to behavioral problems\ -- 3 items.\ 4. Pain.\ - feel afraid or scared, feel sad or blue, feel angry, trouble sleeping, worry about what will happen.\ - e.g., Child is limited a lot in school work or activities with friends due to behavioral problems.\ 5. Behavior (getting along).\ 3. Social Functioning -- 5 items.\ 4. Role limitations due to physical problems -- 2 items.\ 6. Temperament & Mood.\ - trouble getting around, other kids not wanting to be friends, teased, doing things other peers do, hard to keep up when playing with others.\ - e.g., Child is greatly limited in school work or activities with friends due to physical health.\ 7. General Health.\ 4. School Functioning -- 5 items.\ 5. Bodily pain -- 2 items.\ 8. Change in Health. - hard to concentrate, forget things, trouble keeping up with schoolwork, miss school because not well, miss school for doctor appointments. - e.g., Child has extremely severe, frequent and limiting body pain.\ 6. General behavior -- 6 items.\ - e.g., Child very often exhibits aggressive, immature, delinquent behavior.\ 7. Mental health -- 5 items.\ - e.g., Child has feelings of anxiety and depression all the time.\ 8. Self-esteem -- 6 items.\ - e.g., Child is very dissatisfied with abilities, looks, family/peer relationships and life overall.\ 9. General health perceptions -- 6 items.\ - e.g., Parent believes child\'s health is poor and likely to get worse.\ 10.Change in health -- 1 item.\ - e.g., Child\'s health is much worse now than 1 year ago. ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- \Role Emotional and Role Behavioral are separate scales in PF98 but combined into one in the PF50 and PF28. ::: Table [4](#T4){ref-type="table"} summarizes the major characteristics of four orthopedic-specific measures. The PODCI \[[@B80]\] was designed specifically as a very comprehensive measure of musclo-skeletal outcomes associated with pediatric orthopedic problems. ASK was designed to measure children\'s activities in terms of both capacity and performance \[[@B81]\], and it assesses domains not covered in detail by other instruments \[[@B82]\]. The FAQ walking scale provides the most complete measure of walking abilities. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Major Characteristics of Orthopedic-Specific Systems ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Instrument* PODCI ASK FAQ walking scale ------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------ ------------------------------------------------ Full Name Pediatric Outcomes\ Activities Scale for Kids Gillette Functional\ Data Collection\ Assessment Questionnaire:\ Instrument Functional Walking Scale Origin 1997 1996 1993 Measurement constructs Musculoskeletal outcomes Physical disability in terms of capacity or performance dimensions Complete range of functional walking abilities Age range (years) 2 -- 18 2 -- 18 2 + \# Items 108 30 1 \# Domains; and Domains 8\ 9\ 1\ Comorbidity, Upper Extremity & Physical Function, Transfers & Basic Mobility, Sports & Physical Function, Pain, Treatment Expectations, Happiness, Satisfaction with Symptoms. Personal Care, Dressing, Eating & Drinking, Miscellaneous, Play, Locomotion, Standing Skills, Stairs, Transfers. Walking. Overall Score Yes, Global Function, mean of sub-scales for domains 1 to 4 above Yes, additive and not weighted Yes Sub-scale Scores Yes, additive No No Completion Time (minutes) 10 -- 20 5 -- 12 \< 2 Spanish Language version No No No Assessment Perspective Patient &/or Parent (Diagnostic, complications and aims section by clinician) Patient Parent ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ::: The choice of existing measures is based on a process of elimination considering the relative strengths of each instrument and the complementarities among measures. Neither the SF-36 nor the EQ-5D is valid for use in adolescent patients with orthopedic problems \[[@B83]\]. A review of measurement of HRQL in children by Eiser & Morse \[\[[@B84]\]; see also \[[@B85],[@B86]\]\] identified HUI and CHQ and PedsQL as the only 3 generic measures that fulfill all specified review criteria: established reliability and validity; suitable for self- and proxy-report; and brief (\<30 items). PODCI outperformed CHQ physical functioning scale for orthopedic patients \[[@B87]\]. However, PODCI has considerable problems with missing data, especially in upper extremity function and physical function and sports scales for children ages 2 to 5 years, associated with the use of \"too young\" response options \[[@B88]\]. ASK is reported to be more sensitive to change in disability levels than HUI \[Young N, personal email communication to W Furlong 2002-02-18\]. The FAQ walking scale provides the most complete assessment of functional walking abilities, especially at the upper end of the scale \[[@B89]\]. In summary, there are few measures available for assessing subjects less than 5 years of age and even fewer for subjects less than 2 years of age. Most relevant measures are available in self-complete format only. Preliminary recommendations for the NF1-CTD study were that PedsQL be used as the generic health profile, HUI be the multi-attribute preference-based measure of HRQL utility scores for children age 5 years and older and that CHSCS-PS be the measure for children age 2 through 4 years, ASK be the measure of activity limitation, FAQ walking scale be the measure of walking ability, and that a small feasibility study of these instruments be completed with a convenience sample. Feasibility study ----------------- A pilot feasibility study surveyed 8 NF1 patients using HUI and FAQ walking scale measures. Questionnaires were completed by 6 NF1 patients and 3 parents. The combined HUI and FAQ walking scale questions took respondents an average of 13 minutes (range, 9--20 minutes) to complete. The patients were 11 to 50+ years old and had health problems ranging from mild to severe. One patient with tibial dysplasia and 2 patients with scoliosis were included. HUI data were collected from 5 patients, 2 parents and both the patient and parents in one case. Health problems were reported in 7 of the 8 HUI3 attributes (vision, speech, ambulation, dexterity, emotion, cognition, and pain; no problems with hearing were reported). The attributes associated with the most morbidity, as assessed using HUI3 single-attribute utility scores \[[@B60]\], were pain (mean score = 0.81), speech (0.94), cognition (0.94), and emotion (0.94). For the 7 patients having complete data, 5 had two or more HUI2 and HUI3 attributes at less than full function. On the conventional utility scale in which being dead = 0.00 and in perfect health = 1.00, the HUI3 scores ranged from 0.45 to 1.00. The mean HUI3 score, 0.73, is similar to the mean score of 0.77 for adults with arthritis \[[@B90]\]. FAQ walking scale data were collected from 4 patients (1 of the 5 survey patients did not answer the question), 2 parents and both the patient and parents in one case. Five patients were reported to be at Level 10 (walks, runs, and climbs on level and uneven terrain and does stairs without difficulty or assistance), one patient to be at Level 8 (walks outside the home for community distances, is able to get around on curbs and uneven terrain in addition to level surfaces, but usually requires minimal assistance or supervision for safety), and one patient at Level 6 (walks more than 15--50 ft. outside the home but usually uses a wheelchair or stroller for community distances or in congested areas). In summary, the feasibility study showed that the HUI and FAQ walking scale questions were acceptable to patients\' families and that results, especially for HUI, reflected the large variability in HRQL of the sample of patients. Choice of measures for illustrative study ----------------------------------------- No single measure will provide sufficient data to address all the important study objectives. A set of measures is required. The set of measures should provide complementary data of health status and preference-based scores of HRQL. Redundancy in measurement is reduced, and efficiency of measurement is increased, by selecting the most comprehensive generic measures and then supplementing these measures with the most appropriate set of specific measures. It is recommended that HUI be selected as the comprehensive generic measure for ten reasons: a\) it includes both generic health profile and preference-based scoring systems; b\) the preference-based scoring systems are well-validated; c\) it is the most comprehensive, compact and efficient of these types of systems; d\) it includes many of the most important domains in the context of NF1-CTD; e\) it is applicable for all people age 5 years and older; f\) well-developed data collection questionnaires are available to match the study design criteria; g\) HUI results facilitate integrating effects of morbidity and mortality, and cost-utility economic evaluations; h\) it has been used successfully in a variety of studies of musculoskeletal problems; i\) population norm data are available; and j\) a closely-related health status system, the CHSCS-PS, is available to assess children 2 through 4 years of age. The HUI will provide a broad set of measures for comparisons with other populations and for estimating HRQL on a general scale such that dead = 0.00 and perfect health = 1.00. As a generic measure, HUI also has the ability to capture side effects and the effects of co-morbidities. However, these broad measures may not be responsive to small but important changes in health status. Therefore, HUI should be complemented by a set of instruments focused on pediatric, orthopedic and walking issues. The PedsQL 4.0, a pediatric generic health profile, should also be included in the set of measures because: a\) it includes domains, social and school function, which complement HUI and CHSCS-PS domains; b\) it is appropriate for children ages 2 through 18 years; c\) it is not overly burdensome in terms of data collection; d\) patient and parent assessment questionnaires are available; and e\) it can be interviewer-administered to facilitate data collection by telephone, if necessary. Two specific measures should also be part of the set of instrumentation: the ASK and the FAQ walking scale. ASK is an orthopedic-specific instrument which has been shown to cover the most important domains in the context of musculoskeletal disorders, including the impact of limb lengthening surgery experienced by many children with tibial dysplasia. ASK is also attractive because it provides overall summary scores for both performance and capability measures, and is only moderately burdensome to complete. Walking ability is one of the most important aspects of health that is frequently compromised in NF1-CTD patients, and the FAQ walking scale is the most complete scale of functional walking ability currently available and it only requires asking one question. CHQ is not recommended because it does not add much to the set of recommended measures and it is burdensome to complete. PODCI is not recommended because it is very burdensome to complete, it has been reported to have major problems with \"missing data\", and the system for collapsing questionnaire responses into summary scores is not well validated. Children ages 2 to 5 years of age should be assessed by their parents using three questionnaires: the CHSCS-PS (12 questions), the PedsQL (23 questions); and the FAQ walking scale (1 question). It is expected that all three of these questionnaires can be completed in an average of 15 minutes. Children and adolescents ages 5 to 17 years of age should be assessed by their parents using four questionnaires: the HUI (15 questions), the PedsQL (23 questions), the FAQ walking scale (1 question), and the ASK (30 questions). These four questionnaires are expected to be completed in an average of 20 to 30 minutes. Children and adolescents older than 11 years should provide self-assessments using four questionnaires: the HUI (15 questions), the PedsQL (23 questions), the FAQ walking scale (1 question), and the ASK (30 questions). On average, it is expected that respondents will complete all four questionnaires in 20 to 30 minutes. Conclusions =========== This paper highlights reasons why patient-focused measures of FHS and HRQL should be considered important tools in the field of orthopedic surgery. It has also noted that there is increasing competition for scarce health-care resources, that allocation decisions about these resources are being informed by evidence based on patient-focused health measures, and that these measures are being under-utilized by the orthopedic surgery community. The orthopedic community faces numerous obstacles in utilizing FHS and HRQL measures. One major obstacle is that the multitude of existing measures makes it difficult to decide which measures may be appropriate for a specific application. A second obstacle is that most of the information about FHS and HRQL measures is not reported in the orthopedic literature. A third obstacle is that usually no one measure can capture all the important aspects associated with a specific orthopedic issue. The framework outlined in the paper provides guidance for selecting appropriate FHS and HRQL measures. The framework guides orthopedic investigators to combine their basic study criteria, including objectives and clinical context, with key criteria for FHS and HRQL measures from the published literature. The results in this paper identify some major sources of information about health measures, identify some of the most widely used measures of FHS and HRQL, and provide summaries of key characteristics for selected measures in three major taxonomical classes: generic preference-based multi-attribute systems; generic pediatric health profile systems; and orthopedic-specific systems. It is clear that there are many important differences among measures both within and across taxonomical classes. All measures are not equal. There are sound factors for making judgements about which measures are most appropriate for a given application. A process of appraisal and elimination was used to select one measure from each taxonomical class for inclusion in the NF1-CTD study illustrative example, and a pilot study of the most readily available selected measures confirmed the feasibility of their use in a small sample of NF1-CTD patients. The paper shows that a set of relevant, valid, reliable, responsive and practical patient-focused health measures for use in an orthopedic study can be readily identified and selected from the published literature and information available on the worldwide web. We encourage orthopedic researchers to use the framework to identify and select appropriate patient-focused health measures in their future studies. Conflict of Interest ==================== W. Furlong and D. Feeny have a proprietary interest in Health Utilities Inc. which distributes copyright Health Utilities Index (HUI^®^) instrumentation and provides methodological advice on the use of HUI. Authors\' contributions ======================= All authors were involved in critical review of drafts for intellectual content and have given final approval to the version submitted for publication. W Furlong was responsible for much of the overall design, acquisition of data, and initial drafting. R Barr and D Feeny made important contributions to the analysis and interpretation of results. S Yandow conceived the idea of presenting the information in a published manuscript. Acknowledgments =============== We are pleased to acknowledge funding from a Shriner\'s Foundation Planning Grant and support of the following members of the Planning Grant team: Dr. John Carey in the Department of Pediatrics at the University of Utah School of Medicine and member of the medical staff at Intermountain Shriners Hospital for Children for his role as Principal Investigator of the Shriner Foundation Planning Grant; and Dr. Jan Friedman and Patricia Birch in the Department of Medical Genetics at the University of British Columbia for providing the HUI and FAQ walking scale feasibility study data reported in this paper. The granting agency played no role in the design, interpretation, or analysis of the work reported here and have not reviewed or approved of this manuscript. Dr. James Wright\'s review comments identified some important points that we used to improve the paper.	PubMed Central	2024-06-05T03:55:52.609552	2005-1-12	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548287/", "journal": "Health Qual Life Outcomes. 2005 Jan 12; 3:3", "authors": [ { "first": "William", "last": "Furlong" }, { "first": "Ronald D", "last": "Barr" }, { "first": "David", "last": "Feeny" }, { "first": "Suzanne", "last": "Yandow" } ] }
PMC548288	Because bone marrow and adipocytes are derived from the same stem cells, it is not surprising to find so many inflammatory peptides expressed in fat tissue. Many of these are classical inflammatory peptides derived from components of the adipose tissue and all are known also as fat derived peptides (FDP). With aging, there is a linear accumulation of adipose cells and percent of body fat increases. This increased body fat is characterized by increased visceral adiposity \[[@B1]\] and occurs despite the decreased subcutaneous fat and progressive sarcopenia typical of aging \[[@B2]\]. Visceral adiposity has been associated with greater risks for age-related diseases \[[@B3]\]. In addition, fat infiltration typical of aging occurs in many organs including liver and bone marrow. As adipose tissue accumulates throughout the body and in other organs, it is possible that this hyperplastic adipose tissue over expresses FDP. The metabolic syndrome is a common disorder consisting of a cluster of abnormalities including insulin resistance, dyslipidemia, and hypercoagulability and is associated with increased risk for cancer, Alzheimer\'s disease, type-II diabetes and atherosclerosis \[[@B4]\]. It is also associated with increased fat mass and increased inflammatory peptides. The obesity epidemic of the rapidly growing aging population makes understanding underlying relationship between adiposity, chronic inflammation and the metabolic syndrome essential. Increased inflammatory peptides are being studied as possible modifiable markers of the increased risk predictors of disease and possibly the underlying link between obesity and the poor clinical outcomes seen with the metabolic syndrome. More specifically, C-reactive protein (CRP) is the most well established inflammatory cytokine in the clinical setting but there are other inflammatory cytokines including IL-6, leptin, TNF-α, and other (non-cytokine) FDP, such as PAI-1, adiponectin and resistin, which may play a role in the pathogenesis, and/or serve as markers of risk in the metabolic syndrome. The fact that many of these peptides are derived from adipose tissue leads us to the question of whether adipose tissue itself is the underlying pathophysiological link between obesity and the poor clinical outcomes associated with the metabolic syndrome. We will provide a brief overview of some of the peptides associated with the metabolic syndrome. Cytokines with a potential role in the metabolic syndrome ========================================================= TNFα, leptin, and IL-6 are examples for cytokines that may have a role in the metabolic syndrome. TNFα, previously known as lymphotoxin and cachetin, is believed to be involved in the wasting that occurs during acute and chronic illness and malignancy. In the basal state TNFαis directly proportional to fat mass and has been shown to be involved in the development of insulin resistance \[[@B5]\]. In-vitro studies have demonstrated that TNFαdecreases the insulin receptor tyrosine phosphorylation, and down regulates several steps in the insulin signaling pathway \[[@B6]-[@B9]\] while neutralizing agents for TNFαhave been shown to improve insulin resistance. \[[@B10]\] Thus, TNFα is not only a classical cytokine but may be causal in the insulin resistance of the metabolic syndrome of aging. Leptinis a peptide derived from adipose tissue and like other cytokines acts through a cytokine receptor. It is expressed and secreted in direct proportion to fat mass. Leptin exerts is effect predominantly through receptors in the hypothalamus but it may also have peripheral actions \[[@B5]\]. Leptin serves as a marker of energy sufficiency by rapidly decreasing during starvation and weight loss. \[[@B11]\] With obesity, leptin levels are increased in proportion to fat mass, but its activity to decrease appetite seems reduced. Leptin appears to have an important role in energy regulation but no apparent role in development of inflammation. IL-6is another cytokine derived from adipose tissue. Its expression and circulating levels correlate directly with obesity, and weight loss will lower circulating levels. Elevation of circulating IL6 is a predictor of the development of cardiovascular disease and diabetes \[[@B12]\]. Infusion of IL6 results in hyperlipidemia, hyperglycemia and insulin resistance in experimental models. \[[@B13]\] Additionally, IL6 decreases the expression adiponectin, an \'anti-diabetic\' cytokine. \[[@B14]\]IL-6 plays a role in the development of insulin resistance and may directly cause induction of CRP. Other inflammatory cytokines such as IL-1, IL-8, IL18, Serum Amyloid A, have been shown to be increased with obesity and may have a yet undetermined role in the syndrome. These cytokines are other examples of inflammatory markers which do not have a clear role in the causation of systemic inflammation. Non-cytokine Fat Derived Peptides with a role in the metabolic syndrome ======================================================================= Adiponectinis highly expressed in adipose tissue, and is the one non-cytokine FDP that is protective from inflammation. Unlike most FDP, circulating levels are inversely proportional to obesity and therefore tend to be low in obesity. Adiponectin levels increase with weight loss and with use of insulin sensitizing drugs. \[[@B15]\] Adiponectin administration has been shown to improve insulin sensitivity. \[[@B16]\] Low levels of adiponectin have been linked to inflammatory arthrosclerosis in humans.\[[@B17]\] Animal models have shown that low adiponectin levels increase smooth muscle proliferation in response to injury, increase free fatty acids levels and cause insulin resistance.\[[@B18]\] The pro-diabetic and pro-atherogenic effects of low adiponectin levels seen in the metabolic syndrome provide a link between inflammation and obesity. Plasminogen activator inhibitor type-1 (PAI-1)is the primary inhibitor of fibrinolysis and is highly expressed in adipose tissue. Levels of PAI-1 are elevated in acute conditions such as deep venous thrombosis, and chronic conditions such as obesity, the metabolic syndrome of aging and diabetes. PAI-1 levels are correlated with adiposity and significantly overexpressed in the adipose tissue of obese compared to lean animals. \[[@B19]\] Levels are decreased by weight loss and drugs that improve insulin sensitivity \[[@B20]\]. The relationship of PAI-1 to obesity provides a potential link between the metabolic syndrome and hypercoagulabilty. Angiotesinogen (AGT)is a peptide that is produced in the liver and in adipose tissue. The strong correlation between obesity and hypertension implies that adipose tissue may play a role in blood pressure regulation and in fact there is a correlation between circulating AGT levels and obesity/hypertension \[[@B21]\]. Animal studies have shown that overexpression of AGT results in hypertension while under expression of AGT results in decreased blood pressures \[[@B22]\]. Resistinis a peptide which is elevated in obesity and appears to play a role in glucose homeostasis in rodents. In experimental models, resistin induces hepatic insulin resistance while anti-resistin antibodies have the opposite effect \[[@B23]\]. In humans, the role of resistin is less clear and it is not known what role it has glucose homeostasis or whether it directly relates to adipose tissue mass. The role of resistin in pathogenesis of inflammation is also unclear. Markers of inflammation or markers of obesity? ============================================== Low grade inflammation is a predominant feature in the metabolic syndrome of aging and seems to be linked to the development of diabetes and poor vascular outcomes. We have briefly named several cytokines and other FDP that are generally increased with fat mass. Although many of these FDP have a role in metabolic homeostasis, many seem to lack distinct role in inflammatory pathogenesis. While many FDP have roles in vivometabolism, we suggest that some levels of cytokines are increased because of the hyperplastic characteristic of adipose tissue, and their levels are better serve as marker of adipose tissue hypertrophy, rather than having a causal role in aging. Thus, whether aging is inflammatory state or whether it is a state associated with increased inflammatory marker is subject for further studies.	PubMed Central	2024-06-05T03:55:52.615828	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548288/", "journal": "Immun Ageing. 2005 Jan 21; 2:1", "authors": [ { "first": "Eric", "last": "Rudin" }, { "first": "Nir", "last": "Barzilai" } ] }
PMC548289	Introduction ============ Celso-Ramon Garcia (Figure [1](#F1){ref-type="fig"}), who departed this world in 2004 at the age of 82, was a remarkable human being who did much to shape the specialty of reproductive medicine as it is known today. His research yielded important discoveries that continue to influence the daily lives of millions of women and men around the globe. Reviewing his career and his accomplishments is like looking through a kaleidoscope -- elements of greatness and the purest embodiment of the medical arts are brought together and with a small twist transformed into a new vision. Hard work and determination brought him from a relatively modest but academically-oriented Spanish immigrant family to medical training in community hospitals in Brooklyn, and then into the elite circles of East Coast academic medicine. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Celso-Ramon Garcia, M.D. (1922--2004) ::: ![](1743-1050-2-2-1) ::: Background ========== Garcia was raised speaking Spanish, but his English diction and elocution were so honed by his teachers in grammar and high school that hardly a trace of accent was left to be detected by the Boston Brahmans he would later encounter. Along the way his interests took seemingly improbable turns, yet there was a clear rationale that was beyond the imagination of the average physician. Indeed, his work presaged several important developments in the field of reproductive medicine. Garcia began his career with an interest in obstetrics, but ended up conducting clinical research on contraception that on more than one occasion was nominated for the Nobel Prize in Medicine or Physiology. He was revered as a meticulous abdominal infertility surgeon, yet he embraced the fledgling field of endoscopy and endoscopic surgery. While being celebrated for his surgical technique, he promoted research into the psychological aspects of infertility. Towards the end of his career he became interested in sexuality in the menopause, bringing his career through the full cycle from obstetrics, to family planning, to infertility to reproductive aging. His accomplishments in each of these spheres were well recognized by his peers who chose him President of the American Association of Planned Parenthood Physicians, Chairman of the Board of Directors of Planned Parenthood of America, Founding President of the Society of Reproductive Surgeons, and President of the American Society of Reproductive Medicine and bestowed upon him the Scientific Leadership Award from the Global Alliance for Women\'s Health, a non-governmental organization of the United Nations. Early years =========== Garcia\'s course to greatness was based on a diverse set of skills that he used creatively throughout his career. He majored in chemistry at Queens College where he revealed a remarkable aptitude for analysis of organic compounds. During his undergraduate studies, Garcia served as a teaching fellow in chemistry at New York University and authored two papers on qualitative and quantitative analysis. These are the earliest public manifestations of his passion for rigor and precision, which subsequently guided his clinical practice and his scholarship. After receiving his baccalaureate degree he entered medical school at the Long Island College of Medicine (now SUNY-Downstate) and graduated in 1945. He completed a rotating internship at the Norwegian Hospital in Brooklyn and then served two years in the U.S. Army Medical Corps in Fairbanks Alaska and then the Phoenixville General Army Hospital outside of Philadelphia. Garcia started a residency in pathology at Cumberland Hospital in Brooklyn where he was introduced to Sam Lubin, who became his friend and mentor. Lubin\'s work focused on uterine pathology and contractility and this sparked Garcia\'s interest in obstetrics and gynecology. With Lubin, Garcia would later publish papers on the effects of meperidine on labor. Further studies and research ============================ After a year of pathology residency and a year as a research fellow, Garcia transferred to the obstetrics and gynecology program at Cumberland Hospital. With a pathologist\'s keen eye and an appreciation of tissue biology/healing, Garcia was well-equipped to excel in the art of reproductive surgery. In the operating rooms at Cumberland Hospital Garcia met Shirley Stoddard, the newly appointed Director of Surgical Nursing; she was to become his wife and lifelong companion. After completing residency, Garcia explored openings in academia but few opportunities were available. One exception was an advertisement for assistant professor of obstetrics and gynecology at the newly established medical school at University of Puerto Rico, for which Garcia applied and was accepted in 1953. Garcia\'s research during residency, mentored by Lubin, had focused on obstetrical issues -- something that he could have easily developed as a junior faculty member at the University of Puerto Rico. However, Garcia was about to embark on the first of several sharp turns that would lead him into seemingly improbable arenas. His arrival in Puerto Rico fortuitously coincided with the time for the first large scale clinical trials of the oral contraceptive pill. Within a year of Garcia\'s arrival, Gregory Pincus (director of the Worcester Foundation for Experimental Biology in Shrewsbury, Massachusetts) was in Puerto Rico looking for sites to conduct these trials. It was there that Pincus was first introduced to Garcia. Pincus had an uncanny ability to sense talent; he always surrounded himself with the brightest people. Pincus, undoubtedly impressed by Garcia\'s fund of knowledge in organic chemistry, training in pathology, and of course, the fact that he was bilingual in addition to his training in obstetrics and gynecology, promptly recruited Garcia to oversee his important research program in Puerto Rico. Pincus would subsequently introduce Garcia to John Rock, who arranged for Garcia to be named Sydney Graves Fellow in infertility and gynecology at the Free Hospital for Women in Brookline, Massachusetts. There, Garcia continued to work on oral contraceptives but also pursued clinical work in infertility. Although the Graves fellowship no longer exists, contemporary reproductive medicine was developed by its incumbents. Rock had an interesting manner of selecting these fellows. He usually interviewed candidates over breakfast in his hotel room at the annual meeting of the American College of Obstetricians and Gynecologists. He could identify the smart ones within a minute, and had little patience for individuals who were not well-spoken. The diction and elocution drilling Garcia had endured and his insistence on precision brought him through with flying colors. Garcia quickly became indispensable to Rock. Garcia moved to Boston, where he held assistant and instructor faculty posts at Harvard Medical School. During this time, Garcia also commuted to Puerto Rico to manage the oral contraceptive trials. Rock had a thriving infertility practice and Garcia did much of the surgery. Garcia\'s contributions to Rock\'s clinical practice were, however, underappreciated and when it came time for Garcia to be proposed for a full-time position at the Free Hospital for Women, the job did not materialize. This scenario is not unfamiliar to practitioners in our field, and it elicited the obvious reaction: Rock and Garcia pulled up stakes and took their hugely lucrative practice to the Faulkner Hospital in Boston. But after a short interval, the Rock/Garcia partnership was persuaded to return to the Free Hospital where an appropriate appointment was arranged for Garcia. Mrs. Stanley McCormick, a benefactor of remarkable vision who had provided initial funds to Pincus and colleagues at the Worcester Foundation to begin studies on oral contraceptive steroids, helped establish the Rock Reproduction Study Center at the Free Hospital campus, greatly facilitating research and training. The Worcester Program and Massachusetts General Hospital years ============================================================== By 1960, Pincus had recruited Garcia to the Worcester Foundation as a senior scientist and director of the training program in Physiology of Reproduction. Sponsored by grants from the Ford Foundation and National Institutes of Health, this was the first training program of its kind. The discipline of reproductive biology as we know it today had its roots in the Worcester program. Although Garcia was appointed chief of the infertility clinic at Massachusetts General Hospital in 1962, he continued to visit Puerto Rico to perform follow-up examinations on women who had participated in the oral contraceptive trials in gratitude for their role and with genuine concern for potential untoward long-term effects. That was a personal mission, not a mandated component of the study protocol. This post-trial surveillance helped identify rare side effects that ultimately led to product reformulation and lowering of the steroid content of oral contraceptives. Garcia subsequently carried out groundbreaking studies on patient acceptance of oral contraceptives, recognizing that future developments in family planning must address the social and demographic aspects, including cultural differences in preferences for methods. The technologies that gave men and women the ability to control their own reproduction through contraception and infertility treatments are arguably among the most important medical advances of the 20^th^century. This is in good part Garcia\'s legacy \[[@B1]-[@B3]\]. While his career embraced the full spectrum of reproductive medicine and surgery, undoubtedly Garcia\'s most significant role was as co-developer of the first oral contraceptive approved by the U.S. Food and Drug Administration. Important medical advances like \"the pill\" or IVF do not come without controversy, societal baggage, and rhetoric. The pill\'s creators fully understood these implications, and knew such peripheral distractions would take away from the significance of their work. Consequently, there was no phone call from Sweden despite several nominations to the Nobel committee. But it is best to look back on the history of the development of the oral contraceptive with awe and wonder. Indeed, the commercial availability of oral contraceptives was also important because it established a formal approval and regulatory framework for pharmacologic agents of an entirely new class: drugs designed to be used by healthy people for long periods of time. It provided a clear understanding of how post-marketing surveillance can lead to product reformulations that reduce side effects without reducing efficacy. It yielded an understanding of how sociology and demography must be incorporated into the development of pharmaceuticals and the practice of medicine. Garcia the peerless surgeon =========================== Garcia maintained his connections with Rock and Rock\'s family of protégés, one of whom (L.M.) later lured him and other Rock/Pincus trainees, Edward Wallach and John V. Kelly, to the Department of Obstetrics and Gynecology at the University of Pennsylvania. Garcia spent the rest of his career at Penn developing the specialty of reproductive surgery, particularly microsurgical approaches. Here again came the totally un-expected. Garcia was at his best when confronted with the most challenging open abdominal procedures, which always ended with the pelvis in a pristine state, not a drop of blood, all surfaces meticulously re-peritonealized, no risk of inflammation or adhesion. Garcia\'s novel regimen of intraperitoneal corticoids and antihistamines (an early anti-adhesion therapy) was merely icing on the cake. The two-stage tuboplasty procedure was celebrated both locally and internationally. Yet this master of the open abdomen quickly recognized the potential of minimally invasive surgery, establishing one of the first endoscopic surgery programs in USA after a brief sabbatical with his colleague, Professor Kurt Semm in Kiel, Germany. During his career, Garcia authored numerous textbook chapters on surgery of the ovaries, tubes and uterus that guided a generation of gynecologists in reconstructive surgery. He was also an early advocate of laser and electrosurgical methods, as well as microsurgical techniques designed to minimize tissue trauma. When Garcia\'s residents and fellows under-performed, he could not mask his displeasure, yet when they excelled he could not hide his joy. His concern for the outcomes of a rapidly evolving medical specialty led him to develop one of the first electronic infertility data registries \[[@B4]\]. In 1968, Garcia made yet another change in tack, developing an interest in psychological aspects of infertility. While this may have puzzled colleagues who knew Garcia only by reputation as a surgeon, those of us who worked with him knew well his holistic commitment to medicine and his devotion -- unprecedented even in the current era -- to his patients and their families. He had a profound understanding of the human frailties that emerge during the rigors of infertility treatment. One of us (J.F.S.) assisted Garcia in the care of an attorney\'s wife who underwent a two-stage tuboplasty. The husband demanded to be present during all medical examinations of his wife, he tape recorded all sessions, and refused to pay for Garcia\'s services, despite the fact that she conceived within three months after the hoods were removed from her reconstructed tubes. Garcia did not press the attorney for payment -- he knew that the treatment course was exceptionally arduous with considerable post-surgery discomfort. When this same lawyer called eight months later asking if Garcia would serve as an expert witness in a plaintiff\'s medical malpractice case, Garcia thanked him for his call and politely said his schedule would not permit it, never mentioning the unpaid bill. He forgave, but he did not forget. Final years and retirement ========================== Later in his career, Garcia and his colleagues undertook studies on the menopause that presaged the current interest in female sexual dysfunction. In retrospect, this was a logical extension of his long-standing interest in the social and psychological aspects first of contraception and family planning, and later of infertility. Garcia published on perimenopausal sexuality, and reported the first studies the relationship between sex steroid levels and sexual behavior. Garcia also recognized the need for multidisciplinary collaboration in the care of women, particularly in the post-reproductive years, and co-authored an early self-help manual for menopausal women \[[@B5]\]. The Women\'s Wellness Program he established at the Hospital of the University of Pennsylvania opened years ahead of similar facilities elsewhere. Garcia gracefully retired from practice to devote time to his wife and family. This life transition, seamlessly executed, surprised many who were familiar with Garcia\'s intensity. But there should have been no surprise. Garcia demanded the highest technical standards from residents and fellows in the operating room, the same standards that he held for himself. He would not carry on if he could not perform to those standards, and he would not neglect his wife who needed increasing medical attention. Epilogue ======== Throughout his professional life, Celso-Ramon Garcia was a major force driving innovation in clinical care, and ultimately the evolution of the field of reproductive medicine. Let all who follow in the arena of reproductive endocrinology honor his contributions, appreciate the unique attributes that made him a great physician and educator, and gain insight from a truly remarkable career. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JLS and LM contributed equally to this work and its revisions.	PubMed Central	2024-06-05T03:55:52.616593	2005-1-26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548289/", "journal": "J Exp Clin Assist Reprod. 2005 Jan 26; 2:2", "authors": [ { "first": "Jerome F", "last": "Strauss" }, { "first": "Luigi", "last": "Mastroianni" } ] }
PMC548290	Introduction ============ It is estimated that more than 110 million Africans live in areas prone to epidemics of malaria. Populations in these areas are infrequently challenged by malaria and, therefore, do not fully develop acquired immunity. As a result, the disease remains life threatening to all age groups. The impact of malaria epidemics could be greatly reduced by timely detection or, ideally, by prediction and prevention through vector control and deployment of appropriate drugs \[[@B1]\]. Rainfall is recognized as one of the major factors influencing variability in malaria transmission in warm semi-arid and desert-fringe areas of the Sahel, the Greater Horn and Southern Africa. Explosive epidemics may occur in these regions after excessive rains, usually with a lag-time of several weeks during which time mosquito vector populations and malaria infections increase rapidly. Epidemics can be especially severe among communities stressed by recent periods of drought and poor food security. Recent research has found that rainfall estimates would be able to provide useful epidemic early warning information, even in highland-fringe settings, such as those in Kenya and Ethiopia, where temperature is also an important limiting factor for the development of the malaria parasite \[[@B2]-[@B4]\]. Consequently, rainfall monitoring forms one of the essential elements for the development of integrated Malaria Early Warning Systems for sub-Saharan Africa, as outlined by the World Health Organization \[[@B5],[@B6]\]. During a meeting of the Roll Back Malaria Technical Resource Network on Prevention and Control of Epidemics (RBM-TSN) it was decided that immediate benefit could be realized from the routine availability of a simple indicator of changes in epidemic risk in these regions of marginal transmission \[[@B7]\]. The indicator was to be based on the difference between current rainfall (derived from meteorological satellite estimates) and the expected (average) for the particular time of the year; results were to be made available on the internet in a frequently updated format. The purpose was to provide timely alerts to malaria control programme staff and RBM partners working in areas of increased epidemic risk \[[@B8]\]. The following article provides an update on existing online rainfall monitoring resources and a description of a new tool that has been developed to address remaining needs of the malaria control community Discussion ========== Following discussion among members of the RBM-TSN a consensus map of epidemic risk zones was produced \[[@B7]\]. The map, shown in Figure [1](#F1){ref-type="fig"}, was used as a mask to exclude areas where malaria transmission is considered absent or endemic, as opposed to epidemic. This mask is based purely on climatic constraints to malaria transmission, and does not yet account for areas in the northern and southern margins of the continent where control has eliminated malaria risk. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Epidemic risk zones in Africa (adapted from \[1\]). ::: ![](1475-2875-4-6-1) ::: The epidemic risk map was then combined with rainfall anomaly data (i.e., the difference between observed rainfall and the expected, i.e. average, rainfall for a particular time of the year) to provide a simple indicator of changes in risk in epidemic prone areas. Figure [2](#F2){ref-type="fig"} illustrates the resulting dekadal (i.e., \~10-daily) rainfall anomaly maps, which are updated approximately every 10 days, and have been available in experimental form through the Africa Data Dissemination Service (ADDS) since June 2002. The ADDS is an operational part of the Famine Early Warning Systems Network (FEWS NET) which is maintained by the United States Geological Survey (USGS) and supported by the U. S. Agency for International Development (USAID). The maps can be accessed at: <http://igskmncnwb015.cr.usgs.gov/adds/> under \"RFE Anomaly -- Malaria\", where the anomaly map for the most recent dekad is displayed along with documentation on how the map is produced. The existence of this online monitoring resource was publicized and their use and validation by control services and researchers was encouraged \[[@B8]\]. The rainfall estimate data underlying these maps was tested against laboratory-confirmed malaria incidence figures for selected districts in Southern Africa, where they showed a good association \[[@B9]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Rainfall Anomalies in Zones with Malaria Epidemic Potential: 21--31 December 2004 ::: ![](1475-2875-4-6-2) ::: In the year following the launch of the ADDS dekadal rainfall anomaly maps, WHO commissioned field visits to a number of epidemic prone countries to evaluate whether the National Malaria Control Programmes were aware of this resource and how useful they considered it may be for their efforts. Sudan, Uganda, Niger, Mali and Burkina Faso received field visits. In general, all of the control programmes had been aware of the rainfall anomaly maps, but only those in Uganda and Sudan had monitored them regularly during the previous year. The control programmes in the Sahelian countries did not agree with the epidemic risk zone used in the mask because their recent experience was that epidemic outbreaks had occurred beyond the northern boundary of the epidemic risk zone. This was also partly true in Sudan where epidemic outbreaks have been known to occur along the Nile River margins in the northern half of the country. Uganda\'s malaria control programme, however, had found the maps to be reasonably accurate and a useful monitoring resource. Further dialogue with malaria control programmes in West Africa and Southern Africa also raised the point that a single dekadal rainfall anomaly map could raise an alert; when in fact the rainfall levels were not abnormally high -- but just 10 days earlier than \'normal\'. This suggested that additional information about the temporal distribution of rainfall was necessary. In order to respond to these issues, USGS and WHO-HealthMapper agreed to collaborate on the development of the dekadal anomaly maps in a format which could be downloaded, viewed and archived by surveillance staff directly in HealthMapper, a basic mapping and surveillance software developed by WHO\'s Communicable Disease Surveillance and Response Department <http://www.who.int/csr/mapping/tools/healthmapper/healthmapper/en/>. In addition to the most recent map, it is possible to download the dekadal maps for the previous six months and begin to construct a seasonal time series. The integration of the rainfall anomalies maps within HealthMapper also allowed the users to improve their analyses by combining ancillary data related to malaria directly on top of the rainfall anomalies maps for their country. Figure [3](#F3){ref-type="fig"} provides an example for Niger. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### 10 daily rainfall anomaly map for Niger viewed in the HealthMapper ::: ![](1475-2875-4-6-3) ::: Staff working at the International Research Institute for Climate Prediction (IRI) have since developed a web-based Malaria Early Warning System (MEWS) interface that enables the user to gain a broader contextual perspective of the current rainfall season by comparing it to previous seasons and climatological averages. The interface is in the IRI Data Library and takes the form of an online \'clickable map\' of Africa: <http://iridl.ldeo.columbia.edu/maproom/.Regional/.Africa/.MEWS/>. It displays the most recent dekadal rainfall map (Figure [4](#F4){ref-type="fig"}) over which national and district administrative boundaries and the epidemic risk zone can be overlaid (in this case as a guide rather than an absolute mask which may have excluded districts of local interest). These visual features can be toggled on or off and the user can zoom in to any region for more clarity. The user can \'zoom\' into a more localized region of interest. Dekadal rainfall can be spatially-averaged over a variety of user-selected areas, including administrative districts and 11 × 11 km, 33 × 33 km, 55 × 55 km and 111 × 111 km boxes. Upon the selection of this sampling area and a specific location of interest (by a click on map at the location of interest), four time-series graphs are generated (Figure [5](#F5){ref-type="fig"}). These time-series provide an analysis of recent rainfall with respect to that of recent seasons and the overall climatology. A description of the time-series figures, the data used and its source is also provided. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### MEWS \'Clickable Map\' for Rainfall Monitoring: 21--31 December 2004 ::: ![](1475-2875-4-6-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Summary information on current rainfall/seasonal development and expected rainfall for location of interest. ::: ![](1475-2875-4-6-5) ::: Conclusions =========== Access to frequently-updated rainfall information is an important requirement for the development of integrated early warning systems for malaria and other climate sensitive diseases \[[@B6],[@B10]\]. These operational rainfall monitoring tools have been developed primarily for application in warm semi-arid regions where rainfall anomalies are the main determinant of epidemic outbreaks, however they may also be an important information source for highland-fringe epidemic settings. They are offered as an experimental resource for testing within MEWS applications in Africa -- and may be modified in future in response to user feedback and further evaluation. While it is recognized that much of Africa does not (yet) have easy access to the internet, email is becoming more prevalent and it is now relatively easy for regional support centres, such as the WHO-Intercountry Programme for Malaria Control in Southern Africa, to prepare bulletins with malaria relevant climate data for distribution by email and courier to district health teams in epidemic prone areas as part of an overall MEWS process \[[@B11]\]. Acknowledgements ================ We would like to thank Charles Delacollette and other members of the Roll Back Malaria Technical Support Network on Epidemic Prevention and Control for their inputs during the development of these monitoring tools. We would also like to acknowledge Madeleine Thomson (IRI) and Jean Pierre Meert (WHO) for the field evaluations in Mali, Niger and Burkina Faso. The technical assistance provided by John del Corral (IRI) is also greatly appreciated.	PubMed Central	2024-06-05T03:55:52.618675	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548290/", "journal": "Malar J. 2005 Jan 21; 4:6", "authors": [ { "first": "Emily", "last": "Grover-Kopec" }, { "first": "Mika", "last": "Kawano" }, { "first": "Robert W", "last": "Klaver" }, { "first": "Benno", "last": "Blumenthal" }, { "first": "Pietro", "last": "Ceccato" }, { "first": "Stephen J", "last": "Connor" } ] }
PMC548291	Background ========== HCMV is a member of the betaherpesvirus family \[[@B42],[@B48]\]. Other members of this family include human herpesvirus 6 (HHV-6), and human herpesvirus 7 (HHV-7), and all are widely distributed in human populations. During productive replication, the 230 kilobase pair (kbp) viral genome replicates by a rolling circle mechanism, which generates long head-to-tail concatemers that are cleaved to unit length and packaged in capsids. The state of the HCMV genome during latency remains unidentified and is likely to be circular and extrachromosomal \[[@B6]\]. The HCMV genome has been detected in cells within the hematopoietic lineage as early as CD34+ progenitors and up through differentiated macrophages \[[@B23],[@B29],[@B38],[@B54]\]. Defective HSV viruses created by high multiplicity serial passage of virus stocks have been described on numerous occasions and have been characterized in detail at the molecular level \[[@B13],[@B18],[@B31],[@B43],[@B52],[@B67]\]. Naturally occurring defective HSV viruses and laboratory derived HSV amplicon vectors are composed of head-to-tail tandem reiterations of subgenomic regions containing a functional origin of DNA replication (Ori~S~or Ori~L~) and a DNA cleavage/packaging signal \[[@B3],[@B4],[@B30],[@B57],[@B60]-[@B62]\]. These two cis-acting functions can be relatively small ranging from ca. 90--150 base pairs (bp) for the ori and ca. 250--300 bp for the asequence. The functional HCMV oriLyt is much more complex than either of the HSV oris; the HCMV oriLyt consists of multiple direct and inverted repeats and extends over at least 1500 bp \[[@B1],[@B2],[@B24],[@B37]\]. HCMV is unique among the herpesviruses in not having an origin binding protein homolog that is required for DNA replication \[[@B45]\]. The HCMV asequence varies in size from ca. 550 bp to 762 bp, however, the conserved pac-1 and pac-2 cis-elements which determine the sites for cleavage of replicated viral DNA are present \[[@B15],[@B28],[@B58],[@B64],[@B65]\]. In contrast to HSV, HCMV does not efficiently produce defective virus genomes, this difference may be related to the distinct biology of the two viruses \[[@B45]\]. However two reports described the identification of what may potentially be HCMV defective viruses created by serial high multiplicity passage \[[@B47],[@B59]\]. These reports characterized HCMV defectives as very large subgenomic DNA molecules, in some cases extending over two thirds of the genome. In addition to these replication defective HCMV viruses, a recent report by Borst et al. 2003 \[[@B7]\], described the construction of an HCMV amplicon. In this report we further utilized the HCMV amplicon for gene delivery to human CD34+ cells. HCMV infects cells of the hematopoietic lineage \[[@B34],[@B38],[@B39],[@B55],[@B68]\]. Viral genomes can be found in CD34+ cells from seropositive individuals and granulocyte-macrophage progenitors and differentiated macrophages can be infected experimentally \[[@B56]\]. We were interested in determining whether the tropism of HCMV can be exploited to construct defective HCMV virus vectors (amplicons) targeted to hematopoietic stem cells. The general feasibility of such an approach for other cell types has been shown using other herpesviruses, e.g. HSV, EBV, and HHV-7 \[[@B20],[@B25],[@B26],[@B30],[@B35],[@B49],[@B70],[@B71]\]. Methods ======= Cells and virus --------------- HCMV Toledo (passage 8, from Dr. S. Plotkin, Aventis Pasteur, Doylestown, PA), and HCMV TownevarRIT (passage 134, from Dr. Plotkin via Dr. Ed Mocarski, Stanford University), were propagated in human fibroblasts (HF) cultured in Dulbecco\'s modified Eagle\'s medium supplemented with 10% fetal calf serum (JRH Biosciences, Lenexa, Kans.). Recombinant HCMV, RC2.7EGFP, expressing enhanced green fluorescent protein (EGFP) (Clonetech, Palo Alto, CA), under the control of the major early 2.7 promoter, was constructed by cotransfection of plasmid pEAG2.7EGFP with a set of overlapping cosmid clones derived from HCMV Toledo (G.M. Duke, unpublished data). Plasmid constructions --------------------- Plasmid pON205 (Spaete and Mocarski, 1985), contains the Towne strain asequence, was obtained from Ed Mocarski (Stanford University). pEAG2.7EGFP was derived by cloning the EGFP gene from plasmid pEGFP-N2 (Clonetech, Palo Alto, CA) between the EagI and SmaI site of the β2.7 gene taken from Toledo (G.M. Duke, unpublished data). HCMV amplicon plasmid Tn9-8 was derived by inserting the 6 kpb DraI fragment of TownevarRIT (corresponding to nucleotides 91,166 -- 95,909 relative to AD169) (Figure [1B](#F1){ref-type="fig"}), spanning the HCMV oriLyt into the EcoRI site of pON205. Tn9-8 was partially sequenced by single-cycle and multicycle dideoxy-nucleotide chain termination method of Sanger et al., \[[@B51]\]. The plasmids designated Tn9-8GF5 and Tn9-8GF7 incorporating the EGFP gene with the HCMV Major Immediate Early (MIE) promoter and SV40 poly A sequence was isolated as a 2,334 bp NsiI fragment from plasmid pEGFP-N2 (Clonetech, Palo Alto, CA), and cloned into the HCMV amplicon Tn9-8 at the PstI site in both orientations. The gptgene in Tn9-8-gptwas derived by cloning a PCR fragment from Escherichia coliDH5α using the primer pairs 5\'CTGCAGCTAGTCTAGACTGGGACACTTCACATGAGC3\'and 5\'CTGCAGCTATGTATCTAGAGCCAGGCGTTGAAAAGATTA3\'. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Schematic representation of amplified region near oriLyt of the HCMV genome. A. Restriction enzyme map of minimal oriLyt and adjacent region of heterogeneity (block). B. Region of heterogeneity shown as a dimer. Arrow indicates junction of the repeat segment. The number 91,166 to the left of the restriction map corresponds to the nucleotide position of the AD169 genome (EMBL accession number X17403). C. Autoradiograph of Southern blot utilizing a minimal oriLyt probe, pON2623 (Kemble et al., 1996). Monomers and dimers are depicted with one and two arrows, respectively. Trimers and tetramers can be seen in the TownevarRIT viral stock. ::: ![](1479-0556-3-1-1) ::: Generation of viral stocks containing amplicons ----------------------------------------------- Plasmid DNA was transfected by CaPO~4~precipitation of approximately 4 μg of Tn9-8 amplicon DNA. The Tn9-8 DNA was transfected into approximately 1 × 10^6^passage 16 human fibroblast (HF) cells. At 24 hours post transfection, the cells were infected with CMV Towne at a multiplicity of infection (MOI) of 5 plaque forming units (PFU) per cell. Fresh medium was added to cells four days after infection and cells were harvested at 6 to 7 days post infection as described previously (Spaete and Frenkel, 1982). Virus stocks are prepared by three freeze-thaw cycles. Serial passages of amplicon-containing viral stocks on fresh HF cells were superinfected with CMV Towne as a helper virus at a MOI of 1. Southern blot analysis ---------------------- Viral DNAs were digested with restriction enzyme, electrophoresed in 0.8% agarose gels, transferred to Hybond-N+ nylon membranes (Amersham Corp.), (Maniatis et al., 1989), and immobilized with a UV Crosslinker 1000 (Hoefer Scientific Instruments, San Francisco, CA). DNA on the membrane was probed with fluorescein-labeled pUC9 DNA using conditions previously described (Spaete and Mocarski, 1985). Isolation of CD34^+^cells and infection with CMV ------------------------------------------------ The isolation of cord blood CD34+ stem cells was carried out by All Cells Inc. (Berkeley, CA) using CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech, Auburn, CA). The positive selection of the CD34+ cells was carried out using hapten-conjugated antibody to CD34+ followed by anti-hapten antibody coupled to MACS Microbeads. The magnetically labeled cells are enriched on positive selection columns in the magnetic field. The purity of the CD34+ population was \>95% as analyzed by flow cytometry. The purified CD34+ cells were suspended in Iscove\'s modified Dulbecco\'s Minimal Essential Medium containing 5% fetal bovine serum. 2 × 10^5^CD34+ cells were used for each infection with TN9-8GF5 amplicon containing stocks, RC2.7EGFP virus, CMV Towne virus, or uninfected cell control. The cells mixed with virus were centrifuged at 500 × g for 10 mins at room temperature and were then placed in 37°C water bath for one hour. Following this the cells were cultured in 6-well cell culture plates (Costar) for 18--72 hours. At the end of the incubation the cells were harvested for CD34 staining. EGFP expression and immunostaining for flow cytometric analysis --------------------------------------------------------------- Amplicon containing viral stocks prepared from passage 1 were used to infect HF or human CD34+ cells maintained in 12 well culture plates. At 24 hour intervals post infection, the wells were observed for EGFP expression with a Nikon TE2000 microscope under UV illumination. Immunostaining for CD34+ cells was done using Phycoerythrin (PE)-conjugated anti-CD34 antibody (Becton Dickinson, San Jose, CA). Infected or control cells were incubated with 20 μl of PE-labeled anti-CD34 antibody for 45 minutes at room temperature and subsequently were washed twice with PBS containing 0.1% BSA. The cells were directly analyzed for EGFP and CD34+ staining on a FACSCalibur instrument (Becton Dickinson, San Jose, CA), at 18 and 36 hours post infection. Results ======= In order to exploit the natural tropism of HCMV for cells of the hematopoietic lineage, in a nonlytic manner, an HCMV amplicon i.e. a plasmid containing the HCMV oriLyt and asequences was constructed. Theoretically, due to the large size of the HCMV genome, an amplicon derived from this virus should be able to carry the large DNA inserts and be capable of efficient introduction into hematopoietic cells by infection. Heterogeneity at oriLyt ----------------------- During analysis of cosmid clones of HCMV strain Towne, sequence heterogeneity was observed in the EcoRI E fragment of Towne that was not present in the Toledo strain \[[@B27]\]. The EcoRI E region spans in part the complex oriLyt region \[[@B1],[@B2],[@B24],[@B37]\]. Sequences in a 1.2 kbp repeat fragment were shown to give rise to the heterogeneity observed at this locus in the Towne genome (Figure [1](#F1){ref-type="fig"}). The coordinates of a single repeat unit starts at nucleotide 94,561 relative to the AD169 sequence and end at nucleotide 95,807 \[[@B10]\]. This segment can repeat at least three times in Towne strains from different passage histories (Fig. [1](#F1){ref-type="fig"}). This heterogeneity is different from the 189 bp repeat region previously described for the Towne strain oriLyt which occurs near the BamHI sites in Figure [1](#F1){ref-type="fig"} (nt 93337--93525 relative to AD169), \[[@B11],[@B12]\]. Since Towne replicates to relatively high titers in cell culture, it was deemed advantageous to incorporate this heterogeneity in the origin containing sequences to be used in the amplicon construct. Construction of the HCMV amplicon --------------------------------- As a test of the feasibility of the system, an HCMV amplicon was constructed which incorporated the two cis-acting functions required for the propagation of the defective virus genomes in the presence of helper virus (Figure [2](#F2){ref-type="fig"}). HCMV amplicon plasmid Tn9-8 was derived by inserting the 6 kpb DraI fragment of Towne (corresponding to nucleotides 91,166--95,909 relative to AD169) \[[@B10]\] (Figure [1B](#F1){ref-type="fig"}), spanning the HCMV oriLyt into the EcoRI site of pON205. The resulting amplicon was designated Tn9-8 (Figure [2](#F2){ref-type="fig"}), and was partially sequenced to verify its structure. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Schematic representation of the HCMV amplicon plasmids Tn9-8gpt, Tn9-8GF7 and Tn9-8GF5. The EGFP expression cassette was cloned in two orientations in the unique PstI site in Tn9-8. The gptexpression cassette was cloned between the unique PstI and HindIII sites. ::: ![](1479-0556-3-1-2) ::: Generation of viral stocks containing amplicons ----------------------------------------------- As a test of the ability of this construct to function as an amplicon, plasmid Tn9-8 was transfected in human fibroblast (HF) cells, and subsequently infected with HCMV Towne strain at an MOI of 5 to provide helper virus replication functions. Seven days later, infected cells were harvested, sonicated, and viral stocks were prepared for passage to fresh HF cells. Fresh HF cells were infected with the progeny of the transfection/infection and incubated for 7 days. The DNA from these infected cells was harvested (designated passage 1), restricted with HindIII and DpnI, and Southern blotted \[[@B36]\]. Southern blot analyses of DNA demonstrated that Tn9-8 was susceptible to digestion with DpnI, consistent with replication of the plasmid in bacteria (Figure [3](#F3){ref-type="fig"}, lane 2). In contrast, Tn9-8 in infected cells was resistant to DpnI demonstrating that it had replicated in eucaryotic cells (Figure [3](#F3){ref-type="fig"}, lanes 3--5). This observation is consistent with replication and packaging of Tn9-8 into infectious virions. These results demonstrate that foreign DNA sequences, exemplified by the plasmid pUC9, can be introduced into defective genomes that are packaged and propagated in serially passaged virus stocks. To examine whether these results were the consequence of amplicon replication and packaging or integration of the amplicon plasmid into the helper virus, DNA prepared from passage 1 infected cells was digested with ClaI, XbaI, AflII and DpnI. These enzymes digested the Towne helper virus DNA to fragments no larger than 13.6 kbp but do not cut within Tn9-8. The amplicon DNA was significantly larger than 23 kbp consistent with the amplicon being replicated as a concatamer (Figure [4](#F4){ref-type="fig"}). This result indicated that the high molecular weight DNA containing plasmid sequences was packaged independently and was not integrated into helper virus. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Southern blot analysis of passage 1 of HCMV amplicon DNA probed with plasmid pUC9. Lane 1. Plasmid Tn9-8 linearized with HindIII serves as a marker for correct migration of monomeric repeats. Lane 2. Plasmid Tn9-8 restricted with HindIII and DpnI as a control for non-replicating DNA. Lanes 3--5. Infected cell DNAs restricted with HindIII and DpnI. The signal in lanes 3--5 (arrow) demonstrates authentic replication and packaging of amplicon Tn9-8 in eucaryotic cells. ::: ![](1479-0556-3-1-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Southern blot analysis of high molecular weight HCMV amplicon DNA at passage 1 probed with plasmid pUC9. DNA prepared from passage 1 infected cells was digested with ClaI, XbaI, AflII and DpnI, Southern blotted and probed with plasmid pUC9. The samples are from those shown in Fig. 3, lanes 3 and 5. The high molecular weight DNA containing plasmid sequences (arrow) demonstrates the major hybridizing species migrating slower than the 23 kbp lambda DNA HindIII digest indicated as the marker on the left of the autoradiograph. ::: ![](1479-0556-3-1-4) ::: Packaged defective viral genomes derived from Tn9-8 or a derivative containing a selectable marker (Tn9-8-gpt), were serially passaged in HF cells. Defective viruses could be detected at passage 3 when probed with plasmid pUC9; however, the copy number appeared to diminish upon serial passage (not shown). Selection with mycophenolic acid on Tn9-8-gptamplicons did not enhance recovery. Rescue of monomeric repeat units in bacteria -------------------------------------------- Concatemeric DNA was prepared from passage 2 and 3 virus stocks containing the defective virus genomes (Tn9-8-gpt), digested with PstI and HindIII, respectively, in order to analyze monomeric repeat units. The HindIII-digested DNA was circularized by ligation and used to transform E. colibacteria to analyze structure and to demonstrate shuttle vector capability between eucaryotic and bacterial hosts. A number of plasmids prepared from the rescue attempt had a restriction enzyme pattern indistinguishable from the input (Fig. [5A](#F5){ref-type="fig"}, lanes 1 and 2). Other plasmids however exhibited the expected restriction pattern consistent with a head-to-tail amplification of the asequence (lanes 3 and 4). Digestion with NaeI produced a fragment of the predicted size of a unit length asequence (762 bp), and this product hybridized with an asequence specific probe (PstI-SgrAI fragment from Tn9-8), (Figs. [2](#F2){ref-type="fig"} and [5B](#F5){ref-type="fig"}, lanes 3 and 4). This type of amplification has been readily seen in restriction enzyme digested DNA preparations of parental genomes of both HSV and HCMV \[[@B31],[@B40],[@B41],[@B58],[@B64],[@B69]\] and has also been observed in HSV amplicons using Southern blot hybridizations \[[@B15]\]. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Tn9-8-gptwas rescued from concatamers following serial passage in HF cells. (A) Tn9-8-gptwas passaged in HF cells and monomer units were recovered by linearizing concatemeric DNA from serial passage 3 with HindIII and cloning in bacteria (lanes 2 and 4) and also by linearizing passage 2 DNA with PstI and cloning in bacteria (lane 3). Lane 1 represents the unpassaged clone for comparison. Following rescue in bacteria, DNA was prepared and cut with NcoI. Fragments were separated on an agarose gel and visualized with ethidium bromide staining (left panel). The gel was transferred to a nylon membrane and probed with an asequence specific probe (right panel). (B) Fragments from an NaeI digest were also separated on an agarose gel and visualized as in panel A. The gel was transferred to a nylon membrane and probed with an asequence specific probe. Lane 5 shows a 100 bp ladder. Lanes 3 and 4 show a ca. 800 bp fragment that hybridizes to asequences. ::: ![](1479-0556-3-1-5) ::: Expression of heterologous genes in an HCMV amplicon ---------------------------------------------------- To demonstrate that the HCMV amplicon could be used as a vector system to support the expression of a foreign gene, EGFP under the transcriptional control of the HCMV major immediate early (MIE) promoter was used as test reporter gene. Two resulting amplicon plasmids designated Tn9-8GF5 and Tn9-8GF7 both expressed EGFP following transfection of HF cells in the absence of helper virus, as expected (not shown). Packaged amplicons were generated by introduction of Tn9-8GF5 into cells and infecting with HCMV 24 hours later at an MOI of 5. Transfection-derived viral stocks were passaged onto fresh HF cells supplemented with Towne helper virus at an MOI of 1. Viral stocks prepared from passage 1 were used to infect HF cells and grown on 12-well tissue culture plates. A limited number (ca. 0. 1%) of brightly fluorescing cells could be seen by microscopic examination at 24, 72 and 96 hours post-infection (Figure [6](#F6){ref-type="fig"}). This demonstrates that a foreign gene can be expressed in the context of a HCMV amplicon viral stock in infected HF cells. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Fluorescent Microscopic Analysis of TN9-8GF5 amplicon infected cells. Human fibroblast cells (HF) or human cord blood CD34^+^cells were infected with TN9-8GF5 amplicon-containing stocks, or mock infected. Cells were observed at different time-points 24, 72 and 96 hrs post infection with TN9-8GF5 amplicon under the fluorescent microscope (Nikon TE2000 microscope). EGFP expressing fluorescent cells were observed in the TN9-8GF5 amplicon infected human fibroblast cells or human CD34+ cells at different time-points. Control uninfected cells were negative (not shown). ::: ![](1479-0556-3-1-6) ::: To test the utility of the HCMV amplicon in gene therapy or gene delivery, we used packaged amplicons in viral stocks to infect and deliver an expressed gene into human CD34+ progenitor cells. Viral stocks containing amplicons carrying EGFP under the transcriptional control of the HCMV major immediate early (MIE) promoter prepared from passage 0 and passage 1 were used to infect CD34+ cells derived from cord blood. Starting at 24 h after infection, CD34+ cells were examined for EGFP expression by fluorescent microscopy. EGFP expression was observed in TN9-8GF5 amplicon-infected CD34+ cells starting at 24 h post-infection. The cells remained positive for EGFP expression for more than 96 hrs, at which point the cells were terminated (Figure [6](#F6){ref-type="fig"}). In a separate experiment, at 36 hours post infection, the cells were stained with PE-labeled anti-CD34 and analyzed for the CD34 marker and EGFP expression. EGFP expression was observed in the CD34+ population in the TN9-8GF5 amplicon (0.3%, 0.1%) (Figure [7a](#F7){ref-type="fig"}, &[7b](#F7){ref-type="fig"}) or the CMV-EGFP virus (0.6%) (Figure [7c](#F7){ref-type="fig"}) at 36 hours post infection. The CMV Towne control-virus infected cells or uninfected CD34+ cell control were negative for EGFP expression (Figure [7d](#F7){ref-type="fig"}, &[7e](#F7){ref-type="fig"}). A small population (7--12%) of the cells lost expression of the CD34+ marker upon in vitroculture. EGFP expression was also observed in a CD34(-) population infected with either TN9-8GF5 amplicon containing viral stocks (0.8%, 0.1%) or with the CMV-EGFP virus, RC2.7EGFP (3.6%) (Figure [7a, 7b](#F7){ref-type="fig"} &[7c](#F7){ref-type="fig"}). The CMV Towne infected cells or uninfected control cell also had a significant CD34 negative population but were negative for EGFP expression (Figure [7d](#F7){ref-type="fig"}, and [7e](#F7){ref-type="fig"}). These results clearly demonstrate that CD34+ cells can be infected with replication competent or incompetent CMV vectors expressing a foreign gene. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Flow cytometry analysis of human cord blood CD34^+^cells infected with CMV amplicon containing stocks, virus, or uninfected cell control. TN9-8GF5 amplicon (a,b), CMV-EGFP (RC2.7EGFP) virus (c), CMV (Towne) infected (d), or control uninfected human cord blood CD34 cells (e,f), were stained 36 hours post-infection with PE-antiCD34 antibody (a-e), or were left unstained (f), and were analyzed for two-color cytometry analysis using a FACS Calibur instrument. The dot-plots are generated using Cell Quest software and reveal the EGFP^+^cells populations. Numbers in the upper right and lower right quadrants indicate percentage of the EGFP^+^CD34^+^and EGFP^+^CD34^-^cells respectively. A frequency lower than 0.01% is considered negative. ::: ![](1479-0556-3-1-7) ::: Conclusions =========== We have shown that a replication-defective virus vector system that is derived from HCMV is capable of delivering and expressing foreign genes in infected primary cells including progenitor stem cells such as human CD34+ cells. Further improvement and optimization of the system offers the potential to deliver gene-based therapies to multipotent cells. Advantages for use of the HCMV amplicon --------------------------------------- Foremost among the advantages of the vector system we have described is the potential ability to efficiently infect and deliver genetic information to hematopoietic stem cells (CD34+) and other dividing and non-dividing cell types which may support HCMV infection \[[@B34],[@B38],[@B39],[@B55],[@B68]\]. Genetic hematological disorders such as thalassemias and sickle-cell anemia and other hemaglobinopathies could therefore be targeted for therapy with this strategy. Another potential advantage for the system is that vector DNA could possibly be maintained as an episome with minimal concern for the potential consequences of random integration of vector DNA (i.e. activation of oncogenes or inactivation of tumor suppressor genes). In order to insure efficient segregation as an episome, the EBV latent replication origin, oriP, and the transactivator, EBNA-1, could be added as was previously shown for another hybrid herpesvirus vector \[[@B71]\]. However such a modification may not be necessary because HCMV genomes appear to be carried continuously in cells of hematopoietic origin in infected individuals. Yet another potential advantage as with other herpesviral vectors, is that the HCMV vector system should have the capacity for very large inserts. Infection of CD34+ cells with HCMV ---------------------------------- The infectivity of CD34+ cells from seropositive and seronegative subjects with HCMV has been tested both in vivoand in vitro\[[@B53]\]. Furthermore, hematopoietic stem cells are also reported as a site for HCMV latency. Efficient transduction of human CD34+ cells with retroviral and non-viral vectors has been unsatisfactory due to the lack of maintenance of high levels of expression of the transgene following engraftment of the engineered cells \[[@B16]\]. The HCMV MIE promoter may not be the right promoter for optimal expression in a CD34+ cells, since it has been shown that in the context of a lentiviral-based gene transfer system this promoter appeared to function less efficiently due to a cell-type specific expression defect \[[@B16]\]. The approaches to improving the efficiency of gene transfer into human cells have focused on improving gene delivery vectors and optimizing ex vivoculture conditions, which preserve the developmental properties of the stem cells \[[@B14],[@B22]\]. Umbilical cord blood is recognized as a rich source of hematopoietic CD34+ stem cells \[[@B33]\]. In our experiments we used cord blood derived CD34+ cells for infection with HCMV amplicon containing stocks or HCMV-EGFP virus. However, bone marrow derived CD34+ cells have also been shown to be infectable in vitrowith HCMV \[[@B34]\]. Gentry & Smith \[[@B21]\], reported a progressive loss of primitive cell properties including a reduction of CD34 expression upon in vitroculture of cord blood derived CD34+ cells. In a separate study, cord blood derived CD34+ cells cultured with IL-3 in vitroshowed a progressive decline of the CD34+ population and more differentiated cells originating in the CD34(-) population \[[@B9]\]. In our experiments with \>95% pure cord blood derived CD34+ cell population, a loss of CD34 expression in a small percent population (9--12%) of stem cells upon in vitroculture has been observed. EGFP expression was also seen in the CD34(-) population (Fig [7a, 7b](#F7){ref-type="fig"}, &[7c](#F7){ref-type="fig"}). It is possible that HCMV infection of CD34 cells could induce cell differentiation and loss of primitive properties including reduction of CD34 expression. Further studies of HCMV infection in CD34 cells will help in defining whether CD34+ infected cells undergo cell differentiation by increased expression of other markers such as CD33, CD38, HLA-DR or cytokines. It is also relevant to note here that HCMV virus carries homolog sequences for HLA-related and cytokine-related molecules and infection can induce cellular cytokines \[[@B5],[@B8],[@B19],[@B32],[@B44],[@B46],[@B50],[@B66]\]. The HCMV amplicons contain only the cis-acting ori and packaging sequences, and have no structural gene sequences. However, amplicon containing viral stocks are a mixture with HCMV replication competent helper virus. HCMV induced cell-differentiating effect, if any, might be minimized using a helper virus-free amplicon system. In this regard, it should be possible to test a number of strategies to prepare helper virus-free stocks \[[@B17],[@B63]\]. These preparations would be useful for therapeutic applications in immuno-compromised patients. Competing Interests =================== The authors of this publication are supported financially by salary and shares of MedImmune Inc., during the completion of this work. A patent application has been filed with the United States Patent Office relating to the content of this manuscript. The authors have assigned all rights of ownership to MedImmune Inc. The authors declare that they have no other competing interest. Authors\' Contributions ======================= KM carried out the expression analysis in human fibroblasts and CD34 cells. MNP & GMD generated amplicon stocks and MNP did the \'a\' sequence analysis. GWK provided critical intellectual input. RRS provided the original idea and experimental design as well as cloning of the amplicon, generation of amplicon stocks and Southern analysis.	PubMed Central	2024-06-05T03:55:52.620132	2005-1-26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548291/", "journal": "Genet Vaccines Ther. 2005 Jan 26; 3:1", "authors": [ { "first": "Kutubuddin", "last": "Mahmood" }, { "first": "Mark N", "last": "Prichard" }, { "first": "Gregory M", "last": "Duke" }, { "first": "George W", "last": "Kemble" }, { "first": "Richard R", "last": "Spaete" } ] }
PMC548292	Background ========== As health care professionals begin to understand the importance of quality of life (QL) and emotional and social well-being in the treatment and progression of cancer, it has become standard practice, and in fact has been described as a bioethical imperative, to include QL assessment in all oncology clinical trials \[[@B1]-[@B7]\]. This is particularly the case in Phase I trials for novel therapeutics, where in many cases the patients admitted have exhausted other treatment options and likely face death within months under best supportive care \[[@B8]\]. A Phase I trial is the first test of a new therapeutic in humans and aims to establish a maximum tolerated dose, to evaluate dose limiting toxicity, and to examine the drug\'s pharmacology. There is generally little chance of clinical disease response and relatively high potential risk of toxicity in this type of trial. For patients in this situation, quality of life remains perhaps the most important variable to consider in the evaluation of the new treatment \[[@B9],[@B10]\]. We report here on the initial QL of patients enrolled in the Reolysin Phase I trial, the first clinical usage of the reovirus in humans with cancer. The reovirus as a possible treatment for cancer attracted a great deal of media attention when the journal Science, in 1998, published very encouraging results showing tumor regression in animal models \[[@B11]\]. Further work has established the reovirus as a potential therapeutic in the treatment of brain, colorectal, ovarian and breast cancer cell lines \[[@B12],[@B13]\]. A small sample of patients with different types of primary cancers that had exhausted other treatment options, all of whom had subcutaneous tumors that were easily palpable and injectable with Reolysin, were enrolled in the current trial. Due to the nature of the population under study, it seemed especially important to use instrumentation that was flexible enough to allow these very ill patients to identify what was important to each of them as individuals, as well as to collect data from standardized instruments that would allow direct comparisons with other patient populations. Thus, a battery of tests to measure both individualized and standardized QL, distress levels, and spirituality was selected. Important aspects of QL were measured, including physical, psychological, emotional, social, and spiritual functioning, with emphasis on mood states and psychopathology. Expectations and hopes regarding participation in the trial were also elicited during a semi-structured interview. The specific instruments used included an interview-based subjective measure of QL, the Schedule for the Evaluation of Individual Quality of Life -- Direct Weighting (SEIQoL -- DW)\[[@B14]\]. The SEIQoL-DW is a relatively new measure and unique in QL measurement in that it elicits from patients their own self-generated list of the \"five most important domains of QL\" for them, rather than asking about pre-set areas. After patients identify their most important domains, they rate how well each domain is for them currently, and how important overall in their lives each domain is. This instrument and its predecessor, the SEIQoL \[[@B15]\], have been used in oncology in several published studies \[[@B16]-[@B18]\]. The SEIQoL differs from the SEIQoL-DW in that a procedure called judgement analysis is used in the SEIQoL to arrive at the relative importance of each of the domains for each patient. This process is much more time consuming, abstract and complicated than the direct weighting procedure used in the SEIQoL-DW. While the SEIQoL procedure has been deemed overly burdensome for some patients with both early and advanced cancer \[[@B16],[@B17]\], primarily due to the judgement analysis portion of the procedure, the SEIQoL-DW has been found to be acceptable and practical to use in a validation study of patients on Phase I clinical trials \[[@B18]\]. Another study of the SEIQoL-DW found that patients with advanced cancer were good judges of their own QL, and able to complete the interview with little difficulty \[[@B17]\]. In past studies, the most important areas of QL that patients on Phase I clinical trials identified were equally health and family \[[@B18]\]. However, in a sample of advanced cancer patients family concerns were consistently identified as more important than health issues \[[@B17]\]. The patient population in the Campbell & White study \[[@B18]\] was not defined except by their participation in Phase I trials, whereas the Waldron & O\'Boyle sample \[[@B17]\] all had advanced incurable cancer. It may be the case that as illness severity progresses, concerns are directed toward domains that offer more hope. Other studies that have used the SEIQoL found family, health and finances to be the top three areas in men with early stage prostate cancer \[[@B16]\], and similarly family, health, marriage and leisure/hobbies were most important to a group of cardiac patients \[[@B19]\]. Thus, although there does seem to be some consistency in the areas nominated by diverse patient groups, differences in the relative importance of the areas are commonly found. The other QL questionnaire used in this study is the very widely used European Organization of Research and Treatment of Cancer (EORTC) Quality of life Questionnaire (QLQ C-30), a 30 item standardized self-administered questionnaire that taps into important domains of QL, including physical, psychological, emotional and social functioning. This was included to allow comparisons with a well-known and validated standardized quality of life questionnaire with set domains and subscale scores. Another study at our Centre directly compared the SEIQoL to the EORTC QLQ C-30 in a sample of early stage prostate cancer patients \[[@B16]\]. The authors compared the domains nominated by the patients to those included on the EORTC QLQ C-30, and concluded that although there was substantial overlap on some items, many items identified by patients were not included on the standardized questionnaire. The area of spirituality was also of interest in this population of very ill people, as issues of death and dying are often accompanied by questioning in the realm of spirituality. The Spiritual Health Inventory \[[@B20]\] was used for this purpose, as it measures self-acceptance, relationships with others, and hope, which may be an important factor as patients participate in the trial. In terms of depression, anxiety and other psychiatric symptoms that are frequent in cancer patients, the Brief Symptom Inventory (BSI)\[[@B21]\] was used to broadly assess many areas of psychopathology, and the Beck Depression Inventory (BDI)\[[@B22]\] to focus in more detail on depressive symptomatology. Both of these instruments are widely used in the oncology literature and thus there are many published reports with values that can be used for comparison purposes. Previous work with patients in Phase I trials has found that patients\' expectations going into trials are generally more optimistic than oncologists\'. Patients with incurable malignancy in a Phase I trial estimated a greater potential for benefit from the experimental therapy than did oncologists \[[@B10]\]. They also estimated that the experimental therapy had less potential for toxicity than the standard treatment. The oncologists estimated the potential for toxicity on both treatments to be about equal, and lower, than did patients. Patients in Phase I trials for new drugs, when asked why they had agreed to try the new treatment, cited the potential for helping their disease to be the number one reason for participation \[[@B9]\]. Indeed, despite cautious words from the medical staff, it is not surprising that patients with a disease resistant to all other treatments might hope for at least a slowing of their disease progression with an experimental treatment. Thus, the preliminary QL of such patients, as measured in the current study, may be inflated by these hopes. Other studies looking at the effects of participation in Phase I trials on QL have found either no detrimental effects of participation \[[@B8]\], or enhancement of QL over the course of the trial compared to a control group that received supportive care \[[@B23]\]. The purpose of the current study was to investigate individualized QL in a group of patients with metastatic incurable cancer participating in a Phase I trial of a highly media lauded new therapeutic, and investigate their expectations regarding the trial. Areas of importance and SEIQoL Index scores for these patients will be compared with those of patients in other studies, and compared to their own scores on the standardized QL and psychological measures assessed. Relationships between the different measures will also be explored. Methods ======= Subjects -------- Patients were recruited as specified in the protocol: \"A Phase I Clinical Trial to Evaluate Dose Limiting Toxicity and Maximum Tolerated Dose of Intralesional Administration of REOLYSIN for the Treatment of Histologically Confirmed Malignancies\". All patients had histologically confirmed evaluable palpable tumors of any histological type that had failed to improve on existing standard therapy. The injectable lesion was required to be between 1 and 10 cm^2^, and accessible and measurable for delivery of an intralesional injection. Patients were required to have a life expectancy of at least 12 weeks and a ECOG performance status of ≤ 3 and must not have received active cancer treatment for least 21 days prior to entrance onto the trial. Adequate organ reserve in terms of bone marrow, hepatic, renal and cardiac functions was required. Patients on immunosuppressive therapy or alternative/complimentary/unproven systemic or local therapies were ineligible. Procedures ---------- After patients had been referred to the above mentioned trial, but prior to being definitively accepted (pending complete assessment of inclusion/exclusion criteria), patients met with a psychologist (LC) for the assessment protocol. At that time they were interviewed concerning their expectations about their health, both without any further conventional treatment and with the potential experimental treatment. They then completed the interview-based individualized QL interview, followed by the quantitative questionnaires as detailed below. All 16 patients followed the same procedures. Instruments ----------- ### Demographics Form Demographic information including age, education, marital status, occupation and current employment status was obtained on a form created for this study. Medical history including type of illness, dates of first diagnosis and subsequent relapses and site of metastases were collected, and later verified from patient charts. ### Expectations Interview Patients were asked three questions in a semi-structured interview: How do you feel about potentially being a part of this trial? How do you see your disease progressing without any further conventional treatment? Once on the trial, how do you see your disease progressing? Short answers to these questions were recorded verbatim at the time of the interview. Quality of Life --------------- ### Schedule for the Evaluation of Individual Quality of Life -- Direct Weighting (SEIQoL -- DW) \[[@B14]\] This schedule takes the form of a semi-structured interview, in which the investigator first describes quality of life as an individually defined construct, then elicits from the patient their own five most important domains of QL, rather than asking them about pre-set areas. Patients are asked to think of what areas of life determine their own happiness, or quality of life. After patients identify their most important domains (\"cues\"), they rate the quality of each domain currently in their lives by drawing a bar graph on a 100 mm scale from worst possible to best possible. This is called the \"level\" of the cue. Finally, they rate how important each domain is overall in their lives using a direct weighting disk. This disk consists of five overlapping different colored laminated disks that can be rotated around a central point to form a pie chart. Each piece represents one of the five chosen domains. The patient manipulates the disk until the proportion of each piece making up the pie represents the relative importance of that domain in their lives (\"weight\"). The weight value of each cue is calculated by determining the percentage of the overall pie that each piece covers by reading off a larger backing disk that is labeled with a 0--100 scale around the pie. Then by multiplying the level of each domain by its weight and summing the product for all five items, a summary score representing overall subjective quality of life can be calculated. This is called the SEIQoL Index Score. More detailed descriptions of the procedures are available in other publications \[[@B14],[@B18],[@B24]\]. ### The European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 Quality of Life Questionnaire \[[@B25]\] This 30-item questionnaire includes five functional domains of quality of life: physical function (5 items), emotional function (4 items), cognitive function (2 items), social function (2 items) and role function (2 items). There are also several symptom scales: fatigue (3 items), pain (2 items), nausea and vomiting (2 items), and one item each for dyspnea, sleep disturbance, appetite, constipation, diarrhea and financial difficulties. Finally, two items assess global quality of life. The questionnaire shows high internal consistency, and overall reliability and validity of the survey has been demonstrated in international clinical trials with cancer patients of heterogeneous diagnoses including lung cancer \[[@B26]\]. Distress -------- ### Beck Depression Inventory (BDI) \[[@B22]\] This 21-item questionnaire gives a global score on depressive symptoms, and norms are available for many different populations, including cancer patients. Higher scores represent more depressive symptoms. ### Brief Symptom Inventory (BSI) \[[@B21]\] A general mental health measure of 58 questions which provides scores on nine dimensions of psychopathology or psychological distress: somatization, obsessive-compulsive, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, and psychoticism. Three global scores can be calculated: the Global Severity Index (GSI), the Positive Symptom Total (PST) and the Positive Symptom Distress Index (PSDI). The GSI was used as the global score in this study. Spirituality ------------ ### Spiritual Health Inventory (SHI) \[[@B20]\] This instruments defines spiritual health as the capacity to transcend oneself and meet three basic needs; the need for self-acceptance, the need for relationships with others and/or a supreme being, and the need for hope. These three factors accounted for 71% of the variance in validity studies. A single total score is calculated by summing all items. The possible range of scores is 31--155, with higher scores indicating higher levels of spiritual health. The 31-item questionnaire takes very little time to complete. Results ======= Subjects -------- Demographic characteristics and disease variables of participants are presented in Table [1](#T1){ref-type="table"}. Patient \#10 was registered in the trial but too ill to complete any of the questionnaires or the interview. Therefore no data for this patient is included in the study. The remaining 16 patients who provided data all had metastatic disease that was considered incurable, 6 men and 10 women. The largest patient group consisted of five women who had metastatic breast cancer, followed by three patients with malignant melanoma. Patients ranged in age from 32 to almost 76 years old, with a median age of 53 years. They had been diagnosed with cancer for a median of 3.3 years (range 0.5--26.9 years) before entrance to the study. They had on average 16 years of education (range 12--25), and therefore represented a highly educated group. At the time of analysis, nine of the patients had died, at a median of 136 days from the time of the interview (range 21--664 days). The remaining seven were still alive, a median of 242 days from the time of the interview (range 207--709 days). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographic and Disease Characteristics ::: Patient Number Gender Age (Years) Cancer diagnosis Years since first diagnosis Location of metastases Days from interview to death ---------------- -------- ------------- ------------------------------------------ ----------------------------- ---------------------------------------------------- ------------------------------ 1 Female 45 Mucoepidermoid carcinoma (head and neck) 14.7 Lymph nodes 28 2 Male 50 Squamous carcinoma (head and neck) 0.7 Lymph nodes Alive-709 3 Female 54 Anaplastic thyroid carconoma 10.8 Lungs/liver/bone 21 4 Female 47 Malignant Melanoma 12.7 Eye/breast/liver/chest wall/lung 664 5 Female 47 Metastatic Breast carcinoma 6.6 Chest wall/bone 241 6 Female 60 Metastatic Breast carcinoma 2.7 Chest wall /lungs /retroperitoneum Alive-529 7 Male 32 Soft tissue sarcoma 0.5 Right lower extremity 96 8 Male 56 Neuroendocrine islet cell tumor 2.4 Liver/face/neck/ Scalp 44 9 Female 56 Metastatic Breast carcinoma 2.7 Chest wall/ left supraclavicular skin/ intrabdomen 142 11 Male 42 Malignant melanoma 6.4 Liver/lung/spleen Alive-368 12 Male 64 Klatskin\'s tumor 1.4 Abdomen 171 13 Female 76 Metastatic Breast carcinoma 26.9 Lung/bone/liver 136 14 Female 55 Malignant melanoma 1.8 Axilla /lung /liver Alive-242 15 Female 48 Metastatic breast carcinoma 2.8 Neck /Chest Wall /Brain Alive-242 16 Female 46 Soft tissue sarcoma 3.9 Lung/skin/breast/ retroperitoneal Alive-227 17 Male 70 Squamous carcinoma (head and neck) 9.7 Head/neck Alive-207 ::: Expectations Interview ---------------------- Interviews were conducted with all of the 16 patients. To the question \"How do you see your disease progressing without further conventional treatment?\", nine of the patients indicated they felt it would get worse and they would eventually die of their disease. This was stated in different ways: Nothing else left\...getting gradually worse\...terminal -- could be months, could be years\...wouldn\'t go very well. The other seven patients offered more hopeful or neutral prognoses: don\'t think about it -- stay positive\...still hopeful and optimistic\...other things available still\...would still take chemo, hope it would work\...wouldn\'t ever give up on hope\...faith\...not sure, unknown. When asked how they felt about being in the trial, most patients indicated feeling excited, fortunate, grateful and hopeful. One indicated that they felt scared as well as hopeful, not knowing what to expect, and one said they felt like a guinea pig. To the question \"Once on the trial, how do you see your disease progressing?\", ten of the patients mentioned hoping for the tumor to shrink, for a remission, or for some extension of life. Five patients mentioned hoping for a cure, to be cancer free. One patient just mentioned hoping to help others, and four others said that although they were hoping for personal benefit, if it didn\'t help them it might help others in the future. In general, patients were hopeful yet philosophical about the trial. The 54-year old woman with melanoma captured these sentiments with her comments: It feels hopeful. Maybe it won\'t help me -- I won\'t be disappointed. It might help others down the road. It\'s on the frontier -- exciting. If it works it\'s a bonus. I don\'t totally expect anything. It may extend life. Just day by day carry on. EORTC QLQ C-30 -------------- Quality of life scores on the EORTC QLQ C-30 are presented in Table [2](#T2){ref-type="table"}. All 16 patients completed the questionnaire. On the functional scales, where higher scores indicated better functioning, scores ranged from a low of 55 on social functioning, to a high of 76 for cognitive functioning, on a scale of 1--100. The overall global QL rating was 57. On the symptom scales, where higher scores indicate more symptomatology, scores ranged from a low of 14 (nausea and diarrhea) to a high of 42 on pain and fatigue. The next most prevalent symptoms were sleep problems and appetite loss. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### EORTC Scores ::: Functional Scales (Higher scores = higher function) Mean SD ----------------------------------------------------- --------------------- ----------------------------------- Physical Function 63.75 32.02 Role Function 62.50 38.73 Emotional Function 75.52 18.12 Cognitive Function 76.04 24.32 Social Function 55.21 32.04 Global Quality of Life 57.22 21.79 Symptom Scales (Higher scores = more symptomatic) Fatigue 41.67 25.82 Nausea 14.58 14.75 Pain 41.67 25.82 Dyspnea 22.92 26.44 Sleep 35.42 30.96 Appetite 33.33 32.20 Constipation 25.00 28.55 Diarrhea 14.58 17.78 Finances 29.17 26.87 ::: Correlations between subscales are presented in Table 6 (additional file [1](#S1){ref-type="supplementary-material"}). The subscales of role function, cognitive function, global QL, fatigue and appetite loss were significantly related to seven other subscales each. Social functioning was associated with scores on six other subscales. Finances, diarrhea, and sleep were unassociated with any other subscales, and pain was associated only with dyspnea. All significant correlations were in the expected directions. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### SEIQoL Items ::: Patient Number Item 1 Item 2 Item 3 Item 4 Item 5 ---------------- -------------- --------------- -------------------- -------------------- ------------ 1 Children Spouse Religion Physical Fitness Finances 2 Family Exercise Nature Computer Work 3 Spouse Children Friends Activities Father 4 Family Friends Dog Gardening Fun 5 Pain Control Finances Health Energy Activities 6 Travel Health Family and Friends Spouse Activities 7 Children Family Mobility Hope Work 8 Work Family Finances Health Activities 9 Family Grandchildren Friends Travel with Spouse Finances 11 Family Friends Active at Home Work Finances 12 Spouse Friends Family Belief Art 13 Activity Grandchildren Sewing Gardening Travel 14 Family Faith Positivity Activities Friends 15 Work Recreation Mobility Family Friends 16 Spouse Children Family Faith Exercise 17 Family Spouse Work Family tree Religion Mean Level 70.9 68.4 64.6 56.9 62.5 SD 31.3 26.0 32.0 32.1 27.1 Mean Weight 0.25 0.23 0.19 0.20 0.16 SD 0.08 0.08 0.06 0.08 0.09 ::: Psychological Scores -------------------- Scores on the BDI, BSI and SHI are presented in Table [3](#T3){ref-type="table"}. Fifteen of the 16 patients completed the questionnaires. Scores on the BDI averaged 11, in the moderate range of depressive symptomatology. On the BSI, scores ranged from a low of 0.17 on paranoid ideation, to a high of 0.91 on the obsessive-compulsive subscale. The overall global severity index was 0.55. These scores are higher than those of the general population, but quite a bit lower than those of psychiatric outpatients \[[@B21]\]. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Psychological Scores ::: Mean SD ---------------------------------------- ---------------------- ----------------------------------- BSI Somatization 0.74 0.56 BSI Obsessive Compulsive 0.91 0.63 BSI Interpersonal Sensitivity 0.38 0.36 BSI Depression 0.76 0.62 BSI Anxiety 0.50 0.40 BSI Hostility 0.21 0.27 BSI Paranoid Ideation 0.17 0.17 BSI Psychoticism 0.16 0.17 BSI Global Severity Index 0.55 0.36 Beck Depression Inventory Total Score 11.40 9.46 Spiritual Health Inventory Total Score 118.33 16.02 ::: Patients scored an average of 118 on the SHI. The most highly endorsed items on the scale of 1--5, where 1 corresponds with the heading \"not at all\", and 5 with \"very much\", were the following: \"I believe other people accept me even with my faults\" (4.5); \"I actively participate in decisions concerning my health care\" (4.5); \"I believe my nurses and doctors care about me\" (4.3); \"My life has a purpose\" (4.2); \"I feel accepted and forgiven despite some past actions\" (4.0). The lowest scores were on the following items: \"I wonder if God is angry with me\" (1.1); \"I feel angry with others\" (1.3); \"I feel a need to be forgiven for some of my thoughts and feelings\" (1.7); \"I worry about life after death\" (1.9); \"I feel angry with myself\" (1.9); and \"I feel out of touch with my own feelings and with others\" (1.9). SEIQoL ------ All 16 patients completed the SEIQoL. The average time taken was 13.5 minutes, range 5--30 minutes. Areas identified by patients as the most important in determining their overall quality of life are presented in Table [4](#T4){ref-type="table"} by patient, along with average levels and weights associated with each cue by order of identification. As can bee seen, most patients identified family, children, or spouse as the single most important factor in determining their current quality of life. The average level of each of the five cues ranged from 57--71 on a scale of 0--100, where 100 was the best possible state for that cue. The weights assigned to the cues varied from 16% to 25%, a fairly narrow range, with those cues identified earlier in the process generally being assigned higher importance. The overall index scores, which take into account both the level of the cue and its weight, were an average of 69, SD 20.5 and ranged from 27--100. The frequency of nomination of different cues as any of the five domains is presented in Table [5](#T5){ref-type="table"}. All but one patient mentioned some family relationship as one of the five domains, while some patients nominated several different specific family relationships. This was followed by the general ability to participate in chosen activities (e.g. exercise, recreation, travel, gardening, sewing). Seventy-five percent of the patients nominated some type of activity in their top five. The next most frequent category was friends, endorsed by 44% of the patients. This was followed equally by health (mobility, fitness, energy), faith (religion, belief, hope), and work, with 38% of the patients nominating each category. Finances were nominated by 31% of the patients, followed by several items that were mentioned by only one person each and warranted separate categories. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Frequency of Cue Nomination ::: Cue N (out of 16) \% --------------------------------------------------------------- ----------------------------- ---------------------------------- Family (Children, Spouse, Grandchildren, Parent, Family Tree) 15 93.8 Activities (exercise, gardening, sewing, recreation, travel) 12 75 Friends 7 43.8 Health (mobility, physical fitness, energy) 6 37.5 Faith (religion, belief, hope) 6 37.5 Work 6 37.5 Finances 5 31.3 Pet 1 6.3 Computer 1 6.3 Pain Control 1 6.3 Art 1 6.3 Fun 1 6.3 Positivity 1 6.3 Nature 1 6.3 ::: The internal validity of each of the cues was assessed by performing regressions of the combination of each cue level and its weight onto the total index scores. The resulting R^2^values ranged from .19--.76, median .47, mean .50. The highest R^2^value was for the first cue generated, and the lowest value was associated with the fourth cue. This is much lower than in previous reports \[[@B16],[@B17]\] and may constitute reason to pause before attributing high levels of credence to the validity of all of the cues in influencing overall QL. Two examples of cues, cue levels and cue weights are illustrated in Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}. Figure [1](#F1){ref-type="fig"} illustrates the responses of patient \#1, a 45 year old woman with mucoepidermoid cancer of her head and neck region who died four weeks following the interview. The most important areas to her were health, followed by children, spouse, religion and finally finances. This profile is unusual for this group in that most patients, if they nominated health as a cue at all, did so later in the process. She indicated that the areas that were going the best were finances and religion, followed by children, spouse and health. This combination of things not rated as going very well in some important areas resulted in an index score of 59 on the SEIQoL This is quite a bit higher than her global QL score on the EORTC of just 25. Another example is patient \#2 (figure [2](#F2){ref-type="fig"}), a 50 year-old male with head and neck cancer who, as of this writing, has been alive for 709 days following the interview. For him, things were going well in the areas most important to him; family, work and computers. This resulted in an index score of 81, consistent with his global QL score on the EORTC of 75. These examples illustrate that the SEIQoL scores are related to overall QL scores, and suggest that they may be related to health status as well. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Patient \#1: Cues, Levels and Weights ::: ![](1477-7525-3-7-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Patient \#2: Cues, Levels and Weights ::: ![](1477-7525-3-7-2) ::: Correlations between measures ----------------------------- Correlations between the SEIQoL index and scores on the other measures are presented in Table 6 (see additional file [1](#S1){ref-type="supplementary-material"} -- Carlson Table 6.doc). The index score was significantly positively correlated with the global QL score (r = .53, p \< .05), and negatively associated with the symptoms of nausea (r = -.58, p \< .05), pain (r = -.53, p \< .05) and appetite loss (r = -.59, p \< .05) on the EORTC QLQ C-30. Scores on the BDI were positively associated with appetite loss (r = 0.56, p \< .05) and fatigue (r = 0.56, p \< .05), not surprising since these are both symptoms of depression assessed by the BDI. Depression scores were also negatively related to social functioning (r = -.79, p \< .01) and global quality of life (r = -.69, p \< .01), indicating that people who endorsed more depressive symptoms also tended to report lower social functioning and lower overall QL. Higher scores on the Global Severity Index of the BSI were associated with worse emotional (r = -.55, p \< .05), cognitive (r = -.58, p \< .05) and social (r = -.69, p \< .01) functioning, as well as worse global QL (r = -.70, p \< .01) and more sleep disturbance on the EORTC. Higher scores on the spiritual health inventory were associated with lower scores on the global severity index of the GSI (r = -.54, p \< .05) and less nausea (r = -.63, p \< .05). Significant correlations between days to death and psychological scores were found on two instruments in the nine patients who had passed away at the time of writing. The BDI total score was negatively correlated (r = -.75, p \< .05), and the EORTC global QL score was positively correlated (r = .77, p \< .05) with time to death. This indicates an association between higher levels of depression and lower global QL at the time of the interview, and fewer days to death. Discussion ========== This study is the first to use the SEIQoL-DW instrument to assess individualized QL in a population of patients with advanced cancer participating in a Phase I clinical trial. Of note is that the instrument was easy to use in this population and acceptable even to those patients who were quite ill. Replication of the wide range of individual differences in the cues chosen and the weights associated with each cue was seen in this group. The areas of QL that were nominated by patients as the most important factors in the determination of their overall QL were primarily family relationships, the ability to participate in pleasurable activities, and friendships. Only 38% of this sample mentioned health or health-related domains such as mobility, fitness and energy as one of the five cues. This is in contrast to other samples of cancer patients where over 70% of patients nominated health as an important domain. For example, health was nominated by 73% of cancer patients in phase I trials (not necessarily advanced cancer) \[[@B18]\], 87% of men with early stage prostate cancer \[[@B16]\], and 70% of patients with advanced incurable cancer (these patients were not on trials) \[[@B17]\]. The commonality between these studies is that family was consistently nominated as the most frequent domain. In terms of the index scores, our average of 69 was higher than that of the group of advance incurable cancer patients (mean = 58) and the patients participating in Phase I trials (mean = 61). It was comparable to the men with early stage prostate cancer (mean = 71), but the relatively small range of mean scores among these studies is notable. Construct validity is supported in that those patients whom one might expect to have higher QL (i.e. less ill patients), indeed reported higher QL. That the scores in the current sample were more comparable to the early stage prostate cancer patients than the incurable cancer patients may speak to the hope patients were feeling regarding the potential of the reovirus treatment. In terms of the index scores for other illness populations, patients prior to hip arthroplasty scored 59, after the surgery scores increased to 69 \[[@B27]\], on par with the cancer patients in this study (mean 69). Another study of hip replacement patients found scores of 62 prior to surgery, with improvements to 71 following surgery \[[@B28]\], a similar improvement pre- to post-surgery. Severely disabled multiple sclerosis patients scored 61 \[[@B29]\], patients with ALS had a higher mean index of 76 \[[@B30]\], and cardiac patients scored a high index of 82 after myocardial infarction or coronary artery bypass graft surgery and prior to beginning cardiac rehab \[[@B31]\];. Interestingly, only 24% of the ALS patients nominated health (disease progression) as a cue. Thus, although the areas of importance varied by individual in all these studies, the resultant index scores seem to demonstrate some consistency across similar populations. Another indication of the construct validity of the SEIQoL-DW is its high correlation to global QL scores on the EORTC QLQ C-30. This speaks to the validity of the self-generated items, as the sum of the products of their importance and current status was associated with the overall global assessment of QL on standardized domains. Predictably, our patients had a global QL on the EORTC of 57, much lower than the 77 reported in a large normative community sample \[[@B32]\]. Scores on all five functional scales and symptom scores were all also worse in this population, not surprising considering the extent of their disease status. However, they did have the same global QL scores compared to a large sample of patients with advanced malignancy from 12 institutions in 10 countries (both 57), but scored higher on most of the other functional scales than this group (Physical function: 64 vs. 60; Role function: 63 vs. 50; Emotional function: 76 vs. 50; Cognitive function: 76 vs. 63; Social Function: 55 vs. 50) \[[@B33]\]. The greatest differences in favor of the patients in this trial were seen on emotional, cognitive and role function. Interestingly, lower global QL scores on the EORTC and higher depression scores on the BDI were associated with a shorter time to death in those patients who had already passed away, an association that has been reported in other studies \[[@B33]-[@B35]\]. In fact, in an international sample of 411 patients with advanced malignancy, similar to the current group, the single-item global QL scale remained independently prognostic of death in a proportional hazards model stratified on diagnostic category, after allowing for performance status and age, and, among solid tumor patients, metastatic site \[[@B33]\]. Patients who were in terminal care from a large sample from 12 oncology outpatients departments around the United States scored an average BSI Global severity index score of .93. Those who were under symptom control scored .80, whereas lower scores were seen for patients in active therapy (.59), adjuvant therapy (.60) or no current therapy (.65)\[[@B36]\]. Our sample mean of .55 is quite low in comparison and most comparable to patients in active therapy. An even larger sample of over 4000 patients of all disease sites and stages of illness from Johns Hopkins Oncology Centre reported an average global severity index very similar to our patients, at .54 \[37\]. This seems to indicate that our sample is reporting less psychopathology than would be expected of patients in similar disease states, but comparable levels to cancer patients in general. They also scored an average of 11 on the BDI, which indicates mild depressive symptomatology. The spirituality scores of this group average 118. This is very similar to a group of lung cancer patients who had a mean of 120 \[[@B20]\]. Analysis of the individual items showed that these patients endorsed feeling well supported by the medical team and quite peaceful and accepting of themselves, and well accepted by others. They reported not feeling angry at themselves or others, or worried about life after death. The spirituality scores were correlated with the global severity index of the BSI, indicating that those who felt more spiritually at ease also endorsed fewer symptoms of psychopathology. In summary, these patients in a Phase I trial of a promising novel therapeutic were easily able to complete the SEIQoL-DW interview as well as a battery of other psychological questionnaires. They reported feeling excited and hopeful about the trial, with about two-thirds hoping for disease regression, and another third optimistically hoping for a cure. However, most acknowledged that although they hoped for the best they were realistic in their expectations. The individuality of QL as defined by each person was reinforced in this group, as many different cues were nominated as important and variable weights were assigned to the same cues. Consistent with reports from other seriously ill groups, health status received less focus than other aspects of life, primarily family relationships and activities. Overall QL on standardized measures and psychological status was generally better than other seriously ill patient groups, but comparable to cancer patients in general. QL and depression scores were related to time until death in those patients who had passed away. Authors\' contributions ======================= LC wrote the research proposal and ethics applications, conducted the interviews with the patients, designed the database, conducted the statistical analysis, and wrote the first draft of the paper. BB is the department head for Psychosocial Resources, and in conjunction with LC conceived of and designed the study. He also made the linkages with medical oncology to facilitate the study. DM is the oncologist and principal investigator of the Phase I Reovirus trial, and recruited all the patients. All authors reviewed and edited the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Table 6: Correlations between scores on the SEIQoL, BDI, BSI, SHI and EORTC QLQ C-30 ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ Dr. Linda Carlson was a Terry Fox Postdoctoral Research Fellow of the National Cancer Institute of Canada during the time the study was run. She is currently a Canadian Institutes of Health Research New Investigator. Special thanks to our research nurse Ms. Sally Lim for her tireless efforts in assuring the smooth running of the study, and to Ms. Jodi Cullum for data management.	PubMed Central	2024-06-05T03:55:52.622805	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548292/", "journal": "Health Qual Life Outcomes. 2005 Jan 27; 3:7", "authors": [ { "first": "Linda E", "last": "Carlson" }, { "first": "Barry D", "last": "Bultz" }, { "first": "Donald G", "last": "Morris" } ] }
PMC548293	Background ========== In a recent study, University of Toronto researchers identified the \"Top 10 Biotechnologies to Improve Health in Developing Countries\" \[[@B1]\]. The study underscores the importance of harnessing new technologies to improve global health and development, a belief that is gaining widespread acceptance. For instance, the overall goal of the United Nations Millennium Project\'s Science and Technology Task Force is to address how science and technology can be leveraged to help countries achieve the Millennium Development Goals (MDGs) \[[@B2]\]. Its mission is guided by the understanding that most of the MDGs cannot be reached without a strong contribution from science and technology. The potential contribution of genomics and biotechnology to these goals has also been demonstrated \[[@B3]\]. However, these technologies are most beneficial to countries that have the scientific capacity to absorb and use them. The aim of the Inter-Academy Council on Science and Technology Capacity (IAC) was to develop a global strategy for promoting capacities in science and technology and the first report of the Council was recently presented to UN Secretary General Kofi Annan \[[@B4]\]. There is a wide range of scientific capacity and health system development across the Eastern Mediterranean Region (EMR) \[[@B5]\], which consists of 21 countries. Some countries in the region have taken the initiative in biotechnology by establishing regulations and encouraging private sector involvement. In other countries of the region, there is a serious lack of scientific capacity not only to conduct research and development in biotechnology but even to absorb the benefits of biotechnology and apply them to help meet the health and socio-economic needs of the population \[[@B6]\]. According to UNDP\'s Arab Human Development Report 2003, research and development in the Arab world represents less than 0.2% of Gross National Product (GNP). Fewer than one in 20 Arab university students were pursuing scientific disciplines, compared, for instance, to one in five in South Korea \[[@B7]\]. There is a risk that, as the genomics revolution gathers momentum, the imminent genomics divide between developed and developing countries will increase unless urgent action is taken to reverse the trend \[[@B8]\]. In order to assess the potential of genomics to address health needs in the Region, the World Health Organization-Eastern Mediterranean Regional Office (WHO-EMRO) and the University of Toronto Joint Centre for Bioethics jointly organized a Genomics and Public Health Policy Executive Course, held September 20^th^--23^rd^, 2003, in Muscat, Oman. This 4-day course/workshop was sponsored by WHO-EMRO with additional support from the Canadian Program in Genomics and Global Health. The overall objective of the Genomics Policy Executive Course was to familiarize participants with the potential of genomics and related biotechnologies to address health needs and to collectively address the question of how best to harness genomics to improve health in the region (table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Objectives of the Genomics Policy Executive Course ::: -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- To familiarize participants with the current status and implications of health genomics/biotechnology, and to provide information relevant to public policy on health genomics/biotechnology To provide frameworks for analyzing and debating the policy issues and related ethical questions in health genomics/biotechnology, and to help understand, anticipate and possibly influence the legal and regulatory frameworks under which health biotechnology industries will operate, both nationally and internationally To begin developing an opinion-leaders network across different sectors (industry, academic, government, NGOs) by sharing perspectives and building relationship To formulate recommendations for future policy and strategic directions at the regional, national and individual levels. -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: Methods ======= We based the program and designed the sessions and lecture topics of this course on prior courses held in Nairobi, Kenya in March 2002; in Toronto, Canada in May 2002; and in Kumarakom, India in January 2003. The sessions and presenters of the course are shown in table [2](#T2){ref-type="table"}. The course participants and facilitators for each session were identified through a combination of recommendations from field experts in the region and from WHO\'s network of experts. People identified to participate in the course included scientists from academic institutions and industry, industry executives, regulatory officials, representatives from the legal sector, and media. The participants were carefully chosen in an attempt to represent a wide range of interests relevant to the emerging area of genomics and to have geographical, discipline and gender balance. They included scientists from academic institutions and industry, industry executives, legal and regulatory officials, WHO representatives and the media. In total there were 51 participants, from 13 countries of EMR. We drew as many of the faculty as possible from the region. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Program ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- [Saturday, 20 September 2003]{.underline} ----------------------------------------------- ------------------------------------------------------------------------------------------------------------------------- 08:00 -- 08:30 Registration 08:30 -- 10:30 Session I:\ Opening Address\ H.E. Dr Ali Bin Moosa, Minister of Health, Oman\ Dr H. A. Gezairy, Regional Director, EMRO\ Introduction and Course Overview\ Team from University of Toronto Session Chair: Dr Ali Jaffer Suleiman, Ministry of Health, Oman 10:30 -- 11:00 Coffee break 11:00 -- 12:30 Genomics: Scientific Developments\ Professor Riad Bayoumi, Sultan Qaboos University 12:30 -- 14:00 Lunch Break 14:00 -- 15:30 Session III:\ WHO Report on Genomics and World Health\ Professor Alexander Capron, Director, Department of Ethics, Trade and Human Rights, WHO/HQ 15:30 -- 16:00 Coffee break 15:45 -- 16:30 Session IV:\ Top 10 Biotechnologies for Improving Health in Developing Countries Professor Abdallah S. Daar, University of Toronto [Sunday, 21 September 2003]{.underline} 08:30 -- 09:00 Golden Nuggets (previous day\'s summary)\ Dr Peter A. Singer, University of Toronto 09:00 -- 10:30 Islamic Perspective on Stem Cells, Cloning, Genetic Engineering, ..etc\ Dr Mohammed Al Bar, King Fahd Medical Research Centre, Saudi Arabia 10:30 -- 11:00 Coffee break 01:00 -- 12:30 Intellectual Property Rights\ Professor Richard Gold, McGill University, Centre for Intellectual Property Policy 12:30: -- 14:00 Lunch Break 14:00 -- 15:30 Business Models\ Mr Khalil Ahmed, Managing Director, Shantha Biotech, India\ Dr Peter A. Singer, University of Toronto 15:30 -- 16:00 Coffee break 16:00 -- 17:30 Group Work [Monday, 22 September 2003]{.underline} 08:30 -- 09:00 Golden Nuggets (previous day\'s summary)\ Dr Peter A. Singer, University of Toronto 09:00 -- 10:30 Innovation Systems\ Dr Peter A. Singer, University of Toronto 10:30 -- 11:00 Coffee break 11:00 -- 12:30 Regulatory Systems and Related Issues\ Dr D.C. Jayasuriya, Director, UNAIDS, Pakistan\ Dr Anwar Nasim, Chairman, National Council of Biotechnology, Pakistan 12:30 -- 13:00 TRIPS and Pharmaceutical Issues in Public Health.\ Dr. Abdel Aziz Saleh Special Advisor to the Regional Director for Medicine, WHO/EMRO 13:00 -- 14:00 Lunch Break 14:30 -- 15:30 Public Engagement\ Mr Ehsan Masood, Scidev.net, United Kingdom 15:30 -- 16:00 Coffee Break 16:00 -- 17:30 Group Work [Tuesday, 23 September 2003]{.underline} 08:30 -- 09:00 Golden Nuggets (previous day\'s summary)\ Dr Peter A. Singer, University of Toronto 09:00 -- 10:00 Opinion Leaders Network\ Dr Peter A. Singer, University of Toronto, Dr Tara Acharya, University of Toronto 10:00 -- 10:30 Break 10:30 -- 12:30 Group Work 12:30 -- 14:00 Lunch break 14:00 -- 15:30 Group Presentations and Discussion 15:30 -- 16:00 Coffee Break 16:00 -- 17:30 Recommendations, Concluding Remarks and Closure of the Workshop\ Dr Peter Singer, University of Toronto ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: The sessions dealt with a wide range of relevant topics, starting with recent scientific advances in genomics and stem cell research, followed by discussions on business models in genomics and biotechnology, intellectual property rights and regulatory frameworks, public engagement and an internet-based opinion leaders\' network. The presentations were designed to be interactive and foster active discussion from, and among, the participants, and each presentation was followed by a moderated discussion period. Early in the course, the attendees were placed into one of five study groups -- these groups were carefully designed to capitalize on the diverse backgrounds of the participants. Each group was assigned the task to on the deliberate the key question \"How best to harness genomics and biotechnology to improve the health of the people in the Eastern Mediterranean Region?\"The groups met frequently to discuss the presentations, and each participant was also provided a course reader with additional literature on the lecture topics (table 3). The overall task of these study groups was to draw upon the course material and their own experiences and propose a set of recommendations for genomics and biotechnology policy in the region. On the last day of the meeting each group presented these recommendations. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Course Readings ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Bhutta ZA (2002) Ethics in international health research: a perspective from the developing world.\ Bull World Health Organ.;80(2):114--20. Review. Bloom BR & Trach D (2001) Genetics and Developing Countries. BMJ;322:1006--7. Capron AM (2001) Stem Cells: Ethics, Law and Politics. Biotech Law Report;5:678--699. Collins FS, Green ED, Guttmacher AE, Guyer MS; US National Human Genome Research Institute. (2003) A vision for the future of genomics research.\ Nature.24;422(6934):835--47. Daar AS, Thorsteinsdóttir H, Martin DK, Smith AC, Nast S, Singer PA. (2002) Top ten biotechnologies for improving health in developing countries. Nat Genet.;32(2):229--32. Gold ER (2003) SARS genome patent: symptom or disease? Lancet.;361(9374):2002--3. Juma C & Konde V (2002). The New Bioeconomy: Industrial and Environmental Biotechnology in Developing Countries. Geneva, Switzerland: United Nations, July 2002. Lundvall B, Johnson B, Andersen EA, Dalum B (2002) National systems of production, innovation and competence building. Research Policy;31:213--231 Nasim A (2000) Ethical Issues of the Human Genome Project: An Islamic Perspective inBioethics in Asia. Eubios Ethics Institute Eds: N Fujiki and DRJ Macer p. 209--214 Pang T & Weatherall D (2002) Genomics and Global Health. BMJ;324:p.1051--52 Singer PA, Daar AS (2001) Harnessing genomics and biotechnology to improve global health equity. Science;294(5540):87--9. Sulston J (2003) Beyond release: the equitable use of genomic information.\ Lancet.;362(9381):400--2. Thorsteinsdóttir H, Daar AS, Smith RD, Singer PA (2003) Genomics -- a global public good?\ Lancet.;361(9361):891--2. Time to Unite Islam and Science. Nature. 2003 Mar 13;422(6928):99. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: In order to assess the level of interest in the formation of an email-based opinion leaders\' network to continue discussion among the participants following the course, a brief survey was conducted on the participants\' internet access and their expectations of the network. Results ======= Dr Peter A. Singer, Director of the University of Toronto Joint Centre for Bioethics, described the overall aim of the course \"How to best harness genomics to improve health in the region\", as well as the 4-day program. Dr Ali Jaffer Suleiman of the Ministry of Health in Oman acted as chairperson of the meeting. Professor Riad Bayoumi of Sultan Qaboos University highlighted new scientific developments that have resulted from the genomics revolution, such as proteomics; mapping of single nucleotide polymorphisms (SNPs) to understand human genetic variation and its relationship with disease; gene expression chips to monitor differential gene expression and identify drug targets; and bioinformatics as a new field that combines biology, mathematics, statistics and computer programming to mine large-scale biological data. Professor Alexander Capron, Director, Department of Ethics, Trade and Human Rights at WHO, described the 2002 report of the World Health Organization \"Genomics and World Health\" \[[@B9]\]. He called attention to the recommendations from the report -- these include improving technical cooperation between WHO and its member states (e.g. assessing the health impacts of genomics research to support informed priority setting; capacity building for genomics research and biotechnology in developing countries; development of ethical review structures and bioethics capacity). Professor Abdallah Daar described a recent study, conducted by the University of Toronto Joint Centre for Bioethics\' Canadian Program on Genomics and Global Health, to identify the ten most promising biotechnologies for improving health in developing countries in the next five to ten years. These technologies offer guidance to those who can influence the direction of R&D in developing countries and challenge common assumptions about the relevance of biotechnology for these countries, as shown by the mapping of the Top 10 Biotechnologies onto the UN Millennium Development Goals \[[@B10]\]. However, to foster biotechnology in developing countries it is essential to build capacity (among researchers, politicians, legislators, entrepreneurs, etc). Professor Al Bar of King Fahad University, Saudi Arabia, illustrated Islamic perspectives on genetic testing, cloning, recombinant DNA technology and other genomics-related technologies. He showed that while Islam and science have always been aligned through history, today\'s religious leaders in the region must take the initiative to develop and formulate recommendations for genomics. Dr. Peter Singer described the concept of innovation systems and presented some observations of other countries\' innovation systems and their successes. One definition of a national system of innovation (NSI) is the \"network of institutions in the public and private sector whose activities initiate, import, modify and diffuse new technologies\" \[[@B11]-[@B13]\]. The application of NSI to developing countries is a fairly recent concept. The Canadian Program on Genomics and Global Health is currently conducting studies of the NSI of 7 developing countries: Brazil, China, Cuba, Egypt, India, South Africa and South Korea \[[@B14]\]. Despite the identification of factors that foster innovation systems, there is no one model that guarantees success -- each country follows its own unique path. Mr. Khalil Ahmed, Managing Director of Shantha Biotechnics, described the successes of this biotechnology company based in Hyderabad, India. Shantha was established in 1993, in part with seed money from the Government of Oman. Today, it is the first Indian company to receive WHO certification for a recombinant hepatitis-B vaccine Shanvac-B™, paving the way for UNICEF to buy 8.5 million doses for distribution globally. Hepatitis B vaccine is currently priced internationally as high as \$8--10 per dose, while Shantha is selling it at \$2 per dose \[[@B15],[@B16]\]. Professor Richard Gold of McGill University\'s Centre for Intellectual Property Policy described the basics of intellectual property rights, patents and copyright issues, as well as policy options for developing countries in the international context. He outlined the characteristics of international agreements, which offer considerable flexibility to countries on how to apply patent laws to genomics and biotechnology. Developing countries have a number of options, such as compulsory licensing and research exemptions, to deal with property rights. Dr Jayasuriya, Director of UNAIDS in Pakistan described ways in which legal systems can facilitate best use of biotechnology. It is essential that legal reform keep up with the rapidly evolving science of genomics. Health law can facilitate best use of biotechnology -- by providing for fast-track approval of biotechnology products; reducing import duties on health interventions; and allowing multiple channels of procurement and distribution to improve access and optimize prices. Dr Anwar Nasim, Chairman of Pakistan\'s National Council of Biotechnology, described the role of regulations both to promote useful biotechnologies and limit their risks for human health and the environment. A national-level regulatory body could provide guidelines for the use and release of biotechnology products, conduct biosafety reviews and risk assessments and formulate feedback mechanisms to improve the system through experience. The National Commission of Biotechnology of Pakistan, established in November 2001, focuses on biotechnology regulatory issues in health, agriculture, environment and industry. Similar commissions could be set up in other countries of the region and their interaction could further regional cooperation in biotechnology. Mr. Ehsan Masood of Scidev.net pointed out that active public engagement based upon knowledge can stimulate action to improve public health. Public engagement is far greater in today\'s world than it has ever been in the past. This is because of several factors including; a) the growing implications of research on public health, b) the increased awareness in civil societies to invest in health care and research, and to influence policy change and action, c) development and access of information technologies. On the last day of the course the five participant groups, who had been assigned the task to on the deliberate the key question \"How best to harness genomics and biotechnology to improve the health of the people in the Eastern Mediterranean Region?\", were invited to present their findings. The main points of these group presentations are summarized in the recommendations that emerged from the course. Discussion ========== The session discussions and group presentations underscored the urgent need for action to create the enabling environments (at regional, national and individual levels) for research and development in genomics and biotechnology. The issue of awareness of biotechnology among political leaders to garner support at the highest level was raised early on in the course, and was reinforced throughout the course discussions. The participants felt strongly that political commitment is crucial to the advancement of biotechnology in the region. Political leadership is a critical factor in raising the profile of science in developing countries. A prominent example in the Eastern-Mediterranean Region is that of the Sultan Bin Mohammed Al-Qassimi, Shaikh of the Arab emirate of Sharjah. He has made a dedicated effort to change the face of science in the Gulf \[[@B17]\]. For example, in an attempt to attract Arab scientists working abroad to return to work in the Gulf, and more specifically to Sharjah, he has built two universities, six museums and established a science foundation in just a decade. He is also actively engaged in creating an environment of regional cooperation. The course attendees recommended that WHO-EMRO should take the responsibility to engage political leaders in the region\'s national governments. The participants shared experiences of the importance of government support for biotechnology -- in Egypt there appears to be relatively strong support for biotechnology by the government, with biotechnology activity in academic and other research institutions as well as in the private sector. In Iran, considerable advances have been made in private and academic research centres, leading to a number of biotechnology products that are soon to reach the market, as well as well-respected scientific journals such as the Iranian Journal of Biotechnology \[[@B18]\]. Other countries such as Pakistan, Tunisia, Lebanon, and Morocco are making steady gains in biotechnology, some in conjunction with their powerful public health sectors and others as a result of their strong scientific bases. It will also be crucial to involve, inform and engage religious leaders in the region in order to promote genomics and biotechnology for improving public health. In doing so, it is worthwhile to note that Islam and science have always been aligned through history and it is well-established that Islam has historically contributed tremendous achievements to the advancement of science. \[[@B19]\] A recent article argues that the Muslim world has neglected to pay attention to contemporary ethical issues in science and technology \[[@B20]\]. The author calls for the establishment of an independent Islamic bioethics panel to advise Islamic governments and communities. Other measures, such as training Muslim bioethicists, incorporating biomedical issues into school curricula and educating the community about such issues are also recommended. The active participation and involvement of organizations like COMSTECH was recommended to give support and guidance to WHO-EMRO\'s efforts. COMSTECH was established by the Islamic Summit in 1981 and includes among its objectives the building of indigenous capabilities in the fields of science and technology, promotion and continuing cooperation and coordination in scientific and technological areas of is member states and creation of effective institutional structure for planning research, development and monitoring of scientific and technological activities. COMSTECH has already launched networks across the region for exchange of information, and has valuable experience in the promotion of cooperation and coordination amongst the member states in science and technology activities in high technology areas \[[@B21]\]. A major discussion point at the workshop was the effective assessment of existing capacity in the region. Several participants shared disappointment over the relative lack of participation of the region in global science and technology advances, citing, for instance, poor representation of the region at international scientific conferences. Many felt the need to evaluate and assess the level of scientific activity and capacity in individual countries in the region, in order to identify, among other things, strengths and weaknesses, entry points for countries, opportunities for regional collaboration, and areas for improvement. The workshop attendees felt it important to carry out this type of survey of each country\'s innovation system as a prerequisite to revising and revamping national and regional genomics policy. The factors identified by the participants as important for assessment in this survey -- scientific capacity within public and private sector, private enterprise, religious and political leadership and intellectual property rights -- can be considered to be part of the National System of Innovation (NSI). This proposed survey of NSI in the region can help to identify factors for successful growth of biotechnology sectors. Similarly, the recently completed study by the Canadian Program on Genomics and Global Health of the health biotechnology innovation systems of Brazil, China, Cuba, Egypt, India, South Africa and South Korea may also help to identify some of these factors \[[@B22]\]. Workshop participants from Egypt, Iran, Lebanon, Pakistan, and Tunisia contributed to the discussion of NSI with descriptions of components of their countries\' biotechnology innovation systems. In Egypt, there is increased recognition of the importance of intellectual property rights in fostering innovation. The Minister of Health has taken a keen interest in improving linkages between sectors to enhance inter-sectoral communication. The president himself was instrumental in the establishment of the Mubarak City for Scientific Research and Technology (MCSRT), which is currently engaging in collaborative projects with the United States and with China. Private sector development is of high priority, as indicated by government support of companies such as Vacsera, which is now making recombinant insulin in Egypt \[[@B23]\]. One successful story from Egypt is the development by scientists at the Agricultural Genetic Engineering Research Institute in Giza of a powerful bio-pesticide based on a new strain of Bacillus thuringiensis\[[@B24]\]. In Iran, the trade embargo has led to a shortage of funds, but has in some ways helped to strengthen the innovation system through the need for self-reliance. Iran and Cuba have reached an agreement for cooperation and transfer of technology to produce hepatitis-B vaccine, interferon-α, streptokinase, and erythropoietin. Iran has developed several products based on recombinant DNA technology, and the country is also actively building research collaboration networks. Pakistan has had some successes in agricultural biotechnology and made some advances in bioethics. Although there are powerful institutes in place, the country needs to continue to strengthen facilities, resources and human capital. There is a need to bring products to the public. Lebanon has a well-developed healthcare industry, and has developed expertise in genetic testing, bioethics as well as intellectual property rights. Tunisia has strong research infrastructure and has made advances in genetic counseling, cytogenetics, and diagnosis of genetic diseases, along with regulation and legislation. However the pharmaceutical industry is not able to optimize the potential of public sector research, for which regional cooperation would be valuable. The participants identified strengthening the linkages between academia and the private sector as key to strengthening capacity in genomics. One example of a concerted effort to improve academic-private sector links is Jeddah BioCity, a research facility with close ties to the King Faisal Specialist Hospital and Research Center. Founded by Sultan Bahabri, head of King Faisal Specialist Hospital and Research Center in Jeddah and other Saudi scientists, this private venture plans the construction of state of the art biotechnology laboratories and companies and is intended to make Saudi Arabia a world leader in biotechnology. As highlighted by the 2004 report of the UN Commission on Private Sector and Development report \[[@B25]\], the process of commercialization for development involves the dissemination and facilitation of knowledge flows between public and private sectors of both developed and developing markets. The report recommends action in both the public and private spheres, and emphasizes the linkages between these spheres, recognizing the importance of cooperation and partnerships to achieve goals. With stronger public-private linkages, the private sector in developing countries will be able to help provide new genomics-based technologies at affordable prices. Given that over the last few decades, market forces have driven the R&D agenda of pharmaceutical companies based in the North to de-emphasize the health concerns of developing countries, it is becoming increasingly important to develop indigenous capacity in private enterprise. The successes of biotechnology firms in developing countries such as India and Egypt were seen as a source of inspiration by the course attendees. The attendees observed that the key to effective participation in the genomics revolution is building scientific capacity, and expressed serious concern over the region\'s limited capacity to absorb and utilize genomics and biotechnology. Critical to capacity-building is access not just to technology but, more importantly, to scientific knowledge. One point-of-entry that was greeted with enthusiasm by the participants was bioinformatics, which is typically less-resource intensive compared with other genomics-related sciences, such as sequencing and proteomics. According to the report on the \"Top 10 technologies to improve health in developing countries\" countries of EMR can take advantage of genomic data and apply the power of bioinformatics to local health problems without having to invest heavily in the technologies used to produce them, A dedicated Genomics and Health Research Fund for the region may help to break down financial barriers to encourage scientific research and development in the region. The participants expressed enthusiasm for setting up National Biotechnology Commissions to address genomics policies at the national level and to contribute to the development of the above-mentioned national biotechnology strategy. A National Commission on Biotechnology (NCB) has been set up in Pakistan. The overall goal would be to help devise regulations to facilitate innovation in biotechnology. The NCB could help coordinate national biotechnology activities and policy, with activities including evaluation of biotechnology capacity (see above) to priority setting and public engagement. Accordingly, the NCB should have broad cross-sectoral representation in order to minimize the negative effects of inter-institutional rivalry. One of the main goals of the NCB (or equivalent national agency), following the above-mentioned national biotechnology survey, will be to help develop and adopt a national biotechnology strategy, perhaps as part of a long-term science and technology policy. Other important objectives are discussed below. At the outset of the conference, participants voiced their concern about the low level of awareness (which goes beyond just publicawareness) of biotechnology and genomics in the region, and that this lack of awareness may be the biggest barrier to advances in genomics. Public awareness and engagement can help change the pace of research, leading to increased opportunities for greater societal involvement for improved health care. Media can be used as a catalyst to raise community awareness for social beneficence, equity and justice in health care. Recently, in May 2004, science journalists from across the region met in Cairo to discuss the hurdles they face in science reporting. The hurdles identified at this meeting included bureaucracy and poor access to scientific research taking place in the region \[[@B26]\]. The meeting concluded with the creation of a provisional network of Arab science journalists that will aim to provide its members with training, skills and contacts, as well as promote the coverage of scientific issues from a development perspective. Similarly, public health specialists and scientists should also be encouraged to engage actively disseminate evidence based and correct information in disease prevention and control through media. While it was agreed that science should be permitted to march forward, the participants emphasized that ethics, regulations and laws must keep up with the science \[[@B27]\]. For example, an important focus area of the NCB is that of intellectual property rights. The participants agreed that developing countries must build capacity and knowledge to choose the best policy options to benefit from the international patent regime. Compulsory licensing under specific circumstances may be a good option for developing countries and national laws should permit compulsory licensing \[[@B28]\]. The forces of globalization must balance forces of protectionism, especially by the developed world, and both national and regional regimes should respect international patent law to take advantage of globalization. If developing countries are involved in international research collaborations, they should ensure that they obtain patents. In view of this, it is essential for developing countries to build capacity and training in patent law and learn how to formulate effective patents, and the NCB could play a key role in this effort. Regional cooperation in science was given a boost this year, with the establishment of a network of science academies of the Organisation of Islamic Conference \[[@B29]\] at a meeting organized by the Third World Academy of Sciences. This formal network aims to provide the partner states with mutual support and supports discussion of the scientific aspects of common problems. It could help to build a unified approach to capacity building in science and technology within member states. Health advances in developing countries have lagged behind those in the developed world. The rapid advance in genomics research in developed countries compared with the relatively slow progress of genomics R&D in developing countries threatens to create a North-South genomics divide in the coming years, which may enhance existing health inequities. With the appropriate emphasis on its health needs, incentives for public-private R&D partnerships, and a sound set of regulatory policies, the Eastern Mediterranean Region may well reap the benefits of genomics and biotechnology. The overall goal of the Genome Policy Executive Course, a WHO-University of Toronto initiative, was to help provide the impetus for cross-sectoral dialogue on genomics and health policy in the region. The internet-based opinion leaders\' network is expected to foster dialogue to help achieve the objectives outlined by the participants of the course. The participants and the organizers of the course felt strongly that the recommendations formulated at the course must be shepherded by individuals. People felt that progress could be achieved if individuals make a significant and concerted effort to ensure that these recommendations are fulfilled. One way to spur action and maintain the momentum generated by this course is by coordinating the participants into a network within which they can continue to interact and share information and experiences. This internet-based network will be moderated in order to streamline discussions. A number of short-term projects are envisioned that could be coordinated by various expert members of the network. The results of the survey (table [4](#T4){ref-type="table"}) administered to the participants during the course suggested that 80% of them had reliable access to internet and would be willing to spend 1--2 hours a week taking part in the discussion. The main objectives of the network, as identified by the participants in the survey, would be dissemination of information, exchange of ideas, maintaining inter-connectivity, consensus building through wide participation, and influencing policy and media. The network is now established and is being used by the participants to exchange information. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Opinion leaders\' network survey results in brief ::: Goals of network • Dissemination of information ---------------------------- ----------------------------------------------------------------------------------------------------------------------------- Access • 41 (79%) no access issues -- reliable connectivity from work • 3 needed some assistance with email access (internet connection at work; compensation for access; help to post responses) Obstacles to participation • 47 (92%) identified lack of time due to professional responsibilities • 98% of those with connectivity willing to dedicate 1 hour a week to the network • 3 people identified lack of connectivity as a barrier ::: Conclusions =========== The meeting concluded with a set of recommendations for the EMRO and Member States (table [5](#T5){ref-type="table"}) \[[@B30]\]. The recommendations were developed through consensus among the participants. The process of consensus development involved the following steps: (i) the recommendations were drafted based on the group presentations, (ii) any recommendations which the participants did not support were deleted (iii) recommendations that were missing but deemed to be important were added (iv) the final list was scrutinized to sharpen language and consolidate points where possible. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Recommendations ::: --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- [Recommendations for the Eastern Mediterranean Regional Office]{.underline} The workshop recommends that the Regional Director EMRO may be requested to address the governments at the highest level for actively considering the proposals of this workshop and for giving priority attention to genomics for health and health biotechnology. The political leadership may be provided effective advocacy material, with special reference to its link with poverty alleviation, public health objectives, and need for transfer (and internalization) of technology. EMRO and Organization of the Islamic Conference Standing Committee for Science and Technology (COMSTECH), and possibly other groups should provide coordination and networking among national biotechnology bodies (see below) and coordinators to exchange information, expertise, training, and Regional cooperation in production and utilization of health biotechnology. EMRO, in collaboration with member states and their national biotechnology bodies, should coordinate a national survey/inventory/situation analysis/needs assessment of health biotechnology innovation systems, including scientific and management capacity, government policies, legislation and regulations, intellectual property policies, private sector activity, and strengths/weaknesses, opportunities and threats. EMRO, in collaboration with COMSTECH and member states, should develop a proposal/feasibility study for a Regional Genomics and Health Research Fund emphasizing both peer-reviewed research and capacity strengthening. [Recommendations for Member States]{.underline} Each member state should create an effective National Commission on Genomics, Biotechnology and Health, if this function has not otherwise been established, including a coordinator who will serve as the focal point for this activity. The membership should be multisectoral and include youth, women, and civil society. The focus should include ethical issues. Based on evidence from the national survey described above, governments of member states should develop and adopt, at the highest level, a national biotechnology strategy. The National Commission on Biotechnology should develop programs of public awareness and engagement. Important \"publics\" here include media and religious leaders as well as the public at large. The discussion should include ethical issues. The National Commission on Biotechnology should encourage academic institutions including schools and universities, to include health biotechnology topics within their curricula and create specialized programs and degrees where appropriate. There should be particular emphasis on ICT and bioinformatics. The National Commission on Biotechnology, in collaboration with the relevant ministries, should develop a plan to integrate genetic and genomics products (including diagnostics, vaccines, therapies, and other genomic priorities), within the health system and public health programs. The emphasis should be on accessibility and equity to improve the health of the poor. [Recommendations for Individuals]{.underline} There is a need for strong personal commitment to strengthen the initiative on genomics and biotechnology to improve health and well-being of people in the EMRO Region. Workshop participants, as well as other concerned individuals, should are therefore encouraged to actively engage in the implementation of these recommendations. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: The participants felt the need for EMRO to: a\) Request regional governments and policy makers at highest level to give priority to genomics for health and health biotechnology b\) Develop linkages with Organization of Islamic Conference Standing Committee for Science and Technology (COMSTECH) and other international partners to build Regional networking and cooperation for developing and utilizing health biotechnology. c\) In collaboration with Member States undertake a national survey/situation analysis/needs assessment of health biotechnology innovation systems including resource capacities, government policies (legislation, regulations intellectual property policies and private sector activity. d\) In collaboration with COMSTECH initiate a research grant for applied (health) genomics and biotechnology The participants agreed that each member state would benefit from creating effective National Commissions on Biotechnology (NCB) to develop national biotechnology strategies. NCB should; a) develop national priorities, programmes and guidelines aimed at raising public education and awareness and b) collaborate with civil sectors to develop plans to integrate genetic and genomic products (including diagnostics, vaccines, therapies and other genomic priorities within the health systems and public health programmes and c) build capacities for utilization and access of health biotechnology to the needy and e) ensure ethical safeguards against unwanted harm and exploitation and improve equity to improve health of the poor. The participants also felt that there was a need for strong personal commitment at individual level by experts and key actors to engage actively in the implementation of the recommendations to strengthen the initiative on genomics and biotechnology to improve the health and well-being of people in EMR. One concrete outcome of the workshop that may contribute to strengthening capacity in the region is the joint agreement between WHO/EMRO and the University of Toronto Joint Centre for Bioethics to provide scholarships to the Master of Health Sciences Program at the JCB. Furthermore, there seems to be growing need to establish a Regional Health Biotechnology Network for EMR, and WHO-EMRO is now planning to hold a Regional meeting to discuss this in Iran in July 2004. WHO-EMRO has initiated follow-up of these recommendations. Ministers of Health from the region, at the 50^th^Regional Committee Meeting held in October 2003, urged Member States to establish national bodies for genomics and biotechnology to formulate strategic vision for creating public awareness and for developing biotechnology for equitable health care in the region. Recently, a paper on harnessing genomics and biotechnology for public health was presented at the EM 28th Regional Consultative Committee Meeting (RCC) held in Cairo in April of this year. The recommendations in the paper were derived from those developed at the workshop. The paper will be presented at this year\'s Regional Committee Meeting to be held in October 2004. The methods and recommendations outlined in this paper demonstrate progress in bringing genomics and biotechnology to the forefront of science policy in developing countries. Our procedure has now led to the formation of three regional networks -- the two previous ones being the African Genome Policy Forum, which encompasses participants from 10 African nations, the Indian Genome Policy Forum. We have also now held a course for Latin America and the Caribbean in association with PAHO and the UN University in Venezuela May 23--26 2004, and a similar network is being created of participants of that course. The next one will be held in the Southeast Asian region, most likely based in Hong Kong. These regional genome policy networks will provide models to establish a Global Genome Policy Forum. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= TA drafted the manuscript; PAS and ASD conceived of the course, and all authors participated in its design and coordination. All authors revised the manuscript for critical content and approved the final draft. Acknowledgements ================ We would like to thank the course participants and members of the EMRO Genome Policy Forum for their insightful comments and suggestions. Funding was obtained from WHO-EMRO and the Canadian Program on Genomics and Global Health (CPGGH). CPGGH funds for this course derived primarily from Genome Canada and the International Development Research Centre (Canada). A full list of CPGGH funders is available at <http://www.geneticsethics.net>. ASD receives support from the McLaughlin Centre for Molecular Medicine and PAS is supported by a Distinguished Investigator award from the Canadian Institutes of Health Research.	PubMed Central	2024-06-05T03:55:52.627325	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548293/", "journal": "Health Res Policy Syst. 2005 Jan 21; 3:1", "authors": [ { "first": "Tara", "last": "Acharya" }, { "first": "Mohammed Abdur", "last": "Rab" }, { "first": "Peter A", "last": "Singer" }, { "first": "Abdallah S", "last": "Daar" } ] }
PMC548294	Background ========== Medawar et al. \[[@B1]\] showed almost half a century ago that rodents injected at birth with splenocytes from genetically different donors could accept transplants from that donor as an adult. These milestone experiments guided the notion that the introduction of antigens during neonatal life leads to tolerance and that the immune system functions by making a distinction between self and nonself. For some years, Matzinger et al. have persevered on the hypothesis that tolerance is not an intrinsic property of the newborn immune system \[[@B2],[@B3]\]. For example, many studies have shown that neonatal exposure to antigen may prime T cells and induce both Th1 and Th2 cells \[[@B4]-[@B7]\]. Moreover, Adkins et al. have demonstrated that although neonates develop compartmentally distinct primary responses to antigen immunization (mixed Th1/Th2 in lymph nodes and Th2 in spleen), after rechallenge the elicited secondary response is always of the Th2 type \[[@B7],[@B8]\]. They have also proved that even in the lymph nodes, the Th2 function persists for a prolonged period after a single immunization, and that animals initially immunized as neonates are impaired in their capacity to develop the expected Th1 memory effector function observed in adults \[[@B9]\]. The biased immunogenic neonatal immunity may be attributable to factors associated with antigen presentation such as type of antigen-presenting cell, accompanying adjuvant and the nature, concentration and in vivo availability of the antigen \[[@B5],[@B10]-[@B13]\]. Resting T cells need two signals to be activated; signal 1 from TCR binding to MHC/peptide and signal 2 (co-stimulation) from a professional phagocyte, such as a dendritic cell or a macrophage. Tolerance is associated to a lack of co-stimulation that usually occurs when antigen is encounter by a non-professional phagocyte, or by professional phagocytes in a non-APC tissue (lymphoid tissue, skin, etc)\[[@B14]\]. In this study, we have evaluated the effect of adoptive transfer of DCs from adult and neonatal mice infected with L. (L.) mexicana, and from healthy adult and neonatal mice. As in the L. majormouse model, we have shown that infection with L. (L.) mexicanastrain MHOM/BZ/82/BEL21, generates a Th1 response associated to protective immunity in C57BL/6 mice, and a Th2 response related to non-healing disease in BALB/6 mice \[[@B15]\]. Leishmaniasis is an excellent model to study the extremes of host/parasite relationships, particularly the diversity of the immune response associated to the genetic background of the host. In addition, mice can reproduce the distinct clinical forms observed in humans \[[@B16],[@B17]\]. These models have been particularly important to show that skin-derived DCs including Langerhans cells play an important role in cutaneous leishmaniasis, where they can transport Leishmaniaantigens to the lymph nodes and induce specific immune responses \[[@B18]-[@B24]\]. Moll et al. have also shown that Langerhans cells may act as reservoirs sustaining parasite-specific stimulation of T memory cells, thus protecting animals from reinfection \[[@B25]\]. Results and Discussion ====================== Establishment of a L. (L.) mexicanainfection model in neonatal BALB/c mice ---------------------------------------------------------------------------- The progress of L. (L.) mexicanainfection in neonatal BALB/c mice, after the inoculation with 5 × 10^4^, 1 × 10^5^, 2 × 10^5^or 5 × 10^5^promastigotes was determined by measuring the footpad thickness during 6 weeks. All 4 experimental groups developed lesions. Mice that received 1 × 10^5^, 2 × 10^5^and 5 × 10^5^promastigotes respectively, showed a significant increase (p ≤ 0.05) on footpad thickness starting from the second week, reaching a maximal value on the sixth week of evaluation (Fig. [1A](#F1){ref-type="fig"}). This increase in footpad thickness was much greater (p ≤ 0.05) in the group inoculated with 5 × 10^5^promastigotes, with lesions appearing from the first week (Fig. [1A](#F1){ref-type="fig"}). Moreover, this experimental group presented a similar evolution to that observed in L. (L.) mexicana-infected adult BALB/c mice inoculated with 1 × 10^6^promastigotes (Fig. [1B](#F1){ref-type="fig"}). The statistical analysis using a Wilcoxon matched-pairs signed-ranks test of the percentage increase from the starting footpad thickness in both neonatal and adult BALB/c mice infected with 5 × 10^5^and 1 × 10^6^promastigotes, respectively, showed a significant (p ≤ 0.05) two-tailed value and a very significant Spearman correlation (r = 1.000, p = 0.0014). The starting footpad thickness in neonatal and adult BALB/c mice was 1.67 mm and 1.85 mm, respectively. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Progression of L. (L.) mexicanainfection in neonatal BALB/c mice. A. Footpad thickness of adult mice infected with 1 × 10^6^promastigotes (■), non-infected mice (○), neonatal mice infected with 5 × 10^4^promastigotes (△), neonatal mice infected with 1 × 10^5^promastigotes (▲), neonatal mice infected with 2 × 10^5^promastigotes (□) and neonatal mice infected with 5 × 10^5^promastigotes (●). B. Percentage increase from the starting footpad thickness in both neonatal (□) and (■) adult BALB/c mice infected with 5 × 10^5^and 1 × 10^6^promastigotes, respectively. ::: ![](1475-9292-4-2-1) ::: We used 5 × 10^5^promastigotes as the optimal concentration for L. (L.) mexicanainfection in all the subsequent experiments including the infection control group. This neonatal murine model of L. (L.) mexicanainfection used half the numbers of promastigotes previously described to infect adult BALB/c mice \[[@B16]\]. A significant Spearman correlation attested that our neonatal model was comparable to the adult model of infection. Although infected neonatal mice have a statistically similar clinical outcome that infected adult mice, we ignore whether these mice have similar level of infection and therefore similar concentrations of antigens carried over by the transferred DCs, however, looking at the present results one can speculate that DCs from mice infected during neonatal life induced tolerance probably due to a high parasite burden, and not a lack of adjuvancity since DCs from healthy neonatal mice were able to partially protect against Leishmaniainfection. Other studies have shown a similar pattern of Th2-biased immune response in other models of neonatal infection \[[@B7],[@B11]\]. We also observed that even after the inoculation of considerable numbers of parasites, neonatal mice differed significantly from adult mice in their percentage increment from the starting footpad thickness, suggesting a functional impairment of the primary immune response. This may be explained, first by the fact that in BALB/c mice carry a point mutation in the Nramp1 (natural-resistance-associated macrophage protein) gene that allows the mRNA degradation of macrophage activation genes, increasing susceptibility to Leishmaniainfection \[[@B26]\]. Susceptibility associated with the dominant expression of the costimulatory molecule CD86 (B7-2) and the subsequent generation of the Th2-mediated response \[[@B27]-[@B31]\]. Second, the proof that murine naïve neonatal T cells, unlike adult T cells, express a Th2 phenotype and are highly deficient in Th1 functions \[[@B32],[@B33]\]. Morphological and immunophenotypic characterization of murine splenic dendritic cells ------------------------------------------------------------------------------------- DCs obtained by our purification method showed characteristic dendritic cell morphology, and a 97% purity as determined by CD11c immunostaining and flow cytometry. A minor fraction of about 3.5 % expressed CD3 and NK1.1 (Fig. [2](#F2){ref-type="fig"}). The expression of CD11c, MHC-II and CD86 molecules was detected by immunocytochemistry, thus demonstrating that these cells showed characteristics of functionally mature DCs. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Frequency distributions of purified dendritic cells labelled CD11c-FITC showing 97.17% purity (right), and FITC-isotype control (IgG1) (left). The information shown is from a single cell isolation procedure, representative of various separate experiments. ::: ![](1475-9292-4-2-2) ::: Splenic DCs were isolated for our adoptive transfer experiments since they are mobile antigen-presenting cells that migrate to peripheral lymph organs where they stimulate naive T cells, thus initiating primary T cell responses \[[@B34]-[@B36]\]. Further, splenic DCs have been isolated by standardized procedures based on the high expression of CD11c and the lack of CD205 \[[@B37]\]. Progression of the infection in neonatal recipient BALB/c mice after adoptive transfer of dendritic cells from the distinct experimental groups ----------------------------------------------------------------------------------------------------------------------------------------------- The adoptive transfer of 1 × 10^3^, 1 × 10^5^or 1 × 10^6^DCs from adult BALB/c mice infected during adulthood with L. (L) mexicanaon neonatal recipient mice modified the course of infection, showing a delayed lesion growth from the second week onward (Fig. [3](#F3){ref-type="fig"}) as compared with the infection control group. This reduction in footpad thickness was dependent of DC numbers, since at the highest concentration of 1 × 10^6^, lesions were smaller than those observed with 1 × 10^3^and 1 × 10^5^DCs from the fifth week onward (Fig. [3](#F3){ref-type="fig"}). At the seventh week of infection, lesion size decreased by 40% after the adoptive transfer of 1 × 10^6^DCs, whereas in animals inoculated with 1 × 10^5^and 1 × 10^3^DCs the decrease was of 33% and 22%, respectively. In contrast, the adoptive transfer of 1 × 10^5^or 1 × 10^6^DCs from adult BALB/c mice infected during neonatal life with L. (L) mexicanafail to modify the course of infection of neonatal recipient BALB/c mice as compared with infection control animals (Fig. [4](#F4){ref-type="fig"}). However, those mice receiving 1 × 10^6^DCs showed a significant reduction in lesion growth (p ≤ 0.05) on weeks 2, 3 and 4. This effect disappeared from the fifth week onwards (Fig. [4](#F4){ref-type="fig"}). Moreover, the adoptive transfer of 1 × 10^5^or 1 × 10^6^DCs from healthy adult BALB/c mice modified the course of infection in neonatal recipient mice, showing a delayed and significant decrease (p ≤ 0.05) in lesion growth from the second week of infection (Fig. [5](#F5){ref-type="fig"}). This reduction in footpad thickness was dependent on DC numbers, since at 1 × 10^6^lesions were significantly (p ≤ 0.05) smaller than those observed in mice transferred with 1 × 10^5^DCs, which also initiated their lesions on the third week (Fig. [5](#F5){ref-type="fig"}). At the seventh week of infection, lesion size decreased by 30% after the adoptive transfer of 1 × 10^6^DCs and by 10% in mice receiving 1 × 10^5^DCs. similarly, the adoptive transfer of 1 × 10^3^or 1 × 10^5^DCs from healthy neonatal BALB/c mice modified the course of infection of neonatal recipient mice, showing a delayed and significant decrease (p ≤ 0.05) in lesion growth from the second week of infection. This reduction in footpad thickness was very similar in both tested concentrations (Fig. [6](#F6){ref-type="fig"}). At the seventh week of infection, lesion size decreased by 35% in both groups. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Progression of infection in neonatal recipient BALB/c mice after adoptive transfer of DCs from adult BALB/c mice infected during adulthood with L. (L) mexicana. Footpad thickness of neonatal mice infected with 5 × 10^5^promastigotes (■); neonatal mice transferred with 1 × 10^6^(○;), 1 × 10^5^(△) and 1 × 10^3^(□) DCs and subsequently infected with 5 × 10^5^promastigotes. ::: ![](1475-9292-4-2-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Progression of the infection in neonatal recipient BALB/c mice after adoptive transfer of DCs from adult BALB/c mice infected during neonatal life with L. (L) mexicana. Footpad thickness of neonatal mice infected with 5 × 10^5^promastigotes (■); neonatal mice transferred with 1 × 10^6^(○) and 1 × 10^5^(△) DCs and subsequently infected with 5 × 10^5^promastigotes. ::: ![](1475-9292-4-2-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Progression of the infection in neonatal recipient BALB/c mice after adoptive transfer of DCs from healthy adult BALB/c mice. Footpad thickness of neonatal mice infected with 5 × 10^5^promastigotes (■); neonatal mice transferred with 1 × 10^5^(○) and 1 × 10^6^(△) DCs and subsequently infected with 5 × 10^5^promastigotes. ::: ![](1475-9292-4-2-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Progression of the infection in neonatal recipient BALB/c mice after adoptive transfer of DCs from healthy neonatal BALB/c mice. Footpad thickness of neonatal mice infected with 5 × 10^5^promastigotes (■); neonatal mice transferred with 1 × 10^5^(△) and 1 × 10^3^(◇) DCs and subsequently infected with 5 × 10^5^promastigotes. ::: ![](1475-9292-4-2-6) ::: Disease progression was substantially decreased after transferring cells from adult BALB/c mice infected during adulthood with L. (L) mexicana, healthy adult BALB/c mice and healthy neonatal BALB/c mice. The reduction in these 3 groups was statistically significant (p ≤ 0.05) as compared with the infection control group. This reduction in footpad thickness was absent or considerably diminished in mice receiving DCs from adult BALB/c mice infected during neonatal life with L. (L) mexicana(Fig. [7](#F7){ref-type="fig"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Progression of the infection in neonatal recipient BALB/c mice after the adoptive transfer of DCs from the different experimental groups. Footpad thickness of neonatal mice infected with 5 × 10^5^promastigotes (■); neonatal mice transferred with 1 × 10^6^DCs from adult BALB/c mice infected during adulthood (◇), adult BALB/c mice infected during neonatal life (○), healthy adult BALB/c mice (●) and 1 × 10^5^CDs from healthy neonatal BALB/c mice (△) and subsequently infected with 5 × 10^5^promastigotes. ::: ![](1475-9292-4-2-7) ::: Our results showed that the preceding intraperitoneal adoptive transfer of DCs diminished the progression of L. (L.) mexicanainfection in neonatal BALB/c recipient mice. These results contrast with those of Moll and Berberich \[[@B38]\] showing that only intravenous administration of antigen-pulsed Langerhans cells, but not intradermal or intraperitoneal inoculation, induced resistance against Leishmaniainfection. In this study, the observed protection depends on the quantity and provenance of the transferred DCs, since the effect was more evident with high cellular numbers of DCs from adult BALB/c mice infected during adulthood and healthy neonatal mice, where lesions were about 40% smaller than in the infection control group. DCs from these two groups have the intrinsic capacity to induce protective or resistant immune responses very early in life. That neonatal DCs appear to be more protective, on a per cell basis, than adults DCs is a very striking result since only Dadaglio et al.\[[@B39]\] have shown that neonatal DCs are as effective as adult DCs in expressing MHC and costimulatory molecules; taking-up, processing and presenting antigens to T cells inducing CTL responses in vivo. Others have shown that neonatal DCs are not fully functional \[[@B40],[@B41]\]. Also, animals receiving DCs from healthy adult mice showed a slightly but significantly reduced protection from that observed with DCs from adult mice infected during adulthood and healthy neonatal mice. Various studies have shown that epidermal DCs in aged skin are reduced significantly compared with young skin in mice and humans \[[@B42]-[@B48]\]. This cellular reduction may be the consequence of a decreased production in the bone marrow of DC progenitors or alternatively, these stem cells may be less responsive to cytokine and chemokine signals required for their homing to the skin \[[@B49]-[@B51]\]. Our results favor the latter hypothesis, since the same numbers of transferred DCs from healthy neonatal or adult mice induced a somewhat different disease outcome. More notable was the observed absence of a protective effect in mice receiving DCs from adult BALB/c mice infected with L. (L) mexicanaduring neonatal life. This result confirmed recent studies by Adkins et al. showing that animals initially immunized as neonates are unable to develop the expected Th1 memory effector function observed in adults \[[@B9]\]. These investigators proposed that in neonates, the spleen is the primary site of tolerance induction to self-antigens whereas the lymph nodes are the sites of immune responsiveness to foreign antigens. The initial and transitory protection observed at the greatest concentration of DCs from adult mice infected during neonatal period, suggests impairment in their accessory functions specifically in those associated with signal 2 and signal 3. Signal 2 comprises co-stimulatory factors essential for the clonal expansion of T cells and signal 3 involves in situ properties of DCs such as tissue interaction and migration where cytokines, chemokines and extracellular matrix components are crucial \[[@B36]\]. Conclusions =========== Our results show that tolerizing DCs from animals initially immunized as neonates play a key role in the attenuation of Th1 responses. The present results may have a considerable epidemiological impact on leishmaniasis, where infection at early stages of life may impose a tolerogenic state that favors the development of visceral or diffuse cutaneous leishmaniasis, both characterized by Th2-type responses. In this study, we have shown that intraperitoneal adoptive transfer of splenic DCs is able to surpass the genetic bias of the mice, allowing the development of an immune response that modifies the progression of L. (L.) mexicanainfection. Methods ======= Animals ------- Adult (6 weeks) and neonatal (about 24 hour newborn) female BALB/c mice (Taconic, Germantown, NY, U.S.A.) were raised in the Animal House of the Instituto de Biomedicina, under appropriate conditions of temperature, water and feeding. Specific Antibodies ------------------- The following rat monoclonal antibodies were used to isolate and characterize dendritic cells: CD19 (B cells, clone IBL-2), MOMA-2 (Macrophages/Monocytes), CD45R (B and NK cells; clone RA3-6B2), CD3 (T cells; clone KT3), CD11c (dendritic cells and other leukocytes, clone N418), CD19 (clone 6D5) conjugated to phycoeritrine (PE), NK1.1 (clone PK136) conjugated to PE, Macrophages-Monocytes (MOMA-2) conjugated to fluorescein isothiocyanate (FITC), CD3 (clone KT3) conjugated to FITC. All were purchased from Serotec Ltd. (Oxford, United Kingdom) except CD205 (dendritic cells, clone NLDC-145, DEC205) donated by Georg Kraal, Vrije Universiteit, Amsterdam, The Netherlands; I-A^d^(MHC-II, clone AMS-32.1) and CD86 (B7.2, clone GL1) purchased from BD Pharmigen (San Diego, USA). Parasite culture and isolation of L. (L.) mexicanapromastigotes ----------------------------------------------------------------- Amastigotes of Leishmania (Leishmania) mexicana(MHOM/BZ/82/BEL21) were extracted from footpad nodules of hamsters infected a month earlier with 1 × 10^6^amastigotes. The nodules were aseptically dissected out and washed in phosphate-buffered saline (PBS, pH 7.4) with 100 U/ml penicillin and 100 μg/ml streptomycin, and finely cut and ground in a Petri dish containing cold PBS. The suspension was filtered through a sterile sieve to remove large debris. These parasites were cultured on blood agar base (Sigma-Aldrich, St. Louis, U.S.A.) at room temperature for 7 days (the stationary growth phase) to obtain infective promastigotes. For an enriched population of parasites, free of erythrocytes and cellular debris, 100 μl of that sample were cultured in 2 ml Schneider\'s insect cell culture medium (Sigma-Aldrich, St. Louis, U.S.A.) for one week at room temperature. Promastigotes were isolated after 3 washes with sterile PBS and centrifugation at 1000 g at 4°C for 15 min. Pellets were resuspended in 1 ml of sterile PBS. Viable parasites were counted by trypan blue exclusion. Parasite concentration was adjusted to 5 × 10^4^, 1 × 10^5^, 2 × 10^5^and 5 × 10^5^per μl to be used in the different experimental groups. Experimental infection with promastigotes of L. (L.) mexicana --------------------------------------------------------------- A similar pattern of L. (L.) mexicanainfection to that established in adult mice \[[@B52]\] was determined in neonatal BALB/c mice. Neonatal BALB/c mice (n = 12) were inoculated subcutaneouslly into the left hind footpad with 5 × 10^4^, 1 × 10^5^, 2 × 10^5^, or 5 × 10^5^promastigotes suspended in 10 μl sterile PBS, applied with a tuberculin syringe (29-gauge needle) connected to a stepper repetitive pipette (Tridak, Danbury, U.S.A.). For comparison, adult BALB/c mice were infected the standardized optimal parasite load of 1 × 10^6^promastigotes of L. (L.) mexicana\[[@B52]\]. The course of infection was evaluated weekly for 6 weeks, measuring the experimental left footpad using a dial gauge caliper (Mituyoto N° 7300, U.S.A.). Isolation and purification of dendritic cells --------------------------------------------- DCs from adult and neonatal BALB/c mice were isolated from the spleen. Under sterile conditions, spleens were minced on a metallic mesh with RPMI-1640 (Life Technologies, Rockville, U.S.A.) supplemented with 10% of decomplemented fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol and 100 U/ml penicillin (complete RPMI-10). The cell suspension was filtered on a nylon sieve and transferred to 15 ml centrifuge tubes (Corning Life Sciences, Acton, U.S.A.) and spun at 250 g at 4°C for 10 min. Viable cells were counted by trypan blue exclusion. Cell concentration was adjusted to 1 × 10^7^cells/ml in complete RPMI-10 and 8 ml plated in tissue culture flasks. The flaks were incubated for 2 hr at 37°C in a 5% CO~2~incubator (NuAire, Inc., Plymouth, U.S.A.), allowing DCs to adhere. Non adherent cells were carefully removed and placed in sterile 50 ml centrifuge tubes and spun at 250 g, 4°C for 10 min. Adherent cells were covered with 10 ml complete RPMI-10 and incubated as before for 16--18 hours, allowing DCs to detach. After gently washing the surface of the flasks with a plugged Pasteur pipette and complete RPMI-10, pools of the eluted cells were placed in sterile 15 ml centrifuge tubes and spun at 250 g, 4°C for 10 min. For each tube, the cell pellet was resuspended in 6 ml complete RPMI-10. This volume was carefully layered over a 3 ml NycoPrep™ density gradient (Nycomed Pharma AS, Torshov, Norway) and centrifuged at 600 g, 20°C for 20 min. Mononuclear cells were removed from the interface ring using a Pasteur pipette, transferred to a sterile 15 ml centrifuge tube and spun down in 10 ml complete RPMI-10 at 400 g, 20°C for 15 min three times. The final pellet was resuspended in 1 ml of cold (4°C) Hanks balanced salt solution supplemented with 10% decomplemented FBS and 2 mM HEPES. Cells were quantified and viability assessed by trypan blue exclusion. The final purification stage consisted of an immunomagnetic negative selection of DCs. The cell suspension obtained above was incubated under continuous agitation at 4°C for 1 hour, with primary rat anti-mouse monoclonal antibodies recognizing B and T lymphocytes, NK cells and monocytes/macrophages (1.5 μg/ml antibody per 1 × 10^6^cells). After incubation, cells were washed three times in Hanks centrifuging at 250 g, 4°C for 10 min. The pellet was resuspended in 1 ml cold Hanks in a sterile 15 ml centrifuge tube and incubated under continuous agitation at 4°C for 1 hour with a secondary sheep anti-rat IgG polyclonal antibody coupled to magnetic microspheres (Dynabeads^®^M-450, Dynal Biotech Inc., Lake Success, U.S.A.) at a 7:1 sphere/target ratio. Non-dendritic magnetic-coated cells were removed by positive selection in three sequential depletions using a magnetic gadget (Dynal MPC^®^Dynal Biotech Inc., Lake Success, U.S.A.) at 4°C for 6 min. Characterization of dendritic cells ----------------------------------- DC purity was determined by flow cytometry and immunocytochemistry. For flow cytometry, 1 × 10^5^cells were suspended in PBS (1% FBS) and incubated with 10 μl primary monoclonal antibodies directly coupled to PE or FITC recognizing T and B lymphocytes, NK cells and monocytes/macrophages. DCs were characterized by an indirect method using primary monoclonal antibodies to CD11c and a secondary antibody, hamster anti-rat IgG1conjugated to FITC (clone MARG1-2, Serotec Ltd., Oxford, United Kingdom). The incubations were carried out in the dark at 4°C for 45 min, followed by 3 washes and centrifugation at 250 g, 4°C for 10 min. The cell pellet was resuspended in 500 μl PBS and the percentage of labeled cells determined in a flow cytometer (FACScan, Becton Dickinson, Franklin Lakes, U.S.A.). The control consisted of an antibody of irrelevant specificity conjugated to FITC. For immunocytochemistry, 1 × 10^5^cells were suspended in PBS (1% FBS) and spun down at 50 g in a Cytospin (Shandon Inc., Pittsburg, U.S.A.). Sample slides were hydrated in PBS, fixed in fresh acetone for 5 min. and sequentially incubated for 90 min with primary rat monoclonal antibodies to CD11c and CD205, biotinylated goat anti-rat IgG (50 μg/ml) (Vector Laboratories, Burlingame, U.S.A.) for 45 min., and Vectastain^®^Elite ABC kit (Vector Laboratories, Burlingame, U.S.A.) at 1:100, 30 min. Five-minute washes with PBS were done between incubations. The reactions were developed for 3 minutes in Vector^®^NovaRed™ substrate. The slides were then washed and counterstained with methyl green. Omissions of the primary antibody and incubation with an antibody of irrelevant specificity at the same protein concentration were used as controls. Adoptive transfer of dendritic cells ------------------------------------ DCs were isolated from the spleens of the following donor groups: a) Adult BALB/c mice infected during adulthood with L. (L.). mexicana(n = 4); b) Adult BALB/c mice infected during neonatal life with L. (L.). mexicana(n = 4); c) Healthy neonatal BALB/c mice (n = 4); d) Healthy adult BALB/c mice (n = 4). The infection control group consisted of neonatal BALB/c mice infected with 5 × 10^5^promastigotes of L. (L) mexicana. DCs from the 4 experimental groups were adjusted to 1 × 10^3^, 1 × 10^5^, or 1 × 10^6^cells/ml in sterile PBS for intraperitoneal transfer to neonatal recipient BALB/c mice. Cells, at the mentioned concentrations, were injected in 20 μl volumes using a tuberculin syringe (29-gauge needle) connected to a stepper repetitive pipette (Tridak, Danbury, U.S.A.). After sixteen hours of adoptive transfer, neonatal recipient BALB/c mice were infected with 5 × 10^5^promastigotes of L. (L) mexicana. Isolation of DCs and adoptive transfer experiments were done in duplicates. Statistical analysis -------------------- The results were expressed as mean ± standard error of the mean (SEM). Each experimental group consisted of 4--5 individuals. Comparisons between groups were made with Student t test and Welch t test for unpaired samples. Any value of p ≤ 0.05 was considered significant. All tests were performed using GraphPad InStat 3.02 (GraphPad Software, San Diego California USA, <http://www.graphpad.com>). List of abbreviations ===================== APCs: antigen-presenting cells DCs: dendritic cells TCR: T-cell receptor Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= LVP carried out most of the experimental work and drafted the manuscript. JC developed the experimental design, carried out part of the experimental work and drafted the manuscript. NLD participated in the in experimental design and evaluated the progression of infection in the mice. FJT conceived the study, participated in the experimental design and coordinated the work. All authors read and approved the final manuscript. Acknowledgements ================ We are grateful to Becky Adkins for insightful discussions and critical appraisal of the manuscript; Marian Ulrich for reading and commenting on the manuscript; Martin A. Sanchez for excellent scientific consultation on the flow cytometry information. We would like to acknowledge the personnel from the Animal House of the Instituto de Biomedicina and the Flow Cytometry Facility of Banco Municipal de Sangre in Caracas. This work was supported by Fondo Nacional de Ciencia, Tecnología e Innovación (FONACIT) Proyecto S1- 98000041 and Millennium Scientific Initiative Grant 4572VE, UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, European Commission INCO Programme, and CDCH- Universidad Central de Venezuela.	PubMed Central	2024-06-05T03:55:52.631328	2005-1-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548294/", "journal": "Kinetoplastid Biol Dis. 2005 Jan 25; 4:2", "authors": [ { "first": "Loida V", "last": "Ponce" }, { "first": "José", "last": "Corado" }, { "first": "Nilka L", "last": "Díaz" }, { "first": "Felix J", "last": "Tapia" } ] }
PMC548295	Background ========== Peritoneal adhesions resulting from surgical injury are often associated with pelvic pain, bowel obstruction and infertility \[[@B1]\]. Epidemiological studies conclude that 30 to 35% of all hospital readmissions are associated with adhesion associated complications, of which 4.5 to 5.1% are directly related to adhesions \[[@B2]\]. Mechanisms of adhesion formations are not completely known. It is also not clear why adhesion form in some patients and not in others. Therefore, deciphering genetic components that signal adhesion formation may help diagnose adhesion-prone patients prior to surgery. Needless to mention that such information will facilitate finding ways to prevent post-surgical adhesion formation. Parietal and visceral peritoneum that surfaces the intraperitoneal organs is covered by a layer of squamous epithelial cells, the mesothelium. The submesothelial layer consists of fibroblasts, macrophages and blood vessels. Surgical abrasion to the peritoneum releases mesothelial cells, macrophages, fibroblasts, and blood containing cytokines and several cell types at the site of injury. Coagulation of blood creates a fibrinous mass between injured surfaces. In some patients fibrinolysis of clot followed by proliferation of mesothelial cells covers the wound. In others, failure of fibrinolysis followed by proliferation and migration of fibroblasts into the proteinous mass generates fibrous tissues of adhesion. Consequently, the process of adhesion formation include inflammatory response, fibrin deposition, cell-proliferation, -differentiation, -migration, -death, angiogenesis, extra cellular matrix (ECM) turnover regulated by cytokines, hypoxia, genetic and epigenetic factors \[[@B3]\]. Recent studies illustrate roles of peritoneal fibroblasts in adhesion development \[[@B4]-[@B10]\]. It is also proposed that fibroblasts from the chronic wounds migrate into the fibrin deposit; secrete ECM proteins causing wound contraction and scar formation \[[@B11]\]. The migration of fibroblasts may be coordinated by TGF-β1 mediated interactions of integrin receptors \[[@B10]\] with the RGD sequence of the fibrin, fibrinogen and fibronectin at the fibrin clot \[[@B12]\]. Additional cytokines and the hypoxic condition at the site of injury may also influence peritoneal fibroblasts to attain a phenotype supporting formation of adhesion tissue. This change in the phenotype of fibroblasts may be induced by changes in expression pattern of several genes during the process of adhesion development. Therefore, identifying differences in the global gene expression pattern between normal and adhesion fibroblasts may provide additional clues to the mechanisms by which normal fibroblasts attain the adhesive, proliferating and migratory phenotype required for the formation of fibrous tissues of adhesions. In the present study, we compared gene expression patterns between adhesion and normal peritoneal fibroblasts using GF211 gene filters (Research genetics) containing 4325 randomly selected known genes. Furthermore, we confirmed the expression pattern of genes of interest by a semiquantitative RT-PCR method and examined possible contribution/s of TGF-β1 and hypoxia in the transformation of normal peritoneal fibroblasts into an adhesion phenotype. Methods ======= Peritoneal-tissue collection, fibroblast-isolation and culture -------------------------------------------------------------- Tissues were collected at the initiation of surgery and after the entry into the abdominal cavity of female patients (25--50 years) undergoing laparatomy for pelvic pain as described earlier \[[@B4]\]. All patients gave informed written consent for the tissue collection, which was conducted under a protocol approved by the Wayne State University Institutional Review Board. Normal parietal peritoneal tissues were collected from these patients from the anterior abdominal wall, approximately midway between the umbilicus and symphyses pubis, and lateral to the midline incision. Peritoneal tissues from adhesions, that were at least 3 inches away from the site of normal tissue collections, were also collected from the same patient. The peritoneal fibroblasts were isolated and separated from mesothelial cells by a differential centrifugation procedure that is briefly described earlier \[[@B4]\]. The isolation of fibroblasts from mesothelial cells were also verified by the RT-PCR detection of Collagen type I, Matrix metalloproteinase-2 (MMP-2) and Transforming growth factor-β3 (TGF-β3) \[[@B13]-[@B15]\]. The primary cultures were maintained in a humidified incubator (37°C, 5% CO~2~) for 3 days in DMEM (Life Tech.) supplemented with 10% fetal bovine serum (Life Tech.) and antibiotics (Penicillin and Streptomycin 50 U/ml; Life Tech.). The monolayer of cells were passaged in trypsin-EDTA solution (Life Tech.). Cells at 3--5 passages were cultured in serum free medium in 75 cm^2^flasks (Fisher Scientific, Pittsburgh, PA) to 75% confluency prior to studies. Gene expression pattern in the fibroblasts from adhesion and normal peritoneum ------------------------------------------------------------------------------ Total RNA was isolated from monolayer of fibroblasts at 12 h of culture in serum free medium using Trizol reagent (Invitrogen Inc.). Human Gene Filters (GF211; Research Genetics, Inc., Huntsville, AL) containing 4325 known human cDNA spots were used for the identification of differentially expressed genes between adhesion and normal fibroblasts from human peritoneum. Method suggested by the manufacturer was strictly followed. In brief, 10 μg of total RNA from monolayer cultures of fibroblasts were subjected to cDNA synthesis in presence of 10 μl ^33^dCTP (10 mCi/ml; ICN Radiochemicals, Irvin, CA). Radiolabeled cDNA was separated from the free nucleotides using Bio-Spin 6 chromatography column (Bio-Rad Laboratories). Gene filters were labeled as adhesion or normal fibroblasts and washed with 0.5% sodium dodecyl sulfate (SDS) prior to prehybridization. Individual membrane was transferred to separate roller tubes of the hybridization oven (Fisher Scientific, Inc., Pittsburg, PA), each containing MicroHyb hybridization solution (Research Genetics) supplemented with Human Cot-1 DNA (Life Technology) and Poly dA (Research Genetics). The membranes were rotated at 10 RPM and at 42°C for 2 h. Radio-labeled cDNA prepared from adhesion and normal fibroblasts total RNA was denatured by heating in a boiling water bath for 3 min. The denatured probes were then injected into the prehybridization solution containing respective membrane. The membranes were hybridized with respective probe for 18 h at 42°C. The hybridization solution was then replaced with washing solution (2 × SSC containing 1% SDS). The temperature of the oven was raised to 50°C and RPM of rotors was increased to 15. Membranes were washed for 20 minutes when washing solution was replaced with a batch of fresh and prewarmed (50°C) washing solution. Washing was continued for additional 20 min. A third wash was performed with 0.5 × SSC solution containing 1% SDS at 55°C for 15 minutes. Membranes with cDNA spots facing up were covered with Saran wrap and exposed to phosphor screen (Kodak) for overnight. The screen was scanned with a Phosphor Imager (Storm System; Amersham Biosciences Corp., Piscataway, NJ). After acquisition of signal intensities from the normal and adhesion fibroblasts of one patient, filters were stripped according to protocol and subjected to gene filter experiments with the RNA samples from a second patient and images were scanned. Tiff images obtained from normal and adhesion fibroblasts of two patients were analyzed using Pathway 4 software (Research Genetics) for the identification of differentially expressed genes between the normal and adhesion fibroblasts of each patient. Relative abundance of selected genes in the fibroblasts from adhesion and normal peritoneum ------------------------------------------------------------------------------------------- Steady-state levels of mRNA of selected genes that are known to have a role in cellular adhesion, proliferation, migration, apoptosis and demonstrating different expression levels between adhesion and normal fibroblasts in the gene filter experiments were verified further by a previously described semiquantitative RT-PCR method \[[@B16]\]. Total RNA (1 μg) from the monolayer culture of adhesion or normal fibroblasts was subjected to reverse transcription as described earlier. Complementary DNA (100 ng) was subjected to PCR amplification of the cDNA of interests in a 25 μl reaction mixture containing 50 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl~2~, 0.2 mM dNTP, 0.5 U Taq Polymerase (all from Life Technology, BRL) and 1 μM each of sense and antisense primers. Primer sequences were determined using Primer3 software from the Internet <http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi>. The control primers (sense5\'-ggaggttcgaagacgatcag-3\' and antisense5\'-cgctgagccagtcagtgtag-3\') were expected to provide an amplicon of 509 bp from human 18S ribosomal subunit cDNA (gi: 337376). Accession numbers of genes of interests are provided in Table [2](#T2){ref-type="table"} and nucleotide sequences of primers and expected size amplicons are provided in the Table 3. Each PCR cycle consisted of a hot start at 95°C for 1 min, followed by melting at 95°C for 30 sec, annealing at 58°C for 1 min and extension at 72°C for 1 min. At the end an extension reaction at 72°C for 10 min was performed. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Genes differentially expressed in the adhesion fibroblasts and known to have roles in cell-adhesion, -proliferation, -migration, -differentiation and -death. ::: Accession Number Definition Fold Change Functions ------------------------------ ---------------------------------------------------- ----------------- --------------- ----------------------------------------------------------------------------------------------------------------- *Increased in Intensity* gi:17986276 Collagen, type IV alpha 2 2.4 2.7 See Discussion gi:4506760 S100 calcium-binding protein A10 2.3 2.7 See Discussion gi:6679055 Nidogen 2 6.4 7.1 See Discussion gi:14250074 Transmembrane 4 superfamily member 1 3.7 2.6 See Discussion gi:4758081 Chondroitin sulfate proteoglycan 2 3.4 3.2 See Discussion gi:187538 Metallothionein 1E 4.3 4.0 See Discussion gi:4336324 Small membrane protein 1 2.4 2.6 Cell viability \[54\] gi:17738299 Cyclin-dependent kinase inhibitor 2A 2.0 2.0 Cell proliferation \[55\] gi:16359382 Nuclear receptor subfamily 4, group A 1.6 2.1 Antagonizes TNF-α induced apoptosis \[56\] gi:40353726 Synaptopodin 2.9 2.4 Actin cytoskeleton dynamics \[57\] gi:23398519 Vasodilator-stimulated phosphoprotein 1.5 2.1 Enhances actin based cell motility, Cytoskeltal dynamics \[58\] gi:28329 α-Smooth muscle actin 3.0 3.2 Myofibroblast transformation \[44\] gi:14574570 Bcl-2 related gene bfl-1 1.6 1.4 Anti apoptotic; inhibitor of p53 induced apoptosis gi:796812 p53 tumor suppressor 1.5 1.6 Cell cycle arrest and apoptosis \[52\] *Decreased in Intensity* gi:184522 Insulin-like growth factor binding protein 3 3.2 2.3 See Discussion gi:4504618 Insulin-like growth factor binding protein 7 2.3 2.0 Growth suppressing factor \[59\] gi:28610153 Interleukin 8 3.2 2.6 Inhibits fibroblast migration, delays wound healing, reduces wound contraction \[60\] gi:4504982 Lectin, galactoside-binding, soluble 3 \[galectin) 3.0 3.0 Tumor-suppressive and pro apoptotic \[61\] gi:12803916 Gap junction protein, beta 1, \[Connexin 32) 1.8 2.2 Tumor suppressive and Proapoptotic \[62\] gi:14589894 Cadherin 5, type 2, VE-cadherin \[vascular\] 2.3 1.7 Down regulation associates with tumor metastasis, Initiates endothelial-mesenchymal transdifferentiation \[63\] gi: 16198356 Lactotransferrin 2.2 2.1 Inhibits growth of malignant tumors. Elevated by high level of estrogen \[64\] gi:21619838 Lipocalin 2, Oncogene 24p3 3.3 2.5 Proapoptotic \[65\] gi: 23273645 Calponin 1, basic, Smooth muscle cell 1.7 2.5 Inhibits smooth muscle cell contraction and Tumor Suppressive \[66\] gi:40225461 RAP1A, member of RAS oncogene family 1.8 1.6 Inhibits cell proliferation \[67\] gi:4507112 Synuclein-gamma 1.5 1.3 Expression reduced in carcinoma \[68\] \* Adhesion/Normal peritoneal fibroblasts values of gene expression intensity from patient 1 (P1) and 2 (P2). ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### PCR primers, amplicon size and expression ratios of genes between adhesion and normal peritoneal fibroblasts ::: Transcripts Primer Sequence (5\' to 3\') Amplicon size (base pairs) Adhesion/Normal Ratio Gene Filter\* Adhesion/Normal Ratio (RT-PCR)\\ ----------------------------------------------------------------- ------------------------------ ---------------------------- ------------------------------------- ------------------------------------ ------ 18S Ribosomal Subunit Sense ggaggttcgaagacgatcag 509 (No spot) 0.9 Antisense cgctgagccagtcagtgtag Collagen type IV alpha 2 chain(COL4A2) Sense caccatgcccttcctgtact 351 2.6 2.3 Antisense ttgcattcgatgaatggtgt S100 Calcium binding protein A10 (S100A10) Sense cacaccaaaatgccatctca 389 2.5 2.1 Antisense cttctatgggggaagctgtg Nidogen 2 (NID2) Sense gcttacgaggaggtcaaacg 500 6.8 2.9 Antisense ttcacccggaaggtattcag Transmembrane 4 superfamily member 1 (TM4SF1) Sense tcgcggctaatattttgctt 500 3.2 1.9 Antisense gcctccaagcactccattta Chondoitin sulfate proteoglycan 2 (CSPG2) Sense gaaccaaattatggggcaga 400 3.3 3.0 Antisense ctcccaatccttcgtcgata Insulin-like growth factor binding protein 3 precursor (IGFBP3) Sense gggtaggcacgttgtaggaa 603 -2.8 -2.8 Antisese gtgaggctggctaagaatgc Metallothionine (hMT-Ie) Sense cagagggtctctgggtttca 400 4.2 3.3 Antisense gccccatgtcctctcactaa \* Average intensity of Adhesion/Normal peritoneal fibroblast gene expression data from patient 1(P1) and 2 (P2) presented in Table 2. Minus (-) sign indicate fold decrease in intensity in the adhesion fibroblasts. \\ Ratios of Adhesion/Normal mean values from 4 patients. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Expression profiles of genes in the adhesion vs. normal peritoneal fibroblasts and the effects of TGF-β1 or hypoxia on the expression level of genes in the normal peritoneal fibroblasts ::: Transcripts Adhesion/Normal fibroblasts(Gene Filter & RT-PCR) TGF-β1 Effects(RT-PCR) Hypoxia Effects(RT-PCR) ----------------- ------------------------------------------------------- ---------------------------- ----------------------------- COL4A2 ↑ ↑ ↑ S100A10 ↑ ↑ ↑ NID2 ↑ --- ND TM4SF1 ↑ --- ND CSPG2 ↑ ↑ --- IGFBP3 ↓ --- ND hMT-Ie ↑ ↑ ↑ ↑ = Up regulation (p \< 0.05); ↓ = Down Regulation (p \< 0.05); ---= No Change ND= Not determined ::: Initially cDNA of interests were amplified from normal peritoneal fibroblasts at different (25 to 35) PCR cycles. PCR products were subjected to agarose gel electrophoresis. Molecular weight marker (100 bp DNA ladder; Life Technology) were also loaded in adjacent lanes. DNA in the gel were stained with 1:10,000 dilution of SYBR Green I dye (Molecular Probes, Inc., Eugene, OR) and photographed using a DC 120 Kodak digital camera (Eastman Kodak, Rochester, NY) for the verification of size and analysis of band intensity using Image J software <http://rsb.info.nih.gov/ij/>. Band intensities were plotted to determine the linearity of PCR reactions for the amplification of target transcripts. Target cDNA were amplified by PCR from normal and peritoneal fibroblasts at specific PCR cycle within its linear range of amplification. Total RNA samples from normal and adhesion fibroblasts of 4 patients (included RNA from normal and adhesion fibroblasts of two patients used for the gene filter experiments) were used for the RT-PCR experiments. Optical densities obtained from amplicons of 4 patients (1 normal and 1 adhesion fibroblast RNA sample per patient) were used to derive mean ± standard error of mean values representing relative levels of each mRNA species in normal and adhesion fibroblasts. Effects of TGF-β~1~or hypoxia on gene expression pattern -------------------------------------------------------- Effects of TGF-β1 or hypoxic conditions on the steady state levels of specific gene transcripts in the normal peritoneal fibroblasts were also studied to examine the possibility of adhesion causing factors potentiating the gene expression pattern in the normal fibroblasts similar to adhesion fibroblasts. Normal peritoneal fibroblasts were cultured in six well culture plates (FALCON). When confluent, monolayer of cells in culture were exposed to 1 ng/ml TGF-β~1~(Sigma Chemical Company, St. Louis, MO) or hypoxia (2% Oxygen) for 24 h. Control plates were cultured for the same duration in absence of TGF-β1 or hypoxia. Total RNA was isolated from the control, TGF-β1 and hypoxia treated cells and subjected to RT-PCR reactions as described above to determine relative levels of 18S ribosomal subunit and gene specific transcripts in the control and treated cells. RT-PCR experiments were conducted twice with the normal peritoneal fibroblasts isolated from 3 patients to have six control, six TGF-β1 treated and six hypoxia exposed amplicons. This included normal fibroblasts from one new patient and two patients that were used exclusively for RT-PCR experiments for the confirmation of gene array data. Optical densities of amplicons from six control or treated cells per mRNA species were used to derive the mean ± standard error of mean values for comparison. Statistical analysis -------------------- Band intensity value of each RT-PCR experiment (normal, adhesion or treated fibroblasts) was used to derive Mean ± Standard error of Mean using Statview 4.5 software (Abacus Concepts, Berkley, CA). Differences between Means were tested for significance by one-way analysis of variance with the specific post hoc test using the same software to compare differences in the steady state levels of different mRNA species. Results ======= Expression pattern of genes between adhesion and normal peritoneal fibroblasts ------------------------------------------------------------------------------ Hybridization intensities of radio labeled cDNA from normal or adhesion fibroblasts from both the patients were different when analyzed using Pathway software. Comparison of hybridization intensities from individual gene spots between normal and adhesion fibroblast RNA (Figure [1](#F1){ref-type="fig"}) demonstrated that the expression levels of \~4% genes were \>1.5 fold different. BLAST search of the accession number of genes from the list provided by the manufacturer showed that genes with altered expression level between normal and adhesion fibroblasts are reported to be involved in cell adhesion and migration; transformation, transcription, translation and growth factors as well as cytokines and signaling molecules. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Images depicting radioactive signals from GF211 filters hybridized with radiolabeled cDNA.Gene filters were hybridized with ^33^P labeled cDNA from normal peritoneal fibroblasts or fibroblasts from adhesion tissue. Unbound signals were washed and relative radioactive signal intensities were detected using a Phosphoroimager as described in the Methods. A.Tiff images of radioactive signals from individual gene spots of filters hybridized with normal (above) and adhesion fibroblasts, both isolated from Patient 1. B.Scatter plot showing signal intensities from normal peritoneal (Intensity I) and adhesion (Intensity II) fibroblasts. Dotted lines indicate a two fold changes in hybridization intensities from the median (solid line). ::: ![](1477-7827-3-1-1) ::: Gene filter data from two patients showed similar expression pattern of collagen type 1 (alpha 2), Collagen type III (alpha 1), fibronectin 1, Matrix metalloproteinase-1 (MMP-1), Transforming Growth Factor beta-1 (TGF-β1), TGF-β2 and tissue plasminogen activator as reported earlier using multiplex PCR technique (Table-[1](#T1){ref-type="table"}). Signal intensities representing TGF-β3 (gi:22531293), TGF-β III Receptor (gi:26251745), VEGF-A (gi:2565322), VEGF-B (gi:39725673) and VEGF-C (gi:19924300) expression levels were respectively 1.6, 1.5, 1.9, 1.3 and 1.3 fold (average values from two patients) lower in the adhesion compared to normal fibroblasts. No spots for antiapoptotic bcl-2 and proapoptotic bax were present in GF211 filters. Signal intensities representing anti apoptotic molecule bcl-2 related gene bfl-1 (gi: 14574570) and pro-apoptotic molecule p53 (gi:796812) were higher (Table [2](#T2){ref-type="table"}) in adhesion compared to normal fibroblasts. Expression levels of proapoptotic molecule bad (gi: 14670387) and bak1 (gi: 33457353) were not different between normal and adhesion fibroblasts. A list of additional genes that are differentially expressed between normal and adhesion fibroblasts and known to be involved in apoptosis as well as cell adhesion, proliferation and migration are listed in Table [2](#T2){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Ratios of signal intensities from adhesion and normal peritoneal fibroblasts detected from gene filters representing relative expression level of genes in patient 1 (P1) and 2 (P2). ::: Gene Accession Number P1 (A/N) P2 (A/N) Reference\* ----------------------------- ---------------------- -------------- -------------- ----------------- Collagen Type I (alpha 2) gi:48762933 1.4 1.5 \[4,15,53\] Collagen Type III (alpha 1) gi:15149480 2.0 1.7 \[15\] Fibronectin 1 gi:53791222 1.5 1.2 \[4,15\] MMP-1 gi:13027798 1.6 1.4 \[4\] TGF-β1 gi:10863872 1.4 1.7 \[4,15\] TGF-β2 gi:339549 1.5 1.3 \[4\] tPA gi:2161467 -1.5 -2.0 \[8\] Minus (-) sign represents lower signal intensity in adhesion (A) compared to normal (N) fibroblasts (gene filter data) \* Citations reporting expression levels of respective genes in fibroblasts from normal human peritoneum and adhesion using multiplex PCR technique ::: Semiquantitative RT-PCR experiments (Figure [2](#F2){ref-type="fig"}) conducted to verify expression pattern of specific genes from the gene filter experiments that were not studied earlier in the peritoneal fibroblasts confirmed higher expression (p \< 0.05) of Collagen type IV (alpha 2) chain (COL4A2), S100 Calcium binding protein A10 (S100A10), Nidogen 2 (NID2), Transmembrane 4 superfamily member 1 (TM4SF1), Chondroitin sulfate proteoglycan 2 (CSPG2) and Metallothioneine (hMT-Ie) in adhesion compared to normal fibroblasts. The semiquantitative RT-PCR experiments also confirmed lower expression levels of Insulin-like growth factor binding protein 3 precursor (IGFBP3) mRNA in the adhesion compared to normal peritoneal fibroblasts. Transcript levels of 18S ribosomal subunit estimated by RT-PCR method was not significantly different between fibroblasts isolated from normal and adhesion peritoneum (Figure [2](#F2){ref-type="fig"} and Table [3](#T3){ref-type="table"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Relative abundance of specific mRNA species in the normal and adhesion fibroblasts.Genes differentially expressed between the normal and adhesion fibroblasts, as identified by gene filter experiments, were amplified by the RT-PCR technique at 26 PCR cycle. PCR products (20 μl) were subjected to electrophoresis, stained with fluorescent dye, photographed and optical density determined as described in Methods. A: Representative gel showing amplicons from normal (odd lane numbers) and adhesion (even lane numbers) fibroblasts. Lanes 1&2, 3&4; 5&6; 7&8; 9&10and 11&12show RT-PCR products from COL4A2; NID2; CSPG2; S100A10; 18S ribosomal subunit and TM4SF1 mRNA respectively. Lanes 13&14; 15&16and 17&18show RT-PCR products from 18S ribosomal subunit, IGFBP3 precursor and MET-1e mRNA respectively. L:Lanes loaded each with 7 μl of 100 bp DNA ladder. The 600 bp band of the ladder is shown by arrow head. B. Histogram showing mean and standard error of mean values of optical densities derived from amplicons of specific genes (x axis) from normal (empty bars) and adhesion (filled bars) fibroblasts isolated from 4 patients as described in Methods. \Significantly different (p\< 0.05) between normal and adhesion fibroblasts. ::: ![](1477-7827-3-1-2) ::: Effects of TGF-β1 or hypoxia on the expression levels of specific genes in the normal peritoneal fibroblasts ------------------------------------------------------------------------------------------------------------ Exposure to TGF-β~1~or hypoxic conditions for 24 h altered expression levels of specific genes in the normal peritoneal fibroblasts as evidenced by semiquantitative RT-PCR. Transcript levels of COL4A2, S100A10, CSPG2 and hMT-Ie were up regulated by TGF-β1 in the normal peritoneal fibroblasts (Figure [3](#F3){ref-type="fig"}), whereas transcript levels of NID2, TM4SF1, and IGFBP3 were not altered by TGF-β1 treatment. Hypoxic conditions elevated expression levels of COL4A2, S100A10 and hMT-Ie transcripts in the normal peritoneal fibroblasts (Figure [4](#F4){ref-type="fig"}). Transcript levels of CSPG2 were not significantly altered by hypoxia. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effects of TGF-β1 on the steady state levels of specific mRNA species in normal peritoneal fibroblasts.Normal peritoneal fibroblasts were cultured for 24 h in absence or presence of TGF-β1 and total RNA from cells were examined for the steady-state levels of different mRNA species by semiquantitative RT-PCR technique as described in Methods. A. Representative gels showing amplicons generated by RT-PCR from specific gene transcripts (denoted on the left of the panel) from control (lanes 1, 2 and 3) and TGF-β1 (lanes 4, 5 and 6) treated cells. Complementary DNA for all genes except IGFBP3 precursor was amplified at 26 PCR cycles. IGFBP3 precursor transcripts were amplified at 25 cycles. LLane loaded with 100 bp DNA ladder. BHistogram showing mean and standard errors of mean of optical densities from amplicons representing specific mRNA species (x axis). The RT-PCR experiments were conducted twice from normal and peritoneal isolated from 3 patients to obtain OD values of six amplicons from control (empty bars) or treated (shaded bars) fibroblasts statistical analysis. \ Significantly different from control conditions at p\< 0.05. ::: ![](1477-7827-3-1-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effects of hypoxia on the steady state levels of specific genes in normal peritoneal fibroblasts.Normal peritoneal fibroblasts were cultured for 24 h in normoxic and hypoxic conditions and total RNA from cells were examined to determine the steady state levels of specific transcripts as described in Methods. Complementary DNA for all genes was amplified at 26 PCR cycles. Histogram showing mean and standard errors of mean of optical densities of amplicons representing specific mRNA species (x axis) from control (empty bars) or hypoxia exposed cells (shaded bars) from 3 patients. The RT-PCR experiments were conducted twice to obtain OD values of six amplicons from normoxic or hypoxic fibroblasts for statistical analysis. Images of gels with amplicons from cells treated with hypoxia are not shown. \* Significantly different from control conditions at p\< 0.05. ::: ![](1477-7827-3-1-4) ::: Discussion ========== We present evidence that the expression pattern of large number of genes differ between the fibroblasts isolated from adhesion tissues and normal human peritoneal supporting the notion that adhesion fibroblasts may attain a different phenotype following peritoneal injury. Genes that displayed altered expression levels in this transition included those involved in cell proliferation, differentiation, signaling molecules, transcription and translation factors, proteolysis and cytokines. Results indicate that fibroblasts from adhesion tissue may perceive and respond to external and internal cues differently than those residing in normal human peritoneum. We attempted to decipher the functional consequences of altered gene expression pattern in the adhesion fibroblast to further elucidate the mechanism of adhesion formation and point to additional ways adhesion development may be restrained. Expression pattern of genes in the fibroblasts from normal and pathological sites are shown to be different also in earlier studies \[[@B17]\]. More relevant to the present study are the reports \[[@B4],[@B8]\] on the mRNA levels of human type I collagen (alpha 2), fibronectin 1, MMP-1, TIMP-1, TGF-β1, TGF-β2, IL-10, PAI-1, tPA and COX-2 in adhesion and normal peritoneal fibroblasts from humans estimated by multiplex PCR technique. Gene filter data from two patients also showed similar pattern of collagen, type 1 (alpha 2), fibronectin 1, MMP-1, TGF-β1, TGF-β2 and tPA mRNA levels in the normal and adhesion fibroblasts (Table [1](#T1){ref-type="table"}). Expression pattern of TIMP-1, IL-10, PAI-1, COX-2 in the adhesion and normal peritoneal fibroblasts as reported earlier \[[@B4],[@B8],[@B9]\] could not be verified by gene filter experiments because GF211 filters do not have spots representing these genes. Even so, similarities in the expression pattern of many genes between two patients (Tables 1--3) and those reported earlier using multiplex PCR technique \[[@B4],[@B8]\] validate our findings. The semiquantitative RT-PCR experiments conducted to verify expression pattern of specific genes recorded from gene filter experiments show that mRNA levels of COL4A2, S100A10, nidogen-2, TM4SF1, CSPG2, MT-1e and IGFBP3 precursor indeed differ between normal and adhesion fibroblasts. Even though expression levels of these transcripts were significantly different between normal and adhesion fibroblasts, only minor variations in the optical densities of amplicons were recorded within normal or adhesion tissues of patients of different age groups. This indicate that age dependent differences in the expression levels of genes in the fibroblasts from normal or adhesion tissues may tend to attain a relatively similar expression levels when in culture. Despite the fact that our study focused on the steady state levels of mRNA species and not on translation or posttranslational events, analysis of the functional consequences of altered expression of encoded proteins from the literature as referred below indicated that changes in the pool of these mRNA species may lead to the transformation of normal peritoneal fibroblasts into a specialized phenotype during the healing process. COL4A2 is a major structure-defining component in all basement membranes \[[@B18]\] and forms a framework for the ordered aggregation of additional molecules like laminin, heparin sulphate proteoglycans, and nidogen \[[@B19]\]. Relatively higher levels of COL4A2 observed in the adhesion fibroblasts may enhance synthesis of basement membrane in the tissues of adhesions. As COL4A2 gene is up regulated during malignant transformation and tumor vessel proliferation \[[@B20]\], it is anticipated that up regulated levels of COL4A2 in the adhesion fibroblasts may aid to the formation of adhesion tissue by increasing proliferation of adhesion fibroblasts and supporting new vessel formation for the nourishments of growing tissue. S100A10 proteins interact with Annexin A2 forming a heterotetrameric structure AIIt; that dock into the cell membrane promoting F-actin reorganization and cell migration \[[@B21]\]. AIIt also colocalizes with uPAR and plasminogen in the cells \[[@B22]\]. Heightened levels of S100A10 may enhance migration of adhesion fibroblasts by changing F-actin dynamics and influencing Cathepsin B and plasminogen machinery \[[@B23]\]. S100A10 also interacts with cytosolic phospholipase A2, inhibits its activity and decreases synthesis of archidonic acid \[[@B24]\]. Therefore, increase in S100A10 levels in the adhesion fibroblasts may deplete intracellular levels of archidonic acid and Prostaglandin E2 (PGE2) that are known to inhibit cell proliferation, collagen I synthesis, contraction of ECM and fibroblast migration \[[@B25]\]. Nidogen-2 (entactin-2) interacts with laminin1 P1, collagen I, collagen IV, perlecan and fibulin-2 in the extracellular space and stabilizes the basement membrane. It also interacts with α6β1 and α3β1 integrin receptors on cells \[[@B26]\]. Relatively higher levels of nidogen-2 secreted by adhesion fibroblasts in the extracellular space may strengthen the basement membrane and enhance integrin mediated adhesion and migration of fibroblasts into the growing tissue of adhesion. TM4SF molecules (tetraspanins) play important roles in cell migration and in the generation of complexes with integrins functionally relevant for cell motility, tumor progression and wound healing \[[@B27]\]. It is proposed that tetraspanins can influence cell migration by (i) modulating integrin signaling and integrin-mediated reorganization of the cortical actin cytoskeleton; (ii) regulating compartmentalization of integrins on the cell surface or (iii) directing intracellular trafficking and recycling of integrins \[[@B27]\]. Therefore, heightened intercalation of TM4SF1 in the cell surface of adhesion fibroblasts may facilitate their integrin-mediated migration into the developing tissues of adhesion. Versicans (CSPG2) are also known to influence α4β1 and α2β1 integrin mediated invasion of melanoma cells \[[@B28]\]. Higher CSPG2 in the fibroblasts of adhesion tissues may assist in the integrin-CSPG2 mediated migration of peritoneal fibroblasts to the site of injury and increase the number of fibroblasts by enhancing proliferation and decreasing apoptosis as evidenced in other cell types \[[@B28],[@B29]\]. Veriscan interacts with hyaluronan and CD44 and increase the viscoelastic nature of the pericellular matrix, creating a highly malleable extracellular environment that supports a cell-shape change necessary for cell proliferation and migration \[[@B30]\]. Because MT-1e transcripts are detected in cell types that have undergone myoepithelial differentiation \[[@B31]\], significant differences in the MT-1e mRNA levels between adhesion and normal peritoneal fibroblasts indicate that fibroblasts in the adhesions are at different state of differentiation compared to normal peritoneum. Molecules including IL-1; IL-6, TNF-α, EGF, bFGF, glucocorticoids, LPS, and estrogen that promote post surgical adhesion formation \[[@B32]-[@B34]\] directly or indirectly increase MT-1 transcripts and proteins in several tissues and cell types \[[@B35]\]. Therefore, it is likely that these molecules may increase adhesion formation by augmenting MT-IE levels which in turn may increase proliferation, reduce cell death and confer invasiveness of adhesion fibroblasts \[[@B36]\]. Contrary to increase in the above mentioned mRNA species in the adhesion fibroblasts, steady state levels of IGFBP3 precursor transcript were found to be lower. Because IGFBP-3 is known to inhibit cell growth by sequestering IGF, its decreased level may enhance proliferation of adhesion fibroblasts \[[@B37]\]. Reduced levels of IGFBP3 mRNA are reported in the tumorigenic cells \[[@B38]\]. Therefore, reported lower incidence of pelvic adhesion formation in the primates on anti-estrogenic therapy \[[@B32]\] could be due to the antiproliferative effects of anti-estrogens mediated in part, by IGFBP-3 \[[@B39]\]. IGFBP-3 also induces growth inhibition and apoptosis \[[@B40]\]. Decrease in the levels of IGFBP-3 in the adhesion fibroblasts may promote adhesion development both by increasing proliferation and reducing apoptosis at the site of injury. Our attempts to examine the regulatory roles of TGF-β1 and hypoxia, factors known to promote adhesion development \[[@B3]\], on the expression pattern of specific genes show that not all changes in the gene expression pattern between the normal and adhesion fibroblast are the function of these factors (Figure [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}; Table [4](#T4){ref-type="table"}). Our data show that while mRNA levels of COL4A2, S100A10 and MT-1e are elevated by both TGF-β1 and hypoxia in the human peritoneal fibroblasts, the mRNA levels of only CSPG2 is influenced by TGF-β1. Moreover, transcript levels of nidogen-2, TM4SF1 and IGFBP3 mRNA were not influenced by TGF-β1. Based on these results we hypothesize that genes that are not influenced by TGF-β1 and hypoxia in the peritoneal fibroblasts may be influenced by factors such as interleukins and TNF-α that are also known to play role in adhesion formation. Alternately, TGF-β1 and/or hypoxia may influence actions of these genes at the post transcriptional level without altering transcript levels. TGF-β1 induced up regulation of integrin α5, αv and α6 subunits in the normal human peritoneal fibroblasts without altering mRNA levels \[[@B10]\] is consistent with this possibility. It is also possible that TGF-β1 and hypoxia may alter expression of these genes in mesothelial and other cell types following peritoneal injury. Likewise lower levels of VEGF transcripts in adhesion fibroblasts may be compensated by its higher levels in other cell type required for angiogenesis during adhesion formation \[[@B3]\]. Detected lower intensity of VEGF-A isoform in the adhesion fibroblasts may also be due to the fact that spots representing this isoform do not distinguish different VEGF-A splice variants that are known to be up or down regulated during adhesion formation \[[@B16]\]. It is known that a new phenotype of fibroblasts is induced during wound healing. These fibroblasts, termed- myofibroblasts, express higher levels of α-smooth muscle actin and vinculin-containing fibronexus adhesion complexes \[[@B41]\]. Fibroblasts isolated from adhesion tissues express higher levels of α-smooth muscle actin transcripts compared to normal peritoneal fibroblast (Table-[2](#T2){ref-type="table"}) \[[@B42]\] and TGF-β1 induces formation of adhesion complex in these cells \[[@B10]\]. These observations in addition to the known roles of TGF-β in the development of post surgical adhesions \[[@B43]\] and transformation of fibroblasts into smooth muscle α-actin expressing myofibroblasts \[[@B44]\] imply that this cytokine may influence transformation of normal fibroblasts into a phenotype similar to myofibroblasts in the developing tissues of adhesion. Therefore, hindering this transformation may reduce adhesion formation. For instance, augmenting E prostanoid 2 (EP2) receptor pathways may be a way to reduce the incidence of adhesion formation because prostaglandin E~2~(PGE~2~) is shown to inhibit TGF-β1 induced expression of α-SMA, production of Collagen I and the transformation of fibroblasts to myofibroblasts via EP2 signaling \[[@B45]\]. Additionally, adhesion formation may be reduced by P311(PTZ17) and Interferon γ treatments, which inhibits TGF-β1 induced myofibroblast transformation \[[@B46],[@B47]\]. During the course of normal wound healing, myofibroblasts disappear, possibly by apoptosis \[[@B48]\]. In contrast, when there is abnormal wound healing, myofibroblasts persist \[[@B49]\]. Data obtained in our study also indicate that adhesion fibroblasts may resist apoptosis due to anti apoptotic effects mediated by increased hMet1-e and CSPG2 levels and down regulation of IGFBP3. They may also attain a high proliferating nature due to up regulation of S100A10 and CSPG2 genes, and down regulation of IGFBP3 (Table [2](#T2){ref-type="table"}). Higher proliferating and reduced apoptotic nature of adhesion fibroblasts derived from altered ratio of bcl-2 and bax expression is suggested in an earlier study \[[@B5]\]. It is apparent now that higher proliferative and reduced apoptotic nature of adhesion fibroblasts in human as reported earlier \[[@B5]\] could also derive from altered expression of hMet1-e, CSPG2, S100A10, CSPG2, IGFBP3, and the Bfl-1 that inhibits p53-induced apoptosis and is induced by cytokines TNF-α and IL-1β \[[@B50]\]. This altered phenotype of adhesion fibroblasts, acquired during the healing process, may lead to the accumulation of extra number of cells at the site of peritoneal injury resulting fibrosis and scar formation. Of note, one of the pivotal differences between wounds that proceed to normal scar compared with those that develop hypertrophic scars or fibrosis may be a lack of or reduced cell death \[[@B51]\]. Therefore, excess fibroblasts at the site of peritoneal wound healing may divert the normal process of healing towards fibrosis and adhesion. The elevated levels of p53 in the adhesion fibroblasts during this disarray, as evident from the gene filter data (Table [2](#T2){ref-type="table"}), may guard against its transition towards malignancy \[[@B52]\]. Conclusions =========== It is evident from our study that steady state levels of several genes are different between adhesion and normal peritoneal fibroblasts in human and that adhesion development may be a function of several genes. Changes in the functional interdependence of these genes at the site of injury may transform normal peritoneal fibroblast into cell type/s with altered phenotype. These cells- designated as adhesion fibroblasts may mimic previously known myofibroblasts and are highly proliferative. These cells resist apoptosis and secrete ECM molecules to renovate basement membrane. With changed expression pattern of cell surface molecules these cells may respond to intracellular signaling for migration over the fibrin clot. This altered nature of adhesion fibroblasts therefore may play a major role in the formation of the fibrous mass of adhesion-tissue that bridges adjacent and injured peritoneum. Blocking changes in the expression or function of genes necessary for this transformation of normal peritoneal fibroblasts may curtail adhesion formation. This could be achieved by the application of PGE~2~, EP~2~blockers, interferon γ, P311and applying measures to induce apoptosis in the peritoneal fibroblast at the site of injury. The obvious question -- \"how to maintain apoptosis at a desired level for normal peritoneal healing?\" however, remains to be answered. List of Abbreviations ===================== Collagen type IV (alpha 2) chain (COL4A2), Nidogen 2 (NID2), Chondroitin sulfate proteoglycan 2 (CSPG2), S100 Calcium binding protein A10 (S100A10), Transmembrane 4 superfamily member 1 (TM4SF1), Metallothioneine (hMT-Ie), Insulin-like growth factor binding protein 3 precursor (IGFBP3), Transforming growth factor (TGF), Prostaglandin E2 (PGE2), Urokinase Plasminogen activator receptor (uPAR), Annexin 2 and S100A10 complex (AIIt), tissue Plasminogen Activator (tPA), Plasminogen Activator Inhibitor (PAI), Cyclooxigenase (COX), Matrix metalloproteinase (MMP), Tissue Inhibitor of Metalloproteinase (TIMP), Interferon γ (IFN-γ), IL (Interleukin). Authors\' Contributions ======================= GMS and MPD were responsible for the isolation of peritoneal fibroblasts from normal peritoneum and adhesion tissues as well as establishing hypoxia chambers. MPD provided patient information and valuable suggestions during writing the manuscript. UKR performed microarray and semiquantitative RT-PCR experiments, analyzed the data and wrote the manuscript. Acknowledgment ============== U. K. Rout thanks Research Genetics (Invitrogen Corporation) for the license to use Pathway Universal Software and acknowledges a Department of Obstetrics and Gynecology, WSU Grant support for this study.	PubMed Central	2024-06-05T03:55:52.633596	2005-1-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548295/", "journal": "Reprod Biol Endocrinol. 2005 Jan 10; 3:1", "authors": [ { "first": "Ujjwal K", "last": "Rout" }, { "first": "Ghassan M", "last": "Saed" }, { "first": "Michael P", "last": "Diamond" } ] }
PMC548296	Review ====== This paper reviews the growth of the Pharmaceutical Benefits Scheme (PBS) during 2002--03; concerns about the sustainability of the Scheme, the government\'s response, a potential new threat that emerged and issues that remain to be tackled. The growth and sustainability of the PBS ---------------------------------------- From March 2003 to March 2004, a total of \$5.8 billion was spent on prescription medicines subsidised under the PBS. Of this, \$4.89 billion (84%) was paid by the Commonwealth, the remaining \$0.91 billion through patient co-payments \[[@B1]\]. In comparison, in 2002--03, the Commonwealth spent \$7.24 billion on public hospital services \[[@B2]\] and \$8.17 billion on medical and diagnostic services (through Medicare benefits) \[[@B3]\]. Although the PBS is the smallest of these components of Commonwealth expenditure, it has the highest average annual growth rate over the last decade (around 12% pa), compared to 6% pa for public hospital services, and 5% pa for medical services. At these rates, by 2011 the Commonwealth would be spending more on subsidised pharmaceuticals than it would spend on either public hospital or medical services, and by 2022, more on pharmaceuticals than both public hospital and medical services together. Such projections make the sustainability of the PBS a major concern, especially given an aging population and the continued introduction of useful (but increasingly expensive) new medicines \[[@B4]\]. While the growth rate of the PBS has slowed over the last two years (10% during March 2002--03 and 8% from March 2003--04) the past history of PBS expenditure shows considerable fluctuations over the years. These fluctuations are caused by expensive but valuable new drugs coming onto the Scheme, more cost-effective generic drugs replacing older drugs whose patent has expired and administrative changes, such as increased patient co-payments, transiently reducing usage. The government\'s response -------------------------- In 2003, as in 2002, the government proposed a 27% increase in PBS patient co-payments and safety-net thresholds in order to transfer more of the cost of the PBS from the government to consumers. Once again this measure was rejected by Labor and other opposition parties in the Senate because of concern that such increases would impact on equitable access to necessary medicines \[[@B5]\]. Regardless, the government continued to argue that without increased patient contributions (and patient restraint) the PBS would become unsustainable. By mid 2004, the Labor party was faced with an impending Federal election and had serious trouble costing its tax and spending promises. As a consequence, Labor abandoned their previous principled stand in the Senate of blocking the government\'s proposed increase in PBS copayments and safety-net thresholds \[[@B6]\] arguing that they needed the additional \$1.1 billion to spend on election promises \[[@B7]\]. They also stated that, if returned to government, a substantial proportion of the \$1.1 billion might be achieved through administrative reforms to the PBS and savings achieved by the use of cheaper generic drugs as expensive drugs moving off patent. Not surprisingly, consumer and public health groups were appalled with this Labor \"back-flip\" while the Greens and Democrats said the decision was a disgrace \[[@B8]\]. Labor said the decision was difficult but necessary. Several other measures were introduced by the government in order to improve the community\'s understanding of PBS processes and costs. From June 2003, all recommendations of the Pharmaceutical Benefits Advisory Committee (PBAC) to list, not list or defer a decision to list a medicine on the PBS were made publicly available on the PBS website \[[@B9]\]. Unfortunately, only summary information was provided; commercial-in-confidence concerns of pharmaceutical manufacturers precluded making more detailed information available, such as cost-effectiveness data, on which PBAC based its decision. From 1 August 2003 the full cost of PBS medicines appeared on medicine labels if the price was greater than the co-payment. The full cost included what the consumer has paid and the amount that is paid through the PBS. The aim was to help people understand what medicines really cost and how the PBS helps make medicines affordable for all. In addition, the government commissioned a \$24 million advertising campaign that emphasised that patient responsibility was, \"the prescription for a healthy PBS\". Critics noted that by neglecting to inform the public that pharmaceutical marketing and inappropriate prescribing habits of doctors also produced pressures on the PBS, the campaign missed an opportunity to initiate a more balanced and constructive debate about the viability of the PBS \[[@B10]\]. During the year under review, pharmaceutical reforms designed to stop PBS cost-shifting in Victorian public hospitals were evaluated \[[@B11]\]. The reforms were a joint initiative of Victorian Department of Human Services (DHS) and the Australian Government Department of Health and Ageing (DoHA). Since the early 1990s there had been increasing cost pressures on State and Territory funded public hospitals. Their response included restricting drug supplies to discharged patients, often to only two or three days of treatment. Patients then needed to see their GP to obtain a PBS prescription to cover their needs. The effect was to \"cost shift\" pharmaceutical supplies from the State and Territories to the Australian Government. The reforms trialed in Victoria allowed public hospital doctors to write PBS prescriptions for both outpatients and discharged inpatients. They also allowed PBS access to a group of cancer chemotherapy drugs for use by day-admitted patients and outpatients. The qualitative evaluation undertaken was generally positive although it noted the reforms had increased the administrative work of both doctors and pharmacists. There was also concern that PBS rules (designed for general practice) were not always appropriate for specialised public hospitals. The Society of Hospital Pharmacists of Australia supported the need to modify PBS procedures to take into account public hospital expertise but also noted the need for more integration of medicines funding \[[@B12],[@B13]\]. Subsequently, the Victorian reforms are being implemented in other States and Territories. Programs promoting the quality use of medicines (QUM) were further developed throughout 2002--03. Specific programs were coordinated by the National Prescribing Service (NPS), Australian Divisions of General Practice and the Pharmacy Guild of Australia. The NPS was set up in 1999 with government funding but with an independent Board of Directors in order to provide unbiased educative activities to assist health practitioners (and more recently consumers) to use medicines wisely. Evaluation of NPS activities has consistently shown that spending money on targeted QUM interventions can save considerably more money on the PBS by reducing inappropriate prescribing. It was estimated that NPS activities during the period 1 July 2000 to 30 June 2002 generated PBS savings in the range of \$55.6 million to \$83.9 million through the following prescribing intervention programs: antibiotics in primary care; peptic ulcer management; management of dyspepsia; COX-2 selective NSAIDs; managing hypertension; and managing dyslipidaemia \[[@B14]\]. In 2003, the NPS received an additional allocation of government money to provide educational material about drugs newly listed on the PBS (the RADAR project). The latter was in response to considerable evidence that intensive pharmaceutical promotion at the time of PBS listing was associated with drugs being prescribed for broader indications than those indicated in the PBS listing (causing so-called PBS \"leakage\" or \"blow-outs\") \[[@B15]\]. However, the 2003 NPS educational budget of \$12.5 million needs to be compared with the estimated \$1.0 billion promotional budget of the Australian pharmaceutical industry \[[@B16]\]. The Enhanced Divisional Quality Use of Medicines (EDQUM) program was a 1999--2000 Federal Budget initiative, originally announced as the Incentives for Quality Prescribing (IQP) program. The program offered Divisions of General Practice (Divisions) a proportion of monies saved if Divisional QUM activities improved prescribing and lowered PBS costs. The Divisions were allowed to use any savings made for a range of primary health care activities. The program evolved significantly due to feedback from the medical profession. It was implemented on a pilot basis in thirteen Divisions on 1 July 2002. Under the program, Divisions were encouraged to invest their own resources in a range of drug utilisation data collection and/or education related activities. Activities were implemented in close consultation with the NPS and focused on one or more of the following target drug groups: antibiotics, peptic ulcer drugs and cardiovascular drugs. An evaluation of the pilot EDQUM project was undertaken in early 2004 \[[@B17]\]. Barriers to implementation included perceptions that the program was primarily focused on reducing pharmaceutical costs to government; limited capacity of existing prescribing software systems to extract drug utilisation data; and the need for Divisions to take a commercial risk in developing their EDQUM initiatives (due to the absence of up-front funding) which limited their capacity to systematically implement a comprehensive range of strategies. Program achievements included the development of a wide range of shared resource material \[[@B18]\] and the creation (by some Divisions in association with software vendors) of data extraction tools. The latter have allowed a small number of practices to gain access to comprehensive information from which further initiatives to improve quality or change practices can evolve. The evaluation report recommended that standards should be established for prescribing software so that comparable data could be extracted from different systems to facilitate comparison of individual prescribing practice with evidence based guidelines. It also noted the difficulties of attributing any cost-savings in the PBS to Divisional activities. Following this report, the Government supported a four year extension to the EDQUM program in the 2003--2004 Budget. The Third Community Pharmacy Agreement between the Government and the Pharmacy Guild of Australia (1 July 2000 to 30 June 2005) also provided a range of QUM activities over the year in question including medication reviews of problem patients (conducted at the request of GPs), quality care pharmacy programs and the provision of consumer medicine information \[[@B19]\]. A potential new threat that emerged ----------------------------------- In 2003--2004 the PBS became caught up in negotiations concerning the Australia-United States Free Trade Agreement (AUSFTA). This saga has been extensively reported elsewhere \[[@B20],[@B21]\]. The government remained adamant that the AUSFTA provisions concerning the PBS were benign and would also increase the transparency of PBAC decision-making. Others were concerned that the AUSFTA contained major concessions to the US pharmaceutical industry that undermined the egalitarian principles and operation of the PBS and had the potential to increase the costs of medicinal drugs to Australian consumers. Time will tell who is right. Issues still to be tackled -------------------------- The EDQUM project highlighted the needs for software standards in order to extract comparable drug utilisation data from different prescribing systems. The need for prescribing software standards has also been raised in connection with three other issues of relevance to the PBS: pharmaceutical promotion, independent therapeutic information and drug-drug interaction checking. The uptake of computers by Australian general practitioners (GPs) was stimulated by the Australian government in 1999. A one-off grant of around \$10,000 was offered to those practices that purchased a computer, acquired internet connectivity (an E-mail address) and promised to use computer prescribing software to write the majority of their prescriptions. This increased the numbers of GPs writing prescriptions with the aid of a computer from around 50% in 1999 to more than 90% in 2004 \[[@B22]\]. Legible, printed prescriptions have been one of a number of positive outcomes of this initiative. However, new problems emerged. One software vendor (Health Communication Network Ltd.) became the dominant market leader because its business model relied on pharmaceutical promotion to heavily subsidise the cost of GPs purchasing and updating its prescribing software (Medical Director™). This business model facilitated software uptake but also resulted in advertisements for the latest and most expensive drugs appearing on the computer screen at the time of prescribing (and elsewhere). GPs using this software package were shown to prescribe more antibiotics per patient than those who wrote \'scripts manually. It was suggested that this may have been due to default settings in the software automatically writing in the maximum number of repeat prescriptions allowed.\[[@B23]\] Another default option in this software was the automatic production of a, \"Do not substitute generic drugs\" message on the prescription. The latter was eventually changed by the government amending regulation 19(5) of the National Health (Pharmaceutical Benefits) Regulations 1960 \[[@B24]\]. However, the issue of pharmaceutical promotion in prescribing software has yet to be tackled. Pharmaceutical promotion has never been allowed on government supplied \'script pads. It is hard to understand why it was allowed on the computerised equivalent. Pharmaceutical promotion distorts the information flow to physicians by selectively promoting the benefits of the latest and most expensive drugs. It provides minimal information about drug side-effects, contra-indications and opportunity costs. Cost-effective generic drugs are rarely promoted and non-drug solutions usually not at all. Pharmaceutical promotion has clearly been shown to influence physician\'s prescribing \[[@B25]\] and has resulted in cost-blow outs on the PBS due to \"leakage\" of prescribing away from cost-effective indications approved by PBAC \[[@B26]\]. In addition, pharmaceutical promotion in prescribing software, occurring at the time of physician decision making, is likely to be much more influential than promotion in medical journals, gimmicks and give-ways. As a consequence, several medical and consumer organisations have advocated further amendment of the National Health (Pharmaceutical Benefits) Regulations 1960, Part V, Regulation 19, to prohibit prescribing software from displaying pharmaceutical advertisements. Government intervention is also required to ensure that key national resources of objective therapeutic information, such as the Australian Medicines Handbook and Therapeutic Guidelines, are incorporated in prescribing software. The provision of objective therapeutic information is an important strategy of the QUM component of Australian National Medicinal Drug Policy \[[@B27]\]. Ironically, while both the Australian Medicines Handbook and Therapeutic Guidelines have been converted into electronic formats they are not yet included in computerised prescribing software. The problems have included arguments between software vendors and guideline producers over who should pay for the integration and a lack of defined standards for electronic information representation and interfacing. More recently, the NPS RADAR project has shown the way forward. RADAR provides independent information to health professionals about medicines that have a new or a changed listing on the PBS. RADAR drug monographs have recently been incorporated in four leading GP prescribing packages using an open standard interface. This project has moved ahead because the Australian government provided financial support to both the NPS and software vendors to enable the RADAR integration to take place. Following a workshop on electronic decision support, HL7 Australia has presented a work plan to the Australian Health Information Council Electronic Decision Support Steering Committee that would build on the RADAR project by incorporating the Australian Medicines Handbook and Therapeutic Guidelines into clinical software in a standard manner \[[@B28]\]. However, this plan has yet to proceed because of a current review of E-Health policy and reorganisation of its governance \[[@B29]\]. The third area of prescribing software requiring government intervention is standards for drug-drug interaction checking. The NPS tested four popular GP software packages by entering a common set of elderly patients on multiple medications \[[@B30]\]. This revealed very different behaviour by different software packages; some missed serious drug-drug interactions, others produced numerous trivial and clinical unimportant alerts. GPs noted that the latter behaviour caused them to turn off all alerts \[[@B31]\]. There is an urgent need for standards concerning acceptable drug-drug interaction detection &/or external assessment of prescribing software, another item on the HL7 Australia work plan. Conclusions =========== The PBS remained in the media and policy spotlight during 2003--04. While the growth rate of the PBS has slowed during the year under review the sustainability of the Scheme remains an ongoing concern. One strategy adopted by the government was to transfer more of the cost of medicines to consumers through higher PBS co-payments and increased safety-net thresholds. However, such measures can result in higher costs elsewhere if poorer patients forgo necessary medicines and end up being hospitalised with uncontrolled disease. Cost-shifting (and patient inconvenience) was reduced by allowing State and Territory public hospitals limited access to the PBS but these reforms also showed the need for changes in the PBS to make it more suitable for hospital practice and the desirability of further integrating health funding systems. Educational strategies focusing on the quality use of PBS medicines were successfully pursued but would benefit from increased funding. In addition, there was an exploratory attempt to focus the attention of Divisions on PBS costs by rewarding them with a moiety of any money saved by their members through more cost-effective prescribing. However, the difficulties experienced by the EDQUM project in extracting useful drug utilisation data from computerised prescribing systems highlighted the need for prescribing software standards as did other problems with such software. Information communication technology and information management (ICT/IM) has the potential to allow individual health practitioners, Divisions and governments to compare what is being done with what is recommended best-practice, highlight major discrepancies, and provide targeted education and appropriate incentives to reduce the gap. However, as the events of 2003--04 show, this potential is unlikely to be realised if the development of clinical computer systems is left solely to market forces. Competing interests =================== Dr. Harvey is a past Board member of Therapeutic Guidelines Limited, Co-Chair of HL7 Australia\'s Decision Support Technical Committee, a Councillor of the Australia Consumer\'s Association and a member of the Australian Labor Party.	PubMed Central	2024-06-05T03:55:52.638067	2005-1-12	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548296/", "journal": "Aust New Zealand Health Policy. 2005 Jan 12; 2:2", "authors": [ { "first": "Ken J", "last": "Harvey" } ] }
PMC548297	Background ========== Ovaries are the primary source of estrogen. In ovariectomized rats, the production of estrogen is shifted from ovary to a number of extragonadal sites \[[@B1],[@B2]\]. Simpson developed the intriguing concept of extragonadal aromatization, i.e. androgens, particularly androstenedione produced primarily in the adrenal glands, can be converted (aromatized) into estrogens at extraglandular sites, including the mesenchymal cells in adipose tissue and skin, osteoblasts and chondrocytes in bone, vascular endothelial, aortic smooth muscle cells as well as numerous sites in brain \[[@B2],[@B3]\]. Estrogen synthesised within these sites is biologically active in a paracrine or intracrine fashion, although it may escape the local metabolism and enter the circulation \[[@B2],[@B3]\]. Estrogen production in extragonadal sites is dependent on an external source of C19 androgenic precursors \[[@B4]\]. Circulating levels of testosterone (T) and androstenedione as well as dehydroepiandrosterone (DHEA) and DHEA sulphate (DHEAS) become extremely important in terms of providing adequate substrate for estrogen biosynthesis in extragonadal sites. It is now recognized that much of the physiology of androgens is consistent with the concept that T functions as a circulating pro-hormone. In the postmenopausal woman, circulating T levels are an order of magnitude greater than circulating estrogen levels, which by itself suggests the importance of T for maintaining local estrogen levels in extragonadal sites \[[@B2]\]. 65--75% of the circulating T is formed peripherally from androstenedione, DHEA and DHEAS secreted by the adrenals, which form a large precursor reservoir that is available for conversion to T and finally to estrogen \[[@B2]\]. Thus in the species of rat, it is still unknown that whether it is the same situation of T in extragonadal aromatization. We have observed that the release of corticotrophin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus of the ovariectomized rats increased significantly compared with the intact rats \[[@B5]\]. And more interestingly, the hypothalamic CRH expressions showed a gradual elevation along with time after ovariectomy (submitted data), which suggested that the activity of hypothalamus-pituitary-adrenal axis (HPAA) might promote. These observations lead us to hypothesize that more androgens from adrenal cortex might gradually be converted into estrogens by extragonadal aromatase in the rats along with time after ovariectomy. To test the hypothesis, we measured the blood concentrations of estradiol (E~2~) and T as well as the aromatase expressions in adipose and liver tissues in the ovariectomized rats. Methods ======= Animals ------- Female Sprague-Dawley rats (180--200 g), with regular 4-day estrus cycles were purchased from Medical Experimental Animals Centre of Fudan University (Shanghai, China). The animals were housed under laminar flow in an isolated room with controlled temperature and at a 12 /12 (light /dark) schedule. Forty-eight of them underwent ovariectomy with ether anaesthesia, which were then divided randomly into six groups: 1 month after ovariectomy (OVX1M), OVX2M, OVX3M, OVX4M, OVX5M, OVX6M. The corresponding control groups were: intact 1 month (INT1M), INT2M, INT3M, INT4M, INT5M, INT6M. All experimental procedures involving the use of animals were conducted in accordance with NIH Guidelines and were reviewed and approved by the Animal Use and Care Committee for the Fudan University. Tissue collection and preparation --------------------------------- At the time of sacrifice, the vaginal cytology of each rat was first examined. The tissues of the rats were collected respectively, and those of the intact control animals, during the period of diestrous. All the operations were carried out at 4°C. The liver tissues, subcutaneous abdominal (SA) adipose tissues and the adrenals were excised, and then snap-frozen in liquid nitrogen, and stored at -80°C. The preparation of the microsomal pellet was accordance with the report by Hiroshi \[[@B6]\]. Total tissue RNA was extracted using \'TRIzol Regent\' (Biobasic Inc, Canada), and the purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis before analytical procedures. Semiquantitative RNA analysis ----------------------------- To compare the level of adrenal cytochrome P450 17α hydroxylase/lyase (P450c17), liver and SA adipose P450arom expressions after different treatments, PCR methologies were adapted to provide a semiquantitative measure of mRNA levels. Primers were synthesized based on published reports \[[@B7],[@B8]\]. Table [1](#T1){ref-type="table"} summarizes the anticipated size of PCR products, sense and antisense sequences, and locations of primers. Total RNA (2 μg) was transcribed in reverse, in a final volume of 20 μl, using 200IU M-MLV reverse transcriptase in the presence of 25 pmol downstream primer (Sangon Inc), 0.5 mM deoxy-NTP and 20IU Rnasin (from Promega) for 60 min at 42°C before heat denatured for 5 min at 95°C. The cDNAs obtained were further amplified by PCR using 25 pmol of upstream primer (Sangon Inc). We first determined the linear range of amplification of cDNA using each of the primer sets, and then chose an appropriate amplification cycle within this range for each cDNA species. For P450arom and P450c17, we used 35 PCR amplification cycles, and 20 cycles for β-actin gene expression. Each PCR reaction underwent an amplification regimen characterized by (P450arom: 1 min at 94°C, 1 min at 60°C, 2 min at 72°C; P450c17: 1.5 min at 72°C, 2 min at 56°C, 4 min at 72°C) with Taq DNA polymerase (3U per tube) and 2.2 mM magnesium chloride (from Promega) in a final volume of 50 μl. To check the presence of DNA contamination, RT-PCR was performed on 2 μg of total RNA without M-MLV reverse transcriptase (negative control). An internal control (water instead of RNA) for each RT-PCR was performed to investigate RNA contamination of the mixture. For each sample 5 μl of the PCR amplification products were analysed on 2% agarose gels and stained with ethidium bromide. The intensities of the bands were evaluated using the Image Master Software (SYDR-1990, SYNGENE, U.S.A.). The RT-PCR products were extracted and purified from agarose gel by Golden Beads Gel Extraction kit (Sangon Inc., China) and sequenced using radioactive dideoxychain terminating method (Sangon Inc., China). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Properties of oligonucleotide primers used for PCR. ::: ---------------------------------------------------------------------------------------------------------- Target mRNA Size of PCR products (bp) Sense (S) or antisense (AS) Primer sequence (location) ------------- --------------------------- ----------------------------- ---------------------------------- P450c17 299 S\ 5\'-TCATCAAGAAGGGAAAAGAA-3\'\ AS 5\'-TGAAGCAGATAGCACAGATG-3\' P450arom 289 S\ 5\'-GCTTCTCATCGCAGAGTATCCGG-3\'\ AS 5\'-CAAGGGTAAATTCATTGGGCTTGG-3\' β-actin 550 S\ 5\'-AAGCAGGAGTATGACGAGTCCG-3\'\ AS 5\'-GCCTTCATACATCTCAAG TTGG-3\' ---------------------------------------------------------------------------------------------------------- ::: Western blotting ---------------- A 50 μg sample of the microsomal protein was loaded into each lane along with a prestained protein size marker (Bio-Rad Laboratories, Inc., Hercules, CA), electrophoresed on a 10% SDS-polyacrylamide gel at 18 V/cm, and electroblotted onto a polyvinylidene difluoride membrane (Micron Separations, Westboro, MA) using a wet electroblotter. After blocking in fat-free milk, incubation was conducted with the antiaromatase antibody (1:200; Boster Biological Technology LTD., China) and β-actin antibody (1:3000) at room temperature in 18°C for 4 h in TBS-T solution (20 mM Tris, 137 mM NaCl, and 0.1% Tween-20, pH 7.6). After extensive washing, blots were incubated with AP-labelled goat antirabbit antiserum (Sino-American Biotechnology Co., China) for 60 min at room temperature in 18°C and developed using NBT/BCIP detection system (Amersham Pharmacia Biotech). The intensities of the bands were evaluated using the Image Master Software (SYDR-1990, SYNGENE, U.S.A.), and values were normalized to β-actin immunoreactivity in each sample and expressed as percent of the control. Specificity of the aromatase immuno-staining was determined by preincubation of antiserum for 24 h at 4°C with varying concentrations of aromatase, with the primary antibody omitted to identify non-specific staining as well. RIA of blood estrogen and testosterone concentrations ----------------------------------------------------- At the time of sacrifice, the blood samples (0.8 ml) of the rats were collected from tail veins respectively, and the corresponding intact controlling animals, during the period of diestrous. The plasma was separated by centrifugation and stored at -70°C until assayed. Concentration of blood hormones were determined by double-antibody RIA kits purchased from the National Atomic Energy Research Institute (Beijing, China.). The samples were assayed in duplicate, and all the subjects\' samples were assayed together. The sensitivity of the kit was 0.8 pg/ml (testosterone) and 1.4 pg/ml (estrogen), the intra- and interassay coefficients of variation, 3.7--8.0% and 4.74--7.7%. Statistical analysis -------------------- All data are presented as means ± S.E.M. Statistical analysis was performed on raw data using two-way analysis of variance (ANOVA), with the significance set at p \< 0.05 and p \< 0.01 in two-tailed testing chosen. Results ======= Vaginal cytology of the animals ------------------------------- The epithelial cells were stained by haematoxylin-eosin (HE). The intact rats (INT1M, 2M, 3M, 4M, 5M, 6M) showed regular 4-day estrus cycle change. The cyclic change disappeared in the ovariectomized (OVX1M, 2M, 3M, 4M, 5M, 6M) rats. A few of mature vaginal epithelia were observed in the smears of the OVX5M and OVX6M rats, and the percent of mature epithelia increased significantly in the OVX6M rats (p \< 0.05) (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The percent of mature vaginal epithelia and the blood concentrations of E~2~and T of the rats ::: Groups The percent of mature vaginal epithelia Blood E~2~level (pg/ml) Blood T level (pg/ml) -------- ----------------------------------------- ------------------------- ----------------------- ---------------- 1M INT (15.6 ± 0.88)% 56.60 ± 14.13 27.92 ± 1.74 OVX (0.19 ± 0.019)%\* 4.06 ± 1.36\* 18.91 ± 2.53\* 2M INT (15.8 ± 0.91)% 59.12 ± 11.23 26.39 ± 2.04 OVX (0.17 ± 0.015)%\* 8.94 ± 1.78\* 18.03 ± 2.20\* 3M INT (14.8 ± 0.90)% 58.25 ± 13.93 28.86 ± 2.19 OVX (0.20 ± 0.025)%\* 16.72 ± 3.71\* 20.21 ± 1.96\* 4M INT (15.1 ± 0.86)% 59.35 ± 10.35 26.99 ± 2.14 OVX (0.19 ± 0.020)%\* 28.10 ± 8.88\^\#^ 20.89 ± 2.69\ 5M INT (16.4 ± 1.01)% 63.34 ± 15.85 27.03 ± 1.93 OVX (0.26 ± 0.034)%\* ^\#^ 28.97 ± 6.61\^\#^ 21.35 ± 2.33\ 6M INT (16.8 ± 1.06)% 59.15 ± 13.34 29.36 ± 2.56 OVX (0.29 ± 0.027)%\* ^\#^ 31.05 ± 6.61\^\#^ 21.54 ± 2.09\ \p \< 0.05 vs corresponding intact; \# p \< 0.05 vs OVX1M ::: Blood concentrations of estrogen and testosterone ------------------------------------------------- The blood E~2~concentrations decreased significantly in the OVX1M, 2M and 3M (p \< 0.01) compared with those in the INT1M, 2M and 3M. The concentrations in OVX4M, 5M and 6M groups increased significantly (p \< 0.05) compared with OVX1M, though still lower than INT4M, 5M and 6M. There were no disparities between the INT1M, 2M, 3M, 4M, 5M and 6M groups (Table [2](#T2){ref-type="table"}). The blood T concentrations decreased significantly in the OVX groups compared with corresponding intact controls (p \< 0.05). Though there were slightly increases in the OVX3M, 4M, 5M and 6M compared with OVX1M, no statistical significances were detected (Table [2](#T2){ref-type="table"}). RT-PCR analysis: Effects of ovariectomy on adrenal P450c17, liver and SA adipose P450arom mRNA levels ----------------------------------------------------------------------------------------------------- Comparison of the amplified PCR fragments with rat P450c17 and ovary aromatase cDNA sequences revealed 100% homology (data not shown). Densitometric analysis of the mRNA concentration using target product/β-actin was expressed as the mean with SEM. The ratios of liver P450arom to β-actin in the OVX1M, 2M, 3M, 4M and 5M groups were lower than the corresponding intact controls (p \< 0.05). The ratio increased significantly in the OVX6M compared with OVX1M (p \< 0.05), and no difference was detected between OVX6M and INT6M (Fig [1](#F1){ref-type="fig"}). The ratios of SA adipose P450arom to β-actin in the OVX1M, 2M, 3M, 4M and 5M groups appeared no significant changes compared with those in the corresponding intact control, and the ratio in the OVX6M was higher than that in all other groups (p \< 0.05) (Fig [2](#F2){ref-type="fig"}). The ratio of adrenal P450c17 to β-actin in the OVX1M decreased significantly compared with that in the INT1M (p \< 0.05), and those in the OVX4M and OVX5M increased slightly, with statistical disparities between OVX4M and INT4M, or OVX5M and INT5M. In the OVX6M, the ratio increased markedly, and was higher than OVX1M (p \< 0.05) (Fig [3](#F3){ref-type="fig"}). However, no changes occurred on the ratios of adrenal P450c, liver and SA P450arom among INT1M, 2M, 3M, 4M, 5M and 6M(Fig [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"},[3](#F3){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### RT-PCR analysis of liver P450arom mRNA expressions of the rats.The upper picture shows the gel electrophoresis of the RT-PCR products for the liver P450arom. Densitometric analysis of the mRNA concentration using PCR product/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. \ p \< 0.05 vs corresponding intact controls, \# p \< 0.05 vs OVX1M. ::: ![](1477-7827-3-6-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### RT-PCR analysis of SA adipose P450arom mRNA expressions of the rats.The upper picture shows the gel electrophoresis of the RT-PCR products for the SA adipose P450arom. Densitometric analysis of the mRNA concentration using PCR product/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. \* p \< 0.05 vs corresponding intact controls, \# p \< 0.05 vs OVX1M. ::: ![](1477-7827-3-6-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### RT-PCR analysis of adrenal P450c17 mRNA expressions of the rats.The upper picture shows the gel electrophoresis of the RT-PCR products for the adrenal P450c17. Densitometric analysis of the mRNA concentration using PCR product/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. \* p \< 0.05 vs corresponding intact controls, \# p \< 0.05 vs OVX1M. ::: ![](1477-7827-3-6-3) ::: Western blot analysis: Effects of ovariectomy on liver and SA adipose P450arom protein levels --------------------------------------------------------------------------------------------- Densitometric analysis of the protein concentration using aromatase/β-actin was expressed as the mean with SEM. The ratios of liver P450arom to β-actin in the OVX groups were lower than the corresponding intact controls (p \< 0.05). But the ratio increased significantly in the OVX6M compared with OVX1M (p \< 0.05) (Fig [4](#F4){ref-type="fig"}). The ratios of SA adipose P450arom to β-actin in the OVX1M and OVX6M increased significantly compared with those in the INT1M and INT6M (p \< 0.05). And the ratios in the OVX2M, 3M, 4M and 5M produced no disparities compared with corresponding intact controls (Fig [5](#F5){ref-type="fig"}). No disparities were detected among INT1M, 2M, 3M, 4M, 5M and 6M (Fig [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}). No immunoreactive bands detected in the samples when using antiserum after preabsorption with excessive antigens and omission of the primary antibody. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Western blot analysis of liver P450arom expressions of the rats.The upper picture shows the Western blot analysis of the liver aromatase P450. Densitometric analysis of the protein concentration using aromatase/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. \* p \< 0.05 vs corresponding intact controls, \# p \< 0.05 vs OVX1M. ::: ![](1477-7827-3-6-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Western blot analysis of SA adipose P450arom expressions of the rats.The upper picture shows the Western blot analysis of the SA adipose aromatase P450. Densitometric analysis of the protein concentration using aromatase/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. \* p \< 0.05 vs corresponding intact controls, \# p \< 0.05 vs OVX1M. ::: ![](1477-7827-3-6-5) ::: Discussion ========== The most interesting findings in the present study were that the circulating E~2~levels increased gradually along with time after ovariectomy in the rats. If ovary is not the source of estrogen in ovariectomized rats, then the question arises as to the origin of the estrogens found in the circulation. Extragonadal aromatization has been in a general sense recognized, although its significance is only becoming to be appreciated, as will be explained further. It has been reported that aromatization in the adipose tissue is not negligible under normal and pathological conditions \[[@B9]\]. Due to the highest conversion of C19 precursor such as androstenedione to estrogen was observed in adipose tissue obtained from the subcutaneous abdominal wall but not from intraperitoneal cavity \[[@B10]\], our research focused on the aromatase activity of the SA adipose tissues. In the present results, increased aromatase protein and mRNA expressions were observed in the OVX6M rats\' SA adipose tissue. Hemsell and co-workers \[[@B11]\] first addressed the significance of adipose tissue as a major source of estrogen production, i.e. there is a progressive increase in the conversion efficiency with advancing age, and the increase of estrogen production as a function of obesity \[[@B12]-[@B14]\]. The body fat of ovariectomized rats increases significantly \[[@B15]\], and the mesenchymal cells from the SA adipose tissue could be active, which might be closely related to the higher aromatase expression in the OVX6M. Yet, only in the OVX6M was there an associated significant increase of the aromatase expression in the SA adipose tissue, but not in other OVX groups, which by itself suggested that the elevated expression of aromatase in the OVX6M was not closely implicated in the obesity of the rats. We have also observed that the content of hypothalamic CRH increased along with time after ovariectomy in the rats, with a significant elevation in the OVX6M (submitted data). It has been reported that CRH may regulate the expressions of extragonadal aromatase via CRH receptor-II \[[@B16]\], which may help us understand the involved mechanism of the enhanced SA adipose aromatization and the eventual increased E~2~in the OVX6M rats, but further studies are earnestly needed. However, in the present results, it\'s confusing that the protein but not mRNA expression of SA adipose aromatase in the OVX1M showed an elevation compared with that in the INT1M. This might provide evidence indicating the inconsistency on the levels between transcription and translation of the aromatase gene. The factors possibly involved in the regulation of aromatase expression are still poorly understood \[[@B17]\]. Many studies have been performed to assess the possible dependence of the enzyme on androgens, though the data available are conflicting \[[@B17],[@B18]\]. The situation is complicated by the fact that, in postmenopausal women, only about 25% of circulating T is derived by direct secretion from the ovaries \[[@B2]\]. The rest is formed largely from circulating precursors derived from the adrenal cortex. In the present study, the blood concentrations of T in the rats were detected. In the OVX groups there were decreases of the T level compared with corresponding intact ones, and no significant changes occurred along with the time after ovariectomy. These might be implicated in the less importance of T in the extragonadal aromatisation in rats than in women. Nevertheless, it\'s the first time to present the dynamic changes of T levels in the rats after ovariectomy. But further explorations are still needed on the way by which the aromatase enzyme expression is activated in ovariectomized rats. The microsomal enzyme cytochrome P450c17 is an important regulator of steroidogenesis. The enzyme has two functions: 17alpha-hydroxylase and 17,20-lyase activities. These functions determine the ability of adrenal glands and gonads to synthesize sex steroids (17,20-lyase activity) \[[@B19]\]. Its activity is abundant in testis, and lesser in ovary, and low levels of P450c17 activity in adrenals \[[@B20]\]. It is well known that adrenal is the principle organ to secrete sexual hormones except ovarian in females \[[@B21]\]. In the present study, due to the T levels did not show significant change along with the time after ovariectomy, the mRNA expressions of P450c17 were semi-quantified by RT-PCR to indirectly report the androgen synthesis in the adrenals. Excitedly, the ratio of adrenal P450c17 to β-actin in the OVX4M and OVX5M increased slightly, and in the OVX6M, the ratio increased markedly, which was higher than OVX1M. These suggested that the androgen synthesis activity of adrenal might be enhanced, and the more androgens might secrete from adrenal cortex. Though it has been reported that the splanchnic tissue is a minor site for extragonadal aromatization of androgens \[[@B22]\], there is a significant conversion of androstenedione to estrone by liver tissues \[[@B15]\]. In adult liver homogenates, C19 norsteroid (19-nortestosterone; NT) is readily aromatized to estrogens \[[@B23],[@B24]\]. The present results showed that the aromatase expressions (mRNA and protein) of the liver tissue in the OVX1M, 2M, 3M, 4M and 5M groups were lower than the corresponding intact controls, and in the OVX6M the ratio increased significantly compared with that in the OVX1M. On one hand, after ovariectomy, the diminution of C19 precursor from ovaries for aromatization may induce the decreased expressions of aromatase in the OVX rats compared with the gonad-intact rats in our results. On the other hand, the results suggested that aromatization of liver tissue enhanced six months after ovariectomy. However, in the present study, the aromatase expressions in the OVX rats did not show consistent changes between the SA adipose and liver tissues, which may suggest that the role of SA adipose and splanchnic tissues in the extraglandular aromatisation might be different. Nonetheless, in the final analysis, our results suggested that both the SA adipose and liver tissues contributed to the extragonadal aromatisation to promote the circulation estrogen concentrations. Conclusions =========== Both the subcutaneous abdominal adipose tissues and the liver tissues contributed to the extragonadal aromatization to promote the circulating E~2~levels in the rats along with time after ovariectomy; the adrenal compensation might also be activated naturally. Authors\' contributions ======================= Hong Zhao and Zhanzhuang Tian designed the study, performed the studies and the statistical analysis, and drafted the manuscript. Junwei Hao performed the animal experiment. Boying Chen conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Acknowledgements ================ This project was financed by the Shanghai Acupuncture Research Centre (No. 03DZ19554-3) and Shanghai Medical Health Bureau (the Key Project 20003 on Traditional Chinese Medicine).	PubMed Central	2024-06-05T03:55:52.640138	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548297/", "journal": "Reprod Biol Endocrinol. 2005 Jan 21; 3:6", "authors": [ { "first": "Hong", "last": "Zhao" }, { "first": "Zhanzhuang", "last": "Tian" }, { "first": "Junwei", "last": "Hao" }, { "first": "Boying", "last": "Chen" } ] }
PMC548298	Background ========== World Health Organization published the Global Tuberculosis Control Report (2003) on \'World Tuberculosis Day\' where India is ranked number one in the world for high incidence of smear positive cases of pulmonary (about 0.9 million) and extrapulmonary TB (about 0.2 million) every year \[[@B1]\]. Undoubtedly, TB itself has reemerged in India as a serious problem since 1985 with the advent of HIV/AIDS \[[@B2]\]. However, cutaneous TB is still rare in countries like India (0.10%), Hong Kong (0.07%) and Madrid (0.14%) \[[@B2]\] and it manifests either as a true bacterial invasion or as a tuberculid (hypersensitivity reaction) with primary focus elsewhere. Evidences of bacterial invasion are found in lupus vulgaris, the most common manifestation (55%) in patients with cutaneous TB (1975--95) in northern India \[[@B2]\], primary chancre, tuberculous verrucosa cutis, scrofuloderma, tuberculous cutis orificialis and tuberculous cutis miliaris disseminates \[[@B3]\] whereas erythema induratum (Bazin disease) and lichen scrofulosorum are tuberculid lesions \[[@B2]\]. Moreover, atypical mycobacteria such as M. kansasii, M. scrofulaceum, rather than M. tuberculosisare the most common etiological agents for cutaneous TB in HIV/AIDS and other immunocompromised patients. WHO defines acquired drug resistance as the isolation of drug-resistant M. tuberculosisfrom a patient who has been treated for TB for 1 month or longer \[[@B4]\] and primary drug resistance as the isolation of drug-resistant strain from a patient without a history of previous treatment. The selection of M. tuberculosiswith mutations conferring resistance to antitubercular drugs may result from poor management including the prescription of incorrect regimens and non-compliance with treatment. We report a rare case of reinfection with same but MDR M. tuberculosisin a polymyositis patient on immunosuppressive therapy resulting in multiple cutaneous abscesses and miliary TB. Case Presentation ================= A 23-year-old polymyositis patient with suggestive clinical symptoms, muscle biopsy, enzyme assay, needle EMG and NCV test was on immunosuppressive drugs such as corticosteroids at 1 mg/kg/day for 3 to 4 weeks, tapered gradually over a period of 10 weeks to 1 mg/kg every alternate days and methotrexate at 7.5 mg weekly with gradual increase to 25 mg weekly for a period of 1 year when he experienced slightly improved muscle power. He had single cutaneous abscess 6 months later on the right side of the neck with no sinus formation and cervical and axillary lymphadenopathy. He was HIV-negative with normal CXR and there was no AFB in three consecutive (spot-early morning-spot) sputum samples (induced in two occasions). However, the aspirated pus from cutaneous abscess and biopsy of the axillary lymph node showed plenty of M. tuberculosisas culture was confirmed by niacin and nitrate reduction tests, rapid bacteriophage assay (FAST PlaqueTuberculosis kit, BIOTEC Laboratories Ltd, UK) and RFLP analysis (figure [1](#F1){ref-type="fig"}) using species-specific probes for devR(Differentially expressed virulent gene) \[[@B5]\]. In RFLP analysis, the devRgene (Gene bank nucleotide sequence accession no. U22037) encodes a response regulator that is part of a two-component signal transduction system of M. tuberculosis. The authenticity of the amplified product was established by hybridization of immobilized PCR products to an internal oligonucleotide, devR1, mapping within the devRgene. The chromosomal DNA was prepared by chloroform-isoamyl alcohol DNA extraction and 4.5 μg of DNA from the isolate was restricted with PvuII. Separation of PvuII-restricted DNA by electrophoresis, Southern blot hybridization with a 513-bp PCR probes for devR(devRf, 5\' GGTGAGGCGGGTTCGGTCGC 3\'; devRr, 5\' CGCGGCTTGCGTCCGACGTTC 3\') and chemiluminescence detection were done according to the standard method recommended for the DNA fingerprinting of M. tuberculosis\[[@B6]\]. Histopathologically there were nonspecific necrosis with disintegrating polymorphs, very few granulomatous cells and no epithelioid cells in the biopsy specimen. USG of kidney, liver or spleen revealed no abscess. The strain was susceptible to the 4 first line drugs \[isoniazide (H), rifampicin (R), ethambutol (E) and pyrazinamide (Z)\] by BACTEC 460TB system. Treatment with 4-drug regimen (H = 300 mg/d, R = 450 mg/d, E = 800 mg/d and Z = 1.5 g/d) for 8 months (2EHRZ/6HR) \[[@B7]\] with first 2 months supervised showed clinical improvement. However, the patient returned 6 months later with multiple cutaneous abscesses, mainly on his back, left thigh and left arm and miliary mottling in CXR. He denied any irregularity in antituberculosis therapy. All immunosuppressive drugs (corticosteroids and methotrexate) were discontinued as he was readmitted. Isolates from 2 of 3 consecutive sputum samples and aspirated pus samples from all 5 completely drained abscesses were reconfirmed as the identical M. tuberculosisstrain by phage assay and RFLP analysis (figure [1](#F1){ref-type="fig"}). The isolate was resistant to 4 first-line drugs by BACTEC 460TB system and to 9 drugs, tested into Loewenstein Jensen media (Hi-media, Mumbai, India): H (1 μg/ml), H (10 μg/ml), R (20 μg/ml), R (50 μg/ml), E (2 μg/ml), E (10 μg/ml), Z (50 μg/ml, at pH 5.5), streptomycin (5 μg/ml), streptomycin (50 μg/ml), paraaminosalicylate (2.5 μg/ml), cycloserine (30 μg/ml), amikacin (700 μg/ml) and ciprofloxacin (12.5 μg/ml). Treatment was continued with H (5 mg/kg/d), R (10 mg/kg/d), Z (30 mg/kg/d), E (15 mg/kg/d), kanamycin (15 mg/kg IM 5 times weekly) and sparfloxacin (500 mg/d) for 18 months with first 2 months under supervision. A slow but complete clinical, microbiological and radiological cure after 1.5 year was followed up for another 16 months. Discussion ========== India is an endemic country for TB with an estimated 20,000 infectious cases and 1--3.3% of new cases of MDR TB every year \[[@B8]\]. Infections are common with more than one strain of M. tuberculosisduring the same episode (multiple infection), in different lesions (multiple infection) or during successive episodes (reinfection) in HIV-positive as well as -negative individuals \[[@B9]\]. In this study identical strains of M. tuberculosiswere isolated at 6 months interval, but contrary to the first, the strain in the second episode was resistant to all the first line and most of the second line antitubercular drugs. As far as could be ascertained, this is the first case of isolation of MDR strain of M. tuberculosisfrom a HIV-negative but immunosuppressed patient who had an infection with the same, but susceptible strain 6 months before. As evident from the study, the chance of exogenous reinfection with the same but drug-resistant strain from some undetected source within the endemic community is the most likely explanation. This explains the acquisition of resistance against all those drugs, to which the patient was not exposed earlier. Reinfection though occurs usually after first two to five years in immunocompetent hosts, may progress to active disease at any time after treatment has been discontinued and even during treatment for active tuberculosis \[[@B10]\]. Moreover, an ongoing tuberculous infection and simultaneous immunosuppressive therapy might significantly divert the immune response, thereby increasing the overall susceptibility to \'superinfection\' \[[@B10]\] with the same but MDR strain. The chances of \'simultaneous infection\' \[[@B11]\] with both the strains (susceptible and MDR) could be another possibility. Drug-susceptibility testing could discriminate simultaneous infection with different susceptibility profiles if individual colonies from the isolate were tested whereas in our case the sensitivity pattern of the susceptible strain might have been obtained at first attempt. Even RFLP and phage typing were not enough to determine simultaneous infections or reinfection (superinfection). Endogenous reactivation which is higher than the rate of exogenous reinfection in endemic countries \[[@B12],[@B13]\] with multi- \"drug resistance in previously treated case\"\[[@B14]\] might be a remote possibility. The history of regular medication itself might be notoriously misleading in patients with multidrug resistance \[[@B14]\]. However, it is difficult still to explain the acquisition of multidrug resistance of the strain in endogenous reactivation in such a short period against those drugs, to which the patient was not exposed. Any switch in specimens or cross-contamination of cultures in laboratories were unlikely since no other sample to switch or contaminate was identified. Niacin test (95% positive \[[@B15]\]) and nitrate reduction test (97% positive \[[@B15]\]) differentiated M. tuberculosisfrom other mycobacteria in M. tuberculosiscomplex whereas RFLP and phage typing excluded atypical mycobacteria, like M. kansasiiand M. scrofulaceum. The overall acuracy of the drug susceptibility test ranges between 84--100%, since mycobacteria often clump \[[@B16]\]. We tried thorough vortexing with glass beads to obtain homogenous inoculum suspensions. It was a rare case of miliary TB of non-reactive type \[[@B17]\] with MDR M. tuberculosisinvasion of the skin without any muscle involvement and sinus formation as the biopsy of the axillary lymph node showed non-specific necrosis containing disintegrating polymorphonuclear leukocytes and enormous number of AFB. This type is more often seen with severe HIV infection than in patients with immunosuppressive therapy \[[@B18],[@B19]\], where the liver and the spleen are most commonly involved followed by the lung, the bone marrow and the kidney \[[@B17]\], granulomas and epithelioid cells are lacking and CXR shows inconspicuous diffuse mottling. In our case, there was no other organ involvement. Moreover, the rapid skin and lung involvement despite adequate antituberculosis therapy suggests that the tubercle bacilli were somehow protected from the drugs in the cutaneous and subcutaneous tissue (\'paradoxical expansion of disease during therapy\' \[[@B20]\]) either due to polymyositis-associated subcutaneous calcification or due to obstruction in the small arteries with the large mass of bacilli leading to necrosis and abscess formation in the surrounding areas. This is probably the first reported case of cure where all first and most of the second line drugs (a total of 9 antitubercular drugs) were found to be resistant. However, the 6-drug antituberculosis therapy might not be considered as the only reason for cure, since 4 first line drugs showed high-level resistance and chances of cross-resistance between closely-related aminoglycosides, amikacin and kanamycin and quinolones, sparfloxacin and ciprofloxacin could not be ignored \[[@B21],[@B22]\]. Discontinuation of immunosuppressive therapy with a reconstitution of the immune system and complete surgical drainage of the abscesses might have proved to be an important adjunct to the treatment. Conclusions =========== Severe immunosuppression may lead to disseminated TB such as miliary TB or other rare types of extra-pulmonary TB such as cutaneous abscesses. Follow-up of patients is important, and if response to treatment is poor, adherence to treatment, drug resistance and other possible reasons such as continuation of immunosuppressive therapy should be considered. In this case intervention by drainage of abscesses, discontinuation of immunosuppressive treatment and possibly long-term treatment with additional second line antituberculosis drugs eventually lead to cure. Abbreviations ============= AFB -- Acid fast bacilli AIDS -- Acquired immunodeficiency disease syndrome CXR -- Chest radiograph DNA -- Deoxyribonucleic acid EMG -- Electromyography HIV -- Human immunodeficiency virus MDR -- Multidrug resistant M. tuberculosis- Mycobacterium tuberculosis NCV -- Nerve conduction velocity PCR -- Polymerase Chain Reaction RFLP -- Restriction-fragment length polymorphism TB -- Tuberculosis UK -- United Kingdom USG -- Ultrasonography WHO -- World Health Organisation Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= CM conceived the study, carried out the case study and follow-up, in the clinical as well as microbiological aspects, formatted the study design, performed sensitivity of the organism, participated in the FAST plaqueassay and molecular identification method and drafted the manuscript. AG carried out FAST plaqueassay and molecular identification method. AA participated in the design of the study and acted as an overall supervisor. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/4/63/prepub> Acknowledgements ================ We acknowledge the cooperation on behalf of the patient and his relatives and the helpful attitude on behalf of our clinician friends during the study. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The PCR amplified product for devRgene (513 bps) of M. tuberculosis. (From left to right) Lane M; 100 bp ladder Banglore Genei (India), Lane 1; Negative control, Lane 2; M. tuberculosisH37Rv, Lane 3; Patient\'s isolate of sensitive M. tuberculosisstrain (on first admission), Lane 4; Patient\'s isolate of MDR M. tuberculosisstrain (on readmission), Lane 5; M. kansasii, Lane 6; M. scrofulaceum. ::: ![](1471-2334-4-63-1) :::	PubMed Central	2024-06-05T03:55:52.642409	2004-12-23	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548298/", "journal": "BMC Infect Dis. 2004 Dec 23; 4:63", "authors": [ { "first": "Chiranjoy", "last": "Mukhopadhyay" }, { "first": "Ankita", "last": "Garg" }, { "first": "Archana", "last": "Ayyagari" } ] }
PMC548299	Background ========== Lipoprotein lipase (LPL) hydrolyzes triglycerides in very low-density lipoproteins and chylomicrons \[[@B1]\]. Tissue-specific regulation of LPL activity is a major mechanism to distribute lipids among tissues according to the physiological needs \[[@B2]\]. Current information indicates that in adipose tissue, the regulation is post-translational and occurs by a shift of the lipase protein towards an inactive form under the influence of another gene with short-lived message and product \[[@B3]\]. This information derives from in vivoexperiments. To study the underlying mechanism an in vitromodel is urgently needed. Experiments with isolated adipocytes do not seem to bring out the mechanism and the in vivoexperiments indicate that it is the extracellular LPL that is the target for the regulation \[[@B4]\]. We have therefore explored the possibility to use tissue explants and report our experiences in this paper. The results support the view that it is the extracellular enzyme that is being regulated. Unfortunately the preparation of tissue explants seems to trigger a proteolytic response in the tissue. Results ======= LPL activity and mass decreased when explants of rat adipose tissue were incubated ---------------------------------------------------------------------------------- In the first set of experiments we incubated explants of rat adipose tissue and followed LPL mass and activity (Figure [1](#F1){ref-type="fig"}). LPL mass decreased by more than 50% in three hours. The decrease then continued so that after six hours only around 20% of the original mass remained. With tissues from fed rats, in which most of the LPL protein is in the catalytically active form, the LPL activity decreased in parallel to LPL mass. In tissues from fasted rats, in which most of the LPL protein is in the catalytically inactive form, the decrease was less steep for LPL activity than for LPL mass. The inset in Figure [1](#F1){ref-type="fig"} shows that there was a delay of about two hours before the decrease of LPL mass accelerated. There was no difference between tissue from fed and fasted rats. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Changes in LPL mass and activity during incubation of adipose tissue explants.Epididymal adipose tissue was dissected from fed and 24 h-fasted rats, cut into small pieces and rinsed in cold PBS. About 100 mg tissue was then incubated at 37°C for the indicated times as described in the Methods section. LPL mass (triangles, panel A) and activity (circles, panel B) in tissue explants from fed (filled symbols, ● and ▼) or fasted (open symbols, ○ and ▽) rats. Values at the start of the incubations were for LPL mass: 17.4 ± 1.1 and 17.1 ± 2.5 ng/μg DNA and for LPL activity: 6.3 ± 1.8 and 3.5 ± 0.7 mU/μg DNA, in tissues from fed and fasted rats, respectively. The inset shows a separate experiment where incubations were stopped at shorter times. Only LPL mass was followed in that experiment. Some of the data points for fed and fasted rats fall on top of each other. Values are mean ± SEM for five parallel incubations. ::: ![](1471-2121-6-4-1) ::: We tried variations in technique and several different incubation media in experiments such as those in Figure [1](#F1){ref-type="fig"}. There was some variation in the absolute values but the results were in principle the same, a rapid decline of LPL mass and activity. The rate at which LPL mass decreased was similar with tissue explants from fed and fasted rats, and LPL activity roughly followed LPL mass in tissues from fed rats. One possible explanation could be that LPL was released from the tissue into the medium. The amounts of LPL mass or activity that appeared in the medium were, however, small (Table [1](#T1){ref-type="table"}). To test whether lipase might have adsorbed to the plastic dishes these were rinsed out with warm SDS solution, but only small amounts of LPL protein were recovered (Table [1](#T1){ref-type="table"}). Hence it is clear that there was a loss of LPL mass from the system. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Changes in LPL mass during incubation. Explants of rat adipose tissue were incubated for three hours as in Figure 1. The tissue explants and the medium were recovered and then the vessel was rinsed out with warm (\~80°C) SDS solution. This was then suitably diluted with Triton X-100 to match the composition of the medium used for the ELISA. Mean ± SEM of five parallel incubations. ::: LPL mass (ng/g tissue) ------------------ ------------------------ ------------------ Before incubation After incubation Adipose tissue 1882 ± 13 665 ± 11\* Culture medium 26 ± 6 Washing solution 12 ± 3 Total 1882 ± 13 702 ± 8 \* \* P \< 0.001 compared with before incubation ::: Another possibility was that the cells lost their ability to produce LPL. Isolated adipocytes, incubated under the same conditions as the tissue explants, released LPL activity (Figure [2](#F2){ref-type="fig"}) and mass (data not shown) to the medium, while cellular activity (Figure [2](#F2){ref-type="fig"}) and mass (data not shown) increased slightly. Total LPL activity in the system increased by about 60% during four hours of incubation. When cycloheximide was added, the release of LPL to the medium was virtually abolished and cellular LPL activity decreased with time. Total LPL activity in the system decreased by almost 70%. This demonstrates that the cells depend on synthesis of new LPL protein to sustain LPL activity and secretion to the medium. Adipocytes isolated from tissue explants that had been incubated for three or six hours as in Figure [1](#F1){ref-type="fig"} retained the ability to release LPL to the medium (not shown). Hence, the loss of LPL activity that occurred when tissue explants were incubated was not due to a loss of LPL production within adipocytes. In these experiments we also noted that the release of LPL from adipocytes was similar whether the cells were isolated from fed or fasted rats (not shown). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### LPL activity in cells and in medium during incubation of isolated adipocytes, and the effect of heparin.Adipocytes (from 180 -- 200 g rats) were incubated under the same conditions as used for the tissue explants in Figure 1 without (filled symbols) or with (open symbols) 0.1 mg/ml cycloheximide. ●, ○ -- adipocytes; ▼, ▽ -- medium. Mean ± SEM for five wells at each time. ::: ![](1471-2121-6-4-2) ::: Is the loss of LPL mass an intra- or extracellular event? --------------------------------------------------------- Heparin release is often used to assess the LPL activity of tissues and releases mainly extracellular LPL \[[@B4]\]. Figure [3](#F3){ref-type="fig"} shows that in fresh explants of adipose tissue a substantial fraction of tissue LPL could be released by heparin. More LPL was released from tissues of fed rats (Figure [3](#F3){ref-type="fig"}). When the tissue explants were incubated for three hours before the heparin challenge, much less LPL was released and after six hours virtually no LPL was released (Figure [3](#F3){ref-type="fig"}). This suggested that the decrease of LPL affected mainly the extracellular enzyme. To test this hypothesis we isolated adipocytes from tissue explants after three or six hours of incubation (Figure [4](#F4){ref-type="fig"}). The LPL activity and mass in the adipocytes was the same when the cells were isolated from tissue explants that had been incubated for three or six hours as when they were isolated from fresh tissue explants. Hence, it was extracellular LPL (calculated as the difference between tissue total and adipocytes) that accounted for the rapid decrease of tissue LPL during incubation. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Changes in the amount of heparin-releasable LPL mass during the incubation.Conditions as in Figure 1 but at the designated times, the explants of adipose tissue were transferred to new medium containing heparin and incubated for a further 45 min. Values are means ± SEM for five parallel incubations. Black bars represent explants from fed rats; grey bars represent explants from fasted rats. ::: ![](1471-2121-6-4-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### LPL activity and mass within and outside the adipocytes.Conditions as in Figure 1 but adipose tissue explants from 180--200 g rats were used to get enough material to isolate adipocytes. At the end of the incubation some of the tissue explants were incubated with collagenase and adipocytes were isolated as described in the methods section. Filled symbols -- fed rats; open symbols -- fasted rats. Panel A shows LPL mass. ▲, △ -- tissue, ▼, ▽ -- adipocytes. Panel B shows LPL activity. ■, □ -- tissue, ●, ○ -- adipocytes. Values are means ± SEM for five parallel incubations. Some of the data points for fed and fasted rats fall on top of each other. ::: ![](1471-2121-6-4-4) ::: Is the LPL protein degraded? ---------------------------- These results indicated that the decline of LPL mass occurred through proteolytic cleavage of the extracellular enzyme. To test this hypothesis, we included a cocktail of protease inhibitors in the medium used for incubation of tissue explants. This slowed down the decrease of LPL mass (Figure [5A](#F5){ref-type="fig"}). Chloroquine had no effect (Figure [5B](#F5){ref-type="fig"}), indicating that the degradation did not occur in lysosomes. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Effect of protease inhibitors on the decrease of LPL mass during incubation of tissue explants.Conditions as in Figure 1. A cocktail of protease inhibitors (panel A) or chloroquine (final concentration 150 mM, panel B) were included in the medium of some of the incubations. The tissue was from fed rats. Similar results (not shown) were obtained with tissue from fasted rats. ○ -- without protease inhibitor, ● -- with protease inhibitor. Mean ± SEM for five parallel incubations. ::: ![](1471-2121-6-4-5) ::: To further study the proteolytic process, ^125^I-LPL was added to the incubations. TCA soluble material appeared in the medium demonstrating that proteolytic degradation took place (Figure [6](#F6){ref-type="fig"}). We noted that some of the TCA precipitable material became associated with the tissue explants suggesting, binding and/or uptake of the lipase. To explore if the degradation required that the lipase was taken up into cells in the tissue we incubated the labelled lipase in conditioned medium. Analysis by SDS-PAGE showed that several fragments were formed (Figure [7](#F7){ref-type="fig"}). Addition of either of two non-specific MMP inhibitors, Captopril or GM6001, prevented the degradation almost completely. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Fate of ^125^I-LPL added to the incubation.Conditions as in Figure 1 but ^125^I-LPL was added to the medium. ● -- TCA precipitable radioactivity in medium, ▲ -- TCA precipitable radioactivity in the tissue explants, ■ -- TCA soluble radioactivity in medium. Mean ± SEM for five parallel incubations. ::: ![](1471-2121-6-4-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Analysis by SDS-PAGE of the cleavage of ^125^I-LPL in conditioned medium and the effect of an MMP inhibitorExplants of adipose tissue from fed rats were incubated as in Figure 1 for four hours and the medium was collected. ^125^I-LPL was then incubated at 4°C (to minimize the risk of conformational changes) in this medium with or without the MMP inhibitor GM6001 (5 μg/ml). Lane 1 -- fresh medium, lane 2 -- conditioned medium, lane 3 -- conditioned medium + inhibitor. The lower band in lane 1 is a proteolytic fragment of LPL that is always present in preparations of the enzyme from bovine milk \[23\]. ::: ![](1471-2121-6-4-7) ::: Discussion ========== The objective for this study was to find an in vitrosystem to study the mechanism for down-regulation of LPL activity in rat adipose tissue that occurs on food deprivation. Isolated adipocytes have been tried in several laboratories \[[@B5]-[@B9]\], but the differences with nutritional state are rather small. This is true whether one measures LPL within the cells or the rates at which the cells secrete LPL to the medium. These observations are repeated here. The lack of difference within adipocytes indicates that other cell types are needed and may in fact be responsible for the pronounced down-regulation of extracellular LPL activity that occurs on food deprivation \[[@B3],[@B4]\]. We therefore tried to use tissue explants, which have proved valuable in other studies of adipose tissue \[[@B10]\]. Our results show that degradation of the enzyme was a major process when explants were incubated. The degradation occurred extracellularly; LPL mass and activity in the adipocytes did not change during incubation for up to six hours. Added ^125^I-LPL was degraded and this was prevented by addition of MMP inhibitors to the medium. There must be damage of cells when the tissue is cut into small pieces, and there is probably some degree of hypoxia during incubation of the pieces. Cultured explants have, however, been widely and successfully used to explore various aspects of adipose tissue biology (\[[@B10]\] and references therein). In preliminary experiments we found that the explants retained their ability to take up glucose, to synthesize proteins and to secrete leptin. Adipocytes isolated from the explants after several hours of incubation had the same LPL activity as adipocytes from fresh tissue (Figure [4](#F4){ref-type="fig"}). This indicates that the cells produced LPL at an essentially unchanged rate, since the LPL activity in adipocytes decreased rapidly when protein synthesis was inhibited by cycloheximide (Figure [2](#F2){ref-type="fig"}). Hence, the rapid decrease of LPL when explants were incubated was not due to a general loss of functionality, but reflected specific processes leading to degradation of extracellular LPL. Our results are in line with observations made already in the sixties. There was evidence from chromatographic separations for at least two different forms of LPL in rat adipose tissue \[[@B11]\]. Cunnigham and Robinson found that incubation of fat pads from fed rats resulted in a rapid loss of LPL activity until a low activity, stable to prolonged incubation, was attained \[[@B12]\]. In contrast, the LPL activity of isolated fat cells was stable to prolonged incubation. The concept of stable and unstable forms of the lipase can now be interpreted as a reflection of extracellular lipase that is exposed to proteolysis, and intracellular lipase that is protected from the extracellular proteases. The degradation of LPL in conditioned medium was almost completely abolished by the two non-specific MMP inhibitors that we tested. We have not characterized the proteolytic activity further but note that adipose tissue produces at least two MMPs, 2 and 9 \[[@B13]\]. The loss of LPL mass during incubation of tissue explants was relatively slow during the first two hours and then accelerated. It is likely that the tissue trauma and/or the loss of blood circulation triggered an activation of the MMP system. It has been shown that primary culture of human adipose tissue explants dramatically alters adipocyte gene expression \[[@B14]\]. It is of interest to note that LPL activity does not decrease during perfusion of fat pads \[[@B15]\], whereas it does decrease when whole fat pads are cut out and incubated in vitro\[[@B12]\]. Two pathways for turnover of adipose tissue LPL have been demonstrated so far. One is dissociation of the lipase, perhaps after loss of catalytic activity, into the blood and degradation in the liver \[[@B16]\]. Release of LPL into blood from adipose tissue has been directly demonstrated by measurement of arterio-venous difference in man \[[@B17]\]. This pathway can, however, not operate in tissue explants. Another pathway, demonstrated with cultured fat cells, is endocytosis and degradation in lysosomes \[[@B18]\]. This pathway did not seem to contribute significantly in the present system since the rate at which LPL mass decreased was not affected by chloroquine. The present findings suggest a third pathway, extracellular proteolysis in the tissue. Conclusions =========== The rapid decrease of LPL that occurs when adipose tissue explants are incubated engages only the extracellular enzyme. The adipocytes continue to produce and secrete the enzyme and intracellular LPL remains essentially constant for at least six hours. The decrease in extracellular LPL is due to proteolytic cleavage/degradation of both active and inactive forms of the enzyme. The effects of inhibitors indicate, but do not prove, that the degradation is mediated by MMPs. It appears that this process is accelerated in the tissue fragments compared to intact tissue. Methods ======= Animals ------- Male Sprague-Dawley rats were from Möllegaard Breeding Center (Ejby, Denmark). Unless otherwise stated, the rats were 23 days old and weighed around 60 g. After transport to Umeå they were allowed to acclimatize for seven to ten days by which time they had reached a weight of approximately 120 g. The rats were kept in a well ventilated, temperature (21°C) and humidity (40--45%) controlled room with free access to a standard laboratory chow (Laktamin AB, Stockholm) and tap water. The light in the room was on between 6 a.m. and 6 p.m. In experiments where the rats were to be fasted, food was withdrawn from the cages at 6 a.m. and a grid was placed at the bottom of the cages to prevent coprophagia. The adipose depot used in all experiments was the periepididymal one. The rats were killed by decapitation. Animal experiments were approved by the animal ethics committee in Umeå. Materials --------- Cycloheximide, bovine serum albumin (BSA), the MMP inhibitors GM6001 (Galardin) and Captopril, chloroquine and collagenase were from SIGMA (St. Louis, MO, USA). Protease inhibitor cocktail tablets \"Complete Mini\" were from Roche Diagnostics, Mannheim, Germany. Heparin was from Lövens (Malmö, Sweden). Substrate for the LPL activity assay was ^3^H-labelled triolein in Intralipid (10%) kindly prepared by Pharmacia-UpJohn (Stockholm, Sweden). Parker medium (Parker 199) was from SBL (Stockholm, Sweden). ^125^I-LPL was prepared as before \[[@B19]\]. All other reagents were of the highest commercial grade possible. Assays ------ LPL was extracted from tissues by homogenization in a Tris-HCL buffer (pH 8.2) containing detergents and protease inhibitors as described \[[@B20]\]. The homogenate was centrifuged for 15 min at 3000 rpm after which the intermediate phase (between the floating fat droplets and the pellet) was used for assay of LPL activity and mass. In most cases the extract was kept on ice and assayed within a few hours. Under these conditions LPL activity is stable. In some cases the extracts were frozen and kept at -70°C for later assay. LPL activity was measured as described previously \[[@B20]\]. Briefly, two μl of tissue homogenate (triplicate samples) was incubated for 60 min at 25°C with substrate in the presence of ten μl heat-inactivated serum from fasted rats (as source of apolipoprotein CII) and 6% BSA. The total volume was 200 μl. After termination of lipolysis by addition of organic solvents, the fatty acids were extracted and counted for radioactivity. One mU of lipase activity represents one nmol of fatty acids released per minute. LPL mass was measured with an ELISA as described \[[@B20]\]. The chicken antibodies used recognize both active and inactive forms of the lipase \[[@B21]\]. Briefly, three different dilutions of tissue homogenate were incubated in microtiter plate wells previously coated with affinity-purified chicken anti-LPL IgG. Detection was mediated via the 5D2 monoclonal antibody (a kind gift by Dr John Brunzell, University of Washington, Seattle) followed by a peroxidase conjugated anti-mouse IgG antibody. Absorbance at 490 nm was measured in a Spectramax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). DNA content was assayed using Labarca\'s method \[[@B22]\]. In vitroincubation of adipose tissue -------------------------------------- Epididymal adipose tissue was dissected out from fed or 24 h fasted rats. The tissue was cut into small pieces (5 mg or less). A total of about maximal 100 mg tissue pieces were immediately put into culture plates. Each well contained 15 ml of Parker Medium 199 supplemented with 2% BSA, 0.5% casein hydrolysate, 10 mM glucose and adjusted to pH 7.4. Incubations were at 37°C and 5 % CO~2~: 95 % O~2~with continuous gentle shaking motion in a Cellstar Incubator (Queue Systems, Asheville, Canada). After incubation, the tissues were prepared for measurement of LPL activity and mass as described above. In some experiments samples of the medium were taken for assay of LPL activity and/or mass. In some experiments adipocytes were prepared after collagenase treatment of the tissue pieces as described \[[@B4]\]. To measure heparin releasable LPL (HR-LPL), adipose tissue explants were incubated with heparin (final concentration was 50 IU/ml) for 45 min at 37°C. Statistics ---------- Student\'s t-test was used for analysis of the data. Authors\' contributions ======================= GW carried out the experiments and participated in their design and in writing of the manuscript, TO participated in the design of the study and drafted the manuscript. GO conceived of the study and coordinated the work. All authors read and approved the final manuscript. Acknowledgements ================ This study was funded by the Swedish Medical Research Council Projects no. 727, 12203 and 12663.	PubMed Central	2024-06-05T03:55:52.643966	2005-1-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548299/", "journal": "BMC Cell Biol. 2005 Jan 25; 6:4", "authors": [ { "first": "Gengshu", "last": "Wu" }, { "first": "Gunilla", "last": "Olivecrona" }, { "first": "Thomas", "last": "Olivecrona" } ] }
PMC548300	Background ========== The coalescent is a very versatile stochastic model of the genetic variation in a set of sequences sampled from a population. It allows to accommodate a wide range of assumptions about rates and modes of evolution, and of population history \[[@B1]-[@B5]\]. As the observed sequence data are positively correlated due to common ancestry, coalescent theory also provides a framework for understanding the relationship between a population\'s history and its genealogy. For instance, it has long been noted that genealogies of samples taken from exponentially growing populations tend to be \"star-like\" with short branch lengths near the root of the tree. In contrast, the inter-node distances in genealogies from constant-size populations typically are much more evenly spaced. Thus, coalescent theory quantifies the imprint that demographic development of a population leaves in the data. While the original theory was outlined for constant population size \[[@B1],[@B2]\], Slatkin and Hudson \[[@B6]\] soon developed a coalescent model for the case of an exponentially growing population. Subsequently, a general approach allowing arbitrary population size variation through time was presented by Griffith and Tavaré \[[@B7]\]. Therefore at least in principle the coalescent model provides a basis for statistically inferring the demographic historyas a function of time from the sampled sequences \[[@B3],[@B8]-[@B12]\] or, alternatively, from the corresponding inferred genealogies \[[@B13]-[@B15]\]. In practice, however, application of coalescent theory to this problem has been restricted to very simple demographic scenarios such as constant size, exponential or logistic growth. Only recently methods have emerged that attempt the completely non-parametric estimation of the demographic function from the data. Polanski et al. proposed an approach based on pairwise distances \[[@B16]\], hence generalizing the method by Slatkin and Hudson \[[@B6]\]. Pybus et al. \[[@B14]\] presented the \"skyline plot\" method that uses a step-function to approximate the population history obtained from an estimated genealogy. This method was subsequently refined to the \"generalized skyline plot\" \[[@B17]\] which is essentially a regularized version of the classic skyline plot. If the population size is truly constant through time the generalized skyline plot estimate of population size collapses to the phylogenetic coalescent estimator proposed by Felsenstein \[[@B13]\]. The advantage of the skyline plot over the method suggested by Polanski et al. \[[@B16]\] is that it takes into account the genealogical relationship among the sequences. This helps to decrease bias and improves the efficiency of the resulting estimator compared to methods based on summary statistics and pairwise distances \[[@B13]\]. Unfortunately, the skyline plot approach also has several deficiencies. First, it is unclear how to extend the approach to allow multiple genealogies as input. This is important in order to accommodate phylogenetic error, and to allow non-parametric inference of population history in coalescent approaches that take all possible genealogies into account \[[@B7]-[@B10]\]. Second, and perhaps more critical, the (generalized) skyline plot only provides a population size trend rather than a realistic estimate of population size changes, as by construction the population function is modeled by a step function. Moreover, the change-points of this function are fixed at the inter-nodes of the underlying tree. In this paper we propose a novel framework to non-parametric estimation of the demographic history. This approach relies on Bayesian reversible-jump MCMC inference \[[@B18]\] to obtain a smooth population size function from a given set of genealogies. The new method not only renders many deficiencies of the classic and generalized skyline plot obsolete but it is also computationally efficient, with running times of the algorithm for typical data in the order of minutes on standard PC hardware. The framework has been implemented in the computer language R \[[@B19]\] and incorporated in the R package APE \[[@B20]\]. The remainder of the paper is organized as follows. In the next section we describe the mathematical and statistical theory of the new framework. Subsequently, we apply the method to simulated and biological sequence data and discuss the results. In the last section we briefly outline possible further extensions and related directions of research. Results ======= Background in coalescent theory ------------------------------- ### Basic model In a pan-mictic population with constant effective population size N~e~, where every individual has a single parent, the waiting time w~n~until any two of nsampled lineages coalesce is exponentially distributed with rate ![](1471-2148-5-6-i1.gif)\[[@B1],[@B2]\]. For nsequences there are therefore n- 1 intervals I~n~, I~n-1~,\..., I~2~with rates r~n~, r~n-1~,\..., r~2~and interval lengths w~n~, w~n-1~,\..., w~2~. With ![](1471-2148-5-6-i2.gif) we denote the time until all samples have reached the most recent common ancestor. The coalescent model implies that the waiting time to the next coalescent event follows an inhomogeneous Poisson-process with a hazard rate r~n~that varies in time tbecause of the change in the number of lineages. Thus, it is straightforward to also include variable population size in the coalescent simply by using the hazard rate ![](1471-2148-5-6-i3.gif). From standard theory in survival analysis \[[@B21]\] it follows that the corresponding density for the waiting times is given by ![](1471-2148-5-6-i4.gif) where τ~i~is the time at the beginning of the interval I~i~. This is exactly the distribution from the variable population size coalescent ![](1471-2148-5-6-i5.gif) as developed in \[[@B7]\]. The coalescent model can be further expanded to diploid populations \[[@B22]\] or to include other effects like selection, recombination or geographical structures \[[@B4]\]. In this paper, however, we focus solely on the coalescent/survival model given by Eq. 2. ### Estimation of population size If the waiting times w~i~are known Eq. 2 can be used directly to estimate N~e~(t). This is typically done by maximizing the likelihood ![](1471-2148-5-6-i6.gif) assuming a simple parametric model for the population size change. For constant population size this has been done in \[[@B13]\], for more complicated scenarios such as logistic growth see, e.g., \[[@B14]\]. In a typical setting, however, the waiting times are themselves estimated from sequence data. In this case the total likelihood function will be a weighted sum of the likelihoods for all possible waiting times, so that in effect the w~i~are marginalized out in favor of the actually observed data. In practice exact calculation of this sum is prohibitive, hence one relies on approximating MCMC methods \[[@B8]-[@B10]\]. As a shortcut to avoid these computationally very expensive procedures one may also substitute the \"true\" waiting times by those obtained from inter-node distances of a single estimated gene tree (see, e.g., \[[@B23]\] for an overview of relevant likelihood-based tree inference methods) and proceed as above. Note that the resulting plug-in approximation ignores the uncertainty from estimating the w~i~in the inference of demographic parameters. However, this is justifiable if the phylogenetic error is much smaller than the error introduced by the coalescent. This will be the case if sequences are sufficiently long and the substitution rate is comparatively high (a typical example would be virus data). For non-parametric estimation of population size, Pybus et al. suggested the \"skyline plot\" \[[@B14]\]. This method assumes a piece-wise constant function for the population size N~e~(t) and allows population size changes only at the beginning and end of an interval I~i~. The estimated effective population size ![](1471-2148-5-6-i7.gif) in interval I~i~according to the skyline plot is given by the simple relation ![](1471-2148-5-6-i8.gif) This is the maximum likelihood estimate under the assumed model of fixed change-points. The \"generalized skyline plot\" subsequently introduced by Strimmer and Pybus \[[@B17]\] reduces the over-fitting present in the classic skyline plot by applying a simple form of regularization: adjacent intervals that alone are likely to have high stochastic noise are pooled together (cf. Fig. [2b](#F2){ref-type="fig"} and [2d](#F2){ref-type="fig"}). Choice of an optimal grouping of intervals (i.e. model selection) is performed by employing a second-order variant of the Akaike criterion \[[@B24]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Comparison of prior and posterior demographic functionsTop row: Bayesian inference using a prior demographic function with constant mean and constant variance (a 95% confidence band is indicated by showing the 2.5% and 97.5% quantiles). Bottom row: Bayesian inference using the \"skyline plot\" prior function. ::: ![](1471-2148-5-6-2) ::: A Bayesian non-parametric approach to estimating demographic history -------------------------------------------------------------------- ### Outline In this paper we present a non-parametric approach to infer population size changes in time that overcomes the limitations of previous approaches. More specifically, we develop a non-parametric Bayesian estimator for the function N~e~(t) conditioned on observed or sampled inter-node distances w~n~, w~n-1~,\..., w~2~by determining the posterior distribution P(N~e~(t)\\|w~n~, w~n-1~,\..., w~2~). In order to sample the non-parametric demographic function from this posterior we use the reversible jump Markov chain Monte Carlo (rjMCMC) algorithm \[[@B18]\]. As a result, we obtain for any given time tboth a point estimate ![](1471-2148-5-6-i9.gif) -- here we choose the posterior median -- as well as the associated credible interval (e.g., the lower and upper 2.5% quantiles). If the considered inter-node distances w~n~, w~n-1~,\..., w~2~are fixed and obtained from a single estimated tree, the resulting method is already directly applicable to phylogenetically informative data such as viral sequences (this is the focus of this paper). However, sampling of non-parametric demographic functions can also be combined in a conceptually straightforward fashion with sampling of trees, as outlined below. ### Bayesian inference using reversible jump MCMC In a nutshell, Bayesian inference of a parameter xconsists of updating its prior distribution P(x) to a posterior distribution P(x\\|D) that takes account of the information in the observed data D. The relative evidence of the data for different values of xis summarized in the likelihood L= P(D\\|x) that accordingly plays a central role in the computation of the posterior via Bayes\' theorem ![](1471-2148-5-6-i10.gif) For most realistic problems the posterior distribution cannot be computed analytically, in particular if xis a high-dimensional vector. Instead, one utilizes computational procedures to efficiently draw random samples from the posterior. This in turn allows computation of summary statistics such as the median or the upper and lower 2.5% quantiles. Markov chain Monte Carlo (MCMC) is one particularly useful sampling algorithm as it doesn\'t require calculation of the sum (or integral) in the nominator of Eq. 4. Briefly, sampling via MCMC is done by constructing a Markov chain with the possible combinations of parameter values as \"states\", and the desired posterior as its stationary distribution. These properties can be guaranteed by following certain rules for accepting or rejecting proposed new parameter values. Here we use the Metropolis-Hastings-Green method, i.e. the reversible jump MCMC algorithm \[[@B18]\], that has the advantage of not only allowing changes in the parameters values but also in the dimension of the parameter vector itself. Specifically, if xis the initial state, and ![](1471-2148-5-6-i11.gif) a proposed new state with proposal density ![](1471-2148-5-6-i12.gif), then the acceptance probability according to Green \[[@B18]\] is ![](1471-2148-5-6-i13.gif) where ![](1471-2148-5-6-i14.gif) is the likelihood ratio P(D\\|![](1471-2148-5-6-i11.gif))/P(D\\|x), ![](1471-2148-5-6-i15.gif) is the prior ratio P(![](1471-2148-5-6-i11.gif))/P(x), ![](1471-2148-5-6-i16.gif) is the proposal ratio q(![](1471-2148-5-6-i11.gif))/q(x), and ![](1471-2148-5-6-i17.gif) is the determinant of the Jacobian resulting from the potential change of dimension of the parameter vector. Accordingly, for the application of MCMC to infer the functional form of demographic history a variety of components need to be specified: • a suitable parameterizationof the estimated function N~e~(t) • the likelihood function, • a prior distributionfor each considered variable, and, • rules to construct the Markov chain(i.e. acceptance probabilities). In the following sections we now describe each of these elements in detail. For further general information on the statistical and mathematical background of the MCMC algorithm we refer to the many excellent monographs on this topic (e.g., \[[@B25]\]). ### Parameterization of N~e~(t) In our suggested procedure we approximate the sampled demographic history N~e~(t) by a piecewise linear function. This spline of first order degree consists of a first node at position a~0~= 0 and height h~0~, followed by kinternal supporting nodes at (a~1~; h~1~), (a~2~;h~2~),\..., (a~k~; h~k~), and a terminal node at ![](1471-2148-5-6-i18.gif) with height h~k+1~Hence, the spline is defined for all t∈ \[0, T\], and for any given kthe it contains kfree position parameters and k+ 2 free height parameters. Note that, unlike in the skyline plot, we do not constrain the change-points a~1~,\..., a~k~to lie on the grid points defined by the inter-node distances w~i~. Moreover, we also allow that the number of internal nodes kchanges during sampling of the population function from the posterior. Hence, kis technically a hyper-parameter that controls the roughness of the resulting spline. As will be clear from the outline of the MCMC algorithm below, note that the final point estimate ![](1471-2148-5-6-i9.gif) obtained from posterior sampling will be a mixture of linear splines (i.e. a smooth and possibly nonlinear function) rather than a single spline. ### Likelihood function The likelihood Lemployed in our procedure is the product of the densities of the waiting times between subsequent coalescence events, i.e. ![](1471-2148-5-6-i19.gif). This function depends via Eq. 2 on the effective population size N~e~(t), and hence indirectly on the spline parameters a~i~, h~i~and k. Because N~e~(t) is represented by a linear spline, calculation of the likelihood can be done in a computationally efficient fashion. ### Prior distributions #### Number of change-points Following \[[@B18]\] we employ a truncated Poisson-distribution as the prior distribution for k, i.e. ![](1471-2148-5-6-i20.gif) where cis a normalizing constant to ensure that P(k) is a proper distribution. For the hard upper limit of the number of change-points we use k~max~= 30. The parameter λacts as a smoothing parameter, set in a typical analysis to about λ= 0.1 - 1.0. As an alternative to using a fixed λwe also suggest a hierarchical Bayes approach where λis drawn from a Gamma distribution ![](1471-2148-5-6-i21.gif) with some shape parameter aand scale parameter b(for instance, a≈ 0.5 and b≈ 2 so that E(λ) = ab≈ 1 and Var(λ) = ab^2^≈ 2). #### Positions We assume that the internal nodes of the spline are a prioriuniformly distributed in the interval \[0, T\]. As a simple trick to avoid very small inter-node distance we generate 2k+ 1 random variables, and set the change-points a~j~= z~\[2j\]~for j= 1,\..., k. The corresponding joint density is ![](1471-2148-5-6-i22.gif) with a~0~= 0 and a~k+1~= T. #### Heights As prior distributions for the heights h~i~we assume a Gamma distribution Gamma(h~i~\\|α~i~, β~i~) (9) which ensures that sampled heights are always positive. The parameters α~i~and β~i~determine the a priorimean and variance of height h~i~. More generally, one can also allow fully time-dependent prior parameters α(t) and β(t). This is particularly advisable if the population size is known in advance to be subject to large changes in time. In a strict Bayesian approach, the choice of the prior distribution for the heights is completely external to the observed data. One simple possibility would, e.g., be to assume an arbitrary constant for the mean and variance. However, we recommend to follow a more pragmatic \"empirical Bayes\" route and to use the data at hand (or some other related data set) to obtain an informed guess about the prior heights. For example, an assumed constant population size as prior mean could be estimated using the method by Felsenstein \[[@B13]\]. Another possibility is to employ the skyline plot as a prior mean estimate (this is the default in our program). However, note that in practice the actual choice of prior height distribution seems to matter only little for estimating the posterior demographic function (see Figure [2](#F2){ref-type="fig"} and the section on simulated data below). Only when there are few coalescent events per unit of time will the posterior estimate of the demographic function be dominated by the prior. ### Construction of the Markov chain There are four different possibilities to change the state defined by the parameters c~i~, h~i~, and kof the spline describing the effective population size N~e~(t): 1\. varying the position of a change-point (i.e. internal node), 2\. changing the height at a certain change-point, 3\. generating a new change-point (\"birth\" step), and 4\. deleting an existent change-point (\"death\" step). Let η~k~, π~k~, b~k~, and d~k~the probabilities of the four moves given k, with η~k~+ π~k~+ b~k~+ d~k~= 1. In order to satisfy the requirement of detailed balance in the corresponding Markov chain the probabilities of birth and death steps (b~k~and d~k~) need to be synchronized \[[@B18]\]. This can be achieved, e.g., by setting ![](1471-2148-5-6-i23.gif) and ![](1471-2148-5-6-i24.gif) where cis chosen so that b~k~+ d~k~\< 0.9 for all k. Next, we describe the individual procedures to propose and accept one of the above four moves as implemented in our program. #### Height change First, a height h~j~is selected out of the k+ 2 existing heights with probability ![](1471-2148-5-6-i25.gif). Second, a new height ![](1471-2148-5-6-i26.gif) is generated by ![](1471-2148-5-6-i26.gif) = h~j~exp(z), where zis a uniformly distributed random variable on ![](1471-2148-5-6-i27.gif). Third, the new height is accepted with probability ![](1471-2148-5-6-i28.gif) where αand βare from the prior distribution and ![](1471-2148-5-6-i14.gif) denotes the ratio of the likelihood of the new state ![](1471-2148-5-6-i11.gif) (with modified height) and the likelihood of the current state x. #### Position move First, a change-point a~j~is chosen randomly with probability ![](1471-2148-5-6-i29.gif). Second, its new position ![](1471-2148-5-6-i30.gif) within \[a~j-1~, a~j+1~\] is determined by drawing from the corresponding uniform distribution. Third, ![](1471-2148-5-6-i30.gif) is accepted with probability ![](1471-2148-5-6-i31.gif) #### Birth step First, the position a\* of the new change-point is found by uniformly drawing from (0, L), and let the neighboring nodes left and right of a\* have positions a~j~and a~j+1~. Second, the corresponding new height h\* is generated by randomly disturbing the current height N~e~(a\) on the position a\ according to N~e~(a\) + zN~e~(a\) where zis a uniformly distributed random variable on the interval ![](1471-2148-5-6-i27.gif). Note that the birth step increases the dimension of the parameter vector from 2k+ 2 to 2k+ 4 as a new change-point and a new height are generated. The corresponding acceptance probability of the birth step is computed according to Eq. 5 with likelihood and prior ratios as above, and with proposal ratio ![](1471-2148-5-6-i32.gif) and Jacobi determinant ![](1471-2148-5-6-i33.gif) #### Death step This is the inversion of the birth step and consists of removing a change-point. First, a\* chosen from a~1~,\..., a~k~with probability ![](1471-2148-5-6-i29.gif). Second, the corresponding height h\* is also removed from the vector of spline parameters. The acceptance probability for the death step is ![](1471-2148-5-6-i34.gif) where the proposal ratio and the Jacobi determinant is the same as for the birth step. ### Computation of estimated N~e~(t) and associated confidence intervals In order to obtain an estimate of the effective population size in time we now proceed as follows. First, the Markov chain is started with an initial state that corresponds to a completely flat demographic function, i.e. N~e~(t) = c, where c is some rough estimate of population size, and k= 0. Second, 100,000 repeats of the MCMC algorithm are performed, of which the first 5,000 are ignored to allow for a \"burn-in\" period. Third, the remaining samples are thinned out by a factor of 1:50 to remove auto-correlation. As a result, 1900 independent samples from the joint posterior of the spline parameters a~i~, h~i~and kare obtained. Subsequently, in order to obtain a point estimate ![](1471-2148-5-6-i9.gif) and associated confidence bands we compute the distribution of the effective population size at a number of fixed equidistant time points t~1~, t~2~,\..., t~1000~∈ \[0, T\]. Finally, we report as summary statistics the corresponding median and the lower and upper 2.5% quantiles. ### Extension to multiple genealogies In this paper we have introduced non-parametric sampling of demographic histories assuming a fixed underlying genealogical tree (or equivalently, a fixed set of inter-node distances w~n~, w~n-1~,\..., w~2~.) However, in our approach -- unlike previous non-parametric methods such as the skyline plot -- it is also conceptually straightforward to incorporate phylogenetic error. This can be done by joint sampling of trees and demographies according to the following simple algorithm: 1\. Given sequence data D, sample a tree G\* with clock-like branch lengths (see, e.g, refs. \[[@B8],[@B9],[@B11],[@B12],[@B26]\] for suitable methods). 2\. Use the method described in this paper to sample the demographic function conditioned on the inter-node distances ![](1471-2148-5-6-i35.gif) from G\. 3\. Repeat steps 1 and 2 to obtain the posterior distribution for the population size function, now conditioned on Drather than on some given w~n~, w~n-1~,\..., w~2~. Note that each sampled tree may have a different depth ![](1471-2148-5-6-i36.gif). This means that the interval \[0, T\] for the prior (and posterior) height distribution has to be set in advance (and independent of the T\). For the case of 0 \<t\<T\* sampling of heights then proceeds as described above, while for T\* \<t\<T-- the region with no data from a given sampled tree -- the heights are simply drawn from the respective prior distribution. Discussion ========== In order to test the potential of the proposed reversible jump MCMC algorithm we first applied it to synthetic data simulated according to various demographic scenarios. Subsequently, we reanalyzed two viral data sets from Central Africa and Egypt. Computer program ---------------- The proposed framework has been implemented by us for the case of a single underlying genealogy. The program is written in the statistical computer language R \[[@B19]\] and is incorporated in recent versions of the R package APE \[[@B20]\]. To install the APE package, simply run the R program, and enter at the R prompt install.packages(\"ape\") This downloads the APE package from the Internet. The proposed reversible jump MCMC approach is implemented in the function \"mcmc.popsize\" of which an extensive description along with examples can be obtained online by typing library(\"ape\") help(mcmc.popsize) into the R command window. The APE package also includes routines for plotting the inferred population function (e.g., all figures in this paper were prepared with APE). Note that the use of this R program is only valid if the phylogenetic error is low -- this is typically the case when the evolutionary rate is high and the available sequences are long (e.g. viral data). If the phylogenetic error is not negligible compared to the coalescent error, please use software such as BEAST \[[@B27]\]. Simulated data -------------- In the simulation setup we followed Pybus et al. \[[@B14]\] and Strimmer and Pybus \[[@B17]\]. Specifically, we performed simulations assuming constant population size (N~e~(t) = 100) as well as exponential population growth (N~e~(t) = l000e^-t^), using 25 and 100 sampled lineages, respectively. To estimate the population size function we employed the proposed MCMC algorithm and the classic and generalized skyline plot. In the former the smoothing parameter λwas drawn from the hierarchical model with default parameters (a= 0.5 and b= 2). Figure [1](#F1){ref-type="fig"} shows the results from a typical run of the simulations. The top row illustrates the case of constant population size, whereas the bottom row demonstrates exponential growth. On the left in Figure [1](#F1){ref-type="fig"}, top row, the true underlying constant population size is shown (the thick dashed line), together with the estimate provided by the classic skyline plot. On the right, this is contrasted with the estimate obtained by using our reversible jump MCMC algorithm. Clearly, the median of the posterior distribution of N~e~(t) is a very good point estimator of the true demographic history. In addition, the 95% confidence band is also automatically obtained by the MCMC method. Interestingly, it can be immediately seen that the uncertainty in N~e~(t) increases with a growing distance from the present. This simply reflects the fact that near the root of the tree for constant population size there are only few coalescent events. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Simulated dataTop row: Example of a simulation with constant population size: (left) true demographic history (dashed line) and estimate obtained with the classic skyline plot; (right) point estimate obtained with rjMCMC and 95% confidence band. Bottom row: Example with exponential population growth: (left) true population growth and classic skyline plot; (right) results from rjMCMC approach. ::: ![](1471-2148-5-6-1) ::: In Figure [1](#F1){ref-type="fig"}, bottom row, an example for a simulation with an exponentially growing population is shown. As for the constant population, the rjMCMC algorithm is capable of recovering the original population size function (shown as thick dashed line) complete with confidence bands, whereas the skyline plot contains a large amount of stochastic noise, and only provides a rough exploratory picture of the population size changes. In Figure [2](#F2){ref-type="fig"} the influence of the choice of prior demographic function on the final posterior estimate is investigated using further simulations of an exponentially growing population. The left column depicts the prior distributions (specifically the 2.5%, 50% and 97.5% quantiles for each time point) for two typical cases: a constant prior function (= constant population size with constant variance), and the \"skyline plot\" prior function (= time dependent piecewise- constant population size and variance). The right column of figure [2](#F2){ref-type="fig"} presents the corresponding posterior distributions as obtained with the present rjMCMC approach. The results for both cases are very similar. This indicates that there is sufficient signal in the data to make the posterior demographic function (almost) independent from the choice of prior distribution. Note that only near the left and right end of the investigated time intervals there are some slight differences. These can be explained by the lack of data points near the borders. HIV-1 in Central Africa ----------------------- Next, we applied our method to infer the demographic history from a set of HIV-1 sequences from Central Africa. These data was originally used by Vidal et al. \[[@B28]\] who examined the genetic diversity of HIV-1 type M in this region. Further detailed analysis can be found in Rambaut et al. \[[@B29]\] and Yusim et al. \[[@B30]\]. Here we use the reconstructed phylogeny of Yusim et al. with which Strimmer and Pybus also estimated the demographic history by means of the generalized skyline plot \[[@B17]\]. Figure [3](#F3){ref-type="fig"} shows the result of the analysis with the reversible jump MCMC algorithm compared with the predictions from the classic and generalized skyline plots. As in Yusim et al. \[[@B30]\] an evolutionary rate of 0.0023 substitutions per year was assumed to convert the time axis into units of years. The first row of Figure [3](#F3){ref-type="fig"} displays the tree of Yusim et al. \[[@B30]\] and the corresponding classic skyline plot. The latter exhibits a large amount of noise, nevertheless the main demographic signal is clearly visible in the graph. In contrast, in Figure [3c](#F3){ref-type="fig"} (second row) the effective population size as estimated by the rjMCMC algorithm is displayed. The thick line shows the median and the thin lines the 95% confidence interval. Especially in the middle part of the figure, where most of the data is located, the confidence interval is very narrow, indicating a stable estimation of the demographic history. Also note that for this data the average number of change-points in the MCMC run was k= 9.25, i.e. the estimated effective degree of freedom is much less than that implicitly assumed in the classic skyline plot. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### HIV-1 in Central AfricaTop row: a) underlying genealogy; b) classic skyline plot. Bottom row: c) population size function estimated with rjMCMC and corresponding 95% confidence band; d) comparison rjMCMC versus generalized skyline plot. ::: ![](1471-2148-5-6-3) ::: A comparison with the generalized skyline plot \[[@B17]\] is shown in Figure [3d](#F3){ref-type="fig"}. This demonstrates that the generalized skyline plot, in contrast to its classic cousin, provides a very good noise-reduced approximation to the demographic history as estimated by the reversible jump MCMC approach. However, especially near the present the step function employed in the generalized skyline plot leads to unrealistic jumps in the population size that are not present in the smooth estimate provided by the proposed MCMC method. HCV in Egypt ------------ In Egypt 10%-20% of the general population are infected with the Hepatitis C virus (HCV) \[[@B31]\]. This endemicity seems mainly to be caused by percutaneous medical procedures such as needle injections that took place during a countrywide health campaign between 1964 and 1982 against schistosomiasis. In order to investigate this phenomenon blood samples were obtained from various regions of Egypt and used to study the epidemic history of Hepatitis C. For instance, Tanaka et al. \[[@B32]\] analyzed the molecular evolution of HCV genotype 4a. Specifically, they utilized 47 sequences (AF217800-AF217812 \[[@B31]\] and AB103424-AB103457 \[[@B32]\]) from the NS5B region of the HCV subtype 4a to reconstruct the respective phylogeny, and subsequently applied the skyline plot method to infer the demographic history. We repeated their analysis with the reversible jump MCMC approach developed in this paper. We downloaded the sequence data from the HCV sequence database \[[@B33]\] and inferred the corresponding maximum-likelihood genealogy using the TREEFINDER program \[[@B34]\]. This tree is depicted in Figure [4a](#F4){ref-type="fig"}, next to the demographic history estimated from it by the classic skyline plot (Figure [4b](#F4){ref-type="fig"}). In the bottom of the figure we show the estimated population size function and its 95% confidence bands as obtained by our rjMCMC method (Figure [4c](#F4){ref-type="fig"}) and we also compare our results with those of the generalized skyline plot (Figure [4d](#F4){ref-type="fig"}). For the generating the time axis in these plots we assumed an evolutionary rate of 0.00045 substitutions per year. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### HCV in EgyptTop row: a) underlying reconstructed genealogy; b) classic skyline plot. Bottom row: c) population size function estimated with rjMCMC and corresponding 95% confidence band; d) comparison rjMCMC versus generalized skyline plot. ::: ![](1471-2148-5-6-4) ::: Generally, the star-like shape of the inferred tree already is indicative of exponential growth. This is confirmed by both the skyline plot as well as by our analysis (Figure [4d](#F4){ref-type="fig"}). Moreover, it can be seen that around 1940 the growth rate increased (i.e. the slope of N~e~(t) in the log-plot changes). Near the present, the rate decreased again. Also note the broadening of the confidence interval since 1940 which reflects the sparsity of available observations. This implies that the claim of Tanaka et al. \[[@B32]\] that the demographic history recently changed back to constant population size after an exponential growth is not firmly backed by the data. For further biological analysis of the HCV data we refer to Pybus et al. \[[@B35]\]. Conclusions =========== We have presented a new approach to non-parametric inference of demographic history from an inferred genealogy. This method is based on reversible jump MCMC sampling of the population size function N~e~(t). Unlike its predecessors, the classic and generalized skyline plots, it returns a smooth and realistic estimate of the demographic history and thus overcomes the constraints due to assuming a step function. Moreover, it automatically provides confidence limits. Nevertheless, the procedure is still computationally fast and can be run on any standard PC hardware. In our examples we demonstrated the advantage of non-parametric estimation of demographic history. Parametric estimation always assumes a certain functional form of population growth which may lead to problematic statements (cf. the HCV data set), in particular if the confidence bands of the estimated function N~e~(t) are not taken into account. From the methodological point of view, model selection via rjMCMC has the advantage that the effective dimension, i.e. the degree of smoothing, is automatically chosen in a data-driven manner. There is only one parameter (λ) that controls the a prioridegree of smoothing, and this is adjusted accordingly by the investigated data. In addition, a further advantage of our MCMC approach is that -- in contrast to the skyline plot -- at least in principle it is straightforward to incorporate it in more general MCMC sampling schemes that also take account of the uncertainty in the genealogy. During the referee process we have learned that the authors of the software package BEAST \[[@B27]\] have developed a similar non-parametric method to Bayesian coalescent inference of population history (A.J. Drummond et al., in preparation). We plan to work with Dr. Drummond to make available in BEAST joint sampling of sampling of demographic histories and of trees. This would combine the present rjMCMC approach and the method developed by Drummond and colleagues. Authors\' contributions ======================= This paper summarizes the main results from a master\'s thesis of R.O. supervised by K.S. and L.F. Accordingly, K.S. and L.F. jointly devised the project and R.O. carried out all analyses and simulations. All authors participated in the development of methodology. R.O. and K.S. wrote the manuscript. All authors approved of the final version. Acknowledgements ================ We are grateful for financial support from the Deutsche Forschungsgemeinschaft (DFG) in the Emmy Noether program (R.O. and K.S.) and from the SFB 386 (L.F). We thank G??nter Ra??er and Leonhard Held for valuable comments and discussion and Juliane Sch??fer for critical reading of the manuscript.	PubMed Central	2024-06-05T03:55:52.646601	2005-1-21	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548300/", "journal": "BMC Evol Biol. 2005 Jan 21; 5:6", "authors": [ { "first": "Rainer", "last": "Opgen-Rhein" }, { "first": "Ludwig", "last": "Fahrmeir" }, { "first": "Korbinian", "last": "Strimmer" } ] }
PMC548301	Background ========== The mammalian myocardium contains several cell types, of which the cardiac myocytes (CM) make up most of the heart\'s mass. Although a small proportion of CM in the adult myocardium remains mitotic, most CM lose the capacity to undergo cell division shortly after birth \[[@B1]\]. In the adult heart, approximately 70% of the cells is represented by non-myocytes, most of which belong to the fibroblast compartment. In this study, we investigate how the stress response of cardiac cells to increased mechanical stress (MS) can be compared to genotoxic stresses induced by two DNA damaging agents, ionising radiation (IR) and ultraviolet radiation (UV). In cardiac cells, MS is increased during excessive pressure or volume load of the heart, as seen in hypertensive and valvular heart disease. This results in an adaptive growth response leading to structural and functional cardiac changes, including CM hypertrophy and hyperplasia of fibroblasts, to compensate for the increased workload \[[@B2]\]. In vitro cyclic stretch of rat cardiac cells has been shown to be an appropriate model for cellular changes that occur during overload of cardiac muscle in vivo \[[@B3]\]. Mechanical signals may be transferred to the nucleus of cells through integrin receptors, cytoskeletal filaments and nuclear scaffolds \[[@B4]\] and through ion channels, ion exchangers and hormone receptors \[[@B5]\]. Radiation induced heart disease (RIHD) has been recognised as a late adverse effect of thoracic radiotherapy if the heart was situated in the radiation field \[[@B6]\]. IR induces the formation of reactive oxygen species that react with different components of the cell, thereby inducing macromolecular lesions. IR can activate several signal transduction pathways, involving growth factor receptors, death receptors and DNA damage sensing proteins \[[@B7]\]. Primary fibroblasts in culture are known to go into senescence shortly after IR \[[@B8]\], after which terminal differentiation of these cells is induced \[[@B9]\]. Cultures of CM do not demonstrate cell death, nor a loss of function upon a single dose of 10,000 rad (\~100 Gy) \[[@B10],[@B11]\]. The density of DNA damaging events after UVC, which include helix-distorting photolesions, is three orders of magnitude higher than the density of DNA damage that occurs after IR \[[@B12]\] and sufficient to block cellular DNA replication and transcription. Signal transduction after UV is mediated via components of the cellular membrane, involving growth factor receptors, and via DNA damage sensing proteins \[[@B7]\]. UV is known to induce cell cycle arrest and apoptosis in several cell types \[[@B13],[@B14]\]. The aim of the study was to identify and compare differentially expressed genes (up-regulated or down-regulated when compared with untreated controls) in cardiac cells in response to MS, IR or UV. To this purpose, cultures enriched for ventricular CM or fibroblasts were exposed to one of the three stressors. Differentially expressed genes were identified using Affymetrix GeneChips. Several statistical methods have been developed to analyse Affymetrix gene expression microarrays. We used a method based on Linear Modelling on Probe Level \[[@B15]\] to describe the signal of every perfect match (PM) probe. Because overall changes in the expression of functionally related genes are more informative than the expression pattern of single genes, genes in microarray studies can be assigned to functional groups \[[@B16]\]. In the present study, such an approach was used to classify genes, based on biological function or on the role of a gene product in common intracellular pathways. Results ======= Accuracy of the linear model ---------------------------- As an example for the accuracy of the linear model that was used to describe the PM probe signals, Figure [1A](#F1){ref-type="fig"} shows a dot plot of all PM probe signals as calculated by the linear model, against the actual PM probe signals determined from fibroblasts after UV. These data were used to calculate correlation coefficients (R^2^) between the signals calculated by the model and the actual PM signals obtained. Figure [1B](#F1){ref-type="fig"} shows the distribution of R^2^values for fibroblasts after UV. The majority of probe-sets have a correlation coefficient ≥ 0.90, indicating that the model used fitted the data accurately. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Accuracy of the linear model. Dot plot of all PM probe signals as calculated by the linear model, against actual PM probe signals determined from fibroblasts after UV (A). The data presented in Figure 1A were used to calculate correlation coefficients (R^2^) between the signals calculated by the linear model and the actual PM signals of fibroblasts after UV (B). ::: ![](1471-2164-6-6-1) ::: Numbers of differentially expressed genes ----------------------------------------- A Series entry (accession number GSE2032) at Gene Expression Omnibus (GEO), a public gene expression database of NCBI \[[@B17]\], gives access to all microarray data generated in this study. Figure [2](#F2){ref-type="fig"} represents the numbers of genes with a unique LocusLink ID that were up-regulated or down-regulated (q \< 0.005) in CM-enriched cultures/fractions and cultures of fibroblasts after one of the three stressors. When using these criteria, the numbers of differentially expressed genes (up-regulated or down-regulated) after UV were considerably higher than after IR or MS. After each of the three stressors more genes were up-regulated in CM-enriched cultures/fractions than in cultures of fibroblasts. Conversely, higher numbers of down-regulated genes were determined in fibroblasts. These differences were most pronounced after MS (Figure [2A](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Numbers of differentially expressed genes. Numbers of differentially expressed genes (q \< 0.005) with a unique LocusLink ID in cultures of fibroblasts (dotted) and CM-enriched cultures after MS (A), in cultures of fibroblasts (dotted) and CM-enriched fractions after IR (B), or in cultures of fibroblasts (dotted) and CM-enriched fractions after UV (C). Overlapping parts of the circles represent genes that show differential expression both in CM-enriched cultures/fractions and cultures of fibroblasts. ::: ![](1471-2164-6-6-2) ::: Based on information available at NettAffx™ \[[@B18]\], extended with standard textbooks and recent literature, genes with a unique LocusLink ID were assigned to several functional groups. Subsequently, the status of each gene within a functional group was determined in both cell populations (CM-enriched cultures/fractions and cultures of fibroblasts). Individual probe-sets within these functional groups and their q-value after the three stressors are listed in table 1 (see [additional file 1](#S1){ref-type="supplementary-material"}). In this table, down-regulated genes are distinguished from up-regulated genes by a minus-sign in front of their q-value. Figure [3](#F3){ref-type="fig"} shows the percentage of genes in a functional group that are differentially expressed after MS, IR or UV. In accordance with Figure [2](#F2){ref-type="fig"}, the highest percentages of genes were differentially expressed after UV (on average 39.4% for both cell populations), followed by IR (13.0%). In general, the percentages of differentially expressed genes were lowest after MS (8.3%). Several functional groups, including heat shock proteins and genes involved in cholesterol biosynthesis, showed high proportions of differentially expressed genes with a hypergeometric probability P \< 0.005. These functional groups were considered to have significantly high percentages of differentially expressed genes. On the other hand, in the group of genes encoding for ion channels and exchangers, low percentages of differentially expressed genes with a hypergeometric probability P \< 0.005 were determined, both after IR and UV. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Percentages of differentially expressed genes. Percentage of total number of genes within functional groups that are differentially expressed in CM-enriched cultures/fractions and cultures of fibroblasts after MS, IR or UV. Numbers between brackets represent total numbers of genes within a functional group. For example, of the 22 MAPkinases and phosphatases found to be represented by the array, 27% were differentially expressed in CM-enriched fractions after UV. AA: amino acid. \Hypergeometric probability P \< 0.005 ::: ![](1471-2164-6-6-3) ::: Percentages of up-regulated genes --------------------------------- Figure [4](#F4){ref-type="fig"} shows the percentages of differentially expressed genes that were up-regulated after IR or after UV. In several functional groups, including p53 target genes and genes involved in mitosis, a significant percentage of differentially expressed genes was up-regulated (hypergeometric probability P \< 0.005). Other functional groups, including cytoskeletal components and genes involved in cholesterol biosynthesis mainly had down-regulated genes. In all 25 functional groups, the hypergeometric probability P of the percentage of up-regulated genes after MS was not below the pre-set threshold of 0.005. Therefore, no significant percentages of up-regulated genes were determined in any of the functional groups after MS (data not shown). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Percentages of up-regulated genes. Percentage of changed genes that were up-regulated per functional group after IR or UV. Numbers between brackets represent numbers of differentially expressed genes in CM-enriched fractions and cultures of fibroblasts, respectively. For example, of the 8 MAPkinases and phosphatases that were differentially expressed in CM-enriched fractions after UV, 63% were up-regulated. \Hypergeometric probability P \< 0.005 ::: ![](1471-2164-6-6-4) ::: General stress response genes ----------------------------- Probe-sets that showed an up-regulation or down-regulation after more than one stressor are listed in table 2 (see [additional file 2](#S2){ref-type="supplementary-material"}). The overlap in responsive genes between IR and UV was larger than the overlap between MS and one of the radiation types. Both after IR and UV, several genes that are known to play a central role in the radiation response of cells, including p21, GADD153 and mdm2, were up-regulated. These genes were not up-regulated after MS. A striking large proportion of genes encoding cytoskeletal components were down-regulated both after IR and UV. Validation of microarray results by semi-quantitative PCR --------------------------------------------------------- Gene expression changes detected using microarrays were validated by semi-quantitative PCR, using RNA from CM-enriched cultures at 24 hours after MS. The increased gene expression of Tenascin C and biglycan were confirmed with PCR in two independent experiments. In these two experiments, Tenascin C gene expression increased 1.59 and 1.64 times, respectively. Biglycan gene expression increased 1.42 and 1.25 times, respectively. Figure [5](#F5){ref-type="fig"} shows a representative result of the Tenascin C PCR. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Representative result of the Tenascin C PCRTotal RNA was isolated from CM-enriched cultures at 24 hours after MS or control treatment. After cDNA synthesis, semi-quantitative PCR was used to determine Tenascin C gene expression. Lane 1: smart ladder; lane 2: negative control; lane 3: positive control; lanes 4 and 5: CM-enriched after control treatment; lanes 6 and 7: CM-enriched after MS. ::: ![](1471-2164-6-6-5) ::: Discussion ========== In this study, a linear model was used to describe GeneChip PM probe signals and to determine effects of three types of stressors on gene expression in two neonatal rat heart cell populations, consisting of CM that are known to be terminally differentiated cells and fibroblasts that still have the capacity to undergo mitosis. Several studies have shown a good correlation between Affymetrix GeneChip data and RT-PCR \[[@B16]\] or Northern-blot \[[@B19],[@B20]\]. In a previous study neonatal rat CM-enriched cultures and cultures of neonatal rat cardiac fibroblasts were irradiated with a single dose of 8.5 Gy and some mRNA transcripts were quantified by competitive PCR \[[@B21]\]. In accordance with the present study, no significant changes in gene expression of transforming growth factor-β1 (TGF-β1), fibroblast growth factor-2 (FGF-2) and collagen type I were determined at 24 h after IR in cultures of cardiac fibroblasts. Moreover, no significant IR-induced changes were determined in gene expression of atrial natriuretic peptide (ANP) in CM-enriched cell populations in both the latter and the present study. On the other hand, CM-enriched cultures showed a reduced TGF-β1 expression (by PCR) at 24 h after 8.5 Gy, which was not observed in the present study. This might be due to differences in experimental design between the two studies, as in the former study CM-enriched cultures were obtained by incubation with bromodeoxyuridine to prevent fibroblast proliferation. Also, during and after irradiation, the cells were incubated in higher serum concentration than used in the present study. Here, differences between the two cell populations are observed in the predominant type of gene expression response, i.e. up-regulation versus down-regulation. After all three stressors, differentially expressed genes were mostly up-regulated (q \< 0.005) in CM-enriched fractions, while in cultures of fibroblasts the majority of changed genes were down-regulated. After MS, these differences were most pronounced. Paracrine signalling is involved in the response of CM and cardiac fibroblasts in co-cultures subjected to MS \[[@B22]\]. In the CM-enriched cultures used in this study, remaining fibroblasts might stimulate gene transcription of the CM, resulting in higher numbers of up-regulated genes. It cannot be excluded that differences in level of toxicity between the three stressors applied in this study caused differences in gene expression levels. Of the three stressors examined in this study, both IR and UV are known to induce oxidative stress. Accordingly, both stressors induced high numbers of up-regulated genes involved in anti-oxidative processes. The high percentages of up-regulated genes encoding heat shock proteins in both cell populations after IR and UV might indicate oxidative stress induced protein damage in these cells. In comparison, after MS only few genes encoding heat shock proteins were up-regulated in either cell population. Heinloth et al. (2003) proposed a model for the regulation of several gene expression patterns after IR and UV in human dermal fibroblasts, based on microarray analysis. The general view that p53 plays a central role in signal transduction after IR and UV was confirmed in their study. Moreover, IR down-regulated the expression of genes involved in mitosis. UV, on the other hand, induced the expression of genes involved in protein degradation and the MAPK pathway \[[@B23]\]. The latter data are in accordance with the data of the present study, showing that UV did affect genes participating in the MAPK pathway (although not significantly) in both cell populations, but IR did not. Moreover, in CM-enriched fractions, IR resulted in a down-regulation of a large percentage of genes involved in mitosis. Several genes, involved in cell cycle regulation and mitosis are expressed in foetal cardiac myocytes and down-regulated in adult myocytes, in accordance with their terminal differentiation \[[@B24],[@B25]\]. Due to the close relation between foetal and neonatal cells, an expression of mitotic genes in the neonatal CM used in this study can be expected. The high numbers of up-regulated genes involved in ubiquitination and protein degradation in both cell populations after UV are also in accordance with the study of Heinloth et al. (2003) and with other studies on cultured cells after UV \[[@B20]\]. These results might reflect the need of cells to replace molecules that were damaged by UV irradiation, although the proteasome is also proposed to play a role in several cellular processes, including DNA-repair, cell cycle regulation and cell survival after irradiation \[[@B26]\]. In CM-enriched cultures, MS led to a relatively high proportion of up-regulated genes that are involved in cholesterol biosynthesis. Among these genes, expression of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase, which forms the starting enzyme of the mevalonate pathway and is considered to be the key enzyme in cholesterol biosynthesis, was up-regulated. This suggests that in neonatal rat cardiac myocytes cholesterol biosynthesis is stimulated after MS. Neonatal rat heart myocytes that undergo hypertrophy in culture show an increased biosynthesis and intracellular accumulation of cholesterol \[[@B27]\]. Moreover, the mevalonate pathway has been proposed to play a role in Ras activation in neonatal rat cardiac myocytes subjected to MS in culture, leading to hypertrophy \[[@B28]\]. In vivohypertrophy of the heart is also associated with elevated myocardial cholesterol contents \[[@B29]\]. An increased cholesterol biosynthesis in cardiac myocytes that undergo MS might therefore accompany a hypertrophic response of these cells. However, of the three foetal genes that are known to be re-expressed in hypertrophic myocytes, i.e. smooth muscle alpha-actin, ANP and beta-myosin heavy chain, only the last gene was up-regulated at the time point of investigation. In contrast to MS, IR and UV led to a down-regulation of genes involved in cholesterol biosynthesis in CM-enriched fractions. In previous studies, alterations in cholesterol contents of cardiac myocytes and fibroblasts were associated with alterations in protein to DNA ratios, levels of several enzymes including ATPases and phosphatases, and diffusion rates of membrane proteins \[[@B30]-[@B32]\]. Moreover, a decrease in beating rate observed in aging cultures of CM was opposed by increased membrane amounts of cholesterol \[[@B33]\]. The role of cholesterol biosynthesis in the cellular functions of cardiac cells after MS, IR or UV needs further investigation. Higher numbers of differentially expressed genes involved in ECM formation, including genes encoding collagens, fibronectin and laminin, were determined in CM-enriched cultures after MS than in cultures of fibroblasts. As mentioned before, paracrine signalling between CM and remaining fibroblasts might play a role in cellular responses in these CM-enriched cultures. Alterations in expression of genes involved in ECM formation might originate from remaining fibroblasts after stimulation by CM. Interestingly, MS does not affect the expression of these genes in cultures of fibroblasts, which suggests that paracrine signalling from CM is necessary for alterations in expression profiles of genes involved in ECM formation in fibroblasts. The down-regulated genes encoding cytoskeletal components in cultures of fibroblasts after IR and UV, including myosin and troponin, are mostly CM-specific. Therefore, these genes are likely to originate from remaining CM in the fibroblast cultures, although their numbers were low (3--5%). The extremely low numbers of differentially expressed genes encoding for ion channels and exchangers might be explained by a low number of cardiac cell type-specific genes within this functional group. Conclusions =========== MS, IR and UV mainly induce stress-specific and cell-type specific gene expression profiles in neonatal rat CM-enriched cultures and cultures of neonatal rat heart fibroblasts. Functional groups that show significant percentages of differentially expressed genes suggest that certain cellular pathways are activated after one or more stresses. Methods ======= Cell culturing -------------- The experiments were performed with permission of the local committee on animal experiments, installed by the University of Leiden according to the Dutch law. Cardiac cells were isolated from ventricles of neonatal rat hearts, and cultured as described before \[[@B14],[@B34]\]. By applying a pre-plating method, cultures enriched for CM and cultures of fibroblasts were obtained. After 2 days of culturing with 5% horse serum (HS), culture media of CM-enriched cultures were replaced by media containing 2.5% HS. One day later, these cultures were subjected to UV, IR or MS. Three days after isolation, cultures of fibroblasts were subcultured for 6 days in medium containing 10% foetal bovine serum (FBS). Then, culture medium was replaced by medium containing 2.5% FBS. One day later, confluent cell cultures were subjected to UV, IR or MS. Irradiation and cyclic stretch ------------------------------ Cells were subjected to a single dose of 8.5 Gy of X-rays at room temperature, using a 6 MV accelerator (SL 75-5 Philips), operated at a dose rate of 8 Gy min^-1^. During irradiation, sham-irradiated control cells were kept at room temperature. After irradiation, cell cultures were maintained at 37°C for 24 h. In another experiment cells were irradiated with 10 J/m^2^UVC at room temperature at a dose rate of 0.2 J/m^2^per s. Before irradiation, medium was collected and cells were rinsed with PBS. Following irradiation, growth medium was returned to the cultures and the cells were incubated for an additional 24 h. Control cells received a similar treatment except for UV irradiation. To subject cells to cyclic stretch, CM-enriched cells (54 ± 5% CM) and fibroblasts (95 ± 2% non-myocytes) were cultured in 6-well Flex I culture plates (Flexcell, Hillsborough, NC, USA), coated with collagen I. Medium was changed to medium containing 2.5% serum and after 24 h plates were placed in the Flexercell Strain Unit FX-2000^®^(Flexcell) in which the frequency and magnitude of stretch were regulated by a computer-controlled vacuum pump. The apparatus applied an equiaxial cyclic stretch of 20% elongation to the wells at a frequency of 60 cycles/min (1 Hz). Stretch was applied for 24 h. Control cells were grown in identical culture plates and incubated in the same incubator as the stretched cultures, but were not mounted in the Flexercell Strain Unit. Cell harvesting after irradiation --------------------------------- Twenty-four hours after IR or UV, non-irradiated and irradiated CM-enriched cultures were trypsinised and subjected to centrifugal elutriation \[[@B35]\]. The proportion of CM in each elutriation fraction was determined by flow cytometric analysis of myosin expression, as described before \[[@B35]\]. After elutriation of IR-exposed cultures, CM-enriched fractions contained 74 ± 3% CM and after elutriation of UV-exposed cultures, CM-enriched fractions contained 85 ± 3% CM. Because of the high purity of the fibroblast cultures (95 ± 2% non-myocytes), no centrifugal elutriation was applied on these cells. CM-enriched fractions and cultures of fibroblasts were used for RNA isolation. Cell harvesting after MS ------------------------ After undergoing MS or control treatment for 24 h, cells were collected from CM-enriched cultures and from cultures of fibroblasts by trypsinisation. The proportion of CM in the cell cultures was determined by flow cytometric analysis of myosin expression as described before \[[@B35]\]. CM-enriched cell cultures, containing 54 ± 5% CM, and fibroblast cultures, containing 95 ± 2% non-myocytes, were used for RNA isolation. RNA isolation and labelling --------------------------- RNA was isolated applying an RNeasy^®^kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer\'s instructions. Ten to 14 μg of total RNA from CM-enriched fractions and 7.5 to 14 μg of total RNA from fibroblast cultures was used for labelling. Per experiment, the quantities of total RNA from irradiated and control cells were equal. cDNA was synthesised using the Gibco BRL Superscript system (Invitrogen, Carlsbad CA, USA). Briefly, single stranded cDNA was synthesised using Superscript II reverse transcriptase and T7-oligo(dT)24 primers at 42°C for 1 h. Double stranded cDNA was obtained by using DNA ligase, DNA polymerase I and RNAse H at 16°C for 2 h, followed by T4 DNA polymerase at 16°C for 5 min. After clean up with a phase lock gel (Qiagen), ds-cDNA was used for in vitrotranscription (IVT). cDNA was transcribed using the BioArray HighYield^®^RNA transcript labelling kit (Enzo Lifesciences, Pharmingdale NY, USA) in the presence of biotin-labelled ribonucleotides, or using the MEGAScript T7^®^kit (Ambion, Austin TX, USA), in the presence of biotin-labelled CTP and UTP. In each experiment, cDNAs from control cells and stressed cells were labelled simultaneously using the same labelling kit. After clean up with a RNeasy kit (Qiagen), the biotin-labelled IVT-RNA was fragmented in a buffer containing 40 mM Tris-acetate (pH 8.1), 100 mM potassium-acetate and 30 mM magnesium-acetate, at 94°C for 35 min. Hybridisation of IVT-RNA ------------------------ RG-U34A arrays (Affymetrix, Santa Clara CA, USA) were used, representing \~7000 transcripts, of which 6399 had a unique LocusLink identification number (ID) at the time of data analysis. Biotin-labelled IVT-RNA was hybridised to the arrays at 45°C for 16 h according to the manufacturer\'s instructions. After hybridisation, the arrays were washed in a GeneChip Fluidics Station 400 with a non-stringent wash buffer at 25°C followed by a stringent wash buffer at 50°C. After washing, the arrays were stained with a streptavidin-phycoerythrin complex. After staining, intensities were determined with a GeneChip scanner, controlled by GeneChip software (Affymetrix). The intensities were background corrected using gcrma \[[@B36]\] and normalized at the probe level by VSN \[[@B37]\]. Data analysis ------------- For each stressor, three independent experiments were performed. To describe the signal of every PM probe, the following linear model was used: signal \~P + T + E + TE, based on a method described before \[[@B15]\]. In our model, the symbols P, T, E and TE represent the effects of probe, treatment, experiment per stressor (1, 2, or 3) and the interaction between treatment and experiment, respectively. Subsequently, analysis of variance was applied on the treatment effect to determine the p-value for each probe-set. The p-values were corrected for multiple testing using the Benjamini Hochberg step-up procedure \[[@B38]\] which yielded q-values for the false discovery rates (FDR). The FDR level of control was set to 0.005, equivalent to selecting genes with a q-value\<0.005 (keeping up- and down-regulated genes separate by the sign of the treatment coefficient). Some genes are represented by more than one probe-set. A gene was determined to be differentially expressed when at least 50% of the probe-sets representing this gene showed significant up-regulation or down-regulation. To determine significant proportions of differentially expressed genes within functional groups, the hypergeometric probability P was calculated. P \< 0.005 was considered significant. To determine the accuracy of the linear model that was used to describe PM signals, R^2^was calculated for every probe-set, using the following standard formula: R^2^= 1-ΣR~i~^2^/Σ(signal~i~-mean signal)^2^, where R~i~= observed-fitted value for signal~i~and mean signal = the mean of observed signal~i~. To compare data on differentially expressed genes with microarray data in literature on species other than rat, RESOURCERER of the Institute of Genomic Research \[[@B39]\] was used. RNA isolation and semi-quantitative PCR --------------------------------------- Total RNA isolation, cDNA synthesis and semi-quantitative PCR were performed as described before \[[@B40]\]. In short, total RNA was isolated using an RNeasy^®^kit (Qiagen). Following DNAse (Amersham Pharmacia Biotech, Uppsala, Sweden) treatment, cDNA was synthesised using M-MLV reverse transcriptase (Life Technologies, Rockville, MD, USA) and oligo(dT) primers (Amersham). Semi-quantitative PCR was performed, using the following primers and annealing temperatures: Tenascin C sense: CGA CAG TTT TGT TAT CAG GAT CAG, Tenascin C antisense: GGC ACA TAA GTA ATC CGG AAA T, 60°C; Biglycan sense: CAA CAA CCC TGT GCC CTA CT, Biglycan antisense: GGT GT GCT TCT TTG CTT CC, 65°C. A competitive PCR was performed on GAPDH to correct for differences in cDNA concentrations. After agarose gel electrophoresis, intensities of PCR product bands were determined by Scion image analysis software (Scion Corporation, Frederick, MD, USA). Authors\' contributions ======================= MB performed experiments, developed and analyzed functional groups of genes and prepared the manuscript. CGCW performed experiments, developed and analyzed functional groups of genes and participated in discussions. HV and AL participated in discussions and gave textual advice. PS performed microarray normalization and data analysis. JW and LHFM participated in discussions. AAZ participated in discussions, suggested the assignment of genes to functional groups and gave textual advice. All authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 q-values of genes within functional groupsTable 1. Excel-file containing the 25 functional groups of genes used in this study. Of every gene, Affymetrix probe-set IDs and q-values after the three types of stress are listed. Down-regulated genes are represented by a minus-sign in front of their q-value. q-values \< 0.005 are marked red (in case of up-regulation) and green (in case of down-regulation). ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 General stress response genesTable 2. Excel file containing Affymetrix probe-set IDs, LocusLink ID and gene description of all probe-sets that showed an up-regulation or down-regulation after two or more stresses. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This study was financially supported by the Netherlands Heart Foundation (grant no. 97.176), the Interuniversity Research Institute of Radiopathology and Radiation Protection (grant no. 9.0.13) and the European Union. The authors would like to thank Rebecca Esveldt-Van Lange, Cindy Schutte-Bart and Minka Bax for skilful technical support.	PubMed Central	2024-06-05T03:55:52.649799	2005-1-18	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548301/", "journal": "BMC Genomics. 2005 Jan 18; 6:6", "authors": [ { "first": "Marjan", "last": "Boerma" }, { "first": "Caroline GC", "last": "van der Wees" }, { "first": "Harry", "last": "Vrieling" }, { "first": "J Peter", "last": "Svensson" }, { "first": "Jan", "last": "Wondergem" }, { "first": "Arnoud", "last": "van der Laarse" }, { "first": "Leon HF", "last": "Mullenders" }, { "first": "Albert A", "last": "van Zeeland" } ] }
PMC548302	Background ========== Bacterial strains belonging to the Enterobacteriaceaespecies Escherichia coliare common as commensals in the intestinal flora of humans and warm-blooded animals \[[@B1]\]. Typing systems for identification of related E. colistrains were developed in the early 1940ies when it became evident that certain E. colistrains were agents of infantile gastroenteritis \[[@B2]\]. In 1944, Kauffmann established the method of serological typing for E. coliO- (lipopolysaccharide) and H-(flagellar) antigens which allowed the grouping of E. colistrains according to their O:H-types (serotypes) \[[@B3]\]. Serotyping proved to be widely useful for identification of enteropathogenic E. coli(EPEC) strains from stools of diarrhoeic infants \[[@B4],[@B5]\] and is successfully employed for characterization of pathogenic E. colistrains from both humans and animals \[[@B2],[@B3]\]. The genetic analysis of E. colipopulations by multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST) allowed the detection of clonal types of strains which carry specific virulence markers and are associated with disease in humans \[[@B6],[@B7]\]. It was shown that the O:H serotype is a good marker for identification of strains belonging to clonal types of pathogenic E. coli\[[@B6],[@B7]\]. At present, 176 O- and 53 H-antigens are described for E. coliwhich can occur in different combinations in wildtype isolates of strains \[[@B2],[@B3],[@B5],[@B8]\]. However, the large number of O- and H-antisera which are needed for E. coliserotyping and the laborious typing procedure restricts its usage to a few reference laboratories. Therefore, alternative typing methods were developed including molecular characterization of genes coding for the O- and H-antigens in E. coli\[[@B9]-[@B12]\]. Typing of fliCgenes by PCR was successfully employed for characterization of human pathogenic O157:H7 and O26:H11 strains \[[@B13],[@B14]\]. Analysis of the nucleotide sequence of fliCgenes coding for flagellar antigens H7 and H6 revealed large sequence similarities between strains sharing the same H-type but different O-types \[[@B15],[@B16]\]. Moreover, molecular typing of the fliCgene allows H-typing of non-flagellated (non-motile) E. coliisolates which cannot be analyzed for their flagella with H-specific antisera \[[@B14],[@B15],[@B17]\]. The flagellar type H4 is frequently occurring in E. colibelonging to many different O-groups including strains of shiga toxin-producing E. coli(STEC) and extraintestinal pathogenic serotypes \[[@B18]-[@B20]\], (K.A. Bettelheim, The VTEC table, May 2003, <http://www.microbionet.com.au/vtectable.htm>). Moreover, a cryptic fliC-H4 gene was described to be present and to be spontaneously expressed in E. colistrain P12b (O15:H17) which carries another type of flagella called H17 which is not encoded by the fliCgene \[[@B21],[@B22]\]. Therefore, we became interested in the genetic variability of flagellar H4 genes in E. colistrains belonging to different O-serogroups and pathotypes. We have used the published nucleotide sequence of the fliCgene present in the E. coliH4 reference strain U9-41 (accession AB028472) to develop a PCR which allows discrimination between the fliC-H4 gene variants. Nucleotide sequence analysis of the fliCgene was performed on other E. coliH4 strains that either showed deviations in the PCR analysis or were reported to harbour allelic types of the H4-fliCgene \[[@B9]\] or revealed differences in the agglutination reaction compared to the reference strains. To study the expression of different flagellar H4 types we have cloned their corresponding fliCgenes and have introduced them into the laboratory E. coliK-12 strain JM109 \[[@B23]\]. Expression of recombinant flagella was demonstrated by serotyping and by immuno electron microscopy. Results ======= Serological detection of the flagellar H4 antigen in different E. coliwildtype host strains --------------------------------------------------------------------------------------------- E. colireference strains U9-41 (O2:K1:H4) and P12b (O15:H17) \[[@B3]\] and the E. coliK-12 strain JM109 (O-rough:H48) (this study), which was used for expression studies were investigated for inhibition of motility in swarm-agar containing 1:600 dilutions of either H4, or H48-antiserum (see Methods). U9-41 and P12b were fully inhibited for their motility in the presence of H4 antiserum derived from strains U9-41 or from P12b but not in the presence of H48 specific antiserum. The E. coliK-12 strain JM109 was not inhibited for motility by H4 antisera but by H48 antiserum. These findings indicate that H4 antisera specifically inhibited the motility of E. coliH4 strains and that the antigenically different flagellar type H17 was not expressed or lost in our P12b isolate, similar as previously described with spontaneously arising variants of P12b \[[@B24]\]. We became interested if other E. coliH17 strains would also carry the genes for expression of H4 type flagella as it was described for P12b \[[@B21],[@B22],[@B24]\]. For this, we have investigated five additional E. coliH17 strains (872-69, 107-74, 305-78, 870-69 and 327-01) for their serological reaction with different H4 specific antisera and all strains showed specific positive reactions (Table [1](#T1){ref-type="table"}). H-serotyping performed with strains from the collection of the Robert Koch-Institute revealed 88 E. colistrains which agglutinated with H4 antisera and the results obtained with 10 representative strains are shown in Table [1](#T1){ref-type="table"}. All strains agglutinated with both, H4~U9-41~and H4~P12b~antiserum, but were not agglutinating with antisera made against other H-antigens (data not shown). Differences in agglutinating titers between H4~U9-41~and H4~P12b~antisera were not more than twofold with either strain indicating that both sera were similar for their specificity (Table [1](#T1){ref-type="table"}). The strains P7d, E1541-68, 107-74 and 305-78 showed significantly lower agglutination titers with H4 antisera than did the reference strains U9-41 and P12b, which had been used for production of H4 typing sera, respectively (Table [1](#T1){ref-type="table"}). These findings prompted us to compare all the H4 strains with all other E. coliH-types for polymorphisms in the fliCgene by HhaI digestion of fliC-PCR products as described in the Method section. HhaI-RFLP typing of fliCgenes in E. coli -------------------------------------------- PCR products obtained with primers fliC-1 and fliC-2 were digested with HhaI to obtain H-serotype specific RFLP patterns from reference strains for the 53 different E. coliH-serotypes \[[@B3],[@B9]\]. HhaI-RFLP typing of E. coli fliCgenes coding for flagellar types H1 to H56 revealed individual patterns corresponding to the different H-serotypes (Fig. [1](#F1){ref-type="fig"}). Flagellar antigens H3, H17, H35, H36, H44, H47, H53, H54 and H55 were reported to be not encoded by fliCbut by other genes (flkA, fllA, flmAand others) in the corresponding E. colistrains and the fliCHhaI patterns obtained from these strains do therefore not correspond to their H-serotypes \[[@B21],[@B25]-[@B27]\]. HhaI-RFLP patterns were found conserved among strains sharing the same H-serotype independent of their O-antigen as previously described \[[@B9]\] (data not shown). An exception was found for H-serotypes 2, 8, 18, 19, 21, 33 and 47 in which single strains possessed different HhaI-RFLP patterns when compared with the corresponding H-type reference strain (data not shown). The HhaI-RFLP patterns of different H-types were distinguishable from each other (Fig. [1](#F1){ref-type="fig"}). The results from HhaI-RFLP typing corresponded with the earlier reports showing that the fliCgene in strain P12b codes for flagella of serotype H4. The relationship between fliCgenes present in the different E. colistrains was further investigated by nucleotide sequencing. Nucleotide sequence analysis of the fliCgenes present in representative E. coliH4 strains --------------------------------------------------------------------------------------------- To obtain the entire coding region of the fliCgene in E. colistrains oligonucleotide primers fliC-5 and fliC-6 were deduced from the fliC-H4 chromosomal region and applied to amplify the corresponding chromosomal regions from E. colistrains U9-41 (O2:K1:H4), P12b (O15:H17), U1-41 (O5:K4:H4), P7d (O68:H4), C107-74 (O15:H17) and E1541-68 (O154:H4) as listed in Table [1](#T1){ref-type="table"}. These strains were taken as flagellar type H4 representatives according to previously published results \[[@B3],[@B9]\] and according to the H-serotyping performed in this study. The analysis of the coding sequences of the fliCgenes present in these strains revealed in all cases a length of 1050 bp. As a control, the fliCgene of the strain U9-41 was sequenced and found to be identical to the previously deposited fliCsequence (accession AB028472). The identity of the fliC-H4 sequences ranges from 97,6 % to 100 %. The greatest sequence difference to the fliCgene coding sequence of strain U9-41 was found in the fliCgene of strain P7d (exchange of 20 nucleotides), whereas the fliCsequences of strains U1-41 and U9-41 were identical to each other. All deduced flagellins have a length of 349 amino acids. The greatest deviation in the primary structure to the FliC protein of the reference strain U9-41 was again observed for the FliC protein of strain P7d (exchange of 9 aa in a stretch of 349 aa). Fig. [2](#F2){ref-type="fig"} shows the alignment of the deduced FliC proteins of all investigated strains. The FliC protein of strain U1-41 is 100% identical to that of strain U9-41 and therefore not shown. Similarities of the deduced amino acid sequences of the FliC proteins (flagellins) are summarized in Table [2](#T2){ref-type="table"}. PCR based detection of fliC-H4 specific DNA sequences ------------------------------------------------------- Amplification of the fliCgenes present in E. colistrains U9-41 and P12b with primers fliC-1 and fliC-2 resulted in the generation of a 953 bp internal PCR product with both strains (Fig. [3](#F3){ref-type="fig"}, lanes 4+5). The nucleotide sequences of this stretch of DNA of strains U9-41 and P12b were compared for restriction enzymes which cut at different sites in the fliC-H4~U9-41~and fliC-H4~P12b~sequence. HhaI was found to cut at identical sites confirming the results from HhaI RFLP typing (Table [3](#T3){ref-type="table"}). In contrast, enzymes HpaII and MboI were found to generate each different restriction fragments from PCR products of the fliC-H4~U9-41~and fliC-H4~P12b~gene, respectively. Both enzymes were taken for RFLP typing of amplified fliCgenes (primers fliC-1 and fliC-2) from 88 E. colistrains which showed agglutination reactions with H4 antisera (Table [3](#T3){ref-type="table"}). Eighty-six of the 88 strains showed HpaII and MboI restriction profiles which corresponded to the patterns obtained with strain U9-41 (O2:K1:H4) (Table [3](#T3){ref-type="table"} and Fig. [3](#F3){ref-type="fig"}). Exceptions were made by strains P7d (O68:H4) and P12b (O15:H17) which showed individual restriction patterns which differed from all other H4 strains investigated in this study. (Table [3](#T3){ref-type="table"}). Cloning and expression of fliC-H4 genes in the E. coliK-12 strain JM109 --------------------------------------------------------------------------- In order to study the functional expression of the fliC-H4 genes in a different genetic background we cloned the corresponding PCR products of strains U9-41 and P12b into the vector pLITMUS38 as described in the Methods section. The fliCcoding regions were inserted downstream of the lacZpromoter of pLITMUS38 and the fliCrecombinant plasmids were transformed into the laboratory E. coliK-12 strain JM109 \[[@B23]\]. JM109 was serotyped as O-rough:H48, and it showed the same HhaI-RFLP fliC-pattern as the E. colireference strain P4 (O16:H48) \[[@B3]\] (Fig. [1](#F1){ref-type="fig"}). The functional expression of the cloned fliC-H4 genes in the JM109 derivative strains TPE1976 (fliC-H4~U9-41~clone) and TPE1978 (fliC-H4~P12b~clone) was analyzed by tube agglutination with H4 and H48 antisera, respectively. The strains TPE1976 and TPE1978 showed agglutination with H48 and H4 sera whereas the parental JM109 strain reacted only with H48 serum (Table [4](#T4){ref-type="table"}). To find out if the flagellins of the fliCrecombinant plasmid carrying JM109 strains were co-assembled in all flagella or assembled separately we studied the parental strain JM109 and its fliC-H4 derivative TPE1978 by IEM (Fig. [4B--G](#F4){ref-type="fig"}). Both strains were found to express 2-4 flagella per cell which appeared morphologically typical as long helical filaments (diameter 18 nm, length up to 20 μm) (Fig. [4A](#F4){ref-type="fig"}), \[[@B29]\]. The reaction of H48 antiserum with bacteria followed by detection of adsorbed antibodies with anti-rabbit-IgG coupled with 10 nm immuno-gold particles showed a specific and homogeneous labelling of flagella present on the surfaces of JM109 (Fig. [4C](#F4){ref-type="fig"}) and of TPE1978 (Fig. [4B](#F4){ref-type="fig"}). When H4 serum was used, only flagella of strain TPE1978 became immuno-labelled (5 nm gold particles) (Fig. [4D](#F4){ref-type="fig"}) and no labeling was observed with JM109 (Fig. [4E](#F4){ref-type="fig"}), confirming H-serotyping results. Sequential double labeling experiments with H48 and H4 antibodies resulted in staining of all flagella on the surfaces of JM109 (Fig. [4G](#F4){ref-type="fig"}) and TPE1978 (Fig. [4F](#F4){ref-type="fig"}). However, both 5 nm (H4 label) and 10 nm (H48 label) gold particles were only bound to flagella of TPE1978 (Fig. [4F](#F4){ref-type="fig"}) whereas the flagella of JM109 were exclusively labelled with 10 nm gold particles which indicates the H48 antigen (Fig. [4G](#F4){ref-type="fig"}). These results demonstrate that both H48 and H4 flagellins are co-assembled in the flagella made by the fliC-H48/H4 genes in strain TPE1978. Discussion ========== The fliCgenes of representative E. colistrains for the 53 different H-types were recently investigated and compared for their nucleotide sequences \[[@B21]\]. Among the H-type reference strains which were not analyzed for their complete fliCgene sequences, were the reference strains for the E. coliH4 (U9-41) and H17 (P12b) flagellar antigen \[[@B21],[@B22]\]. In this work, we performed a complete characterization of the fliC-H4 genes from different E. colistrains with primers which were generated on the basis of the previously published fliCsequence (accession AB028472) of the E. coliH4 reference strain U9-41. The comparison of the fliCgene sequence encoding H4 flagella in strain P12b with the fliCsequences of five other E. coliH4 or H17 strains revealed a high similarity on the DNA and on the amino acid level (Fig. [2](#F2){ref-type="fig"} and Table [2](#T2){ref-type="table"}). We established a PCR/RFLP typing assay for genotypic investigation of clinical E. coliisolates which reacted with H4 antisera. Genotyping of 88 E. colistrains comprising 20 different O-serogroups (Table [3](#T3){ref-type="table"}) revealed that 86 of the strains gave RFLP patterns with HhaI, MboI and HpaII which were indistiguishable from the prototype fliC-H4 gene and only two strains showed alternative patterns. The detection of some genetic variants in the fliC-H4 gene of E. colistrains studied here points to a sequence diversity similar as described previously for the fliCgenes of E. coliH6 and H7 strains \[[@B15],[@B16]\]. These results correspond to previous findings indicating that the H4 antigens in different E. colityping strains are not fully identical \[[@B20],[@B24],[@B28]\]. It was suggested earlier that the strain P12b encodes two flagellins, H4 and H17, which are subject to phase variation for their expression \[[@B22]\] and mutants of P12b could be isolated which expressed only the H4 antigen \[[@B24]\]. Recent studies have demonstrated that the fliCgene of strain P12b encodes flagellar type H4 and it was suggested that the gene for the H17 flagellin is encoded by a locus outside fliC, however the gene responsible for the flagellar type H17 was not identified in the study \[[@B21]\]. In our study, cultures of strain P12b were fully inhibited for motility and swarming in the presence of H4 antiserum but not in the presence of H48 antiserum which was used a non-specific control. This result indicates that H17 type flagella were not expressed or lost from our P12b isolate, similar as previously described with mutant strains of P12b \[[@B24]\]. The functional expression of the cloned fliC-H4~U9-41~and fliC-H4~P12b~genes in the genetic background of E. coliK-12 strain JM109 which shows flagellar serotype H48 confirmed their coding capacity. Since we found coexpression of cloned flagellins with the parental H48 flagellin, we became interested in the composition of flagella made by the fliCrecombinant plasmid carrying JM109 strains. Electron microscopy of flagella from JM109 and from the fliC-H4~P12b~recombinant plasmid carrying strain revealed no differences between JM109 and TPE1978 showing both a typical helical organization of normal sized flagella on their surface (Fig. [4](#F4){ref-type="fig"}). Immuno-gold staining of bacteria which were prior incubated with H48 and H4 antisera revealed high specificity of the rabbit antisera for the flagellar structure (Fig. [4B--G](#F4){ref-type="fig"}). Single (Fig. [4 B+D](#F4){ref-type="fig"}) and double labelling (Fig. [4 F](#F4){ref-type="fig"}) experiments with different sized gold markers demonstrated that all flagella present on the surface of TPE1978 were labelled after incubation with H48 and H4 antiserum. Our results indicate that both H48 and H4 flagellins are coassembled in the flagella made by the fliC-H4~P12b~recombinant plasmid carrying strain. This finding is surprising in view of the molecular weight size differences found between H48 and H4 flagellin. To our knowledge, the assembling of two different flagellins in the same filament has not yet been demonstrated before. The H48 flagellin of E. coliK-12 (accession AE000285) is a 51.3 kDa protein consisting of 498 amino acids and thus much larger than the 36.3 kDa H4 flagellin which is composed of 349 amino acids. However, both flagellins share conserved N- and C-terminal sequences which are known to be involved in the structural assembly of flagella \[[@B29]\]. H48 and H4 flagellins differ largely for their central regions which are not involved in flagellar assembly and function but which contain flagellar antigenic epitopes \[[@B29]\]. We were able to show that the introduction of an isolated fliCgene in E. colican change the antigenic properties of the flagella made by this strain. Horizontal transfer of fliCgenes may contribute to the diversity of flagellar serotypes by recombination within E. colirecipient strains. The mammalian host immune system is the driving force for continuous selection of new flagellar antigens in E. coli. Published data indicate that both mutation and recombination events in the fliCgene have taken place in the evolution of E. coliflagellar antigens \[[@B15],[@B16]\]. Conclusions =========== Our fliCsequence data have shown that the flagellar type H4 which is present in E. colistrains of clinical importance covers several genetic variants which are closely related to each other. We have shown for the first time that flagellins of different molecular size are expressed and coassembled into functional flagella in a laboratory E. coliK-12 strain. Methods ======= Bacterial strains ----------------- The reference strains used for production of antisera for O- and H-typing of E. coliwere obtained from the International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen, Denmark and are described elsewhere \[[@B3]\]. Origin and serotype data on five additional strains with flagellar type H17 are listed in Table [1](#T1){ref-type="table"}. A laboratory collection of 88 E. coliisolates originating from humans and animals was investigated by PCR/RFLP typing for fliC-H4 genes. These strains were previously investigated for the O-types and for production of Shiga-toxins (Stx) and were isolated in different countries between 1941 to 2002 \[[@B3],[@B19],[@B30]\] (Table [3](#T3){ref-type="table"}). The E. coliK-12 strain JM109 is described elsewhere \[[@B23]\]. Production of E. coliO and H-specific antisera ------------------------------------------------ Rabbit antisera against the different O- and H-antigens of E. coliwere prepared according to ∅rskov and ∅rskov \[[@B3]\]. Antisera for typing of flagellar antigens H4 were produced with reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) \[[@B3]\]. Our strain P12b was found to express its fliCencoded H4 antigen and produced flagellar type H4 (this work). Motility inhibition test ------------------------ Expression of flagella and swarming of E. colistrains was tested by inoculating bacteria in tubes containing 10 ml portions of swarm-agar (L-broth + 0.3% agar) as described \[[@B3]\]. Inhibition of motility of E. colistrains in the presence of flagellar-specific antiserum (H4 and H48) was tested in swarm-agar containing a 1:600 dilution of the respective antiserum. Cultures which were inhibited for motility were observed over two weeks for possible switch to motility by phase variation. Serological typing of H-antigens of E. coli --------------------------------------------- H-serotyping was performed as described \[[@B3]\]. In brief, bacteria were grown in tubes containing 10 ml 0.3% semi-solid LB-agar \[[@B23]\] for two to three passages. Highly motile bacteria were transferred in LB-medium, incubated 6h at 37°C and inactivated by addition of 0.5% formaldehyde in solution. Agglutination reactions were performed in two fold dilutions of 0.5 ml portions of serum in phosphate-buffered saline pH 7.4 (PBS) \[[@B22]\] with 0.5 ml formalized bacteria in glass tubes which were incubated for 2h at 50°C. Agglutination tests were read by eye immediately after incubation as described \[[@B3]\]. PCR-typing of fliC genes -------------------------- The oligonucleotide primers fliC-1 (5\' CAA GTC ATT AAT AC(A/C) AAC AGC C 3\') and fliC-2 (5\' GAC AT(A/G) TT(A/G) GA(G/A/C) ACT TC(G/C) GT 3\') were used for amplification of internal parts of fliCgenes present in the E. colireference strains as described \[[@B9]\]. The PCR was performed for 25 cycles at 94°C for 60 sec, 55°C for 60 sec and 72°C for 120 sec \[[@B9]\]. PCR products of sizes varying between 950 to 2500 bp were obtained with E. colireference strains for 53 different H-types \[[@B3]\] (Figure [1](#F1){ref-type="fig"}). Amplified DNA was digested with HhaI and the resulting restriction fragments were compared on 2% agarose gels. Restriction enzymes HpaII and MboI were used for characterization of fliC-H4 specific PCR products. Gel images were stored digitally and analyzed with BioNumerics software, version 2.5 (Applied Maths, Kortrijk, Belgium) for similarity (Dice, complete linkage) (Fig. [1](#F1){ref-type="fig"}). Nucleotide sequence analysis of fliCgenes ------------------------------------------- Two primers deduced from the published fliC-H4 sequence (AB028472) were used for the amplification of the entire fliC-H4 coding regions. The PCR was performed for 30 cycles: 30 sec at 94°C, 60 sec at 58.1°C and 90 sec at 72°C with primers fliC-5 (5\'-TGA GTG ACC AGA CGA TAA CAG GG-3\') and fliC-6 (5\'-GGA CGA TTA GTG GGT GAA ATG AGG-3\') and yielded a 1243 bp product. PCR products were purified with the QIAquick™ PCR Purification Kit (Qiagen, Hilden, Germany) and used for sequencing. Sequencing reactions were carried out using the dye terminator chemistry (PE Applied Biosystems, Darmstadt, Germany) and separated on an automated DNA sequencer (ABI PRISM^®^3100 Genetic Analyzer). The sequences were analysed using the Lasergene software (DNASTAR, Madison,WI, USA) and the Mac Vector software (Oxford Molecular Group, Campell, CA, USA) to assemblings and alignings. Nucleotide sequence accession numbers ------------------------------------- The nucleotide sequence of the genomic region of E. colistrain P12b (O15:H17) with a size of 1234 bp containing the fliCgene for flagellin has been submitted to EMBL data library under accession number AJ515904. The coding sequences of the different fliCgenes from the following strains have been assigned the following accession numbers: AJ605764 for strain U1-41 (O5:K4:H4), AJ605765 for strain P7d (O68:H4), AJ605766 for strain C107-74 (O15:H17) and AJ536600 for strain E1541-68 (O154:H4). The origin of the strains is listed in Table [1](#T1){ref-type="table"}. Molecular cloning of fliCgene PCR products -------------------------------------------- PCR products encompassing the complete coding sequences of the fliC-H4 genes were obtained from genomic DNA prepared as described \[[@B9]\] of E. colistrains U9-41 and P12b using primers fliC-5 and fliC-6. The amplification products were inserted into the vector pLITMUS38 (New England Biolabs, Beverly, MA, USA) digested with EcoRV. The orientation of the the insert PCR products was determined by using commercially available sequencing primers LITMUS forward 28/38 and LITMUS Reverse 28/38 (New England Biolabs). Immuno electron microscopy (IEM) of E. coliflagellar antigens --------------------------------------------------------------- Motile cultures of E. colistrains were produced by repeated passage on semi-solid agar followed by growth in L-Broth as described above. Cultures carrying recombinant pLITMUS38 plasmids were grown in the presence of 100 μg/ml ampicillin. IEM was performed using fresh, non-formalized cultures of motile bacteria. For IEM, aliquots of respective bacterial cultures were diluted 1:2 in PBS pH 7.2 and adsorbed onto glow-discharge treated 400 mesh grids coated with Pioloform and carbon (Wacker Chemie, Munich, Germany) \[[@B31]\]. Grids with adsorbed bacteria were preincubated for 30 min at room-temperature with blocking buffer (0.1 % bovine serum albumine (Sigma, Deisenhofen, Germany) in PBS). Rabbit anti-H48- and anti-H4 hyperimmune sera were diluted 1:1000 in blocking buffer. After conditioning, specimens were incubated for 30 min at room temperature on droplets of the specific, unlabelled antibodies. Non-bound antibody was removed by washing the grids twice for 10 min on blocking buffer. Immuno-specifically bound primary antibodies were detected using anti-rabbit-IgG-gold 5 or -gold 10 nm conjugates (British Bio Cell International Ltd, Cardiff, UK). The conjugates were diluted 1:20 in blocking buffer and reacted for 30 min at room temperature. Unbound conjugate was removed by a sequence of washing steps (two times with blocking buffer for 5 min each; once with PBS for 3 min and a final wash with double destilled water for 3 min) at room temperature. Before negative staining with 1 % uranyl acetate (pH 4.0--4.5), the grids were washed rapidly on 4 droplets of double destilled water. The preparations were analyzed with an EM 10 electron microscope (Zeiss-LEO, Oberkochen, Germany) at an accelerating voltage of 80 kV. To look for different antigenic determinants expressed on the flagella of strains JM109 or TPE1978, double immuno-labelling was performed using H48 and H4 antisera sequentially and two anti-rabbit-IgG-gold conjugates with different sized markers for the detection of the bound primary unlabelled rabbit antibody. Both E. colistrains were incubated with rabbit anti-H4 (1:1000) followed by anti-rabbit-IgG-5 nm gold and two washing steps on droplets of blocking buffer for 10 min. The samples were subsequently incubated with anti H48 antibody (1:1000) followed by incubation with anti-rabbit-IgG- 10 nm gold. Removal of surplus conjugate and negative staining were performed as detailed above. Author\'s contribution ====================== LB conceived of the study and carried out PCR genotyping and coexpression studies. ES carried out sequence determination and alignments and construction of recombinant plasmids. SZ and SK performed serological assays and PCR genotyping. CS performed analysis of fliCrecombinant plasmid carrying strains. AM and HG developed the IEM methodology and HG contributed to the data analysis. All authors participated in review and preparation of the final manuscript. Acknowledgements ================ We are grateful to Ida and Frits ∅rskov and to Flemming Scheutz (International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen, Denmark) for donating E. coliserotype reference strains. We thank Bärbel Jungnickel for excellent processing of the electron micrographs. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### HhaI digested fliCPCR products obtained with primers fliC-1 and fliC-2 from E. colireference strains for 53 different expressed H-types as indicated at the right side. Similarity of restriction fragment patterns was calculated with BioNumerics software and is indicated by the dendrogram on the left side. The flagellar antigens of strains encoding H-types H3, H17, H35, H36, H44, H47, H53, H54 and H55 are not encoded by fliCbut by other genes (flkA, fllA, flmAand others) in the corresponding E. colistrains and the fliCHhaI patterns obtained from these strains do therefore not correspond to their H-serotypes \[21, 25, 26, 27\]. ::: ![](1471-2180-5-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Alignment of the deduced FliC (flagellin) sequences from E. colistrains representing the flagellar antigen H4 and its genetic variants: U9-41 (accession BAA85081); C107-54 (accession CAE53943), E1541-68 (accession CAD60547); P12b (accession CAD56695); and P7d (accession CAE53942). ::: ![](1471-2180-5-4-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Electrophoretic separation of restriction enzyme digested fliCPCR products (primers fliC-1 and fliC-2) of E. colistrains U9-41 and P12b on 2% agarose: Lanes: 1+8= molecular weight standard; 2 = U9-41 (HpaII); 3 = P12b (HpaII); 4 = U9-41 (undigested); 5 = P12b (undigested); 6 = U9-41 (MboI); 7 = P12b (MboI). Sizes of restriction fragments are listed in Table 2. ::: ![](1471-2180-5-4-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### \(A) Low power micrograph of E. colistrain TPE1978 cells showing the density of the bacterial samples used for indirect IEM and the presentation of flagella (bar length = 1 μm) (B) IEM of strain TPE1978 flagella after incubation with rabbit flagellar H48 antiserum (1:1000) and detection of bound antibody by anti-rabbit-IgG- 10 nm gold (1:20), bar length = 100 nm. (C) Strain JM109 flagella after incubation with rabbit flagellar H48 antiserum and detection of bound antibody by anti-rabbit-IgG- 10 nm gold. (D) Strain TPE1978 flagella after incubation with rabbit flagellar H4 antiserum (1:1000) and detection of bound antibody by anti-rabbit-IgG- 5 nm gold. (E) Strain JM109 flagella after incubation with rabbit flagellar H4 antiserum and detection of bound antibody by anti-rabbit-IgG- 5 nm gold. (F) Double-labeling IEM of strain TPE1978 after sequential incubations with rabbit flagellar H4 antiserum and anti-rabbit-IgG 5 nm gold, followed by rabbit flagellar H48 antiserum detected by anti-rabbit-IgG- 10 nm gold. Both, 5 nm and 10 nm gold markers are bound at comparable amounts over all flagella present on the bacteria. (G) Double-labeling IEM of strain JM109 after sequential incubations with rabbit flagellar H4 antiserum and anti-rabbit-IgG- 5 nm gold followed by rabbit flagellar H48 antiserum and anti-rabbit-IgG- 10 nm gold. Only H48 specific (10 nm) gold particles are bound to the flagella of JM109. ::: ![](1471-2180-5-4-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Agglutination titers with H4 antisera derived from strains U9-41 and P12b ::: Strain reported serotype agglutination with antisera^a^ ---------- ------------------- -------------------------------- ------- U9-41 O2:K1:H4^b^ 12800 12800 U1-41 O5:K4:H4^b^ 25600 25600 P7d O68:H4^b^ 1600 3200 E1541-68 O154:H4^b^ 3200 1600 P12b O15:H17^c,e^ 6400 12800 872-69 O20:H17^d,e^ 12800 12800 107-74 O15:H17^d,e^ 3200 3200 305-78 O15:H17^d,e^ 3200 3200 870-69 O20:H17^d,e^ 6400 12800 327-01 O77:H17^d,e^ 12800 12800 a\) reciprocal value of agglutination titers with antisera (the same results were obtained in two separate experiments). H4~U9-41~and H4~P12b~indicates the H4 antisera produced with strains U9-41 and P12b, respectively. b\) E. colistandard test strain \[3-5\] c\) E. colistandard test H-test strain \[3-5\], able to produce spontaneously a variant expressing flagellar antigen H4 and containing a fliC-H4 gene \[21, 22\] d\) strains and serotype data provided from Flemming Scheutz, Statens Seruminstitut, Copenhagen, Denmark e\) all H17 strains were shown to carry a fliC-H4 genotype and expressed flagellar H4 antigens (this work). ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Amino acid identitity/divergence between the deduced FliC proteins of the six investigated E. colistrains (aligned length 349 aa, no gaps). ::: percent identity -------------------- ---------- ------------------ -------- --------- ---------- -------- -------- U9-41 U1-41 C107-74 E1541-68 P12b P 7d Percent divergence U9-41 \\\* 100.0 99.7 99.4 98.6 97.4 U1-41 0.0 \\\* 99.7 99.4 98.6 97.4 C107-74 0.3 0.3 \\\* 99.1 98.3 97.1 E1541-68 0.6 0.6 0.9 \\\* 98.0 98.0 P12b 1.4 1.4 1.7 2.0 \\\* 96.6 P 7d 2.6 2.6 2.9 2.0 3.5 \\\* ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### PCR/RFLP typing of fliCgenes in E. coliH4 strains with restriction enzymes HhaI, HpaII and MboI ::: DNA fragments (bp) obtained by enzymatic digestion of the 953 bp PCR product obtained with primers fliC-1 and fliC-2 ------------------------------------ ---------------------------------------------------------------------------------------------------------------------- --------------------------- ---------------------- fliC-genotype (prototype strain) HhaI HpaII MboI fliC-H4 (U9-41)^a^ 362, 304, 104, 66, 50, 29^d^, 20^d^, 16^d^2^d^ 296, 240, 225, 159, 33^d^ 429, 313, 182, 29^d^ fliC-H4 (P7d)^b^ 362, 304, 104, 66, 50, 29^d^, 20^d^, 16^d^,2^d^ 296, 273, 225, 159 429, 313, 182, 29^d^ fliC-H4 (P12b)^c^ 362, 304, 104, 66, 50, 29^d^, 20^d^, 16^d^,2^d^ 296, 273, 225, 159 495, 458 a\) PCR/RFLP patterns found in strains U9-41, U1-41, E1541-68, C107-74 and in 82 E. colistrains which reacted with H4 antisera. The serotypes of all 86 strains are: O2:K1 (4 strains), O5 (2), O7 (1), O8 (1), O13 (1), O15 (2), O20 (2), O50 (1), O60 (1), O77 (1), O78 (1), O99 (3), O111 (9), O113 (32), O114 (5), O117 (1), O119 (1), O141 (3), O154 (1), O176 (1), O181 (1), O-untypable (9), O-rough (3). Forty-two of the strains belonging to O-groups O60, O113, O114, O141 as well as O-rough and O-untypable strains produced Shiga-toxins. b\) RFLP patterns found with strain P7d (O68:H4) c\) RFLP-patterns found with strain P12b (O15:H17) d\) fragments smaller than 50 bp are not detectable on 2% agarose gels (Fig. 3). Exact fragment sizes were calculated on the basis of nucleotide sequence analysis for each of the restriction enzymes used ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### H-Agglutination reaction of fliC-H4 recombinant plasmid carrying E. coliK-12 strains ::: Strain serotype or fliCrecombinant JM109 derivative agglutination with H-specific antisera^a^ ---------- ------------------------------------------------ ------------------------------------------- ---------- ---------- U9-41 O2:K1:H4 \<200^b^ 12800 12800 P12b O15:H17 \<200^b^ 6400 12800 JM109^c^ O-rough:H48 12800 \<200^b^ \<200^b^ TPE1976 JM109 (pLITMUS38-fliC-H4~U9-41~)^d^ 12800 12800 12800 TPE1978 JM109 (pLITMUS38-fliC-H4~P12b~)^e^ 12800 6400 12800 a\) reciprocal value agglutination titers with antisera (the same results were obtained in two separate experiments). H4~U9-41~and H4~P12b~indicates the H4 antisera produced with strains U9-41 and P12b, respectively. b\) no agglutination with start serum dilution 1:200 c\) E. coliK-12 \[23\]. d) fliC-H4 gene cloned from strain U9-41 e\) fliC-H4 gene cloned from strain P12b :::	PubMed Central	2024-06-05T03:55:52.652675	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548302/", "journal": "BMC Microbiol. 2005 Jan 24; 5:4", "authors": [ { "first": "Lothar", "last": "Beutin" }, { "first": "Eckhard", "last": "Strauch" }, { "first": "Sonja", "last": "Zimmermann" }, { "first": "Stefan", "last": "Kaulfuss" }, { "first": "Christoph", "last": "Schaudinn" }, { "first": "Andrea", "last": "Männel" }, { "first": "Hans R", "last": "Gelderblom" } ] }
PMC548303	Background ========== Persistent atrial fibrillation (AF) is difficult to treat. In the absence of class I or III antiarrhythmic drugs sinus rhythm is maintained in only 30--50% of patients during the first year after Direct Current electrical cardioversion (ECV)\[[@B1],[@B2]\]. Furthermore, even following an aggressive approach with repeated ECVs and use of prophylactic drugs, arrhythmia-free outcome is still poor: only 39% of patients maintain sinus rhythm during two years of follow-up\[[@B1],[@B2]\]. Notwithstanding the recent results of AFFIRM and RACE showing no beneficial effect of rhythm control over rate control a rhythm control strategy may be indicated in severely symptomatic patients and those with a tachycardiomyopathy\[[@B3]\]. In recent years research has focused on the atrial remodeling processes that are induced by AF itself and that trigger the arrhythmia to become sustained: \"AF begets AF\"\[[@B4]\]. One of the remodeling processes induced by AF is fibrosis. Fibrosis causes dispersion of conduction, which, in its turn is related to inducibility of AF\[[@B5]\]. The renin-angiotensin system seems to play an important role in the development of fibrosis in heart failure. It was shown that pre-treatment with enalapril may attenuate atrial fibrosis and conduction abnormalities in a canine model of heart failure, and the occurrence of AF in patients with left ventricular dysfunction \[[@B5]-[@B7]\]. A recent experimental study showed that angiotensin II blockers may prevent electrical remodeling when started before start of AF\[[@B8]\]. In the present study we report on the effects of ACE inhibition on the outcome of ECV and the prevention of early recurrences after ECV of persistent AF. Methods ======= One hundred-seven consecutive patients with persistent AF, defined as the presence of AF for at least 24 hours were included in this study\[[@B9]\]. ECV was performed according to a previously described step up protocol\[[@B10]\]. Successful ECV was defined as the presence of sinus rhythm for at least 4 hours after ECV. No difference was made between unsuccessful ECV due to shock failure or due to an immediate recurrence of AF (within 2 minutes after successful ECV). ACE pre-treatment was defined as use of ACE inhibitors before onsetof the current AF episode. Most patients on ACE inhibitors used these drugs for hypertension or congestive heart failure. To make sure that all patients were not completely remodeled at the very moment of start of the current episode of AF (and thereby verifying the fact that they were treated with ACE inhibitors before the process of electrical remodeling started), only patients with at least 1 month sinus rhythm before the current episode of AF were included in this study. Duration of AF was determined as precisely as possible by previous electrocardiograms, 24-hours Holter registrations, and by the patient\'s history. None of the patients were on class I or III antiarrhythmic drugs neither at the moment of ECV nor during follow-up. Statistical analysis -------------------- Quantitative variables were compared between groups using a two-tailed t-test for normally distributed variables or a Wilcoxon two-sample test for skewed distributed variables. For qualitative variables (categorical or ordered), group differences were evaluated using a Fisher\'s exact test or a Chi-square test. Accordingly, baseline characteristics are given in mean ± SD, median and range (min-max) or percentages. To determine the predictive factors for successful ECV, an univariate logistic regression analysis was performed using the relevant baseline predictors. Variables with a p-value \< 0.20 were selected for the multiple logistic regression analysis to derive a model with statistically significant predictors, by using a backward selection method. All p-values are two-sided and a p-value of \< 0.05 was considered statistically significant. SAS version 6.12 (Cary, NC) was used for all statistical evaluations. Results ======= Baseline characteristics are listed in table [1](#T1){ref-type="table"}. Twenty-eight patients (26%) were treated with ACE inhibitors beforestart of the current episode of AF (pre-treated patients). In the latter group, ECV was successful in 96% while in the group of patients not pre-treated with ACE inhibitors before start of the current AF episode only 80% had a successful ECV (p = 0.04). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Baseline characteristics ::: All Successful ECV Unsuccessful ECV P ----------------------------------- --------------- ---------------- ------------------ ------ N = 107 N = 90 N = 17 Duration in days, median (range) current episode 115(71--175) 113 (61--175) 126 (81--189) ns total AF duration 144 (95--232) 142 (86--213) 160 (102--340) Ns Number of previous ECVs, N(range) 1 (0--5) 1 (0--5) 1(0--3) Ns Underlying heart disease \* (%) Left ventricular dysfunction 21% 25% 0% 0.02 Hypertension 30% 32% 17% ns Valve disease 19% 19% 22% ns Coronary artery disease 22% 21% 28% ns Lone AF 16% 16% 15% ns Echocardiography (mm ± SD) LA parasternal long axis 47 ± 8 47 ± 9 47 ± 8 ns LA 4 chamber view long axis 67 ± 9 67 ± 10 67 ± 6 ns LA 4 chamber view short axis 46 ± 8 46 ± 9 43 ± 5 ns RA 4 chamber view long axis 62 ± 8 62 ± 8 63 ± 7 ns LVEDD 50 ± 8 50 ± 8 50 ± 7 ns LVESD 35 ± 10 35 ± 10 33 ± 9 ns Medication Beta blocker 49% 50% 48% ns Calcium channel blocker 36% 36% 37% ns Digoxin 47% 46% 49% ns ACE pretreatment 26% 30% 6% 0.04 Diuretics 31% 31% 29% ns Angiotensin receptor blocker 1% 1% 0% ns \* more then 1 entity per patient AF = atrial fibrillation, ECV = electrical cardioversion, LA = left atrium, RA = right atrium, LVEDD = left ventricular end diastolic diameter, LVESD = left ventricular end systolic diameter ::: In general, patients treated with ACE inhibitors before start of the current episode of AF had more evidence for heart disease than those who were not pre-treated. Prevalence of hypertension (46% versus 24%, respectively, p = 0.03), and left ventricular dysfunction (40% versus 14%, respectively, p = 0.01) was significantly higher in the pre-treated group in comparison to those who were not pre-treated with ACE inhibitors. (Table [2](#T2){ref-type="table"}) Furthermore, there was a trend towards a higher prevalence of coronary artery disease in the pre-treated group compared to the not pre-treated group (36% versus 18%, respectively p = 0.07). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Baseline characteristics divided in ACE pre-treatment and no ACE pre-treatment. ::: No ACE pre-treatment ACE-pre-treatment P ----------------------------------- ---------------------- ------------------- ------ N = 79 N = 28 Duration in days, median (range) current episode 121 (74--178) 96 (59--135) ns total AF duration 161 (105--247) 117 (63--160) ns Number of previous ECVs, N(range) 1 (0--5) 1(0--3) ns Underlying heart disease \* (%) Left ventricular dysfunction 14% 40% 0.01 Hypertension 24% 46% 0.03 Valve disease 19% 21% ns Coronary artery disease 18% 36% 0.07 Lone AF 17% 14% ns Echocardiography (mm ± SD) LA parasternal long axis 46 ± 7 50 ± 10 0.03 LA 4 chamber view long axis 66 ± 9 67 ± 12 ns LA 4 chamber view short axis 45 ± 8 49 ± 10 ns RA 4 chamber view long axis 61 ± 8 64 ± 7 ns LVEDD 49 ± 7 54 ± 8 0.01 LVESD 34 ± 9 38 ± 12 ns Medication Beta blocker 51% 47% ns Calcium channel blocker 35% 39% ns Digoxin 45% 51% ns Diuretics 29% 34% ns Angiotensin receptor blocker 1% 0% ns \* more then 1 entity per patient AF = atrial fibrillation, ECV = electrical cardioversion, LA = left atrium, RA = right atrium, LVEDD = left ventricular end diastolic diameter, LVESD = left ventricular end systolic diameter ::: An additional 21 patients received ACE inhibitors afterstart of the current episode of AF because of either insufficiently treated hypertension or left ventricular dysfunction newly documented with echocardiography. Success of ECV in these patients was comparable to patients who were not treated with ACE inhibitors at all (80% and 82%, respectively). Use of beta adrenergic receptor blockers, calcium channel blockers or digoxin, started before the present episode of AF or not, did not influence success of ECV. After 1 month of follow-up 49% of the pre-treated patients compared to 50% of those who were not pre-treated with ACE inhibition were still in sinus rhythm (Table [3](#T3){ref-type="table"}, Figure [1](#F1){ref-type="fig"}). ::: {#T3 .table-wrap} Table 3 All No ACE-pretreatment ACE-pretreatment P ----------------------------- ----- --------------------- ------------------ ------ Successful ECV (%) 84% 80% 96% 0.04 SR at 1 month follow-up (%) 50% 50% 49% ns ECV = electrical cardioversion, SR = sinus rhythm ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Effect of pretreatment with ACE-inhibitors before start of AF. ::: ![](1471-2261-5-3-1) ::: Multivariate analysis showed that pre-treatment with ACE inhibitors and a smaller left atrial size were independent predictors of successful ECV (OR = 5.8, C.I. 1.3--26.1 and OR = 5.6, C.I. 1.2--25.3, respectively). Discussion ========== Main results ------------ This post-hoc retrospective analysis shows that use of ACE inhibitors before the onset of AF enhances acute ECV outcome but it does not improve maintenance of sinus rhythm. Furthermore, when ACE inhibitiors are instituted later, i.e. after the start of AF, it does no longer improve success of ECV. Effect of ACE-inhibition on structural remodeling ------------------------------------------------- ACE inhibitors reduce the incidence of AF in patients with left ventricular dysfunction\[[@B7],[@B6]\]. This may be related to the protective effects of ACE inhibitors, which help to maintain atrial integrity and attenuate fibrosis\[[@B5]\]. In the present study pre-treatment with an oweveHowACE inhibitor (i.e. use of ACE inhibitors before onset of the arrhythmia) improved acute outcome of ECV. In view of the above this suggests that ACE inhibitors may prevent or diminish AF induced structural remodeling. These clinical findings are compatible with experimental findings showing that ACE-inhibition could attenuate heart failure induced atrial functional remodeling and fibrosis in dogs\[[@B11]\]. In atrial tissue from AF patients an ACE-dependent increase of activated extracellular signal-regulated kinase (Erk) type 1 and 2 was found, which may contribute to the development of atrial fibrosis during AF\[[@B12]\]. Effect of ACE-inhibition on electrical remodeling ------------------------------------------------- In 1995 it was shown that AF induces several electrophysiological changes, called electrical remodeling\[[@B4]\]. Experimental data show that ACE inhibition prevents short-term (\< 2 hours) tachycardia-induced atrial electrical remodeling\[[@B8],[@B13]\]. However, enalapril could not attenuate or prevent the long-term (7 days) effects of tachycardia on remodeling\[[@B8]\]. Several studies have investigated the role of calcium channel blockers on electrical remodeling. In these studies it was also shown that although there was a short-term prevention of electrical remodeling, calcium channel blockers did not have a long-term protective effect on electrical remodeling \[[@B14]-[@B16]\]. Effect on AF recurrences ------------------------ Madrid et al. investigated the role of the angiotensin II type 1 receptor antagonist irbesartan in amiodarone treated patients\[[@B17]\]. Persistent AF patients were randomized to either treatment with amiodarone alone or treatment with amiodarone in combination with irbesartan. Drug treatment was started at least 3 weeks before cardioversion but afterthe start of AF. No differences were found in electrical cardioversion outcome between the two treatment groups which is in contrast to the present study. This may relate to the fact that all patients were in AF at the very moment of start of irbesartan. Two months after cardioversion it appeared that patients treated with irbesartan and amiodarone had a significantly lower AF recurrence rate compared to amiodarone alone treated patients, 15% versus 37%, respectively (p = 0.007). This difference was maintained during a median follow-up of 254 days. According to their figure 2, the benefit of irbesartan (in combination with amiodarone) was mainly achieved by reduction of recurrences after the first two weeks after cardioversion. An earlier study on the role of ACE inhibitors in patients with heart failure and AF showed a trend towards more patients maintaining sinus rhythm after ECV when instituted on lisinopril in comparison to patients not treated with lisinopril\[[@B18]\]. In the present study no difference in recurrence rate could be found between patients with and without ACE-pretreatment. Limitations of the study ======================== This was a non-randomized and post-hoc analysis. This implies that all findings can only be used to generate hypotheses. However, this is the first clinical study showing that ACE-inhibition initiated before start of AF enhances direct ECV outcome. Only a very small amount of patients was on angiotensin receptor blockers. Whether the effect of pretreatment with angiotensin receptor blockers on cardioversion outcome would be the same as the effect of pretreatment with ACE inhibitors could not be investigated in this study. However, in this context the results of the study of Madrid et al. are encouraging \[[@B17]\]. Conclusions =========== Pretreatment with ACE-inhibitors significantly improves acute outcome of ECV when initiated before the onset of AF but it does not lead to better maintenance of sinus rhythm. When ACE inhibitiors are, however, instituted later, i.e. after the start of AF, it does no longer improve success of ECV. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= TVN carried out the study and drafted the manuscript, HC designed the study, and interpreted the data, MB participated in the draft of the manuscript and interpreted the data, DVV participated in the draft of the manuscript and interpreted the data, IVG participated in the design of the study and interpreted the data. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2261/5/3/prepub>	PubMed Central	2024-06-05T03:55:52.656246	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548303/", "journal": "BMC Cardiovasc Disord. 2005 Jan 24; 5:3", "authors": [ { "first": "Trudeke", "last": "Van Noord" }, { "first": "Harry JGM", "last": "Crijns" }, { "first": "Maarten P", "last": "van den Berg" }, { "first": "Dirk J", "last": "Van Veldhuisen" }, { "first": "Isabelle C", "last": "Van Gelder" } ] }
PMC548304	Background ========== Clostridium difficileinfection may be a compounding factor in ulcerative colitis (UC) with data suggestive of a role in disease exacerbation or initiation by the organism in a subset of patients \[[@B1]\]. Adjunctive antibiotic therapy, either broad-spectrum or gram positive-specific, has shown limited efficacy in UC \[[@B2],[@B3]\]. However, sporadic reports in which significant therapeutic potential was achieved \[[@B4]-[@B6]\] suggest that a distinct subclass of patients with an infectious-associated phenotype may exist. Although there have been an increasing number of case reports on ulcerative colitis complicated by cytomegalovirus infection \[[@B7]-[@B9]\], there are few with conclusive evidence of infection with Clostridium difficile. Herein, we report a case of steroid-resistant UC with profound rheumatologic involvement immediately preceded by C. difficileinfection in which remission of UC symptoms was twice achieved in response to adjunct metronidazole and vancomycin therapy. The patient remained symptom-free without supportive therapy for a four-year period. Case presentation ================= A 32-year-old white male presented with nausea, vomiting, crampy lower abdominal pain and diarrhea. His initial lab values were hemoglobin 15.6 g/dl (14--18 g/dl), hematocrit 45.8% (40--54%), sodium 131 mEq/l (135--146 mEq/l), potassium 3.9 mEq/l (3.5--5.5 mEq/l), bicarbonate 24 mEq/l (22--24 mEq/l), chloride 91 mEq/l (95--112 mEq/l), BUN 37 mg/dl (7--25 mg/dl), serum creatinine 1.6 mg/dl (0.7--1.4 mg/dl), and normal liver function tests. He was treated with intravenous fluids, Ciprofloxacin and anti-emetics, to which the diarrhea resolved in 2--3 days. After one month he had recurrent watery diarrhea accompanied with rectal pain, bleeding and vomiting. He also developed fever of 101.6 F. The physical exam was remarkable for decreased bowel sounds and moderate lower abdominal tenderness. There were no masses or organomegalies palpable. The rectal exam was remarkable for tenderness and heme-positive mucus. The remainder of the physical exam was uneventful. Stool samples from the initial visit were positive for Clostridium difficilecytotoxin, and depicted many WBCs (neutrophils 99% and eosinophils 1%), suggesting an infectious etiology. Stool sample was negative for giardia lamblia, salmonella, shigella, campylobacter, aeromonas and plesiomonas. There was no predominance of staphylococcus, yeast, or pseudomonas. The patient was initially started on Ciprofloxacin (500 mg bid.) for 3 days without symptom resolution. Metronidazole was added (250 mg tid.), but intolerance of the patient to metronidazole with exacerbations of nausea prompted the replacement to Vancomycin (250 mg qid) in conjunction with Ciprofloxacin for 7 days. During this initial treatment period, the patient characteristically developed migratory polyarticular joint pain and swelling involving the shoulders, elbows, fingers, hips and knees. Lack of response to antibiotics suggested an autoimmune disease as a contributing factor. At this time a sigmoidoscopy and pinch biopsy revealed diffuse acute and chronic inflammation with cryptitis, mucin depletion, and glandular foreshortening and branching suggestive of UC (Fig. [1](#F1){ref-type="fig"}). The patient was treated with Asacol (400 mg bid.) in conjunction with metronidazole (250 mg qid). After 7 days of combined therapy with continued weight loss and no improvement, prednisone (60 mg qid.) was added, resulting in cessation of GI symptoms and moderate improvement in joint pain and swelling. After 7 days of continued improvement, the antibiotic and Asacol therapy were stopped. The patient was maintained on high dose steroids for 14 days. However, fever, gastrointestinal, and joint symptoms recurred upon tapering of prednisone to 20 mg a day. When symptoms recurred, a stool culture was negative for infectious agents including Clostridium difficileantigen, confirming an autoimmune component to disease pathology in this patient. Prednisone was increased to 30 mg per day at which point gastrointestinal symptoms resolved but joint swelling and pain continued. Over the course of 3 months prednisone tapering was attempted three additional times, with each resulting in symptom recurrence. After 3 months of treatment, the patient experienced an acute dental abscess and was prescribed a combination vancomycin (250 mg tid) and metronidazole (250 mg tid). During this period, the patient was able to gradually reduce prednisone dosage without symptom recurrence. Antibiotics were continued for 30 days during which time prednisone was tapered completely. Symptoms did not recur and the patient remained symptom-free without further therapeutic intervention for 4.5 years. After this prolonged period of remission of UC symptoms, a second episode similar to the first occurred. The patient once again presented with bloody diarrhea, nausea, vomiting, and joint pain. Stool cultures were Clostridium difficilepositive by culture and cytotoxin. Once again the patient was treated with 60 mg daily of prednisone and metronidazole (250 mg tid). Symptoms did not resolve, and vancomycin (125 mg tid) was added, resulting in a significant improvement in symptoms. After 2 weeks of combined therapy metronidazole treatment was stopped and symptoms recurred within a week. Metronidazole was once again added, leading to resolution of symptoms. Combined antibiotic and steroid therapy continued for 2 months during which time prednisone was tapered completely and a second remission of UC symptoms was induced. Discussion ========== The patient was remarkable in several regards. The onset of UC and spondyloarthropathy coincided with C. difficileinfection. While various therapeutic regimens of prednisone, vancomycin and metronidazole preceded a negative C. difficiletoxin test, remission of UC symptoms could only be induced by the combination of these three medications. C. difficiletoxin tests have shown varying efficacy in diagnosing pseudomembranous colitis. It has been demonstrated that toxin-negative patients with C. difficilepositive stool culture may still have symptomatic pseudomembranous colitis \[[@B10]\]. Lynch et al showed that 46% of stool specimens from patients test negative by cytotoxin but positive by culture \[[@B11]\]. Likewise, others have shown that some cases of C. difficileinfection cannot be detected by the cytotoxin assay and suggested that the organism has the ability to vary toxin production \[[@B12]-[@B16]\]. The bacterium also tends to persist as an antibiotic-resistant vegetative spore, which explains the high frequency at which the symptoms recur following treatment. Since toxin-negative patients have presented with C. difficilerelated diarrhea, it is reasonable to assume that the organism may exist undetectably in a symptomatic patient, but especially in a patient with inflammatory bowel disease (IBD). The elusiveness of the bacterium in diagnosis of C. difficileinfected IBD patients was confirmed in 2001 by Markowitz et al. They demonstrated that single-toxin assays failed to identify C. difficileinfection in approximately 40% of pediatric IBD patients and in a case report where a toxin A positive, toxin B negative variant was detected \[[@B14],[@B17]\]. It has also been shown that IBD patients have a higher incidence of C. difficileinfection than in healthy controls \[[@B18]\]. When considered together with the case reported here, this information leads us to the hypothesis that a small subclass of UC pathology may actually have an infectious etiology resulting from chronic C. difficileinfection. IBD has been shown to be more prevalent in industrialized nations, where antibiotic treatment has its longest history \[[@B19]\]. The increase in IBD prevalence parallels rising antibiotic use over the last 50 years \[[@B20]\]. If an undetected antibiotic-resistant enteric pathogen is responsible for inducing or exacerbating IBD, then widespread antibiotic use may have contributed to the increased prevalence of IBD in these industrialized nations. This case is especially interesting considering H. pyloriand its recently discovered tendency to adhere to glycoconjugates expressed in inflamed gastric mucosa \[[@B21]\]. In light of this new information, it is tempting to speculate that C. difficilecould similarly bind to inflamed colonic tissue in this subclass of UC patients. Further research in this area could yield valuable new data. The potential utility of combined therapy is further suggested by the fact that antimicrobial resistance among C. difficile strains to metronidazole and with intermediate resistance to vancomycin is emerging in countries like Hong Kong (where one of 100 C. difficile isolates was resistant to metronidazole) and Spain (where 9% of 469 clinically significant C. difficile isolates were resistant to metronidazole, particularly in isolates recovered from HIV-positive patients and few patients also had intermediate resistance to vancomycin) \[[@B22]-[@B24]\] It is interesting to note that no resistance was found to metronidazole or vancomycin among C. difficile strains from isolates in patients from UK, Germany, Brazil, Poland or Kuwait \[[@B25]-[@B29]\]. The Public Health Laboratory Service (PHLS) Anaerobe Reference Unit (ARU) has also not been successful in detection of metronidazole resistance in any of their over 1000 C. difficile isolates tested \[[@B24]\]. The impact of drug resistance should be considered if long-term treatment is utilized in patients. However, a complicating factor in this context is that for metronidazole and vancomycin, minimum inhibitory concentration susceptibility breakpoints are usually set for isolates causing systemic infections that are based upon antimicrobial levels in blood (serum) and not on the levels in the intraluminal area, where higher drug concentrations can be achieved \[[@B30]\]. Conclusions =========== Recurrent relapses could only be suppressed in this patient by the combination therapy of corticosteroids, metronidazole and vancomycin. Our observations suggest that this combination therapy may be effective to confront and induce remissions of UC symptoms in patients with C. difficiletoxin-positive refractory autoimmune symptomatologic UC as opposed to the use of a single agent. An opportunistic C. difficileinfection commonly results from the use of broad-spectrum antibiotics like quinolones. The frequent use of these antibiotics in treating IBD suggests that these patients could develop C. difficileinfection during treatment, thereby exacerbating symptoms and preventing remission of UC symptoms. It may therefore be practical to probe for C. difficileinfections more meticulously in patients with IBD. Competing interests =================== The author(s) declare they have no competing interests. Authors\' contributions ======================= All authors contributed equally to this work. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-230X/5/3/prepub> Acknowledgements ================ Written consent was obtained from the patient for the publication of patient\'s details. This work was supported by grants NIH 1 P20 RR15577, and NIH 1 P20 RR16478 from the National Institutes of Health, and a grant from FAIR, the Fund for Arthritis and Inflammatory Research Inc. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Colonic mucosal inflammation in ulcerative colitis with loss of goblet cells and neutrophilic infiltrate (magnification ×100) ::: ![](1471-230X-5-3-1) :::	PubMed Central	2024-06-05T03:55:52.658539	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548304/", "journal": "BMC Gastroenterol. 2005 Jan 24; 5:3", "authors": [ { "first": "Jonathan", "last": "Miner" }, { "first": "M Monem", "last": "Gillan" }, { "first": "Philip", "last": "Alex" }, { "first": "Michael", "last": "Centola" } ] }
PMC548305	Background ========== In 1943 Kanner coined \"infantile autism\" (autism derived from Greek autos, or self) after observing 11 children, mostly boys, on the basis of their social isolation. Autism, often referred to as autistic disorder or infantile autism, is a complex behavioral disorder, which, by definition, develops prior to age three. Autism is defined completely on the basis of impairments in social interaction and communication, repetitive and stereotypic behaviors. Recent research has examined autistic traits in a population of twins and found that social impairment actually follows a unimodal distribution without a clear demarcation to separate cases of the disorder \[[@B1]\]. For this reason, future discussion of autism may be referred to as autism spectrum disorder (ASD) \[[@B2]\]. For most children, the onset of autism is gradual; however, about 30% have a \"regressive\" onset. Fifty to seventy percent of children with autism are defined as mentally retarded by nonverbal IQ testing. Seizures develop in about 25% of children with autism. About 25% of children who fit the diagnostic criteria for autism at age two or three years subsequently begin to talk and communicate, and by six or seven years blend to varying degrees into the regular school population. The remaining 75% continue to have a life-long disability requiring intensive parental, school, and societal support. There are no biologic markers, therefore the standard criteria, compiled by the American Psychiatric Association Manual of Psychiatric Diseases, 4th edition (DSM-IV), is the primary diagnostic reference used in the United States for autism. The causes of autism can be divided into \"idiopathic,\" which comprises the majority of cases, and \"secondary,\" in which an environmental agent, chromosome abnormality, or single gene disorder can be identified. About 5--10% of individuals with autism can be diagnosed with secondary autism; the remaining 90--95% have idiopathic autism. About 30% of children with idiopathic autism have complex autism, defined by the presence of dysmorphic features or microcephaly or a structural brain malformation \[[@B3]\]. About 70% of children with idiopathic autism have essential autism, defined as the absence of physical abnormalities. A recent CDC case-finding study in Brick Township, New Jersey reported prevalence at 40 per 10,000 for autism \[[@B4]\]. While the latest epidemiologic study from the United Kingdom utilizing specialized visiting nurses who monitored child health and development at seven months, 18 to 24 months, and three years of age reported a prevalence rate of 16.8 per 10,000 for autism \[[@B5]\]. The sex ratio for autism has been estimated at 4 males:1 female \[[@B6],[@B7]\]. Social cognition and communication in autism may be related to dysfunction in the amygdala, hippocampus, and related limbic and cortical structures. The cerebellum may also form part of a distributed neuronal network responsible for social cognition and communication. Serotonin is the neurotransmitter implicated in autism \[[@B8]\] The single gene disorders in which secondary autism is observed include fragile X syndrome, tuberous sclerosis, phenylketonuria, Rett syndrome \[[@B3],[@B9]\], Sotos, Neurofibromatosis I, Joubert syndrome \[[@B10]\], and Smith-Lemli-Opitz syndrome \[[@B11]\]. Risk to sibs of idiopathic cases is 75 times greater than the prevalence in the general population \[[@B7]\] and higher concordance for autism among monozygotic (60--90%) than dizygotic (0--10%) twins \[[@B12]\] argue for a genetic predisposition to idiopathic autism. Multiple independent whole genome scans and chromosomal abnormalities studies have pointed out several candidate regions on chromosomes 2q, 3q, 7q, 6, 13q, 15q, 16p, 17q and sex chromosomes. These regions possess candidate genes that have been screened for mutations or association with autism \[[@B13]\]. However, a clear involvement of a major susceptibility gene (or genes) in autism remains far from clear. The results from linkage studies and the drop in the concordance rates between monozygotic and dizygotic twins suggests that the genetic etiology of autism is certainly heterogeneous (different genes in different families), polygenic (more than one affected gene per individual) with epigenetic influences and allelic heterogeneity (different variants in the same gene may lead to different patterns of genetic disease) \[[@B13]-[@B15]\]. To gain further insight into secondary autism we have compiled data on patients with autistic traits tested for fragile-X syndrome using molecular methods and chromosome abnormalities using cytogenetic analysis and FISH. Methods ======= A search was initiated using the key word autism in the indication field of the Genzyme Genetics, Orange laboratory database. For each case the electronic data and/or files were reviewed. The referral center, age, sex, karyotype, fluorescence in situ hybridization (FISH) and fragile X results were extracted and tabulated. G-banded metaphases were prepared using standard procedures and FISH was performed using the protocol provided by the manufacturer of the commercial probes (Vysis Inc., Illinois, MI). Fragile X test: Isolated DNA was tested by both Southern blot analysis and Polymerase chain reaction (PCR) for the size and methylation status of the CGG repeat expansion within the FMR-1 gene. Southern blot analysis was performed with the probe StB12.3 on EcoR1 and Eag1 digested DNA. PCR products were separated by acrylamide gel electrophoresis and detected with a CGG repeat probe. Results ======= Patients with autistic traits were referred by physicians to our laboratory for genetic testing. The clinical diagnostic criteria applied were not specified on the test request forms. A total of 433 patients with an indication of autism were sent to our laboratory for genetic diagnosis. The median age was 4 years. Sex ratio was 4.5 males to 1 female \[354:79\] (Table [1](#T1){ref-type="table"}). A chromosome abnormality was found in 14/421 \[3.33 %\] cases. A Fragile-Xq27.3 was diagnosed in 7/316 \[2.2 %\]. Chromosome abnormalities are summarized in Table [2](#T2){ref-type="table"}: The aberrations were: 4/14 \[28%\] supernumerary markers from cs 15 \[[@B3]\] and cs 2 \[[@B1]\] (Fig [1a](#F1){ref-type="fig"}); 4/14 \[28%\] deletions of 2q37.3, 3p25, 12q21.2q23.3 and 13q13.2q14.1(Fig [1b](#F1){ref-type="fig"}); 1/14 \[7%\] duplication of 15q11.2q13; 3/14 \[21%\] inversions of 10p11.2q21.2, 17q23q25 (de novo) and 14q11.2q33 (mosaic) (Fig [1c](#F1){ref-type="fig"}); 2/14 \[14%\] translocations, one balanced t(1;14) and one unbalanced der(14;18) (Fig [1d](#F1){ref-type="fig"}). The 3/4 marker/ring had a 15 centromere signal by FISH and had either nil, one or two signals for D15S11, that demarcates the involvement of the critical region of Prader Willi/Angelman syndrome (Table [2](#T2){ref-type="table"}). Fluorescence in situ hybridization was performed on 23 of 433 patients for reasons other than to characterize a cytogenetic abnormality, 3 to rule out Williams-Beuren Syndrome, 5 for DiGeorge syndrome, 7 for proximal duplication of D15S11, 6 for subtelomere rearrangements, 1-for subtelomere rearrangement & proximal duplication D15S11 and 1 for duplication D15S11, Smith-Magenis Syndome, and DiGeorge Syndrome. All 23 FISH tests were negative (Table [3](#T3){ref-type="table"}). Fragile X (Table [4](#T4){ref-type="table"}): The mutations detected were: A full mutation (fM) and abnormal methylation in 3 \[43 %\] and mosaic mutations with partial methylation of variable clinical significance in 3 \[43%\]. The mosaic size mutations were: an fM \[200--900 repeats(r)\] / deletion mutation \[30 r\] with partial methylation. The faint bands for the full mutation and deletion mutation may be a reflection of the sample quality (Fig [2](#F2){ref-type="fig"}, Lane 5). Two size mosaics had permutation (pM) \[150 r\]/fM \[400 r\] (Fig [2](#F2){ref-type="fig"}), and pM \[155 r\]/fM \[800 r\] with normal and abnormal methylation. A premutation mosaic female carrier with an atypical EcoR1 and Eag1 pattern and a typical BssH1 pattern gave 2.8, 3.0, 5.2--5.4 Kb bands, the Eag1 pattern suggested a DNA sequence change \[14%\]. PCR gave reproducible bands corresponding to 29, 65, 80 repeats and a faint band for 39 repeats. Intermediate mutation (45--54 r) was found in 5 males and 2 females. Discussion ========== Chromosomal causes of secondary Autism Spectrum Disorder (ASD) -------------------------------------------------------------- About 1.7 % to 4.8 % of individuals with ASD have chromosome abnormalities, including unbalanced translocations, inversions, rings, and interstitial deletions and duplications (Table [5](#T5){ref-type="table"}). The chromosome abnormalities that have been reported on more than one occasion are duplication of 15q, deletions of 18q, Xp, 2q and the sex chromosome aneuploidies 47,XYY and 45,X \[[@B16]\]. A recent FISH subtelomere study found one out of ten unselected patients with ASD had a subtelomeric 2qter deletion \[[@B17]\]. In our experience 7/7 ASD patients were negative for subtelomeric rearrangements. Children with Down syndrome have autism more commonly than expected. The incidence was at least 7 % in one study \[[@B18]\]. This finding suggests that chromosome abnormalities may lower the threshold for the expression of autism. In our study (4/14 = 28 %) and in other surveys, the common recurring chromosomal abnormality was duplication of the proximal 15q region (Table [5](#T5){ref-type="table"}). About 1% of individuals with ASD have a chromosomal duplication in the Prader-Willi/Angelman region of proximal 15q \[\[[@B19],[@B20]\], present study 4/421\[0.95%\]\]. The duplicated region q11.2q13 is on the maternal chromosome 15 in autistic patients \[[@B21]-[@B23]\]. Most commonly, this is a supernumerary isodicentric 15q chromosome detectable by routine cytogenetic studies or, less commonly, an interstitial duplication of the region detected by FISH analysis for the SNRPNgene region. These two chromosome abnormalities have only subtle effects on the physical phenotype. Supernumerary isodicentric 15q chromosomes are de novooccurrences. Duplication of proximal 15q may result from segregation of a parental chromosome translocation or an interstitial 15q duplication. An abnormal gene dosage within 15q11.2-q13 might cause susceptibility to autism. The 15q11-q13 region is shown schematically in Figures [3](#F3){ref-type="fig"} &[4](#F4){ref-type="fig"}. Chromosome 15q11.2-q13 has gained support as an autism candidate region on the basis of the association of maternally derived chromosomal duplications of this region with an autistic phenotype \[[@B20],[@B24]-[@B27]\] and genetic evidence for linkage and allelic association in the same interval in chromosomally normal autism families \[[@B28]-[@B34]\]. The maternal specificity of chromosome 15 duplications in autism suggests a genomic imprinting effect. There are multiple imprinted genes in 15q11.2-q13, and two neurodevelopmental disorders exhibiting opposite patterns of genomic imprinting have been mapped to this region \[[@B35]-[@B37]\]. Interstitial deletion of 15q11.2-q13 specific for the paternal chromosome is the most frequent cause of Prader-Willi syndrome (PWS; MIM 176270), whereas maternal-specific deletion of the same common interval results in Angelman syndrome (AS; MIM 105830). The converse of Angelman Syndrome is observed in autism, that is a maternal duplication. The four causes of Angelman syndrome are 1) maternal deletion of 15q11.2q13, 2) paternal UPD15 3) mutations in UBE3A 4) mutations leading to imprinting errors of this region. A population-based study showed a high rate of ASD in AS \[[@B38]\]. But, a mutation was not identified in the UBE3A putative promoter or coding region in 10 idiopathic ASD patients \[[@B39]\]. Lack of expression of the maternally expressed UBE3A gene in the brain is thought to be the cause of AS. Since patients with deletions compared to other types of AS mutations have a more severe phenotype, suggests the involvement of additional gene losses, such as GABAA receptor gene cluster \[[@B40]\]. Transcripts increased in patients with duplications 15q11.2q13 are maternal UBE3A \[[@B41]\], maternal ATP10C \[[@B40],[@B42]\] and other transcripts including antisense transcripts that could regulate gene expression \[[@B43]\] and may contribute to the duplication phenotype. Therefore, over expression of genes in 15q11-q13 probably confers ASD risk. Region proximal to D15S11 is considered to have no phenotypic effect. However, one of our patients had a marker 15 negative for D15S11, therefore, some duplications of 15, proximal to D15S11 and the autism candidate region may also influence susceptibility to autistic traits. Initial studies to characterize the phenotype of 15q11.2q13 duplication patients have found variation among affected people including mental retardation, motor coordination problems, seizure disorder, and impairments in attention, communication, and social function (some but not all with ASD or attention deficit hyperactivity disorder (ADHD)) \[[@B44],[@B45]\]. It appears there may be a parent-of-origin effect on the linkage and association signals in this region of UBE3A and ATP10C \[[@B46],[@B32],[@B33]\]. Further studies across data sets, and rigorous evaluation of potential functional effects of associated alleles, and a thorough assessment of haplotype transmission within ATP10C and neighboring genes would be conclusive. The majority of linkage and association data point to the GABRB3gene, which is one of a cluster of γ-aminobutyric acid (GABA) receptor subunits that map to the distal, apparently nonimprinted segment of the duplicated region (Fig. [3](#F3){ref-type="fig"}). Although a number of groups have detected genetic effects at GABRB3in independent autism populations \[[@B29]-[@B31]\], other studies have failed to replicate these observations \[[@B47]-[@B49]\]. Yardin et al 2002 \[[@B50]\] recommended a systematic screening by FISH, of chromosome region 15q11.2q13 in cases with autistic-like syndrome A deletion of 2q37.3 was identified by FISH in a patient with autism and macrocephaly in our study. Wolff et al 2002 \[[@B51]\] also reported an autistic patient with a 2q37.3 deletion detected using subtelomere probes. Whole genome screens have suggested several chromosomal regions that are potentially associated with a susceptibility gene for autism. \[[@B52]-[@B57]\]. Three studies revealed positive linkage to 2q \[[@B52],[@B53],[@B57]\] and a third study demonstrated linkage to distal 2q in a subset of patients with autism and delayed onset of phrase speech \[[@B53]\]. Genomic scans are limited by the number of loci that are assessed; therefore, not all areas may be equally represented. It is important to note that telomeric regions may have increased meiotic recombination and may be under-represented in these types of analyses. Thus, the FISH approach is an important correlative study in the search for susceptibility genes. Macrocephaly in ASD: most children with autism are born with normal head circumference and about 20% meet the criteria for macrocephaly \[[@B58]\]. The increased rate of growth in head circumference appears to be most dramatic in the first year of life and corresponds to increased growth of the cerebral cortex as measured by MRI \[[@B59]\]. A de novo deletion of chromosome 3, del(3)(p25), was found in one case with ASD and development delay in our patient pool. A deletion of 3q region was found by Konstantareas & Homatidis 1999 \[[@B60]\]. Genome wide scan found a major susceptibility locus at 3q25-27 and there was also allelic association in families with autism spectrum disorder originating from a subisolate of Finland \[[@B61],[@B62]\]. Animal models and linkage data from genome screens implicate the oxytocin receptor at 3p25-p26 \[[@B61],[@B62]\]. To date, there have been no reports of 12q deletions in patients with ASD, to the best of our knowledge. For the first time we report an interstitial deletion 46,XY,del(12)(q21.2q23.3) in a patient with ASD, development delay, mental retardation, multiple congenital abnormality, and family history of Down syndrome. 46,XY,del(13)(q13.2q14.1)de novo was found in one of our patients\' with ASD. Hyperserotonemia in autism is one of the longest-standing biochemical findings. The serotonin 2A receptor gene (HTR2A) on chromosome 13q14q21 is a primary candidate gene in autism. Converging data from recent genome screens also implicates the genomic region containing HTR2A \[[@B63]-[@B66]\]. Correlation of HTR2A disruption or deletion in our case with a 13q13.2q14.1 deletion and other 13q deletion in ASD patients would complement genome screen data. The recent physical mapping of the serotonin 5-HT(7) receptor gene (HTR7) to 10q23 \[[@B67]\] raises the question if the inversion inv(10)(p11.2q21.2) present in our patient, which is considered a normal familial variant could have long range influence on HTR7 and susceptibility to autism in some cases. So far there has been no observed association or link between chromosome 14 and ASD. Also, the mosaic inversion inv(14)(q11.2q33) \[3/20\] found in one of our patients is considered a cultural artifact when seen in 1or 2 cells. In this study, a patient with inv(17)(q23q25)de novo, had ASD, hypotonia and developmental delay. Chromosome 17q shows association with autism by genome wide scans and in linkage studies \[[@B57],[@B66],[@B68]\]. There is also interest in 17q since serotonin transporter gene (SERT) has been mapped to17q11-q12 \[[@B69]\]. We report a patient with 18p deletion due to an unbalanced translocation between 14 and 18. Majority of the reported cases with autism involve deletion of 18q \[[@B70]-[@B72]\]. A deletion of the 18p-arm (at band 11.3) in about 50% cells and 50% of the cells with a duplication of the long arm in peripheral blood was described in a mildly obese girl with DSM-III-R autistic disorder and moderate mental retardation \[[@B73]\]. Another preschool girl with selective autism and a deletion of Chromosome 18p11.l has also been described \[[@B74]\]. She had communication problems consistent with a diagnosis of autism. However, in the area of reciprocal social interaction she was a little less deviant than most children and had no major behavior problems typical of autistic disorder. Linkage and association studies have suggested at least 2 candidate loci, one on the short and the other on the long arms of chromosome 18 \[[@B75]\]. One of the breakpoints in the balanced translocation, 46,XX,t(1;14)(q25;q31.2) was 1q25, in the present study. A recent report, links D1S1675 that maps to chromosome 1q24 with autism \[[@B76]\] using obsessive-compulsive behaviors as a restricting criterion for the analysis. The proximity of our breakpoint and D1S1675 may be coincidental or causal. Although no cases with X- rearrangements were identified, in this study, the literature on abnormal X chromosome and autism is discussed as it has been cited in multiple cases. An autistic (ICD-10) woman had a translocation, t(X;8)(p22.13;q22.1) \[[@B77]\], a boy with \"autistic disorder\" had duplication of Xp22 \[[@B78]\], and a de novo Xp22.3 deletion was observed in 3 autistic females \[[@B79]\]. Further, mutations in cell adhesion genes NLGN4 on Xp22.3 and NLGN3 on Xq13 are reported in patients with autism \[[@B80]\]. Therefore, subtle Xp rearrangements have to be considered in the cytogenetic assessment of ASD patients Fragile X syndrome ------------------ The typical clinical picture in FRAXA includes mental retardation, macro-orchidism, large ears, and prominent jaw. Within neurons, the FMR protein (FMRP) interacts with mRNA and ribosomes, suggesting a role in regulating protein synthesis \[[@B81]\]. FMRP is heavily synthesized in dendritic spines in response to synaptic activity, and abnormal dendritic spine size and shape have been noted in FRAXA patients and fmr1 knockout mice \[[@B82]\]. These abnormalities may correspond to an abnormal postsynaptic response that weakens synaptic connections \[[@B83]\]. A multicenter study in Sweden \[[@B84]\] found fragile X in 13 of 83 boys (16%) with infantile autism but in none of 19 girls with infantile autism. Klauck et al. (1997) \[[@B85]\] concluded from molecular genetic studies of 141 patients from 105 simplex and 18 multiplex families that an association of autism with fragile X is nonexistent and that the Xq27.3 region is not a candidate for autism. Stoll (2001) \[[@B86]\] presented 11 children under the age of 8 years and the difficulties in diagnosis of fragile X syndrome at this age. The author concluded on the importance of fragile X DNA test for all children with mental retardation, autism, or significant developmental delay without a clear etiology. Whereas only a few percent of children with autism have fragile X syndrome, at least half of children with fragile X syndrome have autistic behaviors, including avoidance of eye contact, language delays, repetitive behaviors, sleep disturbances, tantrums, self-injurious behaviors, hyperactivity, impulsiveness, inattention, and sound sensitivities. The frequency of the fragile X syndrome among individuals with autism was ascertained up to 1993 using cytogenetic method. The incidence ranged from 12.7%--1.6%. However, these studies had different criteria for classifying positive cases. The differences were: the number of metaphases analyzed ranged from 20 to 100 and the cut-off range from 1% to 4% metaphases with a fragile X \[[@B84],[@B87]-[@B94]\]. Using molecular analysis the incidence of fragile X syndrome was 5% (1/20) \[[@B95]\], 3.3%(1/30) \[[@B96]\], 12% (3/25) \[[@B97]\] and the present study 2.2 % (7/316). The difference in incidence between the present (2.2%) and previous studies (3.3% -- 12%) may be due to small sample size in the other studies or clinical criteria for selection of patients or both. ### Mosaicism for FRAX mutation: Forty-three percent of FRAX patients had a mosaic mutation in our study. The prevalence of males who carry a full mutation and a permutation is 15--20% \[[@B98]-[@B102]\] among the affected individuals. Nolin et al 1994 \[[@B103]\] analyzed a group of affected fragile X males by Southern blotting and found 41% (61/148) to be mosaic. This observation of 41% is significantly higher than previous reports 15--20%. The difference could be technical modifications, which permitted the identification of faint premutation bands in some patients. The higher percentage (43 %) of affected males with mosaicism in our study suggests that the occurrence of such individuals may be frequent in patients with ASD. The degree of mental retardation seemed not to be influenced by the presence of premutation alleles in some of the cells and a full mutation in the rest of the cells \[[@B104]\]. While Merenstein et al 1996 \[[@B105]\] study suggests there may be some variation of clinical expression in fragile X males with a full mutation and permutation. It has been hypothesized that these mosaic cases should show higher levels of functioning than those who have only the inactive full mutation gene, but previous studies have provided negative or equivocal results. In one study, the cross-sectional development of communication, self-care, socialization, and motor skills was studied in 46 males with fragile X syndrome under age 20 years as a function of two variables: age and the presence or absence of mosaicism. The rate of adaptive skills development was 2--4 times greater in mosaic cases versus full mutation cases. FMR1 protein (FMRP) levels was shown to correlate with IQ, even in mosaic males for 38% of the IQ variance \[[@B106],[@B107]\]. There was also a trend for cases with autism to be more prevalent in the full-mutation group \[[@B108]\]. However, we found a high incidence (43 %) of mosaic FRAX mutation in autistic patients, which requires confirmation in other FRAX studies of large cohorts of ASD patients. ### Deletion mutation The molecular mechanism of the FRAX is based on the expansion of a CGG repeat in the 5\' UTR of the FMR1 gene in the majority of fragile X patients. The instability of this CGG repeats containing region is not restricted to the CGG repeat itself but expands to the flanking region as well. de Graaff et al 1996 \[[@B109]\] described four unrelated fragile X patients mosaic for both a full mutation and a small deletion in the CGG repeat containing region. Sequence analysis of the regions surrounding the deletions showed that both the (CGG)n repeat and some flanking sequences were missing in all four patients. The 5\' breakpoints of the deletions were found to be located between 75--53 bp proximal to the CGG repeat. This suggests the presence of a hot spots for deletions in the CGG repeat region of the FMR1 gene and emphasizes the instability of this region in the presence of an expanded CGG repeat \[[@B110]-[@B113]\]. All reported cases had fragile X phenotype (which may be an ascertainment bias), and the deletion was usually a faint band suggesting a recent mutation in a small population of cells. Our case had an unusual Southern band pattern, consistent with the presence of both a full mutation (3.7 and 5.8--7.9 kb; 200--900 r)and a deleted (2.8 kb; 30 r) mutation with partial methylation. Since immunohistochemical staining or Western blot analysis was not performed to assess the FMRP production it precludes clinical correlation. A 6-year old mosaic permutation female carrier had 29,65,80,39 repeats. The autism spectrum disorder in our patient may be due to diminished translational efficiency in FMRP production. Tassone et al (2000) \[[@B114]\] \[have shown FMRP in 61% and 70% lymphocytes in two females 91/2 years and 33 years of age with 103/33, 180/30 repeats and IQ of 49 and 90, respectively. The first patient has physical, cognitive, and behavioral features of the fragile X phenotype but FMRP was in the normal range, while the second patient also had FMRP level in the normal range was treated for depression and has a history of ovarian cyst, premature menopause at 27 years, and a hysterectomy at 31 years. She also experienced social anxiety, panic attacks, mood swings, and mild obsessive compulsive behaviors. Johnston et al. (2001) \[115\] recently observed that emotional problems, including depression and interpersonal sensitivity, were more likely to occur in carrier females with \>100 repeats. Austism spectrum disorder has been observed mostly in males with permutations \[[@B114],[@B116],[@B117]\]. The mosaic permutation in our female patient was 80 repeats as the largest expansion which is still below 100 repeats found in the affected cases in literature. Clinical correlation in our permutation mosaic case is hindered by the lack of FMRP studies and even so, the tissue distribution of mosaicism and FMRP profile would be the necessary phenotype determinant. The significance of intermediate alleles found in 7/316 (Average age 4.36 years) of our patients requires further exploration on larger independent samples as they may raise the threshold for important developmental disabilities and/or physical features \[[@B116]\]. The limitations of our retrospective study were a lack of uniform clinical criterion for inclusion and limited behavioral diagnostic information for purposes of dissecting the phenotype. However, for autism diagnosis it may be reliable, as shown in a recent study \[[@B118]\] which examined the UK General Practitioner Research Database (GPRD) and found the diagnosis of autism among general practitioners in the UK had a high positive predictive value. Conclusions =========== In our experience, the incidence of chromosome (3.33%) and fragile-X (2.2%) abnormalities totals to 5.53% in a population of patients with an indication of autism sent for genetic testing. Since, 28% percent of chromosome abnormalities detected in our study were subtle; a high resolution cytogenetic study for ASD patients with a scrutiny of 15q11.2q13, 2q37 and Xp23.3 region should be standard practice. The higher incidence of mosaic fragile-X mutations with partial methylation in our ASD population vs incidence of mosaicism in reported populations with fragile X syndrome \[50% vs 15--40%\], suggests that faint bands and variations in the Southern band pattern may occur rather frequently in fragile X positive autistic patients. The mosaic FRAXA mutation with normal and abnormal or partial methylation may also enhance the threshold for autism spectrum disorder. Careful analysis and high quality gels are required to rule out the type of mutation in patients with autistic traits. FRMP estimates in positive cases would be an extremely useful adjunct especially in mosaic cases. Future studies are necessary to corroborate the high incidence of mosaicism and their role in ASD. Competing interests =================== The author(s) declare that they have no competing interests. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2350/6/3/prepub> Acknowledgements ================ I thank Bernice Allitto, Deborah Anaya for their help and everyone who contributed to this database. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Chromosome abnormalities in patients with autistic traits (A) 4 markers: \[multiple copies\] derived from chromosome 2 \[1\], 15 \[3\] with nomenclature (B) 1 duplication of chromosome 15 with arrow denoting the region involved (C) 3 partial deletions (right homolog)(multiple copies) of 3p25, 12q21.2q23.3 & 13q13.2q14.1 with the ideogram, the arrows denote the deleted region. (D) 3 inversions (the right homolog) (multiple copies), inv(10)(p11.2q21.2), inv(14)(q11.2q32), inv(17)(q24.2q25.3) with arrows on the ideogram showing the inverted region (E) 2 translocations (partials in 2 copies, chromosomes involved (right) and their normal homolog (left)) one apparently balanced t(1;14)(q25;q31.2) and one unbalanced der(14;18)(q10;q10). The ideogram with arrows show the breakpoints ::: ![](1471-2350-6-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Mosaic fragile X mutations: Southern blot using probe StB12.3 on EcoR1 and Eag1 (methylation-sensitive enzyme) digested DNA. Lane 1 -- permutation carrier, Lane 2 & 3 -- normal female, Lane 4 -- normal male, Lane 5 -- an ASD male mosaic with fm (3.7 and 5.8--7.9 kb, 200--900 r)(arrows)/a deletion mutation (2.8 kb faint band, 30 r)(arrow). By PCR a 30 repeats and \>200 repeats were amplified. Lane 6 -- an ASD male mosaic fm (6.4 kb, 400 r)/pm (3.3 kb, 150 r). ::: ![](1471-2350-6-3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Chromosome 15q11-q13 region showing the autism candidate region. A schematic representation of the 15q11-q13 interval duplicated in autism cases and deleted in Prader-Willi/Angelman syndrome is shown. IC denotes the position of the 15q imprinting center. Loci corresponding to previous reports of linkage and association are indicated by dark and light arrowheads, respectively, below the map. (Adapted with permission from Sutcliffe J et al article \"Dense linkage disequilibrium mapping in the 15q11-q13 maternal expression domain yields evidence for association in autism\' in Molecular Psychiatry (2003) 8, 624--634) ::: ![](1471-2350-6-3-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### The map location of D15S11 probe used to characterize the markers ::: ![](1471-2350-6-3-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographics of the ASD patients tested ::: Demographics ------------------------------------------------------ Patients with ASD referred for genetic testing = 433 Median age = 4 years Sex ratio = 4.5 males to 1 female ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The chromosome abnormality found in 3.33 % (14/421) patients with an indication of autism ::: Sl \# Age Sex Clinical Indication Chromosome Result FISH Result Fragile X Result ----------- --------- --------- --------------------------------------------------------- ----------------------------------------------- --------------------------------------------------- ---------------------- Marker 4/14 = 28% 1 6.1 M Autism 47,XY,+mar\[27\]/46,XY\[3\] .ish der(2)(D2Z+) NRM 2 5 F Autism 47,XX,+mar .ish der(15)(D15Z+,D15S11++, GABRB3+) NT 3 3 M Autism 47,XY,+mar de novo .ish i(15)(q11.2)(rRNA++,D15Z+,D15S11-) NT 4 5.1 F Autism, MR mos47,XY,+r\[15\]/46,XX\[15\] .ish der(15)(rRNA+,D15Z+,D15S11+,GABRB3+). NT Duplication 1/14 = 7% 5 16.75 F Autism 46,XX,dup(15)(q11.2q13)de novo .ish dup(15)(q12)(D15S11++,GABRB3++)de novo NT Deletion 4/14 = 28% 6 6.5 F Autism, DD, MR, macrocephaly .ish del(2) (q37.3)(D2S447-) NT 7 6.6 F Autism, DD 46,XX,del(3)(p25) de novo NT 8 11.7 M Autism, DD, MR, multiple congenital abnormality, h/o DS 46,XY,del(12)(q21.2q23.3) .ish 12(wcp12x2) NRM 9 3.6 M Autism 46,XY,del(13)(q13.2q14.1)de novo .ish 13q13(D13S6x2),13q14(RBx2) NT Inversion 3/14 = 21% 10 2.8 F Autism 46,XX,inv(10)(p11.2q21.2)\* NT NRM 11 3.5 M Autism Mos46,XY,inv(14)(q11.2q33)\[3\]/46,XY\[17\]\* NT NRM 12 3.25 M Autism, hypotonia, DD 46,XY,add(17)(q23) or inv(17)(q23q25)de novo .ish inv(17)(q24.2q25.3)(wcp17x2,MPOx2,D17S928x2) NT Translocation 2/14 = 14% 13 2.7 F Autism 46,XX,t(1;14)(q25;q31.2) NT NT 14 3.3 M Autism 46,XY,der(14;18)(q10;q10) NT NRM DD = developmental delay, MR = mental retardation, DS = Down syndrome, NT = not tested NRM = normal \inv(10) is a normal familial variant and inv(14) is a frequently observed artifact of culture ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Summary of FISH cases ::: Sl \#* Age in years Sex Clinical Indication Karyotype FISH FRAX ----------- ------------------ --------- ---------------------------------------------- --------------- -------------------------------------------------------------------- ---------- 1 5.25 M Autism 46,XY Normal ish 7q11.23(ELNX2) 2 7 M Autism, FTT, MR, DD 46,XY Normal ish 7q11.23(ELNX2) NRM 3 M Autism 46,XY Normal ish 7q11.23(ELNX2) NRM 4 3.6 M Autism, DD, hypocalcemia, cardiac defect, MR 46,XY Normal ish 22q11.2(TUPLEx2) 5 6.3 M Autism, DD, hypotonia 46,XY Normal ish 22q11.2(TUPLEx2) 6 3.5 M Autism not ordered Normal-ish 22q11.2(TUPLEx2) 7 M Autism 46,XY Normal-ish 22q11.2(TUPLEx2) 8 M Autism 46,XY Normal-ish 22q11.2(TUPLEx2) NRM 9 6.5 M Autism, DD, MR 46,XY Normal, ish 15q12(D15S11x2) NRM 10 11.6 M Autism, DD 46,XY Normal, ish 15q12(D15S11x2) NRM 11 3.5 M Autism 46,XY Normal, ish 15q12(D15S11x2) NRM 12 3 M Autism 46,XY Normal, ish 15q12(D15S11x2) 13 2 M Autism, DD,MR 46,XY Normal-ish 15q12(D15S11x2) NRM 14 2.5 F Autism, DD,MR 46,XX Normal ish 15q12(D15S11) NRM 15 3.6 M Autism, DD, patch hyperpigmentation 46,XY Normal, ish 15q12(D15S11x2) NRM 16 3.5 M Autism, DD, seizures 46,XY Normal- subtelomere panel NRM 17 3.5 F Autism, DD 46,XX Normal-subtelomeres NRM 18 4.6 M Autism, DD 46,XY Normal-subtelomeres NRM 19 8.6 M Autism, DD 46,XY Normal-subtelomeres NRM 20 M Autism 46,XY Normal-subtelomeres NRM 21 2.7 M Autism 46,XY Normal-subtelomeres ABN 22 16 M Autism, DD, dysmorphic features, MR 46,XY Normal, subtelomeres Normal, ish 15q12(D15S11x2) 23 17.5 F Autism 46,XX Normal ish 15q11.2q13(D15S11x2), 17p11.2(SMSx2), 22q11.2(TUPLE1X2) M = male, F = female, NRM = normal, ABN = abnormal, DD = development delay, MR = mental retardation, FTT = failure to thrive. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### The details of Fragile X mutations found in 2.2 % (7/316) patients with a clinical indication of autism ::: Sl \# Age In years Sex Clinical indication FRAXA Result Methylation Chrom Result FISH Result ----------- ------------------ --------- ------------------------- ---------------------------------------------------------------------- ------------------------------- ------------------ ----------------- Full mutation 3/7= 43% 1 4.5 M Autism ABN-Full mutation (400,533,667repeats \[r\]) abnormal methylation 46,XY NT 2 5 M Autism ABN-Full mutation (600--1100 r) abnormal methylation 46,XY NT 3 4.5 M Autism, DD ABN- Full mutation (267--933 r), abnormal methylation 46,XY NT Mosaic mutation 3/7 = 43% 4 2.3 M Autism ABN-size mosaic Full mutation \[800 r\]/ premutation \[155 r\] Normal & abnormal methylation 46,XY NT 5 1.7 M Autism ABN-size mosaic Full mutation \[400 r\]/ premutation \[150 r\] Normal & abnormal methylation 46,XY Normal-sub tel 6 8.5 M Autism, DD ABN-mosaic Full mutation \[200--900 r\]/ deletion mutation \[30 r\], partial methylation 46,XY NT Mosaic Premutation carrier 1/7 = 14% 7 6 F Autism ABN- premutation mosaic \[29,65,80,39(light band)r\] 46,XX NT Av 4.36 years 2F, 5M Intermediate mutation 7 cases with 45--54r DD = developmental delay, NT = not tested ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Summary of reported population studies to assess the frequency of chromosome abnormalities (excluding fragile sites, polymorphisms and single cell abnormalities) ::: Study Subjects karyotyped Chromosome abnormality Clinical diagnosis ---------------------------------------- --------------------- --------------------------------------------- -------------------------------------------------------------------------------------------------- Konstantareas & Homatidis 1999 \[123\] 127 6 \[4.7%\] Diagnosed using the clinical criteria of autistic disorder by DSM-III (1983--1989) 46,XY,inv(2)(p11q13)pat,3q+ 47,XY,+mar 47,XY,+mar 47,XX,+13 47,XX,+inv dup(15)(pter→ q13::q13→ pter) 47,XY,+der(15)(pter→ q15::p11→ pter)de novo Gillberg & Wahlstrom 1985 \[124\] 46 2 \[4.3%\] Diagnosed using the American Psychiatric Association (1980) criteria DSM-III 47,XY,+21 47,XYY Lauritsen 1999 \[122\] 145 4 \[2.8%\] Cases with psychotic symptoms before 2--3 years or beginning at 2--3 or later between 1969--1993 47,XX,+mar,?t(13;22) 46,XX,t(9;10)(p23;q23.1) 46,XY,inv(10)(p11.21;q21.2)mat 46,XY,t(7;12)(q21.4;q15)de novo Li 1993 \[121\] 104 5 \[4.8%\] Diagnosed using the American Psychiatric Association (1980 & 1987) guidelines, DSM-III & III-R 47,XY,+21 46,XY/47,XY,+21 \[12/88\] 46,XY,t(5;6)(q13;p23)de novo 46,X,inv(Y)(p11q11) 46,fra(X)(q27.3),inv(Y)(p11q11) Weidmer-Mikhail 1998 \[120\] 59 1 \[1.69 %\] DSM-III-R (1991--1995) Tetrasomy 15 Ritvo 1990 \[6\] 233 9 \[3.9%\] DSM-III (1984--1988) 6-trisomy 21 Partial trisomy 8 Deletion 9p 46,XX,t(5q;11q)pat Wassink 2001 \[119\] 278 13 \[4.7 %\] DSM-III,-III-R & -IV (1980--1999) 46,XX,del(8)(p23) MR, diaphragmatic hernia, hemivertebra, 2-vessel umbilicus, strabismus 47,XX,der(14)t(14;?)(q22;?) MR, abnormal facies & palate, failure to thrive 46,XX,dup(15)(q11.2;q13) MR, abnormal EEG, precocious puberty 2--46,XY,del(15)(q11.2q13) Mild MR Moderate MR & Seizures mat47,XX,+mar de novo 47,XX,+mar McCune-Albright syndrome Microcephaly, abnormal facies 47,XY,+del(15)(q22) MR 46,XY,del(16)(q13q22) MR, Seizures, failure to thrive, abnormal facies, webbed neck macrocephaly, syndactyly 46,XY,add(17)(q23) MR & abnormal facies 2--47,XX,+21 MR, heart murmur, esotropia, recurrent pneumonia. MR1VSD, pneumonia and seizures 46,XY,add(22)(q13) Macrocephaly and failure to thrive Present 421 14 \[3.3%\] Physician referrals to a genetic lab. 1995--2003 March :::	PubMed Central	2024-06-05T03:55:52.659769	2005-1-18	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548305/", "journal": "BMC Med Genet. 2005 Jan 18; 6:3", "authors": [ { "first": "Kavita S", "last": "Reddy" } ] }
PMC548306	Background ========== In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery. \[[@B1]-[@B4]\], and in situproduction of anti-infective protectants \[[@B5]\] and microbicides \[[@B6]\]. A common approach to genetic manipulation of bacteria is based on the use of plasmid expression vectors since these recombinant molecules can be introduced into bacterial cells by a variety of genetic techniques such as natural transformation, artificial transformation, transduction, conjugative mobilization, and electroporation \[[@B7]-[@B9]\]. However, the major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. Certainly, this has consequences when using recombinant bacteria in vivowhere their replication occurs in the absence of selection. An alternative approach is to integrate recombinant DNA molecules into the bacterial chromosome since this method allows increased in vivostability of the genetic constructs. Therefore a lot of efforts have focused on the development of efficient expression systems based on chromosomal integration of expression cassettes \[[@B10],[@B11]\]. Naturally transformable bacteria represent a convenient model, since heterologous DNA can be easily integrated into their chromosomes, whereas genetic manipulation of non-transformable bacteria is more difficult and relies mainly on electroporation and conjugative mobilization of foreign DNA molecules. We have previously described a genetic system based on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable streptococci \[[@B12],[@B13]\]. A series of transposon insertion vectors containing two regions of homology with Tn916\[[@B14]\] have been created in order to manipulate both naturally transformable and non-transformable Gram-positive bacteria carrying Tn916\[[@B12]\]. The aim of this work was to select a strong promoter to improve this genetic system making it suitable for expression of single-copy recombinant genes in a broad spectrum of Gram-positive bacteria. Results and discussion ====================== Promoter selection by chromosomal integration --------------------------------------------- To select resident promoters from the genome of Streptococcus gordonii, we performed a random ligation of streptococcal DNA to a promoterless catgene, conferring resistance to chloramphenicol (Cm). The ligation mixture was used to transform the naturally transformable S. gordonii«Challis» strain V288 and transformants were selected for Cm resistance. Chromosomal DNA flanking the promoterless catgene provided the homology for the random integration of catinto the chromosome during transformation (Fig. [1](#F1){ref-type="fig"}). 71 Cm-resistant (Cm^R^) transformants were isolated, presumably as a result of transcriptional fusions of streptococcal promoters to the promoterless catgene. Eighteen Cm^R^transformants were selected for further characterization. The strategy commonly used to select promoters is based on cloning random chromosomal DNA fragments in a promoter probe vector upstream of a promoterless reporter gene. However, integrating the promoterless reporter gene (cat) directly into the streptococcal chromosome, allowed us to select resident chromosomal promoters expressing catafter in vivotranscriptional fusion at a single locus on the chromosome. This is preferable when looking for promoters to express heterologous genes integrated into the chromosome in a single copy. In vivoanalysis of promoter strength was determined in the eighteen selected transformants by measuring the minimum inhibitory concentration (MIC) of Cm. Fifteen transformants exhibited a MIC of 16 μg/ml, whereas the remaining three transformants exhibited a MIC of 8 μg/ml. The fifteen strains with higher MIC were tested for the stability of the chloramphenicol-resistance phenotype: after 50 generations of growth without selection, bacterial cultures were plated on non-selective medium and at least 200 colonies were picked and tested for Cm resistance. The stability of the resistance phenotype varied considerably among the different transformants (data not shown). Three strains (GP214, GP215 and GP216) showed a 100% stability and were chosen for further analysis. Cloning of a promoter from S. gordonii ---------------------------------------- The structure of the integrated catgene in GP214, GP215 and GP216 was analyzed by Southern blot. Only GP215 showed to have a single catcopy integrated into the chromosome, whereas in GP214 and GP216 integration occurred at multiple sites (data not shown). In order to clone the regions flanking catintegration site in GP215, the chromosome of this strain was cut with TaqI whose recognition sequence is absent inside the catgene. The derivative fragments were ligated to pBLUESCRIPT, and the ligation mixture was used to transform Escherichia colicells; transformants were then selected for Cm^R^. All transformants analyzed for plasmid content showed to carry a plasmid of the same size. One of these transformants (GP334) was selected for further analysis and the transforming plasmid was named pVMB5. By restriction analysis we showed that pVMB5 contains a 2.2 kb TaqI insert where a 600 base pairs (bp) streptococcal DNA fragment was cloned upstream of the catgene. This DNA fragment was stably maintained in E. coli, where it retained its promoter activity, conferring Cm^R^to E. coli. This is of considerable interest since it is known that very often cloning streptococcal promoters in a high copy number plasmid results in the failure of that plasmid to be established \[[@B15]\]. Sequence analysis ----------------- The streptococcal DNA upstream of the catgene in pVMB5 was sequenced (GenBank accession number: U74080). Analysis of the sequence revealed the presence of an open reading frame (ORF1), preceded by a typical ribosome binding site (RBS) (Fig. [2](#F2){ref-type="fig"}). A BLAST search with the partial sequenced genome of S. gordonii<http://www.tigr.org/index.shtml> showed that ORF1 represents the 5\'-end of a gene encoding the first 48 amino acids (aa) of an uncharacterized protein conserved in bacteria and whose function is unknown. As indicated in figure [2](#F2){ref-type="fig"}, this truncated streptococcal protein is translationally fused to the N-terminus of CAT. Sequence analysis also revealed that the first 122 bp of the cloned streptococcal fragment belong to the 3\'-end of a gene encoding a putative acetyltransferase (ORF2). To obtain information about the chromosomal region containing the 600 bp streptococcal DNA fragment cloned in pVMB5, we looked throughout the whole contig sequence and we found out that upstream ORF2, and partially overlapping with it, there is an ORF encoding a DltD horthologue (dltD), a protein involved in D-alanine incorporation into lipoteichoic acid (LTA) \[[@B16]\], whereas downstream ORF1, there is a gene encoding a Tmp7 transmembrane protein (Fig. [2](#F2){ref-type="fig"}). Identification of the transcriptional start site ------------------------------------------------ To identify the sequences responsible for the observed promoter activity, the 5\'-end of the cat-specific mRNA was mapped by primer extension with a specific primer. Total RNA was isolated from E. coliGP334 (containing pVMB5) and S. gordoniiGP215. In both strains, the position of the 5\'-end of the mRNA was located at the same purine residue at position 423 of the 600 bp region of streptococcal DNA, 26 nucleotides upstream of the ORF1 translational start site (Fig. [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}). Putative -35 and -10 sequences closely resembling the consensus E. coliσ^70^and Bacillus subtilisσ^43^\[[@B17]\] recognition sequences TTGACA (-35) and TATAAT (-10) could be identified. The -35 region was TTGCAA, and the -10 region was TAGAAT. The spacing between the -35 and -10 region was 17 bp and was thus similar to the spacing in B. subtilis(17 to 19 bp) and E. coli(16 to 18 bp) promoters. Moreover, a TG nucleotide pair was found 1 bp upstream of the -10 region; such a structure is typical of Gram-positive bacteria promoters \[[@B18]\]. Based on this information, we concluded that indeed we isolated a streptococcal promoter which was designated PP. The spacing between the 5\'-end of the mRNA and the -10 hexanucleotide was only 2 bp, which is unusually short. A second putative -10 region could be identified in the TATGAT hexanucleotide (Fig. [2](#F2){ref-type="fig"}), whose distance from the 5\' end of the mRNA is 7 bp. However, since this sequence is not preceded by the TG nucleotide pair typical of Gram-positive promoters, and is separated from the -35 region only by 12 bp, we suppose that this -10 region of PP is probably not active. Insertion vectors to express heterologous proteins -------------------------------------------------- We have previously developed a genetic system based on the use of Streptococcus pyogenessurface fibrillar M6 protein, as a partner for the construction of translational fusions to deliver foreign proteins on the surface of S. gordonii\[[@B10]\]. To determine the possibility of using PP for chromosomal single copy heterologous gene expression in Gram-positive bacteria, we first generated a transcriptional fusion of PP with a promoterless gene encoding the M6 protein (emm6) in pSMB47, a suicide vector capable of integrating heterologous DNA into the conjugative transposon Tn916via homologous recombinantion \[[@B12]\]. The recombinant plasmid was named pSMB139 (see Methods) (Fig. [4](#F4){ref-type="fig"}). To construct a Tn916insertion vector that could be used to express translational fusions between M6 and any heterologous proteins under the PP promoter control, a 900 bp AvrII-HindIII fragment internal to emm6in pSMB139 was replaced by a 390 bp AvrII-HindIII fragment of pSMB55 containing a multiple cloning site \[[@B10]\] (Fig. [4](#F4){ref-type="fig"}). The resulting vector, named pSMB148 (Fig. [4](#F4){ref-type="fig"}), allows to create translational fusions of heterologous proteins between the first 122 N-terminal aa and the last 140 aa of the M6 protein, which provides sequences necessary for cell wall anchoring. pSMB148 was used to create two derivative vectors in which the emm6gene was fused respectively with a DNA sequence encoding 339 aa of the chicken ovalbumin (OVA) (pSMB156), and a DNA sequence encoding 458 aa of the tetanus toxin fragment C (TTFC) (pSMB288) (see Methods). A schematic representation of the recombinant proteins is shown in Fig. [5A](#F5){ref-type="fig"}. Expression of recombinant proteins in Gram-positive bacteria ------------------------------------------------------------ S. gordonii, B. subtilis, and Enterococcus faecaliswere three Gram-positive hosts used to analyze the capability of PP to direct transcription of the heterologous genes expressing M6, M6/TTFC and M6/OVA recombinant proteins. Expression of M6 in S. gordonii --------------------------------- pSMB139, bearing a transcriptional fusion of PP with emm6, was introduced by natural transformation in S. gordoniiGP201, a strain with a single copy of Tn916integrated into the chromosome. One of the transformants (GP1241), in which the integrative suicide vector drove the integration of the PP-emm6fusion into Tn916, was isolated and analyzed for M6 protein expression. Envelope fractions (containing surface associated proteins) prepared from equal amounts of cells grown to mid-log, early and late stationary phase, were analyzed by Western-blotting with an anti-M6 monoclonal antibody. Multiple bands could be detected in fractions of cells grown to mid-log and early stationary phase, whereas no band was detected in the fraction of cultures grown to late stationary phase (Fig. [6A](#F6){ref-type="fig"}). The intensity of the signal was higher during the mid-log growth phase suggesting that either PP is more active during exponential growth or that the M6 protein is being degraded during stationary phase. The presence of multiple reactive bands of molecular masses close to the hypothetical size of M6 (predicted molecular weight, 49 kDa) is probably due to the fact that coiled-coil proteins like M6 run at aberrant sizes on denaturing gels \[[@B19]\]. Expression of M6/TTFC in B. subtilis -------------------------------------- Competent cells of B. subtilisGP800.2, containing one copy of Tn916integrated into the chromosome, were transformed with the insertion vector pSMB288, bearing the transcriptional fusion PP-emm6/ttfc. One transformant, GP848, was chosen for further studies. A culture of GP848 was grown to mid-exponential phase and analyzed by Western-blotting for the presence of recombinant M6/TTFC. As shown in Fig. [6B](#F6){ref-type="fig"}, two reactive bands could be detected in the envelope fraction. The lower band, indicated by an arrow, corresponds to the mature protein (predicted molecular weight, 82 kDa), while the upper band probably represents an unprocessed form (predicted molecular weight, 86.4 kDa). Expression of M6/OVA in E. faecalis ------------------------------------- Using a previously described genetic system \[[@B12]\], we constructed a derivative of the E. faecalisstrain OG1SS \[[@B20]\] expressing the recombinant M6/OVA protein under PP control. pSMB156, bearing the PP-emm6/ovafusion, was first introduced in the Tn916containing B. subtilisGP800.2 by natural transformation, to obtain a recombinant conjugative transposon containing the transcriptional fusion. The recombinant transposon was then transferred by conjugation into E. faecalisOG1SS. Transconjugants were detected at a frequency of 4 × 10^-10^transconjugants/recipient. One of them (GP431) was analyzed for cell-surface expression of M6/OVA by flow-cytometric analysis using an anti-ovalbumin polyclonal antibody. The presence of recombinant M6/OVA on the surface of GP431 was clearly demonstrated by the increase of the fluorescence intensity in this strain, as compared to the parental control OG1SS (Fig. [7](#F7){ref-type="fig"}). Conclusions =========== We have isolated and characterized a promoter from the chromosome of S. gordonii, and demonstrated that it can be used to direct expression of heterologous genes in different Gram-positive bacteria when integrated in a single copy into the chromosome. This promoter, together with the genetic system based on suicide vectors able to integrate into conjugative transposons, represents a useful tool for the stable manipulation of a broad spectrum of Gram-positive bacteria. Methods ======= Bacterial strains, plasmids and growth conditions ------------------------------------------------- All strains and plasmids used in this work are listed in Table [1](#T1){ref-type="table"}. E. colistrains DH5α and HB101 were cultured in Luria-Bertani (LB) broth. For maintenance of plasmids, ampicillin (100 μg/ml), chloramphenicol (20 μg/ml) or erythromycin (100 μg/ml) was added to the growth medium. Streptococcal strains were cultured in Brain Heart Infusion medium (BHI, Difco) or Tryptic Soy Broth (TSB, Difco) in the presence of chloramphenicol (5 μg/ml), erythromicin (100 μg/ml) or streptomycin (500 μg/ml) whenever required. Transformation of naturally competent cells of S. gordoniiV288 and GP201, scoring and genetic analysis of transformants was carried out as already described \[[@B21],[@B22]\]. B. subtilisstrains were grown in LB broth with erythromicin (3 μg/ml) when it was required. Competent cells of B. subtilisGP800.2 were prepared and transformed according to described procedures \[[@B23]\]. Agar (1.5%) was added to LB, BHI or TSB to obtain solid media. All cultures were incubated at 37°C. DNA manipulation ---------------- Total DNA preparation of S. gordoniiwas performed as previously described \[[@B21]\]. Plasmid DNA was prepared using the Qiagen Plasmid Kit (Qiagen) according to the manufacturer\'s instructions. All recombinant techniques were performed following standard procedures \[[@B24]\], using E. coliHB101 or DH5α as a host. DNA restriction enzymes were obtained from Roche and used according to the manufacturer\'s instructions. Promoter selection by chromosomal integration --------------------------------------------- A 1.6 kb HindIII-BamHI fragment of plasmid pKT \[[@B25]\], containing a catgene, was ligated with random chromosomal DNA fragments of S. gordoniiV288 previously cut with both HindIII and BamHI. The initiation codon of catwas 34 bp downstream of the HindIII site, therefore HindIII cleavage would leave catpromoterless and preceded by an intact ribosome binding site. The ligation mixture was introduced in S. gordoniiV288 and transformants were selected for Cm^R^. MIC determination ----------------- The MIC of chloramphenicol for S. gordoniiwas determined following standard procedures \[[@B26]\]. Construction of recombinant vectors ----------------------------------- A 1648 bp fragment containing the emm6gene (encoding M6, a fibrillar surface protein of S. pyogenes) was amplified by PCR from plasmid pVMB20 \[[@B22]\] using the oligonucleotides 5\'-AT[GGATCC]{.underline}AT[CATATG]{.underline}GCTAAAAATAACACGAAT-3\' (upstream primer, containing a BamHI site and introducing a NdeI site at the ATG translation initiation codon) and 5\'-GCAT[GTCGAC]{.underline}CATAATCATTAAATGTATCTCAT-3\' (downstream primers containing a SalI site). This 1648 bp PCR fragment was digested with BamHI and SalI and cloned in pVA891 \[[@B27]\] previously digested with BamHIand SalI, resulting in plasmid pSMB89. A 451 bp region containing the streptococcal promoter PP was amplified from pVMB5 using the following primers: 5\'-CGA[GGATCC]{.underline}TTTA[ATCGAT]{.underline}ACTCATG-3\' (upstream primer, containing a BamHI and a ClaI site) and 5\'-CCG[CATATG]{.underline}GTTCTCCTTTTTATTTGT-3\' (downstream primers containing a NdeI site). After digestion with BamHI and NdeI, this PCR product was inserted between the BamHI and NdeI site of pSMB89 to obtain a transcriptional fusion of PP with emm6. The resulting plasmid was named pSMB128. This plasmid was first cut with BamHI, treated with Klenow enzyme to generate blunt ends, and finally cut with SalI to obtain a 2.0 kbfragment containing PP-emm6fusion. This fragment was gel-purified and ligated to the suicide integrative plasmid pSMB47 \[[@B12]\] previously cut with HindIII, treated with Klenow enzyme to generate blunt ends, and finally cut with SalI. The resulting plasmid was named pSMB139 (Fig. [4](#F4){ref-type="fig"}). To introduce a multiple cloning site in the emm6gene contained in pSMB139, the 900 bp AvrII-HindIII fragment internal to emm6was replaced with the 390 bp AvrII-HindIII fragment of emm6from pSMB55 \[[@B10]\] (Fig. [4](#F4){ref-type="fig"}). The resulting plasmid was named pSMB148. A 1016 bp DNA region encoding 339 aa of the chicken ovalbumin (OVA) (Gene Bank accession number: V00383) was amplified with the following primers: 5\'-CT[AGATCT]{.underline}GACAGCA CCAGGACAC-3\' (upstream primer containing a BglII site) and 5\'-TA[AAGCTT]{.underline}TAGGGG AAACACATCTG-3\' (downstream primer containing a HindIII site). After digestion with BglII and HindIII, this segment was introduced in pSMB148 previously digested with BglII and HindIII. The resulting plasmid, named pSMB156, contained a translational fusion of M6 with OVA. To create a translational fusion of M6 with the tetanus toxin fragment C (TTFC) a 1374 bp BglII-HindIII fragment encoding 458 aa of TTFC was isolated from pSMB158 \[[@B28]\] and cloned between the BglII and HindIII sites of pSMB148. The resulting plasmid was named pSMB288. Western-blot analysis --------------------- Preparation of S. gordoniiand B. subtiliscell envelope fractions (representing the protoplast surface containing the cell membrane together with cell wall fragments associated to the protoplasts) was performed as already described \[[@B29],[@B30]\]. The monoclonal antibody 10B6 \[[@B31]\] diluted 1:1000 was used to detect the presence of M6 protein. M6/TTFC fusion protein was visualized with an anti-TTFC rabbit serum (Calbiochem-Novabiochem Corporation) diluted 1:1000. Flow-cytometric analysis ------------------------ Flow-cytometric analysis of E. faecaliswas performed as already described \[[@B28],[@B32]\] using an anti-OVA rabbit serum diluted 1:300 \[[@B29]\]. DNA sequence determination -------------------------- The promoter containing fragment cloned in pVMB5 was sequenced by dideoxy chain termination method \[[@B33]\] as already described \[[@B34]\]. Denatured plasmid DNA was used as template. RNA isolation ------------- Total RNA was isolated from a 50 ml cell culture of S. gordoniiand E. coligrown to late exponential phase (OD~590~≌ 0.5). Cells were harvested by centrifugation at 6000 × gat 4°C and lysed according to the following procedures. E. colicells were first resuspended in hot (100°C) lysis buffer (50 mM Tris/HCl pH8, 1 mM EDTA, 1% SDS) and lysed by boiling the suspension for 5 minutes. S. gordoniicells were resuspended in lysozyme buffer (25 mM Tris/HCl (pH8), 10 mM EDTA, 50 mM glucose) and subjected to three cycles of freezing in liquid nitrogen and thawing at 52°C. After incubating with 0.2 mg/ml of lysozyme for 30 min at 37°C, one volume of hot (100°C) lysis buffer (100 mM Tris/HCl (pH8), 2 mM EDTA, 2% SDS) was added, and complete lysis was obtained by boiling cells for 5 minutes. After boiling, all lysates were cooled on ice for 5 min and total RNA was purified using the SV Total RNA Isolation System (Promega). Primer-extention analysis ------------------------- Primer extention analysis was performed with a synthetic oligonucleotide 5\'-GTTCTTTACGATGCC-3\' (position 47 to 61 relative to the initiation of the catgene). Two pmol of the oligonucleotide, labeled with \[γ-^32^P\] ATP (3000 Ci/mmol, Amersham) using T~4~polynucleotide kinase (New England Biolabs), were precipitated with 10 μg of RNA and the pellet was resuspended in 8 μl of Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase buffer (50 mM Tris/HCl (pH8.3), 8 mM MgCl~2~, 10 mM DTT). The mixture was heated at 65°C for 3 min, cooled rapidly at -80°C for 1 min and then transfered on ice until it was completely thawed. 1 μl of a 3.75 mM deoxynucleoside triphosphate solution and 10 U of M-MuLV Reverse Transcriptase (New England Biolabs) were added to the RNA-primer hybrid. The reaction mixture was incubated at 48°C for 30 min and terminated with 10 ml of stop solution (95% formamide, 20 mM EDTA pH8.0, 0.05% bromophenol blue, and 0.05% xylene cyanol FF). The reverse transcriptase reactions were analyzed by electrophoresis on a 6% polyacrylamide-7 M urea gel with sequencing reaction obtained with the same primer used as size standards. Conjugation ----------- Conjugation experiments were performed on solid media as previously described \[[@B12]\]. Authors\' contributions ======================= RP, characterization of promoter, engineering of S. gordonii, writing of manuscript. TM, engineering of B. subtilisand E. faecalis MRO, participation in experimental work, data evaluation RM, participation in experimental work, data evaluation, writing of manuscript GP, design and coordination of the study, data evaluation, direct supervision of experimental work, writing of manuscript All authors read and approved the final manuscript. Acknowledgements ================ The work was supported in part by the European Commission grant QLK2-CT2000-00543 to GP, a grant from MIUR (COFIN 2002) to GP and from MIUR (FIRB RBAU01X9TB) to MRO. The authors wish to thank Claudio Gualerzi for help and advice with the primer extention analysis. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **In vivotranscriptional fusion by integration of a promoterless catgene into the chromosome of a naturally transformable streptococcus*. A promoterless catgene is ligated in vitroto random chromosomal fragments, using a restriction site a few base pairs upstream of its translation initiation codon (a). The ligation mixture is then used to transform the recipient strain (b), and the homologous sequences allow chromosomal integration of the promoterless catgene (c). By this process, the catgene is integrated into the chromosome between two direct repeats. Since some of the chromosomal fragments contain promoters (P), it is possible to obtain expression of the promoterless catgene after in vivotranscriptional fusion with resident chromosomal promoters. ::: ![](1472-6750-5-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Schematic representation of the S. gordoniilocus containing PP promoter. DltD orthologue, an ORF encoding a putative acetyltransferase (ORF2), ORF1, the gene encoding a Tmp7 transmembrane protein, and the catgene are indicated by arrows. Dashed arrows designate that the ORF is only partially represented in the scheme. The 600 bp TaqI-HindIII fragment including PP promoter and cloned upstream of the catgene in pVMB5 is indicated by a dotted box. Nucleotide sequence of the PP promoter region (332 bp) is reported inside the box. The transcriptional start site determined by primer extention analysis is marked with an asterisk. Proposed -35 and -10 regions are underlined. A second putative -10 sequence is overlined. ORF1 putative ribosome binding site (RBS) is boxed. ATG initiation codon of catis in bold characters and the sequence of HindIII site is in italic letters. The complete sequence of the 600 bp cloned fragment is available on GenBank (Accession number: U74080). T, TaqI site; B, BamHI site; H, HindIII site ; loop, putative transcriptional terminator. ::: ![](1472-6750-5-3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Primer extension analysis of the PP promoter. Localization by primer extension of the transcriptional start site of the catmRNA specified by the PP promoter in E. coliGP334 (lanes 1--2), and S. gordoniiGP215 (lanes 3--4). The sequence of the region upstream of the catgene in pVMB5 was used as standard. The A residue, complementary to the T at position 423, indicated by an arrow, represents the transcriptional start site used in both strains. ::: ![](1472-6750-5-3-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Construction of the insertion plasmid pSMB148. pSMB139 was constructed by introducing a 2.0 kb fragment, containing a transcriptional fusion of PP promoter with emm6, in the Tn916insertion vector pSMB47 (see Methods). pSMB148 is a derivative of pSMB139 in which a 900 bp AvrII-HindIII fragment was replaced with a 390 bp AvrII-HindIII fragment from pSMB55 containing a multiple cloning site \[10\]. ::: ![](1472-6750-5-3-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Schematic representation of recombinant M6, M6/TTFC and M6/OVA expressed on the surface of S. gordonii, B. subtilisand E. faecalis*. The 458 aa protein TTFC (white bar) and the 339 aa protein OVA (light gray bar) were fused with the first 122 N-terminal aminoacids and the last 140 C-terminal aminoacids of M6 (dark gray bar). The predicted molecular weight of M6, M6/TTFC and M6/OVA is 49 kDa, 82 kDa and 66.9 kDa respectively. ::: ![](1472-6750-5-3-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Western blot analysis of recombinant S. gordoniiand B. subtilisstrains expressing M6 protein and M6/TTFC fusion protein*. (A) S. gordoniienvelope fractions. Lane 1 through 3, GP1241 expressing M6 under the control of PP promoter. Lane 1, GP1241 harvested after overnight growth. Lane 2, GP1241 harvested after early stationary phase. Lane 3, GP1241 harvested after exponential phase. Lane 4, recipient strain GP201 (negative control). Lane 5, GP231 (positive control). Blot was developed with anti-M6 monoclonal antibody 10B6. (B) S. gordoniiand B. subtilisenvelope fractions. Lane 1, S. gordoniiGP204 (negative control). Lane 2, S. gordoniiGP1253 expressing M6/TTFC (positive control). Lane 3, B. subtilisrecipient strain GP800.2 (negative control). Lane 4, B. subtilisGP848 expressing M6/TTFC under the control of PP promoter. Blot was developed with anti TTFC rabbit serum. Molecular weight markers are shown in the right side of panels. ::: ![](1472-6750-5-3-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Flow-cytometric analysis of E. faecalisexpressing M6/OVA. (A) OG1SS, recipient strain not expressing M6/OVA. (B) GP431, recombinant strain expressing M6/OVA on the surface. Bacterial cells were treated with anti-OVA rabbit serum and than with FITC-conjugated goat anti-rabbit IgG. xaxis, arbitrary units (a.u.) of fluorescence intensity (log~10~); yaxis, relative cell number. ::: ![](1472-6750-5-3-7) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Strains and plasmids used in this work ::: Strain* Relevant genotype^a^ Source/reference --------------- ------------------------------------------------------------------------ ---------------------- S. gordonii V288 Recipient in transformation \[35\] GP201 ΩTn5253, Sm^R^, Cm^R^, Tc^R^ \[13\] GP204 str-204, Sm^R^ \[13\] GP214 pKT random in V288, Cm^R^ This work GP215 pKT random in V288, Cm^R^ This work GP216 pKT random in V288, Cm^R^ This work GP231 emm6, Er^R^, Sm^R^ \[21\] GP1241 ΩTn5253(ΔtetM::pSMB139) Er^R^, Sm^R^, Cm^R^, Tc^S^ This work GP1253 emm6.1::TTFC \[28\] E. faecalis OG1SS Recipient in conjugation, Sm^R^, Sp^R^ \[20\] GP431 ΩTn916, Er^R^, Tc^S^(Δ tetM::pSMB156) This work B. subtilis GP800.2 ΩTn916, Tc^R^ \[12\] GP847 ΩTn916, Er^R^, Tc^S^(Δ tetM::pSMB156) This work GP848 ΩTn916, Er^R^, Tc^S^(Δ tetM::pSMB288) This work Plasmid Description Source/reference pKT Ap^R^, Cm^R^ \[25\] pBluescript Ap^R^ Stratagene pVMB5 pBluescript::PP-cat, Ap^R^, Cm^R^ This work pVMB20 pBluescript::emm6.1::ermC, Ap^R^, Er^R^ \[21\] pVA891 Cm^R^, Er^R^ \[27\] pSMB47 pVA891 derivative containing DNA sequences from Tn5253, Cm^R^, Er^R^ \[12\] pSMB89 pVA891::emm6, Cm^R^, Er^R^ This work pSMB128 pVA891::PP-emm6, Cm^R^, Er^R^ This work pSMB139 pSMB47::PP-emm6, Cm^R^, Er^R^ This work pSMB148 pSMB47::PP-emm6/55, Cm^R^, Er^R^ This work pSMB156 pSMB47::PP-emm6/55::ova, Cm^R^, Er^R^ This work ^a^Sm, streptomycin; Cm, chloramphenicol; Tc, tetracyclin; Er, erythromycin; spectinomycin; Ap, ampicillin. :::	PubMed Central	2024-06-05T03:55:52.665499	2005-1-14	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548306/", "journal": "BMC Biotechnol. 2005 Jan 14; 5:3", "authors": [ { "first": "Roberta", "last": "Provvedi" }, { "first": "Tiziana", "last": "Maggi" }, { "first": "Marco R", "last": "Oggioni" }, { "first": "Riccardo", "last": "Manganelli" }, { "first": "Gianni", "last": "Pozzi" } ] }
PMC548319	Background ========== Because of its intense oxidative metabolism, the brain consumes a high fraction of total oxygen generating large amounts of reactive oxygen species \[[@B1],[@B2]\]. Although brain antioxidant defenses function properly during most of human life, a number of neurodegenerative processes which involve redox-active iron accumulation become evident with age \[[@B3]-[@B5]\]. Iron is a pro-oxidant that in the reductive intracellular environment catalyses hydroxyl radical formation through the Fenton reaction \[[@B6]\]. At present, the crucial components of the iron homeostasis machinery have been identified. Thus, current efforts should be directed to the understanding of the mechanisms that regulate cellular iron levels and antioxidant defenses. This is of primary importance for the development of strategies to ameliorate iron accumulation and oxidative damage in neurons. In vertebrates, cellular iron levels are post-transcriptionally controlled by the activity of iron regulatory proteins (IRP1 and IRP2), cytosolic proteins that bind to structural elements called iron-responsive elements (IREs). IREs are found in the untranslated region of the mRNAs of the major proteins that regulate cellular iron homeostasis: the transferrin receptor, involved in plasma-to-cell iron transport, and the iron-storage protein ferritin. IRP2-/- mice are born normal but in adulthood develop a movement disorder characterized by ataxia, bradykinesia and tremor \[[@B7]\]. IRP1-/- mice are normal with slight misregulation of iron metabolism in the kidney and brown fat \[[@B8]\]. Thus, IRP2 seems to dominate the physiological regulation of iron metabolism whereas IRP1 seems to predominate in pathophysiological conditions. Iron is internalized into cells by the import transporter DMT1. Four DMT1 isoforms have been identified that differ in both the N-and the C-termini \[[@B9]\]. Two of the isoforms have a 3\' iron responsive element (IRE) in their mRNA. Additional variation is given by exons 1A and 1B in the 5\' end. Expression of DMT1 in response to iron availability follows a pattern similar to transferrin receptor \[[@B10]\], but its control by the IRE/IRP system is not clear \[for review see \[[@B11]\]\]. A new iron transporter, IREG1, also known as ferroportin or MTP1, was recently described \[[@B12],[@B13]\]. The protein is expressed mainly in enterocytes and macrophages \[reviewed in \[[@B14]\]\]. In enterocytes IREG1 is responsible for iron efflux during the process of intestinal iron absorption, while in Kupffer cells IREG1 mediates iron export for reutilization by the bone marrow \[[@B15]\]. The presence of both DMT1 and IREG1 has been described in neurons, glioma cells and astrocytes \[[@B16]-[@B18]\]. The presence of IREG1 in neurons opens the possibility that they may be able to down-regulate intracellular iron concentration through its expression. In this study we examined iron homeostasis in SH-SY5Y neuroblastoma cells and hippocampal neurons. We found that iron accumulation killed a large proportion of cells, but a sub-population became resistant to iron accumulation developing an adaptative mechanism intended to decrease intracellular iron content. Results ======= Iron accumulation and cell death -------------------------------- Iron accumulation was determined in SH-SY5Y cells grown to confluence and then cultured for two days in media containing from 1.5 to 80 μM iron (Figure [1A](#F1){ref-type="fig"}). Total cell iron increased with increasing extracellular iron, reaching a plateau at 40--80 μM Fe (Figure [1B](#F1){ref-type="fig"}). The observed increase in cell iron was accompanied by increases in the labile iron pool (Figure [1C](#F1){ref-type="fig"}). Iron accumulation indeed caused loss of cell viability, with hippocampal neurons demonstrating higher sensitivity than SH-SY5Y cells to iron treatment (Figure [2](#F2){ref-type="fig"}). Nevertheless, a sub-population of cells survived to high iron concentrations. It was of interest to inquire into the processes underlying this adaptation, since they could help to understand iron accumulation observed in a number of neurodegenerative diseases. Consequently, we characterized the components of the iron homeostasis machine during the process of iron accumulation. Ferritin and DMT1 regulation ---------------------------- Ferritin, the main iron-storage protein in mammalian cells, is considered the first line of defense against iron overload. Increasing iron from 1.5 to 5 μM produced a robust 4-fold increase in cell ferritin content (Figure [3A](#F3){ref-type="fig"}). Further increases in iron induced additional increases in ferritin. At 80 μM extracellular iron, ferritin increased 11-fold compared to the basal 1.5 μM iron condition. In molar base, ferritin increased more than iron. The iron to ferritin mol : mol ratio decreased from 1500 at 1.5 μM Fe to 400 at 10 μM Fe to 200 at 80 μM Fe (Figure [3B](#F3){ref-type="fig"}). We further characterized iron homeostasis in SH-SY5Y cells by examining the expression of the iron importer DMT1 (Figure [4](#F4){ref-type="fig"}). A 3.5-fold decrease in DMT1 protein expression was observed when iron increased from 1.5 to 80 μM. The presence of DMT1 even at high iron concentration explains the sustained iron uptake observed at 80 μM Fe \[[@B19]\]. Thus, DMT1 activity persisted even under conditions of iron accumulation that preceded cell death. IREG1 expression and functionality ---------------------------------- Given that the presence of IREG1 in the central nervous system has been reported \[\[[@B16]\]\], it was of interest to examine if it participates in neuronal iron homeostasis. Western blot analysis revealed that SH-SY5Y cells expressed anti-IREG1 reactive bands of 65.3 and 122.1 KDa molecular weight (Figure [5A](#F5){ref-type="fig"}). Densitometric analysis revealed a 10-fold increase in the 122.1 KDa band in the 1.5 to 80 mM Fe range while the 65.3 band had a minor increase (Figure [5A](#F5){ref-type="fig"}). Both bands were eliminated if the antibody was incubated with the immunogenic peptide before the assay (Figure [5B](#F5){ref-type="fig"}). A similar pattern was obtained with an independent anti-IREG1 antibody (the kind gift of Dr. David Haile). Thus, it is most likely that the 65.3 and 122.1 KDa bands correspond to the monomer and dimer of IREG1. The stability of the 122.1 KDa band was dependent of the concentration of b-mercaptoethanol in the sample buffer since increasing b-mercaptoethanol produced a shift in the 122.1 KDa /65.3 KDa band ratios (Figure [5C](#F5){ref-type="fig"}). It is possible that in neuronal cells Ireg1 tends to form S-S bridged dimers resistant to the electrophoresis conditions. The presence of IREG1 in SH-SY5Y neuroblastoma cells and hippocampal neurons was further documented by immunocytochemistry. IREG1 was detected in both types of cells, with a predominantly cytosolic distribution pattern (Figure [6](#F6){ref-type="fig"}). The levels of IREG1 expression were directly proportional to the amount of iron in the culture. Thus, it was determined by two independent methods that IREG1 expression in neuronal cells increased with cell iron content. Efflux of iron from neurons has never been reported. In view of the presence of IREG1, we tested whether SH-SY5Y cells actually had an iron efflux function. To that end, iron efflux from cells pre-cultured for 2 days with varied iron concentrations was determined by atomic absorption spectrometry. This method was preferred to the use of radioisotopic iron since the latter could underestimate a putative iron efflux because of isotope dilution with the pre-existing iron pool (see Figure [1B](#F1){ref-type="fig"}). SH-SY5Y cells had discrete but measurable iron efflux activity (Figure [7A](#F7){ref-type="fig"}). The iron efflux rate increased markedly in the 20--80 μM Fe range (Figure [7B](#F7){ref-type="fig"}). Interestingly, the efflux rate correlated closely with the presence of the 122.1 KDa band, while the correlation between efflux activity and the 62.5 KDa band was weaker (Figure [7C](#F7){ref-type="fig"}). Discussion ========== The number of neurological diseases associated with iron accumulation in the brain underlines the need for increased knowledge of the mechanisms of brain iron homeostasis. In this study we show that iron accumulation by SH-SY5Y neuroblastoma cells and hippocampal neurons resulted in cell death of part of the population, while another fraction survived by adapting the expression of iron homeostasis proteins. Iron content increased significantly as a function of Fe in the culture up to 20--40 μM Fe, increasing very little thereafter up to 80 μM Fe. Cell iron increase was accompanied by increased ferritin content. The increase in ferritin more than compensated for the increase in iron. Iron to ferritin mol ratios of 1500, 260 and 190 were obtained for 1.5, 20 and 80 μM Fe in the culture media. Thus, the IRE/IRP system of SH-SY5Y cells over-responded to iron accumulation in terms of ferritin expression. Despite the increase in ferritin, the LIP increased between 1.5 and 80 μM Fe. This finding clearly indicates that in SH-SY5Y cells the level of labile iron is a function of total iron, even in the presence of ample ferritin supply. It is possible that ferritin-stored iron contributes to the LIP each time that ferritin undergo lisosomal degradation. Iron accumulation was accompanied by a marked decrease in DMT1 expression. Nevertheless, some DMT1 persisted even at 40--80 μM iron. The persistence of DMT1 at high iron concentrations could underline the continuous iron uptake observed under these conditions \[[@B19]\]. This is curious because at 40--80 μM Fe cells were dying. Sustained DMT1 expression points to the inability of neuronal cells to shut-off iron uptake and the need for additional defense mechanisms to prevent iron-mediated cell death. The discovery of increased IREG1 expression in response to cell iron accumulation is a major break-through in the understanding of cell survival under conditions of iron accumulation. Total IREG1, and especially a putative IREG1 dimer, increased markedly in the 20--80 μM Fe range. Thus, in SH-SY5Y cells IREG1 is up-regulated by increased cell iron. Expressed IREG1 was functional since it associated with increased iron efflux activity. Iron efflux activity in astrocytes \[[@B18]\] and neurons (this work) indicate that iron efflux from brain cells is a dynamic process, and highlights the importance of iron transporters as determinants of iron accumulation. The regulation of IREG1 expression is unknown but seems to be cell-specific. In enterocytes, IREG1 expression is induced by iron deficiency \[[@B13]\] while in macrophages iron increases IREG1 expression \[[@B20]\]. The findings reported here indicate that in neuronal cells IREG1 has a macrophage-like regulation. This is certainly the case for cells in the 40--80 μM range that survived to iron accumulation. IREG1\'s predominantly cytosolic distribution pattern is similar to that of Kupffer cells \[[@B12]\]. Again, this distribution points to macrophage-like behavior of neuronal IREG1. In examining brain biopsies from Alzheimer\'s patients an intriguing question arises: Why do some neurons die or present evident signs of degeneration while others in the vicinity show a normal phenotype? Extrapolating on the data presented here, it is tempting to hypothesize that surviving neurons induce IREG1 expression while sick neurons do not. Nevertheless, at present we cannot exclude that other regulatory molecules may play a pivotal role under these conditions. Conclusions =========== Hippocampal neurons and SH-SY5Y cells displayed an active system to regulate iron content. Nevertheless, this system was unable to block iron accumulation which resulted in death of part of the cell population. Another fraction of the cell population developed an adaptative mechanism that includes decreased expression of the import transporter DMT1 and increased expression of ferritin and the efflux transporter IREG1. The finding that neurons regulate the expression of functional IREG1 opens new avenues for the understanding and possible treatment of iron-related neurodegenerative processes. Methods ======= Antibodies and immunodetection ------------------------------ Antibody D-1, prepared against the C-terminal end of the IRE-containing isoform of DMT1 was used as described previously \[[@B10]\]. Additionally, a rabbit polyclonal antibody against peptide CGPDEKEVTKENQPNTSVV, corresponding to the consensus sequence of human, rat and mouse carboxyl-terminal sequence of IREG1, was obtained from BioSonda, Chile <http://www.biosonda.cl>. Western analysis ---------------- Cell extracts, cells were prepared treating cells with lysis buffer (50 μl per 1 × 106 cells of 10 mM MOPS, pH 7.5, 3 mM MgCl2, 40 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 0.5 μg/ml aprotinin, 0.7 μg/ml pepstatin A, 5% glycerol, 1 mM dithiothreitol, 0.1% Triton X-100). The mixture was incubated for 15 min on ice and centrifuged for 10 min at 5,000 × g. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay. The supernatant was stored at -70°C. For Western analysis, 30 micrograms of protein from each sample were boiled in Laemmli sample buffer for 5 min and subjected to SDS-PAGE on a 7.5% acrylamide gel. Proteins were transferred to nitrocellulose membrane and blocked for 1 hr at 25°C with 5% nonfat dry milk in blocking saline (20 mM Tris, 0.5 M NaCl, 0.05% Tween-20). Membranes were incubated with primary antibody overnight at 4°C, rinsed with blocking saline and incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody for 1 hr at 25°C. Transferred proteins were detected with a peroxidase-based chemiluminiscence assay kit (SuperSignal, Pierce Chem. Co., Rockford, IL). Chemiluminiscence was detected using a Molecular Imager FX device (Bio-Rad, Hercules, CA). The bands were quantified by densitometry using the Quantity One (Bio-Rad) software. Cell culture and iron challenge ------------------------------- Human neuroblastoma SH-SY5Y cells (CRL-2266, American Type Culture Collection Rockville, MD), were seeded at 1 × 105 cells in 2-cm^2^plastic wells and cultured in a 5 % CO~2~incubator in MEM/F12 medium supplemented with 10 % fetal bovine serum and 5 mM glutamine. The medium was replaced every two days. Under these conditions, doubling time was about 48 hours. After 8 days in culture, the culture reached a steady-state number of cells. At this time, cells were challenged with iron for the next two days as described \[[@B19]\]. In brief, low-iron culture media was supplemented with either 1, 5, 10, 20, 40 or 80 μM Fe^3+^as the complex FeCl~3~-sodium nitrilotriacetate. Cell viability was quantified by the MTT assay (Molecular Probes, OR) following the manufacturer\'s instructions. This model of iron loading attempts to replicate neuronal iron accumulation that occurs during life \[[@B4]\]. Hippocampal neurons were prepared from E18.5 rat embryos \[[@B21]\]. Neurons were plated over poly-L-lysine coated cover slips at 100,000 cells/cm^2^. Cultures were maintained in 10% bovine serum until 3 hours after plating, when the culture medium was replaced with medium containing B27 supplement \[[@B22]\]. After 3 days in culture, the cells were challenged with iron as described above. Labile iron pool ---------------- The intracellular labile or reactive iron pool of neuroblastoma cells was determined as described \[[@B23],[@B24]\]. The increase in fluorescence after the addition of SIH chelator is directly proportional to the iron labile pool, i.e., iron in complexes with affinity constant \< 10^6^. Immunocytochemistry ------------------- Cells grown in cover slips were sequentially fixed with 2% and 4% parafolmaldehyde (PFA) in Eagles\' MEM, and then washed three times with phosphate-buffered saline (PBS). The fixed cells were permeabilized with Triton-X-100 (0.2%) in PBS at room temperature for 3 min and blocked with defatted milk (10%) in PBS for more than 1 h. The cells were incubated with anti-IREG1 antibody (1:500) overnight at 4°C, washed with PBS and then incubated with Alexa-546-conjugated goat anti-rabbit IgG. The labeled cells were observed with a Zeiss LSM 510 Meta confocal laser scanning microscope. Data analysis ------------- Variables were tested in triplicates, and experiments were repeated at least twice. Variability among experiments was \<20%. One-way ANOVA was used to test differences in mean values, and Turkey\'s post-hoc test was used for comparisons (In Stat program from GraphPad Prism). Differences were considered significant if P \< 0.05. Authors\' contributions ======================= MTN conceived of the study, participated in its design and coordination and drafted the manuscript. PA performed the experiments with hippocampal neurons, did the ferritin assays and participated in the analysis and interpretation of data. MN optimized the immunocytochemistry detection of IREG1, performed the confocal microscope observations and participated in the analysis and interpretation of data. VT did the Western blot, labile iron pool and viability assays and contributed to the discussion of the results. MA set up the method to determine total Fe concentration, did the sample and control measures of iron and participated in the analysis and interpretation of data. All coauthors participated in refining the text. Acknowledgements ================ We are indebted to Dr. David Haile for supplying anti-IREG1 antibody. This work was supported by project P99-031 of the Millennium Institute for Advanced Studies in Cell Biology and Biotechnology, Santiago, Chile. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *Total and labile iron in the progressive iron accumulation modelA:*SH-SY5Y cells were grown for 8 days and then challenged for 2 days with different concentrations of iron (range 1.5--80 μM). B:total iron content determined by mass spectrometry. Increased level of iron in the culture media produced increased levels of intracellular iron. Data is mean ± SD of 4 independent determinations. C:The calcein-sensitive iron pool increased at 80 μM Fe compared with 7 μM Fe. Representative tracings are shown. ::: ![](1471-2202-6-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Cell viability upon iron load.SH-SY5Y cells and hippocampal neurons were subjected to the progressive iron accumulation protocol described in Figure 1A. Viability in the cell cultures was determined by the MTT method. Representative curves from 4 (SH-SY5Y cells) or 3 (hippocampal cells) independent experiments are shown. ::: ![](1471-2202-6-3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Ferritin contentA:ferritin was determined in extracts from cells cultured for 2 days with different iron concentrations as described in Figure 1A. Data is mean ± SD from 4 independent determinations. B:iron : ferritin molar ratio. Total iron and ferritin total data were from Figure 1B and 3A, respectively. Ferritin synthesis response was larger that iron accumulation, an indication of a very active IRE/IRP system. ::: ![](1471-2202-6-3-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Changes in DMT1 expressionUpper panel:DMT1 from cells cultured for 2 days with different iron concentrations was determined by Western blot. A continuous decrease in DMT1 expression was observed in the 1.5--80 μM Fe range. Shown is one of three similar experiments. Lower panel:densitometric analysis of the DMT1 bands shown in Figure 4A. ::: ![](1471-2202-6-3-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### IREG1 expression increases upon increasing iron exposure. IREG1 from cells cultured for 2 days with different iron concentrations was determined by Western blot. A shows results of one of two similar experiments. Bands of 65.3 and 122.1 KDa were evident. The side panel shows the densitometric analysis of the bands. The 122.1 KDa band increased in the 1.5 to 40 mM Fe range. B: Both the upper, 122.1 KDa, band and the lower, 65.3 KDa, band diminished when the antibody was pre-incubated with peptide CGPDEKEVTKENQPNTSVV, prior to Western blot of 30, 20 and 10 mg of cell extract. C: treatment of 80 mM Fe extracts with increasing b-mercaptoethanol. A decrease in the 122.1 KDa band with increased b-mercaptoethanol was apparent. ::: ![](1471-2202-6-3-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Immunocytochemistry determination of IREG1.Hippocampal neurons and SH-SY5Y cells, labeled with rabbit anti-IREG1 antibody and with Alexa-546-conjugated goat anti-rabbit IgG, were imaged in a confocal microscope. Shown are representative fields of cells cultured at different iron concentrations. Note the preferentially cytosolic distribution of IREG1. ::: ![](1471-2202-6-3-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Kinetics of iron efflux from SH-SY5Y cells.SH-SY5Y cells were cultured for 2 days with varied amounts of iron as described in Figure 1A. The culture medium was changed to fresh DMEM and the kinetic of iron exit from the cells was followed by determining the concentration of iron in the medium by absorption spectrometry (A). Data is means ± SD from 3 experiments. The efflux rate, obtained from the slope of the curves in Figure 7A, was plotted against iron concentration during the 2-day culture period (B). In C**, the efflux rate was plotted against the density of the 65.3 KDa or 122.1 KDa bands. To that end, the 65.3 KDa or 122.1 KDa bands shown in Figure 5 were quantified by densitometry and paired with the efflux rates determined in Figure 7A at the same iron concentrations. A good correlation between efflux rate and the presence of the 122.1 KDa band was observed. ::: ![](1471-2202-6-3-7) :::	PubMed Central	2024-06-05T03:55:52.668422	2005-1-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548319/", "journal": "BMC Neurosci. 2005 Jan 24; 6:3", "authors": [ { "first": "Pabla", "last": "Aguirre" }, { "first": "Natalia", "last": "Mena" }, { "first": "Victoria", "last": "Tapia" }, { "first": "Miguel", "last": "Arredondo" }, { "first": "Marco T", "last": "Núñez" } ] }
PMC548382	Background ========== A common idea in animal ecology is that adverse or stressful environmental conditions facilitate the evolution of social behaviour \[[@B1]\]. The formation of groups across a huge number of animal taxa is thus considered to have broad implications for the benefit of individuals, including mate finding, the efficient location and use of resources, thermoregulation, energetic benefits and defence against natural enemies or competitors \[[@B2],[@B3]\]. Basic proximate prerequisites for communal behaviour are cues indicating the location of conspecifics and the ability to receive and process information regarding these cues, which in turn induce inter-individual attraction \[[@B3]\]. Because the costs and benefits of communal behaviour typically vary with environmental conditions, the degree to which individuals are mutually attracted is regulated by signals indicating the presences of predators, food availability, etc. \[[@B4]\]. In the vast group of insects that depend on ephemeral resources, such as decaying plant tissues, dung and carrion, aggregation in the immature stages across resource patches is the result of the choice of a female to lay batches of eggs and/or to aggregate with conspecifics \[[@B5]-[@B8]\]. In studies of Drosophilaas an ecological model system, one benefit that females flies seem to achieve by this spatial aggregation is that larval survival probability to the adult stage is highest at intermediate densities \[[@B9],[@B10]\], indicating the existence of so-called Allee effects \[[@B11]\]. Competing filamentous fungi co-occurring with Drosophilalarvae on the same patches have been demonstrated to cause high rates of mortality when larvae feed solitarily or in small groups, whereas larger groups are able to hamper mould growth \[[@B12]\] (Fig. [1](#F1){ref-type="fig"}), which in turn increases larval survival \[[@B9],[@B13],[@B14]\]. Although the mechanisms leading to mould suppression are not fully understood, physical damage of the fungal tissue from the feeding (shovelling food with the mouth hooks) and locomotor (crawling and digging) behaviour of the fly larvae \[[@B15]\] seems to be the major cause of the repression of mould growth \[[@B12],[@B14]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The effect of larval density on mould growth.The effect of Drosophilalarval density (a. one larva, b. 5 larvae, c. 10 larvae) on the growth of Aspergillus niger. Patches (2.5 cm diameter) contained standard Drosophilarearing medium. Photographs were taken 10 days after infection with fungal spores. Spores and fly larvae were simultaneously transferred to the patches. Whereas one larvae did not significantly hamper mould development (a), five and ten larvae caused a substantial reduction in fungal growth (b) or even entirely suppressed fungal development (c). (unpublished study) ::: ![](1742-9994-2-2-1) ::: Given the relationship between spatial oviposition patterns, Allee effects and the suppression of mould, spatial aggregation in Drosophilacan be interpreted as an adaptive behaviour against competing fungi on larval feeding sites in order to enhance offspring survival. These ecological interrelationships might set conditions for facilitating social behaviour in the fly larvae because, at the level of larval behaviour, a more efficient strategy that might control the rapid establishment of noxious fungi would be to exert physical stress directly on fungal colonies. Thus, larvae should display an assortative behaviour on the site on which fungi are growing, rather than moving randomly and independently of each other across a resource patch, by which the fungal tissues might only incidentally be destroyed. In the present study, I have provided groups of Drosophila melanogasterMeigen (Diptera, Drosophilidae) larvae with fungal-infected (2-day-old colonies of Aspergillus nigervan Tieghem) and uninfected (control patches) (F-C treatment) food patches and examined whether the distribution of larvae across the patches is driven by fungal infection. In comparison with this naturally occurring heterogeneity in patch quality, I have also studied the distribution of fly larvae when they were offered only infected (F-F treatment) or uninfected (C-C treatment) food patches in order to test for the existence of grouping behaviour in two types of homogenous larval environment. If grouping is irrelevant under the given experimental setting, no deviation from the regular larval distribution across the food patches would be expected, i.e larvae should distribute themselves across patches in order to minimise larval competition for food \[[@B16]\]. Although Drosophilais a thoroughly studied model organism in foraging biology \[[@B17],[@B18]\], knowledge about social interactions between the insect larvae is surprisingly limited. This is intriguing because drosophilids are also model organisms in spatial ecology in which Drosophilacommunities are characterised by strong intraspecific aggregation across patchily distributed substrates (e.g. decaying plant tissues) \[[@B19]-[@B21]\]. The lack of knowledge concerning social interactions among larvae and its possible role in competition with filamentous fungi have provided the specific impetus of the present study. Results ======= Larval aggregation in the F-C treatment (Δpl) ----------------------------------------------- The proportion of larvae on fungal-infected patches minus the proportion on uninfected patches, Δpl, was used as a measure of the way in which Drosophilalarvae distributed themselves between the two types of food patches in the F-C treatment (see method section for details). The number of larvae in both food patches (LARVAE) and the experimental day (DAY) did not influence Δpl(Table [1](#T1){ref-type="table"}). The estimated intercept for Δplwas significantly different from zero (GLM d.f.= 1, mean square= 4.475, F= 20.13, P\< 0.0001, N= 35), the positive value for Δpl(Fig. [2a](#F2){ref-type="fig"}; intercept estimate: 0.3576 ± 0.0797, t= 4.49, P\< 0.001) indicating the aggregation of larvae on fungal-infected sites (see method section). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of LARVAE and DAY on larval aggregation in the F-C treatment. Analysis of variance for the effect of the number of larvae in both food patches (LARVAE) and experimental day (DAY) on Drosophilalarval distribution between fungal-infected and uninfected food patches (F-C treatment). ::: Explanatory variable *d.f.* Mean square **F-value P -------------------------- ------------ ----------------- --------------- --------- LARVAE 1 0.0361 0.15 0.7042 DAY 3 0.0470 0.19 0.9018 Error 30 0.2462 ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Larval aggregation in the heterogeneous (F-C) and two types of homogeneous (F-F and C-C) larval environment.*(a) Δpl(where Δpl= proportion of larvae from the fungal-infected patch -- proportion of larvae from the uninfected patch) as a measure of larval aggregation in the F-C treatment (Δpl= 0: no effect of fungal-infected patches on larval distribution behaviour; Δpl\> 0: aggregation of larvae on fungal infected patches; Δpl\< 0: larvae avoid fungal colonies). (b) \\|Δpl\\| as a measure of the general tendency of Drosophilalarvae to aggregate with conspecifics in the heterogeneous environment (F-C) and two types of homogeneous environment (F-F and C-C). Because larval aggregation in the F-C treatment was measured independently of the patch type (see Methods), \\|Δpl\\| is larger than Δpl(2a). (F: fungal-infected patches, C: uninfected control patches) ::: ![](1742-9994-2-2-2) ::: Comparison of larval aggregation in the F-C, F-F and C-C treatment (\\|Δpl\\|) ------------------------------------------------------------------------------ \\|Δpl\\|, the absolute value of Δpl, was used as a measure of the general tendency of Drosophilalarvae to aggregate with conspecifics in the heterogeneous environment (F-C) and the two types of homogenous environment (F-F or C-C). By using \\|Δpl\\|, aggregation in the F-C treatment was quantified independently of whether a food patch was infected with fungi or not. With regard to all three larval environments, the estimated intercepts for \\|Δpl\\| were significantly different from zero, and hence indicate larval aggregation (Table [2](#T2){ref-type="table"}). Within each treatment LARVAE and DAY had no effect on \\|Δpl\\| (Table [3](#T3){ref-type="table"}). In comparison with the homogenous environments (F-F and C-C treatment), the F-C treatment induced stronger larval aggregation (Fig. [2b](#F2){ref-type="fig"}, Table [4](#T4){ref-type="table"}). Moreover, there is a statistical trend of LARVAE influencing fly larval aggregation (Table [4](#T4){ref-type="table"}). This was due to differences in LARVAE as a function of TREATMENT (GLM d.f.= 2, mean square= 0.0134, F= 3.34, P= 0.0393, N= 105). Significantly fewer larvae were found to be feeding in both patches in the C-C treatment (8.89 ± 1.64 SE) than in the F-C (9.43 ± 0.95 SE) or the F-F treatment (9.46 ± 0.61 SE). However, LARVAE within one type of environment had no effect on larval aggregation (Table [3](#T3){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The general tendency to aggregate with conspecifics (\\|Δpl\\|) in the heterogeneous (F-C) and two types of homogeneous (F-F and C-C) larval environment. Test of the effect of intercept as the only explanatory variable for the general tendency of Drosophilalarvae to aggregate with conspecifics (measured as \\|Δpl\\|, see text for details) in three types of larval environment (F-C, F-F or C-C). Whereas \\|Δpl\\| = 0 and no explanatory power of intercept would indicate a regular distribution of larvae across the food patches, \\|Δpl\\| \> 0 and a significant effect of intercept indicates larval aggregation in one of the experimental food patches (see also Fig. 2b). Note that, in contrast to Δpl(Fig. 2a), \\|Δpl\\| measures larval aggregation in the F-C treatment independently of whether a food patches was fungal-infected or not. For each type of larval environment an individual test was performed, with N= 35 for each treatment. ::: Parameter estimate* ------------------------ -------------------------- --------------- ---------- Larval environment Intercept ± SE **t-value P** F-C 0.51 ± 0.05 10.55 \<0.0001 F-F 0.37 ± 0.05 8.13 \<0.0001 C-C 0.35 ± 0.04 9.24 \<0.0001 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### The effect of LARVAE and DAY on the general tendency of Drosophilalarval aggregation (\\|Δpl\\|) under three environmental conditions. Analysis of variance for the effect of LARVAE and DAY on Drosophilalarval distribution between food patches in three different larval environments (F-C, F-F or C-C). ::: Larval environment Explanatory variable *d.f.* Mean square **F-value P** ------------------------ -------------------------- ------------ ----------------- --------------- --------- F-C LARVAE 1 0.2120 2.63 0.1154 DAY 3 0.0931 1.15 0.3433 Error 30 0.0807 F-F LARVAE 1 0.0682 1.03 0.3177 DAY 3 0.1132 1.71 0.1855 Error 30 0.0661 C-C LARVAE 1 0.0329 0.66 0.4236 DAY 3 0.2090 4.18 0.4998 Error 30 0.0400 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### The effect of the larval environment on the general tendency of Drosophilalarvae to aggregate with conspecifics (\\|Δpl\\|). Mixed model analysis of variance for the tendency to aggregate with conspecific larvae in D. melanogasterin three types of larval environment (F-C, F-F or C-C). Larval aggregation was measured as \\|Δpl\\|, the absolute value of Δpl(see Methods). TREATMENT (F-C, F-F or C-C) and LARVAE were fixed main effects, whereas experimental day (DAY) was a random factor. DAY is nested within TREATMENT and was used as the error term in testing the effect of TREATMENT. ::: Explanatory variable *d.f.* Mean square **F-value P** -------------------------- ------------ ----------------- --------------- --------- TREATMENT 2 0.4168 5.08 0.0302 LARVAE 1 0.2237 3.44 0.0670 TREATMENT (DAY) 9 0.0831 1.28 0.2603 Error 92 0.0651 ::: Discussion ========== On the background of ecological interactions between insects and filamentous fungi on ephemeral resources, the experiment presented in this study was designed to test for social attraction in Drosophilalarvae, an attraction that I hypothesised to be advantageous when larvae are confronted with noxious moulds. The results demonstrate that the fly larvae significantly aggregated on food patches on which young fungal colonies were growing (F-C treatment, Fig. [2a](#F2){ref-type="fig"}). Moreover, when provided with a homogeneous environment (F-F or C-C treatment), larvae displayed significant aggregation across the two food patches (Fig. [2b](#F2){ref-type="fig"}). In comparison, however, aggregation was significantly enhanced when larvae had the choice between a mould-free and a mould-infected site (Fig. [2b](#F2){ref-type="fig"}). Thus, the results suggest that grouping behaviour in Drosophilalarvae involves both mutual attraction between group members \[[@B2]\] and the attraction of individuals to the same environmental stimulus \[[@B22]\], i.e. cues emitted by the fungi. Group formation in eusocial insects and those living in groups for part or most of their lives is often mediated by pheromones, e.g. cuticular hydrocarbons that induce attraction between individuals \[[@B22],[@B23]\]. Chemical communication is also widespread in drosophilid behaviour, including those associated with spatial aggregation in adult flies \[[@B24]-[@B26]\]. Because several receptors on the cephalic lobe of Drosophilalarvae have gustatory, mechanosensory and olfactory functions \[[@B27]\], both chemical and physical cues (e.g. substrate vibrations caused by larval movements) might be involved in mutual attraction. However, the mechanisms leading to grouping and group cohesion in Drosophilalarvae are unknown. Interestingly, strong social attraction (communal digging) between Drosophilalarvae is present in third-instar larvae, a behaviour regulated by a peptide neuromodulator (Drosophilaneuropeptide F, dNPF) \[[@B28]\]; this shows striking similarities to the correlation of social feeding and the expression of a neuropeptide Y receptor homologue (NRP-1) in Caenorhabditis elegans\[[@B29]\]. Moreover, neurons that detect aversive environmental stimuli have been demonstrated to induce social feeding in C. elegans\[[@B30]\], thus providing support for the proposed relationship between environmental stress and social behaviour \[[@B31]\]. In contrast to C. elegans, the intimate communal digging behaviour in old third-instar Drosophilalarvae does not occur in the context of food foraging behaviour but is part of the post-feeding phase prior to pupation \[[@B28]\]. Whereas downregulation of dNPF expression coincides with social behaviour in old and non-feeding Drosophilalarvae, higher levels of dNPF expression in younger larvae seem to suppress strong larval aggregation and communal digging behaviour \[[@B28]\]. Therefore, it remains to be seen whether similar neural regulatory mechanisms are involved in earlier developmental stages of Drosophilalarvae with respect to social affinity related to foraging for food and attraction to fungal competitors. With regard to the proximate causes of attraction to mould-infected sites, fungal-borne volatiles such as CO~2~or ethylene \[[@B32]\] might be perceived by fly larvae and might guide them to young mould colonies. A general reason for the formation of social groups seems to be an adaptive response to stressful environmental conditions \[[@B1],[@B31]\]. As outlined in the introduction, filamentous fungi co-occurring with drosophilids on larval feeding sites can impede fly larval development; indeed, larval aggregations have been shown successfully to suppress mould growth \[[@B12]\] (Fig. [1](#F1){ref-type="fig"}). Consequently, the presence of fungi may indicate stressful ecological conditions that initiate attraction towards fungal patches and enhance the mutual attraction of Drosophilalarvae (Fig. [2](#F2){ref-type="fig"}). In connection with the benefits that accrue from mould suppression, the present study demonstrates that the larval-driven inhibition of fungal development is not a mere by-product attributable to the maternal decision to aggregate eggs across patches but is the consequence of a positive response of individuals to conspecifics. Because adult density-dependent oviposition choices influence a larva\'s food quality and its susceptibility to natural enemies \[[@B33]\] or abiotic stress, as well as its probability of coming into contact with intra- and interspecific competitors, the present study demonstrates the possibility of adaptive behavioural relationships between the well-known adult gregariousness in Drosophilaand communal behaviour in the immature stages. Further investigating this kind of behavioural adult-offspring correlations would strongly contribute to our understanding of the evolutionary costs and benefits of spatial aggregation in insect communities \[[@B34]\]. Conclusion ========== The study presented here demonstrates that fungal-infected food patches (1) attract first-instar Drosophilalarvae and (2) enhance group foraging behaviour. Because the larval-driven reduction in mould growth \[[@B12]\] has been shown to improve the chance of larval survival to the adult stage \[[@B9],[@B13],[@B14]\], social attraction to fungal-infected sites may reflect an adaptive behavioural response. In connection with the maternal behaviour of aggregating eggs across substrate patches, the condition-dependent mutual attraction of larvae can be discussed as a communal defence behaviour against competing mould. Given that filamentous fungi seriously deteriorate the developmental conditions for insect larvae, mould may constitute one important ecological factor that has, at least in the larval stages, facilitated social attraction in Drosophila. Thus, this study highlights the largely unappreciated role of inter-kingdom competition as a potentially important driving force in the evolution of insect behavioural traits. In general, the group formation of Drosophilalarvae in response to well-defined ecological conditions might be an interesting model system for the study of proximate and ultimate aspects of social biology. Methods ======= Experimental set-up ------------------- I experimentally analysed the grouping behaviour of Drosophilalarvae using a D. melanogasterstrain that originated from wild animals caught in 2003 near Kiel, northern Germany (approx. 54° N, 10° E). Flies had been reared for 18 generations on standard Drosophilamedium (30 gram corn meal, 30 gram sugar, 30 gram brewer\'s yeast extract (Leiber, Germany), and Nipagin) under constant environmental conditions (photoperiod of 16 hours and a temperature of 22°C). In order to obtain first instar larvae, a population of approx. 300 individuals (five to seven days old) were offered a 10 cm Petri dish containing a hard Agar medium (22 gram Agar, 90 cm^3^sugar beet syrup and 9.5 cm^3^Nipagin (10% in 95% ethanol) per 500 cm^3^water), on which they were allowed to oviposit for a period of 16 to 18 hours, including a period of darkness. Subsequently, flies were removed and the eggs were incubated at 22°C over a 16-hour photoperiod. After 24 hours, almost all larvae had hatched from the eggs and were then isolated from the medium by washing them off the Agar plate onto fine-meshed gauze with water. These larvae were used in the experiments. I used the same medium as for fly rearing (without adding the antifungal agent Nipagin) to simulate an uninfected or a fungal-infected larval environment as follows: aliquots of 3.5 cm^3^hot medium were transferred to each of two small pots (10 mm diameter, 5 mm high) that were glued to the bottom of a Petri dish (45 mm diameter, 13 mm high). Within one Petri dish, the larval food patches were placed at a distance of approx. 10 mm and surrounded by an Agar layer (5 mm high), so that the surfaces of the Agar and the food patches were at the same level. After the food patches had cooled down, one patch was provided with 1 μl of water containing approx. 800 conidiospores of the fungus A. niger(F treatment) and, as a control, the second patch was provided with spore-free water (C treatment). In addition to this F-C treatment, I simultaneously prepared arenas in which both patches were infected with spores (F-F) or both patches remained uninfected (C-C). The arenas were immediately sealed with lids and incubated under the aforementioned conditions. Two days later, the fungal spores had germinated and tiny translucent hyphal colonies were visible. A group of ten Drosophilalarvae were transferred, with a fine brush, to each arena at a distance of approx. 10 mm from each of the two food patches. Subsequently, the arenas were sealed with lids and stored at 22°C in an illuminated incubator. In order to avoid any systematic effects on larval distribution behaviour that might be caused by the position of the light tubes, the arenas of all three treatments were randomly arranged in the incubator. After six hours, the number of larvae in each food patch was recorded. Preliminary experiments had shown that this time period was sufficient to obtain final larval distribution patterns across the two patches, which remained nearly constant until the next day. Five to ten replicates for each treatment were simultaneously prepared at four different days. Statistical analysis -------------------- Based on the number of larvae that were found to be feeding in both patches, I calculated the proportion of larvae in each patch. Larvae that were not found on any of the food patches were ignored. However, I tested for the effect of the number of larvae in both patches (LARVAE) on the degree to which larvae aggregated across the food patches (see below). To obtain a measure of the degree of larval aggregation across the two food patches in the F-C treatment, the proportion of larvae on the fungal-infected patch in each arena was reduced by the proportion of larvae on the uninfected patch, yielding Δpl. Δpl= 0 would indicate no effect of the presence of fungal-infected and uninfected food sites on larval distribution patterns. Whereas Δpl\> 0 would indicate aggregation on fungal-infected sites and thus larval attraction to fungal colonies, Δpl\< 0 is expected if larvae avoid fungal colonies and aggregate on uninfected patches. Subsequently, I used the absolute values of Δpl, \\|Δpl\\|, that were obtained in all three types of treatment (F-C, F-F and C-C) in order to compare the tendency to aggregate with conspecific larvae in the heterogeneous larval environment (F-C) with larval aggregation in two types of homogeneous environment (F-F or C-C). Note that, because the absolute value of Δplcan only be equal to or larger than zero, \\|Δpl\\| measures larval aggregation in the F-C treatment independently of whether a food patch was fungal-infected or not. I applied the GLM procedure provided by SAS version 8.2 to test if Drosophilalarvae aggregated on fungal infected food patches, i.e. if Δplis significantly larger than 0 (see above). For this only the intercept was tested as an effect in the statistical model \[[@B35]\]. The result of the parameter estimate for the intercept are given. Before this test, I verified that LARVAE and experimental DAY did not affect Δpl(Table [1](#T1){ref-type="table"}), which justifies the removal of these variables from the full model (backward elimination of non-significant variables) \[[@B36]\]. The same procedure was applied to test for the general tendency of larval aggregation (measured as \\|Δpl\\|) under different environmental conditions (see Table [2](#T2){ref-type="table"} to [4](#T4){ref-type="table"}). To analyse the effect of TREATMENT (F-C, F-F or C-C) and LARVAE on the general propensity of Drosophilalarvae to aggregate across the experimental food patches (\\|Δpl\\|), I used the aforementioned GLM procedure with a RANDOM statement to account for possible effects of experimental DAY on larval distribution patterns. In this model, TREATMENT and LARVAE were fixed main effects. Since five to ten replicates for each treatment were prepared at four different days, DAY was considered as a categorical random factor. DAY is nested within TREATMENT and was used as the error term in testing for the effect of TREATMENT \[[@B37]\]. The results of the tests of hypotheses for mixed model analysis of variance are shown in Table [4](#T4){ref-type="table"}. Acknowledgments =============== Frank Kempken and Hanna Schmidt provided spores of A. niger. Denise Olbrich, Katherina Habekost and Saskia Heppner are acknowledged for help with data collection and preparation of experimental arenas. I thank Jacqui Shykoff and two anonymous reviewers for their valuable comments on an earlier version of this paper.	PubMed Central	2024-06-05T03:55:52.670567	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548382/", "journal": "Front Zool. 2005 Jan 27; 2:2", "authors": [ { "first": "Marko", "last": "Rohlfs" } ] }
PMC548501	Background ========== Prostate cancer is the most common cancer and the second leading cause of cancer related deaths in men in the United States with an estimated 230,110 new cases and 29,500 deaths in 2004 \[[@B1]\]. Initial treatment for prostate cancer is usually androgen-ablative therapy, radiotherapy or radical prostatectomy and the patients respond to androgen-ablative therapy in the beginning of treatment. However, many patients eventually fail this therapy and die of recurrent androgen-independent prostate cancer and metastasis \[[@B2]\]. Up to 30% of men undergoing radical prostatectomy will also relapse, often as a result of micrometastatic cancer present at the time of surgery \[[@B3]\]. The efficacy of cytotoxic chemotherapy for treatment of hormone-refractory prostate cancer has been tested in clinical trials. In general, response rates of \<10% were observed in single-agent studies \[[@B2]\]. Thus, there is a tremendous need for the development of mechanism-based targeted strategies for treatment of prostate cancer. Taxotere, a member of taxane family, is semi-synthesized from an inactive taxoid precursor extracted from the needles of the European yew, Taxus baccata. Its known basic mechanism of action is that it binds to tubulin and deranges the equilibrium between microtubule assembly and disassembly during mitosis \[[@B4]\]. Stabilization of microtubules by Taxotere impairs mitosis and exerts an anticancer effect in tumors \[[@B4]\]. Taxotere has shown clinical activity in wide spectrum of solid tumors including breast, lung, ovarian, prostate cancers, etc \[[@B5],[@B6]\]. In metastatic breast, lung, and ovarian cancer, randomized trials have shown that Taxotere-containing therapies are superior to or as effective as established standard chemotherapeutic regimens and are often associated with an improved safety profile \[[@B6]\]. Clinical trials have also found that weekly Taxotere in patients with metastatic hormone-refractory prostate cancer is associated with improvements in clinical benefit response and quality of life \[[@B7],[@B8]\]. Thus, Taxotere is currently considered to be among the most important anticancer drugs in cancer chemotherapy. In addition to its effects on microtubules, Taxotere also induces apoptosis with down-regulation of bcl~XL~and bcl-2, and upregulation of p21^WAF1^and p53 \[[@B9],[@B10]\]. We have previously reported that Taxotere down-regulates some genes for cell proliferation, mitotic spindle formation, transcription factors, and oncogenesis, and up-regulates some genes related to induction of apoptosis and cell cycle arrest in prostate cancer cells, suggesting the pleiotropic effects of Taxotere on prostate cancer cells \[[@B11]\]. Capecitabine is an orally administered systemic prodrug of 5\'-deoxy-5-fluorouridine (5-DFUR or Furtulon) which is converted to 5-fluorourasil (5-FU) \[[@B12]\]. Capecitabine is readily absorbed from the gastrointestinal tract. In human and animals, carboxylesterase hydrolyzes much of Capecitabine to 5\'-deoxy-5-flurocytidine (5-DFCR). Cytidine deaminase, an enzyme found in most tissues including tumors, subsequently converts 5-DFCR to 5-DFUR. The enzyme, thymidine phosphorylase (dThdPase), then hydrolyzes 5-DFUR to the active drug 5-FU both in vivoand in vitro. After being converted to 5-FU, Capecitabine inhibits essential cellular biosynthetic processes and is incorporated into DNA to inhibit normal bioprocess function of the cell \[[@B13]\]. Capecitabine has shown anti-tumor activity in various cancers including prostate cancer \[[@B14]-[@B16]\]. 5-FU-based chemotherapy improves overall and disease-free survival of patients with cancer. However, response rates for 5-FU-based chemotherapy are low and a large number of tumors eventually becomes resistant to 5-FU \[[@B13],[@B17]\]. Clinical trials showed that chemotherapeutic combination with Taxotere and Capecitabine resulted in improved objective response rate and overall survival without a significant increase in the treatment related adverse effects in metastatic breast cancer and advanced non-small cell lung carcinoma \[[@B18],[@B19]\]. However, the molecular mechanism(s) of action of Taxotere and Capecitabine have not been fully elucidated. In this study, we utilized high-throughput gene chip, which contains 22,215 known genes, to determine the alternation of gene expression profiles of hormone insensitive (PC3) and sensitive (LNCaP) prostate cancer cells exposed to Taxotere and Furtulon. The purpose of this study was: 1) to identify novel genes that have key roles in cancer cell killing and resistance induced by Taxotere and/or Furtulon; 2) to test whether similar genes are altered by Taxotere and Furtulon; 3) to test whether combination therapy alters genes that may reflect better treatment outcome or may provide information whether combination therapy could be antagonistic; 4) finally to provide molecular information for further mechanistic investigation and future clinical application. Methods ======= Cell culture and growth inhibition ---------------------------------- PC3 (ATCC, Manassas, VA) and LNCaP (ATCC) human prostate cancer cells were cultured in RPMI-1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in a 5% CO~2~atmosphere at 37°C. Taxotere (Aventis Pharmaceuticals, Bridgewater, NJ) was dissolved in DMSO to make 4 μM stock solution. Furtulon (Roche, Palo Alto, CA) was dissolved in PBS to make 100 mM stock solution. For growth inhibition, PC3 and LNCaP cells were treated with Taxotere (1, 2, and 4 nM), Furtulon (50, 100, and 200 μM), or 1 nM Taxotere plus 50 μM Furtulon for one to three days. Control PC3 and LNCaP cells received 0.01% DMSO or 0.1% PBS for same time points. After treatment, PC3 and LNCaP cells were incubated with MTT (0.5 mg/ml, Sigma, St. Louis, MO) at 37°C for two hours and then with isopropyl alcohol at room temperature for one hour. The spectrophotometric absorbance of the samples was determined by using ULTRA Multifunctional Microplate Reader (TECAN, Durham, NC) at 595 nm. The concentrations of Taxotere and Furtulon used for our in vitrostudies are easily achievable in humans, suggesting that our experimental results are relevant for human applications. The experiment was repeated three times and a ttest was performed to verify the significance of cell growth inhibition after treatment. Microarray analysis for gene expression profiles ------------------------------------------------ PC3 and LNCaP cells were treated with 2 nM Taxotere, 110 μM Furtulon, or 1 nM Taxotere plus 50 μM Furtulon for 6, 36, and 72 h. Total RNA from each sample was isolated by Trizol (Invitrogen, Carlsbad, CA) and purified by RNeasy Mini Kit and RNase-free DNase Set (QIAGEN, Valencia, CA) according to the manufacturer\'s protocols. cDNA for each sample was synthesized by Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) using the T7-(dT)~24~primer instead of the oligo(dT) provided in the kit. Then, the biotin-labeled cRNA was transcripted in vitrofrom cDNA by using BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY), and purified by RNeasy Mini Kit. The purified cRNA was fragmented by incubation in fragmentation buffer (40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc) at 95°C for 35 min and chilled on ice. The fragmented labeled cRNA was applied to Human Genome U133A Array (Affymetrix, Santa Clara, CA), which contains 22,215 human gene probes, and hybridized to the probes in the array. After washing and staining, the arrays were scanned. Two independent experiments were performed to verify the reproducibility of results. Microarray data normalization and analysis ------------------------------------------ The gene expression levels of samples were normalized and analyzed by using Microarray Suite, MicroDB™, and Data Mining Tool software (Affymetrix, Santa Clara, CA). The absolute call (present, marginal, absent) and average difference of 22,215 gene expressions in a sample, and the absolute call difference, fold change, average difference of gene expressions between two or several samples were normalized and identified using these software. Statistical analysis of the mean expression average difference of genes, which show \>2 fold change, was performed using a ttest between treated and untreated samples. Clustering and annotation of the gene expression were analyzed by using Cluster and TreeView \[[@B20]\], Onto-Express \[[@B21]\], and GenMAPP \[[@B22]\]. Genes that were not annotated or not easily classified were excluded from the functional clustering analysis. Real-time RT-PCR analysis for gene expression --------------------------------------------- To verify the alterations of gene expression at the mRNA level, which appeared on the microarray, we chose representative genes (Table [1](#T1){ref-type="table"}) with varying expression profiles for real-time RT-PCR analysis. Two micrograms of total RNA from each sample were subjected to reverse transcription using the Superscript first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA) according to the manufacturer\'s protocol. Real-time PCR reactions were then carried out in a total of 25 μL reaction mixture (2 μl of cDNA, 12.5 μl of 2X SYBR Green PCR Master Mix, 1.5 μl of each 5 μM forward and reverse primers, and 7.5 μl of H~2~O) in SmartCycler II (Cepheid, Sunnyvale, CA). The PCR program was initiated by 10 min at 95°C before 40 thermal cycles, each of 15 s at 95°C and 1 min at 60°C. Data were analyzed according to the comparative Ct method and were normalized by actin expression in each sample. Melting curves for each PCR reaction were generated to ensure the purity of the amplification product. Western blot analysis --------------------- We also conducted Western Blot analysis to verify the alterations of genes at the level of translation for selected genes with varying expression profiles. The PC3 and LNCaP cells were treated with 1 and 2 nM Taxotere or 50 and 110 μM Furtulon for 24, 48, and 72 hours. After treatment, the cells were lysed in 62.5 mM Tris-HCl and 2% SDS, and protein concentration was measured using BCA protein assay (PIERCE, Rockford, IL). The proteins were subjected to 10% or 14% SDS-PAGE, and electrophoretically transferred to nitrocellulose membrane. The membranes were incubated with anti-cathepsin C (1:200, Santa Cruz, Santa Cruz, CA), anti-p16 (1:200, Santa Cruz, Santa Cruz, CA), anti-IKKα (1:100, Santa Cruz, Santa Cruz, CA), anti-p21^WAF1^(1:500, Upstate, Lake Placid, NY), anti-Bax (1:10000, Trevigen, Gaithersburg, MD), anti-survivin (1:200, Alpha Diagnostic, San Antonio, TX), anti-CDC2 (1:200, Santa Cruz, Santa Cruz, CA), anti-cyclin A (1:250, NeoMarkers, Union City, CA), anti-cyclin B (1:200, Santa Cruz, Santa Cruz, CA), anti-cyclin E (1:250, NeoMarkers), and anti-β-actin (1:10000, Sigma, MO) primary antibodies, and subsequently incubated with secondary antibody conjugated with fluorescence dye. The signal was then detected and quantified by using Odyssey infrared imaging system (LI-COR, Lincoln, NE). Results ======= Cell growth inhibition ---------------------- MTT assay showed that the treatment of PC3 and LNCaP prostate cancer cells with Taxotere, Furtulon, or lower concentration of Taxotere plus Furtulon resulted in dose and time-dependent inhibition of cell proliferation (Figure [1](#F1){ref-type="fig"}), demonstrating the inhibitory effect of Taxotere and Furtulon on the growth of PC3 and LNCaP prostate cancer cells. Regulation of mRNA expression by Taxotere and Furtulon treatment ---------------------------------------------------------------- Microarray analysis showed that the alterations of gene expression were occurred as early as 6 hours of Taxotere and/or Furtulon treatment, and were more evident with longer treatment (Table [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). Clustering analysis based on gene function showed down-regulation of some genes for cell proliferation and cell cycle progression (cyclin A, cyclin F, CDC2, CDK2, etc), transcription factors (transcription factor A, ATF5, TAF1131L, FOXM1, etc), and oncogenesis (GRO oncogene, BRCA1 associated RING domain, tumor-associated nuclear protein p120, etc) in Taxotere and/or Furtulon treated prostate cancer cells (Table [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). In contrast, Taxotere and/or Furtulon up-regulated some genes that are related to the induction of apoptosis (GADD45A, GADD45B, etc), cell cycle arrest (p21^CIP1^, VDUP1, BTG, etc), and tumor suppression (Table [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). Combination treatment with Taxotere and Furtulon also altered expression of some genes (CDC27, CDK9, p18, IKKα, etc) that showed no change in mono-treatment (Table [4](#T4){ref-type="table"} and [5](#T5){ref-type="table"}), suggesting the synergic effects of combination treatment on some genes. Taxotere and Furtulon also up-regulated some genes (S-100P, ALDH1A3, casein kinase, annexin, etc) responsible for chemotherapeutic resistance, suggesting the induction of cancer cell resistance to these agents (Table [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). Taxotere and Furtulon also showed differential effects on PC3 cells with alteration of metastasis-related genes and on LNCaP cells with down-regulation of survivin, cyclin B & E, CDC2, CDC25, and specifically AR by Furtulon, suggesting their effects mediated by both AR-independent and dependent pathways (Table [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). Target verification by real-time RT-PCR and western blot -------------------------------------------------------- To verify the alterations of gene expression at the mRNA level, which appeared on the microarray, we chose representative genes with varying expression profiles for real-time RT-PCR and Western Blot analysis. The results of real-time RT-PCR for these selected genes were in direct agreement with the microarray data (Figure [2](#F2){ref-type="fig"}). The same alternations of gene expression were observed by real-time RT-PCR analysis, although the fold change in the expression level was not exactly same between these two different analytical methods. The results of Western Blot analysis were also in direct agreement with the microarray and real-time RT-PCR data (Figure [3](#F3){ref-type="fig"} and our earlier report \[[@B11]\]). These results support the findings obtained from microarray experiments. Discussion ========== It has been known that Taxotere binds to microtubules while Capecitabine is incorporated into DNA, inhibiting the bioprocess in cancer cells \[[@B4],[@B13]\]. However, the precise molecular mechanisms for inhibiting cancer cell growth by Taxotere and/or Capecitabine have not been fully elucidated. From gene expression profiles of Taxotere and/or Capecitabine treated prostate cancer cells, we found that these chemotherapeutic agents caused alterations in the expression of many genes related to the control of cell proliferation, apoptosis, transcription, translation, cell signaling, oncogenesis, and angiogenesis (Figure [4](#F4){ref-type="fig"}), although the cellular target of Taxotere or Capecitabine appears to be different. It has been well known that CDCs regulate the molecules related to the cell cycle initiation and progression and that cyclins associate with cyclin-dependent protein kinases (CDKs) and CDCs to control the process of cell cycle \[[@B23],[@B24]\]. The CDK inhibitors including p21^WAF1^, p16^INK4A^, and p18^INK4C^have been demonstrated to arrest the cell cycle and inhibit the growth of cancer cells \[[@B23],[@B24]\]. Our results showed that Cyclins (cyclin A2, cyclin E2, cyclin F, cyclin B1), CDK2, CDC2, and other cell growth promotion genes (pescadillo, spermidine synthase, mitotin) \[[@B25]-[@B27]\] were down-regulated in Taxotere and/or Furtulon treated prostate cancer cells, while CDK inhibitor p21^WAF1^and other growth inhibitor genes (BTG2, VDUP1, anti-proliferative B-cell translocation gene 1) \[[@B28],[@B29]\] were up-regulated, suggesting that Taxotere and/or Furtulon inhibited the growth of prostate cancer cells through the arrest of cell cycle and the inhibition of cell proliferation (Figure [4](#F4){ref-type="fig"}). The down-regulation of CDC27, CDK9, EGF, and FGF12B, and up-regulation of p16^INK4A^and p18^INK4C^were also observed in combination treatment but not in mono-treatment, suggesting the synergic effect of combination treatment. These observations are novel in Taxotere and/or Furtulon treated prostate cancer cells. Induction of apoptosis by chemotherapeutic agents also leads to the inhibition of cancer cell growth. It has been reported that Taxotere is able to induce apoptosis by caspase-3 dependent or independent cell death mechanism \[[@B30]\]. Capecitabine may induce apoptosis through Fas/FasL or Bax/Bcl-2 pathway \[[@B31],[@B32]\]. From gene expression profile, we found that Taxotere and/or Furtulon increased level of growth arrest and DNA-damage-inducible alpha (GADD45A), GADD45B, p53 regulated PA26 nuclear protein (PA26), and p53-induced protein 11 (PIG11), all of which are related to the induction of apoptotic processes. GADD45A and GADD45B have been known to promote apoptosis and regulate G2/M arrest \[[@B33]\]. PA26 is a target of the p53 tumor suppressor and a member of the GADD family with the properties of inducing apoptosis \[[@B34]\]. PIG11 as a downstream target of p53 is also involved in the apoptotic processes \[[@B35]\]. The combination treatment also showed down-regulation of negative regulator of programmed cell death ICH-1S and Bcl-2-associated transcription factor, which was not occurred in mono-treatment. The induction of apoptosis mediated by GADD45A, GADD45B, PA25, and PIG11 could be another molecular mechanism by which Taxotere and/or Furtulon inhibit the growth of prostate cancer cells. We also found that Taxotere and/or Furtulon inhibited the expression of transcription factors (FOXM1, ATF5, TFAM, TAFII31L), translation factors (EIF1A, EIF5A), oncogene (GRO1, GRO3, BRCA1-associated protein, tumor-associated nuclear protein p120), and heat shock protein, and up-regulated the genes for differentiation (prostate differentiation factor). These results are novel, and suggest the beneficial effects of Taxotere and/or Furtulon on the inhibition of cancer cell growth and oncogenesis. It is important to note that Taxotere and/or Furtulon also up-regulated the expression of some genes which are known to induce cell resistance to chemotherapeutic agents and to favor cell survival. Among these genes, calcium-binding protein S100P has been found to be highly expressed in cells which develop acquired resistance to anti-tumor agents \[[@B36]\]. The overexpression of aldehyde dehydrogenase 1 (ALDH1) has also been detected solely in classical multidrug resistance cancer cells \[[@B37],[@B38]\]. It has been reported that Annexin-I, casein kinase 1, and cisplatin-resistance associated protein expressions modulate drug resistance in tumor cells \[[@B39],[@B40]\]. The up-regulation of these molecules by Taxotere and/or Furtulon could induce cell resistance to chemotherapeutic agents. Also, Taxotere and/or Furtulon were found to up-regulate the expression of Notch 3, angiopoietin, activating transcription factor 3, which could favor cell survival \[[@B41]-[@B43]\]. Further in depth mechanistic studies are needed to address these issues. The investigation on overcoming these unbeneficial effects with other agents must be devised, which is ongoing in our laboratory. Taxotere showed no effect on AR expression while Furtulon down-regulated AR expression in LNCaP cells, suggesting that the combination could be superior in AR-positive cells. The genes altered by Taxotere and/or Furtulon with respect to the control of cell growth, apoptosis, transcription, oncogenesis, and metastasis in androgen insensitive PC3 cells are different from that in androgen sensitive LNCaP cells, suggesting that the effects of Taxotere and Furtulon may be mediated by both AR-dependent and independent signaling pathways. We observed up-regulation of tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2, and protease inhibitor 3 in Taxotere and/or Furtulon treated PC3 cells, suggesting that Taxotere and/or Furtulon may exert anti-metastatic effect. However, we also observed increase in the expression of MMP1, MMP9, cathepsin B, uPA, and tPA in Taxotere and Furtulon treated PC3 cells, therefore, more experimental studies are needed to reveal the overall effect of Taxotere and Furtulon on metastatic processes. These results were not observed in androgen sensitive LNCaP cells, suggesting difference in effects that could be mediated through different cell signal transduction pathways. Conclusions =========== In conclusion, Taxotere and/or Furtulon directly and indirectly caused changes in the expression of many genes that are critically involved in the control of cell proliferation, apoptosis, transcription, translation, oncogenesis, angiogenesis, metastasis, and drug resistance (Figure [4](#F4){ref-type="fig"}). These findings could provide molecular information for further investigation on the mechanisms by which Taxotere and Furtulon exerts their pleiotropic effects on prostate cancer cells. These results could also be important in devising mechanism-based targeted therapeutic strategies for prostate cancer, especially in devising combination therapy for drug resistant prostate cancers. However, further in-depth investigations are needed in order to establish cause and effect relationships between these altered genes and therapeutic response in prostate cancer cells. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= FHS designed the study and prepared the manuscript. YL carried out cell growth inhibition, microarray and Western Blot analysis and drafted the manuscript. MH participated in the design of the study. RL and SHS carried out real-time PCR. JE prepared Furtulon reagent. All authors read and approved the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/7/prepub> Acknowledgements ================ This work was partly funded by a grant from Aventis Pharmaceuticals (awarded to F.H.S.). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Effects of Taxotere and/or Furtulon on the growth of PC3 and LNCaP Cells. PC3 and LNCaP prostate cancer cells treated with Taxotere and/or Furtulon resulted in a dose and time-dependent inhibition of cell proliferation (\* : p\< 0.05, n = 3) ::: ![](1471-2407-5-7-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Real-time RT-PCR showing the altered expression of specific genes in Taxotere and Furtulon treated PC3 and LNCaP cells. (C: control; T+F: Taxotere and Furtulon combination treatment.) ::: ![](1471-2407-5-7-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Western Blot analysis showing the altered expression of specific genes in Taxotere and Furtulon treated PC-3 or LNCaP cells. (C: control; T: Taxotere treatment; F: Furtulon treatment; T+F: Taxotere and Furtulon combination treatment.) ::: ![](1471-2407-5-7-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effects of Taxotere and Furtulon on cell cycle, apoptosis, and other pathway related gene expression analyzed and visualized by GenMAPP software integrated with cDNA microarray data. A: PC3 cells. B: LNCaP cells. (positive value: increase in fold change; negative value: decrease in fold change; A: PC3 cells; B: LNCaP cells) ::: ![](1471-2407-5-7-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The primers used for real-time RT-PCR analysis ::: ------------------------------------------------------------------ Genes Primer Sequence PCR Product ----------- ------------------------------------ ----------------- ATF5 ctc ctc ctt ctc cac ctc aa\ 103 bp gcc gac ttg ttc tgg tct ct Cyclin A2 aat cag ttt ctt acc caa tac\ 127 bp ctg atg gca aat act tga Fas/Apo-1 caa aag tgt taa tgc cca agt\ 187 bp gca gtc tgg ttc atc cc FOXM1 gcc aca ctt agc gag acc c\ 189 bp atc aca agc att tcc gag aca GADD45 cgc ctg tga gtg agt gc\ 154 bp ctt atc cat cct ttc ggt ctt IGFBP2 atg ggc gag ggc act t\ 189 bp cag ctc ctt cat acc cga ctt uPA ggg agc aga gac act aac gac t\ 108 bp ctc aca gcc cac aca aca ag Ki-67 ccg ggc tcc atc atc t\ 148 bp ctc cag acg cca aaa taa gac p21^WAF1^ ctg gag act ctc agg gtc gaa\ 98 bp gga tta ggg ctt cct ctt gga p27^KIP1^ cgc tcg cca gtc cat t\ 187 bp aca aaa ccg aac aaa aca aag PIR cac tag ccc tcc atc ctc tac\ 151 bp ggg tct gcc aat gct tct MMP1 gct ttc ctc cac tgc tgc t\ 146 bp aac ttg cct ccc atc att ctt STK6 tca gcg ggt ctt gtg t\ 162 bp ctc ttt tgg gtg tta ttc agt Survivin cca ctg ccc cac tga gaa c\ 118 bp acc gga cga atg ctt ttt atg TRIP13 tct ggc agt gga caa gca gtt\ 136 bp tgg gag acg gct gtg tgg GAPDH ctg cac cac caa ctg ctt ag\ 222 bp ttc agc tca ggg atg acc tt β-actin cca cac tgt gcc cat cta cg\ 99 bp agg atc ttc atg agg tag tca gtc ag ------------------------------------------------------------------ ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Fold change of genes in PC3 cells exposed to combination treatment or mono-treatment with Taxotere or Furtulon ::: Gene Taxotere Furtulon Taxotere+Furtulon ----------------------------------------------------------------------- -------------- -------------- ----------------------- ------ ------ ------ ------ ------ ------- *Cell cycle and apoptosis* NM\_001237.1 cyclin A2 (CCNA2) NC -1.1 -2.0 -1.1 -1.7 -3.0 -1.1 -1.4 -2.5 BE407516 cyclin B1 -1.1 -1.1 -2.3 NC -3.0 -6.1 -1.1 -3.0 -5.3 NM\_001761.1 cyclin F (CCNF) -1.3 -1.3 -2.5 -1.5 -2.5 -2.6 -1.6 -1.6 -2.5 NM\_014303.1 pescadillo homolog 1 NC -1.5 -2.3 1.3 -1.9 -2.6 -1.1 -2.1 -3.5 NM\_003132.1 spermidine synthase (SRM) NC -1.5 -2.5 -1.1 -1.3 -2.5 -1.1 -1.1 -2.3 NM\_000389.1 cyclin-dependent kinase inhibitor 1A (p21, Cip1) 1.7 1.0 2.6 1.9 2.6 3.5 NC 1.7 3.5 NM\_003914.1 cyclin A1 (CCNA1) 1.3 2.3 2.8 NC 1.5 2.5 NC NC 2.0 L49506.1 cyclin G2 -2.1 2.0 2.6 -2.8 2.3 2.3 NC 3.7 2.5 NM\_004354.1 cyclin G2 (CCNG2) -1.9 3.0 3.2 NC 2.5 2.3 1.0 4.6 3.5 AL535380 B-cell translocation gene 1, anti-proliferative 1.1 1.7 3.5 NC 1.4 2.8 -1.5 1.6 2.6 NM\_006472.1 upregulated by 1,25-dihydroxyvitamin D-3 (VDUP1) 1.3 2.1 -2 1.0 2.1 2.3 1.0 2.3 NC NM\_015675.1 growth arrest and DNA-damage-inducible, beta (GADD45B) 1.2 2.5 6.5 1.6 1.5 2.6 1.0 NC 3.5 AF087853.1 growth arrest and DNA damage inducible protein beta NC 2.0 4.6 1.3 1.2 2.3 NC NC 3.2 AF078077.1 growth arrest and DNA-damage-inducible protein GADD45beta 1.5 2.0 5.3 1.1 1.2 2.1 NC 1.4 2.8 *Transcription and translation* NM\_007111.1 transcription factor Dp-1 (TFDP1) NC -1.6 -3.2 -1.3 -1.1 -2.0 NC -1.1 -2.0 NM\_012068.2 activating transcription factor 5 (ATF5) NC -2.1 -3.5 NC -1.5 -2.1 -1.3 -1.1 -2.6 NM\_012251.1 transcription factor A, mitochondrial (TFAM) 1.3 -1.5 -2.5 NC -1.6 -2.0 -1.1 -2.0 -3.7 AF220509.1 transcription associated factor TAFII31L NC -2.3 -3.2 -1.3 -1.3 -2.3 NC -1.2 -5.7 AA393940 eukaryotic translation initiation factor 5A -1.3 -1.5 -3.0 -1.6 -2.0 -3.5 -1.1 -1.1 -2.0 NM\_001674.1 activating transcription factor 3 (ATF3) 1.4 1.9 14.9 1.5 2.1 3.5 1.1 2.6 4.9 *Oncogenesis and other* NM\_001511.1 GRO1 oncogene (GRO1) 2 -1.5 -3.0 1.7 1.2 -2.0 NC NC -4.0 NM\_002090.1 GRO3 oncogene (GRO3) 2.8 -3.0 -4.0 2.5 -1.4 -6.5 NC -2.8 -13.9 NM\_005754.1 Ras-GTPase-activating protein SH3-domain-binding protein NC -1.7 -2.8 NC -2.0 -2.5 NC -2.6 -4.6 NM\_000026.1 adenylosuccinate lyase (ADSL) NC -1.3 -2.5 NC -1.4 -2.6 NC -1.6 -3.0 M80261.1 apurinic endonuclease (APE) -1.1 -1.4 -3.0 -1.1 -1.9 -2.6 NC -2.1 -3.7 D13413.1 tumor-associated 120 kDa nuclear protein p120 -1.1 -2.0 -3.2 -1.7 -1.7 -2.5 -2.3 -1.1 -3.0 AI743685 methionine aminopeptidase; eIF-2-associated p67 NC -2.6 -2.0 1.5 -1.5 -3.2 NC -2.3 -2.8 NM\_002634.2 prohibitin (PHB) NC -1.4 -3.0 NC -1.9 -2.1 NC -1.5 -2.6 NM\_002546.1 osteoprotegerin 1.5 -1.3 -3.0 1.2 -2.1 -3.0 NC -2.8 -5.3 AF003934.1 prostate differentiation factor mRNA -1.5 4.3 26.0 -1.7 4.9 16.0 NC 4.3 12.1 NM\_000177.1 gelsolin (amyloidosis, Finnish type) (GSN) NC 1.5 2.8 NC 2.0 3.5 NC 1.9 2.3 *Invasion and metastasis* NM\_003254.1 tissue inhibitor of metalloproteinase 1 (TIMP1) NC 2.0 2.5 NC 2.6 3.7 NC 2.6 3.7 NM\_003255.2 tissue inhibitor of metalloproteina(TIMP2) NC 1.6 2.5 NC 2.3 3.5 NC 1.6 3.5 NM\_002638.1 protease inhibitor 3 (PI3) 1.2 2.0 2.0 1.1 3.7 5.7 1.0 3.0 4.6 NM\_005562.1 laminin, gamma 2 (nicein (100 kD) 1.3 1.5 2.0 1.1 2.5 3.7 NC 2.3 3.7 NM\_001908.1 cathepsin B (CTSB) NC 1.7 3.0 NC 1.7 2.8 1.1 1.3 2.5 NM\_002658.1 plasminogen activator, urokinase (PLAU) NC 2.3 3.5 NC 2.8 2.3 1.0 2.1 1.9 NM\_000930.1 plasminogen activator, tissue (PLAT) 1.1 2.5 4.6 NC 5.7 9.8 NC 3.2 4.9 NM\_002421.2 matrix metalloproteinase 1 (MMP1) NC 1.4 4.3 NC 4.9 18.4 NC 5.7 13.0 NM\_004994.1 matrix metalloproteinase 9 (MMP9) 1.1 1.7 2.0 1.0 1.9 2.0 NC 2.5 2.1 NM\_000435.1 Notch homolog 3 (NOTCH3) NC 2.0 2.6 -2 2.3 3.2 NC 3.5 3.5 *Resistance to chemotherapeutic agents* NM\_000499.2 cytochrome P450, subfamily I (CYP1A1) 1.4 3.0 4.0 NC 3.5 5.3 1.2 9.2 10.6 NM\_006697.1 cisplatin resistance associated (CRA) 1.0 1.7 2.0 -1.3 2.0 2.6 NC NC 2.6 NM\_005980.1 S100 calcium-binding protein P (S100P) -1.2 4.9 24.3 1.1 10.6 27.9 NC 6.5 19.7 NM\_002961.2 S100 calcium-binding protein A4 (S100A4) 3.0 3.7 5.3 1.0 5.7 11.3 2.8 6.5 10.6 NM\_020672.1 S100-type calcium binding protein A14 (LOC57402) NC 1.5 2.0 NC 2.1 2.6 1.1 1.6 2.1 NM\_001894.1 casein kinase 1, epsilon (CSNK1E) NC 1.7 3.2 NC 2.0 2.6 1.1 1.7 3.2 NM\_000700.1 annexin A1 (ANXA1) NC 1.9 2.3 NC 2.0 2.3 NC 1.6 2.1 The genes in this list showed a \>2 fold change in expression in at least one time point in both mono and combination treatment. NC: No change; Negative value: Decrease; Positive value: Increase. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Fold change of genes in LNCaP cells exposed to combination treatment or mono-treatment with Taxotere or Furtulon ::: Gene Taxotere Furtulon Taxotere+Furtulon -------------------------------------------------------------------- -------------- -------------- ----------------------- ------ ------ ------ ------ ------ ------- *Cell cycle and apoptosis* NM\_001786.1 cell division cycle 2 (CDC2) -1.2 -7.5 -12.1 -1.1 -2.8 -1.6 NC -4.0 -14.9 D88357.1 mRNA for CDC2 delta T -1.1 -6.1 -14.9 NC -2.8 -1.6 -1.1 -4.3 -13.0 NM\_001237.1 cyclin A2 (CCNA2) -1.2 -5.7 -12.1 NC NC -2.1 -1.1 -8.6 -13.9 NM\_004702.1 cyclin E2 (CCNE2) 1.6 -5.3 -3.2 NC -2.3 -1.7 -1.7 -4.9 -8.0 AF112857.1 cyclin E2 splice variant 1 mRNA 1.2 -3.5 -3.2 NC -2.0 -1.6 -1.4 -7.0 -8.6 NM\_001761.1 cyclin F (CCNF) -1.5 -2.5 -3.7 -1.1 -1.1 -2.0 -1.4 -2.3 -2.8 AB012305.1 cyclin-dependent kinase 2 -1.7 -3.5 -5.3 -2.0 -2.1 -2.1 NC -2.8 -3.5 U30872.1 mitosin mRNA -1.2 -4.6 -6.1 NC -2.1 -1.3 NC -3.2 -7.0 NM\_000389.1 cyclin-dependent kinase inhibitor 1A (p21, Cip1) 1.3 9.8 8.6 1.0 2.0 2.0 1.0 8.0 7.5 NM\_006763.1 BTG family, member 2 (BTG2) 1.5 6.1 6.5 1.1 1.7 2.1 1.4 5.3 3.7 NM\_006472.1 upregulated by 1,25-dihydroxyvitamin D-3 (VDUP1) 1.0 3.5 3.5 1.0 1.5 2.6 1.0 2.3 3.0 NM\_001924.2 growth arrest and DNA-damage-inducible, alpha 1.4 7.5 7.5 NC 1.9 2.6 1.1 7.0 7.5 BC003637.1 Similar to DNA-damage-inducible transcript 3 1.0 2.6 2.6 1.1 2.1 2.5 NC 2.6 2.8 NM\_014454.1 p53 regulated PA26 nuclear protein (PA26) 1.1 4.0 3.0 1.1 1.4 2.6 1.1 3.7 3.7 NM\_004701.2 cyclin B2 (CCNB2) -1.1 -8 -13.9 NC 1.3 -1.3 -1 -4.9 -19.7 NM\_001255.1 CDC20 (cell division cycle 20) -1.9 -90.5 -147 NC 1.6 -1.9 -1.6 -13 -157 NM\_021873.1 cell division cycle 25B (CDC25B) -1.1 -1.6 -2.5 NC NC -1.4 -1 -2.3 -3.25 NM\_001827.1 CDC28 protein kinase 2 (CKS2) -1.1 -4.6 -6.5 NC 1.6 -1.6 -1.2 -3.5 -5.7 NM\_001168.1 survivin (BIRC5) -1.1 -27.9 -294 NC NC -1.6 NC -6.9 -181 *Transcription, translation, oncogenesis, angiogenesis, other* NM\_021953.1 forkhead box M1 (FOXM1) -1.2 -13.9 -42.2 -1.1 -1.2 -2.0 NC -2.8 -45.3 NM\_000465.1 BRCA1 associated RING domain 1 (BARD1) -1.3 -1.9 -2.0 NC -1.1 -2.1 -1.2 -2.6 -4.9 NM\_003368.1 ubiquitin specific protease 1 (USP1) NC -2.6 -1.6 NC -2.0 -1.4 NC -2.1 -1.9 NM\_006716.1 activator of S phase kinase (ASK) -1.2 -6.5 -5.3 -1.2 -1.3 -2.0 -1.1 -4.6 -11.3 NM\_003246.1 thrombospondin 1 (THBS1) NC -2.0 -3.5 -1.9 -1.4 -2.6 -1.1 -1.6 -4.6 NM\_001147.1 angiopoietin 2 (ANGPT2) 3.5 3.5 8.0 1.7 5.3 8.0 -0.5 4.9 2.8 AF187858.1 angiopoietin-2 isoform-1 NC 2.6 4.3 NC 1.5 3.5 NC 2.0 2.0 NM\_000435.1 Notch (Drosophila) homolog 3 (NOTCH3) 1.3 3.0 2.8 1.0 2.3 2.1 NC 2.1 3.0 NM\_001674.1 activating transcription factor 3 (ATF3) NC 1.5 2.5 NC 1.7 2.5 NC 2.6 4.3 NM\_003158.1 serinethreonine kinase 6 (STK6) -1.7 -11.3 -9.8 1.1 1.2 -1.7 -1.3 -8.6 -26 NM\_003600.1 serinethreonine kinase 15 (STK15) -1.2 -3.7 -8.6 NC NC -1.6 -1.3 -4 -6.1 AF162704.1 androgen receptor mRNA NC 1.3 -1.6 1.2 1.3 -2.1 -1.1 -1.6 -2 *Resistance to chemotherapeutic agents* NM\_000693.1 aldehyde dehydrogenase 1A3 (ALDH1A3) NC 4.0 3.2 1.0 1.1 2.1 1.2 4.9 4.3 NM\_005980.1 S100 calcium-binding protein P (S100P) 1.0 5.7 4.0 1.5 2.6 3.0 NC 2.6 2.6 The genes in this list showed a \>2 fold change in expression in at least one time point in both mono and combination treatment. NC: No change; Negative value: Decrease; Positive value: Increase. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Comparison of difference in gene expression between combination treatment and mono-treatment in PC3 cells ::: Gene Taxotere+Furtulon ----------------------------------------------------------------------------------- ----------------------- ------ ------ *Decrease in combination treatment, No change or increase in mono-treatment.* NM\_001261.1 cyclin-dependent kinase 9 (CDC2-related kinase) (CDK9) -1.3 -1.2 -2.3 NM\_016507.1 CDC2-related protein kinase 7 (CrkRS) NC -1.6 -2.3 AF080157.1 IkB kinase-a (IKK-alpha) NC -2.0 -1.6 U62296.1 transcription factor NF-YC -1.1 -1.7 -2.3 BC005003.1 nuclear transcription factor Y, gamma -1.1 -2.0 -2.1 BC001771.1 transcription factor IIF NC -1.6 -2.3 AI434345 activating transcription factor 1 NC -2.5 -2.6 BC001173.1 eukaryotic translation initiation factor 3 NC -1.3 -2.0 U78525.1 eukaryotic translation initiation factor (eIF3) NC -1.4 -2.0 NM\_001814.1 cathepsin C (CTSC) NC -1.3 -2.0 NM\_003377.1 vascular endothelial growth factor B (VEGFB) -2.1 NC NC AF035620.1 BRCA1-associated protein 2 (BRAP2) -2.3 NC NC AF035620.1 BRCA1-associated protein 2 (BRAP2) -2.3 -2.0 -1.4 NM\_005346.2 heat shock 70 kD protein 1B (HSPA1B) -1.1 -2.1 -1.1 BC000478.1 heat shock 70 kD protein 9B NC -1.7 -2.3 NM\_014278.1 heat shock protein (hsp110 family) (APG-1) -1.2 -1.3 -2.0 BC002526.1 Similar to heat shock protein, 110 kDa -1.1 -1.4 -2.1 *Increase in combination treatment, No change or decrease in mono-treatment.* NM\_000077.1 cyclin-dependent kinase inhibitor 2A (p16, inhibits CDK4) NC 1.7 2.1 NM\_001262.1 cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) NC 2.1 2.0 J03202.1 laminin B2 chain mRNA NC 1.3 2.0 BG164365 microtubule-associated protein 1B NC 2.3 2.0 NM\_000594.1 tumor necrosis factor, member 2 NC 2.5 1.6 NM\_001065.1 tumor necrosis factor, member 1A NC 2.0 1.9 BC000125.1 Similar to transforming growth factor, beta 1 -1.2 1.9 2.1 NM\_005649.1 transcription factor 17 (TCF17) NC 1.1 2.3 NM\_005923.2 mitogen-activated protein kinase kinase kinase 5 (MAP3K5) NC 1.6 2.1 NM\_005204.1 mitogen-activated protein kinase kinase kinase 8 (MAP3K8) NC 2.3 4.3 NM\_000785.1 cytochrome P450, subfamily XXVIIB NC 2.1 1.5 NM\_002960.1 S100 calcium-binding protein A3 (S100A3) NC 2.5 2.5 NM\_002962.1 S100 calcium-binding protein A5 NC 8.6 10.6 NC: No change; Negative value: Decrease; Positive value: Increase. The genes in this list showed a \>2 fold change in expression in at least one time point in combination treatment. ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Comparison of difference in gene expression between combination treatment and mono-treatment in LNCaP cells ::: Gene Taxotere+Furtulon ----------------------------------------------------------------------------------- ----------------------- ------ ------- *Decrease in combination treatment, No change or increase in mono-treatment.* NM\_001256.1 cell division cycle 27 (CDC27) NC -1.6 -2.0 NM\_001963.2 epidermal growth factor (beta-urogastrone) (EGF) NC -3.5 -1.9 NM\_004113.2 fibroblast growth factor 12B (FGF12B) NC -4.3 -1.2 U13022 negative regulator of programmed cell death ICH-1S (Ich-1) NC -2.3 -2.0 AF249273.1 Bcl-2-associated transcription factor short form mRNA NC -1.7 -2.3 NM\_001071.1 thymidylate synthetase (TYMS) NC -1.4 -2.6 AF005068.1 breast and ovarian cancer susceptibility protein (BRCA1) NC -4.6 -17.1 NM\_012068.2 activating transcription factor 5 (ATF5) NC -2.0 -2.8 NM\_021809.1 TGF(beta)-induced transcription factor 2 (TGIF2) NC -1.6 -2.1 NM\_001412.1 eukaryotic translation initiation factor 1A (EIF1A) NC -1.2 -2.3 NM\_002758.1 mitogen-activated protein kinase kinase 6 (MAP2K6) NC -1.7 -2.1 *Increase in combination treatment, No change or decrease in mono-treatment.* NM\_006034.1 p53-induced protein (PIG11) NC 6.1 7.5 NM\_000227.1 laminin, alpha 3 NC 1.7 2.0 NM\_000094.1 collagen, type VII, alpha 1 (COL7A1) NC 1.4 6.5 NM\_016437.1 tubulin, gamma 2 (TUBG2) NC 1.4 2.1 NM\_000853.1 glutathione S-transferase theta 1 (GSTT1) NC 2.0 2.1 NC: No change; Negative value: Decrease; Positive value: Increase. The genes in this list showed a \>2 fold change in expression in at least one time point in combination treatment. :::	PubMed Central	2024-06-05T03:55:52.673268	2005-1-18	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548501/", "journal": "BMC Cancer. 2005 Jan 18; 5:7", "authors": [ { "first": "Yiwei", "last": "Li" }, { "first": "Maha", "last": "Hussain" }, { "first": "Sarah H", "last": "Sarkar" }, { "first": "James", "last": "Eliason" }, { "first": "Ran", "last": "Li" }, { "first": "Fazlul H", "last": "Sarkar" } ] }
PMC548502	Background ========== Many chromosomal regions have been shown to be linked or associated to asthma and asthma-associated traits in humans \[[@B1]\]. More recently, asthma genes have been identified on chromosomes 2 \[[@B2]\], 13 \[[@B3]\], 14 \[[@B4]\], and 20 \[[@B5]\]. The investigation of the genetic aetiology aims at the improvement of preventive strategies, diagnostic tools and therapeutic alternatives \[[@B6]\]. These final steps have not yet been reached or are even in sight, while the reasons for this delay are unclear. The mainstay of all genetic studies has been genome-wide linkage scans in families with at least two asthma-affected siblings. Based on a previous analysis of a genome-wide scan of asthma \[[@B7],[@B8]\] with inconsistent chromosomal findings to earlier studies, we decided to expand the initial sample with additional families by the same core protocol for clinical examination and using the same set of microsatellite markers \[[@B7],[@B8]\]. The increased the number of identically pheno- and genotyped families could be used to define sub-phenotypes, which may be a promising strategy to explain the aetiological heterogeneity observed so far. Relevant clinical subsets may be defined by different age of onset, different disease course by degree of severity, extrinsic (allergic sensitization detectable) and intrinsic (no allergic sensitization detectable, symptoms often during infections of the upper respiratory tract) asthma type, and house dust mite allergy (HDM), as well as genetic background as judged by geographical origin of parents (table [1](#T1){ref-type="table"}). We hypothesized that the restriction to a smaller well-defined sample would reduce heterogeneity and improve the power of detecting linkage. Linkage regions should show higher lod scores than in the total sample and lead from phenotype subgroups to a genotypic dissection. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Phenotypes of the 201 families included. ::: Phenotype Definition No. of families ---------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------- --------------------- Early onset asthma occurrence of asthmatic symptoms for one child before the age of 2 years and for his or her sib pair at least before the age of 4 years 67 Extrinsic asthma all asthmatic children are positive for at least one SPT or specific IgE 134 HDM SPT positive at least 3 family members with a positive skin prick test (SPT) for HDM 33 HDM RAST positive at least 3 family members with a positive result of serum specific IgE for HDM 42 Severe asthma asthma severity index (see table 3) for one asthmatic child is 4 or 5 and for his or her sib pair 3, 4 or 5 56 Seasonal variation:\"Winter-Type\" one affected child suffers from asthma attacks in the winter half-year and his or her sib pair at least not solely in the summer half-year 39 Seasonal variation:\"Summer-Type\" one affected child suffers from asthma attacks in the winter half-year and his or her sib pair at least not solely in the summer half-year 35 German nationality both parents are of German descent 170 ::: Methods ======= Clinical evaluation ------------------- 97 families consisting of at least two children with confirmed clinical asthma were collected during the first stage of the German genome scan \[[@B7]\]. We now recruited another set of families during a period of 18 months. Trained staff from 3 university hospitals as well as 6 pediatric pulmonary practices carried out an identical phenotyping procedure as described previously \[[@B7]\]. This procedure contained detailed interviews of every family member, skin prick tests (SPT) of frequent allergens, blood samples (for IgE and allergen-specific IgE (RAST) measurements, eosinophil count), peak flow tests for a period of 10 days and dust collection at patients\' homes. SPT and RAST assays included several pollens, animal furs, mould, and house dust mite allergens (ALK-SCHERAX, Hamburg, Germany). The ethics commission of \"Nordrhein-Westfalen\" approved all study methods and informed consent was obtained from all parents and children. Children with premature birth, low birth weight and any severe pulmonary disease other than asthma were excluded as prematurity (and or associated low birth weight) may be a major non-genetic risk factor for the development of pulmonary symptoms. Another 4 families were excluded after genotyping because of Mendelian errors. The additional 104 families comprised 452 individuals (table [2](#T2){ref-type="table"}). Their 244 children had a mean age of 10.8 years. 75 families contributed 2 children, 22 families 3 children and 7 families 4 children). In total, the 201 families had 465 children; 255 were male, 210 female, and 413 had physician-diagnosed asthma. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Clinical characteristics of the 201 families (867 participants) that also include non-affected siblings of the core families. Descriptors of categorical variables include n/total sample in the first row and percent in the second row. Descriptors of continuous variables include mean values in the first row and standard deviation in the second row. ::: -------------------------------------------------------------------------------------------- Parents Children ---------------------------------- -------------- -------------- ------------- ------------- Stage 1 Stage 2 Stage 1 Stage 2 sex females 97/194\ 104/208\ 96/221\ 114/244\ 50.0% 50.0% 43.4% 46.7% Asthma diagnosis 33/194\ 60/208\ 200/221\ 213/244\ 17.0% 28.8% 90.5% 87.3% D.pter skin \> = 3 mm 68/193\ 58/206\ 111/217\ 107/242\ 35.2% 28.2% 51.2% 44.2% D.far skin \> = 3 mm 57/193\ 48/207\ 101/217\ 88/242\ 29.5% 23.2% 46.5% 36.4% D.pter. CAP class \>1 51/183\ 43/147\ 124/217\ 102/198\ 27.9% 29.3% 57.1% 51.5% D.far. CAP class \>1 46/183\ 39/147\ 122/217\ 100/198\ 25.1% 26.5% 56.2% 50.5% age (years) 39.8\ 40.9\ 11.0\ 10.8\ +/-5.7 +/-5.5 +/-4.2 +/-3.4 onset asthma (years) not reliable not reliable 3.8\ 4.3\ +/-2.8 +/-3.1 height (cm) 172.3\ 172.3\ 146.9\ 156.3\ +/-9.0 +/- 9.9 +/-19.2 +/-18.1 weight (kg) 76.0\ 78.0\ 41.5\ 47.4\ +/-14.9 +/-16.6 +/-17.0 +/-16.6 ln(Ige) (kU/L) 4.1\ 4.1\ 5.0\ 5.0\ +/-1.7 +/-1.6 +/-2.0 +/-1.8 ln(eosinophil) (count / mm3) 0.3\ 0.7\ 1.1\ 1.1\ +/-1.7 +/-1.5 +/-1.6 +/-1.6 FEV1 (ml) 3.594\ 3.464\ 2.312\ 2.861\ +/-804 +/- 957 +/-883 +/-1.061 -------------------------------------------------------------------------------------------- ::: The trait \"asthma\" was based on the clinical diagnosis as described earlier \[[@B7]\]. \"lnIgE\" was used for analysis as a quantitative variable and categorized by cut-off point of 100 kU/l. Furthermore, the following phenotype subsets (see also table [1](#T1){ref-type="table"}) were selected from the total study sample: ### Early onset asthma We created a subset of 67 families with one child having asthma symptoms before the age of 2 years and another child having asthma symptoms before the age of 4 years. In about half of the families within this subtype, both affected children had asthmatic symptoms before the age of 2 years. We were of course dependent on the recollection of the parents -- but keeping in mind that the state of health of the children is of high priority in those families who joined our study and the period of time is quite clear, the data seemed valuable to us. ### Extrinsic/intrinsic asthma sample The group of families with atopic (\"extrinsic\") asthma was large, consisting of 134 families all with at least one positive skin prick test reaction or one increased specific IgE. ### Positive Dermatophagoides farinae skin prick test and increased specific IgE in serum Almost all family members participated in a skin prick test (SPT). We selected an \"HDM SPT positive\" group of 33 families with at least 3 family members showing a skin prick test reaction ≥ 3 mm. House dust mite allergy is quite common in asthmatic patients, and a known trigger factor for asthma attacks. Some studies have successfully used the combination with asthma as lead trait \[[@B9],[@B10]\]. As the functional relevance might be slightly different in study participants with serum antibodies, another subgroup was based on the measurement of HDM-positive specific IgE at concentrations ≥ 0.35 kU/l, which resulted in a group of 42 families. ### Asthma severity Several interview questions related to asthma symptoms, diagnostic findings and therapy. We used information about asthma attack frequency, actual medication and emergency hospital visits for a severity index with 5 levels. This index is based on subjective patient information, current/previous therapy and influence on quality of life (table [3](#T3){ref-type="table"}). \"Severe Asthma\" was seen in 56 families where one sib had at least grade 4 and another grade 3, 4 or 5. \"Moderate Asthma\" with one sib of maximum grade 2 and another with maximum grade 3 was defined in the same way. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Asthma severity grade definition. ::: Severity grade Frequency and percentage in asthmatic children Clinical criteria -------------------- ---------------------------------------------------- ----------------------- ------ ------ 1 29 (7.2%) none none none 2 118 (29.3%) none none yes none yes none 3 164 (40.8%) none yes yes once/month yes none 4 72 (17.9%) once/month yes yes at least once/month yes none 5 19 (4.7%) at least once/month yes yes ::: ### Seasonal variation of asthma attacks All participants were asked to specify the months when asthma attacks occurred. 39 \"winter-type\" families had one affected child with symptoms only in the cold months and his or her sibs not a fully \"summer-type\". In contrast 35 \"summer-type\" families were found with one affected child with summer attacks and his or her sibs not purely \"winter-type\". Attacks year-round usually were seen with severe disease only. Sporadic attacks usually did not have any seasonal preferences while both the intrinsic, usually infection-related \"winter-type\", as well as the extrinsic, hay fever related \"summer-type\", appeared to be concordant within families. This observation may already support the hypothesis of different genes in different subtypes. ### German sample In 170 of 201 families, both parents were of German descent (\"German\"). In this subgroup, we tried to reduce the genetic heterogeneity by leaving out the remaining 31 families, of which 5 were from Sweden and 5 from Turkey. DNA analysis ------------ DNA was isolated from peripheral white blood cells using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer\'s recommendation. For genotyping we used almost the same microsatellite marker as in the first scan \[[@B7],[@B8]\]. The final analysis included 408 markers of which 364 were autosomal and 7 were based on the X-chromosome, all included in the previous scan as well as a set of 37 previous fine mapping markers. For the baseline marker set, the mean distance was 10 cM, with an average marker-information content of 0.87 and a mean heterozygosity of 0.79. Marker amplification was performed in microtiter plates, either in 96- (Peltier Thermal Cycler PT-225, MJ Research, Waltham, MA) or in 384-well format (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA). Fragment analysis of PCR pools was conducted on an ABI 3700 DNA sequencer and genotypes were scored using GENESCAN and GENOTYPER (ABI) software. In the second scan, 98% of all possible genotypes could be unequivocally determined. Statistical analysis -------------------- Genotyping data were transferred to a SQL 2000 database, pre-checked with previously developed routines and exported in ASCII format for multipoint linkage analysis with MERLIN 0.9.3 \[[@B11]\]. Allele frequencies were estimated from the (unrelated) parental alleles. As different primers were used in some assays between the first scan in 1997 and the second scan in 2002, we adjusted the original allele size before replacing the allele size with its respective order. This procedure led to comparable allele frequency distributions in both scans. For ordering of all markers we used the Marshfield comprehensive human genetic linkage map that was slightly modified according to the marker order given by Golden Path 13 and expressed intermarker distances to Haldane cM. Error detection as implemented in MERLIN was then applied to discard unlikely genotypes from the analysis. Finally, P-values for qualitative traits were derived from the Kong and Cox method based on the score statistic S\_all \[[@B12]\]. For the quantitative trait ln(IgE) a variance component was applied including only age and sex as covariates. Results ======= Figure [1](#F1){ref-type="fig"} shows the linkage results for asthma, total IgE and all other subgroups. Chromosomes are arranged by number from p-ter to q-ter with distance in centimorgans on a linear scale. Table [4](#T4){ref-type="table"} reports all loci with p-values below 0.01 in at least two adjacent markers for one phenotypic subgroup. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Multipoint linkage results for all traits. Chromosomes are arranged with increasing numbers with orientation from p-ter to q-ter. Distance is given in centimorgans (cM) on a linear scale. A = asthma all, B = asthma German families, C = extrinsic asthma, D = HDM RAST positive, E = HDM SPT positive, F = severe asthma, G = early onset asthma, H = winter-type, I = summer-type, J = LnIgE continuous, K = LnLgE categorical. ::: ![](1471-2466-5-1-1) ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Linkage results in 201 core families with at least 2 children having asthma. Weak linkage (p \< 0.01) and suggestive linkage (p = 0.0007) is indicated in bold. ::: GDB PIC Chr. cM (Marshfield) Asthma All Asthma German Extrinsic Asthma HDM RAST +ve HDM SPT +ve Severe Asthma Early Onset Winter-Type Summer-Type Ln(IgE) continuous Ln(IgE) categorical ---------- -------- ------ ----------------- ------------ --------------- ------------------ -------------- ------------- --------------- ------------- ------------- ------------- -------------------- --------------------- D1S478 0.8404 1 48.53 0.03 0.03 0.04 0.5 0.9 0.12 0.3 0.13 0.8 0.002 0.0011 D1S234 0.8559 1 55.1 0.07 0.08 0.04 0.3 0.9 0.12 0.2 0.2 0.8 0.0002 0.00009 D1S255 0.7710 1 65.47 0.005 0.04 0.0015 0.04 0.4 0.03 0.15 0.7 0.2 0.002 0.005 D1S197 0.8285 1 76.27 0.007 0.06 0.002 0.12 0.4 0.09 0.07 0.5 0.11 0.08 0.2 D1S484 0.8050 1 169.68 0.08 0.12 0.012 0.004 0.003 0.3 0.3 0.08 0.3 0.007 0.013 D1S431 0.9008 1 182.35 0.12 0.13 0.02 0.03 0.04 0.5 0.3 0.3 0.2 0.005 0.005 D1S2815 0.9201 1 188.85 0.06 0.09 0.012 0.06 0.07 0.3 0.3 0.2 0.03 0.0008 0.0007 D1S238 0.8722 1 202.73 0.4 0.7 0.4 0.2 0.1 0.3 0.8 0.2 0.6 0.004 0.003 D1S2655 0.8976 1 216.82 0.7 0.9 0.6 0.2 0.2 0.2 0.9 0.7 0.7 0.05 0.007 D2S2374 0.9039 2 54.96 0.03 0.07 0.04 0.011 0.008 0.04 0.02 0.8 0.4 0.3 0.5 D2S2328 0.9545 2 61 0.02 0.04 0.014 0.007 0.007 0.008 0.04 0.8 0.06 0.14 0.4 D2S2294 0.9617 2 64.84 0.02 0.03 0.02 0.003 0.02 0.003 0.5 0.8 0.02 0.13 0.4 D2S2298 0.9585 2 65.94 0.02 0.02 0.02 0.003 0.02 0.002 0.06 0.8 0.014 0.11 0.3 D2S2113 0.9331 2 88.15 0.3 0.5 0.3 0.2 0.4 0.008 0.13 0.7 0.3 0.5 0.5 D3S1597 0.8199 3 29.92 0.7 0.7 0.4 0.6 0.9 0.4 0.4 0.6 0.8 0.009 0.006 D3S1286 0.9021 3 41.56 0.4 0.3 0.3 0.2 0.3 0.13 0.2 1 0.4 0.02 0.007 D3S1300 0.8748 3 80.32 0.12 0.3 0.2 0.09 0.0014 0.3 0.5 0.3 0.8 0.4 0.09 D3S1285 0.7673 3 91.18 0.07 0.08 0.2 0.07 0.0008 0.2 0.2 0.13 0.6 0.5 0.5 D4S1607 0.8843 4 183.63 0.3 0.3 0.3 0.07 0.05 0.2 0.005 0.07 0.6 0.4 0.3 D4S1535 0.8938 4 195.06 0.05 0.07 0.013 0.009 0.03 0.03 0.006 0.007 0.11 0.07 0.09 D4S2924 0.8934 4 199.93 0.06 0.09 0.007 0.002 0.03 0.02 0.009 0.02 0.2 0.08 0.15 D5S426 0.8990 5 51.99 0.006 0.006 0.03 0.008 0.14 0.04 0.002 0.007 0.2 0.09 0.4 D5S418 0.9101 5 58.55 0.015 0.03 0.06 0.004 0.2 0.012 0.007 0.012 0.4 0.02 0.07 D7S484 0.9204 7 53.5 0.5 0.7 0.2 0.4 0.5 0.5 0.4 0.5 0.9 0.02 0.009 D7S528 0.9481 7 57.79 0.2 0.4 0.014 0.14 0.3 0.3 0.5 0.5 0.6 0.0012 0.0007 D7S510 0.9783 7 59.93 0.14 0.4 0.02 0.3 0.3 0.2 0.6 0.6 0.4 0.0009 0.0013 D7S485 0.9688 7 60.68 0.1 0.3 0.01 0.3 0.3 0.13 0.6 0.7 0.4 0.0007 0.0004 D7S2548 0.9596 7 62.28 0.2 0.4 0.05 0.4 0.5 0.12 0.7 0.7 0.3 0.003 0.002 D7S691 0.9403 7 63.67 0.08 0.2 0.02 0.4 0.5 0.08 0.6 0.6 0.3 0.003 0.007 D7S2506 0.9313 7 69.56 0.2 0.4 0.13 0.9 0.8 0.14 0.7 0.7 0.3 0.007 0.011 D7S663 0.9049 7 78.65 0.06 0.05 0.08 0.3 0.4 0.2 0.5 0.3 0.14 0.0007 0.001 D7S669 0.8351 7 90.42 0.08 0.05 0.04 0.4 0.2 0.13 0.5 0.5 0.02 0.0014 0.0012 D9S257 0.9630 9 91.87 0.09 0.06 0.3 0.3 0.4 0.4 0.4 0.4 0.004 0.5 0.5 D9S283 0.9681 9 94.85 0.015 0.01 0.1 0.2 0.3 0.2 0.11 0.1 0.002 0.2 0.3 D9S1796 0.9745 9 97.53 0.02 0.014 0.07 0.3 0.2 0.05 0.07 0.14 0.006 0.07 0.13 D9S1781 0.9565 9 99.4 0.015 0.007 0.05 0.14 0.2 0.11 0.03 0.03 0.003 0.07 0.15 D9S1851 0.9708 9 103.42 0.02 0.008 0.04 0.06 0.2 0.2 0.05 0.02 0.003 0.3 0.5 D9S1786 0.9825 9 104.48 0.013 0.006 0.03 0.04 0.11 0.13 0.05 0.02 0.002 0.4 0.5 D9S176 0.9798 9 105.02 0.02 0.009 0.05 0.09 0.1 0.2 0.14 0.03 0.005 0.5 0.5 D9S1690 0.9709 9 106.63 0.06 0.04 0.2 0.2 0.14 0.4 0.4 0.13 0.007 0.5 0.5 D9S1784 0.9667 9 111.99 0.11 0.04 0.2 0.07 0.2 0.3 0.6 0.4 0.008 0.5 0.5 D10S547 0.8313 10 29.15 0.14 0.2 0.4 0.8 0.8 0.6 0.3 0.2 0.9 0.007 0.005 D10S191 0.8726 10 37.9 0.4 0.5 0.2 0.3 0.5 0.8 0.5 0.3 0.8 0.008 0.012 D11S922 0.9045 11 2.11 0.2 0.4 0.4 0.3 0.02 0.13 0.3 0.9 0.06 0.0005 0.0006 D11S902 0.8133 11 21.47 0.3 0.3 0.5 0.9 0.3 0.3 0.5 0.6 0.5 0.006 0.003 D11S935 0.7997 11 45.94 0.5 0.4 0.4 0.4 0.4 0.4 0.6 0.9 0.05 0.003 0.04 D11S968 0.8094 11 147.77 0.003 0.007 0.05 0.11 0.3 0.04 0.12 0.6 0.2 0.0005 0.0002 D12S355 0.8343 12 74.58 0.07 0.08 0.06 0.003 0.04 0.4 0.2 0.012 0.6 0.3 0.4 D12S1684 0.9339 12 86.4 0.02 0.015 0.05 0.002 0.05 0.9 0.15 0.2 0.4 0.08 0.4 D12S1667 0.9727 12 92.89 0.05 0.08 0.2 0.003 0.04 0.8 0.15 0.08 0.4 0.03 0.2 D12S81 0.9707 12 94.44 0.08 0.12 0.2 0.005 0.02 0.8 0.3 0.09 0.3 0.013 0.2 D12S351 0.9822 12 95.56 0.02 0.013 0.05 0.002 0.0003 0.5 0.3 0.05 0.3 0.0009 0.02 D12S95 0.9785 12 96.09 0.012 0.007 0.04 0.002 0.0002 0.5 0.2 0.06 0.4 0.0012 0.02 D12S327 0.9757 12 97.78 0.02 0.008 0.07 0.008 0.0009 0.4 0.05 0.13 0.2 0.04 0.2 D12S1716 0.9590 12 101.45 0.04 0.005 0.2 0.02 0.006 0.4 0.06 0.2 0.2 0.011 0.06 D12S1706 0.9696 12 104.12 0.12 0.015 0.3 0.07 0.06 0.6 0.3 0.4 0.2 0.007 0.2 ::: On [chromosome 1]{.underline}, two linkage areas for total IgE could be found at about 55 cM (p = 0.00009) and at 188 cM (p = 0.0007), which reached the threshold for suggestive linkage (p = 0.0007) \[[@B13]\]. On [chromosome 2]{.underline} at about 64 cM (p = 0.002) evidence was found for a locus for severe asthma and for house dust mite sensitive asthma. Early onset, together with HDM, is represented both on [chromosome 4]{.underline} at about 195 cM (p = 0.002) and on [chromosome 5]{.underline} at about 55 cM (p = 0.002). Three loci for total IgE regulation were found on [chromosome 7]{.underline} (65 cM, p = 0.0004) and chromosome 11 (2 cM, p = 0.0005; 147 cM, p = 0.0002), all with suggestive linkage according Kruglyak-Lander. On [chromosome 9]{.underline} at about 104 cM (p = 0.002) there is evidence for a locus linked to allergic rhinitis plus asthma (\"summer-type\"), having an effect mainly in families of German descent. Lastly, a locus showing suggestive linkage could be identified on [chromosome 12]{.underline} (95 cM, p= 0.0002) for house dust mite sensitised asthmatic patients. Discussion ========== Several linkage regions in asthma families could be found in this extended sample, although again no linkage with P \< 0.003 could be found with any marker for asthma. Asthma is apparently of such a heterogeneity that even investigating affected sib pairs from more than 200 families failed to yield significant results. Nevertheless, the quantitative trait IgE in these families, both as a discrete and continuous variable, led to three suggestive linkage findings on chromosomes 1, 7, and 11, which have all been discussed in prior publications (table [5](#T5){ref-type="table"}). ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Comparison of asthma loci ::: Chr. Position This study (2004) Bradley (2002) \[14\] Cookson (2001)\[15\] Daniels (1996) \[16\] Dizier (2000) \[17\] Laitinen (2001) \[18\] Malerba (1999) \[19\] Ober (1998. 1999, 2000) \[20-22\] Xu J (2000, 2001) \[23, 24\] Xu X (2001) \[25\] Yokouchi (2000) \[9\] CSGA (1997) \[26\] --------------- -------------------- ----------------------- ---------------------- ------------------------- ---------------------- ------------------------ ----------------------- ----------------------------------- ------------------------------ -------------------- ----------------------- -------------------- 155cM IgE Asthma Asthma, Atopy Asthma Hispanic population 1188cM IgE AD Slope IgE 264cM severe asthma HDM 350cM IgE HDM AD Loose Asthma 4195cM early onset HDM Slope HDM and Asthma 555cM early onset HDM Slope BHR Asthma 765cM IgE IgE, Slope, Eosinophils SPT Asthma, IgE 9104cM German summer type Asthma symptoms, Atopy 112cM IgE IgE SPT Asthma Hispanic population Asthma 11147cM IgE IgE 1295cM HDM Eosino-phils, IgE Asthma Asthma IgE, Asthma HDM and Asthma Asthma ::: Phenotypic dissection --------------------- Moreover, the strategy of phenotypic dissection proved to be quite successful, though the sample size of subsets seems to be very small. The size of the sample in a linkage study is known to be one of the most important factors for a significant finding \[[@B1]\], making the artificial limitation of subsets a double-edged decision. Clearly, there is a remarkable phenotypic heterogeneity of asthmatic diseases but -- as these families represent different genetic influences-limitation of the sample size did not affect statistical properties as significance values were smaller compared to the whole study sample. In particular, the subgroups segregated by high disease severity, sensitisation by HDM, early onset and population origin attained better lower significance levels than the main trait (table [4](#T4){ref-type="table"}). So far, results were not adjusted with the Bonferroni correction for multiple testing as this would have been too conservative for this explorative investigation. Subgroups are often closely related and highly overlapping. Nevertheless, there might be a problem with multiple testing and the criteria for suggestive linkage in a genome scan on a single phenotype \[[@B13]\]. Sample recruitment with restricted phenotypic requirements proved to be successful for some other traits as well. Early onset definitions have been used for cancer studies and metabolic disorders to increase the underlying genetic component \[[@B27],[@B28]\]. Separation in sub-samples of ethnically diverse populations has also been conducted \[[@B26]\], but higher disease activity scores \[[@B29]\] seemed to give an advantage in other fields also. Other Asthma Genome Scans ------------------------- Recently, evidence was shown for asthma genes on chromosomes 2 \[[@B2]\], 13 \[[@B3]\], 14 \[[@B4]\], and 20 \[[@B5]\]. Our study does not support linkage loci in those regions. As the region on chromosome 13 was responsible for only 10% of the observed IgE serum level variability, this small effect may be easily overlooked in another study. A lack of replication does not necessarily mean an error in the primary study. Most likely, genetic heterogeneity is the cause, where the proportion of patients with a particular gene variant is different between studies. A polygenic model of the inheritance mode of asthma with minor effects by single genes could explain the highly diverse association and linkage results thus far. Frequent replication, however, may point toward lead genes. Most regions listed in table [4](#T4){ref-type="table"} have already been described and some of the traits are supported by the phenotypes proposed in our analysis. Table [5](#T5){ref-type="table"} summarizes previously published asthma/atopy loci in approx. 20 cM distance that would be consistent with our findings. Both loci on chromosomes 4 and 5 are supported by the phenotypes of early onset, HDM and \"winter-type\", which might relate to the clinical picture of chronic obstructive bronchitis. Phenotypes described by other studies include lung function variation and HDM, which is supported by our data. The \"atopy\" locus (defined by SPT and RAST) on chromosome 3 confirms linkage studies of atopic dermatitis (AD) \[[@B14]\] and might be the genetic link between both diseases. The locus on chromosome 12 with suggestive linkage for HDM in our study is known from many other reports. Also our first scan showed weak evidence for linkage in this region at D12S351 (p = 0.01) \[[@B7]\], whereas the best result for the HDM subset lies now about 0.5 cM apart at D12S95 with p = 0.0002. These consistent findings are probably based on frequent alleles playing an important role in extrinsic asthma. Linkage results for SPT and RAST for HDM were quite similar, but the HDM SPT positive group -- which is a smaller group-, reached the suggestive linkage level on chromosome 12. SPT, even more than RAST, might represent the individual\'s allergic disposition. On chromosome 2, we found a locus in families with severe asthma that has not yet been described in other studies. A p-value of 0.007 has already been found in our first genome-wide scan \[[@B7]\] for D2S2298 and our main trait asthma. The same marker for severe asthma in both samples improved to a p-value of 0.002. Severe asthma is rather rare, and a disease-aggravating allele on chromosome 2 could be easily overlooked in a heterogeneous asthma sample. Severe asthma may be a lethal disease, while a severe-asthma gene would be of high importance for intensified treatment. The German \"summer-type\" locus on chromosome 9 is also not frequent in other studies. This might be due to the fact that many, but not all, genes are necessary for a common trait. Our first asthma scan has already shown linkage in this region with a p-value of 0.007 for D9S1784 \[[@B7]\], which now improves to p = 0.002 for the \"summer-type\" subset at D9S1786, about 7.5 cM apart. Conclusions =========== We conclude that this phenotypic dissection is a useful tool to detect linkage in a heterogeneous disease like asthma because some of the sub phenotypes reached even better significance values than the main trait. We show that the precision of the phenotype can be more effective than expanding the sample size only. Unfortunately, large sample sizes are needed to assure at least moderate sample size in subsets. Grouping by early onset or disease severity could be applied to almost every complex disease, but for a more specific dissection, clinical expert knowledge is required. A prior analysis of clinical data can help to identify symptom clustering, which -if consistent within families- can reduce genetic heterogeneity. Competing interests =================== The author(s) delcare that they have no competing interests. Authors\' contributions ======================= MW, JA, and CS were involved in the study design, JA organised the sample collection, conducted the genetic analysis, and prepared the manuscript, MW organized funding and supervised the study. YAL and PN organised and helped with genotyping and revised the article critically for important intellectual content. SL, FR and MW carried out the statistical analysis and made substantial contributions to its conception and design, CS, DB, FF, HJ, JK, AK, AS, MS, WW, PW assisted in the recruitment of families and examined patients and therefore made substantial contributions to the acquisition of data, asthma severity grades are part of the thesis of CS. GS was responsible for the IgE analysis. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2466/5/1/prepub> Acknowledgements ================ We thank all participating families for their help as well as the members of the clinical centres for their work. We wish to thank Guido Fischer for data handling, Liane Thaller for secretarial assistance, Margret Bahnweg, Bettina Wunderlich, Regina Pospiech and Inka Szangolies for excellent laboratory work; Michelle Emfinger and Leah Bajc for proof-reading the manuscript. The project was funded by the Deutsche Forschungsgemeinschaft DFG WI621/5-1, DFG FR1526/1, DFG KN378/1-1, GSF FE 73922, GSF FE 73915, BMBF 07ALE087 and the National Genome Network 01GS0122.	PubMed Central	2024-06-05T03:55:52.681341	2005-1-5	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548502/", "journal": "BMC Pulm Med. 2005 Jan 5; 5:1", "authors": [ { "first": "Janine", "last": "Altmüller" }, { "first": "Corinna", "last": "Seidel" }, { "first": "Young-Ae", "last": "Lee" }, { "first": "Sabine", "last": "Loesgen" }, { "first": "Dieter", "last": "Bulle" }, { "first": "Frank", "last": "Friedrichs" }, { "first": "Heidemarie", "last": "Jellouschek" }, { "first": "Julika", "last": "Kelber" }, { "first": "Angela", "last": "Keller" }, { "first": "Antje", "last": "Schuster" }, { "first": "Michael", "last": "Silbermann" }, { "first": "Wolfgang", "last": "Wahlen" }, { "first": "Peter", "last": "Wolff" }, { "first": "Gerhard", "last": "Schlenvoigt" }, { "first": "Franz", "last": "Rüschendorf" }, { "first": "Peter", "last": "Nürnberg" }, { "first": "Matthias", "last": "Wjst" } ] }
PMC548503	Introduction ============ Health reforms that aim at increasing efficiency, quality and users\' satisfaction need to take into consideration human resource issues, because the health sector is labor-intensive and the performance of health systems depends on qualified and motivated workers \[[@B1]-[@B4]\]. At the same time, the support of the workforce is crucial to ensure successful implementation of reforms. In Latin America, the need to improve the performance of the workforce had been pointed out in many health sector assessments conducted in the 1970s and 1980s by the United States Agency for International Development (USAID), the World Bank (WB), other agencies and independent researchers. (See for Argentina \[[@B5]-[@B7]\], for Bolivia \[[@B8]-[@B10]\], for Brazil \[[@B11]\], for Chile \[[@B7],[@B12]\], for Colombia \[[@B12]-[@B14]\], for Costa Rica \[[@B15]\], for the Dominican Republic \[[@B16]-[@B18]\], for Ecuador \[[@B19],[@B20]\], for El Salvador \[[@B21]\], for Guatemala \[[@B22],[@B23]\], for Mexico \[[@B12],[@B24]-[@B26]\], for Nicaragua \[[@B27]\], for Panama \[[@B28],[@B29]\], and for Uruguay \[[@B7]\].) From these reports and studies, and notwithstanding the differences among the countries in the region, we can summarize the problems present during the 1970s and 1980s as follows: • The skill mix of health personnel was often inadequate to meet the needs of the communities, and highly qualified staff often performed tasks that could be conducted by less-trained providers. The health systems of the region were characterized by an excess number of medical specialists and insufficient numbers of other professionals such as primary care providers, nurses, pharmacists, public health specialists, epidemiologists, health economists, accountants, social workers, administrators, communication experts, planners, health educators, nutritionists, physical therapists and sanitary engineers; • There was an over-concentration of qualified health personnel in hospitals and urban centers, coupled with shortages in poor neighborhoods and rural areas; • A large majority of physicians held at least two jobs, one in the government and one in the private sector. In countries with fragmented health systems, physicians could even have three jobs: they worked part time for the social security institute, they worked for the ministry of health, and also held a private practice. Dual or triple employment generated conflicts of interests; physicians used the public sector to draw patients for their private practice, and their productivity in the public post was low and absenteeism high; • Human resources management systems were weak, largely due to dispersal of accountability: in many countries the terms and conditions of employment were under the control of the public service commission or the ministry of finance, and the education of human resources was under the control of the ministry of education or the private sector. Ministries of health did not have any input in determining the types and number of persons to be trained, and their involvement in hiring and managing the health workforce was limited. Health managers handled relations with the labor unions, had some limited supervisory roles, ensured organizational adherence to recruitment policies, and were responsible for some training. • Salary increases were generally based on years of service. In the majority of countries, central labor unions negotiated working conditions directly with governments and signed collective agreements that left administrators with little room to compensate workers according to performance; • Personnel decisions (hiring and promotion) were too often guided by favoritism, political dictates, and nepotism; • Health professionals were insufficiently committed to the public system due to the conflict of interests mentioned above, poor personnel management systems and the perception that wages were low; • The medical profession strongly dominated the definition of health sector policies and the regulation of the conditions of practice of all health professions; • Communication between providers and patients was poor, and providers and service users had very different social and cultural backgrounds. In countries with Amerindian-speaking populations, providers did not speak their languages; • The regulation of training institutions and conditions of practice was weak; • The training of health promoters and other auxiliary personnel such as dental assistants, midwives, laboratory technicians, equipment maintenance and repair technicians, and pharmacy clerks was poor or non-existent, thus their performance was poor. According to the literature reviewed, these conditions led to low productivity and efficiency; inadequate equipment; shortages of supplies and drugs; unmotivated and inadequately trained staff; questionable quality of care; and low users\' satisfaction. By the mid-1970s, the need to reform the human component of the health services was very urgent, and the urgency increased with the severe economic downturn that countries of the region suffered during in the early 1980s. The size of the Latin America health labor force (about nine million \[[@B30]\]) implied that reformers attempting to resolve the human resources problems mentioned above needed to dedicate a large amount of time and resources to it. This paper reviews the impact of the health reforms carried out under the leadership of the World Bank. Data come from a review of the literature including the leading Latin American and non-Latin American journals, monographs, documents found in ministries and reform offices, technical reports, papers presented at conferences and fieldwork carried out by the authors between 1980 and the present in Bolivia, Colombia, Costa Rica, Dominican Republic, Ecuador, El Salvador, Honduras, Mexico and Peru. The World Bank\'s neoliberal health sector reforms ================================================== In the early 1980s, with a few exceptions, countries in the region entered a severe economic downturn. The International Monetary Fund and the World Bank required -- as a condition for new lending and for refinancing the debts -- reduction of public spending. The impact of structural adjustment programs on health services was severe and compromised the ability of governments to maintain the physical infrastructure of the facilities, provide the necessary supplies and equipment, and maintain competitive salaries for the workforce. The World Bank took advantage of this situation to provide loans to the ministries of health and social security funds. Together with the loans, the Bank offered guidelines for the reorganization of the health services according to the ideological economic principles held by the institution. The World Bank had started its activities in the health sector in the early 1970s, and by the early 1990s it was the world\'s largest health sector lender. In 1999, the total accumulated value of health, nutrition, and population loans worldwide amounted to USD 16.8 billion (in 1996 dollars) \[[@B31]\]. By the late 1980s, the Bank placed health financing at the center of its health policy dialogue with borrowers. In 1987 the Bank proposed four changes \[[@B32]\]: to impose user fees at government facilities; introduce insurance or other risk coverage systems; use nongovernmental resources more effectively; and decentralize planning, budgeting and purchasing for government health services \[[@B32]\] A few years later, it looked at the roles of the government and the market in the health sector, and described the main components that have guided most World Bank-led health reform efforts \[[@B33]\]. These principles were reiterated in 1997 \[[@B34]\]. The underlying principles of the Bank-led reforms include the belief that the private sector is more efficient than the public sector, and that decentralized administrative units are better-equipped to respond to the needs of the population than centralized governments. The Bank also proposes to limit government financing to a basic package of services, to be determined for each country by cost-effectiveness studies and the country\'s ability to pay. Services included in the basic package are made available at no cost to the indigent population; governments and the rest of the population subsidize them. In the Bank\'s health reform model, the role of the state is limited to that of a regulator of the health care market. The United States health system is closer to the World Bank model than the national health services or social security funds of other industrial nations. The World Bank-led reforms did not include strategies to correct the human resources problems presented earlier, even if some of them had been identified by Bank-sponsored studies; the World Bank reformers believed that market forces would resolve them. As we will see, the contrary occurred and privatization and decentralization had some negative consequences for the workforce. The reliance on the market also hid the structural problems that needed to be taken into account at the time reformers designed human resource policies. The design and implementation process which were characterized by secrecy, generated dissatisfaction among health workers. We have organized our analysis of human resources during the reforms under five categories: resistance of the workforce to the implementation of a market-oriented health care model; faulty implementation processes; inadequately trained personnel in managerial positions and in service delivery; institutional and legal dimensions including insufficient and faulty information, lack of financial resources, and civil service statutes; and weak regulatory legislation to ensure the quality of professionals and the performance of the sector. Resistance to the implementation of a market-oriented health care model ======================================================================= The neoliberal health reforms intended to change the values that had inspired the Latin American systems and the relations between the government and the health workforce. Thus, health would no longer be considered a right; only the insured would be entitled to receive a broad array of services. Health workers would lose the protection they had enjoyed as public servants and become part of a flexible workforce (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Resistance of the workforce to a market-oriented health care model ::: ---------------------------------------------------------------------------------------------------------------------------------- a\. Health is not a right, and the reformed system is no longer based on the principles of solidarity and access to health care. b\. Health workers become part of a flexible workforce and are encouraged to compete, instead of collaborate, among themselves. c\. Worker unions lose power to influence the system and negotiate work conditions of behalf of affiliates. d\. The reform is an abdication of the responsibilities of the government to protect the population. e\. Physicians fear losing their professional autonomy. ---------------------------------------------------------------------------------------------------------------------------------- ::: Humans tend to resist change, but opposition to health reform was compounded by conflicts with the value system that inspired it. Most Latin American constitutions recognize health as a right and most governments had adopted Alma Ata\'s primary health care principles as their strategy to promote Health for All. Health policies were based on the principle of solidarity and fostered cooperation among health workers and also between them and other workers in related sectors such as education and agriculture \[[@B35]\]. It was assumed that health workers would do the best for their clients out of a sense of personal duty \[[@B4]\]. Human resource changes were needed to implement Alma Ata, but the budget reductions required by the World Bank caused a significant deterioration of working conditions. As their purchasing power worsened, health workers intensified undesirable behaviors to increase their income. These included levying illegal fees, diverting patients from the public sector to their private clinics \[[@B15]\], using public supplies and equipment for personal profit \[[@B36]\], and reducing their productivity in the public sector \[[@B15]\]. As indicated, Latin American physicians have supplemented their income from the public system by means of their private practice. Physicians value the stability and fringe benefits of a public post, but as the public delivery system deteriorated in many countries, private clients became their main source of revenue. By the late 1980s in most Latin American countries (with the exception of Argentina, Costa Rica, Cuba, Dominican Republic, Guatemala and Panama), more than half of the health expenditures, including the cost of medicines, occurred in the private sector \[[@B37]\], and in most countries of the region, personnel expenditures accounted for about 60% to 70% of the public health budget \[[@B38]\]. One of the objectives of the neoliberal reforms was to create a more flexible labor force by decreasing the number of tenured public employees and increasing the number of temporary personnel. This change threatened: the job security of the civil service, a very important dimension in countries with little political stability and where workers are frequently exposed to arbitrary political removals; the providers\' income, which now would depend on their ability to compete for contracts and clients; and that which workers expected from their employer (recognition, opportunities for self-actualization and promotion). The promoters of health reforms failed to acknowledge that in a politically unstable region, civil service tenure was necessary to maintain an efficient, productive, and loyal labor force. Predictably, these threats triggered the opposition of professional associations and unions, led to strikes, and lowered productivity during the reform process. Health workers of most countries of the region are unionized \[[@B39]\]. The unions protect the workers from the politicization of the appointments and promotions, and their leaders regularly engage in collective bargaining with the managers of the public sector. The stability provided by the civil service status facilitates the formation of strong union leadership: union leaders remain in office longer than policymakers. Labor unions anticipated that the decentralization and privatization of the sector would have a negative impact on their membership and their bargaining power. Labor unions in Bolivia, Ecuador, El Salvador, Mexico, Nicaragua, Peru and Venezuela expressed their opposition to the reforms on the grounds that they were an excuse for governments to relinquish their constitutional responsibility to ensure access to care and would lead to dramatic changes in work conditions \[[@B39]\]. In some countries, such as El Salvador and Mexico, unionized workers successfully stalled or delayed the implementation of the reform. Analysts of the health reform policies agree with the workers\' concerns. Segall \[[@B4]\] asserts that a market system does not nurture the service ethic that should characterize the workforce and leads providers to adopt self-seeking behaviors instead of working in the interests of the patient or the community. Others worry that the uncertainties generated by the reform, the stress and demands added to the workforce and the misalignment between the values of the workers and those of the reformed system are very detrimental to the workers\' motivation \[[@B1],[@B40]\]. According to Rigoli and Dussault \[[@B41]\] the unions\' fears were justified; union membership appears to have decreased in recent years. In addition, health professionals strongly opposed the idea of having their professional autonomy limited by professional administrators who would force them to adhere to diagnostic and treatment protocols based on economic principles rather than on technical criteria. The cool reception that Mexican professional associations offered to foreign insurance companies and health maintenance organizations is a good example of this concern \[[@B42]\]. Institutional and legal constraints =================================== The large majority of countries in Latin America do not have accurate information on the numbers and distribution of the health workforce. There is no centralized entity responsible for gathering information on the health personnel practicing in the country or in the different geographical regions, and even when only public sector workers are examined, the numbers collected at different administrative levels differ (people may move to a different location, and regional or local governments hire additional staff without reporting to the central authorities). The reforms worsened the possibility of having accurate information. If the system is decentralized and the majority of providers are in the private sector, the sources of information from which human resources data will need to be collated multiplies. The challenge may be even greater if each agency gathers the information differently, and without this basic information it is very difficult to engage in human resources development planning (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Institutional and legal dimensions ::: ------------------------------------------------------------------------------------------------------------------------- a\. Lack of accurate information on the availability of human resources and their distribution. b\. Civil service status limits the capacity of managers to change the working conditions of personnel. c\. Decentralizing human resources is expensive: homologation of salaries and benefits; hiring of additional personnel. d\. Decentralized governments have limited ability to manage personnel to respond to the needs of the population. ------------------------------------------------------------------------------------------------------------------------- ::: Civil service laws and negotiated agreements with worker unions have limited the freedom of public sector managers to reorganize the workforce; managers had to \"administer the rules\" \[[@B43]\] issued elsewhere. For instance, managers could not change the status of public sector employees to flexible contract workers, dismiss employees, increase workloads or change the working schedule. They may also have had difficulties rewarding their employees because the salaries and the conditions of employment were set outside the health sector by the public employment commission, the ministry of labor, or the ministry of finance or their corresponding decentralized entities. Reformers did not foresee these limitations. Managers overcame these constraints by hiring additional personnel under flexible contracts. In Brazil there are now about 15 different types of contracts for public sector employees; a significant expansion of the use of temporary workers and contracts without fringe benefits has been reported in Argentina \[[@B44]\], Colombia \[[@B45]\], Ecuador \[[@B2]\], El Salvador \[[@B30]\], Panama and Peru \[[@B2]\]. Funds for the temporary positions have different origins, including extrabudgetary government allocations, revenues generated through user fees, or savings from positions left empty due to retirements or administrative leaves. Transferring human resources to other politico-administrative levels was more difficult than anticipated. Kolehmainen-Aitken \[[@B46]\] and Homedes and Ugalde \[[@B47]\] have suggested that this is the most complex part of the decentralization process. The fear of transfers caused discontent and anxiety. On the other hand, managers did not want to absorb everybody who was transferred \[[@B46]\]. The decentralized personnel felt insecure about the new reporting mechanisms and the new managers\' expectations, and vulnerable to political crossfire. Decentralizing human resources is expensive. Prior to decentralization -- in countries such as Bolivia, Colombia and Mexico -- local or regional governments frequently hired additional personnel under different pay scales and benefit packages than those used by the central government. After decentralization, each decentralized administrative unit had to set pay scales, fringe benefits, performance assessments and reward systems for its workers. Because it was not possible to lower the salaries and reduce the benefits of workers with higher salaries and benefits -- generally federal/national workers -- it was necessary to bring the salaries and benefits of all the workers to the level of those who had the most generous package. Higher salaries and benefits represent an increase in the fixed costs of the system for an indefinite period. Moreover, the creation and/or strengthening of the decentralized administrative structures are often not accompanied by a decrease in the number of federal employees, further raising the costs of personnel to the system \[[@B48]\]. In Mexico, from 1985 to 1987 the cost of transferring federal employees to 14 states was 140,000 million pesos (approximately 452 million US dollars) \[[@B49]\], and in Colombia the World Bank indicated that the cost of transferring state and departmental health personnel to Cali\'s Municipal Health Secretariat was prohibitive \[[@B50]\]. Decentralization has been promoted under the assumption that the proximity of decision makers to the communities facilitates providing services more in accordance with the needs of the community. But the decentralized structures\' ability to respond to local needs has been constrained by: the civil servants they inherited, who are often inadequate in terms of skill mix and geographical distribution; the conditions of employment imposed by other government sectors and the unions; and the local elite, who do not have in mind the health needs of the communities and who lobby to have relatives and friends hired by local health authorities. In several countries of the region, decentralization has broadened the urban-rural inequities in the distribution of personnel and in the quality of services \[[@B51]-[@B54]\]. Rich decentralized units are able to offer better working conditions and attract qualified personnel from poorer municipalities, a sort of brain drain. Similarly, the development of the private sector also tends to drain qualified resources from the public sector \[[@B43]\]. Inadequately trained personnel ============================== Countries considered the human resource implications of reform only when they faced resistance from the unions or realized they did not have the financial and human resources required to implement the reform \[[@B55]\]. The health reform plan in Costa Rica recognized the ambiguity of the policies and procedures related to human resources but did not modify them. As planned, incentives were established to increase workers\' productivity and the use of short-term contracts. These two strategies ended up triggering greater grievances on the part of public sector employees \[[@B43]\]. The human resources units of the health ministries and the regional and local administrations were inadequately prepared for the reform (Table [3](#T3){ref-type="table"}). Traditionally they had had a narrow scope of responsibilities, often limited to managing the relationship with the unionized workforce, ensuring compliance with national/provincial policies on recruitment and deployment of personnel, and organizing continuing education activities \[[@B56]\]. The personnel attached to these departments generally had limited or no human resources development training \[[@B30],[@B57]\]. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Inadequately trained personnel ::: ----------------------------------------------------------------------------------------------------- a\. Human resources units are not adequately staffed, especially to manage change. b\. Lack of management experts, especially experts in insurance systems and contract managers. c\. Insufficient numbers of people trained in primary health care and public health related fields. d\. Training centers unable to produce personnel to operate the reformed health system. ----------------------------------------------------------------------------------------------------- ::: The tasks required by the reform overstretched the capacity of these units. Included among these tasks were: the development of new organizational structures, defining new job descriptions, reassigning responsibilities and designing new reporting systems, establishing performance evaluation methods and assisting all decentralized units to carry out similar duties in their jurisdictions. The decentralization of personnel often unveils rivalries and discontent among personnel who feel unfairly treated. All these issues need to be addressed, negotiated and resolved; and most human resources units were ill-prepared to lead the process \[[@B56]\]. For example, when the health system in Bolivia was decentralized, the salaries for several workers in Santa Cruz were delayed for months. Costa Rica introduced performance-based contracts between the Social Security Fund (Caja Costarricense de Seguro Social) and the health units, but according to a senior executive of the Ministry of Health, the targets were set at minimum levels and some units decreased their level of production \[personal communication with the authors\]. The mismatch between the abilities of the workforce and the needs of the system documented in the 1980s still prevails. The lack of coordination between training institutions and employers is at the root of the problem, along with weaknesses in the regulation of health professions and the dominance of physician\'s groups on the policy making process. According to Bach \[[@B56]\], shortages of personnel trained in disciplines such as primary health care, health economics, public health, health communication, health education, nutrition, and environmental engineering continue to severely limit the possibilities for improving the quality and efficiency of the health care system. Only in very few instances has reform included the human resources development activities to address these issues. For example, Costa Rica trained multifunctional teams for their lowest-level facilities and promoted the training of general doctors instead of specialists \[[@B58]\]. The National Autonomous University of Mexico modified its curriculum to promote family medicine; it introduced public health concepts and interdisciplinary experiences around issues related to promoting the health and wellbeing of the elderly and protecting workers from occupational hazards \[[@B59]\]. In most countries, managerial positions were traditionally given to physicians with little or no management training \[[@B55]\]. The neoliberal reforms require managers and staff with experience in specific dominions such as insurance, capacity to write contracts and enforce them, ability to monitor performance, knowledge of performance-based reimbursement systems, and expertise in health services research to evaluate progress. Decentralization to the state and municipal levels generates the need for even more managers, and countries that have given autonomy to hospital executives to manage their human and financial resources face additional challenges. With the exception of Chile, countries in the region had limited expertise in private health insurance \[[@B60]\]; Costa Rica, the Dominican Republic, Mexico and Venezuela engaged expensive foreign technical assistance to develop performance-based contracts and management information systems. The promoters of the reforms recognized the need for good managers but, because they had heavily criticized the public sector and questioned its role, it was difficult to recruit qualified staff into managerial positions and, in turn, it was difficult for those recruited to motivate and retain qualified staff \[[@B56],[@B57]\]. Most health reform projects included management training. The Pan American Health Organization \[[@B61]\] evaluated 15 such training programs and concluded that the training did not change the performance of the systems and that for only two projects was management capacity improved. The reasons for the failure included: difficulties in recruiting trainers; inability of the local universities to respond to the needs of the projects; inappropriate selection of training participants; conflict between project units and the ministries of health; political interference; and the absence of a human resource development plan. Theoretically the deficiencies in formal training can be corrected with supervision and continuing education activities, but this did not occur in Latin America. The authors of the report emphasize the need for countries to develop comprehensive human resources development plans to ensure the efficacy and sustainability of the training programs. After having spent millions of dollars in training and infrastructure, the region\'s capacity to manage contracts is still limited \[[@B62]\], and when contracts are in place, they are expensive to administer and the legal system is insufficiently developed to enforce them. In Colombia, most public hospitals were unable to compete with the private sector and are now bankrupt \[[@B63]\]. Poor management in decentralized entities has been considered one of the main reasons for the decentralization failure \[[@B64],[@B65]\]. Faulty reform implementation process ==================================== Countries in the region used a top-down approach to define and implement reform. The implementation was often led by a handful of top health executives, newly created reform offices, the political elite and international agencies, which in turn contracted for the technical assistance of international consultants and universities closely aligned with the neoliberal ideology of the World Bank. In general, there was little interest in involving professional associations, unions or even local universities in this process \[[@B2],[@B56]\] (Table [4](#T4){ref-type="table"}). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Faulty reform implementation process ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------ a\. Lack of involvement of professional associations and labor unions in the definition of the reform. b\. Secrecy surrounding definition of the reform raises suspicions among those responsible for implementing it and predisposes them to resist the changes. c\. Lack of transition plan. ------------------------------------------------------------------------------------------------------------------------------------------------------------ ::: In Costa Rica the labor unions were involved initially in the reform, but their influence was undermined by the World Bank and the Inter-American Development Bank, which adopted a more closed, centralized decision-making style \[[@B58]\]. According to a top executive of the Ministry of Health in El Salvador, the group preparing the reform operated in secret, and in his view, the secretive process was desirable because if health workers had participated they would have created obstacles to its implementation \[[@B66]\]. Indeed, it appears likely that the labor unions and professional associations in this country and many others would not have approved neoliberal reforms. Some authors have offered a different interpretation of the exclusion of the workforce and assert that it was the complexity of the human resources issues and the need to involve many players (the ministries of education, labor, finance and health, and professional associations and unions) in their solutions that led reform promoters to ignore the labor force and other stakeholders \[[@B30]\]. Regardless of the underlying motivation, the secrecy that surrounded the process of defining and implementing the reforms produced rumors, confusion and hostility against the reform among civil servants and professional groups \[[@B30],[@B39],[@B67],[@B68]\]. The objectives and processes by which reforms were introduced were never made clear; and often the reforms were perceived as responding more to ideological concerns of international organizations, in particular the International Monetary Fund and the World Bank, than to the needs, resources and sociopolitical realities of the countries \[[@B57]\]. The president of the Medical Association of El Salvador said that his association had tried to obtain information about the reform for over a year and that all his knowledge was based on \"rumors and guesswork that led nowhere\" \[[@B66]\]. In addition, health reformers did not consider the strategies and resources needed during the transition and early stages of implementation; such as allocating new financial resources and establishing clear communication channels. In addition, as discussed in the previous section, the reforms failed to adequately train managers who could lead the transition to a new management system \[[@B40]\]. Inadequate regulatory system to ensure high-quality training and health providers ================================================================================= The need for regulation increases in health systems where the private sector plays a prominent role. In Latin America it was not until the early 1990s, largely as a consequence of the health reforms, that health policy makers became aware of the urgency to regulate the health system. A regulatory system includes adequate regulations, the institutions to enforce them, and a judicial system that ensures a timely response in the event of conflict. Enforcing regulations is important to guarantee that trained personnel provide safe and adequate services (Table [5](#T5){ref-type="table"}). ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Inadequate regulatory framework to ensure the quality of professionals and the performance of the sector ::: --------------------------------------------------------------------------------------------------------------------------------------------------------------- a\. Limited quality controls in training institutions. b\. Physician-dominated field that precludes other professional groups from being recognized as health care providers within the official health care system. c\. Limited accreditation of health care professionals. --------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: Prior to the reforms, the regulatory systems were poorly developed and, when they existed, they were not tailored to the needs of consumers and enforcement was very limited \[[@B69]\]. For instance, the licensing of providers was a purely bureaucratic formality with no assessment of qualifications. The patronage and bossism observed in many countries were further expressions of regulatory deficiencies \[[@B70]\]. The number of medical schools has grown spectacularly in the last two decades and, in most countries, there were no mechanisms to ensure the quality of the training institutions or to test the abilities of the graduates. The number of medical schools in Chile grew by 68% between 1992 and 2000, in Peru the growth was also by 68%, in Argentina by 61% and in Brazil by 21%. The growth occurred mainly in the private sector \[[@B71]\]. Costa Rica had no private universities until the 1970s; now there are 70, several of which train health professionals \[[@B72]\]. By its very nature the regulation of the health profession relies heavily on the opinions of professionals and especially on physicians, who in turn place a great value on their autonomy and have had little interest in responding to social and political demands \[[@B55]\]. Medical associations have traditionally opposed health reforms and have had a very strong influence on health policy-making \[[@B29],[@B66],[@B73],[@B74]\]. The dominance of physicians has alienated other professions such as therapists, nurses, pharmacists, optometrists and psychologists \[[@B75]\]. For example, in Chile the government proposed to train and use more optometric technicians, but medically trained ophthalmologists opposed the proposal. After a long negotiation process involving ophthalmologists, optometric technicians, insurance companies, universities, parliamentary representatives and consumers, an agreement was reached. The four-year trial period allows optometric technicians to expand their scope of practice while medical schools take in more ophthalmologists for training \[[@B58]\]. The regulation of the health professions has a long way to go in Latin America, and it is probably impossible to establish sustainable regulatory mechanisms in the absence of political and judicial reforms; for the system to work, it needs to free itself from political interference \[[@B70]\]. Some argue that the separation between professional associations and licensing bodies \[[@B76]\] must be increased, and there is general agreement that the perspective of the general public \[[@B75]\] must be included. Consequences of the health reform on human resources ==================================================== Bach \[[@B56]\], Brito et al. \[[@B77]\], Dussault and Dubois \[[@B78]\], Rigoli and Dussault \[[@B41]\] have identified human resources issues as the main obstacle for the success of the reforms. The neoliberal health reforms did not solve the workforce problems that had previously been identified, and created additional ones that have had a negative impact on the health systems (see Table [6](#T6){ref-type="table"}). ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Consequences of the health reform on human resources ::: ----------------------------------------------------------------------------------------------------------------- a\. Working conditions have worsened, and talented workers migrate to the private sector or to other countries. b\. The motivation of workers has deteriorated. c\. Productivity and quality may have deteriorated. d\. The uneven ratio of specialists to primary physicians has not changed. e\. The uneven distribution of personnel (hospital and urban bias) persists. f\. Corruption has not decreased. ----------------------------------------------------------------------------------------------------------------- ::: The implementation of the reforms has been uneven in the Latin American region. Technical, logistical, political and financial problems have surfaced everywhere. While most countries decentralized, a few -- such as Colombia and Chile -- managed to significantly expand private insurance and, with the exception of Brazil, very few have engaged in large contracts with private sector providers. The most salient feature has been significant changes in hiring modalities. In Brazil there are 15 different types of contract arrangements \[[@B30]\] and in Peru the need to expand service coverage led to hiring 10 000 health professionals (physicians, nurses and technicians) between 1992 and 1996 under temporary contracts without social security; by the late 1990s about 12% of the health workers did not have social security \[[@B77]\]. Health workers in Ecuador have suffered wage reductions, in Mexico with decentralization the states have increasingly hired temporary workers \[[@B47]\], and in Argentina there has been a rise in precarious contracts, even fraudulent ones, such as full-time jobs under the label \"autonomous professional\" \[[@B30]\]. Another important result of the reform is the surge of multiple jobs, particularly in Argentina, Brazil, El Salvador, Panama, Peru, Uruguay, and to a lesser degree Chile \[[@B30]\]; that has caused stress and dissatisfaction among physicians. A survey of nurses conducted in Argentina, Brazil, Colombia and Mexico \[[@B44]\] revealed that the reforms increased stressful conditions at work, job dissatisfaction, insecurity from flexible contracts, malpractice concerns, inter-institutional migration, and new bureaucratic tasks for which nurses were not trained. The nurses specifically mentioned that they needed to do more work in less time with fewer staff; and complained about excessive paperwork, including billing, and about having less time for direct patient care than before the reform. One of the nurses who participated in the study said \"Patients may feel that we really don\'t care that much about them, because we just don\'t have enough time to spend with them and really know what is going on \[[@B44]\].\" In a different survey, nurses who had gone through reform restructuring held more negative perceptions of patient care than those who had not; and they also expressed a higher desire to unionize \[[@B41]\]. Furthermore, according to the Tavistock Group \[[@B79]\] \"cooperation throughout a health care system can produce better outcomes and much greater value for individuals and for society. Such cooperation requires agreement across disciplinary, professional and organizational lines about the fundamental ethical principles that should guide all decisions in a truly integrated system of health care delivery.\" If this statement is correct, by fragmenting the system through privatization and decentralization, and by introducing competition among health professionals, the neoliberal health reforms have compromised the quality of care. Similarly, one of the basic principles of neoliberal reforms -- that the efficiency of the system will increase by using flexible contracts and rewarding productivity -- is not supported by the data. The World Bank conducted an evaluation of civil service reforms in 15 countries and concluded: \"None of the cases reviewed so far have revealed any empirical evidence that the Civil Service Reform and related Technical Assistance Loans have succeeded in fostering the needed change in work attitudes, ethics and organizational culture that could lead to greater efficiency/productivity in the civil service\" \[[@B1]\]. Research has also uncovered problems with using performance-based payment schemes. For example, in health it is often difficult to establish who is responsible for the outcome. Costa Rica implemented a pilot project in Barva de Heredia in which physicians received an incentive based on productivity, but this was not extended to the nurses and the other clinic staff \[[@B80]\]. The project increased the costs to the system without increasing the productivity of physicians or the quality of care, and as a result the government halted its plans to extend the model to other health facilities. Health providers can also manipulate the information to maximize their benefits rather than the well-being of the patients; and substandard working conditions rather than workers\' action may be responsible for a poor outcome \[[@B81]\]. Mexican providers opposed a malpractice evaluation system because they did not want to be held liable for errors due to equipment deficiencies and lack of supplies \[[@B42]\]. Health workers in Costa Rica feared that the focus on productivity compromises the commitment to patients \[[@B58]\] and discourages the provision of services that require extra time, such as health education \[[@B2],[@B55]\] and mental health counseling. Establishing a valid and reliable merit-pay system is extremely complicated; placing too much emphasis on material rewards may displace more intrinsic motivators such as the pleasure of doing good, or caring for the patient. Bennet and Franco \[[@B1]\] even suggest that loyalty to the organization may decrease as the worker becomes aware of more lucrative opportunities with other employers. This could have serious consequences. Attracted by NGOs and the higher salaries of private hospitals, the most talented public servants could leave the public sector. Others raised questions about the sustainability of these strategies. In Brazil, productivity-based payment systems resulted in increased productivity, but the increase was not sustained over time and created competition among workers who were expected to collaborate \[[@B82]\]. For an interesting discussion on incentive systems and motivation in a different context, see Le Grand \[[@B83]\]. There is a belief among neoliberal economists that private sector workers are more productive and less corrupt than public employees. Recent hospital studies confirm that because of fear of termination, absenteeism is less frequent among non-tenured physicians hired through short-term contracts than among civil servants, but short-term contracts have not increased commitment to the institution \[[@B84]\]. Corruption continues to be pervasive in both private and public hospitals, and productivity differences between the private and public hospitals have not been documented \[[@B85]\]. Costa Rica has attempted to reduce the waiting lists by contracting for the provision of services with private groups, under the condition that the recipient of the contract is someone not working for the clinic that makes the referral, a condition that is often violated \[[@B72]\]. Decentralization can also be seen as a transfer of financial responsibilities from central government to local authorities, which has the potential to affect wages and job stability \[[@B39]\] and increase inequity. Poor local authorities cannot compete with the conditions of employment offered by wealthier municipalities and have difficulties in attracting personnel. In a decentralized system it is more difficult to structure career ladders, especially for workers who choose to locate in rural areas \[[@B81]\]; and decentralization can also exacerbate forms of patronage and political domination. The experience in many countries has proven that it is more difficult to resist the politicization of decision-making when health managers interact with local political leaders without central controls \[[@B46],[@B57],[@B70]\]. In decentralized health systems, particular attention needs to be paid to establishing good coordination among those responsible for the vertical program at the central level, the decentralized administrative units, and the clinical staff. The absence of good coordination may result in health workers\' reporting to two supervisors: the person responsible for the vertical program and the supervisor of the health facility or region, as is the case in Mexico \[[@B47]\]. In sum, in the majority of Latin American countries, the neoliberal reforms have not made the health delivery model more responsive to the needs of the community; have not increased the productivity of health workers; have had a negative impact on working conditions and staff motivation; appear to have further compromised the quality of care; and have had a limited impact on the capacity to regulate the health professions and training institutions. Discussion ========== More recent studies suggest that many of the old workforce problems remain unresolved \[[@B56],[@B70],[@B86],[@B87]\]. Even the World Bank, which promoted the reforms, has finally recognized \[[@B88]\] that the neoliberal strategies are not having the desired impact. The Bank questions the performance of the private sector and highlights the need to find the institutional arrangements and policies that best respond to local conditions and resources. Health reform provided a perfect opportunity to promote and encourage workforce improvement. Prerequisites for the progress of such processes are political will, effective relationships between the educational and service-providing institutions, and the open collaboration of professional groups. However, the reforms had the opposite effect. The neoliberal orientation challenged the use of conventional regulation strategies because, by encouraging professionals to seek their own interest instead of the interests of society as a whole, it questioned whether society and regulatory bodies could continue to trust and have faith in the criteria expressed by health professionals. Human resources account for the lion\'s share of health budgets, and poor performance had been identified as the main constraint to efficiency, quality of services and user\'s satisfaction. The values that guided the neoliberal reform and the privatization and decentralization initiatives worsened the problems affecting the Latin American workforce and added new challenges. The reform implementation process was also responsible for the failures. The promoters of the neoliberal health reforms underestimated the importance of involving professional organizations and unions in the planning and implementation of the reform efforts, raising suspicion and resistance among organized health workers. In addition, the reforms were designed in secret and implemented using a top-down approach. The only significant advance in human resource development in the last 10 years is the increased interest in strengthening the capacity to regulate training institutions and practitioners. Solving the problems that affect human resources is no easy task, and probably the solutions are different for each country; ignoring the problems and hoping that the market will resolve them is a recipe for failure. Managing change is very complex; embarking on reform without having secured the collaboration of the workforce and ensuring the availability of sufficient qualified staff is irresponsible. The exclusion of the organized labor force from reform discussions is indefensible; the collaboration and motivation of the health workers is essential to the reform, and the leaders of the organized groups can assist in informing and aligning the workforce \[[@B40],[@B89]\]. Policy changes requiring a different skill mix of health personnel require careful planning because there is a significant time lag between deciding that there is a need to train additional professionals and having them available. For example, a 10% rise in the number of students in a medical school produces only a 2% increase in the supply of doctors 10 years later \[[@B78]\]. Most countries did include a training component, but as mentioned earlier it was insufficient, and part of the problem was that the reform implementation was rushed, without the benefit of field-testing the underlying theories, gathering evidence on the appropriateness of the strategies, or learning from the reform experience. One thing that reforms could have done was to support interventions that, independently from the reform, some countries had designed to overcome workforce weaknesses. Some interventions were national programs, others were pilot projects, and there were also experimental projects. The following are examples of these autochthonous interventions. To solve the urban-rural gap and improve equity, most ministries of health in the region created the obligatory rural health service known as pasantía or servicio social that requires physicians to spend one year in a rural health center prior to graduation or immediately after receiving their degree \[[@B35]\]. A few social security institutes have also found ways to reduce health inequities; such is the case of the Mexican and Ecuadorian Institutes of Social Security (IMSS and IESS). IMSS organized COPLAMAR, an extensive primary and secondary care program for poor rural populations, which brought general practitioners and specialists to rural areas. The Ecuadorian program known as Seguro Social Campesino offered primary care services for rural dwellers and, when needed, hospitalization at the Ecuadorian Social Security Institute; this program aimed at reducing the rural-urban gap in a country of which 70% of the population then lived in the rural areas \[[@B90]\]. Mexico created a training program for traditional midwives who worked in dispersed rural populations; the objective of the program was to enhance the quality of their services and reduce maternal and infant mortality \[[@B91]\]. The Ministry of Health of the Dominican Republic, in an effort to increase health equity, trained and deployed more than 5 000 health promoters in rural centers. The promoters periodically visited every rural household to monitor infant growth, promote nutrition and sanitation, and assist in immunization campaigns \[[@B35]\]. Costa Rica attracted the world\'s attention with the Open Walls Hospital, a program that required the specialists of a regional hospital to schedule -- when needed -- weekly visits to dispersed rural populations. The program also intended to convey the message to specialists that they were not different from other health workers and had an obligation to serve poor rural dwellers even when doing so would involve personal inconvenience \[[@B80]\]. To enhance the professional status of primary care practitioners, reduce referrals, and improve quality of care, IMSS created many positions in the specialty of family physician, forcing the medical profession to recognize the status of the new specialty. Colombia\'s Ministry of Health sent nurses to a graduate health education program taking advantage of a US fellowship program in order to diversify the human resource composition of the Ministry. Some of these projects were successful; this was the case of family physicians and COPLAMAR in Mexico. However, the first 14 Mexican states that decentralized in the 1980s dismantled the program, transferring it to the states\' departments of health to create the state health system, and the quality of rural health deteriorated rapidly \[[@B92]\]. Others function poorly, as is the case of the compulsory year of social service in all the countries where it was established. Similarly, the health promoters program in the Dominican Republic suffered from insufficient training, lack of continuous education, absence of efficient supervision, and poor remuneration, comments that can be extended to all health promoter programs of the region. The Seguro Social Campesino has suffered from inadequate financing, and plans to extend it to the entire rural population have been placed on permanent hold. Other programs were discontinued because of indifference and lack of support from policy makers. Thus, the Hospital without Walls ceased after all Ministry of Health hospitals were transferred to the Social Security Institute. Due to budgetary problems, the Colombian Ministry of Health did not employ the health educators on their return from graduate school. The health reforms would have provided a perfect opportunity to support and strengthen many of these and other autochthonous interventions. A proper course of action would have been to evaluate the projects that had been developed locally, identify their strengths and weaknesses, and establish their viability and sustainability. Through trial and error and with appropriate resources and incentives, many of them are likely to be more effective and less costly than foreign programs invented by those who hardly know the realities of developing countries and are inspired by ideological principles and questionable economic theories. There is much more that needs to be done to improve the training and management of human resources for health, and very often the solutions depend on the collaboration of a wide range of stakeholders such as those who produce health workers, those who employ them, those who pay for their services, those who negotiate working conditions and those who define the standards of professional practice. It is no easy task and can be successfully accomplished only if there is strong political will, if there is openness and trust among all stakeholders, and if sufficient resources and time are allocated to this effort. Most countries of the region have the capacity to find appropriate solutions to the problems they are facing. Dussault \[[@B55]\] argued that change is possible only on the basis of shared values, and as we have seen, the values that inspired the neoliberal reform did not coincide with those expressed in the Latin American Constitutions and in the primary health care principles that had guided the development of the health sector during the 1970s and 1980s. As Segall mentioned \[[@B4]\], it is important to recover the spirit of cooperation among health providers, and there is a need to take explicit steps to raise their motivation and patient-centered behavior. Without a motivated workforce, all other efforts to change the system may be even counterproductive. Policy makers and administrators will have to explicitly identify strategies that foster collaboration, inner motivation and work ethics, and this may require abandoning the market orientation of the neoliberal reforms and embracing the values that inspired the primary health care movement. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= Both authors have contributed equally to the design, data collection, data analysis, drafting and completion of this article. Acknowledgements ================ We are grateful to Peter Lloyd-Sherlock and René Leyva for their constructive comments on earlier versions of this manuscript.	PubMed Central	2024-06-05T03:55:52.690971	2005-1-19	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548503/", "journal": "Hum Resour Health. 2005 Jan 19; 3:1", "authors": [ { "first": "Núria", "last": "Homedes" }, { "first": "Antonio", "last": "Ugalde" } ] }
PMC548504	Background ========== In their 1986 paper Israel, Rosenberg, and Curtin \[[@B1]\] gave a sort of rallying call for researchers to consider the analytical potential for multiple cause of death data collected by the United States National Center for Health Statistics (NCHS). Beginning with the implementation of the Eighth revision of the ICD in 1968, the NCHS developed and employed several computer systems to automatically select the underlying cause for each death certificate and to produce multiple cause of death data \[[@B2]\]. The resulting multiple cause of death datasets by year are made publically available through the NCHS website. Acknowledgment of the potential for multiple cause of death data analysis is increasing in other countries as well \[[@B3],[@B4]\]. For example, the Australian Bureau of statistics point out that using multiple cause of death data allows researchers to: more comprehensively understanding and track death due to chronic disease which do not often appear as the underlying cause of death (e.g. Alzheimer\'s, diabetes, pneumonia), to provide better documentation on multi-morbid associations and the strength of associations between conditions which led to death (for example by examining the frequency of associations between diseases such as diabetes and ischaemic heart disease), and to assist in identifying problems with the process of recording and coding cause of death information \[[@B4]\]. Multiple cause of death data has been used to look at trends in certain diseases, e.g. HIV \[[@B5],[@B6]\] and lung disease \[[@B7]\], but despite its availability, surprisingly few studies have looked at it broadly. Indeed there is no annual standard summary tabulation report of the multiple cause of death data put out by NCHS. This may be due in part to the overwhelming amount of information that arises when combinations of causes of death are considered. There are an enormous number of complex combinations which could be summarized and perhaps it is not clear what tables may be of general interest. The purpose of this article is to examine some straightforward relationships of multiple causes of death as a function of factors available on the death certificate (demographics of decedent, place of death, type of certifier, disposal method, whether an autopsy was performed, and year of death). Using all death certificates issued by the state of Minnesota between 1990 and 1998 (326,332 deaths), the current study documents the relationship between these factors and the associated frequency of reporting of multiple causes of death as well as the associated frequency that a cause is considered underlying (after data processing) given that it is mentioned on the death certificate. The implication being that differences found are either due to actual differences in causes of death in these groups or due to systematic biases in the reporting of causes of death, or a combination of both. The study will not be able to discern which is the cause but hopes to contribute at the very least by providing an example of the potential relationships which can be examined with the rich multiple cause of death data. Methods ======= Data source ----------- The data used are from the Minnesota Department of Health Mortality Database and include entries from 326,332 individual death certificates, which represent all deaths occurring in Minnesota during the period of 1990--1998. Record axis codes (those codes which have been completely data processed) are used for all analyses in this paper rather than entity axis codes. A brief description of the entity and record axis coding is given here. The translation of causes of death listed on the death certificate (see Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"} for the actual certificate) to the codes used for statistical analysis goes through many steps. As seen in Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}, the medical information which focuses on the sequence of medical conditions that resulted in death is provided in a two-part format. Part I is for the conditions which directly lead to death, and Part II is for other conditions which contribute to death but are not directly related to the immediate cause of death \[[@B1]\]. The underlying cause of death is defined as the \"(a) the disease or injury which initiated the train of events leading directly to death, or (b) the circumstances of the accident or violence which produced the fatal injury\" \[[@B8]\]. The entity axis codes represent what is actually written on the death certificate by the certifier expressed in terms of ICD codes including an indicator of which line the code came from and which position on the line it came from (if more than one code was listed per line). While the conditions listed in Part I should form a causal sequence initiated by the underlying cause listed on the lowest line, errors in properly completing the form occur regularly and a reselection of the underlying cause of death is done nationally 30--40% of the time. The decision to reselect an underlying cause other than that listed on the lowest used line in Part I is governed by a set of rules developed by WHO as part of the periodic revision of the International Classification of Disease \[[@B9]\] and is incorporated, along with a complex set of decision tables, into the Automated Classification of Medical Entities (ACME) software. The record axis codes represent a further processing of the entity axis codes to be consistent with the underlying cause data and more amenable to statistical tabulation and analysis. The record axis codes distinguish the ICD code selected as the underlying cause of death and lists all additional causes of death mentioned but does not distinguish them in terms of their ordering or original location on the death certificate. For more detail on entity and record access codes, see \[[@B10]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### US standard certificate of death.Line 27 Part I and Part II are where the causes of death are listed. ::: ![](1471-2288-5-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### This figure displays the backside of the certificate of death. Details are given for filling out specific lines. ::: ![](1471-2288-5-4-2) ::: Using the record axis codes, we have for each death record: one underlying cause of death and up to 14 non-underlying causes of death (with no distinction of importance given amongst the non-underlying). When we refer to a cause of death and do not want to distinguish if it is underlying or non-underlying we will refer to it as a \"mentioned\" cause of death. In addition to listing one underlying and up to 14 non-underlying causes of death, each death certificate also contains information about the demographics of the deceased, including age, gender, race, marital status, and educational attainment. Also, other conditions related to the death are recorded -- place and time of death, who completed the death certificate, if autopsy has been performed and type of body disposal. Minnesota Laws and guidelines govern the process for who and how a death is certified under different circumstances in Minnesota. For example when an unattended death occurs (e.g. at a persons residence) a medical examiner\'s investigator must arrive at the scene. The medical examiner will contact the last attending physician asking about past medical history of the decedent and most likely cause of death. When an attending physician has seen the decedent within 90 days and the death is natural, jurisdiction is usually given to the physician to certify the death. Sudden or unexpected deaths due in part to any factor other than natural disease must be referred to the medical examiner\'s office. Autopsies are performed at the discretion of the medical examiner but can also be performed for any death at the request of the immediate family. The underlying and non-underlying causes of death derived from the death certificate, in this study, are coded according to the 9^th^revision of International Classification of Disease (ICD). The specific ICD9 codes are grouped into standard reporting of cause of death categories resulting in a total of 107 different causes of death. In this study, individuals are dichotomized as having multiple causes of death (i.e. at least one non-underlying cause) or not having multiple causes of death (i.e. only having an underlying). In addition, because heart disease is the leading cause of death and diabetes is a good example of a disease which often shows up as a non-underlying cause of death, this research investigates two sub -populations: 1) Individuals that have ischemic heart disease (ICD-9: 410 -- 414) mentioned as a cause of death (n = 79,833), and 2) Individuals that have diabetes mellitus (ICD-9: 250) mentioned as a cause of death (n = 27,181). For both sub-populations, a dichotomous variable is created to indicate whether the mentioned disease is coded as the underlying cause of death or not. Data analysis ------------- Descriptive statistics including total numbers and proportion of all deaths (n = 326,332) in each of the covariate categories are reported as well as proportions of people within each covariate category who have multiple causes of death. In order to examine the association between each covariate and the dichotomous outcome of multiple causes of death, logistic regression is used to mutually adjust each factor for the others. 95% confidence intervals of odds ratios are reported. Trends in multiple cause of death reporting across time are investigated graphically. Similarly, descriptive statistics including total numbers and proportions will be presented for the two sub-populations with ischemic heart disease (n = 79,833) or diabetes mellitus (n = 27,181) mentioned either as underlying or non-underlying cause of death. Logistic regression is used and 95% confidence intervals are reported to examine factors that are associated with each of these diseases being reported as underlying cause of death rather than non-underlying. Results ======= Overall, 68.9% of the 326,332 deaths from 1990--1998 had at least one non-underlying cause of death in addition to the underlying cause (i.e. have multiple causes). There was a noticeable decreasing trend of reporting multiple causes of death over the 9 year period from 1990 to 1998 with 74.0% in 1990 consistently dropping down to 64.8% in 1996 and remaining around 66% until 1998. Table [1](#T1){ref-type="table"} presents the marginal percentage of individuals in each demographic and death related category as well as the proportions and adjusted odds ratios of having multiple causes of death by each of the covariates. The youngest (\<25) and oldest (85+) age groups had the lowest and highest percent of multiple causes of death (61.7% and 71.8%, respectively). Interestingly, the age group from 45--64 did not have higher odds of having multiple causes than the young (\<25) group. The percentage of men with multiple causes of death reported was slightly higher (1%) than that of women. Individuals over 25 years old with less education had a higher percentage of multiple causes of death (71.3%) compared to those with higher education (66.6%). The most pronounced difference with respect to demographics was found in race categories, with Native American having the highest percentage of multiple cause of death (74.5%), compared to 68.9% of white. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Percent of all deaths (n = 326,332) by each covariate. Probability of reporting multiple causes of death given covariate, marginal percent by category, and adjusted odds ratios of reporting multiple causes of death given covariates. ::: % of death by categories % with multiple COD by categories Odds Ratio (95% CI)^1^ --------------------- ------------------------ ------------------------------ --------------------------------------- ---------------------------- DEMOGRAPHIC Age 0--24 3.0 61.7 1 25--44 4.6 69.0 1.38(1.31--1.45) 45--64 13.3 62.1 1.01(0.97,1.06) 65--84 47.8 69.4 1.41(1.35,1.47) 85+ 31.3 71.8 1.58(1.51,1.65) Sex Female 50.3 68.6 1 Male 49.7 69.3 1.11(1.09,1.13) Race White 96.4 68.9 1 Black 1.7 67.5 1.15(1.078,1.22) Native American 0.9 74.5 1.54(1.41,1.69) Asian 0.5 65.0 1.04(0.94,1.16) Hispanic 0.5 66.6 1.13(1.01,1.26) Education^2^ High school 32.4 68.1 1 Below High School 44.3 71.3 1.03(1.01,1.05) Above High School 23.3 66.6 0.94(0.92,0.96) Marital Status Married 41.0 67.4 1 Single 12.7 68.0 1.08(1.05,1.11) Widowed 38.5 71.1 1.09(1.07,1.11) Divorced 7.8 68.2 1.12(1.08,1.15) DEATH RELATED Autopsy No 77.8 69.0 1 Yes 9.4 74.4 1.57(1.52,1.62) Unspecified 12.8 64.7 0.85(0.83,0.87) Certifier Physician 82.0 69.2 1 Coroner 12.6 70.2 1.23(1.19,1.26) Osteopath 1.4 75.3 1.27(1.183,1.37) Other/Unknown 4.1 57.8 0.61(0.59,0.63) Disposal method Burial 76.3 70.1 1 Donation 0.3 69.7 1.02(0.89,1.17) Removal 1.5 47.1 0.35(0.33,0.37) Cremation 20.5 66.8 0.94(0.93,0.96) Unknown 1.4 Death place Hospital Inpatient 35.2 73.4 1 Residential 19.1 58.7 0.48(0.47,0.49) Nursing home 35.5 70.6 0.77(0.76,0.79) ER 5.5 65.5 0.64(0.61,0.66) Unknown 4.7 ^1^Odds ratios are odds of having multiple causes of death given particular category as compared to odds in reference category. Odds ratios are obtained from logistic regression and are mutually adjusted for all other variables. ^2^Education is listed only for population where age\>25. ::: For places of death, hospital in-patient (73.4%) and nursing home (70.6%) had the highest probability of reporting multiple causes of death, and residence had the lowest percentage (58.7%) (Table [1](#T1){ref-type="table"}). Between different types of body disposal methods, \"removal\", which refers to moving the body outside of the US, had the lowest percentage (47.1%) of reporting multiple cause of death. For deaths that had autopsy performed, there was an increased odds of 1.57 that multiple causes of death would be reported. In terms of different types of medical examiners, the difference was less than 1% (69.2% vs. 70.1%) marginally between physician and coroners, the two most frequently seen types of examiners, but examining this difference across time (Figure [3](#F3){ref-type="fig"}) found an interesting interaction effect in the trend. The physicians showed a decrease in multiple cause of death reporting while the coroners stayed constant or slightly increased over the decade. Table [2](#T2){ref-type="table"} provides reference for the 25 leading underlying causes of death and leading mentioned causes of death in this dataset. It also lists the leading causes of death which occur on death certificates only reporting an underlying cause of death with no non-underlying. The top four causes based on only one cause certificates are the same as the overall top four causes. But it is interesting that the fifth leading cause in this category is \"Symptoms and ill-defined conditions\" which typically are assigned as the underlying cause only if the sole cause listed. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Interaction between certifier and year. ::: ![](1471-2288-5-4-3) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Based on Minnesota death records (n = 326,332) from 1990--1998. Top 25 causes of death ranked by underlying and any mention cause of death. Top 25 causes of death for only those deaths where only one cause was listed (i.e. n = 101,423 deaths). ::: ranked by underlying cause number of deaths with underlying cause \% all deaths rankeded by any mention number of deaths with cause mentioned \% all deaths ranked by cause when only one cause mentioned number of deaths with cause as only one cause \% of deaths with only one cause mentioned ---- --------------------------------------------------------- ---------------------------------------- --------------- ----------------------------------------------------------------- --------------------------------------- --------------- ----------------------------------------------------- ----------------------------------------------- -------------------------------------------- 1 Ischemic Heart Disease 61540 0.1886 Other diseases of the Heart 88502 0.2712 Ischemic Heart Disease 13753 0.1356 2 Cerebrovascular Disease 26413 0.0809 Ischemic Heart Disease 79833 0.2446 Other diseases of the Heart 8862 0.0874 3 Other diseases of the Heart 22566 0.0692 Cerebrovascular Disease 43885 0.1345 MN of Trachea, Bronchus & Lung 8157 0.0804 4 MN of Trachea, Bronchus & Lung 18476 0.0566 Symptoms & ill-defined conditions 42196 0.1293 Cerebrovascular Disease 7602 0.0750 5 Pneumonia -- except newborn 12465 0.0382 Other mental disorders 38565 0.1182 Symptoms & ill-defined conditions 6459 0.0637 6 Other COPD 11114 0.0341 Pneumonia -- except newborn 31312 0.0960 Other mental disorders 3494 0.0345 7 Other mental disorders 10120 0.0310 Diabetes mellitus 27181 0.0833 MN of Breast 3360 0.0331 8 Diabetes mellitus 7959 0.0244 Hypertension without heart disease 26036 0.0798 MN of Intestine, not rectum 3218 0.0317 9 MN of Intestine, not rectum 7388 0.0226 Other COPD 25872 0.0793 Pneumonia -- except newborn 3131 0.0309 10 Diseases of the arteries, veins & pulmonary circulation 7181 0.0220 MN of Trachea, Bronchus & Lung 19896 0.0610 MN of Other & unspecified sites 3042 0.0300 11 MN of Breast 6646 0.0204 MN of Other & unspecified sites 19386 0.0594 MN of Prostate 2380 0.0235 12 Symptoms & ill-defined conditions 6488 0.0199 Diseases of the arteries, veins & pulmonary circulation 19180 0.0588 Other COPD 2332 0.0230 13 Transportation accidents -- Motor Vehicle 5809 0.0178 Other diseases of the digestive system 17943 0.0550 MN of Pancreas 2313 0.0228 14 Other diseases of the digestive system 5777 0.0177 Pneumoconiosis & other resp. Diseases 17728 0.0543 Diseases of arteries, veins & pulmonary circulation 1758 0.0173 15 Other disease of the Nervous System and Sense Organs 5714 0.0175 Other disease of the Nervous System & Sense Organs 16799 0.0515 Other Neoplasms of lymphatic & Hematopoietic tissue 1701 0.0168 16 MN of Prostate 5669 0.0174 Chronic and Unspec. Nephritis & renal failure & renal sclerosis 16065 0.0492 Alzeimer Disease 1536 0.0151 17 MN of Other & unspecified sites 5504 0.0169 Arteriosclerosis 13879 0.0425 Other disease of the Nervous System & Sense Organs 1477 0.0146 18 Suicide 4435 0.0136 Transportation accidents -- Motor Vehicle 10367 0.0318 Residual, Undefined 1300 0.0128 19 MN of Pancreas 4136 0.0127 Medical complications & misadventures 10299 0.0316 MN of Brain, other nervous 1292 0.0127 20 Residual, Undefined 4135 0.0127 MN of Intestine, not rectum 9116 0.0279 Leukemia, & Aleukemia 1271 0.0125 21 Pneumoconiosis & other resp. Diseases 4028 0.0123 Septicemia 8790 0.0269 Perinatal conditions 1206 0.0119 22 Accidental falls 4010 0.0123 Suicide 8740 0.0268 MN of Ovary, Fallopian tube, Broad ligament 1124 0.0111 23 Alzeimer Disease 3757 0.0115 MN of Breast 8704 0.0267 Other diseases of the digestive system 1079 0.0106 24 Other Neoplasms of lymphatic & Hematopoietic tissue 3584 0.0110 Other genito-urinary disease 8382 0.0257 Suicide 990 0.0098 25 Leukemia, & Aleukemia 3440 0.0105 MN of Prostate 8369 0.0256 MN of Kidney 955 0.0094 ::: The results presented so far explored how covariates may be correlated with multiple causes of death being reported. The following results pertain to the conditional probability that a particular cause of death (ischemic hear disease or diabetes) is selected as underlying given that it is mentioned. Results for the subpopulations with ischemic heart disease or diabetes mentioned are shown in Table [3](#T3){ref-type="table"} and Table [4](#T4){ref-type="table"}, respectively. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Population with Ischemic Heart Disease mentioned on death certificate (N = 79833). Marginal percent by category, conditional percent with ischemic heart disease as underlying given that it is mentioned by category and odds ratios of ischemic heart disease being reported as underlying cause when it is mentioned given covariates. ::: % of deaths by category % with heart disease as underlying Odds Ratio (95% CI)^1^ --------------------- ------------------------ ----------------------------- ---------------------------------------- ---------------------------- DEMOGRAPHIC Age 0--44 1.7 80.0 1 45--64 12.4 81.8 1.2 (1.03,1.36) 65--84 53.0 76.1 0.84 (0.74,0.95) 85+ 32.8 76.8 0.87 (0.77,1.00) Sex Female 45.2 76.3 1 Male 54.9 77.8 0.95(0.91,0.99) Race White 97.9 77.2 1 Black 0.8 71.8 0.78(0.66,0.94) Native American 0.7 69.7 0.55(0.45,0.66) Asian 0.3 77.2 1.12(0.81,1.56) Hispanic 0.3 72.2 0.79(0.59,1.06) Education^2^ High school 29.1 76.3 1 Below High School 49.1 77.9 1.07(1.03,1.11) Above High School 21.8 76.5 1.02(0.97,1.07) Marital Married 44.9 77.5 1 Single 8.2 79.7 1.25(1.16,1.33) Widowed 40.0 75.9 1.04(0.99,1.09) Divorced 6.9 77.9 1.13(1.05,1.22) DEATH RELATED Autopsy NO 77.8 76.9 1 Yes 10.5 78.0 0.91(0.86,0.97) Unspecified 11.7 77.8 1.12(1.06,1.18) Certifier Physician 79.5 75.5 1 Coroner 15.2 84.3 1.34(1.25,1.42) Osteopath 1.5 80.9 1.25(1.07,1.46) Other/Unknown 3.8 81.0 1.23(1.13,1.35) Disposal method Burial 78.9 77.3 1 Donation 0.3 75.3 0.74(0.56,0.98) Removal 1.6 85.3 1.74(1.48,2.05) Cremation 18.0 66.8 0.95(0.91,0.99) Unknown 1.2 Death place Hospital Inpatient 36.1 72.9 1 Residential 20.1 90.5 1.83(1.74,1.93) Nursing home 27.9 71.2 0.83(0.79,0.87) ER 11.4 83.2 1.67(1.39,1.97) Unknown 4.5 ^1^Odds ratios are odds of having ischemic heart disease as underlying rather than contributing given particular category as compared to odds in reference category. Odds ratios are obtained from logistic regression and are mutually adjusted for all other variables. ^2^Education is listed only for population where age\>25. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Population with Diabetes mentioned on death certificate (N = 27181). Marginal percent by category, conditional percent with diabetes as underlying given that it is mentioned and odds ratios of diabetes being reported as underlying cause when it is mentioned given covariates. ::: % of deaths by category % with diabetes as underlying given that it was mentioned Odds Ratio (95% CI)^1^ --------------------- ------------------------ ----------------------------- --------------------------------------------------------------- ---------------------------- DEMOGRAPHIC Age 0--44 2.3 51.7 1 45--64 13.2 34.5 0.51(0.43,0.60) 65--84 59.5 27.7 0.37(0.32,0.43) 85+ 24.9 28.4 0.39(0.33,0.45) Sex Female 51.7 30.2 1 Male 48.3 28.3 0.92(0.86,0.97) Race White 95.4 28.9 1 Black 2.0 36.5 1.19(0.98,1.43) Native American 1.5 41.0 1.42(1.136,1.76) Asian 0.5 27.2 0.88(0.59,1.30) Hispanic 0.7 36.6 1.33(0.978,1.82) Education^2^ High school 31.2 30.1 1 Below High School 47.8 28.0 0.99(0.93,1.06) Above High School 21.0 30.8 1.03(0.96,1.11) Marital Married 43.6 27.8 1 Single 8.5 34.3 1.25(1.13,1.38) Widowed 40.2 29.0 1.09(1.01,1.16) Divorced 7.7 33.5 1.11(0.99,1.23) DEATH RELATED Autopsy No 83.3 28.3 1 Yes 5.5 22.8 0.70(0.62,0.80) Unspecified 11.3 39.4 1.49(1.37,1.62) Certifier Physician 85.0 29.7 1 Coroner 10.0 24.2 0.69(0.62,0.77) Osteopath 1.5 30.4 1.03(0.81,1.29) Other/Unknown 3.5 34.3 1.23(1.08,1.42) Disposal method Burial 79.2 28.6 1 Donation 0.3 25.0 0.84(0.51,1.39) Removal 1.1 37.0 1.46(1.143,1.87) Cremation 18.3 31.4 1.06(0.99,1.14) Unknown 1.1 Death place Hospital Inpatient 34.8 25.2 1 Residential 18.8 30.9 1.29(1.19,1.41) Nursing home 37.4 32.7 1.57(1.47,1.68) ER 6.4 28.3 1.15(1.02,1.29) Unknown 2.6 ^1^Odds ratios are odds of having diabetes as underlying rather than contributing given particular category as compared to odds in reference category. Odds ratios are obtained from logistic regression and are mutually adjusted for all other variables. ^2^Education is listed only for population where age\>25. ::: Table [3](#T3){ref-type="table"} gives the odds ratio of ischemic heart disease being selected as underlying cause of death when it was mentioned as a cause, given the covariates main effect. Overall 77.1% of the time that heart disease was mentioned as a cause, it was selected as the underlying cause of death. The 45--65 year age group had the highest probability of heart disease being codes as underlying when it was mentioned (81.8%). Males had a slightly lower probability than females to have heart disease as underlying cause of death when it was mentioned on the death certificate. Furthermore, Blacks and Native Americans were less likely to have heart disease coded as underlying cause of death when it was present on the certificate as compared to Whites. Individuals that had an autopsy performed were less likely (0.91 odds ratio) to have ischemic heart disease selected as underlying when it was mentioned. If a physician is the death certifier, the probability of selecting heart disease as underlying cause of death is relatively the lowest when compared to coroner, osteopath and other and unknown certifiers. Amongst body disposal methods, the probability for heart disease to be reported as underlying cause was the lowest if bodies were donated (OR = .7 with \"burial\" as baseline category), and highest if bodies were removed (OR = 1.7). Finally, for those individuals who had heart disease mentioned on their death certificate, patients who died at a residence (not a nursing home) were most likely to have ischemic heart disease selected as the underlying cause of death (90.5% or an OR= 1.8 compare to hospital in-patient). Unlike Ischemic heart disease, only 29.3% of deaths with diabetes mentioned on the certificate had it selected as the underlying cause of death. While only 2.3% of deaths with diabetes mentioned occurred in the youngest age group (0--44 years), (Table [4](#T4){ref-type="table"}) this group has a much larger probability of having diabetes be the underlying cause compared to non-underlying (51.7% reported as underlying). Men were less likely to have diabetes selected as underlying when it is mentioned on the certificate than women. Blacks and Native Americans both have significantly higher odds (OR = 1.2 and 1.4, respectively) of diabetes being the underlying cause given that it was mentioned as compared to Whites. The role of autopsy is that it was less likely diabetes was reported as underlying (OR = 0.7) when one was performed than if one was not. Moreover, if a coroner was the death certifier, diabetes was less likely to be reported as underlying. An increase in the reporting of diabetes as underlying was found for deaths that were removed. Finally, deaths occurring outside of the hospital inpatient setting all show increased odds of diabetes being selected as the underlying cause of death when it has been mentioned. We also examined what other leading causes of death showed up as underlying when ischemic heart disease or diabetes was mentioned on the certificate. As mentioned above, 77.1% of the individual with ischemic heart disease mentioned on their death certificate had it reported as the underlying. The second most common underlying cause of death when ischemic heart disease was mentioned was, in fact, diabetes (3.4% of the time underlying), followed by cerebrovascular disease (2.7% of the time underlying), then pneumonia (1.5% of the time underlying). When we focus on the population that has diabetes mentioned on the death certificate, as mentioned above 29.3% of the time diabetes is selected as the underlying, and the second most common underlying cause selected is ischemic hear disease at 25.4%, followed by cerebrovascular disease at 7.75% then followed by Other diseases of the heart 2.9%. Discussion ========== Distinct differences in the frequency of multiple causes of death were found across time, age, race, disposal method and place of death. Definitive explanations for the differences cannot be given based on this study, but it is of interest to consider plausible explanations which may motivate further investigation. The increased reporting of non-underlying causes of death as the age of the decedent increases is likely due to actual increases in co-morbidity with age, hence would be explained by actual differences in the causes of death. The differences found in reporting of multiple causes of death for the other factors may be partly due to systematic reporting biases. According to the NCHS All Mortality Altas \[\[[@B11]\], p. 3\], the quality of cause of death determination in the US is affected by the accuracy and completeness of information -- from medical diagnosis to final coding and processing of underlying cause of death. Although since 1968 the automated selection of the underlying cause of death has helped to reduce coding and processing errors, the completeness and accuracy of the information supplied on the certificate and the decedent\'s medical diagnosis remain as potential sources of error. If the certifier enters only one underlying cause and no other causes, then that cause will have to be selected as the underlying and there will not be multiple causes of deaths for that record. It is interesting to note that \"Symptoms and ill-defined conditions\" is the 5^th^most commonly reported cause of death to be the only cause of death listed on the certificate. This reporting of it as the only cause of death pushes it up to be the overall 12^th^leading cause of death. If almost any other cause would be listed simultaneously on the death certificate, this code would not end up as underlying. The decreasing trend in reporting multiple causes over the decade may be reflective of a gradual change in the procedures of death certification. It would be of interest to consider this trend across different states and longer periods of time including shifts from one ICD coding system to the next. Previous literature offers various plausible explanations to what contributes to the inaccuracy of reporting causes of death. The cause of death reported on the death certificates depends on a person\'s disease history that leads to death \[[@B12]\]. If a person dies after a long, well-characterized illness, the cause of death on the certificate is likely to be more accurate than a sudden or unobserved death. Also, when lack of adequate information on the decedent\'s disease history, the more narrowly characterized the cause of death on the certificate, the more likely it is to be in error. If we assume that reporting multiple causes on the death certificate can be considerd a proxy for level of familiarity of the death certifier with the patient, we would expect that a death which occurs in a hospital or nursing home would be more likely to have multiple causes reported, possibly due to a better documentation of disease history. On the other hand, death at the ER and in particular at the person\'s residence, which is conceivably often sudden should show a much lower percentage of multiple cause of death reporting. Analysis results from this current study match such speculations, supporting the argument that a good understanding of disease history is crucial. Still another result that supports this conclusion is the fact that performing autopsy, which gain better understanding of the disease condition, increased the probability of reporting multiple cause of death. Gender and race can also play a role in the accuracy of reporting. Lloyd \[[@B13]\] showed that positive predictive value of the death certificate tended to be lower in women than in men. Although no large differences were seen between men and women with respect to frequency of multiple causes, there was a higher percentage of multiple causes reported for Native Americans. It is conceivable that the high percentage of multiple cause of death observed for Native Americans might be associated with the geographic factors of concentrated residence and the unique practices of local clinics. Moreover, results (not shown) indicate differences exist across counties of Minnesota in the reporting of multiple causes of death ranging from 50% to 80%. These results support suggestions for better standardized training for physicians and coroners. Similar to the case of reporting multiple cause of death, the selection of ischemic heart disease and diabetes as underlying compared to non-underlying differs across the several factors considered. The implications of these differences across demographics are that mortality rates would be differentially affected when underlying cause of death is used compared to any mention cause of death. For example, for diabetes we might conclude that diabetes is being under-reported in Whites compared to Blacks, Native Americans and Hispanics if only underlying cause of death were considered since the proportion of diabetes as underlying to mentioned is substantially lower in Whites. This is not to say there is any inaccuracy in the way it is being coded but it points out where multiple cause of death reporting will provide a different perspective than underlying. Nevertheless, studies have shown the sensitivity and positive predictive value of the death certificate are particularly poor with regard to stroke and diabetes \[[@B14]\]. Furthermore, Lloyd \[[@B13]\] concluded that physicians may use coronary heart disease as a \"default\" cause when facing some unknown cause of death cases. The fact that individuals with autopsy performed have lower probability of having heart disease selected as underlying when it is mentioned might suggest that heart disease is often over-assigned as the default disease when no further medical details are available. This is further demonstrated by the very high ratio of ischemic heart disease being coded as the underlying compared to non-underlying cause of death when the death occurred at the person\'s residence. As mentioned in the introduction, one limitation of this research is the fact that there is no outside panel of experts who decide independently what the true causes of death are for each decedent, thus whether the associations we found are due to actual differences or reporting bias cannot be discerned. Therefore, this study cannot provide sensitivity or specificity per se, but it aims to identify factors that are associated with variability in reporting multiple cause of death and that perhaps contribute to inaccuracy in reporting underlying cause. Conclusions =========== There is much to be learned from multiple cause of death data. It provides ways of looking at mortality data that go well beyond the typical examination of underlying cause of death. Future research is needed to understand further what the greatest concerns are about the accuracy of reporting causes of death. Multiple cause of death data have the potential to help point out potential concerns in the accuracy as well as provide a more complete picture of mortality for causes which are frequently not recorded as the underlying cause of death. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= MW conceived of the study and wrote most of the manuscript. JH performed the statistical analysis and wrote part of the manuscript. JO provided access to the data and collaboration regarding processes underlying data collection. DD provided details about cause of death reporting and collaboration regarding processes underlying data collection. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2288/5/4/prepub>	PubMed Central	2024-06-05T03:55:52.696435	2005-1-17	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548504/", "journal": "BMC Med Res Methodol. 2005 Jan 17; 5:4", "authors": [ { "first": "Melanie M", "last": "Wall" }, { "first": "Jinzhou", "last": "Huang" }, { "first": "John", "last": "Oswald" }, { "first": "Diane", "last": "McCullen" } ] }
PMC548505	Background ========== Obesity is the most common health problem in developed countries \[[@B1]\]. It is a chronic disease and should be treated as such. Its prevalence is increasing worldwide \[[@B2]\]. In the United States, it is estimated that 64% of the adult population is either overweight or obese with a body mass index (BMI; kg/m^2^) above 25 \[[@B3]\]. The rate of obesity is increasing \[[@B4]\] and has risen by more than 75% in the USA since 1980 \[[@B5]\]. In 2001, the prevalence of obesity (BMI ≥ 30) was 20.9% vs 19.8% in 2000, an increase of 5.6% \[[@B6]\]. In Israel, according to a survey of the Nutrition Department of the Ministry of Health, 55% of adult (ages 25--64) women and 59% of adult men have a BMI above 24.9 \[[@B7]\]. Obesity is associated with increased prevalence of many serious chronic diseases such as diabetes mellitus, hypertension, dyslipidemia, and coronary heart disease \[[@B8],[@B9]\]. It may be responsible for approximately 300,000 deaths in the USA per year \[[@B10]\]. In the Nurses Health Study, the 14-year mortality rate for women with a BMI greater than 32 was more than double that of women with a BMI of less than 19 \[[@B11]\]. Obesity now ranks second only to smoking as a cause of preventable death but, soon, obesity may surpass smoking as the leading cause of preventable death in the USA \[[@B12]\]. In the USA, 19% of deaths from coronary disease and 62% of deaths from diabetes can be attributed to obesity \[[@B13]\]. The risk of death from all causes increases in moderately and severely overweight men and women of all age groups \[[@B14]\]. Diet and exercise have limited effectiveness on long-term maintenance of weight loss \[[@B15]\]. Within five to seven years, 95% of all patients regain the lost weight or more \[[@B16]\]. Pharmacotherapy in combination with a reduced energy diet improves long-term efficacy \[[@B17]\]. Loss of 5--10% of their initial body weight substantially improves the health of obese patients and modifies their cardiovascular risk factors \[[@B8],[@B18]\]. Despite growing information on the pathophysiology of obesity and its high prevalence, obesity and obesity-related diseases are still under-diagnosed and untreated by family physicians \[[@B19]\]. Most family physicians cite lack of time, resources, reimbursement from insurance companies, or knowledge of effective interventions as significant barriers \[[@B20]\]. The intervention of primary physicians during a ten minute physician/patient encounter and telephone consultation with a community dietitian resulted in a significant decrease in the weight of patients \[[@B20]\]. Recently, several physicians\' organizations have issued recommendations for treating obesity to family physicians, including instructions in nutrition, physical activity and medications. Such recommendations were based on a number of studies that proved the effectiveness of family physician weight-reduction programs, when based on the readiness of patients to make necessary lifestyle changes and use of appropriate techniques to increase the willingness of the patient to make necessary changes \[[@B21]-[@B24]\]. The purpose of this study was to examine if more efficient and effective weight-reducing treatment can be given in the family doctor setting. The study compare a non-pharmacological intervention with drug intervention (orlistat) and compare regular management with more intensive family physician based management.. Methods ======= Study design ------------ The study was conducted in three primary care clinics in an urban area in central Israel. The family physicians who took part in this study participated in 80 hours CME course dealing with obesity treatment in Israel. The patients were divided into three groups according to their choice. Patients in groups A and B were treated with orlistat at 120 mg TID. Orlistat (Xenical ^®^) is a lipase inhibitor for obesity management that acts by inhibiting the absorption of dietary fats. The patients in group A received in addition a personal reduced-energy diet and met with a family practitioner and a clinical dietitian once every two weeks. The personal diet was created according to the daily schedule and preferred foods of the individual, emphasizing low-fat foods. The patients received instructions regarding the importance of physical activity and, at each meeting, realistic intermediate goals for achieving two or three small changes in eating habits and physical activity, based on the patient\'s desires, were determined. The obstacles to change and ways to overcome them were discussed. Support, based on improvements in the patient\'s health parameters such as an improvement in a blood test, was given. Some of the patients helped with self-criticism, by keeping food and physical activity diaries and by grading their own goal accomplishments. Patients were taught how to resist temptation and reward themselves for success. We also recruited the support and encouragement of the patient\'s family. At each meeting, the time of the next meeting was determined and it was emphasized that the most important thing for the patients to do was to attend the meetings, even if their goals were not achieved. Group B patients were treated with 120 mg orlistat t.i.d., a general formulated reduced-energy diet and follow-up by the family physician once every four weeks for weighing and prescription renewal. In groups A and B, patients were asked at each meeting if any side effects of orlistat appeared. Patients in group C were given a personal low-calorie diet, designed according to their preferences, and followed-up by a clinical dietitian once a month. The prescribed daily caloric intake was equal in the three groups and was 1200 calories per day for women and 1500 calories per day for men. Before the intervention, every patient received an explanation of the three treatments, the importance of reducing weight, and how excess weight affects their health. All were instructed about the recommended rate of weight loss and a final weight reduction goal of at least 5% of their initial weight within half a year was established. Patients who achieved the 5% reduction goal before the end of the study could choose either to stop the intervention or to complete the study period. Patients -------- Obese (BMI \> 30) patients of either sex or patients with a BMI above 27 plus two or more cardiovascular risk factors, aged 20--75 years, were eligible for the study. Patients were excluded if they were pregnant or lactating or if they had any contraindication against using orlistat (chronic malabsorption syndrome, cholestasis, pancreatic disease). Before the intervention, patients underwent an initial screening that included recording of a complete medical history, measurement of vital signs, body weight and height, and calculation of the BMI. Laboratory analysis included a lipid profile. The readiness of the patient to receive treatment was assessed. At the beginning of the intervention, all participants were in the third stage of readiness (\"the preparation stage\") according to the Transtheoretical Model of Behavior Change. An adverse event of any dose of orlistat was considered serious if it resulted in death, was life-threatening, required hospitalization, or resulted in significant disability. Efficacy measures ----------------- The main measure was weight loss. Each patient was weighed during each meeting. The primary endpoint was reduction of at least 5% of the initial weight during the study period (six months). Achievement of this goal was defined as successful treatment. Another measure was improvement in the lipid profile. The second lipid profile was done to half of the patients, only those who had dyslipidemia in the first profile had been offered a second profile. Statistical analysis -------------------- Data on patients\' background and weight-reduction results between groups were compared using the Chi square test. Continuous data of the groups, i.e., measurements at the beginning of the study and continuous background data, were compared using one-way tests (ANOVA). When significant statistical differences were found among the three groups, the Tukey Post Hoc test was used to examine the statistical difference between each group of two. ANCOVA was also used and, if there was a difference amongst the groups, the significance (for the measured parameter) was checked using the Bonferonni technique. Results ======= Two hundred and twenty-five patients participated in the study. Their demographic characteristics, history of cardiovascular disease, and initial lipid profile are shown in Table [1](#T1){ref-type="table"}. There were no significant differences between groups in average age, initial BMI or gender of the participants. The average length of follow-up and the number of meetings varied among groups. There were no cases of significant side effects that required stopping orlistat treatment of any of the participants. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Participants\' demographic data, initial lipids profile, by treatment group ::: ------------------------------------------------------------------------------------------------------------------ Group A Group B Group C P\* value -------------------------------------------------------- ----------- ----------- ----------- --------------------- Number of patients 62 112 51 Age (years +/- SD) 47.3 ± 11 46.8 ± 12 51 ± 9.6 NS Gender (% female) 71 74 61 NS Ischemic heart disease (%) 0 4 0 NS Hypertension (%) 44 51 27 P \< 0.05 Diabetes mellitus (%) 9 18 20 NS Dyslipidemia (%) 16 38 66 P \< 0.001 Initial body weight Initial body mass index (BMI; kg/m^2^+/- SD) 33 ± 3.8 34 ± 4.4 31 ± 3.6 P \< 0.01\ 34 \> 31(B \> C) Initial triglycerides (mg/dl, +/- SD) 170 ± 53 184 ± 49 255 ± 205 P \< 0.01\ 170 \<255\> 183\ (A \< B \> C) Initial low density lipoproteins (LDL; mg/dl, +/- SD) 150 ± 30 156 ± 36 152 ± 44 NS Initial high density lipoproteins (HDL; mg/dl, +/- SD) 42 ± 7.0 44 ± 6.7 47 ± 14.9 NS Average length of treatment (weeks, +/- SD) 13 ± 12.0 9 ± 4.7 23 ± 12 P \< 0.001\ 23 \> 13 \> 9\ (C \> A \> B) Number of meetings with physician/dietitian (+/- SD) 4.3 ± 2.0 3.5 ± 1.5 5.2 ± 2.9 P \< 0.001\ 5.2 \> 3.5 (C \> B) ------------------------------------------------------------------------------------------------------------------ NS = Not significant. Group A -- Orlistat, a personal reduced-energy diet and a meeting with a family practitioner and a clinical dietitian once every two weeks. Group B -- Orlistat, a general formulated reduced-energy diet and follow-up by the family physician once every four weeks. Group C -- a personal low-calorie diet and follow-up by a clinical dietitian once a month. \* When significant statistical differences were found among the three groups, we examined the statistical difference between each group of two. ::: Patient-reported adverse events in the orlistat-treated groups were all related to the gastrointestinal tract. The most commonly reported events were flatulence with discharge (9.2%), fatty or oily stool with increased defecation (9.2%), feeling of fullness in the stomach (4.6%), and constipation (1.7%). There were no statistically significant differences in side effects between groups A and B. In group A, 11 patients (17.7%) stopped the treatment for the following reasons: cost of the medication (47%), lack of time (33%), or dissatisfaction (20%). In comparison, ten patients (8.9%) in group B stopped the treatment, mainly because of the cost of the treatment (65%) or low motivation (23.5%). The reasons for stopping treatment significantly differed between groups A and B (p= 0.03). The percentage of patients who attained their weight reduction goals was largest in group A where patients received orlistat and intense follow-up, in addition to a personally-designed diet (Fig. [1](#F1){ref-type="fig"}). Changes in lipid profiles are shown in Table [2](#T2){ref-type="table"}. The treatment resulted in a significant reduction in triglyceride levels in all groups, a significant reduction of low density lipoproteins (LDL) in groups A and B and no significant difference between initial and final high density lipoproteins (HDL) in any group. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Weight loss by treatment group\* ::: ![](1471-2296-6-5-1) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Changes in lipid profile during treatment according to treatment group^1^(paired sample ttest) ::: Group A Group B Group C -------------------------------------------------- ----------- ----------- ----------- Number of patients^2^ 32 55 28 Initial triglycerides (mg/dl, +/- SD) 170 ± 53 184 ± 49 255 ± 205 Final triglycerides (mg/dl, +/- SD) 139 ± 43 153 ± 35 165 ± 60 Delta (+/- SD) -31 ± 21 -31 ± 25 -90 ± 187 P value (pre-post) \<0.001 \<0.001 0.01 Initial low density lipids (LDL; mg/dl, +/- SD) 150 ± 30 156 ± 36 152 ± 44 Final LDL (mg/dl, +/- SD) 129 ± 28 143 ± 32 147 ± 34 Delta -21 ± 26 -12 ± 16 -5 ± 34 P value (pre-post) \<0.001 \<0.001 NS Initial high density lipids (HDL; mg/dl, +/- SD) 42 ± 7.0 44 ± 6.7 47 ± 14.9 Final HDL (mg/dl, +/- SD) 43 ± 6.6 45 ± 6.7 48 ± 15.3 Delta (+/- SD) 0.9 ± 2.9 0.8 ± 3.3 1.4 ± 8.8 P value (pre-post) NS NS NS NS = Not significant. ^1^See footnotes to Table 1 ^2^Patients with pre and post lipid profile analyses ::: Patients in Group A reduced 5.12 kg (range of 5--8 Kg.) of their initial body weight, patients in Group B reduced 7.8 kg (range 10--12 Kg.) from their initial body weight and patients in Group C reduced 3.12 kg (range 5--6 Kg.) of their initial body weight. Discussion ========== Obesity is a worldwide problem. Treatment of a patient for obesity involves two processes: evaluation of the severity of the obesity and general health condition of the patient, and management which includes guidance in how to gradually reduce weight and maintain the new weight together with imparting healthy lifestyle habits and keeping track of improvement. In this study, we examined weight-reduction techniques that can be carried out in the setting of a community family practice. Intense treatment, combining frequent counseling by the family physician and a dietitian with medications (group A), resulted in the best weight reduction and lipid profile improvement in the short period of this study, as was reported earlier in special weight reduction clinics \[[@B25]-[@B27]\] The effectiveness of interventions in primary care setting are controversial \[[@B28]-[@B30]\]. Beermann et al \[[@B28]\] in a community survey of 792 patients found that Orlistat was not prescribed according to the approved indication in the majority of cases. The dropout rate was high and most patients had minor gain from the treatment. Linne et al \[[@B29]\] noted that success rate of Orlistat in primary-care practice is limited by failure to follow prescribing recommendations. A simple questionnaire to 70 patients revealed that in many cases the referral physician had not observed basic rules and regulations, nor given appropriate information on Orlistat use. Hauptman et al \[[@B30]\] in a study conducted in seventeen primary care centers in the United States. The study indicates that orlistat is an effective adjunct to dietary intervention in the treatment of obesity in primary care settings. There are many advantages to a program involving cooperation between the family physician and the dietitian. The family physician is acquainted with the patient for a longer time than a dietitian. The physician is familiar with the patient\'s health condition, medications taken by the patient, the patient\'s environment and lifestyle and can recommend a treatment suitable to the patient\'s personality and lifestyle. The family physician is knowledgeable about weight-reducing drugs and possible side effects. The patient trusts and has confidence in the family doctor. Together with the dietitian, a personal diet appropriate to the drug treatment can be created. Obesity is a chronic disease. The physician and the patient must recognize that obesity treatment is a prolonged process that extends a lifetime. Since family physicians are usually familiar with their patients as well as with the patient\'s family and environment for many years, family physicians know what changes the patient can achieve. They can recruit family members to support the necessary lifestyle changes. Also, because of their training, family physicians are the most suitable professional to holistically treat obesity and its complications. There were several limitations in our study. One limitation was the non-random division of patients into groups A, B and C. It is possible that the patients who chose drug treatment differed from those who chose a diet only. Perhaps they were more ready for the weight-reduction process and to expend money for the drug (in Israel, there is a patient co-payment for medications). The rate of women amongst the patients seeking treatment was higher than the rate of men, as in other reports \[[@B26],[@B27]\]. Hence, we assume that our study represented the segment of the population more prone to try dieting and weight-reduction programs. Ways to increase the number of men participating in weight-reduction programs must be found. The study periods for groups A and B (which received orlistat) were shorter than for group C, partly because there is significant co-payment for orlistat and partly because the targeted weight had been achieved earlier. The drug was well tolerated with minimal gastrointestinal side effects. The patients were treated by family physicians that were orientated toward and trained in weight reduction. Results might have been less successful with other physicians. Hence, family physicians should be trained in the subject by increasing both their knowledge and their treatment skills. Healthcare funds must recognize the importance of weight reduction so that they will allocate the necessary additional time and resources of their physicians, clinics and multi-field staff. This study evaluated only the weight reduction period. Long-term results and whether or not the patient maintained the lifestyle change for a long period were not examined. Further studies should examine the best program for maintaining the new weight. In conclusion, this study showed that within the setting of the family practice it is possible to carry out an effective program of weight reduction and achieve significant weight loss. The addition of orlistat can further improve results. We believe that by providing family physicians with the proper tools, similar success can be achieved in many more clinics. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= AF and SP conceived and designed the study, participated in the collection, analysis and interpretation of data and drafted the manuscript. SV Participated in the statistical analysis, interpretation of data and draft of the manuscript. MS and EF participated in the design of the study, data collection and interpretetion. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2296/6/5/prepub> Acknowledgements ================ None	PubMed Central	2024-06-05T03:55:52.702473	2005-1-29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548505/", "journal": "BMC Fam Pract. 2005 Jan 29; 6:5", "authors": [ { "first": "Amiel", "last": "Feigenbaum" }, { "first": "Shmuel", "last": "Pasternak" }, { "first": "Efrat", "last": "Zusk" }, { "first": "Miri", "last": "Sarid" }, { "first": "Shlomo", "last": "Vinker" } ] }
PMC548506	Background ========== Familial Juvenile Hyperuricemic Nephropathy (FJHN) (McKusick 162000) is an autosomal dominant disorder characterized by hyperuricemia, decreased urinary excretion of urate and the development of progressive chronic interstitial nephritis. Renal impairment usually appears between 15 and 40 years of age, leading to end-stage renal disease (ESRD) within 10 to 20 years \[[@B1]\]. A candidate gene for FJHN has been positioned in 16p11.2-12, together with evidence for genetic heterogeneity \[[@B2],[@B3]\]. The candidate gene was later found to map within the same genetic interval as MCKD2, a locus responsible for medullary cystic kidney disease, therefore suggesting that FJHN and MCKD2 are 2 facets of the same disease \[[@B1]\]. The marked thickening of tubular basement membranes observed in FJHN closely resembles the renal histological findings of the nephronophthisis-medullary cystic kidney disease complex (NPH-MCKD). Diseases of this group share the macroscopic feature of cyst development at the corticomedullary border of the kidney and the renal histological triad of tubular basement membrane disintegration, tubular atrophy with cyst development and interstitial cell infiltration with fibrosis \[[@B4]\]. Within this complex, clinical entities can be distinguished based on the mode of inheritance, the age of onset for ESRD and the presence of extra-renal involvement. For the recessive forms of the disease 4 different genes have been cloned. The NPHP1 gene \[[@B5],[@B6]\] and NPHP4 \[[@B7]\] are responsible for juvenile forms of NPH, while NPHP2 \[[@B8]\] and NPHP3 \[[@B9]\] account for, respectively, the infantile and adolescent forms. MCKD, the autosomal dominant disorder that presents in early adulthood, is usually accompanied by the detection of corticomedullary cysts on imaging studies. MCKD1 maps to 1q21 and remains to be cloned, while MCKD2 has been positioned in 16p12. Mutations in the uromodulin/Tamm-Horsfall protein coding UMOD gene located within the critical interval of FJHN and MCKD2 at 16p11.2-12 were recently identified in FJHN and MCKD2 families \[[@B10]\], therefore providing definite evidence that MCKD and FJHN are allelic disorders. Meanwhile, a mutation cluster in exon 4 of UMOD was reported for both diseases \[[@B11],[@B12]\]. We here describe a novelheterozygous missensemutation in affected individuals from a Portuguese FJHN family that also displays corticomedullary cysts on ultrasound examination. This mutation, c.920A→C, resides in exon 5 and is the fourth reported outside exon 4. Case presentation ================= Case report ----------- The proband is an affected female who was first evaluated at the age of 24, when she presented with a gout attack and hyperuricemia. At age 27 she was told having renal failure. However, a renal biopsy was not performed. At age 44, serum creatinine was 2.8 mg/dl and ultrasound imaging detected numerous renal cysts. Renal disease slowly progressed and the patient reached ESRD when she was 49 years old. Her father died at the age of 55 from ESRD and suffered from hyperuricemia and gout. The proband\'s only son is also affected. At the age of 18 years he had a gout attack. On the initial evaluation, serum uric acid was 15 mg/dl and serum creatinine 1.6 mg/dl. Renal cysts were also detected on ultrasound examination. Mutation analysis ----------------- Informed consent was obtained from tested individuals. Genomic DNA was isolated from peripheral blood leucocytes and the coding region of the UMOD gene was screened for mutations by direct sequencing of PCR products. We used a set of primers previously described \[[@B10]\] except in exons 4 and 5, for which different additional internal sequencing primer were designed based upon sequences from GenBank (accession numbers NT\_024776.6 and M17778). Results ------- A novelheterozygous missensemutation, c.920A→C, was detected (Figure [1](#F1){ref-type="fig"}) and found to segregate with the disease in this family. The mutation results in a Lys to Thr at position 307. This mutation was not detected in any of the 100 control chromosomes tested. In fact, no polymorphism affecting the translation of uromodulin was detected in 100 control chromosomes in a previous report \[[@B10]\] and we are, therefore, excluding the possibility of this allele being a mere polymorphism. In addition, the affected mother was found to be homozygous for the T allele in the common T to C transition synonymous polymorphism at codon C174. Discussion ---------- The UMOD gene has 12 exons and codes for the 640 amino-acid uromodulin, a glycsoyl phosphatidylinositol (GPI) anchored protein that accounts for the primary structure of the 85-kD Tamm-Horsfall glycoprotein (THP). THP is the most abundant protein in the urine and, in normal kidney, uromodulin expression is restricted to the thick ascending limb (TAL) and distal convoluted tubule. Urinary excretion occurs by proteolytic cleavage of the GPI counterpart at the luminal surface of TAL. It has been suggested that one of THP major role is of an urinary anti-adherence factor preventing type 1 fimbriated E coli from binding to the urothelial receptors \[[@B13]\]. The majority of the mutations so far published are clustered in exon 4, between codons 52 and 282, and most are missensemutations affecting cysteine residues (Table [1](#T1){ref-type="table"}). Exon 4 contains 3 calcium binding epidermal growth factor (cbEGF)-like domains, between residues 31 and 148. A fourth potential cbEGF-like domain extends from amino-acids 281--336, throughout exon 5. They contain 6 conserved cysteine residues responsible for the protein\'s tertiary structure, as a result of intramolecular disulfide bonding. It has been hypothesized that protein misfolding, consequence of mutations in these cbEGF-like domains, may affect uromodulin intracellular trafficking and lead to cellular protein accumulation and apoptosis \[[@B14]\]. The release of cells debris and uromodulin aggregates in the interstitium could stimulate an inflammatory response and, in addition, be responsible for tubular obstruction and medullary cyst formation. It has been proposed that hyperuricemia in these patients is secondary to a reduced TAL sodium reabsorption with volume contraction and a compensatory increase in proximal urate reabsorption. The role of hyperuricemia in the chronic interstitial nephritis remains to be clarified, since the deposition of sodium urate crystals in the medullary interstitium does not occur in these patients. The mutation c.920A→C here reported, replaces the basic amino-acid Lys for the uncharged polar Thr at position 307 (p.K307T), being the fourth mutation described in exon 5. The affected residue locates within the fourth cbEGF-like domain and is immediately preceded by a highly conserved cysteine residue. Conclusions =========== It has been referred that exon 4 sequencing should become the first diagnostic test in patients with chronic interstitial nephritis with gout or hyperuricemia, even in the absence of a family history \[[@B11]\]. In view of the increasing number of mutations in exon 5 being identified, we now recommend that exon 5 should be included in the initial sequencing effort, since otherwise nearly 12% of UMOD mutations can be missed. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JC was responsible for the study design and drafted the manuscript. JC and CC carried out the molecular analysis. AG collected the clinical data. JR participated in the study design. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2350/6/5/prepub> Acknowledgments =============== This work was supported by a grant from Roche Farmacêutica Química, Portugal. We thank all members of the affected family for their participation. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### a)Sequence of the index patient with exon 5 c.920A→C heterozygous missensemutation. b)The same mutation sequenced with a reverse primer. ::: ![](1471-2350-6-5-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### UMOD mutations reported in the literature. ::: Mutation exon reference --------------------------------------------------- ------ ---------------- c.156T→G; p.C52W 4 \[15\] c.176A→C; p.D59A 4 \[11\] c.230G→A; p.C77Y 4 \[16\] c.278\_289del/insCCGGCTCCT; p.V93\_G97del/insAASC 4 \[12\] c.307G→T; p.G103C 4 \[10\] c.334T→C; p.C112R 4 \[11\] c.376T→C; p.C126R 4 \[16\] c.383A→G; p.N128S 4 \[16\] c.403T→A; p.C135S 4 \[15\] c.443G→A; p.C148Y 4 \[10\] c.444T→G; p.C148W 4 \[14\] c.449G→C; p.C150S 4 \[14\] c.509G→A; p.C170Y 4 \[11\] c.529\_555del; p.H177\_R185del 4 \[10\] c.553C→G; p.R185G 4 \[18\] c.553C→A; p.R185S 4 \[11\] c.563\_661del; p.E188\_L221del 4 \[11\] c.584G→T; p.C195F 4 \[15\] c.605G→C; p.W202S 4 \[15\] c.610C→G; p.R204G 4 \[11\] c.649T→C; p.C217R 4 \[10\] c.649T→G; p.C217G 4 \[11\] c.665G→C; p.R222P 4 \[11\] c.668G→A; p.C223Y 4 \[17\] c.674C→T; p.T225M 4 \[11\] c.674C→A; p.T225K 4 \[12\] c.707C→T; p.P236L 4 \[15\] c.744C→G; p.C248W 4 \[12\] c.764G→A; p.C255Y 4 \[16\] c.844T→C; p.C282R 4 \[11\] c.898T→G; p.C300G 5 \[16\] c.920A→C; p.K307T 5 present report c.943T→C; p.C315R 5 \[14\] c.950G→A; p.C317Y 5 \[14\] :::	PubMed Central	2024-06-05T03:55:52.705154	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548506/", "journal": "BMC Med Genet. 2005 Jan 27; 6:5", "authors": [ { "first": "Joaquim", "last": "Calado" }, { "first": "Augusta", "last": "Gaspar" }, { "first": "Carla", "last": "Clemente" }, { "first": "José", "last": "Rueff" } ] }
PMC548507	Background ========== In the last few years, exciting advances in the biology and molecular genetics of the development of Plasmodiumparasites in their mosquito vectors \[[@B1],[@B2]\] have led to the creation of transgenic mosquitoes that are partially resistant to malaria infection \[[@B3]\], bringing the efforts to control malaria with the techniques of transgenesis a major step forward \[[@B4],[@B5]\]. A crucial aspect of these advances is, of course, the fact that the mosquito\'s genetic make-up determines, at least partly, its resistance to malaria infection \[[@B6],[@B7]\], giving hope for the possibility that key genes controlling resistance may be identified. This hope has been reinforced by the recent identification, in a rodent model of malaria, of several mosquito immune genes that affect parasite development \[[@B8],[@B9]\]. Unfortunately, several aspects of the current knowledge make it difficult to estimate the relevance of such laboratory-based studies to control malaria in natural conditions \[[@B10]\]. One of the crucial aspects is that most studies on the genetics of resistance have considered the response of a mosquito line or colony to a single malaria genotype, while any malaria control programme based on the release of resistant mosquitoes in highly endemic areas can be effective only if mosquitoes are resistant to all the genotypes of the parasite \[[@B11]\]. Because of the limited genetic variation in laboratory colonies compared to natural populations of mosquitoes \[[@B12]\] and the large diversity of natural populations of malaria parasites \[[@B13]\], it is currently far from clear whether this will be possible. One potential problem of the genetic diversity in natural populations could be that, as in many other host-parasite systems (e.g. plant-fungus \[[@B14]\], snail-schistosome \[[@B15]\], bumble-bee-trypanosome \[[@B16]\], Daphnia-bacterium \[[@B17]\]), the outcome of the interaction is determined by an interaction between host and parasite genotypes. In systems governed by such genotype by genotype interactions, individual hosts are resistant to only a portion of the parasite genotypes and, reciprocally, individual parasites can infect only particular host genotypes \[[@B18]\]. In other words, no parasite is best at infecting all hosts, and no host is best at resisting all parasites, so that the success of infection depends on the specific combination of the two opponents. Despite its potentially important role for malaria epidemiology and control, such a genetic specificity of host-parasite compatibility between malaria parasites and their insect vectors have never been investigated. This study examines the potential for genotype by genotype interactions in the combination that is most important for the epidemiology of malaria -- Plasmodium falciparumand Anopheles gambiae. Malaria genotype by mosquito genotype interactions were tested with a standard procedure of quantitative genetics from measurements of the resistance of different genetic backgrounds of the mosquito A. gambiae, a major malaria vector in sub-Saharan Africa, to different isolates of the human malaria parasite P. falciparum. The parasite isolates were obtained from naturally infected children in western Kenya that harboured gametocytes, the infective stage of the parasite. The genetic backgrounds of mosquitoes were \'mosquito families\' generated as the F1 offspring of single egg-laying females. Each mosquito family was challenged with each parasite isolate, and all mosquitoes were simultaneously fed on the blood of the gametocyte carriers via membrane-feeding. This basic design was repeated three times throughout three successive experimental blocks that involved different families and isolates, giving a total of 18 mosquito families, 11 parasite isolates, and 62 specific interactions. As in previous studies \[[@B7]\], the resistance of mosquitoes was quantified with the proportion of blood-fed females that developed oocysts and with the number of oocysts. The genotype by genotype interaction on mosquito resistance was estimated according to standard quantitative genetic methods as the interaction effect in a statistical analysis between the parasite isolate and the mosquito family \[[@B17],[@B19]\]. These methods are based on the idea that sibs are genetically more similar that non-sibs. Therefore, partitioning the variance of any trait (e.g. number of oocysts) among families (individuals sharing a mother) and within groups of sibs give an indication of the extent to which the trait has a genetic basis \[[@B20]\]. Methods ======= Mosquitoes ---------- The mosquitoes used in this study came from a colony that had been established in 2001 from A. gambiae s.s. caught in the area surrounding Mbita, a small village on the shore of Lake Victoria in Suba District (western Kenya). These mosquitoes had been initially adapted to feed on a Parafilm^®^membrane, and then maintained in standard insectary conditions using a rabbit as a blood source for routine maintenance. Females of the colony were blood-fed on a rabbit and allowed to lay eggs in individual vials. Immediately after hatching, each larva was individually placed in one well of a 12-well plate with three mL of filtered lake water. They were fed daily on a standard diet of Tetramin^®^fish food (0.06 mg per larva on day 0; 0.12 mg on day 1; 0.24 mg on day 2; 0.36 mg on day 3; 0.48 mg on day 4; 0.6 mg on the following days). Adults were kept in an insectary and supplied with a 6% glucose solution and cotton soaked with distilled water. The temperature and humidity in the insectary followed the daily environmental fluctuations. So that mosquito age at pupation did not affect the success of infection, only females that pupated seven days after hatching were used. The wing length of the mosquitoes, measured from the tip (excluding the fringe) to the distal end of the allula with a precision of 0.02 mm, was used as an indication of body size \[[@B21]\]. Where both wings could be measured, the mean of the two lengths was used. Gametocyte carriers ------------------- P. falciparumcarriers were recruited from the two- to 10-year old children in the rural area around Mbita, from December 2003 to January 2004. Finger-prick blood samples were collected and thick blood smears were air-dried, stained with 8% Giemsa during 15 minutes, and examined microscopically for the presence of P. falciparum. Children with asexual parasitemia (\>1,000 parasites/μL) were immediately treated with sulfadoxine-pyrimethamine according to national guidelines. Asymptomatic gametocyte-positive children were recruited for the study after their parents or guardians had signed an informed consent form. The Kenyan and the United States National Institute of Health ethical review committees approved this recruitment procedure. Experimental infections ----------------------- For logistic reasons, the experiment was repeated three times, and within each experimental block infections were done simultaneously. For each of the three blocks, P. falciparumisolates were collected from gametocyte carriers that had been identified one or two days before, and used to feed the mosquitoes on the same single day (block 1: December 14, 2003; block 2: January 23, 2004; block 3: January 28, 2004). The gametocyte densities were assessed just before blood withdrawal on a blood smear (as described above) by counting against 500 leukocytes, and converted to numbers of parasites per μL by assuming a standard leukocyte count of 8,000/μL. Although gametocyte densities in the venous blood and the peripheral finger-prick blood might differ, potential differences were assumed to be proportional among the isolates. A sample of five mL of venous blood was collected from each gametocyte carrier in a heparinized tube, 400 μL of which were stored at -20°C for further parasite genotyping. So that the importance of human factors such as transmission-blocking immunity \[[@B22]\] was reduced, the blood was centrifuged at 37°C for three minutes at 2,000 gand the autologous serum was replaced with the same volume of a pool of AB serum from two French blood donors without any malaria exposure (the same pool of AB serum was used for all experimental blocks). The mixture was used to feed mosquitoes, which had been starved for 12--16 h before blood feeding, with a standard membrane-feeding system \[[@B23]\]. For each mosquito family, i.e. each group of mosquitoes that was derived from the eggs of a single female, equal groups of three-day old females were randomly chosen and fed separately with each isolate. Depending on the size of the family, each feeding cage contained between four and 15 females. Mosquitoes were allowed to take a blood meal for 40 minutes, after which unfed and partially fed mosquitoes were discarded. Seven or eight days after the infective blood meal, mosquitoes were dissected and their midguts were stained with 2% mercurochrome in distilled water in order to detect the presence and number of oocysts by light microscopy. Microsatellite genotyping ------------------------- P. falciparumDNA was extracted from the blood samples using the QIAamp DNA blood kit following the manufacturer\'s instructions (Qiagen, CA). The isolates were typed using the semi-nested PCR method slightly modified from a previous study \[[@B24]\] (details are available upon request to PD) and the markers used \[[@B25]\] and their GenBank accession number in parenthesis are as follows: PJ2 (G37826), UIDG (G37823), Polyα (L18785), TA60 (AF010556), ARA2 (G37848), Pfg377 (L04161), PfPK2 (X63648), TA87 (AF010571), TA109 (AF010508). The microsatellite PCR products were size-genotyped using a standard size Genescan 500 LIZ on an ABI Prism 310 Genetic Analyser (PE Applied Biosystems, CA). Data analysis ------------- Only those mosquito families in which at least four individuals had been fully fed and had survived infection with each isolate were included in the analyses. The likelihood that a mosquito had been infected was analysed with a nominal logistic analysis. The intensity of infection was analysed with an analysis of variance (ANOVA). In this analysis, the square root of the number of oocysts was used, so that the assumptions of the statistical tests (in particular, normality of the residuals) were satisified. As the study was run in three successive experimental blocks, both analyses included the effect of block, and the effects of family, isolate and their interaction. The effect of wing length was also included as a potential confounder \[[@B26]\]. As different families and isolates were used in each experimental block, the factors family, isolate and their interaction were nested within block. Block, family and isolate were considered as random factors. Results ======= The three successive experimental blocks involved three, five and three parasite isolates, and nine, four and five mosquito families, respectively. A third (151) of the 455 mosquitoes of the study were infected by P. falciparumoocysts, and the number of oocysts in infected mosquitoes ranged from one to 97 (mean 11.0, median 3). The prevalence and the number of oocysts differed among blocks (block effect, Table [1](#T1){ref-type="table"}), and were lower in larger mosquitoes (wing length effect, Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Statistical analysis of the effects of mosquito family and parasite isolate on the success of infection. The proportion of infected mosquitoes (a, nominal logistic analysis) and the square root of the number of oocysts (b, ANOVA) were analysed as a function of the mosquito family, the parasite isolate, and their interaction. In both analyses, the mosquito\'s wing length was included as a confounder. As the study was run in three experimental blocks using different families and isolates, the factors family, isolate and their interaction were nested within block. Block, family and isolate were considered as random factors. ::: \(a) Proportion infected \(b) Intensity of infection -------------------------------- ------ -------------------------- ----------------------------- ---------------- ------- --------- Source d.f. χ^2^ P Sum of Squares F P Experimental Block 2 85.5 \<0.001 265.2 2.40 0.155 Wing Length 1 3.1 0.029 6.4 7.14 0.008 Family (within Block) 14 3.8 0.927 29.5 0.66 0.800 Isolate (within Block) 8 15.5 0.482 462.5 19.40 \<0.001 Family\Isolate (within Block) 34 127.2 \<0.001 110.2 3.59 \<0.001 Error (for analysis b) 395 356.8 ::: Family by isolate interaction ----------------------------- While the crude variation among families (averaged across all parasite isolates) was substantial (mean infection rate ranging from four to 83%; mean number of oocysts ranging from 0.1 to 16.4), most of this variation was due to differences among blocks (Fig. [1](#F1){ref-type="fig"}), so that there was no evidence that families differed in the overall proportion of infected individuals or in the intensity of infection (family effect, Table [1](#T1){ref-type="table"}). Similarly, most of the crude differences among isolates (averaged across mosquito families) were due to differences among blocks. Thus, isolates did not vary in the proportion of mosquitoes they infected (ranging from four to 94%), although they did vary in the number of oocysts they produced (median ranging from zero to 35) (isolate effect, Table [1](#T1){ref-type="table"}). More importantly in the context of our study, while neither the families of mosquitoes nor the parasites differed in their average responses to all of their partners, the interaction between mosquito family and parasite isolate (an estimation of the genotype by genotype interaction) had a highly significant effect on the likelihood and the intensity of infection (family by isolate effect, Table [1](#T1){ref-type="table"}). Thus, no parasite isolate was most infectious to every host genotype. Rather, isolates that were most infectious on one host tended to be less infectious than the other isolates on other hosts (Fig. [1](#F1){ref-type="fig"}). Similarly no host genotype was most resistant to every parasite isolate (Fig. [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Graphic representation of the mosquito family by parasite isolate interactions underlying (a) the probability and (b) the intensity of infection. Each point represents the proportion of infected mosquitoes (in a) or the mean of the square root of the number of oocysts (in b) for a given combination of family and isolate. The families are indicated on the x-axes, and are separated into the three experimental blocks of the study with vertical lines. Different colours represent different isolates (squares: isolates containing two clones; circles: isolates containing three clones), and the lines connect points representing the same isolate. Crossing lines give an indication of family by isolate interactions. ::: ![](1475-2875-4-3-1) ::: Genetic characterization of P. falciparumisolates --------------------------------------------------- While the quantitative genetic analysis of the data gives an adequate representation of the genetic basis of the mosquito\'s resistance \[[@B20]\], the use of natural isolates may complicate the interpretation, as (i) they do not necessarily consist of different malaria clones and (ii) isolates often contain several clones in areas where transmission is high \[[@B27],[@B28]\]. However, genotyping the blood samples at nine microsatellite markers showed that the isolates differed. The overall genetic diversity was high, ranging from four to 15 allelic variants per locus. Each isolate had an allelic pattern that differed from all other isolates at, at least, one locus (data not shown), showing that the isolates were genetically distinct. Using the maximum number of alleles at a single locus as a conservative estimate of the number of clones, each isolate was found to contain either two or three distinct clones of P. falciparum(Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Description of P. falciparumisolates. The number of gametocytes per 500 leukocytes, converted to numbers of parasites per μL (assuming a standard leukocyte count of 8,000/μL) and the maximum number of alleles at a single locus found for 9 microsatellite markers (a conservative estimate of the number of clones) are given for each isolate. ::: Experimental block Isolate Gametocyte density (parasites/μL) Number of clones -------------------- --------- ----------------------------------- ------------------ 1 A 176 3 1 B 32 3 1 C 32 2 2 D 32 3 2 E 16 2 2 F 16 3 2 G 32 3 2 H 32 2 3 I 48 2 3 J 16 2 3 K 16 3 ::: Potential confounding effects ----------------------------- Separate analyses of the data for the two numbers of clones (two or three) ensured that the number of clones contained in each isolate did not confound the interpretation. The effect of the mosquito family by parasite isolate interaction on the likelihood of infection was significant in both cases (two clones: P= 0.050; three clones: P= 0.003) and the effect of the interaction on the number of oocysts was significant in one of the cases and showed a tendency in the other case (two clones: P= 0.219; three clones: P= 0.008). In addition, differences in gametocyte density between isolates (Table [2](#T2){ref-type="table"}) may be expected to bias infection success \[[@B23]\]. There were sufficient data to analyse the effects of the mosquito family by parasite isolate interaction separately for the isolates with 16 or 32 gametocytes/μL (i.e. one or two gametocytes per 500 leukocytes). At both gametocyte densities, the interaction significantly influenced the probability of infection (16 gametocytes/μL: P\< 0.001; 32 gametocytes/μL: P\< 0.001) and the number of oocysts (16 gametocytes/μL: P\< 0.001; 32 gametocytes/μL: P\< 0.001). In conclusion, the two potential confounders -- number of clones per isolate and gametocyte density -- had no qualitative influence on the results of the analysis. Discussion ========== While the specificity of mosquito-malaria interactions at the species level is well documented \[[@B29]\], the present results are the first experimental evidence of the genetic specificity of mosquito infection by malaria parasites at the intraspecific level. This finding corroborates an earlier study, where a mosquito line selected for resistance to malaria infection varied considerably in its response against different Plasmodiumspecies and strains \[[@B6]\]. The present study goes one step further by suggesting that mosquito resistance to malaria is at least partly determined by the specific interaction between its own and the parasite\'s genotype. This idea is supported by two other studies showing that, in a strain of mosquitoes selected to resist infection by a wide variety of malaria species, different genetic loci are involved in the responses against different Plasmodiumparasites \[[@B30],[@B31]\]. The present study suggests, moreover, that the genes conferring resistance to a particular parasite depend on the genetic background of the mosquito. The specificity of host-parasite interactions is often postulated to occur at the level of parasite recognition. While the molecular mechanisms of Plasmodiumrecognition by mosquitoes are still largely unknown, a group of thioester-containing proteins (TEPs) represents a promising family of candidate recognition molecules. One of them, the complement-like protein TEP1, has recently been shown to bind to and mediate the killing of the rodent malaria parasite P. bergheiby the mosquito A. gambiae\[[@B9]\]. Moreover, two allelic variants of the TEP1gene are associated to susceptible and refractory strains of A. gambiae\[[@B9]\]. It is therefore tempting to speculate that this protein may be involved in the specific recognition of particular malaria genotypes by the insect\'s immune system. The mosquito genotype by parasite genotype interactions shown in this paper may help to understand some puzzling aspects of the epidemiology of malaria. Thus, even in areas with intense transmission, the probability that a mosquito becomes infected is generally low \[[@B26],[@B32]\]. Furthermore, the probability of infection is generally low even when, as in our study, mosquitoes fed on a blood-meal known to contain infectious gametocytes \[[@B33],[@B34]\]. This could have several explanations: mosquitoes fail to pick up infective gametocytes, transmission-blocking immunity in the human hosts prevents the parasite\'s development within the mosquito \[[@B22],[@B35]\], or parasites are cleared by mosquitoes that mount a sufficiently effective immune response \[[@B2]\]. The present results indicate an additional reason: that many parasites are incompatible with many of the mosquitoes in a natural population. The epidemiological consequences of mosquito genotype by malaria genotype interactions are perhaps most obvious in the context of malaria control with mosquitoes transformed to be resistant against malaria. The strong genetic specificity of compatibility between parasite isolates and individual insect vectors suggests that most studies on the mechanisms underlying the resistance of mosquitoes against Plasmodiummight be misleading for the development of malaria control strategies. Indeed, most of these laboratory-based studies focus on the response of one mosquito line or colony against a single parasite strain and thus do not represent the genetic diversity of mosquitoes and parasites in natural populations \[[@B12],[@B13]\]. However, any malaria control programme based on the release of mosquitoes harbouring \'resistance genes\' is unlikely to be effective if resistance is expressed against only a subset of the parasite genotypes of the local population. Indeed, as parasites facing resistant mosquitoes will be under strong selective pressure to avoid mosquito defence mechanisms, genotypes that are eliminated by the resistance genes might be replaced rapidly by genotypes that cannot be controlled. Thus, before a possible release of transgenic mosquitoes, it will be crucial to ensure that the transformed mosquitoes are resistant to all of the parasite genotypes in the local population. This reinforces the idea that any release of genetically modified mosquitoes for reducing transmission of mosquito-borne diseases must be preceded by studies that have moved from the laboratory to the field \[[@B10]\]. Conclusions =========== This study demonstrated that the resistance of an anopheline mosquito to P. falciparum*development, a major component of its vector competence, varies considerably between different combinations of parasite isolates and individual, genetically variable, vectors. Optimal transmission may thus require some specific compatibility between the insect\'s and the parasite\'s genotypes. This result has important consequences for the epidemiology of malaria. Overall, it suggests that conclusions from a particular subset of mosquito and malaria genotypes will not necessarily hold for other combinations of genotypes. Therefore, field studies taking into account the full diversity of mosquito and parasite populations are necessary to reach valid conclusions concerning the technologies developed in laboratories for the control of malaria. Authors\' contributions ======================= LL participated in the design and the coordination of the study, carried out the fieldwork, participated in the molecular analysis, performed the statistical analysis, and wrote the manuscript. JH participated in the fieldwork. PD carried out the molecular analyses and helped to draft the manuscript. LCG supervised and coordinated the fieldwork. JCK conceived and designed the study, performed the statistical analysis, and wrote the manuscript. Acknowledgments =============== The authors thank J. Kongere, D. Oulo, L. Omukuba, J. Wauna, S. Orao, S. Ombonya and S. Otieno for their assistance during the fieldwork in Mbita, F. Renaud for helpful discussions, O. Kaltz for statistical advice, and two anonymous reviewers for their comments on a earlier version of the manuscript.	PubMed Central	2024-06-05T03:55:52.706672	2005-1-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548507/", "journal": "Malar J. 2005 Jan 11; 4:3", "authors": [ { "first": "Louis", "last": "Lambrechts" }, { "first": "Jean", "last": "Halbert" }, { "first": "Patrick", "last": "Durand" }, { "first": "Louis C", "last": "Gouagna" }, { "first": "Jacob C", "last": "Koella" } ] }
PMC548508	Background ========== This is the second presentation from a project for the International Programme on Chemical Safety (IPCS) evaluating the possible use of transgenic animal mutagenicity assays in toxicity testing and mechanistic research. Part I, preceeding this article, discussed comparison of effects of chemicals using certain transgenic assays with results using the bone marrow micronucleus test. The assessment of the potential genotoxicity of chemicals in vivois important for both the verification and confirmation of intrinsic mutagenicity and for establishing the mode of action of chemical carcinogens. Although the present trend is to reduce animal testing, in vitrodata must be confirmed by testing in in vivoconditions which take into account whole animal processes like absorption, tissue distribution, metabolism and excretion of the chemical and its metabolites, and overall toxicity \[[@B1]\]. In the mid 1980s, the mouse spot test \[[@B2]\] was suggested as a complementary in vivotest to the bacterial mutagenicity assay for detection of mutagenic substances and as a confirmatory test for the identification of carcinogens \[[@B3]\]. The mouse spot test has been used to assess a number of chemicals (see e.g. [Additional file 1](#S1){ref-type="supplementary-material"}, see separate file). It is at present the only in vivomammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484 \[[@B4]\]). However to achieve an acceptable sensitivity, a large number of animals are necessary and it is therefore an expensive type of test and seldom used. More recently assays using transgenic animals have been developed for testing in vivogene mutagenicity. The two transgenic mouse models with the best data base available are the lacImodel (commercially available as the Big Blue^®^mouse), and the lacZmodel (commercially available as the Muta™ Mouse). The present study compares the results of in vivotesting of a number of chemicals using the mouse spot test and compares it with results from these two transgenic mouse models. Descriptions of test systems ---------------------------- ### Mouse spot test In the spot test, mouse embryos which are heterozygous for different recessive coat colour genes, are treated in uteroat gestation day 9--11 with the test substance. The exposed embryo at gestation day 10 contains about 150--200 melanoblasts and each melanoblast has 4 coat colour genes under study \[[@B2],[@B5]\]. The in uteroexposure may result in an alteration or loss of a specific wild-type allele in a pigment precursor cell resulting in a colour spot in the coat of the adult animal. The frequency of spots is compared with the frequency in sham-exposed controls \[[@B2],[@B4]\]. In the mouse spot test there are 4 possible mechanisms by which the recessive coat-colour alleles can be expressed: 1) gene mutation in the wild-type allele, 2) deficiency (large or small) of a chromosomal segment involving the wild-type allele, 3) nondisjunctional (or other) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) \[[@B5]\]. Gene mutagenic but also clastogenic effects are detected by this test system. ### Transgenic mouse models The transgenic mutation test systems the lacImodel (Big Blue^®^mouse), and the lacZmodel (Muta™ Mouse) are described in detail in the preceding article: Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test Methods ======= Data presented in this documentation are the results of an extensive literature research. Concerning data on transgenic mouse assays only primary literature was used. Data on the mouse spot test were extracted from reliable reviews on this item or from primary literature. For all other data informations from secondary literature or data banks were used. Results and Discussion ====================== Comparison of the mouse spot test with transgenic mouse model systems --------------------------------------------------------------------- In the literature search chemicals have been identified that had been tested using the spot test andthe Muta™ mouse assay (n = 20) or the Big Blue^®^mouse assay (n = 9) or both transgenic mutation assays (n = 8). The results (including references) are given in [Additional file 1](#S1){ref-type="supplementary-material"}. The results on 15 out of 20 substances (2-acetylaminofluorene, acrylamide, benzo\[a\]pyrene, 1,3-butadiene, cyclophosphamide, ethylmethanesulfonate, N-ethyl-N-nitrosourea, N-methyl-N\'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, 4-nitroquino-line-1-oxide, N-nitrosodiethylamine, N-nitrosodimethylamine, procarbazine, 4-acetylaminofluorene and N-propyl-N-nitrosourea) showed agreement between the Muta™ mouse and the mouse spot test. No agreement was seen with 5 out of 20 substances (4-acetylaminofluorene, 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), hydrazine, mitomycin C, trichloroethylene). The positive results obtained with the Big Blue^®^mouse assay agreed with results in the mouse spot test for 7 out of 9 substances (2-acetylaminofluorene, benzo\[a\]pyrene, 1,3-butadiene, cyclophosphamide, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, N-nitrosodimethylamine); one (di-(2-ethylhexyl)phthalate) was negative in both test systems and only one (methyl methanesulfonate) showed no agreement between the two test systems. With two exceptions, 4-acetylaminofluorene and N-propyl-N-nitrosourea (discussed later), all of the tested substances showed also clearly positive results in in vitrogene mutation assays (exception of 1,3-butadiene, negative results) and in the majority of in vivostudies on this endpoint. Further they induced carcinogenic effects in long-term studies on mice. Although no data on carcinogenicity on mice is available on N-propyl-N-nitrosourea, this substance might also be included in the category mentioned above, since carcinogenic effects were reported in rats \[[@B113]\] and in vitrogene mutation assays revealed clearly positive results. The following substances did [not]{.underline} show agreement between results in the mouse spot test and transgenic mouse assays or negative results were reported in both test systems (see [Additional file 1](#S1){ref-type="supplementary-material"}). These are therefore discussed in more detail here; for references see Additional file [1](#S1){ref-type="supplementary-material"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of the Muta™ mouse assay and the Big Blue^®^mouse assay for predicting mouse carcinogenicity in comparison with the mouse spot test ::: Term\# Calculation\* for the mouse spot test Calculation\* for Muta™ and/or Big Blue^®^mouse combined \\ ------------------------- --------------------------------------- --------------------------------------------------------------- Sensitivity 84% (16/18) 79% (15/18) Specificity 0 (0/0) 0 (0/0) Positive predictability 100% (16/16) 100% (15/15) Negative predictability 0 (0/2) 0 (0/3) Overall accuracy 84% (16/18) 79% (15/18) \# Sensitivity = % of carcinogens with a positive result in the specified test system (STS) Specificity = % of noncarcinogens with a negative result in the STS Positive predictivity = % of positive results in the STS that are carcinogens Negative predictivity = % of negative results in the STS that are noncarcinogens Overall accuracy = % of chemicals tested where STS results agree with the carcinogenicity results \: carcinogens with genotoxic and nongenotoxic mechanisms were considered but not substances without data on carcinogenicity; only data on mice were used \\: judged as positive in transgenic assays if positive in one of the two test systems For methylmethanesulfonate, the weak positive results were judged as positive. Trichloroethylene was not included in the calculation (inconclusive results in the mouse spot test). ::: ### 4-Acetylaminofluorene This substance showed mutagenic activity in the Muta™ mouse assay \[[@B19]\] but negative results in the mouse spot test \[[@B12],[@B13]\]. No data on carcinogenicity are available on 4-acetylaminofluorene. However, data on two in vitrotest systems indicated gene mutagenic activity supporting results in the transgenic assay \[[@B15]-[@B18]\]. ### 2-Amino-3-methylimidazo(4,5-f)quinol (IQ) IQ is mutagenic in the Muta™ mouse assay \[[@B28]\] but negative results were obtained in the mouse spot test \[[@B29]\]. This negative result in the mouse spot test is in contrast to all other in vivogene mutation assays on rodents and insects which revealed positive results \[[@B27]\]. Furthermore, gene mutagenic activity was detected in in vitrotest systems and carcinogenic effects were observed in long-term studies on mice \[[@B27]\]. The results in the Muta™ mouse assay are in accordance with these data. ### Di-(2-ethylhexyl)phthalate Negative results in the mouse spot test \[[@B51]\] are in agreement with the negative Big Blue^®^assay \[[@B11]\]. Furthermore no gene mutagenic or questionable activity was reported in in vitrotests and in tests on Drosophila. Carcinogenic effects were obtained in studies on mice but nongenotoxic mechanisms are presumed. ### Hydrazine This substance induced mutagenic effects in the mouse spot test \[[@B72]\] but negative results were observed in the Muta™ mouse assay \[[@B71]\]. Other in vivoas well as in vitrotest systems revealed gene mutagenic effects \[[@B70]\]. Increased tumor incidences were observed in carcinogenicity studies on mice. Overall, the mouse spot test but not the Muta™ mouse assay reflects data on genotoxicity and carcinogenicity. However, a single exposure was used in the Muta™ mouse assay \[[@B71]\]. Studies on other in vivogenotoxicity endpoints have shown generally negative results after single exposure but genotoxic activity after repeated application, for example the mouse bone marrow micronucleus assay was positive \[[@B20]\]. It is possible that positive results may be found using another experimental design in the Muta™ mouse assay e.g. repeated exposure. ### Methyl methanesulfonate Only weak mutagenic effects were observed in the Muta™ mouse \[[@B19],[@B57],[@B75]-[@B77]\] and negative results in the Big Blue^®^mouse \[[@B63]-[@B65],[@B78]\]. In the mouse spot test this carcinogenic substance is mutagenic \[[@B3]\] as well as in other gene mutation assays in vitroand in vivo\[[@B73],[@B74]\]. However, there is evidence that the chromosome mutagenic activity is detectable at much lower doses than the gene mutagenic activity. Tinwell et al. \[[@B19]\] have shown in Muta™ mice a weak gene mutagenic effect in the liver but no effect in the bone marrow. The same dose induced in these animals a significant increase in bone marrow micronuclei indicating clear clastogenic activity. However, the transgenic mutation assay is less suitable for detection of these effects \[[@B1]\]. ### Mitomycin C No mutagenic activity was observed in the Muta™ mouse assay after single application and ambiguous results after repeated exposure \[[@B93]\] but positive results were obtained with the mouse spot test \[[@B2],[@B3]\] and other gene mutation assays in vitroand in vivowith this carcinogenic substance \[[@B90]-[@B92]\]. The reason for this discrepancy is similar to that presumed for methyl methanesulfonate above. Clastogenicity in bone marrow but no gene mutagenic activity in liver and bone marrow has been shown in the same animals in the Muta™ mouse assay combined with a micronucleus assay \[[@B93]\]. However, using another experimental design for detection of gene mutations in the Muta™ mouse assay (dose level up to the MTD, repeated exposure) positive results might be obtained. ### Trichloroethylene Also with this carcinogenic substance, no mutagenicity was detected in the Muta™ mouse assay \[[@B117]\], the mouse spot test was positive \[[@B3]\], but this result is possibly related to contaminations with epoxides \[[@B116]\]. Further in vitroand in vivoassays on gene mutation resulted in weak positive, questionable, or negative effects \[[@B116]\]. Results in chromosome mutation assays are equivocal. However, a further (simple) reason for this discrepancy between the Muta™ mouse assay and the mouse spot test might be that the MTD was not reached in the Muta™ mouse assay presented by Douglas et al. \[[@B117]\]. In general, from the studies on genotoxic carcinogens given above, the results do not seem to give a preference for either the spot test or transgenic mouse model system. However, considering the mechanisms of action of specific substances there is some evidence, that the mouse spot test detects gene mutations as well as chromosome mutations whereas the transgenic mouse assays are restricted to gene mutations. Evidence for this hypothesis has been shown with the examples methyl methanesulfonate, mitomycin C, and trichloroethylene. In the mouse spot test, there are four possible mechanisms by which the recessive coat-colour alleles can be expressed (see introduction) including gene and chromosome mutations. Although the chromosome mutations have to survive several mitoses to cause the expression of the recessive allele \[[@B118]\], there is evidence that also predominantly clastogenic substances might result in a positive mouse spot test. In contrast, the transgenic mutation assays detected point mutations and maximal small deletions and insertions \[[@B1]\]. Predictivity of the transgenic animal assays and the mouse spot test for carcinogenicity ---------------------------------------------------------------------------------------- The sensitivity, specificity and predictivity of carcinogenicity for the transgenic mouse model (Muta™ mouse assay and the Big Blue^®^mouse assay combined) and the mouse spot test are documented in Table [1](#T1){ref-type="table"}. Data on 18 substances (see [Additional file 1](#S1){ref-type="supplementary-material"}) are available on carcinogenicity in mice [and]{.underline} mutagenic effects in transgenic mice as well as mutagenic effects in the mouse spot test (trichloroethylene not included because of inconclusive results in the mouse spot test). Although the data pool is not sufficient for a comprehensive comparison, there is some indication, that no significant differences were detectable between the two test systems. Advantages and disadvantages of both test systems ------------------------------------------------- ### Sensitivity of the test system In comparison to models using endogenous genes like the target genes in the mouse spot test, the spontaneous mutant frequency in transgenic animals is relatively high. This might be due to the fact that bacterial DNA is the target gene (high methylation rate) and/or the transgene is silent and no transcription related repair occurs as in endogenous genes which are more efficiently repaired \[[@B1]\]. However, comparing the number of cells and genes at risk at the time of exposure, the mouse spot test is numerical inferior to the transgenic mouse mutation assays. In the mouse spot test, the exposed embryo at gestation day 10 contains about 150--200 melanoblasts and each melanoblast has 4 coat colour genes under study \[[@B2],[@B5]\]. In the transgenic Big Blue^®^mouse, for example, 30--40 copies of the target gene (the constructed λLIZα shuttle vector) are integrated on chromosome 4 of eachcell of the animal \[[@B1]\]. ### Other factors To achieve an acceptable sensitivity, a large number of animals are necessary in the mouse spot test. Many pregnant dams have to be in one treatment group to get a sufficient number of surviving F1-animals, since the test substance may induce maternal and developmental toxicity. Fahrig \[[@B2]\] suggested that 30--40 pregnant mice are needed per treatment group for evaluation of spots in the progeny. At least 150 F1-mice are recommended for the concurrent vehicle control \[[@B5]\] and at least two dose groups are used (OECD guideline 484 \[[@B4]\]). Therefore, the mouse spot test is an expensive type of in vivotest. In contrast, in transgenic mutation assays ca. 20 animals (3 dose groups and 1 concurrent vehicle control group in laboratories which already established this test system) are recommended per species and gender \[[@B119]-[@B121]\]. In the mouse spot test the discrimination between spots of mutagenic and non-mutagenic origin may be problematic \[[@B2]\]. A comparison of both test systems is presented in Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Advantages and Disadvantages of mouse spot test compared to the transgenic Big Blue^®^and Muta™ mouse assays ::: Mouse spot test ^\[2-5\]^* Transgenic mouse mutation assay^\[1,\ 122\]^ ---------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ [Age restriction]{.underline} Exposure restricted to embryos at gestation day 9--11 Usually less than 3 months [Toxicokinetics and metabolism]{.underline} Restrictions in toxicokinetics: test substance reaches the fetal melanoblasts after administration to the dams and absorption of the test substance itself or the toxic metabolites via the placenta No further barrier like the placenta after absorption and distribution [Target tissue]{.underline} Restricted to melanoblasts No tissue restriction; analysis of mutagenic potency in different organs [Type of mutation]{.underline} Detects 1) gene mutation, 2) large or small deletions, 3) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) Detects 1) gene mutation, 2) small deletions or insertions [Dependency of effects on application route]{.underline} Only systemic effects can be detected; no application route specific effects For different routes systemic as well as local mutagenic effects can be detected [Target gene/cell]{.underline} 4 genes per cell in ca. 200 melanocytes Ca. 40 (Big Blue) or ca. 80 (Muta™ mouse) copies of the transgene per nucleus of each cell of the organismk [Number of animals]{.underline} Animal consuming test system Not more than 5 animals per gender per dose necessary [Specificity of test system]{.underline} Discrimination between spots of mutagenic and non-mutagenic origin may be problematically Identifying and isolating mutated genes with a high specificity [Characterisation of mutations by molecular methods]{.underline} Less suitable for identification of mutations in DNA analysis due to size of the genes detection of the \"molecular signature\" of a particular mutagen by DNA sequence analysis with standardized methods [Possibility of parallel investigation of several genetic endpoints]{.underline} No combination with other genotoxic endpoints possible The transgenic mouse assay can be combined with other in vivogenotoxic endpoints in the same animal: e.g. micronuclei, chromosomal aberration, unsheduled DNA synthesis, sister chromatid exchange [Endogenous versus foreign target gene]{.underline} The mouse spot test shows an in situ end point (expression of the target genes) Target genes are integrated parts of foreign DNA and consequently no \"normal\" mutational target [Costs]{.underline} Expensive type of in vivotest Less expensive ::: Conclusions =========== Although the mouse spot test is a standard genotoxicity test system according to the OECD guidelines, this system has seldom been used for detection of somatic mutations in vivoin the last decades. This is partly due to considerations of cost effectiveness and number of animals needed for testing but also for toxicological considerations. The usefulness of the mouse spot test in toxicology is limited by restrictions in toxicokinetics, sensitivity, target cell/organ, and molecular genetics. From the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations in vivo. Author\'s contributions ======================= UW was the main author. The other authors were involved in the discussions, writing small parts of text and in final preparation of the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Results in the transgenic mouse assay versus mouse spot test ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This paper is based on work performed by the authors in preparation of an Environmental Health Criteria document on \'Transgenic Animals in Mutagenicity Testing\' for the International Programme on Chemical Safety (IPCS). However, opinions expressed in this paper are the sole responsibility of the authors. We acknowledge the financial support of the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety.	PubMed Central	2024-06-05T03:55:52.709135	2005-1-27	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548508/", "journal": "J Carcinog. 2005 Jan 27; 4:4", "authors": [ { "first": "Ulrich", "last": "Wahnschaffe" }, { "first": "Annette", "last": "Bitsch" }, { "first": "Janet", "last": "Kielhorn" }, { "first": "Inge", "last": "Mangelsdorf" } ] }
PMC548509	Background ========== Injuries have been recognised as a major public health problem in both developed and less developed countries \[[@B1]\]. It is generally acknowledged that this problem is growing rapidly in sub-Saharan Africa \[[@B2]\]. Studies in Tanzania show that injuries are an important cause of death among adults \[[@B3]\], and accounted for 12% of all admissions at the national hospital in the country\'s largest city \[[@B4]\]. Studies on the magnitude of injuries and the groups at risk, have been conducted world-wide, and especially in developed countries. Hospital based studies, which are commonly reported from developing countries, presumably provide a representative picture of the prevalence and incidence of serious injury, but only a partial picture of the circumstances in which injuries occur. Given the limited access to hospital care in poor countries, however, data based on health facility data are not likely to be representative. In contrast, population-based studies are costly and rarely carried out particularly on topics such as injury, which are not high on the public health agenda in developing countries at present. Several studies have been conducted in high-income countries to examine factors associated with injury morbidity \[[@B5]-[@B7]\]. In developing countries, a number of population-based studies on nonfatal injuries have been done \[[@B8]-[@B14]\]. In order to understand the circumstances and risk factors associated with nonfatal injuries, we conducted a community-based study in an urban and a rural location of Tanzania. We describe injury patterns in both settings. We also investigate demographic and socioeconomic factors associated with nonfatal injuries. Methods ======= Study area ---------- The survey was conducted in Dar es Salaam city (an urban area) and Hai District (a rural area). These areas are part of a health and demographic surveillance system carried out by the Adult Morbidity and Mortality Project (AMMP) in six districts in Tanzania from 1992 to 2004. One aim of the project was to measure rates and causes of morbidity and mortality. At the time this investigation was carried out, the areas were being prospectively monitored through repeated censuses to ascertain the resident population at risk. Deaths were recorded through an active reporting system and probable cause of death determined by a validated verbal autopsy \[[@B15],[@B16]\]. Dar es Salaam city lies on the east coast of Tanzania. The AMMP demographic surveillance population was situated in two of the city\'s three municipalities. These areas contained 8 \'branches\' (an urban administrative unit) and covered 63,330 persons in 15,269 households living in urban and peri-urban neighbourhoods. Hai District lies on the South-Western slopes of Mount Kilimanjaro in Northern Tanzania. The AMMP demographic surveillance area in Hai covered 51 out of 61 villages in the district and around 62% of the total district population (159,906 persons in 40,238 households). Agriculture, livestock keeping and commercial mining are the main economic activities there. Details of the study population have been described elsewhere \[[@B15],[@B16]\]. Sampling procedure ------------------ In the urban surveillance area, an initial cluster sample of 500 households was randomly selected. Because initial data collection yielded fewer injuries than expected in the first two enumeration areas, the sample size was increased to 2,000 households with the difference made up of households selected at random from the remaining six surveillance branches. Thus, the final sample under-represented the first two branches. Information was sought on all individuals residing in the selected households. A two-stage cluster sampling method was adopted in selecting the rural sample. In the first stage, using existing AMMP data on mortality and poverty, six out of 51 villages were selected to represent different levels of socio-economic status and injury mortality. A random sample of 2,000 households was obtained from the selected villages in the second stage. All individuals in the selected households were included in the survey. Ethical clearance and informed consent -------------------------------------- Ethical clearance for this study was given by the Tanzania Commission for Science and Technology and the Regional Committee for Medical Research Ethics in Norway. Informed verbal consent was sought from each family. For children below age 15, parents or guardians were interviewed; for adolescents aged 15 to 18, consent was obtained from both the parent and the child. Data collection --------------- The survey tool was translated into Swahili, back translated into English, and pre-tested before use in the field. Two questionnaires were used in the study. Questionnaire 1 was a screening form used to identify whether a household member had an injury in the past one year that resulted in losing one or more days of \'normal\' activity (e.g. not being able to work or go to school). The head of household or any other responsible person was interviewed to obtain information about the household members. Variables included were age, sex, relationship to head of household, level of education, religion, marital status and occupation. Questionnaire 2 was used to record the circumstances in which the injury occurred. Some of the variables included were: month and year when the injury occurred; cause of the injury; place of occurrence; activity at time of injury; length of disability; and health facility use. Efforts were made to interview the injured person if an adult, otherwise we interviewed an informed member of the injured person\'s household. The number of days with restricted activity (disability days) was considered as a measure of severity of injury. Poverty was assessed at the household level using data from the 2000--2001 National Household Budget Survey and variables from AMMP data. The measure of poverty used was a predicted value of monthly consumption expenditure per adult equivalent for each household included in the study \[[@B17],[@B18]\]. Statistical analysis -------------------- Data analysis was done using STATA (version 7). Bivariate analyses were performed by cross tabulations and the chi-squared test was used to test for homogeneity. In the case of multiple injuries, the most recent injury episode was considered in all the analysis. Multiple logistic regression was used to examine the influence of socio-demographic and socio-economic factors on the risk of being injured, controlling for potential confounding variables. Odds ratios are reported with 95% confidence intervals. Tests for trend in associations over groups defined by other factors were performed where appropriate. Preliminary analysis indicated that a long recall period underestimated annual injury rates, with the effect being greater for injuries resulting in \<30 disability days while the rates for injuries resulting in 30 or more disability days were quite stable\[[@B19]\]. We therefore categorized severity of injury as \'minor\' if resulting in less than 30 days of lost activity and \'major\' if resulting in 30 or more days of lost activity. This kind of categorization has also been used in the Ghana study \[[@B13]\]. The category for major injuries is less likely to include actual minor injuries, and therefore constitutes a well-defined small group. However, the category for minor injuries might include some injuries that were actually severe. About 37 (7.3%) individuals who sustained an injury reported between 15 and 21 disability days, with only 3 reporting 22 to 29 days of restricted activity. Poverty quintiles were classified as most poor, very poor, poor, less poor, or least poor in terms of socioeconomic status. Adjustment for clustering was performed with standard STATA commands for analysis of survey data. Results ======= Data were gathered on a total of 15,223 individuals residing in 3,653 households. The urban sample included 8,188 individuals while the rural sample included 7,035 persons. The response rates were 89% and 92% for the urban and rural areas respectively. The rural sample had a larger proportion of individuals aged 44 years and above (24%) compared to the urban area (11%). This was comparable to national averages as reported by the 2002 national census in Hai (17%) and Dar es Salaam (10%)\[[@B20]\]. Educational status was higher in the urban area. Of the total sample, 509 persons reported to have sustained an injury in the past one year preceding the survey, representing an injury incidence of 32.7 per 1,000 persons per year (95% CI= 29.9 -- 35.7). The incidence for all, minor and major injuries was 24.5, 16.4 and 8.1 per 1,000 persons per year for Dar es Salaam, and 42.5, 32.8 and 9.7 for Hai district. The mean age of the injured was 27.6 years (standard deviation 20) and 62% were males. Almost all injuries were unintentional (96%). On average, 14 days of normal activity were lost per person because of an injury. In Dar es Salaam, the most common cause of injury reported in both males and females was transport injuries, followed by falls and cuts (Table [1](#T1){ref-type="table"}). In Hai, cuts ranked first, followed by falls and transport injuries. The proportion of individuals who sustained transport injuries in the urban area was four times higher than in the rural area (33.0% vs 7.6% respectively; p \< 0.001). Cuts and stabs accounted for 49% of the injuries in Hai compared to only 18% in Dar es Salaam (p \< 0.001). There was no statistical difference in the distribution of injury categories between males and females in the urban area (p = 0.37). In the rural area, males and females differed with respect to the most common causes of injuries (p \< 0.001), with transport injuries experienced almost exclusively by males, and cuts being more frequent in females. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Cause of nonfatal injury by sex in the urban and rural areas ::: Cause of injury Total Males Females ----------------------------- ----------- ----------- ------------- ----------- ----------- ----------- *Urban (Dar es Salaam)* *Total* *206* *100* *136* *100* *70* *100* Transport injuries 68 33.0 43 31.6 25 35.7 Falls 56 27.2 35 25.7 21 30.0 Cuts/stabs 38 18.5 26 19.1 12 17.1 Burn 12 5.8 6 4.4 6 8.5 Struck by object 12 5.8 10 7.4 2 2.9 Animal bites 4 1.9 3 2.2 1 1.4 Assault 7 3.4 4 2.9 3 4.3 Other 9 4.4 9 6.7 0 0 *Rural (Hai)* *Total* *303* *100* *177* *100* *126* *100* Transport injuries 23 7.6 22 12.4 1 0.8 Falls 83 27.4 49 27.7 34 26.9 Cuts/stabs 149 49.2 77 43.5 72 57.1 Burns 18 5.9 8 4.5 10 7.9 Struck by object 11 3.6 11 6.2 0 0.0 Animal bites 6 1.9 4 2.3 2 1.6 Assault 3 0.9 3 1.7 0 0 Other 10 3.3 9 5.1 1 0.8 ::: As expected, the cause of injury varied by age (Figure [1](#F1){ref-type="fig"}). In the urban area, transport injuries were most common among adults aged 15 years and above while burns were common among children under 5 years. Cuts and stabs ranked second as a cause of injury among the 5 to 14 year olds. In Hai, cuts were the commonest cause of injury in all age groups except among 0--4 year olds where burns and falls were most frequent. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### 1a: Cause of injury by age in Dar es Salaam (urban area) 1b: Cause of injury by age in Hai (rural area) ::: ![](1471-2458-5-11-1) ::: Major injuries accounted for 33% and 23% of all injuries in Dar es Salaam and Hai respectively (Table [2](#T2){ref-type="table"}). The percentage of transport injuries resulting in a major injury was 41% and 30% in the urban and rural areas respectively. Of cuts or stabs in the rural area, 13% were major whilst in the urban area, 21% of the cuts were major. More than one third of the falls were categorised as major injuries in both areas. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Major injuries as a percentage of all injuries (≥ 30 disability days) by cause and sex in the urban and rural areas ::: ----------------------------- ------------------------- --------------------------- ----------- ------------- Cause of injury No. of all injuries Both sexes Males Females Percent of major injuries *Dar es Salaam (Urban)* Total 206 33.0 28.7 41.4 Transport injuries 68 41.2 34.9 52.0 Falls 56 35.7 28.6 47.6 Cuts/stabs 38 21.1 23.1 16.7 Burn 12 33.3 16.7 50.0 Struck by object 12 33.3 30.0 50.0 Other 20 20.0 30.8 0 *Hai (Rural)* Total 303 22.8 23.7 21.4 Transport injuries 23 30.4 27.3 100.0 Falls 83 38.6 36.7 41.2 Cuts/stabs 149 12.8 13.0 12.5 Burns 18 16.7 25.0 10.0 Struck by object 11 45.5 45.5 0 Other 19 15.8 10.0 28.6 ----------------------------- ------------------------- --------------------------- ----------- ------------- ::: After controlling for age, sex and education, persons residing in Hai were 1.7 times as likely to have had an injury in the past one year as compared to those residing in Dar es Salaam (Table [3](#T3){ref-type="table"}). Males had a higher risk of being injured than females. Those with primary education only had an increased risk of having an injury compared to their counterparts who had no formal education. Children aged 5 to 14 had slightly higher odds of sustaining a minor injury compared to adults, while for major injuries adults aged 45 years and above were at an increased risk (p \< 0.01 comparing trends with age for minor and major injuries). After adjusting for age, sex, education and area, there was no significant association between poverty and risk of nonfatal injury. Male sex turned out to be the only significant risk factor for major injuries. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Adjusted odds ratios (OR) for all, minor (\<30 disability days) and major (≥ 30 disability days) injuries by demographic and socio-economic factors ::: Factors Total All injuries Major injuries Minor injuries ----------------------------------------- ------------ ------------------ --------------------- -------------------- --------------------- --------- --------------------- Sample No. OR^a^(95% CI) No. OR^a^(95% CI) No. OR^a^(95% CI) Area Dar es Salaam (Urban) 8188 206 1.0 68 1.0 138 1.0 Hai (Rural) 7035 303 1.66 (1.37 -- 2.02) 69 1.09 (0.75 -- 1.58) 234 1.94 (1.54 -- 2.44) p \< 0.001 p = 0.65 p \< 0.001 Age\* 0--4 1720 49 1.28 (0.83 -- 1.99) 13 0.85 (0.38 -- 1.91) 36 1.53 (0.91 -- 2.57) 5--14 3711 140 1.23 (0.98 -- 1.56) 29 0.88 (0.56 -- 1.40) 111 1.37 (1.04 -- 1.79) 15--44 7140 212 1.0 59 1.0 153 1.0 45+ 2651 108 1.25 (0.97 -- 1.59) 36 1.57 (0.99 -- 2.47) 72 1.12 (0.84 -- 1.50) p = 0.20 p = 0.10 p = 0.11 Sex Females 7844 196 1.0 56 1.0 140 1.0 Males 7379 313 1.75 (1.46 -- 2.12) 81 1.57 (1.11 -- 2.21) 232 1.81 (1.46 -- 2.25) p \< 0.001 p \< 0.01 p \< 0.001 *Education\\*** None 3546 97 1.0 30 1.0 67 1.0 Primary 9674 362 1.50 (1.09 -- 2.06) 92 1.02 (0.59 -- 1.74) 270 1.77 (1.21 -- 2.59) Secondary+ 2001 50 1.18 (0.77 -- 1.82) 15 0.79 (0.37 -- 1.71) 35 1.41 (0.84 -- 2.36) p = 0.01 p = 0.67 p \< 0.01 *Poverty quintiles (n = 12320)\\\** 1 (Most poor) 2471 95 1.13 (0.82 -- 1.56) 22 1.05 (0.56 -- 1.96) 73 1.15 (0.80 -- 1.66) 2 2462 78 0.92 (0.66 -- 1.29) 21 1.01 (0.52 -- 1.97) 57 0.89 (0.61 -- 1.32) 3 2466 81 0.96 (0.69 -- 1.32) 23 1.11 (0.61 -- 2.01) 58 0.91 (0.62 -- 1.32) 4 2462 72 0.86 (0.61 -- 1.22) 25 1.24 (0.67 -- 2.29) 47 0.74 (0.49 -- 1.12) 5 (Least poor) 2459 83 1.0 20 1.0 63 1.0 p = 0.49 p = 0.96 p = 0.19 \* 1 missing subject, \\ 2 missing subjects ^a^Adjusted odds ratios from logistic regression models that included all variables in table except poverty \\\* Adjusted for age, sex, area and education p-value for test of heterogeneity ::: We investigated associations between different factors and some of the most frequent causes of injury (Table [4](#T4){ref-type="table"}). Rural residents were significantly less likely to have transport injuries compared to urban dwellers (OR = 0.39; 95% CI 0.23 -- 0.66). Children aged between 5 and 14 years were less likely to sustain transport injuries compared to adults aged 15--44 years. Males had a significantly increased risk of having transport injuries compared to females. However, household consumption expenditure was not associated with risk of transport injuries. In the rural area, the commonest type of transport involved was bicycle (52%) while in the urban area cars and trucks (46%) and commercial buses (22%) were frequently involved (Table [5](#T5){ref-type="table"}). In Dar es Salaam, 41% of those involved in transport injuries were pedestrians who were struck by motor vehicles or bicycles, whereas in Hai, the largest proportion of those with a transport injury were vehicle occupants (52%) followed by cyclists (35%). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Adjusted odds ratios (OR) for transport injuries, falls and cuts or stabs by demographic and socio-economic factors ::: Factors Total Sample Transport injuries Falls Cuts/stabs ----------------------------------------- ------------------ ------------------------ --------------------- ---------------- --------------------- --------- --------------------- No. OR (95% CI)^a^ No. OR (95% CI)^a^ No. OR (95% CI)^a^ Area Dar es Salaam (Urban) 8188 68 1.0 56 1.0 38 1.0 Hai (Rural) 7035 23 0.39 (0.23 -- 0.66) 83 1.56 (1.09 -- 2.25) 149 4.27 (2.96 -- 6.15) p \< 0.001 p = 0.01 p \< 0.001 Age\* 0--4 1720 5 0.52 (0.14 -- 1.97) 15 2.21 (0.98 -- 4.98) 5 0.35 (0.12 -- 0.99) 5--14 3711 8 0.28 (0.13 -- 0.63) 51 2.44 (1.58 -- 3.79) 54 1.12 (0.77 -- 1.62) 15--44 7140 60 1.0 41 1.0 80 1.0 45+ 2651 18 1.01 (0.56 -- 1.83) 32 1.98 (1.22 -- 3.21) 48 1.19 (0.82 -- 1.74) p \< 0.01 p \< 0.001 p = 0.07 Sex Females 7844 26 1.0 55 1.0 84 1.0 Males 7379 65 2.66 (1.64 -- 4.29) 84 1.62 (1.14 -- 2.30) 103 1.38 (1.03 -- 1.85) p \< 0.001 p \< 0.01 p = 0.03 *Education\\*** None 3546 10 1.0 30 1.0 23 1.0 Primary 9674 67 1.76 (0.63 -- 4.93) 96 1.56 (0.90 -- 2.69) 151 1.61 (0.98 -- 2.66) Secondary+ 2001 14 1.10 (0.34 -- 3.56) 13 1.50 (0.67 -- 3.38) 13 1.03 (0.48 -- 2.21) p = 0.15 p = 0.27 p = 0.05 *Poverty quintiles (n = 12320)\\\** 1 (Most poor) 2471 15 1.18 (0.56 -- 2.49) 19 0.67 (0.36 -- 1.22) 41 1.36 (0.81 -- 2.28) 2 2462 12 0.90 (0.41 -- 2.01) 24 0.85 (0.48 -- 1.51) 35 1.14 (0.68 -- 1.91) 3 2466 12 0.93 (0.43 -- 2.01) 24 0.84 (0.48 -- 1.49) 27 0.87 (0.51 -- 1.48) 4 2462 13 0.99 (0.47 -- 2.11) 21 0.76 (0.41 -- 1.39) 26 0.86 (0.50 -- 1.50) 5 (Least poor) 2459 14 1.0 27 1.0 30 1.0 p = 0.96 p = 0.74 p = 0.31 \* 1 missing subject, \\ 2 missing subjects ^a^Adjusted odds ratios from logistic regression models that included all variables in table except poverty \\\* Adjusted for age, sex, area and education p-value for test of heterogeneity ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Vehicles involved in crashes causing traffic injuries in the urban and rural area ::: Type of vehicle Dar es Salaam (n = 68) Hai (n = 23) --------------------- ---------------------------- ------------------ Car/truck 46 26 Bus 22 9 Bicycle 16 52 Motorcycle 13 4 Train 3 \- Cart \- 9 ::: Our results show that rural residents were 1.6 times as likely as urban dwellers to experience a fall resulting in an injury. Falls were more likely in children aged below 15 years and adults 45 years and above (Table [4](#T4){ref-type="table"}). They were reported to occur mainly in and around homes in the urban area (Table [6](#T6){ref-type="table"}). In the rural area, outside home, on the roads and farms were reported to be the most frequent places of occurrence for falls. ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Place of injury by area and cause ::: Place of injury All injuries Falls Cuts ---------------------------------- ------------------ ----------- ---------- ------ ------ ------ Home Inside 16.9 12.5 23.2 6.0 13.2 8.1 Outside 24.3 24.8 41.1 31.3 39.5 24.8 Workplace/factory 8.3 2.3 3.6 3.6 23.7 1.3 Farm 0 33.3 0 22.9 0 53.0 On the road 38.8 19.5 5.4 25.3 15.8 6.7 Recreation area including sports 9.7 1.9 21.4 2.4 7.9 1.3 School 1.9 4.6 5.4 8.4 0 4.0 ::: Table [4](#T4){ref-type="table"} shows that rural inhabitants had a four fold risk of experiencing injuries due to cuts or stabs compared to urban residents (OR = 4.27; 95% CI = 2.96 -- 6.15). It was noted that 68% (102/149) of injuries due to cuts in the rural area were related to agricultural activities, of which 81% occurred in adults aged 15 years and above. The farm and outside homes were where the injuries occurred most (Table [6](#T6){ref-type="table"}). In the rural area, about 50% of children aged 5--14 years were injured when working on farms or around their homes and one third of the injuries were related to play. In the urban area, most (70%) of the children aged 5--14 years were injured while playing. Burns accounted for about 6% (30/509) of all injuries in both areas. Children aged less than five years were 8 times as likely to sustain injuries due to burns as adults aged 15 to 44 years (OR = 8.58; 95% CI = 1.73 -- 42.5). Although the magnitude of association appears to be large, the estimate was based on small numbers (16 and 5 injuries respectively; table not shown). Discussion ========== In this study, we found major differences between urban and rural residents with respect to cause and severity of injury and the circumstances in which they occurred. This has great implications in setting priorities when planning for intervention strategies. Transport injuries formed the most common injury category in the urban area. The low risk of transport injury in the rural areas is probably a reflection of the relatively lower level of motorization in this mainly agricultural area. However, they will often have more serious consequences than other types of injury. A number of studies have reported similar findings. In a study from Pakistan, farmers were found to be at a lower risk of traffic injury than labourers and vendors \[[@B11]\]. A study from Bangladesh found a low incidence of traffic injury in a rural population \[[@B14]\]. Our data revealed that in the rural area, bicycle injuries predominated while in the urban area motorized vehicles accounted for a large proportion of transport injuries. Bicycles play a very important role in rural areas of Tanzania as a means of transport. In the urban area, most of the transport injury victims were passengers on public transport and pedestrians. Previous studies from developing countries have also reported the dominance of pedestrians, passengers of commercial vehicles and cyclists as vulnerable road users to transport injuries \[[@B10],[@B11],[@B21]\]. A hospital-based study conducted in an urban area in Tanzania reported that 42% of those subjected to transport injuries were pedestrians \[[@B4]\]. Strategies for prevention of transport-related injuries should take into account the local patterns. Cuts and stabs by instruments such as axes and machetes constituted the most frequent injury category in the rural area, which is due to the fact that rural residents engage in agricultural activities using unprotected equipment. Cuts and stabs also contributed significantly among children aged 5 to 14 years with farm work being the common activity in the rural setting while play was the main contributing factor in the urban area. As emphasized in other studies, there is a need for safe space for play among children. In addition, the issue of a working child in developing countries like Tanzania needs to be addressed. Falls were also a significant contributor among young children and older adults. A better understanding of the circumstances in which falls occur would assist in planning for fall-prevention programmes in Tanzania. Injuries due to accidental poisoning were infrequently reported in both areas and hence grouped under \'others\'. It is possible that people were not comfortable reporting such events. In addition, near drowning did not feature among the causes of nonfatal injuries although the urban area has a port. Except for transport injuries which were the commonest cause of fatal and nonfatal injuries in the same surveillance areas, the distribution of nonfatal injuries differed essentially from those of fatal injuries reported for 1992--1998 in the same surveillance areas \[[@B22]\]. In addition to transport injuries, the commonest causes of injury death were suicide, assault, accidental poisoning and drowning while the main causes of nonfatal injuries were falls, cuts and burns in these settings. A significant risk factor for injury turned out to be the place of residence. The likelihood of self reported injury was 66% higher for rural residents compared to urban residents. Similar findings have been reported from other studies \[[@B13],[@B23]\]. However, a study from Uganda found a high incidence of injury in the urban setting \[[@B12]\]. For severe injuries, we observed similar rates in the urban and rural areas. The main explanation is that transport injuries are more common and more severe in the urban areas whereas cuts and stabs are less common but more severe than in the rural area. Age is an important risk factor for many injuries but its influence varies between specific injury groups. Our findings show that adults aged 15--44 years are at a high risk of transport injuries. This has great economic impact since these are people in their most productive years and the injuries impose a considerable burden on their families and the society as a whole. Children below 15 years were at greater risk of injuries due to falls. This may be due to high risk environments such as lack of proper play facilities. This is in keeping with studies from other developing countries whereby falls among children have been reported to be a common cause of injury seen in hospitals \[[@B24]\]. As expected, males were found to have an increased overall risk of injury which was more pronounced for transport injuries. A possible explanation may be that men spend more time on the roads than females and therefore they are more prone to high risk behaviours or unsafe road practices \[[@B25]\]. Socio-economic status has been documented to be an important determinant of injury, although the effect depends on the socio-economic indicator considered, the cause and severity of injury \[[@B7]\]. We found no significant relationship between income poverty and nonfatal injuries. This is consistent with findings from other studies \[[@B26],[@B27]\]. Persons with primary education were at a greater risk of injuries than those with no formal education. This finding conforms to results from a previous study done in India \[[@B28]\]. The findings of this study are subject to a number of limitations. The information is based on self-reported data elicited through interviews, which is subject to recall bias \[[@B19],[@B29]\]. A 12 month recall period was used in this study in order to include as many injuries as possible. Although long recall periods underestimate injury rates, they can be useful in investigating associations between injuries and different risk factors. Initial multiple logistic regression analysis was done using injuries reported in the three months prior to the interview only. We found that the pattern of the associations was similar to that based on all injuries except for area of residence where the size of the effect was stronger when a short recall period was used (results not shown). Other studies have demonstrated that relative risks are not affected when a long recall period is employed \[[@B30]\]. In a previous report, we found that memory decay was greater in the rural area than in the urban area \[[@B19]\]. Therefore, the actual difference in rates between the urban and rural area may have been underestimated. Intentional injuries such as assaults and domestic violence are probably underreported since they would not be adequately captured in such a survey. This may lead to injury rates being underestimated. In this study, a clinical injury severity assessment was not possible. Disability days were used instead as a measure of severity of injury. One should be careful in generalizing the findings to other urban and rural settings of the country. Dar es Salaam is in many ways different from other urban areas of Tanzania and Hai is a relatively wealthy rural area. Furthermore, it might be difficult to generalize the findings to the surveillance areas due to the selection process that was employed. However, from our knowledge of the areas, we see no obvious reasons indicating that our samples should be very different from the urban and rural surveillance areas. Despite its limitations, this study has generated information that could be useful for targeted prevention at the local level. Conclusions =========== This study, the first of its kind in Tanzania, describes the patterns of nonfatal injuries and associated socio-demographic and socio-economic factors. It has attempted to identify specific groups of individuals as having a greater risk of experiencing certain types of injuries. This information is important for raising the level of awareness among policy makers and the public in general since the problem of injury receives little attention in most of the developing world including Tanzania. It is also useful in setting priorities for cause-specific prevention strategies. More detailed qualitative studies are required, however, on sensitive events such as assault. A nationally representative sample is also essential to measure the health burden due to nonfatal injuries. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= CM designed and conducted the study, performed statistical analysis, wrote the initial draft and revisions of the manuscript after consultation with other authors. IH, AN and GK participated in the design of study and revision of the manuscript. PS and YH participated in study design and co-ordination, and in revision of manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/11/prepub> Acknowledgements ================ The Norwegian State Educational Loan fund and the Norwegian Research Council funded this work. The Centre for International Health in University of Bergen carried out this research in collaboration with the Adult Morbidity and Mortality Project (AMMP) of the Tanzanian Ministry of Health. We thank David Whiting and Gregory Kabadi for the coordination and conduct of the survey. We also thank Dr. Deo Mtasiwa and Dr. Gabriel Masuki for providing assistance during data collection. The efforts of our research assistants are greatly acknowledged.	PubMed Central	2024-06-05T03:55:52.711079	2005-1-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548509/", "journal": "BMC Public Health. 2005 Jan 28; 5:11", "authors": [ { "first": "Candida", "last": "Moshiro" }, { "first": "Ivar", "last": "Heuch" }, { "first": "Anne Nordrehaug", "last": "Åstrøm" }, { "first": "Philip", "last": "Setel" }, { "first": "Yusuf", "last": "Hemed" }, { "first": "Gunnar", "last": "Kvåle" } ] }
PMC548510	Background ========== As a barrier and immune organ, the gastrointestinal tract, lung and skin play a key role in protecting the body from a hostile environment \[[@B1]\]. The low incidence of infection at normal epithelial surfaces reflects the presence of innate, broad-spectrum antimicrobial defense mechanisms \[[@B2]\]. Host defense peptides (HDPs) of the innate immune response play an important role in the protective barrier function of the epithelia \[[@B3]\]. Host defense peptides have been isolated from diverse organisms, including plants, insects, bacteria and vertebrates \[[@B4]\]. Several classes of mammalian peptide antibiotics have been ascribed pivotal roles in innate immunity \[[@B5]\]. Among these are various cysteine-rich peptides such as defensins \[[@B6],[@B7]\] and the more structurally diverse cathelicidins \[[@B8]\]. Produced as precursors, they require proteolytic processing to liberate the mature functional antimicrobial peptide. Cathelicidins contain a conserved N-terminal cathelin domain, and a structurally diverse C-terminal domain that possesses the peptide\'s antimicrobial activity. Rabbit CAP18 was the first cathelicidin precursor described, and its mature peptide has broad-spectrum antimicrobial activity \[[@B9]\]. Cathelicidins have since been identified in many other species including hCAP18/LL37 in humans \[[@B10]\], protegrins in swine \[[@B11]-[@B13]\], CRAMP in mice \[[@B14],[@B15]\] and SMAP29 in sheep \[[@B16]\]. Many of these peptides demonstrate extremely broad-spectrum antimicrobial activity, including Gram positive and Gram negative bacteria and fungi \[[@B4],[@B15]\]. In addition, they achieve bacterial killing much more rapidly than any commercially available antibiotic \[[@B17]\]. Recently, a new family of synthetic, α-helical HDPs called \"ovispirins\" was described \[[@B18]-[@B20]\]. Although some of these modified peptides had similar antimicrobial activity of naturally occurring peptides, they manifested appreciable cytotoxicity. We have demonstrated recently that variants of Ovispirin, the so called Novispirin peptides, displayed more favorable toxic/ therapeutic ratios in vitroand broad spectrum activity in infected rat burn model \[[@B21],[@B22]\]. Some of these peptides are induced at epithelial surfaces in response to invading organisms \[[@B23]-[@B25]\]. Many HDPs kill microorganisms by causing membrane permeabilization, although not necessarily as their sole mode of action \[[@B26]\]. Some HDPs also direct chemotaxis, promote wound healing, angiogenesis and contribute to adaptive immunity by mobilizing memory T cells and immature dendritic cells \[[@B25],[@B27]\]. Recent studies have also demonstrated antitumor activity after treatment with HDPs \[[@B28]\]. In addition, several antiviral activities were reported. Recently it has been demonstrated that rabbit neutrophil peptide alpha-defensin NP1 protects cells from infection with HSV-1 and 2 \[[@B29]\]. Other studies revealed that human neutrophil peptide HNP1 to 3 and Theta-defensins also inhibit HSV infection although by different mechanisms \[[@B30]-[@B32]\]. The ancestral human theta-defensins retrocyclin blocked HSV attachment \[[@B33]\]. The inhibition of adenovirus replication by the antimicrobial peptide awaits identification of a mechanism of action \[[@B30]\]. Anti-HIV activity of defensins were first reported 1993 by Nakashima and coworkers \[[@B34]\]. The alpha-defensins exhibited anti-HIV activity on at least two levels: directly inactivating virus particles; and affecting the ability of target CD4 cells to replicate the virus \[[@B35]-[@B37]\]. Binding to gp120 of HIV-1 and inhibition of HIV entry has also been identified as the mechanism of inhibition of HIV infection by theta defensins \[[@B38]\]. Due to their inhibitory effect on HIV-1 replication and due to an association of a single-nucleotide polymorphism in a beta defensin gene human beta-defensins might also play an important role in host defense against HIV-1 \[[@B39]\]. The antibacterial activity of HDPs is largely mediated by pore formation leading to permeablization of the bacterial membrane. Although some selectivity for bacterial membranes has been described, the lipid membrane of enveloped viruses might also be a target of antimicrobial peptides \[[@B32],[@B40]\]. This might allow development of antiviral effector molecules for topical application against a broad spectrum of enveloped viruses. Targeting host cell-derived membrane components might be a particularly interesting approach to inhibit viruses that rapidly develop resistance to compounds directed against viral proteins such as HIV. Therefore, we screened a panel of different natural occurring and designer HDPs for Env-independent inhibition of HIV infection at an early step in the viral replication cycle. Results ======= Given the potential cytotoxicity of HDPs, it was important to discriminate between modulation of cell metabolism and direct antiviral effects. We were concerned that HDPs could modulate host cell metabolism without affecting cell viability as assayed for example by standard MTT assays. We therefore decided to use immunodeficiency virus-based vectors transferring the luciferase gene to determine both, the HDP antiviral activity and modulation of cell metabolism. The luciferase activity of target cells, stably transduced with the lentiviral vector prior to HDP treatment should reveal any effect of HDPs on cellular transcriptional and translational efficacy. Treating cells with HDPs during infection with the lentiviral vector and comparison with results obtained with the stably transduced cells should then allow identifying HDPs that inhibit early steps in the viral replication cycle. To validate the luciferase-based assay for modulation of cell metabolism, 293 cells were stably transduced with a lentiviral vector transferring the luciferase gene. Incubation of transduced cells with increasing concentrations of Protegrin-1 and LL37 led to a dose-dependent inhibition of luciferase activity (Fig. [1](#F1){ref-type="fig"}). Comparison to the MTT test revealed a similar dose response curve, although the MTT test might be less sensitive at lower concentrations of the HDPs. Testing cell proliferation by a BrdU incorporation assay revealed a threshold level above which proliferation is strongly reduced. To allow side by side evaluation of cytotoxic and antiviral effects of HDPs with the same read-out the luciferase-based assay was used in most subsequent experiments. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Comparison of different cytotoxicity assays.293A target cells stably transduced with the luciferase gene were incubated for 48 hours in the indicated concentrations of LL37 or Protegrin-1. Viability, cell proliferation and cell metabolism of parallel cultures were assessed by a standard MTT assay, Brd-U incorporation and the luciferase assay, respectively. Values are expressed as percentage of the values obtained from cultures without HDPs. The mean and the standard deviation of triplicates are given. ::: ![](1742-4690-2-2-1) ::: A panel of HDPs containing members of the major classes of antimicrobial peptides were analyzed for inhibitory effects against lentiviral vectors. Given the variability of the viral envelope protein, we focused on identifying Env-independent inhibitory activities by using VSV-G pseudotyped lentiviral vectors. All antimicrobial peptides used in this study, human cathelicidin LL37, recombinant human β-Defensin-2, porcine Protegrin-1 (PG-1), fungal Plectasin and Novispirin G10, inhibited gram positive and gram negative bacteria revealing antimicrobial activity in the expected range (Table [1](#T1){ref-type="table"}) and confirmed bioactivity of the peptides used. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Summary of the radial diffusion assay results comparing host defense peptides with a clinically used antibiotic (Ampicillin) ::: M E C (μg/ ml) ------------------ ---------------- -------------- ------------ ------------- ------------ ------------- S. aureus 0,91 ± 0,04 4,74 ± 0,1 9,13 ± 0,5 2,42 ± 0,4 4,2 ± 0,4 10,50 ± 0,2 S. epidermidis 4,48 ± 0,2 13,09 ± 0,6 8,96 ± 0,2 8,20 ± 0,5 2,8 ± 0,02 ND E. faecalis 4,46 ± 0,3 13,61 ± 0,7 \- 10,60 ± 0,7 4,3 ± 0,3 28,85 ± 0,6 P. aeruginosa 3,3 ± 1,1 12, 28 ± 0,5 6,87 ± 0,8 \> 128 1,56 ± 0,4 ND E. coli 2 ± 0,1 11,66 ± 1,5 3,66 ± 0,3 79,10 ± 0,3 1,63 ± 0,3 18,9 ± 0,9 A. baumanii 5,01 ± 0,2 13,13 ± 0,2 3,19 ± 0,5 \> 128 4,5 ± 0,02 ND All clinical isolates from human wounds showed significant sensitivity to HDPs. ND: not determined. MEC: minimal effectory concentration. MEC \> 128 means that the tested bacteria is not susceptible to the drug tested. Depicted data consists of 3 individual experiments; each condition was performed in quadruplicates ::: The inhibitory effect against the lentiviral vector was determined by preincubation of the vector with human cathelicidin LL37, recombinant human β-Defensin-2, porcine PG-1, fungal Plectasin and Novispirin G10 for 30 minutes in increasing concentrations of peptide prior to the addition of vectors with antimicrobial peptide to the target cells. Stably transduced target cells were incubated in parallel with the HDPs to detect effects on cell metabolism. Luciferase activities were determined 2 days after infection. All HDPs led to a reduction in luciferase activity of cells transduced with the lentiviral vector (Fig. [2A--E](#F2){ref-type="fig"}), but most of them also reduced the luciferase activity of stably transduced target cells. Specific inhibition of early steps of infection was only seen for the cathelicidin LL37 and PG-1. As an additional control for the specificity of inhibition, a non-enveloped adenoviral vector transferring the luciferase gene was also incubated with the same panel of HDPs. None of the HDPs led to a dose-dependent inhibition of early steps of the adenoviral replication cycle (Fig. [2F--J](#F2){ref-type="fig"}). Two-fold serial dilutions were used to determine 50% inhibitory concentrations (IC~50~) of LL37 and PG-1, resulting in IC~50~s of approximately 30 μg/ml and 16.8 μg/ml, respectively (Fig. [3](#F3){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Inhibitory activity of HDPs against lentiviral and adenoviral vectors.Percent luciferase activity of 293A target cells transduced in the presence of the indicated amounts of HDP with the VLΔBH lentiviral vector (A to E) or an adenoviral vector (F to J) both transferring the luciferase gene is shown. Modulation of cell metabolism was investigated in parallel by incubating 293A target cells stably transduced with a luciferase gene with the indicated amounts of HDP. The luciferase activity is expressed as percentage of the luciferase activity of cells cultured in the absence of HDP. The mean and the standard deviation of triplicates are given. ::: ![](1742-4690-2-2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### 50% inhibitory concentrations of LL37 and Protegrin-1.Two-fold serial dilutions were used to determine the IC~50~s of LL37 (A) and Protegrin-1 for the VLΔBH vector (B). Modulation of cell metabolism was investigated in parallel by incubating 293A target cells stably transduced with a luciferase gene with the indicated amounts of HDP. The luciferase activity is expressed as percentage of the luciferase activity of cells cultured in the absence of HDP. The mean and the standard deviation of triplicates are given. ::: ![](1742-4690-2-2-3) ::: In initial attempts to characterize the mechanism of inhibition of lentiviral vector infectivity, LL37 and PG-1 were added at different time points during infection: both HDPs were either preincubated with the vector preparation for 30 minutes prior to addition to the cells or the HDPs and the vector were added simultaneously to the cells (Fig. [4A+B](#F4){ref-type="fig"}). In addition, cells were first infected with the lentiviral vector for two hours prior to addition of the HDPs. LL37 and PG-1 exerted the strongest inhibition after preincubation of HDPs and the lentiviral vectors, indicating a direct effect on infectivity of the vector particles. However, adding LL37 and PG-1 two hours after incubation of cells with the lentiviral vector also led to a stronger reduction of luciferase activity than observed after incubation of cells stably transduced with the luciferase gene suggesting a second target in the infection cycle that is affected by LL37 and PG-1. To further discriminate between direct inhibitory effects on vector particles and effects mediated by potential HDP cell interactions lentiviral vector particles were first incubated with LL37 and PG-1 for 30 minutes and then added either undiluted or at a 1:10 dilution to the target cells. Due to a 10-fold lower HDP concentration in the latter cultures, vector infectivity should be reduced to a lesser extent, if inhibitory effects are mediated by cellular targets. Comparable dose-dependent inhibition curves (Fig [4C,D](#F4){ref-type="fig"}) of diluted and undiluted vectors demonstrate that the inhibitory effects of LL37 and PG-1 depend on the HDP concentration during preincubation of the vector particles and not on the HDP concentration during subsequent cell culture. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Time and concentration dependent inhibition of lentiviral vectors by LL37 and Protegrin-1.The lentiviral vector VLΔBH transferring the luciferase gene was either preincubated with 200 μg/ml of LL37 (A) or 50 μg/ml of Protegrin-1 (B) for 30 minutes (-30) or added simultaneously (0) with LL37 and Protegrin-1 to 293A target cells. Target cells were also preincubated for 120 minutes with the lentiviral vector prior to addition of LL37 and Protegrin-1. Two days after infection luciferase activities were determined as percentage of luciferase activities of cells cultured in the absence of HDPs. Cells stably transduced with the luciferase gene were also cultured in the presence and absence of LL37 and PG-1 to determine the effect of HDPs on the cell metabolism. The mean and the standard deviation of triplicates is given. A lentiviral vector transferring the GFP gene (HIV-CSCG) was incubated for 30 minutes at the indicated concentrations of LL37 (C) or Protegrin-1 (D). The vector was then added directly to 293A target cells (undiluted) or after a 1:10 dilution in medium lacking the HDPs. The number of GFP positive cells at each HDP concentration is given as percentage of GFP-positive cells of cultures transduced with diluted and undiluted vectors in the absence of HDPs. The mean and standard deviation of triplicates are shown. ::: ![](1742-4690-2-2-4) ::: The lentiviral vector used in this study had been pseudotyped with the G protein of vesicular stomatitis virus (VSV-G). To evaluate whether LL37 and PG-1 directly target VSV-G or a lentiviral protein, the inhibitory effect of these HDP against the lentiviral vector was compared side by side to their effect on a retroviral vector containing the amphotropic MLV Env for entry into target cells. The dose dependent reduction in the luciferase activity in the target cells was very similar in cells transduced with the lentiviral or the MLV vector (Fig. [5](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Comparative analysis of inhibition of lentiviral and retroviral vector infectivity.Percent luciferase activity of 293A target cells transduced in the presence of the indicated amounts of HDP with a lentiviral vector (VLΔBH) or a retroviral vector (pRV-172) both transferring the luciferase gene is shown. Modulation of cell metabolism was investigated in parallel by incubating 293A target cells stably transduced with a luciferase gene with the indicated amounts of HDP. The luciferase activity is expressed as percentage of the luciferase activity of cells cultured in the absence of HDP. The mean and the standard deviation of triplicates of two independent experiments are shown. ::: ![](1742-4690-2-2-5) ::: The inhibition of HIV vectors containing the HIV-1 envelope by LL37 and PG-1 were studied on P4CCR5 cells expressing CD4 and coreceptors. IC~50~s of 25 μg/ml and 14 μg/ml were observed for LL37 and PG-1, respectively (Fig [6A,B](#F6){ref-type="fig"}), while only minimal inhibitory effects on cell proliferation were detected at these concentrations. Inhibition of early steps of wild type HIV-1 infection by LL37 and PG-1 was also evaluated on P4CCR5 indicator cells, which produce beta-Galactosidase upon expression of the viral tat gene after infection. The BrdU incorporation assay was used to evaluate modulation of cell function. A dose dependent reduction of the titer of HIV-1 on P4CCR5 cells was observed for both HDPs. However, the IC~50~of LL37 was approximately 3-fold higher than the IC~50~previously determined for the lentiviral vectors resulting in a narrow gap between antiviral and antiproliferative effects of LL37. In contrast, the IC~50~of PG-1 was below 10 μg/ml, while inhibitory effects on cell proliferation were not observed up to concentrations of 50 μg/ml. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Inhibitory effects of Protegrin-1 and LL37 on HIV-1 Env mediated vector entry (A, B) and HIV-1 infection (C,D).A lentiviral vector transferring the GFP gene (VGΔBH-SIN) was incubated at increasing concentrations of LL37 (A) or Protegrin-1 (B) prior to transduction of P4CCR5 cells. The vector titer is given as percentage of the titer of the vector incubated in the absence of HDPs. Wild type HIV-1 was incubated with increasing concentrations of LL37 (C) and Protegrin-1 (D). The virus titer was subsequently determined on P4CCR5 indicator cells and is expressed as percentage of the titer of the untreated HIV-1 virus stock. The toxicity of LL37 and Protegrin-1 was determined in parallel using the BrdU incorporation assay. ::: ![](1742-4690-2-2-6) ::: Discussion ========== From the panel of five HDP studied, the cathelicidin LL37 and PG-1 were found to specifically inhibit lentiviral and retroviral vector, but not adenoviral vector infectivity. The strongest inhibition was seen if the lentiviral vectors were preincubated with LL37 and PG-1. This suggests that these HDPs directly interacted with the vector particles, which is consistent with our observation that inhibition was dependent on the HDP concentration during preincubation of the vectors with HDPs, but not on the HDP concentration during infection of the cells. Since lentiviral vectors and retroviral vectors were inhibited to a similar degree although they do not share any viral protein, the target for the HDP on the particles is probably cell-derived. This could either be the lipid membrane derived from the cell, which surrounds the vector particles or cellular membrane proteins that are frequently incorporated in lentiviral and retroviral particles during budding \[[@B41]\]. A permeabilizing effect of LL37 and PG-1 on the viral particles would be consistent with our data, but other mechanisms of inhibition cannot be excluded. While the inhibitory effect of PG-1 was also detected with wild type HIV-1 on P4CCR5 cells, LL37 inhibited HIV-1 to lesser degree then the lentiviral vectors. Due an IC~50~of 88 μg/ml against wild type HIV-1, it is questionable whether LL37 concentrations are sufficiently high at mucosal membranes to play a role in host defense against HIV-1. Conclusions =========== Modulation of cell metabolism was generally seen at concentrations of HDPs exceeding 50 μg/ml, while the MEC of the antibacterial activity ranged from 1 to 10 μg/ml. This might leave a sufficient window for therapeutic intervention of bacterial infection. However, for the treatment of HIV-1, the therapeutic window of LL37 and PG-1 is rather narrow. It should also be noted that the HDP-induced modulation of cell metabolism and cytotoxicity can be cell type dependent. Therefore, increasing the selectivity of HDPs for early steps in the viral replication cycle seems to be necessary for further development of the human cathelicidin LL37 and the porcine Protegrin-1 as antiviral agents for systemic or topical applications. Methods ======= Preparation of vectors transferring the luciferase or GFP genes --------------------------------------------------------------- To generate lentiviral vector particles transferring the luciferase gene, a codon-optimized (Geneart GmbH, Regensburg, Germany) HIV-1 gag-polexpression plasmid (Hgp^syn^) \[[@B42]\] and a VSV-G expression plasmid (pHIT-G) \[[@B43]\] were used to package the SIV-based vector VLΔBH. This vector contains the luciferase gene replacing the GFP gene of VGΔBH \[[@B44]\].5 μg of Hgp^syn^, 2 μg of pHIT-G and 5 μg of VLΔBH were transiently cotransfected by the CaPO~4~coprecipitation method into 293T cells as previously described \[[@B45]\]. An HIV vector construct containing the GFP reporter gene (HIV-CSCG) \[[@B46]\] was also used to prepare lentiviral vector particles by cotransfection with Hgp^syn^, pcTat \[[@B47]\], pcRev \[[@B47]\] and pHIT-G or pSVIIIenv3-2, an HIV-1 envelope expression plasmid \[[@B48]\]. The MLV vectors were prepared by cotransfection of pHIT-456, pHIT-60 and pRV-172 \[[@B49]\]. Two days after transfection, the supernatants were cleared from cellular debris by low speed centrifugation (10 minutes, 1000 × g) and filtration through 0.2 μm filters from Roth (Karlsruhe, Germany). Aliquots were stored at -80°C. Construction and Production of Ad.OW126 Vector ---------------------------------------------- Beginning with a first generation E1- and E3-deleted adenoviral vector, we generated a replication-competent adenoviral vector Ad.OW126, which harbors in the E1 region the firefly luciferase cDNA (subcloned from pGEM-Luc (Promega, Madison, WI)), a IRES element \[[@B50],[@B51]\], and an Ad5 E1A ΔE1B-55K gene. The entire expression cassette is driven by the human CMV-IE promoter in parallel to the transcriptional orientation of the adenovirus E1 gene products and terminated by the bovine growth hormone polyadenylation site. The expression cassette was flanked upstream by the Ad5 packaging sequence and downstream by the Ad5 pIX. The Ad.OW126 vector was generated by in vitroligation \[[@B52]\] to H5dl327 (kindly provided by T. Shenk, Princeton University, Princeton, NJ), utilizing the unique Bst1107 I restriction site. The vector was propagated in 293 cells and purified by two rounds of CsCl density centrifugation \[[@B53]\], dialyzed (Slide-A-Lyzer, Pierce, Rockford, IL) against 1500 ml of PBS with 1 mM MgCl~2~and 10% glycerol four-times (1 hour each) at 4°C, and stored at -80°C until use. The concentration of the vector was determined by measuring absorbency at 260 nm \[[@B54]\], and the infectious titer was determined by plaque assay on 293 cells \[[@B55]\]. The ratio of infectious to non-infectious virus particles was approximately 1:80. Generation of 293A cells stably transduced with a luciferase gene ----------------------------------------------------------------- To stably transduce the luciferase gene into 293A cells, a self-inactivating version of VLΔBH, VLΔBH-SIN similar to VGΔBH-SIN \[[@B44]\] was packaged by cotransfection with Hgp^syn^and pHIT-G. 293A cells were plated in 24 well plates at a density of 50.000 cells / well and transduced with 200 μl of VLΔBH-SIN vector for two hours. Two days after plating cells were transferred to one well of a six well plate and transduced again with 1 ml of VLΔBH-SIN vector. Cells were subsequently expanded resulting in 293-Luc cells. Luciferase assay ---------------- The supernatant of infected 293A or 293-Luc cells, cultured in 96 well plates was removed and cells were lysed in 50 μl of cell lysis buffer (Promega, Pittsburgh, PA). 20 μl of the cell lysates were used in the firefly luciferase assay system of Promega as described by the manufacturer. Each single value of the triplicates was expressed as percent of the mean of triplicates of control cultures infected with the same vector in the absence of HDPs and the mean and the standard deviation of the percent values was calculated for each triplicate. Host defense peptides --------------------- The antimicrobial peptides (human LL37, porcine PG1-1, mutants from the ovine SAP29: Novispirin G10 and fungal Plectasin) used in this study were prepared by solid phase synthesis and purified by RP-HPLC. Recombinant human β-Defensin-2 was produced by a molecular farming approach in transgenic potato tubers and purified by perfusion chromatography (data not shown). The peptides (≥ 98% pure) were dissolved in 0.01% acetic acid and used for all in vitroand in vivostudies. Potential endotoxin contamination was monitored with the chromogenic Limulusamoebocyte lysate assay (BioWhittaker, Walkersville, MD) using Escherichia coliendotoxin (supplied with the kit) as the standard. Endotoxin levels for the peptides were not detectable. Bacteria -------- The following strains were used in this study: Gram-negative strains: Acinetobacter baumannii(ATCC 19606), Escherichia coli(ATCC 25922) and Pseudomonas aeruginosa(ATCC 27853) Gram-positive strains: Staphylococcus aureus(ATCC 25923), Staphylococcus epidermidis(ATCC 12228) and Enterococcus faecalis(ATCC 29212). All bacterial strains were analyzed with API test strips (BioMerieux, Hazelwood, MO) to confirm identity and aliquots were stored frozen in 50 % skim milk at -80°C. Bacteria were grown overnight in trypticase soy broth (Becton Dickinson, Franklin Lakes, NJ) at 275 rpm and 37°C. An aliquot of the resulting stationary phase cultures was then transferred to 20 ml of trypticase soy broth and incubated at 37°C for 2.5 hours to reach log phase. This subculture was transferred to a 50 ml conical polystyrene tube and centrifuged for 10 min at 4°C at 880 g. The bacterial pellet was washed once with chilled phosphate buffered saline, pH 7.4, and resuspended in 5 ml of the same cold buffer. One milliliter was removed to measure its optical density at 620 nm. The bacterial concentration was calculated from the following formula: CFU/ml = OD~620~× 2.5 × 10^8^. Growth Inhibition Assay ----------------------- To monitor bacterial growth inhibition in vitroa radial diffusion assay was performed as previously described \[[@B56]\]. Briefly, the underlay agar consisted of 1% agarose (A-6013, Sigma Chemical, St. Louis, MO) and 0.3 mg/ml trypticase soy broth (TSB) powder in 10 mM sodium phosphate with 100 mM NaCl (normal salt medium), pH 7.4. Bacteria (approximately 5 × 10^6^CFU) were mixed with 10 ml of underlay gel (43°C) and immediately poured into square 9 × 9 cm petri dishes. A series of 3 mm wells was punched after the agarose solidified. After appropriate serial dilutions were done, 5 μl of HDP, vancomycin (Abbott Labs, Chicago, IL), gentamicin, ciprofloxacin, or fluconazole (Sigma-Aldrich, St. Louis, MO) were added to the designated wells. Plates were incubated at 37°C for 3 hours. The bacteria-containing layer was covered with a 10 ml overlay of the nutrient rich agar. The overlay agar consisted of 6% (w/v) TSB and 1% agarose in PBS for all assays. After 18 h of incubation at 37°C, the plates were stained with 0.001% Coomassie blue for 10 h. The clear zones (bacterial growth inhibition) around the punched wells indicated antibacterial activity. The diameters of the clear zones were converted into units by subtracting the well diameter and multiplying the difference by 10. Results were plotted using a semi log scale and correlation coefficients and X-intercepts obtained from linear regression analysis. The minimal effective concentration (MEC) corresponded to the X-intercept value. All assays were performed in triplicates and repeated at least once. Cytotoxicity and proliferation Assay ------------------------------------ 293-Luc cells were plated in 96 well plates at a density of 2 × 10^3^cells / well. After 48 hours, 50 μl of MTT-solution (3 mg/ml) was added and incubated at 37° C under 5% CO~2~for 1 hour. After this time medium was removed and 100 μl 0.04 N HCL + 10% SDS was used to dissolve the resulting blue formazan crystals in living cells. The optical density was determined at 550 nm. Each single value of the triplicates was expressed as percent of the mean of triplicates of control cultures infected with the same vector in the absence of HDPs and the mean and the standard deviation of the percent values was calculated for each triplicate. In addition the BrdU Cell proliferation ELISA with chemiluminescence detection (Roche Diagnostics GmbH, Mannheim, Germany) was performed. After 293-Luc cells or P4CCR5 cells were plated in 96 well plates at a density of 2 × 10^3^cells/well BrdU (5-bromo-2\'-deoxyuridine) was added to the cells with a resulting concentration of 10 μM for the last 22 h of the incubation period. After removing the culture medium, the cells were fixed and DNA was denatured in one step with Fixdenat. Thereafter the cells were incubated with Anti-BrdU-POD for 1 h at room temperature. The chemiluminescence detection was measured after automatic injection of substrate solution with a microplate-luminometer (Orion, Berthold detection systems, Pforzheim, Germany). Inhibition of vector infectivity -------------------------------- To determine the effect of HDPs on vector infectivity and cell metabolism, 293A target cells were plated in triplicates into 96-well plates at 2 × 10^3^cells / well. After overnight incubation, the supernatant of the wells were removed and replaced by 25 μl vector preparation and 25 μl HDP at twice the final concentration indicated. 50 μl fresh medium with HDP at the final concentration indicated was added after two hours. One (adenoviral vector) or two (lentiviral and retroviral vector) days after infection, the supernatant was removed and 50 μl of cell lysis buffer (Promega) was added. Lysates were stored at -80°C until determination of the luciferase activity of the extracts. The affect of HDPs on cell metabolism was determined in parallel by plating 293-Luc cells exactly the same way as 293A cells and the luciferase activity was determined after one (as a control to inhibition of adenoviral vector infectivity) or two (as a control of lentiviral infectivity) days of incubation with HDPs. For GFP-expressing vectors, vector titers were calculated from the number of GFP positive cells per well as previously described \[[@B45]\] and the BrdU incorporation assay was used to monitor cytotoxic effects. Inhibition of HIV-1 ------------------- Stocks of HIV-1 were generated by transient transfection of 293T cells with the molecular clone pNL4-3. 25 μl of the virus stock were incubated for 30 minutes at room temperature with 25 μl of LL37 or PG-1 adjusted to twice the final concentration indicated. The mixture was added to P4CCR5 cells plated the day before at a density of 2 × 10^3^cells / well of 96 well plate. 50 μl fresh medium with HDP at the final concentration indicated was added after two hours. Two days after infection, the supernatant was removed and cells were stained by X-Gal. The number of infected cells per well were counted in the microscope. Competing interest ================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= LS, BT and JM performed most of the experiments. LS, OW, ML, EL, HH, HS and KU participated in the experimental design, data interpretation and writing of the manuscript. Acknowledgement =============== Recombinant human β-Defensin-2 was kindly provided by Michael Kleine at Planton GmbH (Kiel, Germany). Novispirin G-10 and Plectasin (WO 03/044049) were kindly provided by Hans-Henrick Christensen and the AMP team at Novozymes A/S (Copenhagen, Denmark). We would like to thank Ralf Wagner for providing Hgp^syn^, Ulrike Blömer for HIV-CS-CG, Joachim Hauber for pcTat and pcRev, Michael Malim for pHIT-G, and Paula Cannon for pHIT456, pHIT60 and pRV172. This work was supported by a grant from the FORUM program of the Ruhr-University Bochum.	PubMed Central	2024-06-05T03:55:52.717455	2005-1-18	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548510/", "journal": "Retrovirology. 2005 Jan 18; 2:2", "authors": [ { "first": "Lars", "last": "Steinstraesser" }, { "first": "Bettina", "last": "Tippler" }, { "first": "Janine", "last": "Mertens" }, { "first": "Evert", "last": "Lamme" }, { "first": "Heinz-Herbert", "last": "Homann" }, { "first": "Marcus", "last": "Lehnhardt" }, { "first": "Oliver", "last": "Wildner" }, { "first": "Hans-Ulrich", "last": "Steinau" }, { "first": "Klaus", "last": "Überla" } ] }
PMC548511	Introduction ============ Migraines and tension-type headaches impose an important burden upon society and the working public. According to the National Headache Foundation, some 45 million Americans suffer from chronic, recurring headaches and 28 million of these suffer from migraine headaches annually \[[@B1]\]. Furthermore, the work force loses approximately 50 billion dollars per year due to absenteeism and medical expenses caused by headaches, with more than 157 million workdays lost each year to migraine sufferers alone \[[@B1]\]. Even though advances have been made with regard to the treatment of acute migraine headaches (i.e., the triptan formulations), many patients often discontinue their migraine interventions due to treatment dissatisfaction \[[@B2]\]. Among individuals seeking treatment for tension-type headaches, the frequency of such headaches is often daily or almost every day \[[@B3]\]. Unfortunately, chronic tension-type headaches are associated with analgesic abuse \[[@B4]\], and are difficult to manage in a primary care setting due to frequent comorbid psychiatric or analgesic use problems \[[@B5]\]. Thus, it is imperative that other methods of treatment be researched and developed in order to increase satisfaction, therapeutic response, and compliance amongst these patients. One novel, but not really new treatment option, is the administration of niacin (nicotinic acid) through intravenous and/or oral routes. Niacin is a well-known over-the-counter (OTC) supplement primarily used for its ability to favorably influence cholesterol levels. Recently, there have been anecdotal reports demonstrating the effectiveness of niacin for aborting acute migraine attacks \[[@B6]\], and for preventing migraine headaches \[[@B7]\]. To assess the therapeutic spectrum of niacin\'s clinical effectiveness, we conducted a systematic review of the literature to examine the evidence of intravenous and/or oral niacin as a treatment for migraine headaches, tension-type headaches, and for headaches of other etiologic types. Methods ======= Literature Search ----------------- We conducted a systematic search of English and non-English language articles in the following databases: MEDLINE (1966--February 2004), AMED (1995--February 2004) and Alt HealthWatch (1990--February 2004). Articles were searched with the key search terms \"Migraine\" combined with the Boolean Operator \"AND \"Niacin\" OR \"Nicotinic Acid.\" Additional searches were conducted with the search terms \"Headache\" and \"Tension.\" To supplement the search, we searched through the references of the articles we found from the databases. Selection of Articles --------------------- To be included in our final review, articles had to report on the use and administration of niacin for migraine or any other types of headache. We included articles assessing original reports in both peer-reviewed and non-peer-reviewed journals. Quality assessment ------------------ We determined the evidence grade of each report found, based on the hierarchy of evidence developed by the Oxford Centre for Evidence Based Medicine \[[@B8]\]. Table [1](#T1){ref-type="table"} displays the hierarchy of evidence. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Grades of Evidence ::: ------- ---------------------------------------------------------------------------------------------------------------------------------- A Systematic reviews of randomized controlled trials and/or randomized controlled trials. B Systematic reviews of observational studies and/or high-quality observational studies including cohort and case-control studies. C Case-series, case-reports and/or poor quality cohort and case-control studies. D Expert opinion without explicit critical appraisal, or based on physiology, bench research or \"first principles.\" ------- ---------------------------------------------------------------------------------------------------------------------------------- ::: Search Results -------------- A total of 14 articles were screened \[[@B6],[@B7],[@B9]-[@B20]\]. Five articles were excluded in total; three because niacin was not the sole therapeutic agent used for the treatment of headache \[[@B16]\], histaminic cephalgia \[[@B17]\], and migraine \[[@B20]\]; and two because the reports were opinion pieces without any objective or subjective data to support the assertions made \[[@B13],[@B19]\]. In total, nine articles were found to meet the inclusion criteria and were included in this systematic review \[[@B6],[@B7],[@B9]-[@B12],[@B14],[@B15],[@B18]\]. Table [2](#T2){ref-type="table"} displays the characteristics of the studies included in this review. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Summary of Articles Demonstrating Niacin\'s Effectiveness for the Treatment of Migraine Headaches, Tension-type Headaches, and Headaches of other Etiologies ::: Reference Condition n Protocol Outcome Evidence Grade --------------- ----------------------------------------------- ------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------- 9 Migraine headaches 21 One initial intramuscular injection (IM) followed by a series of 6 or 8 intravenous (IV) treatments (maximum 50 mg), then regular IM injections (25--50 mg) combined with 50--150 mg of oral administration. 17 of the 21 subjects had a positive response. C: case-series 10 Headaches of different etiologic types 100 100 mg of IV sodium nicotinate or niacin. 75 of the 100 subjects had complete relief. C: case-series 11 Migraine headaches 15 100 mg of IV niacin, and an additional 50--200 mg if necessary to ensure a flushing response of more than 15-minutes. 13 of the 15 subjects had a positive response. The headaches were relieved in 27 of the 31 times when niacin was administered by IV administration. C: case-series 12 Tension headaches 35 22 subjects received 100--200 mg of IV niacin for a total of 53 times. 13 of the 22 subjects had a positive response. The headaches were relieved in 41 of the 53 times when niacin was administered by IV administration. C: case-series 14 Emotional or tension headaches 5 100 mg of IV niacin regularly for 12 weeks combined with a graded schedule of oral dosing, beginning at 300 mg daily, increasing to 900 mg daily, and tapering down to 300 mg daily. All 5 cases of emotional or tension headaches were very responsive to both IV and oral niacin. C: case-series 15 Tension headaches accompanied with depression 50 100 mg of IV niacin regularly for 23 weeks and then continued once every 2 months and as needed. This was combined with a graded schedule of oral dosing, beginning at 300 mg daily, increasing to 900 mg daily, and tapering down to 300 mg daily. In 44 of the 50 subjects the results with niacin therapy were very satisfactory or favorable. C: case-series 18 Migraine headaches 1 300--500 mg of niacin were chewed and allowed to slightly dissolve in the mouth. Resolution of migraine headaches. C: case-report 6 Migraine Headaches 2 500 mg of oral niacin taken at the onset of acute symptoms. In 2 of the 2 subjects, niacin aborted the acute migraine symptoms. In the first subject, niacin resolved the acute attacks in 4 of 4 occasions. In the other subject, niacin resolved the attack on one occasion. C: case-report 7 Migraine headaches 1 375 mg of oral sustained-release niacin twice daily for 1 month, and 375 mg once daily for 2 months. Migraine-free for the first month, and a marked reduction in migraine headaches over the next 2 months. C: Case-report ::: Limitations =========== Article \#1 \[[@B9]\] --------------------- This case series of 21 patients was limited as it was uncontrolled and involved a small number of patients. The methods that were used to evaluate efficacy of the treatment were primarily based upon a subjective questionnaire or medical record. The results would have been more meaningful if all the patients used the questionnaire to evaluate their treatment responses. Finally, although the symptomatic changes were witnessed immediately following niacin injection, the lifestyle recommendations may have had therapeutic benefits as well. Article \#2 \[[@B10]\] ---------------------- This study involved 100 patients having headaches of multiple etiologies. This study was not properly controlled, but did at least provide some comparison against a group of patients that were not administered the flush forms of intravenous niacin. The fact that the control group did not substantially benefit from the intravenous niacinamide lends more therapeutic efficacy to the ability of intravenous sodium nicotinate or niacin to have a marked therapeutic effect. The sample size was sufficient in number (n = 100), but the determination of therapeutic effectiveness was purely subjective and did not include any questionnaire or standardized method of evaluation. Side effects were minimal; 2 patients experienced mild abdominal cramps, 1 patient vomited but was suspected as having a pathologic condition of the stomach, and 1 other patient with migraine vomited forty-five minutes after the injection. A few patients found the treatment to be worse than their headaches. The authors concluded that the relief of headaches seemed to be correlated with the degree of flushing from the sodium nicotinate or niacin, and that this therapy was most useful among the migraine, spinal tap, and idiopathic groups. Article \#3 \[[@B11]\] ---------------------- A number of shortcomings were evident in this article. The first of which was that the study was not properly controlled and did not contain a placebo group or a group of patients acting as self-controls. However, some of the patients were given intravenous treatments on more than one occasion. This procedure helped in determining treatment reliability and reproducibility since niacin appeared to achieve therapeutic benefits on several occasions. There were no standardized methods of evaluating efficacy since the treatment responses were based upon the medical record and subjective reports. The results would have been more meaningful if all the patients used a standardized questionnaire prior to and after each treatment. In addition, there was only one male subject and 14 females in the study. Article \#4 \[[@B12]\] ---------------------- In this study of 35 patients were given treatments of intravenous dihydroergotamine methanesulfonate, intravenous niacin, or oral combination tablets of ergotamine tartrate and caffeine. Although no control group was used in this study, the patients were given multiple treatments on several occasions. This offered an interesting comparison to be made between niacin and other treatments. Since no control or placebo group was included, it cannot be determined if the therapeutic results were due, in part, to chance or placebo effects. The results might have been more meaningful if all the patients were given a standardized questionnaire prior to and after each treatment, and if an objective measure was incorporated to further substantiate patient responses. Article \#5 \[[@B14]\] ---------------------- These cases reported were well described and clearly demonstrated therapeutic responses during the intravenous and oral niacin therapy. The results would have been more reliable if a comparison had been made to a control group or to a similar patient cohort that were not given the same treatments. Even if the patients served as self-controls, and were told to stop the niacin treatment for a specified period of time, more information could have been gained from their therapeutic responses to niacin. Overall, this report of five cases provides an interesting approach to patients having chronic tension headaches and depression. Its value is limited by the difficulty in extrapolating these findings to a greater number of patients. Article \#6 \[[@B15]\] ---------------------- In this study involving 50 patients there was no data that listed pertinent identifying information, treatment response, past treatments, and duration of treatment for each patient. This would have strengthened the report by being more descriptive, and thus more amenable to critical analysis. If a comparison were made to a control group or a similar patient cohort not given the same treatments, more validity could be have been ascribed to this method of treatment. The patients could have also served as their own controls, thus providing more information about the therapeutic responses to niacin. It cannot be determined if the therapeutic results were due, in part, to chance or placebo. No method of evaluating efficacy was mentioned, except that the responses to niacin were \"very satisfactory\" in 44 of 50 cases. The results would have been more meaningful if all the patients were given a standardized questionnaire prior to and after each treatment, and if an objective measure was incorporated to substantiate their responses to niacin. Article \#7 \[[@B18]\] ---------------------- In this report, Hall describes the use of niacin for his migraine headaches remarking that the migraines resolved when intense flushing occurred. According to Hall, niacin\'s benefits and side effects might be due to its ability to release serotonin and histamine from the stomach. There is no reason to doubt Hall when describing his therapeutic response to niacin. However, his report was brief, had no control and was entirely subjective as he was the participant as well as examiner. Article \#8 \[[@B6]\] --------------------- In this report of 2 cases, it was found that in both cases migraines were relieved with oral niacin. The report would have been more rigorous if the patients had acted as their own self-controls. The value of the report is further diminished by the difficulty in extrapolating these findings to a greater number of patients. Article \#9 \[[@B7]\] --------------------- This case report was of a 62-year-old woman with a 40-year history of migraine headaches. The patient never acted as her own self-control, which would have made the findings of the case report more meaningful. The fact that the patient\'s migraine headaches increased in severity after a reduction in dosage does lend more support to niacin as being a migraine preventive agent. The hypothesized increase in serotonin from niacin administration cannot be proven given the limited amount of data contained in the case report. Like all case reports, its value is diminished by the difficulty in extrapolating these findings to a greater number of patients. Discussion ========== The results of this systematic review indicate that niacin may have a therapeutic effect on migraine headaches, tension-type headaches, and headaches of other etiologies. The quality of the evidence at this point, however, is only hypothesis generating, and randomized trials are required to determine the clinical implications of this novel treatment. There are several important limitations to consider in the interpretation of this review. We did not find any randomized or controlled trials of niacin on these headaches. We cannot determine to what extent publication bias has on the results of this review. We are unable to draw clinical inferences on the results of the included studies as they were of low quality and have a low level of external generalizability. Despite these limitations, we attempted to conduct an exhaustive search and included all reports of relevance. Reasons for niacin\'s effectiveness can only be considered hypothetical, and require clarification from future randomized controlled trials. In acute migraine headaches some of the symptoms arise from activation of the trigeminovascular complex. Activation of this complex leads to intracranial vasoconstriction causing the migraine aura, followed by headache due to vasodilation of the extracranial vessels and activation of the perivascular nociceptive nerves \[[@B21]\]. When taken intravenously or orally, niacin causes cutaneous flushing that might abort the acute symptoms of migraine by vasodilating the intracranial vessels, thus preventing the subsequent vasoconstriction of the extracranial vessels. There is evidence that niacin is an effective peripheral vasodilator, but its ability to influence central mechanisms (i.e., cerebral blood flow and cranial hemodynamics) involved in migraine headaches have not been well studied. Niacin causes peripheral vasodilation and cutaneous flushing by inducing the production of prostaglandin D~2~(PGD~2~) in the skin, leading to a marked increase of its metabolite, 9α, 11β-PGF~2~, in the plasma \[[@B22]\]. When niacin is administered orally in amounts of 500 mg or topically via a 6-inch patch of 10^-1^M aqueous methylnicotinate on the forearm, PGD~2~is markedly released in the skin and its metabolite appears in high amounts in the plasma \[[@B22],[@B23]\]. It is not known if PGD~2~causes vasodilation of the intracranial arteries, but niacin\'s ability to abort acute migraine headaches suggests that this might be what is occurring. Old reports cited by Bicknell and Prescott \[[@B24]\], demonstrate that niacin does indeed cause vasodilation of the cerebral and spinal vessels, and that intravenous administration increases the rate of intracranial blood flow in human beings for 20--60 minutes without any significant change in blood pressure. Unfortunately, there have not been more recent reports examining the effects that niacin has upon cerebral blood flow in human subjects. In terms of tension-type headaches, it appears that intravenous niacin is of benefit acutely due to its presumed central vasodilatory properties. Like migraines, part of the underlying pathophysiology of chronic tension-type headaches involves central mechanisms, such as the trigeminal system \[[@B26]\]. Chronic tension-type headaches are also associated with cerebrospinal pressure or intracranial venous pressure (or both) \[[@B26]\]. In fact, tension-type headaches are more similar to migraine headaches than they are dissimilar, in that they seem to progress into migraine headaches due to an escalating pathophysiological process \[[@B27]\]. Thus, niacin might mitigate the acute phase of tension-type headaches through the same hypothesized mechanism of action described earlier. Some of the reports did demonstrate prophylactic benefits when niacin was administered orally every day. It is now recognized that a deficit of mitochondrial energy metabolism (i.e., impaired mitochondrial phosphorylation potential) plays a role in the pathogenesis of chronic migraine headaches \[[@B28]\]. Niacin maintains adequate mitochondrial energy metabolism by increasing substrate availability to complex I \[[@B29]\], and this is how it might function as an effective prophylactic agent for migraine prevention. Two other nutritional agents (riboflavin and coenzyme Q10) augment complex I of the mitochondrial respiratory chain, and have been subjected to clinical trials demonstrating their effectiveness for the prevention of migraine headaches \[[@B30]-[@B32]\]. A deficit of mitochondrial energy metabolism may play a role in the pathogenesis of migraine. Since niacin improves mitochondrial energy metabolism by increasing substrate availability to complex I, it might also be an effective agent for migraine prevention. Niacin might also prevent tension-type headaches by improving mitochondrial energy metabolism within the skeletal muscles, and by increasing blood flow and oxygenation to the skeletal muscles. The overall net-effect could be a reduction in lactic acid concentrations, leading to reduced episodes of muscular tension and soreness. Niacin may reduce lactic acid concentrations since supplemental niacinamide (the amide of niacin) has been shown to reduce blood lactate and pyruvate concentrations by more than 50% in a patient with MELAS (mitochondrial encephalopathy, myopathy, lactic acidosis, and stroke-like episodes) syndrome by the third day of treatment \[[@B33]\]. This possible mechanism might only relate to migraine sufferers, however, since plasma levels of lactic and pyruvic acids were found to be significantly higher in migraine patients compared to patients with tension-type headaches and normal controls \[[@B34]\]. The side effects of intravenous niacin were found to be minimal from the summarized articles. The most common side effects were abdominal cramping, vomiting, and uncomfortable sensations of the skin and burning. A few of the patients described found the treatments to be worse than their headaches. In the articles where blood pressures were evaluated, little-to-no change occurred among the individuals treated with intravenous niacin. In a more recent study, parenteral niacin given to hypertensive and normotensive patients demonstrated a significant decrease in systolic, diastolic, and pulse pressures among the hypertensive subjects \[[@B35]\]. With respect to the oral administration of niacin, very few patients reported side effects. Even though niacin was well tolerated orally, we previously reported in a randomized placebo-controlled trial examining the safety of immediate-release or crystalline niacin, that it can be associated with intolerable side effects \[[@B36]\]. The most common side effects found using 500 mg of immediate-release niacin were unpleasant warmth or flushing, pruritis, chills, tingling, nausea, and vomiting. Approximately 75% of the subjects who were given niacin found it tolerable or difficult to tolerate, but did not indicate that they would never take niacin again. Some 18.2% of the niacin subjects indicated that they found niacin to be intolerable and would never take it again \[[@B36]\]. By contrast, very few of the patients from the summarized articles discontinued treatment due to the side effects of oral niacin. The side effects of greater concern from oral niacin have to do with sustained- or slow-release preparations. These preparations are better in terms of compliance since patients experience less cutaneous flushing with them. However, the use of these preparations have been associated with reversible hepatic toxicity in doses equal to or greater than 1500 mg per day with an acute onset of clinical symptoms of hepatitis in a relatively short period of time (2 days to 7 weeks) \[[@B37]\]. Other reports have demonstrated clinical symptoms of hepatitis when much larger doses of sustained-release niacin (greater than or equal to 3 grams per day) were used for months to years \[[@B38]-[@B42]\]. Sustained-release preparations have a higher incidence of hepatic toxicity in doses comparable to the immediate-release preparations \[[@B43]\], but these important differences are not typically mentioned in reviews of niacin\'s lipid lowering properties. Conclusion ========== Even though niacin\'s mechanisms of action have not been substantiated from controlled clinical trials. It is possible that this agent has a beneficial effect upon migraine and tension-type headaches and the prophylaxis of these headaches. It is imperative that properly designed controlled trials are developed in order to determine niacin\'s true therapeutic effects and adverse effect profile. In terms of its ability to abort acute migraine headaches, a placebo-controlled trial of parenteral niacin and sumatriptan seems to be worthy of consideration. In terms of prophylaxis, a placebo-controlled trial of oral niacin and riboflavin or coenzyme Q10 also seems worthy of investigation. Competing interests =================== Dr. Jonathan Prousky is associated with Swiss Herbal Remedies, Ltd., a company that sells nutritional supplements including niacin. They had knowledge of this manuscript and have not seen this manuscript. Authors\' contributions ======================= JP wrote the manuscript. DS contributed to the text, revised the results section, and assisted with the tables.	PubMed Central	2024-06-05T03:55:52.720346	2005-1-26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548511/", "journal": "Nutr J. 2005 Jan 26; 4:3", "authors": [ { "first": "Jonathan", "last": "Prousky" }, { "first": "Dugald", "last": "Seely" } ] }
PMC548512	Background ========== Recent reports using murine models of allergic asthma have shown that the Th2 type cytokine IL-13 may play a critical role in the pathogenesis of asthma, either by regulating airway inflammation, mucus hyper-secretion or airway hyper-responsiveness \[[@B1]-[@B5]\]. Evidence suggests that the potential role of IL-13 in asthma may come from its aptitude to directly interact with airway resident cells, such as epithelial cells or airway smooth muscle (ASM) cells, as shown by the ability of IL-13 to stimulate a set of different pro-asthmatic genes including inflammatory cytokines such as thymus and activation-regulated chemokine (TARC), eotaxin, monocyte chemotactic protein-1 (MCP-1) as well as growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) \[[@B6]-[@B10]\]. The ability of IL-13 to modulate ASM responsiveness to G-protein coupled receptor (GPCR) agonists, either by increasing contractile agonist-evoked calcium responses \[[@B11]\], and/or by impairing ASM responsiveness to β2-adrenoceptor stimulation \[[@B6]\], may also explain, at least in part, the putative role of IL-13 in allergen-associated BHR reported in animal models \[[@B1]-[@B4]\]. Previous reports have shown that other cytokines such as tumor necrosis factor alpha (TNFα) or interleukin (IL)-1β, may also participate in airway hyper-responsiveness by modulating ASM responsiveness to contractile GPCR agonists \[[@B12]-[@B14]\]. These data strongly support the current concept that cytokine modulation of ASM, an effector cell thought to solely regulate bronchomotor tone \[[@B12]\], may play an important role in the development of airway inflammation and bronchial hyper-responsiveness, the two main features of asthma. The molecular mechanisms by which IL-13 induces \"pro-asthmatic responses\" in ASM have not been clearly established. Identifying the expression profile of \"pro-asthmatic\" genes activated by IL-13 in ASM cells may therefore provide new insight into the design of novel therapeutic approaches for asthma. Using complementary molecular approaches, we investigated the effect of IL-13 on the transcription of \"pro-asthmatic\" genes in human airway smooth muscle cells (HASMC). The effect of IL-13 was compared to that of IL-13R130Q, a naturally occurring isoform resulting in a change from glutamine to arginine residues in the coding region that is associated with high serum IgE levels \[[@B15]\]. Interestingly, no report has yet investigated whether both IL-13 and IL-13R130Q share the same or have different biological activities. We found that both IL-13 and IL-13R130Q stimulate the same set of important genes that encode for proteins which may be clinically relevant for regulating airway hyper-responsiveness, airway inflammation and airway remodeling, key characteristics of asthma. Methods ======= Cell Culture ------------ Human tracheas were obtained from lung transplant donors, in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was removed under sterile conditions and the tracheal muscle was isolated. The muscle was then centrifuged and resuspended in 10 ml of buffer containing 0.2 mM CaCl~2~, 640 U/ml collagenase, 1 mg/ml soybean trypsin inhibitor and 10 U/ml elastase. Enzymatic dissociation of the tissue was performed for 90 min in a shaking water bath at 37°C. The cell suspension was filtered through 105 μm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham\'s F12 medium (Gibco BRL Life Technologies, Grand Island, NY) supplemented with 10% FBS (HyClone, Logan, UT) 100 U/ml penicillin (Gibco), 0.1 mg/ml streptomycin (Gibco), and 2.5 μg/ml fungizone (Gibco). Aliquots of the cell suspension were plated at a density of 1.0 × 10^4^cells/cm^2^. The cells were cultured in Ham\'s F12 media supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin and this was replaced every 72 h. Human ASM cells in subculture during the second through to fifth cell passages were used because, during these cell passages, the cells retain native contractile protein expression, as demonstrated by immunocytochemical staining for smooth muscle actin and myosin \[[@B16]\]. Unless otherwise specified, all chemicals used in this study were purchased from Sigma/Aldrich (St. Louis, MO). RNA isolation ------------- Total cellular RNA was isolated from IL-13 (50 ng/ml), IL-13R130Q (50 ng/ml) or control treated HASMC using the RNeasy mini kit (Qiagen, Inc. Valencia, CA) as per manufacturer\'s instructions. The IL-13 was purchased from R&D Systems (Minneapolis, MN) and the IL-13R130Q was generated in house at Centocor Inc. The quality and quantity of RNA was assessed using the Agilent 2100 Bioanalyzer (South Plainfield, New Jersey). Samples that demonstrated high quality (ratio of 28S rRNA and 18S rRNA is greater than 1.7) were submitted for microarray analysis. Microarray Processing --------------------- A complimentary DNA (cDNA) microarray, or DNA chip (Target B), containing a total of 8159 human gene cDNA clones from Research Genetics (IMAGE consortium, Huntsville, AL), Incyte Genomics (Santa Clara, CA) and internal sources was used in this study. All clones have been verified by DNA sequencing and are printed as 2 independent spots on a given chip. Duplicate chips were used for each RNA sample. Non-linear normalization between duplicate chips allowed each clone to be averaged to a single intensity value for each RNA sample. RNA amplification, probe synthesis and labeling, cDNA chip hybridization and washing were performed as described previously \[[@B17]\]. Agilent Image Scanner was used to scan the cDNA chips (Agilent Technologies, Palo Alto, CA). Fluorescence intensity for each feature of the array was obtained by using ImaGene software (BioDiscovery, Los Angeles, CA). Microarray data analysis ------------------------ In this study, fifty one-color cDNA microarrays were used to profile gene expression in human airway smooth muscle cells from 3 donors stimulated with IL-13, or its variant IL-13R130Q at 2 time points (6 hr and 18 hr). Untreated samples from the same group of donors were used as control. The samples being analyzed are listed in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Summary of number of samples from each donor and treatments ::: Time Donor Untreated IL-13 IL-13R130Q ------- ----------- ----------- ------- ------------ 6 hr Donor 1\* \- \- \- Donor 2 1 2 2 Donor 3 1 2 2 18 hr Donor 1 1 2 2 Donor 2 1 2 2 Donor 3 1 2 2 \The samples from Donor 1 at the 6 hr time point were not included due to poor quality of RNA. ::: Purified cDNA probes were hybridized to two microarrays, each containing two spots for each cDNA. Raw intensity data from the cDNA arrays were first normalized within each sample. Linear normalization and then nonlinear normalization was performed within each sample. Outlier intensity data points (greater than 1.4 fold away from the median of replicate measurements) were identified and removed from the data sets. The average intensity was generated by calculating the arithmetic mean of nonoutlier intensity values. The averaged intensity for each clone was further normalized across all samples. Chip-to-chip normalization was performed by dividing the averaged intensity of each clone by the 50.0^th^percentile of all measurements in that sample. The intensity of each clone was then normalized to the median intensity of that clone in the untreated control group. The normalized intensity was then log transformed. Using Partek Pro™ 5.1, sources of variance, such as batch effects, were identified by Principle Component Analysis (PCA) and appropriate factors were taken into account in the Analysis of Variance (ANOVA). ANOVA was performed to identify the genes that were differentially expressed by cytokine stimulation. Treatment (IL-13 and IL-13R130Q), time (6 hour and 18 hour), and donor (1, 2, and 3) were the three main effects considered in ANOVA. P-value cutoff was 0.05. Benjamini and Hochberg false discovery rate (FDR) was performed for multiple testing correction. After comparing the gene lists from IL-13 and IL-13R130Q treatments, it was clear that these two treatments resulted in the regulation of the same set of genes. Subsequently, samples from these two treatments were combined and regarded as replicates in ANOVA. Furthermore, outliner samples in the data set were detected by PCA and removed to improve the detection power. As an alternative approach, fold change comparisons (cutoff = 1.5 fold) between a treated condition and the control were carried out within each donor by using GeneSpring™ 6.2 \[[@B18]\]. A gene was considered as reliably detected in a given condition if more than half of the replicates representing the same condition had a raw expression intensity of more than 50, CV smaller than 25%, and raw intensity being generated from 2 or more of the duplicate spots representing the clone. A pair-wise comparison between a treatment and its untreated control was performed only on the genes that were reliably detected in at least one condition of the pair. The genes that showed at least 1.5 fold differential expression in two or more donors were identified for each cytokine treatment at a time. Reverse Transcription and Real Time PCR --------------------------------------- 1 μg of total RNA from each of the IL-13 (50 ng/ml) or IL-13R130Q (50 ng/ml) or control treated HASMC were used for the reverse transcription (RT) reaction. The RT reaction was performed as per protocol using TaqMan^®^RT reagents (Applied Biosystems) at 37°C for 120 min followed by 25°C for 10 min. Forty ng of cDNA per reaction were used in the Real Time PCR using the ABI Prism^®^7900 sequence detection system (Foster City, California). In the presence of AmpliTaq Gold DNA plolymerase (ABI biosystem, Foster City, California), the reaction was incubated for 2 min at 50°C followed by 10 min at 95°C. Then the reaction was run for 40 cycles at 15 sec, 95°C and 1 min, 60°C per cycle. Assays-on-Demand™ primers and probes (Applied Biosystems) were used in the PCR. The Real Time PCR data was analyzed using the standard curve method. Flow Cytometry -------------- Flow cytometry was performed as described previously with slight modifications \[[@B19]\]. Briefly, adherent cells treated with IL-13 for 24 hr were washed with PBS, detached by trypsinization (2 min, 37°C) and then washed with Ham\'s-F12 (10% FCS) media, centrifuged, and transferred to microfuge tubes (1.5 ml). Cells were incubated with anti-IL-13Rα2 (5 μg/ml, Santa Cruz Biotech) antibody followed by 1 hr incubation with a fluorescein isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch Laboratories; West Grove, PA). In parallel experiements, cells were incubated with the FITC-conjugated mouse anti-VCAM-1 antibody (2 μg/ml, Santa Cruz Biotech) for 1 h at 4°C. The cells were then centrifuged and resuspended in cold PBS in microfuge tubes. Samples were then analyzed using an EPICS XL flow cytometer (Coulter, Hialeah, FL). VCAM-1 and IL-13Rα2 levels were expressed as the increase in mean fluorescence intensity over un-stimulated cells. Results ======= IL-13 regulates gene expression of HASMCs ----------------------------------------- IL-13 may exert its deleterious effects in asthma by directly altering gene expression in airway resident cells such as epithelial cells or ASM cells \[[@B5]-[@B7],[@B20]\]. In order to determine which genes are regulated by IL-13 in airway smooth muscle cells, we employed the cDNA microarray technology. We also wanted to ascertain if the effect of IL-13R130Q, a naturally occurring isoform of IL-13 and associated with high serum IgE levels \[[@B15]\], was any different than IL-13 in terms of modulating gene expression. The concentrations of IL-13 (10--100 ng/ml) used in our study were shown previously to stimulate gene expression in human ASM cells \[[@B7],[@B8],[@B10]\], although the in vivorelevance of these particular concentrations remains unknown. Three donors were used and two types of analyses were carried out (Fold change analysis; Statistical Analysis). Both IL-13 and IL-13R130Q generated a similar expression profile i.e., genes regulated by IL-13 were the same as those regulated by IL-13R130Q at the 1.5 fold cutoff. Table [2](#T2){ref-type="table"} lists genes of interest that were identified from analyzing the data and divides them into one of three categories. Genes involved in all three characteristics of asthma (airway inflammation, remodeling and bronchial hyper-responsiveness) were identified. Of particular interest are vascular cellular adhesion molecule (VCAM)-1, Tenascin C, IL-13Rα2 and Histamine Receptor H1. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Summary of genes up regulated by IL-13 and IL-13R130Q. ::: Category Gene(s) Fold change ------------------------------------ -------------------------------- ---------------- Airway Inflammation* Adhesion Molecules VCAM-1 ↑ 2 fold ALCAM Selectin P ligand Laminin B1 Chemokines Chemokine Ligand 2 Chemokine Ligand 11 Chemokine Ligand 26 Chemokine Ligand 27 Cytokine receptors IL-13 Rα2 ↑ 1.6 fold Interleukin 1 receptor Airway Remodeling Extracellular matrix Tenascin C ↑ 2 fold Tenascin R Collagen Type I Collagen Type VI Collagen Type III Fibulin 1 CD44 Cell proliferation Pim-1 eEF1A Cytokines PDGFC Retinoic acid Receptor Interferon beta 1 Bronchial Hyper-responsiveness Cytoskeletal constituants Vimentin Tropomyosin 1 Tropomyosin 2 Actin Calcium regulators Phospholipase D Calreticulin hGIRK1 TRPC4 TRPC6 Sphingosine kinase 1 Rho GDP dissociation inhibitor FKBP1A Receptor Histamine H1 receptor ↑ 1.3 fold The fold changes correspond to the genes in bold. ::: Real Time PCR validation ------------------------ TaqMan™ Real Time PCR was used to validate VCAM1, IL-13Rα2, Tenascin C and Histamine Receptor H1. As shown in Figure [1A](#F1){ref-type="fig"}, VCAM1 was upregulated between 2 and 2.5 fold upon IL-13 or IL-13R130Q treatment at the 6 and 18 hour time points in both donors. This is comparable to the microarray data (Table [2](#T2){ref-type="table"}). In Figure [1B](#F1){ref-type="fig"}, IL-13Rα2 mRNA is upregulated with IL-13 or IL-13R130Q. However, the upregulation is more pronounced at the 18 hour time point compared to 6 hour. In Figure [2A](#F2){ref-type="fig"} Tenascin C is upregulated with IL-13 and IL-13R130Q and in Figure [2B](#F2){ref-type="fig"}, Histamine Receptor H1 shows an upregulation of about 1.5 fold in both donors at both time points and with both treatments. Again, this is comparable to the microarray data (Table [2](#T2){ref-type="table"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Real Time PCR (Taqman^®^) analysis showing the level of A) VCAM1 B) IL-13Rα2 upon treatment of ASM from two donors with IL-13 or IL-13R13Q for 6 or 18 hrs. The quantity of each gene is normalized to 18S and relative to the untreated sample. Values shown are mean ± standard deviation from an n = 6. ::: ![](1465-9921-6-9-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Real Time PCR (Taqman^®^) analysis showing the level of A) Tenascin C and B) Histamine receptor H1 upon treatment of ASM from two donors with IL-13 or IL-13R13Q for 6 or 18 hrs. The quantity of each gene is normalized to 18S and relative to the untreated sample. Values shown are mean ± standard deviation from an n = 6. ::: ![](1465-9921-6-9-2) ::: Validation of VCAM-1 and IL-13Rα2 at the protein level ------------------------------------------------------ In order to validate the modulatory effect of IL-13 on VCAM-1 and IL-13Rα2 genes at their protein level, flow cytometry was performed to confirm the up regulation of VCAM-1 and IL-13Rα2 in HASMC by IL-13. As shown in Figure [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}, IL-13 (10--100 ng/ml, 24 hr) differentially stimulates the expression of VCAM-1, with levels increasing in a dose-dependent manner, while IL-13Rα2 levels were identical at 10, 30 and 50 ng/ml. At 100 ng/ml IL-13, VCAM-1 and IL-13Rα2 levels were significantly increased by 20% and 35% over basal, respectively (n = 3, p \< 0.05). Increases in VCAM-1 and IL-13Rα2 proteins by IL-13 nicely correlate with changes in mRNA levels (Figure [1A](#F1){ref-type="fig"} and Figure [1B](#F1){ref-type="fig"}), suggesting that the IL-13 regulates the expression of inflammatory proteins via transcriptional mechanisms. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effect of IL-13 on VCAM-1 expression. ASM cells were incubated with the indicated concentrations of IL-13 for 24 hr. VCAM-1 expression was assessed by flow cytometry as described in Methods. Values shown are mean ± SEM and are significantly different from basal, n = 3 different experiments. \P \< 0.05 significant from untreated cell. ::: ![](1465-9921-6-9-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effect of IL-13 on IL-13Rα2 expression. ASM cells were incubated with the indicated concentrations of IL-13 for 24 hr. IL-13Rα2 expression was assessed by flow cytometry as described in Methods. Values shown are mean ± SEM and are significantly different from basal, n = 3 different experiments. \P \< 0.05 significant from untreated cell. ::: ![](1465-9921-6-9-4) ::: Discussion ========== Recent evidence using various experimental approaches such as gene-deficient mice, soluble inhibitors or transgene overexpressing IL-13 in the airways have highlighted the critical role of IL-13 in the pathogenesis of allergic asthma, possibly due to its ability to regulate goblet cell metaplasia, mucus hypersecretion and airway hyper-responsiveness \[[@B1],[@B3],[@B21]\]. Few reports that used microarray analyses applied to cultured human ASM cells demonstrated that IL-13 differentially regulates a number of important genes that are relevant for the pathogenesis of asthma \[[@B7],[@B10]\]. Although the functional relevance of such gene microarray analyses remains yet uncertain, these studies strongly suggest that IL-13 may be involved in the pathogenesis of asthma by directly modulating physiological responses of the ASM. Compared to the previous gene microarray reports \[[@B7],[@B10]\], we did confirm the physiological relevance of the microarray data using two different experimental approaches. At least for four different genes, VCAM-1 (an adhesion protein), IL-13Rα2, Histamine H1 receptor (a G protein-coupled receptor) and Tenascin C (an extracellular matrix glycoprotein), there was a close correlation between the data obtained from gene microarray with those obtained by real time PCR analyses. In addition, we showed that IL-13 stimulates the expression of VCAM-1 and IL-13Rα2 at the protein level, showing the physiological relevance of the gene array data. It is interesting to note that no difference in gene expression profile were noticeable between cells exposed to IL-13 or IL-13R130Q, an IL-13 polymorphism recently found to be associated with elevated serum and allergen-specific IgE \[[@B15],[@B22]\]. Our report is the first to suggest that this particular IL-13 polymorphism is equally effective as IL-13 in the transcriptional regulation of the genes examined in the present study. Our present study further supports the concept that IL-13 regulates the expression of different \"pro-asthmatic\" genes that are potentially important in the regulation of all three key features of asthma, i.e., airway inflammation, airway remodeling and bronchial hyper-responsiveness (for details see Table [2](#T2){ref-type="table"}). Previous reports using cultured ASM cells also demonstrated that IL-13 can stimulate the expression of other pro-inflammatory proteins, such as eotaxin \[[@B8],[@B23],[@B24]\], TARC \[[@B9]\] or VEGF \[[@B25]\] and tenascin \[present report and \[[@B10]\]\]. Upregulation of tenascin C and R, glycoproteins that contribute to extracellular matrix structure \[[@B26]\], may play an important role in airway remodeling, a characteristic of chronic asthma. The stimulatory role of IL-13 on VCAM-1 may be important in asthma since VCAM-1 has been regarded as a key player in the development of airway inflammation \[[@B27]\]. The ability of IL-13 to increase the expression of different G-protein coupled receptors (GPCR), such as Histamine H1 \[present report and \[[@B10]\]\] or CysLT1 receptor \[[@B28]\] represents one potential mechanism by which IL-13 promotes airway hyper-responsiveness to GPCR agonists previously described both in vivo\[[@B1],[@B3],[@B21]\] or in vitroin isolated airways preparations \[[@B11],[@B29],[@B30]\]. Additional studies are needed to determine whether IL-13 also modulates ASM responsiveness to Histamine. The receptor complex by which IL-13 regulates cellular function comprises the IL-13Rα1, which binds IL-13 and forms a complex with the IL-4Rα to initiate signal transduction via the JAK/STAT6 pathway \[[@B31]\]. IL-13Rα2, the other cell surface protein binds IL-13 with high affinity but the complex is not functionally active. One previous report using transgenic mice showed that overexpressing IL-13 in the airways induced a marked increase in both IL-13Rα1 and IL-13Rα2 mRNA levels, mostly in epithelial cells and macrophages \[[@B32]\]. Our study is the first to show that the expression of IL-13Rα2 is also transcriptionally increased by IL-13 in ASM cells. Although the functional significance of such regulation remains unknown, it is possible that the newly induced IL-13Rα2 could function as a decoy receptor to limit IL-13 signaling in ASM cells. Additional studies are needed to further support this hypothesis. Conclusions =========== These data further support the hypothesis that gene modulation by IL-13 in ASM may be essential for the events leading to the development of allergic asthma. Additional studies are clearly needed to define the transcriptional regulation of the different \"pro-asthmatic\" genes by IL-13, which may lead to novel therapeutic approaches for the treatment of allergic asthma. Authors\' contributions ======================= FS, LL, YA and RAP participated in the conception and design of the study. FS coordinated the study and along with YA drafted the manuscript. OT performed the RNA isolation and flow cytometry experiments. CH and KL performed the microarray data analysis. MB performed the Real Time PCR experiments. DG and BA proof read the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ This study was supported through grants from the National Institutes of Health (RO1-HL55301 to RAP and 2RO1-HL064063 to YA), and American Lung Association grant RG-062-N (YA). Yassine Amrani is a Parker B. Francis Fellow in Pulmonary Research.	PubMed Central	2024-06-05T03:55:52.722811	2005-1-20	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548512/", "journal": "Respir Res. 2005 Jan 20; 6(1):9", "authors": [ { "first": "Farhat", "last": "Syed" }, { "first": "Reynold A", "last": "Panettieri" }, { "first": "Omar", "last": "Tliba" }, { "first": "Chris", "last": "Huang" }, { "first": "Katherine", "last": "Li" }, { "first": "Michelle", "last": "Bracht" }, { "first": "Bernard", "last": "Amegadzie" }, { "first": "Don", "last": "Griswold" }, { "first": "Li", "last": "Li" }, { "first": "Yassine", "last": "Amrani" } ] }
PMC548513	Background ========== Obesity is becoming increasingly common and is recognized as a major public health problem worldwide \[[@B1]-[@B3]\]. In the UK, the overweight and obese population increased by almost 15% between 1980 and 1992. Similar increases have been noted in many countries, including the USA \[[@B4]\], Sweden \[[@B5]\] and the Netherlands \[[@B6]\]. In Israel, 55% of females and 58% of males aged 25--64 were reported to be overweight or obese (Body Mass Index \>25 kg/m^2^) in a Mabat survey \[[@B7]\] in 2000. Guidelines published in 1996 for the management of obesity recommended setting a modest weight loss and weight maintenance, rather than a targeted ideal weight, as goals \[[@B8]\]. Lately, pharmacological therapies have been proposed as adjuncts to diet and lifestyle changes to improve long-term weight loss \[[@B9]\]. The World Health Organization declared obesity an epidemic in developed countries. Awareness of physicians of their important role in defusing the obesity epidemic has increased. Yet, the number of family practitioners (FP) treating obesity problems continues to be low \[[@B10]\]. Many chronic health problems are exacerbated by unhealthy behaviors and harmful environmental conditions. From the psychological perspective, healthful lifestyles and environmental conditions may yield large health benefits. The widespread adoption of a healthier lifestyle rather than medical technologies has resulted in a substantial decline in premature mortality and morbidity \[[@B11]\]. The media plays a major role in informing the public about health risks. Efforts to convince people to adopt health practices that prevent disease have relied heavily on persuasive communications in health education campaigns \[[@B11]\]. Health benefits are accelerated by community-wide efforts to reduce habits that impair health \[[@B12]\]. Self-efficacy refers to the belief in one\'s ability to organize and execute the courses of action required to produce a given attainment \[[@B11]\]. Such beliefs influence the courses of action people choose to pursue, how much effort they put into a given endeavor and how long they will persevere in the face of obstacles and failure. If people believe they have no power to produce results, they will not attempt to effect changes. Self-efficacy is not a fixed ability that one has or lacks in one\'s behavioral repertoire. Rather, it is a thinking process, a generative capability in which cognitive, social, emotional and behavioral sub-skills are organized and effectively orchestrated to serve innumerable purposes. Self-efficacy is concerned not with the number of skills one has, but with what one believes one can do with the skills under a variety of circumstances. Efficacy beliefs operate as a key factor in a generative system of human competence \[[@B11],[@B13]-[@B15]\]. Self-efficacy is an important contributor to performance accomplishments, whatever the underlying skills might be \[[@B11]\]. The greater a person\'s efficacy beliefs, the greater the academic challenges one sets for oneself and the greater their intrinsic interest \[[@B16]\]. Personal efficacy beliefs influence the level of interest in occupational pursuits even when the influence of ability is removed \[[@B17],[@B18]\]. A sense of personal efficacy is constructed through a complex process of self-persuasion. Efficacy beliefs are the product of cognitive processing of diverse sources of information conveyed inactively, vicariously, socially and physiologically. Once formed, efficacy beliefs contribute to the quality of human functioning \[[@B11]\]. Self-efficacy is measured by the strength of a subject\'s beliefs in the ability to execute requisite activities. Social cognitive theory distinguishes among three basic processes of personal change: the adoption, general usage and maintenance over time of new behavioral patterns \[[@B19]\]. Efficacy beliefs affect each of these phases \[[@B11]\]. Self-efficacy conceptualizes a person\'s perceived ability to perform on a task as a mediator of performance on future tasks \[[@B11],[@B13]\]. A change in the level of self-efficacy can predict a lasting change in patients\' behavior if there are adequate incentives and skills \[[@B13]-[@B15]\]. People\'s beliefs that they can motivate themselves and regulate their own behavior play a crucial role in whether they even consider acting. Thus, practitioners who judge themselves incapable of treating obesity do not even try. Obesity has not been considered a disease till recent years. Physicians did not treat obesity unless they were asked to do so by patients. To our knowledge and after a literature search, no intervention studies have yet been conducted to change the self-efficacy of FPs in Israel towards treating obese people. Among other courses for which physicians usually receive credits towards their annual professional training, a course was offered by the Israeli Academic Medical Council. The course was initiated by the Israeli Association of FPs and recommended by the Medical Professional Journal of FPs in Israel. Registration in the course was open to all FPs. The present research was a preliminary study. This being the case, the first group of FPs who attended the new course participated in it. Though that group contained a small number of subjects, the importance of investigating a new program contribution to FPs self-efficacy for future research was evident. The objectives of the course were to enrich the knowledge of FPs with up-to-date information on obesity and to raise their motivation to treat it. The study objective was to determine if an interactive course would raise the self-efficacy of FPs to treat obesity. It was hypothesized that the self-efficacy of FPs would be enhanced as a result of participating in an interactive obesity-treatment course. Methods ======= Subjects -------- Twenty-nine FPs (62% female) chose to participate in the course along with other Continuing Medical Education (CME) courses. All participants work as FPs in public health care clinics throughout the country. Design ------ This study was based on a one group, pre-course -- post-course test design, without a control group. It was accompanied by qualitative interviews to validate the results of the data analysis. Research tools -------------- The strength of the efficacy beliefs of the FPs to treat obesity was estimated by using a five point scale Likert type questionnaire containing nine items (Table [1](#T1){ref-type="table"}). The subjects were asked how confident they were in the ability to treat obesity across problem situations. The levels of confidence were as follows: 1. not at all confident, 2. somewhat confident, 3. moderately confident, 4. very confident, 5. completely confident. The questionnaire was filled out before and after the course. The items were generated from the criteria on the domain map that was constructed on the basis of theoretic analyses of knowledge accumulated in the domains of self-efficacy and health behavior change \[[@B11],[@B13]-[@B15]\] and consultation with experts. The researchers used the analytic induction method for the theoretic analysis of knowledge. The map\'s main criteria were: obesity -- a serious disease; up-to-date clinical and behavioral knowledge; processes of change, e.g: decision making, planning, monitoring and behavior controlling; resilience; lack of time and social involvement. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Original domain map criteria and the sequence of criteria in the questionnaire regarding the self-efficacy beliefs of family practitioners to manage obesity ::: Original sequence of criteria Sequence in the questionnaire ----------------------------------------------------------------------------------------------- ------------------------------- Efficacy to treat a problem of high priority 1 Efficacy to give up-to-date and correct information 7 Efficacy to persuade, support and help patients make decisions 3 Efficacy to make patient plan behaviors and situations 6 Efficacy to make patient monitor his behavior 4 Efficacy to make patient control behaviors and situations 9 Efficacy to treat obesity regardless of previous failure or unsuccessful experiences 2 Efficacy to treat obesity regardless of lack of time 5 Efficacy to bring about involvement of other people in the patient\'s behavior change process 8 ::: The Alpha Cronbach for the reliability of the tests was α = 0.88 for the pre-course test and α = 0.90 for the post-course test (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Pre-course (α = 0.88) and post-course (α = 0.90) scale mean, SD, item total correlation, and α if item deleted (n = 29) ::: Item no. Mean SD Item total correlation α, if item deleted ------------- ------ ------ ------------------------ -------------------- Pre-course 1 0.77 0.13 44.03 0.88 2 0.61 0.15 43.62 0.87 3 0.53 0.16 43.40 0.86 4 0.59 0.16 42.83 0.86 5 0.74 0.15 39.85 0.87 6 0.84 0.15 41.39 0.87 7 0.44 0.13 46.61 0.88 8 0.77 0.16 39.89 0.86 9 0.72 0.14 39.36 0.85 Post-course 1 0.67 0.16 20.10 0.88 2 0.75 0.16 20.67 0.88 3 0.67 0.16 20.11 0.88 4 0.46 0.14 21.17 0.90 5 0.59 0.15 19.82 0.90 6 0.72 0.15 20.29 0.88 7 0.68 0.15 21.50 0.89 8 0.71 0.16 19.21 0.88 9 0.81 0.16 19.99 0.88 ::: The following aspects of structure validity were examined: (a) for the content aspect, the items matched the domain concept map. Final version was rewritten on the basis of experts\' and researchers\' comments. The items were finally presented in an nonsequential order to make subjects think while reading the questions and not relate to earlier answers; and (b) for the substantive aspect, FPs were interviewed regarding their self-efficacy to assure that the questions were clear and did not require rewording. The interview was a 30 min. open interview. A physician was given open questions such as: \"Describe your feelings and thoughts of efficacy to treat obesity\" or \"How the self-control lectured help your efficacy perceptions\". The subject was free to speak openly on the issue. The interview was actually a verbal reflection of thoughts and feelings of the subjects. It was recorded and later on transcribed by the researchers. The interview was analyzed by the constant comparative qualitative method of analysis \[[@B20],[@B21]\]. Every sentence said by the subject in the interview was considered a unit to be taken into account for the content analysis. The present study design used paired t-test in order to compare pre and post strength of efficacy beliefs for treating obesity. Procedure --------- Participants answered the questionnaire before the start of the course and at the end of the last session, and a few days later participated in an open interview. The course was interactive and contained 12 clinical and psychological lectures given by experts in all subjects relating to obesity (Table [3](#T3){ref-type="table"}). Every lecture was followed by a workshop. The program consisted of six monthly sessions. Each session (from 17:00 to 21:00) started with two medical lectures followed by a 90 min workshop, and ended with a panel discussion. In the first workshop FPs said they did not address the problem of obesity unless they were asked to do so by patients. Even if they wanted to relate to it they would feel uncomfortable with it, as if that was none of their business. They reported between 1--3 obesity treatments a day. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Table of contents of the interactive course: \"Monitoring and treating obesity by the family practitioner\" ::: ---- ------------------------------------------------------------------------------------------------------------------------------------------------ 1 Obesity -- the epidemiological perspective 2 The pathogenesis and metabolic factors of obesity 3 The clinical approach of the family physician to the obese patient 4 Nutrition, diet and treatment of overweight 5 Self-control and behavior modification -- diagnosing and managing the stages of change: the trans-theoretical model, the self-regulation model 6 Physical activity and exercise, lifestyle and energy expenditure 7 Drug treatment of obesity 8 Surgical treatment of obesity 9 The metabolic syndrome 10 The mechanism of hypertension in obesity 11 The diabetic obese patient 12 Infertility of obese women ---- ------------------------------------------------------------------------------------------------------------------------------------------------ ::: FPs filled in clinical report forms and presented the cases they treated and tools they used. The cases were discussed during the course and feedback was given by colleagues and experts. The course supplied the FPs with knowledge, skills and psychological tools such as: food and physical activity diaries, decision making tools, self-evaluation tools, self-report tools, self-monitoring tools, persuading tools, stimulus control tools and counter-conditioning tools, to treat obesity. Medical knowledge and skills were not examined. The course attempted to raise the self-efficacy of FPs by creating a supportive atmosphere, providing feedback, recalling successful experiences, learning from models, bringing patients into the workshops, discussing sociological and psychological problems and allowing reflection of thoughts and emotions. Studies have shown that reflection enhances metacognitive processes such as: self-monitoring, self-evaluation, self-reaction and attribution \[[@B11],[@B22]-[@B25]\]. Since self-appraisal of efficacy is a form of metacognition and efficacy beliefs are structured by experience and reflective thoughts, we viewed reflection on obesity issues and on FPs self-efficacy as a forethought process so that the mental process the FPs went through had an effect on the processing of their efficacy appraisals. We expected their appraisals to go through a change \[[@B13]-[@B15]\]. Results ======= The differences between FP pre and post course efficacy appraisals was tested by paired t-test (Table [4](#T4){ref-type="table"}) and significant differences were found (Effect size .317). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Difference between pre and post course efficacy beliefs to treat obesity ::: Item no. Mean pre-course Mean post-course t df N ---------- ----------------- ------------------ ---------- ---- ---- 1 3.93 4.48 2 3.86 4.48 3 4.21 4.41 4 3.48 4.17 5 3.00 3.79 6 3.34 4.07 7 4.17 4.55 8 3.31 4.31 9 3.41 4.10 -3.606\* 28 29 \(significant 1-tailed) p\< 0.0005 ::: All subjects reported enhanced efficacy beliefs to treat obesity. Negative feelings towards the course were not said or written. Yet, efficacy belief enhancement of the group differed among the items (see Table [4](#T4){ref-type="table"}). Three FPs left the course right after the first meeting claiming a lack of time in learning and dealing with new perceptions and no time to treat the family holistically. They also said that the time slot of the course was inconvenient. Some FPs presented cases which had failed to bring about a change in the patient\'s behavior. Those cases were discussed and the FPs were given strategies and tips for further treatment. In addition, the interviews resulted in reflection by the FPs on the process of their self-efficacy. Coded sentence units of the interviews were constantly compared while evidence accumulated. Nine main criteria were generated through that systematic analysis. These criteria matched the questionnaire items. The four criteria that received the most evidence formed four core constructs. The first was the contribution of the course to the FPs. FPs appreciated the importance of up-to-date information, the various treatment perspectives, and the skills they acquired. Knowledge was a precondition for change. FPs talked about the practical tools and tips they acquired. This is illustrated in the example: \"It is hiswill and actions and ourhelp\". The second criterion was the efficacy to persuade patients to treat obesity. FPs described how they could persuade patients to treat obesity in a variety of ways. The third criterion was the FP performance reports. FPs presented their clinical report forms and described how they successfully used the new knowledge, skills, tools and tips they acquired in the course to manage obesity in their clinics. The fourth criterion was efficient time management. Time management scored the lowest on the pre-course questionnaire. Whenever the issue was raised during the course, FPs claimed that time was the main obstacle in treating obesity. By the end of the course, a significant change in their perception of time was noted. Prior to the beginning of the course, only 1--3 clinical report forms on obesity treatment were reported by the FPs; at the end of the course, that number increased to 8--37. After having taken the course, obesity problems were not ignored anymore. Table [5](#T5){ref-type="table"} shows the criteria listed in the questionnaire, and the criteria generated from coded sentence units mentioned during interviews. The criteria derived from the interviews are illustrated by characteristic examples. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Questionnaire criteria, criteria coming out of interviews, and examples ::: -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Questionnaire criteria Criteria coming out of interviews Examples from interviews --------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 1\. Efficacy to treat a problem of high priority a\. Course contribution: motivation to learn, and to work harder \- \"I used to treat obesity as something that needed cosmetics now I know it\'s a disease that needs a cure\...and it is my job\"\ - \"I\'ve a lot more to learn\...I wish I had more courses like this one\...It\'s very important\...I\'m ready to work hard\...to do a lot more\...\"* 2\. Efficacy to give up to date precise information b\. Course contribution: enriched important knowledge, treatment perspectives, skills, practical tools and tips \- \"Now I have the tools, a lot of information\...now I have the right perspective\...I shouldn\'t be angry with him but supportive\...with the tools it\'s much easier\...It\'s hiswill and actions and ourhelp. I feel I know how to help him\...the course was helpful\" 3\. Efficacy to persuade, support and help patients make decisions c\. Efficacy to persuade patients to treat obesity \- \"Today I\'m more patient\...I know when it\'s the right moment to bring up the issue\...and I know how to do it\"\ - \"I can use emotions to persuade him\"\ - \"Before taking the course, I wasn\'t self-confident enough to do it, now I feel free to talk about this\...I can show him statistics on obesity\...\" 4\. Efficacy to make patients plan behavior and situations d\. Efficacy to make patients plan, monitor and control health behavior \- \"Since he is aware of his expectations I can make him plan or act\...\"\ - \"under my guidance he can see what\'s right and what\'s wrong\... and he can change things\...\"\ - \"when he understands the process he can initiate behavior\" 5\. Efficacy to make patients monitor behavior 6\. Efficacy to make patients control behavior and situations 7\. Efficacy to treat obesity regardless of previous failures or unsuccessful experiences e\. FP performance reports \- \"What you taught in the course works!\"\ - \"Now that I know that taking small steps is better than expecting a dramatic weight loss -- it is easier\...and the patient is happier.\ I now treat 10 obese people\...\"\ - \"I now treat 8 people, before taking the course, I did not treat any\"\ - \"I have 9, I was given feedback by a patient\... she said: I lost two pounds, thank you!\...you are great\...\" 8\. Efficacy to treat obesity regardless of lack of time. f\. Efficient time management \- \"I\'ll tell you my secret, everybody starts work at 8.00, I com at 7.00.\ I\'m ready to do it, I want to succeed, after all, it is my job\"\ - \"I make a double appointment for an obese patient\...\"\ - \"If I treat obesity now, I save time, I won\'t have to treat other diseases in the future\"\ - \"I keep thinking about how to be more efficient, I have to do something about the time!\"\ - \"I know that when I want something I find the time\" 9\. Efficacy to bring about other people\'s involvement in the patient\'s behavior change process g\. Better done with someone\'s help \- \"I tell her, doing it alone is too difficult. You should bring your husband or a friend to the next visit\" h\. FP weight loss \- \"I lost weight as a result of the course\" i\. Enhancing efficacy beliefs by reflection, feedback and supportive climate \- \"Did you hear the FPs\' reaction to my presentation?\...Wow!\... that was great\...I understood I had great success\"\ - \"I was given feedback by patients, now I know I can!\"\ - \"The best thing that happened to me in this course is that it made me think\"\ - \"I could open myself to talk about things that I hadn\'t done before\...I think it\'s because of the warm climate\" -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: Discussion ========== The purpose of the study was to discover whether participation in an interactive course would result in a change in the efficacy beliefs of FPs to treat obesity. Results show a significant difference between pre and post course beliefs. The increase was satisfactory (\>4 in a scale of 1--5) and the results of the qualitative analysis indicate that the criteria derived from the interviews matched those of the questionnaire. When speaking openly, FPs addressed the same issues that made up the domain theoretical concept map. Several researchers have suggested that quantitative efforts in the study of self-efficacy should be complemented by qualitative studies aimed at gaining a deeper understanding of attitudes and emotions \[[@B22]-[@B24]\]. The criteria obtained from the interviews not only matched the criteria in the questionnaire but also enriched our knowledge with a deeper understanding of the attitudes and thoughts of the FPs about the course and the way they looked at obesity management after the course. All sentence units of the interviews were taken into account for content analysis. The study showed that acquisition of knowledge and skills enable a person to meet personal standards of merit that tend to heighten beliefs of personal efficacy \[[@B11]\]. In judging their efficacy, individuals necessarily unite personal agency with means. They act on beliefs of how well they can use prescribed means \[[@B10]\]. The FPs felt the course equipped them with appropriate knowledge and means for treating obese patients. This was expressed in many sentence units. Another important psychological role in all phases of behavior change was the efficacy to persuade patients to treat obesity. The FPs\' presentations of clinical report forms and descriptions of success to manage obesity in their clinics support research findings that efficacy beliefs predict both intentions and behavior \[[@B10]\]. The FPs challenged time management as efficiently as they could, which is another important change in the perception of their role as FPs. The impact of the questions analyzed in this study extends beyond the issues asked by the questionnaire. New insights were gained through qualitative analysis: FPs analyzed the process they experienced during the course. They described how their efficacy beliefs were enhanced through reflection, feedback and the supportive climate of the course. Studies have shown that reflection enhances self-efficacy processes since self-appraisal of efficacy is structured by experience and reflective thought \[[@B25]\]. FPs reported that reflection on thoughts and emotions helped them construct their beliefs. Feedback regarding the quality of one\'s work progressively raises perceived efficacy, which, in turn, predicts subsequent performance. This is illustrated in studies of self-regulated productivity \[[@B26]\]. The feedback the FPs received from their colleagues and the experts on the quality of their reported experiences enhanced their efficacy beliefs. The FPs feedback on the course showed that the workshops contributed most to their self-efficacy. FPs explained that they had got the chance to \"bring the clinic into the workshop\", to discuss their performance, to get feedback on it, and to be stimulated to reflect on the treatment and on themselves as physicians. Incentives for mastering activities contribute to the growth of interest and perceived efficacy\[[@B10]\]. The credit points FPs received for their professional training served as an incentive that fostered performance accomplishments. The FPs reported on an increased number of obesity treatments which was an important gain of this educational program. There were no other absences except for the 3 who had left after the first session. At the end of the course FPs requested that the course be continued. In summary, an effective program of widespread change in health practices includes four major components. The first is informational and intended to increase the physician\'s awareness and knowledge of the subject. The second involves development of skills needed to translate informed concerns into effective action. The third is aimed at building a robust sense of self-efficacy to support the exercise of control in the face of difficulties that inevitably arise. This is achieved by providing repeated opportunities for guided practice and corrective feedback in applying the skills in simulated situations that people are likely to encounter. The final component involves creating social support for desired changes. The present program contained all these components. The limitations of the study were the small number of participants and the reliance on one motivated group of FPs. These limitations result from the fact that this was the first course organized by the Israeli Association of Family Physicians on the subject of obesity. It was important to study the effect of the program on FPs self-efficacy for future continuing education and research. We recommend studying the effect of interactive courses on the lifestyle and weight loss of FPs. Future research should consider randomized samples from larger courses and analyses of the correlation between course success and actual FPs performance, FPs\' self-efficacy and treatment outcomes e.g: BMI change, Lipids and BP. We also recommend studying the differences in obesity treatment outcomes and in general health feelings between patients whose FPs attended obesity courses and patients whose FPs did not. Practical recommendations for Continuing Medical Education planners would be to focus on workshops rather than on lectures, to enhance those processes the FPs felt less efficacious to go through and to have guidance of Endocrinology experts\' as well as Psychology and Education specialists to improve communication with patients and their families in an effort to enhance motivation to loose weight. It is also recommended to bring patients to the workshops to reflect on the treatment they have received. Competing interests =================== The author(s} declare that they have no competing interests. Authors\' contributions ======================= SK and AF conceived and designed the study, participated in the collection, analysis and interpretation of data and drafted the manuscript. SV Participated in the statistical analysis, interpretation of data and draft of the manuscript. SP participated in the design of the study, data collection and interpretation. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6920/5/4/prepub> Acknowledgment ============== Prof. Elliot Berry, Director of the Department of Human Nutrition and Metabolism of the Hebrew University-Hadassah Medical School of Jerusalem, is acknowledged for his helpful comments.	PubMed Central	2024-06-05T03:55:52.725076	2005-1-29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548513/", "journal": "BMC Med Educ. 2005 Jan 29; 5:4", "authors": [ { "first": "Sara", "last": "Katz" }, { "first": "Amiel", "last": "Feigenbaum" }, { "first": "Shmuel", "last": "Pasternak" }, { "first": "Shlomo", "last": "Vinker" } ] }

PMC547904

Background ========== Infection with *Chlamydia pneumoniae*(Cpn) usually causes acute respiratory tract infections \[[@B1]\]. Chronical infection with Cpn may also contribute to formation and progression of atherosclerotic lesions apart from the classical risk factors such as hypertension, hypercholesterolemia and hyperlipidemia \[[@B2]\]. Cpn has been extensively studied in the context of atherosclerosis \[[@B3]\] because atherosclerosis and cardiovascular disease are the leading causes of death in the United States, Europe and much of Asia \[[@B4],[@B5]\]. The obligate intracellular bacterium has been detected in atherosclerotic lesions by immunohistochemistry, polymerase chain reaction and electron microscopy \[[@B6]-[@B8]\] and has also been cultured from atheromatous plaques \[[@B9],[@B10]\]. On the cellular level smooth muscle cells and macrophages in the intima have been found to be infected with Cpn \[[@B11],[@B12]\]. In general terms, if the inflammatory response does not effectively neutralize or remove the offending agents, such as Cpn, it can continue indefinitely resulting in the progression of the disease \[[@B2]\]. *Chlamydiae*exhibit a unique developmental cycle with two morphological distinct infectious and reproductive forms: the elementary- and the reticulate body. The life cycle proceeds for 48 -- 72 h and ends with pathogen release that may damage the host cells \[[@B13]\]. Infection with this pathogen is accompanied by cytoplasmic alterations and damage of the host cells \[[@B12]\]. A balance between pro- and anti-apoptotic influences by *Chlamydia*can be postulated. On one hand it has been shown that *Chlamydia psittaci*and *Chlamydia trachomatis*can induce apoptosis *in vitro*\[[@B14],[@B15]\]. On the other hand established cell lines infected with Cpn or *Chlamydia trachomatis*were protected from apoptotic cell death induced by various stimuli \[[@B16],[@B17]\]. Most of the studies investigating pro- and anti-apoptotic activity of Cpn were performed in tumor cells or established cell lines. Since the character of cell death is affected by host cell type and *Chlamydia*strain it is of relevance to determine cell death in *Chlamydia*infected primary cells \[[@B18]\]. As regards the role of Cpn in atherosclerosis we need to understand death promoting and inhibiting capacities of Cpn in primary cultures of vascular cells. Human aortic smooth muscle cells (HASMC) play an important role in the development of atherosclerotic lesions \[[@B19]\]. Therefore, we sought to clarify the nature of cell death induced by Cpn in HASMC. Apoptosis and necrosis represent two extreme morphologically defined forms of cell death \[[@B20]-[@B23]\]. Recently the hybrid term aponecrosis was introduced describing the incomplete execution of the internal apoptotic pathway and the following necrotic degeneration \[[@B24]\]. Whether smooth muscle cell death is apoptotic, necrotic or even aponecrotic in nature would predictably influence the inflammatory response in the plaque. Necrotic cells releasing their contents provoke inflammation, whereas in apoptosis dead cells are ingested by neighboring cells and, therefore, do not provoke an inflammatory response \[[@B25]\]. In order to characterize the fate of HASMC following infection with Cpn we analyzed cell death by morphological and biochemical methods. Results ======= Various morphological characteristics of bacterial infection in HASMC --------------------------------------------------------------------- In order to examine the morphological characteristics of Cpn infection HASMC as well as the tumor cell line HEp-2 were infected with Cpn-K6 and Cpn-VR1310, respectively, and stained after 24 h, 48 h and 72 h with anti-Cpn-MOMP antibody. According to the staining protocol intra- and extracellular localization of Cpn could be distinguished (Figure [1](#F1){ref-type="fig"}). Already after 24 h, but also after 48 h and 72 h, HASMC as well as HEp-2 cells were infected with numerous bacteria. Round or oval shaped smooth bordered intracellular inclusions were found in tumor cells (Figure [1E](#F1){ref-type="fig"}) as well as HASMC (Figure [1B,D](#F1){ref-type="fig"}). In contrast to tumor cells in primary HASMC Cpn were found also as single or rarely as irregular shaped, aggregated immunoreactive spots (Figure [1A, C](#F1){ref-type="fig"}, [4D](#F4){ref-type="fig"}). Quantification of morphology in HASMC revealed that after 72 h all cells were infected with at least one chlamydial spot (100% infection rate). Approximately 25% of the cells carried one or multiple inclusions. The different morphological characteristics in HASMC (inclusions, single and aggregated spots) could also be discriminated 48 h post infection. The ratio of cells carrying inclusions versus cells carrying spots was much higher in HEp-2 cells compared to HASMC irrespective of the infectious dose (Figure [2A](#F2){ref-type="fig"}). This ratio did not considerably vary between different Cpn strains K6 and VR1310 or as a function of the number of bacteria used for infection. Spots were usually much lower in number and not as prominently observed in HEp-2 cells compared to HASMC (Figure [2C](#F2){ref-type="fig"}). In HASMC the number of spots increased with higher infectious doses while at the same time the number of inclusions remained constant (Figure [2B](#F2){ref-type="fig"}) in contrast to Cpn infections of HEp-2 cells where more inclusions were formed at higher infectious doses without an increase of spots. Cpn lyse HASMC in a dose-dependent manner ----------------------------------------- To compare the lytic capacity of different Cpn isolates and to identify a possible time course of host cell death during the chlamydial infectious cycle, LDH release of HASMC was analyzed at 24, 48 and 72 h post infection. HASMC were inoculated with two different Cpn isolates: Cpn-VR1310 and Cpn-K6 at serial 2-fold dilutions ranging from 1 to 32 inclusion forming unit (IFU)/cell (Figure [3](#F3){ref-type="fig"}). Both isolates were found to be free of *Mycoplasma spec*. contamination as assessed by PCR. LDH release of mock treated HASMC or of cells inoculated with heat inactivated bacteria was comparable to the spontaneous LDH release of uninfected cells (data not shown). Both Cpn isolates lysed HASMC in a strictly dose dependent fashion at all time points analyzed. Cpn mediated host cell lysis at 48 h post infection is shown in figure [3A](#F3){ref-type="fig"}. Data obtained from 24 h and 72 h post infection were similar and consistent with the displayed data subset. The levels of HASMC lysis induced by infection with 8 IFU/cell of Cpn-K6 (0.9% ± 3) and Cpn-VR1310 (18.6% ± 8) indicate large differences in the lytic capabilities of both Cpn isolates. Comparison of Cpn-induced LDH release at different time points (24, 48 and 72 h) demonstrated a constant increase in *Chlamydia*-specific HASMC lysis indicating continuous triggering of cell death throughout the whole infectious cycle (Figure [3B](#F3){ref-type="fig"}). In order to reach the same amount of HASMC lysis (27% ± 2) 4 IFU/cell of Cpn-VR1310 and 32 IFU/cell of Cpn-K6 respectively were required. This suggests that for a given degree of HASMC lysis either high amounts of a lytically less active isolate or lower amounts of a lytically more active isolate are required. Cpn infection of HASMC causes nuclear chromatin condensation as well as extensive damage of organelles and cell membrane ------------------------------------------------------------------------------------------------------------------------ In order to discriminate between apoptotic and necrotic cell death of infected HASMC ultrastructural analysis by transmission electron microscopy (TEM) was carried out. As a control apoptosis was induced by treatment with the well described kinase inhibitor staurosporin. Untreated HASMC had an elongated nucleus rich in euchromatin with one or two well-defined nucleoli (Figure [4A](#F4){ref-type="fig"}). The cytoplasm was scattered by numerous narrow-spaced profiles of rough endoplasmic reticulum (rER), a small Golgi complex and single elongated mitochondria. In Cpn infected cultures a variable number of HASMC harbored large inclusions containing many infectious elementary bodies and metabolically active reticular bodies (Figure [4B](#F4){ref-type="fig"}). The nucleus of these cells was slightly rounded but chromatin structure as well as morphology of organelles was unchanged. A large number of infected HASMC without Cpn inclusions were destroyed (Figure [4C](#F4){ref-type="fig"}). The round shaped nuclei showed a distinct condensation of heterochromatin and condensed nucleoli. Cell organelles including mitochondria and rER profiles were dilated and cell membranes were completely disrupted. Detection of single bacteria by TEM was possible only on rare occasions since distorted organelles in dying cells were barely distinguishable from Cpn. Apoptotic HASMC treated with staurosporin showed a distinct condensation of chromatin (Figure [4D](#F4){ref-type="fig"}) and nuclei were fragmented frequently; damage of cell membranes and swelling of organelles appeared to a lower extent than in Cpn infected HASMC. In general the overall ultrastructural morphology of HASMC infected with spot-like Cpn infection suggests a hybrid form of cell death: it shares chromatin condensation with apoptosis and damage of organelles and membranes with necrosis. Spot-like infection induces aponecrotic morphology in HASMC ----------------------------------------------------------- The combination of chromatin staining by DAPI with labeling of DNA strand breaks via TUNEL staining and assessment of membrane integrity by NHS-biotin staining allows a distinction between necrosis as well as early and late phases of apoptosis using confocal laser scanning microscopy \[[@B23]\]. Cells dying by necrosis exhibit NHS-biotin labeling of the cytoplasm but not chromatin condensation or TUNEL staining of nuclei. Cells in early phases of apoptosis display nuclei with condensed or fragmented chromatin, with or without TUNEL staining, and are surrounded by NHS-biotin negative cytoplasm due to intact cell membranes. In late phases of the apoptotic cell death TUNEL positive nuclei occur together with a NHS-biotin positive cytoplasm. HASMC were infected with Cpn for chosen periods of time or were inoculated with mock isolates or treated with staurosporin. Cells infected with Cpn-VR1310 (Figure [5E, F](#F5){ref-type="fig"}) showed an identical staining pattern as cells infected with Cpn-K6 (Figure [5A -- D](#F5){ref-type="fig"}). Mock inoculated cells displayed round or oval shaped nuclei with finely dispersed chromatin visualized by DAPI staining and were always TUNEL negative. NHS-biotin was bound exclusively to the cell surface indicating intact membranes (Figure [5G](#F5){ref-type="fig"}). In Cpn infected cultures a variable labeling pattern of HASMC was observed. Cells with Cpn inclusions always displayed normal chromatin staining and no TUNEL or NHS-biotin labeling (Figure [5A](#F5){ref-type="fig"}). HASMC with spot-like infection either contained an unaltered nucleus and NHS-biotin negative cytoplasm (Figure [5B](#F5){ref-type="fig"}) or showed a TUNEL positive nucleus with condensed chromatin embedded in NHS-biotin stained cytoplasm (Figure [5C, E](#F5){ref-type="fig"}). In HASMC displaying both Cpn spots and aggregates the nucleus was always labeled by TUNEL and the cytoplasm by NHS-biotin (Figure [5E, F](#F5){ref-type="fig"}). Cpn-infected HASMC never showed membrane damage in combination with an unaltered nucleus, representing necrotic cell death. Cpn-infected HASMC displaying TUNEL positive nuclei and damaged membranes could be detected at 24, 48 and 72 h post infection. 8 h post infection was the earliest time point when TUNEL positive nuclei could be detected, suggesting induction of apoptosis during the whole life cycle of Cpn (not shown). TUNEL-positive HASMC were found even at infectious doses of 1 IFU / cell for Cpn-VR1310 and 8 IFU / cell for Cpn-K6 and the amount of TUNEL-positive HASMC increased strictly dependent on the chlamydial dose used for infection (Figure [5I](#F5){ref-type="fig"}). Again, both Cpn strains used in this study elicited the same features of cell death in HASMC according to the infection morphology. Identical to the lytic capacity (Figure [3A](#F3){ref-type="fig"}) of these strains, K6 had to be used at higher IFU compared to VR1310 to generate a similar number of cells with TUNEL-positive nuclei and NHS-biotin positive cytoplasm. In contrast to HASMC neither Cpn-K6 nor Cpn-VR1310 induced a concentration dependent increase in TUNEL positive nuclei in HEp-2 cells and numbers of TUNEL positive nuclei remained below 10% (not shown). In staurosporin treated cells the rounded nuclei were fragmented or exhibited chromatin that was condensed marginally, whereas NHS-biotin labeling was predominantly negative (Figure [5H](#F5){ref-type="fig"}). Just a minority of the cells revealed a condensed TUNEL-positive nucleus and at the same time were also NHS-biotin positive reflecting late stages of apoptotic cell death. Cpn infection induces phosphatidylserine externalization in HASMC ----------------------------------------------------------------- HASMC were infected for 48 h with Cpn-K6 or -VR1310 in 2-fold dilutions ranging from 8 to 386 IFU/cell or from 1 to 32 IFU/cell, respectively. As controls cultures were inoculated with mock isolates or with heat inactivated bacteria (data not shown) and analyzed for annexin-V binding and propidium iodide uptake. Uninfected as well as Na-azide treated HASMC served as negative and staurosporin-treated cells as positive controls for apoptosis (Figure [6C](#F6){ref-type="fig"} upper right and upper left panel). Control cultures inoculated with mock lysate (Figure [6](#F6){ref-type="fig"}) or heat inactivated bacteria and untreated cells showed always less than 20% annexin-V single positive cells. This proportion of annexin-V positive cells represented background staining, probably due to the experimental procedure. Upon infection with 128 IFU/cell of Cpn-K6 for 72 h the number of single annexin-V positive cells increased to more than 50% (Figure [6B](#F6){ref-type="fig"}). Upon Cpn infection annexin-V and propidium iodide double-positive staining reached 10% as compared with 4% in mock-inoculated and 3% in untreated HASMC (not shown). In staurosporin treated cultures the amount of both annexin-V single and double positive cells increased strongly (Figure [6](#F6){ref-type="fig"} upper left panel) indicating a continued induction of classical apoptotic cell death. The amount of annexin-V single positive HASMC infected with Cpn increased linearly in a dose- (Figure [6A](#F6){ref-type="fig"}) and time- (Figure [6B](#F6){ref-type="fig"}) dependent manner. Cpn-VR1310 again induced phosphatidylserine externalization at much lower doses compared to Cpn-K6. Neither mock inoculation (Figure [5A, B](#F5){ref-type="fig"}[6C](#F6){ref-type="fig"} lower left panel), nor inoculation with heat inactivated bacteria (data not shown), induced apoptosis. This indicates that infection with living bacteria is required for induction of cell-death. Taken together, the dose- and time dependence between infection and phosphatidylserine externalization were consistent with induction of early apoptotic events. *Chlamydia*infection in HASMC induces caspase-3 independent cell death by dissipation of mitochondrial membrane potential ------------------------------------------------------------------------------------------------------------------------- After induction of apoptosis an enzymatic cascade is activated leading to the disassembly of the cell \[[@B23]\]. Key enzyme in this cascade is caspase-3. However, in some cases apoptosis can proceed without involvement of caspase-3 \[[@B26]\]. In order to investigate the role of caspase-3 HASMC were infected with Cpn-K6 128 IFU/cell or Cpn-VR1310 32 IFU/cell, respectively, for 48 h (24 h and 72 h not shown) or treated with 1 μM staurosporin for 12 h to induce apoptosis. Samples were analyzed for caspase-3 activation by measuring enzyme activity after lysing the cells. No activity of caspase-3 in Cpn infected HASMC (Figure [7A](#F7){ref-type="fig"}) could be detected. In contrast distinct caspase-3 activity was found in staurosporin-treated cells. A major role in the induction and regulation of cell death is played by mitochondria. Pro-apoptotic factors result at mitochondria in the dissipation of the mitochondrial membrane potential and release of cytochrome c. After infection of HASMC with Cpn-K6 or Cpn-VR1310 cells were loaded with TMRE (tetramethylrhodamineethylester) \[[@B27]\], a dye, that is only taken up into mitochondria with an intact mitochondrial potential \[[@B28]\]. Cells with intact membranes excluding propidium iodide were analyzed by flow cytometry. The number of TMRE negative cells strictly increased with the chlamydial dose whereas blocking of chlamydial protein synthesis by addition of chloramphenicol completely abrogated this effect (Figure [7B](#F7){ref-type="fig"}). Moreover, labeling of Cpn-K6 infected or mock treated cells for cytochrome c and mitotracker red revealed distinct loss of cytochrome c staining of the mitochondria in the infected population (Figure [7D](#F7){ref-type="fig"}) but not in mock treated cells (Figure [7C](#F7){ref-type="fig"}). Taken together, the data suggest *Chlamydia pneumoniae*induce caspase-independent apoptosis-like cell death in infected HASMC by involving mitochondrial membrane dissipation resulting in the release of cytochrome c. Discussion ========== In our in vitro study cell death was only induced in HASMC by infection with viable Cpn but not after incubation with heat inactivated or chloramphenicol treated bacteria. Additionally, different Cpn isolates showed various lytic activities towards host cells that correlated to different reproduction rates in tumor cells as measured by real time PCR \[[@B29]\]. A reproductive infection would seem to be a prerequisite for cell death induction in HASMC. The observation that heat inactivated or chloramphenicol treated bacteria never caused changes of the host cells underlines that cell death induction is not a bystander effect of a potential cytopathic chlamydial component such as the heat stable LPS. It can be concluded that HASMC death depends on internalization of the *Chlamydia*and is associated with the suppression of bacterial replication and productive infection. The necessity for a reproductive infection was also shown for *Chlamydia psittaci*which induced caspase 3 independent apoptosis in macrophages and epithelial cells only after reproductive infection along with bacterial protein synthesis \[[@B15]\]. Cell death induction was never found in cells harbouring inclusions which normally are viewed as the manifestation of a reproductive infection. HASMC formed inclusions with a relatively low frequency compared to tumor cells. In contrast, spots resulting presumably from single bacteria were found frequently illustrating a different infection morphology of Cpn in these primary vascular cells. Currently no data are available regarding metabolism, protein synthesis and reproduction or functional relation with host cells of these single, spot-like bacteria. From our data it only can be concluded that these single bacteria are sensitive to chloramphenicol which inhibits protein synthesis in Cpn and abrogates cell death induction. Cell death seems to result from an interaction of the host cell with these single bacteria as it is ongoing for extended times. Cpn induced cell death in HASMC did not fulfill all criteria for apoptosis or classical necrosis. Condensation of chromatin, positive TUNEL staining, externalization of phosphatidylserine, inhibition of TMRE labeling and loss of cytochrome c immunoreactivity in mitochondria were clear markers for apoptosis. But early damage of cell membrane and organelles as found by TEM and NHS-biotin labeling indicated necrotic type of cell death in Cpn infected cells. According to previous classifications Cpn mediated cell death belongs to apoptosis-like cell death \[[@B30]\] or can be termed aponecrosis due to characteristic ultrastructural features \[[@B24]\]. Cpn induced cell death in HASMC having features of necrosis and apoptosis is in agreement with a study employing mouse embryonic fibroblasts infected with *C. trachomatis*which died through a combination of necrosis and apoptosis \[[@B18]\]. If cell death induction by Cpn in HASMC is caspase-3 independent the question arises about alternative pathways that are involved. Mitochondria are important regulators of cell death and loss of mitochondrial permeability transition is an important indication for apoptosis \[[@B31]\]. Here we show by TMRE labeling dissipation of the mitochondrial potential of *Chlamydia*infected cells. This effect could be inhibited by blocking bacterial protein synthesis using chloramphenicol. Moreover, cytochrome c was released from mitochondria of infected HASMC as indicated by loss of immunorecactivity. The important role of mitochondria in *Chlamydia*induced cell death has also been shown in tumor cell lines infected with *Chlamydia psittaci*\[[@B14]\]. Pro-apoptotic Bcl-2 family members like Bax were activated and apoptosis induction occurred during the whole chlamydial life cycle. Apoptosis induced by *Chlamydia psittaci*was blocked in cells over expressing Bax inhibitor and anti-apoptotic Bcl-2 \[[@B14]\]. Bax^-/-^mouse embryonic fibroblasts were shown to be resistant to *Chlamydia muridarum*-induced apoptosis and fewer bacteria were recovered after two infection cycles. This suggests that Bax-dependent apoptosis may be used to initiate a new round of infection \[[@B32]\]. In a second study, *Chlamydia trachomatis*infected mouse fibroblasts showed a higher level of apoptosis than *Chlamydia*infected HeLa cells as measured by annexin V / propidium iodide double labeling \[[@B18]\]. This not only shows that Bax activation could reflect a stress response in infected cells \[[@B33]\] but that primary cells show a different stress response to *Chlamydia*infection than tumor cells. Beside Bax other factors like apoptosis-inducing factor (AIF) may be involved in caspase-independent cell death \[[@B34]\] but they have never been investigated in *Chlamydia*-induced cell death. AIF which is upregulated and translocated from the mitochondria to the cytosol may play an important role in induction of caspase-3 independent cell death \[[@B34]-[@B36]\]. Injection of AIF into the cytoplasm induces similar chromatin condensation coupled to phosphatidylserine exposure as we found in Cpn infected HASMC \[[@B37]\]. Future experiments will have to show if AIF, Bax or other Bcl-2 family members are involved in Cpn induced HASMC death. There exist numerous studies showing that Cpn and *Chlamydia trachomatis*prevent cells in vitro from undergoing apoptosis \[[@B16],[@B17],[@B38]-[@B44]\]. However, these experiments were performed on established cell lines. Our study shows that in primary cultures of cells such as HASMC various types of Cpn infection occur. Spot like infection resulted in aponecrosis whereas cells with Cpn inclusions appeared to be prevented from cell death. Apparently Cpn in inclusions have evolved strategies to inhibit cell death presumably to complete the developmental cycle. About possible mechanisms can be speculated: it has been shown that Cpn inserts components of a type-III secretion system into the inclusion membrane \[[@B45]\] and releases factors into the host cell \[[@B46]-[@B48]\] that affect host cell metabolism. Cpn secretes proteins that translocate from the inclusion to the cytoplasm \[[@B47],[@B49]\]. CADD, a secreted protein from *Chlamydia trachomatis*, has been shown to interact with death domains and co-localizes with Fas. Recombinant CADD has been shown to induce caspase dependent apoptosis in several tumor cell lines \[[@B50]\]. Therefore, other yet unidentified proteins from *Chlamydia*could interact with the host cell death machinery and inhibit or promote cell death. Currently, it is unclear how the observed morphology of Cpn in HASMC and the induced aponecrotic cell death affects the progression of atherosclerosis. Cpn infection of HASMC was accompanied by phosphatidylserine externalization which might result in the ingestion of dying cells by macrophages. Macrophages will then either stop further progress of Cpn mediated damage or in turn become activated and thereby contribute to inflammation in the atherosclerotic vessel \[[@B51],[@B52]\]. Early membrane damage found in the aponecrotic HASMC may provoke or assist inflammation in the atherosclerotic vessel due to release of cellular constituents. Finally damage of vascular smooth muscle cells by Cpn infection might lead to plaque instability \[[@B19]\]. Conclusions =========== Cpn infection of cultured HASMC results in cell death that can be termed aponecrosis due to the sharing of apoptotic and necrotic features. This form of cell death occurs just in cells bearing single or aggregated bacteria but not in cells with inclusions. Aponecrotic HASMC may in vivo assist chronic infection in the vascular wall supporting the progression of atherosclerosis. Methods ======= Chlamydial organisms, cells, antibodies and reagents ---------------------------------------------------- Cpn-VR1310 and Cpn isolate Kajaani 6 (Cpn-K6) were kindly provided by G. Christiansen (Institute of Microbiology and Immunology, University Aarhus, Denmark) and M. Puolakkainen (Haartman Institute, University of Helsinki, Finland), respectively. HEp-2 cells obtained from ATCC (Manassas, USA) were cultivated in MEM-Eagle\'s (Gibco BRL, Invitrogen, Basel, Switzerland) and HASMC purchased from Clonetics (Verviers, Belgium) were maintained in DMEM supplemented with 10% FCS, 2 mM glutamine, all from Gibco BRL, Invitrogen, and 10 μg/ml neomycin (Sigma Chemicals, Buchs Switzerland). Cells and chlamydial organisms were tested for *Mycoplasma spec*. contamination by PCR \[[@B53]\] and 4\',6-Diamidin-2\'-phenylindol-dihydrochloride (DAPI) (Roche, Mannheim, Germany) staining and were mycoplasma free. Anti-Cpn-MOMP monoclonal antibody was from DAKO (Zug, Switzerland); anti-cytochrome c antibody raised in sheep was from Sigma, FITC-labeled monoclonal anti-*Chlamydia*-LPS antibody from Biorad (Redmont, USA). Donkey anti-mouse polyclonal antibody was obtained from Jackson Immuno Research (Baltimore, USA) and staurosporin from Sigma Chemicals. Chlamydial culture and infection of HASMC ----------------------------------------- Cpn was cultured according to an established protocol \[[@B54]\]. Briefly, confluent HEp-2 cells were inoculated with Cpn at 1 IFU/cell in medium, centrifuged at 1000 × g for 1 h and grown for 72 h in medium containing 10% FCS, 2 mM glutamine, 10 μg/ml neomycin and 2 μg/ml cycloheximide (Sigma). Subsequently, cells were harvested, disrupted by sonification and bacteria were purified on a discontinuous renografin (Sigma Chemicals) gradient as previously described \[[@B55]\]. Non infected HEp-2 cells were subjected to the same harvesting / purification procedure and referred to as mock controls. In order to determine the chlamydial titer (IFU/ml), confluent HEp-2 cells grown on cover slips were infected with serial 2-fold dilutions of purified bacteria. After 72 h cells were fixed, stained with anti-*Chlamydia*-LPS antibody, inclusions were counted under a fluorescence microscope and the titer was calculated. HASMC were seeded into 6-well plates (Nunc, Roskilde, Denmark) at a density of 2.4 × 10^4^cells/cm^2^one day prior to infection. Infection with chlamydial organisms was performed in DMEM using titers between 8 and 386 IFU/cell which were added to HASMC, centrifuged onto the cells for 1 h at 1000 × g. Subsequently, the supernatant was replaced by DMEM containing 10% FCS, 2 mM glutamine and 10 μg/ml neomycin. In some experiments chloramphenicol 0.1 mg/ml was added analysis or cells were treated with 1 μM staurosporin for 14 h. Cells were grown at 37°C in the presence of 5% CO~2~until they were analyzed. Determination of infection morphology, intra- versus extracellular staining of Cpn ---------------------------------------------------------------------------------- Live HASMC and HEp-2 cells were incubated with anti-Cpn-MOMP antibody (1:50) in PBS + 2% BSA for 1 h at room temperature (RT). After washing and fixation with 2% paraformaldehyde + 3% sucrose in PBS at RT, cells were incubated for 1 h with donkey anti-mouse-FITC antibody, diluted 1:100 in PBS + 2% BSA. Bacteria located extracellularly are stained exclusively green by this step. Subsequently, cells were lysed with 0.1% Triton-X 100 for 1 min at RT, rinsed with PBS + 2% BSA for 20 min, and incubated with anti-Cpn-MOMP antibody (1:50) followed by Texas Red-labeled donkey anti-mouse antibody (1:200) in 2% BSA in PBS. The second staining step identifies both intracellular and extracellular bacteria. Counting of cells with inclusions or spots as well as the number of inclusions and spots were performed on volume data obtained using a laser scanning microscope (SP2, Leica, Heidelberg, Germany) followed by image analysis using the software package Imaris (Bitplane, Zurich, Switzerland). Determination of cytotoxicity by lactate dehydrogenase release (LDH) -------------------------------------------------------------------- LDH release of HASMC was assessed using a cytotoxicity detection kit (Roche) following the manufacturer\'s instruction. In brief, HASMC were seeded in a 96-well plate (NUNC) at a density of 2.4 × 10^4^cells/cm^2^, infected with Cpn between 2 and 32 IFU/cell and cultivated for 24 h, 48 h and 72 h. For cell death analysis 100 μl of cell-free supernatant was harvested, mixed with dye solution, incubated for 20 min and absorption was measured at 490 nm. Percent *Chlamydia*-specific lysis was calculated according to the formula: ((experimental value -- spontaneous release)/(maximum release -- spontaneous release) × 100. Spontaneous release corresponded to untreated HASMC and infected cells lysed additionally with Triton-X 100 showed the maximal release. Transmission electron microscopy (TEM) -------------------------------------- Cells were fixed in 50 mM sodium cacodylate buffer pH 7.3 containing 2% glutaraldehyde and 0.8% paraformaldehyde and postfixed with 1% OsO~4~in 50 mM sodium cacodylate buffer, pH 7.3 dehydrated in an ethanol series and embedded into epon (Catalys). Ultrathin sections of 50 nm were contrasted with uranyl acetate and lead citrate and studied with a CM 100 transmission electron microscope (Phillips, Leiden, Netherlands). TUNEL-, NHS-biotin-, Cpn- and DAPI-staining for analysis by confocal microscopy ------------------------------------------------------------------------------- Cells were trypsinized, washed in PBS and incubated with 0.1 mg/ml NHS-biotin (Pierce, Rockford, USA) in PBS for 30 min on ice, fixed with 3% paraformaldehyde + 2% sucrose in PBS and cytospinned onto glass slides. Samples were permeabilized with 0.1% Triton-X 100 in PBS for 1 min at RT and stained for DNA strand breaks using terminal transferase (Roche), incorporating dUTP-FITC (Roche) overnight at 37°C as previously described \[[@B56]\]. Subsequently, cells were incubated with anti-Cpn-MOMP antibody (1:50) and detected with donkey anti-mouse Texas Red antibody (1:200) in 2% BSA in PBS for 1 h, followed for 45 min by 0.5 μg/ml streptavidin-Cy5 (Jackson Immuno Research) detection of NHS-biotin. Nuclei and DNA of bacteria were stained with 1 μg/ml DAPI in PBS for 10 min. Samples were then embedded in fluorescence mounting medium (Dako) and analyzed using a confocal laser scanning microscope (SP2, Leica, Heidelberg, Germany). Annexin-V/propidium iodide staining for flow cytometry ------------------------------------------------------ Phosphatidylserine exposure and propidium iodide incorporation was assessed using the annexin-V-FLUOS staining kit (Roche). In brief, supernatants of HASMC cultures were collected and mixed with trypsinized cells. Samples were washed in PBS followed by 20 min staining with annexin-V / propidium iodide staining solution and analyzed by flow cytometry (Becton Dickinson, Basel, Switzerland). Determination of caspase-3 activity ----------------------------------- 0.2 × 10^6^HASMC consisting of floating and adherent cells were lysed at 4°C in cell lysis buffer (20 mM PIPES, pH 7.2, 100 mM NaCl, 1 mM EDTA, 10 mM DTT, 0.1% CHAPS, 10% sucrose), subjected to three freeze-thaw cycles and centrifuged at 14000 × g for 10 min. Protein content of supernatants was measured using the micro BCA protein assay reagent kit (Pierce). Cell lysates containing 40 μg protein were incubated with DEVD-*p*-nitroanilide (0.8 mM) in lysis buffer and absorption was measured every 5 min in a spectrophotometer at 405 nm for 6 h at 37°C. Caspase-3 activity was calculated as the initial ascending slope of absorption depicted as arbitrary units. TMRE and propidium iodide staining for analysis of mitochondrial membrane potential ----------------------------------------------------------------------------------- Cells were stained with TMRE 100 nM and propidium iodide 2 μg/ml in DMEM with 10% FCS for 30 min at 37°C and 5% CO~2~, collected by trypsinization, combined with detached cells at time of trypsinization and analysed by flow cytometry (Becton Dickinson). Cytochrome c and mitotracker red staining for confocal microscopy analysis -------------------------------------------------------------------------- Cells were incubated in the presence of 50 nM mitotracker red for 30 min, subsequently fixed with 3% paraformaldehyde + 2% sucrose and were cytospinned onto glass slides. Samples were permeabilized with 0.1% Triton-X 100 in PBS for 1 min at RT. Subsequently stained with sheep anti cytochrome c (1 : 2000) in 3 % BSA in PBS for 1 h at 37°C followed by detetion with donkey anti sheep biotin antibody (1:200) in 0.5 % BSA in PBS for 1 h at RT. Cells were labeled with streptavidin Cy-5 (1 : 200) for 1 h at RT and analysed under a confocal laser scanning microscope. Authors\' contributions ======================= CD performed all infections and analyzed morphological markers for cell death. CFM and MKJS performed FACS experiments. DG initiated the study and performed the first infections of vascular cells. MW analyzed viability experiments and provided input for exeriments. PG provided critical intellectual input to the study and organized financial support. UZ established assays for the detection of caspase 3, morphological cell death markers and was leading the study. Acknowledgements ================ We would like to thank to Miriam Erni, Gery Barmettler and Marina Balzer for excellent technical assistance, Michel Le Hir and Jörg D. Seebach for fruitful discussions and Lloyd Vaughan for carefully reading the manuscript. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Diverse morphology of Cpn infection.**HASMC (A, B, C and D) and HEp-2 cells (E) were infected for 72 h with Cpn-K6 at 128 IFU/cell (HASMC; A, B), Cpn-VR1310 at 8 IFU/cell (HASMC; C, D) and Cpn-K6 at 5 IFU/cell (HEp-2; E), respectively. Bacteria localized on the cell surface are stained with FITC (green), *Chlamydia*internally localized are stained with Texas Red (red) and FITC (for detail see material and methods). DNA of bacteria and host cells is stained with DAPI (blue). N = nucleus. Left row: extracellular Cpn. Middle row: extra- and intracellular Cpn. Right row: overlay of left- and middle row and DAPI staining. A, C: spot like infection of a HASMC; note that the majority of bacteria are localized intracellularly B, D: HASMC with a Cpn inclusion. E: Hep-2 cells with Cpn inclusions. ::: ![](1471-2180-5-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Ratio of cells with inclusions versus cells with spots**HASMC and HEp-2 were infected with Cpn-K6 or Cpn-VR1310. Cells carrying inclusions or spots, respectively, were counted 48 h post infection. A: Depicted is the ratio of cells with inclusions versus cells with spots; -◆-: HASMC Cpn-K6 infected, -■-: HASMC Cpn-VR1310 infected, -◇-: HEp-2 Cpn-K6 infected, -□-: HEp-2 Cpn VR1310 infected B: inclusions / cell, C: spots / cell; black bars: HASMC, open bars: HEp-2 cells. The number of inclusions / cell in HASMC remained constant in contrast to the number of spots / cell when increasing the infectious dose, whereas in HEp-2 cells the number of inclusions / cell increases and the spots / cell remain constant. ::: ![](1471-2180-5-2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Lytic capability of different Cpn isolates.**HASMC were infected with two different Cpn isolates. LDH release was measured in triplicates 24 h, 48 h and 72 h post infection and Cpn specific lysis was calculated. The average and standard deviation is plotted. One representative result out of three independent experiments is shown. A: Dose dependent Cpn specific lysis after 48 h revealed different capacities of the isolates tested to lyse HASMC. B: Different amounts of the various Cpn isolates leading to a comparable lysis over time of HASMC. Isolates were used as follows: Cpn-VR1310, 4 IFU/cell -◆-, Cpn-K6, 32 IFU/cell -■- ::: ![](1471-2180-5-2-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Ultrastructure of Cpn infected HASMC.**Cells were either infected with 128 IFU/cell Cpn-K6 for 72 h (B,C) or incubated with staurosporin 1 μM for 16 h (D) or were left untreated (A). A: Untreated HASMC with normal ultrastructure, note the elongated nucleus with finely dispersed euchromatin. B: HASMC with a typical Cpn inclusion. The membrane bound vesicle contains dark (= elementary bodies) and pale (= reticular bodies) stained bacteria. The chromatin structure in the nucleus is unchanged. C: Cpn infected HASMC undergoing cell death. The nucleus is characterized by highly condensed heterochromatin (arrow head) and a compact nucleolus and is surrounded by dilate organelles (arrow). The cell membrane is completely disrupted. D: Staurosporin treated HASMC. The nucleus is partially fragmented containing patchy condensed heterochromatin (arrow heads). ::: ![](1471-2180-5-2-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Light microscopic analysis of the effect of Cpn infection on HASMC.**HASMC were infected with Cpn-K6 128 IFU/cell (A, B, C, D), Cpn-VR1310 32 IFU/cell (E, F) for 72 h or were inoculated with mock lysate (G) or treated with staurosporin 1 μM for 14 h (H). The cells were subsequently stained for TUNEL (green), NHS-biotin (brown), anti-Cpn-MOMP (red) and DNA (blue). A: HASMC bearing a Cpn-K6 inclusion. DAPI stained chromatin structure is normal and TUNEL as well as NHS-biotin labeling are negative. B: Cpn-K6 infected HASMC with multiple chlamydial spots (arrow heads) and normal nuclear structure. C, E: Cpn-K6 (C) or Cpn-VR1310 (E) infected HASMC with spot like infection (arrow heads). The condensed nucleus is TUNEL positive, the cytoplasm is diffusely labeled with NHS-biotin. D, F: HASMC with Cpn-K6 (D) or Cpn-VR1310 (F) in aggregated (arrow) spots and single spots (arrow heads). Note positive TUNEL labeling of the shrunken nucleus and NHS-biotin staining of the cytoplasm. G: Mock inoculated HASMC. Note the fine granular chromatin structure and lack of TUNEL- and cytoplasmic NHS-biotin staining. H: HASMC incubated with staurosporin. The nucleus exhibits a distinct nuclear fragmentation (DAPI staining) but no TUNEL labeling. The cytoplasm is NHS-biotin negative indicating an intact cell membrane. I: Quantification of TUNEL positive nuclei depending on the chlamydial infectious dose 72 h post infection reveals a dose dependent increase of TUNEL positive cells. Cells in 5 random fields with an area of 62\'500 μm^2^were counted and the percentage of TUNEL positive cells was calculated. -▲-: Cpn-K6; -■-: Cpn-VR1310 ::: ![](1471-2180-5-2-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Dose response relationship between bacterial titer and phosphatidylserine exposure in Cpn infected HASMC.**Cells infected with Cpn-K6 in doses between 8 -- 386 IFU/cell or with Cpn-VR1310 in doses between 1 -- 32 IFU/cell were stained with annexin-V / propidium iodide and analyzed by flow cytometry 48 h post infection. Mock inoculated cells are shown as controls. In figure A and B pooled data out of three independent experiments are displayed. A: The amount of single annexin-V positive cells increased dependent on the chlamydial infectious dose (-◆ -: Cpn-K6; -●-: Cpn-VR1310) but not in mock treated (-○-) cells. (\*: significant compared to mock treated cells with p \< 0.001) B: the number of single annexin-V positive cells increased over the time when cells were infected with Cpn-K6 128 IFU/cell (-●-) but not if they were mock treated (-○-) (significant compared to mock treated cells with p \< 0.001)(\#)). C: Dot plots of one representative experiment are depicted to illustrate the amount of annexin V / propidium iodide double labeled cells. Upper left panel: HASMC treated with staurosporin (apoptosis). Upper right panel: HASMC treated with Na-azide (necrosis). Middle left panel: HASMC infected with Cpn-VR1310 at 32 IFU / cell 48 h post infection. Middle right panel: HASMC infected with Cpn-K6 at 128 IFU / cell 48 h post infection. Lower left panel: mock treated HASMC. ::: ![](1471-2180-5-2-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Caspase 3 activity and mitochondrial membrane permeability**A: Caspase-3 activity was assessed in HASMC infected with 128 IFU/cell Cpn-K6 ± Chloramphenicol (CA), loaded with mock isolate or treated with 1 μM staurosporin for 14 h. No caspase-3 activity can be detected in Cpn infected or mock inoculated cells in contrast to staurosporin treated cells. B: HASMC were infected with 4 -- 128 Cpn-K6 (-●-) or 4 -- 32 IFU/cell Cpn-VR1310 (-◆-) and stained by TMRE for detection of mitochondrial membrane potential in combination with propidium iodide. Propidium iodide negative cells were analyzed by flow cytometry for TMRE staining. Infected HASMC treated with 0.1 μg/ml chloramphenicol served as controls (open symbols). Loss of TMRE staining is induced in HASMC infected with viable Cpn but not in cells infected with Cpn treated with chloramphenicol. Mean values with standard error of three independent experiments are shown. C, D: HASMC were infected with Cpn-K6 (IFU 128 IFU/cell, 48 h) and stained for cytochrome c (white). Mitochondria were labeled with mitotracker red (red). Loss of cytochrome c staining is found in mitochondria of HASMC infected with Cpn (D) but not in mock treated cells (C). ::: ![](1471-2180-5-2-7) :::

PubMed Central

2024-06-05T03:55:52.407707

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547904/", "journal": "BMC Microbiol. 2005 Jan 21; 5:2", "authors": [ { "first": "Claudia", "last": "Dumrese" }, { "first": "Christine F", "last": "Maurus" }, { "first": "Daniel", "last": "Gygi" }, { "first": "Mårten KJ", "last": "Schneider" }, { "first": "Michael", "last": "Walch" }, { "first": "Peter", "last": "Groscurth" }, { "first": "Urs", "last": "Ziegler" } ] }

PMC547905

Background ========== Epidemiological studies have shown a significant correlation of blood pressure levels in close relatives and higher concordance values for occurrence of hypertension in monozygotic vs. dizygotic twins, and thus support the idea that genetic factors influence susceptibility to essential hypertension \[[@B1]\]. While recent molecular genetic studies have provided compelling evidence for mutations in at least seven different genes underlying rare forms of monogenic hypertension \[[@B1],[@B2]\], progress in the understanding of the molecular basis of human essential hypertension has been much slower. Hundreds of case-control studies have suggested hypertension-related genetic variants of which only a few if any have tolerated replication analyses; it is possible that common variants of angiotensinogen \[[@B3]\], α-adducin \[[@B4]\] and the G-protein β subunit \[[@B5]\] confer susceptibility to elevated blood pressure in at least some populations. Since 1999, a number of genome-wide linkage studies in families with multiple affected hypertensive members have been published with highly varying results (for review, see \[[@B6]\]). Recent large-scale searches for genes predisposing to hypertension, published as a recent series of articles \[[@B7]-[@B11]\], failed to identify definite linkage of hypertension to any chromosomal locus, although some DNA regions showing suggestive linkage were disclosed. Reasons for these disappointing data were put on the account of the unsuitability of using a single-locus linkage strategy for a multifactorial genetic disease, inherent genetic heterogeneity of essential hypertension, and complex interplay of genetic and environmental factors underlying regulation of blood pressure variation \[[@B12]\]. Disappointments in the previous strategies justify alternative approaches in which a better phenotyping of the study individuals is connected to their targeted molecular genetic characterization. There are several features that collectively make the genes encoding the beta (βENaC) and gamma (γENaC) subunits of the kidney tubular epithelial sodium channel as serious candidates for susceptibility genes of low-renin human essential hypertension. First, gain of function mutations in β and γ ENaC subunits cause Liddle\'s syndrome, a well-known monogenic form of human hypertension associated with low renin activity and low plasma aldosterone level \[[@B13]-[@B15]\]. Second, common βENaC variants occur in increased frequency in hypertensive black individuals \[[@B16]-[@B18]\]. Third, an extensive locus-targeted study on hypertensive family members demonstrated a significant linkage of hypertension to chromosome 16q region harboring both the βENaC and γENaC genes \[[@B19]\]. These data prompted us to carry out a search for common variants of these two genes in Finnish hypertensive patients who were admitted to a special center because of treatment-resistant hypertension and whose renin-aldosterone system was systematically examined. These circumstances provided a group of hypertensive patients, in which secondary forms of hypertension were effectively excluded and who originated from a genetic isolate. Our data suggest that common variants of the ENaC subunits confer susceptibility to human essential hypertension. Methods ======= Patients with hypertension -------------------------- The clinical records of all consecutive patients with hypertension (n = 615) referred to the Hypertension Outpatient Ward, Helsinki University Central Hospital, between 1992--96 were reviewed. Moderate-to-severe hypertension, suspicion of secondary forms of hypertension, or hypertension resistant to drug treatment were causes to the admittance. A letter with request to donate a blood sample for genetic studies on hypertension was sent to those 598 individuals whose address became available in 1998. A total of 399 individuals (67%) of these responded and were subsequently examined at the Hypertension Outpatient Ward in 1998 to 1999. Clinical and family histories were recorded, and venous blood samples taken for DNA analysis. Based on the previous documents and current examinations, altogether 52 individuals were excluded from the present study: clinical records were missing or insufficient in four cases, 22 subjects were considered as normotensive, while 26 were judged to have a secondary form of hypertension. The latter group consisted of the following cases: renal artery stenosis (n = 12), adrenal cortical adenoma (n = 3), hydronephrosis (n = 2), pheochromocytoma (n = 1), IgA glomerulonephritis (n = 1), non-specific chronic glomerulonephritis (n = 1), LED nephritis (n = 1), diabetic nephropathia (n = 1), chronic pyelonephritis (n = 1), hypernephroma (n = 1), fibromuscular dysplasia (n = 1), unspecified renal failure (n = 1). The remaining 347 patients (186 females and 161 males, mean age 49.3 years, SD ± 10.0) comprised our final cohort of patients with moderate-to-severe essential hypertension. Antihypertensive drug treatment was in use in 283 (82%) of the patients (diuretics, 19%; beta-blocking agents, 35%; calcium-channel blockers, 21%; ACE-inhibitors, 33%; angiotensin receptor antagonists, 1%). At least two concomitant drugs were used by 24% of the patients. A flow-chart of the study design is illustrated in Fig. [1](#F1){ref-type="fig"}. Control individuals ------------------- ### Blood donors DNA was extracted from 301 randomly selected healthy blood donors aged 40--50 years (mean, 45 years) visiting the Finnish Red Cross Blood Transfusion Service. Their residences represent the same capital area from which the hypertensive patients originated. ### Normotensive controls These individuals were selected from the participants in the Alpha-Tocopherol, Beta Carotene (ATBC) study \[[@B20]\] using the criteria described previously \[[@B21]\]. In brief, a total of 27271 male smokers (aged 50 to 69 years) with no previous history of myocardial infarction were initially recruited for a cancer prevention trial. DNA samples were available from 70% of the original participants. We picked up all the available blood samples from those fulfilling the following criteria: no known hypertensive disorder, no antihypertensive drugs ever in use, systolic and diastolic blood pressure values ≤ 128 and ≤ 84 mmHg, respectively, at each blood pressure measurement, repeated five times at one-year intervals during a five-year follow-up. We ended up with 175 normotensive men whose mean systolic and diastolic blood pressures were 114.9 (SD ± 5.4) and 73.7 (SD ± 4.3) mmHg, respectively, during this five-year follow-up. The Ethics Review Committee of the Helsinki University Central Hospital approved this study, and all patients and controls gave their informed consent. Laboratory measurements in the hypertensive patients ---------------------------------------------------- The patients were advised to stop using estrogens and spironolactone at least 4 weeks before the tests, diuretics and prostaglandin inhibitors at least two weeks before the tests, and β-adrenergic antagonists and ACE inhibitors at least one week before the test. The only antihypertensive agents permitted at the time of the test were calcium channel blockers. Some of the patients were on oral potassium supplementation because of hypokalemia. The mean baseline blood pressure level at the time of captopril test was 139 ± 16/94 ± 10 mmHg in those without any drugs (n = 79), and 142 ± 16/95 ± 11 mmHg in those with calcium channel blockers (n = 234). Fasting blood samples were taken for determination of serum creatinine, uric acid, cholesterol, potassium, sodium and blood glucose concentrations. Urine samples for determination of the daily (24 h) excretion of potassium and sodium were collected. Most hypertensive patients (n = 298) underwent a test for the responsiveness of serum aldosterone level and plasma renin activity to postural change. To this end, the first blood sample was taken after at least 60 minutes of rest in supine position. After 2 hours of standing and moderate walking, a second blood sample was taken. This test was carried out at the inpatient ward in 220 cases and at the outpatient ward in 78 cases. Urinary electrolyte excretion rates were analyzed in 262 patients (26 ENaC variant carriers and 236 non-carriers) who did not use potassium supplementation. One to three days later, a captopril challenge test (CCT) was carried out as described earlier \[[@B22]\]. This test was carried out in a total of 313 patients, and was performed at the inpatient ward in 229 cases and at the outpatient ward in 84 cases. CCT was started by sitting for at least 30 minutes, followed by oral administration of 50 mg captopril. Blood pressure in the non-dominant arm was measured at 15-minute intervals. Blood samples for the determination of plasma renin activity and serum aldosterone concentration were drawn immediately before and 60 minutes after captopril administration. DNA analysis ------------ Genomic DNA was extracted from peripheral venous blood using standard techniques. For targeted search for ENaC variants postulated to be associated with increased channel activity, we chose to sequence the exons 13 coding for the carboxyterminal domains of βENaC (amino acids 515--640) and γENaC (amino acids 524--649), as well as the 5\'-flanking intronic regions, using oligonucleotide primers, PCR (polymerase chain reaction) conditions and sequencing instruments described previously by us \[[@B21]\]. DNA samples of 27 patients of those 399 initially visiting the Hypertension Outpatient Ward showing the lowest plasma renin activities (median 0.7 μg/L/h at 0 minutes and 0.9 at 60 minutes) and serum aldosterone concentrations (median 236 pmol/L at 0 minutes and 212 at 60 minutes) during CCT were selected for this initial step. Specific PCR-based methods were set up for assaying the three ENaC variants detected during the present study. After PCR of the βENaC fragment, the βENaC -i12 -17CT and βENaC G589S variants could be assayed simultaneously. An aliquot (8 μl) of the PCR product was digested with 3.0 U of *Alu*I (New England Biolabs, Beverly, Massachusetts, USA), followed by analysis of the cleavage products on a 12% polyacrylamide gel. The wild-type (wt) allele results in longest fragments of 266 and 137 bp, while the variant allele produces fragments of 266 and 147 bp for βENaC -i12 -17CT, and 240 and 137 bp for βENaC G589S. For the γENaC V546I variant, 2.0 U of *Sfa*NI (New England Biolabs) was used and the cleavage products were analyzed on a 2% agarose gel. The resulting fragment sizes were 357 and 77 bp for the wild-type allele and 279, 78 and 77 bp for the variant allele. For studies on the possible splicing errors brought about by the βENaC i12-17 variant, we collected lymphocytes from two subjects heterozygous for this variant and one control subject. Total lymphocytic RNA was isolated using Qiagen RNeasy kit (Qiagen, Valencia, California, USA), and first strand synthesis was performed using Superscript system for RT-PCR (Invitrogen Corporation, Carlsbad, California, USA). For gene-specific PCR, we used two sets of primers amplifying a fragment extending from exon 12, either to exon 13 (180 bp) or 141 bp downstream of exon 13 (551 bp). The amplified products were run on a 12 % polyacrylamide gel and visualized by ethidium bromide. The amplified fragments were also sequenced to exclude presence of any splicing defects. Additionally, possible splicing differences between the wild-type and i12-17 variant of βENaC were studied *in silico*using GrailEXP v3.3 (Perceval) exon prediction program \[[@B23]\]. Site-directed mutagenesis and functional characterization of the ENaC variants ------------------------------------------------------------------------------ The human βENaC cDNA and γENaC cDNA cloned into the pBSK-SP6-globin vector were used in construction of the βENaC G589S and γENaC V546I mutations, respectively. Site-directed mutagenesis was performed using the Transformer site-directed mutagenesis kit (Clontech Laboratories, East Meadow Circle, California, USA). Mutagenic primers used were 5\'-cacaccaacttt**A**gcttccagcctg-3\' and 5\'-gctgctctgttgtctgc**A**tcatcgagatcatcgagg-3\' for the G589S and V546I mutations, respectively. The primer 5\'-ccctcgctcg**T**gtgatctggt-3\', which mutates the *Xho*I restriction enzyme site in the pBSK-SP6-globin vector, was used as the selection primer in the mutagenesis reactions. The mutagenic clones were sequenced to confirm the presence of the mutations and to exclude undesired errors during mutagenesis. Healthy stage V and VI *Xenopus*oocytes were injected with mRNAs encoding the β human (h)ENaC wild-type or βG589S hENaC mutant, the γhENaC wt or γV546I hENaC mutant together with the mRNA encoding the αhENaC wt. The total amount of mRNA encoding the three αβγ ENaC subunits was 10 ng. Electrophysiological measurements were taken at 16--24 hours after injection. ENaC activity was assessed by measurement of the amiloride-sensitive current (I~Na~in μA) recorded at -100 mV with a two-electrode voltage clamp amplifier (TEV-200, Dagan Corp.) in a standard solution containing 110 mmol/L NaCl, 1.8 mmol/L CaCl~2~, 10 mmol/L HEPES-NaOH, pH 7.35. The amiloride concentration used was 5 μmol/l in the bath solution. Four batches of oocytes were obtained from different *Xenopus*frogs in which 5 to 7 oocytes were tested for each αβγ ENaC wt and ENaC variants. Statistical analysis -------------------- The renin and aldosterone values were nonnormally distributed, as analyzed using skewness, kurtosis and Kolmogorov-Smirnov tests. Therefore, nonparametric tests (Mann-Whitney\'s U) were used in the statistical analyses, and interquartile (IQ) range and median are used to describe the distributions of target variables. When covariates were included in the analyses, ANCOVA with ranks or logarithm-transformed values of the variables was used. Chi square test, or Fisher\'s exact test if observed frequency in any cell was less than five, were used for the frequency analysis of the variants. Logistic regression was used to obtain age and gender adjusted odds ratios for hypertension in ENaC variant carriers vs. non-carriers. All data was analyzed using statistical SPSS program (version 11.0). Because of relatively small variant group sizes, the primary analyses were performed with all variant groups combined. Secondarily, the variant groups were also compared separately with the wild-type ENaC group. Results ======= Identification of three common ENaC variants and screening for their presence in the three different study groups ----------------------------------------------------------------------------------------------------------------- DNA samples of 27 hypertensive patients with lowest renin activities and aldosterone concentrations were initially selected for targeted search for ENaC variants. The sequencing strategy chosen permits detection of mutations and polymorphisms in the entire coding parts of exons 13 of β and γENaC genes, as well as 26 or 43 nucleotides at the 3\'-ends of introns 12. Three different single-nucleotide substitutions were detected, two in the βENaC and one in the γENaC subunit (Fig. [2](#F2){ref-type="fig"}). Four out of the 27 samples showed a previously unreported substitution of T for C in intron 12 of the βENaC gene (i12-17CT), located 17 nucleotides upstream of the 5\'-end of exon 13. In one DNA sample a single G to A substitution changed the codon 589 of βENaC from GGC to AGC, predicted to result in a substitution of serine for glycine (G589S). This variant has been described previously \[[@B24],[@B25]\]. Upon screening of exon 13 of the γENaC gene for mutations, one sample was detected with a novel point mutation changing codon 546 from GTC to ATC, which results in a substitution of isoleucine for valine (V546I). We next conducted a search for these three ENaC variants in our whole material of patients with essential hypertension (n = 347), normotensive males (n = 175) and randomly chosen blood donors (n = 301) (Table [1](#T1){ref-type="table"}). Altogether, we identified 46 heterozygous carriers of these variant alleles, but no homozygous or compound heterozygous individuals. Their prevalence was significantly different in the three study groups (χ^2^= 15.0, p = 0.0006). Subanalysis of the three groups indicated that the variant allele frequency was higher among the hypertensive patients (9.2%) than in normotensive males (2.9%; p = 0.007) or blood donors (3.0%; p = 0.001), while in the latter two groups it was similar (Table [1](#T1){ref-type="table"}). When frequencies of the individual gene variants in the hypertensive patients were compared to those in the two other groups (normotensive males and blood donors) combined, the βENaC i12-17CT variant was found to occur significantly more often among the hypertensive patients than in other groups (p = 0.001) whereas the differences in the prevalences of βENaC G589S (p = 0.15) and γENaC V546I (p = 0.14) did not reach statistical significance. Clinical characteristics of the variant allele carriers and non-carriers ------------------------------------------------------------------------ Clinical and laboratory data of the hypertensive patients grouped according to their carrier status of the three ENaC variants detected are summarized in Table [2](#T2){ref-type="table"}. There were no significant differences, associated with carrying a variant allele, in the sex, age or BMI of the hypertensive patients, nor their serum creatinine, lipid, potassium or sodium levels. Variant alleles did not seem to associate with cerebrovascular events or diabetes among the hypertensive patients, but small numbers prevent definitive conclusions (Table [2](#T2){ref-type="table"}). Our original study protocol was not designed to disclose health information of the two reference groups (normotensive males and healthy blood donors), and a similar comparison of variant allele carriers and non-carriers in these groups is therefore not feasible. Relation of the variant ENaC alleles to the activity of the renin-aldosterone system ------------------------------------------------------------------------------------ The dynamics of the circulating renin and aldosterone levels in most hypertensive individuals were studied during two challenge tests: during a supine-upright postural test and in response to captopril administration. Baseline plasma renin activity was very similar in the patients with and without variant alleles, whether investigated during the postural test or captopril administration (Table [3](#T3){ref-type="table"}). Plasma renin levels after attainment of upright posture (p = 0.11) and captopril administration (p = 0.12) were not significantly different among carriers and non-carriers of the ENaC variants (Table [3](#T3){ref-type="table"}, Fig. [3](#F3){ref-type="fig"}). Plasma aldosterone levels did not significantly vary according to the ENaC variant carrier status (Table [3](#T3){ref-type="table"}). We also analyzed renin responses (stimulated value minus baseline value) in the two tests according to the ENaC variant carrier status. We found some evidence of a blunted renin response to both postural (p = 0.21) and captopril (p = 0.087) challenge tests in carriers of variant alleles compared to non-carriers, but there was wide interindividual variation in the test results (Fig. [4](#F4){ref-type="fig"}). Use of covariates (urinary sodium excretion, age and BMI) did not cause significant changes in the results of these analyses. We next related the activity of circulating renin-aldosterone system to sodium-potassium homeostasis in ENaC variant carriers and non-carriers. Serum sodium and potassium concentrations in these two groups of hypertensive patients were similar (Table [2](#T2){ref-type="table"}). Urinary sodium excretion rate was not associated with the ENaC polymorphisms studied (data not shown). Urinary potassium excretion rates were not statistically significantly different in patients with (median, 83 mmol/day) and without (median, 79 mmol/day, p = 0.23) ENaC variants (Table [4](#T4){ref-type="table"}). However, when daily potassium excretion (dU-K, in mmol/day) was related to plasma renin activity (in μg/L/h), as mirrored by the renin levels during the postural challenge test, a significant difference was noticed: the median dU-K/renin ratios in the variant carriers vs. non-carriers were 114 vs. 92 when supine (p = 0.29) and 56 vs. 38 when upright (p = 0.034) (Table [4](#T4){ref-type="table"}); the corresponding values for the average (mean of supine and upright) dU-K/renin ratios were 74 and 51, respectively (p = 0.048) (Table [4](#T4){ref-type="table"}). A similar analysis of dU-K/plasma aldosterone ratios demonstrated higher ratios in female variant carriers vs. non-carriers for supine (p = 0.16), upright (p = 0.014) and the average values (p = 0.012), while no significant differences were seen in males. Collectively, these data suggests that hypertensive individuals carrying the ENaC variants tend to excrete increased amounts of potassium in relation to prevailing plasma renin and aldosterone levels. Molecular characterization of the ENaC variants ----------------------------------------------- We tested whether the βG589S or γV546I have any functional impact on ENaC expressed in *Xenopus*oocytes, the most commonly used expression system for ENaC functional studies. When αβγ ENaC subunits were co-expressed to obtain maximal channel activity, neither the βG589S nor γV546I affected ENaC activity as measured by the amiloride-sensitive Na^+^currents. In other words, these data indicate that the current carried by Na^+^ions through ENaC channels present at the cell surface is similar for ENaC wild-type and mutant channels, indicating that the βG589S and γV546I mutations have no detectable functional consequences on ENaC activity, at least when expressed in *Xenopus*oocytes. In order to clarify whether the C-T substitution at position -17 of the intron 12 of the βENaC could affect mRNA splicing, cDNA was synthesized from an RNA fraction prepared from lymphocytes of two hypertensive patients heterozygous for the βENaC i12-17CT mutation and a control individual without this gene variant. Primer pairs for reverse transcription were designed in a way permitting identification of a possible failure to splice intron 12 properly. Regardless of the primer pairs used, similar DNA fragments were generated from the samples of the βENaC i12-17CT carriers and control subject (data not shown). Furthermore, sequence analysis of the amplified DNA fragments revealed the presence of only normally spliced DNA sequence in the βENaC i12-17CT carriers. Furthermore, *in silico*analysis of the βENaC wild-type and variant DNA sequences suggested no differences in exon splicing. However, this analysis is only of predictive value and does not exclude a splicing defect introduced by the variant nucleotide in renal tissue. Discussion ========== The present study indicates that three common variants of the kidney epithelial sodium channel ENaC occur approximately three times more often in patients with moderate-to-severe essential hypertension compared to normotensive males and While direct *in vitro*studies have failed to demonstrate a gain-of-function for these ENaC variants, their association with an increased urinary potassium loss in relation to existing plasma renin activity suggests that in the long run *in vivo*they may result in sodium retention, suppression of renin and aldosterone levels and hypertension. A large number of common and rare polymorphisms of the α-, β-and γENaC have been described in different populations (reviewed in \[[@B26]\] and \[[@B27]\]), but their pathophysiologic role, if any, has remained obscure, at least in the White populations. A systematic search in approximately 500 hypertensive probands, mostly of Caucasian origin, disclosed seven variants of the βENaC and six variants of the γENaC subunit, but no variant, with the possible exception of the βENaC G589S substitution, showed an increased ENaC activity *in vitro*, nor showed cosegregation with hypertension \[[@B24],[@B28]\]. The G589S variant was also identified in a Swedish hypertensive patient \[[@B25]\]. Two amino acid variants, βENaC G589S and γENaC V546I, both occurred with a frequency of about 2% in the hypertensive patients but in only 1% of the background population or normotensive males. The G589 is located in the poorly conserved cytoplasmic carboxyterminal portion of βENaC, 27 amino acids upstream of the functionally important PY motif. Persu et al. \[[@B24]\] identified the same substitution in a hypertensive female with mild hypokalemia and suppressed plasma renin activity. Using measurements of sodium channel activity and amiloride-sensitive sodium flux in *Xenopus*oocytes, these investigators were able to show a borderline 1.3 to 1.5-fold increase in activity for the G589S variant compared with the wild-type subunit. A similar trend was noticed in our experiments (Fig. [5](#F5){ref-type="fig"}). It remains possible that the functional expression of ENaC in *Xenopus*oocytes is not sensitive enough to detect subtle increases in ENaC activity, as it could well be the case for βG589S ENaC variant, and only mutations leading to large changes in ENaC activity are liable to be detected. On the other hand, even minute changes in ENaC may result in significant *in vivo*effects when operating for decades under the influence of unfavorable living habits or variants of other modifier genes promoting salt reabsorption. Accordingly, the βENaC G589S could confer some susceptibility to low-renin hypertension, but more data on untreated patients and families are needed. The γENaC V546I substitution is located in the second transmembrane domain of the ENaC subunit, and it has not been described previously. Seven out of the eight carriers were females, and their renin and aldosterone levels were very similar to those in non-carriers. When expressed *in vitro*in *Xenopus*oocytes, this substitution did not result in an increase in sodium current (Fig. [5](#F5){ref-type="fig"}). It is not possible at present to deduce whether the V546I variant constitutes a pathophysiologically significant allele by itself or merely a genetic marker conferring susceptibility to hypertension. The C→T variant of the nucleotide -17 of intron 12 of βENaC is a novel one and, interestingly, it was present in 4.6% of the hypertensive patients but in only 1% of the 301 random blood donors (p = 0.009) and 175 normotensive males (p = 0.043). Patients with this variant allele displayed the lowest plasma renin levels and responses of all the subgroups examined (Table [3](#T3){ref-type="table"}, Fig. [4](#F4){ref-type="fig"}), but due to large interindividual variation the differences were not statistically significant. This βENaC variant may have remained undetected in earlier studies as they have mostly employed 5\'-PCR primers annealing at the region containing this substitution. Theoretically, a mutation at this site of an intron could affect RNA splicing. We explored this possibility by reverse transcription-PCR experiments of RNA samples from two variant carriers and a control individual, prepared from peripheral lymphocytes known to express βENaC \[[@B29]\]. We could not demonstrate a splicing error, but since homozygous individuals were not available for studies, we may have missed subtle changes. Furthermore, it is not known how well βENaC mRNA splicing in lymphocytes reflects the mechanism in kidney epithelial cells. Another possibility is that the DNA region around the variant nucleotide -17 of intron 12 contains interaction site for regulatory factors affecting transcription of βENaC in tubular cells, or the i12-17CT variant may be in linkage disequilibrium with some yet unidentified mutation present elsewhere in the βENaC or in the closely linked γENaC gene. The fact that we did not find hypokalemia or statistically significant suppression of renin levels in our patients with variant ENaC alleles does not abandon the hypothesis that they act as subtle genes conferring liability to sodium retention and hypertension during lifetime. In fact, even in cases with unequivocal Liddle\'s syndrome due to activating ENaC mutations the penetrance of disease phenotype is variable, with inconstant occurrence of hypertension, hypokalemia and suppressed renin levels from patient to patient \[[@B13],[@B30]-[@B32]\]. This suggests that Liddle\'s syndrome may represent an intermediate between single-gene and complex genetic diseases, necessitating the effect of extrinsic factors, such as substantial salt intake or other modifier genes, to complete the spectrum of syndrome manifestations. It is of particular note that molecular variants resulting in increased ENaC activity may occur outside the cytoplasmic PY motif that long was considered as a critical domain to be affected in Liddle patients \[[@B21],[@B33],[@B34]\]. Our present data are supported by findings of Rayner et al. \[[@B18]\] who recently discovered another βENaC variant (R563Q), which is located in the cytoplasmic domain just adjacent of the cell membrane and was found to be strongly associated with low-renin, low-aldosterone hypertension in a South African black population. Unfortunately, functional characterization of the R563Q variant was not carried out. Previously, another βENaC variant (T594M) was identified in the African Americans \[[@B35]\]. Although initially not linked to elevated blood pressure in the Blacks \[[@B35]\], subsequent studies in a London black population suggested a positive association with hypertension \[[@B16],[@B36]\]. The T594M substitution was reported to result in an increased responsiveness to a cAMP analog due to loss of protein kinase C inhibition of the ENaC \[[@B35],[@B37]\], but other studies have failed to show increased sodium currents in transfected cells \[[@B24]\]. An additional βENaC variant (G442V) present almost exclusively in Blacks has also been suggested to be associated with biochemical alterations compatible with increased ENaC activity in vivo \[[@B17]\]. Our present results and the previous data summarized above suggest that subtle β and γENaC variants do exist in the population that may variably result in elevated ENaC activity, suppression of plasma renin and aldosterone levels, urinary loss of potassium, and elevated blood pressure levels. Individual patients may variably manifest either only one or several of these features, and in some of the variant carriers these parameters may be entirely normal. It will be of interest to test the effectiveness of amiloride in our patients with the βG589S, i12-17CT and γV546I variants as an antihypertensive drug as this specific ENaC antagonist was shown to control blood pressure as well as increase plasma renin, aldosterone and potassium levels in black hypertensive individuals carrying the βT594M allele \[[@B38]\]. There are certain limitations in our study. First, our hypertensive patients represent a highly selected type of patients, since they were recruited by admittance to a specific center focusing on problems in conventional treatment. The clinical study protocol was initially designed for studies on screening for renovascular hypertension in a population, which explains the use of captopril test in the test panel. Unfortunately, urinary aldosterone levels, integrating aldosterone secretion rate over a longer observation period and serving as a valuable marker of Liddle\'s syndrome \[[@B13],[@B31]\], in particular when related to urinary potassium excretion levels \[[@B30]\],were not studied systematically. Our single-point plasma renin and aldosterone measurements may have been liable to incidental variations in their plasma levels, and prevent direct comparison to previous studies relying on urinary aldosterone assays. Second, our normotensive reference population comprised of male patients only. However, we had the advantage of picking up the extreme lowest end, as regards systolic and diastolic blood pressure levels in the absence of any antihypertensive drugs, from a very large material of more than 27000 individuals \[[@B20]\]. Third, due to ethical limitations of the study design, we did not have access to the clinical data of the subjects in the two reference groups (normotensive males and healthy blood donors); it would have been of interest to review the health data of ENaC variant carriers in these two groups. Fourth, our study had limited statistical power for several of the questions asked, particularly when either genetic variant was analyzed alone in carriers versus non-carriers. Therefore, for several of the questions asked in this study these variants may well have only modest effects, too small to be detected using the parameters of the present study. Fifth, pooling of the three genetic variants may represent an oversimplification, as it is uncertain whether these three variants exert similar effects on the various endpoints studied. Finally, our statistical analyses were not corrected for multiple comparisons and therefore some of the results observed in this study could represent chance findings rather than real phenomena. However, this is probably not the case for the observed increased frequency of ENaC genetic variants in hypertensive patients versus normotensive males and blood donors, because this was the primary hypothesis tested and the p values for these comparisons were 0.007 and 0.001, respectively. Conclusions =========== We have demonstrated that almost 9% of Finnish patients with hypertension admitted to a specialized center carry genetic variants of β and γ subunits of the kidney epithelial sodium channel ENaC, a percentage three times higher than that in the normotensive individuals or random healthy controls. Patients with the variant alleles tended to have suppressed renin levels and renin responsiveness to challenging stimuli, and they showed a significantly increased urinary potassium excretion in relation to their renin levels. It will be important to study whether carriers of ENaC variants respond favorably to ENaC blockers (amiloride and triamterene). Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= TH-H collected the clinical material, and participated in the DNA analyses and drafting of the manuscript. KK and TPH designed the study and drafted the manuscript. IT, TT, FF, KH designed the clinical chemical and hormonal assays, and participated in collection of the patient material. HF, HEM and KP participated in DNA analyses and bioinformatics. JV and TK collected the control populations and designed their studies. SS consulted in the statistical analyses and assisted in data handling. IG and LS carried out the electrophysiological studies and participated in drafting of the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2350/6/4/prepub> Acknowledgements ================ We thank Ms Tuula Soppela, Saara Nyqvist, Tarja Pajunen and Susanna Tverin for expert technical assistance. This work was supported by grants from the Finnish Academy, the Finnish Foundation for Cardiovascular Research, the Sigrid Juselius Foundation (to K.K.), and by a grant (3100-059217) from the Swiss National Science Foundation (to L.S.). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Flow diagram of the recruitment of the patients with primary hypertension. ::: ![](1471-2350-6-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Sequence analysis of the variant β and γENaC alleles.**Chromatograms from both sequencing directions, nucleotide substitutions and predicted amino acid changes from three different hypertensive patients are shown. ::: ![](1471-2350-6-4-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Renin values in postural test and after captopril administration.**Individual plasma renin activities at supine, upright, and in response to captopril administration (CCT; 60-minute values) in carriers and non-carriers of the three ENaC variant alleles. The horizontal bars indicate the median renin values in each group. ::: ![](1471-2350-6-4-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Renin responses in postural and captopril tests.**Plasma renin responses (median and interquartile ranges) during the postural and captopril challenge tests (stimulated values minus baseline values in both cases) in carriers and non-carriers of the variant ENaC alleles. ::: ![](1471-2350-6-4-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Channel activity of βENaC G589S and γENaC V546I variants in vitro.**Comparison of channel activity of hENaC wild-type, and the βhENaC G589S and γhENaC V546I variants, when expressed in *Xenopus*oocytes. ENaC activity was measured as amiloride-sensitive Na^+^current. Absolute currents were 5.09 ± 0.98 and 6.75 ± 1.23 μA for ENaC wt in the two series of experiments (number of oocytes given in parentheses). ::: ![](1471-2350-6-4-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### β and γ ENaC variants identified among the three study groups ::: Hypertension n (%) Normotensive males n (%) Blood donors n (%) Adjusted OR (95% CI)^1^ ------------------ -------------------- -------------------------- -------------------- ------------------------- **All variants** 32 (9.2) 5 (2.9)\*\* 9 (3.0)\*\* 3.1 (1.6--6.0) βENaC i12-17CT 16 (4.6) 2 (1.1)\* 3 (1.0)\*\* 4.6 (1.6--13.0) βENaC G589S 8 (2.3) 2 (1.1) 3 (1.0) 2.4 (0.77--7.7) γENaC V546I 8 (2.3) 1 (0.6) 3 (1.0) 2.2 (0.63--7.5) **Non-carriers** 315 (90.8) 170 (97.1) 292 (97.0) \**P*\<0.05 and \*\**P*\<0.01 vs. Hypertension group. ^1^OR for hypertension (versus combined control groups) in ENaC variant carriers vs. non-carriers, adjusted for age and gender. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Demographic and clinical features of the hypertensive subjects, according to their ENaC variant status ::: **Carriers of the ENaC variants** **Non-carriers** -------------------------------- ----------------------------------- --------------------- --------------------- -------------- ------------- βENaC i12-17CT (n = 16) βENaC G589S (n = 8) γENaC V546I (n = 8) All (n = 32) (n = 315) Female/male (n) 9/7 4/4 7/1 20/12 166/149 Age (y) 49.4 ± 8.5 49.3 ± 5.8 50.5 ± 9.0 49.7 ± 7.8 49.3 ± 10.2 BMI (kg/m^2^) 28.0 ± 4.9 28.2 ± 4.8 27.9 ± 3.8 28.0 ± 4.5 27.4 ± 4.9 Serum creatinine (μmol/L) 88 ± 18.9 91 ± 12.8 85 ± 17.1 88 ± 6.7 88 ± 15.1 Serum uric acid (μmol/L) 329 ± 95.3 335 ± 65.6 386 ± 173.2 338 ± 87.1 340 ± 91.2 Fasting blood glucose (mmol/L) 5.3 ± 0.9 5.7 ± 1.1 5.5 ± 0.9 5.4 ± 0.9 5.6 ± 1.1 Serum cholesterol (mmol/L) 5.5 ± 0.8 6.0 ± 1.2 5.3 ± 0.7 5.6 ± 0.9 5.6 ± 1.0 Serum potassium (mmol/L) 4.1 ± 0.3 4.2 ± 0.4 4.2 ± 0.5 4.2 ± 0.4 4.1 ± 0.3 Serum sodium (mmol/L) 141 ± 2.4 140 ± 3.5 138 ± 2.2 140 ± 2.8 140 ± 0.3 Potassium supplementation (n) 1 0 2 3 (9.4%) 31 (9.8%) Cerebrovascular disorder (n) 1 0 0 1 (3.1%) 17 (5.4%) Diabetes (n) 1 1 1 3 (9.4%) 36 (11.4%) Gestational hypertension (n) 3 1 2 6 (18.8%) 44 (14.0%) Data for age, creatinine, uric acid, glucose, cholesterol, potassium and sodium is given as mean ± SD. Carriers of the ENaC variants did not differ significantly from non-carriers. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Plasma renin activity and serum aldosterone concentration during postural and captopril challenge tests ::: **Carriers of the ENaC variants** **Non-carriers** *P-values* ------------------------ ----------------------------------- ------------------ ------------------ ----------------- ----------------- ---------------------------------- βENaC i12-17CT βENaC G589S γENaC V546I All All variants versus non-carriers **Postural test (n)** 15 8 7 30 268 PRA, supine 0.7 (0.4--1.7) 0.7 (0.4--1.0) 0.6 (0.3--1.8) 0.7 (0.4--1.2) 0.8 (0.5--1.4) 0.37 PRA, upright 1.2 (0.8--2.3) 1.5 (0.7--2.9) 1.6 (0.3--2.4) 1.5 (0.7--2.4) 1.9 (1.0--3.5) 0.11 Aldosterone, supine 343 (225--398) 457 (251--592) 358 (238--622) 368 (243--482) 369 (273--474) 0.76 Aldosterone, upright 663 (353--1073) 725 (552--960) 1036 (585--1643) 761 (484--1129) 939 (584--1255) 0.21 **Captopril test (n)** 15 8 8 31 282 PRA, 0 min 1.2 (0.8--2.4) 1.3 (0.5--2.9) 1.3 (0.2--3.4) 1.2 (0.6--2.6) 1.4 (0.7--2.5) 0.53 PRA, 60 min 1.7 (1.0--6.2) 3.0 (0.5--5.7) 2.3 (0.5--8.2) 1.9 (0.8--5.7) 3.6 (1.3--7.7) 0.12 Aldosterone, 0min 554 (302--820) 608 (474--806) 645 (361--1312) 599 (340--845) 618 (431--877) 0.47 Aldosterone, 60min 400 (245--513) 400 (336--472) 356 (261--803) 392 (250--513) 397 (311--539) 0.41 Data is given as median (interquartile range). PRA, plasma renin activity (μg/L/h); Aldosterone, serum aldosterone concentration (pmol/L). Reference values: renin 0.9--2.0 (supine) and 2.0--5.0 (upright), and aldosterone 85--470 (supine) and 220--1000 (upright). ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Urinary potassium excretion and relation to plasma renin and aldosterone levels ::: **Carriers of the ENaC variants** **Non-carriers** *P-values* ------------------- ----------------------------------- --------------------- --------------------- ------------------- ------------------- ---------------------------------- βENaC i12-17CT (n = 14) βENaC G589S (n = 7) γENaC V546I (n = 5) All (n = 26) (n = 236) All variants versus non-carriers dU-K (mmol) 86 (68--112) 85 (66--120) 80 (70--96) 83 (68--102) 79 (65--94) 0.23 dU-K/PRA upright 54 (31--122) 57 (38--180) 97 (43--274) 56 (35--129) 38 (23--84) 0.034 dU-K/PRA mean 67 (37--168) 77 (55--252) 107 (59--321) 74 (46--170) 51 (31--118) 0.048 dU-K/Aldo upright 0.15 (0.06--0.25) 0.10 (0.07--0.15) 0.08 (0.06--0.25) 0.10 (0.07--0.21) 0.08 (0.06--0.14) 0.108 Data is given as median (interquartile range). dU-K, daily urinary potassium excretion; PRA, plasma renin activity (μg/L/h); Aldo, serum aldosterone concentration (pmol/L); PRA mean, average of supine and upright PRA. :::

PubMed Central

2024-06-05T03:55:52.410884

2005-1-20

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547905/", "journal": "BMC Med Genet. 2005 Jan 20; 6:4", "authors": [ { "first": "Tuula", "last": "Hannila-Handelberg" }, { "first": "Kimmo", "last": "Kontula" }, { "first": "Ilkka", "last": "Tikkanen" }, { "first": "Tuula", "last": "Tikkanen" }, { "first": "Frej", "last": "Fyhrquist" }, { "first": "Karri", "last": "Helin" }, { "first": "Heidi", "last": "Fodstad" }, { "first": "Kirsi", "last": "Piippo" }, { "first": "Helena E", "last": "Miettinen" }, { "first": "Jarmo", "last": "Virtamo" }, { "first": "Tom", "last": "Krusius" }, { "first": "Seppo", "last": "Sarna" }, { "first": "Ivan", "last": "Gautschi" }, { "first": "Laurent", "last": "Schild" }, { "first": "Timo P", "last": "Hiltunen" } ] }

PMC547906

Background ========== TRAIL ([T]{.underline}umour necrosis factor [r]{.underline}elated [a]{.underline}poptosis [i]{.underline}nducing ligand) is one of the most promising anti-cancer agent being currently under investigation (for review see \[[@B1]-[@B5]\]). Initially it was shown that TRAIL specifically induces tumour cell apoptosis and tumour regression in nude mice, even when applied as single agent \[[@B6],[@B7]\]. In the meantime rare reports questioned the tumour cell specificity of TRAIL since it was shown that TRAIL induced apoptosis also occurred in normal human liver cells \[[@B8],[@B9]\]. However, subsequentially it was shown that the biochemical preparation of TRAIL rather than TRAIL itself was responsible for the observed toxic effect on hepatocytes \[[@B10]\]. This is strongly supported by the lack of toxicity of TRAIL receptor agonists in humans in first phase I studies \[[@B11]\]. One possible explanation for the tumour specifity of TRAIL could lie in its potential role as a mediator of tumour immune surveillance *in vivo*. In this regard it has been shown that mice lacking TRAIL display a significantly reduced capacity to eliminate syngenic tumour cells in the liver \[[@B12],[@B13]\]. Although TRAIL is a member of the death receptor ligand family certain differences exist to the well characterised ligands TNF ([T]{.underline}umour necrosis factor) or CD95. Most important in this regard is the fact that at least five, instead of only two resp. one, different TRAIL receptors have been identified: The proapoptotic DR4/Trail-R1 \[[@B14]\] and DR5/TRAIL-R2/TRICK2 \[[@B15]-[@B18]\] as well as TRAIL-R3/DcR1/TRID \[[@B16],[@B33],[@B20]\] and TRAIL-R4/DcR2/TRUNDD \[[@B21],[@B22]\], which lack any pro-apoptotic function. The latter were shown to protect cells from TRAIL induced apoptosis by competing with the agonistic receptors for TRAIL binding. In addition, TRAIL-R4/DcR2 is able to induce NF-κB activation, which might upregulate a wide array of anti-apoptotic proteins \[[@B19]-[@B21],[@B23],[@B24]\]. The role of the fifth TRAIL binding protein, the soluble osteoprotegerin (OPG) is still unclear, since it displays only low binding affinity to TRAIL at physiological temperatures \[[@B25],[@B26]\]. In analogy with the signalling events triggered by CD95, multimerisation of the agonistic TRAIL receptors induces the recruitment of the FADD adapter molecule to the receptor, leading to a subsequent autoproteolytic activation of initiator caspase-8 \[[@B27],[@B28]\]. Active caspase-8 in turn triggers the proteolytic activation of downstream caspases including caspase-3. Downstream caspases ultimately degrade a broad range of cellular proteins and apoptosis is finalized (for review see \[[@B29]\]). Up to now, caspase-8 was shown to be the most crucial mediator of TRAIL induced apoptosis \[[@B30],[@B31]\]. However, it has been shown that caspase-10 may act as a surrogate for caspase-8 in some cell systems \[[@B32],[@B33]\]. In contrast to receptor mediated apoptosis, DNA damage triggers apoptosis mainly via mitochondrial death pathways (for review see \[[@B34],[@B35]\]). Key step of mitochondrial apoptosis pathways is the mitochondrial release of pro-apoptotic mediators including cytochrome c. This release is generally controlled by a complex interplay of pro-apoptotic members of the Bcl-2 family namely Bax, Bak, Noxa and Puma. Activation of either of those molecules may occur directly via conformational changes \[[@B36]\] or transcriptional upregulation \[[@B37]-[@B39]\]. Cytochrome c released from the mitochondria triggering the activation of caspase-9 by association with APAF-1 in an ATP dependent manner \[[@B39],[@B40]\]. Caspase-9 subsequently activates the downstream effector caspase cascade including caspase-3 and, in analogy to receptor mediated apoptosis, cell death is finalised. With only very few exceptions \[[@B41]\], apoptosis induction via mitochondrial death pathways is abrogated by anti-apoptotic members of the Bcl-2 family. Anti-apoptotic proteins of the Bcl-2 family interfere with the cytochrome c release from mitochondria on multiple stages \[[@B42]-[@B44]\]. Interestingly, both (death receptor mediated and mitochondrial) pathways are interconnected on several levels. In case of death receptor activation, the propagation of the apoptotic signal is enhanced by caspase-8 mediated activation of Bid \[[@B46]\]. Bid like other BH3-only molecules triggers the release of cytochrome c from mitochondria ultimately resulting in activation of caspase-9 \[[@B45],[@B40]\]. Thus, receptor mediated death pathways are directly connected to mitochondrial death pathways. Vice versa, activation of caspase-9 via the mitochondrial pathway results in secondary activation of caspase-8 and Bid also leading to an amplification of the intracellular death signal \[[@B47]-[@B49]\]. Although TRAIL induces apoptosis when given alone, it has been shown that combination of TRAIL with cytotoxic drugs as 5-fluorouracil, etoposide, paclitaxel, actinomycin C and cisplatin has an even higher apoptotic efficacy \[[@B50]-[@B59],[@B6]\]. Whereas abundant data therefore support the combination of TRAIL with cytotoxic drugs, only limited studies of TRAIL combined with ionising radiation have been performed \[[@B31],[@B33],[@B60]-[@B62]\]. Except for breast and renal cancer, no data supporting the application of TRAIL in the field of radiation oncology are available. Up to now, statistical analysis of a synergistic efficacy have been presented rarely. In addition, potentially harmful effects of a combination on normal cells have not sufficiently ruled out. Methods ======= Chemicals --------- All biochemicals were obtained from Sigma-Aldrich chemicals (Deisenhofen, Germany) unless otherwise specified. Hoechst 33342 was purchased from Calbiochem and dissolved in distilled water as 1.5 mM stock solution. Cell culture ------------ The tumour cell lines MDA-MB 231, HCT-15, Colo 205, NCI H460, FaDu DD and SCC-4 cells were purchased from ATCC (Bethesda, MD, USA). HSF6/ HSF7 fibroblasts, HUVEC and SMC were kindly provided from H-P. Rodemann and R. Kehlbach (Tübingen, Germany). Respectively. All cells were grown in RPMI 1640 medium (Gibco Life Technologies, Eggenstein, Germany) and maintained in a humidified incubator at 37°C and 5% CO~2~. Normal human epithelial cells (HMEC, PrEC, RPTEC, SAEC) and hepatocytes were obtained from Clonetics/Cambrex (Taufkirchen, Germany). Cell culture was performed according to the manufacturer\'s protocols. TRAIL stimulation ----------------- TRAIL induced apoptosis was induced with recombinant human TRAIL/TNFSF10 (R&D Systems, Wiesbaden-Nordenstadt, Germany) in concomitant or sequential application with irradiation. Irradiation ----------- Cells were irradiated with 6 MV Photons using a Siemens Mevatron linear accelerator with a dose rate of 4 Gy per min at room temperature. Quantification of apoptosis induction ------------------------------------- Apoptosis induction was quantified by counting of cells with a characteristic apoptotic morphology after DNA staining with Hoechst 33342. Cells were stained by incubation with Hoechst 33342 at a final concentration of 1.5 μM for 15 min. Microscopy was performed using a Zeiss Axiovert 200 microscope (Carl Zeiss, Jena, Germany) using an excitation wavelength filter of 380 nm. All apoptotic rates were means of at least three independent experiments. The given error bars represent the standard error of the mean from independent measurements of the same cell batch. Westernblotting --------------- Cells (1 × 10^6^) were lysed for 30 min in a lysis buffer containing 25 mM HEPES, 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100, 10 mM EDTA, 10 mM NaF and 125 mM NaCl on ice. After removing insoluble material by centrifugation for 10 min at 12.000 g, 20 μg lysate was separated by SDS-PAGE. Blotting was performed employing a tank blotting apparatus (Biorad, Munich, Germany) onto Hybond C membranes (Amersham, Braunschweig, Germany). Equal protein loading was confirmed by Ponceau S staining (Sigma). Blots were blocked in PBS buffer containing 0.05 % Tween 20 and 5% bovine serum albumin at 4°C over night. Primary antibodies were detected after repeated washings with PBS/Tween 20 (0.05%) of the membrane, using a secondary antibody (anti IgG-AP 1:10.000, Santa-Cruz-Biotech, Heidelberg, Germany) diluted in PBS/Tween and incubated for 3 hours at room temperature and washed three times with PBS/Tween. Detection of antibody binding was performed employing enhanced chemoluminescence (CSPD^®^-Solution Tropix, Applied Biosystems, MA, USA). PARP cleavage was tested using a polyclonal antibodies for cleaved and uncleaved PARP from Boehringer (Mannheim, Germany) in a 1: 1000 dilution. Monoclonal antibodies for caspase 8 were a gently gift from Prof. K. Schultze-Osthoff and used in a 1:45 dilution. β-Actin (Santa Cruz, Heidelberg, Germany) antibody was used in a 1: 5000 dilution. Receptor expression ------------------- Cells (0,2 × 10^6^) were washed twice with PBS and incubated for 30 min with PE-labeled anti-R1/DR4 or -R2/DR5-antibody (R&D Systems, Wiesbaden-Nordenstadt, Germany) at a dilution of 1: 400 in 0,5% FCS/PBS. FACS analyses of superficial receptor expression was performed according to manufacturer\'s protocol with the Quantibrite™ kit from BD (Heidelberg, Germany). Statistical analysis -------------------- Efficacy of the combined modalities were evaluated by the isobolic method \[[@B63]\]. Results ======= Ionising radiation sensitises solid tumour cells to TRAIL induced apoptosis --------------------------------------------------------------------------- Rates of apoptosis induction in response to ionising radiation or TRAIL alone and after combination were determined. Since previous studies on Jurkat T cells or breast cancer cells demonstrated an upregulation of TRAIL receptor R2/DR5 after combined treatment with TRAIL and irradiation \[[@B54],[@B31]\] two different application schedules were tested. TRAIL was either applied directly after cell irradiation or 12 hours later, to allow for receptor upregulation. As shown in figure [1](#F1){ref-type="fig"}, ionising radiation alone (10 Gy) at 48 h induced apoptosis in all cell systems from e.g. 16,0 % in FaDu cells and 34,1% in NCI H460 up to 58,0 % in Colo 205 cells, whereas 0.1 ng/ml TRAIL had a very limited activity in FaDu (3,0%) and Colo 205 cells (14,5%) and up to 30,7 % in NCI H460 tumour cells. In contrast, combination of irradiation (10 Gy) with immediate TRAIL application (0,1 ng/ml) was associated with a much higher apoptotic response (e.g. FaDu 23,3 %, NCI H460 51,9% and Colo 205 80,3%). This effect was even more pronounced when TRAIL was applied 12 hours after irradiation (e.g. FaDu 30,0 %, NCI H460 65,2% and Colo 205 88,0%). In order to test whether the effect of TRAIL and radiation was additive or synergistic an isobologram analysis was performed. Even when applied concomitantly the interaction was synergistic in some cell systems (figure [2a](#F2){ref-type="fig"}, Colo205, HCT 15 and FaDu) but additive in others (MDA MB 231 and SCC-4). When TRAIL was applied 12 hours after irradiation the interaction was synergistic in all but one cell system (figure [2b](#F2){ref-type="fig"}). Processing of caspase 8 and PARP -------------------------------- In order to substantiate the findings on apoptosis induction, caspase activation was verified by analysis of caspase-8 and the processing of the caspase-3 substrate PARP 24 hours after TRAIL application. In keeping with the above results, the most prominent effects were found for Colo 205 and NCI H460 cells with strongly increased caspase-8 and PARP processing after combined treatment. Colo 205 cells were particularly sensitive to sequential application of irradiation and TRAIL, whereas less intensive caspase-8 and PARP processing was found for MDA MB231, SCC4 and FaDu (fig. [3](#F3){ref-type="fig"}). These data correlate well with the kinetics of apoptosis induction as determined by fluorescence microscopy of Hoechst stained cells (fig. [1](#F1){ref-type="fig"}). Irradiation induced regulation of TRAIL receptors ------------------------------------------------- In order to analyse the role of TRAIL receptor regulation in combined therapy with irradiation and TRAIL receptor expression was quantified by flow cytometry using the Quantibrite™ kit system from Becton Dickinson (Heidelberg, Germany). No upregulation of DR4/R1 was found in all tested tumour cell lines. Instead, a subtle downregulation of DR4/R1 after irradiation was observed (fig. [4A](#F4){ref-type="fig"}). Fig. [4B](#F4){ref-type="fig"} demonstrates upregulation of TRAIL receptor DR5/R2 12--18 h after irradiation with 10 Gy in four of six tested cell lines. Colo 205 cells showed the most pronounced receptor upregulation of 196,8%. In HCT-15 and NCI H460 cells an upregulation of R2/DR5 of 118,0% resp. 96,4% could be measured. In MDA MB231 cells and SCC-4 cells no significant upregulation of receptors was found. FaDu cells do not express TRAIL-receptor R2/DR5 and therefore no upregulation of R2/DR5 could be observed after treatment. Combined treatment of TRAIL and ionising radiation do not damage normal tissue ------------------------------------------------------------------------------ As stated above, hardly any data on normal tissue toxicity after combined treatment are available. We therefore analysed the effect of irradiation plus TRAIL on human hepatocytes, fibroblasts (HSF6 and 7), epithelial cells from prostata (PrEC), kidney (RPTEC), breast (HMEC) and small airways (SAEC), endothelial cells from umbilical cord (HUVEC) and small muscle cells (SMC). Normal tissue cells were treated with 10 Gy and a tenfold higher dosage of TRAIL (1 ng/ml) as used for tumour cells. For evaluation of apoptosis strict morphologic criteria as condensation of chromatin and nuclear fragmentation were used. 48 h after combined treatment no relevant sensitisation of normal tissue cells to TRAIL induced apoptosis could be detected in all cultures. Moreover, even preirradiation did not sensitise normal cells to TRAIL induced apoptosis as observed in tumour cells (table [1](#T1){ref-type="table"} and fig. [5](#F5){ref-type="fig"}). Discussion ========== Based on the rationale that radiation and TRAIL induce cell death via distinct but overlapping cell death pathways, tumour cell lines and normal tissue cultures were subjected to either radiation or TRAIL alone or combined with varying application schedules. Our data show that combining radiation with TRAIL induces apoptosis in a significantly higher percentage than either treatment alone. It is important to note that for TRAIL stimulation in our experiments on tumour cells very low concentrations of TRAIL were used (0.1 ng/ml). The pharmacodynamic properties of TRAIL, especially the peak plasma levels in humans, are not known. Since it is likely that toxicity rises with higher doses of TRAIL, the observation of pronounced effects on tumour cell kill at such low doses is particularly intriguing. Any combination of radiation with TRAIL proved to be more effective than either one alone; however depending on dose level and schedule of stimulation less than additive, additive and synergistic effects were detectable. When TRAIL was applied simultaneously with irradiation three of the six cell systems reacted synergistically. In contrast, five of the six cell systems reacted with synergistic effects when TRAIL was given sequentially 12 hours after irradiation. One of the cell lines displayed only less than additive effects. Statistical analysis confirmed, that synergistic effects are more pronounced and occur with greater likelihood after sequential application of TRAIL after irradiation when compared to concomitant treatment schedules. Possible explanations for the positive interaction of TRAIL and DNA damaging agents including ionising radiation are being disputed. Since the sensitisation was associated with upregulation of the DR5 receptor in some experimental settings \[[@B31],[@B54],[@B64],[@B68]-[@B70]\], it is thought that the synergy is based on the increased surface density of the death receptors. Our data support the notion that DNA damage leads to an increased surface expression of the DR5 receptor. However, no tight correlation between receptor upregulation and magnitude of cell kill was observed. It has been suggested, that intact p53 is essential for upregulation of R2/DR5 death receptor expression by ionising radiation \[[@B64],[@B68]\]. However, at least in one of our cell lines (HCT-15) known to harbour a non-function p53 DR5 upregulation was clearly upregulated. Thus, alternative p53 independent pathways for the upregulation of DR5 may exist. This finding is in accordance with data on p53 ^-/-^HCT-116 cells and mouse embryonic fibroblasts showing that NF-κB may also be important for a irradiation induced upregulation of death receptors \[[@B65]\]. Recently, it was shown in overexpression experiments of NF-κB that its subunits c-Rel and RelA regulate expression of cell death molecules in a differential manner. This suggests that RelA, in contrast to c-Rel, acts as a survival factor by inhibiting expression of DR4/DR5 and caspase-8 and up-regulating cIAP1 and cIAP2 \[[@B71]\]. This process depends also on cell type and microenvironment \[[@B54],[@B65]\]. Therefore NF-κB subunits seem to play an ambiguous role in regulation of apoptotic pathways. To know its exact role in radiation induced cell death further research is necessary. Additionally, conflicting data regarding the role of Bcl-2 in death receptor-mediated apoptosis have been provided in the past few years. Interestingly, new data point to a complex relationship between Bcl-2-mediated inhibition of apoptosis and the Bcl-2 protein expression level, the strength and the duration of the death receptor stimulus \[[@B66]\]. Therefore, Bcl-2 expression levels might play a critical role in the modulation of TRAIL sensitivity of tumour cells. Recently, it has been shown that the interaction of 5-FU with TRAIL is strictly Bax but not Bak dependent in HCT 116 cells \[[@B67]\]. Thus, these experiments suggest also a critical role for the proapoptotic Bcl-2 homolog Bax in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signalling cascade. However, the general mechanism of the positive interaction of TRAIL with irradiation remains unclear. It may ultimately turn out, that manifold mechanisms exist and only some mechanisms will be operative in a single cell system \[[@B68]\]. The second part of our experiments the toxicity of TRAIL combined with radiation on normal tissues. In order to be able to draw relevant conclusions for normal tissue cells \[[@B8]\], 10 fold higher doses of TRAIL were used in these experiments. The first important finding is that TRAIL did not induce apoptosis in any of our cell systems including human liver cells. Thus, our experiments confirm the high tumour cell specificity of TRAIL \[[@B6],[@B7]\]. Prior to embarking upon a phase I trial combining TRAIL with irradiation we wished to show a lack of sensitisation to TRAIL by pre-irradiation in normal tissues, compared to tumour cells. Using a wide array of normal cell systems we could not detect any effects of a pre-irradiation on TRAIL sensitivity. Combination of TRAIL with irradiation showed no increase in toxicity over radiation alone. These results are in good accordance with data from Shankar and coworkers showing that TRAIL induced apoptosis rates are only mildly enhanced by a preirradiation of non-malignant human prostate epithelial cells \[[@B68]\]. Conclusions =========== Our data suggest that TRAIL has great potential in cancer treatment, especially in sequential combination with radiotherapy. We did not observe any sensitising effect of the sequential treatment on TRAIL sensitivity in normal tissue cells. Xenograft experiments designed to answer the questions regarding the short and long term efficacy of a combination of radiation with either TRAIL or TRAIL specific antibodies are underway in our laboratory. Abbreviations ============= APAF, apoptosis protease activating factor; FADD, Fas-associated death domain protein; NFκB, nuclear factor κB; PARP, poly-ADP-ribosyl polymerase; TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis inducing ligand. HMEC: human mammary epithelial cells, PrEC: human prostate epithelial cells, RPTEC: epithelial cells of renal proximal tubule, SAEC: small airways epithelial cells, HUVEC: human epithelial cells of umbilical vein, SMC: smooth muscle cells, HSF 6,7: human fibroblasts Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= PM performed FACS-analysis and microscopic evaluation of Hoechst stained cells, analyzed the data, participated in the conception of the trial and participated in the preparation of the manuscript. AS participated in microscopic evaluation of tumour cell apoptosis and accomplished analysis of the resulting data. VJ participated in receptor analysis and microscopic evaluation of Hoechst stained cells. HF carried out Western blotting. PTD participated on the preparation of the manuscript. WB performed isobologramm analysis. CB participated in the conception, design of the study, coordination of the study as well as preparation of the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/5/prepub> Acknowledgements ================ We thank the \"Deutsche Krebshilfe\" for continuous support; grant (10-1764 BeI) to C.B, P.M. W.B. Special thanks to Dr. Stephanie Halene for final revision of and helpful comments on the english manuscript. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Time course of induction of apoptosis in six solid tumour cell lines.**Apoptosis was determined by microscopic evaluation of Hoechst stained cell nuclei 12 to 48 h after treatment with TRAIL 0,1 ng/ml and 10 Gy alone, and after simultaneous and sequential application of combined therapy. Data represent means of three independent experiments; bars ± SD ::: ![](1471-2407-5-5-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Isobolographic analysis of apoptosis induction after combined treatment.**Apoptosis was determined by microscopic evaluation of Hoechst stained cell nuclei 48 h after combined treatment. Envelopes of additivity were calculated with data of 3 independently performed experiments. Datapoints below the curves resemble a synergistic effect, datapoints between the curves demonstrate an additive effect and above the curves a subadditive response of combined treatment. A : tumour cells were treated simultaneously with 10 Gy and 0,1 ng/ml TRAIL. B: cells were irradiated with 10 Gy 12 h prior to treatment with 0,1 ng/ml TRAIL. ::: ![](1471-2407-5-5-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Westernblot analysis of Caspase-8 activation and PARP-cleavage.**Lysates were prepared as described in methods. In six tumour cell lines(Colo 205, NCI H460,, HCT-15, MDA MB 231, FaDu and SCC-4) caspase-8 and PARP cleavage was analysed in untreated cells (lane 1), 24 h after treatment with TRAIL 0,1 ng/ml (lane 2) and 10 Gy alone (lane 3), after simultaneous (lane 4) and sequential application (lane 4) of combined therapy. Every cell line shows a different cleavage pattern according to the rate of apoptosis induction. β-Actin staining was used as loading control. The mapped blots represent each one of three independently performed immunoblots. ::: ![](1471-2407-5-5-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Surface expression of TRAIL receptors after irradiation with 10 Gy in six tumour cell lines**. Quantification of receptor expression was performed 6 to 48 h after irradiation by FACS analyis using the Quantibrite™ evalution system from BD(Heidelberg, Germany) according to manufacturer\'s instructions. Data shown are from one representative experiment (n ≥ 3). A: Cell surface expression of R1/DR4 B: Cell surface expression R2/DR5 ::: ![](1471-2407-5-5-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Microscopic evaluation of normal tissue cells.**Lack of apoptosis was determined by microscopic evaluation of Hoechst stained cell nuclei. Micrographs depict cells of eight different normal tissues, 48 h after irradiation alone and after sequential treatment with 10 Gy 12 h previously to application of 1,0 ng/ml TRAIL. ::: ![](1471-2407-5-5-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Normal tissue toxicity. ::: **Hepatocytes** **HMEC** **PrEC** **RPTEC** **SAEC** **HUVEC** **SMC** **HSF6** **HSF7** ----------------------------------- ----------------- ----------- ----------- ----------- ----------- ----------- ----------- ---------- ---------- **Control** 0,3 ± 0,5 0,3 ± 0,5 0,3 ± 0,5 0 0,3 ± 0,5 0,2 ± 0,2 0,5 ± 0,2 0 0 **TRAIL 1,0 ng/ml** 0,3 ± 0,5 0,5 ± 0,4 0 0 0 0,2 ± 0,1 0,1 ± 0,1 0 0 **10 Gy** 0 0,5 ± 0,5 0 0 0,1 ± 0,2 0,3 ± 0,2 1,5 ± 0,5 0 0 **Simultaneous combined therapy** 0 1,6 ± 0,5 0,7 ± 0,5 0 0 0,3 ± 0,1 1,3 ± 0,9 0 0 **Sequential combined Therapy** 0,3 ± 0,5 0,8 ± 0,5 1,5 ± 1,0 0 0,3 ± 0,5 0,2 ± 0,2 1,2 ± 1,0 0 0 Cells were treated with 1,0 ng/ml TRAIL, 10 Gy, simultaneous combined therapy and with 10 Gy 12 h prior to TRAIL application. Microscopic evaluation of Hoechst stained cell nuclei with strict apoptotic criteria as chromatin condensation and nuclear fragmentation was performed 48 hours after treatment. Table shows percentage of apoptotic cells ± SD (n = 3). :::

PubMed Central

2024-06-05T03:55:52.415486

2005-1-14

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547906/", "journal": "BMC Cancer. 2005 Jan 14; 5:5", "authors": [ { "first": "Patrizia", "last": "Marini" }, { "first": "Angelika", "last": "Schmid" }, { "first": "Verena", "last": "Jendrossek" }, { "first": "Heidrun", "last": "Faltin" }, { "first": "Peter T", "last": "Daniel" }, { "first": "Wilfried", "last": "Budach" }, { "first": "Claus", "last": "Belka" } ] }

PMC547907

Background ========== Obsessive-compulsive disorder (OCD) is a prevalent, chronic and disabling disorder \[[@B1]\]. Controlled pharmacotherapy studies have established superiority of serotonin re-uptake inhibitors (SRI\'s) over noradrenaline reuptake inhibitors and over placebo in OCD and these currently form the cornerstone of pharmacotherapy management \[[@B2]\]. Despite the considerable advances made with the introduction of the SRI\'s into clinical practice, 40--60% of subjects still fail to respond adequately to initial therapy \[[@B3],[@B4]\]. From this it is clear that a need exists to pursue more effective treatments for those with OCD who fail to respond or respond inadequately to SRI\'s. To this end, preliminary evidence supports a role for the addition of atypical antipsychotics to SRIs in OCD. These agents combine serotonin-dopamine antagonism with the advantage of being well tolerated including a low potential for inducing motor side-effects. To date a number of open-label studies have suggested that augmenting SRI\'s with atypical antipsychotics is an effective strategy for treatment-refractory OCD. These include support for risperidone \[[@B5]-[@B7]\], olanzapine \[[@B8]-[@B13]\], and more recently amisulpride \[[@B14]\] and quetiapine \[[@B15]-[@B18]\]. A single open-label study using quetiapine as augmentation showed lack of effect in a small sample using low doses \[[@B19]\]. The outcome of the first controlled study in this area with the antipsychotic haloperidol demonstrated preferential benefit for refractory OCD subjects with co-morbid tic disorder \[[@B20]\]. In two subsequent studies the efficacy of risperidone in SRI refractory OCD has also been reported \[[@B21],[@B22]\]. Interestingly the former study \[[@B21]\], did not replicate the particular advantage for subjects with co-morbid tic disorder. Efficacy has also been shown for quetiapine \[[@B23]\] and olanzapine \[[@B24]\] using similar designs, but the effects on co-morbid tic disorders were not reported. In contrast a recent controlled study using olanzapine failed to demonstrate efficacy over placebo in a six week study \[[@B25]\]. Despite some mixed evidence in this area, in general the available literature appears to support the use of relatively short trials with low doses of antipsychotic agents as augmentation to SRIs. Quetiapine has a particularly interesting profile in that it is the only available antipsychotic with significant 5-HT1D effects and this serotonin receptor subtype has been implicated in OCD \[[@B26],[@B27]\]. Our objective was to examine the effects of quetiapine augmentation in subjects with OCD who had failed to respond adequately to a 12 week trial of an SRI, employing a double-blind, placebo-controlled, six week study design. Methods ======= Patients -------- Forty two subjects aged 18--65 years inclusive were recruited in our multi-centre study comprising five sites in South Africa and one in Canada. Recruitment took place between May 2002 and November 2003. Prior to commencement, all sites in the study received approval from their relevant Research Ethics committees/Institutional review boards and regulatory authorities. All subjects provided written informed consent prior to the commencement of any study-related procedures. Diagnosis was confirmed using the MINI Neuropsychiatric Interview (Version 5.00, 1998) \[[@B28]\] to ensure compatibility with the Diagnostic and Statistical Manual for Mental Disorders, Fourth Edition (DSM-IVTR)\[[@B29]\] criteria for OCD. Subjects with any co-existing Axis I disorder were excluded unless the co-morbid condition was deemed to be secondary to the OCD. Female subjects of childbearing potential were required to use adequate contraception and were not permitted to breastfeed while on the study. Subjects were excluded if they suffered from unstable medical conditions including renal or hepatic insufficiency, epilepsy or had suffered previous brain injury or undergone brain surgery. Taking medication that was deemed likely to interact with quetiapine or any other psychoactive substance was grounds for exclusion. Study Design ------------ All subjects were treated and monitored by investigators for the minimum twelve week duration of SRI-alone treatment phase before inclusion into this study. This was to ensure that patients met criteria for duration of SRI treatment which included at least 6 weeks on the maximum tolerated dose of the relevant SRI. Sample size calculations were based conservatively on similar work in this area \[[@B20],[@B21]\]. Accordingly, for the primary outcome variable (YBOCS), clinically meaningful differences between treatment groups of 6.67 with a standard deviation (SD) of 6 would be detected with a power of 80% at a 5% significance level with a sample size of 14 in each of the treatment groups. The larger sample recruited reflects the anticipation of a 33% drop-out rate in the double-blind treatment phase. A double-blind, randomised, parallel-group six-week augmentation with quetiapine or matching placebo of the SRI to which participants had not responded adequately, was undertaken. Specific SRI\'s, mean doses and dose range are provided in Table [1](#T1){ref-type="table"}. Non-responsiveness to an SRI was defined as either an improvement score on the clinical global impression scale of minimally improved (3) or worse (4,5,6), or less than 25% reduction in Yale Brown Obsessive-compulsive score following twelve weeks of treatment. Inadequate response, as defined above, to at least one SRI administered for a minimum of 12 weeks of which 6 weeks was either at the maximum tolerated dose or alternatively the manufacturer\'s recommended maximum daily dose. SRI doses were maintained at the same level throughout the double-blind treatment phase. For assignment to either quetiapine or placebo groups, we used a computer generated randomization schedule supplied by the sponsoring pharmaceutical company which also packaged the medication. This procedure ensured blinded, balanced allocation to each treatment group across all the study sites. All investigators remained blind to this schedule until closure of the study. No incidents requiring investigators to break the blind occurred through the course of the study. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### SRI\'s used by subjects for failed treatment trial prior to inclusion in the study. ::: **Drug** **N** **Mean maximum tolerated dose (mg/day)** **Median daily dose (mg/day)** **Range** -------------- ------- ------------------------------------------ -------------------------------- ----------- Fluoxetine 13 60 60 60 Citalopram 10 61 60 10 Paroxetine 4 65 60 20 Fluvoxamine 10 290 300 100 Sertraline 1 200 200 0 Clomipramine 2 250 250 0 ::: Treatment --------- At baseline participants were randomly allocated to receive treatment with either quetiapine or placebo using a computer generated schedule and numbered dispensing wallets. A flexible dosing schedule was initiated at 25 mg per day for one week and then doubled weekly to the start of week 4. Based on Clinical Global Impression of Improvement (CGI-I) scores of minimally improved or worse, clinicians were permitted to increase the dose to a maximum of 300 mg per day for the final two weeks of the study. In addition to clinical measures of improvement, clinicians also considered patient tolerability in their decision to adjust doses. Following completion of the treatment phase, subjects were withdrawn from study medication while continuing their SRIs. All subjects were then followed up for any adverse effects. Ratings ------- Patients were assessed by clinicians at baseline and on completion of weeks 2, 4 and 6. Telephonic assessments were performed on completion of weeks 1 and 3. Symptoms of obsessive-compulsive disorder were measured by the same clinician where possible at all study visits using the Yale Brown Obsessive Compulsive Scale YBOCS) \[[@B30],[@B31]\]. A global assessment of severity and improvement was made by clinicians at all assessment points using the Clinical Global Impressions scale of Severity (CGI-S) and Improvement (CGI-I) \[[@B32]\]. Depression was rated using the 10-item Montgomery-Asberg Depression rating scale (MADRS) \[[@B33]\]. For a measure of patient-rated disability we used the Sheehan Disability scale (SDS) \[[@B34]\]. For subjects with tics, frequency and severity were rated using the Yale Global Tic Severity Scale (YGTSS) \[[@B35]\]. Our primary outcome measures for OCD symptoms were (1) the change in YBOCS score from baseline to endpoint and (2) the clinical global impression of improvement (CGI-I) at endpoint. In the final analysis, treatment response was defined as a 25% or greater reduction in YBOCS score and a CGI-I of 1 (very much improved) or 2 (much improved) from baseline to endpoint. Secondary outcome measures included the MADRS, SDS and YGTSS (in subjects with co-morbid tics). Statistical analysis -------------------- Thirty-nine of the forty-two randomised subjects successfully completed the six-week treatment phase. Two subjects withdrew from the study prematurely (Week 1 and Week 4) due to severe levels of sedation. In both of these cases at least one week of study medication had been taken and at least one post-baseline clinical assessment was completed. Both of these subjects were included in the final analysis using data from the last observation carried forward (LOCF). The single subject not included in the efficacy analysis completed the study, but was found not to have correctly fulfilled the study definition of treatment refractoriness and was excluded. Twenty subjects were allocated to the quetiapine arm and twenty-one to the placebo arm. Student\'s t-tests were used to determine any baseline differences in the groups for age, gender, number of previous trials of SSRI\'s, severity of symptoms in relation to OCD, depressive symptoms, and CGI-S. Analysis of variance was undertaken with group and tics as factors. All tests were two-tailed with p-values of less than 0.05 considered significant. Results ======= Study sample characteristics ---------------------------- For the final analysis, our sample comprised 19 men and 22 women. Baseline characteristics of two treatment groups did not differ with respect to age (years) (quetiapine group 33.8(SD 9.66), placebo group 31.81 (SD 12.14); p = 0.57), gender (p = 0.29), number of previous adequate SRI trials (quetiapine 1.55 (SD 1), placebo 1.62 (SD 1.02) (p = 0.83), baseline severity of OCD (CGI-severity, p = 0.47; YBOCS, p = 0.33), depressive symptoms (p = 0.91), patient-rated disability (p = 0.28) or the presence (n = 11, p = 0.66 and severity (p = 0.87) of co-morbid tics. Treatment outcomes ------------------ For the primary outcome measure of severity (YBOCS), quetiapine (p \< 0.0001) and placebo (p = 0.001) augmentation of an SRI significantly improved symptoms of OCD. However quetiapine did not demonstrate significant benefit over placebo at the end of the six-week treatment period (F = .19; p = .636) (Figure [1](#F1){ref-type="fig"}). The mean reduction in YBOCS scores for the combined group was 7.15 points (quetiapine = 7.10; placebo 7.19). Forty percent (n = 8/20) of subjects on quetiapine were classified as responders (YBOCS reduction of \>25% from baseline and CGI-improvement score of 1 or 2) while 47.6% (n = 10/21) of subjects on placebo were classified as responders. A higher number of previous SRI trials for the each treatment group did not correlate with the degree of change on the YBOCS or the response status. Table [2](#T2){ref-type="table"} provides details of individual subject SRI doses, baseline clinical severity ratings and response status. Table [3](#T3){ref-type="table"} provides a summary of baseline and change scores for each of the primary and secondary outcome variables. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **YBOCS change for treatment groups**Quetiapine and placebo groups improved significantly, without significant between group differences (F = 0.19; p = 0.636) ::: ![](1471-244X-5-5-1) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Baseline characteristics of treatment groups ::: Treatment Group **SRI baseline** **SRI Dose baseline (mg/day)** **Previous SRI trials** **Total YBOCS -- Baseline** **Total YBOCS -- Week 6** **Endpoint dose (mg/day)** **% CHANGE** **CGI -- Improvement** **Response status** ----------------- ---- ------------------ -------------------------------- ------------------------- ----------------------------- --------------------------- ---------------------------- -------------- ------------------------ --------------------- **Quetiapine** 1 Paroxetine 60 1 33 29 50 -12.00 3 N/R 2 Citalopram 60 2 25 23 200 -8.00 4 N/R 3 Fluvoxamine 300 1 32 27 300 -16.00 3 N/R 4 Citalopram 70 1 27 25 25 (E/W)\* -7.00 4 N/R 5 Clomipramine 250 2 22 27 100 23.00 5 N/R 6 Paroxetine 60 5 35 32 300 -9.00 4 N/R 7 Fluoxetine 80 3 25 16 300 -36.00 2 R 8 Sertraline 200 1 18 3 50 -83.00 1 R 9 Fluoxetine 20 1 21 20 300 -5.00 4 N/R 10 Fluoxetine 60 1 22 17 300 -23.00 2 N/R 11 Citalopram 60 1 32 17 300 -47.00 1 R 12 Fluoxetine 80 1 32 14 300 -56.00 1 R 13 Fluvoxamine 300 1 30 12 50 -60.00 1 R 14 Fluoxetine 60 1 25 7 50 -72.00 1 R 15 Citalopram 60 1 27 24 200 -11.00 4 N/R 16 Fluvoxamine 300 1 24 12 150 -50.00 2 R 17 Fluvoxamine 200 2 22 22 50 .00 4 N/R 18 Fluvoxamine 300 2 25 22 25 -12.00 4 N/R 19 Fluoxetine 60 2 24 25 25 (E/W)\* 4.00 4 N/R 20 Clomipramine 250 2 27 12 300 -56.00 2 R **Placebo** 1 Fluvoxamine 300 2 32 25 300 -22.00 3 N/R 2 Fluvoxamine 300 1 30 28 300 -7.00 3 N/R 3 Fluoxetine 80 2 34 38 300 12.00 4 N/R 4 Paroxetine 60 1 22 18 300 -18.00 1 N/R 5 Citalopram 60 1 28 26 300 -7.00 4 N/R 6 Fluoxetine 60 5 26 24 300 -8.00 3 N/R 7 Fluoxetine 60 1 23 23 300 .00 4 N/R 8 Fluoxetine 60 1 27 10 300 -63.00 1 R 9 Fluoxetine 40 1 32 18 300 -44.00 2 R 10 Citalopram 60 2 24 23 300 -4.00 4 N/R 11 Citalopram 60 1 35 23 300 -34.00 2 R 12 Fluvoxamine 300 3 22 14 300 -36.00 2 R 13 Citalopram 60 1 28 10 50 -65.00 2 R 14 Fluoxetine 60 1 28 4 50 -86.00 2 R 15 Citalopram 60 1 26 19 100 -27.00 2 R 16 Fluoxetine 60 1 27 12 100 -56.00 1 R 17 Fluoxetine 20 1 26 18 200 -31.00 2 R 18 Fluvoxamine 300 2 23 35 200 52.00 6 N/R 19 Citalopram 60 2 26 9 100 -65.00 2 R 20 Paroxetine 80 1 32 29 300 -9.00 3 N/R 21 Fluvoxamine 300 3 31 25 100 -19.00 2 N/R \*E/W = Early withdrawal ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Summary scores (baseline) and change scores for primary and secondary outcome variables. ::: Quetiapine Placebo -------------------------- ---------------- ---------------- YBOCS (baseline) 26.4 (SD4.6) 27.7(SD3.9) YBOCS (change at week 6) -7.1(SD7.2) -7.2(SD8.4) YBOCS % change -26.9% -26% CGI-Severity (baseline) 5.2 (SD0.8) 5.3 (SD0.8) CGI-Severity (week 6) 4.1 (SD1.4) 4.1(SD1.5) MADRS (baseline) 10.6 (SD 4.8) 10.71 (SD 9.8) MADRS (change at week 6) -2.6 (SD 6.5) -3 (SD 8.3) SDS (baseline) 17.9 (SD 5.3) 19.6(SD4.7) SDS (change at week 6) -5.3(SD5.6) -6.1 (SD4.8) YGTSS (baseline) 24.7 (SD 19.3) 22.6(SD 22.3) YGTSS (change at week 6) -4.5(SD 5.1) -9.4 (SD 14.6) YGTSS % change -18.2% -41.6% ::: Of the 11 subjects with co-morbid tics, six were randomised to quetiapine. Endpoint data was missing for one subject on quetiapine. Of the remaining 10 subjects, 3 (quetiapine n = 2 (33%); placebo n = 1(20%)) were classified as YBOCS responders. The reduction in the YGTSS did not differ significantly between treatment groups with tics (quetiapine -4.5, placebo -9.4; F = 2.8, p = .46). Severity ratings for depressive symptoms (MADRS) were low at baseline (mean 10.6, SD 4.8), showed little change over the study period, and at week 6 remained similar for both groups (quetiapine = 8.2, SD 4.8; placebo = 7.7, SD 6.1). The mean daily dose at week 6 for the quetiapine group was 168.75 mg (SD 120.82) compared to 228.57 mg (SD 99.46) per day for those on placebo. Quetiapine responders (187.5 mg, SD 124.6) did not differ significantly from quetiapine non-responders (156.25 mg, SD 122.1) in their mean daily dose at Week 6 (p = .585). Furthermore, within the quetiapine group, participants receiving ≥ 200 mg/day (10/20 at week 6 demonstrated non-significant differences (F = 6.837, p = .988) and a marginally lower percentage reduction in YBOCS at endpoint (26.7%, SD 20.34) compared to those receiving a dose ≤ 200 mg/day (26.9%, SD 36.24) at endpoint. Tolerability ------------ Quetiapine was generally well tolerated and no serious adverse events (SAE\'s) were reported through the course of the study period. Two patients on quetiapine withdrew from the study due to severe sedation (Week 1 and Week 4) that was judged to be drug related. Otherwise adverse events were in the mild to moderate range and were mostly self-limiting. No subjects on placebo withdrew from the study. Table [4](#T4){ref-type="table"} provides a list of the adverse events and their frequencies in the respective study groups. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Percentage of subjects for each treatment group reporting adverse events ::: Adverse event Quetiapine (%, n) Placebo (%, n) ------------------------ ------------------- ---------------- Sedation 75% (15) 33.3%(7) Dry mouth 15% (3) 0 Headache 15% (3) 38% (8) Fatigue 15% (3) 19% (4) Irritability 10% (2) 4.7% (1) Impaired concentration 10% (2) 0 Dizziness 5% (1) 14.3% (3) Nausea 5% (1) 9.5% (2) Increased appetite 5% (1) 9.5% (2) Delayed ejaculation 5% (1) 0 Weight gain 5% (1) 0 Worsening mood 5%(1) 4.7%(1) Memory difficulties 5%(1) 0 Muscle aches 5%(1) 0 Abdominal tenderness 5%(1) 0 Slurred speech 5%(1) 0 ::: Discussion ========== Our findings indicate that both quetiapine and placebo significantly reduced symptoms in subjects with OCD who had failed to respond adequately to 12 weeks of an SSRI and, that the difference between groups was not significant. Similarly in the subgroup with co-morbid tics, no preferential benefit was noted for quetiapine. Interestingly, the high placebo response was similar to that seen in a recent failed controlled trial of olanzapine \[[@B25]\], but stands in contrast to the positive studies in this area in which low placebo response rates were seen when demonstrating efficacy of quetiapine \[[@B23]\], risperidone \[[@B21]\], and olanzapine \[[@B24]\]. It is likely that features of study design or specific study population characteristics may have contributed to this finding and these are discussed below. First, the duration of a therapeutic trial of an SRI prior to augmentation with an antipsychotic should be of adequate dose and duration. In our study the majority of participants had failed only the single trial of an SRI on which they continued during the study (63.4% mean 1.59). Notably only six weeks of this treatment was required at the maximum tolerated dose. Despite the notion that an optimum trial of pharmacotherapy in OCD is 12 weeks, it may be argued that higher and ultimately effective doses of an SRI had not been maintained for an optimum duration prior to randomization. Given that therapeutic doses of SRIs in OCD are usually on the upper end of the dose range, it seems feasible that the high placebo response rate may reflect a response to SRI\'s once they had been administered at these higher doses for the additional six weeks of the study. It seems possible that the recent study by Shapira et al \[[@B25]\] may have been impacted by similar factors. In a second and related point; the number of previous SRI trials in the subgroup receiving quetiapine did not predict a poorer response to treatment. This effect is probably related to the lack of statistical power to detect these differences in a group in which the low number of previous SRI trials was a distinguishing characteristic. Certainly, previous positive studies in this area have used relatively more refractory groups based on the number of previously failed SRI trials. Taken together with the first point above, we suggest that future work in this area should consider longer periods at maximum tolerated doses of SRI\'s prior to categorisation of subjects as treatment refractory. Third, the use of a slow up-titration resulted in a relatively low mean daily dose being administered for the majority of the study. For instance, a mean daily (week 6) dose of 168 mg/day in the quetiapine group (median 175 mg) had only been achieved for the final two weeks of the study. These doses are comparably low to those used in the negative single-blind study using low dose quetiapine by Sevincok et al \[[@B19]\]. In contrast the positive study using quetiapine by Denys et al \[[@B23]\] employed a more rapid up-titration and a fixed-dose design. This meant that subjects were exposed to 200 mg daily doses that were generally well tolerated, from the start of week 3. The authors of this study were able to show significant YBOCS differences between groups from the end of week 4. Similarly Mc Dougle et al \[[@B21]\], using risperidone, began treatment on 1 mg per day for one week and permitted weekly 1 mg incremental increases for 6 weeks. They found that by the beginning of week 2, most subjects were on or around the mean daily dose for treatment responders (2.2 mg). Despite the significant improvement in the quetiapine group demonstrated in our study, the apparent lack of benefit of doses higher than 200 mg per day may seem surprising, however, we cannot rule out the possibility that administering these higher doses for an adequate duration would have changed the outcome. In addition it must be noted that in our study the quetiapine group reported high rates of sedation (n = 15, 75%) and a 10% (n = 2) rate of premature withdrawal was experienced. As such it seems likely that a more aggressive up-titration schedule might have resulted in even higher rates of withdrawal. By comparison, rates of sedation were equally high, but did not appear to restrict use of the more rapid up-titration in the study by Denys et al \[[@B23]\]. Certainly evidence of efficacy using lower doses has been demonstrated in studies of 6 and 8 weeks duration \[[@B21],[@B23],[@B24]\], and it seems that therapeutically adequate doses should probably be reached earlier than week 4 in a 6 week study. Fourth, the impact of repeated clinical assessments and rating of relatively small changes in clinical severity combined with regular dose increases, may conceivably have increased the placebo response rates resulting from increased optimism, a tendency to over-report improvements and belief that higher doses are more likely to be more effective than lower doses. This may be particularly true for the placebo-treated group that were considerably less likely to report sedation as an adverse event and as such were more likely to have their treatment dose increased at each visit. Our results differ, with respect to placebo response, from a considerable literature that suggests a consistently lower placebo response rate in treatment trials in OCD than in other mood and anxiety disorders. While we believe that the reasons (1--3) discussed above probably provide the main reasons for our finding, the impact of repeated assessments and the potential effect thereof cannot be entirely discounted. Conclusions =========== Despite significant improvement in each of the study groups, response to quetiapine augmentation in SRI non-responders, failed to separate from placebo treated subjects at the end of the six week treatment phase. A number of limitations in study design make comparisons with previous studies in this area difficult and probably contributed to our negative findings. Future work in this important clinical area should address these limitations. Competing interests =================== Dr. Stein has received research grants and/or consultancy honoraria from Astrazeneca, Eli-Lilly, GlaxoSmithKline, Lundbeck, Orion, Pfizer, Pharmacia, Roche, Servier, Solvay, Sumitomo, and Wyeth. Authors\' contributions ======================= PDC drafted manuscript and analysed data, BV protocol design, SS protocol design, JEM drafting of manuscript, statistical design, data acquisition, MVM protocol design, DJS principal investigator, protocol design, drafting of manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-244X/5/5/prepub> Acknowledgements ================ This study was funded by Astra Zeneca The authors wish to acknowledge the contribution of the participating investigators: Dr\'s, Maud, Mohr, Nel, Potgieter, Russouw and Verster

PubMed Central

2024-06-05T03:55:52.417324

2005-1-24

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547907/", "journal": "BMC Psychiatry. 2005 Jan 24; 5:5", "authors": [ { "first": "Paul D", "last": "Carey" }, { "first": "Bavanisha", "last": "Vythilingum" }, { "first": "Soraya", "last": "Seedat" }, { "first": "Jacqueline E", "last": "Muller" }, { "first": "Michael", "last": "van Ameringen" }, { "first": "Dan J", "last": "Stein" } ] }

PMC547908

Background ========== Sprains of the lateral ligaments of the ankle joint account for between 3 and 5% of all emergency department (ED) attendances in the UK \[[@B1]\], with approximately 5600 injuries each day \[[@B2]\]. The injury is painful and incapacitating, and, for all but the most minor injuries, weight bearing is difficult to tolerate. Activities of daily living can be significantly compromised in the first few weeks, and although acute symptoms resolve, persistent symptoms are reported to occur in between 30 to 50% of people \[[@B3]-[@B6]\]. Persistent symptoms include recurrent sprains, instability, swelling, unsightly appearance and pain. Crichton has developed a classification of severity of ankle injuries \[[@B7]\]. • GRADE I -- the ligament is stretched but not torn and the anterior talofibular ligament is usually involved. The anterior draw test is negative. • GRADE II -- the ligaments are partially torn; laxity may be present and there is moderate swelling. • GRADE III -- complete rupture of the ligament resulting in joint instability. The anterior draw test is positive. The focus of this trial is Grade II and III sprains (referred to as severe sprains from this point). Severe sprains have poorer prognosis, and necessitate more intensive management than Grade I sprains (minor sprains). Minor sprains are considered to be self-limiting and require minimal treatment. Recent systematic reviews have highlighted the lack of high quality evidence to support clinicians in managing severe sprains \[[@B2],[@B7]\]. There are few reliable studies describing long-term outcome. Most trials required x-ray evidence of talar tilt or an arthrogram for inclusion and are, therefore, not generalisable to clinical practice in the ED. Current practice ranges from no intervention, physiotherapy, and different types of mechanical support to surgical repair of the ligaments. A recent UK survey of 83 EDs demonstrated the most popular treatments were ice, elevation, Tubigrip^®^support stocking and exercise, each of which was reported as being used in most cases by over 70% of respondents \[[@B8]\]. In addition, over half of responding departments reported that crutches, early weight bearing, and non-steroidal anti-inflammatory drugs were used in most cases. Follow up was used only in selected cases. Practice has been gradually changing from immobilisation of the injured joint to early mobilisation. To aid mobilisation the use of mechanical supports has been suggested. These supports vary in the amount of ankle movement they allow, but all encourage ankle flexion/extension and aim to minimise inversion/eversion that theoretically reduces the risk of further ligament injury. Movement is also proposed as a way of retraining the ankle proprioception which may reduce recurrent injury rate \[[@B9]\]. Other hypothesised benefits of mechanical supports are early restoration of functional mobility, rapid return to usual activities, reduction of pain because the joint is stabilised and protected, and improved quality of life. However, there is also the possibility of discomfort and inconvenience owing to restriction of joint range, and delayed healing, skin and circulatory problems. The present study has two aims 1\. To estimate the clinical effectiveness of three different methods of mechanical support (below knee cast, Aircast^®^ankle support and Bledsoe^®^boot) in comparison to Tubigrip^®^in a\. The recovery of mobility (primary outcome) b\. The recovery of usual occupation c\. Avoidance of residual symptoms including recurrent instability, lasting limitation of physical activity, and need for further medical, rehabilitation or surgical treatment. 2\. To measure the cost of each strategy, including treatment and subsequent health care costs. The NHS National Co-ordinating Centre for Health Technology Assessment has guided the selection of treatments. Tubigrip^®^has been chosen as the reference treatment because it is the cheapest and one of the most commonly used \[[@B10]\]. The Bledsoe^®^boot is lightweight and incorporates a novel design to promote ease of walking. However it is considerably more expensive, and it\'s clinical and cost effectiveness is yet to be proven. The below knee cast is Scotch^®^Cast is commonly used for casting in the NHS \[[@B9]\]. There is a range of lightweight mechanical supports available, and we have selected the Aircast^®^Brace. Methods ======= This pragmatic randomised controlled trial is being run in 6 trusts (covering eight hospitals) across the UK. The trusts are: Birmingham Heartlands and Solihull NHS Trust, North Bristol NHS Trust, Oxford Radcliffe Hospitals NHS Trust, South Warwickshire General Hospitals NHS Trust, University Hospitals Coventry and Warwickshire NHS Trust and the Worcestershire Acute Hospitals NHS Trust. Figure [1](#F1){ref-type="fig"} provides an overview of the trial method and patient journey. Multi-centre Research Ethics Committee approval has been awarded by the Northern and Yorkshire Regional Office and local ethical and research governance approvals have been obtained. Informed consent to participate in the trial and allowing researchers to access hospital records is obtained from all participants. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Study design ::: ![](1471-2474-6-1-1) ::: Study population ---------------- The target population is people attending the ED with a severe sprain of the lateral ligament complex of the ankle. Inclusion criteria ------------------ People who attend with sprain of the ankle who are unable to weight bear, aged 16 years and older, and give informed consent. Weight bearing is used as a proxy for severe sprains as clinical grading is not possible in the acute phase \[[@B7]\]. Flake fractures (\<=2 mm) of the lateral malleolus are included as they are normally treated as soft tissue injuries \[[@B11]\]. Exclusion criteria ------------------ Age less than 16 years old, ankle or other fracture sustained in addition to the sprain (such as to the wrist, head etc). An x-ray is used to exclude fracture where this is clinically indicated and as guided by the Ottawa guidelines \[[@B12]\]. People who can weight bear and those with fractures are ineligible for the trial, and are managed in accordance with normal local practices. Age is used as an exclusion criterion because of the complications involved in the management of epiphyseal injuries (growth plate injuries). Growth plate injuries would not normally be managed using the treatments being tested \[[@B13]\]. Patients are also excluded if they have a contra-indication to any of the four arms of the trial. This is most likely to occur if a patient has a DVT, high risk of DVT or other circulatory disturbance. Other contra-indications include poor skin viability preventing splinting or casting. The decision to exclude on these criteria is made by the attending clinician. The process of identifying participants --------------------------------------- A standard approach to identifying potential participants has been instituted across all participating centres. The approach was designed in consultation with the departments to dovetail with current procedures, and eradicate duplication of clinical and research data collection. The attending clinician (nurse practitioner, physiotherapist or doctor) assesses weight-bearing status. These data are recorded on a form that includes details of the remainder of the clinical examination, including if indicated, x-ray results. At this stage, people able to weight bear are excluded. The attending clinician gives a brief explanation of the trial, an information pack, and arranges an appointment at a follow-up clinic two to three days later. Delay is an accepted practice in the application of mechanical supports, allowing for the resolution of acute swelling. It also filters out patients in whom the diagnosis of severe sprain at initial contact was incorrect, and gives participants sufficient time to consider whether they wish to participate in the trial. A physiotherapist who staffs the follow-up clinic is responsible for checking eligibility and providing a further opportunity for patients to ask questions. If appropriate, the physiotherapist recruits the patients, contacts the randomisation centre, and arranges application of the treatments. Reasons for declining to participate in the trial are recorded. Assessing the generalisability of the results --------------------------------------------- Data on all ankle sprains attending the participating EDs during the recruitment period are collected using a standardised proforma. A copy of this proforma, which is entered into the medical record, is anonymised and passed to the research team. It provides descriptive data on the injury pattern, occupation, age, and sex of patients, including those who are ineligible or decline trial participation at various time points. Completed proformas are audited against ED attendance records to check whether attending clinicians have referred appropriate patients to the follow-up trial clinics. This allows clinicians who are unfamiliar with the trial to be identified and approached individually to be briefed on trial procedures. Treatment allocation -------------------- Randomisation is stratified by centre and provided via a phone-in service, which utilises a computer generated random allocation. Allocation concealment, which shields people who enter patients into a trial from knowing future allocations, is thereby ensured. The randomisation service is independently administered and quality controlled by the Birmingham Cancer Trials Unit. Treatment protocol ------------------ The mechanical supports are fitted to each individual by a trained health professional (physiotherapist, nurse or plaster technician) in the follow-up clinic, to ensure comfort and correct fit. Participants are provided with standardised written and verbal instructions including when to remove the support, encouragement of normal walking with limits of tolerance, simple exercise advice, what to do in the event of experiencing difficulties with the support, and washing instructions. The protocols for duration of support, weight-bearing status, and activity have been determined by the manufacturers recommendations and the results of a national survey of practice completed in the planning phase of the trial \[[@B8]\]. Treatments are applied within three days of the injury, and within an hour of randomisation. Other treatments ---------------- All other treatments are standardised and include the provision of crutches, advice to elevate and pain relieving medications if needed. Withdrawal of the latter treatments would be inappropriate as they constitute normal and accepted care. Physiotherapy is not provided as part of the trial treatment protocol, and is not part of routine care provided in the participating centres \[[@B9]\]. If the participant receives these types of treatment in addition to the trial protocol, this is recorded as an outcome. Baseline and outcome measures ----------------------------- Clinical status is measured at baseline, 4 weeks, 12 weeks and 9 months. The baseline assessment also includes date of birth, sex, body mass index, ethnicity, an assessment of pre-injury abilities including usual levels of mobility, engagement in sporting activity, usual occupation and employment (including hours worked and type of work undertaken) and completion of the baseline version of the outcome questionnaires. The selection of outcome measures is based on a systematic review completed during the preparatory stage \[[@B14]\], and are shown in Table [1](#T1){ref-type="table"}. There is a paucity of information on the psychometric properties of self-completed questionnaires for ankle conditions. The Foot and Ankle Outcome Score (FAOS) is a questionnaire that ascertains functional limitations (including mobility) and the severity of other symptoms after ligament sprains, and has evidence of validity and reliability \[[@B10]\]. Previous versions have been validated against objective tests of ankle function \[[@B15]\]. The Functional Limitations Profile (FLP) is the British version \[[@B16]\] of the Sickness Impact Profile \[[@B17]\]. We are using the FLP occupation and mobility sub-scales to provide more detailed information on the impact of the injuries and treatments including adaptations that occur after the injury. The FLP has not been used in studies of ankle injuries previously. Health related quality of life will be measured using the SF-12 version 1 and EQ-5D \[[@B18]\]. Return to normal occupation and leisure activities will be recorded as the date that people return to work and normal activities. Patients are asked to make a record of significant dates on a calendar to aid recall. The effectiveness of these calendars is being tested in a separate study. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Outcome measures ::: **Outcome** **Outcome measure** --------------------------------------------------------------------------------- --------------------------------------------------------------------------- Functional limitations and severity of symptoms after ligament sprains Foot and Ankle Outcome Score (FAOS) Impact of injuries and treatments including adaptations that occur after injury Functional Limitations Profile (FLP) occupation and mobility sub scales Health related quality of life Short Form 12 version 1 (SF-12 v1) Health related quality of life EuroQol (EQ-5D) Health economics Resource Use Questionnaire Return to normal occupation and leisure activities Date of return to work and normal activities recorded on a trial calendar ::: Timing of follow-up ------------------- The mechanical supports are likely to have maximal impact and benefit during the first three months of recovery, and this time period is defined as the primary time point. We anticipate that some participants will still be wearing the mechanical support at 4 weeks. The natural time course of recovery of ankle sprains is for functional limitations to stabilise between 3 and 9 months and it is expected that the difference between treatments will narrow in the longer term as the majority of people will recover \[[@B3],[@B5],[@B6]\]. Participants will be followed to 9 months to ensure that there are no longer-term complications from injury or their treatments. Follow-up and masking --------------------- Follow-up data are collected by postal questionnaire. We have implemented an intensive approach to follow-up. Participants are mailed a follow-up questionnaire, and if a response is not received within one week, they are telephoned to ensure they have received the questionnaire. A second questionnaire is mailed if necessary. If there is still no response, the participant is contacted one week later, and a core set of outcomes are collected over the telephone. All follow-up data are collected and analysed by individuals who are independent of the recruitment, randomisation, baseline assessment and are masked from the treatment provision. This level of masking will be maintained until final analysis of the data has been completed. The only exception to this rule will be if the data monitoring committee require unmasked data, and in this circumstance, only the independent Chairperson will be aware of assignments. Quality assurance ----------------- Trial procedures are audited at regular intervals to ensure compliance with the protocol. Maintaining trial profile and competence in trial procedures is challenging in the ED, because of a high turn-over of staff and a diversity in the staff groups dealing with injuries in different hospitals. To combat these difficulties, training is provided on cycles that coincide with staff rotation, as well as various other time points (e.g. in-service training). Data analyses ------------- The analysis will be conducted as intention to treat. An analysis of all people who completed the trial will be undertaken, and in addition, a sensitivity analysis will be undertaken to assess the range of potential biases that could result from loss to follow up or withdrawal. Numerical and graphical summaries of all the data will be compiled, including a detailed description of missing data at the clinic visit, questionnaire and individual level. Logistic and log-linear multinomial regression models will be used to provide estimates of the recovery rates and the prevalence of residual symptoms, with confidence intervals. As there are multiple centres and repeat assessment, random effect (or hierarchical) models will be used to investigate the components of variation. The common current and cheapest therapy is Tubigrip^®^, so each of the three other treatments will first be compared with it. Any treatments found to be more effective than Tubigrip^®^will be compared with each other. Regression modelling will allow an assessment of factors that might indicate the appropriate choice of treatment. As these analyses are pre-specified, issues of multiple comparisons are minimal. The sensitivity of the above analyses to missing data at various levels will be assessed and quantified using modern statistical methods for incomplete multivariate data \[[@B19]\]. Economic analysis ----------------- Severe sprains may have a range of direct cost consequences across primary and secondary health care, they may also have cost consequences for patients themselves in terms of their personal expenditure and return to work. The costing study will seek to adopt a broader societal perspective, including patient costs, when estimating differences in the cost of resources used in the four arms of the trial. The economic analysis will compare resource use (costs) with any measurable changes in health outcomes (benefits). The health outcomes of the four technologies will be measured in terms of changes in specific disease parameters and validated measures of health-related quality of life (HRQL). Any uncertainties in the cost and outcomes data will be incorporated into a sensitivity analysis. The cost of each mechanical support protocol will be determined through observation and will include staff time, overheads, equipment and transport; plus follow-up visits to hospital, GP surgeries, physiotherapists or others. The consequences for patients in terms of GP and hospital visits, including travel expenses and time off work, and expenditure on aids or private practitioner input are obtained from structured resource use questions added to patient follow-up questionnaires. Further treatments recorded include pain relieving medications, anti-inflammatory and other topical agents, bandages, supports or footwear. Patients are also asked to distinguish whether these are NHS-funded or private treatment paid for by the individual or private provider. Patient self-reported information on service use has been shown to be accurate in terms of intensity of use of different services \[[@B20]\]. Hospital notes and records will also be audited for information on service use. The cost of primary and hospital services will be estimated from a variety of sources, including the finance departments of the trial hospitals and services concerned and national sources \[[@B21],[@B22]\]. Differences in resource use and outcomes are ascertained in follow-up questionnaires at 12 weeks and 9 months. The appropriate technique of economic evaluation will depend on the results of the study \[[@B23]\]. The simplest eventuality would be where the least expensive intervention is found to be better on at least one outcome measure and no worse on any other i.e. dominant. Another is where two interventions have the same outcomes in which case cost-minimisation analysis will be used to compare the two. However, where an intervention is clearly better in terms of outcome but is also more costly, a different approach is required. One accepted method is to compare the different interventions in terms a single outcome measure identified as clinically important for the condition being treated. The primary outcome meets this requirement. Therefore, the costs per unit improvement in mobility will be used to provide an estimate of overall cost-effectiveness; average and incremental (relative to reference treatment) cost-effectiveness ratios will be estimate for the different treatments used. However, this approach does not allow comparison with other types of intervention/condition combinations and also does not consider the value (utility) of differences in improvement in health status. In the present study, the EQ-5D will be used to generate such utility scores that can be compared to costs. A cost-utility analysis will estimate outcomes in terms of differences in quality-adjusted life years (QALYs), representing the period of life subsequent to a health care intervention weighted or adjusted for the quality of life experienced by the patient during that period, and compare these with cost for the four interventions \[[@B23]\]. The cost-utility analysis will present the incremental cost of the extra benefit gained both in summary form in terms of incremental cost per QALY, and also using a \'disaggregated\' approach where the extra costs are presented alongside the HRQOL dimensions such as pain. Sample size ----------- Although there was a paucity of published data available to inform the sample size estimate at the outset of the trial, the preliminary phase of the study has enabled us to make a more accurate estimation of the sample size required. The presented estimate is based on a standard sample size calculation for a two-sample t-test with equal variances and a significance level of 0.05, using the variance estimated from an ANOVA of the 4-week data (n = 100). The minimal clinically important difference is set at 10 percent, which represents a small to moderate effect size. A target of 600--650 participants will allow us to detect clinically important outcomes at 4 and 12 weeks. We are powered \>90% to detect differences of 10 percent in the primary outcomes, and have sufficient power to detect differences in a range of secondary outcomes at 80% power. To account for the possibility of loss to follow-up, the estimate includes an inflation of 20%. However, the trial should be able to report whether any of the treatments have a sustained negative effect on outcome at 9 months. We are taking a pragmatic approach to estimating the sample size, and the Data Monitoring Committee are reviewing assumptions underlying the calculation at 6-monthly intervals. Qualitative sub-study --------------------- A purposive sample of randomised patients will be interviewed to obtain qualitative data on the patients\' experiences of the various treatments in a semi-structured interview. Patients will also be asked for their views on their willingness to pay a deposit for the boots/splints to encourage their return. Up to 20 patients will be interviewed using a semi-structured format. The data will be analysed using thematic content analysis. Conclusions =========== We have described the protocol and conduct of a large scale UK randomised controlled trial of mechanical supports for the management of acute severe ankle sprains. There has been a paucity of good quality research conducted to date, which may be partly explained by the challenges implicit to studying acute events within EDs \[[@B24],[@B25]\]. These include a very short window of opportunity in which patients satisfy the criteria of acute injury and reliance on attending clinical staff to identify and approach for inclusion potential participants who are often in a state of discomfort. The trial is currently in the middle of the recruitment phase and is running well. Trial procedures have been well received by both patients and clinicians. Much effort has gone into maintaining the profile of the trial and disseminating trial procedures across all disciplines of ED staff (e.g. doctors, nurses, reception staff and plaster technicians). This has been paramount to the success of the trial. By referring potential patients to the follow-up trial clinics ED personnel, with no direct involvement in the trial, play a vital role in the trial process. The intense follow-up has produced good completion rates. Using a pragmatic approach to sample size estimation was useful, in particular, re-estimation after an adequate run in period, and is recommended where little prior data exists. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= SEL wrote the original protocol, secured funding, act as co-principal investigator and lead in the writing of this manuscript. MWC wrote the original protocol, secured funding and act as co-principal investigator. JRD and SW contributed to the development of the protocol and securing funding. JLH and AS contributed to the development of the protocol and securing funding and wrote the statistical and economic aspects of the protocol respectively. RAN is responsible for the day to day co-ordination of the trial, has contributed to refining the protocol and outcome measure package. JLM revised the protocol and the sample size calculations. MC refined the economics section of the protocol and is providing economic input. EJW is responsible for the data management and trial support. All authors have contributed to and approved the final manuscript. CAST research team ================== Lamb SE, Cooke MW, Nakash RA, Withers EJ, Hutton JL, Marsh JL, Szczepura A, Wilson S, Clark M, Dale JR, MacNamara A, Kendall J, Skinner D, Sakr M, Kelly C, O\'Byrne G, Morrell R, Dunn M, Bateman S, Kempson S, Hooker F, Parker N, Noakes E, Vaux N, Doughty G, Nichols V, Ritchie C, Sabine E. Trial Steering Committee ------------------------ Professor Bill Gillespie, Professor Sallie Lamb, Dr Matthew Cooke, Professor Jeremy Dale, Professor Jane Hutton, Dr Jen Marsh, Professor Ala Szczepura, Dr Sue Wilson, Rachel Nakash, Vicki Staples. Data Monitoring Committee ------------------------- Dr Janet Dunn, Professor Damian Griffin, Pat Overton-Brown. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2474/6/1/prepub> Acknowledgements ================ This project is funded by the NHS R&D Programme Health Technology Assessment Programme (project number 01/14/10). The views and opinions expressed herein are those of the authors and do not necessarily reflect those of the Department of Health. Lisa Craven for data entry and patient follow-up. Ellen Jørstad for assisting with preparing this manuscript for publication. Thanks to Braun Medical, Sheffield and Aircast Ltd Partnership for their product supply. Thanks to clinicians, reception staff and plaster technicians at all trial centre sites.

PubMed Central

2024-06-05T03:55:52.421033

2005-1-13

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547908/", "journal": "BMC Musculoskelet Disord. 2005 Jan 13; 6:1", "authors": [ { "first": "SE", "last": "Lamb" }, { "first": "RA", "last": "Nakash" }, { "first": "EJ", "last": "Withers" }, { "first": "M", "last": "Clark" }, { "first": "JL", "last": "Marsh" }, { "first": "S", "last": "Wilson" }, { "first": "JL", "last": "Hutton" }, { "first": "A", "last": "Szczepura" }, { "first": "JR", "last": "Dale" }, { "first": "MW", "last": "Cooke" } ] }

PMC547909

Background ========== Nicotinic acetylcholine receptors (nAChRs) are the most studied members of the cys-loop family of ligand-gated ion channels (LGICs) which also contains γ-aminobutyric acid (GABA) receptors, glycine receptors and 5-HT~3~receptors \[[@B1]\]. Distinct subtypes of nAChRs mediate, for example, fast synaptic transmission in the brain and at neuromuscular junctions \[[@B2]\]. All are believed to be pentameric assemblies of various combinations of different subunits (α, β, γ/ε, δ), some of which exist as multiple isoforms \[[@B3]\]. nAChRs are important targets for novel analgesics as well as new drugs being devised for Alzheimer\'s disease and schizophrenia \[[@B4],[@B5]\]. Mutations in nAChRs are associated with certain forms of epilepsy \[[@B6],[@B7]\] and several congenital myasthenias \[[@B8]\]. There is a substantial and growing body of physiological, pharmacological, genomic structural and modeling data on receptors formed from these subunits. The volume and diversity of these data present severe challenges for their efficient storage and interpretation. Here we describe a relational database, initially relating to the α7 subunit, whose aim is to provide an easy to use, extensible web-based interface to access functional data and relate it back, wherever possible, to structural data. Construction and content ======================== The database is currently limited to nAChR α7 subunits. This makes the initial task of populating the database manageable. To allow 3^rd^level normalization, the principal information prototype stored in the database is referred to as an \"experiment event\". An experiment event is a collection of simultaneous measurements and their associated measurement conditions. For example, two drugs tested against the wild type α7 and a mutant under the same experimental conditions would be described by 4 (2 drugs × 2 sequences) experiment events. If the identical measurements were repeated in, say, calcium-free saline, this would yield 8 experiment events. Using this prototype a unique set of experimental conditions is associated with a single set of data. Therefore, the database tables (see Figure [1](#F1){ref-type="fig"}) have been designed around the central experiment table \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. Quite often, several real experiments are performed within any one publication. We wanted to capture all of that data. Thus, all the data for one experiment that was reported have been stored (non-redundantly) as a separate entity. The database is managed by the MySQL relational database management system. PHP scripts query the MySQL system and transform the data into HTML pages for serving to the client. The database currently aims to store molecular information rather than whole-organism genetic information which can be sourced from elsewhere, see for example \[[@B9],[@B10]\]. A typical set of experiments reported in one paper might concern the properties of one mutation at a particular position and its associated changes in physiology and/or pharmacology. All of this data is held in the database. By use of appropriate alignments (either stored within the server or uploaded) the positions of these data can be highlighted on a homology model based on the acetylcholine binding protein AChBP \[[@B11]\]. The model is viewable by either using the Chime plugin, or by setting the browser to use Rasmol as a helper application. User instructions for configuring different browsers are provided on the server, as well as the list of combinations of operating system and browser that we have tested to date. For any database, data integrity will ultimately determine its usefulness. The initial population of the database has been carried out by the authors, but it is desirable and practical in the long term to have a procedure whereby any laboratory can submit data. To help ensure reliable deposition procedure, each depositor will have a unique identifier (thus making the data accountable). A depositor will submit their data which is then tabulated and presented as a form which the depositor is then asked to confirm as being correct. Only when this confirmation is received is the data committed to the database. Although this will reduce some entry-based mistakes, the database will nonetheless need to be curated to maintain integrity. The database does not attempt to duplicate the functionality of other databases. For example we store a local alignment of α7 subunits taken directly from pfam \[[@B12]\], but we do not perform any alignments ourselves. Such tools are already available on numerous web-servers and in any case the user may wish to upload their own alignment. Wherever possible, we link to a well-established resource, as is the practice in for example, SWISS-PROT \[[@B13]\]. Utility and discussion ====================== Access to the database is through various routes. The first route is by a very simple search string (e.g. citation = Smith\*). The user is then presented with a page informing him of the number of hits and asking what information should be displayed from those hits. The second is a simple incremental search in which the user applies criteria sequentially until he chooses to examine the results. As the query is built, the number of hits returned by the query in its current state is shown. The third search method is also incremental but via the use of a set of tabbed pages which divide the information into intuitive categories such as pharmacology and physiology. Additionally, the user can make choices about alignments used and even upload their own. As the query is built, summary information of its status is also displayed. The fourth method to access the data is provided by a \'fast lane\' route which provides more experienced users with a quicker and more direct route to the data of interest. The user formulates and constructs a query offline and then uploads it as a simple ASCII file. A sample query form and details of the format are presented in the supplementary information. The result of a query is presented in tabular form. Only the information requested is actually presented with the option for further or refined searches. In addition to this, the results of any position matches can be displayed via the homologous 3-dimensional structure of the AChBP. This is automatically available if the user selects the alignments available from the server. If the user uploads their own alignment the model will only be produced if AChBP was included. The server streams a Rasmol script or spawns a Chime window depending on browser configuration. In addition to these query routes, the user is also able to browse the contents of the database. The current database has been designed such that it could easily be extended to include other nAChR subunits and then on to other members of the cys-loop ligand-gated ion channel family. We are currently pursuing this aspect. Such expansion will allow trends that link structure to function in this receptor family to be more readily identified. The database is very complementary to some existing resources, in particular the LGICdb \[[@B14],[@B15]\]. This database contains sequence, phylogenetic data and sets of coordinates, but does not attempt to store all aspects of individual experiments as we report here. However, this could be used in conjunction with our database to for example explore equivalent positions in related receptors using sequence and phylogenetic information stored therein. Our approach is somewhat similar to that employed by the ProTherm database \[[@B16]\] which aims to store thermodynamic data for mutant and wildtype proteins \[[@B17]\]. Although currently there are no entries common to both databases, it is envisaged that thermodynamic data reported for either the α7 nAChR receptor or related channels will appear in the ProTherm database and can thus be used in conjunction with the A7DB to help illustrate for example how loss of function might be related to protein stability. Furthermore, the similarity in design of these databases should make cross-interrogation possible in the future. We should state that the main difference between A7DB and ProTherm is that instead of thermodynamic data we store pharmacological and physiological data and in that sense it is closer to the voltage-gated potassium channel database \[[@B18],[@B19]\]. Finally, the A7DB is also complementary to the Protein Mutant Database \[[@B20]\] where mutations across many proteins are stored but the searching and data tabulation is primarily text-based. This might be particularly useful if another mutation had been reported in a different protein that bound a similar ligand, for example in acetylcholine also binds to acetylcholinesterase, which we do not store data for. Conclusions =========== We have shown here how the collation and careful storage of experimental data pertaining to one sub-family (the α7 nAChR sub-family) of the ligand-gated ion channels can be assembled into a useful resource. However, the real power of the database will be when it is combined with machine-learning technologies to explore complex relationships between sequence, structure and function \[[@B21]\]. We believe that this resource will grow in usefulness as the amount of data increases. For example, during the construction of this database, atomic coordinates were released for the transmembrane region of the nAChR from *Torpedo marmorata*\[[@B22]\]. Its development is particularly timely given these recent structural developments which allow three-dimensional information to be correlated with function derived from an ever-increasing body of experimental data. Availability and requirements ============================= The database is freely available at: <http://www.lgics.org/a7db/> Authors\' contributions ======================= The project was conceived and technical aspects managed by PCB. The coding and design was done by SB. MSPS and DBS provided critical evaluation of the design and construction process. DBS, LP, AKJ and LB populated the database. All authors have approved the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Supplementary information. This doc file provides a complete description of the table entries and also provides a description of the uploadable query file. Further information about the online help is also provided. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ SDB, MSPS and PCB are supported by the Wellcome Trust. AKJ, LB, LP and DBS acknowledge support from The Medical Research Council of the UK. AKJ also acknowledges support of a grant to DBS from Dupont. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The A7DB database scheme. All the information pertaining to an experiment is stored within a single table called \"experiment\". The sequence table stores all the wildtype sequences for the different species contained within the database. The third table is the admin table which makes entries and changes traceable. A full description of each of the entries is supplied as supplementary information. ::: ![](1471-2202-6-2-1) :::

PubMed Central

2024-06-05T03:55:52.423613

2005-1-20

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547909/", "journal": "BMC Neurosci. 2005 Jan 20; 6:2", "authors": [ { "first": "Steven D", "last": "Buckingham" }, { "first": "Luanda", "last": "Pym" }, { "first": "Andrew K", "last": "Jones" }, { "first": "Laurence", "last": "Brown" }, { "first": "Mark SP", "last": "Sansom" }, { "first": "David B", "last": "Sattelle" }, { "first": "Philip C", "last": "Biggin" } ] }

PMC547910

Background ========== The worldwide spread of the problem of infections due to Gram-negative bacteria such us *Pseudomonas aeruginosa*and *Acinetobacter baumannii*resistant to most classes of antimicrobial agents has made the medical community to rethink of colistin, an old, basically abandoned for the last two decades antibiotic. Intravenous colistin (polymyxin E) has been used in the industrialized countries during the last years mainly in patients with cystic fibrosis infected with *Pseudomonas aeruginosa*resistant to other available antimicrobial classes \[[@B1]\]. Excluding this population, the agent has been infrequently used during the last two decades because of major concerns about nephrotoxicity and neurotoxicity \[[@B2]\]. Several reports during the early years of use of the medication, mainly in the decade 1960 to 1969 left the medical community with the impression that the medication is very toxic \[[@B3],[@B4]\]. However, the recent experience of several clinicians worldwide with the use of the medication has not verified the old reports about the serious or common toxicity of colistin \[[@B5]-[@B8]\]. We analysed data of a group of patients who received colistin for more than four weeks to investigate further the concerns about the toxicity of the medication. Methods ======= Design of the study-patient selection ------------------------------------- Patients who received intravenous colistin from 1/October/2000 to 31/January/2004 at \"Henry Dunant\" Hospital, a 450-bed tertiary care center in Athens, Greece, were identified by the pharmacy electronic databases. All patients who were found to have received intravenous colistin for more than four weeks continuously, or in two courses of colistin but with no more than seven days interruption between the courses were included in this study. Administration of intravenous colistin in two courses for more than four weeks in each course were studied as two different units in the analysis of toxicity, if there was more than one month colistin-free period between the two courses. One milligram of the colistin formulations used is approximately equal to 12.500 IU (Colomycin, Forest Laboratories^®^, Kent, UK or Colistin, Norma^®^, Athens, Greece). The study was approved by the Institutional Review Board (IRB) of the hospital. Definitions of infections ------------------------- Diagnosis of pneumonia required two or more serial chest radiographs with at least one of the following: new or progressive and persistent infiltrate, consolidation, cavitation, or pleural effusion. In addition, patients must have had fever \>38°C with no other recognized cause, or abnormal white blood cell count \[leukopenia (\< 4000 WBC/mm^3^) or leukocytosis (= 12.000 WBC/mm^3^)\], and at least two of the following: new onset of purulent sputum or change in character of sputum, increased respiratory secretions or increased suctioning requirements, new onset or worsening of cough or dyspnea or tachypnea, rales or bronchial breath sounds, or worsening gas exchange \[[@B9]\]. Bacteremia required either growth of a recognized pathogen from one or more blood specimen cultures or at least one of the following signs or symptoms: fever (\>38°C), chills, or hypotension *and*at least one of the following: a) common skin contaminant (e.g., diphtheroids, *Bacillus*sp., *Propionibacterium*sp., coagulasenegative staphylococci, or micrococci) grown from two or more blood cultures drawn on separate occasions or b) common skin contaminant (e.g., diphtheroids, *Bacillus*sp., *Propionibactαium*sp., coagulase-negative staphylococci, or micrococci) grown from at least one blood culture from a patient with an intravascular line and physician- instituted antimicrobial therapy \[[@B9]\]. Infections at other body sites or fluids, such as urinary tract infections and surgical site infections were defined based on guidelines from the Centers for Disease Control and Prevention \[[@B9]\]. Clinical specimens of all body sites were considered when defining the etiologic microbiologic agent of the infection. Data collection-entry --------------------- Data for several variables including demographic and clinical information as well as results of laboratory and imaging tests were collected and entered in a computer database. All available results of renal function tests (serum creatinine, urea, creatinine clearance, urinalysis), liver function tests (SGPT, SGOT, alkaline phosphatase, γ-GT, bilirubin), creatine phosphokinase (CPK) during the course of colistin treatment and at hospital discharge were collected. Data analysis ------------- Creatinine values at the beginning of colistin treatment were compared with the maximum value of creatinine during therapy as well as with the creatinine value at the end of treatment using a non-parametric test (Wilcoxon). The data analysis was performed using SPSS and S-plus software. Results ======= Study population ---------------- During the period from 1/October/2000 to 31/January/2004, 152 patients received intravenous colistin. Data about the efficacy of the medication were analysed and reported elsewhere \[[@B5]\]. In this report, we present detailed data regarding the toxicity of colistin in a subgroup of 17 patients who received more than four weeks of intravenous colistin. Table [1](#T1){ref-type="table"} describes various characteristics of this group of patients including demographic and clinical data, such as comorbidity, site of infection, and responsible pathogens. Two patients received two prolonged courses of intravenous colistin each with more than one month colistin-free period between the courses. Subsequently, there were 19 courses of prolonged colistin for the analysis of toxicity. Among these 19 courses, the administration of intravenous colistin was without interruption in 15 courses, while there was interruption of colistin administration in 2 courses for two days, interruption in 1 course for 3 days, and interruption in 1 course for 4 days. The cause for the interruption of colistin in these cases was the attending physicians\' attempts to obtain cultures without the confounding influence of antimicrobial treatment in patients with puzzling continuing symptoms of infection. The mean duration of colistin administration (± SD), for each course of colistin therapy was 43,4 days (± 14,6 days). The mean daily dosage ± SD for each course was 4.4 million IU (352 mg) ± 2.1 million IU (168 mg) and the mean cumulative dosage of colistin ± SD for each course was 190.4 million IU (15.232 mg) ± 91.0 million IU (7.280 mg). The mean daily dosage for a patient with body weight of 70 kg was 62.857 IU/kg (5 mg/kg). For patients with impaired renal function, dosage adjustments were done mainly after consulting the ICU director or the Infectious Disease specialists of the hospital, based on the following protocol: if serum creatinine level was 1.3--1.5 mg/dl, 1.6--2.5 mg/dl or = 2.6 mg/dl, the dosage of colistin administered was 2 million IU (160 mg) every 12 hours, 24 hours, or 36 hours, respectively. Patients who were on dialysis treatment received 1 million IU (80 mg) of colistin after dialysis. Colistin was given for long duration in patients mainly in the ICU setting, because of persistence of infections due to Gram-negative bacteria sensitive only to colistin or bacteria sensitive also to antibiotics of other classes that were previously administered (based on in-vitro susceptibility test results) but failed. Subsequently, no other therapeutic strategy was considered better than the continuation of colistin (as combination therapy or monotherapy) because of failure of previous antibiotic regimens and the lack of other therapeutic options. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographic and clinical features of patients managed with prolonged intravenous colistin for infections caused by multidrug-resistant Gram-negative bacteria. ::: **Characteristics of patients** **Median (range) n (%)** ---------------------------------------------------------- -------------------------- **Demographic** Age, years \[median (range)\] 51 (18 -- 79) Sex, male 12/17 (70%) **APACHE II score** On admission to ICU \[median (range)\] 14 (7 -- 35) On 1^st^day of colistin treatment \[median (range)\] 14 (6 -- 22) **Comorbidity** Malignancy 2/17 (11%) Hemodialysis 2/17 (11%) Urogenital disorders 3/17 (18%) Heart dysfunction 5/17 (29%) Diabetes mellitus 5/17 (29%) Lung dysfunction 5/17 (29%) Liver failure 1/17 (6%) Hematological disorders 3/17 (18%) Neurological disorders 8/17 (47%) Neuropathy/myopathy 4/17 (23%) Trauma 7/17 (41%) **Transfusion** 15/17 (88%) **Prior hospitalization** 12/17 (70%) **Prior surgery** 14/17 (82%) Elective 9/14 (64%) Emergency 5/14 (36%) **Prosthetic material** 11/17 (64%) **Catheters/Invasive devices** Tracheostomy 14/17 (82%) CSF drainage 5/17 (29%) **Prior medications** Prior antibiotic use 17/17 (100%) Prior antifungal use 8/17 (47%) Anti-tumor treatment 0/17 (0%) Cortisone treatment 9/17 (53%) **Duration of hospitalization (days)**\[median (range)\] 152 (29 -- 591) **Duration of stay in ICU (days)**\[median (range)\] 70 (22 -- 134) **Site of infection\*** Pneumonia 13/19 (68%) Bacteremia 1/19 (5%) Urinary tract infection 2/19 (11%) Meningitis 2/19 (11%) Surgical site infection 1/19 (5%) **Pathogens†** *Pseudomonas aeruginosa* 12/20 (60%) *Acinetobacter baumannii* 5/20 (25%) *Klebsiella pneumoniae* 2/20 (10%) *Enterobacter cloacae* 1/20 (5%) **Mortality** 7/17 (41%) \* The analysis of the site of infection was based on 19 episodes of infection in 17 patients. **†**The analysis of the responsible pathogens was based on 20 isolates from 19 episodes of infection in 17 patients \[two pathogens were isolated in one episode of pneumonia (*Klebsiella pneumoniae + Acinetobacter baumannii*mixed infection)\]. ::: The analysis of the responsible pathogens was based on 20 isolates from 19 episodes of infection in 17 patients (two pathogens were isolated in one episode). In 2 episodes of infection, both caused by *Pseudomonas aeruginosa*strains, the minimum inhibitory concentrations (MICs) were not available. In the remaining 10 *Pseudomonas aeruginosa*isolates, the MICs to colistin ranged from = 0.5 mg/l to 2 mg/l. In vitro susceptibility testing for the 5 isolated *Acinetobacter baumannii*strains revealed MICs to colistin = 0.5 mg/l, for the 2 *Klebsiella pneumoniae*strains MICs were 0.5 mg/l and 2 mg/l, and for the *Enterobacter cloacae*strain MIC was 1 mg/l. The all-cause in-hospital mortality was 41,2% (7 out of 17 patients). Clinical cure and improvement, defined as resolution and partial resolution, respectively, of presenting symptoms and signs of the infection by the end of colistin treatment was observed in 14 of the 19 episodes of infection (73.7%) \[cure 10/19 episodes (52.6%), improvement 4/19 episodes (21.1%)\]. Unresponsiveness, defined as persistence or worsening of presenting symptoms and/or signs of the infection during intravenous colistin administration, was observed in 5 of the 19 episodes of infection (26.3%). All patients had been admitted to the ICU at some time during their hospitalisation. In 12 courses out of the 19 analyzed the whole colistin therapy was administered in the ICU, in 1 course it was administered in a hospital ward, and in 6 courses it was given both in the ICU and a hospital ward. Renal toxicity -------------- Figure [1](#F1){ref-type="fig"} depicts the box plots of serum creatinine and blood urea values of patients on the first day of colistin, the maximum values during the course of administration of colistin, and the values at the end of the treatment with colistin (one patient, who was on hemodialysis treatment prior to and during the colistin course, was removed from the analysis of the comparisons of the creatinine and urea values). The median value of baseline creatinine at the beginning of colistin therapy for the 18 prolonged courses of colistin was 0,6 mg/dl. Compared to this value, there was a slight increase of the median of values of creatinine at the end of the colistin course by 0,1 mg/dl, without statistical significance (p = 0,67). The median of the maximum values of creatinine observed was 0,85 mg/dl and was found to be statistically different from both the median of start (p \< 0,001) and end of treatment values (p \< 0,001). The maximum absolute increase of creatinine, compared to baseline, observed in an episode was 1,4 mg/dl. Only one patient had an increase of more than 50% of the baseline creatinine level to a value higher than 1.3 mg/dl at the end of colistin treatment. For the subgroup of 12 patients with 14 courses of prolonged colistin administration, with normal serum creatinine at the initiation of treatment, the median serum creatinine values at baseline and at the end of colistin treatment were 0,60 mg/dl and 0,55 mg/dl, respectively. In addition, the median maximum value of serum creatinine during the course of colistin treatment was 0,80 mg/dl among this group of patients. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The distribution of serum creatinine and blood urea levels on the 1^st^day of colistin treatment (start), at the peak value (max), and the end of colistin treatment (end) in all studied courses of prolonged treatment with colistin. (The horizontal line within the boxes represents the median creatinine or blood urea baseline value at the 1^st^day of colistin treatment. \"Shaded\" boxes in the figures represent the distribution of the laboratory values contained between the 25^th^and 75^th^percentiles (the interquartile range). Dotted lines (whisker lines) extend from the box, down and up, to the minimum and maximum values of the distributions that are not outliers (i.e. that are within 1.5 times the interquartile range); these values of the distribution are shown as \"brackets\". Any value, which is an outlier, is drawn as a \"horizontal line\"). ::: ![](1471-2334-5-1-1) ::: At the initiation of colistin administration the median value of baseline blood urea for the 18 courses was 42 mg/dl. During the 18 prolonged courses of colistin administration there was a slight increase in blood urea values; the median maximum value was 60 mg/dl. However, at the completion of colistin therapy median blood urea value returned to 41 mg/dl. During the 19 courses of colistin administration, other potentially nephrotoxic drugs were given. Specifically, aminoglycosides were co-administered in 12 courses \[median duration of co-administration (± SD) in these 12 courses 12,5 (± 16,8) days\], teicoplanin in 10 \[median duration of co-administration 16,0 (± 9,0) days\], vancomycin in 8 \[median duration of co-administration 23,0 (± 11,2) days\], amphotericin in 4 \[median duration of co-administration 8,0 (± 9,0) days\]. Four patients had history of chronic renal dysfunction, with only one of them receiving hemodialysis treatment prior to admission. This patient continued the hemodialysis during his treatment with colistin and died due to sepsis. For the other 3 patients the baseline, the maximum, and the end-of-therapy creatinine values (mg/dl), were 1,8/2,1/2,1 for the first patient, 3,7/3,7/2,4 for the second patient and 2,4/3,8/2,3 for the third patient. Only one patient had acute renal failure prior to colistin use, which was due to severe haemorrhage during surgery. This patient received 13 courses of hemodialysis. One day after the last course, colistin therapy had to be initiated for the management of *Acinetobacter baumannii*pneumonia, sensitive only to colistin and gentamicin. Apart from a slight elevation of serum creatinine (from 2,3 mg/dl to 2,7 mg/dl) on the 5^th^day of treatment no other sign of nephrotoxicity was observed during a 34-day administration of colistin (the 9^th^day creatinine returned to 0,7 mg/dl). Neuromuscular toxicity ---------------------- No apnea or other evidence of neuromuscular blockade was noted in any of these patients who received prolonged treatment with colistin. Four patients were clinically diagnosed to have polyneuropathy and/or myopathy before or during colistin treatment. In the first patient the polyneuropathy symptoms appeared while she was on her 25^th^day of colistin treatment and became worse on the 27^th^day. From then on, and although colistin was continued for 11 more days, the symptoms gradually subsided. No confirmatory electromyographic testing was performed. The second patient was transferred from another ICU where he was diagnosed as having myopathy due to sepsis. Fifteen days after his admission, and while his myopathy was improving, therapy with colistin was initiated; the treatment did not affect the gradual improvement. An electromyography performed on his 21^st^day in our ICU, showed mild axial sensory-motor polyneuropathy and myopathy. He received colistin for a total of 52 days and made full recovery from his neuropathy/myopathy during the course of colistin administration. The third patient developed ICU polyneuropathy one week prior to colistin administration and, again, recovered gradually from it while he was on colistin treatment. The fourth patient was transferred from another ICU, where he had developed ICU polyneuropathy. Only this patient had moderate worsening of the neuropathy while he was receiving colistin. Despite that, he received colistin for a total of 35 days for a respiratory infection with persisting *Pseudomonas aeruginosa*sensitive only to colistin. His neuropathy improved gradually after the end of colistin treatment. Thus, our assessment regarding these 4 patients who experienced polyneuropathy and/or myopathy is that colistin therapy was probably associated with the development of neurotoxicity only in one of the 4 patients. Liver and biliary tree toxicity ------------------------------- Data for the possible hepatobiliary toxicity of colistin were collected. Both laboratory and clinical findings were taken into consideration. In subgroup analyses of patients for whom data were available, no substantial changes on liver function tests was found, as described in Figure [2](#F2){ref-type="fig"} (data for SGPT, alkaline phosphatase, direct and indirect bilirubin are not shown). Three of our patients were found to have increased levels of hepatic and cholestatic enzymes during colistin administration. One of them was found to have acute cholecystitis, the second had severe inflammatory systemic reaction (SIRS), and the third had transient elevation of transaminases while he was receiving anti-epileptic medications together with colistin. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The distribution of liver function tests \[SGOT (AST = aspartate aminotransferase), γ-GT, and total bilirubin\] on the 1^st^day of colistin treatment (start), at the peak value (max), and at the end of colistin treatment (end) in studied courses of prolonged treatment with colistin. (The horizontal line within the boxes represents the median creatinine or blood urea baseline value at the 1^st^day of colistin treatment. \"Shaded\" boxes in the figures represent the distribution of the laboratory values contained between the 25^th^and 75^th^percentiles (the interquartile range). Dotted lines (whisker lines) extend from the box, down and up, to the minimum and maximum values of the distributions that are not outliers (i.e. that are within 1.5 times the interquartile range); these values of the distribution are shown as \"brackets\". Any value, which is an outlier, is drawn as a \"horizontal line\"). ::: ![](1471-2334-5-1-2) ::: Conclusions =========== The main finding of the study is that no significant deterioration of renal function was found in patients with baseline normal serum creatinine value during prolonged administration of intravenous colistin. In addition, in the group of patients with pre-existing chronic renal dysfunction (not on hemodialysis) the administration of the medication for a long duration did not influence further their renal function. The observed slight increase in the serum creatinine values at the end of treatment (0,1 mg/dl increase in the median creatinine values from the start to end of colistin administration) cannot be attributed only to the prolonged colistin administration as other factors such as concomitantly administered potential nephrotoxic agents, and sepsis per ce may have had an effect. In addition, apnea or other evidence of neuromuscular blockade was not observed in any of the 17 patients who received prolonged treatment with colistin. Early experience with colistin revealed a high incidence of toxicity, mainly nephrotoxicity. In 1969, Ryan et al. reported the fatal case of a previously healthy 4-year-old child with persistent fever after appendectomy who died due to cardiac arrest \[[@B10]\]. The child had received colistin intramuscularly due to the growth of *Pseudomonas aeruginosa*and *E. coli*strains from the excised specimen. This patient developed acute tubular necrosis (confirmed by autopsy) and apnea requiring intubation and mechanical ventilation. However, in this case the dosage of colistin used was 30 mg/kg of body weight every six hours (120 mg/kg/day) whereas the recommended dose is 2,5 -- 5 mg/kg/day. One year later, several reports showed high renal adverse reaction rates after colistin administration \[[@B2],[@B4],[@B11]\]. In many of them the dosage used was also higher compared to the recommended, i.e. 6,3 mg/kg/day in one report, and 26 mega units in another administered as 10 mega units by intramuscular injection, 10 mega units by intravenous injection and 6 mega units by inhalation (no details are provided in the report about the ratio of IU of colistin per mg). In addition, dosage adjustments in patients with renal dysfunction were not always followed, as well as there were also other factors which perhaps contributed to toxicity, such as the co-administration of other medications with potential toxicity, including anesthetic drugs and muscle relaxants. However, recent data from published reports do not corroborate this finding \[[@B14]\]. Notably, one study conducted in 35 patients with ventilator-associated pneumonia due to multidrug-resistant *Acinetobacter baumannii*, who received either intravenous colistin (21 patients) or imipenem (14 patients), showed an almost double increase in nephrotoxicity rates among the patients in the imipenem group compared to colistin group \[[@B12]\]. In addition, Stein et al. recently reported their experience from 3 patients with orthopedic infections who received colistin for 3 -- 6 months without developing any adverse effect requiring discontinuation of the treatment \[[@B13]\]. The conclusion in the majority of the published studies during 1960 -- 1970 was that colistin must be used in serious infections where other antimicrobial agents have failed, always with close monitor of renal function, and with precaution in the co-administration of other possible nephrotoxic or neutotoxic agents \[[@B2],[@B4]\]. Subsequently, the considerable difference regarding reporting toxicity of colistin between old and recent studies deserves an explanation. First, the formulations of currently used colistin may be more purified compared with the old ones. The experience that was accumulated from the use of vancomycin may support this possibility. It is known that the concerns about serious and/or common nephrotoxicity of vancomycin overwhelmed the older literature. Second, fluid supplementation and supportive treatment of severely ill patients has been improved nowadays. Third, higher doses of colistin were administered to patients during the first years of its use. Our study is not without limitations. It is a retrospective study with the inherited problems related to this study design, including difficulties in the determination of colistin associated sensory neurotoxicity. In addition, the number of studied patients is relatively small and there is no control group. However, our study offers insight related to interesting experience from a group of patients who received prolonged treatment with colistin for various infections, mainly as a salvage treatment of persisting and serious infections. In conclusion, intravenous colistin seems to be a safe therapeutic intervention for serious infections due to multidrug-resistant Gram-negative bacteria even when it is administered for a prolonged duration (more than four weeks). More studies will be needed to further clarify the correct use of this old antibiotic in the 21^st^century. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contribution ====================== MEF and AM conceived the idea; MR, IAB, KR, and SKK collected the data; all authors contributed in the writing and preparation of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/5/1/prepub>

PubMed Central

2024-06-05T03:55:52.425243

2005-1-10

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547910/", "journal": "BMC Infect Dis. 2005 Jan 10; 5:1", "authors": [ { "first": "Matthew E", "last": "Falagas" }, { "first": "Michael", "last": "Rizos" }, { "first": "Ioannis A", "last": "Bliziotis" }, { "first": "Kostas", "last": "Rellos" }, { "first": "Sofia K", "last": "Kasiakou" }, { "first": "Argyris", "last": "Michalopoulos" } ] }

PMC547911

Background ========== Enterococcal meningitis is an uncommon disease accounting for only 0.3% to 4% of cases of bacterial meningitis which is nevertheless associated with a high mortality rate. It has been described most frequently in patients with neurosurgical conditions (i.e. head trauma, shunt devices, or cerebrospinal fluid leakage), although it can also occur as a \"spontaneous\" infection complicating remote enterococcal infections such as endocarditis or pyelonephritis \[[@B1]\]. *Enterococcus faecalis*and *Enterococcus faecium*are the two species most frequently isolated during the course of meningitis (76%--90% and 9--22% respectively). *Enterococcus casseliflavus*, first considered as a subspecies of *E. faecium*, is a motile enterococcus that produces a yellow pigment in agar and often has a VanC phenotype determining an intrinsic low level resistance to vancomycin. It has been implicated in a wide variety of infections in humans, especially immunocompromised hosts, but to the best of our knowledge it has never been associated to meningitis \[[@B2]-[@B5]\]. We describe here a case of enterococcal meningitis caused by *E. casseliflavus*that was believed to originate from the gut in an old patient with bowel erosions. Case presentation ================= A 77-year-old Italian female presented for evaluation of fever, stupor, diarrhea and vomiting of 3 days duration. She had no urinary symptoms. There was no history of head injury nor of previous surgical procedures. She had been suffering from rheumatoid arthritis for 30 years, for which she was being treated with steroids and methotrexate; other medical problems were insulin-dependent diabetes and moderate renal failure. On admission, she was febrile (temperature, 38.0°C), alert but not oriented to time and place. Her neck was stiff, and she had a positive Kernig\'s sign. A CT brain scan showed an increase in subarachnoid space and in the volume of the ventricular system. Laboratory examinations revealed a white blood cell count of 15,100/μL with 70% neutrophils and 23% lymphocytes. Results of urinalysis were unremarkable. Cultures of blood and urine were drawn and subsequently resulted negative. The patient\'s cerebrospinal fluid (CSF) was opalescent with a glucose concentration of 14 mg/dl, a protein level of 472 mg/dl, and a white cell count of 200/μL with 95% polymorphonuclear leukocytes and 5% lymphocytes. Gram staining of CSF revealed no organisms. Pending the culture results the patient was empirically treated with intravenous meropenem, cotrimoxazole, acyclovir and dexamethasone. Culture of CSF yielded *E. casseliflavus*that was identified using the Vitek-2 system (bioMérieux-Vitek) on the basis of 6.5% NaCl tolerance, bile-esculin hydrolysis, and growth rate at 45°C, arginine hydrolysis, methyl-a-D-glucopyranoside testing, and acid production from ribose, motility testing, and yellow pigmentation testing. The isolate was sensitive to penicillin, ampicillin, ampicillin-sulbactam, imipenem, teicoplanin, tetracyclines and linezolid; it exhibited intermediate sensitivity to vancomycin (MIC, ≥8 μg/mL), trimethoprim-sulfamethoxazole (MIC, ≥10 μg/mL), levofloxacin (MIC, 4 μg/mL), norfloxacin (MIC, 8 μg/mL), ciprofloxacin (MIC, 2 μg/mL) and quinupristin-dalfopristin (MIC, 2 μg/mL); it was resistant to clindamycin (MIC, 4 μg/mL) and showed high resistance to gentamicin, streptomycin and kanamycin (MIC, ≥2000 μg/mL). The patient became afebrile 48 hours after the beginning of antibiotic therapy with rapid improvement of her mental status and disappearance of meningeal signs (within 36 hours). Once the organism was identified (4 days later), trimethoprim-sulfamethoxazole and acyclovir were discontinued and ampicillin-sulbactam (3 g every 6 hours) was added. After 2 weeks of antibiotic therapy the patient was discharged in good health with sterilization of the CSF culture. An echocardiogram revealed no vegetations whereas a colonoscopy examination showed two ulcerative lesions associated with two polyps, oedema and multiple punctuate erosions. Enterococcal infections of the central nervous system are quite rare and according to a MEDLINE search of the English literature only three cases of CNS infection by a motile *Enterococcus*identified as *E. gallinarum*have been previously documented; they occurred in patients with ventriculoperitoneal shunts for hydrocephalus \[[@B6],[@B7]\]. *E. casseliflavus*and *E. gallinarum*are responsible for 1--2% of all enterococcal infections and are characterized by the fact that they possess intrinsic low-level vancomycin resistance \[[@B8]\]. The VanC-1 ligase is specific for *E. gallinarum*, and the VanC-2/3 ligase is specific for *E. casseliflavus*\[[@B9]\]. Organisms with resistance to VanC remain susceptible to teicoplanin. This naturally occurring vancomycin resistance has not been shown to be transferable, and the related genes are chromosomally encoded in the members of these species \[[@B4],[@B9],[@B10]\]. Despite the intrinsic low-level vancomycin resistance exhibited by *E. casseliflavus*it is important to remember that most strains are susceptible to penicillin and ampicillin. Combination therapy of ampicillin with an aminoglycoside such as gentamicin or streptomycin is considered the standard therapy of enterococcal meningitis due to ampicillin-susceptible strains \[[@B1]\]. Meropenem is not superior to ampicillin for therapy of enterococcal infections and most species of *E. casseliflavus*are beta-lactamase negative. There was not therefore a clear indication for the use of combination therapy with meropenem and a beta-lactamase inhibitor in the case reported. Our patient had gastrointestinal signs and symptoms, and colonoscopy revealed multiple erosions, which probably were the portal of entry of *E. casseliflavus*. In fact, enterococcal meningitis may appear as a complication of diverse gastrointestinal diseases such as enterocolitis, peritonitis, abdominal surgery, or bowel carcinoma. A case of bowel erosions as the portal of entry of enterococcal meningitis has been previously reported \[[@B11]\]. Several studies have demonstrated that *E. gallinarum*and *E. casseliflavus*colonize the gastrointestinal tracts of both hospitalized individuals and nonhospitalized healthy ones \[[@B8],[@B12],[@B13]\]. Therapy with various antimicrobial agents, including cephalosporins and vancomycin, may play a role in increasing colonization with these organisms. Edlund et al. reported a significant increase in the emergence of *E. gallinarum*and *E. casseliflavus*in healthy subjects who were administered oral vancomycin \[[@B14]\]. Our patient was not taking any antimicrobials before hospital admission. *E. gallinarum*and *E. casseliflavus/flavescens*are part of the normal stool flora of the general population; this has perhaps impacted the ability of researchers to detect specific risk factors \[[@B8]\]. Although our patient had significant underlying conditions this case of meningitis was mild and had most of the typical features of \"spontaneous\" enterococcal meningitis (community-acquired infection, severe underlying diseases, and immunodepression). The clinical significance of the enterococci that are intrinsically resistant to vancomycin has not been fully established yet. Infection with *E. gallinarum*and *E. casseliflavus*has been associated with high mortality, but it is difficult to attribute their mortality directly to infection or to the underlying conditions of the patient. Conclusions =========== This case is the first report of *E. casseliflavus*meningitis and it is the fourth documented so far with a motile *Enterococcus*species. Awareness of infection of central nervous system with *Enterococcus*species that possess an intrinsic vancomycin resistance should be increased. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= IC and RDL carried out the clinical study of the patient and conceived of the study. GS and GBC carried out the microbiologic studies. AT and AC carried out the clinical study of the patient, conceived of the study and drafted the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/5/3/prepub> Acknowledgements ================ Written consent was obtained from the patient for publication of study

PubMed Central

2024-06-05T03:55:52.427698

2005-1-14

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547911/", "journal": "BMC Infect Dis. 2005 Jan 14; 5:3", "authors": [ { "first": "Chiara", "last": "Iaria" }, { "first": "Giovanna", "last": "Stassi" }, { "first": "Gaetano Bruno", "last": "Costa" }, { "first": "Rita", "last": "Di Leo" }, { "first": "Antonio", "last": "Toscano" }, { "first": "Antonio", "last": "Cascio" } ] }

PMC547912

Background ========== Unilateral vocal fold paralysis is symptomatic when it results in failure of the mobile vocal fold to approximate the paralyzed vocal fold during adduction. Despite the lack of movement, the paralyzed vocal fold will often contact the contra lateral mobile vocal fold permitting adequate glottic closure \[[@B1]\]. Medialization laryngoplasty is a common procedure used to restore glottic competence. This procedure was popularized by Ishiki and initially used in patients with vocal fold paralysis. In recent years, the indications for this procedure have expanded to include most forms of glottic incompetence, including the use of bilateral medialization in mobile vocal folds for vocal fold bowing and atrophy \[[@B2]\]. Although medialization is now widely accepted, the choice of implant material is still a subject of controversy. The ideal vocal fold injection material would be readily available, have excellent biointegration with no or minimal immunologic response, has an excellent biomechanical in vivo match to the injection site tissues, and is deliverable through a fine-gauge needle. Such a material does not presently exist. Teflon (Polytetrafluoroethylene) was the first modern material used for vocal fold injection. Long-term results, unfortunately, have shown an unacceptably high rate of granuloma formation associated with Teflon injection. This results in a gradual degradation of the injected material by foreign body giant cells and fibroblastic proliferation in the location of the implant \[[@B3]\]. Autologous tissues such as fat or fascia are well described. These tissues have the benefit of medializing the vocal fold with minimal tissue reaction \[[@B4],[@B5]\]. Collagen injection has been used with some success, its major drawback is the need for serial injections in some cases \[[@B2]\]. Recently, an expanded polytetrafluroethelyne implant (e PTFE, Gore-Tex^®^) has been advocated as an implant material for medialization of the vocal fold \[[@B6]\]. This material has been widely used in cardiac- and vascular surgery and soft tissue augmentation with minimal complications \[[@B2]\]. Cashman et al published a histological study of changes in the rabbit larynx in response to Gore-Tex implantation. They found evidence of a foreign body giant cell reaction and a thin rim of fibrous tissue surrounding the implant. Although the fibrous capsule invaginated between the folds of the implant, it did not appear to grow into the implant itself. The implant was secure in the soft tissue with no migration or spontaneous extrusion noted \[[@B2]\]. Case presentation ================= A 64-year-old female patient underwent transcervical excision of a right vagal neurofibroma. This was complicated by right vocal fold paralysis with dysphonia and aspiration. Six months later, the patient underwent fat injection for medialization of the right vocal fold. After an initial improvement, the patient suffered a recurrence of the aspiration, chest infections and dysphonia. The patient subsequently underwent right medialization using Gore-Tex implant (Gore Thyroplasty Device, Medtronics, Xomed^®^) with cricothyroid approximation. Videofluroscopy and Speech assessment confirmed an improvement in aspiration and speech. Six months later, her symptoms recurred with subsequent aspiration pneumonia. A decision was made after lengthy counseling to proceed with a laryngectomy 13 months following the implantation of Gore-Tex. Figure [1](#F1){ref-type="fig"} shows the Gore-Tex in the laryngectomy specimen. Histopathology -------------- Light microscopy revealed a mild foreign-body giant cell granulomatous reaction with some associated fibrosis (Figures [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}). The granulomatous response was limited to the periphery of the Gore-Tex and although it closely followed the profile of the material it did not encroach into or significantly break up the material. However, some fibrosis penetrated a short way into the Gore-Tex in a few areas. The giant cells were present mainly in a multinucleated form. Figure [4](#F4){ref-type="fig"} shows the Gore-Tex strip under crossed Polaroids showing birefringence of the implanted material. Birefringent fragments were only very occasionally seen within isolated giant cells. The giant cell response was mainly seen in areas where the end of a Gore-Tex strip formed a sharp corner. In one area a very small focus of metaplastic bone formation was seen. There was no significant lymphocytic, neutrophilic inflammation or vascular ingrowth. Discussion ========== Gore-Tex has been used as an effective implant for medialization laryngoplasty in the management of paralytic dysphonia and to a lesser extent aspiration \[[@B6],[@B7]\]. Animal studies involving rabbits and a porcine model have demonstrated host tolerance of the implant, with no evidence of granuloma formation, resorption, extrusion or migration of the implant \[[@B2],[@B8]\]. However, adverse responses to Gore-Tex in humans requiring implant removal from the lips, face, nose and larynx have been reported \[[@B9]-[@B12]\]. To date, there have been no reports describing the histological changes in a human laryngectomy specimen with a Gore-Tex implant. Conclusions =========== Our findings are consistent with the animal models confirming that Gore-Tex implantation does not result in a significant granulomatous reaction in the human larynx over a 13-month period. Moreover, there is no evidence of resorption or infection. Further, the lack of lymphocytes in association with the granulomas indicates that there is no significant immunological hypersensitivity. Histologically, the slight permeation by connective tissue is similar to that seen in Gore-Tex vascular and cardiac implants. The degree of the slight giant cell response appears to be dependent on the profile of the material; a sharp edge incited more of a response than a flat surface. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= All authors equally contributed towards the background research and writing of the paper. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6815/5/1/prepub> Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Laryngectomy specimen showing Gore-Tex (arrow) as a pale nodule in the paraglottic space lying beneath an intact mucosa. ::: ![](1472-6815-5-1-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Low power magnification showing broad interlacing bands of Gortex material (white arrow) surrounded by mild foreign-body giant cell granulomatous reaction with some associated fibrosis (black arrow). ::: ![](1472-6815-5-1-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### High power magnification showing only a small number of multinucleated foreign-body type macrophage giant cells (arrowhead) associated with the periphery of the Gortex bands (arrow). There is little evidence of breakdown of Gore-Tex. ::: ![](1472-6815-5-1-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Gore-Tex under crossed polaroids showing birefringence (arrow). ::: ![](1472-6815-5-1-4) :::

PubMed Central

2024-06-05T03:55:52.428587

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547912/", "journal": "BMC Ear Nose Throat Disord. 2005 Jan 21; 5:1", "authors": [ { "first": "K", "last": "Rajkumar" }, { "first": "HS", "last": "Khalil" }, { "first": "M", "last": "Elloy" }, { "first": "E", "last": "Sheffield" }, { "first": "DL", "last": "Baldwin" } ] }

PMC547913

Background ========== In the post-genomic era, biologists encounter a flood of information derived mainly from microarray experiments. The blessing of this wealth of information is accompanied by a great difficulty in identifying the biologically significant findings, which are often embedded in irrelevant information. Currently, there are several approaches to deal with this problem. One approach is to identify a category of genes which is overrepresented in the microarray output. This approach can be carried out using the Gene Ontology project (GO) which describes gene products in terms of their associated biological processes, cellular components and molecular functions \[[@B1]\]. The advantage of this approach is that it can be easily automated and thus can be used for quick screening of large outputs. On the other hand, this approach limits the analysis to the structure of the GO project and thus does not support the desire of many researchers to customize their analysis. A second approach involves searching the literature for information about each of the genes on the list. Although this approach is comprehensive, it suffers from many downsides: it is time consuming; there is no systematic way to integrate the information learned about each gene; usually one gets distracted with seemingly interesting comparisons early on during the literature search and thus does not give the genes at the end of the list the same weight that was given to genes that appear at the top of the list; there are multiple names and symbols for each gene and thus it is hard to extract the literature information for any particular gene since each author may refer to it differently. A third approach entails curated databases that have gathered all the known information pertaining to each gene. This approach is limited by the quality of the curation process. For example for studying the yeast *Saccharomyces cerevisiae*, there are excellent curated databases, such as the Yeast Proteome Database \[[@B2]\] and the Saccharomyces Genome Database \[[@B3]\], which contain all the known information about each gene. On the other hand in other organisms the curation procedure is at a less advanced stage and thus the information contained in the curated databases is still partial. We have developed an analysis tool that combines the advantages of all the mentioned approaches and overcomes some of the disadvantages. Our tool (MILANO -- Microarray Literature-based Annotation) uses an automatic search of literature databases for performing custom annotation of the list of genes obtained from a microarray output. This is done by generating dynamic annotations for genes, built according to terms provided by the researcher. The program receives as input a list of gene identifiers obtained from any microarray experiment and a set of custom search terms. The program expands each gene identifier to its informative synonyms and searches literature databases for co- occurrences of every gene on the list with each of the custom terms. The program\'s output is an annotation table with the numbers of publications for each gene-term combination (hit-counts). This novel annotation format can be easily used within a web browser or a spreadsheet program to quickly identify genes within the list that are related to the terms provided by the researcher, and may be easily extended, as every hit-count in the annotation is a hyperlink to the query\'s results. The great advantage achieved by this method over standard static annotations, such as Gene Ontology (GO) annotations, is that the annotations are generated based on terms provided by the researcher, and therefore help in addressing the specific scientific question the researcher is pursuing. The program is able to search two literature databases, GeneRIF \[[@B4]\] and Medline \[[@B5]\]. GeneRIF contains \~90,000 short summaries of curated articles relevant to known genes. An initial search of the microarray results against the GeneRIF database provides results within minutes and is easily evaluated, thereby providing immediate insights to the microarray results. This search is followed by a comprehensive Medline search via Pubmed, allowing the identification of more subtle biological insights. To demonstrate the power of this strategy, we have analyzed a list of 148 genes affected by over-expression of p53 \[[@B6]\]. Our analysis assisted in retrieving from the list 11 known p53 targets, which are all the known targets in the list, and in identifying within the p53-affected genes a subset of putative p53 target genes that are known to be involved in apoptosis (43 genes), in cell cycle arrest (21 genes), and in Cancer (48 genes) as shown in Figure [3](#F3){ref-type="fig"}. This example demonstrates the usefulness of our tool in narrowing down microarray results to a small list of genes involved in a specific biological activity. Implementation ============== Web Interface ------------- MILANO is accessed through a familiar web form (Fig [1A](#F1){ref-type="fig"}). A CGI (Common Gateway Interface)-based Perl \[[@B7]\] program is executed on submission, which creates the combined Boolean searches for the requested databases. The user can decide whether to provide gene symbols directly, or provide LocusLink/Gene numbers, which are expanded to synonyms as described below. Results, formatted as an HTML table, are displayed immediately on-screen for GeneRIF searches and sent by e-mail for Pubmed searches. Synonym expansion ----------------- Gene aliases are collected from the LocusLink database file, downloaded from the NCBI ftp server \[[@B8]\]. We use an awk \[[@B9]\] program to extract gene symbols, aliases and product names. The alias collection is then processed by a Perl program that removes symbols that are shorter then three characters or that appear in a 23,000-word English dictionary, enhanced for scientific terms. This database is stored in a fashion than enables us to extract processed aliases for a gene by its LocusLink number. Pubmed searches --------------- Pubmed searches are performed by a Perl program which uses the NCBI eutilities esearch web service for accessing the Pubmed database \[[@B10]\]. There are limitations on when and how often we can query the NCBI server, so we integrated into the program a mechanism that makes sure that is does not make more than one query every three seconds. The Generic NQS (Network Queuing System) \[[@B11]\] ensures that jobs that include more than 100 queries run only between 9 p.m. and 5 a.m. ET. GeneRIF searches ---------------- The GeneRIF collection is automatically downloaded weekly from the NCBI ftp server \[[@B12]\], and processed by a Perl program to include gene symbols from the synonym expansion database into every GeneRIF. The database is then indexed by a database server (SRS 7.1.3, Lion Bioscience AG), which provides a query interface for counting and displaying GeneRIF entries. Results ======= Expanding the search terms -------------------------- One of the major problems in the automation of literature searches is the ambiguity in gene names \[[@B13]\]. Multiple names are used in the literature for any specific gene and thus it is not straightforward to define the Medline query that will find most of the relevant information on a gene. In order to overcome this problem we used the LocusLink database \[[@B14]\] to expand any gene symbol to all its synonyms. We also included in the expanded form the gene product name since many genes are mentioned in the literature by their product name and not by one of their symbols (for example most of the citations for the beta actin gene can be found by searching the Medline with the term \"beta actin\" and not with its official symbol \"*ACTB*\"). Although expansion of the search terms is a useful tool to increase the number of articles retrieved for each gene it also adds many irrelevant articles due to the fact that some of the gene symbols are not informative as Medline query terms. For example one of the aliases of the gene aquaporin\_1 is *CO*, a term that is mostly mentioned as an abbreviation for Carbon mono-oxide, and one of the aliases of the gene CD36\_antigen is *FAT*, which is found in over 100,000 articles, unrelated to CD36. In order to diminish this problem we filtered out from the list of gene symbols any term that was shorter than three characters and any term that is an English word. In order to check our name expansion strategy we conducted a Medline search for 16862 well-known human genes (all the genes that have an NM number indicating the identification of their full length mRNA), using three search strategies: using only the official symbol for each gene (Symbol), using the official symbol together with all its aliases and the gene product (Expanded) and using only the informative terms (Filtered). Using the Expanded search allowed the identification of literature information on about \~1900 additional genes over a query using the official symbol only (Table [1](#T1){ref-type="table"}). Using the Filtered search terms allowed this addition without adding significantly to the number of queries that returned non-reasonable results. In addition to expanding the number of genes that were found in the literature, the Filtered search terms also increased the number of articles found per gene (from an average of 198 articles per gene found by searching with the symbol alone to an average of 451 articles per gene when searching with the filtered terms). These results indicate that our gene name expansion strategy achieves a higher percentage of relevant literature for each gene while limiting the addition of irrelevant information. Conducting automatic literature searches ---------------------------------------- After expanding the search terms, MILANO performs an automatic search of literature databases, and retrieves the number of hits each query returned. MILANO performs Boolean searches in which one can search for co-occurrence of each of the primary terms (the expanded gene name) with user defined secondary terms (Figure [1](#F1){ref-type="fig"}). The program\'s output is a table (Figure [2](#F2){ref-type="fig"}) containing the number of publications for each gene-term combination (hit-counts). This table could serve as an annotation table, because the number of publications reflects the relationship between the genes to the secondary term used. For example a gene that has a role in DNA damage will appear in more articles about \"DNA damage\" or \"gamma irradiation\" than unrelated genes. In order to assist in further evaluation of the results, we have built the annotation table such that each number in the table is a hyper-link to the literature database and thus clicking on it will perform this specific search again and will open a window containing the actual abstracts found by this combination of search terms. Literature databases supported by the program --------------------------------------------- The MILANO program can search two databases (Figure [1](#F1){ref-type="fig"}) -- the full Medline database, currently containing more than 12,000,000 references, and the GeneRIF database that contains more than 90,000 short summaries of curated articles relevant to known genes. There are several advantages in using the GeneRIF database over the full Medline: the searches are quick and the results are obtained within minutes; each article is summarized by a sentence or two, reducing the amount of information that needs to be read; the curation procedure extracts from the papers only the information relevant to the gene, minimizing the cases in which two terms appear in the same abstract but are not related to each other; the GeneRIF entries are based on the full text of the articles and not only on the abstracts. However, since the curation procedure is an on-going process, the coverage of this database is only partial and thus information is missing and can be found only by performing a Medline search. For that reason our tool allows a combined search strategy in which both databases can be searched simultaneously. The GeneRIF database provides results within minutes and is easily evaluated, thereby providing immediate insights to the microarray results. In parallel a comprehensive Medline search can be done. Although this search takes longer and its results obtained by email, it allows the identification of more subtle biological insights. P53 --- To demonstrate the power of our literature-based annotation strategy, we analyzed a list of 148 genes affected by over-expression of p53 \[[@B6]\]. This list of genes was obtained by microarray experiments and nicely demonstrates the difficulty of microarray analysis since it contains many putative p53 target genes and their relevance to p53 cellular activity is not clear. Our first aim was to identify the known p53 target genes that were affected by p53 overproduction in this experiment. By using specific secondary terms, we were able to trim down the list of 148 genes to a much shorter list of genes highly enriched for known p53 target genes (Figure [3A](#F3){ref-type="fig"}). In order to evaluate the number of target genes that were missed by our annotation strategy, we manually compiled a list of all known p53 target genes, \~60 genes. Eleven of these 60 genes were represented in the list of genes affected by over-expression. Our automatic annotation strategy found all of them. Moreover, the use of MILANO reduced the amount of articles per gene from an average of 2088 articles per gene in the initial list to 56 articles per gene in the limited list (Figure [3B](#F3){ref-type="fig"}). The p53 example also demonstrates the usefulness of searching the GeneRIF curated database in which the use of the secondary term p53 allows filtering out most of the irrelevant genes without losing any known target gene (Figure [3A](#F3){ref-type="fig"}). P53 is involved in apoptosis, cell cycle arrest and cancer. It is interesting to find out which of the genes affected by p53 is involved in these processes. Using MILANO we easily identified genes known to be involved in these processes (Figure [3C](#F3){ref-type="fig"}), which helped the process of analyzing the microarray data. Comparison with other tools --------------------------- Recently, few literature mining tools has been developed, using a similar approach to the one presented here \[[@B15]-[@B17]\], however all of them suffer from the problem of inappropriate use of primary search terms. In order to demonstrate the advantage of using MILANO over the other tools, we have performed a comparative analysis of all these tools by looking at their performance on the 11 known p53 target genes described above. The software were run with these 11 genes as the primary search terms and \"P53\" as the secondary term and reported the number of occurrences of those terms. The results that are summarized in table [2](#T2){ref-type="table"} demonstrate that MILANO-GeneRIF search was the only method that revealed connections between all 11 genes and p53 and that the MILANO-Medline search gave the most comprehensive search results. PubMatrix \[[@B15]\] does not expand the primary search terms and thus it misses many literature occurrences. This problem is best demonstrated by its poor performance on the CDKN1A gene which is one of the most studied targets of p53. The synonym expansion methods used by MicroGENIE \[[@B16]\] improved the results regarding the CDKN1A gene, but missed the SFN gene completely, and gave non-informative synonyms to XRCC5 and TRAF4 (\"Ku\" and \"TNF\" respectively). BEAR GeneInfo \[[@B17]\] did not perform synonym expansion correctly for CDKN1A, and gave non-informative synonyms for PCNA and TRAF4 (\"cyclin\" and \"h. mln62 mrna\" respectively). When we attempted to analyze the full data set of 148 genes, some of the compared tools failed to give results due to errors. Discussion ========== MILANO is a simple and intuitive literature search tool. It allows automatic Medline and GeneRIF searches followed by a quick survey of the results. Using this tool dramatically reduces the time needed to query literature databases. Moreover, due to its systematic nature, it assists in treating the 1^st^and the 100^th^query in an unbiased manner. The MILANO program uses all the published information for the annotation of each gene according to its co-occurrence in the literature with a user defined secondary search term. These features of MILANO makes it especially suitable for analyzing microarray results, since it can be used to annotate the results with terms defined by the user and not limited by preset terms such as the GO terms based annotation. We have demonstrated the power of our program by the analysis of a list of 148 genes that were deregulated in cells that overproduced the p53 tumor suppressor gene \[[@B6]\]. Frequently one of the first tasks in microarray data analysis is to determine the overlap between new results and results expected based on the literature. For example in analyzing the list of genes induced by over expression of p53 one expects to find known p53 target genes. Thus, we applied our automatic literature search tool in order to answer this question. We found that use of this tool dramatically shortens the time needed for such an analysis by allowing the researcher to focus on a relatively small subset of potential target genes and by reducing the amount of literature relevant to each gene (Figure [3](#F3){ref-type="fig"}). Our tool was also found useful in automatically sorting the target genes into functional groups. Based on the knowledge of p53 cellular functions we defined secondary search terms that fit p53\'s main activities -- apoptosis and cell cycle arrest \[[@B18]\]. Using these terms allowed the quick identification, from the primary list, of a subset of genes that were not known to be involved in those processes and thus may be interesting for further research (Figure [3C](#F3){ref-type="fig"}). Several literature mining approaches have been developed to integrate multiplex biological datasets into the context of published medical literature. A good example of such an approach is the PubGene program \[[@B19]\], which searches for literature co-occurrences of gene names in order to build a network among the genes. PubGene is useful for quickly realizing and viewing known relationships between genes, but it does not assist in annotating gene lists. To this end one needs an automatic literature searching tool that allows the use of flexible secondary terms with which co-occurrences are counted. Recently such tools have been built. PubMatrix \[[@B15]\] allows automatic Boolean searches to be performed on Pubmed using any list of primary and secondary terms. This tool carries out the search on the exact terms entered by the user thus in order to apply it to the analysis of microarray data, one has to translate each of the enriched spots to a name suitable for a Medline search. Two other tools -- microGENIE \[[@B16]\] and BEAR GeneInfo \[[@B17]\] uses a very similar approach but in order to make it more compatible to microarray analysis, they allow the use of gene identifiers as input and provides the needed translation to gene names. During the translation the gene name is expanded to include its synonyms. All of these tools have improved the ability of researchers to quickly use the published literature to annotate lists of genes. However, they suffer from the limitations of any literature data search tool; the ambiguity of gene names and the partial information that can be retrieved by limiting the literature searches to abstracts \[[@B13]\]. MILANO\'s aim is to further improve the literature based automatic annotation approach by adding two essential features that address these limitations: Smart synonyms -------------- Each gene symbol is expanded to all its aliases, while removing non-informative terms, and the gene product name is added to the query. This addresses the synonym problem, while omitting many of the irrelevant results, thus reducing the polysemy problem (words with multiple meanings). The advantage of our synonym expansion scheme over the existing tools is demonstrated by the comparison presented in table [2](#T2){ref-type="table"}. The GeneRIF database -------------------- In contrast to the existing tools, MILANO is able to search not only the Medline database, but also the GeneRIF database, which contains short summaries of articles relevant to known genes. The curation of GeneRIF is done by the National Library of Medicine\'s MeSH indexing staff, who have advanced degrees in the life sciences and use the full text of articles for the indexing process \[[@B4]\]. Using this database reduces the limitations of relying only on abstracts and aids in finding only relevant information about each gene. Nevertheless, the GeneRIF database suffers from the problems of all manually curated databases; it is partial and contains mistakes and biases introduced by the curation team. However, our ability to identify all of the p53 target genes within a group of p53-affected genes by using the GeneRIF database alone (Figure [3](#F3){ref-type="fig"}) demonstrates that, at least for well annotated genes, using such a database may be the ideal solution for annotating microarrays results. The quality of GeneRIF-based annotation depends on the amount of information entered for each gene in the GeneRIF database, which for many genes is insufficient (data not shown). However, its performance will improve as more information is incorporated into this database and we believe that in the future it will become the preferred annotation tool. Meanwhile, we recommend using MILANO for performing combined searches; searching the GeneRIF database provides quick results and searching the full Medline database allows a broader view that is not limited by the curation procedure. Conclusions =========== We present MILANO <http://milano.md.huji.ac.il>, a literature mining tool that can help in annotating microarray results in light of all available literature using experiment-specific terms. In designing MILANO we focused on the accuracy of the search results by providing two novel features: i) Expansion of gene names to include in the literature searches all their informative synonyms, while removing non-informative synonyms; ii) Searching two literature databases -- Medline and GeneRIF. While Medline encompasses all the literature and provides the most comprehensive results, it also contains many irrelevant articles. GeneRIF provides a subset of Medline articles that are relevant to known genes and thus avoids most of the irrelevant results often found in Medline searches. The usefulness of MILANO is demonstrated by the automatic analysis of a list of 148 p53 target genes. The use of literature mining dramatically reduced the time and effort required for a task such as identifying the known p53 target genes within this list. A search in GeneRIF immediately discovered the full list of target genes, with no false hits. Availability ============ All software and databases are freely available and may be executed online at our web site: <http://milano.md.huji.ac.il>. The author will provide data, scripts and programs used on demand. We encourage users to install the software on their own servers, as we provide no assurance to the privacy or accuracy of the results. Authors\' contributions ======================= RR designed and programmed the software. IS managed the project and drafted the manuscript. Acknowledgments =============== We would like to thank Zahava Siegfried for critical readings of the manuscript. R.R. was supported by a fellowship from the Sudarsky Center for Computational Biology <http://www.cbc.huji.ac.il>. This research was supported by grants from the Association for International Cancer Research (AICR) and by the F.I.R.S.T. (Bikura) program of the Israel Science Foundation (Grant No. 4103/03). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The MILANO data input page <http://milano.md.huji.ac.il>. ::: ![](1471-2105-6-12-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### An example of a result of MILANO search using a short list of gene symbols that were expanded by the program to include all their informative synonyms versus p53 related terms. All reported numbers are hyperlinked and will initiate a new search for that specific term combination. ::: ![](1471-2105-6-12-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Analysis of a list of genes affected by p53 overproduction.**A. The number of genes remaining after filtering the p53-affected genes with terms intended to reveal known p53 targets. B. Average number of articles per gene in the different queries. C. Venn diagram depicting the different functions of p53 affected genes as reflected by a GeneRIF search. ^1^Search term is \"p53 AND (target OR transcriptional OR activation OR repression)\" ::: ![](1471-2105-6-12-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Summary of Medline hit counts for all the full length mRNA genes (16,862 genes) using different search strategies. ::: Type of primary term^a^ Positive results^b^ Non reasonable results^c^ Articles per gene^d^ ------------------------- --------------------- --------------------------- ---------------------- Symbol 10,045 20 198 Expanded 12,028 140 817 Filtered 11,910 22 451 ^a^The Medline search was conducted using three searching strategies: Symbol refers to a search in which each gene was represented by its official symbol; Expanded refers to searches in which each gene was represented by the gene symbol, all its synonyms and the official gene product name; Filtered refers to searches in which non informative names were filtered out of the expanded list. ^b^Number of queries that returned at least one result. ^c^Number of queries that returned more than 33,000 results. We used 33,000 as a rough estimate of non reasonable results based on the fact that some of the most investigated genes, like p53, appear in less than 33,000 abstracts. ^d^The average number of abstracts per gene counting only genes that appeared at least once and did not appear in more than 33,000 abstracts. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Comparative analysis of literature mining tools. Eleven known p53 target genes were analyzed using five methods. The numbers represent the number of reoccurrences of each gene with the term \"P53\". ::: **Gene Id** **Primary Symbol** **MILANO -- GeneRIF**^a^ **MILANO -- Medline^a^** **PubMatrix^b^** **BEAR GeneInfo^a^\[17\]** **MicroGenie^c^** -------------------- -------------------- -------------------------- -------------------------- -------------------- ---------------------------- -------------------- [1026]{.underline} CDKN1A [74]{.underline} [4180]{.underline} [49]{.underline} [50]{.underline} [3058]{.underline} [1647]{.underline} GADD45A [7]{.underline} [281]{.underline} [21]{.underline} [45]{.underline} [313]{.underline} [2810]{.underline} SFN [2]{.underline} [25]{.underline} [25]{.underline} [25]{.underline} 0 [3486]{.underline} IGFBP3 [1]{.underline} [42]{.underline} [36]{.underline} [42]{.underline} [36]{.underline} [355]{.underline} TNFRSF6 [25]{.underline} [740]{.underline} [418]{.underline} [43]{.underline} [707]{.underline} [4583]{.underline} MUC2 [2]{.underline} [29]{.underline} [29]{.underline} [29]{.underline} [29]{.underline} [4609]{.underline} MYC [21]{.underline} [1715]{.underline} [1715]{.underline} [1715]{.underline} [1715]{.underline} [5111]{.underline} PCNA [13]{.underline} [1269]{.underline} [1173]{.underline} [3671]{.underline} [1173]{.underline} [59]{.underline} ACTA2 [1]{.underline} 0 0 0 0 [7520]{.underline} XRCC5 [1]{.underline} [52]{.underline} [42]{.underline} [42]{.underline} [583]{.underline} [9618]{.underline} TRAF4 [2]{.underline} [1]{.underline} [1]{.underline} [1570]{.underline} [326]{.underline} ^a^The search was performed with LocusLink ids as the primary search terms. ^b^The search was performed with the primary gene symbols as the primary search terms. ^c^The search was performed with UniGene ids as the primary search terms. :::

PubMed Central

2024-06-05T03:55:52.429338

2005-1-20

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547913/", "journal": "BMC Bioinformatics. 2005 Jan 20; 6:12", "authors": [ { "first": "Ran", "last": "Rubinstein" }, { "first": "Itamar", "last": "Simon" } ] }

PMC547914

Background ========== In the last quarter century, more than five hundred published manuscripts contained experiments using the popular rat crypt cell lines IEC-6 and IEC-18 originally established by Quaroni and Isselbacher in 1978 and 1981 \[[@B1],[@B2]\]. These lineages have stood the test of time as being two of the most reliable and phenotypically well-preserved gastrointestinal cell lines to date. In our lab, we utilize the IEC-18 lineage because it is more robust in serum-restricted conditions and, in our experience, it is more suitable for correlation with distal intestinal crypt and enterocyte physiology than cell lineages derived from colonic tumors. One of the more exciting aspects of IEC-18 cells is their capacity to retain the crypt cell phenotype as well as the continued potential for differentiation/maturation into enterocytes \[[@B3]-[@B5]\]. However, as we have worked with this lineage over the years, the rare but unmistakable morphology of an occasional neuroendocrine-like cell, the suspicious frothiness of a possible paneth-like cell and the rare giant vacuole of a goblet cell precursor have prompted us to wonder how prevalent spontaneous differentiation towards other lineages and/or transformants might be (especially in serum-restricted conditions). This is a fairly crucial experimental question because serum-restricted conditions allow the tightest experimental rigor but may drive IECs towards altered cell fates, potentially introducing uncontrolled experimental confounders. Until recently, we did not have the necessary tools with which to determine more subtle epigenetic divergences in IECs. Our current investigation uses a novel strategy to demonstrate that spontaneous cell fate divergence is not only highly prevalent in IEC-18 cells, but it is fairly uniform and therefore a predictable confounder for gene array studies in this cell line. Results ======= Initial characterization of IEC-18 culture heterotypy using immunolocalization ------------------------------------------------------------------------------ We have recently discovered a novel antigen localization pattern by using anti-carboxyl IGF binding protein-2 (IGFBP-2) antibody (to an antigen which we call C2) to demonstrate a multivesicular pattern in some IEC-18 cells but not in others that are immediately adjacent (illustrated in Figure [1A](#F1){ref-type="fig"}). We note that anti-amino IGFBP-2 showed no staining in IEC-18 cells and that pretreatment with synthetic antigen abolished C2 staining (data not shown), confirming that C2 is a carboxyl fragment of IGFBP-2. For the purpose of this study, we wanted to determine whether or not this heterotypy represented cell fate divergence or systemic pleiomorphism. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Immuno-characterization of IEC-18 cell heterotypy. **A**: Carboxyl IGFBP-2 immunostaining of the sequestered C2 fragment in cells with a preserved crypt cell phenotype but not in others. **B**: F-actin immunostaining (performed without antigen retrieval to visualize dynamic actin filaments) demonstrates intense staining in crypt cells but not in others. **C**: Cells plated at variable density with 10% FBS start out as weakly C2 positive (*i*) but have a progressive loss of C2 immunostaining prior to confluence (*ii-iv*). **D**: Immunolocalization of the C2 antigen demonstrates that both C2 positive (*i*) and C2 negative (*ii*) cells preserve their phenotypes during proliferation. **E-F**: Control immunolocalization using the IGF type 2 receptor (E) as a prelysosome-localized antigen and villin (F) as a cytoplasmic-localized antigen to demonstrate that both intravesicular and cytoplasmic antigens can be evenly detected throughout IEC culture. ::: ![](1471-2121-6-2-1) ::: To further characterize the observed heterotypy, we examined IEC-18 cells with anti-actin antibody in IEC-18 cells without performing antigen retrieval and allowing the detection reaction to proceed until only a limited amount of staining is seen. When used with the right antibody, this technique is a means to visualize dynamic portions of actin filaments, such as stress fibers and the proximal ends of microvilli, because they lack the actin binding proteins that obscure the antigen. If the reaction is allowed to continue, eventually all actin filaments will stain. In the version that we use, the assay is a qualitative assessment of cytoskeletal turnover when we are comparing two adjacent cells (because both cells had exactly the same conditions for detection of actin). Both stress fibers and microvilli are heavily stained and appear to have high turnover rates in cells that have C2 staining, but not in those without it (Figure [1B](#F1){ref-type="fig"}). We note that the punctate dots of filamentous-actin corresponded with small microvilli in C2 positive cells and that the C2 negative cells had a paucity of microvilli on their surface -- which was confirmed by focusing up and down the shafts of the microvilli (data not shown). IEC-18 cells spontaneously lose C2 staining prior to confluence, despite 10% FBS -------------------------------------------------------------------------------- When IEC-18 cells were plated at variable densities in 10% fetal bovine serum (FBS), we saw that all crypt cells were initially positive for C2 staining, but with increasing density there were increasing numbers of C2 negative cells (Figure [1C](#F1){ref-type="fig"}). We also saw that C2 positive cells had increased staining intensity as they approached confluence. This experiment demonstrates two important points: first, that loss of C2 staining begins before confluence and second, that it can occur with comparable prevalence in the presence of FBS as it does in serum free media (SFM). C2 positive cells and C2 negative cells are both capable of phenotype preservation during proliferation ------------------------------------------------------------------------------------------------------- To determine if the C2 positive and negative IEC-18 cells were each capable of phenotype preservation during proliferation, IGFBP-2 stained cells on coverslips were searched for mitotic cells in the stage of cytokinesis and their images were captured by digital photomicroscopy (n = 6 cell pairs for each phenotype). In each case, all daughter cells had the same phenotype as their sister, confirming that both the C2 positive and C2 negative phenotypes are conserved in subsequent cycles of mitosis (representative examples in Figure [1D](#F1){ref-type="fig"}). C2 positive cell abundance is increased in proportion to the efficacy of IGF agonist treatment ---------------------------------------------------------------------------------------------- Because IEC-18 cells grow best in the presence of high dose insulin, we suspected that crypt cell proliferation was dependent upon IGF receptor stimulation. In general, differentiated and benignly transformed epithelial cells are less likely to proliferate upon reaching confluence, so we sought to preferentially drive crypt cell proliferation in a graded fashion using different IGF receptor agonists (Figure [2](#F2){ref-type="fig"}). IGF-II analog, a weak agonist, reduced the crypt cell abundance while NBI 31772 (an agent that displaces IGFs from IGFBPs) increased it significantly and R^3^-IGF-I doubled the number of crypt cells per 10X field (also highly significant) while none of the treatments significantly altered the abundance of C2 negative cells. This strategy allowed us to systematically skew the cell composition and use gene array analysis to determine whether C2 positive cells were epigenetically divergent. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Mean number of C2 positive and C2 negative cells for each treatment condition. C2 positive cells are boxed in gray and C2 negative cells in stripes. Note that IGF-II analog reduces C2 positive cells when compared to SFM, whereas both NBI 31772 and R^3^-IGF-I significantly increase C2 positive and total cell abundance when compared to SFM (\*\*\* = p \< 0.01, \*\* = p \< 0.05, \* = p \< 0.1). In contrast, no treatment significantly altered C2 negative cell abundance. ::: ![](1471-2121-6-2-2) ::: Our gene array methodology identified four candidate genes as potential markers of cell fate divergence in IEC-18 culture ------------------------------------------------------------------------------------------------------------------------- Eleven genes met our criteria for a significant fold change, four were positively correlated with crypt cell abundance and seven were inversely correlated (Figure [3](#F3){ref-type="fig"}). Of these first pass candidates, only six showed the appropriate fold trends across all treatment conditions, consistent with our hypothesis of constitutive expression that could reflect divergent cell fates. Of these six, one was found to have a significant difference between IGF-II analog and SFM, suggesting a direct treatment effect by IGF agonists but not by NBI 31772 (dithiolethione-inducible gene 1). Enzymatic glycosylation-regulating gene is known to be an insulin responsive gene and was also excluded because of the high probability of a direct effect by our IGF agonists \[[@B6]\]. Of the four remaining candidates, one (brain acyl CoA hydrolase) had absolute values that bordered on background levels in the SFM, IGF-II analog and NBI 31772 treatment conditions (defined as 10 arbitrary fluorescent units) and has had a relatively limited characterization in the literature \[[@B7]-[@B9]\]. We saw no obvious means for obtaining or generating an antibody to it and thought it unlikely to be a robust marker at the protein level, in large part because its message has only been found in brain thus far. Another had high homology with the EGF family and is currently a predicted protein based on genomic sequence \[[@B10]\]. However, the two remaining candidate genes we pulled out were well-characterized gut-related proteins (APC and 5-HT2A). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Gene array screen using skewed IEC-18 cell composition to find potential phenotypic markers. Affymetrix rat gene chips were used to compare RNA from SFM and R^3^-IGF-I treated cells to find individual genes with significant fold changes -- defined as greater than two fold more (A) or two fold less (B) with a p-value of less than 0.1 for the purpose of this screen. These were then compared with the fold changes from IGF-II analog and NBI 31772 to look for fold change trends that paralleled the changing cell composition (summarized in C as the percentage of crypt cells). The results are presented as upward arrows (strong positive correlations), upward dashed arrows (weak positive correlations), downward arrows (strong inverse correlations) and downward dashed arrows (weak inverse correlations). The large asterisk points to an example where both direct receptor agonists resulted in a significant difference but NBI 31772 did not, suggesting a direct drug effect rather than an effect of cell composition -- making this gene a less likely candidate. For smaller asterisks, \*\*\* = p \< 0.01, \*\* = p \< 0.05, and \* = p \< 0.1. ::: ![](1471-2121-6-2-3) ::: Immunolocalization for 5-HT2A and APC revealed 2 divergent phenotypes within IEC-18 cell culture ------------------------------------------------------------------------------------------------ Western blots using antibodies against APC and 5-HT2A in IEC-18 cell lysates revealed staining of the appropriate sized band for each (Figure [4](#F4){ref-type="fig"}). In the case of APC, there were several discrete smaller bands which were inversely proportional in abundance to the 300 kD full-sized protein (consistent with proteolytic fragments) when compared across multiple samples (data not shown). In the case of 5-HT2A, there was a single 28 kD band that was faint, suggesting relatively low abundance. Both antibodies were deemed suitable for immunolocalization. Immunolocalization for APC revealed that C2 positive cells also had intense APC staining whereas C2 negative cells either had limited or no APC staining (Figure [5A](#F5){ref-type="fig"}). This finding is in keeping with our gene array analysis and suggests that there is substantial and wide spread divergence of IEC-18 crypt cells away from the crypt cell phenotype and towards an adenoma-like transformation. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Immunolocalization of APC and 5-HT2A in IEC-18 culture. **A**: APC immunostaining parallels that of C2 and is found in crypt cell strands but is absent or scant in adjacent cells. **B-D**: 5-HT2A immunostaining is absent in the majority of cells but is found in a few rare cells. On closer inspection, we found that there seemed to be a progressive increase in staining intensity that correlated with a morphologic transition away from IEC-18 cell morphology and towards that of a neuroendocrine-like cell (illustrated by the numbered circles). ::: ![](1471-2121-6-2-5) ::: Immunolocalization for 5-HT2A demonstrated a second cell phenotype (Figure [5B,C](#F5){ref-type="fig"} and [5D](#F5){ref-type="fig"}). We had previously noted rare neuroendocrine-like morphology characterized by spindle-shaped cells with bipolar, dendritic arbors (unpublished findings), however with 5-HT2A immunostaining we found a mean of 5 positively stained cells per coverslip of confluent cells (n = 6 coverslips of confluent cells in SFM). 5-HT2A is present in high abundance within paneth and neuroendocrine cell types but is absent within small intestine crypt cells *in vivo*\[[@B11]\]. The cells that we observed appeared to be in transition, going from IECs to neuroendocrine-like cell morphology -- with corresponding increases in staining intensity. While still a very low prevalence, the staining was quite intense in this small subset and was not detected in any of the adjacent cells. Double labeling confirms that C2 negative cells have diminished APC abundance ----------------------------------------------------------------------------- Double immunolabeling with overlay technique (using C2 and actin antibodies) demonstrated that C2 negative cells have a paucity of microvilli in comparison to C2 positive cells (Figure [6A](#F6){ref-type="fig"}). Additionally, double labeling with C2 and APC demonstrated that C2 positive cells have uniform APC staining whereas C2 negative cell have variable and overall diminished staining in comparison (Figure [6B](#F6){ref-type="fig"}). These experiments provide an objective demonstration that C2 positive cells retain the IEC phenotype whereas C2 negative cell are undergoing transformation (which is an obligatory step associated with the loss of APC). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Double labeling overlay immunolocalization of C2 with either actin or APC. **A*i***. C2 immunostaining of wet-prepped cells. **A*ii***. C2 immunostaining in the same cells, overlaid with f-actin immunostaining. The proximal cores of actin-stained microvilli bundles located on C2 positive cells are encircled, whereas there are either no or few microvilli on C2 negative cells. **B*i***. C2 immunostaining of wet-prepped cells. **Bii**. C2 immunostaining in the same cells, overlaid with APC immunostaining. The dotted line divides C2 positive cells (below) from C2 negative cells (above). There is consistent APC staining in C2 positive cells, whereas there is variable and comparably reduced APC staining in C2 negative cells. ::: ![](1471-2121-6-2-6) ::: Discussion ========== In this manuscript we have taken advantage of an IEC immunolocalization marker that we call C2 to demonstrate two forms of cell fate divergence within IEC-18 culture. Using a combination of gene array screening and immunolocalization, we found that C2 is lost in over half the cells by the time of confluence and its loss is also associated with a down regulation of the APC gene, decreased APC protein abundance, decreased actin filament turn over, and reduced microvillar density. In short, there is an adenoma-like phenotype that fits with this genotype and this fits well with what is known about the function of the APC protein \[[@B12]-[@B15]\]. In addition, another cell genotype-phenotype correlate was detected by screening out the 5-HT2A gene and visualizing the cells that express its protein, i.e. neuroendocrine-like cells. These findings also fit well with cell phenotypes known to express 5-HT2A in the gut endoderm \[[@B11],[@B16],[@B17]\] and strongly argue against the dogma that IECs persist as a single lineage prior to reaching confluence. While we think our findings have important implications for the existing IEC literature, the more important aspect of this manuscript may be in the methodology we have piloted. In many ways, cell culture, whether primary or immortalized, transformed or not, is by definition a model in flux. The progenitor lineage that a researcher starts with is rarely the hodge podge they end up with after a limited number of passages and it is common, if not expected that epigenetic drift will occur with each cell culture passage. However, what we describe in this manuscript is different in that IEC-18 cells are displaying a uniform trend in cell fate divergence -- a trend that can be modulated with IGF. Many if not most gut epithelial cell lines are IGF (or high dose insulin) dependent for proliferation and thus are potentially vulnerable to this biological confounder. It will remain to be seen if other epithelial cell types have similar behaviors when examined in this fashion. Conversely, there is also a positive light to our findings; IEC-18 cells could be a compelling model for spontaneous adenomatous transformation because these adenoma-like cells are arising from a genetically competent progenitor prior to reaching confluence. To our knowledge, no such model with this property has been previously defined. Our study has notable weaknesses and strengths. First, we have identified two divergent cell fates but have only partially characterized them because we were focused on developing a viable screening methodology (hence the ubiquitous use of the word \"like\" in this manuscript). Second, we are using a rat gene array chip that has approximately 9000 non-EST genes per chip. This is not an exhaustive survey of the rat genome and it is possible that there are other cell fates present in IEC-18 culture that we did not detect. Third, our phenotype assays are based on immunolocalization, which is a semi-quantitative technique with regards to assessing protein abundance. However, in this case we are actually combining cell-to-cell differences in protein abundance with distinguishing morphologic characteristics (e.g. loss of microvilli, flattening, bipolar shape, dendritic arbors, etc.) to delineate the phenotypes between adjacent cells. In short, what we are quantifying, in the case of adenoma-like cells, is the percentage of cells with a given phenotype. For this purpose, blinded immunolocalization is simple, quantitative and exceedingly efficient. The combined methodologies we chose result in a highly accurate technique for assessing divergence and their specificity can be bolstered by comparative studies of co-divergent markers (as we demonstrated with C2 and APC). As for other positives, the methodology is relatively rapid and can detect low prevalence phenotypes (as demonstrated by the anti-5-HT2A antibody). Additionally, we have demonstrated that a transcriptional marker is not required to create an effective screen. What is required, and what should probably prompt a researcher to employ this methodology, is a probe, a phenotype, or a pleiotropism that results in a consistently heterogeneous and quantifiable pattern (as C2 proved to be for us). In closing, we point out one last caveat. Gene array investigation is an evolving science but there remain three potential pitfalls for every new application: experimental design flaws, data integrity issues and biological misassumptions \[[@B18]-[@B20]\]. In this study, we used a well-accepted screening principle (i.e. significant fold changes within individual genes in response to a treatment); we included a paired reference standard for each treatment condition (the SFM control); we increased the screening stringency by adding a requirement for parity in fold trend in accordance with changing cell compositions; and then confirmed our findings by phenotype assays. However, we demonstrated that a small minority of neuroendocrine-like cells were still able to significantly alter the outcome of our gene array screen (a possibility that we had thought to be remote, given our assay\'s stringency). We conclude that even low frequency epigenetic events can be a serious biologic confounder of gene array studies in cell culture. Conclusions =========== We have demonstrated a novel methodology for detecting and characterizing cell fate divergence in cell cultures derived from a common progenitor. The majority of IEC-18 cells are transformed into adenoma-like cells in SFM. IGF agonists reduce the rate of transformation by driving proliferation of the progenitor phenotype but do not prevent it. We also detected a very low incidence of differentiation toward a neuroendocrine-like cell type in these same cultures. Methods ======= Cell culture ------------ Rat ileal epithelial cells (IEC-18 -- American Type Culture Collection, Rockville, MD) from aliquots of passage numbers 6--8 were grown to confluence in DMEM with 10% fetal bovine serum (FBS) and .1% insulin. The time period for complete epithelial cell confluence was 24--48 hours. Confluent cells were incubated for 72 hours in DMEM to establish a serum free period either alone or with 10^-6^M NBI 31772 (Calbiochem, San Diego, CA \[[@B21]\]), 0.5 × 10^-6^M IGF-II analog, or 0.5 × 10^-6^M R^3^-IGF-I (Sigma, St. Louis MO), as a means of boosting crypt cell proliferation. We point out that crypt cells can be driven to proliferate despite confluence whereas more differentiated epithelial cell types are resistant to IGF-driven proliferation upon reaching confluence. Alternatively, IEC-18 cells were diluted to 1/8, 1/4 and 1/2 the original seeding density and plated in DMEM with 10% FBS for 24 hours as a means to evaluate cell fate divergence in the presence of serum. Western blots ------------- Cell lysate samples were collected from 150 mm dishes, washed with PBS and recovered in 700 uL of lysis buffer by scraping the dish. Cells were spun down for 10 minutes in a pre-chilled centrifuge. The supernatant was diluted 4:1 with loading buffer and run on 12 or 15% acrylamide gels. After the gel had been transferred onto a nitrocellulose membrane, 0.5% Ponceau S stain was applied to confirm equitable protein transfer across all lanes. Western blot membranes were incubated in 5% milk block for 1 hour at RT. Goat polyclonal primary antibody for APC and 5-HT2A were applied for 1 hour at RT. Primary antibody was washed off with TBS-Tween and secondary antibody was placed on the membranes for one hour, then washed again. ABC (Elite Series, Vector Labs) was placed on the membrane for an hour and then washed and visualized with SuperSignal chemiluminescent substrate (Pierce, Rockford, IL), each was prepared per the manufacturer\'s instructions. Gene array analysis ------------------- IEC-18 cells were treated and processed as described in C*ell Culture*methods and then utilized to screen for specific cell fate markers. Total RNA was harvested for each treatment condition in triplicate experiments and then used for gene chip analysis per the manufacturer\'s protocol (rat gene chip \# 230A, Affymetrix, Santa Clara, CA). All available non-EST tags were searched within the R^3^-IGF-I data set and those with significantly different fold changes when compared to SFM cells were selected as potential candidates for further analysis (for the purpose of a first pass screen, a significant fold change was defined as a p value \< 0.1 and a greater than two fold increase or decrease). To further refine the list of potential candidates, the fold changes for NBI 31772 and IGF-II analog, which had step-wise reductions in effects upon crypt cell proliferation, were used to determine if there was a positive or inverse trend between a selected gene\'s mRNA abundance and crypt cell abundance. In this way, we sought to select constitutively expressed genes, whose primary differences in abundance would be due to differences in cell composition. Our statistical test for determining significance was a two-tailed paired t-test (provided as part of the standard analysis by the University of Virginia Biomolecular Research Facility and the Dept. of Health Evaluation Sciences \[[@B22]\]). Immunolocalization experiments ------------------------------ Immunolocalization in IEC-18 cells was performed for IGFBP-2 (C2), f-actin, IGF receptor type 2 and villin as well as two proteins that were screened out by our gene array analysis (APC and 5-HT2A) in a minimum of three separate experiments for each. In brief, the cells were fixed in formalin overnight, washed in DIG buffer (4% 1M Tris Base, 6% 5M NaCl, 16% 1M Tris HCl) 5 times then put in blocking solution (1% wt/vol BSA in DIG buffer) for 1 hour at room temperature. This was followed by a one-hour incubation with goat polyclonal primary antibody (all were obtained from Santa Cruz Biotechnology, CA; specifically for the anti-actin antibody the product number was sc-1615) in antibody diluent solution (5% 1M Tris HCl, 1% 1 M Tris Base, 9% NaCl, 3.3% Triton-X 100). Primary antibody was washed off with DIG × 5 and secondary antibody (donkey anti-goat \[Jackson Immunoresearch Labs, West Grove, PA\]) was placed on the slides for one hour. ABC (Elite Series, Vector Labs) was prepared and applied per the manufacturer\'s instructions, washed as above and visualized with DAB solution (Sigma, St. Louis, MO) in parallel. The slides were counterstained with hematoxylin, cover-slipped, photomicrographed and representative images were chosen for publication. With respect to the actin visualization, detection was performed such that colored precipitate was observed under the microscope and stopped when stress fibers and microvillus cores were evident and before the high background of the total cell f-actin began to stain efficiently. In the crypt cell quantification experiments, six 10X images were taken at random, printed on color paper at maximum size, blinded, and all cells were assessed as C2 positive or C2 negative and their means and standard deviations calculated for each treatment condition. C2 double labeling immunolocalization experiments ------------------------------------------------- To objectively test our observations that C2 positive cells have a distinctive colocalization pattern when compared to C2 negative cells, C2 double labeling experiments with f-actin and with APC were performed as overlay experiments using digital microscopy. In brief, C2 immunolocalization was performed as described above except that a grid was drawn on the wet mount slide, allowing digital photomicrographs to be taken at 40X of C2 staining while still covered with stop solution and then a second antibody, either for f-actin or APC was applied and the same process repeated before being counter-stained with hematoxylin and cover-slipped. The same exact images were then re-mapped using the grid and the stored digital images were used for precise position verification. The overlaid double-labeled image was then taken and contrasted to the original image to allow comparison of differential localization of the two antigens. Abbreviations ============= *IGF*, insulin-like growth factor; *IGFBP-2*, IGF binding protein-2; *IGF-II analog*, synthetic truncated IGF-II; *R*^3^-*IGF-I*, synthetic long arginine IGF-I; *NBI 31772*, an alpha-numeric designation for a non-biologic compound that best displaced IGFs from IGF binding proteins in a large bioassay screen; *SFM*, serum-free media; *APC*, adenomatous polyposis coli; *5-HT2A*, serotonin receptor 2A; *C2*, IGFBP-2 carboxyl fragment Authors\' contributions ======================= PG designed the study, participated in the immunolocalization studies, and performed data analyses. JP carried out the immunolocalization, cell count, and Western blot studies. NF maintained the IEC cells lines and expertly provided uniformly confluent cells. PG and JP produced the figures and drafted the manuscript. All authors read and approved the final manuscript. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Western blots of APC and 5-HT2A in IEC-18 cell lysates. Visualization of the appropriate sized band (and break down products) for APC is shown in lane A and 5-HT2A is shown in lane B. ::: ![](1471-2121-6-2-4) :::

PubMed Central

2024-06-05T03:55:52.431908

2005-1-18

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547914/", "journal": "BMC Cell Biol. 2005 Jan 18; 6:2", "authors": [ { "first": "Phillip V", "last": "Gordon" }, { "first": "Jessica B", "last": "Paxton" }, { "first": "Nena S", "last": "Fox" } ] }

PMC547915

Background ========== There is a large number of pharmacological strategies for treating serotonin-related disorders like depression and anxiety. Well-known antidepressant drug effects are blockade of the serotonin (5-HT) and/or norepinephrine (NE) reuptake pumps, direct effects on the 5-HT- and/or NE receptors and inhibition of the monoamine oxidase A (MAO-A) enzyme. Irrespective of which drug or combination of drugs that is used, there is a delay of a few weeks before any therapeutic effect is noticed. This initial lag-period usually is associated with several side-effects, many of which fade away with the appearance of the therapeutic effect. Considering the lag-period needed for the antidepressant effect to emerge, it has been suggested that the antidepressant mechanisms involve secondary molecular neuronal adaptations, rather than being a result of the primary actions of the drugs \[[@B1],[@B2]\]. Some attempts to elucidate the molecular mechanisms involved in such lag-period have been reported, for example both SSRIs and MAO-Is cause desensitization of somatodendritic 5-HT~1A~autoreceptors \[[@B3],[@B4]\]. Furthermore, SSRIs have also, during the initial lag period, been shown to cause desensitization of terminal 5-HT~1B/1D~autoreceptors \[[@B5],[@B6]\]. Gaining more knowledge about molecular mechanisms involved in the initial lag-period may prove important for the discovery of new antidepressant drug targets and also for the knowledge of the mechanisms of action of antidepressants. Transcription factors, with their specific ability to regulate gene expression, have been suggested as prominent novel drug targets \[[@B7]-[@B11]\]. Transcription factor AP-2 is a critical regulatory factor for neuronal gene expression and neuronal development, e.g., in the brainstem \[[@B12]-[@B14]\]. Five different AP-2 genes have been identified, i.e., AP-2α, AP-2β, AP-2γ, AP-2δ and AP-2ε \[[@B15]-[@B19]\]. The isoforms are expressed from different genes and have a molecular weight of around 50 kD. The cis-acting DNA sequences 5\'-(G/C)CCCA(G/C)(G/C)(G/C)-3\' and the palindromic sequence 5\'-GCCNNNGGC-3\' are considered as consensus AP-2 binding sites for all AP-2 proteins \[[@B20]\]. Several genes encoding proteins involved in the brainstem monoaminergic systems have multiple AP-2 binding sites in their regulatory regions \[[@B20]-[@B26]\] indicating an involvement of AP-2 in the expression of these genes. We have recently reported positive correlations between brainstem AP-2α and AP-2β levels and monoaminergic activity in rat frontal cortex \[[@B27]\], indicating a regulatory function of AP-2α and AP-2β not only for neuronal development, but also for neuronal adaptive mechanisms in the adult brain. In two independent studies it was shown that the AP-2β genotype is associated with anxiety-related personality traits \[[@B28],[@B29]\]. The AP-2β genotype has also been linked to binge-eating disorder \[[@B30]\] and to platelet MAO activity \[[@B31]\], which the latter is associated with personality traits. Furthermore, the AP-2β genotype has been associated to CSF-levels of homovanillic acid (HVA) in women \[[@B32]\]. In a previous study, we reported that the levels of AP-2α and AP-2β were decreased in rat whole brain after treatment for 10 days with citalopram, imipramine and lithium, respectively \[[@B33]\]. We have also reported that citalopram changes the levels of AP-2α and AP-2β in rat whole brain in a time-dependent manner, i.e., AP-2 levels were decreased after 7 days of treatment but returned to control levels after 21 days of treatment \[[@B34]\]. Furthermore, AP-2α and AP-2β levels were shown to be increased in rat whole brain after 10 days of treatment with phenelzine \[[@B35]\]. In the present study, we report that neither treatment with citalopram for 1, 7 nor 21 days affect the AP-2α and AP-2β levels in the rat brainstem. Treatment with phenelzine, however, increased the levels of both AP-2α and AP-2β in the rat brainstem after 7 days of treatment, but after 21 days of treatment the levels had returned to control levels. Results ======= The mean relative amounts of AP-2α and AP-2β protein ± standard deviation (SD) for each of the citalopram-, phenelzine- and saline treated animal groups are shown in tables [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}, respectively. No significant differences in the amounts of AP-2α and AP-2β were found between any of the citalopram and saline treated groups. With regard to phenelzine treatment, there was a significant increase in AP-2α levels after 7 days of treatment (2.74 ± 0.57 vs 2.16 ± 0.14: mean relative amount of AP-2α ± SD, phenelzine vs saline, p = 0.037), which returned to control levels after 21 days. A similar result was obtained with regard to AP-2β levels with a significant increase only after 7 days of treatment (2.25 ± 0.25 vs 1.86 ± 0.22: mean relative amount of AP-2β ± SD, phenelzine vs saline, p = 0.017). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Relative amount of AP-2α protein ± SD, for the different animal treatment groups. ::: **day 1** **day 7** **day 21** ---------------- ------------- ---------------- ------------- **saline** 2.29 ± 0.43 2.16 ± 0.14 2.45 ± 0.33 **phenelzine** 2.28 ± 0.61 2.74 ± 0.57 \* 2.54 ± 0.43 **citalopram** 2.35 ± 0.31 2.33 ± 0.49 2.49 ± 0.47 Values are means ± SD, for each group of animals, n = 6. \*p \< 0.05 as compared to animals treated with saline for the same time-period. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Relative amount of AP-2β protein ± SD, for the different animal treatment groups. ::: **day 1** **day 7** **day 21** ---------------- ------------- ---------------- ------------- **saline** 2.00 ± 0.44 1.86 ± 0.22 2.10 ± 0.14 **phenelzine** 2.14 ± 0.61 2.25 ± 0.25 \* 2.00 ± 0.28 **citalopram** 2.16 ± 0.34 2.07 ± 0.47 2.05 ± 0.28 Values are means ± SD, for each group of animals, n = 6. \*p \< 0.05 as compared to animals treated with saline for the same time-period. ::: When comparing the untreated naive animals with the treated animal groups no differences with regard to the levels of AP-2α (untreated animals: 2.49 ± 0.71, mean relative amount AP-2α ± SD) or AP-2β (untreated animals: 2.04 ± 0.64, mean relative amount AP-2β ± SD) were found. Moreover, no differences in the levels of AP-2α and AP-2β were observed between the groups of animals treated with saline for the different time periods. Discussion ========== In two independent studies, we have shown that the levels of AP-2α and AP-2β in rat whole brain were decreased after subchronic (7 or 10 days) treatment with citalopram \[[@B33],[@B34]\]. We have also shown that the AP-2α and AP-2β levels in rat whole brain were increased after treatment with phenelzine for 10 days \[[@B35]\]. For several reasons, we hypothesized that the brainstem should be of particular importance in this regard. Thus, many genes encoding proteins involved in the brainstem monoaminergic systems have binding sites for AP-2 in their regulatory regions. Furthermore, we have previously observed correlations between brainstem AP-2 levels and cortical monoamine activity \[[@B27]\]. The lag-period initially seen before the antidepressant therapeutic effect is obtained also made it interesting to study possible changes in AP-2 levels over time. In the present study, we found that treatment with citalopram for 1, 7 or 21 days did not have any effect on the brainstem levels of AP-2α and AP-2β. Treatment with phenelzine for 7 days, on the other hand, increased the brainstem levels of AP-2α and AP-2β, but after 21 days of treatment the levels had returned to control levels. The phenelzine data presented here, are in line with our previous study showing an increase in AP-2α and AP-2β levels in rat whole brain after 10 days of treatment with phenelzine \[[@B35]\]. The different responses on AP-2 of citalopram and phenelzine are likely to be explained by the different molecular mechanisms of the two drugs. Considerering the specific drug targets for citalopram and phenelzine, respectively, the target for citalopram, 5-HTT, is specific for membranes of the serotonergic system, while the target for phenelzine, MAO-A, is not located in the serononergic neurons \[[@B36]\]. It has been shown that some SSRIs, to some extent, are also able to inhibit MAO-activity \[[@B37]\]. However, all SSRIs, including citalopram, tested had a much higher selectivity for MAO-B than MAO-A and MAO-A, and not MAO-B, is the enzyme considered to be involved in the antidepressant effect \[[@B38]\]. Thus, a MAO-inhibiting effect of citalopram should not be a confounding factor with regard to interpretation of the present results. The fact that citalopram treatment did not affect AP-2 levels in rat brainstem indicates that the decrease in AP-2 levels previously seen in rat whole brain after citalopram treatment, takes place in some other brain region. It has been shown that postsynaptic 5-HT~1A~receptors in hippocampus get an enhanced response to 5-HT after treatment with TCA (that blocks both 5-HT and NE reuptake) for a time-period that corresponds to the time required for initiation of the antidepressant therapeutic effect \[[@B39]\]. An increased 5-HT responsiveness has also been demonstrated for other serotonin receptors than 5-HT~1A~and in other projection areas than hippocampus \[[@B40]\]. Thus, there are reasons to presume that the effect we have seen on AP-2 levels after subchronic citalopram treatment occurs in the 5-HT projection areas rather than in the 5-HT cell bodies in the brainstem. With regard to the increase in brainstem levels of AP-2 after 7 days of phenelzine treatment, it is in line with previous reports that MAO-Is partially enhance the 5-HT transmission by increasing the amount of 5-HT released per action potential \[[@B38]\], an effect which is likely to be regulated by presynaptic mechanisms located in the brainstem. The temporary changes in AP-2 levels after administration of antidepressants (present data and \[[@B34]\]), coinciding in time with the appearance of side-effects, makes it tempting to speculate that those two phenomena are somehow interrelated. In a previous study, we reported higher levels of AP-2α and AP-2β in rat whole brain in untreated naive animals compared to animals treated with citalopram or saline for different time periods \[[@B34]\]. In the present study, however, we did not see any differences in the brainstem levels of AP-2α and AP-2β between naive untreated animals and treated animal groups. This indicates that the changes in AP-2α and AP-2β levels in rat whole brain previously seen in animals treated with citalopram or saline compared to naive untreated animals are located in some other AP-2 containing brain region than the brainstem. As mentioned earlier, we have previously shown that the AP-2β genotype is associated with platelet MAO activity \[[@B31]\]. Thus, it is seems likely that AP-2 is involved in the regulation of the expression of the MAO enzyme. A possible explanation for the elevated AP-2α and AP-2β levels during subchronic phenelzine treatment could be that they are part of a feedback mechanism to counteract the reduction in MAO activity. Conclusions =========== Unraveling of the molecular mechanisms involved in the initial phase of antidepressant treatment is essential for the development of new efficient antidepressant drugs with less side-effects. We find transcription factors, such as AP-2, with ability to regulate expression of specific genes involved in the monoaminergic mechanisms, to be interesting candidates as novel antidepressant drug targets. Methods ======= Animals and treatment paradigms ------------------------------- Adult male Sprague-Dawley rats (10 weeks of age, B&K Universal AB, Sollentuna, Sweden) were housed in groups of three and maintained on a 12 hour light /dark cycle with food and water freely available. Animals were administered phenelzine (n = 18, 10 mg/kg, Sigma, Sweden), or citalopram (n = 18, 10 mg/kg, Lundbeck AB, Helsingborg, Sweden) subcutaneously with daily injections. All drugs were dissolved in saline (NaCl, 9 mg/kg). Saline treated animals (n = 18) received saline injections in the same volume as that given the citalopram and phenelzine treated animals. Each group of animals was treated for 1, 7 or 21 days, respectively. All animals were sacrificed by CO~2~inhalation 24 hours after their last injection. A group of untreated naive animals (n = 6) was sacrificed after 21 days. After sacrifice the brainstem was dissected and nuclear extracts were prepared for measurement of AP-2 levels by Enzyme-Linked Immunosorbent Assay(ELISA). This study was carried out with permission from the local animal ethics committee in Uppsala, Sweden. Extraction of nuclear extracts ------------------------------ Rat brainstem was homogenized in 3 ml buffer A (10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, 0.5 mM PMSF, pH 7.9). The homogenate was incubated on ice for 15 minutes. To this 125 μl Nonidet P40 was added, and the homogenate was centrifuged for 30 seconds at 14000 rpm in 4°C. The pellet was resuspended in 500 μl buffer C 20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF, pH 7.9). Thereafter the tubes were put on a shaker for 15 minutes and centrifuged at 14000 rpm for 5 minutes (4°C). The supernatant i.e. the nuclear protein were aliquoted and stored at -80°C. The protein concentration for all nuclear extracts were determined by the method by Lowry at al. (1951) \[[@B41]\]. The concentration of nuclear extracts were \~8 μg/μl. ELISA measurements ------------------ 96-well microtiter plates were coated (50 μl/ well) with nuclear extracts (10 μg/ml) diluted in 50 mM Carbonate-Bicarbonate buffer pH 9.0. The plates were covered with parafilm and incubated overnight at 4°C. Antigen solution was then removed and 200 μl blocking buffer (PBS, 1 % BSA) was added to each well and the plates were incubated for two hours in room temperature. Following this the blocking buffer was removed and the plates were washed with PBS. Primary antibody (50 μl, goat polyclonal AP-2α and AP-2β, 15 μg/ml respectively, SDS Biosciences, Falkenberg, Sweden) diluted in blocking buffer was then added and the plates incubated overnight at 4°C. After incubation the antibody was removed and the plates were washed three times with Wash buffer I (PBS, 0.05 % Tween-20). Secondary antibody (Donkey anti-goat IgG AP conjugated, SDS Biosciences, Falkenberg, Sweden) diluted 1:350 in blocking buffer, was then added (50 μl) to each well and the plates were incubated for two hours in room temperature. After removal of the secondary antibody the plates were washed three times with Wash buffer I, and once with Wash buffer II (10 mM diethanolamine, 0.5 mM MgCl~2~, pH 9.5). Thereafter, 50 μl substrate (Phosphatase substrate, 5 mg tablets, Sigma, Sweden, diluted in 5 ml Wash buffer II) was added to each well. The reaction continued for 30 minutes and was terminated by adding 50 μl of 0.1 M EDTA, pH 7.5. The plates were analysed in an ELISA reader (Molecular Devices, Thermo Max) at optical density (OD) 405/490. The OD of the AP-2 isoforms for each rat was correlated to a value in a standard curve, where known concentrations of antibody were plotted against optical density. The value form the standard curve was then divided with the concentration of total protein in the nuclear extracts. The quota was used as a value of the relative amount of AP-2α and AP-2β protein. Each rat were analysed twice for accuracy. Statistical analyses -------------------- The statistical comparisons between drug treated and saline treated animals for each time-point were analysed using unpaired t-test. When comparing the groups of untreated animals with all treatment groups we used analysis of variance (ANOVA) and Fisher\'s Protected Least Significant Difference (PLSD). To test if any of the groups of saline treated animals differed in the amounts of AP-2α and AP-2β protein ANOVA and PLSD test were used. All calculations were performed using Stat View 5.0 software (SAS Institute Inc., Cary, NC, USA). Results have been considered statistically significant when p \< 0.05. Authors\' contributions ======================= CB planned the experiments, carried out the molecular and statistical analyses and drafted the manuscript, MD participated in planning the experiments and writing of the manuscript, LO conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Acknowledgements ================ This study was supported by grants from the Swedish Medical Research Council (\#4145), the Swedish Brain Foundation, the AFA foundation, Svenska Lundbecksstiftelsen and Organon.

PubMed Central

2024-06-05T03:55:52.434649

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547915/", "journal": "BMC Pharmacol. 2005 Jan 21; 5:1", "authors": [ { "first": "Cecilia", "last": "Berggard" }, { "first": "Mattias", "last": "Damberg" }, { "first": "Lars", "last": "Oreland" } ] }

PMC547916

Background ========== Brain arteriovenous malformations (AVM) represent a relatively infrequent but devastating source of neurological morbidity in relatively young adults \[[@B1]\]. Prevention of new or recurrent intracranial hemorrhage (ICH) is the primary rationale for treating AVMs. The optimal management of AVMs is not well defined, and the risk of aggressive surgical therapy can be significantly high. There is a subset of AVM patients that are considered to be inoperable due to the location and size of their lesions \[[@B2]\]. To date, there is no clinically available pharmacological treatment of inoperable AVMs to decrease the rate of spontaneous intracranial hemorrhage. Matrix metalloproteinases (MMPs), a family of proteolytic enzymes, degrade extracellular matrix proteins, cell surface molecules, and other peri-cellular substances \[[@B3]\]. Excessive degradation of the vascular matrix by MMPs may result in the destabilization of the blood vessel that potentially leads to weakening of the vessel wall, passive dilatation, and rupture \[[@B4]\]. Previously, we reported increased levels of MMP-9 activity in AVMs that may result in vascular instability associated with growth and bleeding. There is an increasing interest in utilizing MMP inhibitors in treating vascular diseases including abdominal aortic aneurysms. Doxycycline is a clinically available antibiotic agent that possesses non-specific inhibitory effects on various MMPs, and for years it has had a well-established safety record in treating infectious diseases. The primary aim of this study is to demonstrate the feasibility of utilizing doxycycline as an MMP inhibitor to decrease MMP-9 activity in AVMs and potentially decrease the rate of spontaneous hemorrhage. This exploratory investigation supports the concept that further studies be conducted to document the ability of tetracycline and its derivatives to decrease MMP-9 levels in AVM nidal tissue. Such a demonstration could provide a firm rationale for proceeding with clinical trials to test the hypothesis that tetracycline and its derivatives are useful to decrease the rate of spontaneous hemorrhage from AVMs in otherwise untreatable patients or in those awaiting interventional treatment. First, we demonstrate that doxycycline can decrease MMP-9 activity in cultured AVM tissues as a proof of the concept. Further, we present results from a pilot clinical study demonstrating effects of oral doxycycline treatment on MMP-9 expression in AVM tissues. Methods ======= Ex-vivo treatment of cultured AVM tissues ----------------------------------------- After institutional review and informed consent, we obtained AVM specimens after microsurgical resection. AVM nidus was dissected away from any adjacent brain tissue in the operating room and a representative portion of nidus tissue was used for ex-vivo treatment. Ex-vivo culture of AVM tissue was performed using previously described method with modifications \[[@B5]\]. AVM tissues were minced into 1--2 mm fragments and vigorously washed with phosphate buffered saline (PBS). Blood cells dissociated from the tissues were removed by filtration. AVM tissue fragments were placed onto cell culture inserts and immersed in cell culture medium. During the first 24 hours, AVM tissues were incubated in DME H-21 medium containing 10% fetal bovine serum (FBS) to aid in tissue recovery. Following this recovery period, tissue debris and dissociated cells were removed, and AVM tissues were incubated in DME H-21 medium containing 1% FBS for 4 hours. The tissue fragments were equally divided into 12--24 wells. Then AVM tissues were incubated in the medium containing PBS (control) or doxycycline (1, 5, 10 and 100 μg/ml). Each group included 3--5 wells. Size of the tissues dictated a number of treatment groups in each specimen. Medium from each well was collected after 48 hours. Active and total MMP-9 were measured using substrate zymography. Medium was mixed with SDS sample buffer (Invitrogen, Carlsbad, CA, U.S.A.) and separated under non-reducing conditions in a 10% zymogram gel (Invitrogen) containing 0.1% gelatin incorporated as a substrate. Recombinant MMP-2 and MMP-9 proteins (R&D systems) were used as positive controls. After running, the gel was incubated with renaturing buffer (Invitrogen). The gel was then incubated with developing buffer (Invitrogen) overnight at 37°C. The gel was then stained with colloidal blue stain (Invitrogen). Proteolytic bands in the zymogram gels were quantified using Image J Software (NIH). Tissue viability was assessed by measuring the amount of LDH (lactate dehydrogenase) released in the medium according to the manufacturer\'s instructions (Roche, Penzburg, Germany). Some of the tissue fragments were embedded in paraffin for histological assessment. Pilot clinical study -------------------- Fourteen AVM patients received either 100 mg of doxycycline or placebo twice a day for one week, prior to elective AVM resection. During the AVM resection, AVM tissues were collected, and nidus tissues were frozen in liquid nitrogen. Frozen tissues were stored at -80°C until analysis. Clinical data were collected as previously described \[[@B6]\]. The specimens were homogenized and insoluble materials were removed by centrifugation at 3000 rpm for 5 minutes. We used total MMP-9 ELISA kit (R&D) and active MMP-9 ELISA kit (Amersham). Statistical analysis -------------------- Data are presented as mean ± standard deviation. The data for total MMP-9 and active MMP-9 from ex-vivo AVM tissues are presented as a relative expression with control brain samples as 100%. We used ANOVA for comparison, and statistical significance was taken at *P*\< .05. Results ======= Ex-vivo treatment of cultured AVM tissues ----------------------------------------- We collected two AVM tissues for ex-vivo treatment with doxycycline. These patients were not enrolled in the pilot clinical study. AVM-I ----- AVM tissue-I was collected from a 28 y.o. patient with a 29 mm AVM (Spetzler-Martin score 3). The patient had an AVM hemorrhage 236 days prior to the AVM resection. The patient did not receive embolization treatment or radiosurgery. AVM tissue-I was divided into 9 wells and treated with 0, 10, and 100 ng/ml of doxycycline (3 wells for each). Two doxycycline doses were selected to test (1) whether an average doxycycline concentration (10 ng/ml) achieved by standard clinical treatment has any effects on MMP-9 in AVM tissue, and (2) whether a high concentration of doxycycline (100 ng/ml) has any effects of viability of AVM tissues in vitro. There was a gradual increase in LDH release over the course of 8 days, indicating continuous cellular death and a gradual decrease in tissue viability (Figure [1](#F1){ref-type="fig"}). However, there was no difference among different treatment groups. To avoid effects from ongoing cellular death and to ensure tissue viability during the treatment, we chose the first 48 hours to assess the effects of doxycycline on MMP-9 levels in AVM tissues. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Tissue viability assessed by LDH (lactate dehydrogenase) release.**There was a gradual increase in LDH release (mean ± SD) over the course of 8 days, indicating continuous cellular death and a gradual decrease in tissue viability. However, there was no difference among different treatment groups. ::: ![](1471-2377-5-1-1) ::: Tissue integrity was further assessed by examining H&E staining of ex-vivo cultured tissues (Figure [2](#F2){ref-type="fig"}). On day 0 (before the treatment), although cutting and mincing of the AVM tissues had caused minor tissue injury and distortion, a majority of blood vessels were intact and viable. On day 2 (after 48 hours of treatment), blood vessels were still intact and viable, and there was no apparent necrosis or hyalinization of the tissues. However, on day 7, a major part of the tissues, especially tissues surrounding blood vessels were anuclear and hyalinized, indicating that tissues were not intact or viable anymore. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **H&E staining of ex-vivo cultured AVM tissues.**On day 0 (before the treatment), although cutting and mincing of the AVM tissues appeared to cause minor tissue injury and distortion, a majority of blood vessels was intact and viable. On day 2 (after 48 hours of treatment), blood vessels were still intact and viable, and there was no apparent necrosis or hyalinization of the tissues. However, on day 7, a major part of the tissues, especially tissues surrounding blood vessels were anuclear and hyalinized, indicating that tissues were not intact or viable anymore. ::: ![](1471-2377-5-1-2) ::: Doxycycline 10 μg/ml and 100 μg/ml significantly decreased active MMP-9 (control vs doxycycline 10 μg/ml: 100 ± 8 vs 48 ± 16%-control, P \< .05; control vs doxycycline 100 μg/ml: 100 ± 8 vs 59 ± 10%-control, P \< .05) (Figure [4A](#F4){ref-type="fig"}). In addition, there was a significant reduction of total MMP-9 by doxycycline at 10 and 100 μg/ml (control vs doxycycline 10 μg/ml: 100 ± 6 vs 60 ± 16%-control, P \< .05; control vs doxycycline 100 μg/ml: 100 ± 6 vs 61 ± 9%-control, P \< .05). (Figure [4B](#F4){ref-type="fig"}) There was no difference in active MMP-9 and total MMP-9 between doxycycline at 10 μg/ml and 100 μg/ml. A representative zymogram is shown in Figure [3](#F3){ref-type="fig"}. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Representative zymogram showing MMP-9 and MMP-2 standard, control AVM tissues, AVM tissues treated with 10 μg/ml doxycycline, and AVM tissues treated with 100 μg/ml doxycycline.**In AVM tissues, there were proteolytic bands corresponding to pro-MMP-9 (≈97 kDa) and active-MMP-9 (≈88 kDa). ::: ![](1471-2377-5-1-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Active and total MMP-9 levels after 48 hours of ex-vivo doxycycline treatment.AVM-I:**Doxycycline 10 μg/ml and 100 μg/ml significantly decreased active MMP-9 There was a significant reduction of total MMP-9 by doxycycline at 10 and 100 μg/ml There was no difference in active MMP-9 and total MMP-9 between doxycycline at 10 μg/ml and 100 μg/ml. **AVM-II:**Doxycycline 10 μg/ml significantly decreased active and total MMP-9. There was a trend for doxycycline 1 and 5 μg/ml to decrease active and total MMP-9. (mean ± SD) ::: ![](1471-2377-5-1-4) ::: AVM-II ------ AVM tissue-II was collected from a 63 y.o. patient with a 22 mm AVM (Spetzler-Martin score 2). The patients had a history of AVM hemorrhage 167 days prior to the AVM resection. The patient received an embolization treatment 1 day before the AVM resection. The patient did not receive radiosurgery. Using this tissue, we aimed to study effects of doxycycline at more clinically relevant concentrations of doxycycline (1--10 μg/ml). Therefore, AVM tissue-II was treated with 0, 1, 5, and 10 μg/ml of doxycycline for 48 hours. Since the results from AVM tissue-I showed a relatively wide variation in MMP-9 levels in each well among the same treatment group, we increased wells per treatment group from 3 to 4. There was no difference in tissue viability indicated by LDH release among different treatment groups at 48 hours (data not shown). Similar to the AVM-I, doxycycline 10 μg/ml significantly decreased active and total MMP-9 (active MMP-9: control vs doxycycline 10 μg/ml: 100 ± 54 vs 43 ± 15%-control, P \< .05; total MMP-9: control vs doxycycline 10 μg/ml: 100 ± 41 vs 59 ± 21%-control, P \< .05) (Figure [4C](#F4){ref-type="fig"} &[4D](#F4){ref-type="fig"}). In addition, there was a trend for doxycycline 1 and 5 μg/ml to decrease active and total MMP-9. Pilot clinical trial -------------------- 14 AVM patients were enrolled. The trial was originally started as a double blinded randomized trial, and 9 patients were randomized. In an effort to improve recruitment for this feasibility study, we converted to an open label drug design. 10 patients received doxycycline, and 4 patients received placebo. Clinical data are shown in Table [1](#T1){ref-type="table"}. Subjects were informed regarding possible side effects including gastrointestinal symptoms, cutaneous photosensitivity, skin pigmentation, teeth discoloration, and vestibular side effects. To monitor compliance and possible side effects, study subjects were interviewed by phone three times during one-week treatment period. One patient had nausea that was relieved by taking food with the drug. Other side effects were not noted. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of AVM patients enrolled in a pilot clinical study to test effects of oral doxycycline treatment on MMP-9 in AVM lesions. ::: Group Age Gender S-M score\* AVM size\*\* Draining veins History of hemorrhage Number of embolization treatments --------- ----- -------- ------------- -------------- -------------------- ----------------------- ----------------------------------- Dox 50 M 4 38 Superficial & Deep No 1 Dox 48 F 4 30 Deep Yes 0 Dox 52 M 4 42 Superficial & Deep Yes 2 Dox 43 M 3 29 Superficial & Deep No 1 Dox 23 M 4 34 Superficial & Deep No 2 Dox 28 M 4 40 Superficial Yes 2 Dox 56 M 2 10 Superficial No Dox 41 F 3 30 Superficial No 1 Dox 62 M 2 41 Superficial No 1 Dox 42 M 2 2 Superficial Yes 0 Placebo 53 M 2 28 Superficial & Deep No 1 Placebo 17 M 1 18 Superficial No 1 Placebo 48 F 2 13 Deep No 0 Placebo 22 M 3 19 Deep Yes 0 \* Spetzler-Martin score (15), \*\*AVM maximum diameter (10). Dox = doxycycline ::: There was a trend for both total MMP-9 and active MMP-9 levels to be lower in the doxycycline group than in the placebo group (total MMP-9: 2.18 ± 1.94 vs 3.26 ± 3.58 ng/100 μg protein, P = .50; active MMP-9: 0.48 ± 0.48 vs 0.95 ± 1.01 ng/100 μg protein, P = .25) (Figure [5](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Effects of oral doxycycline treatment on MMP-9 in AVM tissues.**There was a trend for both total MMP-9 and active MMP-9 levels to be lower in the doxycycline group than in the placebo group. (mean ± SD) ::: ![](1471-2377-5-1-5) ::: Discussion ========== In this study, we demonstrated the feasibility of doxycycline, a tetracycline derivative, to decrease MMP-9 activity in AVM tissues. First, we demonstrated that a clinically relevant concentration of doxycycline decreased MMP-9 without affecting tissue viability in ex-vivo AVM tissues. Second, there was a trend that oral administration of doxycycline for as short as one week in AVM patients resulted in a decrease in MMP-9 at the target site -- in the AVM nidus. By decreasing MMP-9 activity in AVM tissues, doxycycline may be able to restore the structural stability of AVM blood vessels and modify the clinical course of AVMs. Our data will provide the basis to conduct a clinical study to assess effects of tetracycline derivative treatment on the prevention of hemorrhage from AVMs. MMP-9 is a major enzyme that degrades the vascular extracellular matrix and has been implicated in a number of vascular diseases that involve abnormal angiogenesis and vascular remodeling \[[@B7]\]. MMPs are reported to be increased in cerebral aneurysms, atherosclerotic carotid plaque, and abdominal aortic aneurysm \[[@B8]-[@B10]\]. MMPs are emerging as a potentially new therapeutic target to treat vascular diseases. It has been proposed that pharmacological inhibition of MMPs may stabilize the unstable blood vessels and prevent complications such as vessel rupture \[[@B7]\]. In patients with abdominal aortic aneurysm, doxycycline treatment for one week prior to the repair surgery resulted in decreased MMP-9 and MMP-2 in the wall of the aneurysms \[[@B10]\]. Similar results have been reported in patients with atherosclerotic carotid plaques who received doxycycline for 2--8 weeks \[[@B11]\]. Our data using ex-vivo AVM tissues showed that abnormally high levels of MMP-9 expression in AVM tissues could be reduced by a clinically relevant concentration of doxycycline (i.e. 1--10 μg/ml). In patients with abdominal aortic aneurysm, doxycycline treatment at a conventional dose, 200 mg/day, resulted in a mean plasma concentration of doxycycline at 4.6 μg/ml with a range of 1.3 to 14.4 μg/ml in humans, and there was a corresponding reduction in plasma MMP-9 levels \[[@B12]\]. Similar plasma levels of doxycycline in mice successfully inhibited growth of experimental abdominal aortic aneurysms \[[@B13]\]. We were able to show that doxycycline at 10 μg/ml for as short as 48 hours can reduce MMP-9 expression in AVM tissues. A longer duration of treatment often used in clinical settings may result in a more pronounced reduction in MMP-9 expression in AVM tissues. One of the limitations of the ex-vivo study was the relatively short duration of doxycycline treatment. We chose 48 hours treatment to ensure tissue viability and integrity. Although we chose the doxycycline concentrations based on plasma doxycycline levels achieved by a commonly used therapeutic regimen, it is possible that tissue levels of doxycycline might be significantly lower than those of plasma. Furthermore, functional consequences of the reduction of MMP-9 in AVMs need to be examined in animal models or clinical studies. However, at the present time, there is no animal model that approximates recurrent intracranial hemorrhage from AVMs. There are models that can mimic certain aspects of the AVM phenotype. Hyperstimulation of mouse brain with VEGF using adenoviral transduction causes an increase in capillary density, increased MMP-9 activity, and may, in the appropriate genetic background, result in small vascular malformations \[[@B14]\]. Doxycycline can reduce capillary density and MMP-9 activity in this model \[[@B15]\]. Doxycycline has been shown in other human vascular diseases to reduce MMP levels. For example, Axisa et al. treated patients undergoing carotid endarterectomy with doxycycline or placebo for 2--8 weeks. Although MMP-1, MMP-3, and MMP-9 were reported to be increased, only MMP-1 had a statistically significant reduction by doxycycline; there was a trend for MMP-9 to be decreased by 22%. Baxter et al. used a much longer treatment period, 6 months, in patients with abdominal aortic aneurysm. They reported a gradual reduction of plasma MMP-9 levels by oral doxycycline over 6 month treatment period (baseline vs 3 months vs 6 months: 119 ± 38 vs 84 ± 33 vs 66 ± 24 ng/ml) \[[@B12]\]. To further explore the feasibility of a clinical application of doxycycline in modifying the clinical course of AVMs, we conducted a pilot clinical trial. This trial was primarily designed to test the effects of a short-term treatment with oral doxycycline on the expression of MMP-9 in AVM tissues. Despite the small sample size and short duration of treatment used in this study, there was a clear trend for one week of oral doxycycline treatment prior to surgical resection to reduce expression of both active and total MMP-9 in AVM tissues. These results indicate the feasibility of oral treatment with tetracycline derivatives in reducing MMP-9 activity in AVM tissues. Despite a large difference in the mean values of MMP levels in our pilot data, variance of MMP levels was high. Accordingly, a sample size of 46 in each group would be needed to demonstrate a difference in active MMP-9, assuming an alpha of 0.05 and power of 80%. However, variance may be reduced by utilizing a longer duration of treatment, as suggested by studies in other diseases \[[@B11],[@B12]\]. A clinical study with a modestly larger sample size and longer treatment duration should suffice to verify effects of oral doxycycline on reducing MMP-9 activity in AVMs. There are some further considerations that may account for our high variance that could be avoided in future studies. Surgical AVM tissues consist mainly from nidal tissues, but they may contain intervening astroglial or neuronal tissues that may not express angiogenic factors to the same extent as nidal tissue. Histological examination of each AVM tissue fragments to confirm a predominance of nidal tissues may yield more homogenous tissue and decrease variability in MMP-9 levels. Some of the homogenized samples underwent a several \"thaw-freeze\" processes during the preliminary phase of the experiments, which may have increased variability in MMP-9 levels. Embolization treatment may affect production and activation of MMPs. Our previous study, however, showed no association between MMP-9 and embolization treatment in 37 AVM patients \[[@B16]\]. Based on the experience from this pilot study, future studies can be performed in a more standardized fashion to decrease tissue-to-tissue variability. We used different methods to measure MMP-9 in the medium collected from ex-vivo AVM tissues and homogenized AVM tissues from the pilot clinical trial. We chose zymography, a gel based method detecting enzymatic activity of MMPs, for ex-vivo AVM tissues, because the medium volume that can be used for MMP-9 assay was extremely limited, and our preliminary experiments showed higher sensitivity of zymography compared to ELISA. One of the disadvantages of using zymography is that \"gel-to-gel\" variability in sensitivity makes it difficult to combine the data obtained from multiple gels. On the other hand, ELISA method allows researchers to assay approximately 40 samples at a time when duplicates are used. In addition, establishing a standard curve, ELISA method can express MMP-9 expression values by the absolute unit such as μg/ml or ng/100 μg protein. Therefore, for the clinical pilot trial, in order to quantitatively compare MMP-9 expression among a large number of samples originally planned, we used ELISA to MMP-9 expression. Conclusions =========== In summary, results suggest that doxycycline may have a therapeutic potential to reduce MMP-9 activity in AVM tissues and stabilize blood vessels that are prone to rupture. Our findings may provide a basis for a larger clinical trial to study effects of tetracycline derivatives in preventing AVM hemorrhage. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= TH, a corresponding author, participated in all aspects of the study and manuscript preparation. MMM participated in study design, experiments, analysis, interpretation of data, and manuscript preparation. JFL participated in experiments, analysis and interpretation of data. MTL participated in study design, analysis, interpretation of data, and manuscript preparation. WLY participated in study design, analysis, interpretation of data, and manuscript preparation. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2377/5/1/prepub> Acknowledgments =============== The authors wish to thank Broderick Belenson and Carroll Schreibman for assistance in preparation of the manuscript; Nancy Quinnine, RN and Manju Chopra, MD, for technical assistance; and the members of Center for Cerebrovascular Research and the UCSF AVM Study Project for their continued support. Portions of this work were supported by NIH grants RO1-27713 and K24-NS02091 (WLY), P01-NS44155 (WLY, TH), and American Heart Association Beginning Grant-in-Aid (TH).

PubMed Central

2024-06-05T03:55:52.436360

2005-1-24

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547916/", "journal": "BMC Neurol. 2005 Jan 24; 5:1", "authors": [ { "first": "Tomoki", "last": "Hashimoto" }, { "first": "Melissa M", "last": "Matsumoto" }, { "first": "Jenny F", "last": "Li" }, { "first": "Michael T", "last": "Lawton" }, { "first": "William L", "last": "Young" } ] }

PMC547917

Background ========== Stress can be described as the sum total of all the reactions of the body, which disturb the normal physiological condition and result in a state of threatened homeostasis. Stress is an internationally recognized phenomenon fortified by advancement of industrialization in a demanding civilization. Thus, every individual is likely to face stressful situations in day-to-day life. Stress represents a reaction of the body to a stimulus that tends to alter its normal physiological equilibrium or homeostasis and has been defined as a nonspecific response of the body to any demand imposed on it \[[@B1]\]. Since the introduction of adaptogens, several plants have been investigated, which were once used as tonics due to their adaptogenic and rejuvenating properties in traditional medicine \[[@B2]\]. The drugs of plant origin are gaining increasing popularity and are being investigated for remedies of a number of disorders including antistress (adaptogenic) activity \[[@B3]\]. The initial studies on *Ocimum sanctum*\[[@B4]\], *Withania somnifera*\[[@B5]\] opened a vast area of research and substantial work has been carried out on plants such as *Eleuthrococcus senticosus*and *Panax ginseng*\[[@B6]\]. *Vitis vinifera*(Linn.) (Family: Vitaceae) also called as common grape or wine grape or European grape is one of the fruit crops most widely grown throughout the world \[[@B7]\]. In the indigenous Indian system of medicine (Ayurveda), the aerial parts of *V. vinifera*have been widely used to treat a variety of common and stress related disorders \[[@B8]\]. The composition and properties of grape seeds have been extensively investigated, and reported to have many favourable effects on human health such as the lowering of low-density lipoprotein \[[@B9],[@B10]\], reduction of cardiovascular diseases and cancer \[[@B11]\]. In addition, the seed extracts of *V. vinifera*are reported to have antimicrobial and free radical scavenging properties \[[@B12]\]. In the present investigation, the antistress activity of *Vitis vinifera*was evaluated *in-vivo*, in normal and stress induced rats following a biochemical approach. The antioxidant potential of the extract was evaluated *in-vitro*to support the antistress activity. The plant extract was further evaluated for nootropic activity using conditioned avoidance response in rats. Methods ======= Preparation of extract ---------------------- The riped fruits of *Vitis vinifera*were collected from the Chittor district of Andhra Pradesh, India in the month of October and the seeds were separated from the pulp and shade dried. The dried powdered seed material of *Vitis vinifera*(5 kg) was extracted with boiling water (25 L) for 45 minutes and the filtrate was evaporated under vacuum below 70°C in a vacuum drier to give a final yield of 50 gm. Chemicals used -------------- Vanillylmandelic acid (VMA) and scopolamine butylbromide (SBB) were purchased from Sigma-Aldrich, St. Louis, USA, while ascorbic acid was purchased from Loba Chemie, Mumbai. All other reagents used were of analytical grade. Animals ------- All animal experiments were performed in accordance with our Institutional Animal Ethics Committee and by the animal regulatory body of the government (Regd. No. 516/01/A/CPCSEA). Albino rats of either sex obtained from Ghosh Enterprises, Kolkata were used in the study. They were housed six per cage at a temperature of 22 ± 2°C with 12 h light/ dark cycle under controlled environment. Rats were fed with standard pellet diet (Ratan Brothers, Hyderabad), and water *ad libitum*. Animals were kept for seven days in laboratory for habituation. Antistress activity ------------------- Rats of either sex weighing between 120--150 gm were divided into four groups (I, II, III, IV) each containing six animals. The 24 h urine sample from each group was collected into two different beakers, one containing 5 ml of 10% oxalic acid for the spectrophotometric determination of ascorbic acid at 550 nm \[[@B13]\] and the other containing 0.5 ml of 6 *N*hydrochloric acid for the determination of vanillylmandellic acid (VMA) spectrophotometrically at 360 nm \[[@B14]\]. The experimental protocol was divided into four phases. In the first phase of the experiment, 24 h urine samples were collected in all the four groups and subjected to analysis for both VMA and ascorbic acid and the normal values were recorded for seven consecutive days. In the second phase, the animals in each group were subjected to fresh water swimming stress individually. In this method, rats were forced to swim until exhausted (three to four minutes) in a cylindrical vessel of 60 cm height and 45 cm diameter containing water at room temperature (28°C). Water depth was always maintained at 40 cm. The 24 h urinary levels of VMA and ascorbic acid under stressed conditions were determined again as described above daily for seven consecutive days. The third phase of the experiment consists of administration of *V. vinifera*extract to the same groups of animals after having recovered completely to normal condition. Groups II, III and IV were administered orally with *V. vinifera*(suspended in 2% gum acacia) at daily doses of 100, 200 and 300 mg/kg body weight respectively for seven consecutive days while group I serving as control. The 24 h urine samples were collected and the levels of both VMA and ascorbic acid were determined. The final phase of the experiment consisted of administration of *V. vinifera*extract to the same groups of animals after a recovery period of one week. Groups II, III and IV were administered orally with *V. vinifera*at doses of 100, 200 and 300 mg/kg body weight respectively, one hour prior to the daily induction of stress for seven consecutive days while group I serving as control. The 24 h urine samples were collected and analyzed for VMA and ascorbic acid for seven consecutive days to study the influence of the extract on the stress induced biochemical changes. Nootropic activity ------------------ The Nootropic activity of *V. vinifera*was evaluated by using the conditioned avoidance response (*CAR*) in rats as described by Cook and Weidley \[[@B15]\]. Rats were divided into 4 groups each containing six animals. Groups II, III and IV were administered orally with 100, 200 and 300 mg/kg body weight respectively of *V. vinifera*(suspended in 2% gum acacia) while animals in group I were served as control. After 60 minutes, all the animals were subjected to a training schedule individually by placing inside the perspex chamber of the apparatus. After an accustomed period of five minutes to the chamber, a buzzer was given followed by a shock through the grid floor. The rat had to jump on the pole to avoid foot shock. Jumping on the pole functionally terminates the shock and this was classified as an escape while such jumping prior to the onset of the shock was considered as avoidance. The session was terminated after completion of 60 trials with an interval of 20--30 seconds given for each trial. This procedure was repeated at 24 h intervals until all groups reach 95 to 99% avoidance. After attaining complete training of a particular group, the animals were treated with a single dose of scopolamine butyl bromide (1 mg/kg body weight, i.p.), thirty minutes before the next day dosing. The training schedule was continued further with the daily doses of the extract and vehicle until they returned to normal level from scopolamine induced amnesia. Antioxidant activity -------------------- The antioxidant activity of *V. vinifera*was determined based on its ability to scavenge the hydroxyl radicals \[[@B16]\]. Hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and the extract for hydroxyl radicals generated from the Fe^3+^-ascorbate-EDTA-H~2~O~2~system. The hydroxyl radicals attacks deoxyribose and eventually results in the formation of thiobarbituric acid reacting substances (TBARS). The reaction mixture containing deoxyribose (2.8 mM), ferric chloride (0.1 mM), EDTA (0.1 mM), H~2~O~2~(1 mM), ascorbate (0.1 mM) phosphate buffer (20 mM, pH 7.4) and various quantities of the extracts in a final volume of 1 mL was incubated for 1 h at 37°C. Deoxyribose degradation was measured as TBARS by the method of Ohkawa et al., 1979 and the percentage free radical inhibition was calculated from control. Data and statistical analysis ----------------------------- The results are expressed as means ± standard error of means. Statistical analysis was done using Student\'s paired *t*-test. In all the cases, p \< 0.05 was considered statistically significant. Results ======= Antistress activity ------------------- The urinary data of VMA and ascorbic acid observed in various phases of the experiment are shown in Fig. [1](#F1){ref-type="fig"} and Fig. [2](#F2){ref-type="fig"} respectively. Induction of forced swim stress to the animals produced a significant increase in VMA and decrease in ascorbic acid excretion compared to their respective basal excretion in normal condition. Both the parameters were found to return to their normal levels in three to four days after withdrawal of stress. Daily treatment of *V. vinifera*to the animals under normal condition produced no change in the excretion of VMA and ascorbic acid compared to normal basal levels indicating that *V. vinifera*did not alter excretion of VMA and ascorbic acid in normal condition. Daily administration of *V. vinifera*one hour prior to the induction of stress inhibited the increase in VMA and decrease in ascorbic acid excretion which was manifested during stress alone. The inhibition was found to be significant at all dose levels in a dose dependent manner. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Influence of *Vitis vinifera*seed extract on the 24 h urinary levels of VMA in normal and stress induced rats**. Each bar indicates the mean excretion of six animals. Significant difference from normal condition of the corresponding groups: \*p \< 0.05 Significant difference from stress condition of the corresponding groups: *\#*p \< 0.05 NS- No significant difference from normal condition of the corresponding groups. §No significant difference from stress condition. ::: ![](1472-6882-5-1-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Effect of *Vitis vinifera*seed extract on the 24 h urinary levels of ascorbic acid in normal and stress induced rats**. Each bar indicates the mean excretion of six animals. Significant difference from normal condition of the corresponding groups: \*p \< 0.05 Significant difference from stress condition of the corresponding groups: *\#*p \< 0.05 NS- No significant difference from normal condition of the corresponding groups. §No significant difference from stress condition. ::: ![](1472-6882-5-1-2) ::: Nootropic activity ------------------ The *CAR*of rats administered with the extract of *Vitis vinifera*or vehicle increased gradually to 95% over seven to ten days. The percent avoidance was always higher in the extract treated groups compared to vehicle treated control group. The acquisition (time to achieve 95% *CAR*) for the extract treated groups was quicker and found to be dose dependent. Animals receiving 300 mg/kg body weight of the extract have taken seven days whereas, groups treated with 200 and 100 mg/ kg doses of the extract required eight and nine days respectively to reach the point of acquisition (Fig. [3](#F3){ref-type="fig"}). Administration of scopolamine produced amnesia as seen from reduction in the observed CAR. However, continued treatment of *V. vinifera*produced better retention and recovery in a dose dependent manner than the vehicle treated animals. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Effect of *Vitis vinifera*seed extract on the mean percent of conditioned avoidance response after oral administration in rats**. Scopolamine butylbromide (SBB) was administered thirty minutes before the next day dosing with the extract after attaining complete acquisition. ::: ![](1472-6882-5-1-3) ::: Antioxidant activity -------------------- Degradation of deoxyribose mediated by hydroxyl radicals generated by Fe^3+^/ascorbate/EDTA/H~2~O~2~system was found to be inhibited by *Vitis vinifera*. The extract at quantities of 100, 200, 400 and 800 μg levels scavenged the hydroxyl radicals in a dose dependent manner. Ascorbic acid at concentration of 2500, 5000 and 10000 μg was also found to produce dose dependent inhibition of hydroxyl radicals. The quantity of the extract needed for 50% inhibition was found to be 610 μg (Fig. [4](#F4){ref-type="fig"}). Similar effect was produced by ascorbic acid at a concentration of 4875 μg. (Fig. [5](#F5){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Hydroxyl radical scavenging activity of *Vitis vinifera*in *in-vitro*systems**. Graphical representation of the concentration of *Vitis vinifera*required to inhibit 50 percent of hydroxyl radicals. Each point represents the mean percentage inhibition of six experiments. ::: ![](1472-6882-5-1-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Hydroxyl radical scavenging activity of ascorbic acid in *in-vitro*systems**. Graphical representation of the concentration of ascorbic acid required to inhibit 50 percent of hydroxyl radicals. Each point represents the mean percentage inhibition of six experiments. ::: ![](1472-6882-5-1-5) ::: Discussion ========== Advancement in the understanding of processes leading to explore the reason for stress induced disorders cannot obscure the simple fact that the exhaustion of energy supply still forms the basis that triggers the disorders and collapse of energy metabolism following glucose deprivation in circulation \[[@B17]\]. The desire to control the coping mechanism has led to emergence of science of adaptation, focusing to elucidate the mechanism that may help in modification so that insufficient, excessive and unnecessary responses can be prevented. Literature reports indicate that noradrenaline is released during stressful conditions \[[@B18]\] and metabolized to vanillyl mandelic acid(VMA) peripherally and 3-methoxy 4-hydroxyphenyl glycol (MOPEG) centrally. In the light of such reports, VMA, the major metabolite of sympathetic amines, was taken as indirect biochemical index to represent the increase in peripheral sympathetic activity during stress. In the present study, the increase in the urinary VMA excretion during stress was used as a non-invasive biochemical marker to study the antistress activity of *V. vinifera*. Several studies also indicated that the tissue levels of ascorbic acid decreased on application of stress \[[@B19]\]. Ascorbic acid being a free radical scavenger \[[@B20]\], it is more likely utilized in scavenging the free radicals involved in stress resulting in its decreased urinary concentration and also it has role in the biosynthesis of noradrenaline \[[@B21]\]*i.e*., as a cofactor in the conversion of dopamine to noradrenaline \[[@B22]\]. Based on the above studies ascorbic acid excretion in urine was taken as an indirect biochemical index to indicate the influence of stress on catecholamine synthesis in rats and antistress (adaptogenic) activity of the *Vitis vinifera*extract on prior administration of stress induction. Treatment with *Vitis vinifera*extract along with stress reversed the stress induced biochemical changes i.e., increase in urinary VMA levels and decrease in urinary ascorbic acid levels, in a dose dependent manner. A number of Indian medicinal plants like *Ocimum sanctum*, *Withania somnifera*, *Panax ginseng*etc have been identified for their antistress activity. It was concluded that the antioxidant activity of these plants was partly responsible for their antistress activity \[[@B23]\]. Based on these reports the antioxidant activity of *Vitis vinifera*extract was also done using hydroxyl radical assay method. It was found that *Vitis vinifera*extract has significant good antioxidant activity which was 8 fold more than that of ascorbic acid. The antistress and antioxidant activities were correlated with the nootropic activity of the extract since the role of stress and free radicals have been implicated in the loss of memory, concentration and also in Alzheimer\'s disease \[[@B24],[@B25]\]. The process of nootropic activity involves acquisition, retention and retrieval and is measured using conditioned avoidance response. The acquisition was quicker in the extract treated rats (100, 200, 300 mg/kg body weight) in comparision to control, indicating the involvement of antistress activity of the extract. When challenged with scopolamine butylbromide (1 mg/kg body weight), the amnesia was less in treated group showing better retention and recovery than control group and the *Vitis vinifera*extract was shown to decrease memory loss which could be due to its central cholinomimetic activitiy apart from its free radical scavenging mechanisms. Furthermore, the antioxidant activity of the seed extract provide mechanistic basis in relieving stress by way of combating oxidative damage. Conclusion ========== In conclusion, the present study provides scientific support for the antistress (adaptogenic), antioxidant and nootropic activities of *V. vinifera*seed extract and substantiate the traditional claims for the usage of grape fruits and seeds in stress induced disorders. Further investigations are required to characterize the active constituent(s) responsible for observed activities of the seed extract. Abbreviations ============= VMA: Vanillylmandelic acid CAR: Conditioned avoidance response SBB: Scopolamine butylbromide EDTA: Ethylene diamine tetra acetic acid H~2~O~2~: Hydrogen peroxide Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= SS conceived the study, made substantial contributions in data analysis, data interpretation, writing of the manuscript and in coordination of the experiments. SN made substantial contributions in conceptualization of statistical analyses, drafting the final manuscript and designing the illustrations. RK and SK helped to conceptualize the work. KMB made significant contribution in designing the studies, conducting the experiments, interpretation of the data, and drafting the final manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6882/5/1/prepub> Acknowledgements ================ The Authors are indebted to the All India Council for Technical Education AICTE), Government of India, New Delhi for providing a major financial support to carryout the research work. The authors are also grateful to the Department of Pharmaceutical Sciences Andhra University, Visakhapatnam, India for providing all the facilities to carryout the studies.

PubMed Central

2024-06-05T03:55:52.438202

2005-1-19

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547917/", "journal": "BMC Complement Altern Med. 2005 Jan 19; 5:1", "authors": [ { "first": "Satyanarayana", "last": "Sreemantula" }, { "first": "Srinivas", "last": "Nammi" }, { "first": "Rajabhanu", "last": "Kolanukonda" }, { "first": "Sushruta", "last": "Koppula" }, { "first": "Krishna M", "last": "Boini" } ] }

PMC547972

Introduction {#s1} ============ Due to their ability to interact with fatty acids and other lipids, nuclear hormone receptors (NHRs), including peroxisome proliferator-activated receptors (PPARs), liver X receptor, and farnesoid X receptor, are important regulators of mammalian fat metabolism; accordingly, these small-molecule-gated transcription factors have been favored targets for therapies designed to combat diabetes, obesity, and atherosclerosis \[[@pbio-0030053-b01],[@pbio-0030053-b02],[@pbio-0030053-b03]\]. Although the molecular mechanisms employed by NHRs have been intensively probed, their pleiotropic effects on overall animal physiology are only partially understood. Thus, developing a broader range of experimental systems would be useful for characterizing the fat regulatory networks integrated by NHRs and for revealing the breadth of their physiological influence. The study of invertebrates, such as Caenorhabditis elegans, has facilitated the elucidation and interpretation of biological networks, particularly in the context of the whole animal. However, despite the fact that C. elegans contains 284 NHR genes, compared to only 48 in mammals, there is a notable absence of worm NHRs orthologous to mammalian receptors involved in fat metabolism \[[@pbio-0030053-b04],[@pbio-0030053-b05]\]. In fact, it has been suggested that fat-regulating NHRs, along with several other NHR types, were lost from the C. elegans and Drosophila lineages as these organisms simplified their body plans \[[@pbio-0030053-b06]\]. Conceivably, then, the biological activities associated with these NHRs may also have vanished. Therefore, it is still not clear if worm receptors mediate control over fat metabolism comparable to that of mammalian NHRs. Whether or not C. elegans employs NHRs to regulate fat metabolism, the evolution of the C. elegans NHR family is intrinsically interesting. Only 15 of the 284 worm NHRs are members of the broadly conserved subfamilies found in mammals and other metazoans \[[@pbio-0030053-b04],[@pbio-0030053-b05]\]. The biological activities of most of these "conserved" NHRs have been broadly defined, revealing roles in development, molting, dauer formation, and sex determination \[[@pbio-0030053-b07],[@pbio-0030053-b08]\]. The remaining 269 "divergent" NHRs have thus far been found only in nematodes and are predicted to have originated from repeated duplication of an ancestral gene that also gave rise to the hepatocyte nuclear factor 4 (HNF4) family of receptors \[[@pbio-0030053-b06]\]. Although mammalian HNF4 receptors have been implicated in liver development and glucose homeostasis \[[@pbio-0030053-b09]\], virtually nothing is known about the functions of these 269 C. elegans "nematode-specific" HNF4-like receptors. It will be interesting to determine if these divergent NHRs have adopted novel regulatory functions or whether they carry out physiological tasks similar to those of other metazoan NHRs. Elucidating the physiological responsibilities of even one of the worm HNF4-like receptors will likely advance our understanding of NHR-regulated gene networks and provide insight into the evolution and diversification of the NHR family in nematodes. To this end, we have been systematically investigating the function of HNF4-like receptors in *C. elegans.* In this study, we present a physiological characterization of *nhr-49* (K10C3.6), one of the C. elegans receptor genes most closely related to the mammalian HNF4. Our findings reveal a surprising role for this HNF4-like receptor in the regulation of fat storage and metabolism. Results {#s2} ======= *nhr-49* Is Necessary for Normal Life Span {#s2a} ------------------------------------------ In an RNA interference (RNAi) screen designed to identify the role of C. elegans HNF4-like receptors in worm development and longevity, we found that interference of *nhr-49* resulted in dramatically reduced life span ([Figure 1](#pbio-0030053-g001){ref-type="fig"}A). For further characterization, we obtained a C. elegans strain, *nhr-49(nr2041),* which harbors a deletion in the *nhr-49* gene encompassing part of the DNA binding domain and more than half of the ligand binding domain (LBD); this deletion likely results in complete loss of function \[[@pbio-0030053-b10]\]. At 23 °C, *nhr-49(nr2041)* worms lived only 6--8 d as adults, significantly shorter than the 15 to 18-d life span of N2 wild-type (WT) animals ([Figure 1](#pbio-0030053-g001){ref-type="fig"}A). Although *nhr-49* deletion did not noticeably affect development or fertility, *nhr-49(nr2041)* worms experienced rapid decline in function beginning around day 3 of adulthood, when vacuoles appeared in the intestine and gonad ([Figure 1](#pbio-0030053-g001){ref-type="fig"}B). By days 4 and 5, *nhr-49(nr2041)* animals were significantly smaller than WT, vacuoles were ubiquitous, and there was widespread gonadal necrosis. By days 5--7, the gonad had completely deteriorated, and the worms died shortly thereafter. Thus, *nhr-49* function is not required for development or fertility, but is clearly essential for normal longevity. Even though the increased vacuole formation was consistent with reported aging characteristics \[[@pbio-0030053-b11]\], we have not determined whether the shortened life span of *nhr-49(nr2041)* reflects accelerated aging or an unrelated pathology. ::: {#pbio-0030053-g001 .fig} Figure 1 ::: {.caption} ###### *nhr-49(nr2041)* Animals Have Reduced Life Span and Higher Fat Content \(A) Adult life span of WT (black squares, solid line), *nhr-49(nr2041)* (black circles, dotted line), and *nhr-49* RNAi animals (black diamonds, dashed line). \(B) Nomarksi images of *nhr-49(nr2041)* at days 3, 5, and 7 of adulthood. The arrow in the day 3 image points to a gonadal vacuole that is typical of day 3 worms; the arrow in the day 5 image shows the continued deterioration of oocytes in the gonad, and the arrow in the day 7 image points to the clearing that results from complete gonadal necrosis and collapse. \(C) Nile Red intestinal fat staining of WT and *nhr-49(nr2041).* Each image displays two representative worms from a population of L4 animals. ::: ![](pbio.0030053.g001) ::: *nhr-49(nr2041)* Animals Display Abnormally High Fat Content {#s2b} ------------------------------------------------------------ *nhr-49* was recently identified in a C. elegans genomewide RNAi screen as one of 112 genes that, when knocked down by RNAi, resulted in abnormally high Nile Red fat staining \[[@pbio-0030053-b12]\]. To determine whether deletion of *nhr-49* also yielded a high-fat phenotype, we used a similar Nile Red assay to visualize fat content in *nhr-49(nr2041).* Indeed, we found that *nhr-49(nr2041)* animals stained more brightly with Nile Red than did WT worms ([Figure 1](#pbio-0030053-g001){ref-type="fig"}C). The difference in fat content between WT and *nhr-49(nr2041)* animals was most pronounced in the L3 and L4 stages of larval development; by day 2 of adulthood there was only a slight change in Nile Red staining, suggesting that the effects of *nhr-49* on visible fat content may vary during development (data not shown). *nhr-49* Regulates Genes Involved in Energy Metabolism {#s2c} ------------------------------------------------------ The high-fat phenotype of the *nhr-49(nr2041)* mutant led us to suspect that *nhr-49* might regulate genes involved in energy metabolism. To test this hypothesis, we identified from a survey of the C. elegans genome 65 genes predicted to participate in fatty acid synthesis, β-oxidation, desaturation, elongation, and binding/transport, in addition to 16 genes expected to function in glycolysis, gluconeogenesis, and glucose transport, and eight genes involved in the glyoxylate pathway ([Figure 2](#pbio-0030053-g002){ref-type="fig"}). We then employed quantitative RT-PCR (QRT-PCR) to measure the expression of all 89 of these genes in WT and *nhr-49(nr2041)* mutant worms. For the purpose of our survey, gene expression was measured in all four larval stages (for a complete dataset, see [Table S1](#st001){ref-type="supplementary-material"}). ::: {#pbio-0030053-g002 .fig} Figure 2 ::: {.caption} ###### *nhr-49* Deletion Affects the Expression of 13 Energy Metabolism Genes Putative C. elegans fatty acid, glucose, and glyoxylate metabolism pathways are shown here; each box represents a gene predicted by sequence homology to code for the enzyme indicated in the figure. The fatty acid desaturation elongation pathway is adapted from previously published studies \[[@pbio-0030053-b14],[@pbio-0030053-b17]\]. Mitochondrial localization of β-oxidation enzymes was predicted by TargetP and PSORT (see [Materials and Methods](#s4){ref-type="sec"}); peroxisomal β-oxidation enzymes all contain a carboxy-terminal peroxisomal localization signal. QRT-PCR was used to measure the expression of these 89 genes in WT and *nhr-49(nr2041).* Genes expressed at lower levels in *nhr-49(nr2041)* are shown in red (\>32-fold lower); orange (8- to 32-fold lower); or yellow (2- to 8-fold lower); and genes expressed at higher levels are shown in light green (2- to 8-fold higher) or dark green (\>8-fold higher). Online QRT-PCR data for this analysis are available in [Table S1](#st001){ref-type="supplementary-material"}. ::: ![](pbio.0030053.g002) ::: Our screen revealed that deletion of *nhr-49* significantly altered the expression of 13 genes, including six genes predicted to be involved in fatty acid β-oxidation, three genes involved in fatty acid desaturation, two genes involved in fatty acid binding/transport, and two genes involved in the glyoxylate pathway ([Figure 2](#pbio-0030053-g002){ref-type="fig"}). Similar changes in gene expression were observed when *nhr-49* was knocked down in WT animals using RNAi (data not shown). Overall, *nhr-49* deletion displayed the most dramatic effects on two metabolic pathways, mitochondrial β-oxidation and fatty acid desaturation, as the expression of multiple genes in each of these pathways was significantly compromised by loss of *nhr-49* function ([Figure 2](#pbio-0030053-g002){ref-type="fig"}). In contrast, *nhr-49* deletion had smaller and mixed consequences for genes that participate in peroxisomal β-oxidation and lipid binding/transport. *nhr-49* knockout also marginally reduced expression of two enzymes involved in the glyoxylate pathway, a pathway that facilitates the conversion of fatty acid β-oxidation products to glucose ([Figure 2](#pbio-0030053-g002){ref-type="fig"}). *nhr-49* deletion did not, however, significantly affect the expression of any genes predicted to be involved in glucose metabolism. Thus, it is clear that *nhr-49* is extensively involved in the control of fatty acid metabolism, with a pronounced role in the promotion of mitochondrial β-oxidation and fatty acid desaturation. *nhr-49(nr2041)* Animals are High in Fat Due to Reduced Expression of β-Oxidation Genes {#s2d} --------------------------------------------------------------------------------------- Three genes predicted to participate in mitochondrial β-oxidation, *ech-1* (C29F3.1), F09F3.9, and an acyl-CoA synthetase gene *(acs-2)* (F28F8.2), were expressed at significantly lower levels in *nhr-49(nr2041)* animals throughout development ([Figure 3](#pbio-0030053-g003){ref-type="fig"}A). *acs-2* is predicted to encode a mitochondrial acyl-CoA synthetase, whereas F09F3.9 likely codes for a carnitine palmitoyl transferase, and *ech-1* appears to encode a mitochondrial β-oxidation trifunctional enzyme. Because mitochondrial β-oxidation facilitates the degradation of stored fats for the production of energy, we suspected that *nhr-49(nr2041)* worms might be high in fat due to the reduced expression of these key β-oxidation enzymes. To test this hypothesis, we used RNAi to reduce separately the expression of *acs-2,* F09F3.9, and *ech-1* in WT animals. Indeed, we found that RNAi of either *acs-2* or *ech-1* resulted in worms with elevated Nile Red fat staining ([Figure 3](#pbio-0030053-g003){ref-type="fig"}B). *acs-2* was previously identified for a similar effect on fat storage \[[@pbio-0030053-b12]\]. We conclude that *nhr-49* promotes the expression of three β-oxidation genes, two of which, in WT worms, reduce overall fat storage. Thus, *nhr-49(nr2041)* mutant worms are likely to be high in fat due to the impaired expression of these genes. ::: {#pbio-0030053-g003 .fig} Figure 3 ::: {.caption} ###### Regulation of β-Oxidation Gene Expression by *nhr-49* \(A) QRT-PCR measurement of *acs-2,* F09F9.3, and *ech-1* expression in WT (gray bars) and *nhr-49(nr2041)* animals (blue bars). Expression was measured in all four stages of larval development. Error bars represent standard error of measurement. \(B) RNAi of *nhr-49, acs-2,* or *ech-1* resulted in increased Nile Red fat staining. Each image shows 3 or 4 representative worms from a population of L4 animals grown on plates containing RNAi bacteria and Nile Red fat-staining dye. \(C) *acs-2::gfp* is expressed in many tissues including hypodermis (hyp), intestine (int), body wall muscle (bwm), neurons (neu), and pharynx (pha). ACS-2::GFP localizes to subcellular structures in a pattern similar to what has been reported for mitochondrial proteins \[[@pbio-0030053-b13]\]. \(D) Expression of *acs-2::gfp* was lower in *nhr-49(nr2041)* mutant animals. \(E) Expression of P*~nhr-49~::gfp* promoter fusion in WT animals revealed that *nhr-49* is expressed in multiple tissues, including hypodermis (hyp), body wall muscle (bwm), pharynx (pha), and intestine (int). The animals shown here are genetically mosaic, harboring the P*~nhr-49~::gfp* construct in only a fraction of total cells. \(F) Ectopic expression of *acs-2::gfp* in *nhr-49(nr2041)* was sufficient to reduce Nile Red staining to WT levels. ::: ![](pbio.0030053.g003) ::: ACS-2 Is Expressed in Multiple Tissues and Localizes to Mitochondria {#s2e} -------------------------------------------------------------------- Out of five predicted mitochondrial *acs* genes in *C. elegans, nhr-49* affected only the expression of *acs-2,* suggesting that *nhr-49* may only influence a subset of mitochondrial β-oxidation pathways in worms. To determine if *acs-2* was expressed in a specific set of tissues, we fused the full-length *acs-2* gene and promoter to *gfp (acs-2::gfp);* injection of this construct into WT worms revealed that the ACS-2::green fluorescent protein (GFP) was widely expressed in many cell types, including intestine, hypodermis, pharynx, body wall muscle, and several neurons ([Figure 3](#pbio-0030053-g003){ref-type="fig"}C). Moreover, ACS-2::GFP expression was reduced in all of these tissues in *nhr-49(nr2041)* worms, indicating that the effects of *nhr-49* on *acs-2* expression were widespread ([Figure 3](#pbio-0030053-g003){ref-type="fig"}D). Notably, the expression pattern of *acs-2* overlapped extensively with that of an *nhr-49* promoter/GFP fusion, consistent with the idea that *nhr-49* could control *acs-2* through direct transcriptional activation ([Figure 3](#pbio-0030053-g003){ref-type="fig"}E). As predicted, ACS-2::GFP localized to subcellular structures in patterns similar to those reported for other mitochondrial proteins \[[@pbio-0030053-b13]\] ([Figure 3](#pbio-0030053-g003){ref-type="fig"}C). Overexpression of ACS-2::GFP Is Sufficient to Suppress the High-Fat Phenotype of *nhr-49(nr2041)* {#s2f} ------------------------------------------------------------------------------------------------- Strikingly, overexpression of the ACS-2::GFP fusion was able to suppress the high-fat phenotype of *nhr-49(nr2041)* animals ([Figure 3](#pbio-0030053-g003){ref-type="fig"}F). These data suggest that *acs-2* expression is reduced in *nhr-49(nr2041)* such that it becomes a rate-limiting factor in the consumption of fat; thus, high fat levels can be overcome simply by increasing *acs-2* expression. Overexpression of *acs-2* in WT animals did not affect fat content, however, indicating that WT levels of *acs-2* expression are not rate limiting. These data further support the conclusion that reduced expression of *acs-2* is a significant contributor to the high-fat phenotype of the *nhr-49(nr2041)* mutant. *nhr-49* Modulates Fatty Acid Composition by Promoting Expression of Δ9-Desaturases {#s2g} ----------------------------------------------------------------------------------- In addition to stimulating genes in the mitochondrial β-oxidation pathway, *nhr-49* also promoted the expression of three C. elegans fatty acid desaturases (see [Figure 2](#pbio-0030053-g002){ref-type="fig"}). *fat-5* (W06D12.3) and *fat-7* (F10D2.9) expression was dramatically lowered in *nhr-49(nr2041)* worms (\>30-fold) in all four larval stages, whereas *fat-6* (VZK8221.1) expression was marginally reduced (approximately 2-fold) only in L3 and L4 animals ([Figure 4](#pbio-0030053-g004){ref-type="fig"}A). ::: {#pbio-0030053-g004 .fig} Figure 4 ::: {.caption} ###### Deletion of *nhr-49* Hinders Fatty Acid Desaturation \(A) QRT-PCR measurement of *fat-5, fat-6, and fat-7* expression in WT (gray bars) and in *nhr-49(nr2041)* mutant worms (blue bars). Expression was measured in all four stages of larval development. Error bars represent standard error of measurement. \(B) Relative abundance of individual fatty acid species expressed as percentage of total measured fatty acid. Fatty acids included in total fatty acid measurement but not shown in the figure include C14:0, C15:0, and C17:Δ. Fatty acids were isolated and quantified by GC/MS from WT (dark gray bars), *nhr-49(nr2041)* (blue bars), *fat-7* RNAi animals (red bars), and *nhr-49* RNAi animals (light gray bars). Error bars represent standard error. The fatty acid desaturation/elongation pathway, along with enzymes involved in desaturation and elongation, is shown in the inset; this pathway was adapted from previous studies \[[@pbio-0030053-b14],[@pbio-0030053-b17]\]. ::: ![](pbio.0030053.g004) ::: Studies in yeast have shown that the *C. elegans fat-5, fat-6,* and *fat-7* genes encode Δ9-desaturases, which preferentially convert saturated C16:0 and C18:0 fatty acids to monounsaturated C16:1 and C18:1 fatty acids \[[@pbio-0030053-b14]\]. *fat-5* is a palmitoyl-CoA desaturase, specifically acting on palmitic acid (C16:0), and *fat-6* and *fat-7* are stearoyl-CoA desaturases (SCDs), preferentially functioning to desaturate stearic acid (C18:0). Mammalian SCDs have been shown to be important for maintaining an appropriate level of fatty acid desaturation, vital for modulating membrane fluidity and for controlling lipid metabolism \[[@pbio-0030053-b15],[@pbio-0030053-b16]\]. Furthermore, in *C. elegans,* SCDs are also involved in catalyzing the first step in the synthesis of polyunsaturated fatty acids (PUFAs) \[[@pbio-0030053-b17]\] (see [Figure 4](#pbio-0030053-g004){ref-type="fig"}B inset). Because the expression of these three C. elegans Δ9-desaturases was significantly impaired by *nhr-49* deletion, we used gas chromatography/mass spectrometry (GC/MS) to determine whether overall fatty acid desaturation and PUFA synthesis were altered in *nhr-49(nr2041)* animals. The most significant consequence of *nhr-49* deletion on fatty acid composition was a marked increase in the abundance of stearic acid (C18:0) and a corresponding decrease in the level of oleic acid (C18:1n9) ([Figure 4](#pbio-0030053-g004){ref-type="fig"}B). This result is consistent with a reduction in SCD activity. In mammals, a primary role of SCD is to control the ratio of fully saturated stearic acid to monounsaturated oleic acid (C18:0/C18:1n9), and this ratio has been employed as an indicator of the activity of the mammalian SCD1 \[[@pbio-0030053-b15],[@pbio-0030053-b16]\]. In C. elegans we found that the ratio of stearic acid to oleic acid (C18:0/C18:1n9) was increased from 1.9 in WT animals to 4.3 in *nhr-49(nr2041)* ([Table 1](#pbio-0030053-t001){ref-type="table"}). A similar but less pronounced alteration in the ratio of stearic acid to oleic acid was observed when *nhr-49* was knocked down in WT animals using RNAi ([Figure 4](#pbio-0030053-g004){ref-type="fig"}B and [Table 1](#pbio-0030053-t001){ref-type="table"}). This smaller influence on fatty acid desaturation likely resulted from the fact that *nhr-49* RNAi did not reduce Δ9-desaturase expression as dramatically as *nhr-49* knockout (data not shown). ::: {#pbio-0030053-t001 .table-wrap} Table 1 ::: {.caption} ###### Life Span, Fat Content, and Relative C18:0 Fatty Acid Abundance ::: ![](pbio.0030053.t001) Fat content was determined by Nile Red staining. Life span indicated is the day when half of the worms in a given population expired. Fatty acid abundance is indicated by percent of total fatty acid measured by GC/MS ::: C. elegans SCDs are also important for the synthesis of PUFAs; however, we did not observe any measurable changes in PUFA abundance in *nhr-49(nr2041)* animals ([Figure 4](#pbio-0030053-g004){ref-type="fig"}B). Therefore, despite the dramatic effect of *nhr-49* on SCD activity, *nhr-49* does not appear to play an appreciable role in overall PUFA synthesis. To determine which of the Δ9-desaturases accounted for the influence of *nhr-49* deletion on fatty acid composition, we used RNAi to individually knock down expression of *fat-5* and *fat-7* (because *fat-6* expression is only reduced by approximately 2-fold in *nhr-49\[nr2041\],* we did not examine *fat-6* RNAi animals). Although we did not observe a significant consequence of *fat-5* RNAi on fatty acid desaturation, *fat-7* RNAi altered fatty acid composition in a manner very similar to that of *nhr-49* deletion ([Figure 4](#pbio-0030053-g004){ref-type="fig"}B and [Table 1](#pbio-0030053-t001){ref-type="table"}). *fat-7* RNAi animals displayed a significant increase in the abundance of C18:0 fatty acid, as well as a decrease in the levels of C18:1n9 and C18:2n6 fatty acids. The influence of *fat-7* RNAi on fatty acid composition was stronger than that of *nhr-49* deletion, increasing the ratio of stearic to oleic acid (C18:0/C18:1n9) to 8.0. *fat-7* RNAi animals also exhibited a slight lowering of PUFA abundance. The stronger effect of *fat-7* RNAi on fatty acid composition suggests that *fat-7* expression was reduced to levels even lower than those observed in *nhr-49(nr2041).* Because *fat-7* shares significant homology (approximately 85% nucleotide identity) with *fat-6,* it was possible that *fat-7* RNAi also partially interfered with *fat-6* expression; however, we found by QRT-PCR that the overall levels of *fat-6* mRNA were not noticeably reduced by *fat-7* RNAi. (data not shown). Therefore, we conclude that by strongly promoting SCD expression, *nhr-49* influences fatty acid composition in *C. elegans.* Although it appears that the primary effect of *nhr-49* deletion is a modulation of stearic and oleic acid levels, not PUFA abundance, we cannot rule out the possibility that *nhr-49* influences PUFA synthesis in a more localized manner, such that the changes in PUFA composition are not detectable by our whole-worm analysis. Interference of *fat-7* Expression Reproduces Shortened Life-Span Phenotype {#s2h} --------------------------------------------------------------------------- Using RNAi to knock down the *nhr-49*-dependent genes identified in this study, we found that interference of only *fat-7* resulted in a shortened life-span phenotype similar to that of *nhr-49(nr2041)* (see [Table 1](#pbio-0030053-t001){ref-type="table"}). *fat-7* RNAi animals displayed many of the same characteristics of the *nhr-49(nr2041)* mutant, including widespread vacuole formation and germ line necrosis ([Figure 5](#pbio-0030053-g005){ref-type="fig"}). ::: {#pbio-0030053-g005 .fig} Figure 5 ::: {.caption} ###### *fat-7* Is Necessary for Normal Life Span and Inhibits β-Oxidation \(A) Nomarski images of WT worms subjected to *fat-7* RNAi at days 1 and 3 of adulthood. The arrow in the day 1 image points to vacuole formation in the intestine, and the arrow in the day 3 image points to clearing that results from collapse of the gonad. These characteristics are nearly identical to those observed for *nhr-49(nr2041)* worms (see [Figure 1](#pbio-0030053-g001){ref-type="fig"}B). \(B) QRT-PCR measurement of *acs-2* and *ech-1* expression in WT and *nhr-49(nr2041)* L4 animals grown on control RNAi bacteria (dark gray bars) or on *fat-7* RNAi bacteria (blue bars). Error bars represent standard error of measurement. \(C) RNAi knockdown of *fat-7* expression in WT animals reduced Nile Red fat staining \(D) RNAi of *fat-7* in *nhr-49(nr2041)* also decreased fat staining. ::: ![](pbio.0030053.g005) ::: Interestingly, the effect of *fat-7* RNAi on life span was even more potent than *nhr-49* deletion, reducing adult life span to 3--5 d, as opposed to the 5- to 7-d life span of *nhr-49(nr2041)* (see [Table 1](#pbio-0030053-t001){ref-type="table"}). This is consistent with the finding that SCD activity is more dramatically compromised in *fat-7* RNAi animals than it is in the *nhr-49(nr2041)* mutant. In fact, we observed a striking correlation between *fat-7* activity, as determined by the ratio of stearic to oleic acid in our various mutants, and life span (see [Table 1](#pbio-0030053-t001){ref-type="table"}). WT animals (stearic/oleic = 1.9) lived approximately 16--18 d, *nhr-49 RNAi* animals (stearic/oleic = 2.3) lived approximately 10--12 d, *nhr-49(nr2041)* animals (stearic/oleic = 4.3) lived approximately 6--8 d, and *fat-7* RNAi animals (stearic/oleic = 8.0) lived approximately 3--5 d. Because *fat-7* expression is significantly compromised by *nhr-49* deletion, and because lowered *fat-7* expression can lead to shortened life span, we suggest that the reduced life-span phenotype of *nhr-49(nr2041)* likely reflects, at least in part, diminished *fat-7* expression. Stimulation of *fat-7* by *nhr-49* Feeds Back to Inhibit β-Oxidation {#s2i} -------------------------------------------------------------------- By promoting β-oxidation gene expression, *nhr-49* stimulates fat consumption, and by enhancing *fat-7* expression, *nhr-49* modulates fat desaturation and ensures normal longevity. We next set out to determine whether *nhr-49* regulates these two pathways independently. Although RNAi of *acs-2* or *ech-1* was sufficient to cause a high-fat phenotype, *acs-2* or *ech-1* interference did not significantly impact life span, fatty acid composition, or desaturase gene expression, demonstrating that the effects of *nhr-49* deletion on *fat-7* expression and life span were not a downstream consequence of compromised β-oxidation or increased fat content (see [Table 1](#pbio-0030053-t001){ref-type="table"}). In contrast, RNAi of *fat-7* significantly impacted fat storage and β-oxidation. The effect was paradoxical, however, as *fat-7* interference produced the opposite result of *nhr-49* deletion, causing *reduced* fat content and *increased* expression of genes in β-oxidation, including *acs-2* and *ech-1* ([Figure 5](#pbio-0030053-g005){ref-type="fig"}). As the influence of *fat-7* on the expression of β-oxidation genes is opposite that of *nhr-49,* it is clear that the stimulation of β-oxidation by *nhr-49* does not result from promotion of *fat-7* expression. In fact, by enhancing *fat-7* expression, *nhr-49* indirectly causes inhibition of the very same β-oxidation genes that it is independently stimulating ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). ::: {#pbio-0030053-g006 .fig} Figure 6 ::: {.caption} ###### Model for *nhr-49* Regulation of Fat Storage and Composition By stimulating expression of genes predicted to be involved in fatty acid β-oxidation, *nhr-49* promotes fat consumption; likely by facilitating the flow of fatty acids (FA) into the mitochondrial matrix. Through an independent pathway, *nhr-49* modulates fatty acid desaturation by enhancing the expression of the *fat-7* SCD. Stimulation of *fat-7* is necessary for normal adult life span. Finally, *fat-7* also feeds back to partially inhibit expression of β-oxidation genes. ::: ![](pbio.0030053.g006) ::: Because *fat-7* targets some of the same β-oxidation genes as *nhr-49,* we wondered if the effects of *fat-7* on fat storage might be dependent on *nhr-49;* for example, perhaps *fat-7* represses *ech-1* and *acs-2* expression by blocking the *nhr-49* mediated induction of these genes. However, we found that both *ech-1* and *acs-2* were induced when *fat-7* was knocked down even further in the *nhr-49(nr2041)* mutant using RNAi, indicating that *fat-7* hinders the expression of *ech-1* and *acs-2* through a mechanism that is independent of *nhr-49* (see [Figure 5](#pbio-0030053-g005){ref-type="fig"}B). Furthermore, RNAi of *fat-7* reduced fat content in *nhr-49(nr2041)* worms, demonstrating that the stimulation of fat storage by *fat-7* is also not dependent upon *nhr-49* (see [Figure 5](#pbio-0030053-g005){ref-type="fig"}D). Taken together, our findings demonstrate that *nhr-49* controls two distinct circuits within a gene network: The stimulation of β-oxidation gene expression is independent of *fat-7,* and the promotion of *fat-7* expression is independent of *nhr-49*\'s effects on β-oxidation. However, because *fat-7* expression inhibits the same β-oxidation genes induced by *nhr-49,* the regulation of *fat-7* by *nhr-49* connects these two circuits into a single regulatory network, serving to limit β-oxidation through an apparent feedback mechanism ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). Discussion {#s3} ========== Here we demonstrated that deletion of the *C. elegans nhr-49* gene produced two global phenotypes: shortened life span and high fat content. Using a QRT-PCR strategy, we found 13 genes involved in energy metabolism that depend upon *nhr-49* for expression. The phenotypes of *nhr-49(nr2041)* were attributable to deficiencies in two different metabolic pathways, fatty acid β-oxidation and fatty acid desaturation. The high-fat phenotype is explained by lowered expression of β-oxidation enzymes, and the shortened life-span phenotype results from reduced expression of the *fat-7* SCD. Interestingly, these pathways are linked by an apparent feedback mechanism, as *fat-7* also serves to inhibit the expression of fatty acid β-oxidation genes ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). These results represent the first detailed characterization of a divergent HNF4-like receptor in C. elegans and provide the first description of an NHR-controlled fat-regulatory network in invertebrates. Due to their evolutionary relationship to *nhr-49,* it seems conceivable that many of the C. elegans HNF4-like NHRs might participate in the regulation of fat metabolism. However, we have found that several other worm HNF4-like receptors, including those previously implicated for affecting overall fat storage \[[@pbio-0030053-b12]\], do not significantly impact the expression of fatty acid metabolism genes (M. R. Van Gilst and K. R. Yamamoto, unpublished data). Thus, the regulation of fat metabolism does not appear to be a general mechanism common to all of the divergent NHRs; whether or not *nhr-49* is unique in its control of C. elegans fat metabolism remains to be determined. The mechanism by which *nhr-49* influences fat storage is likely to be through modulation of β-oxidation gene expression ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). Overexpression of *acs-2* alone was sufficient to suppress the high-fat phenotype of *nhr-49(nr2041),* indicating that *acs-2* expression is rate-limiting in *nhr-49* deletion mutants, thus leading to reduced fat breakdown and excessive fat accumulation. Acyl-CoA synthetases activate fatty acids for many different metabolic pathways; in particular, mitochondrial acyl-CoA synthetases are likely to activate fatty acids for transport into the mitochondrial matrix, where the fatty acids are then subject to β-oxidation \[[@pbio-0030053-b18],[@pbio-0030053-b19]\]. Notably, mitochondrial acyl-CoA synthetases are commonly regulatory targets for factors that control fat consumption \[[@pbio-0030053-b18]\]. We propose that by stimulating *acs-2* expression, *nhr-49* likely acts by increasing the flow of fatty acids into the mitochondria for β-oxidation ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). Out of 11 *acs* genes in *C. elegans,* five of which are predicted to be mitochondrial, *nhr-49* specifically affected the expression of only *acs-2,* suggesting that *nhr-49* may be promoting transport of a specific subset of fatty acids into mitochondria. These subsets may differ by fatty acid chain length or tissue distribution. As both *acs-2* and *nhr-49* are expressed in numerous C. elegans tissues, regulation of fat consumption by *nhr-49* may occur throughout the animal. Although *acs-2* is related (with approximately 25% identity) to multiple mammalian acyl-CoA synthetases, mammals contain a particular homolog with approximately 41% identity to the C. elegans ACS-2 protein; this mammalian ACS protein is yet to be characterized. It will be interesting to determine whether the mammalian counterpart of *acs-2* also serves as an important focal point for gene networks that regulate fat utilization in mammals. Interestingly, we also found that *nhr-49* stimulates expression of a carnitine palmitoyl transferase (F09F3.9). Carnitine palmitoyl transferases execute the step immediately downstream of acyl-CoA synthetase in mitochondrial β-oxidation, directly shuttling activated acyl-CoAs into the mitochondrial matrix. Thus, *nhr-49* appears to target multiple enzymes involved in transporting fatty acids across the mitochondrial membrane for β-oxidation ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). RNAi of F09F3.9 did not detectably alter overall fat content, however, suggesting that interference of F09F3.9 expression alone is not sufficient to affect overall fat consumption under the conditions surveyed in our study. In contrast, we found that RNAi of *ech-1,* another β-oxidation gene targeted by *nhr-49,* was sufficient to increase overall fat content; thus it is likely that the reduced expression of *ech-1* also contributes, in some fashion, to the high-fat phenotype of *nhr-49(nr2041).* Thus, we have demonstrated that *nhr-49* strongly promotes the expression of three separate enzymes predicted to participate in mitochondrial β-oxidation. Consequently, we suggest that *nhr-49* plays a prominent role in promoting the breakdown of fatty acids in mitochondria, and that, through control of β-oxidation, *nhr-49* influences overall fat storage. In addition to its effect on β-oxidation, we found that *nhr-49* helps to maintain the balance of saturated and monounsaturated fatty acids. By stimulating the expression of three fatty acid Δ9-desaturase genes, *nhr-49* facilitates the conversion of saturated fatty acids to monounsaturated fatty acids, and consequently, *nhr-49(nr2041)* mutant animals are high in saturated fat and lower in monounsaturated fat. In *C. elegans,* the FAT-6 and FAT-7 SCDs may also be involved in the synthesis of PUFAs \[[@pbio-0030053-b17]\]. However, a dramatic reduction in *fat-7* expression, either by *nhr-49(nr2041)* deletion or by *fat-7* RNAi, did not significantly affect PUFA synthesis. This result suggests that a normal level of FAT-7 expression is not necessary for PUFA synthesis, likely because FAT-6 is the principle enzyme in this pathway, or because FAT-6 is capable of substituting for reduced FAT-7 activity. In either event, we propose that the primary effect of *nhr-49* on fatty acid composition is a modulation of the ratio of C18:0 to C18:1n9 fatty acid ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). However, we cannot rule out the possibility that *nhr-49* and *fat-7* also affect PUFA synthesis in a more localized manner, for example, specific cell types, such that changes in PUFA abundance are not detected in our whole-worm analysis. Our results also indicate that the short life-span phenotype of *nhr-49(nr2041)* is due, at least in part, to reduced expression of *fat-7.* The strong correlation between the increase in C18:0 saturated fatty acid and the shortening of life span is compelling. Several reports in mammals have described changes in fatty acid composition during aging, and it has been argued that an alteration of fatty acid saturation ratios in mitochondrial membranes may affect the rate of aging \[[@pbio-0030053-b20]\]. Additionally, imbalance of fatty acid saturation has also been linked to numerous pathological conditions \[[@pbio-0030053-b15],[@pbio-0030053-b16]\]. Although SCDs have not yet been linked to life span in mammals, studies in C. elegans have identified *fat-7* as one of the targets of the mechanisms that extend longevity through the insulin pathway \[[@pbio-0030053-b21]\]. Thus, it will be interesting to determine if the shortened life-span phenotype of *nhr-49(nr2041)* and *fat-7 RNAi* animals results from an improper ratio of saturated and monounsaturated fats, and if the mechanistic cause of the reduced life span is related to accelerated aging or to another pathology. Although it is clear that *nhr-49* independently promotes the expression of genes in fatty acid β-oxidation and desaturation, we found that downstream components of these pathways do indeed communicate with each other. By inhibiting the expression of genes involved in β-oxidation, including the *nhr-49*-dependent genes *acs-2* and *ech-1, fat-7* works against the actions of *nhr-49,* acting to hinder fat consumption ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). Thus, our results reveal an elegant feedback mechanism: *nhr-49* induces the expression of β-oxidation enzymes---thus promoting fat consumption---yet by independently stimulating *fat-7* expression, *nhr-49* tempers the expression of the very same β-oxidation genes ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). Thus, we suggest that *nhr-49* serves as a key regulator of fat usage, governing pathways that consume fat for energy and partition fat for storage, potentially shifting the balance in response to changing energy needs. Indeed, we have found that in response to starvation, *nhr-49* selectively modulates the expression of these two pathways in order to increase fat consumption (M. R. Van Gilst and K. R. Yamamoto, unpublished data). As the predominant function of *fat-7* is to convert C18:0 to C18:1n9 fatty acid, it is tempting to speculate that one of these two fatty acid species serves as a signal to modulate the feedback effect of *fat-7* on fatty acid β-oxidation ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). In this scenario, C18:0 could be serving as an activator of β-oxidation gene expression, C18:1n9 could be functioning as a repressor, or both ([Figure 6](#pbio-0030053-g006){ref-type="fig"}). Although it seems reasonable to propose that an NHR would communicate this fatty acid signal, we found that the effects of *fat-7* on fatty acid β-oxidation were not dependent on *nhr-49;* hence, one of the other 283 C. elegans NHRs may mediate this effect. Despite its strong sequence similarity with the mammalian HNF4 receptors, the overall biological effects of *nhr-49* on metabolism, fat storage, and life span are remarkably similar to those of the mammalian PPARs. Perhaps most striking is the finding that *nhr-49* and the PPARs, particularly PPARα and PPARδ, positively influence similar genes in multiple metabolic processes, including fatty acid β-oxidation, fatty acid desaturation, and fatty acid binding/transport \[[@pbio-0030053-b22],[@pbio-0030053-b23]\]. Moreover, knockout of PPARα or PPARδ can lead to high-fat phenotypes \[[@pbio-0030053-b23],[@pbio-0030053-b24]\], demonstrating that the physiological effects of these receptors on fat storage are similar to those of *nhr-49.* The regulation of SCDs by *nhr-49* also parallels the regulation of the SCD1 gene by PPARα. In mammals, the Δ9-desaturase SCD1 is a lipogenic enzyme that is controlled by several key regulators of fat storage, including PPARα, leptin, and sterol response element-binding protein \[[@pbio-0030053-b15],[@pbio-0030053-b25],[@pbio-0030053-b26]\]. When SCD1 expression is left unchecked, as occurs in leptin knockout mice, severe obesity can result. Similar to what we found for the *C. elegans fat-7* gene, mammalian SCD1 affects fat storage by inhibiting β-oxidation \[[@pbio-0030053-b27]\]. Thus, because PPARα also promotes fat consumption by stimulating β-oxidation gene expression\[[@pbio-0030053-b28]\], it is clear that PPARα is independently regulating pathways that promote fat consumption and stimulate fat storage. In fact, a similar feedback mechanism to what we have described here for *nhr-49* has been proposed for PPARα and SCD1 \[[@pbio-0030053-b27],[@pbio-0030053-b29]\]. Taken together, these results demonstrate that the multicircuited regulatory networks governing fat usage appear well conserved between *C. elegans nhr-49* and the mammalian PPARα. Although we have not yet determined whether or not the *nhr-49*-dependent genes identified in this study are direct transcriptional targets, each is homologous to known transcriptional targets of the PPARs \[[@pbio-0030053-b22],[@pbio-0030053-b23]\]. Because NHR-49 and the PPARs are clearly regulating similar physiological processes and gene networks, we believe that they are likely to be using similar molecular mechanisms, including direct transcriptional regulation. However, it is possible that *nhr-49* controls fatty acid β-oxidation and desaturation through an indirect mechanism, functioning upstream of the transcription factors that directly act on the *nhr-49*-dependent genes identified in this study. The latter possibility would also be intriguing, as it would demonstrate NHR involvement in fat regulation at a point upstream of a PPAR-like factor. As the physiological functions and regulatory targets of *nhr-49* are highly similar to those of the mammalian PPARs, it seems reasonable to speculate that, like the PPARs, NHR-49 may be regulated by fatty acid ligands. By binding directly to ingested lipids and/or their metabolic products, NHR-49 could serve both as a sensor of intracellular fatty acid levels and an effector, responding to changes in fatty acid composition through modulation of fatty acid β-oxidation and desaturation. Interestingly, the mammalian HNF4 receptors, whose LBDs share 33% identity with the NHR-49 LBD, can bind to several saturated and monounsaturated fatty acids, including stearic and oleic acid \[[@pbio-0030053-b30],[@pbio-0030053-b31]\]; the mammalian PPARs also bind to many types of fatty acids, although they interact preferentially with PUFAs \[[@pbio-0030053-b22]\]. It will be interesting to determine if mammalian HNF4 receptors harbor an undiscovered capacity to regulate fat metabolism in mammals, or if PPARs have adopted their regulatory functions by evolving from an ancestral HNF4 gene that had properties similar to *nhr-49* and the modern PPARs. Alternatively, the PPARs and *nhr-49* may have acquired their activities through convergent evolution. Precise regulation of fatty acid β-oxidation and desaturation is crucial for physiological homeostasis; imbalance in these metabolic pathways in humans can lead to disorders such as diabetes, obesity, atherosclerosis, and accelerated aging. However, although NHRs are important targets for drugs that moderate these metabolic disorders, the NHR-mediated regulatory networks that govern fat usage are notably complex and poorly understood. We showed here that many features of *C. elegans nhr-49* regulation of fat metabolism are closely analogous to those of the mammalian PPARs, key targets for the treatment of metabolic disease. Moreover, our C. elegans studies revealed that *nhr-49,* like PPARα, oversees a branch point for two separate circuits within a network that controls fatty acid consumption and composition. The extent of the functional homology between *nhr-49* and PPARα remains to be determined, that is, whether or not *nhr-49* also functions by binding to fatty acid ligands and directly regulating gene transcription. Nevertheless, our finding that *nhr-49* is a key regulator of fat metabolism in C. elegans represents a considerable advance in our understanding of NHR control of fat storage and composition in invertebrates. Consequently, we suggest that these and future studies in C. elegans will continue to enhance our understanding of the complex roles of NHRs and their ligands on gene networks governing fat physiology. Materials and Methods {#s4} ===================== {#s4a} ### RNAi constructs {#s4a1} All of the RNAi constructs used in the NHR screen were created by cloning full-length NHR cDNAs into the vector L4440 (Andy Fire, Stanford University). The *fat-7* RNAi vector was a gift from Jennifer Watts (Washington State University). All of the other RNAi constructs were obtained from the Ahringer RNAi library \[[@pbio-0030053-b32]\]. ### Life span assays {#s4a2} Approximately 30--40 starved L1 worms were transferred to RNAi bacteria (HT115 transformed with RNAi clone) and life-span assays were carried out at 23 °C as described previously \[[@pbio-0030053-b33]\]. *nhr-49(nr2041)* and WT life-span assays were carried out at 23 °C on OP50 bacteria. ### Nile Red assays {#s4a3} Nile Red fat-staining assays were carried out as described previously \[[@pbio-0030053-b12]\]. ### Selection of fatty acid and glucose metabolism genes {#s4a4} Most of the fatty acid and glucose metabolism candidate genes examined in this study were initially identified using the KEGG pathway database (<http://www.genome.ad.jp/kegg/pathway.html>). Other genes were identified using BLAST searches designed to find C. elegans open reading frames that were highly related to known mammalian glucose and fat metabolism enzymes, or to plant glyoxylate cycle genes. For prediction of β-oxidation enzyme subcellular localization, we used the following online prediction tools. TargetP server version 1.01 (<http://www.cbs.dtu.dk/services/TargetP/>) \[[@pbio-0030053-b34]\] and PSORT (<http://psort.nibb.ac.jp/form.html>). ### Preparation of nematode total RNA {#s4a5} Both C. elegans N2-Bristol (WT) and *nhr-49(nr2041)* were grown at 23 °C on high-growth plates seeded with OP50 bacteria. Gravid adults from 30 10-cm plates were bleached, and embryos were dispersed onto 15-cm nematode growth media (NGM)-lite plates seeded with OP50. The worms were distributed as follows: for L1 harvest, 80,000 embryos/plate; for L2 harvest, 40,000 embryos/plate; for L3 harvest, 20,000 embryos/plate; and for L4 harvest, 10,000 embryos/plate. The remaining embryos were saved for embryonic RNA preparation. Worms were harvested at the appropriate stage, washed twice with M9, and frozen in liquid nitrogen. For RNAi experiments, 10,000 embryos were placed onto NGM-lite plates containing 8 mM IPTG and 100 μg/ml carbenicillin seeded with control bacteria (HT115 transformed with empty L4440 vector) or *nhr-49* RNAi bacteria (HT115 transformed with L4440-*nhr-49*). Worms were harvested at the L4 stage of development, washed twice with M9, and frozen in liquid nitrogen. For RNA preparation, worms were thawed at 65 °C for 10 min, and RNA was isolated using the Tri-Reagent Kit (Molecular Research Center, Cincinnati, Ohio, United States). Isolated total RNA was subjected to DNAase treatment and further purification using RNAeasy (Qiagen, Valencia, California, United States). ### QRT-PCR {#s4a6} cDNA was prepared from 5 μg of total RNA in a 100-μl reaction using the Protoscript cDNA preparation kit (New England Biolabs, Beverly, Massachusetts, United States). Primer pairs (primer sequences available upon request) were diluted into 96-well cell culture plates at a concentration of 3 μM. Next, 30-μl PCR reactions were prepared in 96-well plates. Each PCR reaction was carried out with *Taq*DNA Polymerase (Invitrogen, Carlsbad, California, United States) and consisted of the following reaction mixture: 0.3 μM primers, 1/500th of the cDNA reaction (corresponds to cDNA derived from 10 ng of total RNA), 125 μM dNTPs, 1.5 mM MgCl~2~, and 1X reaction buffer (20 mM Tris pH 8.4, 50 mM KCl). 0.15 μl (0.75 units) of *Taq*DNA Polymerase was used for each reaction. Formation of double-stranded DNA product was monitored using SYBR-Green (Molecular Probes, Eugene, Oregon, United States). All QRT-PCR reactions were carried out and analyzed on a DNA Engine-Opticon 2 (MJ Research, Waltham, Massachusetts, United States). Data were collected using RNA from at least three independent C. elegans growths. To determine the relationship between mRNA abundance and PCR cycle number, all primer sets were calibrated using serial dilutions of cDNA preparations. Primer sets were also calibrated by performing QRT-PCR reactions on serial dilutions of C. elegans genomic DNA. Relative abundance is reported as the mRNA abundance of each gene relative to the mRNA abundance of several control genes, which are expressed at constant levels throughout development. ### GC/MS {#s4a7} Fatty acids were isolated from 10,000 L4 animals grown on a single 15-cm NGM-Lite plate. For WT and *nhr-49(nr2041)* experiments, worms were grown at 23 °C on a lawn of OP50. For RNAi, 10,000 embryos were placed on control bacteria, or on *fat-5, fat-7, ech-1, acs-2,* or *nhr-49* RNAi bacteria. Fatty acid extract was prepared as described previously \[[@pbio-0030053-b17]\]. GC/MS spectra were collected on an HP 6890 gas chromatograph outfitted with a J&W DB-XLB column. The mass spectrometer was an HP MSD 5973 (Agilent, Palo Alto, California, United States) and data were analyzed using Chemstation version A.03.00 software (Agilent). Peaks were assigned using fatty acid standards. ### GFP reporter analysis and ectopic expression of ACS-2::GFP {#s4a8} To make the P*~nhr-49~*GFP plasmid, a genomic fragment including 3.5 kb of the *nhr-49* upstream sequence and 88 bp of the first exon of *nhr-49* was ligated into the L3691 GFP expression vector (a gift of A. Fire). Germ-line transformation was performed following standard procedures \[[@pbio-0030053-b35]\]. P*~nhr-49~*GFP was injected at the following concentrations: 1 ng/μl, 5 ng/μl and 20 ng/μl along with the co-injection marker pRF4 *rol-6(su1006)* injected at 60 ng/μl. At all three P*~nhr-49~*GFP concentrations, we were unable to obtain stably transmitting lines. Animals used for GFP imaging were derived from the F1 generation and were mosaic, hosting the P*~nhr-49~*GFP transgene in only a subset of tissues. To construct *acs-2::gfp,* a 4-kb genomic fragment, including the full-length *acs-2* gene plus 2 kb of upstream sequence, was cloned into the vector L3691. Germ-line transformation was performed using 50 ng/μl of *acs:2::gfp* and 50 ng/μl pRF4 *rol-6(su1006).* For N2/*acs-2::gfp* and *nhr-49(nr2041)/acs-2::gfp,* over ten independent transmitting lines were obtained and analyzed by GFP microscopy. GFP reporter expression was observed using a Zeiss Axiovert S100 fluorescence microscope (Carl Zeiss, Thornwood, New York, United States) under a 40X objective. For rescue experiments, 50--100 ng/μl of *acs-2::gfp* was injected into *nhr-49(nr2041)* and WT animals along with 50 ng/μl of pRF4 rol-6(su1006). Transgenic adults, selected for rolling behavior, were placed on Nile Red plates and assayed for fat content as described above. pRF4 *rol-6(su1006)* was injected into *nhr-49(nr2041)* in combination with unrelated plasmids to demonstrate that the lowered fat content was not a result of hosting a transgenic array or caused by expression of the *rol-6(su1006)* marker gene. Supporting Information {#s5} ====================== Table S1 ::: {.caption} ###### Raw Data for QRT-PCR Screen Data are shown as C~t~, the number of cycles required for amplification of a particular target gene from a cDNA preparation. Changes in gene expression in the *nhr-49(nr2041)* mutant were measured as ΔC~t~. ΔC~t~ are obtained by subtracting *nhr-49*(C~t~) from N2(C~t~) and adjusting for RNA concentration differences with a correction factor (CF). The CF was determined by aligning the data to a set of standard genes. Thus the equation for ΔC~t~ is as follows: Genes were designated as *nhr-49* dependent if the C~t~ was \>1.0 in multiple developmental stages (and was greater than the standard error). *nhr-49* targets are marked with an X. The QRT-PCR results of the major *nhr-49* targets, *acs-2, ech-1, fat-5,* and *fat-7,* were confirmed with a second primer pair. (100 KB XLS). ::: ::: {.caption} ###### Click here for additional data file. ::: We thank Carl Johnson and Nemapharm Pharmaceuticals for the *nhr-49(nr2041)* strain and Trisha Brock and Jennifer Watts for the *fat-7* RNAi vector. We also thank Kaveh Ashrafi, Barbara Meyer, Jennifer Watts, Neil Freedman, Hans Luecke, Marc Diamond, and Holly Ingraham for critical comments on the manuscript, and Jen-Chywan Wang, Ann Sluder, and members of the Yamamoto laboratory for useful discussions. MRVG was supported by a Helen Hay Whitney Fellowship and by a National Institutes of Health grant (NIH K01 DK61361--01). Research support was from NIH. **Competing interests.** The authors have declared that no competing interests exist. **Author contributions.** MRVG and KRY conceived and designed the experiments. MRVG, HH, and AJ performed the experiments. MRVG analyzed the data. MRVG contributed reagents/materials/analysis tools. MRVG and KRY wrote the paper. Citation: Van Gilst MR, Hadjivassiliou H, Jolly A, Yamamoto KR (2005) Nuclear hormone receptor NHR-49 controls fat consumption and fatty acid composition in *C. elegans.* PLoS Biol 3(2): e53. GC/MS : gas chromatography/mass spectrometry GFP : green fluorescent protein HNF4 : hepatocyte nuclear factor 4 LBD : ligand binding domain NGM : nematode growth media NHR : nuclear hormone receptor PCR : polymerase chain reaction. PPAR PUFA : polyunsaturated fatty acid QRT--PCR : quantitative RT-PCR RNAi : RNA interference SCD : stearoyl-CoA desaturase WT : wild-type

PubMed Central

2024-06-05T03:55:52.440163

2005-2-8

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC547972/", "journal": "PLoS Biol. 2005 Feb 8; 3(2):e53", "authors": [ { "first": "Marc R. Van", "last": "Gilst" }, { "first": "Haralambos", "last": "Hadjivassiliou" }, { "first": "Amber", "last": "Jolly" }, { "first": "Keith R", "last": "Yamamoto" } ] }

PMC548128

Background ========== Recent technological advances have led to an explosive growth in high-throughput genomic and proteomic data such as DNA microarrays. The rapid growth in available data has led in turn to a need for novel quantitive methods for analysis. As a consequence of this need, the reconstruction of gene network architectures from DNA microarray expression data has become a major goal in the field of systems biology. An increased understanding of the network architectures and their respective dynamics will enable novel approaches to disease treatments by allowing us, for example, to identify drug targets *in silico*which manipulate the functional outputs of these networks. This process is expected to lead to novel classes of drug based on a network approach to cellular dynamics. Frequently, the gene expression data itself is derived from perturbation experiments such as stress conditions, temperature shifts, and chemical treatments; for example, the widely used yeast cell-cycle datasets of Cho \[[@B1]\] and Spellman \[[@B2]\]. Although these global perturbations are carried out in order to reveal causality between genes, it is not always clear how experiments should be designed so as to reveal as much causality as possible, while both minimising costly experimentation and remaining computationally tractable. A range of computational and mathematical techniques have been adopted in the effort to find a successful gene network reconstruction technique. Reconstruction methods often have to negotiate a tradeoff between intensive (often intractable) computations, and having to perform a large number of costly experiments. Certain progress can be achieved by making simplifications, such as imposing a limit on the number of inputs to each gene, or making steady state assumptions about the system \[[@B3],[@B4]\]. Some techniques described in the literature offer efficient algorithms, but require a large number of experiments, perhaps as many as there are genes \[[@B5]-[@B7]\]. On the other hand, theoretical work on Boolean models has shown \[[@B8]\] that perhaps as few as *O*(*log*(*n*)) experiments (input/output pairs) might be required for *n*genes, but that to infer these relationships requires the use of computationally costly enumeration methods. In this paper, we propose to explore the issue of how perturbation microarray experiments might be designed, and to suggest how such experiments might be optimised so as to maximize inference capability. Logical gene network models have previously been used to investigate gene network robustness \[[@B9]\], perturbation dynamics \[[@B10]\] and evolutionary potential \[[@B11]\], and form the basis of the inference method used in this study. This inference method \[[@B11]\] is similar to others in which networks with a minimal number of connections are reconstructed through enumeration \[[@B12],[@B13]\]. Given the significant speed advantage of integer computation over floating point computation, and that most genes are expected to have few inputs (93% have between 1 and 4 \[[@B14]\]), the method is considered to be adequate for this investigation. In this study, exhaustive evaluation was performed up to a maximum of 4 inputs of both positive and negative sign (see Methods). Enumeration is computationally feasible on an ordinary desktop computer for medium-sized networks (*n*\~ 100), and still tractable for large networks (*n*\~ 1000), though this would require some parallelisation. The global perturbations themselves are simulated by changing the state of each gene at random. A perturbation intensity measure *q*, defines the probability that each gene will change state (see Methods). Results and discussion ====================== A limited number of perturbations significantly improve accuracy ---------------------------------------------------------------- A discrete dynamical model was used to generate time series data from random networks (see Methods). To measure the effect of adding perturbations on inference ability, inference *sensitivity*(defined as true positives/true positives + false negatives, see Methods) was measured against *P*, the number of additional perturbations. Figure [1](#F1){ref-type="fig"} shows the results for predicted solutions with one and two inputs, as well as overall sensitivity. The top graph in figure [1](#F1){ref-type="fig"} shows that overall sensitivity is clearly enhanced by including more perturbation experiments, with lower order solutions (one and two inputs) reaching higher levels of sensitivity. The bottom graph shows the corresponding inverse relationship for the standard deviation of the sensitivity (lower for higher *P*). It should be noted that the algorithm tends to underestimate the number of inputs a gene may have. This is to be expected in genes for which dynamics cannot be informative: for example, consider a gene *i*which has one or more negative inputs, as well as having default value OFF. Since the discrete dynamics for this gene will be the same as if it had no inputs at all (i.e. zero gene expression for *t*\> 0), the presence of the inputs is impossible to infer. This underestimation effect is clear in table [1](#T1){ref-type="table"}, which compares the distribution of inferred solution set sizes (\|*Y*~*i*~\|, see Methods) with the actual solution sizes (i.e. the indegree distribution), and shows that the method is only able to produce roughly half the number of one and two input solution sets that actually exist. The increase in sensitivity with *P*can be explained at least partially, in the following way. Since the time series are discrete, many of the genes may have identical behaviour over time despite having different inputs (i.e. *s*~*i*~(*t*) = *s*~*j*~(*t*) for two different genes *i*and *j*). If we define a \"concatenated\" time series vector ![](1471-2105-6-11-i1.gif) for gene *i*, and then map each gene *i*onto *S*~*i*~, we obtain a many-to-one mapping. As we increase the number of perturbations, we might expect the number of distinct time series also to increase. We define a simple measure to quantify this mapping, *M*= *n*\'/*N*where *n*\' is the number of distinct vectors *S*~*i*~, and *N*is the number of genes. The maximum value of *M*= 1 indicates that the mapping of genes to time series is one-to-one, whereas lower values indicate degenerate mappings. The manner in which *M*increases with the number of perturbations is shown in figure [2](#F2){ref-type="fig"}, and shows how the increase in M reflects the corresponding increase in sensitivity (figure [1](#F1){ref-type="fig"}). Network size and optimal perturbation intensity ----------------------------------------------- The experiments described above were repeated to consider variations in two other parameters: the network size *N*, and the perturbation intensity parameter *q*(roughly, the proportion of genes whose initial expression level is changed in each perturbation experiment -- see Methods). To consider the first case, the minimum number of perturbations *P*\* required to reach a given high accuracy criterion was measured for different values of the network size *N*. The high accuracy criterion was defined as average sensitivity = 0.95 for one-input solution sets (average sensitivity is found using a default value q = 0.5 and averaging for all the sensitivity measurements obtained from 250 random networks). To find *P*\*, we first find the number of perturbations *P*^+^, such that average sensitivity *P*^+^≥ 0.95, and average sensitivity (*P*^+^- 1) \< 0.95. If average sensitivity *P*^+^\> 0.95, we use simple linear interpolation to find the (real) value of *P*\* between *P*^+^and (*P*^+^- 1) for which average sensitivity = 0.95. The resulting values for *P*\* are shown in figure [3](#F3){ref-type="fig"}. Since the relationship is expected to be logarithmic \[[@B8]\], the plot shows *log*(*N*) against *P*\* (logarithms used are base 10). A least squares best fit gives *P*\* ≃ 1.75 *log*(*N*) + 7.02, which, for *N*= 1000, gives *P*\* ≃ 12.26. In order to obtain a measure of variance for *P*\*, we would need to calculate *P*\*-equivalent values for many individual networks separately, then consolidate these values to obtain the relevant statistics. However, because it was only feasible to consider medium-sized networks (20 ≤ *N*≤ 70), and for any such network we often find only a small number of one-input solution sets, such statistics were found to be unreliable. The second case (varying perturbation intensity) suggests an optimal range for *q*. Figure [4a](#F4){ref-type="fig"} shows the inference sensitivity over a range of values for *q*, and figure [4b](#F4){ref-type="fig"} shows the corresponding standard deviation. Again, inference sensitivity for one-input solutions is higher than for two-input solutions, which in turn is higher than overall sensitivity. For one-input solutions, the results show a clear peak for sensitivity close to the range 0.5 \<*q*\< 0.6. Together with a corresponding minimisation of the standard deviation in this interval (though it still remains fairly high in absolute terms), these results suggest that perturbation intensity should be close to this range to optimise inference accuracy. Conclusions =========== A recent analysis of the yeast genetic network has shown that 93% of genes are regulated by between 1 and 4 genes \[[@B14]\]. This suggests that enumerative network reconstruction methods can be useful within computationally feasible limits. Experiments involving large-scale perturbations (such as temperature shifts, chemical stress) are a standard way of obtaining time-series of gene expression data \[[@B1],[@B2]\]. A key result of \[[@B14]\] is that indegree appears to follow an exponential distribution, whereas outdegree follows a scale-free distribution, which has enabled the generation of realistic artificial gene networks used here. A logical model \[[@B11]\] was used to simulate the perturbed expression data. Subsequently, experimental parameters were considered in relation to inference accuracy, namely: a) number of perturbations required, *P*, and b) perturbation intensity, *q*. The inference method itself is most useful for low order inputs, with inference accuracy maximized for predicted single input genes. More accurate methods have been proposed, though these generally require a much larger number of experiments \[[@B5],[@B15]\]. Methods such as the one proposed here, which infer relationships from expression data may well be more successful when used in conjunction with other methods such as promoter analysis \[[@B16],[@B17]\], or when used to drive experimental procedure \[[@B18]\]. Here, the results show that only a relatively small number of perturbations are necessary in order to achieve a substantial inference accuracy, even for large *N*. These relatively modest experimental requirements would presumably imply lower experimental costs. The results also suggest that the perturbations should be calibrated (by changing stress intensity, for example), so as to alter the expression levels of approximately half the genes in each experiment. Generating perturbations which alter the expression level of half the genes at random may be difficult to achieve in practice, though experiments can be designed to come as close to this goal as possible. Even in the absence of optimal perturbations, we hope the simulation approach described here will still serve as a useful tool for planning experiments. Methods ======= Discrete dynamical model ------------------------ For a system of *N*genes, the state of each gene *s*~*i*~(*i*= 1, .., *N*) is represented by the binary values 0(OFF) and 1(ON). Additionally, each gene is assigned a default ON/OFF state *θ*~*i*~∈ {0, 1}. The gene interactions are described by an (*N*× *N*) matrix *C*, composed of elements *C*~*ij*~∈ {-1, 0, +1}, representing the positive(+1), zero(0) or negative(-1) influence of gene *j*on gene *i*. State transitions are calculated as follows: ![](1471-2105-6-11-i2.gif) The state of the *i*th gene at the next timestep, *s*~*i*~(*t*+ 1), is therefore determined by the balance of positive versus negative inputs which are ON at the previous timestep *t*. If the balance is positive, then *u*~*i*~(*t*) \> 0 and the next state will be 1(ON). Similarly, if the balance is negative, then *u*~*i*~(*t*) \< 0 and the next state will be 0(OFF). If *u*~*i*~(*t*) = 0 (indicating either that there are no active input connections, or that they balance out), then the default value *θ*~*i*~determines the next state. This default value needs to be given *a priori*, and for the purpose of this study will be random. Network inference method ------------------------ Assuming we are given the state dynamics *s*(*t*) and the default vector *θ*, the problem is to find the necessary model parameters (*C*) which will reproduce these dynamics. Specifically, a system initialised at *s*(0) should reproduce the given dynamics *s*(*t*) for *t*\> 0. Note that multiple *s*(*t*) expression patterns may be defined, which will be denoted as *s*^*r*^(*t*) for *r*= 0, .., *P*, corresponding to time series with different initial states *s*^*r*^(0). Our problem is to find at least one interaction matrix that will reproduce all given dynamics *s*^*r*^(*t*). The problem of finding an appropriate matrix *C*may be broken up into *N*sub-problems, since in this system, each gene *i*may be solved independently from the others. More precisely, the inputs to gene *i*(i.e. *C*~*i*~, the *i*th row of *C*), can be found independently of the other genes. This reduces the search space from ![](1471-2105-6-11-i3.gif) down to *O*(*N*3^*N*^). Each input *z*^*i*^to gene *i*is represented as an ordered pair (*j*, *g*), *j*∈ {1, .., *N*}, *g*∈ {± 1}, indicating an input from gene *j*of sign *g*. A solution *y*(*i*) for gene *i*is a set of *K*inputs ![](1471-2105-6-11-i4.gif) (with *y*(*i*) = *φ*if *K*= 0). For *K*inputs there are ![](1471-2105-6-11-i5.gif) solutions to evaluate. Starting with *K*= 0 (no inputs), we progress up to a maximum of *K*= 4, exhaustively evaluating all possible solutions for each *K*. However, making a parsimony assumption, if solutions are found for some *K*~*s*~\< 4, the method no longer evaluates for *K*\>*K*~*s*~. Note that the method does not stop as soon as a solution is found, but evaluates all possible solutions for *K*~*s*~. The failure rate (percentage of genes for which no solution was found for *K*≤ 4) never exceeded 3% of the genes in any single network for which reconstruction was attempted. Global perturbations and the perturbation intensity measure ----------------------------------------------------------- The control time series *s*^0^(*t*) is generated by setting *s*^0^(0) = *θ*. The other time series *s*^*r*^(*t*), *r*\> 0 are obtained from initial conditions which are perturbations of *θ*, and correspond to standard experiments such as stress conditions, or chemical treatments. Since, experimental perturbations can usually be modulated in intensity (for example, a temperature shift), this was represented using modulated artificial perturbations. Perturbed initial states *s*^*r*^(0) were generated by randomly changing each state *s*^0^(0) with probability *q*. This means that, on average, there will be *qN*random state differences between each perturbed initial state *s*^*r*^(0), and *θ*. Measuring inference accuracy ---------------------------- Assuming one or more solutions *y*~1~(*i*), *y*~2~(*i*), \... are found for gene *i*, these are consolidated into a solution set, *Y*~*i*~= ∪~*l*~{*y*~*l*~(*i*)}. Note that some information about the solutions has been lost using this approach. For example, a solution set ![](1471-2105-6-11-i6.gif) obtained from a single two-input (*K*= 2) solution: ![](1471-2105-6-11-i7.gif), may be equal to another solution set ![](1471-2105-6-11-i8.gif) resulting from two single-input (*K*= 1) solutions: ![](1471-2105-6-11-i9.gif) with ![](1471-2105-6-11-i10.gif) and ![](1471-2105-6-11-i11.gif). However, this consolidation is convenient in that the solution set is easily compared with the known network structures using standard accuracy measures such as *sensitivity*and *specificity*. Here, accuracy was measured using *sensitivity*, defined as true positives / (true positives + false negatives). The relatively large number of true negatives, makes *specificity*, defined as true negatives / (true negatives + false positives), an uninformative statistic. Here, *true positives*are members of the solution set *Y*~*i*~which are also true inputs (since the networks will be generated artificially, true inputs are known), and *false negatives*are those true inputs which are not members of the solution set *Y*~*i*~. Accuracy statistics were gathered from inferences performed on a large number of medium-sized random networks (20 ≤ *N*≤ 70). Inferences on *R*random networks (each with *N*genes), will produce approximately *RN sensitivity*measurements (slightly fewer due to the nonzero failure rate). Artificial gene network generation ---------------------------------- It appears to be the case in gene networks that indegree follows an exponential distribution, whereas outdegree appears to follow a scale-free distribution. More specifically, for the yeast network, the probability distribution for indegree *k*follows *p*~*k*~\~ *C*~*in*~*e*^-*βk*^with *β*\~ 0.45, whereas the distribution for outdegree follows *p*~*k*~\~ *C*~*out*~*k*^-*τ*^, with *τ*\~ 1 (*C*~*in*~, *C*~*out*~constants) \[[@B14]\]. Here, artificial gene networks \[[@B19]\] were created using the algorithm for generating directed graphs with arbitrary in/out degree distributions described in \[[@B20]\]. The exponential probability distribution for indegree *k*is given by: *p*~*k*~= (1 - *e*^-*β*^)*e*^-*βk*^, where *β*= 0.45 is a constant. Similarly, the power law distribution (including an exponential cutoff term which is both biologically realistic and necessary analytically when *τ*\< 2 \[[@B20]\]) for outdegree *k*is described by: *p*~*k*~= *Ck*^-*τ*^*e*^-*γk*^, where *C*, *γ*, and *τ*= 1 are constants. Since the algorithm begins by generating in/out-degree pairs for each node, we require equal means for both indegree (\<*k*~*in*~\>) and outdegree (\<*k*~*out*~\>). Following \[[@B20]\], we obtain expressions for the mean in/out degree: ![](1471-2105-6-11-i12.gif) Since *β*is given, we obtain a value \<*k*~*in*~\> = 1.76, and fit the free parameter *γ*= 0.436 to obtain \<*k*~*out*~\> = \<*k*~*in*~\> Since the resulting networks are unweighted, non-zero weights (*C*~*ij*~∈ {-1, +1}) are assigned at random with probability 0.5, as in \[[@B19]\]. It should be noted that autoregulatory interactions can be (and indeed were) generated, and that these present no particular problem for the inference method. An example of a network which was used in the analysis is shown in figure [5](#F5){ref-type="fig"}. Authors\' contributions ======================= TM devised and implemented the experiments and drafted the manuscript. RS and AP supervised the project. All authors read and approved the final manuscript. Acknowledgements ================ TM was supported by a grant from the UK Medical Research Council (MRC). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Sensitivity vs. *P.***(a) Sensitivity vs. number of additional perturbations used. (b) The corresponding standard deviation is shown here separately for clarity. The curves represent results for overall (i.e. all solutions) sensitivity, and specific sensitivity for (predicted) one and two-input solutions. Sensitivity is generally lower for higher order of inputs. Accuracy increases significantly with the number of additional perturbations used. The results shown are average values for 250 random networks at each data point. The remaining parameters are fixed: network size *N*= 50, perturbation intensity *q*= 0.5. ::: ![](1471-2105-6-11-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### ***M*vs. *P.***M (the number of distinct \"concatenated\" vectors *S*~*i*~divided by N, the number of genes) increases in value, as the number of perturbations (*P*) is increased. The graph shows curves for three values of perturbation intensity *q*. ::: ![](1471-2105-6-11-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Perturbations required for high accuracy**The minimum number of perturbations (*P*\*) required to reach the high accuracy criterion (average sensitivity = 0.95) for different values of the network size *N*. Each point represents the average value for 250 random networks inferred. This is equivalent to finding the value of *P*for which sensitivity = 0.95 on the one-input curve of figure 1(a) for different values of *N*(figure 1(a) shows *N*= 50). A linear fit is also shown. ::: ![](1471-2105-6-11-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Sensitivity vs. *q.***(a) Average inference sensitivity vs. perturbation intensity *q*. (b) The variance (one standard deviation) is shown here separately for clarity. The results show sensitivity for (predicted) one and two-input solutions being generally higher than the overall case. The results shown are average values for 250 random networks inferred. The remaining parameters are fixed: network size *N*= 50 and *P*= 12. ::: ![](1471-2105-6-11-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Example network.**Example of an artificial gene network with *N*= 50. Positive interactions are shown in black, negative interactions in grey. Note the autoregulatory interaction on the upper right hand side. This diagram was generated using Pajek <http://vlado.fmf.uni-lj.si/pub/networks/pajek/>. ::: ![](1471-2105-6-11-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Solution set sizes Distribution for the inferred solution set sizes, compared to the distribution of indegree in the actual network for the simulations. These statistics were produced from 250 random networks run using the following parameter values: *N*= 50, *P*= 12, and *q*= 0.5. The table illustrates how the algorithm overestimates the number of solutions with zero inputs. ::: \|*Y*~*i*~\| 0 1 2 3 ≥ 4 -------------- ------ ------ ------ ------ ------ inferred 0.57 0.12 0.07 0.05 0.19 actual 0.37 0.24 0.15 0.10 0.14 :::

PubMed Central

2024-06-05T03:55:52.444469

2005-1-19

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548128/", "journal": "BMC Bioinformatics. 2005 Jan 19; 6:11", "authors": [ { "first": "Thomas", "last": "MacCarthy" }, { "first": "Andrew", "last": "Pomiankowski" }, { "first": "Robert", "last": "Seymour" } ] }

PMC548129

Background ========== Systems biology is a data- and knowledge-driven discipline, which heavily relies on automated tools to support the generation and validation of hypotheses. Such tasks aim to provide novel and meaningful views of the functional relationships between biological components at different complexity levels. Over the past seven years hundreds of methods have been reported to analyse these data, with an emphasis on gene expression data classification \[[@B1],[@B2]\]. More recently, the analysis of gene regulatory and protein-protein networks has started to attract contributions from computer and physical sciences \[[@B3]-[@B5]\]. All of these tasks are linked by a need for comparing, classifying and visualising information. The ever-increasing number and sophistication of techniques may represent an obstacle to achieve more meaningful and rigorous data analysis and discovery tasks. One important problem is that users may not have the time and knowledge required to adequately understand the dynamics and operation of several tools. These deficiencies have been reflected, for example, in a lack of sound practices for assessing the statistical significance of results and for selecting the most suitable data sets and classification models \[[@B6],[@B7]\]. On the other hand, the emergence of multiple data sets and prediction models represents an opportunity for developing an integrative data mining paradigm, which is already significantly improving several predictive tasks in systems biology \[[@B5],[@B8],[@B9]\]. The problem domains mentioned above have mainly concentrated on the application of statistical and machine learning models for classification tasks. Emphasis has been placed on the development of supervised and unsupervised classification methods \[[@B2],[@B10],[@B11]\], as well as on the application of statistical tools for assessing the quality of classification results \[[@B12],[@B13]\]. Relatively fewer efforts have been reported on *data visualisation techniques*to support *exploratory analysis*. It has been shown that information visualisation techniques may support predictive data mining applications, including data clustering \[[@B14]-[@B16]\]. These tasks should complement each other in order to achieve higher levels of knowledge integration and understanding. Furthermore, visualisation-based exploratory methods may support: a) the identification of key patterns in the data, and b) the selection of the most adequate models for data pre-processing and/or classification. The former task refers to the recognition of key groups of data, outliers and features based on computationally-inexpensive, user-friendly and robust analyses. Its outcomes may offer guidance to conduct the latter task by gaining a better insight into the high-level structure and relationships found in the data. Such an exploratory, visualisation-based approach may generate useful alternative views for supporting a more intelligent and meaningful application of classification models. One of the traditional approaches to functional genomics information visualisation has been the application of clustering-based visualisation techniques. Such an approach mainly consists of two steps: a) the implementation of a clustering algorithm, and b) the display of the obtained clusters. The resulting clusters may be visualised by generating, for example, dendrograms \[[@B16]\], other hierarchical structures \[[@B17]\] and maps \[[@B14],[@B18],[@B19]\], which highlight or summarise similarity relationships between groups of data. Clustering-based visualisation has become a fundamental tool for analysing gene and protein expression data. Different variations of hierarchical clustering, *Kohonen Self Organising Maps*(SOM) and *Self-Adaptive Neural Networks*(SANN) are relevant examples of techniques belonging to this approach. Their capabilities and applications have been widely reported \[[@B2],[@B15],[@B20]\]. They have been successfully tested on several classification and decision support problems. However, its application to visualisation-driven exploratory analysis is limited by several problems: Many of these techniques are not capable of explicitly and automatically detecting cluster boundaries; some of them are critically sensitive to several learning parameters that need to be selected by the user; some of these solutions were not originally designed to tackle cluster-based visualisation tasks of massive collections of data described by several thousands attributes; and they traditionally require assumptions about the inherent structure of the data, which may not be always possible in exploratory data analyses. Clustering visualisation has also become an important task for the analysis of protein-protein interaction networks. Clustering is a fundamental mathematical property of networks, which allows the identification of key connectivity patterns. Such patterns may be associated with significant functional behaviours and modularity \[[@B3]\]. Moreover, it allows researchers to identify relevant areas for further statistical or experimental analyses. For instance, hierarchical clustering of network nodes (proteins) has been applied to detect functional modules in *S. cerevisiae*\[[@B3]\]. Each node may be represented by a vector of connectivity values that reflects the node\'s interactions with other network members. Graph theoretic approaches have also been applied to detect significant clusters of interconnected proteins \[[@B21]\]. Another important data visualisation approach, which may be applied to clustering-based analysis, comprises the application of *non-linear mapping techniques*. They are based on the idea of transforming the original, *n*-dimensional input space into a reduced, *m*-dimensional one, where *m*\<*n*. These methods are also known as *non-linear projection methods*or *multidimensional scaling*(MDS) methods \[[@B22]\]. They mainly aim to optimize a function, *M*, which reflects key aspects of the distance structure of the original *n*-dimensional space. Thus, these methods aim to preserve such global properties in the transformed, *m*-dimensional space. This principle has been followed by several models including those proposed by Kruskal \[[@B23],[@B24]\] and Sammon \[[@B25]\]. *Principal component analysis*(PCA) \[[@B26]\] is another relevant technique for reducing *n*-dimensional data. In this case the resulting transformation accounts for the greatest variation of the original space, but without preserving the distances observed between the points in the *n*-dimensional space. MDS, including Sammon\'s mapping, applications to gene expression analysis have been reported, which highlight their advantages for supporting the detection of clusters \[[@B27]\]. PCA may not be directly used to visualise clusters. But it may be applied as a pre-processing procedure, and its resulting components may be used as inputs to clustering and supervised classification models \[[@B28]\]. Although widely investigated techniques, such as SOM and Sammon\'s mapping, are usually useful to visualise clusters of high-dimensional data, they present several limitations. For example, the SOM may be highly sensitive to its training parameters, which have to be defined by the user. It also requires the user to define map topologies, and it does not provide automated mechanisms for cluster boundary detection. Its limitations for data exploratory analysis, particularly in relation to data topology preservation, have been stressed in \[[@B29]\]. These and other limitations, as well as adapted solutions, have been discussed in \[[@B14],[@B15]\]. Sammon\'s method may include several data overlaps when the *n*-dimensional input space contains noisy or weakly discriminatory information \[[@B30]\]. Depending of the size of the input data (number of points), the number of learning iterations and computational facilities available, Sammon\'s mapping might be computationally expensive. Empirical analyses have shown that Sammon\'s mapping may easily get stuck at local optima \[[@B30]\]. Moreover, it has been demonstrated that this method may be sensitive to the initialisation scheme applied \[[@B19],[@B31]\]. This paper assesses an alternative, non-linear mapping technique which aims to address key limitations exhibited by traditional methods. Its application to clustering-driven exploratory analysis of gene expression data is investigated. Furthermore, it provides the basis for an interactome network clustering visualisation system. The following section summarises relevant results. Results ======= A relaxation method for non-linear mapping was implemented to visualise relevant similarity relationships in data originating from gene expression and interactome data. Such a method was designed by Chang and Lee \[[@B32]\] to address key limitations observed in methods such as those proposed by Sammon \[[@B25]\] and Kruskal \[[@B23],[@B24]\]. These techniques are related because they aim to achieve a space reduction by preserving the structure of local distances in the data. However, unlike those traditional mapping techniques, the method assessed in this paper adapts a pair of points in the transformed *m*-dimensional space at every processing step, instead of adapting all points at every step. Thus, the term \"relaxation\" is taken from the relaxation method for linear equalities \[[@B32]\]. Chang\'s and Lee\'s method showed to outperform Sammon\'s mapping both in terms of cluster detection effectiveness and computational efficiency. A *mapping iteration*is defined as a complete sequence of adaptation steps involving pairs of points, *p*(*i*,*j*), for each *i*≠ *j*(see Methods for a more detailed description). In this study a point may encode a biological sample described by a gene expression profile (e.g. tumour sample), or a protein described by its interaction profile. Figure [1](#F1){ref-type="fig"} summarises the mapping mechanism of this approach. Analysis of gene expression data -------------------------------- Analyses were performed on three publicly available expression data sets. The first one includes 38 samples from a known leukaemia study \[[@B33]\], which are represented by 50 expression values. The samples are categorised into two classes: *Acute myeloid leukemia*(AML) and *acute lymphoblastic leukemia*(ALL). This data set has been previously validated by several experimental and *in silico*methods. It may be considered as an adequate example for illustrating basic capabilities of clustering algorithms. The second data set includes samples originating from *small, round blue-cell tumours*(SRBCT) \[[@B28]\]. These data consisted of 63 samples categorised into four classes: *Ewing family of tumors*(EWS), *rhabdomyosarcoma*(RMS), *Burkitt lymphomas*(BL) and *neuroblastomas*(NB), which are represented by the expression values of 2308 genes with suspected roles in processes relevant to these tumours. The third data set offers another example of gene expression analysis complexity: A data set consisting of a relatively small number of samples described by thousands of gene expression values, without incorporating pre-processing by feature selection or transformation procedures. This application comprises 20 samples described by 9504 gene expression values for both *normal brain*and a pharmacological model of *Parkinson\'s disease*\[[@B34]\]. The reader is referred to the section of Methods for a more detailed description of the data and their prediction tasks. Several experiments were performed on each data set to assess possible, key advantages and limitations of the mapping approach introduced above. Each experiment requires an input file storing a matrix, **A**, where each entry, *a*~*ij*~, represents an expression value, *j*, for a sample, *i*. The user needs to define only one learning parameter, the number of mapping iterations, as defined above. The output of the algorithm is the projection of the *K*samples represented in **A**on the reduced *m*-dimensional space. Mappings were analysed for *m*= 2 and *m*= 3, which allowed the generation of 2D and 3D visual displays. Experiments were also conducted for several numbers of mapping iterations. The results facilitated a high-level understanding of fundamental similarity relationships between the samples, which are also consistent with previous research. Three important exploratory data analysis tasks were accomplished: The automated, unsupervised detection of clusters relevant to the natural class structure of the data sets; the visualisation of a coherent preservation of local similarity (distance) relationships between samples; and the identification of potential outliers. Figure [2](#F2){ref-type="fig"} depicts mapping results for the leukaemia data. Panels (a) to (d) show results for 0, 1, 10 and 100 mapping iterations respectively. Circles are used to represent the samples. Labels \'0\' and \'1\' refer to ALL and AML samples respectively. In the initialisation of the mapping process (0 iterations) the samples are randomly assigned to positions in the 2D space. After 10 iterations there is a clear indication of separation of samples belonging to different classes. With 100 iterations the ALL samples are clustered on the upper left side of the map, and the AML samples are clustered at the bottom of this map. Such clusters were also distinguished in different experiments. As a consequence of the random initialisation process, clusters may occupy different areas on the resulting maps for different experiments using the same number of iterations. Nevertheless, the system correctly separated samples in different experiments with more than 20 iterations. Figure [3](#F3){ref-type="fig"} shows resulting maps for the SRBCT data with 100 mapping iterations. These data were pre-processed as explained in the section of Methods. EWS, RMS, BL and NB samples are represented by symbols \'1\', \'2\', \'3\' and \'4\' respectively. It suggests that the mapping process was able to identify key similarity relationships. Samples belonging to the same class tend to cluster together. For example, EWS samples are mainly located at the bottom of the map. RMS samples are clustered on its left side. The sample \'1\' (*x*: 0.54, *y*: -1.79) that was located on the left side of Figure [3](#F3){ref-type="fig"} far from the EWS cluster (its natural class) was consistently mapped in this fashion by different experiments. Moreover, this sample was also displayed closer to RMS samples for different experiments, which might suggest a significant relationship between such a sample and the RMS class. Furthermore, a previous study using more sophisticated, supervised learning models showed that this sample may be difficult to correctly classify \[[@B35]\]. This suggests that the map shown in Figure [3](#F3){ref-type="fig"} highlights a potential outlier in the EWS class data. Additional experiments using a smaller number of SRBCT classes (only EWS, RMS, BL) were performed. This was mainly done to explore the possibility of obtaining alternative graphical views of the data. Results are in general consistent with the results produced with 4 classes: Samples belonging to the same class were clustered together. However, these experiments allow a clearer graphical differentiation of classes on the resulting maps. Figure [4](#F4){ref-type="fig"} depicts an example obtained with 100 mapping iterations. For this and other experiments, it was also possible to detect the outlier that was observed in Figure [3](#F3){ref-type="fig"}. Such an EWS sample is located at the top of the map shown in Figure [4](#F4){ref-type="fig"}. SOM-based analyses using the *SOM Toolbox*\[[@B36],[@B37]\] were also implemented to establish comparisons. Figure [5](#F5){ref-type="fig"} shows the *unified distance matrix*(*U-matrix*) and the *label map*(panel on the right side) for a representative result. The section of Methods describes the construction of these maps. The SOM was able to correctly cluster the SRBCT samples. However, these standard SOM visualisation techniques do not provide clear information on sample-to-sample similarity relationships. Moreover, they do not adequately facilitate a direct visualisation of the distribution of samples assigned to each node and their associations. [Additional file 1](#S1){ref-type="supplementary-material"} includes the frequency map, which is another standard SOM visualisation technique, for these results. Figure [6](#F6){ref-type="fig"} shows results obtained by applying Sammon\'s mapping. Symbols \'1\', \'2\' and \'3\' represent classes EWS, RMS and BL respectively. This approach is able to detect class differences between samples. Sammon\'s mapping also isolated the EWS sample suggested above as a possible class outlier (right side of the map). Figure [3](#F3){ref-type="fig"} (near *x*: 1.2, *y*: -1.5) suggests another sample \'1\' as a potential outlier. However, the Sammon\'s mapping did not clearly depict it as a potential outlier because in this case, unlike Figure [3](#F3){ref-type="fig"}, this sample is located closer to class \'1\' samples (Figure [6](#F6){ref-type="fig"}, near *x*: 0, *y*: 0.4). 3D Mapping analyses were also implemented for this SRBCT application. Additional files [2](#S2){ref-type="supplementary-material"} and [3](#S3){ref-type="supplementary-material"} compare the results originating from the relaxation non-linear and Sammon\'s mapping methods. Both methods displayed coherent partitions of the data, which are consistent with the 2D mapping results presented above. Moreover, 3D mappings also suggest relatively strong similarities between EWS and RMS samples because of their proximity on the maps. Figure [7](#F7){ref-type="fig"} displays a relaxation non-linear map of the Parkinson\'s disease model data. Parkinson\'s disease and Normal samples are identified by symbols \'1\' and \'2\' respectively. After 100 mapping iterations, results suggest that the method is able to differentiate between these classes. Parkinson\'s disease samples mainly fall on the upper region of the map (*y*\> 0), and 7 (out of 10) Normal samples are located below that area. Figure [8](#F8){ref-type="fig"} shows SOM-based results, which adequately distinguish between classes. It also indicates that a few Normal samples may be closer to Parkinson\'s samples than to the main group of its own class. Figure [8](#F8){ref-type="fig"} may offer a clearer graphical discrimination between classes. However, the SOM-based results are more dependent on the user\'s selection of an optimum set of learning parameters (see Methods). [Additional file 4](#S4){ref-type="supplementary-material"} shows the frequency map for these results. Although the separation of clusters is less clear, Sammon\'s mapping (Figure [9](#F9){ref-type="fig"}) was in general capable of grouping same class samples. In this figure symbols \'1\' and \'2\' represent Parkinson\'s disease and Normal samples respectively. 3D maps originating from relaxation non-linear and Sammon\'s mapping methods are depicted in Additional files [5](#S5){ref-type="supplementary-material"} and [6](#S6){ref-type="supplementary-material"} respectively. Both techniques offer alternative, but consistent exploratory views of the cluster structure of this data set. Analysis of interactome networks -------------------------------- As a way of promoting alternative, potential applications of the methodology presented, this section illustrates an example of exploratory data analysis of interactome networks. The approach proposed encodes a network as a graph of interconnected nodes. For a network consisting of *N*nodes, the mapping tool (from now on referred to as *interClust*) requires an *N*× *N*matrix, **B**, as the input data. In this symmetrical matrix each element, *b*~*ij*~, represents the *connection strength*between nodes *i*and *j*in the graph. Such values may also be interpreted as *weights*representing the relevance of the interaction between a pair of proteins, e.g.: number of hits observed in interaction experiments or a path distance between two nodes on the graph (indirect interactions). Thus, each row in **B**may be seen as the *connectivity profile*for a node, *i*. That is, the connectivity profile of a node becomes this node\'s coordinates in the *n*-dimensional, input space. An accompanying tool, *inBuilder*, automatically generates such a network representation from a list of pairwise protein-protein interactions and their respective connection strengths predefined by the user. The section of Methods provides more information about design and operation aspects of this approach. Before testing this approach on a real interactome data set, a simple example of a network (Figure [10](#F10){ref-type="fig"}) is used to illustrate its application. In this figure the length of the links does not reflect distances or connection strengths. All of the direct connections are considered equally. This network comprises 25 nodes forming 4 main clusters. In this case a cluster is defined as a compact group of interconnected nodes. Figure [11](#F11){ref-type="fig"} shows the results obtained by applying *interClust*to this network with 20 iterations. This map clearly distinguishes the clusters of the network. Moreover, it preserves local interaction relationships: i.e. if two nodes have similar connectivity profiles in the original space (Figure [10](#F10){ref-type="fig"}), then they are also close to each other in the resulting map. Adequate cluster visualisations were also obtained in experiments with more than 5 iterations. This algorithm was tested on the chromatin interactome in *C. elegans*. It includes 303 proteins and 349 interactions. This data set regroups interactions of proteins involved in transcriptional regulation at the chromatin level. This functional module is of major interest because it is at the crossroads of many biological processes such as development, sex determination, cellular differentiation and proliferation. Its misregulation may have strong consequences such as tumorigenesis or developmental defects \[[@B38]\]. This data set includes both retested \[[@B39]\] and non-retested protein interactions obtained by high-throughput two-hybrid screens (unpublished data). Figure [12](#F12){ref-type="fig"} displays resulting relaxation maps, at different regions and levels of detail, obtained with 100 iterations. Figure [12](#F12){ref-type="fig"} indicates a separation of proteins according to their connectivity patterns. The network encoding scheme and the non-linear mapping algorithm applied distinguished highly-connected proteins (*hubs*) from the other components of the network. Hubs are located in the outer regions of the map. The farther a node is from the centre, the more connections it has. Figure [13](#F13){ref-type="fig"} displays a partial view of a region enriched by hubs, which is well-separated from the main group of proteins. The hubs are also relatively well-separated between them. It reflects the fact that such proteins share very few direct, interacting proteins in common. [Additional file 7](#S7){ref-type="supplementary-material"} depicts some of the hubs automatically isolated by the mapping algorithm, as well as a few nodes located near the centre of the map (F15A2.6, C34E10.5, C14B9.6). These diagrams were drawn using the *InterViewer*tool \[[@B40]\]. A closer examination of a group of proteins located in the outer regions of the map (Figures [12](#F12){ref-type="fig"} and [13](#F13){ref-type="fig"}), for example, reveals that they are involved in key processes such as mitosis and meiosis. This differentiation indicates that such hubs may act as connection components between different biological processes. This is not a surprising finding, but it highlights the capacity of the algorithm to automatically detect some of the most biologically-relevant proteins only on the basis of their connectivity profiles. Seven of these hubs (Y2H9A.1, F56C9.1, F11A10.2, T12D8.7, Y113G7B.23, Y37D8A.9, C53A5.3), for instance, show a significant enrichment of phenotypes obtained by depletion of the transcripts by RNA interference (5.6-fold enrichment compared to genome-wide levels \[[@B41]\]). Moreover, four of these hubs (F56C9.1, F11A10.2, T12D8.7, C53A5.3) are strongly linked to embryonic lethality (*Emb*) by exhibiting a 6-fold enrichment of this property in relation to genome-wide levels \[[@B41]\]. Further analyses on the map suggest that neighboring nodes may reflect functional similarity relationships between the corresponding proteins. One example is illustrated in Figure [14](#F14){ref-type="fig"} (upper area), which focuses on the region surrounding T20B12.2, also known as Tbp-1, a key component of the Polymerase II holoenzyme. In this region it is possible to identify several components of the core Polymerase II enzyme, as well as several related families (histones acetylases and deacetylases, nucleosomes positioning enzymes of the Swi/snf family) that share similar phenotypes. There is a strong enrichment of phenotypes such as embryonic lethality or problems in growth. These phenotypes (like embryonic lethality) are often attributed to proteins with high connectivity. Only eleven (out of twenty seven) proteins examined in this region exhibit wild type phenotype when they are depleted. This cluster shows a 5.4-fold enrichment of phenotypes, a 5.1-fold enrichment of Emb phenotype, and a 33.3-fold enrichment of sterile progeny phenotype in comparison to genome-wide levels \[[@B41]\]. This cluster also exhibits significant associations with sterility, growth defect and problems in larval development. [Additional file 8](#S8){ref-type="supplementary-material"} describes the composition of this representative cluster. Discussion ========== With regard to gene expression data, the relaxation non-linear mapping method was capable to support an automated, unsupervised detection of relevant clusters of samples. Results demonstrated that it may also be useful for the visualisation of local similarity relationships between samples and the identification of potential outliers. In general its performance was comparable to Sammon\'s mapping. One cannot of course expect that a single method would always be able to accurately map different types of high-dimensional data. However, the application of both techniques is recommended as a reliable approach to data exploratory analysis. In this study they offered consistent views of the problems under analysis. SOM, as well as many other cluster analysis techniques, may be more suitable for application after first gaining an adequate insight into the structure and organisation of the data. Such a global understanding may be facilitated through the application of different non-linear mapping methods. Even though a comparison with existing network clustering methods was not implemented, preliminary results suggest that the approach proposed might represent a useful tool for interactome network visualisation and clustering. It distinguished key hubs and facilitated the identification of functionally relevant clusters. It showed that, not surprisingly, most of the hubs detected are essential for the normal development, behaviour and reproduction of *C. elegans*by exhibiting an enrichment of phenotypes obtained from RNA interference. This can be explained by the fact that the partners connected to these hubs are involved in numerous fundamental processes such as mitosis or meiosis. Hence, the modification of the network caused by the absence of a hub may have strong consequences on the topology of the network and the organisation of the cellular processes. Isolation of functional clusters (e.g. chromosome condensation and segregation of the transcriptional core process) is essential to investigate relationships between groups of proteins or modules. Modules playing a role in the same process (e.g. chromosome condensation and segregation during the mitosis) also tended to be interrelated in the clustering analysis. This underlies the fact that these modules are also functionally interconnected and are interdependent to stringently regulate the cellular processes. Without these connections the process may be misregulated and generate aberrant behaviour (i.e. oncogenesis if the mitosis is not well regulated). In this way, interactions that connect these modules are likely to be at the intersection of several biological processes and to regulate the correct succession of events in a cellular process (e.g. condensation of chromosomes before segregation). We do not claim that the tool reported can be used as an interaction prediction technique. The example analysed aims to illustrate the application of exploratory data analysis for detecting regions, which may be biologically meaningful and relevant for assessing the outcomes from protein-protein interaction prediction techniques. Such patterns may be useful for guiding future computational or experimental analyses to validate interaction hypotheses. Meaningful patterns may be associated, for example, with functionally enriched regions, as shown in this paper. Moreover, because of the limitations regarding predictive accuracy and coverage exhibited by existing single-source techniques, it is important to offer user-friendly tools that may help scientists to detect possible, spurious associations. Future research should include a more exhaustive statistical description of all possible hub candidates detected by the mapping process for this and other network examples. Statistical and functional attributes represented by this method should be compared with previous findings from large-scale, comprehensive studies. Such an investigation was not implemented here because it is outside the main scope of this paper. The preservation of local distance structures is an important property to interpret the non-linear mapping techniques studied here. This is the main goal of their data transformation mechanisms. It basically means that the distance between two points, *dm*~*ij*~, in the transformed *m*-dimensional space should be very similar to *dn*~*ij*~(their distance in the original *n*-dimensional data) if *dn*~*ij*~is small. However, if *dn*~*ij*~is relatively large, *dm*~*ij*~is not required to be similar to *dn*~*ij*~. A fundamental difference between the relaxation non-linear and the original Sammon\'s mapping methods is that the former adapts a point-to-point distance at every processing step, instead of adapting all of the distances at every step. For relatively small data sets the computing times required by the Java-based implementation of the relaxation non-linear mapping method were comparable to those obtained from *Matlab*^®^implementations of SOM and Sammon\'s techniques, i.e. in the order of seconds. But for larger data sets, e.g. Parkinson\'s disease data, the non-linear mapping method may run in the order of minutes. This of course depends on the number of mapping iterations and memory resources available. This concern may be addressed by implementing an optimised version of the software, perhaps using another programming language. Another important solution is the implementation of a *frame method*\[[@B32]\], which has demonstrated to improve the computational efficiency of the algorithm without significantly compromising its data structure preservation capabilities. We also intend to expand this and related research within an open-source data analysis and visualisation platform, such as the *TIGR Multiexperiment Viewer*\[[@B42]\]. An important aspect of future research is the adaptation of the relaxation non-linear mapping method to perform tasks beyond exploratory data analysis. A desirable property would be its capacity to generalise solutions for new samples in an incremental fashion. That is, the system should be able to add new samples to a map without having to re-generate it. One possible solution is the application of an artificial neural network to interpolate and extrapolate the mapping as illustrated by Mao and Jain \[[@B43]\]. It is also important to develop hybrid systems to combine the strengths and advantages demonstrated by various mapping techniques \[[@B44]\]. A two- stage approach, for example, represents a feasible solution. In this approach a data set may be firstly partitioned into a set of *Voronoi*spaces using clustering techniques such as *k*-means and SOM, and then independent mapping projections may be performed on each area. It has been suggested that such a hybrid model may be advantageous especially when dealing with massive data sets \[[@B45]\]. Other investigations will involve the assessment and comparison of related techniques \[[@B46],[@B47]\] The mapping algorithm successfully recognised key topological properties and functional relationships in an interactome network based on a graph encoding scheme, which only considers direct interactions. Moreover, it considered all graph connections as equals. Nevertheless, it would be important to implement applications in which the network encoding values, *b*~*ij*~, may also reflect non-direct, shared interactions. This may be done, for example, by defining a graph distance function between network nodes. Another input representation scheme may exploit information relevant to the significance or confidence assigned to the interactions based on experimental evidence. Since cellular networks are organized in a modular fashion, the identification of these modules is crucial to understand relationships between biological processes and offer a higher-order, more accessible representation of the interactomes. The clustering approach proposed in this paper provides a meaningful, simplified representation of complex interactomes. This representation may significantly facilitate exploratory analysis of networks for non-specialists in bioinformatics. This type of analysis is fundamental to detect key network components, such as hubs, which are implicated in many physiological disorders. Identifying these hubs and their associated clusters is also an important step toward the functional annotation of these proteins, as well as for obtaining possible explanations of their involvement in a specific disease. The cluster visualisation tool, *interClust*, may represent a useful technique to analyse other protein-protein interaction networks including a future human interactome. For instance, it may be applied to isolate proteins linked to a human pathology and to associate them with a cluster or functional modules (e.g. the transcriptional core complex of the Polymerase II enzyme). Another important component of future research is the adaptation of network clustering methods to take into account spatio-temporal aspects of interactions based on, for example, microarrays, in-situ hybridization or protein localization data. Non-linear mapping methods may also be applied to support the annotation of unknown proteins. This may be done by assigning a protein to a functional role that is significantly associated with the cluster under consideration. Furthermore, it is of course fundamental to compare this network clustering methodology with existing techniques. Thus, such approaches may aid researchers in the design of further experiments and the selection of more sophisticated bioinformatics analyses. Conclusions =========== This research studied a user-friendly cluster visualisation approach that is able to support the generation of biologically meaningful outcomes. It represents an effective and robust exploratory data analysis technique. Comparisons indicate that applying more than one mapping approach may improve the confidence of results. Moreover, this may facilitate the generation of alternative, meaningful views of the data. Relaxation non-linear and Sammon\'s mapping techniques may be more suitable for exploratory data analysis tasks than SOM. This study did not aim to add another algorithm to the existing collection of supervised and unsupervised classification tools. This methodology is not reported as a competing solution to clustering algorithms. Our study shows how an exploratory data analysis approach based on non-linear mapping can support the identification of relevant, biologically-meaningful patterns. We do not argue that the methodology proposed should necessarily offer more accurate results in relation to existing classification solutions. We recommend this methodology as a first step towards understanding complex data mining problems in functional genomics. Such an exploratory approach may also facilitate the selection of more sophisticated methods and highlight possible, critical features for successfully implementing clustering-based studies. This research indicates that the outcomes originating from an exploratory, pattern visualisation method may be as meaningful as those produced by more sophisticated classification approaches, i.e. SOM. Moreover, the methodology proposed does not require the user to define multiple learning parameters. Exploratory analysis frameworks may facilitate a better insight into a data set before applying more sophisticated, problem-specific classification or predictive models. Such an insight may be achieved by helping users to recognise key features of the underlying structure of the data, detect potential outliers or anomalies and test assumptions about the cluster composition of the data. An adaptation of the relaxation non-linear mapping technique, *interClust*, represents a promising solution to aid researchers to recognise key connectivity and functional patterns in interactome networks. Further research is underway to continue assessing its application to this area. Methods ======= Data ---- The leukaemia data set includes 38 samples originating from \[[@B33]\]. Each sample is represented by 50 expression values. The samples are categorised into two classes: *Acute myeloid leukemia*(AML) and *acute lymphoblastic leukemia*(ALL). The original data sets and experimental protocols can be found at the Broad Institute Web site \[[@B48]\]. For each feature standardisation was applied by subtracting each value from the mean and dividing it by the standard deviation. The SRBCT data consisted of 63 samples categorised into four classes: *Ewing family of tumors*(EWS), *rhabdomyosarcoma*(RMS), *Burkitt lymphomas*(BL) and *neuroblastomas*(NB), which were represented by the expression values of 2308 genes. The dimensionality of the SRBCT expression samples was reduced by applying PCA. It has been shown that the application of PCA is important to facilitate an adequate discrimination of samples in this data set. The 10 dominant PCA components for each case were used as the input to the analysis techniques as suggested by \[[@B28]\], who applied a supervised learning approach to classify the samples after reduction by PCA. Using the raw data (without PCA) the relaxation non-linear mapping was not able to adequately depict differences between the samples. The original data sets and experimental protocols can be found at the National Human Genome Research Institute Web site \[[@B49]\]. The Parkinson\'s disease data include 20 samples described by 9504 gene expression values for both *normal brain*(10 samples) and a pharmacological model of *Parkinson\'s disease*\[[@B34]\]. *M. musculus*was the organism studied in this disease model. The data are available at The Gene Expression Omnibus \[[@B50]\] (accession number GDS22). Additional files [9](#S9){ref-type="supplementary-material"} to [11](#S11){ref-type="supplementary-material"} include, respectively, the leukaemia, SRBCT and Parkinson\'s disease data analysed in this paper. The network of interactions was derived from the chromatin interactome in *C. elegans*. It contains 303 proteins and 349 interactions. It comprises components of the Polymerase II holoenzyme, histones modifying enzymes, nucleosomes positioning proteins and several proteins containing domains known to be essential to this process such as the chromodomain, the bromodomain or the SET domain. This is an early version of the chromatin interactome, which includes a number of retested \[[@B39]\] as well as non-retested interactions determined by a stringent high-throughput two-hybrid screen. It also contains several interologs \[[@B38],[@B51]\]. A combination of experimental and bioinformatic factors (reporter genes used for the phenotypic tests, number of hits per interactions, a blast e-value less than 1E-10, a PHRED score \>20 for 15% of the ISTs (interaction sequence tags) and the frame verification method) were used to provide optimal accuracy. It is known that the two-hybrid approach has the tendency to generate more false positives than the pull-down/Mass spectrometry approach, for example. However, in the data set analysed the rate of false positives is reduced by using more reporter genes (up to 4 genes, unlike traditional large scale two-hybrid screens which commonly use 2 genes). Using 4 reporter genes can reduce the rate of false positives up to 50%. [Additional file 12](#S12){ref-type="supplementary-material"} contains this data set. Algorithms and tools -------------------- The relaxation non-linear mapping algorithm is summarised in Figure [1](#F1){ref-type="fig"} and details on its design are reported in \[[@B32]\]. The adaptation of a pair points, *i*and *j*, in the transformed, *m*-dimensional map is implemented as follows. Given two points, *Pm*~*i*~and *Pm*~*j*~, in the *m*-dimensional map, the adjusted new values, *Pm*~*new*,\ *i*~and *Pm*~*new*~, ~*j*~, are calculated using: ![](1471-2105-6-13-i1.gif) where *dn*~*ij*~and *dm*~*ij*~represent the Euclidean distances between the points, *i*and *j*, in the *n*- and *m*-dimensional spaces respectively. The SOM results were obtained using the *SOM Toolbox*, which is a *Matlab*^®^implementation \[[@B36],[@B37]\]. Each training process consists of two phases. The following parameters were used. Initial learning rates equal to 0.5 (first phase) and 0.05 (second phase). Learning rates were controlled by an inverse-of-time function. The SOM neighborhood radius starts covering one fourth of the map size. The number of training epochs was equal to 10 times the number of map nodes (first phase). For the second phase it was equal to 4 times the number of training epochs in the first phase. A U-matrix depicts the distances between neighbouring map units by displaying a grey scale. In a label map a SOM node represents a class based on a majority voting strategy for the samples associated with this node. In case of a draw, the first class encountered is used. Empty nodes are not labeled. The Sammon\'s mapping analyses were implemented using the SOM Toolbox with 100 mapping iterations, iteration step size equal to 0.2 and the Euclidian distance. For the interaction data set, the tool *inBuilder*was used to transform it into *interClust*input format. The cross-platform tools *interClust*and *inBuilder*are available for academic researchers on request from the authors. Graphical outputs for the relaxation maps were obtained with the proprietary software *Statistica*^©^. [Additional file 7](#S7){ref-type="supplementary-material"} was created using *InterViewer*\[[@B40]\], which is freely available at \[[@B52]\]. Analyses were performed on a PC with a *Pentium*^®^*4*CPU. Authors\' contributions ======================= FA designed the study, implemented the relaxation non-linear mapping algorithm, performed analyses and wrote the manuscript. HW did the SOM and Sammon\'s mapping experiments and helped with the preparation of the manuscript. AC selected the interactome data, interpreted results and helped with the preparation of the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 **SOM frequency map for SRBCT data**It shows the distribution of samples, *X*(*Y*), over each node in Figure [5](#F5){ref-type="fig"}, where *X*represents the class label and *Y*stands for the number of Class *X*samples assigned to the corresponding node. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 **3D visual display originating from relaxation non-linear mapping -- SRBCT data**EWS, RMS and BL samples are represented by symbols \'1\', \'2\', \'3\' respectively. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 **3D Sammon\'s mapping results -- SRBCT data**Symbols \"1\", \"2\" and \"3\" represent classes EWS, RMS and BL respectively. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 4 **SOM frequency map for Parkinson\'s disease data**It shows the distribution of samples, *X*(*Y*), over each node in Figure [8](#F8){ref-type="fig"}, where *X*represents the class label and *Y*stands for the number of Class *X*samples assigned to the corresponding node. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 5 **3D Relaxation non-linear mapping of the Parkinson\'s disease model data**Parkinson\'s disease and Normal samples are identified by symbols \'1\' and \'2\' respectively. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 6 **3D Sammon\'s mapping of the Parkinson\'s disease model data**Symbols \"1\" and \"2\" represent Parkinson\'s disease and Normal samples respectively. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 7 **Examples of key hubs in the interactome**It depicts some of the hubs automatically isolated by the mapping algorithm, as well as a few nodes located near the centre of the map (F15A2.6, C34E10.5, C14B9.6). ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 8 **Description of protein cluster obtained from Figure**[14](#F14){ref-type="fig"} ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 9 **Leukaemia data set analysed in this paper**Log ratios are used to represent the expression levels. The last column shows the class labels. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 10 **SRBCT data set analysed in this paper**Log ratios are used to represent the expression levels. The last column shows the class labels. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 11 **Parkinson\'s disease data set analysed in this paper**Log ratios are used to represent the expression levels. The last column shows the class labels. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 12 **Chromatin interaction network in *C. elegans***The last column shows the connection strength in the graph. All connections are considered equally. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We thank Claude Sardet at the CNRS-Montpellier for supporting AC\'s participation in this collaboration. AC is financially supported by a fellowship from La Ligue Contre le Cancer (ligue départementale de l\'Hérault) and by a grant from la Ligue Contre le Cancer (Equipe labélisée Ligue Nationale contre le Cancer). We thank the anonymous reviewers for their helpful comments and corrections. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Relaxation method for non-linear mapping.**The input to the algorithm is a collection of *K*points described in an *n*-dimensional space. A mapping iteration refers to a complete sequence of adaptation steps involving pairs of points, *p*(*i*,*j*). A point may encode a biological sample described by a gene expression profile (e.g. tumour sample), or a protein described by an interaction profile. ::: ![](1471-2105-6-13-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Visualisation of clusters in leukaemia data for different numbers of mapping iterations.**Panels (a) to (d) show results for 0, 1, 10 and 100 mapping iterations respectively. Circles are used to represent the samples on the maps. Labels \'0\' and \'1\' refer to ALL and AML samples respectively. ::: ![](1471-2105-6-13-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Resulting maps for the SRBCT data.**EWS, RMS, BL and NB samples are represented by symbols \'1\', \'2\', \'3\' and \'4\' respectively. 100 mapping iterations. ::: ![](1471-2105-6-13-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Resulting maps for the SRBCT data considering only 3 classes.**EWS, RMS and BL samples are represented by symbols \'1\', \'2\', \'3\' respectively. 100 mapping iterations. ::: ![](1471-2105-6-13-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **SOM-based analyses on the SRBCT data.**U-matrix and label map (panel on the right side). U-matrix depicts the distances between neighbouring map units using a grey scale. In the label map a node represents a class based on a majority voting strategy for the samples associated with this node. EWS, RMS and BL samples are represented by symbols \'1\', \'2\', \'3\' respectively. [Additional file 1](#S1){ref-type="supplementary-material"} shows its frequency map. ::: ![](1471-2105-6-13-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Sammon\'s mapping analyses on the SRBCT data.**Symbols \"1\", \"2\" and \"3\" represent classes EWS, RMS and BL respectively. ::: ![](1471-2105-6-13-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Relaxation non-linear mapping of the Parkinson\'s disease model data.**Parkinson\'s disease and Normal samples are identified by symbols \'1\' and \'2\' respectively. 100 mapping iterations. ::: ![](1471-2105-6-13-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **SOM of the Parkinson\'s disease model data.**U-matrix and label map (panel on the right side). U-matrix depicts the distances between neighbouring map units using a grey scale. In the label map a node represents a class based on a majority voting strategy for the samples associated with this node. [Additional file 4](#S4){ref-type="supplementary-material"} shows its frequency map. ::: ![](1471-2105-6-13-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### **Sammon\'s mapping of the Parkinson\'s disease model data.**Symbols \"1\" and \"2\" represent Parkinson\'s disease and Normal samples respectively. ::: ![](1471-2105-6-13-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### **Example of a network characterized by a number of clusters.**The length of the links does not reflect distances or connection strengths. All of the direct connections are considered equally. ::: ![](1471-2105-6-13-10) ::: ::: {#F11 .fig} Figure 11 ::: {.caption} ###### **A resulting map after applying interClust to the network shown in Figure 10.**Clusters are identified and local interaction relationships tend to be preserved. 20 mapping iterations. ::: ![](1471-2105-6-13-11) ::: ::: {#F12 .fig} Figure 12 ::: {.caption} ###### **Relaxation map for the chromatin interactome network.**Each panel depicts different regions and levels of detail, obtained with 100 iterations. ::: ![](1471-2105-6-13-12) ::: ::: {#F13 .fig} Figure 13 ::: {.caption} ###### **Partial view of chromatin interactome map.**Its shows a region enriched by hubs, which is well-separated from the main group of proteins. ::: ![](1471-2105-6-13-13) ::: ::: {#F14 .fig} Figure 14 ::: {.caption} ###### **Identification of key functional components based on cluster visualisation.**The region surrounding protein T20B12.2 includes several components of the core polymerase II enzyme, as well as several related families that share similar phenotypes. See [Additional file 8](#S8){ref-type="supplementary-material"} for further descriptions. ::: ![](1471-2105-6-13-14) :::

PubMed Central

2024-06-05T03:55:52.446292

2005-1-20

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548129/", "journal": "BMC Bioinformatics. 2005 Jan 20; 6:13", "authors": [ { "first": "Francisco", "last": "Azuaje" }, { "first": "Haiying", "last": "Wang" }, { "first": "Alban", "last": "Chesneau" } ] }

PMC548130

Background ========== Detection of LOH is one of the most common molecular applications in the study of human diseases, in particular cancer. It is commonly used to examine whether a known tumour suppressor gene is inactivated or to map unknown tumour suppressor gene(s). Detection of LOH not only helps in understanding the molecular mechanisms underlying the development of cancer, but also provides important information useful for disease diagnosis and prognosis. LOH detection is commonly carried out by the analysis of microsatellite markers using an automated DNA sequencer. With the raw data from the sequencer being stored in one file per lane together with corresponding clinical information and patient follow up data, each LOH study \[[@B1],[@B2]\] generates hundred of files that need to be organised and related in a structured format. However, with the increasing number of samples analysed using different software, no tool is currently available to facilitate the extensive and efficient data retrieval and analyses, such as correlation of LOH data with various clinical data sets. We have developed a novel software package: LOH Data Management and Analysis Software (LDMAS) in order to satisfy these needs. LDMAS can retrieve LOH data from automated DNA sequencer platform and clinical data from any patient record system and correlate different data sets according to the user\'s choice. Here we present how LDMAS interfaces to Genotyper software (ABI, Foster City, CA) which is used to determine the presence of LOH, and the patient record system SunQuest (San Francisco, CA), facilitating the identification of LOH markers associated with the development of CIN \[[@B3]\]. CIN show variable clinical behaviour despite morphological homogeneity within each subgroup. Clinically, it is vital to distinguish CIN lesions with different behaviour and identify those likely to persist and progress despite treatment. Implementation ============== System architecture ------------------- LDMAS package is composed of three modules: \(1) MRES (Medical Report Extractor Software) which parses patient report files, extracts the information of interest and organises it into a structured format, applicable to LDAS \(2) LDAS (LOH Data Analysis Software) which obtains LOH data from Genotyper (Applied Biosystems, California) or GDAS (Affymetrix, California) software and correlates it to clinical data obtained from MRES \(3) LDMS (LOH Data Management Software) which is used to gather patients\' clinico-pathological data and extract significant relationship between the various data sets LDAS and LDMS work synergistically to manage and analyse LOH data. The MRES source code for automatic parsing of patient reports is written in C++ using C++ Builder 5.0 (Borland Software Corporation, Scotts Valley, CA), LDAS is written in Visual Basic for Application as an Excel 2000 add-in and LDMS is written as Visual Basic for Application modules embedded within Access 2000 as fully functional software. These modules can be run independently and used for applications other than LOH. LDMAS runs on Microsoft^®^Windows 2000 or Windows XP operating system. Figure [1](#F1){ref-type="fig"} shows LDMAS architecture. Type of input data for LDMAS modules ------------------------------------ The MRES module takes its input from any patient report file containing clinical details such as diagnosis, stage of the disease, treatment and follow up results, parses and formats patient\'s data into a structured format that can be saved as Excel spreadsheet. In this case, data were taken from SunQuest patient record system and MRES converted and produced the data as: \(1) Hospital Number \(2) Hospital ID \(3) Patient Name \(4) Date of Birth \(5) Pathological specimen Number \(6) Date of Diagnosis \(7) Histological diagnosis. The user can manually check the data and use it as template for analysis. Data analysis is carried out using LDAS which obtains LOH data from Genotyper and correlates it to the clinical data obtained from MRES. LDAS obtains data in plain text format and can thus be easily interfaced to any LOH platform generating software such as Genotyper and GDAS. Finally all data are entered into LDMS for storage and intelligent mining of data. Database query results can be exported back to LDAS allowing correlation between LOH and various clinico-pathological parameters such as age, histological grade, treatment modality and their responses, and HPV status as well as carry out multivariate analysis to determine the sensitivity and specificity of the markers involved in the LOH study. A more detailed description of all the modules is provided in LDMAS user\'s guide \[Additional file 1\] and the example below. Advantages of LDMAS ------------------- LDMAS offers several advantages to users. It is user friendly and its architecture is modular allowing versatility of use. It enforces the standardisation of procedures for studies involving large cohorts of individuals. The data is well organized since LDMAS systematically assigns LOH results of each case to its corresponding clinical information. Additionally, LDAS standardizes LOH data analysis implicitly and allows the user to edit the data manually if needed. Microsoft Excel has been chosen to implement LDAS because of its wide use, versatility and convenient statistical analysis features facilitating the implementation of multivariate analysis and correlation testing between LOH and clinico-pathological parameters. Results and discussion ====================== LDMAS application in identification of LOH markers associated with persistence / progression of cervical intraepithelial neoplasia ---------------------------------------------------------------------------------------------------------------------------------- We divided the CIN groups into disease free indicating cases that become CIN free after treatment, and disease persistence/progression indicating cases that develop show progression or persistence of CIN despite treatment. We used LDMAS to retrospectively examine the prognostic value of LOH at 12 microsatellite markers including 10 from 3p14, 3p22-21, 6p21 and 11q23 which are frequently deleted in cervical cancer \[[@B3],[@B4]\], in 164 cases of CIN lesions using archival cytological/histological specimens. LOH was further correlated with high risk HPV infection. Initially MRES was used to automatically parse 4300 patient records and extract clinico-pathological data including age, diagnosis, method of treatment and treatment response during follow up. Out of those, 164 cases with follow up of 3 or more years were chosen for the study and their clinico-pathological information was imported into LDAS. Initially, 71 out of the 164 selected cases were examined for LOH using 12 fluorescent microsatellite markers ran on ABI377 DNA Sequencer. LDAS was then used to identify the microsatellite markers for which LOH was significantly associated with disease persistence/progression of CIN using two tailed student t-test. Figure [2](#F2){ref-type="fig"} generated using LDAS shows that microsatellite markers D3S1300 (3p14.2), D3S1260 (3p22.2), D11S35 (11q22.1) and D11S528 (11q23.3) have the highest LOH in CIN lesions displaying persistence/progression than those who were disease free during follow up \[[@B5]\]. Validation of prognostic markers associated with persistence / progression of CIN --------------------------------------------------------------------------------- To validate this finding, LOH at these four markers was investigated in a further series of 93 cases. Compatible results were obtained from these additional cases. The two sets of data were combined and further compared using LDMS. Methodologies included : 1\) comparison using χ^2^(chi-squared) test of LOH at each of the four microsatellite markers with age, various methods of treatment, different subtypes of HPV infection and between CINs showing disease free or disease persistence/progression. 2\) correlation of LOH data with histological grade of CIN, treatment response and various HPV subtypes. Through such complex analysis, we showed that concurrent LOH at two of the four microsatellite markers could identify 47% of CINs that showed disease persistence/progression with 100% specificity \[[@B5]\]. Furthermore, LOH at D3S1300 was found to be significantly associated with HPV16 infection. Part of this data analysis is supplied in the LDMAS guide \[see Additional file 1\]. More detailed analysis of this study is described in \[[@B5]\]. Algorithm for identifying prognostic disease markers ---------------------------------------------------- Based on the above example, an algorithm can be developed to extract prognostic markers for other diseases. The algorithm can be summarised in the following pseudocode : \(1) Divide the disease in groups according to the pathology staging \(2) Parse patient data from clinical records and use the groups defined in part (1) \(3) FOR each microsatellite marker carry out a two tailed student t-test between the disease groups using LOH data IF t-test p ≤ 0.05 Marker is significant in prognosis of the disease ELSE Marker is not significant in prognosis of the disease \(4) Validate the prognostic markers using χ^2^(chi-squared) test of LOH with clinico-pathological data and correlation of LOH data with histological grade of CIN, treatment response and various HPV subtypes. LDMAS has been implemented using the above pseudocode. Conclusions =========== We have devised an effective algorithm to identify and extract useful markers that can be used to predict the outcome of disease and used the algorithm to successfully identify 4 novel prognostic markers that can be used to predict the outcome of CIN. The algorithm was implemented in a novel software called LDMAS which provides an essential platform for the extraction of useful information from large amount of data generated by LOH studies. Furthermore, LDMAS is used to efficiently store, manage and track the data. Its flexible nature allows the easy manipulation of data facilitating complex analysis as demonstrated in the current study. The various modules of LDMAS can be easily adapted and used with other applications such as high throughput LOH and genotyping using SNPs on Affymetrix^®^GeneChip Mapping arrays and fingerprinting studies. Modules such as MRES can be used independently to parse medical records facilitating extraction of specific clinical information of interest. Additionally, LDMAS can be used to extract clinically useful markers for other diseases. Availability and requirements ============================= The source code and executable files for LDMAS modules as well as user manual including examples from real study data are freely available and can downloaded from our website at : <http://molpath.his.path.cam.ac.uk/bioinformatics/LDMAS.shtml> Additionally examples of input files are provided from our website for users to test the software and assess its functionality. Authors\' contributions ======================= RH designed and developed and implemented LDMAS software and the web site. AE and RH did the experimental work to generate the data necessary to test and validate LDMAS. MD supervised the study and designed the experimental work using CIN biopsies and smears. All authors read and approved the final manuscript. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### LDMAS architecture. ::: ![](1471-2105-6-18-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Example of output from LDMAS. LDMAS generated histogram showing the best set of LOH markers in cervical cancer highlighted by \*. DF indicates Disease Free and DP indicates Disease Persistence/Progression CIN cases. ::: ![](1471-2105-6-18-2) :::

PubMed Central

2024-06-05T03:55:52.450434

2005-1-26

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548130/", "journal": "BMC Bioinformatics. 2005 Jan 26; 6:18", "authors": [ { "first": "Rifat A", "last": "Hamoudi" }, { "first": "Amina", "last": "El-Hamidi" }, { "first": "Ming-Qing", "last": "Du" } ] }

PMC548131

Background ========== Paratuberculosis (Johne\'s disease) is caused by *Mycobacterium avium*subsp. *paratuberculosis*(referred to hereafter as *M. avium*subsp. *paratuberculosis*) and induces a chronic enteritis in ruminants. The disease signs include weight loss, diarrhea, and decreased milk production. In the United States alone the economic burden of Johne\'s disease is estimated at over \$200 million in lost annual revenue to the dairy industry \[[@B1]\]. Prevalence studies in the United States have estimated that between 20 to 30% of dairy herds are infected with *M. avium*subsp. *paratuberculosis*\[[@B2],[@B3]\]. Neonatal calves are most susceptible to infection and are likely to become infected after ingestion of contaminated milk or colostrum \[[@B4],[@B5]\]. During the subclinical stage of infection, the host cell-mediated immune response is robust and appears to control the infection. As the disease progresses from the subclinical to the clinical stage, the cell-mediated response diminishes, and a humoral immune response predominates \[[@B6]\]. Vaccines are not completely protective, but have been reported to reduce fecal shedding and delay the onset of clinical disease \[[@B7]-[@B9]\]. Lipoproteins have long been considered immunomodulators and mycobacteria are especially rich in these post-translationally modified proteins. There are approximately 100 open reading frames identified in the *M. tuberculosis*genome that possess a characteristic amino-terminal acylation motif \[[@B10]\]. The 19-kDa lipoprotein from *Mycobacterium tuberculosis*is immunodominant in both mice \[[@B11]-[@B13]\] and humans \[[@B14],[@B15]\] and has been shown to stimulate CD4^+^T cell proliferation as well as the release of IL-2, IFN-γ, and IL-12 \[[@B16],[@B17]\]. Acylation near the N-terminal portion of the 19-kDa protein is believed to occur at amino acids 19--24 and contributes to its immunogenicity \[[@B12]\]. Furthermore, glycosylation of the *M. tuberculosis*19-kDa protein inhibits innate immune responses, such as the release of TNF-α, IL-6, and IL-10 from macrophages, but does not affect antibody binding \[[@B18]-[@B20]\]. The 19-kDa protein was also shown to induce CD8+ cells to secrete IFN-γ and specifically lyse *M. tuberculosis*-infected monocytes \[[@B21]\] as well as promote neutrophil priming and activation \[[@B22]\]. Finally, B cell epitopes have been described that localize to the linear sequences of amino acids 11--30, 29--47, 61--80, and 140--159, as well as a conformation-dependent epitope at the amino and carboxy-terminal ends because of intramolecular disulfide bonding of cysteine residues \[[@B21],[@B23]\]. Experimental infection and staining of macrophages has shown that the 19-kDa protein is secreted by live *M. tuberculosis*residing within the phagolysosomal compartment \[[@B24]\]. Homologues of the 19-kDa lipoprotein exist in *M. bovis*, *M. avium*, and *M. intracellulare*but are absent from *M. phlei*, *M. smegmatis*, *M. fortuitum*, *M. gordonae*, and *M. leprae*\[[@B25]\]. Despite decades of research, little is known about the *M. avium*subsp. *paratuberculosis*proteins involved in metabolism, cell wall synthesis, macrophage entry and survival, disease pathogenesis, or host immune evasion. However, several antigens have recently been identified and their immunogenicity examined by serodiagnostic and/or lymphocyte stimulation assays \[[@B26]-[@B30]\]. With the genome sequence of *M. avium*subsp. *paratuberculosis*recently defined \[[@B31]\], all proteins produced by this pathogen are now identified and can be characterized. Novel opportunities arising from the genome sequence of *M. avium*subsp. *paratuberculosis*enable us to select and characterize genes of interest. A major goal of this laboratory is to define a complete catalog of immunodominant antigens in *M. avium*subsp. *paratuberculosis*. With the immunostimulatory capabilities of the *M. tuberculosis*19-kDa antigen in mind, the objective of this study was to determine if the 19-kDa protein of *M. avium*subsp. *paratuberculosis*possessed a similar capacity. In this study, the 19-kDa lipoprotein from *M. avium subspecies paratuberculosis*was cloned, expressed, and characterized. In addition, the purified recombinant protein was used to assess cellular immune responses in subclinically infected cattle as well as humoral immune responses in cattle with clinical Johne\'s disease. Results ======= Sequence analysis of the mycobacterial 19-kDa coding region ----------------------------------------------------------- The 19-kDa coding sequence was identified from the *M. avium*subsp. *paratuberculosis*genome project as MAP0261c. Comparison of amino acid sequences from other species of mycobacteria show that this gene product is conserved. Sequence alignment shows the N-terminal half is more variable and the region between amino acids 99 and 123 is highly conserved (Figure [1](#F1){ref-type="fig"}). MAP0261c displays a 95% amino acid identity in *M. avium*, 84% in *M. intracellulare*and 76% in *M. bovis*and *M. tuberculosis*. MAP0261c has a G+C content of 66.2% and encodes for 161 amino acids with a predicted molecular mass of 15.2 kDa. The first 22 amino acids of the *M. tuberculosis*19-kDa protein are hydrophobic and were previously noted to represent a signal peptide that is post-translationally cleaved to expose an N-terminal cysteine \[[@B32]\]. Signal peptidase cleavage analysis of MAP0261c (SignalP3.0; <http://www.cbs.dtu.dk/services/SignalP/>) detected a signal peptide generated from a putative cleavage site between amino acids 34 and 35, to expose an N-terminal serine (Figure [1](#F1){ref-type="fig"}). The SignalP-NN (neural networks) model assigned the highest cleavage probability values to amino acid 35 (C score = 0.324; Y score = 0.439) with the predicted length of the signal peptide being 34 amino acids. This is slightly longer than most signal sequences which range from 18 to about 30 amino acid residues in length \[[@B33]\]. The predicted peptidase cleavage sight for *M. tuberculosis*is between amino acids 21 and 22 and therefore falls within this range (Figure [1](#F1){ref-type="fig"}). Despite a predicted signal peptidase cleavage site, PSORTb analysis software <http://www.psort.org/psortb/> could not predict if the protein was cytoplasmic or membrane located. Cloning and expression of the *M. avium*subsp. *paratuberculosis*19-kDa protein ------------------------------------------------------------------------------- In order to perform immunoassays with purified 19-kDa protein, MAP0261c was amplified from *M. avium*subsp. *paratuberculosis*genomic DNA and cloned into the pMal-c2 expression vector and transformed in *E. coli*. Induced expression resulted in production of a maltose binding protein (MBP)-19 kDa fusion protein that was affinity-purified from *E. coli*lysates. MBP-19 kDa was analyzed by SDS-PAGE (Figure [2](#F2){ref-type="fig"}) to assess yield, purity and size. The predicted mass of MBP alone is 42 kDa, while the predicted mass of the MBP-19 kDa fusion protein is 56 kDa. The purified MBP-19 kDa protein migrated to a position around 50 kDa in SDS-PAGE (Figure [2A](#F2){ref-type="fig"}). Approximately 5 mg of purified protein was easily obtained from a 500-ml broth culture at O.D.~600\ nm~= 0.9. The purified protein was further characterized by immunoblot analysis using a monoclonal antibody (mAb) that detects the MBP affinity tag (Figure [2B](#F2){ref-type="fig"}). Both the fusion protein and MBP alone are detected by the mAb. In addition, the fusion protein is expressed at higher levels under inducing conditions. Immunoblot analysis of the 19-kDa protein ----------------------------------------- The *M. tuberculosis*19-kDa protein is known to be immunodominant, therefore immunoblot analysis was performed to determine if cattle naturally infected with *M. avium*subsp. *paratuberculosis*produce antibodies against the 19-kDa protein. Immunoblots were probed with sera from 9 non-infected and 14 clinically infected cattle. Sera from 8 of 9 non-infected cattle did not react with either MBP or MBP-19 kDa, while 1 non-infected cattle weakly recognized both MBP and MBP-19 kDa protein (data not shown). By comparison, sera from 9 of 14 infected cattle reacted specifically with the 19-kDa protein, but not MBP alone. Antibody reactivity to the 19-kDa protein from 3 clinical cows is shown in Figure [3](#F3){ref-type="fig"}. Sera from the remaining five infected cattle detected both MBP and MBP-19 kDa proteins. This result made it difficult to distinguish if sera from the animal recognized the MAP0261c gene product or if it simply recognized the MBP affinity tag. Collectively, these data suggest that the 19-kDa protein is detectable and immunogenic in cattle with Johne\'s disease. IFN-γ responses of infected cattle ---------------------------------- As an indicator of the cell-mediated responses of infected cattle to the 19-kDa protein, whole blood containing PBMCs from 9 control and 21 infected cows was stimulated with *M. avium*subsp. *paratuberculosis*sonicated whole cell lysate (WCL), MBP, or MBP-19 kDa and IFN-γ production was assayed by ELISA. These cattle were selected from a larger group because they showed no IFN-γ stimulation in response to MBP alone. IFN-γ production in response to WCL stimulation allowed for the segregation of infected cattle into three groups: suspect, positive, and high positive. After subtracting the IFN-γ responses of non-stimulated cells, suspect animals had less than 0.1 absorbance units of IFN-γ production, positive animals had 0.1 -- 0.3 absorbance units of IFN-γ production, and high positive animals had more than 0.3 absorbance units of IFN-γ production. IFN-γ responses by blood mononuclear cells from infected cattle exceeded responses from control cattle for both the WCL and MBP-19 kDa (Table [1](#T1){ref-type="table"}. Significant differences (P \< 0.05) were found between control and high positive groups for both WCL and MBP-19 kDa protein stimulation. However, direct comparisons of the two antigen preps using mononuclear cells from the same animal clearly showed the WCL was a stronger stimulator of IFN-γ production (Table [1](#T1){ref-type="table"}). Discussion ========== A majority of the research on individual mycobacterial proteins has been performed in *M. tuberculosis*, whereas little is known about the *M. avium*subsp. *paratuberculosis*proteome. Indeed, all currently available antigen-based diagnostic tests for Johne\'s disease use an undefined mixture of proteins, such as purified protein derivative (PPD) or WCL, which may not be specific for *M. avium*subsp. *paratuberculosis*. Recent completion of the *M. avium*subsp. *paratuberculosis*genome has already advanced efforts to identify novel antigens \[[@B26],[@B34]\]. Furthermore, the genome will be a critical resource in proteomic studies directed at defining the proteins present in mixtures such as johnin PPD. The present study was performed in order to characterize the *M. avium*subsp. *paratuberculosis*19-kDa protein, as well as assess its immunostimulatory capabilities in cattle. In this study, we show that the 19-kDa protein of *M. avium*subsp. *paratuberculosis*can be readily overexpressed as a fusion protein in *E. coli*. This is not true for many other proteins encoded by *M. avium*subsp. *paratuberculosis*\[[@B34]\] and suggests the putative lipoprotein is not toxic to *E. coli*. Previous studies have suggested the *M. tuberculosis*19-kDa protein undergoes posttranslational modification by the addition of fatty acids to form a lipoprotein \[[@B35]\]. Although not demonstrated directly by our studies, it is possible that the 19-kDa was not posttranslationally modified by the heterologous *E. coli*host. It is unclear whether posttranslational modification of this protein would affect its immunological activity. The recombinant antigen was detected by sera from cattle with Johne\'s disease; however, it was not as strong a stimulator of proliferative T-cell responses as has been reported for its counterpart in *M. tuberculosis*\[[@B36]\]. Furthermore, the 19-kDa protein from *M. tuberculosis*was shown to induce both cellular and humoral immune responses from mice and humans \[[@B11],[@B14]\]. These studies, combined with the present study, may suggest that acylation is more important in cell-mediated immune responses than in the humoral immune response. It is generally accepted that cellular and humoral immune responses of *M. avium*subsp. *paratuberculosis*-infected cattle are biphasic, with IFN-γ responses detected early and antibody responses detected late in infection. However, evidence suggests that an unknown *M. avium*subsp. *paratuberculosis*protein can be detected by antibodies from cattle just 3 weeks after infection \[[@B37]\]. As a measure of cellular immune responses, blood mononuclear cells from infected cattle were stimulated with both the recombinant 19-kDa protein and a whole-cell sonicated lysate of *M. avium*subsp. *paratuberculosis*(WCL), and IFN-γ production was measured. Results from this study suggest that while the 19-kDa protein is a stimulator of IFN-γ production, it is not as potent when compared to WCL. Additionally, we found that a majority (9 of 14) of infected cattle produced antibodies to the 19-kDa protein, as determined by immunoblot analysis. By comparison, sera from the majority of control, non-infected cattle (8 of 9) did not react to the 19-kDa protein. The single non-infected cow that did show reactivity to the MAP0261c gene product may be attributed to exposure of environmental mycobacteria such as *M. avium*subsp. *avium*, which has a similar protein (Figure [1](#F1){ref-type="fig"}). Sera from four clinical cows reacted to the MBP protein, but this reactivity was extremely weak. MBP is found in environmental *E. coli*and likely accounts for reactivity seen in some cattle. In order to avoid potential cross-reactivity with MBP, we attempted to cleave the MBP portion from the MBP-19 kDa fusion protein, but cleavage was not 100% efficient (data not shown). The *M. tuberculosis*19-kDa protein was reported to contain a 21 amino acid N-terminal signal peptide \[[@B22]\], however our SignalP analysis for *M. avium*subsp. *paratuberculosis*identified a putative 34 amino acid N-terminal signal sequence. Furthermore, the computer algorithm PSORTb predicts a putative signal sequence, but it cannot determine if the protein is actually secreted. Antibodies will be produced against the recombinant MBP-19 kDa protein to determine if the protein is secreted by *M. avium*subsp. *paratuberculosis*. Conclusions =========== The results from this study show that the recombinant 19-kDa protein stimulates a weak host immune response in infected cattle. The 19-kDa protein may be used in conjunction with other antigens from *M. avium*subsp. *paratuberculosis*to identify infected cattle but should not be used as a \"stand alone antigen\" in new diagnostic assays. Methods ======= Bacterial strains and culture conditions ---------------------------------------- *M. avium*subsp. *paratuberculosis*strain 19698-1974 (originally isolated in 1974 from a clinical cow housed at the National Animal Disease Center, Ames, IA) was grown in Middlebrook 7H9 liquid media (pH 6.0) supplemented with 10% oleic acid albumin dextrose complex (Becton Dickinson Microbiology, Sparks, MD), 0.05% Tween 80 (Becton Dickinson Microbiology), and 2 mg/ml mycobactin J (Allied Monitor Inc., Fayette, MO). *M. avium*subsp. *paratuberculosis*cultures were grown to log phase at an optical density 540 nm (OD~540~) of 0.4, at 37°C without shaking. *Escherichia coli*DH5α cells were routinely grown in Luria-Bertani (LB) broth or LB agar plates at 37°C supplemented with ampicillin (100 μg/ml) for selection. Cattle ------ The Johne\'s Disease Research Project at the National Animal Disease Center has a repository of sera from cattle that were euthanized with clinical signs of Johne\'s disease, which included shedding, weight loss and diarrhea. Fecal samples from each of these clinical cattle were found to contain more than 100 CFU per gram of feces as determined by colony counts on Herrold\'s egg yolk media (HEYM) agar slants by standard culture methods \[[@B38]\]. Sera from 14 clinical cattle were selected for immunoblot analysis. The National Animal Disease Center also maintains a small herd of non-infected cattle as well as a herd of cattle naturally infected with *M. avium*subsp. *paratuberculosis*. The animals used in IFN-γ experiments were placed in four groups consisting of 9 non-infected healthy cows, 9 suspect subclinical cows (IFN-γ responses \< 0.1), 6 positive subclinical cows (IFN-γ responses 0.1 -- 0.3), and 6 high-positive cows (IFN-γ responses \> 0.3). The non-infected control cows were characterized by repeated negative fecal cultures performed quarterly over a 3- to 5-year period. In addition, these animals were negative on all immunological assays (i.e. ELISA and IFN-γ production) performed during that period. Subclinical cattle (suspect, positive, and high-positive) were characterized by shedding less than 10 CFU/g of feces and were intermittently positive by IFN-γ assays (response \> 0.1) performed quarterly over a 3- to 5-year period. The institutional Animal Care and Use Committee approved all animal procedures described in this study. Comparison of the mycobacterial 19 kDa coding region ---------------------------------------------------- The nucleotide sequences for the 19 kDa coding regions from *M. tuberculosis*, *M. bovis*, *M. avium*and *M. intracellulare*were obtained from the NCBI nucleotide sequence database. The sequences were assembled and compared using MegAlign software (DNASTAR, Inc., Madison, WI). Cloning and expression of the *M. avium*subsp. *paratuberculosis*19-kDa gene ---------------------------------------------------------------------------- A maltose binding protein (MBP) fusion of the *M. avium*subsp. *paratuberculosis*19-kDa sequence (MBP-19 kDa) was constructed using the pMAL-c2 vector (New England Biolabs, Beverly, MA). To amplify the 19-kDa coding region from *M. avium*subsp. *paratuberculosis*, primers were designed directly from the MAP0261c sequence. MAP0261c was amplified with Expand High Fidelity PCR system using the primers 19-kDa-pMal5\' (5\'-GCGC[CAGCTG]{.underline}ACGATCGCGGTCGCGGGCGCGGC-3\') and 19-kDa-pMal3\' (5\'-GCGC[AAGCTT]{.underline}CAGGTCACATCGATCTCGAAC-3\'). The 19-kDa-pMal5\' and 19-kDa-pMal3\' primers contained *Pvu*II and *Hin*d III restriction sites (underlined) for cloning, respectively. This primer set amplified the 19-kDa coding sequence minus the N-terminal 4 amino acids and the C-terminal 3 amino acids. The pMal-c2 vector was digested with *Xmn*I and *Hin*d III and the PCR amplicon was digested with *Pvu*II and *Hin*d III. The two products were ligated overnight at 12°C with T4 DNA ligase (Life Technologies Inc., Rockville, MD), which resulted in an in-frame fusion between the vector-encoded *malE*gene and a majority of the 19-kDa gene. The resulting recombinant plasmid, designated pMal-19-kDa, was transformed into *E. coli*DH5α competent cells. Recombinant clones were selected by plating on LB-ampicillin plates overnight at 37°C. Individual clones were picked and inserts were confirmed by DNA sequencing. The resulting fusion protein was overexpressed and purified by maltose affinity chromatography using amylose resin (New England Biolabs). A detailed expression and purification protocol has been published previously \[[@B39]\]. Expression and purification of the MBP-19 kDa fusion protein was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels either stained with GelCode Blue (Pierce Biotechnology Inc., Rockford, IL) or checked by immunoblot analysis with a monoclonal antibody against MBP developed at the National Animal Disease Center. Expression and purification of the MBP alone (without 19-kDa) was previously described \[[@B26]\]. SDS-PAGE and immunoblotting --------------------------- *E. coli*lysates expressing MBP-19 kDa were prepared as previously described \[[@B40]\]. SDS-PAGE was performed using 12% (wt/vol) polyacrylamide gels. Proteins were electrophoretically transferred onto nitrocellulose membranes (Schleicher and Schuell, Keene, NH) using the Trans Blot Cell (Bio-Rad Laboratories, Hercules, CA) in sodium phosphate buffer (25 mM; pH7.8) at 0.9 amps for 90 minutes. After transfer, the blots were blocked overnight with PBS plus 2% bovine serum albumin (BSA) and 0.1% Tween 20 (PBS-BSA). For immunoblots, serum from cattle with Johne\'s disease was diluted 1:500 in PBS-BSA. Sera were incubated on the blots at room temperature for 2 hours. After 3 washes in PBS plus 0.1% Tween 20, blots were incubated for 1.5 hours in anti-goat peroxidase-conjugated secondary antibody diluted 1:20,000 in PBS-BSA (Pierce Biotechnology Inc.). After secondary antibody incubation, the blots were washed 3 times as described above and were developed for chemiluminescent detection using SuperSignal detection reagents (Pierce Biotechnology Inc.). IFN-γ assays ------------ Blood was collected from the jugular vein of subclinically-infected cattle into sodium heparin vacutainer blood collection tubes. One ml aliquots of whole blood from each animal were plated into 4 wells of 24-well culture plates and cultured alone (non-stimulated) or with 10 μg/ml of *M. avium*subsp. *paratuberculosis*sonicate (WCL), 10 μg/ml of MBP, or 10 μg/ml of MBP-19 kDa. The WCL was prepared by sonication of bacilli and centrifugation exactly as described previously \[[@B37]\]. Blood-antigen mixtures were incubated for 18 hours at 39°C in a 5% CO~2~humidified atmosphere. Plates containing blood-antigen samples were centrifuged at 500 × g for 15 minutes and the plasma was harvested from each well. Plasma samples were frozen at -20°C until being analyzed for IFN-γ concentration by enzyme-linked immunosorbent assay (ELISA) using a commercial kit (Bovigam, BioCor, Omaha, NE) as recommended by the manufacturer. Samples were analyzed in duplicate and were determined to be positive for IFN-γ production if the absorbance of the stimulated sample (WCL, MBP, MBP-19 kDa) was 0.1 units greater than the absorbance of the nonstimulated well for that animal. This classification of IFN-γ positive samples has been previously reported by our laboratory as well as others \[[@B41],[@B42]\]. Statistical analysis -------------------- ANOVA and unpaired t tests were performed to analyze the IFN-γ stimulation data. Analyses were performed to compare average stimulation of control, non-infected cattle to the infected cattle groups (suspect, positive, high positive). Differences were considered significant when P \< 0.05. Authors\' contributions ======================= JFH carried out all the experiments and drafted the manuscript as part of his PhD dissertation. JRS provided advice, participated in its design and coordination and helped edit the manuscript. JPB conceived of the study, participated in its design and helped to draft the manuscript. Acknowledgements ================ The authors gratefully acknowledge the members of the NADC paratuberculosis research project for their assistance during this study. The lab members include: Trudy Tatum, Janis Hansen, Tonia McNunn, and Bart Olthoff. This work was funded by the USDA\'s Agricultural Research Service and CSREES NRI grant 2002-02228 to J. P. B. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Amino acid sequence comparison of the mycobacterial 19 kDa protein**. Non-conserved residues are shaded in black and gaps in amino acid sequence are indicated by hyphens (-). The predicted signal peptidase cleavage site for *M. tuberculosis*is indicated by a single arrow (↓), while the site for *M. avium*subsp. *paratuberculosis*is indicated by a double arrow (⇓). The GenBank accession number and abbreviations are *M. avium*subsp. *paratuberculosis*(M. paratb; [AAS02578]{.underline}), *M. avium*subsp. *avium*(M. avium; [AAB25888]{.underline}), *M. intracellulare*(M. intracel; AAB25885), *M. bovis*(M. bovis; S11234) and *M. tuberculosis*(M. tuberc; NP\_218280). ::: ![](1471-2180-5-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **SDS-PAGE and immunoblot analysis of the MBP-19 kDa fusion protein**. (A) An SDS-PAGE gel showing the noninduced and IPTG-induced *E. coli*protein lysates. Lanes: 1- protein size markers; 2- noninduced *E. coli*MBP-19 kDa; 3- IPTG induced *E. coli*MBP-19 kDa; 4- Affinity purified MBP-19 kDa. (B) Immunoblot probed with a monoclonal antibody to MBP. Lane 1- purified MBP; Lane 2- noninduced *E. coli*MBP-19 kDa; Lane 3- IPTG induced *E. coli*MBP-19 kDa. ::: ![](1471-2180-5-3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **The recombinant 19-kDa protein is recognized by serum from infected cattle**. Affinity purified MBP (lane 1) and MBP-19 kDa (lane 2) were transferred onto membranes in equal amounts and probed with sera from three clinically infected cattle: (A) animal 107; (B) animal 116; (C) animal 167. Reactivity was observed for the 19-kDa protein but not the MBP protein. Size standards are indicated in kilodaltons in the left margin. ::: ![](1471-2180-5-3-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### IFN-γ production by PBMCs in response to WCL and MBP-19 kDa stimulation. ::: **CONTROL WCL** **CONTROL 19 KDA** **SUSPECT WCL** **SUSPECT 19 KDA** **POSITIVE WCL** **POSITIVE 19 KDA** **HIGH POS WCL** **HIGH POS 19 KDA** ---------- ----------------- -------------------- ----------------- -------------------- ------------------ --------------------- ------------------ --------------------- 0 0 0.0294 0.0017 0.1058 0.0056 1.2407 0.9212 0.0623 0.0513 0.0980 0 0.1013 0.7737 1.0966 1.125 0.0072 0.0280 0.0412 0.1036 0.1323 0 0.3719 0.0124 0 0 0.0051 0.0286 0.1909 0.0290 0.3348 0.0925 0 0.0429 0.0259 0.0735 0.1623 0.0245 1.1866 0.7230 0.0560 0.0076 0 0 0.1974 0.0299 2.5995 0.0536 0.0561 0.0183 0.0348 0 0.0066 0.0285 0.0203 0.0071 0.0293 0.0236 0.0268 0.0002 **Mean** **0.0242** **0.0222** **0.0313** **0.0239** **0.1483** **0.1438** **1.1384** **0.4880** :::

PubMed Central

2024-06-05T03:55:52.451743

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548131/", "journal": "BMC Microbiol. 2005 Jan 21; 5:3", "authors": [ { "first": "Jason FJ", "last": "Huntley" }, { "first": "Judith R", "last": "Stabel" }, { "first": "John P", "last": "Bannantine" } ] }

PMC548132

Introduction ============ Apoptosis plays important roles in placentation and embryonic development \[[@B1]\]. The cells undergoing apoptosis have characteristic structural changes in the nucleus and cytoplasm. The nuclear disintegration involves DNA cleavage into oligonucleosomal length DNA fragments \[[@B2]-[@B4]\], and the DNA fragments can be detected by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end-labelling (TUNEL) technique. Expressions of apoptotic regulatory molecules, such as Fas, Fas ligand, P53, and the proteins of Bcl-2 family, have been reported in human placenta \[[@B5]-[@B8]\]. Bcl-2 and P53 are two of the key players in the apoptotic signaling cascades. Bcl-2, a proto-oncogene first discovered in human follicular lymphoma \[[@B9]\], is involved in the inhibition of apoptosis and the survival of a variety of cell types \[[@B10]\]. Bcl-2 protein is located in the membranes of endoplasmic reticulum, nuclear envelope, and mitochondria. Over-expression of Bcl-2 suppresses apoptosis by preventing the activation of caspases that carry out the process. P53 is well known as a tumor suppressor. It is a transcription factor that induces apoptosis mainly through inducing the expression of a batch of redox-related genes \[[@B11]\] and the down-regulating Bcl-2 \[[@B12]\]. The expression of Bcl-2 and P53 human placenta has been studied \[[@B1],[@B13]\]. However, their cellular distribution in the implantation site at early stage of pregnancy has not been reported. Because the monkey and the human share a very similar implantation process in terms of timing, morphological changes, and cell types involved \[[@B14]\], we aimed, in the present study, to investigate the expression, localization of Bcl-2 and P53 in the implantation site of the rhesus monkey, in order to gain some insights to the mechanism of time-dependent apoptosis occurring at the fetal-maternal interface. Materials and methods ===================== Animals ------- Healthy adult male and female rhesus monkeys (*Macaca mulatta*) were purchased from the monkey colony of the Primate Research Center (PRC), Kunming Institute of Zoology (KIZ), Chinese Academy of Sciences (CAS). All experimental procedures were approved by the Animal Ethics Committees of both the Institute of Zoology and PRC. The animals were caged individually and were evaluated daily by visual examination of the perineum for menses, with the onset of menses defined as Day 1 of the menstrual cycle. Adult female monkeys with regular menstrual cycles of approximately 28 days were chosen for this study. Female monkeys on Day 11 of their menstrual cycle were caged with a male monkey of proven fertility from previous mating for 3 days. Vaginal smears were examined the next morning for the presence of sperm. The day when the smear was detected as positive for sperms was designated as Day 1 of pregnancy (D1). The presence of a conceptus was confirmed by ultrasound examination. The monkeys were anesthetized by pentobarbital sodium (3 animals each group), and the uteri were removed surgically from early villous to villous placenta stages: on D17, D19, D28 and D34 of pregnancy respectively and cut into pieces, the specimens were quickly washed in cold phosphate-buffered saline (PBS) to remove adherent blood, then placed in cold 4% paraformaldehyde fixative for 16 h at 4°C and further processed through graded dehydration, clearing and embedding in paraffin for immunohistochemistry and TUNEL assay. Part of the specimen was cryopreserved at -70°C for Western blot analysis. Reagents -------- Primary antibodies including rabbit anti-human P53 (SC-6243) and mouse anti-human Bcl-2 (SC-7382) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Their quality and specificity were confirmed by the result of the Western blot analysis of D34 placental proteins (Figure [1](#F1){ref-type="fig"}). Rabbit anti-human cytokeratin (ZA0070), mouse anti-human actin (ZA0001), and mouse anti-Ki67 (ZM0166) were purchased from Zymed laboratories (San Francisco, CA, USA). Biotin labeled secondary antibodies, alkaline phosphatase (AP) conjugated avidin, horseradish peroxidase (HRP) conjugated goat anti-rabbit IgG, HRP-conjugated horse anti-mouse IgG, and AP substrates \"Vector-red\" were from Vector Laboratories (Burlingame, CA, USA). Digoxigenindideoxy (DIG)-11-dUTP, TdT, blocking reagent, AP-conjugated anti-DIG antibody and 5-bromo-4-chloro-3-indoxyl phosphate/nitro-blue tetrazolium chloride (BCIP/NBT) were purchased from Roche-Boehringer-Mannheim (Mannheim, Germany). Proteinase K was purchased from Merck-Schuchardt (Darmstadt, Germany). Levamisole were purchased from Sigma-Aldrich (St Louis, MO, USA). SuperSignal^®^West Pico substrate was from PIERCE Biotechnology (Rockford, IL, USA). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Western blot analysis of Bcl-2 and P53 for monkey tissue obtained on D34 of pregnancy**. Specific signals of Bcl-2 and P53 proteins were detected. No band was found in the control when antibodies were replaced with normal IgG of the same concentration and origin. ::: ![](1477-7827-3-4-1) ::: Western blot ------------ Western blot was done as previously described \[[@B15]\] with slight modifications to verify the cross-reactive specificity of the antibodies with the monkey tissue. The tissue of implantation sites on D34 of pregnancy was homogenized and the supernatant (50 μg) from centrifugation was run on a 10% SDS-PAGE gel under reduced conditions. After being transferred to the polyvinylidene difluoride membrane, individual lanes were cut and blocked with 5% nonfat milk/PBS for 1 h, followed by incubation at 20°C for 1 h with the primary antibodies (IgG, 0.2 μg/ml) in 5% milk/PBS. The membranes were washed three times, 5 min for each, in 5% milk/PBS and incubated with HRP-conjugated horse anti-mouse IgG (0.2 μg/ml, for Bcl-2) or HRP-conjugated goat anti-rabbit IgG (0.04 μg/ml, for P53) in 5% milk/PBS for 1 h respectively. The membranes were washed in PBS three times 5 min for each, followed by 10 min of incubation with SuperSignal^®^West Pico substrate, then exposed on x-ray film. For negative controls, primary antibodies were replaced with normal IgG of the same concentration and origin. TUNEL ----- Apoptotic cells were identified by using the TUNEL technique \[[@B1],[@B16]\]. The procedure was slightly modified based on Gao et al. \[[@B17]\] as the following. Deparaffinized and hydrated 4 μm sections were first treated with 10 μg/ml proteinase K at 37°C for 20 min, and then subjected to 3\'-end-labelling of the DNA with 1 μM DIG-11-dUTP and 1 U/μl TdT at 37°C for 1 h. The sections were washes three times in Tris buffer, and incubated with blocking buffer (100 mM Tris, 150 mM NaCl, pH 7.5, and 1% blocking reagent) for 30 min at room temperature. Next, sections were incubated with the primary AP-conjugated anti-DIG antibody (1:500 in 1% blocking reagent, 100 mM Tris, and 150 mM NaCl, pH 7.5) at room temperature for 2 h, and then washed with Tris buffer. Staining was developed using the standard substrates NBT (337.5 μg/ml) and BCIP (175 μg/ml). Negative controls were similarly processed with the omission of TdT. Immunohistochemistry -------------------- Serial 4 μm sections of tissue were deparaffinized and rehydrated through degraded ethanol. Antigen retrieval was performed by incubating the sections in 0.01 M citrate buffer (pH 6.0) at 98°C for 20 min followed by cooling at room temperature for 20 min. Non-specific binding was blocked with 5% (v/v) normal goat serum in PBS for 1 h. The sections were incubated with primary antibodies specific for P53 (1 μg/ml), Bcl-2 (2 μg/ml) or Ki67 (2 μg/ml) respectively in 2% goat serum overnight at 4°C. Sections were then washed three times with PBS (10 min each) and incubated with biotinylated secondary antibody (2 μg/ml) at RT for 30 min. 3 × 10 min successive washes were followed by incubation with avidin-AP complex (1:200, RT, 20 min). Sections were developed with standard substrates (337.5 μg/ml NBT and 175 μg/ml BCIP) or Vector Red AP substrates according to the manufacturer\'s protocol after another three washes. Endogenous AP activity was inhibited by supplement of 1 mM levamisole into substrate. The sections stained with Vector Red substrates were counter-stained using haematoxylin. Sections incubated with normal IgG instead of primary antibody served as negative controls. A double immunostaining technique using the antibodies to cytokeratin and actin was performed to localize the extravillous endovascular trophoblast cells. De-paraffinized sections were incubated with 3% H~2~O~2~in methanol for 10 min at room temperature to quench endogenous peroxidase after antigen retrieval treatment as described above. To detect the cytokeratin signal, the sections were washed (3 × 10 min in PBS), blocked for nonspecific signals, incubated sequentially with primary anti-human cytokeratin anbibody (1 μg/ml, RT, 1 h), secondary biotinylated goat anti-rabbit IgG (2 μg/ml, RT, 30 min), and avidin-peroxidase complex (1:200, RT, 20 min), and developed with DAB substrate solution in a similar way as described above. To detect actin signal, the procedure was repeated one more time with anti-human actin antibody (1 μg/ml, RT, 2 h) as primary antibody, AP conjugated horse anti-mouse IgG (1 μg/ml, RT, 40 min) as secondary antibody, and Vector Red developing AP substrate. As a result, the trophoblast cells were labeled brown and the blood vessel wall red. Microscopic assessment ---------------------- Placental samples from three individual monkeys for each group were analyzed. Experiments were repeated at least three times, and one representative from at least three similar results was presented. The mounted sections were examined using a Nikon microscope. For Ki67, the percentages of immunoreactive cells were assessed on at least 2000 cells in each tissue section; For TUNEL, the percentages of positive nuclei were assessed out of at least 2000 nuclei in each tissue section; For assessment of Bcl-2 staining intensity in cells of different compartments, semi-quantitative subjective scoring was performed by three blinded investigators using a 4-scale system with \"-\"= nil; \"+/-\"= weak; \"+\" = moderate; and \"++\" = strong as described by Yue et al. \[[@B18]\]. Results ======= Apoptosis in implantation site of early pregnancy ------------------------------------------------- The TUNEL technique was used to identify cell types that underwent apoptosis in the implantation site of rhesus monkey on D17, D19, D28 and D34 of pregnancy. On D17 and D19, apoptotic nuclei were observed in the syncytiotrophoblast layer covering the basal feet of the anchoring villi (Figure [2 A, B](#F2){ref-type="fig"}, arrowhead) and in the villous stromal cells (Figure [2 A](#F2){ref-type="fig"}, arrow), but not in the cytotrophoblasts. The positive nuclei in the syncytiotrophoblast was only about 0.06%. On D28 and D34, the apoptotic nuclei were present in the syncytiotrophoblast covering the villi (Figure [2 C, D](#F2){ref-type="fig"}), in the villous stromal cells (Figure [2 C, D](#F2){ref-type="fig"}, arrow), in the syncytiotrophoblast layer covering the basal feet of the anchoring villi (Figure [2 E](#F2){ref-type="fig"}), and in the cytotrophoblasts within the cell columns (Figure [2 F](#F2){ref-type="fig"}). On D28, the percentage of TUNEL-positive nuclei in the syncytiotrophoblast was 0.21%. As pregnancy progresses, the percentage increased to 0.34% on D34. In maternal compartment, a lot of apoptotic nuclei were detected in the stromal cells (Figure [2 G](#F2){ref-type="fig"}) and glandular epithelium (Figure [2 H](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Apoptosis detected by TUNEL at the implantation sites of the rhesus monkey on D17 (A), D19 (B), D28 (C, G) and D34 (D, E, F, H) of gestation.**Apoptotic nuclei were stained dark. Arrowhead and arrow in panel A -- D indicated the nuclei of syncytiotrophoblast and villous stromal cells respectively. The insets in C and D showed the positive nuclei under a higher magnification. Note the syncytiotrophoblast layer covering the basal feet of the anchoring villi in E and the cell columns in F. G and H represent the stromal cells and glandular epithelial cells respectively in the endometrium. I was the negative control. St, syncytiotrophoblast; CT, cytotrophoblast; Vi, placental villi. Scale bars represent 50 μm. ::: ![](1477-7827-3-4-2) ::: Proliferative activity in implantation site at early pregnancy -------------------------------------------------------------- Ki67 is a protein expressed in cycling cells from G1 to M phases and is widely used as a roliferative marker (19, 20). As shown in Figure [3](#F3){ref-type="fig"}, Ki67 was expressed in the cytotrophoblasts and the villous stromal cells, but not in the syncytiotrophoblasts. As pregnancy progresses, the percentage of Ki67-positive cytotrophoblast cells lining the villi decreased from more than 85% on D17 to less than 25% on D34 (Panel A-D, and E, F for a higher magnification). However, the cytotrophoblasts at the proximal tip of cell columns remained highly proliferative (more than 70%) at all stages (Panel G and H). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **The proliferating activity revealed by Ki-67 immunostaining at implantation sites of the rhesus monkey on D17 (A, E, G), D19 (B), D28 (C) and D34 (D, F, H) of gestation.**Panels A-D were under a lower magnification. Ki-67 protein was stained red, and nuclei blue. E and F were the placental villi under a higher magnification. G and H were the anchoring villi under a higher magnification. Vi, placental villi. ST, syncytiotrophoblast. CT, cytotrophoblast. Sc, stromal cell. En, endometrium. Scale bars represent 100 μm. ::: ![](1477-7827-3-4-3) ::: Bcl-2 expression in implantation site at early pregnancy -------------------------------------------------------- In order to study the mechanisms of the apoptosis observed at the fetal-maternal interface, the expression of Bcl-2 was investigated by using immunohistochemistry. At the early stages of placentation (D17, D19), Bcl-2 was only detected in the syncytiotrophoblast covering the cell columns (Figure [4, A](#F4){ref-type="fig"} and [4B](#F4){ref-type="fig"}) and the extravillous cytotrophoblast (Figure [4C](#F4){ref-type="fig"}, arrow). At the later stages (D28, D34) it was detected in all the syncytiotrophoblast (Figure [4,D](#F4){ref-type="fig"} and [4E](#F4){ref-type="fig"}), the villous stromal cells (Figure [4F](#F4){ref-type="fig"}, arrow), and the extravillous endovascular trophoblast cells (Figure [4G](#F4){ref-type="fig"}), the fetal origin of these cells were indicated by the anti-cytokeratin antibody staining (Figure [4 G](#F4){ref-type="fig"} inset, brown), and the vascular wall was stained by anti-actin antibody (red). The pattern of Bcl-2 expression in the syncytiotrophoblast was similar to that of the apoptotic nuclei distribution (Figure [2](#F2){ref-type="fig"}). In the maternal compartment, Bcl-2 could be detected in some stromal cells (Figure [4H](#F4){ref-type="fig"}). Notably, the cytotrophoblasts lining the villi, within the cell columns, and the glandular epithelia were negative for Bcl-2 staining. The semi-quantitative expression level of Bcl-2 in different cell types at the various stages was summarized in Table [1](#T1){ref-type="table"}. A gradual increase of Bcl-2 staining was observed in the syncytiotrophoblast as gestation advances. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Immunohistochemical staining for Bcl-2 at implantation sites of the rhesus monkey.**Bcl-2 staining is red, and nuclear counterstain blue. A, villous plancenta on D17. B, villous plancenta on D19. C, extravillous trophoblast cells in the basal plate of D17. D, villous plancenta on D28. E, villous plancenta on D34. F, villous plancenta on D34 under a higher magnification. G, the extravillous endovascular trophoblast cells; in the inset, the fetal origin of these cells was confirmed by anti-cytokeratin antibody (brown), and their position within the vascular wall was confirmed by anti-actin antibody staining (red). H, decidua. I, negative control. Vi, placental villi. ST, syncytiotrophoblast. CT, cytotrophoblast. Sc, stromal cells. Ge, glandular epithelium. Evc, extravillous cytotrophoblast. Scale bars represent 50 μm. ::: ![](1477-7827-3-4-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Semi-quantitative assessment of the immunohistochemical staining of Bcl-2 in the placenta of rhesus monkey. ::: D17 D19 D28 D34 ---------------------------------------------- ----- ----- ----- ----- Syncytiotrophoblast lining the villi +/- +/- ++ ++ Syncytiotrophoblast covering the cell column \+ \+ ++ ++ cytotrophoblast lining the villi \- \- \- \- extravillous cytotrophoblast \+ \+ \+ \+ ::: P53 expression in implantation site at early pregnancy ------------------------------------------------------ The expression profile of P53 was also acquired by using the immunohistochemistry. On D17 and D19, the expression of P53 was only confined to a small number of nuclei in the syncytiotrophoblast (Figure [5,A,B](#F5){ref-type="fig"}). On D28 and D34, its expression was observed not only in the syncytiotrophoblast (Figure [5C,D](#F5){ref-type="fig"}) but also in the nuclei of cytotrophoblasts lining the villi (Figure [5E](#F5){ref-type="fig"}) and within proximal tip of cell columns (Figure [5F](#F5){ref-type="fig"}) where a proliferative activity was high as indicated by Ki67 staining (Figure [3](#F3){ref-type="fig"}). Clustered P53-positive nuclei were seen in the syncytiotrophoblast covering the basal feet of the anchoring villi (Figure [5G](#F5){ref-type="fig"}), coincident well with the strong apoptosis detected by TUNEL (Figure [2E](#F2){ref-type="fig"}). P53 was also expressed in some stromal cells (Figure [5H](#F5){ref-type="fig"}) of the uterine endometrium. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Immunohistochemical staining for P53 at implantation sites of the rhesus monkey on D17 (A), D19 (B), D28 (C, H), and D34 (D, E, F, G) of gestation. P53 was stained dark in nuclei.**A-D were villous placenta under a lower magnification. The inset of panel A shows the staining in the syncytiotrophoblast covering the basal feet of the anchoring villi under a higher magnification. E, staining in villous placenta under a higher magnification. F, staining in cell columns. G, syncytiotrophoblast covering the basal feet of the anchoring villi under a higher magnification. H, the endometrium with arrows indicating stromal cells. ST, syncytiotrophoblast. CT, cytotrophoblast. Scale bars represent 50 μm. ::: ![](1477-7827-3-4-5) ::: Discussion ========== For the first time in present study, we investigated the expression of Bcl-2 and P53 in relation to apoptosis at the fetal-maternal interface of rhesus monkey at the very early stages (D17-D34) of gestation. Villous trophoblasts consist of cytotrophoblasts and syncytiotrophoblast. While cytotrophoblasts possess a brisk mitotic activity during the first trimester of gestation in human, the syncytiotrophoblast is incapable of cell division despite of a metabolic activity \[[@B1]\]. This fact implies that cell proliferation is differently regulated in these two cell types. The reports on the type of trophoblast cells undergoing apoptosis in the first trimester are controversial \[[@B1],[@B21],[@B22]\]. Our results further cleared that the apoptotic nuclei were distributed mainly in the syncytiotrophoblast at the early stages and in the cytotrophoblasts within the cell columns at later stages of pregnancy. In our previous study, Bax expression was found at the Fetal-Maternal Interface of Rhesus Monkey \[[@B17]\]. Bax is a Bcl-2 family member that promotes cell death susceptibility, possibly by countering the effect of Bcl-2 on cell survival through heterodimer interaction. Bax to Bcl-2 \"rheostat\" may be a critical factor in regulating apoptosis in multiplicate tissues. As shown in Figure [6](#F6){ref-type="fig"}, Bax was found expressed in the placenta and glandular epithelium of endometrium and all kinds of cells in placental villi, and no obvious change was observed between different time points from D17 to D34 in placental villi. Therefore, we speculated that Bcl-2 may play a more important role on controlling the apoptosis in placental villi. The diffusive expression of Bcl-2 in syncytiotrophoblast obtained from the first trimester human placenta has been reported recently \[[@B7],[@B23]-[@B25]\]. Our observation on the Bcl-2 expression in syncytiotrophoblast at later stages (D28-D34) agreed well with these data. As shown in this study, although Bcl-2 was expressed, apoptotic nuclei still exsisted in the same region. This phenomenon implies that the expression of Bcl-2 is not sufficient to completely inhibit the apoptosis in the syncytiotrophoblast. Therefore, the role of Bcl-2 here becomes an interesting question. Multiple nuclei sharing the same cytoplasm is a morphological characteristic of syncytiotrophoblast. In such cells, the apoptotic signal may be transmitted from one nuclear to another, and cause a spontaneous abortion. Therefore, the number of nuclei undergoing apoptosis in the syncytiotrophoblast should be limited by some mechanism in order to ensure normal embryo development in normal pregnancy \[[@B1]\]. We speculate that Bcl-2 may be included in this mechanism. The major role of apoptosis-associated Bcl-2 expression in the syncytiotrophoblast might be to limit the nuclear degradation to a special area and inhibit the spread of cell apoptosis signals to the other nuclei sharing the same cytoplasm, thus sustain cell survival in these multi-nucleated cells. Toki et al has also suggested that Bcl-2 might play a major role in avoiding the possible excessive nuclear degradation in syncytiotrophoblast \[[@B26]\]. Further studies, however, are needed to prove this speculation. The immunostaining for Bcl-2 was also detected in part of the extravillous and endovascular cytotrophoblast in our study. These subtypes of cytotrophoblast lost the capacity of proliferation (Ki-67-negative), but they did not undergo apoptosis (negative in TUNEL assay). Therefore, we hypothesize that Bcl-2 may also participate in regulation of the extravillous trophoblast apoptosis by stimulating the cellular survival. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Immunohistochemical staining for Bax at implantation sites of the rhesus monkey on D28.**Bax staining is brown, and nuclear counterstain blue. A, villous plancenta, positive staining was found in all the cells. B, endometrium, glandular epithelium was positive for Bax staining. Vi, placental villi. Ge, glandular epithelium. ::: ![](1477-7827-3-4-6) ::: P53 was partly identified in some nuclei of the syncytiotrophoblast with the same position of apoptotic nuclei, in the basal feet of the anchoring villi in particular, but it is not clear whether the P53 was co-localized with the apoptotic signals. Activation of P53 in some cell types leads to either the cessation of cell growth or apoptosis \[[@B27]\]. Therefore, P53 protein might be related to cell cycle arrest or apoptosis in syncytiotrophoblast during early stage of placentation. Low level of P53 staining was detected in the cytotrophoblasts during the earlier stages of gestation (D17 and D19). However, at the later stages (D28 and D34), the expression was observed predominantly in the nuclei of cytotrophoblasts. The presence of P53 in cytotrophoblast in the primate was consistent with that observed in the human first trimester placenta \[[@B8]\]. Indeed, the TUNEL staining showed that the apoptosis seldom happened in the cytotrophoblast, with the exception of cytotrophoblast at proximal tip of cell columns during later stages of placentation (D28, D34) where a high proliferative activity and P53 expression were detected. This finding supports the hypothesis that a physiological upregulation of the P53 tumour suppressor gene might be a mechanism for controlling excessive trophoblastic proliferation in normal placentation \[[@B26],[@B28]\]. It is known that early pregnancy is unique in its methods of cell proliferation control, the existing data suggest that some growth factors and transcription factors from the embryo and endometrium, such as CSF-1, VEGF, and transcription factors of the helix-loop-helix family, provide at least part of this control \[[@B29]\]. In addition, other studies found maternal age and some diseases, such as diabetes can also influence the apoptotic and proliferative activities in trophoblast cells \[[@B30],[@B31]\]. Further investigations are required to uncover which endocrine event regulates the expression of Bcl-2 and P53. Acknowledgments =============== This study was supported by WHO/Rockefeller Fundation Project (RF96020\#78), the National Science Fundation of China (30270196), Chuang-Xin program of Chinese Academy of Sciences (KSCX-2-SW-201/IOZ-7), and the Chinese \"973\" Program (G1999055901).

PubMed Central

2024-06-05T03:55:52.453898

2005-1-14

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548132/", "journal": "Reprod Biol Endocrinol. 2005 Jan 14; 3:4", "authors": [ { "first": "Peng", "last": "Wei" }, { "first": "Xuan", "last": "Jin" }, { "first": "Xue-Sen", "last": "Zhang" }, { "first": "Zhao-Yuan", "last": "Hu" }, { "first": "Chun-Sheng", "last": "Han" }, { "first": "Yi-Xun", "last": "Liu" } ] }

PMC548133

Background ========== It is well known that cytotoxic factors, such as lipopolysaccharides, derange nitrogen metabolism in hepatocytes and nitric oxide (NO) is involved among the other factors regulating this metabolic pathway \[[@B1]\]. NO is a free radical that is involved in many cellular events. In the biological systems NO has an halflife long lasting few seconds. It is an oxidation intermediate, therefore is both an oxidant and a reducing agent of metabolic products. Its biosynthesis is mainly performed by converting L-arginine to L-citrulline. L-arginine analogues, such as N^g^-nitro-L-arginine methyl ester (L-NAME), act as false substrates and are selective inhibitors of NO synthesis. NO synthase (NOS) is either a constitutive or inducible enzyme. The endothelial isoform (e-NOS) and the neuronal isoform (n-NOS) are constitutive. The inducible form of the enzyme (i-NOS), has the main property to be not regulated by intracellular calcium concentration and Ca^2+^-calmodulin complex, unlike the constitutive form \[[@B2]\]. It is known that iNOS is expressed by many cell types including macrophages, smooth muscle cells and hepatocytes \[[@B3]\]. Hepatocytes have been shown to express large levels of NO following exposure to endotoxins, such as bacterial lipopolysaccharide and/or cytokines, such as tumour necrosis factor-α (TNFα), interleukin-1 \[[@B4],[@B5]\]. NO may posses both cytoprotective and cytotoxic properties, depending on the amount and the isoform of NOS by which it is produced \[[@B6]\]. NO generally mediates beneficial responses, but becomes deleterious when coexistence with enhanced superoxide formation leads to the synthesis of peroxynitrite, a potent oxidant and nitrating agent \[[@B7]\]. According to this hypothesis, authors studied the effect of bradykinin, a proinflammatory mediator kinin, on cell viability and on urea production in cultures of rat hepatocytes. Kinins exert numerous physiological and pathological actions; they partecipate in vascular and cellular events that accompany the inflammatory processes. In pathological states, kinins are thought to be implicated in inflammatory diseases and in haemorrhagic and endotoxic shock \[[@B8]\]. To demonstrate the decrease of cell viability and urea production by bradykinin, the authors studied its effects on NO production. The measurements of NO release from hepatocytes were investigated by using a NO-specific fluorescence indicator, 4,5 diaminofluorescein diacetate (DAF-2DA) \[[@B9]\]. Results ======= Effect of bradykinin treatment on NO production ----------------------------------------------- The amounts of released NO were measured using DAF-2DA, that specifically reacts with the oxidized form of NO, producing the fluorescent triazolofluorescein \[[@B9]\]. NO determination was performed after 2 hours of incubation in the presence of bradykinin (0.01 mM and 0.1 mM). As shown in figure [1](#F1){ref-type="fig"} the treatment with 0.01 mM bradykinin did not produce NO increase compared to control, but 0.1 mM bradykinin increased significantly the NO release. In contrast no appreciable NO release was observed during the same period in hepatocytes cultured with 0.1 mM bradykinin and 1.68 mM L-NAME. Effect of bradykinin treatment on urea production ------------------------------------------------- To evaluate urea synthesis after bradykinin treatment, the hepatocytes were treated with 1 mM NH~4~Cl for 2 h. Figure [2](#F2){ref-type="fig"} shows that only the treatment with 0.1 mM bradykinin significantly decreased urea production and that the treatment with 0.1 mM bradykinin and 1.68 mM L-NAME did not produce a significant urea level decrease in comparison to control. Effect of bradykinin treatment on cell viability ------------------------------------------------ To determine the effects of bradykinin on cell viability, the hepatocytes were exposed to bradykinin (either 0.01 mM or 0.1 mM) for an incubation time of 2 hours. In one experimental series, the cell viability was determined by MTT test after 2 hours of incubation. In a second one, culture medium containing bradykinin was removed and replaced with the same fresh medium at 2 hours after the addition of bradykinin, and then cell viability was measured 24 hours after the end of bradykinin treatment. The MTT test after 2 hours of incubation does not indicate any significant viability difference in treated hepatocyte cultures in comparison to control (figure [3A](#F3){ref-type="fig"}). By MTT test after 24 h (figure [3B](#F3){ref-type="fig"}), a significant lowering of viability is observed in bradykinin 0.1 mM treated hepatocytes in comparison to control. The decrease was significantly reduced by the simultaneous treatment with L-NAME 1.68 mM even if always significantly lower than in control. Cell viability was validated by Trypan blue exclusion test (Table [1](#T1){ref-type="table"}). Discussion ========== The role of NO as mediator of hepatic injury after endotoxic shock remains controversial \[[@B16]\]. Increased NO production in response to cytokines has been demonstrated in cultured hepatocytes \[[@B17]\]. Laskin et al. \[[@B18]\] demonstrated that the induction of acute endotoxemia, caused an increase in NO production in the liver. This was associated with expression of inducible nitric oxide synthase (iNOS) messenger m-RNA in hepatocytes. Also our data showed an increase of NO production after 2 hours treatment of culture with 0.1 mM bradykinin in an arginine supplemented medium, as substrate for the synthesis of NO. The simultaneous treatment with L-NAME, a known inhibitor of NOS, blocked the increase of NO production. In this work we analyzed the urea synthesis after bradykinin treatment. Urea synthesis was decreased after 2 hours treatment with bradykinin 0.1 mM and the simultaneous treatment with L-NAME leaves urea biosynthesis unaltered. These data can be attributed to the control role of arginine in both urea and NO biosynthesis. When NO is synthesized from arginine, by the NOS reaction, cytrulline, an intermediate of urea cycle, is produced. Thus, the urea cycle is bypassed by the NOS reaction \[[@B1]\]. Whether NO exerts cytotoxic or cytoprotective action remains unclear \[[@B6]\]. We also found a significant decrease of viability, at long term, in hepatocytes subjected to bradykinin treatment. The simultaneous treatment of hepatocytes with L-NAME improves cell viability even if control levels are not restored. The data show that the increased NO production plays a role in liver damage induction, that follows the proinflammatory mediator treatment. The hepatocellular injury attributed to NO may be due either to its direct cytotoxicity or its reaction with superoxide to produce the toxic nitrogen metabolite peroxynitrite \[[@B19]\]. Oldenburg et al. \[[@B20]\], demonstrated in other cell types, like cardiomyocytes, that bradykinin caused the increase in reactive oxygen species (ROS) generation. At last, our results show that the increased NO synthesis induces a reduced urea production, that is an index of cell damage. The simultaneous treatment of liver cell cultures with L-NAME decreases NO levels and sustains overall biosynthesis activities and cell viability. Conclusions =========== In summary, we conclude that 0.1 mM bradykinin treatment induces an increase of NO levels and reduction of urea synthesis in the hepatocytes. This increased NO production mediates, after 24 hours, cell toxicity as shown by MTT test. In contrast, the administration of the NOS inhibitor L-NAME protects against cell damage and increases urea levels, suggesting that NO plays a key role in the bradykinin-induced liver damage. Methods ======= Materials --------- Unless otherwise specified, all chemicals were obtained from Sigma (St. Louis, MO, USA). Isolation and culture of rat hepatocytes ---------------------------------------- Hepatocytes were isolated from male rats, Wistar strain, (180 to 200 gbw), by a modification of the method of Seglen \[[@B10]\]. All procedures on the animals were performed according to the CEE directive n. 86/609 on animal experimentation. Rats were anesthetized with diethylether, the pre-perfusion of the liver in situ was performed at a rate of 20--30 ml/min with Ca^2+^-free Hanks balanced salt solution. The liver was then excised and the digestion was carried out by adding 0.05% (w/v) collagenase (type IV) in Hanks balanced salt solution supplemented with CaCl~2~·H~2~O (0.0186 g/L) at a flux rate of 40 ml/min. At this point liver was transferred to a square plate containing 100 ml of RPMI 1640 medium supplemented with 200 mM L-glutamine, 20 ml/L essential amino acid solution and 10 ml/L non-essential amino acid solution, 1% antibiotic antimycotic stabilized solution and 100 μM L-arginine (incomplete medium). The cells were dispersed by gentle distruption with a stainless steel comb. After filtration through 200 μm Nytal mesh, parenchymal cells (hepatocytes) were separated from nonparenchymal cells (endothelial cells, Kupffer cells and stellate cells) by centrifugation at 50 g in Eppendorf Centrifuge 5810R at 4°C for 2 minutes and then washed twice in washing buffer \[[@B11]\]. Then the cells were resuspended in the same medium and filtered through 63 μm Nytal mesh. The viability of the cells was more than 80%, as estimated by trypan blue dye exclusion test \[[@B12]\]. After cell counting the cells were diluited at a concentration of 5 × 10^5^cells/ml with incomplete medium supplemented with 2% fetal calf serum, 0.1 U/ml insulin and 10^-6^M dexamethasone (complete medium). The hepatocytes were then plated in 24 well-plates coated with rat tail collagen at the final cell density of 2.5 × 10^5^cells per well and incubated at 37°C in an humidified atmosphere of 5% CO~2~and 95% air. After 6 hours incubation, the medium was changed and replaced with incomplete medium to remove dead cells. To verify the isolation method efficiency, the acid fosfatase activity per mg of proteins was evaluated. According to literature data, the specific activity of acid fosfatase in nonparenchimal cells is 1,7 folds the same activity in parenchimal cells \[[@B13]\]. Treatment --------- After 24 hours of culture the hepatocytes were exposed either to bradykinin (0.01 mM and 0.1 mM) or bradykinin 0.1 mM supplemented with L-NAME 1.68 mM \[[@B14]\]. Determination of NO from hepatocytes ------------------------------------ DAF-2DA (Alexis Biochemicals, Lausen, Switzerland) was dissolved in DMSO (1 mg/0.45 ml) and diluted to 10 μM in phosphate buffer (0.1 M, pH 7.4). Then the cells were either incubated in phosphate buffer containing 10 μM DAF-2DA, bradykinin (0.01 mM and 0.1 mM) and bradykinin 0.1 mM supplemented with L-NAME 1.68 mM. After 2 hours of incubation in this reaction mixture, the fluorescence from the reaction of DAF-2DA with NO released from hepatocytes was measured with Perkin-Elmer MPF-44B Spectrofluorimeter calibrated for excitation at 495 nm and emission at 515 nm. Results were expressed as a percentage of the fluorescence of the samples in comparison to control. Determination of urea synthesis ------------------------------- To determine the effects of bradykinin on urea production, cells were treated either with bradykinin (0.01 mM and 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM. At the same media 1 mM NH~4~Cl was added. After 2 hours urea levels in the media were measured by spectrophotometric method using Urea Color 2 Kit (Sclavo Diagnostics, Siena, Italia) measuring absorbance at 600 nm and blank sample with the same NH~4~Cl final concentration was used. Urea synthesis was calculated as ng urea per cell per hour. Determination of cell viability ------------------------------- Cell viability was determined by MTT test method \[[@B15]\] and confirmed by Trypan blue exclusion test \[[@B12]\]. MTT (5 mg/ml) was dissolved in RPMI-1640 without phenol red. The solution is filtered through a 0.2 μm filter and stored at 2--8°C for frequent use. To determine the effects of bradykinin on cell viability, cells were either treated with bradykinin (0.01 mM and 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM for a 2 h period. After that cells were used either immediately or after an additional 24 h incubation period in incomplete medium. For the determination of cell viability, the medium has been discarded and MTT solution was added and incubated for 3 hours. At the end of the incubation period the MTT solution was removed and the cells and dye cristals were dissolved by adding dimethylsulfoxide (DMSO). Absorbance was measured at 570 nm in a Shimadzu UV-2100 Spectrophotometer and the results were expressed as a percentage of the absorbance of the samples in comparison to control. Statistical analysis -------------------- At least four independent determinations of each parameter were compared to control using Student\'s T-test. Differences were considered significant when p \< 0.05 was obtained. Authors\' contributions ======================= SS: Fluorimetric analysis and overall statistical analysis of data MG: Director of research MS: Spectrophotometric analysis CR: Primary hepatocyte cultures and characterization All authors read and approved the final manuscript Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Determination of NO release after treatment of hepatocytes with bradykinin. Fluorescence intensity was measured after 2 h incubation: with 10 μM DAF-2DA in basal conditions (white column, reference), in presence of 0.01 mM bradykinin (hatched column), in presence of 0.1 mM bradykinin (crosshatched column) and in presence of 0.1 mM bradykinin with 1.68 mM L-NAME (black column). The excitation wavelength was 495 nm and the emission wavelength was 515 nm. Values, expressed as a percentage of control values, are the means ± S.E.M. (bars) of four independent experiments. ![](1472-6793-5-2-i1.gif) p \< 0,05 compared with control ::: ![](1472-6793-5-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Determination of urea production after treatment of hepatocytes with bradykinin. Urea production was spectrophotometrically determined at 600 nm after 2 h incubation with 1 mM NH~4~Cl in basal conditions (white column), in presence of 0.01 mM bradykinin (hatched column), in presence of 0.1 mM bradykinin (crosshatched column) and in presence of 0.1 mM bradykinin with 1.68 mM L-NAME (black column). Values, expressed as ng urea per cell per hour, are the means ± S.E.M. (bars) of four independent experiments. ![](1472-6793-5-2-i1.gif) p \< 0,05 compared with control ::: ![](1472-6793-5-2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Determination of cell viability in hepatocytes treated with bradykinin. The cell viability was spectrophotometrically determined at 570 nm by MTT assay in hepatocytes incubated in basal conditions (white column), in presence of 0.01 mM bradykinin (hatched column), in presence of 0.1 mM bradykinin (crosshatched column) and in presence of 0.1 mM bradykinin with 1.68 mM L-NAME (black column) for 2 h period. (A) Cell viability determined immediately after. (B) Cell viability determined after an additional 24 h incubation period in incomplete medium Results are expressed as a percentage of control. Values are the means ± S.E.M. (bars) of four independent experiments. ![](1472-6793-5-2-i1.gif) p \< 0,05 compared with control. ![](1472-6793-5-2-i2.gif) p \< 0,05 compared with control and 0.1 mM bradykinin treated cells ::: ![](1472-6793-5-2-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of bradykinin treatment on cell viability ::: Treatment: 2 h+Brad (Viability %) Treatment: 2 h+Brad + 24 h-Brad (Viability %) ------------------------------------ ----------------------------------- ----------------------------------------------- Control 100 ± 10 100 ± 6 0.01 mM Bradykinin 98 ± 12 104 ± 10 0.1 mM Bradykinin 104 ± 8 50 ± 11\* 0.1 mM Bradykinin + 1.68 mM L-NAME 102 ± 10 79 ± 7\*\* Hepatocytes were isolated and cultured in presence and in absence of bradykinin (either 0.01 mM or 0.1 mM) and cotreated with bradykinin 0.1 mM and L-NAME 1.68 mM for a 2 h period. Cell viability was determined by Trypan blue exclusion test either immediately after 2 h or after additional 24 h incubation period in incomplete medium. Results are expressed as a percentage of control. Values are expressed as mean ± S.E.M., n = 4. \*Statistically significant differences (p \< 0,05) from control levels as determined by Student\'s t-test. \*\* Statistically significant differences (p \< 0,05) from control levels and from 0.1 mM bradykinin treated cells as determined by Student\'s t-test. :::

PubMed Central

2024-06-05T03:55:52.455819

2005-1-25

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548133/", "journal": "BMC Physiol. 2005 Jan 25; 5:2", "authors": [ { "first": "Settimio", "last": "Sesti" }, { "first": "Guglielmo", "last": "Martino" }, { "first": "Sergio", "last": "Mazzulla" }, { "first": "Rosa", "last": "Chimenti" } ] }

PMC548134

Background ========== Diabetes mellitus, the prevalence of which is reaching epidemic proportions in many parts of the world, is an increasingly important public health concern. In the United States, diabetes is present in 8% of the adult population, and is associated with a two-fold increase in age-adjusted mortality \[[@B1],[@B2]\]. In addition, there is a high prevalence of chronic medical conditions among subjects with diabetes \[[@B3]-[@B5]\]. For example, in the United States, the prevalence of cardiovascular diseases, stroke and depression in subjects with diabetes was at least twice as high as in subjects without diabetes \[[@B6]-[@B9]\]. Diabetes has detrimental effects on a range of health outcomes including health-related quality of life (HRQoL) \[[@B10]-[@B12]\]. For example, in the Medical Outcomes Study, diabetes was found to impair all dimensions of health except mental health and pain \[[@B13]\]. In a more recent multinational study, diabetes was found to have a notable impact on general health, measured using the Medical Outcomes Short-Form 36 (SF-36) \[[@B14]\]. The magnitude of impact of diabetes on HRQoL was reported to be equivalent to that of having cardiovascular conditions, cancer and chronic respiratory disease \[[@B15]\]. Subjects with diabetes and multiple co-existing chronic medical conditions have poorer HRQoL than those without these conditions \[[@B16],[@B17]\]. For example, subjects with diabetes and co-existing cardiovascular diseases reported significantly lower scores on RAND-36 social functioning, vitality and health-change scales \[[@B16]\]. In another study, subjects with diabetes and co-existing coronary artery disease, peripheral sensory neuropathy and peripheral vascular diseases reported significantly lower scores on several SF-36 scales \[[@B17]\]. In combination, the influence of multiple chronic medical conditions on HRQoL may exhibit an additive, synergistic or possibly subtractive relationship. Assuming that each chronic medical condition results in the lowering of HRQoL, in an additive relationship, the combined effect of two or more chronic medical conditions on HRQoL approximates the sum of the independent effect of each of these conditions, while in a synergistic relationship, the combined effect is greater than the sum of the independent effect of each of these conditions and in a subtractive relationship the combined effect is smaller than the sum of the independent effect of each of these conditions (Figure [1](#F1){ref-type="fig"}). For example, the SF-36 developers \[[@B18]\] reported an additive relationship between hypertension and other co-existing chronic medical conditions on HRQoL, while Gaynes et al. \[[@B19]\] reported synergistic relationships between depression and co-existing arthritis on physical functioning and between depression and co-existing diabetes on role functioning. Several mechanisms could account for these observed synergistic effects. For example, treatment for one medical condition might also adversely affect another pre-existing medical condition, leading to a greater lowering of HRQoL than would occur due to the pre-existing condition alone \[[@B20]\]. Additionally, a medical condition itself (e.g. depression) might adversely affect patient behavior and thus negatively affect treatment outcomes \[[@B21]\]. Although there have not been any reports in the literature, it is theoretically possible that subtractive relationships exist. For example, it was reported that patients undergo changes in their conception of poor levels of functioning, personal values (e.g. changes in life priority) and/or meaning of life in response to their chronic medical conditions, a concept known as response shift \[[@B22]\]. These changes in self-assessment and values may help to cushion the impact of the second medical condition, resulting in a smaller decrement in HRQoL than might otherwise be expected. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### An overview of the possible relationships among multiple chronic medical conditions on HRQoL. \*These relationships (subtractive or synergistic) are discussed in relation to additive relationships. ^†^If net effect is zero, then discussion of subtractive and synergistic relationships do not apply. ::: ![](1477-7525-3-2-1) ::: Two published studies have evaluated the relationship between diabetes and other chronic medical conditions on HRQoL in the general population \[[@B23],[@B24]\]. In the first study \[[@B23]\], diabetes and stroke were found to increase the risks of disability (measured with the Activity of Daily Living and Instrumental Activity of Daily Living scales) among older Mexican Americans in an additive fashion. In the second study, the impact of co-existing obesity, hypertension or diabetes and heart disease on HRQoL among 17,195 U.S. middle- and older-age adults was evaluated \[[@B24]\]. A strong synergistic relationship between heart disease and diabetes on the odds of mobility difficulty (1.8 to 4.0 times), activity of daily living limitations (2.2 to 4.0 times) and poor perceived health (2.1 to 6.8 times) was found, after adjusting for gender, ethnicity, insurance status, history of cancer, and lung diseases \[[@B24]\]. However, there were two important limitations in both studies. First, these studies focused on the physical domains of HRQoL, without assessing the mental and social domains of HRQoL. Second, both studies were conducted among middle-aged and/or elderly adults. Thus, the burden of diabetes and other chronic medical conditions on HRQoL in the rest of the general population with diabetes are not known. Further studies to elucidate the relationship between diabetes and other chronic medical conditions on physical, mental and social domains of HRQoL in the general population are clearly needed. The primary purpose of this study was therefore to evaluate the impact of diabetes and co-existing chronic medical conditions on the physical, mental and social domains of HRQoL in the general population. We also sought to determine if the impact of diabetes and co-existing chronic medical conditions on HRQoL would be additive, synergistic or subtractive. For these purposes, we measured HRQoL using both the SF-36 and the SF-6D. The SF-36 is a comprehensive, generic HRQoL measure that has been extensively validated and allows comparison of HRQoL in subjects with various chronic medical conditions. The SF-6D is a single index utility score derived from the SF-36 which measures health preference, and which is therefore suitable for pharmacoeconomic analyses to assist healthcare resource allocation. We sought to determine the extent to which the SF-6D would reflect the multidimensional information provided by the SF-36. Methods ======= Study design ------------ We analyzed data from a cross-sectional, population-based disproportionately stratified sample of ethnic Chinese, Malays and Indians listed in the 1996 electoral register for the Bishan-Toa Payoh district (representative of the general population) in Singapore from April 1998 to January 1999, details of which have been reported previously \[[@B25]\] and are summarized here. Participants completed either the UK English or Chinese (Hong Kong) SF-36 and the Family Functioning Measure. Eligibility criteria were: age 21 to 65 years on 1^st^January 1998 and ability to read a newspaper in English or Chinese; Chinese, Malay or Indian ethnicity as reflected in the National registration identification card, which reflects parental ethnicity; and status as free-living adults in the community. Persons older than 65 years of age were excluded because of low literacy rates in this age group in Singapore \[[@B26]\]. Fieldworkers visited eligible subjects at their house within 7 days after an introductory letter was sent, invited them to self-administer the SF-36, checked returned questionnaires for completeness and obtained information on ethnicity, socioeconomic status and chronic medical conditions (including diabetes, hypertension, heart disease, lung diseases, musculoskeletal illnesses and mental illnesses) and other potential determinants of HRQoL through a structured interview. Participants were asked to indicate either \"yes\" or \"no\" on a list of chronic medical conditions including diabetes, high blood pressure, heart disease, etc. Instruments ----------- ### The Short-Form 36 Health Survey (SF-36) The SF-36 measures perceived health in the areas of physical functioning (PF), role-physical (RP), bodily pain (BP), general health (GH), vitality (VT), social functioning (SF), role-emotional (RE), and mental health (MH), with higher scores (range 0--100) reflecting better perceived health. The UK English \[[@B27]\] and Chinese (Hong Kong) \[[@B28]\] SF-36 with 4-week recall and metric units of measurement were used in this study (previously validated for use in Singapore) \[[@B25]\]. ### The SF-6D The SF-6D is a six-dimensional health classification system assessing physical functioning (PF), role limitations (RL), social functioning (SF), pain (PN), mental health (MH), and vitality (VT), with 4 to 6 levels per dimension \[[@B29],[@B30]\]. A SF-6D \"health state\" is defined by selecting one level from each dimension. For example, perfect health on the SF-6D would be represented by the health state 111111. A total of 18,000 health states are thus defined. All responders to the original SF-36 questionnaire can be assigned an SF-6D score provided the items used to construct the SF-6D are completed \[[@B29],[@B30]\]. The SF-6D preference-based measure can be regarded as a continuous variable scored on a 0.29 to 1.00 scale, with 0.29 corresponding to the worst health state (i.e. all dimension being at the worst level) and 1.00 corresponding to \"full health\" (i.e. all dimensions being at full functional level). ### Family Functioning Measure The Family Functioning Measure is a 3-item Likert scale assessing the quality of interactions among family members \[[@B31]\], with higher scores (range 0--100) reflecting better family functioning, and has been validated for use in Singapore \[[@B32]\]. Statistical analyses -------------------- Data from participants completing English and Chinese SF-36 versions were pooled to increase the power and representativeness of our study. This approach was supported by previous work demonstrating equivalence of these SF-36 \[[@B33]\] and SF-6D \[[@B34]\] versions in this same study sample. Data were entered into an Excel spreadsheet (Microsoft Corporation, Redmond, Washington) and analyzed using SPSS (SPSS Inc., Chicago, Illinois) software. SF-6D index scores were calculated using a utility function derived from the United Kingdom general population \[[@B29]\], which is currently the only published SF-6D scoring algorithm. Participants with missing scale scores were excluded listwise from analysis. To study the influence of diabetes and other chronic medical conditions on HRQoL, we developed 9 multiple linear regression models, with each SF-36 scale and the SF-6D index score being the dependent variable for a separate model. Each model was built in 3 stages. The first stage assessed the effect of diabetes on HRQoL while adjusting for the influence of sociodemographic factors. The second stage assessed the independent effects of diabetes and a second chronic medical condition on HRQoL while adjusting for the influence of sociodemographic factors. The third stage additionally assessed any interactions between diabetes and this second chronic medical condition on HRQoL. Thus, each model studied the effect of diabetes and one other chronic medical condition. The absence of a significant interaction term would suggest that the influence of two chronic medical conditions on HRQoL scores was additive. The presence of a significant interaction term would suggest that a synergistic or subtractive relationship existed, the former if the coefficient for the interaction term was negative, the latter if the coefficient was positive (since changes in HRQoL scores in the presence of chronic medical conditions were expected to be negative). Ethnicity was coded using dummy variables. Educational level was used as proxy for socioeconomic status. In anticipation that if the number of subjects with a given chronic medical condition was less than 50, the numbers of subjects with this medical condition and diabetes would be very small and not amenable to meaningful interpretation, two chronic medical conditions, stroke and cancer, were thus excluded from analysis because of small numbers of subjects reporting these conditions (n\<50). Two other chronic medical conditions were subsequently excluded from analysis because of small numbers of subjects with diabetes and these conditions (diabetes and lung diseases: n = 27 and diabetes and mental illnesses: n = 9). Results ======= Characteristics of study subjects --------------------------------- Complete data were available from 5,224 of 5,420 subjects in the study, with 196 (3.6%) subjects excluded from analysis (110 because of missing demographic information and 86 because of missing responses to the SF-36). Table [1](#T1){ref-type="table"} shows subjects\' characteristics and distribution of SF-36 scales and SF-6D index scores. Approximately 6% of subjects suffered from diabetes and two-fifths of subjects reported at least 1 chronic medical condition. Prevalence of co-existing conditions in subjects with diabetes ranged from 2.9% (mental illnesses) to 37.2% (hypertension). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of subjects and distribution of SF-36 scales and SF-6D index scores. ::: Subject characteristics (N = 5,224) N (%) unless specified otherwise --------------------------------------------------------------------------------------- ---------------------------------- Mean (SD) age in years 40.3 (11.52) Female gender 2,555 (48.9) Ethnicity Chinese 2,558 (49.0) Malay 1,189 (22.8) Indian 1,477 (28.3) Housing type Lower cost public housing 388 (7.4) Public housing 4,647 (89.0) Private housing 188 (3.6) Years of education ≤ 6 1,266 (24.2) 7--10 2,682 (51.3) \>10 1,276 (24.4) Marital status Married 3,686 (70.6) Single 1,272 (24.3) Divorced/Separated 136 (2.6) Widow 130 (2.5) Working 4,022 (75.0) Smoking 1,045 (20.0) Presence of acute medical conditions\* 3,493 (66.9) Presence of chronic medical conditions Diabetes mellitus 309 (5.9) Hypertension 557 (10.7) Heart disease 125 (2.4) Stroke 32 (0.6) Lung diseases^‡^ 278 (5.3) Cancer 35 (0.7) Musculoskeletal illnesses^‡^ 1,389 (26.6) Mental illness^‡^ 136 (2.6) Prevalence of co-existing chronic medical conditions in people with diabetes mellitus Hypertension 115 (37.2) Heart disease 46 (14.9) Lung diseases^‡^ 27 (8.7) Musculoskeletal illnesses^‡^ 107 (34.6) Mental illnesses^‡^ 9 (2.9) Mean (SD) Family Functioning Measure scores 10.3 (2.75) Mean (SD) SF-36 scores^†^ Physical functioning (PF) 80.3 (23.19) Role-physical (RP) 80.5 (32.94) Bodily pain (BP) 78.3 (21.73) General health (GH) 68.6 (17.21) Vitality (VT) 63.8 (16.98) Social functioning (SF) 80.6 (20.48) Role-emotional (RE) 79.2 (34.98) Mental health (MH) 72.4 (16.70) Mean (SD) SF-6D index scores^†^ 0.77 (0.130) \* Acute medical conditions were self-limiting illnesses and injuries in the preceding one month that might influence SF-36 scores and included acute illnesses (e.g. upper respiratory tract infections, gastroenteritis and headaches), injuries or poor sleep. ^†^Mean scores adjusted for the influence of diabetes mellitus, other chronic medical conditions (and their interactions), socioeconomic status and other factors. ^‡^Lung diseases included asthma and other lung diseases; musculoskeletal illnesses included rheumatism, back pain and other bone or muscle illness; mental illness included depression, anxiety disorder and schizophrenia. ::: The influence of chronic medical conditions on SF-36 scores ----------------------------------------------------------- The influence of chronic medical conditions on individual SF-36 scales is presented in Table [2](#T2){ref-type="table"}. Before adjusting for selected sociodemographic variables known to influence HRQoL (i.e. age, gender, ethnicity and years of education), subjects with self-reported chronic medical conditions (diabetes, hypertension, heart disease and musculoskeletal illnesses) generally reported lower scores for most SF-36 scales when compared to subjects without these conditions, indicating poorer HRQoL. After adjusting for the influence of these sociodemographic variables, chronic medical conditions continued to influence HRQoL, generally to a lesser degree than before adjustment. The influences of heart disease and musculoskeletal illnesses on SF-36 scores were of a similar magnitude and were larger than the influence of diabetes or hypertension. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Influence of diabetes mellitus and other chronic medical conditions on SF-36 scales. ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Unadjusted differences in mean scores ------------------------------------ ----------------------------------------------------------------- -------------- -------------- -------------- -------------- -------------- -------------- -------------- -------------- PF RP BP GH VT SF RE MH SF-6D No chronic medical condition^†^\ 82.0 (22.61) 81.9 (32.16) 80.4 (21.20) 70.0 (16.62) 64.8 (16.43) 81.5 (20.12) 77.5 (36.00) 73.1 (16.35) 0.79 (0.123) (n = 3155) Diabetes mellitus only (n = 309) -7.2 -7.3 -5.8 -3.5 -1.3 -1.8 0.7 0.2 -0.06 Hypertension only\ -4.3 -4.1 -3.4 -3.3 -0.5 0.5 1.0 0.3 -0.05 (n = 557) Heart disease only (n = 125) -9.5 -10.1 -7.3 -5.2 -3.6 -0.8 -4.7 -0.3 -0.09 Musculoskeletal illnesses only^‡^\ -4.7 -3.6 -6.9 -4.4 -3.1 -2.7 -0.8 -2.5 -0.08 (n = 1389) Adjusted differences in mean scores due to medical condition^§^ PF RP BP GH VT SF RE MH SF-6D No chronic medical condition^†^\ 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.00 (n = 3155) Diabetes mellitus only (n = 309) -2.9 \* -3.7 -1.8 -2.36 \* -1.3 -0.1 0.3 -0.2 -0.03 \*\* Hypertension only\ -2.6 \* -3.1 \* -1.5 -1.6 0.1 0.6 -1.0 0.1 -0.03 \*\*\* (n = 557) Heart disease only (n = 125) -5.0 \* -6.5 \* -3.5 -4.0 \* -3.9 \* 0.9 -5.9 -1.0 -0.06 \*\*\* Musculoskeletal illnesses only^‡^\ -3.8\ -3.1\ -6.4\ -3.9\ -2.9\ -3.1\ -3.4\ -2.6\ -0.08\ (n = 1389) \*\*\* \*\* \*\*\* \*\*\* \*\*\* \*\*\* \*\* \*\*\* \*\*\* ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ^†^Mean (SD) scores ^‡^Musculoskeletal illnesses included rheumatism, back pain and other bone or muscle illnesses. ^§^Multiple linear regression, adjusted for influence of ethnicity, age, gender and years of education. \*p \< 0.05, \*\*p \< 0.01, \*\*\*p \< 0.001 Abbreviations: PF = Physical Functioning, RP = Role-Physical, BP = Bodily Pain, GH = General Health, VT = Vitality, SF = Social Functioning, RE = Role -Emotional, MH = Mental Health ::: The influence of chronic medical conditions on SF-6D scores ----------------------------------------------------------- The influence of chronic medical conditions on SF-6D scores is also presented in Table [2](#T2){ref-type="table"}. Subjects with chronic medical conditions similarly reported lower unadjusted mean SF-6D scores. After adjusting for known determinants of HRQoL, lowering of SF-6D scores persisted for all chronic medical conditions, though again to a smaller magnitude. The influences of heart disease and musculoskeletal illnesses on SF-6D scores were of a similar magnitude and were again larger than the influence of diabetes or hypertension. The influence of diabetes mellitus and co-existing chronic medical conditions on SF-36 scores --------------------------------------------------------------------------------------------- Characteristics of subjects with diabetes, with and without other co-existing chronic medical conditions, are shown in Table [3](#T3){ref-type="table"}. Subjects with diabetes only (i.e. no co-existing chronic medical conditions) were generally younger and more likely to be male. There were more Indians with diabetes and co-existing heart disease or musculoskeletal illnesses and more Chinese with diabetes and co-existing hypertension. The distribution of years of education completed was fairly similar in the various ethnic groups. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Characteristics of subjects with diabetes, with and without other chronic medical conditions. ::: ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Co-existing chronic medical conditions among subjects with diabetes -------------------------------------- --------------------------------------------------------------------- --------------- ---------------- ------------------------------- Characteristics No co-existing chronic medical conditions\ Hypertension\ Heart disease\ Musculoskeletal illnesses^†^\ (n = 115) (n = 115) (n = 46) (n = 107) Mean age (years) 48.1 (10.52) 53.7 (8.46) 55.5 (7.95) 53.2 (8.68) Male (%) 64.3 56.5 71.7 54.2 Ethnicity (%) Chinese 23.5 40.0 21.7 36.4 Malays 27.8 22.6 21.7 17.8 Indians 48.7 37.4 56.5 45.8 Questionnaire language (English) (%) 86.1 73.9 91.3 76.6 Education (%) ≤ 6 years 36.5 44.3 30.4 41.1 7--10 years 50.4 42.6 56.5 48.6 \> 10 years 13.0 13.0 13.0 10.3 ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: The impact of co-existing chronic medical conditions on adjusted SF-36 scores of subjects with diabetes is presented in Table [4](#T4){ref-type="table"}. In general, after adjusting for known determinants of HRQoL, the presence of concurrent hypertension, heart disease and musculoskeletal illnesses further reduced SF-36 scores in subjects with diabetes. For example, subjects with diabetes and co-existing hypertension or musculoskeletal illnesses experienced further lowering of physical functioning scores by 2.3 and 3.7 points, respectively. The influence of two chronic medical conditions on SF-36 scores was generally additive, in that statistically significant interaction terms were not present for most comparisons. There were some exceptions in which subtractive effects were observed, mainly clustered within subjects with diabetes and co-existing heart disease (physical functioning, role-physical, social functioning and role-emotional scales) or musculoskeletal illnesses (bodily pain and social functioning scales). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Multiple linear regression models of the influence of diabetes mellitus and a second chronic medical condition (i.e. hypertension, heart disease or musculoskeletal illnesses) adjusted for ethnicity, age, gender and years of education on SF-36 and SF-6D scores. ::: ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Co-existing chronic medical conditions among subjects with diabetes ------------------------------------ --------------------------------------------------------------------- --------------- ------------------------ ------------------------------- No co-existing chronic medical conditions\ Hypertension\ Heart disease (n = 46) Musculoskeletal illnesses^†^\ (n = 115) (n = 115) (n = 107) Physical Functioning Differences in scale scores due to Diabetes mellitus -2.9\* -2.4 -3.5\* -2.8\* 2nd chronic medical condition‡ na -2.3\* -7.9\*\* -3.7\*\*\* Interaction termξ na ns 12.46\*\* ns Role-Physical Differences in scale scores due to Diabetes mellitus -3.7 -3.1 -5.2\* -3.6 2nd chronic medical condition‡ na -2.8 -12.5\*\* -3.1\*\* Interaction termξ na ns 20.1\*\* ns Bodily Pain Differences in scale scores due to Diabetes mellitus -1.8 -1.6 -1.5 -3.8\* 2nd chronic medical condition‡ na -1.3 -3.1 -6.9\*\*\* Interaction termξ na ns ns 7.0\*\* General Health Differences in scale scores due to Diabetes mellitus -2.3\* -2.0 -1.9 -2.2\* 2nd chronic medical condition na -1.3 -3.5\* -3.9\*\*\* Interaction termξ na ns ns ns Vitality Differences in scale scores due to Diabetes mellitus -1.3 -1.4 -0.9 -1.3 2nd chronic medical condition‡ na 0.3 -3.7\* -2.9\*\*\* Interaction termξ na ns ns ns Social Functioning Differences in scale scores due to Diabetes mellitus -0.1 -0.2 -1.3 -1.9 2nd chronic medical condition^‡^ na 0.6 -2.5 -3.4\*\*\* Interaction term^ξ^ na ns 10.2\* 5.4\* Role-Emotional Differences in scale scores due to Diabetes mellitus 0.3 -2.8 -0.9 0.4 2nd chronic medical condition^‡^ na -2.7 -11.8\*\* -3.4\*\* Interaction term^ξ^ na 9.9\* 16.8\* ns Mental Health Differences in scale scores due to Diabetes mellitus -0.2 -1.7 -0.1 -0.1 2nd chronic medical condition^‡^ na -0.5 -0.9 -2.6\*\*\* Interaction term^ξ^ na 5.1\* ns ns SF-6D Differences in scale scores due to Diabetes mellitus -0.03\*\* -0.02\* -0.02\* -0.02\*\* 2nd chronic medical condition^‡^ na -0.03\*\*\* -0.06\*\*\* -0.08\*\*\* Interaction term^ξ^ na ns ns ns ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ^†^Musculoskeletal illnesses included rheumatism, back pain and other bone or muscle illnesses. ^‡^Second chronic medical condition refers to the chronic medical condition other than diabetes mellitus listed in the relevant column (e.g. hypertension, heart disease or musculoskeletal illnesses). ^ξ^Interaction term for diabetes mellitus and second chronic medical condition. \*p \< 0.05, \*\*p \< 0.01, \*\*\*p \< 0.001, ns denotes statistically insignificant. ::: The influence of diabetes mellitus and co-existing chronic medical conditions on SF-6D scores --------------------------------------------------------------------------------------------- The influence of diabetes and co-existing chronic medical conditions on SF-6D scores is presented in Table [4](#T4){ref-type="table"}. Subjects with diabetes and other co-existing chronic medical conditions reported lower unadjusted SF-6D scores than subjects with diabetes only. After adjustment for known determinants of HRQoL, the influence of co-existing chronic medical conditions on SF-6D scores persisted. As before, subjects with diabetes and co-existing heart disease or musculoskeletal illnesses reported the greatest impairment in HRQoL. Diabetes and other co-existing chronic medical conditions, including heart disease, reduced SF-6D scores in an additive fashion. Discussion ========== In this multiethnic, population-based study, we found that subjects with diabetes experienced lowering of HRQoL as compared to subjects without diabetes. The presence of other chronic medical conditions in subjects with diabetes led to further lowering of HRQoL, the effect of which was generally additive. Our findings further underscore the importance of preventing and treating complications of diabetes to prevent further deterioration in HRQoL among subjects with diabetes, and also highlight the need to identify factors that may be modulated to improve HRQoL in these subjects. Our findings are important for several reasons. First, this is the first study showing that the combination of diabetes and a second chronic medical condition may adversely affect the mental domains of HRQoL as measured by the vitality, social functioning and mental health scales of the SF-36 (previous studies having focused on the physical domains of HRQoL) \[[@B23],[@B24]\]. Second, it is reassuring that the effect of diabetes and a second chronic medical condition on HRQoL was in general additive rather than synergistic. Third, given that subjects were drawn from the general population, it is likely that our findings can be readily generalized to the population at large, especially given that this study was conducted in a multiethnic Asian population with one of the highest diabetes prevalence rates in the world \[[@B35]\]. We found that the effect of diabetes on HRQoL was generally mild, with greater impact on the SF-36 scales measuring physical (physical functioning, role-physical, bodily pain, general health, role-emotional (in this study sample)) relative to mental health components (vitality, social functioning and mental health). This was not surprising, given that our subjects were recruited from the general population and were therefore likely to have less severe illness than subjects in hospital or clinic-based studies. Further, other studies have shown that impact of diabetes on HRQoL is intermediate, relative to other chronic medical conditions \[[@B14]\]. For example, Egede \[[@B9]\] reported that the risk of functional disability was lower in subjects with diabetes than subjects with major depression (odds ratio (OR): 2.42 vs 3.00), while Otiniano et al. \[[@B23]\] found that the risk of disabilities in ADL were lower in subjects with diabetes than those with strokes (OR: 2.80 vs 5.55). We also found that with the exception of subjects with diabetes and heart disease, the presence of co-existing chronic medical conditions in subjects with diabetes generally resulted in further significant lowering of HRQoL. Our results are important because they demonstrate that the impact of these co-existing chronic medical conditions in diabetes is not only in increasing healthcare costs \[[@B36],[@B37]\] and mortality \[[@B38]\] but also in increasing the physical and psychosocial burden of diabetes. Given that complications of diabetes constitute the majority of chronic medical conditions commonly present in subjects with diabetes, our findings further underscore the importance of preventing and treating complications of diabetes, and also highlight the need to identify factors that may be modulated to improve HRQoL in these subjects. In our study, the relationships between diabetes and other chronic medical conditions on HRQoL were largely additive. This is in contrast with the study by Gaynes et al. \[[@B19]\], where synergistic effects between depression and diabetes on HRQoL were observed. Instead, we found in general an additive effect with several comparisons showing a subtractive effect on HRQoL (especially in subjects with diabetes and co-existing heart disease). There are several possible explanations for the presence of subtractive effects. One possible explanation is the presence of response shift related to the specific diseases involved. In this study, the subtractive effects were clustered among subjects with diabetes and co-existing heart disease. Diabetes (in particular Type 2 diabetes) is a well-recognized cardiovascular risk factor with subjects with diabetes commonly developing heart disease in the 5^th^or 6^th^decade of life \[[@B40]\], often many years after the diagnosis of diabetes \[[@B41]\] (although heart disease may on occasion be present at or before diagnosis) \[[@B42],[@B43]\]. In contrast, hypertension typically precedes or occurs soon after diagnosis in subjects with Type 2 diabetes, with up to 50% of subjects with diabetes presenting with hypertension at the time of diagnosis \[[@B44]\]. Hence, adaptation to the underlying diabetes may have led to response shift occurring in subjects with diabetes and co-existing heart disease but not with hypertension. A second plausible explanation for this observation is a healthy-responder effect. This would occur if subjects with diabetes and co-existing heart disease were more likely to have passed away or were too sick to participate in the study. Thus the subset of subjects with diabetes and co-existing heart disease who did participate in this study would reflect the healthier end of the spectrum, leading to the observed effect of higher scores for respondents with the two co-existing chronic medical conditions. A third possible explanation is that some conditions are so similar in their effects on HRQoL that having more than one such condition is not particularly problematic to the individual. For example, in our study, subjects with diabetes alone and subjects with heart disease alone experienced lower scores on the physical functioning scale of the SF-36; if these two conditions exerted a similar effect on HRQoL, then a subtractive effect would be expected, as was indeed observed among subjects with diabetes and co-existing heart disease. A secondary objective of this study was to understand the extent to which the single index SF-6D captured information from the multidimensional SF-36. We found that in general, the impact of co-existing chronic medical conditions on the SF-36 was well-reflected in the SF-6D. However, the subtractive effects of diabetes and co-existing heart disease on SF-36 scores were not reflected in SF-6D scores. This suggests that there is some inevitable loss of information associated with a reduction from a multi-dimensional scale to a unidimensional scale. Hence, more studies are needed to evaluate the adequacy of the SF-6D as a summary measure of the SF-36. We recognize several limitations of this study. First, identification of chronic medical conditions was based on self-report which may not be as accurate as physician diagnoses. However, reliability of self-report has been found to be acceptable for conditions that required medical or laboratory diagnostic procedures, including diabetes \[[@B45]-[@B47]\]. Second, although we captured information on subjects with diabetes and co-existing mental illnesses or lung diseases, we had to exclude these because of the small number of subjects. Finally, in this study we did not differentiate between subjects with Type 1 and Type 2 diabetes. However, given that more than 90% of subjects with diabetes are Type 2, this is not likely to affect our findings. Conclusions =========== In conclusion, in this large, multiethnic, population-based study, we found that subjects with diabetes experienced lowering of HRQoL as compared to subjects without diabetes. The co-existence of other chronic medical conditions in subjects with diabetes led to further lowering of HRQoL, the effect of which was generally additive. Finally, we found that the SF-6D is a reasonably good summary measure of the SF-36 although more studies are needed to confirm this observation. List of abbreviations ===================== ADL -- Activities of Daily Living, DM -- Diabetes mellitus, HD -- Heart disease, HRQoL -- Health-related quality of life, HTN -- Hypertension, MS -- Musculoskeletal illnesses, OR -- Odds ratio, SF-36 -- Medical Outcomes Short-Form 36. Authors\' contributions ======================= JT conceived of the study, and participated in its design and coordination. HL Wee and YB Cheung participated in the design of the study and performed the statistical analysis. SC Li and KY Fong participated in the design of the study and its coordination. All authors read and approved the final manuscript. Acknowledgements ================ We would like to thank Dr John Brazier for providing the scoring algorithm for the SF-6D, and Prof PH Feng, Drs ML Boey, KH Leong and Staff Nurse ST Thio for their contributions to the study on which this analysis is based. This study was funded by grants from the Biomedical Research Council of Singapore (03/1/27/18/226) and the National Medical Research Council of Singapore (0160/96).

PubMed Central

2024-06-05T03:55:52.457294

2005-1-12

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548134/", "journal": "Health Qual Life Outcomes. 2005 Jan 12; 3:2", "authors": [ { "first": "Hwee-Lin", "last": "Wee" }, { "first": "Yin-Bun", "last": "Cheung" }, { "first": "Shu-Chuen", "last": "Li" }, { "first": "Kok-Yong", "last": "Fong" }, { "first": "Julian", "last": "Thumboo" } ] }

PMC548135

Background ========== This and the following presentation are part of a project for the International Programme on Chemical Safety (IPCS) evaluating the possible use of transgenic animal mutagenicity assays in chemical toxicity testing and mechanistic research. It was decided to compare the results obtained from those transgenic mutagenicity test systems where considerable data was available, with other in vivo genotoxicity tests: in the first article (Part 1) with the mouse bone marrow micronucleus test and in the second (Part II) with the mouse spot test. The mouse bone marrow micronucleus test is one of several available *in vivo*mammalian test system for the detection of chromosomal aberrations \[[@B1]-[@B5]\]. A documentation of the test procedure and evaluation of results is given in the OECD guideline 474 \[[@B6]\]. This test is routinely used with a widespread acceptance in industry and authorities. Mutagenicity assays using transgenic animals have been developed in particular the *lacI*model \[[@B7]\] (commercially available as the Big Blue^®^mouse), and the *lacZ*model \[[@B8]\] (commercially available as the Muta™Mouse). In this article, available data on the results of mouse bone marrow micronucleus test were compared with results from these two transgenic mouse assays for 39 substances. The advantages and disadvantages of the test systems are discussed. Recently, further transgenic rodent mutation assays have been developed, however, the data base is not sufficient for comparison with other test systems. The mouse bone marrow micronucleus test: principles and procedure ----------------------------------------------------------------- Micronuclei are chromatin-containing bodies in the cytoplasm arising from acentric chromosome fragments or from whole chromosomes that were not incorporated in the daughter nuclei during the last stages of mitosis. Chromosome fragments are associated with the clastogenic (chromosome breakage) activity of the test substance whereas the presence of a whole chromosome is indicative of an adverse effect of the test substance on the mitotic spindle apparatus (aneugenic effects). The difference in size of the micronucleus is therefore an indicator for clastogenicity (small micronucleus) or aneugenicity (large micronucleus). However, the size of the micronucleus is an inaccurate measure. Micronuclei can be distinguished by further criteria, for example by identification of the presence of a kinetochore or centromeric DNA, indicating aneugenic activity. Overall, an increase in micronuclei is an indirect measure of induced structural or numerical chromosome aberrations \[[@B1],[@B2]\]. In the micronucleus test according to the OECD guideline 474, erythroblasts in the bone marrow of mice (or rats) are used as target cells. When a bone marrow erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded. Any micronucleus that has been formed may remain in the otherwise anucleated cytoplasm and can easily been detected. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage \[[@B6]\]. In the last three decades, toxicologists have often used the mouse bone marrow micronucleus test because 1) it is part of the regulatory toxicology in the admission procedure for chemicals and drugs and 2) it has advantages in speed, simplicity, and cost effectiveness in comparison to other *in vivo*systems for testing chromosomal aberrations (e.g. the cytogenetic test). Transgenic mouse models ----------------------- Transgenic mutation test systems contain a foreign gene construct having two essential parts: the transgene containing the *target gene*that serves as target for mutations, and a *shuttle vector*for recovering the target gene DNA from the tissue of the transgenic animal. The transgene is constructed using recombinant DNA technologies. ### *LacI*transgenic mouse model (Big Blue^®^mouse) The proprietary mouse of the Big Blue^®^mutagenesis assay system contains about 30--40 copies of a lambda LIZα shuttle phage vector integrated into its genome at a single locus on chromosome 4. The target site for mutagenesis is the *lacI*gene \[[@B7]\]. The test compound is administered to the mouse either as a single or repeated dose. After the post treatment period for manifestation of the DNA lesions, the tissue of interest is isolated and the DNA extracted. A proprietary lambda DNA packaging extract automatically excises the lambda vector target and packages it into a lambda phage head and the phage is transfected to bacteria. These bacteria are plated on agar indicator plates containing the chromogenic substance (X-gal). The phage transfected bacteria with mutations in the *lacI*gene form blue plaques, whereas bacteria with a nonmutated *lacI*form colourless plaques. The ratio between blue and white plaques is a measure of the mutagenicity. \[[@B7],[@B9]\]. ### *LacZ*transgenic mouse model (Muta™Mouse) The lambda-gt10-*lacZ*shuttle vector for the *lacZ*mouse model contains the entire lacZ target gene \[[@B8]\]. The commercially available *lacZ*mouse model contains about 80 copies of the shuttle vector at chromosome 3. As above, the test compound is administered to the mouse, the genomic DNA is isolated from the tissue of interest, and the lambda genomes are excised by and packed with a bacteriophage packaging extract. The resulting phage particles are then plated on a *lacZ*^-^*E. coli*strain (for transfection) in the presence of the chromogenic substance (X-gal) as indicator. The plaques containing an intact *lacZ*are β-galactosidase active and are blue, whereas plaques containing mutated *lacZ*will be white/colourless. In this original model the ratio between colourless and blue plaques is a measure of the mutagenicity. Due to optical difficulties in the evaluation of plaques, this system has been improved using a selection assay *in lieu*of colour screening whereby only mutant particles form plaques \[[@B10]\]. The number of plaques under non-selective conditions is a measure for the total number of phage-transfected bacteria (intact **or**mutant lacZ gene). The ratio between the number of plaques produced under selective conditions versus the number of plaques under non-selective conditions is a measure of the mutagenicity \[[@B9],[@B10]\]. Methods ======= Data presented in this documentation are the results of an extensive literature research. Concerning data on transgenic mouse assays only primary literature was used. Data on the mouse bone marrow micronucleus assay were extracted from reliable reviews on this item or from primary literature. For all other data informations from secondary literature or data compilation bases were used. Results and Discussion ====================== Comparison of data from the mouse bone marrow micronucleus test and transgenic mouse test ----------------------------------------------------------------------------------------- The authors are aware that a comparison of the transgenic mouse assays with the mouse bone marrow micronucleus test is limited by the fact that different genotoxic endpoints are studied in these two systems. In transgenic mouse assays, point mutations and small insertions and deletions are detected whereas in the mouse bone marrow assay, chromosome breakage leading to light microscopically visible micronuclei resulting from chromosome fragment or micronuclei originated from whole chromosomes are investigated. However, both point mutations and micronuclei may be induced by a single agent, so some overlap of results is to be expected. From the literature, 39 substances were identified with data on the mouse bone marrow micronucleus test **and**the Muta™mouse assay (n = 29) or the Big Blue^®^mouse assay (n = 21) or both transgenic mutation assays (n = 11); see [Additional file 1](#S1){ref-type="supplementary-material"} for references. Agreement between the Muta™mouse and the micronucleus test was seen with 18 out of 29 substances, no agreement with 8 substances and in 3 cases the comparison is inconclusive because of questionable results in the micronucleus assay. With the Big Blue^®^mouse assay, the results obtained with 14 out of 21 substances agreed with results in the mouse micronucleus test, but 6 showed no agreement and another was inconclusive. Most substances included in this comparison are also carcinogenic in long-term assays on mice. No data on carcinogenicity in mice are available for 4-acetylaminofluorene and negative results were obtained with bromomethane and 2,6-diaminotoluene. Carcinogenic substances with positive results in the bone marrow micronucleus and transgenic gene mutation assays ----------------------------------------------------------------------------------------------------------------- ### Studies with Muta™mouse The following 17 substances were gene mutagenic in at least one of the examined organs in the Muta™mouse assay, induced micronuclei in mouse bone marrow and showed positive results in carcinogenicity studies on mice: 2-acetylaminofluorene, 4-aminobiphenyl, benzo(a)pyrene, 1,3-butadiene, chlorambucil, cyclophosphamide, 7,12-dimethylbenz(a)anthracene, ethylmethanesulfonate, N-ethyl-N-nitrosourea, methylmethane-sulfonate, N-methyl-N\'-nitro-N-nitrosoguanidine, N-methyl-N-nitrosourea, 4-nitroquinoline-1-oxide, N-nitrosodimethylamine, procarbazine, quinoline, and urethane. Further studies on chromosome aberration *in vitro*and *in vivo*(see [Additional file 1](#S1){ref-type="supplementary-material"}) supported the results in the micronucleus test, except data on 4-aminobiphenyl (negative micronucleus test in rats) and quinoline (see below). Of the substances tested for mutagenic activity in bone marrow in Muta™mice, 14 gave positive results, only 1,3-butadiene, methylmethanesulfonate, and quinoline were negative, indicating that other organs are more sensitive. 1,3-Butadiene is clearly clastogenic in other studies on chromosome aberration in mice and induced gene mutation in the bone marrow of Big Blue^®^mice (see [Additional file 1](#S1){ref-type="supplementary-material"}). Quinoline, however, gave inconclusive results in other *in vivo*studies on clastogenic effects in bone marrow of rats and mice. ### Studies with Big Blue^®^mouse 12 substances showed gene mutagenic effects in at least one of the examined organs in the Big Blue^®^mouse assay, chromosome mutagenic effects in the mouse bone marrow micronucleus assay and induced carcinogenic effects in long-term assays on mice,: 2-acetylaminofluorene, aflatoxin B1, benzene, benzo(a)pyrene, 1,3-butadiene, cyclophosphamide, 7,12-dimethylbenz(a)anthracene, ethylene oxide, N-ethyl-N-nitrosourea, N-methyl-N-nitrosourea, N-nitrosodimethylamine, urethane. In the Big Blue^®^mouse assay the target organ bone marrow revealed also increased mutation frequencies induced by benzene, 1,3-butadiene, and 7,12-dimethylbenz(a)anthracene. However, negative results in bone marrow were obtained with cyclophosphamide, ethylene oxide, and N-nitrosodimethylamine, indicating that other target organs are more sensitive in the Big Blue^®^mouse assay. All of these 12 substances induced also chromosome aberrations in majority of further *in vitro*and *in vivo*studies. Substances with carcinogenic effects in mice but no agreement between the transgenic mouse assay and the mouse bone marrow assay -------------------------------------------------------------------------------------------------------------------------------- ### Acrylamide The inconclusive result in the bone marrow micronucleus test \[[@B4]\] is contradictory to the Muta™mouse assay \[[@B21]-[@B23]\] which shows mutagenic activity in the target organ bone marrow but also contradictory to other *in vivo*tests on the endpoint chromosome aberration including a cytogenetic test on mice \[[@B4],[@B19],[@B21]\]. Micronuclei were detected in spleen and testis of mice \[[@B4]\]. Overall, using other experimental design the mouse bone marrow micronucleus assay might give clearly positive results. ### 2-Amino-3-methylimidazo(4,5-f)quinoline (IQ) IQ is mutagenic in the liver of the Muta™mouse \[[@B34]\] but negative results were obtained in the mouse bone marrow micronucleus test \[[@B2],[@B35]\]. This negative result is supported by a negative cytogenetic assay on mice. Inconclusive results were obtained in *in vitro*studies on chromosome aberrations but IQ induced micronuclei in rats (see [Additional file 1](#S1){ref-type="supplementary-material"}). This discrepancy might be due to the possibility that a) the liver but not the bone marrow of mice is target organ (the liver but not the blood is target organ in carcinogenicity \[[@B33]\]) or b) IQ is less clastogenic than gene mutagenic in mice. ### ortho-Anisidine The target organ of carcinogenesis in mice (and humans) is the bladder \[[@B36]\]. In the Big Blue^®^assay on mice mutagenic activity was detected in the bladder but not in the liver \[[@B37]\]. As expected, the mouse bone marrow micronucleus test \[[@B4],[@B36]\] gave negative results (also in rats; see [Additional file 1](#S1){ref-type="supplementary-material"}), because the bone marrow is presumably not a target organ of mutagenicity. ### Asbestos crocidolite Local carcinogenic effects were observed in carcinogenicity studies, the lung is the target organ after inhalation \[[@B38]-[@B40]\]. Mutagenic activity was detected in the lung of Big Blue^®^mice after inhalation \[[@B41]\]. As expected, clearly no systemic effects in the bone marrow could be observed in the mouse micronucleus test \[[@B4]\]. ### 2,4-Diaminotoluene The main target organ in carcinogenicity is the liver (also in rats) \[[@B81]\]. Positive results were reported in two Big Blue^®^mouse assays examining the liver \[[@B82]-[@B84]\]. The mouse micronucleus test gave negative results \[[@B4]\], however, systemic effects in the bone marrow are not expected from carcinogenicity studies. Results of other studies on chromosome aberration *in vivo*are inconclusive (see [Additional file 1](#S1){ref-type="supplementary-material"}). ### Hydrazine This substance induced no mutagenic effects in lung, liver, or bone marrow of the Muta™mouse which were target organs in mouse carcinogenicity studies \[[@B119],[@B120]\]. The mouse bone marrow micronucleus test gave positive results after repeated application \[[@B4],[@B119]\]. However, single exposure was used in the Muta™mouse assay \[[@B120]\]. Studies on other *in vivo*genotoxicity endpoints have shown almost negative results after single exposure but genotoxic activity after repeated application, for example in the mouse bone marrow micronucleus assay \[[@B4]\]. Positive results might be expected in the Muta™mouse assay using another experimental design since other *in vivo*as well as *in vitro*test systems revealed gene mutagenic effects. ### Methyl methanesulfonate Only weak mutagenic effects in the liver but no effects in bone marrow were observed in the Muta™mouse \[[@B18],[@B124]\] and negative results in the Big Blue^®^mouse \[[@B111]-[@B113],[@B126]\]. In the mouse bone marrow micronucleus test this carcinogenic substance induced chromosome aberration \[[@B1],[@B127]\]; other *in vitro*and *in vivo*assays clearly supported the clastogenic activity \[[@B121],[@B122]\]. There is evidence that the chromosome mutagenic activity is detectable at much lower doses than the gene mutagenic activity. Tinwell et al. (1998) \[[@B18]\] have shown on Muta™mice a weak gene mutagenic effect in the liver but no effect in the bone marrow. The same dose induced in these animals a significant increase in bone marrow micronuclei indicating clear clastogenic activity. Overall, methyl methanesulfonate is more clastogenic than gene mutagenic. ### Mitomycin C A very similar situation is given with mitomycin C. No mutagenic activity was observed in the Muta™mouse assay in liver and bone marrow after single injection but the same dose induced chromosome aberrations in the bone marrow of the same mice \[[@B142]\]. The mouse bone marrow micronucleus test \[[@B1],[@B47]\] and all *in vivo*and *in vitro*assays on the endpoint chromosome aberration revealed clearly positive results \[[@B139]-[@B141]\]. ### N-Nitrosodiethylamine The main target organ in systemic carcinogenesis is the liver \[[@B146]-[@B148]\]. As expected, mutagenic effects were detected in the liver of the Muta™mouse, but none in the bone marrow \[[@B104],[@B106],[@B149],[@B150]\]. The same mice showed also no micronuclei at these dose levels \[[@B104]\]. Consequently, the mouse bone marrow micronucleus test gave negative results indicating that this is not a target organ of genotoxicity. However, there is also evidence that this substance shows more gene mutagenic activity than clastogenic activity because other *in vivo*studies gave no clear indication for chromosome aberration (see [Additional file 1](#S1){ref-type="supplementary-material"}). ### N-Nitrosodi-N-propylamine There is evidence that this substance shows more gene than chromosome mutagenic activity. Beside local carcinogenic effects in carcinogenicity studies on mice and rats systemic effects were located in the liver (mouse and rats) and in bone marrow (rat) \[[@B160]-[@B163]\]. Several target organs were detected in the Muta™mouse assay including liver and bone marrow \[[@B164]\]. But no chromosome mutagenic activity was recorded in the mouse bone marrow micronucleus test \[[@B4]\]. Further *in vivo*data on this endpoint are not available (see [Additional file 1](#S1){ref-type="supplementary-material"}). ### Phenobarbital Liver tumours were detected in carcinogenicity studies on mice and rats \[[@B165]-[@B167]\]. In the Muta™mouse assay no mutagenic activity was observed although the liver weight of the treated mice increased indicating systemic effects in this organ \[[@B149],[@B168]\]. Two studies are available on the Big Blue^®^mouse, one gave negative \[[@B16]\] and the other weak positive results \[[@B169]\]. Taken together the results in transgenic mice are inconclusive which is in accord with the mouse bone marrow nucleus test (inconclusive results). Inconclusive (*in vitro*) or negative results were obtained in other studies on clastogenicity or other endpoints of genotoxicity (see [Additional file 1](#S1){ref-type="supplementary-material"}). Overall, there is no clear indication of gene or chromosome mutagenic activity in transgenic mouse assays or other genotoxic tests systems. However, genotoxic mechanisms in carcinogenicity cannot be excluded. ### β-Propiolactone This is an alkylating substance with predominantly local carcinogenic effects\[[@B175],[@B176]\]. In the Muta™mouse assay \[[@B131]\] local effects in the stomach were observed in gavage studies plus systemic effects in the liver, but no mutagenic activity was seen in bone marrow of the Muta™mouse \[[@B131]\]. As expected, no increased incidence in micronuclei was detected in the bone marrow of treated mice \[[@B1],[@B4]\] although clastogenic effects were observed in *in vitro*studies, in insects and plants (see [Additional file 1](#S1){ref-type="supplementary-material"}) indicating chromosomal aberration after direct contact with this alkylating substance. ### Trichloroethylene This substance gave clearly positive results in different carcinogenicity studies on mice \[[@B186]\]. Although the target organs of carcinogenicity (including bone marrow) were investigated in the Muta™mouse assay, no mutagenic activity was noted \[[@B187]\]. The positive results in the mouse bone marrow micronucleus test are contradictory to other *in vivo*studies on clastogenicity. However, a further (simple) reason for the negative results in the Muta™mouse assay might be that the MTD was not reached \[[@B187]\]. Overall, further discussion on the mechanisms of carcinogenicity is necessary. ### Tris(2,3-dibromopropyl)phosphate This might be a further example for a substance where genotoxic effects are not related to the target organ bone marrow but induce systemic effects in others. The target organ in oral carcinogenicity studies on mice and rats was the kidney, in mice systemic carcinogenic effects were also seen in lung and liver \[[@B188]-[@B190]\]. In the Big Blue^®^mouse assay, mutagenic activity was detected in the kidney in gavage studies\[[@B46],[@B191]\]. The mouse bone marrow micronucleus assay (i.p. injection)\[[@B4]\] as well as cytogenetic studies on rats and mice were negative \[[@B188]-[@B190]\]. Substance without data on carcinogenicity in mice and differing results in the micronucleus test and transgenic mouse assay --------------------------------------------------------------------------------------------------------------------------- ### 4-Acetylaminofluorene This substance showed mutagenic activity in the Muta™mouse assay \[[@B18]\] but inconclusive results in the mouse bone marrow micronucleus test \[[@B2]\]. No data on carcinogenicity are available on 4-acetylaminofluorene. However, data on two *in vitro*mammalian test systems indicated gene mutagenic activity \[[@B17]\] supporting results in the transgenic assay. Substances without carcinogenic effects in mice and differing results in the micronucleus test and transgenic mouse assay ------------------------------------------------------------------------------------------------------------------------- ### Bromomethane No increased tumour incidences were observed in mice as well as in re-evaluated studies on rats \[[@B58],[@B59]\]. Negative results were obtained also in the examined organs including liver and bone marrow in the Muta™mouse assay. However, DNA methylation in the liver was observed in the same assay even at lower dose levels \[[@B60]\] indicating differences in the sensitivity of these two genotoxic endpoints. The mouse micronucleus test revealed chromosome mutagenic activity \[[@B61]\] although results of other *in vivo*tests are equivocal (see [Additional file 1](#S1){ref-type="supplementary-material"}). ### 2,6-Diaminotoluene No carcinogenic effects were seen in a valid long-term study on mice and rats \[[@B85]\]. In the Big Blue^®^mouse no mutagenic activity was induced \[[@B82],[@B84]\]. However, only the liver was examined and the assays had some limitations (one dose tested, MTD possibly not reached). The mouse micronucleus test revealed chromosome aberrations \[[@B3],[@B47]\] as well as the available *in vitro*studies but no clastogenic activity was detected in a cytogenetic study on rodents (see [Additional file 1](#S1){ref-type="supplementary-material"}). Substances with carcinogenic effects in mice but nongenotoxic mechanisms are presumed ------------------------------------------------------------------------------------- ### Chloroform (German MAK Classification 4 = substances with carcinogen effects where the genotoxic effects are absent or only play an insignificant role) Liver tumours were induced in B6C3F1 mice \[[@B72]\], however no mutagenic effects were detected in the liver of Big Blue^®^mice of the same strain in a valid assay \[[@B74]\]. Accordingly, no increased incidence in micronuclei were observed in the bone marrow of mice \[[@B1],[@B47]\] and no clastogenicity in *in vitro*studies (see [Additional file 1](#S1){ref-type="supplementary-material"}). In contrast, rats showed clastogenic activity in the kidney, but only at toxic dose levels \[[@B73]\]. ### Di-(2-ethylhexyl)phthalate (German MAK Classification 4) This substance induced liver tumours in mice and rats \[[@B86]\]. No increased mutation frequency was seen in the liver of the Big Blue^®^mouse \[[@B16]\] (limited validity, MTD presumably not reached) and no chromosome aberration in the mouse micronucleus test \[[@B4]\]. The majority of *in vitro*and *in vivo*tests revealed also negative results with di-(2-ethylhexyl)phthalate (see [Additional file 1](#S1){ref-type="supplementary-material"}). ### Tetrachloromethane (German MAK Classification 4) An increased incidence of liver tumours were induced in mice and in rats \[[@B185]\]. However, no mutagenic activity in the liver was reported in a Muta™mouse assay although the organ weight increased in the same mice indicating some hepatocellular regeneration \[[@B168]\]. No chromosome aberration was induced in the mouse bone marrow micronucleus test \[[@B4]\]. Also other studies on this endpoint and the majority studies on other endpoint of genotoxicity revealed negative results (see [Additional file 1](#S1){ref-type="supplementary-material"}). Discussion on the comparison of both assays ------------------------------------------- Most carcinogens in [Additional file 1](#S1){ref-type="supplementary-material"} are positive in transgenic mouse assays [and]{.underline} in the mouse bone marrow micronucleus test although different endpoints are studied. This indicates coincidence in both test systems and/or effects of the test substance on different genotoxic endpoints. However, there are several substances (see above for example ortho-anisidine) whose mutagenic (and carcinogenic) potential could not be demonstrated with the mouse bone marrow micronucleus test but with the transgenic mouse assay. This might be due to the fact that the micronucleus test is restricted to the bone marrow as target organ. In contrast, all organs can be examined in transgenic mouse assay for mutagenic activity without any restrictions. A special subgroup of substances should also be mentioned: substances with predominantly local mutagenic/carcinogenic effects and less systemic direction of effects. For these substances the mouse bone marrow micronucleus test is an unsuitable test system. Beside the restrictions on the target organ there is also given the possibility that a substance induces predominantly gene mutations and not or less chromosome aberrations at the same dose level. The example *N*-nitrosodi-*N*-propylamine has shown positive results in different target organs including the bone marrow using the transgenic mouse assay but negative results were shown in the mouse micronucleus test. On the other hand there are also substances for which the transgenic mouse assay is an unsuitable test system. The examples methylmethanesulfonate and mitomycin C have shown that chromosome aberration and not gene mutation is the predominant endpoint at the corresponding dose level *in vivo*. These genotoxic effects are not easily detectable with the transgenic mouse assay which is restricted to the detection of small deletion and insertions in the DNA. However, the negative Muta™mouse assay on mitomycin C has some limitations in the validity of the test system. With increased dose levels (MTD reached) and/or repeated application also gene mutagenic effects might be detected in the transgenic mouse assay. Interestingly, substances with carcinogenic effects induced by nongenotoxic mechanisms gave mainly correct negative results in both test systems although the protocols for the transgenic mouse assays were not optimised except for chloroform. Predictability of the transgenic animal assays and the mouse bone marrow micronucleus test for carcinogenicity -------------------------------------------------------------------------------------------------------------- The sensitivity, specificity and predictive values to cancer for the Muta™mouse assay and the Big Blue^®^mouse assay combined, and the mouse bone marrow micronucleus test are documented in Table [1](#T1){ref-type="table"}. In the present study data on 38 substances were available concerning carcinogenicity in mice [and]{.underline} mutagenic effects in transgenic mice as well as mutagenic effects in the mouse bone marrow micronucleus test ([Additional file 1](#S1){ref-type="supplementary-material"}). The two substances with inconclusive results in the mouse bone marrow micronucleus assay (phenobarbital & acrylamide) were not included in the final calculation data and in the comparison of the micronucleus test versus the transgenic mouse assay. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of the Muta™mouse assay and the Big Blue^®^mouse assay for predicting mouse carcinogenicity in comparison with the micronucleus test ::: Term\# Calculation for the mouse bone marrow micronucleus test Calculation for Muta™mouse and/or Big Blue^®^mouse combined \* ------------------------- --------------------------------------------------------- ---------------------------------------------------------------- Sensitivity 68% (23/34) 82% (28/34) Specificity 0% (0/2) 100% (2/2) Positive predictability 92% (23/25) 100% (28/28) Negative predictability 0% (0/11) 25% (2/8) Overall accuracy 64% (23/36) 83% (30/36) **\#**Sensitivity = % of carcinogens with a positive result in the specified test system (STS) Specificity = % of noncarcinogens with a negative result in STS Positive predictability = % of positive results in the STS that are carcinogen Negative predictability = % of negative results in the STS that are noncarcinogens Overall accuracy = % of chemicals tested where STS results agree with the carcinogenicity results Carcinogens with genetoxic and nongenotoxic mechanisms were considered but not noncarcinogenic substances; only data on mice were used Weak positive results in transgenic mouse assays judged as positive. \*: judged as positive in transgenic assays if positive in one of the two test systems ::: Although the data pool in this document is not sufficient for a comprehensive comparison (low number of examples, especially for specificity and negative predictability; limitations of most transgenic mouse assays with negative results) some differences were apparent between the two test systems. The overall accuracy of the micronucleus test is lower than that of the transgenic mouse assays. This is mainly due to 11 negative results in the micronucleus test system (negative in the micronucleus test but positive in carcinogenicity studies) influencing the terms sensitivity and negative predictability. Three of these negative results in the micronucleus test are obtained with carcinogenic substances \[chloroform, di-(2-ethylhexyl)phthalate, and tetrachloromethane\] for which carcinogenic effects are considered to be of a nongenotoxic mechanism. However, chloroform, di-(2-ethylhexyl)phthalate and tetrachloromethane gave negative results in transgenic mice, so the comparison of both test system is not essentially affected and the evaluation „nongenotoxic" supported. For the other 8 substances out of these 11 with false negative results in the micronucleus test these results are explainable (see above): Ortho-anisidine (mutagenic/carcinogenic effects are restricted to the bladder), IQ (bone marrow presumably not target organ of genotoxicity in mice and more gene mutagenic than clastogenic), asbestos (local genotoxic/carcinogenic effects in the lung), 2,4-diaminotoluene (target organ liver, presumably not bone marrow), N-nitrosodiethylamine (target organ liver, more gene mutagenic than clastogenic), N-nitroso-N-propylamine (presumably more gene mutagenic than clastogenic), beta-propiolactone (mainly local effects and less systemic effects \[in bone marrow\]), tris(2,3-dibromopropyl)phosphate (systemic effects not related to the bone marrow). The term negative predictability is also low in the transgenic mouse assay due to false negative results on six carcinogenic substances; for three of them, hydrazine, mitomycin C, and tetrachloroethylene (detailed treatise in section „direct comparison", see above) genotoxic mechanisms are presumed. For hydrazine (no repeated application) and tetrachloroethylene (MTD not reached) limitations on the experimentel design might be the reason for the negative results. Mitomycin C is clearly more clastogenic than gene mutagenic, and the transgenic mouse with lacI and lacZ is possibly an unsuitable test system. For 3 out of the 6 substances the carcinogenic effects in mice were attributed to nongenotoxic mechanisms: chloroform, di-(2-ethylhexyl)phthalate and tetrachloromethane (see also above), all gave negative results in transgenic mice. Only two substances with negative results in long-term carcinogenicity studies are available in the data pool: bromomethane and 2,6-diaminotoluene. Both gave correct negative results in the transgenic mouse assay (although of limited validity) but false positive results in the micronucleus test (see term specificity). Generally, the differences between the two test systems might be due to the fact that 1) unequal genotoxic endpoints are investigated (chromosome aberration in the micronucleus test versus gene mutation in the transgenic mouse assay), 2) organotrophy of genotoxic effects (especially bone marrow not target organ) might play an essential role and 3) transgenic rodent assay conditions in the different systems may not be optimal for mutation detection. Advantages and disadvantages of both test systems ------------------------------------------------- A comparison of the transgenic mouse assays with the mouse bone marrow micronucleus test is limited by the fact that different genotoxic endpoints are studied in these two systems. In transgenic mouse assays, point mutations and small insertions and deletions are detected whereas in the mouse bone marrow assay, chromosome breakage leading to light microscopically visible micronuclei resulting from chromosome fragment or micronuclei originated from whole chromosomes are investigated. ### Sensitivity of the test system In comparison to other test systems in genotoxicity testing using endogenous target structures the spontaneous mutant frequency in the transgenic mouse assay is relatively high. This might be related to the fact that bacterial DNA is the target gene (high methylation rate) or the transgene is silent and no transcription related repair occurs like in endogenous genes which are more efficiently repaired \[[@B9]\]. In the mouse bone marrow micronucleus test the spontaneous rate of micronuclei is low ranging between 1--3 PCEs with micronuclei per 1000 PCEs. However, frequency of chromosome aberrations is not directly comparable with a gene mutantion frequency. Comparing the target organs and cells at risk at the time of exposure, the mouse micronucleus test is restricted to one target organ, the bone marrow, especially to the erythroblasts. This limitation is not given in transgenic mouse assays: target cells are cells in all organs \[[@B195]\]. ### Considerations of animal welfare Both test systems are similar in the number of animals used for a valid test. The minimal number of mice needed in the mouse bone marrow assay is 25 per gender (3 dose levels, vehicle control, positive control; 5 mice per group) using a treatment schedule with 2 or more applications at 24 h intervals and sampling 18--24 h following the final treatment. In the limit test (for a test substance of low toxicity) only one dose level of 2000 mg/kg bw is necessary (OECD guideline 474, \[[@B6]\]). In transgenic mutation assays ca. 20 animals (3 dose groups and 1 concurrent vehicle control group in laboratories which already established this test system) are recommended per species and gender \[[@B196],[@B197]\]. In terms of animal welfare, it is also desired to merge more than one in vivo genotoxicity assay such as transgenic mouse assay and micronucleus assay using the same animals for both assays. ### Cost effectiveness Due to the simplicity of the mouse bone marrow micronucleus assay and the use of systems for automated analysis, this test is less expensive than the transgenic mouse assay. A comparison both test systems is presented in Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Comparison of the mouse bone micronucleus assay with transgenic mouse models (Muta™mouse and the Big Blue^®^assay) ::: **Mouse bone marrow micronucleus test**\[1,2\] **Transgenic mouse mutation assay**\[9,195\] --------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Type of endpoint** Detects light microscopically visible micronuclei resulting from whole chromosomes or chromosome fragments following chromosome breakage Detects 1) gene mutation, 2) small deletions or insertions **Regulatory use** Widespread acceptance (OECD guideline established since 1983) Not widely used by the industry in toxicological screening; OECD guideline in preparation **Background mutation rate** Spontaneous incidence of micronuclei is low (ca. 0.3%) and almost uniform High spontaneous rate of mutations comparing with other mutation assays **Negative predictivity** low negative predictivity for cancer Low negative predictivity for cancer **Implementation** Simplicity of the test system; easily recognised end-point Higher Complexity of the test system (target cells in mice and expression of mutagenic effects in bacteria; vector system needed) **Toxicokinetics and metabolism** Restrictions in toxicokinetics: test substance or the toxic metabolites may not reach the bone marrow but other target organs No restrictions after absorption and distribution of the test substance **Target tissue** Restricted to erythroblasts in the bone marrow No tissue restriction; analysis of mutagenic potency in different organs; measurement of organotrophic effects **Dependency of effects on application route** Only systemic effects can be detected Systemic as well as local mutagenic effects can be detected **Number of animals** 5 animals per gender per dose recommended 5 animals per gender per dose recommended **Restrictions on the used model** Also some recommendations are given in OECD guideline 474, no limitation concerning species, strain, gender, age of animals, exposure duration Limitations: Muta™mouse assay only 1 species & 1 strain; Big Blue^®^2 species (mouse and rat) but 1 (rat) or 2 strains (mouse); no limitations on other parameters **Costs** Less expensive due to the simplicity of the test system More expensive test system **Molecular mechanism** Mechanisms of the induction of micronuclei originating from chromosome fragments could not be resolved Detection of the \"molecular signature\" of a particular mutagenic substance by DNA sequence analysis with standardised methods **Parallel examination of different genetic endpoints** Combination with other genotoxic endpoints is not recommended but possible if results of the micronucleus test are not influenced and vice versa The transgenic mouse assay can be combined with other *in vivo*genotoxic endpoints in the same animal: micronuclei, chromosomal aberration, UDS, SCE **Type of mutational target** In situ end point Target genes are integrated parts of foreign DNA and consequently no \"normal\" mutational target, no expression UDS: unscheduled DNA synthesis; SCE: sister chromatid exchange. ::: Conclusions =========== In a comparison of the tests available to genetic toxicologists, the results from studies on substances which had been tested in transgenic mutagenicity assays Big Blue^®^mouse and the Muta™mouse were compared with those from the more traditional mouse bone marrow micronucleus test. The transgenic animal mutation assay, which is not yet used in toxicological screening, is not directly comparable with the micronucleus test, because different genetic endpoints are examined: chromosome aberration versus gene mutation. However, from the 39 substances, the majority gave the same positive or negative result in both test systems. The substances where differences occurred were discussed in more detail. The advantages and disadvantages of the transgenic Big Blue^®^mouse and the Muta™Mouse transgenic model compared to the micronucleus test were discussed and both systems were found to have a place in mutagenicity testing and to supplement each other. The transgenic animal assay has, however, some distinct advantages over the micronucleus test in that it is not restricted to one target organ and detects systemic as well as local mutagenic effects. Authors\' contributions ======================= UW was the main author. The other authors were involved in the discussions, writing small parts of text and in final preparation of the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Table: Results in the transgenic mouse assay versus mouse bone marrow micronucleus test ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This paper is based on work performed by the authors in preparation of an Environmental Health Criteria document on Transgenic Animals in Mutagenicity Testing for the International Programme on Chemical Safety (IPCS). However, opinions expressed in this paper are the sole responsibility of the authors. We acknowledge the financial support of the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety.

PubMed Central

2024-06-05T03:55:52.461993

2005-1-17

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548135/", "journal": "J Carcinog. 2005 Jan 17; 4:3", "authors": [ { "first": "U", "last": "Wahnschaffe" }, { "first": "A", "last": "Bitsch" }, { "first": "J", "last": "Kielhorn" }, { "first": "I", "last": "Mangelsdorf" } ] }

PMC548136

Background ========== *Lepidium meyenii*, a traditional Peruvian cruciferous vegetable known as Maca, grows exclusively at altitudes over 4000 m. The hypocotyl, the edible part of the plant, is used as a nutritional supplement and for its enhancing sperm production properties \[[@B1]\]. Maca is presented in different ecotypes according to the colors of its hypocotyls, ranging from white to black \[[@B2]\]. Epidemiological studies have found that consumption of cruciferous vegetables is associated with a reduced risk of prostate cancer \[[@B3]-[@B5]\]. Cruciferous (Brassica) vegetables are broccoli, cabbage, mustard and collard greens, bok choy \[[@B3]\], and members of the genus *Lepidium*\[[@B1],[@B6]\], including Maca. In the many hundreds of cruciferous species investigated, all are able to synthesize glucosinolates \[[@B7]\]. Glucosinolate content in cruciferous vegetables is highly variable, depending of plant age and environmental factors which can cause the broad range of values reported to vegetables of the same variety \[[@B8]\]. This variability may explain differences in epidemiological studies related to protection of intake of cruciferous vegetables against prostate cancer \[[@B3]-[@B5],[@B9]-[@B11]\]. Upon ingestion by humans \[[@B12]\] or rats \[[@B13]\], glucosinolates are converted in isothiocyanates by gut microflora. As alternate mechanism, glucosinolates are catalyzed by dietary myrosinase \[[@B13]\]. Almost all of the mammalian chemoprotective activity from cruciferous is due to these isothiocyanates \[[@B7],[@B14]\]. Therefore, glucosinolates after conversion to isothiocyanates have both antiproliferative and proapoptotic properties in prostate cells \[[@B14]-[@B18]\]. The isothiocyanates formed from aromatic glucosinolates decompose spontaneously to indole-3-carbinol (I3C) \[[@B7]\]. I3C and metabolites induce apoptosis in human prostate cancer cells \[[@B16]-[@B18]\]. The absolute content of glucosinolates in fresh Maca hypocotyls is relatively higher than reported in other cruciferous crops \[[@B19]\]. The most abundant glucosinolates detected in Maca were the aromatic glucosinolates: benzyl glucosinolate (glucotropaeolin) \[[@B19]-[@B21]\]; as a result, it is possible that *Lepidium meyenii*(Maca) may have important effects on prostate. More recently, it has been demonstrated that an integral suspension of *Lepidium latifolium*significantly reduced prostate size and volume in castrated rats where the hyperplasia was induced by steroid treatment \[[@B6]\]. For such reason, the present study has been designed to determine the effect of three ecotypes of Maca on ventral prostate of rats. Methods ======= Animals ------- Adult Holtzman rats were obtained from our Animal House at the Universidad Peruana Cayetano Heredia and used for the present study. The rats were maintained 4--6 per cage at environmental temperature (22°C) with a 12:12 h light/dark cycle. Also they were fed with Purina laboratory chow and tap water *ad libitum*. All animal experiments were conducted in compliance with \"Guide for the care and use of laboratory animals\" of the National Institutes of Health from the USA \[[@B22]\]. The Institutional Review Board of the Scientific Research Office from the Universidad Peruana Cayetano Heredia approved the study. Experimental protocol --------------------- ### Experiment 1: Effect of different ecotypes of Maca on prostate size in rats Rats were treated with vehicle, Yellow Maca, Red Maca or Black Maca for 7 days in dose of 2 g dried Maca hypocotyls/Kg BW. This dose was selected from a previous dose response study \[[@B23]\]. Each Maca treated group included 12 animals, and Control (vehicle) sample size included 35 animals. Maca or vehicle was orally administered using an intubation needle No 18 (Fisher Scientific, Pittsburgh, Pennsylvania). ### Experiment 2: Effect of Red Maca on prostate size and histology in rats treated with testosterone enanthate (TE) Rats were injected (i.m) with 0.1 ml (25 mg) of testosterone enanthate (TE) on day 1 and day 7. Control rats received 0.1 ml oil (im) at day 1 and at day 7. A group treated with TE received also Red Maca (2 g/Kg) for 14 days and another group treated with TE received Red Maca for 42 days. Control rats received vehicle by oral route for 14 or 42 days. Oral treatment (Maca or vehicle) and intramuscular treatment (TE or vehicle) both started on day 1. Preparation of aqueous extract of Lepidium meyenii (Maca) --------------------------------------------------------- The dried hypocotyls of *Lepidium meyenii*were obtained from Carhuamayo, Junin at 4000 m altitude. The ages of different Maca plants were similar. All hypocotyls were obtained at the same time. Irma Fernandez, a Botanist of the Department of Pharmaceutical Sciences at Universidad Peruana Cayetano Heredia, authenticated the identity of the plant by visual inspection. The biological activity of the plant is located in the hypocotyls, which are consumed by natives after natural drying. Traditionally, the dried hypocotyls of Maca are boiled and served as juice. For the present study, the aqueous extract of the hypocotyls was prepared according to the traditional method. First, 500 g of the dried hypocotyls were pulverized and placed in a container with 1500 ml of water, and boiled for 120 minutes. Next, the preparation was left standing to cool, and then it was filtered. Finally, the filtrate containing 333 mg of dry Maca hypocotyls in 1 ml was placed in small vials and kept in 4°C refrigerator. Sacrifice, Blood and sample of tissue ------------------------------------- One day after the last treatment, rats were sacrificed by decapitation. Blood sample was obtained from cervical trunk from rats treated for 7 days with different ecotypes of Maca. Blood samples were centrifuged at 1,000 g, and sera were separated, placed in vials and kept frozen until assayed for sex hormone levels. Ventral prostate was used for histological study. Organ Weights ------------- After animals were sacrificed several organs (testes, epididymis, ventral prostate, seminal vesicles, kidneys, liver, spleen, heart and lungs) were collected, dissected free of fat, and weighed. Measurement of serum estradiol and testosterone ----------------------------------------------- Serum estradiol and testosterone concentrations were measured by radioimmunoassay using commercial kits (Diagnostic Products Co, Los Angeles, USA). The hormone labeled with iodine-125 was used as radioactive marker. Samples were run in the same assay to avoid inter-assay variation. The intra-assay variation was 6.42% for estradiol, and 5.5% for testosterone. Sensitivity of testosterone assay was 4 ng/dl and for estradiol assay was 8 pg/ml. Histological study ------------------ Ventral prostate lobes obtained from experiment 2 were excised and dissected free of fat. Ventral prostates (VP) were immersion-fixed in Bouin\'s fixative. After their dehydration, VP were embedded in paraffin. The tissue blocks were sectioned into 5 um thickness and stained with hematoxylin and eosine (H&E), and then observed under a light microscope. Epithelial height (um) and duct lumen area (um^2^) were measured by sampling 10 random sections per slide in the peripheral region of the ventral prostate. In each duct, 20 cells were measured for epithelial height. All assessments were performed using an axiostar plus microscope (Carl Zeizz, Thornwood, New York, USA). The images were captured by a Moticam2000 (Richmond, B.C, Canada) coupled to a personal computer. Motic image plus 2.0 software (Motic Instruments Inc.) was used for measurements of prostatic epithelial height and duct luminal area and calculated by statistic ANOVA test. Pictures at 50× and 400× magnifications are included. Phytochemistry of Red Maca -------------------------- The phytochemical screening in the aqueous extract of Red Maca was performed using standard phytochemical procedures \[[@B24],[@B25]\]. Maca aqueous extract was lyophilized previously to extraction procedures. After extraction in methanol or ethanol, the presence of alkaloids (Dragendorff reagent; Mayer\'s test), flavonoids (Shinoda test), steroids (Liebermann-Burchard/Thin Layer Chromatography test), anthraquinones (Bornträger reaction), tannins (Gelatin/Ferric chloride test), saponins (Froth test), sesquiterpene lactones (Vainillin test and Ferric hydroxamate test), coumarins (Vainillin test and Ferric hydroxamate test), cardiotonic glycosides (Raymond reagent), and cardenolids (Kedde reagent) were assessed \[[@B24],[@B25]\]. Measurement of infrared (IR) spectra ------------------------------------ IR spectra of lyophilized aqueous extracts of Red, Yellow and Black Maca were measured from 3800 cm^-1^to 650 cm^-1^with an FT-IR spectrophotometer (SPECTRUM2000, Perkin Elmer Ltd., Beaconsfield, England). An overhead-attenuated total refraction (ATR) accessory was equipped as the sample stage for solid samples. All spectral measurements were done at 1 cm^-1^resolutions. Data are presented as absorbance units. Each peak represents the presence of a functional chemical group. Differences in the height of absorbance peaks reflect differences in amount of functional groups. Statistical analysis -------------------- Data were analyzed using the statistical package STATA (version 8.0) for personal computer (Stata Corporation, 702 University Drive East, College Station, TX, USA). Data are presented as mean ± standard error of the mean (SEM). Homogeneity of variances was assessed by the Bartlett test. If variances were homogeneous, differences between groups were assessed by analysis of variance (ANOVA). If F value in the ANOVA test was significant, the differences between pair of means were assessed by the Scheffeé test. If variances were non homogeneous, non parametric tests were used. A value of *P*\< 0.05 was considered statistically significant. Results ======= Red Maca but not Black or Yellow Maca reduced ventral prostate weight in rats ----------------------------------------------------------------------------- Ventral prostate weight was significantly reduced in rats treated for 7 days with Red Maca (P \< 0.05). Black Maca and Yellow Maca did not modify ventral prostate weight (Figure [1a](#F1){ref-type="fig"}). Seminal vesicles weights were not modified by treatment with any ecotype of Maca (Figure [1b](#F1){ref-type="fig"}). Body weight was similar in the control group (415.8 ± 3.3 g, mean ± SEM) and in rats treated with Red (407.5 ± 7.1 g), Yellow (421.5 ± 6.1 g) or Black Maca (426.5 ± 6.8 g). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Ventral prostate (A) and seminal vesicles (B) weights in adult rats treated for 7 days with different ecotypes of Maca (2 g/kg BW). Data are Mean ± SEM \*P \< 0.05 with respect to control value. Number of animals was 35 for controls, 12 for Yellow Maca, 12 for Red Maca and 12 for Black Maca. None: control group receiving vehicle. ::: ![](1477-7827-3-5-1) ::: Maca and serum sexual hormone levels ------------------------------------ Means of serum testosterone and estradiol levels were similar between control group and groups treated for 7 days (Figure [2a,b](#F2){ref-type="fig"}) with Red, Yellow or Black Maca. The group treated with Yellow Maca showed higher serum testosterone levels than the group treated with Black Maca (P \< 0.05). Higher serum testosterone levels in the group treated with Yellow Maca were due to two rats with high serum testosterone levels. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effects of different Maca ecotypes administered for 7 days on serum testosterone (A) or estradiol (B) levels in rats. Data are mean ± SEM. Maca (2 gr/Kg BW) was administered for 7 days. Number of rats was 10 in the control group, 6 in the Red Maca treated group, 6 in the Yellow Maca, and 6 in the Black Maca treated group. P:NS between groups treated with Maca and control rats. ^a^P \< 0.05 with respect to the Yellow Maca treated group. ::: ![](1477-7827-3-5-2) ::: Red Maca reduced ventral prostate weight in rats treated with testosterone enanthate (TE) ----------------------------------------------------------------------------------------- Treatment with TE increased significantly ventral prostate weight almost to double value of the control group (P \< 0.05) (Figures [3a](#F3){ref-type="fig"} and [4a](#F4){ref-type="fig"}). The increase in ventral prostate weight was maintained in high levels up to 5 weeks after last TE injection. Seminal vesicles weight increased 2.5 times that control values (P \< 0.05) (Figures [3b](#F3){ref-type="fig"} and [4b](#F4){ref-type="fig"}). Treatment with only Red Maca for 14 days (P \< 0.05) (Figure [3a](#F3){ref-type="fig"}) or 42 days (P \< 0.05) (Figure [4a](#F4){ref-type="fig"}) resulted in low ventral prostate weight compared with control values. Seminal vesicles values were not affected after treatment with Red Maca for 14 or 42 days (Figures [3b](#F3){ref-type="fig"} and [4b](#F4){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Ventral prostate (A) and seminal vesicles (B) weights in adult rats treated for 14 days with Red Maca. Data are mean ± SEM.TE: rats treated on day 1 and 7 with testosterone enanthate (25 mg each) i.m, Red Maca (2 g/Kg BW) was given orally during 14 days. Rats were sacrificed on day 15. \* P \< 0.05 with respect to vehicle group; ^a^P \< 0.05 with respect to TE group. ^b^P \< 0.05 with respect to the group treated with TE+Red Maca. Number of rats was 13 for the control group, 6 for the Red Maca, 6 for TE, and 6 for the TE plus Red Maca groups. ::: ![](1477-7827-3-5-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Ventral prostate (A) and seminal vesicles (B) weights in adult rats treated for 42 days with Red Maca. Data are mean ± SEM.TE: rats treated on day 1 and 7 with testosterone enanthate (25 mg each) i.m. Red Maca (2 g/Kg BW) was given orally during 42 days. Rats were sacrificed on day 43. \*P \< 0.05 with respect to vehicle group; ^a^P \< 0.05 with respect to TE group. ^b^P \< 0.05 with respect to the group treated with TE+Red Maca. Number of animals was 12 in the control group, 7 in the Red Maca group, 5 in the TE group and 5 in the TE plus Red Maca group. ::: ![](1477-7827-3-5-4) ::: Red Maca administered for 14 days also reduced ventral prostate weight in rats treated with TE (P \< 0.05). The effect was more noticeable after 42 days of treatment with Red Maca (P \< 0.05). After 42 days of treatment with Red Maca, the ventral prostate weight was reduced more than 50% (Figure [4a](#F4){ref-type="fig"}). After 14 or 42 days of treatment with Red Maca, the increased seminal vesicles weights induced by TE was not affected (Figures [3b](#F3){ref-type="fig"} and [4b](#F4){ref-type="fig"}). Ventral prostate weight related to body weight was lower in the group treated for 14 days with Red Maca (0.11 ± 0.01 g/100 g BW) than in controls (0.15 ± 0.006 g/100 g BW) (P \< 0.05). Rats treated with TE plus Red Maca had lower prostate weight related to BW (0.23 ± 0.02 g/100 g BW, P \< 0.05) than rats treated with TE (0.29 ± 0.01) but prostate weight relative to BW was still higher in rats treated with TE plus Red Maca than in controls (P \< 0.05). The same pattern was observed when rats were treated for 42 days with Red Maca (Data not shown). Rats treated with two TE injections showed lower body weight at day 42 after the first injection (P \< 0.05) compared to controls (290.63 ± 30.48 g and 383.60 ± 11.31 g in TE and vehicle treated groups). This low body weight was also observed in the group treated with TE plus Red Maca (319.30 ± 3.08 g) (P \< 0.05). Weights of testes, epididymis, kidneys, liver, spleen, lungs and heart were not affected by treatment with Red Maca for 7, 14 or 42 days (Data not shown). Histological study ------------------ Sections of ventral prostate in the peripheral region of the ductal system after 14 and 42 days of treatment with vehicle (control), Red Maca, ET, or ET plus Red Maca are shown in Figures [5](#F5){ref-type="fig"} and [6](#F6){ref-type="fig"}. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### The effects of Red Maca administered to rats for 14 days on ventral prostatic epithelial height and duct luminal area. **A,B**: Control group; **C,D**: Red Maca treated; **E,F**: TE treated; **G,H**: TE+Red Maca treated. HE stain. Left: ×50 magnification; Right: ×400 magnification. ::: ![](1477-7827-3-5-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### The effects of Red Maca administered to rats for 42 days on ventral prostatic epithelial height and duct luminal area. **A,B**: Control group; **C,D**: Red Maca treated; **E,F**: TE treated; **G,H**: TE+Red Maca treated. HE stain. Left: ×50 magnification; Right: ×400 magnification. ::: ![](1477-7827-3-5-6) ::: At 50× magnification, the number of ducts per field from rats treated with Red Maca for 14 days was slightly higher than in controls (Figures [5a](#F5){ref-type="fig"} and [5c](#F5){ref-type="fig"}). TE reduced the number of ducts per field as a consequence of increase in duct area (Figure [5e](#F5){ref-type="fig"}). Red Maca increased the number of ducts per field in rats treated with TE (Figure [5g](#F5){ref-type="fig"}). At 400× magnification, in Red Maca treated rats (alone or with TE), cell size was decreased and membrane blebbing and nuclear distortion are apparent (Figures [5d](#F5){ref-type="fig"} and [5h](#F5){ref-type="fig"} vs Figures [5b](#F5){ref-type="fig"} and [5F](#F5){ref-type="fig"}). At 50× magnification, compared to control (Figure [6a](#F6){ref-type="fig"}), the treatment with Red Maca for 42 days resulted in high number of ducts per field by Maca effect reducing the lumen area (Figure [6c](#F6){ref-type="fig"}). Treatment with TE resulted in lower number of ducts per field as a result of testosterone induced high luminal area (Figure [6e](#F6){ref-type="fig"}). Treatment with TE plus Red Maca (Figure [6g](#F6){ref-type="fig"}) compared to TE (Figure [6e](#F6){ref-type="fig"}) showed an increase in the number of ducts per field. At higher magnification (400×), it was observed secretory luminal cells lined with a single layer of columnar epithelium in the control group (Figure [6b](#F6){ref-type="fig"}). In specimens treated with only Red Maca, the epithelium of the ventral prostate showed a change from columnar to cuboidal shape (Figure [6d](#F6){ref-type="fig"}). TE caused an increase in proliferation of epithelial cells (Figure [6f](#F6){ref-type="fig"}). Red Maca reduced the epithelium in rats treated with TE (Figure [6h](#F6){ref-type="fig"}). Membrane blebbing and nuclear distortion are apparent in rats treated with Red Maca (Figures [6d](#F6){ref-type="fig"} and [6h](#F6){ref-type="fig"}). Quantitative analyses of epithelial height and luminal area in rats treated for 42 days are presented in Figure [7](#F7){ref-type="fig"}. Rats treated for 42 days with only Red Maca showed lower prostatic epithelial height (P \< 0.05) and duct luminal area (P \< 0.05) than control, TE and TE+Red Maca groups (Figure [7a--b](#F7){ref-type="fig"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Ventral prostatic epithelial height (A) and luminal area (B) in control rats, rats treated with Red Maca (RM) alone, testosterone enanthate (TE) alone or TE+Red Maca. Rats were treated for 42 days. \*P \< 0.05 with respect to control, ^a^P \< 0.05 respect to TE group; ^b^P \< 0.05 respect to TE+ Red Maca. Differences in duct luminal areas were assessed with Mann-Whitney U test. ::: ![](1477-7827-3-5-7) ::: Rats treated during 2 weeks with injections of TE once a week showed higher prostatic epithelial height (P \< 0.05) and duct luminal area (P \< 0.05) at day 42 compared to controls (Figure [7a--b](#F7){ref-type="fig"}). Rats treated with TE plus Red Maca for 42 days showed that prostatic epithelial height and luminal area were similar to those observed in the control group (Figures [7a--b](#F7){ref-type="fig"}). Phytochemistry of Red Maca -------------------------- The phytochemical analysis of the ethanolic extract prepared from lyophilized aqueous extract of Red Maca hypocotyls revealed the presence of alkaloids, steroids, saponins and cardiotonic glycosides, and the absence of flavonoids, anthraquinones, tannins, sesquiterpene lactones and coumarins (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Result of phytochemical screening of extracts of Red Maca. ::: **Tests** **Ethanolic extract** **Methanolic extract** ------------------------------ ----------------------- ------------------------ **Alkaloids** Dragendorff test \+ \+ Mayer\'s test \+ \+ **Flavonoids** Shinoda test \- \- **Steroids** Liebermann-Burchard test \+ \+ **Anthraquinones** Bornträger test \- \- **Tannins** Gelatin/Ferric Chloride test \- \+ **Saponins** Froth test \+ \+ **Sesquiterpene Lactones** Ferric hydroxamate test \- \- Vainillin test \- \- **Coumarins** Ferric hydroxamate test \- \- Vainillin test \- \- **Cardiotonic glycosides** Raymond test \+ NA **Cardenolids** Kedde test NA \- -, test negative; +, test positive; NA, not available ::: The phytochemical analysis of the methanolic extract prepared from lyophilized aqueous extract of Red Maca hypocotyls revealed the presence of alkaloids, steroids, tannins and saponins, and the absence of flavonoids, coumarins, anthraquinones, sesquiterpene lactones and cardenolides (Table [1](#T1){ref-type="table"}). The positive tests were more intense for alkaloids than for the other compounds. The IR spectra of the three Maca\'s ecotypes extracts are shown in Fig. [8](#F8){ref-type="fig"}. The IR spectra of the three ecotypes of Maca in 3800-650 cm^-1^region had 7 peaks, which were at 3291 cm^-1^, 2927 cm^-1^, 1614 cm^-1^, 1406 cm^-1^, 1022 cm^-1^, 924 cm^-1^and 862 cm^-1^. These peaks are due to C-H, OH, amides, amines, carboxylic acids, aromatic, and alkyls groups, respectively. Highest peak values were observed for Red Maca, intermediate values for Yellow Maca and low values for Black Maca. These functional groups correspond among others to benzyl glucosinolate (Figure [9](#F9){ref-type="fig"}). ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Infrared (IR) spectra of lyophilized aqueous extract of three ecotypes of *Lepidium meyenii*(Maca). Data are expressed in absorbance units (A). Wave number is expressed in cm^-1^. IR spectra were measured from 4000 cm^-1^to 650 cm^-1^with a FT-IR spectrophotometer equipped with an ATR apparatus. Highest absorbance values correspond to Red Maca, intermediate values to Yellow Maca and lowest values to Black Maca. Peaks of absorbance are recorded at 3291 cm^-1^, 2927 cm^-1^, 1614 cm^-1^, 1406 cm^-1^, 1022 cm^-1^, 924 cm^-1^and 862 cm^-1^. ::: ![](1477-7827-3-5-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Structure of Glucotropaeolin (Benzyl glucosinolate) ::: ![](1477-7827-3-5-9) ::: Discussion ========== The present study was designed to determine if different ecotypes of *Lepidium meyenii*(Maca), a cruciferous plant that grows exclusively over 4000 m in Peruvian Andes, affect ventral prostate size. It was of great interest to demonstrate that Red Maca reduced significantly ventral prostate weight. This effect was not observed after treatment with Yellow or Black Maca. The effect of Red Maca was specific for prostate, since other organs as testes, epididymis, seminal vesicles, kidneys, spleen, liver, lungs and heart were not affected. It was also demonstrated an effect of Red Maca on rats in which ventral prostate size was enlarged by two injections of testosterone enanthate. In fact, Red Maca administered for 14 or 42 days reduced the effect of TE. At 42 days, the ventral prostate size of rats treated with TE plus Red Maca was similar to that of control rats treated only with vehicle. Epithelial height and luminal areas were proved to be sensitive parameters for the evaluation of androgen effects on prostates \[[@B26]\]. The present study shows that prostatic epithelial height increased after treatment with TE. The same effect has been observed when castrated rats were treated with testosterone \[[@B26]\] suggesting that prostatic epithelial height is androgen dependent. Red Maca was able to reduce the prostatic epithelial height of TE treated rats. This would means that Red Maca interferes the androgen action. Growth of the prostate is a hormone-mediated phenomenon regulated by both androgens and estrogens \[[@B27]\]. However, data showed that Red Maca affect ventral prostate size without affecting serum testosterone or estradiol levels. This is not surprising because previously, it has been published that dietary phytoestrogens may affect prostate size without modify circulating testosterone or estrogen level \[[@B28]\], but affecting the androgen action in the rat prostate \[[@B27]\]. Our data on effect of Red Maca on ventral prostate size in rats previously treated with testosterone enanthate suggest that this cruciferous is acting by interfering the androgen action. Maca is characterized by its higher content on aromatic glucosinolates \[[@B19]-[@B21]\]. Recently, it has been described a metabolite of the aromatic glucosinolates that specifically antagonizes androgen receptor \[[@B18]\]; therefore, it is possible that effect of Red Maca on ventral prostate size may be due in part to an action of glucosinolate metabolites on androgen receptor. However, further studies will be required to clarify mechanism of action of this cruciferous plant. Recently, increasing evidence has been presented suggesting that cruciferous (Brassicas) vegetables may reduce the risk of prostate cancer development \[[@B3],[@B4]\]. The genus *Lepidium*could be an important alternative for treatment of prostate diseases. Other Brassica from the genus *Lepidium*, as *Lepidium latifolium*reduced prostate weight \[[@B6]\] suggesting that cruciferous from the genus *Lepidium*may have important anti-proliferative and proapoptotic effects. In Red Maca treated rats, cell size has decreased and membrane blebbing and nuclear distortion are apparent suggesting a pro-apoptotic effect. It is still unknown the active principle for the effect of Red Maca on ventral prostate and why the action is specific since any other organ was affected. Moreover, the different effects among ecotypes seem to be due to different amount of active metabolites. This study used aqueous extract of dried hypocotyls of *Lepidium meyenii*. In the aqueous extract is possible to find glucosinolates \[[@B7]\] and anthocyanines \[[@B29]\]. Both compounds have antiproliferative and proapoptotic properties in prostate cancer cells \[[@B14]-[@B18],[@B30]\]. As effect was specific for Red Maca and not for Yellow or Black Maca, it is probably that Red Maca has more glucosinolate content than other ecotypes. Results from the infrared (IR) spectroscopy showed that peaks of absorbance were higher for Red Maca, intermediate for Yellow Maca and lower for Black Maca. Each peak reflects specific chemical functional groups. Several functional groups found in the different Maca ecotypes correspond among others to benzyl glucosinolate. In such sense, it is suggested that benzyl glucosinolate content is higher in Red Maca, intermediate in Yellow Maca and lower in Black Maca. In addition to the potential glucosinolates effects on prostate, it is possible that other active metabolites may be acting on prostate. Maca aqueous extracts were further extracted with ethanol or methanol and assessed for different compounds. The compounds found are potential candidates to affect prostate; however, it is difficult at this time to ascertain which specific compound has the prostate effect. In fact, the phyto-chemical screening data showed that aqueous extract of Red Maca has alkaloids, steroids, tannins, saponins and cardiotonic glycosides, all of them may have effects on prostate. Phytochemical study showed that Red Maca was more positive for alkaloids than from other compounds. The alkaloid, (1R,3S)-1-methyltetrahydro-β-carboline-3-carboxylic acid has been reported as a constituent of Maca \[[@B20]\]. This alkaloid acts as antioxidants and free radical scavengers \[[@B31]\]. Beta carbolines are also proapoptotic compounds \[[@B32]\] and they have antitumor activities \[[@B33]\]. Further studies will be required to determine the impact of tetrahydro-beta-carbolines from Maca on prostate. Conclusions =========== Indeed, the data presented here show that Red Maca reduced ventral prostate size in normal adult rats and also in rats treated with testosterone enanthate. Hence, it is proposed that Red Maca may have important implications under pathological conditions of the prostate. Authors\' contributions ======================= GFG conceived of the study participating in its design, coordination, and drafting the manuscript. SM participated in the design of the study and the study of different ecotypes of Maca. JN participated in the biological study with different ecotypes of Maca. GF participated in the phytochemical screening JR participated in the statistical analysis SY participated in the histological study PY participated in the histological study MG participated in the design and analysis of results, and its interpretation. All authors read and approved the final manuscript. Acknowledgements ================ This research was supported by a Grant from Vicerrectorate of Investigation at the Universidad Peruana Cayetano Heredia. We thank to Sharon Castillo for her technical support and Leon Villegas for his assistance on infrared measurement.

PubMed Central

2024-06-05T03:55:52.464882

2005-1-20

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548136/", "journal": "Reprod Biol Endocrinol. 2005 Jan 20; 3:5", "authors": [ { "first": "Gustavo F", "last": "Gonzales" }, { "first": "Sara", "last": "Miranda" }, { "first": "Jessica", "last": "Nieto" }, { "first": "Gilma", "last": "Fernández" }, { "first": "Sandra", "last": "Yucra" }, { "first": "Julio", "last": "Rubio" }, { "first": "Pedro", "last": "Yi" }, { "first": "Manuel", "last": "Gasco" } ] }

PMC548137

Background ========== It is well established in mammals that gonadal steroid hormones are potent negative feedback regulators of the hypothalamic-pituitary-gonadal (HPG) axis. In ranid frogs, the first evidence supporting this notion came from a study on the bullfrog, *Rana catesbeiana*, in which gonadectomy elevated circulating gonadotropins, and estrogen and androgen replacement suppressed this elevation \[[@B1]\]. It was later shown that two gonadal steroids, 17β-estradiol (E~2~) and 5α-dihydrotestosterone (DHT), could directly target the pituitary to modulate the release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in these frogs \[[@B2]-[@B4]\]. Despite the established role of DHT and E~2~as feedback regulators of gonadotropin secretion in ranid frogs, there is still some confusion regarding the exact nature of these feedback effects. For example, in female and juvenile *R. catesbeiana*, DHT suppressed the post-gonadectomy rise in circulating gonadotropins \[[@B1]\], yet it enhanced the responsiveness of the pituitary to gonadotropin-releasing hormone (GnRH) \[[@B1],[@B5]\], suggesting DHT is involved in both negative and positive feedback. On the other hand, in the leopard frog (*R. pipiens*), DHT had no effect on the post-castration rise in gonadotropin in males \[[@B2]\] but modestly stimulated pituitary responsiveness to GnRH, suggesting a role in only positive feedback. Although the effects of E~2~were less variable, some conflicting data also exist. In both *R. catesbeiana*and *R. pipiens*, E~2~consistently inhibited LH and FSH secretion both *in vivo*and *in vitro*, demonstrating a direct and powerful negative feedback effect of E~2~at the level of the pituitary \[[@B1]-[@B3]\]. However, recent studies reported E~2~treatment significantly stimulated the proliferation of primary spermatogonia (I SPG) in the green frogs, *R. esculenta*\[[@B6],[@B7]\], suggesting an additional role of E~2~in the positive feedback of the HPG axis. Results from the previous studies revealed the complex nature in which estrogen and androgen feed back on the reproductive axis, and suggest that the nature of the feedback effects might vary depending on the species, sex, reproductive stage of the frogs used, and the duration of steroid hormones administered. Further, there are still significant gaps in our knowledge regarding how these two steroid hormones feed back on the other two components of the HPG axis, the hypothalamus and the gonad. Therefore, the goal of the present study is to understand how E~2~and DHT impact the HPG axis in gonadally-intact male *R. pipiens*. We will do so by measuring parameters that reflect the function of the HPG axis, including hypothalamic GnRH, pituitary and circulating LH, and spermatogenic activity. Moreover, steroid treatments were administered over two periods, 10 days and 30 days, to examine if the nature of steroidal feedback effects remains consistent over time. These results should allow us to determine if DHT and E~2~act consistently as negative feedback regulators at different levels of the HPG axis. Methods ======= Animals ------- All experimental procedures were conducted in compliance with the animal protocol approved by the Institutional Animal Care and Use Committee at the University of Colorado. Mature male northern leopard frogs, *Rana pipiens*, were obtained from Carolina Biologicals (Burlington, NC) from April to July of 2004. Since the life histories of these animals were not entirely clear, all experiments were conducted within the three-month period to minimize the possible confounding effects of seasonality. As a control, we performed histological analysis on testes of representative frogs from every batch. When testicular histology was compared, little differences were observed among batches of frogs arriving at different times (data not shown). Frogs were kept under a 12L:12D photoperiod and fed live crickets every other day. All frogs were allowed to acclimate to the laboratory environment for at least one week before surgical implant. Surgical Implant ---------------- One-cm silastic capsules containing crystalline cholesterol (Ch: control), DHT, or E~2~were prepared as previously described \[[@B8]\], with some minor modifications. Briefly, silastic tubing (outer diameter = 1.96 mm; inner diameter = 1.47 mm) was filled with 1 cm length of crystalline Ch, DHT, or E~2~(Sigma, St Louis, MO) and sealed on both ends with silicon glue. Ch implant was widely used as a negative control for the experimental treatment of cholesterol-based compounds such as steroid hormones \[[@B8]-[@B11]\]. In a preliminary observation (data not shown), we noted no difference in the testicular morphology in animals that have lost Ch capsules compared to animals that have retained capsules throughout the 30-day period, indicating Ch had minimal effects on the reproduction of *R. pipiens*. Filled capsules were equilibrated in 0.6% saline for at least 48 hours before implant. For implant, frogs were anesthetized by immersion in 0.03% benzocaine. A small incision was made at the base of the left leg and a silastic capsule inserted subcutaneously. The incision was then closed with silk thread. Frogs were monitored for recovery from anesthesia and then returned to their respective holding tanks. Tissue Preparation ------------------ Ten or 30 days after implant, frogs were weighed and sacrificed by quick decapitation using a guillotine. Trunk blood was collected into heparinized tubes, centrifuged, and plasma stored at -70°C until the measurement of LH and steroid hormones by radioimmunoassays (RIAs). Testes were removed, their masses recorded, and immersion-fixed in Bouin\'s fixative overnight. Hypothalami were excised from the brain by four cuts: a coronal cut 1 mm rostral to the optic chiasm, a coronal cut on the caudal border of the optic tectum, and two sagittal cuts along the lateral margins of the median eminence. Hypothalami were flash-frozen on dry ice and stored at -70°C until extraction and the measurement of GnRH by RIA. Pituitary glands were removed, sonicated in 500 μl phosphate-buffered saline (PBS), and stored at -70°C until the measurement of LH by RIA. All carcasses were later inspected for the presence of the silastic capsule in the legs. Animals whose capsules were lost were excluded from data analysis. LH RIA ------ Plasma and pituitary LH levels were measured by a LH RIA developed for the bullfrog (*R. catesbeiana*) \[[@B12]\] and validated for *R. pipiens*\[[@B13]\]. The iodination stock, standard, and antiserum for the RIA were a generous gift of Dr. Paul Licht (University of California at Berkeley). The limit of detection was 0.1 ng/ml. The intra- and inter-assay coefficients of variation were 4.8% and 13.3%, respectively. Pituitary LH levels were normalized for protein content assessed by the Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA). Extraction of Hypothalami and GnRH RIA -------------------------------------- Hypothalamic GnRH was extracted with 1 N HCl from the frozen tissues as previously described \[[@B11]\]. The recovery for hypothalamic extractions, assessed by the post-extraction counting of a known amount of \[^125^I\]GnRH added to representative homogenates prior to extraction, was 86%. GnRH RIA was carried out with an antiserum specific for the mammalian form of GnRH (R1245, provided by Dr. Terry Nett at the Colorado State University) using a protocol described in detail elsewhere \[[@B11],[@B14],[@B15]\]. The intra- and inter-assay coefficients of variation were 7.3% and 5.0%, respectively. Hypothalamic GnRH levels were normalized for protein content determined by the Bradford protein assay. Steroid Hormone RIAs -------------------- E~2~and DHT RIAs were performed using the RIA kits from Diagnostic Systems Laboratories (Webster, TX). These RIA kits have been validated previously for the measurement E~2~and DHT in *R. pipiens*\[[@B11]\]. The limits of detection were 6.5 pg/ml for the E~2~RIA and 4 pg/ml for the DHT RIA. The intra- and inter-assay coefficients of variation were 5.3% and 4.9%, respectively, for the E~2~RIA, and 3.1% and 8.4%, respectively, for the DHT RIA. Both RIAs are highly specific and cross-react minimally with other steroid hormones. Histology --------- After fixation, testes were dehydrated through ascending concentrations of ethanol, defatted in Histoclear, and embedded in paraffin. Thirteen-μm sections were cut on a rotary microtome, mounted on poly-L-lysine-coated slides, and stained with hematoxylin and eosin. Immunocytochemistry (ICC) of Proliferating Cell Nuclear Antigen (PCNA) ---------------------------------------------------------------------- Testes were processed for ICC of PCNA, a cell cycle S phase marker, to identify I SPG and secondary spermatogonia (II SPG) undergoing cell proliferation \[[@B16]\]. Testicular sections, prepared as described above for histological staining, were deparaffinized in Histoclear, rehydrated through descending concentrations of ethanol, and immersed in Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA) for 10 minutes at 90°C. After antigen retrieval, sections were washed with 1% hydrogen peroxide in 0.1 M PBS containing 0.4% Triton × 100 (PBST) for 10 minutes to quench the endogenous peroxidase activity, rinsed 5 times with PBST, and incubated for 48 hours at 4°C in PBST containing a monoclonal anti-PCNA antibody (Santa Cruz Biotechnology, Santa Cruz, CA; 1:500) and 4% normal sheep serum. After incubation, sections were washed with PBST and incubated with a biotinylated sheep-anti-mouse IgG (Jackson Laboratory, West Grove, PA; 1:400), washed, and incubated with the Vectastain ABC reagent (Vector Laboratories) for 1 hour. Sections were washed and the immunoreactivity visualized using diaminobenzidine as the chromagen. After the color reaction, sections were washed, counterstained with hematoxylin, dehydrated through ascending concentrations of ethanol, cleared in Histoclear, and coverslipped. Controls for ICC included the preadsorption of the primary antiserum with 20 μg/ml of recombinant human PCNA (Spring Bioscience, Fremont, CA) and the omission of the primary antiserum. Histological Analysis --------------------- Five to eight testes (each from a different animal) per treatment group were assessed for the following histological parameters: seminiferous tubule diameter, the number of I SPG per tubule, and the number of cysts within each tubule containing II SPG, primary spermatocytes (I SPC), or secondary spermatocytes (II SPC). These germ cells were defined according to Rastogi *et al*. \[[@B17]\]. To score the number of cysts containing II SPG, I SPC, and II SPC, sections stained with hematoxylin and eosin were used. To score the number of I SPG, which were scattered and more difficult to locate, sections processed for PCNA ICC were used. Specifically, PCNA-positive cells that were large, isolated, and located at the periphery of the cysts were scored as proliferating I SPG. This method allowed us to identify more I SPG than if morphological criteria were used alone. Since most spermatids and mature spermatozoa were not confined within the cysts and were therefore difficult to measure, these two germ cell types were not quantified. For each testis, five randomly selected tubules were sampled on a slide that had been coded to conceal the identity of the animal. Histological parameters from five tubules were averaged to give a mean for a single animal. Tubular diameters were measured using a calibrated ocular micrometer. All histological parameters were assessed by one individual blind to the identity of the slides. Statistical Analysis -------------------- Differences among groups were analyzed by the one-way analysis of variance (ANOVA) on log~10~-transformed data followed by the Tukey\'s post-hoc test. Differences were considered significant when *P*\< 0.05. Results ======= Steroid hormone RIAs were performed to monitor circulating steroid hormone levels in Ch- and hormone-implanted animals. For animals implanted for 10 days, circulating E~2~levels were 540 ± 110.5 (Ch group; n = 5) and 1966 ± 13.2 pg/ml (E~2~group; n = 5), and circulating DHT levels were 0.9 ± 0.3 (Ch group; n = 4) and 37.5 ± 4 ng/ml (DHT group; n = 5). For animals implanted for 30 days, circulating E~2~levels were 488 ± 370 (Ch group; n = 5) and 2255 ± 650 pg/ml (E~2~group; n = 9), and circulating DHT levels were 2.1 ± 1.4 (Ch group; n = 4) and 24.6 ± 2.7 ng/ml (DHT group; n = 8). To assess the overall accumulation of GnRH in the hypothalami of control and steroid hormone-treated animals, hypothalami were removed, extracted, and measured for the concentration of GnRH. No significant differences in hypothalamic GnRH concentration were observed among Ch, DHT, and E~2~groups implanted for 10 or 30 days (Fig. [1](#F1){ref-type="fig"}). In animals implanted for 10 days, plasma LH levels were not different among the treatment groups (Fig. [2A](#F2){ref-type="fig"}). However, in animals implanted for 30 days, both DHT and E~2~significantly suppressed circulating LH (Fig. [2B](#F2){ref-type="fig"}). The suppressive effect of E~2~was significantly more potent than DHT, with all E~2~-treated animals having undetectable levels of circulating LH (Fig. [2B](#F2){ref-type="fig"}). In 10-day-implanted frogs, no differences in pituitary LH concentration were observed among the treatment groups (Fig. [3A](#F3){ref-type="fig"}). In 30-day-implanted frogs, E~2~, but not DHT, significantly decreased the accumulation of LH in the pituitary (Fig. [3B](#F3){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Hypothalamic GnRH concentrations in implanted frogs.**Hypothalamic GnRH concentrations in frogs implanted for **(A)**10 days or **(B)**30 days with either Ch, DHT, or E~2~. No significant differences were observed among treatment groups in either 10- or 30-day-implanted animals. Each bar represents mean ± SEM. N = 6--11.+ ::: ![](1477-7827-3-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Plasma LH levels in implanted frogs.**Plasma LH levels in frogs implanted for **(A)**10 days or **(B)**30 days with either Ch, DHT, or E~2~. Each bar represents mean ± SEM. Dissimilar letters indicate significant difference between groups. ND = not detectable. N = 7--10. ::: ![](1477-7827-3-2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Pituitary LH concentrations in implanted frogs.**Pituitary LH concentrations in frogs implanted for **(A)**10 days or **(B)**30 days with either Ch, DHT, or E~2~. Each bar represents mean ± SEM. Dissimilar letters indicate significant difference between groups. N = 7--10. ::: ![](1477-7827-3-2-3) ::: Testicular function was assessed by six parameters: the gonadosomatic index (GSI; \[g testes mass/g body mass\] × 100), the diameter of the seminiferous tubules, and the presence of four germ cell types (I SPG, II SPG, I SPC, II SPC) in the testes. Overall, no differences were observed in any of the six parameters among treatment groups in the 10-day-implanted animals (Fig. [4](#F4){ref-type="fig"}). However, in the 30-day-implanted animals, significant steroid-induced changes in the testes were seen. E~2~significantly depleted the presence of I SPG, II SPG, I SPC, and reduced the GSI (Fig. [5](#F5){ref-type="fig"}), whereas DHT significantly reduced only the presence of II SPG and I SPC (Fig. [5](#F5){ref-type="fig"}). Neither steroid hormone affected the diameter of the seminiferous tubules or II SPC (Figs. [5B, F](#F5){ref-type="fig"}). Representative photomicrographs of testicular histology (Fig. [6](#F6){ref-type="fig"}) showed morphological changes parallel to the quantitative measurements in Figs. [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}. In animals implanted for 10 days, no visible differences in germ cell types were seen among treatment groups; I SPG, II SPG, and I SPC were present equally in the testes of all groups (Figs. [6A, B, C](#F6){ref-type="fig"}). In contrast, 30-day implant with DHT or E~2~resulted in highly pronounced and visible changes in the histology of the testes (Figs. [6D, E, F](#F6){ref-type="fig"}). Whereas Ch-treated tubules contained germ cells of all types (Fig. [6D](#F6){ref-type="fig"}), DHT- and E~2~-treated tubules showed a conspicuous absence of II SPG and I SPC (Figs. [6E, F](#F6){ref-type="fig"}). The loss of I SPG with DHT and E~2~treatments was less visible than the loss of other two germ cell types (Figs. [6E, F](#F6){ref-type="fig"}), a result consistent with the quantitative data (Fig. [5](#F5){ref-type="fig"}). Interestingly, the formation of spermatozoa appeared to be stimulated by DHT and E~2~; in fact, the most prominent germ cells in tubules of DHT and E~2~-treated were the large bundles of mature spermatozoa, which occupied most of the tubular lumen (Figs. [6E, F](#F6){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Testicular function in frogs implanted for 10 days.**Measurements of testicular function in frogs implanted for 10 days with either Ch, DHT, or E~2~. **(A)**Average GSI, **(B)**average diameter of seminiferous tubules, **(C)**number of I SPG, and number of cysts containing **(D)**II SPG, **(E)**I SPC, and **(F)**II SPC were measured from 5--8 animals per treatment group. Each bar represents mean ± SEM. ND = not detectable. No significant differences were observed among treatment groups in any of the parameters measured. ::: ![](1477-7827-3-2-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Testicular function in frogs implanted for 30 days.**Measurements of testicular function in frogs implanted for 30 days with either Ch, DHT, or E~2~. **A)**Average GSI, **(B)**average diameter of seminiferous tubules, **(C)**number of I SPG, and number of cysts containing **(D)**II SPG, **(E)**I SPC, and **(F)**II SPC were measured from 5--6 animals per treatment group. Each bar represents mean ± SEM. Dissimilar letters indicate significant difference between groups. ::: ![](1477-7827-3-2-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Testicular histology of implanted frogs.**Representative testicular histology of frogs implanted with **(A, D)**Ch, **(B, E)**DHT, or **(C, F)**E~2~. **(A, B, C)**Testes from animals implanted for 10 days. **(D, E, F)**Testes from animals implanted for 30 days. Red arrow = I SPG; dark blue arrow = II SPG; light blue arrow = I SPC; yellow arrow = II SPC; green arrow = spermatozoa. Note DHT and E~2~had no visible effects on testes of animals implanted for 10 days **(A, B, C)**. In contrast, DHT and E~2~visibly altered the germ cell composition in animals implanted for 30 days **(D, E, F)**. Note the prominent spermatozoa and the absence of I SPC and II SPG in both DHT- and E~2~-treated testes **(E, F)**. Scale bar = 100 μm. ::: ![](1477-7827-3-2-6) ::: PCNA ICC was conducted to determine the effects of gonadal steroids on the number of proliferating germ cells and to allow for the more consistent identification of I SPG. In animals implanted with Ch, intense PCNA immuoreactivity was observed in both I SPG and II SPG. In addition, the cytoplasm of I SPC was lightly stained (Figs. [7A, D](#F7){ref-type="fig"}), since PCNA was also shown to be expressed during the pre-meiotic S phase and meiotic prophase \[[@B18]\]. All spermatogonia (I SPG and II SPG) identifiable by the hematoxylin counterstain were positive for PCNA. Treatment with DHT (Fig. [7B](#F7){ref-type="fig"}) or E~2~(Fig. [7C](#F7){ref-type="fig"}) did not visibly alter the appearance of cells positive for PCNA in frogs implanted for 10 days compared to the Ch control (Fig. [7A](#F7){ref-type="fig"}). In contrast, in animals implanted for 30 days, a visible reduction in the number of PCNA-positive germ cells was observed in DHT- and E~2~-treated animals (Figs. [7E, F](#F7){ref-type="fig"}) compared to the Ch control (Fig. [7D](#F7){ref-type="fig"}). This reduction was primarily due to the decreased presence of I SPG, II SPG, and I SPC in the testes treated with steroid hormones. In control sections incubated with preadsorbed primary antiserum or no primary antiserum, only background staining was present (data not shown). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **PCNA ICC on testes of implanted frogs.**Representative photomicrographs of PCNA ICC performed on testicular sections of frogs implanted with **(A, D)**Ch, **(B, E)**DHT, or **(C, F)**E~2~. **(A, B, C)**Testes from animals implanted for 10 days. **(D, E, F)**Testes from animals implanted for 30 days. Brown stain = PCNA immunoreactivity. Blue stain = hematoxylin counterstain. Red arrow = I SPG; dark blue arrow = II SPG; light blue arrow = I SPC. Note DHT and E~2~had no visible effects on testes of animals implanted for 10 days **(A, B, C)**. In contrast, DHT and E~2~visibly reduced the presence of PCNA-positive germ cells in animals implanted for 30 days **(D, E, F)**. Σχαλεβαρ = 100 μm. ::: ![](1477-7827-3-2-7) ::: Discussion ========== Under our experimental paradigm, both E~2~and DHT exerted negative feedback effects upon the reproductive axis of the sexually mature male leopard frogs. The manifestation of these inhibitory effects was time-dependent and required treatment duration for longer than 10 days. In frogs implanted for 30 days, E~2~decreased circulating LH to undetectable levels and significantly reduced the presence of I SPG, II SPG, and I SPC. The effects of DHT were less potent, reducing only circulating LH, II SPG, and I SPC. Unexpectedly, the presence of post-meiotic germ cells was either unaffected or stimulated with the DHT or E~2~treatment. These results suggest that along the reproductive axis, the most significant negative feedback effects were upon the pituitary and testes, although a stimulatory effect upon the testes might also exist. All hormone implants elevated circulating steroid hormones to levels above the Ch controls without exceeding the physiological range reported for male ranid frogs. For instance, depending on the reproductive status, the range of circulating E~2~reported for sexually mature male ranid frogs was approximately 100 to 3500 pg/ml, and the range of circulating DHT was approximately 1 to 30 ng/ml \[[@B6],[@B19]\]. Another study \[[@B20]\] reported circulating levels of approximately 10 ng/ml for DHT and 2 ng/ml for E~2~in captive male *R. pipiens*. Thus, circulating E~2~and DHT in the hormone-implanted animals were largely within the high end of the physiological range. DHT and E~2~did not significantly alter hypothalamic GnRH concentration in animals treated for 10 or 30 days. These results were consistent with our previous finding on frogs implanted for 20 days with DHT and E~2~, and speak to the highly stable nature of GnRH peptide accumulation. One should note that this study focused on the mammalian form (Type I) of GnRH because this is the predominant hypophysiotropic form of GnRH in the diencephalon of ranid frogs \[[@B21]\] and the form more sensitive to changes in reproductive status \[[@B22]\]. However, the chicken II (Type II) form of GnRH may also be hypophysiotropic since it binds to pituitary GnRH receptor with high affinity \[[@B23]\], and its presence is detected in the hypothalamic-pituitary portal blood \[[@B21]\]. The ability of steroid hormones to feed back upon the Type II GnRH system in *R. pipiens*is at present unclear and awaits further investigation. Only E~2~treatment for the longer duration (30 days) significantly reduced pituitary LH concentration, although a substantial amount of LH still remained in the pituitary glands of these E~2~-treated animals. Thus, the potent suppression of circulating LH in animals treated with E~2~for 30 days was most likely attributable to the low secretory activity of the pituitary gonadotropes rather than the depletion of LH stores. Somewhat surprising was the inability of E~2~treatment for 10 days to suppress circulating LH. The pituitaries of ranid frogs were shown to be extremely sensitive to the inhibitory actions of E~2~. In *R. pipiens*, *in vitro*exposure of the pituitary to E~2~concentrations as low as 100 pg/ml for only 48 hours significantly suppressed both basal and GnRH-stimulated gonadotropin secretion \[[@B2]\]. Extrapolating from this time course and from our current observation that E~2~implants could elevate circulating E~2~to about 2000 pg/ml, one might expect a substantial decline in circulating LH of these animals after only 10 days of implant, but this was not the case. It is possible that additional *in vivo*mechanisms exist in these frogs to buffer against short-term estrogenic inhibition. Some of these might include changes in the clearance rate of circulating LH \[[@B24]\] and the levels of sex steroid binding proteins \[[@B25]\]. The former could prolong the half-life of LH in circulation; the latter could dampen the inhibitory effects of E~2~by binding to E~2~and decreasing the availability of the bioactive hormone. Another unexpected observation was that DHT treatment for 30 days also suppressed circulating LH. Our results differ from a previous study demonstrating the inability of DHT to suppress post-gonadectomy rise in LH \[[@B2]\] and showed, for the first time, that DHT participates in the negative feedback regulation of gonadotropin secretion in *R. pipiens*. It is at present unclear if reduced circulating LH is a direct consequence of suppressed secretory activity of the gonadotropes or reduced output from the GnRH system. Based on the previous report that DHT had no direct inhibitory effect on pituitary gonadotropin secretion \[[@B2]\], it seems likely that DHT may achieve negative feedback primarily by targeting the GnRH system to suppress GnRH release. This possibility does not conflict with our current observation that DHT lacks an effect on GnRH content. Since GnRH content is a measure of GnRH peptide accumulation which is a result of transcription, translation, mRNA stability, peptide turnover, or release, it is a poor indicator of GnRH release alone. One of the most pronounced negative feedback effects of DHT and E~2~was observed at the level of the testes. In animals implanted for 30 days with these two steroids, a marked loss of several germ cell types (I SPG, II SPG, and I SPC) was observed. Interestingly, although E~2~exerted a greater disruptive effect on spermatogenesis, similar trends of reduction with virtually no qualitative difference were also observed in DHT-treated testes, suggesting similar outcome might be attained with longer DHT exposure. That DHT- and E~2~-induced disruption of spermatogenesis differed only in the degree of severity suggests the disruption occurred primarily via a common pathway, possibly through the inhibition of gonadotropins. In this study, we could not measure circulating FSH because we lack a homologous FSH RIA. However, previous studies have reported that FSH secretion in *R. pipiens*was under the negative control of E~2~\[[@B2],[@B3]\] and possibly DHT \[[@B2]\], since gonadectomy significantly elevated the levels of circulating FSH. The observation that spermatogenic disruption occurred only when circulating gonadotropin was reduced (30 day-implants) further lends support to this hypothesis. Although the roles of FSH and LH in anuran spermatogenesis are not entirely clear, data from other amphibians suggest FSH is essential for supporting the proliferation and survival of spermatogonia \[[@B26],[@B27]\]. Importantly, FSH is required for the completion of the last spermatogonial mitosis, thus the entrance into meiosis and the generation of spermatocytes \[[@B27]\]. On the other hand, LH is specifically required for the stimulation of androgen production in ranid frogs \[[@B28]\] and could be responsible for maintaining high levels of intratesticular androgen required for androgen-dependent stimulation of germ cell formation \[[@B29]\]. Thus, low circulating levels of gonadotropins could be the common pathway leading to defective spermatogenesis in both DHT- and E~2~-treated animals. Although the significant reduction of I SPG in E~2~-treated testes could partially account for the loss of germ cell types that arose from I SPG, this cannot be the sole cause. For example, substantial PCNA-positive I SPG still remained in the testes of frogs implanted with E~2~for 30 days, yet virtually no II SPG remained in the testes of these animals. Similarly, in 30-day-DHT-treated testes, there was no significant decline in I SPG, but II SPG were markedly reduced. These observations indicate a disproportionate loss of II SPG that may have resulted from their failure to survive. These data were consistent with a previous report in the newt that the mitotic penultimate SPG failed to survive when circulating FSH was suppressed \[[@B26]\]. Taken together, we believe that II SPG was the germ cell type most severely affected by steroid treatments, and the loss of II SPG was the most important underlying cause for disrupted spermatogenesis. An interesting observation is that although pre-meiotic germ cells (I SPG, II SPG, and I SPC) were adversely affected by steroid hormone implants at 30 days, meiotic or post-meiotic germ cells (II SPC, spermatids and spermatozoa) appeared unaffected or stimulated. In fact, the most conspicuous germ cells in the seminiferous tubules of 30-day E~2~- or DHT-implanted frogs were mature spermatozoa, which occupied the largest bulk of the tubular lumen. It is possible that low circulating FSH had little influence on the germ cells once they entered meiotic division. Under low circulating LH, spermiation was inhibited, and mature spermatozoa continued to accumulate in the tubule. Another possibility is that E~2~and DHT, while suppressing the presence of pre-meiotic germ cells, actually stimulated the entrance of existing I SPC into meiosis and promoted the survival of post-meiotic germ cells. This possibility was partially supported by the previous observation that testes of frogs treated with DHT for 20 days had fewer II SPG, but significantly more I SPC in the midst of meiotic division \[[@B11]\]. Along the same line of reasoning, it is also possible that DHT and E~2~facilitated the progression of spermatogenesis to the extent that the intermediate germ cell types could no longer be adequately replenished, leaving tubules filled with post-meiotic cells (primarily spermatozoa) and very little else. Regardless, the trend towards reduced GSI in steroid hormone-treated animals, along with the reduced presence of pre-meiotic germ cells, indicate an overall negative effect of these hormones upon the testes. The abundance of spermatozoa in DHT- and E~2~-treated animals nevertheless raised an interesting possibility for the existence of a positive steroidal effect on spermatogenesis. Worth mentioning is the possibility that DHT and E~2~, in addition to affecting spermatogenesis by lowering circulating gonadotropins, have also been shown to act directly upon the amphibian testes. Both androgen and estrogen binding sites were found in the amphibian testes \[[@B30]-[@B33]\]. A number of physiological responses were presumably mediated through these testicular steroid hormone receptors. For instance, E~2~acted directly upon the amphibian testes to suppress androgen secretion \[[@B34]-[@B36]\], stimulate nuclear translocation of c-Fos \[[@B37],[@B38]\], and enhance proliferation of I SPG \[[@B6],[@B37]\]. Similarly, DHT has also been found to directly modulate androgen secretion \[[@B34]\]. Of interest to the present study is the demonstration that E~2~directly stimulated SPG I proliferation in *R. esculenta*\[[@B6],[@B7],[@B37]\]. Under our experimental paradigm, however, such a stimulatory effect was not seen in *R. pipiens*. Whether or not this discrepancy was due to the differences in experimental paradigms or species used is at present unclear. We previously showed that spermatogenesis in the ranid frogs was altered in mature male frogs implanted with DHT and E~2~for 20 days \[[@B11]\]. Specifically, E~2~reduced the presence of II SPG and I SPC, whereas DHT reduced only the presence of the former. However, the study represented only a snapshot in time, so no information was available regarding the progression of events that led to the altered formation of germ cells. Moreover, it was unclear if the treatment with these two steroid hormones for shorter or longer periods could impact the testes differently. Our current results showed that both steroids inhibited circulating gonadotropin and disrupted spermatogenesis progressively in a time-dependent manner, with the longer duration of treatment producing the more pronounced effects. Further, the changes in the testes were qualitatively similar between DHT and E~2~treatments, suggesting declining gonadotropin levels might be the common underlying cause for the disrupted spermatogenesis. These results reflect the highly sensitive nature of the anuran reproductive axis to estrogenic and androgenic modulation. We showed that the continuous exposure of mature frogs to high physiological levels of steroid hormones for a relatively short period could profoundly alter their pituitary and testicular function. In particular, the potency of estrogen hormones raises concerns regarding the potential reproductive disruption that can occur when mature frogs are exposed to short-term and low-level environmental estrogen mimics. Authors\' contributions ======================= PST designed the experiments, analyzed the data, and prepared the manuscript. AEK and JTJ prepared the silastic capsules, implanted the animals, removed the tissues, and performed all the hormone measurements. KBW implanted some animals, prepared all histological samples, counted the germ cells, and performed the PCNA ICC. All authors read and approved the final manuscript. Acknowledgments =============== We thank Dr. Paul Licht for the generous gift of frog LH RIA reagents. This work was supported by NSF IBN-9996398 and NIH HD-042634 to P.-S.T.

PubMed Central

2024-06-05T03:55:52.467464

2005-1-10

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548137/", "journal": "Reprod Biol Endocrinol. 2005 Jan 10; 3:2", "authors": [ { "first": "Pei-San", "last": "Tsai" }, { "first": "Ann E", "last": "Kessler" }, { "first": "Jeremy T", "last": "Jones" }, { "first": "Kathleen B", "last": "Wahr" } ] }

PMC548138

Background ========== Metastases to the umbilicus are universally referred to as Sister Joseph\'s (or Sister Mary Joseph\'s) nodule. Etiology is related to the presence of primary malignant disease in the abdominal cavity or occasionally in the chest and/or breast \[[@B1]-[@B5]\]. Historically, Sister Mary Joseph (1856--1939) was a surgical assistant under the guidance of Dr. William Mayo. She was the first one to note the connection between the umbilical nodule and intra-abdominal cancer. The first case reporting the presence of Sister Mary Joseph\'s nodule was in 1864 by Storer, however; Hamilton Bailey was the first one to use the term \"Sister Mary Joseph\'s nodule. Although skin metastasis is rare and range between 5% and 9%, it is estimated that 1% to 3% of abdomino-pelvic tumors metastasize to the umbilicus \[[@B2],[@B3],[@B5]\]. The most common primary neoplasm is adenocarcinoma (75%), more rarely squamous cell carcinoma followed by undifferentiated tumors or carcinoid can metastasize to umbilicus. In men, gastrointestinal tract (55%) is the most common location of the primary neoplasm that metastasizes to the umbilicus, followed by stomach, colon, rectum, small bowel, and pancreas in a decreasing order \[[@B1]-[@B5]\]. In women, on the other hand, gynecological neoplasms particularly ovarian cancer is the most common primary site, of which serous papillary cystadenocarcinoma (34%) is the most frequent. Further, endometrial carcinoma, cervix, vagina and vulva may also be responsible for the metastasis to the umbilicus. In addition to these the literature reports other but rare sites that could form potential grounds for metastasis to the umbilicus, namely gallbladder, liver, breast, lung, prostate, penis, peritoneum, lymphoma, bladder and kidney. In about 11% of the umbilical metastasis, the origin of the metastasis is unknown \[[@B4],[@B5]\]. Upon recognition of the positive nodules in the umbilicus, physician must consider other potential sources such as endometriosis, melanocytic nevi, fibroma, epithelial inclusion cysts, seborrheic keratosis, pilonidal sinus, keloid, foreign body granulomas, myxoma, omphalitis, abscesses, umbilical hernia and of course primary malignant umbilical tumor (melanoma, squamous and basal cell carcinoma, sarcoma and adenocarcinoma) \[[@B6]\]. In many instances, Sister Joseph\'s nodule is the first and often the only indication of an underlying or occult cancer and/or may indicate recurrence of previously treated malignancy. The presence of umbilical metastasis usually suggests already advanced metastatic process characterized by poor prognosis \[[@B4],[@B5]\]. Therefore, a biopsy of umbilical nodule is recommended which is safe and easy. Obtaining histological description can further direct the clinician to dictate a prompt treatment. In the case of recurrence of the neoplasm, fine needle aspiration cytology may be sufficient to provide definite diagnosis; however, sometimes clinical, cytological, histological, radiological and/or surgical investigations may not be sufficient to identify primary site of the metastasis \[[@B7]\]. Serous papillary cystadenocarcinoma has been documented previously as a cancer responsible for Sister Joseph\'s nodule because of the metastasis to the regional lymph nodes \[[@B6]-[@B9]\]. Herein, we report, to the best of our knowledge, the first case of Sister Joseph\'s nodule in a patient after liver transplantation complicated by serous papillary cystadenocarcinoma. In addition, we review the potential routes by which tumor spread may have occurred and the prognostic significance associated with umbilicus metastasis. Case presentation ================= A 59-year-old Caucasian woman was admitted to our hospital for progressively enlarging but painless mass in the region of the umbilicus. The umbilical mass of approximately 5 cm in diameter and has developed two months after liver transplantation. The nodule was firm with irregular borders, wine-red shade and fixed to the surrounding tissues. The overlying skin was not ulcerated. She never smoked, consumed alcohol or used oral contraceptive pills. The gynecological history was unremarkable. The patient underwent a liver transplantation for end stage liver disease (ESLD) secondary to hepatitis C in mid of 2003. Prior to the transplant, her serum alpha-fetoprotein (alpha-FP) and CEA concentration were measured and both were within normal limits. The transplant procedure and immediate post-transplant period were unremarkable. The post-transplant immunosuppressive regimen consisted of Tacrolimus (6 mg/day) and steroids (prednisone 10 mg/day). Two months after transplantation, during routine follow-up, physical examination revealed multiple subcutaneous satellite nodules in close proximity to the umbilical region and lower abdominal wall. There was bilateral inguinal lymphadenopathy. A plain anterior/posterior chest x-ray was normal. Ultrasound of the abdominal cavity showed a 4 × 4 cm mass below the umbilicus and additional smaller nodules confined to the lower abdominal wall. Paraaortic lymph nodes were not identified. The echogenicity of the mass consisted of alternative hyper- and hypoechoic with poorly defined edges. However, the general condition of the patient was good and the laboratory test showed: hemoglobin level 9.6 g/dl, total bilirubin 0.6 mg/dl, conjugated bilirubin 0.1 mg/dl, Alkaline Phosphates 273 U/l, GGT 40 U/L, AST 36 U/L, and ALT 56 U/L. There was serological evidence of past hepatitis C virus infection. The biopsy of the umbilical mass showed features characteristic for a poorly differentiated serous papillary cystadenocarcinoma. An extended resection of the umbilical and paraumbilical metastasis was performed. In addition, extensive investigation to detect the primary neoplastic origin was initiated. Tumor marker evaluation consisted of: CEA 0.22 \[normal 0--3.7 ng/ml\], alpha feto protein 1.49 (normal: 0.8--9.4 UI/ml), CA125 27.73 (normal: 0--30 UI/ml), CA 19.9 was 8.61 (normal: 0--36.2 UI/ml), CA 15.3 was 22.48 (normal: 0--35 UI/ml). Multiple tumor marker measurements were obtained and each time similar results were reported. A computed tomographic (CT) scan was unable to demonstrate any lesion that could be considered a primary source of the metastasis to the umbilicus. Trans-vaginal ultrasonography showed a normal uterus and ovaries; ultrasonographic examination of thyroid and breast did not show any pathological changes as well. In spite of laborious efforts, the tumor\'s primary site was not found at that time. However, considering the histological results, the umbilicus metastasis could be attributed almost entirely to the ovarian cancer stage 4, since the main primary site in females are the ovaries. In order to prevent further tumor extension and expansion, tacrolimus was reduced from 6 mg/day to 1.5 mg/day. In addition, chemotherapy with Taxol was initiated (50 mg/m^2^I.V) once a week for 16 weeks. To date, after 6 months of follow-up, the patient is doing well, the liver function is normal and tacrolimus serum level is 1 ng/ml. A recent whole body CT-scan showed regression of iliac and inguinal lymph nodes involvement. Discussion ========== Metastases may appear virtually anywhere in the body; however, certain sites are more common then others and umbilical metastasis are unusual sites \[[@B9],[@B10]\]. In majority of cases, the metastatic lesions accompany the symptoms and signs of the primary tumor; however, it can be the first and often the only sign of a carcinoma, although this happens rarely \[[@B10]\]. A variation in vascularity and embryological development makes the umbilicus easy target for metastasis from an intra-abdominal tumor. In fact, during fetal development, the ductus venous (ductus venous Arantii) connects the umbilical portion of the left branch of the portal vein to the inferior vena cava, thus shunting oxygenated umbilical cord blood away form the liver. After birth the duct obliterates and persists as ligamentum venosum or Arantius\' ligament. Because of its functional role, it is commonly believed that the ligament runs from the left branch of the portal vein to the vena cava itself. However, attention to anatomical detail demonstrates that the fibers of the ligament insert either on the left hepatic vein or at the junction between the left hepatic and the middle hepatic vein \[[@B1],[@B11]\]. We know that in cirrhotic patients, the umbilical vein remains open due to portal hypertension. There are numerous potential routes by which a carcinoma can metastasize to the umbilicus in a liver transplant recipient. Malignant carcinomatous cells in the portal venous system may reach the umbilicus by way of a patent umbilical vein \[[@B12]-[@B15]\]. This would be more likely to occur in the presence of portal hypertension and the resulting portal-systemic shunting of blood \[[@B16]\]. Since our patient received liver transplant for cirrhosis secondary to hepatitis C virus infection accompanied by portal hypertension, this could be a possible mode of spread of papillary serous cystadenocarcinoma in our patient. In addition, presence of severe esophageal varicies and large umbilical vein that was viewed by angiography examination before the liver transplantation could have contributed to the spread of tumor. Therefore, in this case, malignant cells could have reached the umbilicus via the umbilical vein. In addition, a pelvic carcinoma of unknown origin may spread via lymph nodes to the umbilical region \[[@B9],[@B17],[@B18]\]. Furthermore, dermal lymphatics are a potential route of spread to the umbilicus and should be considered, since the umbilicus can be reached by retrograde lymphatic flow \[[@B4]\]. Nevertheless, the spread of malignant cells could have occurred via embolization thru the arterial blood supply to the umbilicus. Furthermore, direct implantation of tumor cells may have occurred via direct extension of the neoplasm from the anterior peritoneal surface or via redistribution of the peritoneal fluid flow \[[@B19],[@B20]\]. Finally, cutaneous metastasis to the anterior abdominal wall, may perhaps cause malignant cells to spread in a retrograde direction, namely from the metastatic lesions along the subcutaneous lymphatic vessels to the umbilicus. It is known that the immunosuppression promotes tumor spreading and potentates its expansion, thus immunosuppressive regimen plays a crucial role in the tumor expansion and metastasis. Umbilical metastasis is one of many characteristic signs of extensive neoplastic disease. It suggests advanced distant metastasis and is associated with poor prognosis; mean survival is approximately 10--12 months, although long-term survival has been reported, but only in the presence of solitary metastatic umbilicus nodule \[[@B5]\]. Conclusions =========== Presence of umbilical nodule or nodules requires extensive, meticulous and laborious evaluations since the differential diagnosis for umbilical nodule are many. In our case although, the primary tumor was not found, we treated it as ovarian tumor considering the histological findings and the iliac-inguinal nodes involvement. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= **FP**: conception and design, interpretation of data, drafting the article and revising **EA**: interpretation of data, drafting the article and revising **SDD**: acquisition of data and revising **NM**: interpretation of data, design and coordination and helped to draft the manuscript **GB**: collection of data, design and coordination and helped to draft the manuscript **RM**: collection of data, design and coordination **MM**: acquisition of data and drafting the article **TJ**: drafting the article and interpretation **FR**: acquisition of data and design **MC**: interpretation of data, drafting the article **UV**: drafting the article, revising and supervision ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Preoperative Sister Mary Joseph\'s nodule ultrasonography: 4 × 4 cm mass confined below the umbilicus (arrows). The main lesion is partly hyperechoic and partly hypoechoic with a poorly defined edge. ::: ![](1477-7819-3-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Intraoperative specimen of the umbilical region: Sister Mary Joseph\'s nodule and umbilicus. ::: ![](1477-7819-3-4-2) ::: Acknowledgement =============== Patients consent was obtained for reporting her case.

PubMed Central

2024-06-05T03:55:52.470914

2005-1-14

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548138/", "journal": "World J Surg Oncol. 2005 Jan 14; 3:4", "authors": [ { "first": "Fabrizio", "last": "Panaro" }, { "first": "Enzo", "last": "Andorno" }, { "first": "Stefano", "last": "Di Domenico" }, { "first": "Nicola", "last": "Morelli" }, { "first": "Giuliano", "last": "Bottino" }, { "first": "Rosalia", "last": "Mondello" }, { "first": "Marco", "last": "Miggino" }, { "first": "Tomasz M", "last": "Jarzembowski" }, { "first": "Ferruccio", "last": "Ravazzoni" }, { "first": "Marco", "last": "Casaccia" }, { "first": "Umberto", "last": "Valente" } ] }

PMC548139

1 Introduction ============== The theme of this paper is that recent and proposed changes to the financing and delivery of health services in Australia have focused upon issues of relatively minor significance while failing to address adequately major inequities and system deficiencies. An intriguing question -- not discussed in the paper -- is how such drastic failures could continue year after year with little comment and no decisive policy commensurate with the magnitude of the problems. The political-sociological answer to this question is undoubtedly complex and contentious. The failure may (or may not) be attributed to understandable, even unavoidable obstacles arising from Australia\'s social history. Nevertheless it is important to be aware that these failures exist. The current state of our health services could justifiably be described as a \'silent crisis\'; service delivery is highly inequitable and inefficient; patients are dying unnecessarily and avoidable medical errors are imposing huge financial and human costs on the community. While this occurs, health policy at the political level has focussed upon cost shifting between the States and Commonwealth, between public and private sectors and between the well off and the poorer members of society. Reforms addressing the larger issue have progressed at glacial speed, relative to what is achievable, or they have stalled altogether. In Section 2 there is a brief overview of the economists\' analytical framework in order to introduce two preliminary issues, viz, the role of social values in health system reform and the constraints created by the limited availability of resources. Two commonly made but wrong inferences from this latter constraint are discussed. Section 3 is concerned with recent policy and, in particular, the changes to private health insurance (PHI) which have been introduced since July 1997. In contrast with the *relatively*inconsequential (and possibly negative) impact of these policies, five major problems are outlined in Section 4, each of which has received insufficient or no attention. Policy implications and options for future policy are discussed and highlighted throughout the paper. In the final section I argue that the optimal health system -- entirely public, largely private or one of the myriad combinations between these polar options -- is the system which is most likely to address systemic failures. The most important changes to achieve the optimal system may have less to do with the public-private mix of services, or even funding, than with the extent to which these failures are addressed within any of the conceivable health systems of the future. This, in turn, may depend upon the willingness to create appropriate economic and other incentives. 2 Resources, values and the economic framework ============================================== The discipline of economics provides a framework for the analysis of options which is based upon a comparison of costs and benefits. If social welfare is to be maximised then the logic of the framework must be adopted explicitly or implicitly. The framework focuses attention upon the benefits which might be obtained when resources are used in a particular way, and the benefits which might have been obtained if they had been used somewhere else -- the (opportunity) cost of using those resources in the chosen way. Social wellbeing is maximised when the benefits exceed the (opportunity) costs in every setting and on every margin where choice is possible. While this statement is tautologically true, the focus upon choice highlights two important facts. First, choices generally do exist; the economy is flexible and choices are driven by individual and social preferences. Technical inevitabilities are rarely encountered. Second, and more fundamentally, it is necessary to define \'benefits\'. In principle, the abstract framework is consistent with an almost unlimited number of value systems. For example, in the context of an intensive care department with limited capacity, benefits and costs might be measured by lives saved and lives lost. In this simple example the cost benefit formula would translate into a policy of providing ICU beds to those most likely to live. Social objectives in the health sector are clearly more complex than in most other settings and the nexus between objectives, policies and the optimal health system is more problematical. Nevertheless, an important conclusion from this framework (which needs constant repetition) is that there is not a single \'best\' health system; rather, there are various options which are more or less consistent with different social goals. This conclusion is illustrated in Table [1](#T1){ref-type="table"} using two highly simplified but archetypal social objectives, viz, the egalitarian desire for equal access to health or health services and the social objective of maximising individual choice. As shown, the first of these objectives is more easily achieved through a compulsory public system with defined benefits and constrained choice. The second objective is most likely to be achieved in a less constrained and more competitive private system which responds to individual preferences, as described and generally prescribed by economic theory for less complex markets and social objectives. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The relationship between choice, values and the optimal health system ::: --------------------------------------------------------------------------------------------- **Objectives/Social Values** **Option which maximises likelihood of success** ------------------------------------------ -------------------------------------------------- Equalise access, outcome Universal (monopoly) Public Insurance/Financing Maximise choice; diversity + safety net\ Pure private (competitive) scheme Optimise the max of these 2 objectives Both of the above Mixed public-private scheme --------------------------------------------------------------------------------------------- ::: ***Conclusion 1:**The form of the optimal health scheme depends upon social objectives and disagreement about these translates into differences with respect to the funding and delivery of health services.* While the two objectives in Table [1](#T1){ref-type="table"} are archetypes, they broadly correspond with two important but conflicting \'world views\'; that is, with different ethical beliefs about the appropriate supply and financing of health services. The social values underpinning the competitive market model are well articulated and well labelled. Its \'liberal\' or \'libertarian\' value system emphasises the importance of individual responsibility and freedom of choice. It is the prevailing value system in many aspects of life in a democratic society and there is commonly a presumption that, in the absence of some compelling argument, liberty and choice should be maximised. In economic theory this objective is equivalent to the goal of maximising \'utility\' and the Welfarist theory of \'Social Welfare\'. Even those expounding liberal values, however, generally believe that some constraint upon choice, in the form of compulsory taxation, is justifiable to finance a limited number of public goods and that at least basic medical services should be provided for the medically and financially indigent. With this \'world view\' fairness generally equates with a vertical redistribution of income to help the most needy. In contrast, the value system underpinning the public model is less clearly articulated (at least in Anglo Saxon countries). The financing and provision of services to the entire population is often characterised as \'middle (and upper) class welfare\' and contrasted with the less intrusive \'safety net welfare\' which is all that is required to help those who cannot help themselves. This interpretation of egalitarianism does not, however, correctly represent the values which underpin the public health insurance system. These are nicely described in a report of a commission of enquiry into Canadian Medicare as follows: \'Canadian Medicare is far more than just an administrative mechanism for paying medical bills. It is widely regarded as an important symbol of community, a concrete representation of mutual support and concern\... it expresses the fundamental equality of Canadian citizens in the face of death and disease\... as the Premier of Ottawa pointed out\... \"There is no social program that we have that more defines Canadianism\"\' \[[@B1]\]. The social value or world view embodied in this quotation does not correspond with the simple notion of assistance for the indigent. Rather it corresponds with a desire to \'remove health and health care from the economic reward system\' in the same way as all citizens are, in principle, given equal protection by the law. A close analogy is the desire to have public parks which may be accessed by all members of the community without payment. The objective is not a redistribution of income or the provision of a safety net. Rather, with this view, access to public parks is one of the consequences of belonging to the community; it is a shared benefit and, as such, engenders a feeling of sharing, participation and belonging. The concept is closely linked to the notion of \'social capital\' which \'accumulates\' with an increase in communal sharing and participation. Pay parks are possible, but a fully informed community might reject this option. Its citizens may wish to live in a community where the Arts flourish, where its sportsmen and women are a source of national pride, where parks are free for all citizens, where an acceptable standard of living is guaranteed after retirement and where all citizens have access to the same range of medical services. In European countries the term \'solidarity\' is used to describe this value system. Unfortunately, in English, there is no commonly used and understood word for the concept. (\'Communitarianism\' is the closest translation.) The consequence of this is a degree of confusion in the expression of social values as both sides of the debate attempt to appropriate the word \'equity\' to support their world view. It is clearly desirable that the debate should not be derailed by linguistic ambiguities. ***Conclusion 2:**Health policy should be informed by a careful evaluation of the social values held by different groups of the community with respect to different elements of the health system.* While it is ultimately the responsibility of the government to decide which of these values should be embodied in policy, it is desirable for the government\'s decision to be informed by evidence concerning the community\'s values and the strength of preferences for different values systems by different groups in the community. In his influential book \'The Power of Public Ideas\' Robert Reich emphasises the importance of \'discovery\', or the process of getting the public to articulate these values \[[@B2]\]. To date, neither this nor research into health related social values has been carried out satisfactorily in Australia. ***Conclusion 3:**The choice between public and private funding of health services depends upon social values and, in particular, the strength of liberal-libertarian versus solidarity-communitarian values as they apply to the health sector.* Returning to the first defining characteristic of the economic framework, the emphasis upon choice conflicts with two commonly held technocratic beliefs about the inevitability of particular \'problems\'. First, the common perception that the country cannot, or shortly will not be able to afford health services is unambiguously false, at least in the foreseeable future. In the USA, per capita expenditures are about double the Australian level and the US Health Care Financing Agency (HCFA) has projected a doubling of these expenditures relative to GDP by 2030. This is technically possible. The relevant question is whether or not we obtain commensurate benefits from these expenditures and if, as a society, we chose these benefits in preference to the benefits foregone. Taking an extreme example, if Australians could spend 25 percent of GDP upon health services this option would probably be embraced enthusiastically if it resulted in an illness free life expectancy of 120. Optimal expenditures are entirely a function of the benefits we obtain and are not driven by technological imperatives. ***Conclusion 4:**There is no immediate limit to the optimal level of health expenditures. It is technically possible to increase present expenditures very significantly. The optimal level depends upon the costs and benefits of the various health services. Increased health expenditures should be enthusiastically embraced if they improve health and health related objectives sufficiently. Technocratic arguments asserting the economic impossibility of increased health spending or increased public funding are unambiguously wrong. At best they are based upon unstated political/ideological assumptions and not economic arguments.* A similar argument applies to the second non-problem -- the impossibility of funding health services through public taxation. Arguments of the form \'the Government can\'t afford to pay\' are also unambiguously false. The country which can afford to finance health expenditure from private health insurance can also afford to pay an equivalent amount through taxation and some have argued that PHI is, itself, a form of privatised tax. The government share of the health bill is smaller in Australia than in most developed countries. Likewise, taxation is relatively low. This implies that Australia could significantly raise its level of public funding without exceeding the tax burden which is presently experienced in most comparable countries. More fundamentally, however, the form of financing for health services is flexible and is again a matter of social choice. It is likely that this choice will be influenced by the relative costs and benefits arising from the choice, but in the health sector the known costs and benefits associated with public and private health care are not compelling. Privately funded health care is often a little more expensive, but countries with a strong preference for liberal-libertarian values might sensibly opt for a relatively larger private scheme even if it is more expensive. The principle of paying more for what is wanted should not be controversial! ***Conclusion 5:**The balance between public and private sources of revenue for a health service should be determined premanually by the social philosophy of the country. There are no compelling technical or economic constraints on the freedom of sound choice.* 3 Recent policy issues ====================== Public debate has recently focused upon three \'problems\', namely the high and rising cost of pharmaceuticals, the declining rate of bulk billing by General Practitioners (and particularly amongst health care card holders) and the declining number of people purchasing private health insurance. The comments below do not purport to be an exhaustive analysis of these subjects but are included to contrast the subject matter of the policy debate with the more substantive problems which will be discussed in the following section. Pharmaceuticals and PHI are discussed more fully in Richardson and Segal \[[@B3]\]. Pharmaceuticals --------------- Pharmaceuticals are included in the Pharmaceutical Benefits Scheme (PBS) after a detailed review of their effectiveness and cost effectiveness (see Salkeld et al 1998 for a description \[[@B4]\]). This process does not, by itself, reduce expenditures. Rather, it ensures that drugs whose effectiveness is low in relation to their cost will not be adopted. Expenditures will be lower if the manufacturers of relatively cost ineffective drugs reduce their prices to increase the likelihood of their inclusion in the PBS. However, this may be offset (more or less) if drug companies increase the price of new highly cost effective drugs as they know that the PBAC is aware of their cost effectiveness. Cost effective drugs may also be overused if doctors prescribe them for purposes or at thresholds not tested before their introduction. Partly for this reason the PBAC has sometimes negotiated a price-volume trade-off -- if drug use exceeds the initial expectation then the agreed price is lowered. These measures have not contained pharmaceutical costs. In view of rising drug prices in other countries it is likely that this is primarily or entirely attributable to the high cost of new generation drugs and is not attributable to policy. Nevertheless, expenditures have risen and in recent years copayments have been progressively increased in order to reduce government expenditures through the PBS. However it is not clear that the use of copayments, and particularly in a single sector, will have an overall beneficial effect. First, as demonstrated by the Rand Experiment copayments reduce demand somewhat (elasticities are low but not insignificant) \[[@B5]\]. There is a disproportionate effect upon low income households \[[@B6]\] which, in this case, implies low income non health care card holders. There is little evidence that lower income patients will discriminate between effective and less effective drugs and at least one study suggests that, perversely, the greatest impact will be upon life saving drugs which have a relatively small immediate impact upon symptoms \[[@B4],[@B7]\]. Secondly, relatively larger copayments in one sub-sector violates a fundamental principle for achieving allocative efficiency; viz, a \'level (financial) playing field\' between alternative products/interventions. Violation of this condition increases the likelihood that less cost effective services may be used because of the distorted price signals. Allocative efficiency depends upon relative prices rather than the existence of copayments per se. More specifically, high pharmaceutical prices at the point of service will encourage the use of potentially more expensive interventions including hospital services if a copayment results in the deterioration of a patient\'s health. Thirdly, the effects of copayments on national health expenditures will be relatively small. Wealthy individuals will simply pay the copayment and health care card holders will be largely shielded from them. The most important effect of the copayment is that it will shift costs from the taxpayer to the patient; that is, its major effect will be upon the distribution of income with the healthy-wealthy taxpayer gaining at the expense of the less healthy-less wealthy. In 1960, pharmaceutical costs represented an estimated 22.3 percent of health expenditure. By 2002 they were 13.5 percent of the total. The comparison indicates that the composition of expenditures may vary significantly through time and, taken out of context, focus upon a single sub-sector may be misleading. Cost control requires a full system perspective and there is no optimal level of pharmaceutical expenditures which is independent of the cost of alternative services. The problem is, in part, attributable to the program structure of delivery and financing which treats pharmaceuticals as an independent program as distinct from an input into an overall treatment regime. ***Conclusion 6:**Reliance on copayments for the control of pharmaceutical expenditures is probably an inappropriate policy as it violates an important principle for achieving allocative efficiency. A more global approach to health policy analysis and reform is needed.* Bulk billing ------------ Since the introduction of Medicare the percentage of GP attendances bulk billed rose from 52.5 percent in 1984/85 to a maximum of 80.6 percent in 1996/97, then fell to a low of 66.5 percent in December 2004. The measures discussed below appear to have arrested this trend and by March 2004 bulk billing had risen to 68.7 percent of GP attendances \[[@B8]\]. The chief problem the reforms were designed to overcome was the possibility that the falling level of bulk billing may have jeopardised access to health services by pensioner/health care card holders and other low income households. Importantly, data did not exist to demonstrate that this was, indeed, a problem and that bulk billing or service use by this group had declined by the average or near average percentage. That is, the existence and quantitative significance of the \'problem\' were not documented. The recent \'Medicare Plus\' package introduced a potentially important structural change. Rebates for bulk billed/health care card holders were separated from the rebate to the general public thereby allowing the separate treatment of the two groups. To encourage the bulk billing of the first group the benefit was increased and bulk billing doctors were permitted, for the first time, to direct-bill the Health Insurance Commission for general patients while simultaneously charging an \'over the counter\' extra payment, a measure widely perceived as encouraging an increase in fees as patients will commonly equate the relatively small copayment with the total fee. These measures have two important effects. First, the probable increase in general fees allows the benefit payable for GP bulk billed pensioners/health care card holders to be at a lower level than would occur than without the effective cross subsidy from increased general fees. Secondly, the government can preserve \'equity\' (ie bulk billing the needy) by adjusting only the pensioner/health care card holder benefit, while simultaneously allowing a deterioration of the general rebate. Cost shifting would have been further facilitated by an earlier agreement to allow private health insurers to reimburse medical expenses above the schedule fee as part of an agreement with doctors to remove out-of-pocket costs. But this proposal was rejected by the Senate. In sum, there is now a structure which facilitates cost shifting from the public to the private sector. There are two important caveats to this conclusion. First, a rebate structure which facilities additional cost shifting does not imply that government will use this option in the short or long run. Secondly, the effect of additional cost shifting will be mitigated by an additional element of the Medicare Plus package, namely, the introduction of a \'safety net\' which reimburses 80 percent of out-of-pocket, out-of-hospital costs including billing above the schedule fee once family expenses reach \$1,000 (or \$500 for a health care card holder\'s family). The long run effect of this latter cost off-set is difficult to assess. As reported in the announcement of Medicare Plus the expected cost of the safety net in its first three years would average \$89 million p.a. as compared with likely out of pocket medical expenses of about \$1,400 million. in 2004/05 (extrapolated AIHW) \[[@B9]\]. Even allowing for a probable cost overrun the amount is not large. Nevertheless, the \'safety net\' offers additional protection to those who would be most affected by additional cost shifting; that is, this policy also helps create a structure where equity, defined in terms of need, may be reconciled with increased cost shifting. However, the most significant feature of these changes to the reimbursement formula for medical expenses is that they deal with *relatively*\'small order\' issues. Under Medicare, pensioners/health care card holders with significant health problems have access to emergency and outpatient facilities at public hospitals. Poor access to these will increase inconvenience and queuing and, in an unknown number of cases, result in poorer health. But this more serious outcome is likely to be *relatively*infrequent as the majority of this group have always been bulk billed while others will have satisfactory access to hospital based care. ***Conclusion 7:**Proposed and actual policies towards pharmaceutical expenditures and bulk billing have had a common element. Both represent immediate or potential cost shifting from the government to the public and any net reduction in societal expenditure because of a reduction in patient initiated services will have potentially adverse effects upon the health of the \'near poor\'. However, the financial effects of the policies are relatively small and the indirect adverse effect upon health is likely to be correspondingly small.* Private health insurance (PHI) ------------------------------ The role and regulation of PHI after the introduction of compulsory national health insurance has been ambiguous and anomalous. Since Medicare was introduced as the vehicle for achieving fairness in the financing and delivery of health care it has never been clearly stated why PHI should be closely regulated to achieve fairness in he sub-group of the population which elects to purchase PHI and why it is regulated in a way which inhibits effective competition. But these are the effects of Australia\'s community rating and reinsurance requirements respectively. In the last one and a half decades there has been an ongoing concern that declining levels of private health insurance have had an adverse effect upon the public hospital system and that \'PHI reform\' has been needed to solve this problem. The argument is summarised in the following (constructed) quotation: \'Private health insurance was caught up in a downward spiral caused by the adverse selection identified in the Productivity Commission report \[[@B10]\]. \'Rising premiums triggered by increasing costs induced low risk members to withdraw. Without their cross subsidy of high cost members, premiums were forced to rise again, which further increased premiums, which induced further departures, etc. \"*PHI is primarily purchased to cover the costs of private hospitalisation. Consequently, as PHI declined through time, fewer patients have been able to afford the cost of private hospitals, and this has created an excess demand for public hospital beds. The increasing length of queues is a confirmation of this problem*\". While plausible, the account of history in the second half of this argument is wrong. Between 1985/86 and 1999/00 private hospitals increased their share of admissions by 32.4 percent from 25.9 to 34.3 percent of the total. The share of bed days rose from 21.9 to 28.1 percent of the total or by 28.3 percent \[[@B3]\]. These simple and readily available statistics unambiguously contradict the conventional wisdom propagated by the media and some politicians. It is true that queues in public hospitals have increased, but this is attributable to the increasingly draconian budget caps upon public hospitals throughout the 1990s. That is, queuing in the public sector has been primarily a result of supply side and not demand side factors. (Excess demand for beds cannot explain the closure of wards that occurred in many public hospitals!) The simplicity of the statistics contradicting the conventional wisdom calls into question the analytical capacity of many media commentators (and some independent commentators)! ***Conclusion 8:**Media analysis of the relationship between PHI and queuing in the public system highlights an important system failure; viz, the failure of the media to exercise rudimentary critical skills in their analysis of PHI and hospital queuing.* Even if PHI membership had fallen sufficiently to have reduced the share of private hospital admissions by almost a third, the balance of public and private hospital separations in 1999/00 would have been similar to the share in 1985/86. This suggests that it was the events summarised in the first half of the quotation which were of concern at the government and departmental level and that PHI membership and the public-private mix of health financing and delivery were explicitly or implicitly the real target of health policy. In response to the \'problem\' of falling PHI and the demand side problem for public hospitals (which did not exist!), the government introduced three enduring policy changes. In July 1997 individuals with an income above \$50,000 and families with a combined income above \$100,000 who did not purchase PHI became liable for a 1 percent tax surcharge upon their incomes. In December 1998 a 30 percent rebate on PHI premiums was introduced. (In August 2004 a selective increase in the rebate was foreshadowed); and in September 1999 lifetime community rating was enacted which has the effect of reducing future premiums for those who have held PHI from the age of 30. Discounts apply at any age between 30 and 64; 30 is the age of maximum discount. Failure to enrol by this age will result in a higher premium if PHI is subsequently purchased. The evidence demonstrates that these policies, and particularly the last policy, have succeeded in increasing PHI membership. However it has created an industry which, along with the platypus and echidna could be Australia\'s entrants into the world\'s strange but true contest. Australia would almost certainly win -- even without our egg laying marsupials. First, the surcharge results in a negative price for the wealthy. Individuals and families above the income threshold avoid an increasingly large tax payment as their income rises; that is, if they purchase PHI they will have a greater income at the end of the year than the individuals and families above the threshold who do not buy PHI. This is analogous to supporting the automobile industry by placing a surcharge on wealthy families who fail to buy an Australian car. It would be difficult to find any other support scheme which uses the income tax system to coerce the purchase of a particular product. It would be equally difficult to find a produce where the price is negative. Secondly, (and predating the recent legislation) the use of PHI to cover hospital bills generally results in a greater, not smaller, out of pocket expense. Public hospitalisation is free. Those with PHI commonly pay a copayment. (There is a perverse equity in these two anomalous outcomes. The wealthy are paid to take PHI but financially penalised if they use it!) Third, lifetime community rating also involves a leap of faith. The 30 year old must believe that public policy will remain stable for 50 years -- a belief that should have been challenged when both parties announced changes in the 2004 election campaign. However lifetime community rating also has a bizarre dimension. Insurance is generally purchased to reduce risk and uncertainty. In the health sector these arise because of the risk and uncertainty of ill health and the cost of medical care. Prior to lifetime community rating this uncertainty depended upon possible events in the following 1--3 years. After the change it depended upon the next 1--30 years. Events in 30 years are more uncertain than events in 3 years and, consequently, the legislation increased risk and uncertainty. The perceived risk was amplified by the publicly financed marketing of private insurance. Predictably, people responded to the increased uncertainty by buying more insurance. Thus, to encourage the uptake of insurance the government increased the very thing insurance is designed to reduce, viz, risk and uncertainty. A final anomaly which predates the recent PHI reforms is that private health insurance in Australia leaves the consumer with residual risk. Particularly for ancillary services, most benefits are capped and out of pocket expenditures rise with the price of the service. This pattern of benefits is exactly the opposite of the structure of benefits which will maximise the patient\'s \'expected utility\' \[[@B11]\]. Taken together, the changes introduced since July 1997 have created an extraordinarily complex and perverse set of incentives. The ethical basis of the free market and the liberal-libertarian model is that choices should be determined by individual\'s preferences in relation to real (opportunity) costs. The surcharge subverts this process and coerces choice and the strength of the coercion is based upon economic class. There is no justification for this in the economic theory of the efficient market. Despite this conclusion, it is a legitimate function of government to determine the balance between public and private delivery financing and the distribution of health care costs. As described earlier the preference for private sector funding and provision (*albeit*in the context of compulsory core insurance) is consistent with a legitimate and defensible world view, viz, the liberal-libertarian belief that in a free society individuals should be encouraged to take responsibility for their own lives. In particular, the 30 percent subsidy is the orthodox approach to encouraging an industry which has a special claim for protection and, private health insurance does not compete on a \'level playing field\': it competes with a public sector which is free at the point of service. There are, however, two important caveats. First, and as noted above, the measures taken have destroyed any nexus between potential benefits and the price paid by wealthy individuals. Secondly, the \'product\' is unlike usual insurance where the benefit -- a payment after an adverse event -- does not impinge upon other individuals. PHI is purchased to avoid queues and to select the best possible doctor. With fixed capacity, the avoidance of queues by one person imposes a longer queue on another person -- there is a \'negative externality\'. Selection of the best doctor reduces the access to these doctors by public patients. Consequently, the important debate should be about the \'right\' to purchase preferential care *at the expense of those who do not have PHI*. In a liberal democracy there is a presumption that individuals may spend their own income as they wish. For the individual there is probably no more important context for exercising this \'right\' than in the context of preserving life and its quality. There is, therefore, a head-on-head conflict between the liberal-libertarian \'right\' of the individual to spend his or her income on health care and the communitarian-solidarity based \'right\' of each individual in a community to have equal access to high quality medical care. The latter goal must necessarily be achieved by imposing some constraint upon income-based preferential care to a particular group in the community. In Australia the constraint has been a de facto financial penalty for seeking priority care. Until the advent of engineering of negative prices for the wealthy, the purchase and use of PHI resulted in \'double payment\' for some services, first via taxation and the Medicare Levy and secondly by premium paid for PHI. This \'penalty\' still exists for lower income individuals and households. ***Conclusion 9:**The public debate over Private Health Insurance has been misleading. The contentious issues do not only concern the most effective way of ensuring access to health care (with the erroneous presumption that public monies not spent on health services represents wasted resources). Rather, the contentious issues include the \'right\' or otherwise of the individual to spend their own income on whatever they wish without coercive financial penalties and the consequences for the remainder of the community when one group jumps the queue and has priority access to the most experienced doctors.* Distributional effects of recent policies ----------------------------------------- PHI policy has been consistent with the other policies discussed above in one important respect. The policies are likely to have a relatively small effect upon the *delivery*of health services. Rather, they are about financing care and the public-private balance in the health system. The balance, in turn, affects the distribution of health care costs between different households. Copayments shift costs from the government to patients. As government payments are met by progressive taxation, copayments redistribute the cost of health care from wealthy-healthy taxpayers to the less healthy, less wealthy. Additionally, as copayments have a disproportionate effect upon the use of services by low income households, the proportion of the government subsidy returned to high income households rises as copayments rise. The redistributive effects of PHI are more complex. Low income households which purchase PHI unambiguously pay more for hospital and health services. Their taxes and the Medicare surcharge are unchanged and the purchase of PHI leaves them out of pocket. For higher income households, which are liable for the PHI surcharge, the effects are conceptually ambiguous as they depend upon the assumption made about the surcharge in a counterfactual world in which PHI did not result in a surcharge exemption. The surcharge was created specifically to permit an exemption for those who purchased PHI and the removal of the exemption might therefore be accompanied by the removal of the surcharge. Finally, and as noted earlier, the proposed changes to bulk billing represent a structural change which will facilitate the transfer of medical insurance costs from the public to the private sector. ***Conclusion 10:**Recent and foreshadowed legislative policy initiatives with respect to bulk billing, copayments and PHI, all concern the financing of health services. Recent policy has been introduced with measures to mitigate the impact upon the most needy and, in the case of medical rebates, in a way which accommodates the popularity of bulk billing. Nevertheless, a common feature is that each of the proposed or implemented policies assists with the creation of a structure which facilitates the transfer of expenditure from the public to the private sector. If this occurs, it will reduce the cross subsidy from healthy wealthy to unhealthy less wealthy households. This effect occurs immediately with copayments. The transfers are more complex in the case of PHI.* 4 Five major problem areas ========================== The five issues below have two common elements. First, they are directly concerned with health services and not the distribution of costs and incomes. Second, they have been almost ignored in the public policy debate despite being problem areas where there are quantitatively large inefficiencies and a corresponding potential to increase health outcomes and/or increase the overall cost effectiveness of the system. Efficacy, effectiveness and social objectives --------------------------------------------- In common with all other health systems many, and probably most, of the services provided in Australia have not been adequately evaluated and there is no ongoing process for the elimination of cost ineffective services. In 1987 Chassin et al \[[@B12]\] estimated that between one third and one half of coronary services in his study were \'inappropriate\' in the sense that they had no beneficial, or a detrimental affect upon health. An additional one third to one half of the procedures considered had equivocal benefits. Likewise, Brook \[[@B13]\] estimated that 51 percent of angiography and 42 percent of coronary artery bypass graft procedures were unnecessary. Other studies by the US Health Care Financing Agency and the OECD have likewise concluded that only a small number of services have been evaluated for efficacy \[[@B14]\]. In Australia Segal \[[@B15]\] demonstrated that in the context of diabetes the cost of obtaining a life year varied from \$70,000 (drug therapy) to \$2,400 for behavioural programs. Comprehensive diabetes care was estimated to have a negative cost per life year; ie the program saved life and saved cost. With respect to this issue, the Australian record is comparatively good. It has led the world with the introduction of mandatory economic evaluations for the drugs and services to be subsidised through the PBS and the Medical Benefits Scheme respectively. However the failure of other countries does not indicate that Australian procedures are satisfactory. The overwhelming majority of the services which were accepted before the introduction of mandatory economic evaluations have not been assessed and, with the passage of time, there is a need for the reassessment of services and drugs. A related problem is that drugs or services which are efficacious when used appropriately in a random control trial may be used inefficiently with inappropriate patients who do not have the clinical indications of the patients in the trial. Gold standard medical care requires that cost effective therapy, drawing upon evidence-based medicine, should be the norm and not the exception in medical practice. Cost effectiveness should be determined by a broad ranging comparison of options form across the full range of potential interventions; that is, comparator interventions should take account of substitute services from across the entire health sector. This does not presently occur. Guidelines for the PBS require a comparison between drugs and exclude comparison with lifestyle/behaviour change programs. ***Conclusion 11:**The scale of present evaluation activities is inadequate. In an industry absorbing 9 percent of the GDP -- the country\'s largest industry -- there should be ongoing and large scale evaluation and re-evaluation of the cost effectiveness of the services provided. Evaluation should be based upon a comparison with the full spectrum of substitute services. A failure to do this almost certainly ensures that there will be widespread and significant allocative inefficiency in the level and mix of services.* Implementation of evidence based practice will inevitably be resisted by service providers whose practice and income may be adversely affected. Consequently, provider education for a \'culture change\' is likely to be a slow and ineffective vehicle for change. In contrast, once the desired practice pattern has been determined, financial incentives to encourage this form of practice may be implemented relatively quickly and do not involve direct coercion. The economics literature is replete with examples where such incentives have significantly altered provider behaviour \[[@B16]-[@B19]\]. Australian examples include the use of GP incentives to bulk bill; to undertake rural practice and to increase child immunisation. DRG based financial incentives were spectacularly successful in increasing hospital throughput in Victoria in the early 1990s \[[@B20]\]. ***Conclusion 12:**Reimbursement formula for service provision should include financial incentives at all levels of service delivery for the use of the most cost effective therapies.* The more general principle which should, but does not, permeate the market for health services is that the willingness to pay for services should vary with social objectives, a principle which is successfully incorporated in simple competitive markets when social and individual objectives are identical. As one example where this principle has recently and successfully been used, if society wishes to encourage bulk billing (which clearly benefits patients but lessens provider control over their incomes) then the benefit for services that are bulk billed should be increased relative to the rebate for other services and the differential increased until the target of bulk billing is achieved. Likewise, if society wishes providers to adopt evidence based medicine, hospitals to install clinical pathways, some procedures to be encouraged and others discouraged, then payment for the desired service or process should be increased relative to the reward when these services or activities do not occur. At the level of the individual service, recent research has demonstrated health-related social objectives which are often broader than the minimisation of morbidity and mortality, for example, preferential treatment for particular age groups, particular classes of patients and health benefits \[[@B21],[@B22]\]. Presently these additional \'social preferences\' are ignored. This is unsurprising as the research to identify and measure them is still underway. However, where these preferences are significant and consistent, payments should eventually be adjusted to ensure that they are achieved. ***Conclusion 13:**The payment of service providers should incorporate the principle that society should pay for what it wishes to have in accordance with its social objectives, rather than what it is given. This implies the need for ongoing enquiry into health related social objectives (the numerous objectives loosely grouped under the heading of \'equity\').* Practice variations ------------------- In 1982 John Deeble and I used data from the first full year of compulsory health insurance, (Medibank), to determine the level of service use in each of Australia\'s statistical divisions \[[@B23]\]. An example of the results is shown in Table [2](#T2){ref-type="table"}. Huge variations in service use were detected even between the relatively large geographical units used in the study. Within these units small area variation would have further increased the discrepancies. These differences do not appear to have diminished with time. Thus, for example, Robertson and Richardson \[[@B24]\] found startling variation in the use of well-defined hospital procedures even after standardisation for age, sex and population. In this study data were collected for a two year period for each of Victoria\'s statistical sub-divisions. Procedural rates were expressed as a percentage of the rates which would be expected from the State average service use per age-sex cohort and from the demographic characteristic of the Statistical sub-division (SSD). Results are summarised in Figure [1](#F1){ref-type="fig"}. The bar, lines and circles give an indication of the frequency distribution of the procedure utilisation rates (ie the 25 and 75th percentiles (bars), two standard deviations (lines) and outlying SSDs). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Practice Variations 1976 ::: **Consultations per capita** ------------------------------ ------------- ------------- ----------- **Statistical Division** **GP/(GP)** **Q(Spec)** **Total** Sydney 5.1 2.3 7.4 Tasmania 3.1 1.3 4.4 Darwin 1.1 0.5 1.6 Sydney/Darwin 4.6 4.6 4.6 ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Standardised Rate Ratios for Various Operations in the Statistical Local Areas in Victoria, Compared to the Rate Ratios for All Victoria, Source: Richardson 1999 p198. ::: ![](1743-8462-2-1-1) ::: The results reveal an 8 to 10 fold variation in service use. Part of this is attributable to the random variation that would be expected because of the uncertainty of the episodes of ill health. Using state-wide data the ratio of actual to expected variance was calculated. This is reported in the column of numbers to the left of the bar diagram. For the first procedure, coronary angiography, the observed variance was 13.4 times greater than expected; ie actual variance was 1,440 percent of the expectation. This extraordinary result is reproduced for all of the procedures examined. A second example of uneven service delivery was also published by the same authors. This related to the likelihood of obtaining a high tech procedure -- angiography or revascularisation -- after a heart attack. Results shown in Table [3](#T3){ref-type="table"} indicate that in the 14 days following the heart attack, men and women admitted to a private hospital were 2.2 and 2.27 times more likely to receive angiography than their counterparts at a public hospital. They were 3.43 and 3.86 times more likely, respectively, to undergo revascularisation (coronary artery bypass surgery angioplasty, stent). These discrepancies did not diminish significantly in the following 12 months. The same study identified statistically significant differences in the likelihood of a procedure between men and women, young and old, urban and rural populations. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Ratio: (Likelihood of procedure after admission to a private hospital) ÷ (Likelihood of procedure as a public patient) ::: **Angiography** **Revascularisation** ------------------ ----------------- ----------------------- Within 14 days Men 2.20 3.43 Women 2.27 3.86 Within 12 months Men 2.16 2.89 Women 2.22 2.84 ::: Taken together these studies suggest that there is a highly erratic pattern of service delivery across Australia and between social groups. One of three conclusions is inevitable. Some groups are under-serviced; some groups are over-serviced or both of these problems occur to different sub-groups of the population. This indicates both allocative inefficiency (more health could be obtained with a redistribution of existing resources) and significant inequity for those with poor access to health services or a different form of inequity for those persuaded to undergo procedures where risks exceed likely medical benefits. Government has shown almost no interest in this type of result. There has been recognition of an \'urban-rural\' discrepancy but, 20 years after the demonstration of a more complex pattern than a simple urban-rural dichotomy, remarkably little has been achieved. ***Conclusion 14:**Significant variation in the use of services has been allowed to continue more than two decades after it was identified. Small area variation across Australia almost certainly reflects significant allocative inefficiency and an inequitable access to health services. There appears to have been relatively little interest in this problem.* Lack of coordination -------------------- The health system at present consists of a series of financially independent programs (\'silos\'), which are poorly linked to other health services or programs. There appears to be near universal agreement that a sensible health scheme cannot be built upon the current Federal-State division of financing, responsibilities and powers or upon the existing Commonwealth program structure. In the context of a recent review of hospital care, the Tasmanian Government accepted a recommendation for the pooling of all health and aged care resources \[[@B25]\]. However implementation of the recommendation requires Commonwealth support which may or may not be forthcoming. The fact that a simple solution exists for this most elementary problem but, to date, has not been seriously addressed, represents a fundamental failure of government in Australia. Two case studies are used below to illustrate what gold standard allocative efficiency would imply for the use and coordination of services. They indicate the distance that health services reform must travel in Australia before we have gold standard delivery. The first case study is real. A Seattle-based \'pure\' Managed Care company, Ethix, was asked to establish a health scheme for a small town close to Seattle. Routine surveillance of the medical claims over the first two years of the new scheme highlighted an anomaly. There were excessively large numbers of youths receiving surgery for spinal injuries. Further investigation found that the problem was attributable to a toboggan run on the outskirts of the town which had a tree stump half way down the slope. Youths were crashing into the stump and damaging their spine. The health scheme paid for a bulldozer to remove the stump \[[@B26]\]. Medicare does not pay for bulldozer services. However, in the circumstances described here it should do so. More generally, the vignette illustrates two of the characteristics of gold standard delivery, namely, routine data surveillance to locate problems with any aspect of social or medical behaviour which might be modified to improve health and, secondly, the flexibility of funding which is needed to adopt the most cost effective solution to the identified problems. In contrast, in the Australian health scheme a problem of this sort would not be detected by the health system. It is possible that a similar type of accident could fall under the jurisdiction of an occupational health and safety or traffic accident authority. Otherwise there would be neither the will nor the means to respond. If such a problem was eventually identified the typical response would be accusation, blame shifting and, possibly, litigation. The cause of this problem is related to the practice of Management by Objectives, an aspect of managerialism, which encourages local rather than system-wide thinking. Interestingly, Peter Drucker, one of the early advocates of MBO, in recent years has publishes warnings about its over-use and limitations. ***Conclusion 15:**A key challenge is to establish a single payer (for each person) with flexibility and incentives to purchase the most cost effective services. Services should not be determined by historical program boundaries and rigid budgets. There has been a serious failure by government to address this fundamental issue. The achievement of this relatively simple reform is necessary (but not sufficient) for the achievement of a range of system reforms.* The second case study is a hypothetical scenario constructed by Duckett to illustrate gold standard system coordination and processes \[[@B27]\]. \'A woman with dizziness is concerned about her health. She rings the *State call centre*which advises her to visit her *local health team*. She is able to see the GP quickly who asks her a series of questions from the *relevant research based protocol*and undertakes a clinical examination. The GP emails the results to a local specialist\... who orders some further investigations consistent with the *state research based care path*\... *Advice*of (an) impending admission is automatically *conveyed electronically to the GP and the social worker in the referring health team*. The social worker contacts the hospital to *discuss discharge planning*\... The specialist\... suggest(s) a number of *sources for information about the patient\'s condition*. The *patient contacts the call centre for further information*\... The case is *randomly selected by the hospital audit committee*for quality review. The committee suggests some slight *changes to the state-wide protocol committee*.\' p204. The key elements of this scenario relate to information access and transferral. It illustrates the role of evidence based medicine, routine service review, the adaptation of protocols, universal electronic transfer of all information and the absence of incentives to depart from best practice. Parts of this scenario correspond with Australian practice. But the events in italics would be unusual. It appears to be serendipitous whether a particular problem of a particular person and in a particular part of Australia results in a response which even partially mirrors the gold standard response in this scenario. ***Conclusion 16:**Commonwealth and State authorities should mandate practices which improve the coordination of services. To facilitate this there should be universal use of electronic data systems for patient notes and information transfer. Evidence based protocols and clinical paths should be adopted when available and relevant. Feedback and error learning should be a routine part of the system. All of these desirable features are presently difficult because of the fragmentation of the system which is, in part, attributable to the failure to establish a single funder for all health services received by an individual.* Quality of care --------------- Results from the 1995 \'Quality in Australian Health Care Study\' (QAHCS) suggest that the quality of health care in Australia is a problem which overshadows all others. In the initial study, reported by Wilson et al \[[@B28]\] medical records for more than 40,000 admissions to 28 hospitals in NSW and SA were individually examined to determine whether or not an adverse event (AE) was associated with the admission (prior to or during the episode of hospitalisation). A judgement was made concerning the consequences of the AE and whether or not it might have been avoided. By extrapolating results the authors estimated that about 470,000 admissions were associated annually with an AE and that these would have resulted in 18,000 deaths and 50,000 cases of permanent disability. In a subsequent report Runciman et al \[[@B29]\] estimated that in 50 percent of the AEs in the QAHCS had a high preventability score. Sixty percent of deaths could have been avoided. In this latter study, incidence and not prevalence scores were reported as part of the effort to standardise the methodology with an earlier Harvard Medical Practice Study (HMPS) reported by Brennen et al \[[@B30]\]. This reduced the annual rate of AEs to 10.6 percent of admissions. From the response to these events (discussed below) it appears likely that many have been unable to appreciate the scale of the carnage implied by the report. If the results from the original QAHCS are not discounted then medical errors have been responsible for the death of more Australians per annum than the average annual death rate of Australian soldiers in World War 1 (15,800). Permanent disabilities per annum approximate the annual rate of casualties in The Great War (62,500). Unlike The Great War, the problem of AEs has probably been ongoing for 50 years or more. In Table [4](#T4){ref-type="table"} below the death rate from AEs is compared with mortality rates from other causes which are of particular social concern. To be conservative and to take account of undetected bias, the AE rate reported in a 1995 study is reduced by 50 percent. Preventable deaths are assumed to be 50, not 60, percent of deaths associated with an AE. The resulting, conservative number of deaths from AEs in 1999 were about 40 percent higher than the number of deaths from AIDS, suicide, motor vehicle accident, accidental falls, homicide, drowning and poisoning combined. In Table [5](#T5){ref-type="table"} some equivalent events are listed. The conservative estimate of the unnecessary death rate is about the same as would occur if the Bali bombing occurred every week of the year, year after year. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Perspective: Selected Causes of death, 1999 ::: **Cause** **No of deaths** ------------------------------------------------- ------------------ AIDS 122 Suicide (intentional self harm) 2,492 Motor vehicle accidents 1,741 Accidental falls 520 Homicide 300 Accidental drowning/submersion 278 Poisoning by drugs/medications 1,015 Subtotal 6,468 All deaths from adverse events^(1)^ 9,000 **All preventable adverse events^(2)^** **4,500** \(1) 50 percent of reported estimate \(2) assuming 50 not 60 percent are preventable ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Events equivalent to avoidable AE deaths\* ::: **Cause** --------------------------------------------------------------------------- 1 in 10 customers in restaurants poisoned each year: annual deaths 4,500 13 Jumbo jets crash each year, each with 350 Australian passengers killed 45 Bali bombing type attacks, each with 100 Australians killed each year \'September 11\' every 8 months: only Australians die \*assuming preventable deaths = 25% QAHCS (1995) ::: While those affected by adverse events will, on average, be older and frailer than those who died in Bali or during World War 1, the magnitude of the problem is still staggering. Considering the reaction to the Bali bombing it might have been expected that the publication of the QAHCS would have caused a seismic shock, with the public demanding immediate, comprehensive reform and government passing urgent legislation to mandate any achievable system reform which ameliorated the problem. If the system failures which preceded the Bali bombing warrant a Parliamentary inquiry, equivalent interest might have been expected with respect to a problem responsible for an equivalent number of deaths every week. An effectively unlimited budget might have been approved for the upgrading of quality and safety. In contrast, the actual reaction to the report must constitute one of the more puzzling episodes of Australia\'s social history. At all levels the response was sedate, cautious and incremental and there appears to have been greater concern that our health system might be perceived as unsafe than with the fact that it actually is unsafe. The level of activity in the 5 years following publication of QAHCS suggests that the results may not have been truly believed. But no subsequent study was funded to validate or refute the results. The QAHCS results were not ignored. As summarised in Table [6](#T6){ref-type="table"} an advisory body was immediately established which evolved into the Australian Council for Safety and Quality in Health Care (ACSQHC). Its activities are summarised in five successive annual reports. In addition, the Australian Health Care agreement between the Commonwealth and State governments allocated budgets of \$680 million and \$785 million for quality assurance activities for the periods 1998--2003 and 2003--2008. In each of the States sub-committees and working groups were created which, along with the ACSQHC have resulted in a very large number of reports, publications, some legislation and local initiatives. The ACSQHC alone lists 35 publications \[[@B31]\]. State activities are summarised in ACSQHC, \[[@B32]\]. These initiatives have resulted, *inter alia*, in moves to tighten up hospital accreditation processes; to monitor adverse events more closely; to improve consumer participation in the evaluation of health care; to encourage health professionals to report adverse events; to improve health information technology; to establish practice guidelines, etc. The large number of funded projects are described and listed in ACSQHC, \[[@B33]\]. ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Response to QAHCS ::: ------ ------------------------------------------------------------------------------------------------------------ 1995 QAHCS published 1996 Taskforce established on QAHC 1998 Health ministers ask Advisory Group for report 1998 Interim Report 1999 Report of the National Expert Group \'Actions identified by the taskforce\... need to be implemented at all levels of the health system \...\' 1999 Australian Council for Safety and Quality in Health Care Established ------ ------------------------------------------------------------------------------------------------------------ ::: Despite the high level of activity, the importance, priority and sense of urgency reflected appear more appropriate for an (important) ongoing reform process in an already well-operating system. There is a yawning gulf between this and what the public would undoubtedly demand if our TV and news services were reporting deaths on the scale of the September 11 New York disaster very two months accompanied by about three times this number of injuries. The philosophy of the reform process appears to be summarised by the ACSQHC when it approvingly quotes Berwick as saying that \'there are no quick fixes. We must re-examine all that we do\', p14 \[[@B31]\]. As a description of history the \'no quick fixes\' statement is correct. The 1999 report -- 4 years after the publication of the QAHCS recommended that \'actions identified by the taskforce\... need to be implemented at all levels of the health system\'. By 2001 NSW had passed legislation which, *inter alia*, created the Institute of Clinical Excellence. By 2002 -- 6 years after publication -- the Victorian Quality Council Plan had been established. In 2003 the updated version of this plan set as its goals, *inter alia*, the establishment of a framework, the involvement of consumers and education. Almost 10 years after the report, the ACSQHC called for mandatory participation of all hospitals in a process of assessment \[[@B31]\] and (with possible irony or ill concealed frustration) the Chairman of the ACSQHC argues in the 2004 Annual Report that \'Action must be taken without untimely delay where culpability is clear\', p5 \[[@B34]\]. Between publication of the QAHCS and the Health Ministers\' request for a report in 1995, 13,500 Australian would have died and 37,500 become permanently disabled. By the time the 1999 report was recommending the implementation of various policies at least 18,000 would have died. By the time of the NSW legislation cumulative national deaths would have reached 27,000. The Victorian Strategic Plan to develop a framework was published after the death of at least 31,500. It is scarcely surprising that in 1999 an editorial in the Medical Journal of Australia commented that: \'Welcome though (various initiatives) are, the pace of change nevertheless seems slow given the stark message of the original QAHCS study four years ago\... 50,000 Australians suffer permanent disability and 18,000 die at least in part as a result of their health care\', p404--50 \[[@B35]\]. By 2002 Siddons could still comment that: \'On the 10^th^anniversary of the study year, the most striking outcome has been the paucity of reform currently exhibited at the coalface of tertiary health care\', p823 \[[@B36]\]. The inadequacy of the response is surprising as the evidence suggests that a reduction in AEs would be spectacularly cost effective. The interim report from the national expert group estimated the potential savings from preventable adverse events in 1995/96 would be \$4.17 billion *per annum*. Consequently, expenditures of this amount could be justified if they eliminated the unnecessary AEs or if the cost of the achievable 50 percent reduction in AEs was less than \$2 billion *per annum*. In contrast with the view that little could be done quickly there are a number of examples where, *prima facie*, very significant and effective change could have been/can be rapidly implemented. The chief distinguishing feature of each of the suggestions below is that they involve legislation and regulatory enforcement which appears to be inconsistent with the apparent emphasis upon persuasion and voluntary culture change in many of the present activities. Mandated options include the suggestions below. ***Accreditation***For decades health professionals have believed that a significant number of small hospitals are dangerous. However there has been no decisive action. With full knowledge of the QAHCS results, hospital accreditation remains voluntary in all States except Victoria. There will clearly be self-selection. Low quality hospitals will opt not to seek accreditation and poorly qualified doctors will seek out these hospitals. Universal accreditation could be mandated. Multiple accreditation teams could have the power to randomly inspect hospitals or units within hospitals and close those judged to be dangerous -- as occurs with restaurants with sub-standard hygiene. It is unclear whether or not present accreditation is sufficiently rigorous to reduce avoidable adverse events significantly. There appears little reason why the accreditation process should not itself be reviewed to ensure that credentialed hospitals satisfy rigorous safety standards in their facilities and procedures. ***Doctor Accreditation***Patterns of private practice patterns are already subject to scrutiny in Australia. But the chief purpose is to detect medical fraud. Legislation could require the examination of practices to detect those which deviate significantly from evidence based guidelines constructed by the relevant Royal Colleges. When there is a known relationship between the small number of procedures carried out by a doctor and negative outcomes, as occurs with surgery, critical annual procedure rates may be established which trigger the provision of information to the doctor, the mandatory review of the practice and finally the dis-accreditation of the doctor for the conduct of these procedures. While it is true that some doctors take on the hard cases partial standardisation for case complexity is possible and this problem would obviously be taken into account by those conducting the review. The appropriate systems could be established in 1 to 2 years. ***Mandatory Disclosure and Error Learning***It is not compulsory for hospitals or doctors to register AE and routinely provide feedback to facilitate error learning. This means that the most important vehicle for improving quality and reducing patient risk is not compulsory. The opportunity for error leaning is almost certainly under-utilised in a large number of hospitals and probably ignored by the doctors whose performance most needs monitoring. Legislation might ensure the universality of this critical system reform. The adverse events register could and should be linked to doctors and appropriate threshold levels installed which sequentially trigger information feedback, review, and finally dis-accreditation of the doctor. It is likely that the first of these steps will be sufficient to effect satisfactory change. Despite this, 9 years after publication of the QAHCS the chairman of the ASCQHC notes that \'we have insufficient accurate data for fully appreciating the current size of the multiple causes of this problem \... we need the data from multiple sources, including incident monitoring systems, routine administration data sources and the use of screening tools to practically identify areas that may cause harm.\' (p5) \[[@B34]\]. ***Protection from litigation***The published research on \'high reliability organizations\' suggests that it is wise to separate inquisitorial from punishment processes, such as dis-accreditation. Adverse events are unlikely to be reported if there is a financial incentive to hide the AE. For this reason legislative protection of doctors from the financial outcome of litigation is probably a prerequisite for a successful and comprehensive system of error learning. The consequences for a doctor associated with AE should be based upon medical criteria and uncoupled from the social mechanism for compensating patients. Legal protection alone is unlikely to improve the quality of the information used for error learning. Rather, it should be part of a package of requirements which includes penalties for the failure to report an AE. ***Information transfer***Patient notes are still transferred within hospitals using 19th Century clipboards. It is known that this commonly causes potentially lethal errors. The mandated use of (long available) electronic forms of transmission could alert staff to the risk of inappropriate procedures, the administration of conflicting drugs or the failure to administer a drug. Likewise X-ray films are often misplaced or lost. The consequences may be lethal. Legislation could ensure the use of digital technology to ensure immediate access of results. New wireless technologies make it possible for roving staff -- doctors and other professionals -- to have constant access to text and basic technical data. As with electronic note taking this might take 1 to 2 years to fully install in all Australian hospitals, clinics and nursing homes. ***Hospital systems***Hospital systems in Australia are commonly ramshackle or antique. There are no required pathways or mandatory discharge criteria. There are no internal or external financial incentives for the optimal treatment of patients. These changes are more complex but could be installed comfortably in 4--6 years. ***Conclusion 17:**There is no reason why much of the health system should have missed the IT revolution which has transformed other parts of the community. In relation to the size of the AE problem, the cost of implementing late 20^th^Century information technology throughout the health system is likely to be small relative to the human and financial cost of AEs averted.* ***Minimum staffing requirements***There is no regulation which links on site expertise and the complexity or riskiness of the procedures which may be undertaken in a hospital. For example, it is possible for a hospital to permit significant surgery but have no on-site medical practitioner post-operatively. This potentially lethal practice could be proscribed and minimum staffing ratios implemented within the 1--2 weeks needed to reschedule staff or the location of procedures. It was not until 2003 that the ACSQHC released a paper considering issues of staff rostering, skill mix, staff numbers, staff supervision and team functioning \[[@B33]\]. While endorsing the AMA (voluntary) code of practice, \[[@B37]\] it comments -- almost 8 years after the QAHCS -- that \'responsibility for improving the management of staffing variables cannot (ie should not but still is being) left to individuals. It is a governance responsibility\...\' pii (words in brackets added)\] \[[@B38]\]. ***Queuing***The airline industry operates a highly efficient computerised system of booking and queuing which may be accessed by travel agents throughout the world. By this standard most hospital queuing systems -- if they exist -- are rudimentary. It is possible and desirable for different hospitals in the public hospital system to be interconnected to provide patients or their doctors with available times for treatment city, state or nation-wide. Queuing and scheduling can and should be operated using publicly known criteria. In the USA there is a nation-wide system for matching patients with available organs. (There is, interestingly a prioritisation criterion which, for reasons of equity, assigns compatible organs on the basis of need, not prognosis.) Australia has no such system. ***Conclusion 18:**Queuing should be regulated by a nation-wide prioritising system based upon explicit criteria. This would increase efficiency and, for the patient, increase choice and certainty.* The private sector would have greater difficulty in operating such a system because of the patient\'s attachment to a particular doctor. Private health insurance organisations could, however, offer a similar service to patients who are willing to accept treatment from contracted providers. Australia\'s technologically conservative PHI organisations appear uninterested in these initiatives. ***Information and system audit***The suggestions above and, to date, the majority of the reforms contemplated in reports, represent process measures of success. However, their objective is to reduce adverse errors and for this reason, record analysis of the form conducted by the QAHCS should be an ongoing feature of the system. The QAHCS research was relatively expensive, but these costs are infinitesimal in relation to the importance of the surveillance, the costs, the morbidity and deaths averted. ***Conclusion 19:**A policy of persuasion and culture change is an insufficient response to a problem of the magnitude of the AE epidemic. As a matter of highest priority, legislation should be passed requiring extensive system regulation and reform to reduce the incidence of adverse events. The required changes should be fully funded (or they will not occur) and should include the use of state of the art IT, minimum staffing requirements and system feedback on both individual hospital and individual doctor performance. Evaluation and random audit of all hospitals and health care providers and the reporting of all AEs should be mandatory. Doctors should be given comprehensive protection against the results of successful litigation. The review of doctor performance should be uncoupled from compensation for the patient.* ***Public information***There is no legitimate reason for information relating to hospitals and individual doctors to be withheld from the public. There is a persuasive argument that the public has a right to such information. Additionally public awareness and the likely response from the public to such information are likely to accelerate the pace of reform. There are persuasive reasons for providing information more generally concerning the performance of all parts of the system, including individual care providers. Patient choice in the absence of information is a charade. Failure to forewarn patients that the hospital of their (doctors\') choice has a substandard safety record and that capacity exists in hospitals with a better record could, and arguably should, be regarded as professional negligence. There can be little doubt that, if consulted, the public would overwhelmingly endorse the need for this information. More generally, the provision of information is an effective method for effecting change and it is unlikely that the pace of reform would have been as sedate if the public were properly aware of the safety record of various health care providers. One argument against this option is that the provision of information might result in a loss of confidence in hospitals and doctors. However, the argument that the public should be kept in ignorance to engender unjustified confidence is, at best, dubious and if this ignorance allows an inadequate policy response then it is additionally harmful. In some states of the USA, and most notably New York, severity adjusted mortality rates are available for every hospital and for every doctor. This has not resulted in a significant change in the pattern for public demand but it has galvanised doctors and hospital staff to successfully review and upgrade their procedures \[[@B12]\]. League tables have recently been introduced in England to allow doctors and patients to evaluate the performance of particular hospitals \[[@B39]\]. From late 2004 the performance of individual surgeons will probably be available \[[@B40]\]. ***Conclusion 20:**Information is a key element in achieving system reform and for the efficient operation of all markets, private or public. All institutions and individuals should receive rapid feedback on their performance. Information should be routinely collected and also obtained from random audit of hospitals. In particular, information should be publicly available with respect to the safety record of hospitals and providers of medical care.* ***Financial incentives***As discussed earlier, financial incentives are one some of the most effective, non-coercive ways of achieving desired outcomes. There has been very limited use of this powerful instrument and the financing of medical services has generally been perceived as a reward for providers doing what they select to do rather than as an opportunity for influencing what is done. This is an important missed opportunity. Importantly, financial incentives are non-coercive and avoid the head-to-head conflict between \'clinical autonomy\' and the \'patient\'s right to evidence based medicine\' which may accompany direct regulation. ***Conclusion 21:**Financial incentives should be used flexibly throughout the health system to induce behaviour which minimise AEs.* ***Governance and ownership of the problem***A key theme of this paper has been that government policy has focused almost exclusively upon financial issues and that there has been little concern from within the legislature with health services and health. This is possibly the root cause of the government failure. Activities from within the various bureaucracies have largely been bureaucratic. In its 2004 review of State initiatives the ACSQHC reports that Queensland now has \'a program to develop a workforce culture that values a multi-disciplinary evidence based approach to improvement\'; in 2003 the NT implemented an \'overarching quality committee\'; NSW had \'involved the appointment of patient safety managers\'. The ACT now has a framework which \'provides explicit lines of accountably\'; Victoria now \'has a policy of an open and transparent approach to the provision of information\' etc (p8) \[[@B34]\]. There is no reference to national legislative action to enforce safety. The ACSQHC itself appears frustrated with the scale of the national effort when it comments that \'over the last year with public failures reported in some Australian hospitals it is clear that a small program of national investment and development needs to be matched by the will, skill and capacity of stakeholders\....\' (p5). On the same page it comments that \'Council is actively working to further develop data sources, but cannot do so without continued support by all governments\' (p5). Almost 10 years after the 1995 report the ACSQHC comments that \'future work \... needs to consider what governance and responsibilities are required at all levels of the health care system to ensure the provision of safe health care\' (p84). ***Conclusion 22:**Despite the unnecessary death of between 40,000 and 80,000 Australians since the publication of the QAHCS, Australian government has not taken ownership of the problem of adverse events. As a result, those attempting to effect change have been forced to \'coax and cajole\' Options to \'mandate and enforce\' have been largely ignored.* Disregard of evidence --------------------- The last conclusion relates to the need to generate and disseminate information relating to system safety. The arguments for this apply more generally to health system data. Australia has a wealth of administrative databases which could be employed to investigate and improve system performance. As noted earlier, service use is very uneven across Australia. To the extent that this violates the usual notion of \'equity\' this information should be an important input into policy formulation. It does not appear to be used for this purpose outside NSW. However the quality of the data also presents other opportunities. The effect of different treatments, such as the varying rates of angiography and revascularisation observed between public and private hospitals could be traced through time if record linkage were possible. This would allow an assessment of downstream costs, mortality and morbidity associated with the two patient groups. There are clearly many opportunities for longitudinal research of this sort. A further option which may be piggy-backed on administrative data is the routine provision of information to different groups of patients who have been identified by their service mix. Information of this sort is probably a highly effective way of \'empowering patients\'; that is, enabling patients, and particularly those with a chronic disease, to take greater control of their disease management. These developments have not occurred for a variety of reasons. First, for an \$80,000 million industry, research funding for \'product development and marketing\' -- health services research -- is astonishingly small. In the USA six Federal agencies alone spent \$US 1,658 million in 2002 upon HSR. The agencies and their expenditures are detailed in Table [7](#T7){ref-type="table"}. Significant US funding is also obtained form the US network of Foundations which does not exist in Australia. Benchmarking against the Federal agencies alone, at an exchange rate of \$US 0.70 = \$AUS 1.0 (0.65) and scaling these expenditures down in relation to the size of the US and Australian economies, Australia should be spending about \$AUS 120 million on HSR. Australia does not currently spend a fraction of this amount. As a major initiative, the NHMRC is to provide \$10 million per annum for HSR -- or about 7.7 percent of the US Federal benchmark. ::: {#T7 .table-wrap} Table 7 ::: {.caption} ###### US expenditure upon Health Services Research ::: **Federal Agency** **Expenditure (\$US millions)** --------------------------------------------- --------------------------------- Agency for Health Care Research and Quality 300 National Centers for Health Statistics 127 Extra Mural Prevention Research CDC 18 Centers for Medicare and Medicaid Services 55 Veteran\'s Health Administration 371 National Institute of Health 787 Source: Annual reports ::: A second and possibly related reason is that there is no dedicated instrumentality, similar to the AIHW, which has taken \'ownership\' of the need to provide and periodically to review the need for information generated by HSR. Funding is currently inadequate but also ad hoc. Third, concern over the confidentiality of records has been elevated to such a level that easy and routine data linkage to observe the outcome of different service patterns does not seem to be a possibility. For example, access to Australia-wide, de-identified public hospital records requires the separate consent of all States and Territories as well as the cooperation of the Commonwealth Department of Health or AIHW. Data linkage to determine the consequences of different treatment patterns -- who lives and who dies -- is so difficult that the research is effectively proscribed. It is extremely doubtful that this concern in the bureaucracy over privacy would reflect the preferences of a well-informed population. Patients almost certainly suffer and die because of the interpretation and implementation of our confidentiality laws in a way which seriously inhibits the capacity of the public, researchers and private and public agencies to investigate the outcome of system performance and differences in individual treatments. The bureaucratic fetish with confidentiality does not occur in the USA where the risk of litigation is significantly greater than in Australia. In some states data relating to hospital and doctor mortality rates are regularly published and in California unit record hospital data is available on CD in university libraries. ***Conclusion 23:**Routinely collected administrative data should be fully used to monitor system performance. In particular it should be employed to monitor equity of access to services regularly and to provide disease-related information to population groups identified as having particular needs and interests. A statutorily independent national institute for health services research should be established whose terms of reference require the achievement of these objectives.* 5 Discussion and conclusion =========================== There are various options for the macro reform of the health system and the corresponding reform of financial incentives and the roles and responsibilities of the various players. In particular, Dick Scotton has cogently argued for the adoption of Managed Competition \[[@B41]-[@B43]\]. A partial movement in this direction could be achieved by transferring responsibility for the purchase of health services to the various health regions \[[@B44]\]. There has been no attempt to review this large topic here. Rather, the paper has reviewed the \'micro\' elements of such reform. The most appropriate \'macro\' model for the health system is the model which maximises the likelihood of implementing satisfactory solutions to the numerous problems facing the system including those that have been discussed above. The chief conclusion from this paper is that the \'health care debate\' and recent policy \'reform\' has focussed upon issues which are best described as \'very small order\' as judged by their likely effect upon either health or the cost effectiveness of the health system. They have been primarily concerned with dollars, not health and, more specifically, with the distribution of the cost between the public and private sectors. None of the policies discussed in Section 3 of the paper is likely to increase cost effectiveness. Cost shifting from the PBS to the public creates differential copayments which encourages allocative inefficiency. PHI reforms have created an industry with bizarre financial incentives and is a spectacular example of negative micro economic reform. The common feature of the three policies is that each moves the health system in a direction which is more consistent with the liberal/libertarian world view in which responsibility is transferred to the individual and away from the community. While PHI helps the individual to avoid financial decisions at the point of service, the individual is responsible for the purchase of the insurance and for the payment of (net) premiums. Copayments are the most direct method for shifting responsibility to the individual users of health services. Bulk billing for pensioners/health care card holders preserves the safety net which is needed for the achievement of equity as commonly defined by this world view. Over the longer term the pursuit of these values would redistribute income to the healthy, wealthy and away from the unhealthy unwealthy which is the antithesis of the communitarian/solidarity value system. The vehicle for the transfer is both a decline in community financed expenditures and a corresponding decline in taxation. As discussed in Section 2, decision making with respect to social objectives is the legitimate role of government. However, good economic and social policy seeks to achieve these objectives in a way that is cost effective. The reforms discussed here have not achieved this. In contrast with these policies, the five neglected areas discussed in Section 4 deal with problems which are \'large order issues\' as judged by their likely impact upon health and the cost effectiveness of the health system. Also contrasting with the first group, these issues are relatively \'value neutral\' as judged by either the communitarian or libertarian perspectives. The failure to address satisfactorily these issues is attributable to neglect, not the dominance of a particular ideology. This failure over a very long period itself refects a failure in the governance structure and a failure to identify and act upon opportunities for quantitatively large system improvements. The failure is probably replicated in most other developed countries, reflecting the complexity of the health sector. But benchmarking against similarly impaired systems does not alter the fact that there are opportunities for significantly improving the community\'s health which have been largely ignored. The reasons why this has occurred has not been discussed here and, to a greater or lesser extent, these failures have probably been replicated in most other developed countries, reflecting similar social and technical histories. Competing interests =================== The author(s) declare that they have no competing interests. Acknowledgements ================ The author would like to acknowledge the very helpful suggestions and corrections made by two anonymous reviewers.

PubMed Central

2024-06-05T03:55:52.472431

2005-1-11

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548139/", "journal": "Aust New Zealand Health Policy. 2005 Jan 11; 2:1", "authors": [ { "first": "Jeff RJ", "last": "Richardson" } ] }

PMC548140

Background ========== Oxidative stress is an important factor of acute lung injury. Prolonged exposure to high concentrations of oxygen (hyperoxia) during mechanical ventilation represents a life-saving intervention for critically ill patients. However, it also induces oxidative stress to the lung. The development of therapeutic strategies, aiming to prevent lung injury depends on a better understanding of the underlying pathways of hyperoxia-induced pulmonary damage. Severe, long lasting hyperoxia causes an inflammatory reaction with an influx of inflammatory cells, cell proliferation and hypertrophy, an increase of cytokines, apoptotic activity and subsequent morphologic evidence of lung injury \[[@B1]\]. The first 24 to 48 hrs of oxygen exposure constitute the initiation phase of the pulmonary oxygen toxicity \[[@B1]\]. Even though no morphologic injury has been described during this phase, several changes occur due to the hyperoxic exposure. Highly reactive oxygen species are likely to cause lipid peroxidation, protein and DNA modification that will further cell injury \[[@B2],[@B3]\]. On the other hand, antioxidant enzymes are also induced and may counteract the oxidative stress \[[@B4]-[@B6]\]. Perkowski et al. analysed more then 8700 genes during the early response (0 up to 48 hrs) to hyperoxia in total lung of mice. Out of 385 genes in the lung, 175 showed an increased and 210 a decreased expression \[[@B6]\]. These results indicate that the initiation phase of hyperoxic-induced lung injury already marks a very complex process that is still poorly understood. From previous investigations it may be concluded that in response to oxidative stress, the number of endothelial cells strongly decreases in the post-initiation phase, whereas epithelial cells seem to be relative resistant to oxidative stress \[[@B1],[@B7]\]. In contrast, it has also been shown that in response to hyperoxic ventilation \[[@B8]\], emphysema \[[@B9]\], activation of the Fas/FasL system \[[@B10]\], exposure to donors of nitric oxide or hydrogen peroxide \[[@B11]\], hyperoxia and nitric oxide \[[@B12]\], respiratory distress syndrome \[[@B13]\], and hyperoxia-mediated increase of total lung p53 protein expression \[[@B14]\] alveolar type II cells (TIIcells) are severely damaged, culminating in apoptotic death. TIIcells are functionally highly important epithelial lung cells. They are responsible for the metabolism of alveolar surfactant, serve as progenitor cells of type I pneumocytes, and take part in the inflammatory response of the lung \[[@B15]-[@B17]\]. Thus, damage and apoptotic elimination of TIIcells will severely alter pulmonary function. Following the concept that hyperoxic lung injury is a continuous process, we assumed that appropriate metabolic changes of TIIcells start during the initiation phase. This would then, in response to longer lasting severe hyperoxia or an additional stress, merge in apoptosis in the post-initiation phase \[[@B18]\]. Factors which induce such pro-apoptotic sensitisation of TIIcells in the initiation phase are yet unknown. Elevation of tumour necrosis factor α (TNFα) represents one of the first pulmonary responses to hyperoxia. Pre-treatment of animals with antibodies directed against TNFα reduces hyperoxia-induced lung injury, strongly suggesting a causal relationship between TNFα and hyperoxic lung \[[@B19]\]. TNFα is a classic regulator of cell death by apoptosis or necrosis \[[@B20],[@B21]\]. Cellular response to TNFα is mediated by TNF receptor type I and type II (TNFRI and TNFRII; \[[@B22]\]). Pryhuber et al. \[[@B23]\] studied the contribution of both, TNFRI and TNFRII to hyperoxia-induced lung injury and found that the average length of early survival under hyperoxic conditions is significantly improved in mice that lack the TNFRI (-/-), when compared with wild type or TNFRII (-/-) mice, respectively. However, the blockade of the TNFα receptor function does not protect against pulmonary inflammation and toxicity induced by prolonged hyperoxia \[[@B23]\]. In fact, during prolonged hyperoxia severe lung injury is most likely initiated by additional factors beside TNFα, as the inhibition of TNF receptors does not further affect oxygen-induced mortality \[[@B23]\]. Thus, TNFα and signal transduction via TNFRI seems to be responsible for metabolic changes that regulate the length of survival under short term hyperoxia. In this paper, we tested the hypothesis that TNFα activates caspases in the initiation phase of pulmonary oxygen toxicity in TIIcells without a significant increase in apoptosis. Since apoptosis is modulated by cell-matrix and cell-cell interactions in cultured TIIcells isolated from animals with acute lung injury \[[@B24],[@B25]\]. we used freshly isolated TIIcells for our study. Materials and methods ===================== Hyperoxia --------- Wistar rats (body wt 120 g), each in an individual plastic chamber were continuously gassed with 100 % oxygen for 48 hours. Water and food was available *ad libitum*. Preparation of the bronchoalveolar lavage, alveolar macrophages and TIIcells was carried out as previously described \[[@B26]\]. Immunohistochemistry -------------------- Immunohistochemistry and microscopy were carried out as previously described \[[@B26]\]. The following antibodies were used: Rabbit polyclonal anti-rat TNFα antibody from Biosource Europe (Nivelles, Belgium), rabbit polyclonal anti TNFRI antibody raised against a recombinant peptide (amino acids 30--301) including the extracellular domain of TNFRI (Santa Cruz Biotechnology, Heidelberg, Germany), and anti-active caspase 3 polyclonal antibody was from Promega (Mannheim, Germany). Secondary antibodies conjugated with Alexa 499 and Alexa 594 were from Molecular Probes Europe BV (Leiden, Netherlands). Lung tissue sections were labelled with specific antibodies directed against TNFRI, TNFα, caspase 3, and p180 \[[@B27]\], an integral lamellar body-limiting membrane protein (clone 3C9, Covance/Berkeley Antibody, Richmond, CA, USA). For double staining, the labelled preparations were analysed using a confocal laser scanning microscope (CLSM, Leica Microsystems AG, Wetzlar, Germany), equipped with an argon/krypton laser. Images were taken using a 40 × NA 1.3 oil objective to fluorescent excitation and emission spectra for Alexa 488 (excitation 490 nm, emission 520 nm) and for Alexa 594 (excitation 541 nm, emission 572 nm). With the dual-channel system of the confocal microscope, dual-emission (535/590 nm) images were recorded simultaneously with a scanning speed at 16 s/frame (512 lines). Images were obtained and processed using TCS NT Version 1.5.451 (Leica Microsystems AG, Wetzlar, Germany). As controls, the tissue slides were incubated with the Alexa-labelled second antibodies only. No unspecific binding of the second antibodies occurred (results not shown). For threefold staining (Figure [6](#F6){ref-type="fig"}), fixed lung tissue and freshly isolated TIIcells were incubated in 0.01 M phosphate buffered saline containing 1% (w/v) BSA and 0.3% (w/v) Triton X-100 for 1 hr at room temperature (RT). Detection of lamellar bodies and active Caspase 3 was achieved by incubation with mab 3C9 (20 hrs at 4°C) followed by Alexa 488-labelled goat anti-mouse IgG (2 hrs at RT) and with anti-active caspase 3 followed by Alexa 594-labelled goat anti-rabbit IgG (2 hrs at RT), respectively. Nuclear DNA was stained with 4\',6-diamidino-2-phenylindole (DAPI; Molecular Probes Europe BV, Leiden, Netherlands) for 20 minutes at RT. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Hyperoxia activates caspase 3 in TIIcells. Rats were kept normoxic (control) or subjected to hyperoxia. Lung sections (A) and freshly isolated TIIcells (B) were immunohistochemically threefold stained as described in *Materials and Methods*. Cell nuclei stained light blue (DAPI). TIIcells are distinguishable by the close proximity of their nuclei to green stained lamellar bodies (A; arrows). Active caspase 3 appears red labelled and is predominantly found in the cytosol of TIIcells upon hyperoxic treatment of rats (A and B). Bar 10 μm; ::: ![](1465-9921-6-10-6) ::: Laser scanning confocal microscopy was performed using a ZEISS LSM 510 system with Axiovert microscope (Carl Zeiss Jena GmbH, Jena, Germany) with 40×/1.3 Oil Dic or 63×/1.4 Oil Dic objective, equipped with an argon, helium/neon and violet laser set to 488, 543 and 405 nm, respectively. The multitrack standard FITC/Rhodamine/DAPI configuration was selected. Determination of apoptosis in lung tissue ----------------------------------------- ### TUNEL reaction Sections of rat lung were prepared as described \[[@B26]\]. After deparaffinization and proteinase K-treatment, apoptotic cuts of chromatin DNA were specifically detected by nick end labelling of 3\'-OH DNA ends with fluorescein-dUTP using terminal deoxynucleotidyl transferase. (MEBSTAIN Apoptosis Kit Direct, MBL, Naka-ku Nagoya, Japan). Sections were analyzed using a confocal laser scanning microscope as described above for double staining. Determination of apoptosis in freshly isolated TIIcells ------------------------------------------------------- ### Cell Death Detection ELISA Cytoplasmic histone-DNA fragments were quantified using the Cell Death Detection ELISA (Roche, Mannheim, Germany). ### Flow cytometry We used the TACS™ Annexin V-FITC Detection kit (R&D Systems, Wiesbaden, Germany) to quantify the population of early and late apoptotic cells in percent of total cells. The tests were performed according to the protocols of the manufactures. Determination of caspase activities ----------------------------------- Activities of caspases 3, 8 and 9 were determined in the lysates of 8 × 10^6^TIIcells for each group and for each caspase with the Colorimetric assays from R&D Systems Inc. (Wiesbaden, Germany). When the pro-caspases had to be determined, an aliquot of the lysate (corresponding to 2 × 106 cells) was preincubated with 0.1 μg granzyme (Calbiochem, Bad Soden, Germany; dissolved in 5 μl 0.9% NaCl) for 30 min at 37°C. Determination of NF-κB activation --------------------------------- ### Immunocytochemistry Activation of NF-κB was measured by immunocytochemistry using a monoclonal anti-NF-κB-antibody (MAB3026, Chemicon International, Temecula, USA), that recognises an epitope which includes the nuclear location signal of p65, the DNA binding subunit mainly responsible for the strong gene-inductory potential of NF-κB. Thus, only the activated form of NF-κB was measured. The semiquantitative estimation of NF-κB subunit by confocal microscopy was carried out as recently described in detail \[[@B28]\]. ### Immunoblotting Translocation of NF-κB to the nucleus was assessed as described by Li et al. by immunoblotting of nuclear extracts using a rabbit polyclonal antibody (biomol GmbH, Hamburg, Germany) directed against the p65-subunit \[[@B29]\]. ### Electrophoretic mobility shift assay (EMSA) was employed to detect the activated transcription factor NF-κB. Because this method is based on the binding of the transcription factors to their specific DNA recognition sequences, it is highly specific. Labelling of the NF-κB consensus oligonucleotide and handling of the assay were as described by the manufacturer (Gel Shift Assay Systems, Promega GmbH, Mannheim, Germany). Briefly, 50 micrograms of TIIcell nuclear extract were preincubated in reaction buffer for 10 minutes at RT. A \[32P\]-labelled oligonucleotide (Promega GmbH, Mannheim, Germany) which contains DNA binding sites for NF-κB transcription factors was then added to the reaction mixture and incubated for 20 minutes at RT. The complexes were separated on a 4% polyacrylamide gel that was dried and exposed to autoradiography. The specificity of the DNA-binding protein for the putative binding site was established by competition experiments using unlabelled NF-κB consensus oligonucleotide. Intratracheal application of anti-TNFα antibodies ------------------------------------------------- Wistar rats were lightly anaesthetised by inhalation of ether. The rats obtained intratracheally 50 μg goat IgG (PERBIO SCIENCE, Bonn, Germany) or 50 μg goat polyclonal anti-rat TNFα antibody (Santa Cruz Biotechnology, Heidelberg, Germany) per animal, respectively. Thereafter the rats were kept at hyperoxic conditions as described in hyperoxia. After 48 hrs, the TIIcells were isolated and TNFRI expression and caspase-3 activity were determined as described above. Other methods ------------- For the determination of the TNFα concentration in TIIcells, macrophages, plasma or cell-free bronchoalveolar lavage, we used a commercially available ELISA kit from Biosource (Ratingen, Germany). The determination of different apoptotic markers \[[@B18]\], Western blot analysis \[[@B17]\], mRNA isolation and Real-time quantitative PCR reaction for the determination of the expression of mRNAs in TIIcells was described in detail previously \[[@B26]\]. GSH, GSSG and GSH-reductase were determined by HPLC with subsequent fluorescence detection as previously described \[[@B17]\]. Statistical analysis -------------------- Differences between two groups were assessed using the Student\'s t-test. Probability values \< 0.05 (two-tailed) were considered significant (see legends of Tables and Figures). Results ======= Hyperoxia-induced changes in lung tissue ---------------------------------------- To test the general usefulness of our hypothesis, we first characterised the oxygen-induced changes in lung-tissue-sections. Immunohistochemically, we observed a clear increase in TNFα, TNFRI, and caspase 3 activities in lung tissue of hyperoxic rats in relation to normoxic animals in the initiation phase (Figure [1](#F1){ref-type="fig"}). Albeit the increase of these pro-apoptotic parameters in lung tissue corroborated our hypothesis, this approach does not allow to identify the participating cell types. Furthermore, we tested lung sections for DNA-degradation products using the TUNEL reaction (Figure [2](#F2){ref-type="fig"}). Here, TIIcells are distinguishable from other cells of the lung by their content of lamellar bodies. Lamellar bodies were immunohistochemically labelled in lung sections with an antibody directed against the 180-kDa lamellar body-limiting membrane protein (red, Figure [2](#F2){ref-type="fig"}). Upon hyperoxia, we found sporadically a fragmentation of DNA (green, Figure [2](#F2){ref-type="fig"}), but no co-localisation of the 180-kDa lamellar body-limiting membrane protein and DNA-fragments. The intensity of red lamellar body stain increased in lung sections from hyperoxic rats. This is in good accordance with previously published electron micrographs showing swollen and deformed lamellar bodies after hyperoxia \[[@B30]\]. From these results, we conclude in agreement with the literature \[[@B1],[@B7],[@B25]\]. that sublethal hyperoxia of rats did not induce apoptosis in TIIcells *in vivo*; at least not in the initiation phase. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Hyperoxia increases the expression of TNFRI, TNFα and caspase 3 in vivo. Lung sections of normoxic (control) and hyperoxic rats were prepared as described in *Materials and Methods*and were labelled by single immunofluorescence with antibodies directed against TNFRI, TNFα or active caspase 3 ::: ![](1465-9921-6-10-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### No significant apoptosis was found in TIIcells upon hyperoxia in vivo for 48 hrs. Lung tissue of normoxic rats (left) and of hyperoxic rats (right) were tested for DNA fragmentation by TUNEL reaction as described in *Materials and Methods*. The positive TUNEL reaction is represented by green fluorescence. The presence of lamellar bodies is indicated by red fluorescence. Pseudo-colour blue was used to highlight the contours of lung tissue. Bar: 25 μm. ::: ![](1465-9921-6-10-2) ::: The results indicate that apoptotic parameters as are TNFα content, TNFRI expression and caspase-3 activity increase in lung tissue during the initiation phase but do not induce TIIcell apoptosis *in vivo*. Following our hypothesis, we examined whether TIIcells undergo oxidative stress at our conditions, and whether an increase of TNFα, TNFRI expression, and caspase-3 activity appears in isolated TIIcells. Hyperoxia-induced oxidative stress of freshly isolated TIIcells --------------------------------------------------------------- The determination of cellular GSH, oxidised GSH (GSSG), and the activity of the GSH-reductase showed the oxidative burdening of TIIcells. In response to hyperoxia, the GSSG content significantly increased and the GSH reductase activity significantly decreased (Table [1](#T1){ref-type="table"}). The GSH content in freshly isolated TIIcells has rarely been determined. In our TIIcell population, the GSH content differs in relation to previously published data of freshly isolated TIIcells from rat \[[@B31]\] and rabbit \[[@B32]\] by the factor of about 2 and 4, respectively. These differences might be explained by different methods of TIIcell isolation and by species specificity. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of hyperoxia on GSH reductase activity, and on GSH and GSSG content in TIIcells ::: Control Hyperoxia ------------------------------ ---------------- ----------------- GSH reductase (% of control) 100 76 ± 10\* GSH (μmol/mg protein) 0.022 ± 0.015 0.020 ± 0.008 GSSG (μmol/mg protein) 0.005 ± 0.0015 0.013 ± 0.001\* GSH/(GSH+GSSG) (ratio) 0.81 0.61 Activity of glutathione (GSH) reductase and concentration of GSH and GSSG were determined in freshly prepared TIIcells of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) as described in *Materials and Methods*. Values are means ± standard deviation of n = 3 independent experiments. Asterisk indicates a significant difference to control (*p*\< 0.05). ::: The ratio GSH/(GSH+GSSG) is one of the most sensitive parameters to describe oxidative burdening. Our values are comparable to the values found by van Klaveren et al. in freshly isolated type II cells (0.816 versus 0.815) \[[@B30]\]. This ratio decreased in response to hyperoxia in vitro to 0.74 \[[@B31]\] and in our in vivo approach to 0.61 (Table [1](#T1){ref-type="table"}). With respect to the published data, we conclude that our treatment induced oxidative burdening in freshly isolated type II cells. Hyperoxia activates NF-κB ------------------------- NF-κB activation has been described as an indicator of oxidative stress \[[@B33],[@B34]\]. In response to sublethal hyperoxia, the content of activated NF-κB in TIIcells clearly increased (Figure [3A](#F3){ref-type="fig"}). The picture shows that the larger portion of activated NF-κB seems to be localised in cytosol. The semiquantitative determination of activated NF-κB showed a significant increase (control: 12.7 ± 9.0; hyperoxia: 59.5 ± 11.6; n = 12; p \< 0.01) in the nucleus of TIIcells in response to hyperoxia. In the cytosol, there was also an increase of activated NF-κB (1.6-fold), however, this difference curtly fails the level of significance. Additionally, we detected a translocation of the NF-κB-subunit p65 into the nuclear protein fraction as estimated by Western blot analysis (Figure [3B](#F3){ref-type="fig"}). Hyperoxia increased the content of the p65-subunit in the nuclear protein fraction of TIIcells 1.68-fold (SD 0.58, n = 4) compared to normoxic control, but the difference did not reach significance. Whether the immunoreactive band above the p65-subunit in the Western blot of the hyperoxic group is non-specific or a possible post-translational modification can not be decided. In Figure [3C](#F3){ref-type="fig"} we confirm the hyperoxia induced activation of NF-κB by EMSA of the nuclear protein fractions of TIIcells freshly isolated from control (lanes 1, 2) and hyperoxic (lanes 3, 4) rats. Addition of unlabelled specific oligonucleotide clearly competes with the \[32P\]-labelled probe confirming the specificity of the bands. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Activation of NF-κB in TIIcells and its enrichment in the nuclear protein fraction after hyperoxic treatment of rats. Freshly isolated TIIcells from normoxic (control) and hyperoxic rats were prepared for immunocytochemistry, SDS-PAGE and immunoblotting as described in *Materials and Methods*. A, activation of NF-κB was measured by immunocytochemistry using a monoclonal anti-NF-κB-antibody overlapping the nuclear localisation signal of the p65 subunit in the NF-κB heterodimer. Activated NF-κB in the nucleus and cytosol was quantified as described recently in detail \[28\]. The signal of activated NF-κB in the nucleus increased 4.7 fold in respose to hyperoxia (n = 12; p \< 0.05). Bar: 10 μm. B, the nuclear protein fraction \[52\] of TIIcells was subjected to SDS-PAGE and immunoblotting. The p65 subunit of NF-κB was visualised using a rabbit polyclonal antibody, and its expression was densitometrically estimated. Values of n = 4 independent experiments are given as mean ± SD in arbitrary units (control = 1). C, Electrophoretic mobility shift assay for NF-κB in freshly isolated TIIcells from normoxic (lanes 1, 2) and hyperoxic (lanes 3, 4) rats. Signal competition upon addition of unlabelled oligonucleotide (lanes 2, 4). ::: ![](1465-9921-6-10-3) ::: Hyperoxia increases synthesis and secretion of TNFα by TIIcells --------------------------------------------------------------- The concentration of TNFα significantly increased in response to hyperoxia of rats in plasma, alveolar fluid, lung macrophages and TIIcells (Table [2](#T2){ref-type="table"}) as determined by ELISA. Flow cytometric analysis of the TNFα content in freshly isolated TIIcell preparations from control and hyperoxic rats confirmed the increase of the TNFα concentration in macrophages and TIIcells. In response to hyperoxia, TNFα increased in TIIcells 1.45-fold and in macrophages 1.87-fold compared to control. In TIIcells, TNFα seems to be localised in lamellar bodies (Figure [4](#F4){ref-type="fig"}), whereas cytoplasmic caspase 3 showed no co-localisation with lamellar bodies as expected. The latter result attaches value to the histochemically detected localisation of TNFα in lamellar bodies, because it is unlikely an artefact. The spontaneous secretion of TNFα by TIIcells significantly increased in response to hyperoxia (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Effect of hyperoxia on the TNFα content in different specimen from rat ::: Control Hyperoxia ------------------------------------------------------------------------------ ------------ -------------- Plasma (pg/ml) 106 ± 31 149 ± 11 Macrophages (ng/mg protein) 11.4 ± 3.7 19.4 ± 2.8\* TIIcells (ng/mg protein; n = 6) 18.5 ± 2.1 27.2 ± 6.7\* Bronchoalveolar lavage (pg/ml) 109 ± 1 172 ± 38\* Spontaneous secretion of TNFα by TIIcells (ng × mg cell protein^-1^× hr^-1^) 6.3 ± 1.3 21.2 ± 7.5\* Concentration of TNFα was determined in plasma, macrophages, TIIcells and bronchoalveolar lavage of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) by ELISA as described in *Materials and Methods*. Spontaneous secretion of TNFα was measured in cell-free supernatant upon incubation of freshly isolated TIIcells in DMEM for 30 min at 37°C. TNFα concentrations are given as means with standard deviation of n = 3 independent experiments unless stated otherwise. Asterisk indicates a significant difference to control (*p*\< 0.05). ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### TNFα is localised in lamellar bodies and caspase 3 in the cytosol of TIIcells. After hyperoxia, rat lungs were fixed and the sections were immunohistochemically double labelled as described in *Materials and Methods*. A: bar 50 μm; B: higher magnification of the indicated area of A (arrow); bar 10 μm. By confocal microscopy, TIIcells were identified by the green labelling of lamellar bodies (see Methods). The red labelled TNFα appears yellow (arrow in B) when co-localised in lamellar bodies as shown in two TIIcells in B. ::: ![](1465-9921-6-10-4) ::: Hyperoxia induces the expression of TNFRI and activates caspases in TIIcells ---------------------------------------------------------------------------- Hyperoxia induced an significant increase of TNFRI on TIIcells (4.9-fold ± 2.7, n = 6), whereas the expression of Fas, a member of the same receptor family, did not change (Figure [5](#F5){ref-type="fig"}). TNFRI-mediated action of TNFα depends on the so called \"death domain\" representing a part of the intracellular segment of the receptor protein responsible for the activation of pro-caspase 8. Caspase 8 in turn can activate pro-caspase 3, a feature previously reviewed \[[@B35]-[@B37]\]. We show *in situ*that the activation of caspase 3 actually occurs in TIIcells and that caspase activation as a response to an unspecific stress, e.g. isolation, can be excluded (Figure [6](#F6){ref-type="fig"}). This staining technique excepts cytoplasm and membranes, thus, cells can hardly be delimited. However, TIIcells are identifiable by the immediate proximity of their nuclei to lamellar bodies (green). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Hyperoxia increases the expression of TNFRI but not of Fas in TIIcells. For the expression analysis of TNFRI and Fas the membrane fraction of freshly isolated TIIcells was prepared. TNFRI (A) and Fas (B) were visualised by Western blot technique, and their expression was densitometrically determined. Values of n = 6 independent experiments are given as means ± SD in arbitrary units (control = 1). Asterisk indicates a significant difference to control (*p*\< 0.05). ::: ![](1465-9921-6-10-5) ::: As shown in Table [3](#T3){ref-type="table"}, in TIIcells the activity of caspase 8 and 3 increased in response to hyperoxia, whereas the activity of caspase 9 did not change. Pre-incubation with granzyme activated pro-caspases and resulted in an increase of caspase 8 and -3 activities in control and hyperoxic TIIcells (Table [4](#T4){ref-type="table"}). However, the activation in control cells clearly exceed that in hyperoxic TIIcells, indicating that pro-caspases were already, at least in part, activated in response to hyperoxia. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Effect of hyperoxia on the activity of caspases in TIIcells ::: Control Hyperoxia ------------------- --------- ------------ Caspase 8 (n = 6) 100 143 ± 14\* Caspase 3 (n = 6) 100 168 ± 23\* Caspase 9 (n = 3) 100 108 ± 11 Caspase activity in freshly prepared TIIcells of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) was determined colorimetrically as described in *Materials and Methods*. Values are means ± S.D. given in arbitrary units (control = 100). Asterisk indicates a significant difference to control (*p*\< 0.05). ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Effect of granzyme-treatment on the activity of caspases in TIIcells from normoxic and hyperoxic rats ::: Control Hyperoxia ----------- --------- ------------ ----- ------------ Caspase 8 100 155 ± 7\* 100 110 ± 4\* Caspase 3 100 216 ± 14\* 100 155 ± 10\* Caspase activity in freshly prepared TIIcells of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) was determined colorimetrically as described in *Materials and Methods*. In some experiments, cell lysates were incubated in the presence of granzyme prior to determination of caspase activity (see *Methods*). Values are means ± s. d. given in arbitrary units (values without granzyme = 100) of n = 3 independent experiments. Asterisk indicates a significant difference between granzyme-treated and untreated samples (*p*\< 0.05). ::: Anti-TNFα in vivo prevents hyperoxia-driven increase in TNFRI and in active caspase 3 ------------------------------------------------------------------------------------- In order to demonstrate the causality between hyperoxia and TNFRI-mediated activation of caspases, we attempted to bind TNFα by anti-TNFα antibodies. Table [5](#T5){ref-type="table"} shows that a single intratracheal application of anti-TNFα antibodies immediately preceding hyperoxic treatment prevents the hyperoxic-induced increase of TNFRI expression and caspase-3 activity in freshly isolated TIIcells. These results indicate that both TNFRI expression and caspase-3 activation were induced by TNFα. This corroborates the concept that in a cascade starting by the TNFα/TNFRI-interaction caspase 8 is activated which in turn activates caspase 3. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Effect of intratracheal application of anti-TNFα antibodies on hyperoxic induced parameters of TIIcells ::: Hyperoxia -------------------- ----------- ----------- TNFRI 100 26 ± 19\* Caspase 3 activity 100 64 ± 5\* Rats obtained intratracheal 50 μg goat-IgG (without) or 50 μg goat polyclonal anti-TNFα antibodies (with anti-TNFα antibody) preceding hyperoxic treatment. Hyperoxia of rats was carried out as described in *Material and Methods*. Values are means ± SD of n = 3 independent experiments given in percent (hyperoxia without anti-TNFα antibody = 100). Determination of TNFRI (Western blot) and of caspase 3 activity (colorimetrically) was as described in *Material and Methods*.). Asterisk indicates a significant difference compared to control (*p*\< 0.05). ::: Hyperoxia upregulates genes of TNFRI, TNFα and caspases 3 and 8 --------------------------------------------------------------- The content of individual mRNAs in TIIcells was determined by Real-time PCR. In agreement with microarray analysis in total lung of mice \[[@B6]\] we found that the amount of GAPDH mRNA did not change in the first 48 hrs of hyperoxia (results not shown). Therefore, GAPDH was used as a house keeping gene. In contrast, Ho et al. found a small but significant increase of GAPDH mRNA in lung tissue \[[@B5]\]. This difference may be caused by different base material (TIIcells versus lung tissue) and methodical differences (Taqman versus densitometry of autoradiographs). In response to sublethal hyperoxia the mRNA content of TNFα, TNFRI and caspases increased (Table [6](#T6){ref-type="table"}). The increment of caspase-8 mRNA was not significant, but even a ΔΔc~t~of -1.15 indicates a 2.2-fold increase of mRNA content. ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Effect of hyperoxia on the mRNA content of TNFα, TNFRI, and caspases in TIIcells ::: number of cycles (Δc~t~) increase of mRNA ----------- -------------------------- ------------------ ------- ----- TNFα 3.66 ± 0.66 0.93 ± 0.30\* -2.73 6.6 TNFRI 5.02 ± 0.19 4.44 ± 0.11\* -0.58 1.5 Caspase 8 10.3 ± 0.89 9.15 ± 0.35 -1.15 2.2 Caspase 3 5.33 ± 0.25 4.29 ± 0.48\* -1.04 2.1 The mRNA of three animals per group was isolated and combined. The content of specific mRNA was determined using Real time-PCR. In parallel, GAPDH was determined in each run as internal standard. The values are given as number of GAPDH-corrected cycle (Δc~t~) ± SD of n = 3 determinations. ΔΔc~t~is the difference of the number of cycles between control and hyperoxia; a decrease of Δc~t~indicates an increase of the mRNA, also given as a factor of increase. Asterisk indicates a significant difference to control (*p*\< 0.05). ::: Hyperoxia of rats does not induce apoptosis in freshly isolated TIIcells ------------------------------------------------------------------------ As mentioned above in this section, no increase of apoptosis was detected in TIIcells in response to sublethal hyperoxia by immunohistochemistry (Figure [2](#F2){ref-type="fig"}). To confirm this result, we analyzed biochemical parameters of apoptosis in freshly isolated TIIcells. In agreement with our immunohistochemical results, sublethal hyperoxia of rats did not increase apoptosis in freshly isolated TIIcells (Table [7](#T7){ref-type="table"}). Hyperoxia was without effect on anti-apoptotic Bcl-2, and cytosolic cytochrome C, although the pro-apoptotic Bax increased and the mitochondrial transmembrane potential slightly decreased (Table [7](#T7){ref-type="table"}). In accordance with these results, the activity of caspase 9 did not change. Therefore, activation of caspase 3 seems to be catalysed by caspase 8. ::: {#T7 .table-wrap} Table 7 ::: {.caption} ###### Effect of hyperoxia on apoptotic parameters in TIIcells ::: Control Hyperoxia ---------------------------------- --------- ------------ Bcl-2 (n = 5) 100 96 ± 8 Bax (n = 5) 100 134 ± 28\* Cytochrome c (n = 5) 100 102 ± 15 Mitochondrial membrane potential 100 81 ± 8\* Early apoptotic TIIcells 100 101 ± 26 Late apoptotic TIIcells 100 94 ± 22 Cell death detection ELISA 100 104 ± 13 Several apoptotic parameters were determined in freshly prepared TIIcells of rats exposed to air (control) or oxygen for 48 hrs (hyperoxia) as described in *Materials and Methods*. Values are means ± S.D. given in arbitrary units (control = 100) of n = 3 independent experiments unless stated otherwise. Asterisk indicates a significant difference to control (*p*\< 0.05). ::: Hyperoxia of rats followed by normoxia reduced caspase 3 activity but did not increase apoptosis in TIIcells ------------------------------------------------------------------------------------------------------------ After hyperoxia for 48 hrs, we detected an activation of caspase 8 and caspase 3. Much to our surprise, we found no increase in apoptosis of TIIcells although the activity of these caspases increased. However, apoptosis might occur later than 48 hrs and will not necessarily arise concomitantly with caspase activation. Thus, following hyperoxia the animals were kept for 24 and 48 hrs under normoxic conditions to test TIIcells for appearance of apoptosis at a later time point. During normoxia, caspase 3 activity gradually decreases compared to control, whereas apoptosis did not change significantly as detected by cell death detection ELISA and by the number of early and late apoptotic cells (Table [8](#T8){ref-type="table"}). ::: {#T8 .table-wrap} Table 8 ::: {.caption} ###### Apoptotic parameters upon sublethal hyperoxia of rats followed by normoxia ::: Hyperoxia followed by normoxia for ---------------------------- ------------------------------------ ---------- ---------- Caspase 3 activity 171 ± 18\* 97 ± 16 73 ± 29 Early apoptotic cells 102 ± 13 95 ± 14 109 ± 26 Late apoptotic cells 106 ± 29 125 ± 25 85 ± 17 Cell death detection ELISA 97 ± 23 104 ± 6 95 ± 14 Activity of caspase 3 and parameters of apoptosis detected in freshly prepared TIIcells of rats exposed to oxygen for 48 hrs (hyperoxia) followed by normoxia for 24 or 48 hrs as described in Materials and Methods. Values are means ± S.D. given in arbitrary units (normoxic control = 100) of n = 3 independent experiments. Asterisk indicates a significant difference compared to control (*p*\< 0.05) ::: Discussion ========== Short-time hyperoxia, as used in this experiments represents the initiation phase of lung injury \[[@B1]\]. We started our investigations with the aim to characterise metabolic changes in TIIcells taking place in this phase. On the one hand, these changes should be less complex than in post-initiation phases. On the other hand, therapeutic interventions to avoid or minimise hyperoxia-induced lung injury should focus in particular on this phase, because morphologic injury of lung tissue, inflammation, and death of lung cells originate from here. Furthermore, the metabolic changes in the initiation phase may, at least in part, be still reversible. For the first time, we show that sublethal hyperoxia causes not only an increase in TNFα concentration in lung tissue (as previously published \[[@B38],[@B39]\]), but also in freshly isolated TIIcells and that this increase is combined with an enhanced expression of TNFRI and an activation of caspase 3. It has been widely assumed that macrophages are the source of TNFα in alveolar fluid \[[@B40]\]. Our findings provide evidence that hyperoxic treatment of rats provokes a rise in cellular TNFα not only in alveolar macrophages \[[@B41]\]. We show that the TNFα-gene is up-regulated in TIIcells; in parallel, the TNFα-protein content increased. To the best of our knowledge, our data suggests for the first time that TNFα is localised in lamellar bodies. In the light of this assumption, the secretion of TNFα as a constituent of the lamellar bodies by TIIcells might represent a significant contribution to the increased TNFα-content in alveolar fluid. Whether the small increase of the TNFα concentration in plasma observed by us indicates a beginning systemic inflammation or rather reflects a transfer of TNFα from the alveolar space to plasma, remains open (Table [2](#T2){ref-type="table"}). Cellular effects of TNFα are mediated mainly by its specific receptor, TNFRI. In agreement with our hypothesis, the expression of TNFRI in TIIcells is up-regulated on mRNA and protein level in response to sublethal hyperoxia, while the Fas-expression did not change, although both receptors belong to the same family (Figure [5](#F5){ref-type="fig"}). It may be speculated that a parallel increase of effector and specific receptor always occurs when the cellular metabolism in TIIcells can be affected by an autocrine mechanism. The increase of Fas/Fas-Ligand might be characteristic in post-initiation phases. The intracellular part of the transmembrane TNFRI protein contains the \"death domain\" which is responsible for the activation of caspase 8. This can trigger the path to the final part of apoptosis via activation of caspase 3 \[[@B36]\]. In line with this, we found enhanced levels of caspases 8 and 3, yet caspase 9 was not activated. The latter result is corroborated by the absence of an increased mitochondrial cytochrome C release. The data presented here demonstrate that sublethal hyperoxia of rats did not induce apoptosis in TIIcells despite of the increase in TNFα content, TNFRI expression, and activation of caspase 3. To check whether this pro-apoptotic state in TIIcells is indeed triggered by TNFα and whether it may be reversible, anti-TNFα antibodies were administered intratracheally prior to hyperoxic exposure. We show that this treatment completely prevented hyperoxic-induced increase of TNFRI expression and caspase 3 activation. It is a widely accepted concept that activation of caspase 3 marks the \"point of no return\" in the pathway of apoptotic death of mammalian cells. However, we found that neither in lung tissue nor in freshly isolated TIIcells apoptosis occurs in response to sublethal hyperoxia despite the significant activation of caspases 8 and 3. In other words, the increase of caspase 8 and 3 in response to sublethal hyperoxia did not mark the \"point of no return\" in TIIcells. This interpretation of our results is strongly corroborated by Perfettini and Kroemer \[[@B37]\]. They summarised that caspase inhibition does not avoid but actually encourage death in TNF induced shock, indicating that caspase activation is not basically synonymous with apoptotic cell death. It could be argued that no apoptosis was found in freshly isolated TIIcells because the increase of caspase 3 activity and the increase of apoptotic parameters does not occur concomitantly. However, we found no increase in apoptosis of TIIcells in a normoxic period of 24 and 48 hrs directly succeeding the hyperoxic treatment of animals, whereas the caspase 3 activity gradually decreased. These results indicate that caspase 3 activation in response to sublethal hyperoxia is reversible and does not kill TIIcells essentially. The reason why the activation of caspases did not induce apoptosis of TIIcells is not clear. On the one hand, activation of NF-κB has often been implicated as an anti-apoptotic event \[[@B42]-[@B45]\], and TNFα induces also the expression of anti-apoptotic TNFα-receptor-associated-factors in lung cells and might inhibit by this way TNFα-induced cell death or apoptosis \[[@B46]\]. Therefore, it may be assumed that both NF-κB activation and expression of anti-apoptotic TNFα-receptor-associated-factors arrest the hyperoxia-induced metabolic changes of TIIcells in the pro-apoptotic state. On the other hand, it can not be excluded that the extend of caspase 3 activation (1.68-fold compared to control) is not high enough to induce apoptosis of TIIcells although a 1.8-fold increase of caspase 3 is combined with apoptosis in total lung of a hyperoxia/pneumonia model \[[@B47]\]. We hypothesise that caspase 3 activation is then followed by apoptosis, when its activation occurs *via*strong mitochondrial damage resulting in cytochrome c release and caspase 9 activation in post-initiation phases of pulmonary oxygen toxicity. This concept is supported by recent findings that mitochondrial cytochrome c release is a key event in hyperoxia-induced lung injury \[[@B48]\]. In fact, the mitochondrial membrane potential and Bax significantly changed in TIIcells in the initiation phase of hyperoxic lung injury, but without cytochrome c release or activation of caspase 9. Recently, it has been shown that in response to severe hyperoxia of mice apoptosis and necrosis contribute to an extensive cell death; p53, bax, bcl-x, and Fas increased in mRNA and protein level, but the activity of caspase 3 and caspase 1 did not change \[[@B49]\]. This independence of apoptosis from caspase activities in the lung has also been shown in freshly isolated TIIcells. Previously, we showed that an increase of TIIcell-apoptosis in response to vitamin E deficiency of rats is independent of caspase activation \[[@B18]\]. Furthermore, using p53-deficient and Fas-null mice, Barazzone et al. \[[@B49]\] showed that Fas and p53 activation exhibits no linkage to lung injury in response to severe hyperoxia of mice. Contrariwise, it has also been shown that Fas activation in vitro \[[@B50]\] and in vivo results in TIIcell apoptosis and lung inflammation \[[@B10],[@B51]\]. These results confirm that hyperoxic-induced lung injury is multifactorial, and that the elimination of one factor in the network of factors activated in the post-initiation phases often can not avoid lung injury in response to severe hyperoxia. In summary, TIIcells were not killed in the initiation phase of pulmonary oxygen toxicity. More precisely, its pro-apoptotic sensitisation is the background that, together with an additional stress factor, hyperoxia causes lung injury probably by apoptotic elimination of TIIcells. In agreement with this idea, we showed that the combination of the stress factors hyperoxia and vitamin E deficiency increases TIIcell apoptosis \[[@B18]\]. Taking into account that premature neonates exhibit vitamin E deficiency, it is consequential that ventilation of premature neonates suffering from respiratory distress syndrome with high levels of inspired oxygen amplifies lung injury, which is associated with TIIcell apoptosis \[[@B13]\]. Conclusions =========== In the initiation phase of pulmonary oxygen toxicity, an increase of TNFalpha and its receptor TNFR1 leads to the activation of caspase 8 and 3 in TIIcells. Together with the hyperoxic induced increase of Bax and the decrease of the mitochondrial membrane potential, activation of caspase 3 can be seen as sensitisation for apoptosis. Eliminating the TNFα effect in vivo by anti-TNFα antibodies prevents the pro-apoptotic sensitisation of TIIcells. List of abbreviations ===================== DAPI -- 4\',6-diamidino-2-phenylindole; EMSA -- electrophoretic mobility shift assay; GSH -- gluthatione; GSSG -- oxidised gluthatione; NF-κB -- nuclear factor-κB; TIIcell -- type II pneumocyte; TNFα -- tumour necrosis factor α; TNFR -- TNFα receptor; TUNEL -- terminal transferase dUTP nick end labelling Authors\' contributions ======================= HW carried out the immunohistochemistry, CS performed the mRNA analysis, AT carried out the protein measurements and participated in data analysis, MR applicated anti TNFα antibodies intratracheally, FS participated in the mRNA analysis and the design of the study, FG and BR conceived of the study, participated in its design and co-ordination, analysed the data and wrote the manuscript. All authors read and approved the final manuscript.

PubMed Central

2024-06-05T03:55:52.482954

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548140/", "journal": "Respir Res. 2005 Jan 21; 6(1):10", "authors": [ { "first": "Florian", "last": "Guthmann" }, { "first": "Heide", "last": "Wissel" }, { "first": "Christian", "last": "Schachtrup" }, { "first": "Angelika", "last": "Tölle" }, { "first": "Mario", "last": "Rüdiger" }, { "first": "Friedrich", "last": "Spener" }, { "first": "Bernd", "last": "Rüstow" } ] }

PMC548141

Background ========== Most, if not all, forms of chronic obstruction pulmonary disease (COPD) as well as asthma begin in the small airways. While the pathogenesis of small airway diseases is poorly understood \[[@B1],[@B2]\], it is generally accepted that the fluid and electrolyte transport properties of the epithelia lining these peripheral bronchioles play a crucial role in maintaining normal airway hygiene and patency. Some argue that these fluids are the primary defense because coupled with the ciliated escalator they form the first mechanism for clearing the airway of foreign debris and noxious agents. At the same time, almost nothing is known with certainty about the transport properties of distal airway epithelia or how fluid movements help maintain hygiene. No doubt, the paucity of understanding is due to the inaccessibility and the fragility of the tissue. Most concepts of the mechanisms and functions at this level have been taken from findings in the upper respiratory tract or from the larger cartilaginous ringed structures of the trachea and bronchi \[[@B3]-[@B7]\]. More extrapolations have been made from primary cultures of the same sources \[[@B8],[@B9]\]. Two previously published attempts were made to measure electrolyte transport parameters in isolated segments of small airways dissected from the peripheral airways of sheep \[[@B10]-[@B12]\] and pigs \[[@B13],[@B14]\]. However, in these studies the electrical signals, reflecting underlying transport properties may have been severely muted by tissue trauma during dissection and preparation. For standard electrophysiological studies of epithelia, dissection of the bronchiole would seem mandatory in order to maintain control of solutions on both sides of the epithelium. In order to minimize trauma, however, we attempted to microperfuse small bronchioles (i.d. 0.5--0.8 mm) in the periphery of pig lung without dissection. Unfortunately, since the bronchioles are embedded in a parenchyma of bronchioli and alveoli, this approach sacrifices control of the contra-luminal solution. Nonetheless, under this condition, we now find striking improvements in electrophysiological responses and strong evidence of a highly Cl^-^selective conductance that dominates the electroconductive properties of this epithelium, that is most probably duo to CFTR. Methods ======= Tissue ------ Lungs were excised intact immediately after sacrifice of young pigs (30--60 kg). Lungs were maintained inflated through a ligated plastic tube connected to an aquarium air pump (\~1 L/min) to maintain a positive airway pressure of 10--14 cm-H~2~O. The assembly was wrapped in a plastic bag and transported from the abattoir to the laboratory (\<60 min) in an insulated box chilled with ice. In the laboratory, small pieces of about 0.5 cm^3^were cut from the peripheral lung parenchyma, usually from along the costal diaphragmatic ridge of the lower lobes. In general, the freshest tissue gave the best responses although some tissue responded well after 6--8 hours of storage in a cooled environment. Microperfusion -------------- Under a dissecting microscope, the opening to a small airway was visualized on the proximal cut surface of a small block of lung tissue. The airway opening was then cannulated with a system of two concentric micropipettes \[[@B15]-[@B17]\] with tips fabricated so that the identified open end of the bronchiole could be aspirated into the outer pipette. (Fig. [1](#F1){ref-type="fig"}). Simultaneously, a double barreled inner pipette was inserted into the lumen of the bronchiole for delivering experimental solutions and monitoring electrical potentials through one barrel while constant current pulses were delivered through the other barrel \[[@B18]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Pipette assembly for microperfusing segments of undissected bronchiole. The bronchiole is held in outer large pipette (A) by suction. An inner, septated cannulating pipette provides current passing capacity through one barrel (B~1~) and perfusing fluid to the duct lumen through the opposite barrel (B~2~), which also contains a small cannula pipette (C) that allows changes of perfusing solutions. ::: ![](1465-9921-6-7-1) ::: Solutions --------- The perfused airways were *intact*and therefore remained embedded in the mass of connective tissue and air filled alveoli that normally surround the bronchi *in vivo*. The surrounding parenchymal tissue effectively prevented changing the solution in contact with the serosal surfaces of the airway epithelium, which *in vivo*is the extracellular fluid and in the*intact* preparation could not be readily removed. NaCl Ringer solution is designed to mimic mammalian extracellular fluid. Therefore, we used NaCl Ringer in the bath to establish electrical continuity with the serosal surface of the bronchiolar epithelium during the entire experimental period. The Ringer solution contained in (mM): Na^+^(\~155), K^+^(4.5), Mg^2+^(1.2), Ca^2+^(1.0), PO~4~^3-^(3.5), Cl^-^(152), SO~4~^2-^(1.2), Glucose (5) buffered to pH 7.4 with NaOH. For ion diffusion studies, 150 mM of Cl^-^was replaced with an equivalent amount of gluconate (taken as impermeant), HCO~3~^-^, NO~3~^-^, I^-^, or Br^-^. Luminal solutions perfusing the bronchiolar airway were rapidly changed as needed via a manifold distributing stores of the above solutions through a needle tube to the tip of the perfusing pipette (Fig. [1](#F1){ref-type="fig"}). Agonists were added to solutions (in μM) as needed as forskolin (1), IBMX (100), ionomycin (1), ATP (100), and adenosine (100). Inhibitors were added (in μM) as needed as amiloride (10), NPPB (100), CFTR~Inh~-172 (5) \[[@B19],[@B20]\], GlyH-101 (50) \[[@B21]\] (CFTR~Inh~-172 and GlyH-101 were generous gifts from Dr. A. Verkman, University of California, San Francisco, CA.). Electrical Measurements ----------------------- The basic electrical circuit for recording potentials and conductance during microperfusion has been described previously \[[@B18]\]. The lumen of the bronchiole can be considered as a conductive core of fluid (perfusate) surrounded by an insulating epithelium.Unfortunately, the complex arborizing geometry of the bronchiole make it impossible to calculate the specific conductance of the epithelium from cable analysis as is possible with straight, unbranching tubes like sweat ducts and renal tubules. Thus, in the present protocol, the current pulse induced voltage deflections reflect the total resistance of the preparation and can only be used to compare changes in the epithelial resistance within the same preparation when identical solutions are present in the lumen and bath; e.g., pre- and post-drug application. Although we recorded the total resistance (R~t~) of the system, which includes the summed resistances of the epithelium (including parallel shunts through it) plus the core resistance of the lumen plus the extracellular fluid resistance, the resistance of the epithelium relative to R~t~was small (even after floating the current passing circuit), and therefore changes in the epithelial resistance were obscured. Consequently, the response of TEP\'s was taken as the primary indication of the permeability properties of the epithelium. Temperature ----------- The bathing solution was maintained at 35 ± 2°C. mRNA expression --------------- Total RNA was isolated from the dissected bronchioles of 4 pigs by using RNeasy Mini Kit (QIAGEN Inc. CA). RNA was reversely transcribed using Sensiscript RT Kit (QIAGEN Inc. CA). The resulting first-strand cDNA was directly used for PCR amplification (TaqPCR Core Kit, QIAGEN Inc. CA). The conditions for PCR reactions were as follows: 3 min at 94°C (initial melt); 35 cycles of 1 min at 94°C, 1 min at 55--60°C, 1 min at 72°C and then 72°C 10 min (final extension). For the negative control, RT-PCR was performed in the absence of RT. The PCR products were analyzed by agarose gel electrophoresis stained with ethidium bromide. The primers were constructed on the basis of the published cDNA sequence of CFTR, ENaC, NKCC1 and β-Actin from GenBank. Since the pig gene sequence was not complete, primers were obtained from the human accordant gene, in which highly conserved regions were selected. The pairs of primers for CFTR (accession no. NM\_000492) were sense 5\'-TCCTAAGCCATGGCCACAA-3\' and antisense 5\'-GCATTCCAGCATTGCTTCTA-3\'; sense 5\'-GCCTGGCACCATTAAAGAAA-3\' and antisense 5\'-CTTGCTCGTTGACCTCCACT-3\', which generated a 197-bp and 171-bp CFTR PCR product respectively; for α-ENaC (Z92978) were sense 5\'-CAACAACACCACCATCCAC-3\' and antisense 5\'-TAGGGATTGAGGGTGCAGA-3\', which generated a 225-bp PCR product; for β-ENaC (NM\_000336) were sense 5\'-TGCTGTGCCTCATCGAGTTTG-3\' and antisense 5\'-TGCAGACGCAGGGAGTCATAGTTG-3\', which generated a 277-bp PCR product; for γ-ENaC (X87160) were sense 5\'-TCAAGAAGAATCTGCCCGTGA-3\' and antisense 5\'-GGAAGTGGACTTTGATGGAAACTG-3\', which generated a 237-bp PCR product; for NKCC1 (U30246) were sense 5\'-TCCAGGTAATGAGTATGGTGTCAG-3\' and antisense, 5\'-GTTAAGATGTAGCCACGAAGAGGT-3\', which generated a 205-bp PCR product; and for β-Actin (BC004251) were sense, 5\'-TTCAACTCCATCATGAAGAAGTGTGACGTG-3\' and antisense, 5\'-CTAAGTCATAGTCCGCCTAGAAGCATT-3\', which generated a 312-bp PCR product. All primers showed products closely corresponding to the predicted size for expression of RNA transcripts for these genes. Immunocytochemistry ------------------- The bronchioles were dissected and then fixed in ice-cold 4% formaldehyde buffered in phosphate at 4°C for 3 hours, infiltrated with cryoprotectant (30% sucrose in PBS) overnight, and frozen in OTC medium (Triangle Biomedical Sciences) at -35°C. Sections of 5 μm thickness were cut on a cryostat microtome (Thermo Electron) and mounted on glass slides (Fisher Superfrost Plus). Antigen retrieval was performed using a pressure cooker (10 min in 10 mM citrate buffer, pH 6). To reduce autofluorescence, sections were treated for 20 min with 1.5% sodium borohydride in PBS. Sections were incubated sequentially with blocking solution (30 min), primary antibody (overnight at 4°C), and secondary antibodies conjugated to Alexa Fluor-488 and/or -546 (Molecular Probes). Confocal images were acquired with a Zeiss LSM-510 microscope and assembled using Adobe Photoshop. CFTR was labeled with rabbit antibody R3194 (courtesy of C. Marino), and ENaC with a rabbit antibody against the β-subunit of human ENaC (kindly courtesy of C. Fuller and D. Benos). Tight junctions were labeled with a mouse antibody against the junction-associated protein zonula occludens-1 (Zymed). Nuclei were stained with TO-PRO-3 (Molecular Probes). Statistical treatment --------------------- Differences in mean measurement were assayed by applying the Student T test to paired or unpaired means as appropriate. A probability (P value) of ≤ 0.05 was taken as significantly different. Results ======= Basic electrical properties --------------------------- When the bronchiolar lumen was perfused with NaCl Ringers, which we assumed represented insignificant ion gradients except for a small lumen (145 mM) to serosa (110 mm) Cl^-^gradient. Despite the fact that this small Cl^-^gradient should render the lumen positive, there was a small spontaneous lumen negative potential of about -3 mV (Fig. [2](#F2){ref-type="fig"}; Table [1](#T1){ref-type="table"}). When we applied amiloride (10 μM) to the lumen to block Na^+^conductance (gNa^+^), the TEP decreased slightly, but without statistical significance (Fig. [2](#F2){ref-type="fig"}; Table [1](#T1){ref-type="table"}). We were unable to detect a change in total conductance with amiloride applied to the lumen. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effect of amiloride and Forskolin (Fsk, 5 μM) + IBMX (100 μM) on transepithelial potential (TEP) of bronchiole. In the presence of luminal Cl^-^, the effects of both amiloride and Fsk+IBMX on TEP were almost imperceptible (left side). However, when Cl^-^was substituted with Gluconate to more effectively reveal the Cl^-^conductance, addition of amiloride depolarized TEP and Fsk+IBMX hyperpolarized the TEP (right side), suggesting that the large Cl^-^conductance present in the epithelium mutes (shunts) the smaller changes in conductance occasioned by amiloride and Fsk when isotonic Cl^-^is present bilaterally. NaGlu: Na-gluconate ::: ![](1465-9921-6-7-2) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Amiloride Inhibition ::: NaCl NaCl+Amil NaGlu NaGlu+Amil ------------ ------------ ------------ ------------- ------------- TEP (mV) -3.1 ± 0.6 -2.6 ± 0.7 -57.3 ± 2.7 -43.6 ± 2.7 Δ TEP (mV) \-- +0.5 \-- +13.7 n 25 10 25 16 P value \-- 0.676 \-- \<0.001 ::: Gluconate Substitution ---------------------- However, when we replaced luminal Cl^-^with the impermeant anion, gluconate, the TEP hyperpolarized to as much as -90 mV (Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}). Addition of amiloride (10 μM) to the lumen depolarized the mean TEP of these tissues by an average of 14 mV (Fig. [2](#F2){ref-type="fig"}; Table [1](#T1){ref-type="table"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effect of luminal Cl^-^substitution in a perfused small bronchiole on TEP. The substitution of Gluconate, an impermeant anion, for permeable Cl^-^in the lumen markedly hyperpolarized the TEP. This response indicates a predominant Cl^-^conductance that appears to be constitutively active. ::: ![](1465-9921-6-7-3) ::: Anion Conductance Inhibitors ---------------------------- Under conditions of symmetrical \[Cl^-^\] concentrations, luminal applications of anion conductance inhibitors had virtually no detectable effect on the spontaneous TEP. However, under hyperpolarizing conditions created by Cl^-^substitution in the lumen, NPPB (100 μM), GlyH-101 (50 μM) and CFTR~Inh~-172 (5 μM) significantly depolarized the TEP by 36.7, 20.0 and 8.7 mV (Fig. [4](#F4){ref-type="fig"}; Table [2](#T2){ref-type="table"}). Bumetanide (1 mM) had no effect on the TEP in either the presence or absence of a Cl^-^gradient (not shown). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effect of Cl^-^conductance inhibitors on TEP. In the absence of luminal Cl^-^(Gluconate substitution), two new inhibitors GlyH-101 (50 μM) and CFTR~Inh~-172 (5 μM, re: A. Verkman) showed at least partial inhibition of the Cl^-^:Gluconate anion diffusion potential. NPPB (100 μM) seemed to be the most effective inhibitor in terms of depolarizing the TEP. ::: ![](1465-9921-6-7-4) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Cl^-^Conductance Inhibition ::: CFTR~inh~-172 NPPB GlyH-101 ------------ --------------- ------------- ------------- TEP (mV) -48.6 ± 4.7 -16.5 ± 2.1 -26.6 ± 2.5 Δ TEP (mV) +8.7 +36.7 +20.0 n 3 5 4 P value 0.03 0.001 0.03 ::: Anion selectivity ----------------- We assayed for the relative permeability of several monovalent anions by substituting them for Cl^-^in NaCl Ringer. Amiloride (10 μM) was added in order to block Na^+^transport. For luminal Cl^-^, Br^-^, I^-^, NO~3~^-^, HCO~3~^-^and gluconate, the mean estimated P~x~/P~Cl~were, respectively, 1.0, 0.92, 0.79, 0.65, 0.33 and 0.28 (Table.[3](#T3){ref-type="table"}). When the NaCl Ringer perfusion solution was diluted by 1:2, the potential depolarized by 12 ± 1 mV, indicating Cl^-^permeability exceeded the Na^+^permeability by about 5.4 fold (Fig. [5](#F5){ref-type="fig"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Anion selectivity sequence of the perfused bronchiole. The sequence of the bronchiole (upper data) roughly fits the known sequence of CTFR in other tissues (lower data taken from literature; cf. text) ::: Airway Anion selectivity: **Cl^-^** ≈ **Br^-^** **\>** **I^-^** **\>** **NO~3~^-^** **\>** **HCO~3~^-^** **\>\>** **Gluconate** --------------------------- -------------- ------- ----------- -------- ----------- -------- -------------- -------- --------------- ---------- --------------- Transepithelial TEP : \~0 -1.9 -5.1 -9.2 -19.5 -44.8 Estimated P~x~/P~Cl~: 1.0 0.92 0.79 0.65 0.33 0.28 CFTR anion selectivity: **NO~3~^-^** **≈** **Br^-^** **≈** **Cl^-^** **\>** **I^-^** **\>** **HCO~3~^-^** **\>\>** **Gluconate** Estimated P~x~/P~Cl~: 1.1 1.1 1.0 0.39 0.014 \~0 ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### NaCl dilution diffusion across the bronchiolar epithelium on TEP. The hyperpolarization of the TEP with diluted NaCl (75 mM) indicates that Cl^-^must be significantly more permeable through the epithelium than Na^+^. ::: ![](1465-9921-6-7-5) ::: Agonists -------- When we added forskolin (5 μM) plus IBMX (100 μM), adenosine (100 μM), ATP (100 μM) or ATP (100 μM)+adenosine (100 μM) to the perfusate to activate CFTR gCl^-^in the presence of isotonic Cl^-^concentrations and in the absence of a hyperpolarizing gradient, the TEP did not change perceptibly (not shown), but in the presence of the Cl^-^gradient, the TEP hyperpolarized significantly to all agonists; the response appeared to be increased when both ATP and adenosine were added together (Fig. [6](#F6){ref-type="fig"}; Table [4](#T4){ref-type="table"}). In order to observe the maximum effect on the activation of Cl^-^conductance, amiloride (10 μM) was present in all luminal perfusates to block ENaC Na^+^conductance. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Effect of Cl^-^conductance agonists on TEP: Applying Fsk (5 μM)+IBMX (100 μM), adenosine (100 μM), ATP (100 μM) and ATP (100 μM)+adenosine (100 μM) to the perfusate to activate CFTR gCl^-^in the presence of a hyperpolarizing Cl^-^gradient, the TEP hyperpolarized significantly; however, the response appeared to be increased when both ATP and adenosine were added together. In order to observe maximum effect on activation of Cl^-^conductance, amiloride (10 μM) is present in all luminal perfusate above to block Na^+^conductance. ::: ![](1465-9921-6-7-6) ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Cl^-^Conductance Agonists ::: Fsk+IBMX ATP Adenosine ATP+Adenosine Ionomycin ------------ ------------ ------------- ------------- --------------- ------------- TEP (mV) 53.0 ± 2.8 -51.0 ± 0.8 -55.8 ± 6.7 -52.0 ± 6.5 -40.1 ± 7.5 Δ TEP (mV) -26.0 -5.5 -5.5 -6.3 0.4 n 13 4 5 6 5 P value \<0.001 0.007 0.003 0.011 0.836 ::: RT-PCR ------ Two sets of different primers for CFTR as well as primers for α-, β-and γ-ENaC, NKCC1, and β-Actin all produced products of predicted sizes for each of the corresponding mRNAs (Fig. [7](#F7){ref-type="fig"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### RT-PCR bands for CFTR (lanes 2 &3), α,β,γ subunits of ENaC (lanes 5,6,7 respectively), and NKCC1 (lane 9) and β-Actin (lane 10). Two independent sets of primers were used to detect CFTR. The presence of CFTR, ENaC, and NKCC1 may indicate that the epithelium has both absorptive and secretory functions. β-Actin was used as a housekeeper marker for control. Size ladders in 100 bp increments with lowest band equal to 100 bp (lanes 1,4,8). ::: ![](1465-9921-6-7-7) ::: Immunocytochemistry of CFTR --------------------------- Immunoreactive CFTR antibody (gift of C. Marino) was detected in the apical domains of the bronchiolar epithelia in a continuous border of the bronchiole. The tight junctions were labeled simultaneously and appeared as punctate areas within the border staining for CFTR consistent with the expected location for these structures (Fig. [8A](#F8){ref-type="fig"} and inset). Negative controls consisted of omission of primary antibodies and showed no labeling of the antibody in any tissue (Fig. [8B](#F8){ref-type="fig"}). ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Immunocytochemical localization of CFTR in bronchiole epithelium. A.). Antibody R3194 against CFTR prominently labels (green) the apical margin of epithelial cells lining the bronchiole cut in cross section. Inset: high magnification view of epithelial cells showing demarcation of apical margin by tight junction-associated protein ZO-1 (red). B.) Control serial section stained without primary antibody. ::: ![](1465-9921-6-7-8) ::: Discussion ========== The airways are extraordinarily specialized conduits for air to and from the alveoli for gas exchange. They must remain moist in order to remain flexible and to effectively filter air before it enters the delicate tissues of the alveoli. Hence, a principal function of the airway epithelia is to provide and service a continuous layer of aqueous fluid on the airway surfaces. For decades, the upper airways, trachea, and large bronchi have served as models for the entire tracheobronchial epithelial function \[[@B14],[@B22],[@B23]\], and yet it is known that there are distinct regional differences progressing from nares to alveoli. For example, anatomically, the upper airways and bronchi are characterized by submucosal glands that secrete fluid directly into the airway, but are absent in the peripheral small airways \[[@B24]\]; \[[@B25]\]. Likewise, the trachea and larger airways are kept patent by cartilaginous rings whereas the peripheral airways are generally held open (but may collapse) by internal retractile forces of the lung parenchyma \[[@B26]\]. Similarly, the cell populations change. The epithelium of the upper respiratory tract changes from ciliated, pseudostratified columnar to simple cuboidal cells in the smaller airways where the proportion of Clara cells increase and that of ciliated cells decrease \[[@B27]\]. Functionally, there are differences as well. For example, in humans and in dogs, the spontaneous transmural electrical potential appears to become less negative (lumen: blood) from upper to lower airways \[[@B13],[@B28],[@B29]\]. Differences in airway surface fluid composition may also exist \[[@B30]\]. The functions of the lower peripheral airway epithelium is poorly defined and understood because this tissue is inaccessible and relatively unamenable to standard physiological techniques for study. Nonetheless, a few courageous attempts to unravel these mysteries have been made. About ten years ago, Ballard \[[@B13],[@B14]\] and Al-Bazzaz \[[@B10]\] isolated and perfused small airways and bronchioles from pig and sheep, respectively. Ballard perfused porcine airways of about 1 mm diameter and reported a mean spontaneous TEP potential difference in bilateral NaCl Ringer solution of about -3.4 mV and in lumen Cl^-^free solution of about -16 mV \[[@B14]\]. Al-Bazzaz perfused ovine bronchioles of about 250 μm diameter and reported a mean spontaneous TEP of -2.5 mV in bilateral NaCl Ringer and of about -4.2 mV mean TEP in lumens perfused with Cl^-^free Ringers \[[@B11]\]. In all of these studies, it has been difficult to ascertain to what degree the electrical responses were muted or altered by trauma to the tissue during dissection because similar measurements are not possible *in vivo*. We, too, found similar, relatively small TEP voltages in microdissected porcine airways. Undissected bronchioles ----------------------- Therefore, in order to maximally preserve, and minimally traumatize the airway epithelium, we avoided dissecting the bronchial structure and microperfused the airways imbedded in lung parenchyma. We found that the spontaneous TEP in bilateral Ringer solution was about -3 mV, slightly more negative than reported earlier. But in striking contrast to the previous studies (including our own dissected preparations), we found that the bi-ionic TEP with Cl^-^free solutions in the lumen was as much as -90 mV (mean: -57 mV; Table [1](#T1){ref-type="table"}; Fig. [2](#F2){ref-type="fig"}). These differences almost certainly reflect differences in trauma to the airway. Because of the extremely complicated morphology that arises from arborization of the airway in route to hundreds of alveolar acini, it is impossible to physically remove the surrounding tissues (very small bronchioles, respiratory bronchioles, blood vessels, and alveolar sacs) without breaking or tearing smaller \"branches\" from the \"tree\" of airways. Both Ballard and Al-Bazzaz attempted to patch these breaks by micro-suturing obviously dangling limbs; unfortunately, only larger branches are amenable to such heroic attempts and many smaller, even microscopic transepithelial openings, must remain. In order for an epithelium to reflect its *in vivo*electrical properties *in vitro*, it is fundamental that the integrity of the epithelial sheet be conserved because TEP measurements depend on separation of charge. Breaks, holes, or tears in the barrier inescapably create electrical shunts, which, by allowing simultaneous back leak of epithelial current, prevent separation of charge. Even though the individual cells or groups of cells of the epithelia function and respond physiologically, the transepithelial voltage signals will be erroneously muted or lost through such shunts. In the present case, it appears that perfusion of the bronchiole without dissection from surrounding parenchyma, minimizes trauma induced shunts and allows detection of a much more complete and robust electrical signal. Unfortunately, the price for this preservation of signal is the inability to control the contra luminal solution. Keeping in mind that the undissected bronchiole is embedded in a mass of air filled alveoli together with numerous other elements of the tissue at this level, it is easy to see that changing the bath solution can have no acute effects on the composition of the contra luminal solution at the basilateral membrane of the epithelium. Consequently, we did not change the bath solution and assumed that normal Ringer solution is sufficiently similar to extracellular fluid *in vivo*to avoid introducing significant free solution diffusion potentials or altering the physiological composition of the native fluid present in the extracellular compartment of the in tact preparation. Cl^-^Conductance ---------------- The simple fact that substituting Cl^-^in the lumen spontaneously created a large lumen negative transmural potential (Figs. [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}; Table [1](#T1){ref-type="table"}) immediately indicates that the small airway is characterized by a dominant constitutively active anion selective conductance. The fact that imposing a putative 1:2 NaCl diffusion gradient across the epithelium resulted in -12 mV hyperpolarization indicates that the intact bronchiole epithelium is at least 5 times more permeable to Cl^-^than to Na^+^(Fig. [5](#F5){ref-type="fig"}). The facts that this Cl^-^conductance is immediately evident upon initial perfusion without any prior agonist stimulation and that additions of agonists did not significantly hyperpolarize the bronchiole perfused with 150 mM NaCl and hyperpolarized bronchioles perfused with Na Gluconate only by about 20--25% demonstrates that the Cl^-^conductance is constitutively open under these conditions (Fig. [6](#F6){ref-type="fig"}). That is, from the first moment of measurement after cannulation, the large Cl^-^conductance is present (Figs. [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"} and [5](#F5){ref-type="fig"}). There was no need to activate PKA or wait for the transmural potential to develop even though the addition of forskolin, adenosine, and ATP appeared to increase Cl^-^conductance (Figs. [2](#F2){ref-type="fig"}, [6](#F6){ref-type="fig"}; Table [4](#T4){ref-type="table"}). In secretory cells where secretory activity is usually an acute, temporal event, CFTR is assumed to remain closed until activated by PKA and ATP. However, in the human sweat duct, also a rich source of CFTR, where the transport function is exclusively absorptive and where CFTR is thought to be the only anion conductance through the tissue, CFTR appears with a large Cl^-^conductance at the first moment of perfusion and measurement, indicating a constitutively open state for the CFTR channel in this tissue as well. It is well established that CFTR can be regulated in the classic sense by PKA phosphorylation and ATP in the sweat duct, but the cytoplasm must first be \"rinsed\" of small solutes by permeabilizing the basilateral membrane \[[@B31]\]. CFTR ---- Since obstructive airway disease in Cystic Fibrosis arises in the small airways \[[@B32]-[@B34]\] and CF is known to be due to mutations in the CFTR gene that expresses a Cl^-^channel, we asked if this conductance could be due to CFTR. We found several lines of evidence that are consistent with CFTR being responsible for the Cl^-^conductance. First, the anion selectivity sequence, excepting NO~3~^-^, is grossly the same as that for CFTR (Table [3](#T3){ref-type="table"}); i.e., Cl^-^≈ Br^-^\> I^-^\> NO~3~^-^\> HCO~3~^-^\> Gluconate, and compares favorably with that of CFTR: SCN \> NO~3~^-^\> Br^-^\> Cl^-^\> I^-^\> HCO~3~^-^\> F^-^\> ClO~4~^-^\> gluconate \[[@B35]-[@B37]\]. Second, it is well known that CFTR is activated characteristically by cAMP mediated protein kinase A, which is driven pharmacologically by forskolin and IBMX. Here, we see (Figs. [2](#F2){ref-type="fig"}, [6](#F6){ref-type="fig"}; Table [4](#T4){ref-type="table"}) that these agonists routinely elevate the bionic Cl: gluconate diffusion potential by an average of -22 mV (n = 13). Similarly, ATP and adenosine may activate CFTR in airway epithelia since these agonists can elevate cAMP via purinergic receptors. \[[@B38]-[@B40]\]. In contrast, when we applied ionomycin to elevate intracellular Ca^2+^, there was no effect (not shown), suggesting that since CFTR is not sensitive to Ca^2+^mediated activation either, a.) the conductance is due to CFTR, b.) other Ca^2+^activated Cl^-^conductances must be fully constitutively activated or not present, or c.) luminal application of the ionophore drug does not increase intracellular Ca^2+^effectively. Third, CFTR is known to be inhibited by NPPB, CFTR~Inh~-172, and GlyH-101\[[@B19],[@B21]\]. These inhibitors had varying, but consistently inhibitory effects on the TEP (Fig. [4](#F4){ref-type="fig"}; Table [2](#T2){ref-type="table"}) indicating inhibition of Cl^-^conductance in the airway epithelium. However, the fact that none of the inhibitors appeared to completely inhibit the transepithelial potential might argue that another anion channel conductance is present. Two points diminish this argument. First, if the tissue is actively transporting, anion channel inhibitors will not completely ablate the TEP because that component of potential generated by the basilateral K^+^emf and the apical Na^+^emf should not be blocked and should be reflected across the epithelium in the TEP. Secondly, even when the CFTR Cl^-^channel has been isolated from active transport components, anion channel blockers do not usually completely block its conductance in native tissue \[[@B41]\]. Moreover, recently, in contrast, to our results, GlyH-101 was reported to be ineffective in blocking the Cl^-^conductance of pig nasal airways \[[@B42]\]. It is not known whether CFTR is present in the nasal epithelium of pigs, but biochemically, we found markers for conserved regions of expressed CFTR RNA from reverse transcriptase polymerase chain reactions (Fig. [7](#F7){ref-type="fig"}) in bronchioles dissected free of parenchyma post perfusion. Appropriate bands for CFTR protein in lysates of frozen peripheral lung tissue were also observed with affinity purified antibodies (J. Riordan, personal communication), and as shown in (Fig. [8](#F8){ref-type="fig"}) CFTR localized immunocytochemically, specifically to the apical surface of the bronchiolar epithelia. These data strongly suggest that CFTR is a part of, or probably is, the primary source of the anion conductance in small airways. Transport Function ------------------ Not only does the bronchiole resemble the sweat duct with respect to exhibiting a constitutively active Cl^-^channel that responds to activation of PKA, but both are also apparently insensitive to ionomycin. They both show equally large bionic diffusion potentials for Cl^-^and are several fold more permeable to Cl^-^than to Na^+^, and yet both have very low transmembrane potentials in bilateral isotonic Ringer solutions \[[@B43]\]. Both are incompletely inhibited by anion channel blockers \[[@B20],[@B41],[@B44]\], and both are sensitive to amiloride. On the basis of these observations and by analogy with the sweat duct, it is tempting to propose that the bronchiole at least in its basal state is a constitutively absorptive epithelium. Conclusions =========== We have found that the epithelium of terminal airways of the pig appears to express an anion permeability that constitutively dominates the electroconductive properties of this zone of the airway. Its anion selectivity sequence is similar to that expected for CFTR, and its activity can be enhanced by forskolin/IBMX or decreased by anion channel blockers known to inhibit CFTR. RT-PCR amplification products and specific antibodies identify CFTR in this tissue. The small airway appears to share a number of properties with the human sweat duct and may, by analogy, belong to a class of highly absorptive epithelia. Abbreviations ============= CFTR: Cystic Fibrosis Transmembrane Conductance Regulator ENaC: Epithelial Na^+^Channel NKCC: Na^+^-K^+^-2Cl^-^Cotransporter TEP: Transepithelial Potential PKA: Protein Kinase A Acknowledgments =============== This work was supported by CFRI, the Nancy Olmsted Trust, and the USPHS- NIH 5R01 DK51899-04 and DE 14352. We gratefully acknowledge the assistance of Ms. Joanne Albrecht, Ms. Suchi Madireddi, and Mr. Kirk Taylor. We sincerely thank Dr. Chris Marino (University of Tennessee, Memphis) for the CFTR antibody and Dr. Allen Verkman (UCSF) for supplying the Cl- conductance inhibitors.

PubMed Central

2024-06-05T03:55:52.486980

2005-1-17

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548141/", "journal": "Respir Res. 2005 Jan 17; 6(1):7", "authors": [ { "first": "Xiaofei", "last": "Wang" }, { "first": "Christian", "last": "Lytle" }, { "first": "Paul M", "last": "Quinton" } ] }

PMC548142

Background ========== Sickle cell disease is a common debilitating hematologic disease occurring in 1 in 650 African Americans. Lung disease is a major cause of cardiopulmonary disability and mortality \[[@B1],[@B2]\]. Progressive restrictive lung disease related to recurrent episodes of acute chest syndrome may develop with advancing age \[[@B3]\]. Acute chest syndrome is a clinical manifestation triggered by pathological processes including infections, fat embolism, infarction and bronchospasm \[[@B4]\]. Nearly 70% of patients with acute chest syndrome are hypoxemic (as measured by pulse oximetry \< 90% or PO~2~\< 80 mmHg by blood gas analysis) \[[@B5]\]. Hypoxemia causes sickle hemoglobin to gel, inducing red blood cell sickling and vaso-occlusion within pulmonary blood vessels. Asthma is the most common chronic disease of childhood occurring in 6% of the general population. Moreover, the African American population has the highest prevalence (8%), emergency department (ED) visits, hospitalizations and risk of mortality of any ethnic population in the United States \[[@B6]\]. Reversible airway obstruction, airway inflammation and nonspecific bronchial hyperresponsiveness are the hallmarks of asthma. Exacerbations of asthma result in mucous plugging, bronchoconstriction with decreased air exchange, ventilation-perfusion mismatch and subsequently hypoxemia. Aggressive treatment with oxygen, bronchodilators and oral corticosteroids are recommended for symptomatic relief of acute episodes. The African American population is at significant risk for the occurrence of both diseases simultaneously but little is known of the affect of asthma on individuals with sickle cell anemia. In this study, we examined hospitalized children with sickle cell disease with concomitant asthma establishing whether there was an increased rate of acute chest syndrome or other complications compared to patients with sickle cell disease without asthma in those presenting with vaso-occlusive pain crises. Methods ======= We performed a 5-year retrospective chart review of 48 children with asthma and sickle cell disease and compared them to a control group of 48 children with sickle cell disease alone. The 48 children with sickle cell and asthma represented all patients with complete medical records with both diseases that were cared for at our center. These children had no hospitalizations for sickle cell crises at other facilities. Children in the control group were matched for age, hemoglobinopathy and gender. Sickle cell disease was determined by high performance liquid chromatography and isoelectric focusing analysis and grouped into phenotypes of hemoglobin SS or hemoglobin SC. Children were included in the asthma group if they had medical record documentation of a discharge diagnosis of asthma (ICD-493) and had been prescribed asthma medications. Vaso-occlusive pain was defined as pain that could not be explained by injury or infection requiring hospital admission and treatment with intravenous pain medication. The criteria for a diagnosis of acute chest syndrome included respiratory distress, hypoxemia, and a new infiltrate on chest x-ray that required hospitalization and a transfusion of packed red blood cells. A cerebral vascular accident diagnosis was based on the new onset of an acute neurological syndrome with a focal neurological finding on examination associated with ischemic changes (images compatible with stroke) on a brain MRI or computed tomography scan. Inpatient admissions were recorded from March 1997 to March 2002 for episodes of acute chest syndrome, vasoocclusive pain crises, cerebral vascular accidents, total blood transfusions, exchange transfusions and chronic transfusions (monthly blood transfusions). Exclusion criteria included any child who had incomplete documentation of their hospital records or those who had moved into or out of the Milwaukee area during the 5-year study period. Six children were excluded because they moved from Milwaukee. The study was approved by the Investigational Review Board. Statistical Methods ------------------- The Mann-Whitney test was used to evaluate differences between the incidents of vasoocclusive pain crises, acute chest syndrome, total blood transfusions and cerebral vascular accidents. Chi-squared analysis was used to evaluate the number of exchange and chronic transfusions. Statistical significance was given to p values 0.05 or less. Results ======= All subjects in the asthma group had asthma recorded in their medical record with symptoms consistent with asthma. All had been prescribed albuterol; while 28 (58%) had been prescribed controller medications (24 inhaled corticosteroids, 12 inhaled cromolyn sodium, and 5 leukotriene modifier). Most patients had been prescribed combination therapy with more than 1 controller medication or had been switched from 1 controller to another. There was no reliable means to confirm adherence to the prescribed asthma drug regimen. Seventeen (35%) subjects had not had prior ED [or]{.underline} hospital admissions for asthma. Of these children, 13 (76%) had been prescribed only albuterol. Twelve (25%) had at least 1 ED visit [and]{.underline} at least 1 hospital admission for asthma. Twenty-six (54%) subjects had only hospital admissions for asthma, while 15 children had only been treated in the ED for acute asthma. Of 14 children with a severity category documented, 9 had mild intermittent asthma, 1 each with mild persistent and moderate persistent asthma, and 3 with severe persistent asthma. No patient had documented bronchoprovocation with methacholine and only 3 had documented spirometry with 1 consistent with airway obstruction, 1 with reversibility of airway obstruction, and one with poor technique and unreliable results. Two patients (age 3) were too young for spirometry. Four subjects had been seen in the Asthma and Allergy clinic for consultation and the diagnosis of asthma reaffirmed. Twenty-one (44%) subjects had a primary family member with asthma. Only 5 children were born preterm (27, 33, 34, 36, and 37 weeks gestation) and none were diagnosed with bronchopulmonary dysplasia. The cases and controls were well-matched for age, gender and type of hemoglobinopathy (Table [I](#T1){ref-type="table"}). The cases (21 males and 27 females) consisted of 42 children with HgbSS and 6 with HgbSC. The control group (17 males and 31 females) included 41 children with HgbSS and 7 with HgbSC. The age of the children ranged from 3 to 18 years old with a mean age of 10.1 years (median 10 years) for the case patients and 10.3 years (median 10 years) for the control children. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Subject Demographics ::: **Patients with Sickle Cell & Asthma** **Patients with Sickle Cell Disease** -------------- ---------------------------------------- --------------------------------------- **Males** 21 17 **Females** 27 31 **Mean Age** 10.1 years 10.3 years **HgbSS** 42 41 **HgbSC** 6 7 ::: Patients with sickle cell disease and asthma had significantly more episodes of acute chest syndrome, cerebral vascular accidents, blood transfusions and chronic transfusions as compared to the control group (Table [2](#T2){ref-type="table"}). Admissions for vaso-occlusive pain crisis and exchange transfusions were not statistically significant between groups. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Results in children with sickle cell disease with and without asthma ::: **Patients with Sickle Cell & Asthma (n = 48)** **Patients with Sickle Cell Disease (n = 48)** **Statistical Significance** ------------------------------------------- ------------------------------------------------- ------------------------------------------------ ------------------------------ **Admissions for Acute Chest Syndrome** 90 58 P = 0.03 **Number of Cerebral Vascular Accidents** 10 2 P = 0.05 **Total Blood Transfusions** 432 226 P = 0.01 **Chronic Blood Transfusions** 13 4 P = 0.04 **Vaso-occlusive Pain Crises** 248 223 P = 0.52 **Exchange Blood Transfusions** 9 4 P = 0.21 ::: No patient with SC disease in either group had a history of a cerebral vascular accident. Only 1 patient with SC disease had acute chest syndrome, while 3 children in the asthma group had acute chest syndrome with one child experiencing 2 episodes. Discussion ========== Despite the high prevalence of these two diseases that affect the African American population, there is paucity of research investigating patients with concomitant asthma and sickle cell disease. In this study, we discovered that children with both asthma and sickle cell disease are significantly more likely to develop severe complications of sickle cell disease including acute chest syndrome and cerebral vascular accidents compared to children with sickle cell disease alone. The significance of these findings relates to the hypothesis that appropriately aggressive treatment of asthma in children with sickle cell disease may diminish the frequency of pulmonary complications. Patients with reactive airway disease and sickle cell disease have a lower transcutaneous oxyhemoglobin saturation \[[@B7]\]. The lower transcutaneous oxyhemoglobin saturation increases sickling of red blood cells, causing subsequent gelling and vaso-occlusion in multiple organs leading to numerous complications. A high prevalence (73%) of airway hyperreactivity to cold-air challenge occurs in children with sickle cell anemia even in the absence of clinical symptoms of asthma \[[@B8]\]. Cold air or other provocative challenge tests had not been performed in our group of patients. Our results are in agreement with an abstract that showed 18 children with both asthma and sickle cell disease had increased hospital admissions for pain crises and acute chest episodes compared with patients with only sickle cell disease \[[@B9]\]. Our results are unique because we assessed for an increased frequency of transfusions and cerebral vascular accidents in a larger group of patients. Cerebral vascular accidents affect 10% of the children with sickle cell disease often with devastating long term implications. The Cooperative Study of Sickle Cell Disease reported that recent and recurrent episodes of acute chest syndrome are risk factors for cerebral vascular accidents \[[@B10]\]. Our findings of increased cerebral vascular accidents in patients with sickle cell disease and asthma may be due to their increased and recurrent episodes of acute chest syndrome. The mechanism linking stroke and acute chest syndrome is unknown but may be related to hypoxemia due to pulmonary disease. Hypoxemia has been reported to increase adhesion of red blood cells to endothelium \[[@B11]\]. Likewise, increased red blood cell adhesion to pulmonary endothelium has been associated with bone marrow vaso-occlusive crises due to hypoxia, cytokine expression and fat embolization \[[@B12]\]. Whether hypoxemia secondary to asthma affects red blood cell and pulmonary/cerebral endothelium adhesion characteristics is unknown. Our study demonstrates that pediatric patients with sickle cell disease and asthma are more likely to require acute and chronic blood transfusions. It is not surprising that these patients needed more frequent transfusions since treatment for cerebral vascular accidents and severe acute chest syndrome is blood transfusions. Although SS type of sickle cell anemia is more severe than SC type, the small numbers of patients with SC prevent us from making broad conclusions regarding the degree of risk based on hemaglobinopathy. Two studies have demonstrated increased airway hyperreactivity with reversibility in patients with sickle cell disease without known asthma \[[@B8],[@B13]\]. Thirty-five percent of sickle cell patients had evidence of lower airway obstruction and 78% of these reversed with bronchodilator. Even in 30% of those with normal lung function, 30% had a positive response to bronchodilator. Therefore, even methods to determine the presence bronchial hyperresponsiveness are not sufficient to discriminate asthma from acute chest syndrome. Whether this hyperreactivity is due to asthma or is secondary to the pathophysiology of sickle cell disease is still unclear. Additional studies are needed to confirm that sickle cell disease is associated with the development of reversible airway obstruction. If the association is valid, the effects of routine use of anti-inflammatory controller agents prophylactically or therapeutically would deserve investigation. A randomized, placebo-controlled trial of 43 episodes of acute chest syndrome in 38 children revealed that intravenous dexamethasone prevented clinical deterioration in mild to moderately severe episodes of acute chest syndrome \[[@B14]\]. Mild and moderately severe acute chest syndrome was defined as respiratory distress, and normal mental status without pulmonary infiltrates or arterial hypoxemia. The study excluded children with an exacerbation of reactive airways disease. If patients with sickle cell disease are at increased risk of airway inflammation and obstruction, successful treatment with intravenous steroids may have been due to aggressive treatment of underlying asthma. Limitations of our study include those inherent in a retrospective analysis including selection bias, measurement bias and confounding factors. The children who received transfusions for acute chest syndrome likely represent the more severe form of disease. In contrast, those patients deemed to have asthma were primarily being treated as if they had mild to moderate asthma. Only 3 had been diagnosed with severe persistent asthma and none were on daily or every-other-day oral steroids. Importantly, even individuals with mild intermittent asthma can experience severe asthma exacerbations. Other sources of potential error that deserve recognition include the diagnosis of asthma (whether over-diagnosed or under-diagnosed), medication compliance and unknown admission to other hospitals. Although a strict definition of asthma was lacking, the history gleaned from the medical records supported an asthma diagnosis. Most patients with asthma are not cared for by asthma specialists and frequently the diagnosis is made on clinical grounds without formal pulmonary function testing. Additionally, spirometry was not routinely performed during ED and hospital admissions. Although other EDs and hospitals in the metropolitan area evaluate, treat, and admit pediatric patients with asthma exacerbations, the concomitant diagnosis of sickle cell disease likely prompted evaluation at the children\'s hospital. Conclusions =========== In this small series, children with a history of asthma and sickle cell disease developed acute chest syndrome and cerebral vascular accidents more frequently than children with sickle cell disease without asthma. The implications of this retrospective study are wide ranging and should lead to further prospective investigations. Whether aggressive asthma therapy in patients with sickle cell disease and asthma reduces the incidence of serious complications is unknown. The potential gains are far reaching and could make enormous impacts on the morbidity, quality of life and mortality of many patients. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= MEN conceived the study, participated in its design and coordination and drafted the manuscript. JL participated in data collection. MCZ participated in coordination and manuscript preparation. JPS participated in design, coordination and manuscript preparation. KJK participated in design and coordination.

PubMed Central

2024-06-05T03:55:52.490770

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548142/", "journal": "Clin Mol Allergy. 2005 Jan 21; 3:2", "authors": [ { "first": "Mark E", "last": "Nordness" }, { "first": "John", "last": "Lynn" }, { "first": "Michael C", "last": "Zacharisen" }, { "first": "Paul J", "last": "Scott" }, { "first": "Kevin J", "last": "Kelly" } ] }

PMC548143

Introduction ============ Estrogen is considered to elicit different growth responses in various tissues through binding to the estrogen receptor (ER) α and ERβ \[[@B1]-[@B3]\]. The modulation of estrogen responsive gene transcription by the ER is termed a \"*genomic*\" action of estrogen. In contrast, \"*non-genomic*\" effects of estrogen are characterized by a rapid onset of action within seconds to minutes after hormone exposure through post-translation modification of signaling proteins. ER-mediated signaling pathways are considered to support the growth of normal, preneoplastic and neoplastic cells \[[@B1]-[@B3]\]. In contrast to the classical genomic pathways of estrogen action that occur over the course of several hours or days, recent studies have shown evidence of a rapid signaling pathway mediated by cell surface ERs and non-genomic estrogen-induced signal transduction pathways which contribute to cell proliferation \[[@B4]\]. Recently, we have identified that estrogen stimulated the growth of HEK 293 cells in an ER-independent manner given that this cell line does not contain ER (unpublished Singh KP, Venkat S, Roy D). These findings suggest that in addition to ER mediated actions, other factor(s) must be involved in the stimulation of cell growth by estrogen. More recently, mitochondria have been implicated in the control of cell proliferation \[[@B5]\]. For instance, the mitochondrial peripheral benzodiazepine receptor (PBR) has been implicated in the regulation of human breast cancer cell proliferation \[[@B6]\]. Similarly, we have demonstrated that mitochondria can modulate the expression of nuclear cell cycle genes and human breast tumor growth \[[@B7],[@B8]\]. For instance, the growth of estrogen-dependent and estrogen-independent cells, is inhibited by controlling mitochondrial biogenesis \[[@B8]\]. In this paper, we critically review the role of mitochondria in the growth of estrogen-dependent cancer and non-cancer cells. Mitochondria are important targets of estrogen action. The cross-talk between the cell nucleus and the mitochondria appears to control estrogen-induced signaling involved in the apoptosis, proliferation, and differentiation of both normal and malignant cells. Mitochondria through its interaction with the cytoskeleton, export of cleaved signaling peptides, or generation of ROS appears to transduce signals to the nucleus for the activation of transcription factors involved in the cell cycle progression of estrogen-dependent cells. The understanding of the regulation of mitochondrial biogenesis by estrogenic compounds would open a new way to better understand steroidal and non-steroidal estrogen action at the cellular level. Estrogen actions at the mitochondria ------------------------------------ Besides apoptosis, respiration, and oxidative phosphorylation; mitochondria also control ion homeostasis and the synthesis of heme, lipids, amino acids, and nucleotides. Steroidogenesis is also controlled by mitochondria. Estrogen biosynthesis-related enzymes, 3 β-hydroxysteroid dehydrogenase and aromatase, have been demonstrated in the mitochondria of ovarian tumor epithelial cells \[[@B9]\]. ### Estrogen transport to mitochondria Although estrogen synthesis occurs in the mitochondria, exogenously added estrogen is also transported to this organelle. For instance, *in vivo*exposure of ovariectomized rats to tritiated 17-β-estradiol (E2) showed with increasing time, the translocation of this hormone from the plasmalemma mainly to the mitochondria (75%) rather than the nuclei in liver, adrenal gland, and spleen tissues \[[@B10]\]. The lipophilic property of E2 allows this molecule to easily diffuse into lipid bilayers. Since mitochondria are enriched with lipids, the organelle has the ability to act as an estrogen-sink within the cell. Although passive diffusion of estrogen into the mitochondria exists, a rapid delivery of E2 via receptor-mediated endocytosis from the plasma membrane to the mitochondria has been reported as a potential new pathway in HepG2 cells \[[@B11]\]. The uptake of E2-BSA from the medium by HepG2 cells occurred as early as 30 min. post-exposure and the ligand could be viewed in organelles that resembled vesiculated mitochondria found in steroid producing cells of the adrenal cortex and testes \[[@B11]-[@B13]\]. ### Estrogen effects on mitochondria morphology The impact of estrogen on mitochondrial morphology has previously been reported in the human breast cancer cell line MCF7. Transmission electron microscopy (TEM) revealed that an 8 day treatment of MCF7 cells with E2 (10 nM) resulted in large, clear mitochondria \[[@B14]\]. These alterations in mitochondria structure were observed as early as 2 days after treatment with a physiologically relevant dose of estrogen. The delamellated cristae of E2 treated mitochondria resemble an early anaerobic state of mitochondria development seen in embryonic rats and primates in which the cell depends on glycolysis \[[@B15]\]. However, it is not known whether the reported estrogen induced morphological changes had an affect on mitochondrial function. ### Estrogen receptor localization in mitochondria The function of estrogen at the mitochondria is not clear, however, recent studies have identified ERα and ERβ within the mitochondria implicating its role in the regulation of mitochondrial genome transcription. Subcellular fractionation of rabbit ovarian and uterine tissue revealed isoforms of ERα and ERβ in the mitochondrial enriched fraction as detected by western Blot analysis using ER specific antibodies \[[@B16]\]. More recently, ERβ was shown to be localized in the mitochondria of human lens epithelial cells (HLE-B3), human heart, rat primary neuron and primary cardiomyocyte, and in a murine hippocampal cell line HT-22 \[[@B17],[@B18]\]. Western blot analysis using polyclonal antibodies against human ERα and -β showed both ERs present in purified mitochondria isolated from the human breast cancer cell line MCF7 \[[@B19]\]. Using immunohistochemistry with confocal microscopy and immunogold electron microscopy, ERα and ERβ were identified in the MCF7 mitochondrial matrix. Mitochondrial ERα and ERβ were reported to account for 10% and 18%, respectively, of total cellular ER-α and -β when MCF7 cells were treated with E2. Treatment of MCF7 with E2 (10^-8^M and 10^-9^M) significantly increased the mitochondrial level of ERα and ERβ by 2.5-fold. ### Estrogen influence on mitochondrial gene expression An increasing body of evidence has shown that mitochondrial transcription is enhanced by estrogen treatment. For instance, a 16-fold increase in cytochrome oxidase II (CO II) mRNA is reported in the GH4C1 rat pituitary tumor cell line when treated for 6 days with E2 (0.5 nM) \[[@B20]\]. The mitochondrial gene for subunit III of cytochrome oxidase (CO III) is induced as early as 3 h following a single dose of E2 in the hippocampus of ovariectomized female rats \[[@B21]\]. Other mitochondrial transcripts have also been reported to increase in the human hepatoma cell line, HepG2, and rat hepatocytes when exposed to ethinyl estradiol (EE). A 40 h exposure to an EE concentration ranging from 0.5 to 10 μM resulted in a 2- to 3-fold induction of CO I, CO II, and NADPH dehydrogenase subunit 1 (NADPH-DH1) mRNA \[[@B22]\]. E2 (20 μM), although less potent than EE, showed a similar effect of induction from 1.5- to 1.8-fold in mitochondrial transcripts CO I, CO II, and NADPH-DH1 when treated for 12 h. The E2 catechol metabolite 4-OH-E2 caused a greater response in CO I and CO II transcript levels as compared to E2 after 24 h of treatement with a dose of 10 μM. The mitochondrial gene for ATP synthase subunit 6 (ATPase 6) was also elevated in female rat liver tissue exposed to EE (5 μg/day) for 42 days. An increase in the transcript level of COX7RP (cytochrome c oxidase subunit IV-related protein) was reported after a 6 h E2 (100 nM) treatment in MCF7 cells \[[@B23]\]. It is not known whether the COX7RP transcript is translated to a functional protein in the mitochondria, but the study proposed that COX7RP may represent a regulatory subunit of cytochrome c oxidase that modulates a high state of energy production in estrogen sensitive target tissues. More recently, a 12 h E2 (0.3 μM) treatment of MCF7 cells was demonstrated to enhance the mitochondrial transcript levels of CO I approximately fourfold and CO II\~2.5-fold \[[@B19]\]. The mechanism of estrogen-induced mitochondrial gene transcription is not clearly understood. The involvement of estrogen responsive elements (EREs) and/or the ER may be a possible mechanism in the increase of these mitochondrial transcripts. Sequences with partial similarity to the ERE consensus sequence, (AGGTCANNNTGACCT), have been reported in the mouse mitochondrial genome \[[@B24]\]. These partial EREs were detected in genes CO I and CO II which may account for the observed increases in these two transcripts in rat GH4C1 pituitary cells and rat hepatocytes \[[@B20],[@B22]\]. Other genes in which the various EREs were detected include 12S rRNA, 16S rRNA, tRNA-gln, cytochrome oxidase b, unidentified reading frame (URF) 4, URF5, and the D-loop region \[[@B24]\]. In the human mitochondrial genome, we identified partial or ERE 1/2 sites in the D-loop region, CO II, tRNA-met, 12S rRNA, 7S rRNA, URF1, and URF5 (unpublished Felty Q and Roy D). The presence of these partial EREs in the mitochondrial genome may lend support to a novel ER signal transduction pathway. A mechanism of ER translocation into the mitochondria and ER binding to mitochondrial EREs remains unclear. Using electrophoresis mobility shift assay (EMSA) and plasmon resonance analysis, the recombinant human ERα- and ERβ-containing mitochondrial proteins were demonstrated to specifically bind putative EREs in the mtDNA D-Loop, and this ER binding was enhanced by E2 treatment and inhibited by ICI 182780 \[[@B19]\]. Based on this evidence, it is biologically plausible that ER mediates mitochondrial transcription in the same manner as the glucocorticoid receptor (GR) which is translocated into the mitochondria and binds glucocorticoid response elements (GRE) after treatment with glucocorticoid \[[@B25],[@B26]\]. Whether estrogen-induced mitochondrial transcription participates in the development and growth of estrogen dependent breast cancer is not known. Long-term stilbene estrogen (diethylstilbestrol = DES) treatment of Syrian hamsters produced tumors in the kidney with a 5- to 10-fold higher transcript level of CO III than age-matched control kidneys \[[@B27]\]. Estrogen and the electron transport chain ----------------------------------------- Besides transcription of mitochondrial genes, estrogen has also been demonstrated to effect mitochondria at the protein level. Estrogen has been demonstrated in several studies to inhibit mitochondrial respiratory complex I, II, III, IV, and mitochondrial ATP synthase (F~0~F~1~-ATPase) \[[@B28]-[@B31]\]. Several studies have reported estrogen specific inhibition of mitochondrial respiratory proteins, but it is not clear whether estrogen can modify mitochondrial proteins at the post-translational level. However, there is a report that E2 increased the phosphorylation of a 76 kDa protein in the mitochondrial fraction of the rat corpus luteum \[[@B32]\]. The presence of protein kinases within the mitochondria together with evidence for estrogen-induced phosphorylation of mitochondrial proteins suggest that estrogen may regulate mitochondrial respiratory physiology at the post-translational level \[[@B33]\]. Besides a direct interaction with mitochondrial proteins, estrogen may indirectly effect the electron transport chain (ETC) through an increase of membrane fluidity. Given that thyroid hormone increases mitochondrial membrane fluidity \[[@B34]\] and that physiologic concentrations of estrogen can alter the fluidity of human red blood cell membranes \[[@B35]\], it is likely that a low dose of estrogen may facilitate electron transfer by increasing respiratory protein interactions through more membrane fluidity. Thus, the rate of electron transfer may increase as a consequence of more frequent collisions/interactions between respiratory chain complexes and electron carriers. The three proton pumps of the ETC depend on electron flow to generate the mitochondrial membrane potential (ΔΨ~m~). In human neuroblastoma cells, the following ER ligands: tamoxifen (30.2 μM), clomiphene (10.6 μM), and nafoxidine (2.8 μM); were reported to modulate ΔΨ~m~while E2 did not change ΔΨ~m~at concentrations up to 100 μM \[[@B36]\]. Assuming that estrogen can effect the fluidity of the inner mitochondrial membrane, a rise in ΔΨ~m~could result from an increased rate of electron transfer. The formation of ROS in mitochondria is reported to occur at high ΔΨ~m~\[[@B37],[@B38]\] and therefore suggests that estrogen modulation of ΔΨ~m~may be a possible mechanism for the generation of ROS. Whether physiologic concentrations of estrogen can increase ΔΨ~m~in target tissues is not clear, but these lines of evidence suggest that estrogen may modulate ΔΨ~m~in a dose- and tissue-specific manner. This effect is significant because estrogen-induced mitochondrial reactive oxygen species (ROS) may participate in cell signaling. In the following section, we provide a detailed review of the effects of E2 on mitochondrial respiratory complexes. ### NADH dehydrogenase (complex I) The effect of natural estrogens and synthetic estrogens on the mitochondrial ETC has been demonstrated in several studies. Human NADH dehydrogenase (complex I) is the largest respiratory chain complex consisting of 7 mitochondrial genome encoded subunits and more than 41 subunits encoded from the nuclear genome. This integral membrane protein is located within the mitochondrial inner membrane and the matrix. Two electrons enter the ETC from the oxidation of NADH by ubiquinone (CoQ) at complex I which is coupled with proton movement across the inner membrane from the matrix to the intermembrane space. Natural estrogens, 17-α-estradiol, E2, and estrone, at concentrations of approximately 10 μM were demonstrated to inhibit mitochondrial electron transport in homogenates of rat uterus, liver, and skeletal muscle \[[@B39]\]. The synthetic estrogen DES was also reported to inhibit electron transfer from complex I to CoQ at a half-maximal inhibitory concentration range of 0.2--2.6 μM \[[@B28]\]. Although DES at a dose of 20--30 μM could inhibit electron transfer by 90% it was much less effective than rotenone and piericidin A which could inhibit electron tranfer by 90--95% at a dose of 30--50 nM due to a tighter binding affinity to complex I. The researchers postulated that at relatively low doses, DES reversibly inhibits electron transfer at complex I. Additionally, DES displayed specific binding to a site in which rotenone and piercidin A bind to complex I. Since photoreactive analogues of rotenone have been reported to label complex I \[[@B40]\], these results implicate estrogen specific binding to respiratory complex I. Another class of compounds, phytoestrogens, which are found in our diet can also inhibit the activity of complex I. Phytoestrogens, genistein (found in soy beans) and resveratrol (found in red wine), can inhibit the activity of complex I, but are considered less active than rotenoid compounds \[[@B41]\]. There are very few reports that have investigated co-treatment of estrogen with mitochondrial inhibitors of oxidative phosphorylation (OXPHOS). Complex I inhibitors rotenone, piericidin A, and amytal have been used in co-treatment with DES and/or estrogens to elucidate the site of estrogen action on electron transfer \[[@B28],[@B39]\]. In MCF7 cells, it was demonstrated that co-treatment with rotenone (10 nM) and E2 (10 nM) strongly inhibited ornithine decarboxylase activity by 86% \[[@B42]\]. More recently, a study reported that treatment with 10 μM of 2-methoxyestradiol (2-Me) induced apoptosis in Ewing sarcoma cells through hydrogen peroxide (H~2~O~2~) production \[[@B43]\]. Since a 2 h prior treatment of rotenone (6 μM) inhibited 2-Me induced apoptosis and H~2~O~2~production, it was suggested that the H~2~O~2~source was the mitochondria. Although the biological significance of estrogen interaction with complex I is unknown, evidence of estrogen inhibition of electron transfer support a novel role of estrogen in the formation of mitochondrial ROS. ### Succinate dehydrogenase (complex II) Human succinate dehydrogenase (complex II) is a membrane bound protein located on the matrix side of the inner mitochondrial membrane. Complex II consists of 4 subunits all encoded from the nuclear genome. Two electrons enter the mitochondrial ETC from the oxidation of FADH~2~by CoQ at complex II. Although previous studies have reported complex I and cytochrome bc~1~reductase (complex III) as the major sites of ROS generation, ubiquinone radicals have been reported to contribute to basal levels of ROS at complex II \[[@B44]\]. Physiologically relevant ROS generation supported by the complex II substrate succinate occurs at complex I through reversed electron transfer \[[@B45]\]. Research studies of the protective effect of estrogen on the brain use the complex II inhibitor, 3-nitroproprionic acid (3-NPA) to model the condition of ischemia. Although the mechanism of estrogen neuroprotection is not clearly understood, estrogen has been proposed to modulate cerebral energy/glucose metabolism. Both 17-β-E2 and 17-α-E2 have been demonstrated in vivo to reduce ischemic brain damage induced by middle cerebral artery occlusion in ovariectomized rats \[[@B46],[@B47]\]. To model an in vitro state of interrupted energy metabolism as seen in cerebral ischemia and chronic neurodegenerative disease, the human neuroblastoma cell line SK-N-SH was treated with 3-NPA (10 mM) \[[@B48]\]. This study demonstrated that pretreatment of SK-N-SH with E2 (2 μM) restored ATP levels to 80% at 12 h as compared to the control cells treated with 3-NPA alone. Whether mitochondrial function can be preserved with a physiological concentration of estrogen is not clear as this study used a high dose (2 μM) which can be cytotoxic in certain tissues. The maintenance of ΔΨ~m~and ATP levels by estrogen when faced with 3-NPA toxicity was proposed to be due to the antioxidant effect and/or ATP increasing effects. At the 2 μM E2 dose an antioxidant effect is likely because estrogen is reported to be an effective neuroprotective antioxidant in the micromolar dose range \[[@B49]\]. However, another possibility may be due to an estrogen-induced increase in complex II activity which would overcome inhibition by 3-NPA as succinate dehydrogenase activity has been reported to increase in the brain of E2 treated rats \[[@B29]\]. Estrogen has been shown to cause multiple effects at the level of complex II that include the inhibition of electron transfer, maintenance of ΔΨ~m~and ATP levels, and the enhancement of succinate dehydrogenase activity. ### Cytochrome bc~1~reductase (complex III) The human cytochrome bc~1~reductase (complex III) is an integral membrane protein located in the inner mitochondrial membrane consisting of 10 subunits encoded from the nuclear genome and 1 subunit encoded from the mitochondria. Complex III transfers electrons from ubiquinol (CoQH~2~) to cytochrome c which is coupled to proton movement across the inner membrane. The synthetic stilbene estrogen, DES (10 μM--50 μM), is reported to inhibit complex III at the site of electron flow from ubiquinone to cytochrome c~1~\[[@B50]\]. The ER ligand tamoxifen is also reported to inhibit electron transfer at the site of complex III in isolated rat liver mitochondria \[[@B30]\]. The phytoestrogen resveratrol has been reported to bind both ER α/β, increase expression of estrogen responsive genes, and stimulate cell proliferation of MCF7 and T47D breast cancer cells \[[@B51],[@B52]\]. Interestingly, resveratrol has been shown to inhibit complex III activity (20%) by competition with ubiquinol (CoQH~2~) and preserve mitochondrial function by its action on complex III in isolated rat brain mitochondria \[[@B53],[@B54]\]. Besides the inhibition of electron transfer at complex III, the phytoestrogen genistein (50 μM) induced mitochondrial permeability transition (MPT) in isolated rat liver mitochondria. Since estrogen can inhibit electron transfer at complex III, the potential of estrogen to modulate the formation of mitochondrial ROS exists at multiple respiratory complexes. ### Cytochrome c oxidase (complex IV) In humans, the mitochondrial cytochrome c oxidase (Complex IV) is located in the inner membrane and catalyzes the transfer of electrons to oxygen with water being the final product of the reduction reaction coupled with proton movement across the inner membrane. The mitochondria genome encodes 3 subunits of complex IV while the other 10 subunits are encoded from the nuclear genome. The effect of estrogen and ER ligands on complex IV has been reported to increase and decrease enzymatic activity. During the oestrus cycle in rats, E2 decreased complex IV activity in brown adipose tissue, which suggests a role for E2 in the modulation of oxidative capacity \[[@B55]\]. The ER ligand tamoxifen was reported to restore complex IV activity to normal levels in disrupted rat liver mitochondria \[[@B56]\]. In rat liver mitochondria, tamoxifen was demonstrated to have a biphasic effect on complex IV in which a low concentration of 10--15 μM increased enzyme activity while a higher concentration of 50 μM inhibited activity by 50% \[[@B30]\]. Although the biological effect of estrogen actions on complex IV remains to be elucidated, it has been suggested that estrogen modulation of complex IV activity may increase energy production in estrogen sensitive tissues \[[@B23]\]. Furthermore, it has been proposed that ΔΨ~m~and ROS formation may be controlled by a hormone mediated reversible phosphorylation of complex IV \[[@B57]\]. Thus, the possibility for estrogen control of ROS formation by modulating complex IV activity may provide a mechanism of estrogen-induced redox signaling by the mitochondria. ### Mitochondrial ATP synthase (F~0~F~1~-ATPase/Complex V) Mitochondrial ATP synthase (F~0~F~1~-ATPase/Complex V) is composed of two distinct parts: 1) the F~1~-ATPase portion which protrudes into the matrix and synthesizes ATP when protons pass through it down their electrochemical gradient. 2) the F~0~-ATPase which forms a transmembrane proton channel through the inner membrane. F~0~F~1~-ATPase is encoded by 2 mitochondrial genome encoded subunits and 14 nuclear encoded subunits. F~0~F~1~-ATPase like respiratory chain complexes I-IV represents another enzyme in the mitochondrial inner membrane that is sensitive to estrogen. Inhibition of rat liver F~0~F~1~-ATPase by DES (10 μM) has been demonstrated and the F~0~portion was reported to contain a distinct binding site for DES \[[@B58]\]. This specific binding site for the F~0~portion has made DES a unique probe for the rapid isolation of functional F~0~from rat liver mitochondria \[[@B59]\]. Using E2-BSA conjugates, a 23 kDa estrogen binding protein was identified in rat brain mitochondria \[[@B60]\]. The 23 kDa protein was identified as the oligomycin-sensitivity conferring protein (OSCP) which forms the stalk region between F~0~and F~1~subunits of F~0~F~1~-ATPase in mitochondria. The OSCP was proposed to be the specific site on F~0~F~1~-ATPase where estrogen modulates ATPase activity. Differential effects of estrogen on F~0~F~1~-ATPase activity in isolated rat heart, liver, and brain mitochondria were observed when treated with E2, DES, and resveratrol \[[@B31]\]. E2 (13 nM) stimulated F~0~F~1~-ATPase activity in the heart by 10%, but not in the liver and brain. The phytoestrogen resveratrol (13--15 μM) inhibited F~0~F~1~-ATPase activity in the heart and liver while lower doses (133 pM--1.3 nM) stimulated F~0~F~1~-ATPase activity in the liver by 10%. Both phytoestrogens resveratrol (19 μM) and genistein (55 μM) can inhibit F~0~F~1~-ATPase activity in rat brain mitochondria \[[@B61]\]. In rat heart, liver, and brain the mitochondrial F~0~F~1~-ATPase activity was inhibited by DES (6.7 μM) 61%--67% \[[@B31]\]. In rat brain mitochondria, 17α-estradiol and E2 partly inhibited F~0~F~1~-ATPase activity at low concentrations of 15 nM and 3.4 nM, respectively, while 17α-E2 preserved mitochondrial function altered by the stress of anoxia-reoxygenation \[[@B62]\]. Although these studies of estrogen effects on F~0~F~1~-ATPase have been shown in isolated mitochondrial preparations, F~0~F~1~-ATPase inhibition by E2 was demonstrated to occur with intact human osteolclastic FLG 29.1 cells \[[@B63]\]. These studies of F~0~F~1~-ATPase activity demonstrate that estrogens and estrogen-like compounds possess cell-type and dose-specific effects on mitochondrial function. Modulation of mitochondrial ROS by estrogens -------------------------------------------- Within the cell, mitochondria are considered to be a major source of ROS, which include superoxide anion (O~2~^•-^), H~2~O~2~, and the hydroxyl free radical (^•^OH) \[[@B64]-[@B66]\]. Since mitochondria consume 85% of the oxygen used by the cell, the mitochondrial ETC generates a substantial amount of intracellular ROS \[[@B67]\]. As electrons pass through the mitochondrial ETC, some electrons leak out to molecular oxygen (O~2~) to form O~2~^•-^which is dismutated by manganese superoxide dismutase (MnSOD) to form H~2~O~2~\[[@B64],[@B68]\]. During mitochondrial respiration, 2% of the electron flow is reported to result in the formation of H~2~O~2~\[[@B66]\]. However, lower values of free radical leak were reported in the range of 0.4%--0.8% for heart mitochondria respiring on physiological concentrations of succinate (\<0.5 mM) \[[@B37]\]. In support of these findings, intact rat skeletal muscle, heart, and liver mitochondria were reported not to produce measurable amounts of ROS when respiring on complex I and complex II substrates \[[@B69]\]. In addition, this study reported a lower estimate of electron flow (0.15%) that contributed to H~2~O~2~production under resting conditions. These results suggest that mitochondria produce low levels of ROS that can be effectively scavenged by the cell\'s antioxidant defenses at resting conditions. It is this point, the low basal level of ROS produced by the mitochondria at rest, which makes mitochondrial ROS ideal signaling molecules since its contribution to the intracellular level of ROS is not at so high a level to induce oxidative stress; instead, a low oxidant level provides a physiologically safe window for redox signaling which allows the cell to regulate mild to moderate oxidative changes and critically respond to them by activating cellular processes such as proliferation and differentiation rather than triggering cell death. ### Characteristics of mitochondrial ROS Mitochondria are a predominant source of ROS in most cell types with unique characteristics that may allow it to participate in growth signal transduction. First, mitochondria are unique because they are a regulatable source of ROS in response to external stimuli. For example, cortical neurons exposed to N-methyl-D-aspartate (NMDA) were reported to couple a rise in intracellular calcium with mitochondrial O~2~^•-^production \[[@B70]\]. Tumor necrosis factor alpha (TNF-α) is another example of stimulated mitochondrial generation of O~2~^•-^in L929 cells and this ROS generation is coupled to the cytokine by the TNF-α receptor \[[@B71],[@B72]\]. Few other examples exist of mitochondria producing ROS in response to external stimuli, but more recently integrins (cell surface receptors that interact with the extracellular matrix) were reported to modulate mitochondrial ROS production for signal transduction \[[@B73]\]. Although signal pathways involved in triggering mitochondrial ROS remain largely unknown, it has been proposed that mitochondria participate in integrin signaling in a nonapoptotic manner, which leads to gene expression and cell differentiation. Mitochondrial ROS can enter the cytosol as either H~2~O~2~or O~2~^•-^where it can participate in redox signaling. Within the mitochondria, MnSOD can dismutate O~2~^•-^to H~2~O~2~which is a highly diffusible signaling molecule that can exit the mitochondria. In addition to H~2~O~2~, O~2~^•-^was demonstrated to be released by mitochondria to the cytosol via the voltage-dependent anion channels (VDACs) \[[@B74]\]. In regard to turning the mitochondria ROS signal off, cellular antioxidant defenses such as SOD, catalase, and glutathione peroxidase easily degrade ROS, which terminates the signal. Therefore, mitochondrial ROS fulfill the prerequisites of a 2^nd^messenger since they are short-lived (rapidly generated and degraded), produced in response to a stimulus, highly diffusible, and ubiquitously present in most cell types. Mitochondria are highly dynamic structures capable of changing their shape (by elongation, branching, swelling) and their location inside a living cell \[[@B75]\]. It is becoming clear that the morphological, functional, and genetic differences (heteroplasmy) that exist within the mitochondria population may reflect a division of labor within the cell. Mitochondria have been reported to be morphologically heterogeneous and unconnected within individual cells \[[@B76]\]. Pancreatic acinar cells were reported to contain distinct groups of mitochondria classified by their cellular location that included perinuclear, subplasmalemmal, and perigranular mitochondria \[[@B77]\]. In light of this finding, the highly diffusible H~2~O~2~generated by mitochondria may become a specific signaling molecule as a function of location. For example, perinuclear mitochondria may generate H~2~O~2~that only transduces signals to the nucleus or the subplasamlemmal mitochondria may only activate signal cascades of plasma membrane origin. Additionally, perinuclear, subplasmalemmal, and perigranular mitochondria were independently activated by intracellular calcium signals in their immediate environment, which supports distinct calcium functions for each type of mitochondria. It is significant that mitochondria can create subcompartments or \'microzones\' within the cytoplasm because signal transduction depends on the close proximity of substrates and effector molecules to be an efficient process. In addition, given the presence of other endogenous ROS sources besides the mitochondria such as NADPH oxidase, peroxisomes, cytochrome p450, xanthine oxidase, cyclooxygenase, lipooxygenase, and γ-glutamyl transpeptidase \[[@B78]\]; and because ROS is involved in a variety of signal cascades, understanding how mitochondrial ROS is activated at the right place and at the right time is vital in understanding the organelle\'s role in signal transduction. Compartmentalization has already been reported to play a key role in redox signaling and we consider this attribute when describing the mitochondria as a signal transducer \[[@B79]\]. In adult cells, mitochondrial clustering functions to create steep gradients of low molecular weight species such as O~2~, ATP, and pH resulting in specialized microzones that may facilitate signal specificity \[[@B80]\]. In the cytosol, the volume occupied by mitochondria in cells is highly variable and ranges from 15% to 50%. Based on volume, mitochondria compose a significant compartment within the cytosol that harbors signaling molecules. H~2~O~2~produced within the mitochondria is highly diffusible in contrast to O~2~^•-^, which cannot diffuse through membranes making it easily compartmentalized. Thus, mitochondrial generated O~2~^•-^may be kept separated from the cytosol until an appropriate stimulus releases it through VDACs. Another route for O~2~^•-^release may be through the mitochondrial permeability transition pore (MPTP) as low molecular weight compounds up to molecular weight 1500, can be exchanged between the mitochondrial matrix and the cytosol via this pore \[[@B81]\]. Since the MPTP is reported to reversibly open/close naturally in intact cells without resulting in apoptosis, mitochondrial signaling molecules could be exchanged with the cytosol by the transient \'flickering\' (open/closing) of the MPTP in response to certain stimuli \[[@B82]\]. In addition to location and compartmentalization, protein scaffolds mediate the selective activation of the mitogen activated protein kinase (MAPK) signaling pathway which raises the question of whether mitochondria may also act as a protein scaffold for signaling complexes \[[@B83]\]. A-kinase anchoring protein (AKAP) is reported to tether protein kinase A (PKA) to the mouse mitochondrial outer membrane \[[@B84],[@B85]\]. What makes AKAPs unique is their ability to simultaneously bind multiple signaling enzymes such as other kinases and phosphatases \[[@B86]\]. This multivalent scaffold has been described as a \'transduceosome\' capable of integrating signals from multiple pathways \[[@B87]\]. Whether these multivalent scaffolds exist on mitochondria is not clear at this time, but these signaling complexes could be a mediator of signals between the mitochondria and the nucleus during cell division. For example, AKAP84/121 has been demonstrated to concentrate on mitochondria in interphase and on mitotic spindles during metaphase transition alluding to its role in the cell cycle \[[@B88]\]. In addition to AKAP, a mitochondrial signaling complex has been reported to activate MAPKs. The PKCε can form signaling complexes with ERKs, JNKs, and p38 MAPKs in the murine heart \[[@B89]\]. Activated PKCε was shown to increase phosphorylation of mitochondrial ERK and p38 MAPKs. Whether the anchoring of PKC to mitochondria depends on AKAP is not known at this time. ### Mechanisms of estrogen-induced mitochondrial ROS Studies of the mitochondrial ETC have reported only two ROS forming sites, the FMN group of complex I and the ubiquinone site in complex III \[[@B45],[@B90]\]. The topology of ROS production, on which side of the mitochondrial inner membrane O~2~^•-^is produced, was reported to occur on the cytosolic side by complex III and on the martix side of the inner membrane by complex I \[[@B69]\]. Estrogen is known to act as either an antioxidant or pro-oxidant depending on the concentration \[[@B91]\]. Whether physiological concentrations of estrogen can stimulate mitochondrial ROS at complex I and complex III is not clear since most studies have been performed with cytotoxic doses. In the following section, we provide evidence for potential mechanisms of estrogen induced mitochondrial ROS. Electrons feed into the mitochondrial ETC at complex I and complex II. At complex I and complex II, ubiquinone or co-enzyme Q~10~(CoQ) oxidizes NADH and FADH~2~, respectively. CoQ functions as a mobile electron carrier within the mitochondrial inner membrane and transfers 2 electrons from both NADH and FADH~2~to complex III \[[@B92]\]. CoQ is known to offer protection from heart disease by increased ATP production and antioxidant actions \[[@B93]\]. It is a highly lipophillic compound due to its structure which includes an isoprenoid tail of usually 10 isoprene units in length, hence the designation Q~10~. Other than its role as an electron carrier and antioxidant, CoQ is also reported to act as a pro-oxidant. Although the pro-oxidative action of CoQ within the mitochondria is a matter of debate, O~2~^•-^formation occurs from a single electron transfer from ubisemiquinone to molecular oxygen. Exogenously added CoQ has been demonstrated to enhance O~2~^•-^generation in isolated respiratory complex I and III \[[@B94]\]. Further evidence in support of CoQ redox-cycling come from a study that demonstrated H~2~O~2~derived from decomposing O~2~^•-^was inhibited after the removal of CoQ \[[@B90]\]. Upon the re-addition of CoQ, H~2~O~2~was detected again which indirectly demonstrated that the O~2~^•-^may originate from CoQ. Although some reports make a case for O~2~^•-^formation by CoQ redox cycling in mitochondria, arguments against its role as a source of O~2~^•-^come from a study which demonstrated that O~2~^•-^formation did not occur in a water-free nonpolar reaction system that mimics the lipophilic nature of the inner mitochondrial membrane \[[@B95]\]. However, pretreatment of the membrane with toluene which increased its permeability to protons provided conditions in favor of O~2~^•-^formation by CoQ. Thus, it was proposed that under certain pathological conditions in which the inner mitochondrial membrane is protonated, CoQ becomes a significant source of O~2~^•-^\[[@B96]\]. In line with this result, a study demonstrated that CoQ (100 μM) enhanced the release of H~2~O~2~from mitochondria in the presence of antimycin A (2 μM) and to a lesser extent with Ca^2+^(10 μM) \[[@B97]\]. The antimycin A and Ca^2+^pretreatment was thought to induce a leaky inner mitochondrial membrane thereby allowing protons to interact with CoQ and enhance ROS production by redox cycling. Interestingly, CoQ shares similar characteristics to catechol metabolites of estrogen. The catechol metabolites 2- and 4-OH-E2 contain hydroxyl groups that can be oxidized to semiquinones, which in the presence of molecular oxygen can be further oxidized to quinones with the formation of O~2~^•-^\[[@B98]\]. Since CoQ undergoes reduction/oxidation (redox) reactions which result in the radical semiquinone intermediate (semiubiquinone) and quinone, it is biologically possible for catechol estrogens to participate in shuttling electrons and to act as a pro-oxidant like CoQ. Given that MCF7 cells treated with the o-quinone form of estrogen and NADPH produce significant amounts of H~2~O~2~in the mitochondrial subfraction \[[@B99]\]; and that mitochondrial enzymes catalyze redox reactons of stilbene estrogen in the mitochondria \[[@B100]\], the capacity of catechol estrogens to redox cycle within mitochondria suggests that these metabolites could facilitate mitochondrial ROS formation. Assuming that estrogen is a weak electron carrier compared to CoQ, it may have a tendency to leak electrons to molecular oxygen instead of transferring its electrons to complex III. The phytoestrogen and dietary flavinoid quercetin is reported to act as a pro-oxidant. Foods of plant origin contain flavinoids known to act as antioxidants or pro-oxidants depending on the concentration and metal chelates which catalyze the oxidative process \[[@B101]\]. Estrogen is similar to some flavinoids with respect to inhibition of the mitochondrial respiratory chain at complex I and complex II \[[@B102],[@B103]\]. Based on these reports, the formation of O~2~^•-^by estrogen at the level of electron transport could be one mechanism of increased mitochondrial ROS. Mitochondrial Ca^2+^, \[Ca^2+^\]~m~, accumulation is reported to promote the generation of ROS \[[@B104]\]. For example, an increase in \[Ca^2+^\]~m~was reported to stimulate H~2~O~2~production by rat brain mitochondria in the presence of rotenone \[[@B105]\]. Using confocal microscopy, we have shown a time-dependent increase in \[Ca^2+^\]~m~with E2 (100 pg/ml) treatment of MCF7 cells \[[@B106]\]. Another study reported an increase in \[Ca^2+^\]~m~with E2 (10 ng/ml) treatment of hippocampal neurons \[[@B107]\]. The mechanism for these increases in \[Ca^2+^\]~m~is not clear, however, the inhibition of Na-dependent Ca^2+^efflux from mitochondria was reported to increase calcium retention in E2 (1 nM) treated synaptosomal mitochondria \[[@B108]\]. There is some evidence which suggests allosteric inhibition of a respiratory complex may be a mechanism for hormone induced ROS formation. Allosteric inhibition of the respiratory complex IV (COIV) or cytochrome c oxidase is reported to occur by cAMP-dependent phosphorylation; and this inhibition is turned-off by Ca^2+^-activated dephosphorylation \[[@B109]\]. A study proposed that a hormone stimulated increase of cellular Ca^2+^may activate a mitochondrial protein phosphatase which dephosphorylates cytochrome c oxidase. In turn, cytochrome c oxidase is activated which results in a rise of ΔΨ~m~and ROS \[[@B57]\]. Interestingly, the ER ligand tamoxifen (15 μM) showed a slight stimulatory effect on cytochrome c oxidase \[[@B30]\]. Since estrogen is capable of increasing \[Ca^2+^\]~m~, it is possible for estrogen to signal the formation of mitochondrial ROS through a similar mechanism. The inhibition of respiratory complex I is known to favor ROS generation. Rat brain mitochondria that respired on complex I substrates produced a substantial level of ROS when inhibited with rotenone concentrations as low as 20 nM \[[@B110]\]. Since estrogen is known to inhibit respiratory complex I, we speculate that complex I interactions with the hormone could favor ROS production in a manner similar to rotenone. The phytoestrogen genistein is another flavinoid besides quercetin that acts like a pro-oxidant at the level of mitochondria. Genistein (50 μM) treatment of rat liver mitochondria was shown to increase ROS formation through interaction with respiratory complex III which resulted in the opening of the membrane transition pore \[[@B111]\]. Besides hormone interactions with respiratory enzymes, post-translational modifications such as phosphorylation-/-dephosphorylation that affect the activity of mitochondrial proteins should also be considered in ROS generation. The cAMP-dependent protein kinase is reported to phosphorylate 6-, 18-, 29-, 42-kDa mitochondrial proteins in bovine heart and phosphorylate the human 18-kDa subunit which promotes the activity of complex I \[[@B112]-[@B115]\]. Since estrogen is reported to stimulate cAMP-dependent protein kinase activity in hippocampal neurons, it raises the possibility for estrogen to induce cAMP accumulation in mitochondria \[[@B116],[@B117]\]. If estrogen increased cAMP levels within mitochondria, then cAMP-dependent phosphorylation of mitochondrial respiratory complexes may modulate ΔΨ~m~and/or \[Ca^2+^\]~m~in favor of ROS generation. Several isoforms of the enzyme nitric oxide synthase (NOS) are reported to exist which include inducible-(i), endothelial-(e), neuronal-(n), and mitochondrial-(mt) NOS. Estrogen has been reported to induce various isoforms of NOS. The activity of eNOS is modulated by estrogen in human aortic endothelial cells, uterine artery, heart, and skeletal muscle \[[@B118],[@B119]\]. Estrogen has also been shown to stimulate protein expression of nNOS in human neutrophils and the transcription of iNOS in rat macrophages \[[@B120],[@B121]\]. These effects are not limited to E2 because the estrogen-like chemical bisphenol A and the phytoestrogen resveratrol are also reported to stimulate NO synthesis \[[@B122],[@B123]\]. These lines of evidence demonstrate a significant role of estrogen compounds in the modulation of NOS and NO. In regards to redox signaling, a NO-dependent inhibition of cytochrome c oxidase has been proposed to generate O~2~^•-^which is dismutated into the membrane permeable second messenger H~2~O~2~\[[@B124]\]. Since estrogen is capable of inducing NOS activity and expression, we postulate that an estrogen induced rise in NO could participate in a similar manner whereby generating mitochondrial H~2~O~2~. Interestingly, NO induced mitochondrial biogenesis has been demonstrated in several cell lines, which include brown adipocytes, 3T3-L1, U937, and HeLa cells \[[@B125]\]. We have shown that estrogen can influence mitochondrial biogenesis (data unpublished) and postulate that estrogen-induced NO could be one possible mechanism. More specifically, since mtNOS activity is dependent on Ca^2+^, we propose that an estrogen-induced rise in \[Ca^2+^\]~m~could stimulate mtNOS activity ultimately leading to the generation of ROS via NO-dependent inhibition of cytochrome c oxidase \[[@B126]\]. Estrogen-induced growth of cells in relation to mitochondria ------------------------------------------------------------ The rapid stimulation of intracellular ROS by platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and nerve growth factor (NGF) suggests that this underlying mechanism of cell growth may be shared with other growth factors including estrogen \[[@B127]\]. Exogenous addition of low concentrations of H~2~O~2~and/or O~2~^•-^has been demonstrated to stimulate cell growth in a variety of cell types including muscle cells, fibroblasts, amnion cells, prostate cancer cells, and aortic endothelial cells \[[@B78]\]. The molecular signaling mechanism that initiates ROS production by mitochondria is not clear, however, other cell processes besides apoptosis may be coupled to this signaling event. Tumor necrosis factor alpha (TNF-α) induces gene expression via mitochondrial respiratory chain dependent activation of NF-κB, AP-1, JNK, and MAPKK \[[@B73]\]. The proliferative response of endothelial cells to hypoxia was demonstrated to be initiated upstream by mitochondrial ROS which activated the MEK/ERK pathway \[[@B128]\]. Although other endogenous ROS sources besides mitochondria such as NAD(P)H oxidase exist, mitochondria will be the focus of this paper for the following reasons: (*i*) mitochondria are the principal source of intracellular ROS in epithelial cells. (*ii*) the growth of adenocarcinomas occur in tissue of epithelial cell origin. A characteristic of rapidly dividing cancer cells is their capacity to produce significant amounts of intracellular ROS, which has been implicated in the promotion of accelerated cell cycle activity in neoplastic cells. Mitochondria have long been suspected to play a role in the development and progression of cancers. The ROS molecules H~2~O~2~and NO have been demonstrated to stimulate mitochondrial biogenesis, a process that depends on the flow of molecules into and out of the organelle \[[@B125],[@B129]\]. Since mitochondrial proteins are encoded in two separate genomes (mitochondria and nuclear genome), biogenesis is a coordinated effort in which mitochondria transmit signals to the nucleus and vice versa. The question of how mitochondria transmit these signals in the process of cell proliferation has risen from reports of its involvement in cell growth. Cerebral granular cells isolated from newborn rats with high mtNOS activity were reported to exhibit maximal proliferation rates which depended on NO and H~2~O~2~levels. In addition, MnSOD displayed an increased pattern of activity similar to mtNOS \[[@B130]\]. NO has been proposed to inhibit cytochrome c oxidase in favor of O~2~^•-^production and therefore MnSOD may dismutate O~2~^•-^generated by NO-dependent inhibition into the signaling molecule H~2~O~2~. Ethinyl estradiol, E2, and estrogen catechol metabolites at a dose of 0.25 to 5 μM are reported to increase mitochondrial O~2~^•-^in cultured rat hepatocytes and HepG2 cells \[[@B131]\]. Although the biological significance of the estrogen-induced mitochondrial O~2~^•-^is not known at this time, ROS has been demonstrated to modulate ER protein expression in various cell lines. Treatment of human breast cancer cells MCF7 and T-47D with H~2~O~2~(2.5 μM) increased the protein level of ERβ \[[@B132]\]. In addition, PMA (100 ng/ml) treatment increased the expression of ERβ in the macrophage cell line J774A.1. Evidence for the involvement of redox signaling with estrogen-induced cell proliferation has been demonstrated in several studies. Liposomes containing SOD or catalase inhibited *in vitro*estrogen-induced proliferation of Syrian hamster renal proximal tubular cells \[[@B133]\]. The cytokines IL-1β and TNF-α are known to cause the release of O~2~^•-^from human fibroblast cells. Co-treatment with an inhibitor of IL-1β and TNF-α synthesis, pentoxifylline, inhibited stilbene estrogen-induced increase in myeloperoxidase activities, 8-hydroxydeoxyguanosine (8-OHdG) formation, mutations in the testicular genome, and prevented estrogen-induced testicular preneoplastic lesions \[[@B3]\]. Recently, we have shown that estrogen-induced stimulation of macrophage cells and MCF7 cells in part occurs through ROS \[[@B134],[@B135]\]. We have also observed inhibition of estrogen-induced MCF7 cell growth by ROS scavengers such as N-acetylcysteine, ebselen, and catalase (unpublished Singh M, Felty Q and Roy D). ROS can modulate effector molecules such as PKC, p53, extracellular regulated kinase (ERK), nuclear factor-κB (NF-κB), and the c-fos/c-jun heterodimer (AP-1); and these effector molecules are known to participate in growth signal transduction \[[@B136]\]. Therefore, estrogen-induced production of mitochondrial ROS may activate cell growth in estrogen-sensitive tissues. Oxidative stress has been shown to affect mitochondrial proteins of chronically estrogenized Syrian hamster kidney. A decrease in thiol/sulfhydryl groups was reported in the mitochondrial fraction at a preneoplastic stage of carcinogenesis \[[@B137]\]. Estrogen-induced oxidative stress may be responsible for these post-translational modifications in mitochondrial proteins. This finding is significant in the context of cell signaling because redox reactions involving cysteine thiol groups transduce signals by breaking or forming protein dithiol/disulfide bridges \[[@B138]\]. Since estrogen can induce mitochondrial ROS, we infer that the oxidation of thiols in response to estrogen converts the oxidative stress to a change in protein function involved in cell growth. Oxidative stress modifies mitochondrial matrix protein thiols \[[@B139]\]. Similarly, thiols on protein subunits 51-kDa and 75-kDa of NADH dehydrogenase (complex I) have been reported to form mixed disulfides with glutathione (glutathionylation) in response to mitochondrial oxidative stress. This post-translational modification was reversible and correlated with an increase in mitochondrial O~2~^•-^production \[[@B140]\]. Evidence in support of a ROS signal transduction pathway originating from complex I comes from a study which reported that the mitochondrial complex I inhibitor, rotenone, blocked ROS mediated signaling. Interestingly, this study demonstrated that a co-treatment of rotenone (10 nM) and E2 (10 nM) inhibited ornithine decarboxylase activity by 86% in MCF7 cells \[[@B42]\]. Since ornithine decarboxylase activity is a marker for cell growth, it appears that a signal transduction pathway for estrogen-induced cell growth may originate from the mitochondria assuming that rotenone inhibition is specific to complex I. The antitumor arotinoid, mofarotene (Ro 40-8757), has been demonstrated to down-regulate mitochondrial encoded NADH dehydrogenase subunit 1 (MtND1) expression in breast cancer cell lines MDA-MB-231, ZR-75-I, and MCF7 \[[@B141]\]. Since MtND1 has been reported to form part of the rotenone-binding site in complex I \[[@B40]\], the absence of MtND1 may remove an important site of estrogen action in mofarotene treated cells and may account for the anti-proliferative effects of this compound. Whether these protein interactions and/or modifications can occur as a result of estrogen exposure remains to be investigated. From these investigations, we infer that estrogen mediated cell growth via mitochondrial generated ROS signaling molecules may exist and merits future exploration to address this novel pathway. The mitochondrial thioredoxin system has been demonstrated to play a role in cell cycle progression. In general, the two antioxidant oxidoreductase enzymes thioredoxin (Trx) and thioredoxin reductase (TrxR) that compose the system modulate signal transduction properties of ROS by the reduction of intracellular disulfides. Trx acts as a protein disulfide reductant for ribonucleotide reductase and several transcription factors including p53, NF-κB, and AP-1 \[[@B142]\]. Once oxidized the active disulfide site is reduced by TrxR re-generating the reductant form of Trx. Enzyme isoforms Trx-2 and TrxR2 are reported to exist in the mitochondria. A biological role for TrxR2 in cell growth was demonstrated in HeLa cells using a dominant negative form of TrxR2 (TrxR2DN) \[[@B143]\]. An increase of G~1~to S phase transition, cell growth, and transcription of cell cycle genes was induced by TrxR2N expression. TrxR2DN expression was suggested to increase intracellular H~2~O~2~, which in turn signaled cell proliferation. Although it is not clear whether estrogen can modulate the H~2~O~2~levels of mitochondria, estrogen (10 nM--100 nM) treatment of primary human endometrial stromal cells *in vitro*show an increase in Trx protein and mRNA which implicate Trx involvement in cell growth and differentiation of estrogen responsive tissue \[[@B144]\]. Alterations in cellular redox status by increased expression of TrxR2 have been suggested to play a role in the growth of hepatocellular carcinomas \[[@B145]\]. Whether estrogen can signal cell growth through Trx2 and/or TrxR2 is not known, but these findings suggest that estrogen may modulate signal transduction of mitochondrial derived ROS via the thioredoxin system. Transduction of estrogn-induced mitochondrial signals to nucleus ---------------------------------------------------------------- Mitochondrial ROS fulfill the characteristics of a 2^nd^messenger since they are short-lived (rapidly generated and degraded), produced in response to a stimulus, highly diffusible (H~2~O~2~), and ubiquitously present in most cell types. It is not known whether mitochondrial ROS like H~2~O~2~are involved in signaling pathways that control estrogen-induced cell proliferation. In this section, we provide evidence in support of redox signaling pathways of mitochondrial origin, which may be involved in cell cycle progression of estrogen-dependent cells. ### Redox sensor proteins and transcription Protein kinases are known to participate in phosphorylation signal cascades, however, zinc finger domains contained in some proteins may allow them to participate in redox signaling networks. Zinc finger structures within a protein consist of at least two zinc-coordinated thiolates. Upon oxidation zinc is released from the protein, which converts cysteine thiol groups to disulfide. A conformational change in the protein may result in either its activation or inhibition. There are several protein kinases such as a-raf and PKC that contain zinc finger domains. In addition, zinc finger domains are also found in hormone receptors such as the GR and ER \[[@B146]\]. Both PKC and c-raf have been demonstrated to be redox activated at the zinc finger domain \[[@B147],[@B148]\]. For instance, ROS can trigger the release of zinc ions from PKC which results in its activation. Another protein kinase, c-raf, known to participate in the MAPK signal cascade was also demonstrated to be redox activated at the zinc finger domain. The mitochondrial localization of protein kinases src, Akt, a-raf, and PKC is evidence that this subcellular compartment harbors oxidant sensitive proteins that may facilitate cross-communication between redox and phosphorylation networks \[[@B33],[@B89]\]. Although the role of the protein kinase a-raf in the mitochondria is not clear, a-raf mRNA is highly expressed in normal murine tissues such as the epididymis, ovary, kidney, and urinary bladder \[[@B149]\]. In Hela cells, epidermal growth factor rapidly (2 min.) and transiently activated a-raf, which in turn phosphorylated the MAP kinase activator MEK1 \[[@B150]\]. Therefore, mitochondrial ROS may activate MAPK signaling via a-raf. It is interesting to note that E2 can stimulate the phosphorylation of a-raf and cell cycle progression in MCF7 cells \[[@B151]\]. Whether the estrogen induced phosphorylation of a-raf depends on ROS is not known. Mitochondrial PKCδ and PKCε could also activate the raf/MEK/ERK pathway or directly activate MAPKs, respectively \[[@B152],[@B153]\]. Rapid effects of estrogen have been demonstrated to mediate the DNA binding activity and phosphorylation of transcription factors. E2 treatment of rat adipocytes doubled AP-1 DNA binding and phosphorylated CREB protein within 15 min \[[@B154]\]. The redox sensitive protein Akt is known to phosphorylate an upstream kinase, IKKα, which stimulates the degradation of Iκ-B \[[@B155]\]. Estrogen-induced mitochondrial ROS may stimulate Akt leading to the degradation of Iκ-B and activation of the transcription factor NF-κB. Whether estrogen treatment can activate Akt via mitochondrial derived ROS is not clear, however, phosphorylation and translocation of Akt to the mitochondria was demonstrated when cells are treated with estrogen \[[@B156]\]. Given that E2 can stimulate mitochondrial ROS generation; ER, src, a-raf, Akt, and PKC are targets of oxidative stimuli localized at the mitochondria; and the transcription factors AP-1, NF-κB, and CREB are stimulated by oxidants \[[@B127],[@B131],[@B157]\]; it is possible that estrogen specific effects at the level of mitochondria can activate these transcription factors. Based on these studies we postulate that estrogen-induced mitochondrial ROS stimulates oxidant sensors a-raf, Akt, or PKC, which in turn activate transcription factors such as NF-κB, CREB, or AP-1 via the MEK/ERK pathway resulting in the transcription of cell cycle genes containing DNA responsive elements for NF-κB, CREB, or AP-1 and ultimately estrogen-induced cell proliferation (Fig. [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Hypothetical scheme outlining three E2-induced signaling pathways from mitochondria. (*i*) E2 binding to a plasma membrane receptor and/or mitochondrial respiratory complexes generates ROS which leads to kinase activation. (*ii*) E2-induced rise in mitochondrial calcium leads to the activation of calcium-dependent proteases which process signal peptides, in turn responsible for kinase activation. (*iii*) E2-induced cytoskeleton modifications by mitochondria leads to kinase activation. Increased kinase activity results in the activation of transcription factors responsible for cell cycle progression. ::: ![](1477-3163-4-1-1) ::: ### Estrogen and mitochondrial-cytoskeleton interactions Mechanical signals associated with cytoskeletal tension generation and cytoskeleton restructuring are a requirement for anchorage dependent cells to pass through the late G~1~restriction point \[[@B158]\]. Since these cytoskeleton dependent effects on the G~1~checkpoint are independent from the MAPK signaling pathway, a new question rises of whether mitochondria can modulate cell growth by interacting with the cytoskeleton. Mitochondria are reported to be associated with three major cytoskeletal structures, which include microtubules, microfilaments of actin, and intermediate filaments \[[@B159]\]. Mitochondrial tubulin and microtubule associated proteins (MAPS) are reported to bind to porin or VDAC a component of the permeability transition pore \[[@B160]\]. The association of the cytoskeleton with VDAC could be biologically significant because the actin filament severing and capping protein gelsolin has been reported to modulate ΔΨ~m~by its interactions with VDAC \[[@B161]\]. Epithelial cell spreading results from the binding of integrins to the extracellular matrix which depends on the actin cytoskeleton \[[@B162],[@B163]\]. The survival of epithelial cells depends on this interaction with the extracellular matrix, which if disrupted leads to a specific form of apoptosis called anoikis \[[@B164]\]. Actin filaments are necessary to cluster integrin receptors and proteins linked to their cytoplasmic domain into focal adhesion complexes \[[@B165]\]. These focal adhesion complexes provide a direct link between the extracellular matrix and the actin cytoskeleton. Anchorage-independent growth is a property of cancer cells, which may depend on the mitochondria based on evidence from the following studies. Long-term exposure of cells to ethidium bromide, an intercalation agent which inhibits mtDNA replication, results in the depletion of mitochondria. Mitochondria-depleted (ρ°) brain and breast tumor cells have been shown to lose their ability for anchorage-independent growth \[[@B166]\]. In addition, human ρ° cell lines derived from ovarian carcinoma, cervical carcinoma, and osteogenic sarcoma were demonstrated to be non-tumorigenic or poorly tumorigenic when administered subcutaneously to nude mice \[[@B167]\]. Taken from these reports is the interesting possibility that cancer cells maintain tension on the cytoskeleton via the contraction and expansion of mitochondria instead of binding to the extracellular matrix. Actin assembly and disassembly is regulated by the protein gelsolin. Since gelsolin is reported to prevent apoptotic mitochondrial changes by binding and closing VDAC, perhaps other diverse functions are modulated by interactions between the mitochondria and cytoskeleton. More recently, it was demonstrated that mitochondrial ROS production is stimulated by integrin induced changes \[[@B73]\]. Although integrin receptors are linked to the actin cytoskeleton, it is not clear whether the signal that is transduced to the mitochondria occurs via the cytoskeleton. Furthermore microtubules have also been implicated in the biogenesis of mitochondria based on the inhibition of mitochondrial mass increase and mtDNA replication caused by the microtubule-destabilizing drug colchicine and the estrogen metabolite 2-methoxyestradiol in mammalian cells \[[@B168]\]. Mitochondria biogenesis is reported to occur in the G~1~phase of the cell cycle but also starts in the late S phase \[[@B169]\]. Although mitochondria were not reported to be an upstream signaling event for generating cytoskeleton tension, physical effects of mitochondria such as shape changes and stretching or contraction could generate tension based on its association with the cytoskeleton. Changes in cytoskeleton tension may be mediated by mitochondria in response to estrogen. For instance, transmission electron microscopy (TEM) showed an increase in mitochondria size of MCF7 cells treated with E2 \[[@B14]\]. This change in mitochondrial size may generate mechanical forces that, in turn, may transduce a signal to the nucleus. Another possibility is that estrogen stimulated mitochondrial ROS may affect the elasticity of the actin network. Actin filaments are a prevalent feature of the cytoskeleton, which partially determine the overall mechanical strength of a cell. Thiol oxidation forms disulfide-bonded actin dimers resulting in interfilament cross-links \[[@B170]\]. Estrogen-induced oxidative stress has been recently reported to oxidatively modify cysteine residues of proteins \[[@B137]\]. Thus, estrogen-induced mitochondrial ROS could stimulate the formation of actin dimers that modulate cytoskeleton tension which in turn transduces a signal to the nucleus (Fig. [1](#F1){ref-type="fig"}). ### Mitochondrial proteolysis and peptides as signals The mitochondrial protein cytochrome c is known to be released to the cytosol where it initiates a signal for apoptosis. Given the role of cytochrome c the existence of other mitochondrial protein signaling molecules is a likely possibility that could mediate a diverse number of cellular processes including cell growth. It has been shown that mitochondria have the capability to export mitochondrial-matrix proteins to other cellular compartments such as the nucleus, peroxisome, endoplasmic reticulum, and secretory vesicles \[[@B171]\]. For example, mitochondrially transmitted factors (MTFs) are peptides derived from mitochondrial encoded proteins that are presented on the cell surface as minor histocompatability antigens. MTFs are derived from the mitochondrial encoded NADH dehydrogenase subunit 1 gene in murine and humans while rat MTFs are derived from the mitochondrial encoded ATPase 6 gene \[[@B172],[@B173]\]. Although the synthesis and cell surface expression of MTFs was inhibited by the mitochondria specific protein synthesis inhibitor chloramphenicol, it is not clear whether post-translational modifications of mitochondria proteins are also responsible for MTFs; given that chloramphenicol is also an inhibitor of proteolysis in rat liver mitochondria \[[@B174]\]. Thus, mitochondria may serve as a subcellular compartment of proteolysis that generates signaling peptides that are exported to the cytosol. It is possible that proteolysis plays a significant role in mitochondrial protein processing because the chemical rhodamine 6G (R6G) inhibits matrix catalyzed processing of rat-liver mitochondrial precursors which include iron-sulfur protein, cytochrome c~1~, and core protein I of the cytochrome bc~1~complex; the α and β subunits of F1 ATPase and subunit IV of cytorochrome oxidase \[[@B175]\]. The molecular mechanism of R6G inhibition of protein processing was not identified, but it was proposed to be due to an interaction between R6G and a matrix protease. The import of proteins into mitochondria has been investigated in great detail while the process of export is minimally explored. A novel ATP-binding cassette (ABC) transporter, Mdl1, located in the inner mitochondrial membrane of yeast is required for the export of mitochondrial peptides with a molecular mass of 2100 to 600 daltons generated by proteolysis \[[@B176]\]. It was suggested that the export of peptides from the mitochondria may allow the mitochondria to communicate with its environment. This novel mode of communication may exist based on studies that demonstrate mitochondrial specific cleavage and export of cytokines. The cytokine IL-1β is localized in the mitochondria of LPS stimulated human peripheral blood monocytes and the 31 kDa form of IL-1β is reported to be cleaved into the 17 kDa mature active form within the mitochondria upon exposure to the HIV coat glycoprotein 120 \[[@B177],[@B178]\]. Since macrophages secrete IL-1β by the ABC transporter, it is possible that proteins up to 17 kDa may be exported from the mitochondria \[[@B179]\]. In addition to IL-1β, mitochondrial Trx protein may also participate in a similar protein export mechanism. A truncated form of Trx, Trx80, is reported to be a potent mitogenic cytokine, however, it is not known whether Trx80 is derived from mitochondrial Trx \[[@B180]\]. Based on these reports it appears that cleaved proteins may be released as growth factors in response to an estrogen-induced rise in mitochondrial calcium that activates proteolysis. Thus, cleaved signaling peptides from the mitochondria may stimulate cell growth in an autocrine manner (Fig. [1](#F1){ref-type="fig"}). Several proteases are known to exist in the mitochondrial matrix such as ATP-dependent human Lon protease, ATP-dependent human Clp proteinase chain P (hClpP), and Ca^2+^dependent neutral protease \[[@B181]-[@B183]\]. Since endogenous proteolysis is a mechanism that regulates cell cycle progression, we postulate that E2-induced rise of mitochondrial calcium can activate calcium dependent proteases such as calpeptin and possibly hClpP. Once activated mitochondrial matrix proteins could be cleaved into bioactive forms that are exported to the cytosol. The heterogeneous association between ERα cleavage products and regulatory proteins has been suggested to play a role in physiological or pathological processes \[[@B184]\]. Low molecular weight ERα isoforms (\~35--28 kDa) have been identified in mitochondria \[[@B16]\]. The 44-kDa protein related to the nuclear RXRα receptor is reported to be enzymatically cleaved and imported into the mitochondrial matrix \[[@B185]\]. It may interact with mitochondrial proteins or bind the organelle genome. Once in the cytosol mitochondrial clevage products may also regulate the function of various enzymes. For example, a truncated ERα 46-kDa protein in human endothelial cells mediated an acute activation of eNOS in response to a 15 min E2 (30 nM) treatment \[[@B186]\]. A significant finding from this report is that the truncated ERα 46-kDa mediates acute responses of estrogen rather than transcriptional responses in endothelial cells. Recently, it was reported that physiologic concentrations of E2 (\<10 nM) induced NOS1 and activates the cGMP signal transduction pathway leading to sustained expression of Trx and MnSOD in human SH-SY5Y cells \[[@B187]\]. Thus, the acute activation of mtNOS by ERα cleavage products is yet another interesting possibility for redox signaling. Conclusion ========== In summary, mitochondria are a major target of estrogen. It appears that mitochondria through its interaction with the cytoskeleton, export of cleaved signaling peptides, and/or generation of ROS may transduce signals to the nucleus for the activation of transcription factors, such as, AP-1, NF-κB, and CREB involved in the cell cycle progression of estrogen-dependent cells. These interactions between estrogen and mitochondria merit furture investigations, which may shed new light on the role of mitochondria in cell growth. Acknowledgements ================ This work was partly supported by the NIEHS grant ES10851 to DR; financial support to QF through NCI Cancer Prevention and Control Training Grant (CA 4788).

PubMed Central

2024-06-05T03:55:52.492207

2005-1-15

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548143/", "journal": "J Carcinog. 2005 Jan 15; 4:1", "authors": [ { "first": "Quentin", "last": "Felty" }, { "first": "Deodutta", "last": "Roy" } ] }

PMC548144

Background ========== The establishment of successful pregnancy requires a profound reorganization of uterine tissues. Rapid growth and differentiation of the endometrial stroma is the earliest and most striking event in pregnancy. Differentiation of stromal cells leads to the formation of unique cells, termed decidual cells, which differ greatly from the original stromal cells \[[@B1]\]. The decidua is an important component in the maternal recognition of pregnancy and can be induced by either the implantation of the blastocyst or by artificial stimuli. An interesting feature is that -- the growth and differentiation of the endometrial cells -- occurs differently in different regions of the uterus \[[@B1]\]. Mesometrial decidual cells are formed after the antimesometrial decidua and their death takes place after antimesometrial cell degeneration. Regression of the decidual cells appears to be controlled by an intrinsic cell death program of apoptosis, which takes place after day 10 of pregnancy in the antimesometrial region first, followed by the mesometrial region \[[@B2]\]. Prostaglandin F~2α~can induce different biological actions at the beginning and at the end of pregnancy. PGF~2α~, Prostaglandin E~2~(PGE~2~), and 6-keto-PGF~1α~are produced by the pregnant uterus \[[@B3]\]. An increase of uterine PGE~2~and PGF~2α~is observed on day 5 of pregnancy, allowing the decidualization process to take place. When embryo access to the uterus is impaired, production of prostaglandins (PGE~2~and PGF~2α~) is suppressed \[[@B4]\]. During postimplantation, PGE2 returns to the original preimplantation levels, but PGF~2α~decreases \[[@B4]\]. Whereas PGF2α contributes to the process of decidualization, implantation and recognition of pregnancy. An increase in PGF~2α~over certain values can terminate pregnancy \[[@B6]\]. A high level of PGF~2α~is known to induce inhibition of implantation, alteration of embryo development, and induction of luteal regression \[[@B5]\]. During infection, an inflammatory response mediated by cytokines can be generated \[[@B7]\]. This release of PGF~2α~can induce premature uterine contraction and premature labor \[[@B8]\]. Depending on which stage of pregnancy an increase of PGF~2α~secretion occurs, it can alter implantation, induce abortion or even embriolethality \[[@B7],[@B8]\]. Because an increase in PGF~2α~levels after implantation can be detrimental for the progression of pregnancy, the aim of this investigation was to determine whether PGF~2α~affects directly decidual cells and leads to disturbance in the expression of genes crucial for decidual survival. Methods ======= Animal model ------------ Pseudopregnancy was induced by mating Holtzman Sprague Dawley female rats with vasectomized male rats. The day a vaginal plug was found was designated day 1 of pseudopregnancy. Decidualization of the uterine endometrium was induced by scratching the antimesometrial surface with a hooked needle on day 5 of pseudopregnancy under ether anaesthesia. Animals were housed in a controlled environment (22--24°C) and kept under controlled conditions (lights on; 0500--1900 hrs) with free access to standard rat chow and water. The University of Illinois at Chicago animal care and use committee approved the animal care and handling. Primary decidual cell culture and hormone treatment --------------------------------------------------- For each experiment 15 pseudopregnant rats were used. Decidual cells were isolated as previously described \[[@B9]\]. Cells were pooled and seeded into six-well plates (1.4 × 10^6^cells/ well). They were allowed to attach 3--4 hrs before washing in PBS to remove erythrocytes. Cells were incubated in RPMI 1640 without phenol red (Mediatech, Whashington DC), containing 1% CD-FBS (HyClone Laboratories Inc, Logan, UT), and treated with or without high levels (5 μM \[[@B10],[@B11]\]) of PFG~2α~(Sigma, St. Louis, MO) for 12 hr). After incubation, the cells were harvested in cold PBS, quick frozen in liquid nitrogen and kept at -80 C until RNA isolation. Gene identification by cDNA array --------------------------------- Total RNA was isolated from primary decidual cells (PDC) by using a RNAII isolation kit (Clontech, Palo Alto, CA), following the manufacture\'s instructions. RNA isolated from wells treated with either PGF~2α~or vehicle were pooled independently and subjected to cDNA array. cDNA probes were generated from 4 μg of total RNA in a reverse transcriptase reaction using a mix of dATP, dTTP and dGTP (Takara Biomedicals, Shiga, Japan) plus \[α^32^P\]-dCTP (Amersham, MO), and a mixture of primers specific to each gene present in the array. All probes used had 5 to 10 × 10^6^cpm and the difference between control and experimental probes in each assay was less than 10%. cDNA were hybridized to an Atlas Rat 1.2 Array (\#7854-1) nylon membrane (Clontech, CA). Hybridization and post-hybridization washes were performed according to the manufacturer\'s protocol. The signal was scanned with a phosphorImager (Molecular Dynamics, CA) after 48 h of exposure. Control and experimental RNA were always processed in parallel. Data analysis ------------- Spot intensities from scanned membranes were analyzed using the AtlasImage 1.5 software (Clontech, CA). Grids were orientated manually and adjusted to ensure optimal spot recognition using AtlasImage\'s fine tuning tools, discarding spots with dust or locally high background. The software makes the analysis for background correction and normalization versus housekeeping genes. It also calculate the ratio and indicates which genes has a ratio \> 2 (up regulated), or ratio \< 0.5 (down regulated) or 0.5 \< ratio \< 2 (equal expression). Gene expression data were normalized using the Sum method included in the Atlas Image software. Data points where the expression was not greater than two standard deviations were discarded. For the final analysis, data points were the averages from triplicates and any non-reproducible data were discarded. The relative RNA expression with differences between control and treatment being higher or equal to 2 and lower than 0.5 were considered significantly different \[[@B12]\]. Results ======= Effect of PGF~2α~on gene expression in primary decidual cells ------------------------------------------------------------- From the total genes available in the rat 1.2 membrane arrays, only 20% of the genes were detected in rat primary decidual cells. Sixty genes were significantly down regulated, whereas only six genes were up regulated by PGF~2α~(Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}). PGF~2α~-receptor gene expression was similar in both control and treated groups. On the other hand, no differences in the expression of housekeeping genes such as ribosomal proteins and β-actin could be detectedin control and PGF~2α~treated cells (data not shown). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Representative cDNA expression array using mRNA obtained from rat primary decidual cells treated with either PGF~2α~or vehicle. ::: ![](1477-7827-3-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### cDNA expression array of rat primary decidual cells treated with PGF~2α~or vehicle. ::: ![](1477-7827-3-3-2) ::: Effect of PGF~2α~on the expression of genes related to the extracellular matrix (ECM) ------------------------------------------------------------------------------------- The majority of genes whose expression was affected by PGF~2α~in primary decidual cells are genes involved in the regulation of the ECM. Genes such as gelatinase A (MMP2), cathepsin L, tissue inhibitor metalloproteinases 2 (TIMP2) and 3 (TIMP3), plasminogen activator inhibitor1 (PAI1), tissue type plasminogen activator (tPA), urokinase plasminogen activator (uPA), endothelin 1, calponin, carboxypeptidase D and calponin acidic were down regulated. The opposite effect was observed for prostromelysin 53 kDa (proMMP3), plasma proteinase I alpha and alpha 1 antiproteinase -- all of which were significantly up regulated by PGF~2α~(Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of PGF~2α~on the expression of genes related to the extracellular matrix (ECM). ::: **Gene Bank number** **Swiss protein number** **Name** **Function** **Ratio** **% decrease** **Fold increase** ---------------------- -------------------------- ---------------------------------------------- -------------------- -------------- ---------------- ------------------- U65656 P97581 Gelatinase A Metalloproteinase 0.04 ± 0.02 87.53 Y00697 P07154 Cathepsin L Cysteine protease 0.09 ± 0.05 91.09 L31884 P30121 Tissue inhibitor metalloproteinase 2 (TIMP2) Protease inhibitor 0.05 ± 0.01 95.54 U27201 P48032 Tissue inhibitor metalloproteinase 3 (TIMP3) Protease inhibitor 0.04 ± 0.01 91.95 X63434 P29598 Urokinase type Plasminogen activator (uPA) Serine protease 0.14 ± 0.09 94.86 M24067 P20961 Plasminogen activator inhibitor (PAI) Protease inhibitor 0.09 ± 0.05 91.68 M2367 P19367 Tissue type plasminogen activator (tPA) Serine protease 0.17 ± 0.02 89.90 X71071 Q08290 Calponin Cytoskeleton 0.08 ± 0.004 92.50 U06755 P37397 Calponin, acidic Cytoskeleton 0.24 ± 0.13 76.76 U62897 O35850 Carboxypeptidase D Carboxypeptidase 0.24 ± 0.14 76.48 M64711 P22388 Endothelin 1 Hormone 0.34 ± 0.2 77.48 X02601 P03957 Polypeptide, 53 kDa, growth factor induced Metalloproteinase 2.54 ± 1.04 3 J03552 P14046 Plasma proteinase inhibitor α1-inhibitor III Protease inhibitor 3.64 ± 0.64 4 M32247 P17475 α1 antiproteinase Protease inhibitor 2.45 ± 0.44 3 Ratio = net intensity of PGF~2α~normalized/net intensity of control normalized, and represent the mean ± SEM of each ratio from separate experiments. Ratio \> 2 = up regulation; ratio \< 0.5 = down regulation; 0.5 \< Ratio \< 2 = equal expression. ::: PGF~2α~regulation of genes related to the proteasome protein component system ----------------------------------------------------------------------------- Messenger RNA encoding proteasome Iota, proteasome component C2, proteasome subunit RC7-I, 26-S proteasome regulator and proteasome component C3 were down regulated by PGF~2α~. Only proteasome component C9 was up regulated after PGF~2α~treatment (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### PGF~2α~regulation of genes related to the proteasome component system. ::: **Gene Bank number** **Swiss Protein number** **Name** **Function** **Ratio** **% decrease** **Fold increase** ---------------------- -------------------------- ---------------------------- --------------------- ------------- ---------------- ------------------- D10755 P34062 Proteasome Iota Proteosomal protein 0.07 ± 0.04 93.64 M29859 P18420 Proteasome component C2 Proteosomal protein 0.31 ± 0.15 78.28 U77918 Q63569 26-proteasome component C3 Proteosomal protein 0.39 ± 0.08 61.94 J02897 P17220 Proteasome component C3 Proteosomal protein 0.31 ± 0.02 59.02 D21799 P40307 Proteasome subunit RC7-I Proteosomal protein 0.18 ± 0.01 82.62 X55986 P21670 Proteasome subunit C9 Proteosomal protein 2.32 ± 0.72 3 ::: PGF~2α~regulation of genes involved in trafficking and signal transduction -------------------------------------------------------------------------- The mRNAs encoding for 14-3-3ε and z/δ proteins, and Annexin IV were down regulated in PDC after PGF~2α~treatment (Table [3](#T3){ref-type="table"}). PGF~2α~down regulated the expression of serum glucocorticoid kinase (SGK), casein kinase I, extracellular signal-regulated kinase (ERK-1), male germ cell-associated kinase (MAK), and Wee tyrosine kinase. PGF~2α~also significantly inhibited Crk-associated kinase (CAS), calcium calmodulin kinase II (CAMKII) and IV (CAMKIV), phospholipase C1, and protein phosphatases such as phosphatase 2A and protein tyrosine phosphatase 1B (Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### PGF~2α~regulation of genes encoding proteins involved in trafficking and signal transduction. ::: **Gene Bank number** **Swiss protein number** **Name** **Function** **Ratio** **% decrease** **Fold increase** ---------------------- -------------------------- ------------------------------------------------- ------------------------- ------------- ---------------- ------------------- D17615 P97286 14-3-3 z/δ protein Traficking protein 0.14 ± 0.07 86.07 M84416 P29360 14-3-3 Epsilon protein Traficking protein 0.29 ± 0.15 71.63 L01624 Q06226 Serum Glucocorticoid kinase (SGK) Kinase 0.25 ± 0.12 93.49 L07578 Q06486 Casein kinase I δ Kinase 0.29 ± 0.15 72.18 M61177 P21708 Extracellular signal-regulated kinase 1 (ERK1) Kinase 0.29 ± 0.13 66.67 D29766 Q63766 Crk-associated kinase Kinase 0.09 ± 0.06 87.16 M35862 P20793 Male germ cell-associated kinase (Mak) Kinase 0.20 ± 0.11 78.6 D31838 Q63802 Wee tyrosine kinase Cell cycle kinase 0.23 ± 0.12 69.47 M20637 P10688 Phospholipase C δ1 Phospholipase PI kinase 031 ± 0.15 68.24 J04503 P20650 Protein phosphatase 2C α Phosphatase 0.18 ± 0.03 58.87 L12385 P26438 Protein Phosphatase 2A Phosphatase 0.38 ± 0.13 65.91 L13408 P15791 Ca^+2^/Calmodulin dependent kinase II δ subunit Kinase 0.34 ± 0.12 72.12 M63334 P13234 Ca^+2^/Calmodulin dependent kinase IV Kinase 0.29 ± 0.15 70.90 M33962 P20417 Protein Tyrosine phosphatase 1B Phosphatase 0.21 ± 0.08 79.77 D38224 P55260 Annexin IV (ANX4) Exocytosis protein 0.14 ± 0.08 86.34 L24810 Q64572 Ca^+2^/Calmodulin Phosphorylase B Kinase 5.06 ± 2.06 5 ::: PGF2α effect on the expression of genes associated with G-proteins, growth factors, chemokines and cytokines ------------------------------------------------------------------------------------------------------------ The 5-hydroxytryptamine receptor 2A (HTR2A), adenosine A2A and A2B receptors (ADORA2A and ADORA2B respectively), guanine nucleotide-binding protein G α3 subunit (GN-BPG α~3~), guanine nucleotide-binding protein α stimulating (GN-BP α stimul.), Rab 11A and guanine nucleotide-binding protein α12 subunit (GN-BP α~12~) gene expression were down regulated by PGF~2α~(Table [4](#T4){ref-type="table"}). The mRNA expression of growth factors such as bone morphogenetic protein 4 (BMP4), ADP ribosylation factor 5 and 6 (ADPRF5 and 6 respectively), glia maturation factor β (GMFB), and transforming growth factor β I (TGF-βI) decreased after PGF~2α~treatment as well (Table [1](#T1){ref-type="table"}). Interferon inducible protein (Table [4](#T4){ref-type="table"}) and leukocyte common antigen (data not shown) were the only cytokine and chemokine related genes found significantly down regulated in response to PGF~2α~. Conversely, PGF~2α~stimulated the expression of basic fibroblast growth receptor factor 1 (bFGRF1) (Table [4](#T4){ref-type="table"}). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Effect of PGF~2α~on the expression of genes associated with G-proteins, growth factors, chemokines and cytokines. ::: **Gene Bank number** **Swiss protein number** **Name** **Function** **Ratio** **% decrease** **Fold change** ---------------------- -------------------------- ------------------------------------------------------ -------------------------- -------------- ---------------- ----------------- X52498 P17246 Transforming Growth Factor β1 Growth factor 0.33 ± 0.2 72 M60921 P27049 Antiproliferative B-cell translocation gene 2 (BTG2) Growth factor 0.28 ± 0.15 51 X61381 P26376 Interferon-induced protein Chemokine 0.25 ± 0.14 56 L11586 Q64604 Leukocyte common antigen Intracell. phosphatase 0.07 ± 0.02 83 Z22607 Q06826 Bone Morphogenetic protein 4 (BMP4) Growth factor 0.34 ± 0.17 78 L12384 P26437 ADP Ribosylation factor 5 Intracellular transducer 0.02 ± 0.008 67 L12385 P26438 ADP Ribosylation factor 6 Intracellular transducer 0.18 ± 0.07 83 Z11558 Q63228 Glia maturation factor β (GMFB) Growth factor 0.17 ± 0.07 83 M30705 P14842 5-hydroxytryptamine receptor 2A (HTR2A) G proteins 0.06 ± 0.03 93 S47609 P30543 Adenosine A2A receptor (ADORA2A) G proteins 0.13 ± 0.08 87 M91466 P29276 Adenosine A2B receptor (ADORA2B) G proteins 0.17 ± 0.07 83 M20713 P08753 Guanine nucleotide-binding protein G α3 subunit G proteins 0.22 ± 0.14 78 M17525 P04894 Guanine nucleotide-binding protein α stimulating G proteins 0.24 ± 0.13 75 M75153 P24410 Rab-11A G proteins 0.18 ± 0.11 82. D85760 Q63210 Guanine nucleotide-binding protein α12 subunit G proteins 0.15 ± 0.09 85 D12498 Q04589 Basic Fibroblast Growth Receptor factor 1 Growth factor 2.32 ± 1.07 3 ::: Discussion ========== Levels of PGF~2α~in the uterus need to be under tight control to avoid interference with the establishment and progression of pregnancy. Pathophysiological elevations in PGF~2α~lead to excessive uterine contractions. Therefore, PGF~2α~production must be avoided to maintain a quiescent uterus \[[@B13]\]. In this paper we present initial data demonstrating that elevated PGF~2α~can target the decidua, an endocrine tissue whose integrity is fundamental for the success of implantation and for the progression of pregnancy. Our results demonstrate that elevated PGF~2α~down regulate the expression of decidual genes related to the proteasome protein component system, those involved in trafficking and signal transduction and genes associated with G-proteins, growth factors, chemokines and cytokines. However, interestingly, the main effect of PGF~2α~is related to the turnover and degradation of the ECM which provides mechanical strength to the tissue. Thus, in addition to inducing uterine contractions, PGF~2α~can have a noxious impact on the success of implantation and progression of pregnancy, at least in part by deregulating the turnover of the ECM in the decidua. The largest group of genes down regulated by PGF~2α~is directly or indirectly related with the turnover and degradation of the ECM. Genes such as gelatinase A (MMP-2), TIMP2 and 3 \[[@B14],[@B15]\], cathepsin L \[[@B16]\], carboxypeptidase D are included in different groups of proteases, and can directly affect the degradation of the ECM. Carboxypeptidase D (CPD) or metallocarboxypeptidase D is a 180-kDa protein that contains almost three carboxypeptidase-like domains, a transmembrane domain, and a cytosolic tail. This gene participates in the processing of proteins transiting the secretory pathway \[[@B17]\]. TGF-β is a key factor that favours accumulation of collagen, laminin and fibronectin in the ECM \[[@B17]\]. TGF-β can stimulate PAI-1, inhibiting the protease degradation of the ECM. Other factors included in the plasminogen/plasmin and fibrinolytic system, such as tPA, uPA and PAI-1, can participate in the tissue remodelling of the ECM directly through the binding to specific receptors, and through the transformation of the plasminogen precursor bound to the cell surface to plasmin, which is an active serine protease. Plasmin is able to degrade most of the components of the ECM either directly or indirectly by the activation of MMPs. All of the plasminogen/plasmin factors mentioned previously participate in the process of decidualization \[[@B19]-[@B21]\]. Endothelin-1 is a peptide characterized as a potent endothelial cell-derived vasoconstrictor. It is synthesized as an inactive precursor (preproendothelin) and processed to a mature active form (endothelin) by zinc metalloproteinases. The active form, endothelin-1, promotes synthesis of collagen types I and II by fibroblasts, affects the ECM remodelling and promotes the proliferation of mesangial cells \[[@B21]\]. Endothelin-1 is also associated with neovascularization and regulation of blood flow \[[@B22]\]. This vasoactive factor is also involved in the genesis of endothelial cells behaviour \[[@B22],[@B23]\]. Annexin IV (ANX4, also called Lipocortin) was differentially down regulated by PGF~2α~. This protein belongs to a family of intracellular proteins that binds membrane phospholipids in a calcium-dependent manner. Thus, it can also inhibit phospholipase A2 (PLA2) \[[@B24]\]. ANX4 can also directly bind glycosaminoglycan (GAG) and can be localized not only to the cytoplasm but also the cell surface or the extracellular compartment \[[@B25]\]. It has been proposed that ANX4 could affect the mobilization of different substrates involved in the regulation of the ECM. Moreover, Annexin IV and other annexins can act as ligands for proteoglycans localized on the cell surface, in the ECM, or in secretory granules \[[@B24],[@B25]\]. Calponin (CaP) has three genetic isoforms, h~1~, h~2~and acidic calponin, and can be identified by the individual C-terminal tail sequences. The c-terminal sequences regulate actin association and the cytoskeleton \[[@B26],[@B27]\]. It is known that Cap or basic Cap inhibit the actomyosin ATPase in a calmodulin dependent manner \[[@B27]\]. Basic Cap, by affecting microtubules assembly, can modify the cytoskeleton of the cells, and indirectly, the associated ECM \[[@B27],[@B28]\]. On the other hand, acidic Cap belongs to the family of actin-associated proteins. It can interact with F-actin but not with microtubules, desmin filaments, and tropomyosin as basic Cap does. These properties suggest that acidic Cap is functionally distinct from basic Cap and could affect the ECM in a different manner \[[@B29]\]. Pro-stromelysin, α~1~antiproteinase, plasma proteinase-Iα inhibitor, basic fibroblast growth receptor factor 1, phosphorylase B and proteasome component C9 were the only genes up regulated by PGF~2α~in PDC. Pro-stromelysin is a 53 kDa peptide and a pro-MMP3 precursor and is directly related with the ECM regulation \[[@B30]\]. Plasma proteinase 1α inhibitor belongs to a major group of proteins that includes α2-macroglobulin \[[@B31]\], an important protein secreted by the decidual cells which has a critical role in the control of implantation \[[@B31]\]. Growth factor receptors, such as bFGRF, are part of a multigene family of structurally related factors (FGFs 1 to 9), some of which bind heparin sulphate proteoglycans that are components of the ECM \[[@B32]\]. bFGRFs as well as bFGF are also temporally and spatially present in the pregnant rat uterus \[[@B33]-[@B35]\]. Another member of the TGFβ superfamily expressed in the uterus during early implantation and down-regulated by PGF~2α~was BMP4, a gene which appears to be involved in specific stages of embryo development and is \[[@B36]\]. Proteasome component system proteins, such as proteasome Iota, proteasome component C2, proteasome subunit RC-7 I, 26 S-proteasome and proteasome component C3 were down regulated by PGF~2α~. Proteosomal component systems are the main non-lysosomal proteolytic structures of the cells that participate in the elimination of abnormal proteins, short half-life proteins, and proteins controlling cell cycle \[[@B37]\]. During the process of cell differentiation, the level of proteasome expression and its localization varies. The proteasomal proteins can be intermediaries of ECM by contributing to the modulation of the cell cycle through the induction of proteasomal degradation of cyclin dependent kinase 2. Cell attachment to ECM components, such as fibronectin (FN), does not affect p21 mRNA levels, but the stability of the p21 protein decreased \[[@B36]\]. Kinase activities such as ERKs, calcium/calmodulin kinase II and IV, SGK, and casein kinase can affect the ECM downstream or upstream \[[@B38]\]. On the other hand, ECM can regulate the availability of substrates, as well as factors or effectors downstream or upstream related with different cascades of signal transduction pathways. For example, proteins such as decorin are components of the ECM in many tissues and appear to be involved in matrix assembly \[[@B39]\]. Decorin can cause the rapid phosphorylation of EGF with the concurrent activation of mitogen-activated kinase protein kinase signal pathway. Via TGFβ, decorin can interact with the MAP kinases signal transduction pathways and cross talk with calcium /calmodulin-dependent kinase II \[[@B40]\], which in the cDNA array was down-regulated by PGF~2α~. Crk-associated substrate (CAS) was down-regulated by PGF~2α~and is another gene related to the ECM. Active CAS can modulate changes in cell motility and gene expression by the various MAP kinase cascades, and modify the organization of the actin cytoskeleton \[[@B39]\]. Another gene down-regulated by PGF~2α~was SGK, which is a transcriptionally-regulated serine/threonine protein kinase with 45--55% homology to the catalytic domain of Akt/PKB protein kinase A \[[@B40]\]. Skg is expressed in decidual tissue and can be activated by the phosphoinositide 3-kinase pathway (PI3-Kinase) through PDK1-mediated phosphorylation \[[@B41]\]. Traficking proteins such as 14-3-3 z/δ and ε are involved in the regulation of genes related to the ECM because it can block the activation cascade of signal transduction pathways \[[@B42]\]. Protein phosphatase type 2A (PP2A) was down regulated by PGF~2α~. The first and most important point of control of PP2A is at the transcriptional level. The increase of phosphatase activity corresponded with a decrease in the phosphorylation of cellular proteins in anchorage-dependent cells, but much lesser regulated in anchorage-independent cells \[[@B43]\]. It is well known that members of the proinflammatory cytokine family can induce MMP expression in numerous tissues \[[@B44]\]. Also, cytokines and chemokines, such as interferon inducible protein and leukocyte common antigen, are associated with the local induction of MMP expression in response to proinflammatory cytokines. The indirect action of these molecules through MMPs may aid to generate different changes in the endometrial stroma during maternal recognition of pregnancy \[[@B44]\] We have also compared the pattern of gene expression in rat decidual tissue *in vivo*on day 12 of pseudopregnancy (when the decidua undergoes regression) with that of the PGF~2α~treatment in rat PDC *in vitro*(data not shown). Interestingly, we found a 49% coincidence in the genes that were down regulated in both experimental situations. This coincidence suggests a relationship between the physiological regression and reorganization of the decidual tissue that occurs on day 12 of pseudopregnancy, with the pattern of gene expression in rat PDC after PGF~2α~treatment. On the other hand, many of the genes whose expression was affected by PGF~2α~in the rat PDC, are expressed in the decidua during decidualization and implantation. This suggests a possible connection between the action of PGF~2α~and the physiology of the decidual tissue. Moreover, the ECM is an important component of the decidualization and implantation process. Any modification in its turnover or degradation could affect the formation of the decidua, blastocyst invasion, and the timing of decidual regression and reorganization. It is known that the expression of MMPs/TIMPs plays an important role in the control of implantation. If PGF~2α~silences or decreases the expression of genes related with the systems aforementioned, it also could affect tissue remodelling by directly modifying the proteases involved in this process, or indirectly by affecting the ECM turnover and degradation, important in the accumulation of a spongy mass of tissue around each embryo during decidualization \[[@B45]\]. Most probably, many of PGF~2α~\'s effects on the expression of genes involved in ECM turnover are indirect. Alterations of genes involved in different signalling pathways such as ERK-1, CaMCKII, PLCδ1 and G-protein subunits may impact the expression of a great number of transcripts. In summary, our results show, for the first time, that pathophysiological concentrations of PGF~2α~have a severe impact on the expression of numerous genes associated with the turnover of the ECM in the rat decidua. Future investigation should corroborate the differentially regulated genes, at the level of the message, protein, and in some cases, such as for the metalloproteinases, at the level of activity of the proteins. These data will contribute to the design of future studies on a cluster of gene candidates as targets of PGF~2α~action in this endocrine tissue. Abbreviations ============= TIMP2 = tissue inhibitor metalloproteinases 2; TIMP3 = tissue inhibitor metalloproteinases 3; PAI 1 = plasminogen activator inhibitor 1; uPA = urokinase type; tPA = tissue type palsminogen activator; TGFβ = transforming growth factor β; SGK = serum glucocorticoid kinase; ERK-1 = extracellular signal-regulated kinase-1; MAK = male germ cell-associated kinase; CAS = crk-associated kinase kinase; CamKII = calcium calmodulin kinase II; CamKIV = calcium calmodulin kinase IV; PLC δ~1~= phospholipase C delta 1; BMP 4 = bone morphogenetic factor 4; ADPRF 5 = ADP ribosylation factor 5; bFGFRF1 = basic fibroblast growth factor receptor F1; HTR2A = 5-hydroxytryptamine receptor 2A; ADORA2A = adenosine receptor A2A; ADORA2B = adenosine receptor A2B; GN-BPG α~3~= guanine nucleotide-binding protein G α~3~; GN-BP α stimul. = guanine nucleotide-binding protein α stimulating; GN-BPG α~12~sub. = guanine nucleotide-binding protein α~12~subunit Authors\' contributions ======================= EC and SFG carried out the decidualization, and primary decidual cell culture. EC isolated the RNA isolation, performed the cDNA array assay, the analysis of the results, and drafted the manuscript. GG conceived the study and edited the manuscript. All authors read and approved the final manuscript. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### PGF~2α~has major effects on extracellular matrix (ECM) regulation ::: ![](1477-7827-3-3-3) ::: Acknowledgements ================ This research was supported by NIH grant number NIH HD 12356 and NIH, U54 HD 40093. We thank Dr. CM Telleria and Gil Gibori for constructive reading of the manuscript.

PubMed Central

2024-06-05T03:55:52.497858

2005-1-11

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548144/", "journal": "Reprod Biol Endocrinol. 2005 Jan 11; 3:3", "authors": [ { "first": "Eduardo A", "last": "Callegari" }, { "first": "Susan", "last": "Ferguson-Gottschall" }, { "first": "Geula", "last": "Gibori" } ] }

PMC548145

Introduction ============ Severe acute respiratory syndrome (SARS) is the first new infectious disease of this millennium. SARS has originated from Southern China at the end of 2002 and has a high mortality and morbidity. Within a period of six months beginning at the end of 2002, the disease has affected more than 8,000 people and killed nearly 800 \[[@B1]\]. The disease poses a new threat for respiratory medicine and represents a challenge for antiviral drug development and administration \[[@B2],[@B3]\]. SARS is caused by a novel, SARS-associated coronavirus (SARS-CoV) \[[@B4]-[@B6]\] which has been identified by a World Health Organization (WHO)-led global laboratory network. The first cases of SARS were reported from a hospital in Hanoi, Vietnam, by Carlo Urbani, a WHO scientist who himself died from the disease \[[@B7]\]. After reports from health authorities in Hong Kong on the outbreak of a new form of epidemical atypical pneumonia in public hospitals, the WHO issued a global alert on the disease. During this period, cases of SARS were also reported from China, other Asian countries and even other continents including America (Canada, U.S.A.) and Europe (Germany). Shortly after the initial global alert, the WHO initiated a collaborative multi-center research project on SARS diagnosis, led by eleven principal laboratories in nine countries \[[@B8]\]. Using modern communication technologies to optimize the analysis of SARS tissue samples, it was soon shown that a novel coronavirus is the causative agent of SARS (SARS-CoV) \[[@B4]-[@B6]\]. Due to the death of Carlo Urbani who first identified the new disease, the first isolate of the virus was proposed to be named Urbani strain of SARS-associated coronavirus, but a final terminology has not been proposed so far \[[@B9]\]. Since Koch\'s principles have been shown to be fulfilled by the new pathogen \[[@B10],[@B11]\], it is not necessary to call the virus SARS-associated and the general agreement is now to call it SARS coronavirus (SARS-CoV). Parallel to the progress made in the epidemiology and clinical diagnosis which has recently been demonstrated by numerous case reports, clinical studies and definitions \[[@B1]\], scientists have also revealed basic mechanisms of the underlying causative agent, the SARS coronavirus. As it is crucial for future strategies that SARS is detected in its earliest stages and that therapeutic options are optimized, insights into the molecular mechanism of SARS have to be used to develop new therapeutic strategies and vaccines. While other reviews have focused on the epidemiology, clinical presentation and potential treatment of SARS, the present overview aims to analyze and present the currently available data on molecular mechanisms of SARS. In this respect, it is important to underline that in the present state of no specific drug or vaccine being available, research on molecular mechanism is crucial to identify potential treatment targets. Etiology -------- Prior to the development of therapeutic regimes based on molecular mechanisms of the disease, the causative agent had to be isolated and analysed. Soon after the fast establishment of the international WHO laboratory network, rapid progress was made in the identification process of the causative agent, and it was reported that SARS is most probably caused by a novel strain of the family of coronaviruses \[[@B4]-[@B6]\]. These viruses are commonly known to cause respiratory and gastrointestinal diseases of humans and domestic animals \[[@B12],[@B13]\]. The group of coronaviruses is classified as a member of the order nidovirales, which represents a group of enveloped positive-sense RNA viruses consisting of coronaviridae and arteriviridae \[[@B14]\]. Viruses of this group are known to synthesize a 3\' co-terminal set of subgenomic mRNAs in the infected cells \[[@B15]\]. Origin of the SARS virus ------------------------ Soon after the identification of a new coronavirus as the causative agent of SARS and of a southern Chinese province as the first area of occurrence, animal species of this area have been speculated to be the origin of the SARS-CoV. As analysis of the SARS-CoV genetic sequence revealed large differences to any other currently known coronaviruses in humans or domestic animals \[[@B16],[@B17]\], it was hypothesized that the new virus might originate from wild animals. This hypothesis was supported by a search for coronaviruses in wild animals sold on markets in southern China, which identified the presence of a coronavirus in civet cats. This animal coronavirus was shown to have a sequence identity of more than 99% to the SARS coronavirus \[[@B18]\] with only a limited number of deletions and mutations between both viruses. SARS-CoV has a deletion of 29 nucleotides relative to the civet cat virus, indicating that if there was direct transmission, it went from the animal to man, because deletions occur probably more easily than insertions. Recent reports indicate that SARS-CoV is distinct from the civet cat virus and it has not been answered so far if the civet cat virus is the origin of the SARS-CoV or if civet cats were also infected from other species \[[@B19]\]. Therefore, there are no data available on the possibility of horizontal transmission between animals, and the question whether the jump of the virus from an animal to humans was a single accident or may frequently occur in future with the animals as dangerous reservoirs for future SARS epidemics remains unanswered. So far, the SARS-CoV has been reported to be able to infect not only humans but also macaque monkeys \[[@B11]\], domestic cats, and ferrets \[[@B20]\]. However, transmission of the virus from the domestic cat to man has not been shown. The ability of the SARS-CoV to infect other animal species could point to potential natural reservoirs of the virus. In this respect, coronaviruses are known to relatively easily jump to other species. I.e., the human coronavirus OC43 shares a high degree of genetic sequence homology to bovine coronavirus (BCoV) and it is commonly assumed that it has jumped from one species to the other \[[@B21],[@B22]\]. In the same way, BCoV has been reported to be able to infect humans and cause diarrhea \[[@B23]\]. Whereas the precise mechanisms of these species jumps remain unclear, it is most likely that they represent the results of mutations and epidemiological studies of coronavirus infections in wild animals will therefore be crucial for future understanding and control of new SARS outbreaks. SARS virus taxonomy ------------------- Until the identification of the new SARS-CoV, the coronaviruses have been divided into three subgroups, which differ with respect to their genome \[[@B24]\]. The first group consists of viruses such as the human coronavirus 229E (HCoV-229E), porcine respiratory coronavirus (PRCV), porcine transmissible gastroenteritis virus (TGEV), feline infectious peritonitis virus (FIPV) and feline enteritis virus (FEV) or the canine coronavirus (CCoV). The second group comprises human coronavirus OC43 (HCoV-OC43), bovine coronavirus (BCoV), and mouse hepatitis virus (MHV), and the third group mainly consists of avian species such as the chicken infectious bronchitis virus (IBV). Whereas the SARS-CoV has been shown to cross-react with some group I coronavirus antibodies \[[@B6]\], its genetic sequence does not belong to this group. Within the nucleic acid or protein sequence phylogenetic trees of the coronavirus family, the SARS-CoV has first been located at an equal distance from the second and third group, irrespective of which SARS-CoV RNA region is used for analysis \[[@B6],[@B16],[@B17]\]. Therefore, the SARS-CoV may represent the first member of a new group of coronaviruses (Figure [1](#F1){ref-type="fig"}). However, the taxonomy is still no clear \[[@B19],[@B25]\], and recent studies that focused on the N-terminal domain of the spike protein and on poorly conserved proteins such as Nsp1, matrix protein, or nucleocapsid, have suggested a relation to group II viruses \[[@B26]\]. A similar conclusion can be drawn if the polymerase gene is examined, pointing to an early split-off from the coronavirus group 2 lineage \[[@B27]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Coronavirus classification. The family of coronaviruses belongs to the order of nidovirales and consists of three groups so far. It is still debatable whether the new SARS-CoV should be assigned to group II or to a new fourth group. Group I includes human coronavirus 229E (HCoV-229E), transmissible gastroenteritis virus (TEGV), porcine epidemic diarrhea virus (PEDV), canine coronavirus (CCoV), and feline coronavirus (FIPV). Group II viruses include human coronavirus OC43 (HCoV-OC43), murine hepatitis virus (MHV), and bovine coronavirus (BCoV), and group III species are turkey coronavirus (TCoV), and avian infectious bronchitis virus (IBV). ::: ![](1465-9921-6-8-1) ::: Despite the fact that this new virus most likely jumped to humans from wild animal species, it has remarkably well adapted to the human organism as shown by its high person-to-person transmissibility. SARS virus genome structure --------------------------- The structure of the SARS viral RNA is organized in 13--15 open reading frames (ORF) and contains a total of approximately 30,000 nucleotides \[[@B6],[@B16],[@B17]\]. Recently, 61 SARS-CoV sequences derived from the early, middle, and late phases of the SARS epidemic together with two viral sequences from palm civets were analyzed \[[@B28]\]. Genotypes characteristic of each phase were discovered, and it was found that the neutral mutation rate of the viral genome was constant but the amino acid substitution rate of the coding sequences slowed during the course of the epidemic. The spike protein showed the strongest initial responses to positive selection pressures \[[@B28]\]. Only ORFs exceeding fifty amino acids in translational capacity are considered relevant as they contain the sequences for the structural and functional properties of the virus and are therefore of potential interest for the development for future therapeutic strategies. The comparison of the different SARS-CoV ORFs with those of other coronaviruses reveals a familiar pattern of structural gene arrangement with replicase and protease genes (gene 1a-1b) and the spike (S), envelope (E), membrane (M) and nucleocapsid (N) genes in a typical 5\'- to 3\' order of appearance \[[@B29]\]. The proteins encoded by these genes may be targets for novel treatments. Between these well-known genes, a series of ORFs of unknown function was found: There are two ORFs situated between the spike and the envelope genes and three to five ORFs between the membrane and nucleocapsid genes. Comparison of this gene organization with other known coronaviruses does not indicate a closest proximity to group II coronaviruses. Also, the SARS-CoV genomic sequence does not contain a gene for hemagglutinin-esterase (HE) protein, which is present in the majority of group II coronaviruses. Two-thirds of the SARS RNA is organized in the gene 1a-1b. The sequence of this gene is highly conserved among all coronaviruses \[[@B17]\]. ORFs 1a and 1b encode two polyproteins, pp1a and pp1ab, the latter through a ribosomal frameshifting mechanism. These polyproteins are processed by virus-encoded proteinases, to yield 16 individual proteins. Most potential gene 1a-1b products are fairly well conserved between SARS-CoV and other coronaviruses \[[@B17],[@B29]\]. Many of their functions are unknown but it is suggested that they participate in viral RNA replication, making them potential targets for the development of antiviral compounds. Therefore, research efforts will focus on these proteins. One exception from the overall conservation of SARS-CoV gene 1a-1b is the lack of a sequence coding for PL1^pro^, one of the two papain-like proteinases operating on cleavage sites at the N-terminus of the polyproteins (Figure [2](#F2){ref-type="fig"}). The main proteinase (M^pro^), also called 3C-like protease (3CL^pro^), is responsible for the cleavage of all the remaining proteins encoded by gene 1a-1b \[[@B29],[@B30]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### SARS-CoV genome organization. The structure of the SARS viral RNA is organized into 13--15 open reading frames (ORFs) and contains an overall amount of approximately 30,000 nucleotides. The sequence can be separated into different elements and genomic and subgenomic mRNAs. ::: ![](1465-9921-6-8-2) ::: SARS virus gene expression -------------------------- Apart from gene 1, coronavirus genes are known to be usually expressed from subgenomic mRNAs. They share a common leader sequence at the 5\'-end and initiate at different places in the genome extending toward the 3\'-end of the virus genome \[[@B31]\]. Some ORFs may also be unconventionally translated from a single mRNA. As these uncommon translation mechanisms are not very efficient and the gene products are not very abundant, these ORFs typically encode nonstructural proteins. Whereas the ORFs between the structural protein genes are very heterogeneous among the different coronaviruses and not essential for viral replication, recent studies suggested that deletion of non-essential ORFs may result in a reduced virulence \[[@B32]\]. In agreement with this, some of these non-essential ORFs of the new SARS-CoV genome may be responsible for the high SARS-CoV virulence. So far, five to eight subgenomic mRNAs were found in SARS-CoV-infected cells \[[@B17],[@B27]\]. Thiel and colleagues performed the first detailed study on mechanisms and enzymes involved in SARS-CoV genome expression (Figure [2](#F2){ref-type="fig"}) \[[@B29]\]. They determined the sequence of the SARS-CoV isolate Frankfurt 1 and characterized the major RNA elements and protein functions involved in the genome expression by characterizing regulatory mechanisms such as the discontinuous synthesis of eight subgenomic mRNAs, ribosomal frameshifting and post-translational proteolytic processing. Also, the activities of SARS-CoV enzymes such as the helicase or the two cysteine proteinases (PL2^pro^and M^pro^) were addressed as they are involved in replication, transcription or post-translational polyprotein processing \[[@B29]\]. In conclusion, research in the area of coronavirus gene expression is important to delineate components which directly affect SARS-CoV virulence. SARS virus structural proteins ------------------------------ The structural proteins of the new SARS-CoV are potential targets for new treatment options. The new SARS-CoV only contains the three envelope proteins, spike (S), envelope (E), and membrane (M) but not the hemagglutinin-esterase (HE) protein, which is present in some coronaviruses of the second group. The spike glycoprotein is responsible for the characteristic spikes of the SARS-CoV (Figure [3](#F3){ref-type="fig"}). Intra- and extracellular proteases often cleave the S protein into S1 and S2 domains, with the cleavage process often increasing infectivity of the virus. Molecular modelling has been performed for the S1 and S2 units of the SARS-CoV spike protein \[[@B33],[@B34]\]. The spike proteins of coronaviruses are reported to bind to receptors on their target cells and the domains responsible for receptor-binding are commonly situated in the N-terminal region of S1 \[[@B35]-[@B40]\]. The spikes consist of oligomeric structures, that are formed by heptad repeats of the S2 domain which also represent a fusion peptide sequence. This peptide is responsible for the coronavirus fusion activity. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### SARS-CoV transmission electron microscopy. In the supernatant of SARS-CoV infected cytopathic Vero E6 cells, characteristic virus particles can be found. The diameter of the viruses ranges between 60 nm and 120 nm and the virus shapes are round or oval. There are many protrusions from the envelope which are arranged in order with wide gaps between them. There are also many virus particles in the infected cells present. They often form a virus vesicle with an encircling membrane. A: Higher magnification B: Lower magnification. Scale bars represent 100 nm. Reproduced with permission from Acta Biochimica et Biophysica Sinica 2003, 35(6):587--591 \[126\]. ::: ![](1465-9921-6-8-3) ::: The SARS-CoV has also been reported to cause the formation of syncytia in vivo, but so far only under the condition of cultured Vero cells \[[@B6]\]. The SARS-CoV S protein seems to have most of its characteristics in common with the S proteins of other coronaviruses, but it will be important for the understanding of the SARS-CoV pathogenic properties to identify the exact conditions of membrane fusion, i.e. pH dependency and protease sensitivity, which can increase the infectivity. The envelope and membrane proteins are integral membrane proteins and required for virus assembly \[[@B41]\]. In the case of the murine coronavirus MHV-A59 the coexpression of the E and M proteins but not the S or N proteins is needed for the release of virus-like particles (VLP) \[[@B42]\]. The nucleocapsid and viral core of the SARS-CoV are likely to be formed by the N protein. An interesting feature of the SARS-CoV and other coronaviruses is the resistance against the gastrointestinal fluids despite the lipid composition of their envelope. It has been reported that the SARS-CoV can survive in diarrheal stool for four days and also, patients with SARS often suffer from gastrointestinal symptoms with the virus to be detected in the stool \[[@B4]\]. As the molecular basis for the envelope\'s resistance against acidic environments and gastrointestinal enzymes is unclear, further research has to be carried out in this area which is important for the control of future SARS outbreaks. Evolution of the SARS virus --------------------------- It is unclear when and how novel pathogens such as the SARS-CoV cross the barriers between their natural reservoirs and human populations, leading to the epidemic spread of novel infectious diseases \[[@B43]\]. As with the SARS-CoV, new pathogens are believed to emerge from animal reservoirs and a variety of molecular mechanisms may contribute to the evolution of the viruses or bacteria. Due to the estimated error frequency of 1 × 10 ^-4^for RNA-dependent RNA polymerases \[[@B44]\], RNA viruses such as the SARS-CoV can undergo mutation at a high frequency. The SARS-CoV seems to be relatively genetically stable as the RNA sequences from different SARS patients were quite homogeneous. Even the entire genomic sequences of virus isolates from different continental areas did not differ by more than ten amino acids and it seems that two lineages of the virus can be traced \[[@B45]\]. This obvious contradiction to the high potential error rate of the RNA-dependent RNA polymerase suggests the presence of some proofreading mechanism connected with this enzyme. In fact, a detailed analysis of the SARS-CoV genome by bioinformatics indicates the presence of an exonuclease activity \[[@B27]\]. Next to mutations, a further threat of the SARS-CoV is based on the ability of coronaviruses to undergo RNA recombination at a high frequency \[[@B15]\]. For a variety of other coronaviruses, both recombination and mutation in natural infections have been shown to contribute to the diversification of the coronaviruses. Because of the demonstrated ability of coronaviruses to recombinate, the question whether the SARS-CoV will show a higher frequency of mutations within possible future seasonal changes or in respond to drug treatment is an issue of major concern. It was reported that in the initial phases of the SARS epidemic, the mutation rate was high in the gene for the spike protein, but this stabilized during the middle and final stages of the 2003 epidemy \[[@B28]\]. Thus, the virus had experienced great pressure to adapt to the new host after crossing the species barrier, but has then been optimized \[[@B28]\]. Duration of infection --------------------- Although human coronaviruses are characteristically causing self-limiting short diseases, the question of potential chronic SARS infections is of major importance for a future disease control. If the SARS-CoV is able to cause a chronic persistent infection, chronic carriers may serve as sources for new SARS outbreaks. However, the detection of SARS-CoV in stool of patients for longer periods than 6 weeks after hospital discharge has not been reported so far. Therefore, the danger of chronic carriers may not be relevant. In contrast to common human coronavirus infections with short durations, most animal coronaviruses cause persistent infections. As an example, the feline coronavirus FIPV infects animals which then continue to shed virus for periods reaching up to seven months after infection without carrying disease symptoms \[[@B46]\]. Also, TGEV and MHV tend to cause chronic infections as these viruses may be found in the airways and small intestine (TGEV) or the nervous system (MHV) several months after infection \[[@B47],[@B48]\]. Although the SARS-CoV has jumped to humans it may still have this property of inducing chronic infections. Thus, SARS-CoV RNA was found in patients\' stool specimen more than 30 days after the infections. Clinical picture of SARS ------------------------ The mean incubation period of SARS was estimated to be 6.4 days (95% confidence interval, 5.2 to 7.7). The mean reported time from the onset of clinical symptoms to the hospital admission varied between three and five days \[[@B49]\]. Main clinical features of the disease are in the initial period common symptoms such as persistent fever, myalgia, chills, dry cough, dizziness, and headache. Further, although less common symptoms are sore throat, sputum production, coryza, vomiting or nausea, and diarrhea \[[@B50],[@B51]\]. Special attention has been paid to the symptom of diarrhea: Watery diarrhea has also been reported in a subgroup of patients one week after the initial symptoms \[[@B52]\]. The clinical course of the disease seems to follow a bi- or triphasic pattern. In the first phase viral replication and an increasing viral load, fever, myalgia, and other systemic symptoms can be found. These symptoms generally improve after a few days. In the second phase representing an immunopathologic imbalance, major clinical findings are oxygen desaturation, a recurrence of fever, and clinical and radiological progression of acute pneumonia. This second phase is concomitant with a fall in the viral load. The majority of patients is known to respond in the second phase to treatment. However, about 20% of patients may progress to the third and critical phase. This phase is characterized by the development of an acute respiratory distress syndrome (ARDS) commonly necessitating mechanical ventilation. SARS in adults and children --------------------------- Rapid progress has been made in understanding the clinical presentation of SARS in adults and children \[[@B53]-[@B56]\]. In comparison to adults, SARS seems to be less aggressive in younger children, with no children in one case series requiring supplementary oxygen \[[@B57]\] while in adults, systemic infection as well as respiratory infection may be the rule. SARS is much milder with non-specific cold-like symptoms in children younger than 12 years than it is in adolescents and adults \[[@B58]\]. The reason for the milder clinical presentation of SARS in children is most likely due to differences in developmental stage of the immune system. The course of the disease in teenagers more likely resembles adults in concerning clinical presentation and disease progression \[[@B58]\]. SARS may also develop severe illness requiring intensive care and assisted ventilation in these adolescent patients. The common presenting features are fever, malaise, coryza, cough, chills or rigor, headache, myalgia, leucopaenia, thrombocytopaenia, lymphopaenia, elevated lactate dehydrogenase levels and mildly prolonged activated partial thromboplastin times \[[@B59]\]. The radiographic findings are non-specific: However, high-resolution computed chest tomography in clinically suspected cases may prove to be an early diagnostic aid when initial chest radiographs appeared normal. While rapid diagnosis with the first-generation RT-PCR assay was not satisfactory, improved RT-PCR assays may help to diagnose SARS in early stages. In this respect, a sensitivity approaching 80% in the first 3 days of illness when performed on nasopharyngeal aspirates may be achieved. The best treatment strategy for SARS among children still has to be determined while no case fatality has been reported in children. In comparison to the prognosis in adults, there is a relatively good short- to medium-term outcome. However, it is crucial to emphasize that continued monitoring for long-term complications due to the disease or its treatment is of major importance \[[@B60]\]. Molecular mechanisms of SARS virus pathogenesis ----------------------------------------------- ### Cytocidal mechanisms Coronaviruses are known to exert their effects by cytocidal and immune-mediated mechanisms. In vitro studies using cell culture assays have shown that coronavirus infection commonly results in cytopathic effects such as cellular lysis or apoptosis \[[@B61]\]. Also, the virus can cause cellular fusion leading to the formation of syncytia. These cytopathic effects are caused by steps of the viral replication such as the mobilisation of vesicles to form the viral replication complex \[[@B18]\], leading to the disruption of Golgi complexes \[[@B62]\]. Parallel to results on other coronaviruses, SARS-CoV has been shown to cause cytopathic effects in Vero cells and the formation of syncytia in lung tissues. A further similarity with other coronaviruses seems to be the potential of the SARS-CoV to cause tissue fibrosis \[[@B63]\]. As molecular mechanism for this fibrosis which has been reported for infections with the coronavirus MHV, the N protein has been demonstrated to induce promoter activity of the prothrombinase gene that correlates with fibrin deposition \[[@B64]\]. Immune-mediated mechanisms -------------------------- Next to cytocidal effects, also immune-mediated mechanisms of both the innate and adaptive immune system seem to contribute to the pathogenesis of SARS-CoV infections. In this respect, it has been shown that in MHV infection, T cells and cytokines play an important role in development of the disease \[[@B65]\]. Also, humoral antibodies have been reported to be crucial in infections caused by coronaviruses such as FIPV. Herein, antibodies against the spike protein were shown to be related to the induction of peritonitis \[[@B66]\]. For SARS-CoV infections, it has been reported that there seems to be an inflammatory cell influx consisting in particular of macrophages in the airways, and a massive release of cytokines during the peak of the infection \[[@B67],[@B68]\]. It is therefore crucial that these immune mechanisms are further analysed on the molecular level as it seems appropriate that not only antiviral but also anti-inflammatory strategies are evaluated for a use in the clinical management of future SARS cases. The pharmacotherapy for SARS with anti-inflammatory steroids is controversial and largely anecdotal \[[@B69]\]. It was reported that the initial use of pulse methylprednisolone therapy appears to be more efficacious and equally safe when compared with regimens with lower dosage and should therefore be considered as the preferred steroid regimen in the treatment of SARS, pending data from future randomized controlled trials \[[@B70]\]. A further preliminary, uncontrolled study of patients with SARS, reported that the use of interferon alfacon-1 plus steroids was associated with reduced disease-associated impaired oxygen saturation and more rapid resolution of radiographic lung abnormalities \[[@B71]\]. Mechanisms of target cell specificity ------------------------------------- The most obvious gene which is likely to be a key modifier of SARS pathomechanisms is the spike (S) protein gene. As known for other coronaviruses, it does not only affect viral pathogenesis by determining the target cell specificity but also by other mechanisms. In this respect, a single mutation in the S gene of MHV has significant effects on the viral virulence and tissue tropism \[[@B72]\]. Also, mutations in the S gene led to the emergence of the weakly virulent PRCV from the virulent enteric TGEV \[[@B73]\]. Further potentially important genes are the \'non-essential\' ORFs which show a significant divergence between SARS-CoV and other coronaviruses. In this respect, it was reported that the civet cat coronavirus has a 29-nucleotide deletion leading to a fusion of two non-essential ORFs into one new ORF in the SARS-CoV \[[@B18]\]. It was shown that deletion mutants of \'non-essential\' ORFs of the group 2 mouse hepatitis virus (MHV) leads to a lower virulence without an impact on viral replication \[[@B74]\]. It has to be established if this also applies to \'non-essential\' ORFs of SARS-CoV. Also, other viral gene products such as the M or E proteins may have an impact on the pathogenesis of the disease as they may induce interferon production or apoptosis \[[@B75],[@B76]\]. Molecular targets for antiviral treatment ----------------------------------------- The primary target cells of SARS-CoV infection are respiratory epithelial cells. As the virus can also be detected in stool specimen and patients with SARS often also have gastrointestinal symptoms, epithelial cells of the gastrointestinal tract also seem to be major target cells. Next to these epithelial cells, the SARS-CoV has also been found in macrophages and many other cells as it has been detected in not only in the respiratory tract and stool specimen but also in the blood, liver, kidney and urine \[[@B6]\]. In this respect, pathological examination did not only show changes in the respiratory tract, but also in splenic lymphoid tissues and lymph nodes. Furthermore, signs of a systemic vasculitis were found which included edema, localized fibrinoid necrosis, and infiltration of monocytes, lymphocytes, and plasma cells into vessel walls in the heart, lung, liver, kidney, adrenal gland, and the stroma of striated muscles. There was also thrombosis present in veins. Systemic toxic changes included necrosis and degeneration of parenchymal cells of the lung, heart, liver, kidney, and adrenal gland \[[@B77]\]. It may therefore be concluded that SARS can induce a systemic disease and thereby injuring many other organs apart from the respiratory tract. Target cell receptors --------------------- The SARS-CoV target cell specificity is determined by the spike protein affinity to cellular receptors. In contrast to the all group III coronaviruses and the SARS-CoV for which the receptors have not been finally analyzed, it is known that group I coronaviruses bind to aminopeptidase N (CD13) as receptors \[[@B78]\], while group II coronavirus such as MHV use carcinoembryonic antigen (CEA) as receptor \[[@B79]\]. Recently, it was shown that a metallopeptidase, angiotensin-converting enzyme 2 (ACE2), efficiently binds the S1 domain of the SARS-CoV S protein. SARS-CoV replicated efficiently on ACE2-transfected but not mock-transfected 293T cells. Also, anti-ACE2 but not anti-ACE1 antibodies blocked viral replication on Vero E6 cells, indicating that ACE2 is a functional receptor for SARS-CoV \[[@B80]\] which was also identified by a further study \[[@B81]\]. Recently, the C-type lectin CD209L (also called L-SIGN) was discovered to be a further human cellular glycoprotein that can serve as an alternative receptor for SARS-CoV \[[@B82]\]. The interruption of virus-receptor interactions could be a potential target for future therapeutic strategies (Figure [4](#F4){ref-type="fig"}). In this respect, the receptor-binding S1 domain of the SARS-CoV S protein represents a possible target for new SARS antiviral drugs. Also, antibodies against ACE2, but not inhibitors binding to the active site of ACE2 may be useful for the development of therapeutic strategies. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Potential target sites for therapeutic strategies. In view of the viral life cycle, there are several potential targets for the development of antiviral drugs. Starting from the binding of the virus to the target cell, the spike protein or receptors such as angiotension-converting enzyme 2 (ACE2), cell entry or the different replication steps may be targeted. After replication, virus assembly and exit mechanisms may also be used for antiviral strategies. VLP, virus-like particles. ::: ![](1465-9921-6-8-4) ::: Virus entry ----------- After binding to the receptor, the next molecular step of potential use for the development of anti-SARS drugs is the virus entry into the cells. While most coronaviruses enter their target cells via plasma membrane fusion, a further entry mechanism may be acidic pH-dependent endocytosis \[[@B83]\]. Focusing on these mechanisms, it will be crucial to gain further knowledge about SARS-CoV fusion activity. As a drug development candidate, a putative fusion peptide has good potential (Figure [4](#F4){ref-type="fig"}). Intracellular replication ------------------------- After the binding to a host cell receptor and entry into the cells, the molecular steps of transcription, translation and protein processing display further potential targets for new therapeutic strategies. In this respect, the RNA-dependent RNA polymerases (SARS-CoV RdRp) may be a potential target for a future anti-SARS therapy. A recent study located its conserved motifs and built a three-dimensional model of the catalytic domain \[[@B84]\]. The authors suggested that potential anti-SARS-CoV RdRp nucleotide-analog inhibitors should feature a hydrogen-bonding capability for the 2\' and 3\' groups of the sugar ring and C3\' endo sugar puckering. Also, the absence of a hydrophobic binding pocket for non-nucleoside analog inhibitors similar to those observed in hepatitis C virus RdRp and human immunodeficiency virus type 1 reverse transcriptase seems to be crucial \[[@B84]\]. Also, protease activity is crucial for SARS-CoV RNA replication and protein processing \[[@B29],[@B85]\], and the inhibition of protease function leads to an immediate stop of viral RNA synthesis. Most of the coronaviruses express one major cysteine proteinase, called the main proteinase (M^pro^) or the 3C-like proteinase (3CL^pro^), and two auxiliary, papain-like proteinases (PL1^pro^and PL2^pro^). The latter two are responsible for the cleavage of the viral polyproteins, pp1a and pp1ab, at three sites near the amino-terminus, while the M^pro^processes these proteins at as many as 11 additional sites. Interestingly, SARS-CoV lacks the PL1^pro^\[[@B16],[@B17]\], but it can be assumed that its action is taken over by the PL2^pro^\[[@B29]\]. This is conceivable since operation of the PL2^pro^on PL1^pro^cleavage sites has been shown in IBV and HCoV \[[@B86]\]. Roughly at the position of the PL1^pro^gene in other coronavirus genomes, SARS-CoV displays a domain within ORF1a that lacks any detectable sequence homology and has therefore been named the SARS-unique domain (SUD) \[[@B27]\]. It is not known whether the SUD protein is ever expressed in the life cycle of SARS-CoV but if it is, it may be connected to the high pathogenicity of SARS-CoV compared to other human coronaviruses and, therefore, it may constitute an attractive target for therapeutic intervention. Crystal structures have been determined for the M^pro^s of TGEV \[[@B87]\], HCoV 229E \[[@B85]\], and, more recently, SARS-CoV \[[@B88]\]. They all show a similar overall architecture for the 34 kD enzyme which forms a dimer in the crystals and also at intermediate and high concentrations in solution. The monomer consists of three domains of which the first two are β-barrels with an overall similarity to the 3C proteinases of picornaviruses and to the serine proteinase, chymotrypsin. The third domain is α-helical and was shown to be essential for dimerization \[[@B85],[@B87],[@B88]\]. The active site of the enzyme is located in a cleft between domains I and II and comprises a catalytic dyad of Cys\...His, rather than the catalytic triad common for cysteine and serine proteinases. Anand et al. \[[@B85]\] have synthesized a substrate-analogous hexapeptidyl chloromethylketone inhibitor and bound it to TGEV M^pro^in the crystalline state. The X-ray structure of the complex revealed binding of the P1 glutamine, P2 leucine, and P4 threonine side chains of this compound to the respective subsites in the substrate-binding cleft, in agreement with the pronounced specificity for cleavage by the M^pro^after the substrate sequence (Thr, Val, Ser)-Xaa-Leu-Gln. The structure also showed the expected covalent attachment of the methyl ketone group at P1 of the inhibitor to the catalytic cysteine of the enzyme. In spite of 40% and 44% sequence identity, respectively, to the M^pro^s of HCoV 229E and TGEV, the crystal structure of the SARS-CoV M^pro^revealed some surprises \[[@B88]\]. Within the dimer, one molecule was in the active conformation seen in the other structures, whereas the other one adopted a catalytically incompetent conformation. This enzyme had been crystallized at a pH value of \<6, which in one of the monomers apparently led to the protonation of a histidine residue at the bottom of the S1 specificity pocket. This resulted in major conformational rearrangements leading to the collapse of this binding site for the P1 glutamine residue of the substrate and to a catalytically incompetent conformation of the oxyanion-binding loop. However, when the crystals were equilibrated at higher pH values, their X-ray structures revealed the active conformation for both monomers in the dimer. This pH-dependent activation mechanism allows interesting conclusions to be made for the self-activation of the M^pro^from the viral polyprotein, which probably involves a pH-dependent step. The same hexapeptidyl chloromethylketone inhibitor used by Anand et al. \[[@B85]\] in their crystallographic study of the TGEV M^pro^was employed by Yang et al. \[[@B88]\] to characterize the interaction of the SARS-CoV enzyme with substrate. This was performed by soaking the inhibitor into crystals grown at the low pH. In spite of the inactive conformation of one of the two monomers in the dimer being preserved, the compound was found to bind to it, but with its P1 glutamine side chain pointing towards bulk solvent rather than into the S1 binding site, because of the collapse of the latter. The binding mode of the inhibitor to the active monomer was also somewhat unusual and is not fully understood at present. On the basis of their crystallographic work, Anand et al. \[[@B85]\] found that the binding mode of their hexapeptidyl chloromethylketone inhibitor to the TGEV M^pro^resembled that of AG7088 in complex with its target, the 3C proteinase of human rhinovirus \[[@B89]\], even though the respective target enzymes displayed large structural differences except in the immediate neighbourhood of the active site. AG7088 is in phase II/III clinical studies as an inhalation treatment for the common cold as caused by human rhinovirus. Anand et al. \[[@B85]\] therefore proposed that AG7088 should be a good starting point for the design of anti-SARS drugs, and indeed, the manufacturer of AG7088 confirmed only a few days after their proposal had appeared on-line that the compound was effective against SARS coronavirus in cell culture. AG7088 is now the subject of intensive optimization efforts \[[@B90]\]. Other studies used molecular dynamics simulations of the M^pro^and screened 29 approved and experimental drugs against a model of the SARS CoV proteinase as well as the experimental structure of the transmissible gastroenteritis virus (TGEV) proteinase \[[@B91]\]. It was suggested that existing HIV-1 protease inhibitors, L-700,417 for instance, may have high binding affinities and may therefore provide another good starting point for the future design of SARS-CoV proteinase inhibitors \[[@B92]\]. However, this has to be proved experimentally. Further potential targets are the E and M proteins (Figure [4](#F4){ref-type="fig"}) as they represent the minimum essential components for the assembly of coronaviruses which form the virus-like particles \[[@B41],[@B42]\]. Ultrastructurally, the process of SARS-CoV assembly is most likely localised to the ER-Golgi intermediate compartment \[[@B93]\]. Together with strategies that may focus on the inhibition of virus assembly, the virus exit through secretory pathways is also of interest for the development of new antiviral compounds. With regard to the multitude of potential epithelial target cells, specific endogenous drug delivery systems may also be of relevance. In this respect, the family of peptide transporters consisting of PEPT1 and PEPT2 which are differentially expressed in potentially infected cells of the respiratory tract \[[@B94],[@B95]\], small intestine \[[@B96]\], kidneys \[[@B97],[@B98]\], nervous system \[[@B99]\] and other organs \[[@B100]\], may serve a target for the rational drug design of antiviral drugs. So far, a variety of antiviral drugs or prodrugs such as valacyclovir \[[@B101]\], valganciclovir \[[@B102]\] or the valyl ester of zidovudine \[[@B103]\] have been shown to be transported via these systems and minimal structure requirements for substrate transport have been determined \[[@B104]\]. A further tool which may be used to approach antiviral therapies is the technique of small interfering RNAs (siRNAs). SiRNAs are double-stranded RNAs which lead to a sequence-specific degradation of mRNAs \[[@B105]\]. Recent in vitro studies used six 21-mer siRNAs that were targeted to different sites of the replicase 1A region of SARS-CoV \[[@B106]\]. Monkey kidney cells (FRhk-4) were infected with the SARS-CoV GZ50 strain and transfected eight hours later with the siRNAs. Three of the six siRNAs led to a marked inhibition of virus cytopathic effects and a reduction of virus copies between 85 and 92 %, indicating that siRNAs may have a potency as antiviral treatment options and that the 1A region displays a promising region to suppress virus replication \[[@B106]\]. Vaccines against the SARS virus ------------------------------- As most patients develop an immunity against the SARS-CoV and survive the infection, the possibility of creating an effective and safe vaccine seems to exist \[[@B107]\]. There are several options to develop vaccines against the SARS-CoV \[[@B108]\]. Live-attenuated vaccines ------------------------ Live-attenuated coronavirus vaccines can be generated by deletions in \"group-specific genes\". The deletions of these genes do not change replication properties but attenuate the virus \[[@B109]\]. Examples for the use of live-attenuated vaccines to prevent coronavirus infections are live attenuated IBV vaccines which are used in broiler chickens \[[@B110]\]. For the animal coronavirus infections, live attenuated vaccines have been proven to be significantly more effective than whole killed vaccines, indicating that cell-mediated immunity is a crucial defence mechanism. However, the great threat remains that a vaccine strain can recombine with a circulating wild type strain \[[@B111]\] and without evidence that recombination and reversion of a live-attenuated SARS-CoV to virulence can not occur, it is unlikely that a live attenuated SARS-CoV vaccines will be developed and used. Whole killed vaccines --------------------- Whole killed vaccines are generally safe and easy to generate. In fact, this technique has been applied in veterinary medicine to generate vaccines for BoCV and IBV \[[@B112]\]. Also, an inactivated canine coronavirus vaccine has been produced \[[@B113]\]. A SARS inactivated vaccine was recently developed using the SARS coronavirus (SARS-CoV) strain F69 treated with formaldehyde and mixed with Al(OH)(3) \[[@B114]\]. However, killed vaccines may not protect against different strains of coronaviruses, and live attenuated vaccines have been shown to be more effective than whole killed vaccines in preventing coronavirus animal infections \[[@B115]\]. Recombinant subunit vaccines ---------------------------- Using molecular biology techniques to generate large quantities of recombinant viral proteins, recombinant subunit vaccines, e.g. against the spike protein, are expected to be created relatively easy as shown by two recent studies \[[@B116],[@B117]\]. Eight recombinant human single-chain variable region fragments (scFvs) against the S1 domain of spike (S) protein of the SARS-CoV from two nonimmune human antibody libraries were screened and one scFv 80R efficiently neutralized SARS-CoV and inhibited syncytia formation between cells expressing the S protein and those expressing the SARS-CoV receptor angiotensin-converting enzyme 2 (ACE2) \[[@B117]\]. A recent study used the SARS-CoV spike protein receptor binding domain (aa 318--510) for immunization, which resulted in the induction of effective neutralizing antibodies \[[@B118]\]. However, recombinant subunit vaccines may have a limited ability to protect against SARS-CoV infections in view of the variations which may arise in the viral genome in future outbreaks. Therefore, the approach of recombinant subunit vaccines may have to be supplemented by further vaccine strategies which focus on cell-mediated immunity. Recombinant vectored vaccines ----------------------------- An approach using recombinant vectored vaccines with DNA or a viral vector could be a promising target. The DNA prime and adenovirus or MVA boost approach which is currently analysed for a potential use in the development of HIV vaccines, may also offer a strategy to prevent SARS infections. In this respect, a multi-valent approach which induces both humoral and T cell-mediated host responses seems to be the most attractive strategy. From the field of veterinary medicine, data on this approach are already available: A recombinant fowlpox with the S1 gene of IBV was demonstrated to be relatively protective against IBV \[[@B119]\]. Also, a DNA vaccine was developed which contains the nucleocapsid protein gene of porcine transmissible gastroenteritis virus (PTGV). This vaccine was shown to initiate both humoral and cell-mediated immune host responses \[[@B120]\]. Recently, three murine studies demonstrated that DNA vaccines encoding different SARS-CoV antigens are capable of generating humoral and cellular immunity and may potentially be useful for control of infection with SARS-CoV \[[@B121]-[@B123]\]. However, it was also shown that immunization with modified vaccinia virus Ankara-based recombinant vaccine against SARS is associated with enhanced hepatitis in ferrets \[[@B124]\]. Epitope-based vaccines ---------------------- A further strategy is based on the use of epitopes which can be delivered using viral or DNA vectors. Such an epitope-based strategy for coronavirus vaccination has already been reported \[[@B125]\] and the major advantages is the prevention of a possible vaccine reversion to virulence. A further benefit of this technique is the possibility to eliminate any regions of the viral genomic sequence which be associated with a potential autoimmune effects. The limitation of this approach is mainly based on potential variations. In this respect, epitopes which frequently undergo mutations will not protect against the SARS-CoV infections if used in epitope-based vaccines. If the SARS-CoV evolves as a highly variable virus, it will be crucial to identify highly conserved epitopes of the virus. In summary, the important development of SARS vaccines can be approached using several techniques which should ideally encompass the induction of both humoral and cell-mediated mechanisms. As coronavirus vaccines in animals have partly been reported to cause an enhancement of viral infections \[[@B66]\], a cautious approach has to be followed. A first study has investigated the ability of adenoviral delivery of a codon-optimised SARS-CoV spike protein S1 fragment, membrane protein, and nucleocapsid protein to induce immunity in rhesus macaques. The immunization with a combination of these three Ad5-SARS-CoV vectors and a booster vaccination on day 28 demonstrated antibody responses against the spike protein S1 fragment. Also T-cell responses against the nucleocapsid protein were found and all vaccinated animals displayed strong neutralising antibody responses in vitro. These results indicated that an adenoviral-based vaccine can induce SARS-CoV-specific immune responses in monkeys. Conclusion ========== In summary, the onset of the SARS epidemic in different continents has led to the formation of a successful laboratory network to identify the molecular mechanisms underlying the SARS infection. Next to the development of early diagnostic tests and effective treatment strategies, it is most important to orchestrate research activities which lead to the development of vaccines and antiviral agents, as there is no established therapy to date. Even now in a situation of only a handful of new cases, SARS remains a major global health hazard which may reappear. Acknowledgements ================ Part of this work was supported by grants from the European Commission and the DFG to RH (Hi 611/4-1) and to DAG (Gr 2014/2-1). Support from the Fonds der Chemischen Industrie (RH) and the Deutsche Atemwegsliga (DAG) is also gratefully acknowledged.

PubMed Central

2024-06-05T03:55:52.501415

2005-1-20

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548145/", "journal": "Respir Res. 2005 Jan 20; 6(1):8", "authors": [ { "first": "David A", "last": "Groneberg" }, { "first": "Rolf", "last": "Hilgenfeld" }, { "first": "Peter", "last": "Zabel" } ] }

PMC548146

Background ========== Articular cartilage is composed of extracellular matrix (ECM), the matrix-secreting chondrocyte and water, which all account for the tissue\'s characteristic rigidity as well as its flexibility. These features are necessary in order to warrant life-long survival of the cartilage tissue, especially in the joint where it has to endure pressure forces caused by movement. Chondrocytes arise from a mesenchymal progenitor during development, the same progenitor that gives rise to other mesenchymal cell types including osteoblasts, adipocytes and myocytes. Bone formation can either be endochondral, when chondrocytes mature and calcify to provide a matrix for the invading osteoprogenitors, or intramembraneous involving ossification directly from a mesenchymal ancestor. All these diverse cell types may arise from the same precursor, but are distinguished by specific morphological features and with that, a certain set of characteristic proteins including transcription factors that control their differentiation. Two chondrocyte-specific transcription factors have been identified, Sox9, a member of the SOX-family of transcription factors, and Scleraxis, a member of the basic helix-loop-helix transcription factors \[[@B1],[@B2]\]. However, most of the exclusive markers for cartilage tissue reside in the ECM. The predominant form of collagen in mature cartilage is collagen type IIB, whereas the alternatively spliced collagen IIA is found primarily during development \[[@B3]\]. Aggrecan is the major proteoglycan species in cartilage \[[@B4]\]. The transition of chondrocytes into hypertrophy is distinguished by a change in expression of Cbfa1, the osteoblast-specific transcription factor, which is also switched on during intramembraneous ossification. Distinctive to cartilage, the major collagen molecule in osseus matrix is collagen type I. In contrast, the transcription factors controlling adipogenesis are C/EBPα and PPARγ, which transactivate subsets of genes as a function of either trans-acting factor alone or requiring the co-operative effort of both \[[@B5]\]. C/EBPα is known to bind to and transactivate particularly the promotors of the SCD1, aP2 and the Glut4 genes \[[@B6],[@B7]\], all highly characteristic of the adipocyte phenotype. For decades, the treatment of degenerative cartilage and bone diseases has been a challenge for orthopaedic surgeons due to the apparent inability of cartilage and bone to repair itself. Arthritis, a degenerative joint condition, is one of the most prevalent chronic health conditions in North America. Arthritis can devastate people, but to date there is no effective therapy available and patients can only be helped by surgical joint replacement. An inherent major concern is the limited availability of autografts, which significantly reduces the choice of treatable defects. However, new approaches to cell grafting are being developed in this field: increased yields of cells are achieved by the usage of bioreactors and growth factor administration, such as TGFβ~1~and BMPs \[[@B8],[@B9]\]. Additionally, stem cells are being discovered as a new source of transplantable material. Embryonic stem cells represent a valuable source for cell transplantation since their characteristic features include an unlimited self-renewing capacity and a multilineage differentiation potential \[[@B10],[@B11]\]. In fact, ES-derived glial precursors and cardiomyocytes have been successfully transplanted, integrated and shown to be functionally active in the transplantation site \[[@B12],[@B13]\]. The yield of differentiation of ES cells into an intended lineage can be greatly enhanced by the addition of growth factors or induction substances. Whereas protocols for the differentiation of cardiomyocytes, neuronal cell types, insulin-producing cells or adipocytes from ES cells have been available for many years \[[@B14]-[@B17]\], only recently their differentiation into elements of the skeleton has been reported \[[@B18]-[@B20]\]. Our group has previously shown that vitamin D~3~forces ES cells to undergo osteogenesis \[[@B18]\]. Kramer et al. have reported in 2000 that BMP-2 pushes ES cells to the chondrogenic fate when added during days 3--5 of EB differentiation \[[@B21]\]. Those ES-derived chondrocytes possess a certain plasticity to undergo hypertrophy and calcify \[[@B22]\]. We show here, that prolonged treatment of differentiating ES cell cultures with BMP-2 in synergy with TGFβ~1~, insulin and ascorbic acid leads to improved chondrogenesis *in vitro*. Compared to the brief supplementation described by Kramer et al. \[[@B21]\], the expression of chondrocyte-specific marker genes was highly up-regulated while proteoglycan content revealed an increased chondrocytic yield from 7.26% to 57.03%. As described by other groups \[[@B22]\], ES-derived chondrocytes become hypertrophic and calcify. However, spontaneous calcification did not reach mineralization levels that are found in vitamin D~3~induced ES-derived osteoblasts \[[@B18]\]. Yet, supplementation of chondrocyte-cultures with β-glycerophosphate, ascorbic acid and vitamin D~3~starting at day 20 rescued the osteoblast phenotype. In many differentiations, we also observed an accumulation of lipid droplets and an up-regulation of adipocyte-specific genes. This direction towards adipocyte differentiation varied with the use of specific co-factors, suggesting that in the future, such spurious differentiation may be controlled, once the pathways involved in adipogenesis are better understood. Results ======= Characterization of chondrocyte-like cells derived from ES cells ---------------------------------------------------------------- Embryonic stem cell cultures supplemented with BMP-2, TGFβ~1~, insulin and ascorbic acid show typical morphological changes compared to the untreated cultures. Starting with the fourth week of culture, aggregates consisting of small round cells formed in the supplemented cultures, which stained positive with alcian blue (fig. [1A](#F1){ref-type="fig"}). Little alcian blue staining was seen in control cultures (fig. [1B](#F1){ref-type="fig"}). Polygonal cells, which could also be found in treated cultures, did not stain with alcian blue. Significant immunostaining for the collagen type II (COL II) protein was observed at day 32 in treated cultures corresponding to the active secretion and formation of an extracellular matrix found with chondrocytes. The COL II antibody identified the fibrillary organization of the collagen molecules in the extracellular matrix (fig. [1C](#F1){ref-type="fig"}). Chondrogenic differentiation was confirmed by positive immunostaining for adult proteoglycans (fig. [1D](#F1){ref-type="fig"}), which is detectable in the aggregates identified by alcian blue staining. The distribution was associated with the extracellular matrix similar to that found with the COL II antibody. Staining appeared to be diffuse as extracellular matrix and not individual cells are stained. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Morphology and characteristics of ES-derived chondrocytes after 32 days of culture. (A, B) Determination of proteoglycans in EBs with alcian blue in chondrocyte cultures induced with TGFβ~1~\[2 ng/ml\] and BMP-2 \[10 ng/ml\] from d3--5 of culture and with BMP-2 \[10 ng/ml\], ascorbic acid \[50 μg/ml\] and insulin \[1 μg/ml\] from day 5 onwards (A) compared to control cultures (B). Bar = 8.3 μm. (C, D) Analysis of cartilage-specific matrix proteins in 32 day old EBs induced with the same supplements as in (A, B) by means of immunohistochemistry. Staining with anti-collagen type II (C) and anti-cartilage proteoglycan (D), respectively, both visualized by a secondary AlexaFluor 488 conjugated antibody. Bar = 106 μm. (E) Concentration-dependent effect of BMP-2 on chondrocyte-specific gene expression in 32-day old EBs. Values of cultures supplemented with 10 ng/ml BMP-2 are shown compared to 2 ng/ml as used by Kramer et al. \[2000\], which was set as 1. Values represent means of three independent experiments ± standard deviation, obtained by quantitative RT-PCR analysis. \*\*P \< 0.01; \*\*\*P \< 0.001. (F) Proteoglycan content of EB extracts on day 32. BMP-2 directs increased proteoglycan synthesis in both 2 ng/ml and 10 ng/ml. \*P \< 0.1; \*\*P \< 0.01. Mean ± standard deviation, n = 3. ::: ![](1471-213X-5-1-1) ::: Dependence of gene expression patterns and proteoglycan synthesis on BMP-2 -------------------------------------------------------------------------- Quantitative RT-PCR was used to examine the variability of RNA expression of various cartilage-specific genes in response to BMP-2. Previously, chondrogenesis was induced in ES cells using 2 ng/ml BMP-2 on days three to five of EB formation, when early mesodermal markers such as *Brachyury*and *BMP-4*are expressed \[[@B21],[@B23],[@B24]\]. To improve chondrogenesis, we investigated whether a higher concentration of BMP-2 (10 ng/ml) could increase chondrocyte-specific gene expression. Total RNA was extracted on day 32 and quantitative real-time PCR was carried out. Expression of genes of interest in cultures supplemented with 10 ng/ml BMP-2 was normalized to GAPDH expression and compared to cultures supplemented with 2 ng/ml (fig. [1E](#F1){ref-type="fig"}), which were set as 1. Expression of the small leucine-rich proteoglycans biglycan and decorin was increased 1.6 fold upon supplementation with 10 ng/ml BMP-2 (P \< 0.01). Neither link protein expression nor Sox9 or scleraxis expression were affected by the higher dosage. However, expression of aggrecan and the collagen type II isoforms A and B, which are specific for mature chondrocytes, were significantly increased compared to the low BMP-2 concentration described by Kramer et al. (P \< 0.001). The degree of chondrogenic differentiation under influence of BMP-2 was further quantified by metachromatic detection of proteoglycans (fig. [1F](#F1){ref-type="fig"}). Secreted proteoglycans were extracted on day 32 of culture with guanidine/HCl and combined with dimethylmethylenblue. Based on our measurement of aggrecan, the synthesis of proteoglycan proteins is also increased under the influence of BMP-2. BMP-2 at 2 ng/ml initiated an induction in proteoglycan synthesis of about 2 fold (P \< 0.01), whereas 10 ng/ml BMP-2 increased the proteoglycan content of EBs 2.3 fold (P \< 0.1) compared to non-treated controls. This data shows, that BMP-2 causes the induced synthesis of negatively charged extracellular matrix, characterized by proteoglycans. Additive effect of TGFβ~1~, insulin and ascorbic acid on BMP-2 induced chondrogenesis ------------------------------------------------------------------------------------- Since BMP-2 is believed to play a role in late chondrogenesis \[[@B25]\], we studied the effect of prolonged BMP-2 supplementation beyond day 5 of culture. Additionally, the anabolic effect of insulin and ascorbic acid and the influence of the growth factor TGFβ~1~on BMP-2 induced differentiation was determined quantitatively by PCR analysis at various stages throughout EB differentiation. Table [1](#T1){ref-type="table"} shows the particular combinations of medium supplements used at different culture stages of the \'hanging drop\' protocol used for differentiation, in which day 1--3 represent the hanging drop stage. During days 3--5 the resulting embryoid bodies are cultured in suspension and then plated onto tissue culture treated plastic ware on day 5. Applied concentrations were 10 ng/ml BMP-2, 2 ng/ml TGFβ~1~, 1 μg/ml insulin and 50 μg/ml ascorbic acid. Dexamethasone, which is also known to be a chondro-inducing agent \[[@B26]\], did not evoke a mentionable chondrogenic response in the D3 ES cell line (data not shown). Figure [2](#F2){ref-type="fig"} shows changes in cartilage-specific gene expression under the influence of various differentiation co-factors throughout the 32 days of culture. TGFβ~1~(combination B) evoked a 2-fold increase in collagen II expression for both splice forms. The synergistic effect of both growth factors, TGFβ~1~and BMP-2 (combination C) began to show an increased expressions of aggrecan, link protein and COL IIA compared to cultures that were treated with one supplement alone. Additional supplementation with insulin and ascorbic acid between culture days 3 and 5 (combination D) barely increased aggrecan, link protein and COL II expression, but when given from day 5 onwards enhanced both the BMP-2 and TGFβ~1~effects (supplement combinations E and H). Addition of insulin and ascorbic acid to BMP-2 induced cultures increased collagen type IIA and aggrecan expression minimally to 1.2-fold, link protein and collagen type IIB were induced 2.7- to 2.8-fold compared to BMP-2 alone. The TGFβ~1~response was increased 3- to 4-fold. When cultures were induced with BMP-2 and TGFβ~1~in suspension (d3--5) and supplemented with insulin and ascorbic acid starting on day 5 onwards (combination F), aggrecan, link protein and COL II were up-regulated 10-fold compared to controls. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Combinations of medium supplements used at different culture stages ::: -------------------------------------------------------------------------------------------------- **Supplement combination** **Day 3--5 (hanging drops)** **Day 5 onwards (attached culture)** ---------------------------- ------------------------------ -------------------------------------- **A** BMP-2 **B** TGFβ~1~ **C** BMP-2\ TGFβ~1~ **D** BMP-2\ TGFβ~1~\ insulin\ ascorbic acid **E** BMP-2 insulin\ ascorbic acid **F** BMP-2\ insulin\ TGFβ~1~ ascorbic acid **G** BMP-2\ insulin\ TGFβ~1~ ascorbic acid\ BMP-2 **H** TGFβ~1~ insulin\ ascorbic acid -------------------------------------------------------------------------------------------------- Applied concentrations were 10 ng/ml BMP-2, 2 ng/v ml TGFβ~1~, 1 μg/ml insulin and 50 μg/ml ascorbic acid. The end of the culture period varied with every experiment carried out. ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Changes on cartilage markers in response to various combinations of BMP-2, TGFβ~1~, insulin and ascorbic acid as outlined in table 1. (A) Diagram of expression niveaus of cartilage-specific genes of 32 day old EBs obtained by quantitative RT-PCR and standardized to GAPDH. Untreated control values were set as 1. Mean ± standard deviation, n = 3. \*P \< 0.1; \*\*P \< 0.01; \*\*\*P = 0.001. (B) Proteoglycan content of extracts of 32 day old EBs treated with different combinations of BMP-2, TGFβ~1~, insulin and ascorbic acid (see table 1). Ctrl = control. Means ± standard deviation, n = 3. \*P \< 0.1; \*\*P \< 0.01; \*\*\*P = 0.001. n.d. = not determined. ::: ![](1471-213X-5-1-2) ::: Surprisingly, when BMP-2 supplementation was maintained throughout the culture period (combination G), a dramatic increase of marker gene expression was seen. Aggrecan and COL IIB were up-regulated over 80-fold above controls and link protein and COL IIA expression was increased to 35- and 16-fold, respectively. Here, scleraxis and Sox9 expression behaved conversely. Supplement combination G showed significant decreases of scleraxis and Sox9 expression to 50 and 87% of the control, respectively (P = 0.001). Deferral of the expression of these transcription factors in favour of chondrogenic differentiation characterizes BMP-2 induced differentiation, whereupon chondrogenic processes are furthermore enhanced by TGFβ~1~, insulin and ascorbic acid. The determination of proteoglycan content was used to confirm the results obtained by quantitative RT-PCR for all supplement combinations (fig. [2B](#F2){ref-type="fig"}). TGFβ~1~alone (combination B) did not alter proteoglycan levels compared to controls, and in combination with BMP-2 decreased the proteoglycan content of EBs compared to BMP-2 alone (combination C). Insulin and ascorbic acid did not enhance the proteoglycan synthesis in combination with TGFβ~1~, but caused a 4.2-fold increase in combination with BMP-2 (combination E, P = 0.01). In agreement with aggrecan gene expression, combinations F and G generated a massive 7-fold induction of proteoglycans in EBs (P = 0.001). Genetically manipulated ES cells that express GFP under the control of chondrocyte-specific aggrecan promotor were then used to quantify chondrocyte yield using fluorescence-activated cell sorting. ES cells were differentiated along the chondrocytic lineage using BMP-2 at 2 ng/ml or supplement combination G \[d3--5: TGFβ~1~10 ng/ml, BMP-2 10 ng/ml; d3--32: BMP-2 10 ng/ml, ascorbic acid 50 μg/ml and insulin 1 μg/ml\]. Green fluorescing chondrocytes in both cultures appeared either organized in clusters or scattered as seen in figure [3A](#F3){ref-type="fig"}. The Kramer protocol gave a 7.26 percent yield of chondrocytes, which was increased to 57.03% using our modified protocol (fig. [3B](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Quantification of chondrogenic yield by flow cytometry. (A, B) Genetically modified ES cells expressing GFP from the chondrocyte-specific aggrecan promotor. Green fluorescing chondrocytes appear as clusters of cells within the remaining cell population (A), but are also scattered in the entire population starting at day 28 of differentiation (B). (C) FACS analysis of ES-derived chondrocytes sorted by their GFP expression. Differentiation with BMP-2 only \[2 ng/ml, d3--5\] produced 7.26% GFP expressing cells compared to spontaneously differentiated controls, which contained very low levels of fluorescing cells (1.54%). Prolonged BMP-2 administration together with TGFβ~1~, ascorbic acid and insulin (supplement combination G, see table 1) raised chondrocyte outcome to 57.03%. ::: ![](1471-213X-5-1-3) ::: Kinetic analysis of cartilage-specific gene expression during EB differentiation -------------------------------------------------------------------------------- The degree of chondrogenic differentiation using BMP-2, TGFβ~1~, insulin and ascorbic acid supplementation was then examined over the 35-day culture period using quantitative RT-PCR of various cartilage matrix genes (fig. [4](#F4){ref-type="fig"}). Since the extent of changes in cartilage-specific genes seemed to be dependent on acute (d 3--5) or chronic (d 3--32) application of BMP-2 (supplement combination F versus supplement combination G), the stronger induction by chronic supplementation of BMP-2 was monitored throughout the culture duration. During the first three weeks of the BMP-2 induced differentiation, only minor changes in aggrecan, link protein or collagen type II expression could be detected. Starting with day 26 however, aggrecan expression was up-regulated significantly to 14-fold over control niveaus (P \< 0.1). Reaching day 32, aggrecan, link protein and collagen type II A and B approached peak levels. Consistent with the aggrecan expression profile, link protein expression was found to be significantly up-regulated on days 24--27 (factor 6.3; P = 0.001). Increased values were detectable as early as day 16. On day 26, the quantification of the collagen type IIA and B showed 11- and 24-fold increases respectively. On day 14, splice form B had already reached an 8-fold increase over control values. In contrast, during the early phases of differentiation, between day 5 and 20, collagen type IIA expression was continuously increased 2-fold. Transcripts for biglycan and decorin were detectable throughout all stages of chondrogenic differentiation. With the beginning of the maturation phase in the fourth week of culture (day 21--28), both genes were up-regulated 1.5--2 fold. Transcription factors scleraxis and Sox9 (fig. [4B](#F4){ref-type="fig"}) showed a similar expression profile constantly over the entire culture period. This level did not change significantly during chondrogenic differentiation, but rather decreased compared to controls. Both were already transcribed in day 5 EBs, hallmarking their participation in early differentiation events, where lineage specificity is determined. Additionally, scleraxis transcripts were engaged in later phases of development between days 16--19. Sox9 expression was also increased between days 9--11 and between days 25--27. In conclusion, cultures supplemented with BMP-2, TGFβ~1~, insulin and ascorbic acid express mRNAs of a chondrogenic phenotype, whose expression was time-dependent. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Expression of cartilage-specific genes in the course of EB differentiation induced by BMP-2, TGFβ~1~, insulin and ascorbic acid (supplement combination G, table 1). (A) Aggrecan, collagen type II A and B and link protein, biglycan and decorin. (B) Scleraxis and Sox9. Results show induction factors of expression obtained by quantitative RT-PCR and normalized to GAPDH expression in comparison to corresponding spontaneously differentiated controls, which were set as 1 (mean ± standard deviation, n = 3 independent experiments, 20 EBs each). ::: ![](1471-213X-5-1-4) ::: ES-derived chondrocytes undergo hypertrophy and mineralize ---------------------------------------------------------- During embryo development endochondral ossification occurs in two steps: chondrocytes arise after mesenchymal condensation and become hypertrophic, characterized by expression of collagen type X, and calcification. To test whether this was true for the ES-derived chondrocytes generated with our protocol, we assayed for the Ca^2+^content of the cultures, a measure for mineralization (fig. [5A](#F5){ref-type="fig"}). Calcium was increased 1.2-fold in cells that were supplemented with BMP-2, TGFβ~1~, insulin and ascorbic acid compared to controls (P \< 0.01). Previously, we have described the induction of mineralization in osteoblasts derived from ES cells with vitamin D~3~, which reach maturity at day 32 of culture \[[@B18],[@B27]\]. Here, we observed that the level of mineralization was considerably higher in direct osteoblast differentiation \[11.57 mg/dl\] compared to indirect differentiation using our improved chondrocyte protocol \[3.22 mg/dl\]. We observed, however, that the level of calcium found in VD~3~induced ES-derived osteoblasts could be rescued in the chondrocytes by adding VD~3~on day 20 of differentiation, a time when chondrocytes could be morphologically identified in the cultures (11.18 mg/dl, P \< 0.001). As shown by quantitative RT-PCR, VD~3~treated ES cell derived chondrocytes could alter their expression profile to that of d32 ES cell derived osteoblasts (fig. [5B](#F5){ref-type="fig"}). However, expression of osteocalcin and bone sialoprotein was less than in VD~3~rescued chondrocyte cultures than in VD~3~osteoblast cultures. Interestingly, chondrocyte-specific genes could not be detected in VD~3~osteoblasts and waned in VD~3~rescued chondrocytes. As we have shown previously, ES-derived mineralized osteocalcin expressing osteoblasts can be identified as black appearing cells in phase contrast microscopy \[[@B18]\]. Figure [5C](#F5){ref-type="fig"} shows these black cells in the VD~3~treated cultures. No such cells are visible in control cultures or in ES-derived chondrocyte differentiations. However, by adding VD~3~back in at day 20 to the chondrocytes, mineralization can be detected, although the localization pattern is slightly different. These observations support the hypothesis that VD~3~induces intramembraneous bone formation directly from mesenchymal progenitors while BMP-2 controls endochondral bone formation. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Degree of osteogenesis in cultures treated with chondroinducing medium (BMP, supplement combination G), osteoinducing medium (VD~3~as described in ref. 18) and control ES medium (ctrl). Osteoblast phenotype and mineralization could be increased in chondrocyte cultures by adding VD~3~to chondroinducing supplements at day 20 (BMP+VD~3~). (A) Extent of mineralization as measured by Ca^2+^content. \*\*P \< 0.01; \*\*\*P = 0.001. (B) RT-PCR for chondrocyte-specific and osteoblast-specific marker genes as well as Collagen X as a marker for the hypertrophic state of chondrocytes. (C) Morphology as seen in phase contrast. Black appearing cells were identified as being mineralized osteocalcin expressing osteoblasts previously \[18\]. No such cells can be seen in control (ctrl) and chondrocyte (BMP) but in osteoblast (VD~3~) and rescued osteoblast cultures (BMP+VD~3~). ::: ![](1471-213X-5-1-5) ::: It has been shown by others that the BMP-2 induced alteration in cell fate is both concentration- and time-dependent \[[@B28]\]. Lower concentrations of BMP-2 support chondrogenesis whereas higher concentrations promote osteogenesis. In ES cells, BMP-2 at concentrations of 2 ng/ml, 10 ng/ml and 100 ng/ml did not increase the expression of bone markers with the exception of osteocalcin and osteopontin, which were significantly increased as shown by quantitative RT-PCR (fig. [6](#F6){ref-type="fig"}). In combination with VD~3~(given on days 5--30) however, alkaline phosphatase and Cbfa1 were also significantly up-regulated above controls (5.4- and 4-2-fold, respectively, P \< 0.001). As we noted earlier, BMP-2 induced osteogenesis with or without VD~3~supplementation did not meet the levels that were attained by VD~3~alone, but the late addition of VD~3~on day 20 rescued bone-specific gene expression arguing for an involvement for BMP-2 in endochondral bone formation. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Dependence of genes expressed in cartilage tissue on BMP-2 with and without vitamin D~3~(VD~3~). All cultures contained β-glycerophosphate \[10 mM\] and ascorbic acid \[50 μg/ml\]. Mean ± SD of independent triplicates was quantified by qPCR and is normalized to GAPDH expression. Controls were set as 1. \*\*P \< 0,01; \*\*\*P = 0,001. OCN = osteocalcin, BSP = bone sialoprotein, Cbfa1 = Core binding factor alpha, ALP = alkaline phosphatase, OPN = osteopontin, ONT = osteonectin, Col1 = Collagen type I. Concentrations of BMP-2 up to 100 ng/ml do not lead to osteoblast-specific expression levels that are reached with VD~3~only. 100 ng/ml BMP-2 plus VD~3~given together early during differentiation can significantly up-regulate osteoblast genes. However, when VD~3~is administered later (d20) in combination with BMP-2, osteoblast gene expression is almost restored. ::: ![](1471-213X-5-1-6) ::: Induction of adipocyte differentiation -------------------------------------- During treatment of EBs with various chondrocyte differentiation inducing factors, we noticed accumulation of lipid droplets, which could not be found in untreated controls. Those droplets could indeed be characterized as lipid-containing by means of Oil-Red-O staining (fig. [7A](#F7){ref-type="fig"}). Quantitative real-time PCR analysis revealed a slight up-regulation of adipocyte-specific genes (ADD1, aP2, C/EBPα, GLUT-4, LPL, PPARγ and SCD1) on day 30 in most of the supplement combinations used for induction of chondrocyte differentiation (fig. [7B,C](#F7){ref-type="fig"}). Supplement combination G, which was the most successful for inducing chondrocyte differentiation suppressed adipocyte differentiation. GLUT-4 was most up-regulated in combinations B and C, whereas combinations E and B induced a increase in LPL expression compared to our chondrocyte differentiation protocol. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Characterization of ES-derived adipocytes. (A) Oil-Red-O staining of lipid droplets in ES-derived adipocytes. Bar = 42 μm. (B, C) Influence of BMP-2 alone and in combination with TGFβ~1~, insulin and ascorbic acid (see table 1) on expression of adipocyte-specific genes. Quantitative RT-PCR was performed on 30 day old EBs. Expression of genes of interest was normalized to GAPDH and compared to untreated controls. Values show means ± standard deviations on n = 3 independent experiments. \*P \< 0.1; \*\*P \< 0.01; \*\*\*P = 0.001. ::: ![](1471-213X-5-1-7) ::: Discussion ========== In this study, we demonstrate an improved method for driving ES cells to a cartilaginous fate when stimulated with BMP-2 and TGFβ~1~. In the early phase of differentiation, BMP-2 operates by directing differentiation towards the cartilage lineage and acting on chondroprogenitors. During later differentiation adding mineralizing agents can trigger hypertrophy and mineralization of the ES-derived chondrocytes. In the embryo, maturation of chondrocytes in the process of endochondral ossification follows a timely regulated developmental program, whereby cellular stages can be delimitated molecularly. During ES cell differentiation into chondrocytes, developmental processes follow the same pattern, as judged by the gene expression patterns observed. Chondrocytes, osteoblasts and adipocytes are thought to arise from the same mesenchymal progenitor. Based on our previous observations around VD~3~-induced osteogenesis, we have already hypothesized that during the first 5 days of differentiation mesodermal progenitors develop, which then are susceptible to the VD~3~treatment \[[@B18]\]. Treatment of the cultures with TGFβ~1~at days 3--5 may augment the number of mesenchymal progenitors, as TGFβ~1~is thought to inhibit the proliferation of most cells, but to stimulate some mesenchymal cells such as osteoblasts and chondrocytes \[[@B8]\]. It was not surprising to see adipocytes develop in many of our cultures, as they represent another member of the TGFβ~1~promoted mesodermal lineage. The gain of adipose characteristics in culture is hallmarked by: a) the appearance of cytoplasmic lipid droplets, b) the acquisition of insulin sensitivity with regard to glucose uptake (GLUT4) and c) the expression and secretion of numerous bioactive molecules \[[@B5]\]. All of these characteristics of *in vivo*adipogenesis were met in the EBs treated with BMP-2, TGFβ~1~, insulin and ascorbic acid. Indeed, a future challenge for improving adipogenic cultures will be the discovery of regulatory pathways in adipogenesis. Once identified, such pathways may be antagonized in order to enhance ES differentiation into chondrocytes. During the revision of this paper, a study was published describing that the overexpression of the sox triad, namely Sox9, Sox5 and Sox6, markedly increased chondrocyte marker gene expression (collagen type 2, aggrecan) in ES cells within 3 days \[[@B29]\]. Here, TGFβ~1~, BMP-2, IGF-1 and FGF-2 had no affect on the immediate regulation of these genes. In our differentiation system, Sox9 is elevated very early during differentiation at day 5 (data not shown) underlining its role as an early controller of chondrogenesis. We have shown here that BMP-2 regulates later processes in cartilage development. Marker gene expression levels reached upon overexpression of the Sox trio do not meet the levels we have observed in this study, suggesting that Sox9 may be necessary but not sufficient do direct all progenitors to the chondrocytic lineage. Earlier this year, another study portrayed chondrogenesis in ES cells using encapsulation in alginate \[[@B26]\]. The usage of a 3D culture system led to an increase in Col II and aggrecan expression of about 1.5- to 2-fold above the regular 2D system of plating EBs. Comparing those values to the 80- to 90-fold up-regulation described here, it is clear that three-dimensional signals also need to be incorporated into chondrocyte differentiations to increase differentiation efficiency. Articular cartilage has been refractory to repair following degeneration. Despite its limited capacity to self-repair, cartilage is replete with cells capable of undergoing mitotic division \[[@B30]\]. Current hypotheses suggest that cells may be constrained by their ECM, thus preventing expansion and differentiation or that there is a limit in the bioactive molecules, which support chondrogenesis \[[@B31],[@B32]\]. Engineering bone or cartilage usually requires the handling of autologous cells. Cells are released from the ECM using collagenase and hyaluronidase. However, the small number of available progenitors, within the tissue can be problematic \[[@B33]\]. Moreover, the ability of these harvested cells to proliferate is limited in the elderly, where most degenerative joint disorders occur. The outcome of conventional surgical treatment including joint resurfacing or biological autografts has been unsatisfactory following long-term evaluation \[[@B34]\]. This failure is caused by insufficient repair resulting in the formation of mechanically inadequate resident fibrocartilage. These disappointing results and the limited therapeutic opportunities have led investigators to focus on more appropriate bioregenerative tissue engineering approaches, which could be specifically tailored for a patient\'s needs. Pluripotent ES cells are now being contemplated as a new cell source for tissue engineering since they are the most multifaceted cells amongst all stem cells. While their pluripotency offers a huge potential for cell therapies directed against a wide spectrum of degenerative diseases that are ineffectively treated by traditional approaches, it also poses challenge for controlling developmental fate in the recipient. Understanding the developmental pathways regulating ES cell differentiation, will enable the creation of a cell source that can be manipulated to correct for a particular defect. This study was designed to improve upon the number of differentiated cells needed for the *in vitro*development of functional cartilage as this represents a first critical step in applying ES cells clinically. Additionally, the presented *in vitro*model of chondrogenesis may be useful for *in vitro*embryotoxicity tests \[[@B27]\] as also serve as a new tool in identifying molecular pathways that underlie chondrogenesis. Compared to other *in vitro*models of chondrogenesis, this model incorporates all steps of development beginning with a pluripotent, uncommitted cell and thus might further our understanding of normally unamendable stages of development. Conclusions =========== This study was particularly designed to improve chondrocyte yields from ES cells by investigating the effect of higher BMP-2 concentrations and the complementary effects of TGFβ~1~, insulin and ascorbic acid. By examining gene expression responses and cell sorting for GFP-expressing chondrocytes, we demonstrate that using a higher concentration of BMP-2 in combination with appropriate co-factors, we can significantly enhance ES cell derived chondrogenesis compared to known protocols. In addition, we document that ES-derived chondrocytes behave naturally as they undergo hypertrophy. We show that EBs supplemented with BMP-2 also result in a small amount of adipogenesis *in vitro*. This observation is consistent with knowledge that adipocytes, chondrocytes and osteoblasts arise from the same mesenchymal ancestor. Accordingly, in the future, it will be necessary to understand how to discriminate these populations for cytotherapeutic applications. At this time, the presented *in vitro*model allows the study of mechanisms involved in BMP-2 induced chondrogenesis, osteogenesis and adipogenesis. Methods ======= Cell culture and differentiation of embryonic stem cells -------------------------------------------------------- Cells of the mouse ES cell line D3 (American Type Culture Collection, Rockville, Maryland, USA) were kept in permanent culture as described \[[@B35],[@B18]\] with the additive Leukemia Inhibitory Factor (1000 U/ml, Gibco Life Technologies, Karlsruhe, Germany). Differentiation was initiated in hanging drops. Cells condensed to form EBs, which were transferred on day 3 into suspension culture. At day 5, EBs were plated into 24-well tissue culture primaria plates (Falcon, Heidelberg, Germany). The effects of BMP-2 \[2 or 10 ng/ml\], TGFβ~1~\[2 ng/ml\], insulin \[1 μg/ml\] and ascorbic acid \[50 μg/ml\] in various combinations on the differentiation of chondrocytes were examined. Medium was changed every second day. Alcian blue staining -------------------- Proteoglycans secreted by ES-cell derived chondrocytes were stained with Alcian blue. Cultures were fixed in 2.5% glutaraldehyde, 25 mM sodium acetate, 0.4 M MgCl~2~containing 0.05% Alcian blue for 48 h. Wash steps in 3% acetic acid, 3% acetic acid/25% ethanol and 3% acetic acid/50% ethanol reduced unspecific binding of the dye. Metachromatic test with 1.9-dimethylmethylenblue ------------------------------------------------ Proteoglycan content of differentiated cultures was determined with the DMMB-assay \[[@B36]\]. Proteoglycans were extracted in 4 M guanidin-HCl/0.05 M sodium acetate (pH 5.8) containing 100 mM 6-amino-caproic acid, 10 mM EDTA, 5 mM benzamidine/HCl, 10 mM N-ethylmaleimide, 0.4 mM pepstatin, 1 mM PMSF and 1 μg/ml soy bean trypsin inhibitor for 48 h at 4°C. Non-completely digested cells were separated from the lysate by centrifugation. Lysate was mixed with DMMB reagent (0.16% w/v DMMB in 0.2% formic acid containing 2 mg/ml sodium formate, pH 3.5) and changes in absorption were detected at 535 nm in a spectrophotometer. Concentration of proteoglycans in samples was read against a standard curve of chondroitin sulfate C. Immunofluorescence ------------------ Embryoid bodies were differentiated to the chondrocyte lineage with BMP-2, TGFβ~1~, insulin and ascorbic acid and fixed on day 32 with ice-cold methanol:aceton (7:3) at -20°C for 10 min. For staining with anti-collagen type II (Chemicon, MAB 8887) cultures were digested with pepsin for 15 min at 37°C. Staining with anti-adult proteoglycan (Chemicon MAB 2015) was performed after a 1 h digest with chondroitinase ABC at room temperature. Cells were overlaid with the appropriate dilution of the first antibodies in PBS, 10% FCS at 4°C over night. The corresponding secondary antibody, an AlexaFluor 488 goat anti-mouse IgG (H+L), F(ab\')~2~fragment (A-11006, Molecular Probes, Leiden, The Netherlands) was incubated with the cells for 2 h at room temperature. Cultures were observed in a Leica Fluovert FU fluorescent microscope (Leitz, Wetzlar) with an excitation wavelength of 495 and an emission wavelength of 519 nm. RNA isolation, RT-PCR and real-time quantitative RT-PCR ------------------------------------------------------- Total RNA was isolated from 20 EBs per probe using the RNeasy Midi Kit (Qiagen, Hilden, Germany) according to the manufacturer\'s instructions with on-column DNase I digestion. The amount of RNA was determined using the RiboGreen™ RNA quantitation reagent and kit (Molecular Probes). 275 ng total EB RNA was used as a template for cDNA synthesis with Superscript II (Invitrogen, Paisley, Scotland) as described \[[@B27],[@B35]\] in a total reaction volume of 25 μl. 5 μl aliquots of the first strand reaction were used for amplification performed with specific primers (see table [2](#T2){ref-type="table"}). Primer sequences for osteoblast-specific genes and PCR conditions have been described previously \[[@B18]\]. PCR products were visualized on 3% agarose gels containing 0.1 μg/ml ethidium bromide. Quantitative real-time PCR analysis was performed in an ABI Prism^®^7700 Sequence Detector. The accumulation of reaction products during PCR was monitored by measuring the increase in fluorescence caused by the binding of SYBR^®^Green (Applied Biosystems, Perkin Elmer, Weiterstadt, Germany) to double-stranded DNA. Expression analysis of collagen type II A and B was done in the same PCR run with two differently labelled probes specific for either the A isoform (5\'-CGAGATCCCCTTCGGAGAGTGCTGT-3\'/VIC) or the B isoform (5\'-CCAGGATGCCCGAAAATTAGGGCCAA-3\'/FAM) \[[@B27]\]. Reaction mixtures were set up as suggested by the manufacturer containing either SYBR Green or probes for collagen type II. Following a 10 min Taq Polymerase activating step at 95°C, the reactions were cycled by denaturing at 94°C for 30 s and annealing and elongation for 30 s at the corresponding temperatures (table [2](#T2){ref-type="table"}). Target gene C~T~-values were standardized against GAPDH expression and induction of expression in treated EBs was normalized to control EBs. Primer sequences for murine GAPDH were 5\'-GCACAGTCAAGGCCGAGAAT-3\' and 5\'-GCCTTCTCCATGGTGGTGAA-3\' (T~a~= 60°C). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Sequences and annealing temperatures for the primer sets used in RT-PCR ::: --------------------------------------------------------------------------- **Gene** **Primer sequence** **T~m~in °C** -------------------------- -------------------------------- --------------- **Chondrocyte-specific** Aggrecan 5\'-GATCTGGCATGAGAGAGGCG-3\'\ 61 5\'-GCCACGGTGCCCTTTTTAC-3\' Collagen type II 5\'-GCTGCTGACGCTGCTCATC-3\'\ 60 5\'-GGTTCTCCTTTCTGCCCCTT-3\' Collagen type X 5\'-CAAGCCAGGCTATGGAAGTC-3\'\ 60 5\'-AGCTGGGCCAATATCTCCTT-3\' Link protein 5\'-TTCTGGGCTATGACCGCTG-3\'\ 60 5\'-AGCGCCTTCTTGGTCGAGA-3\' Biglycan 5\'-CATGACAACCGTATCCGCAA-3\'\ 60 5\'-ATTCCCGCCCATCTCAATG-3\' Decorin 5\'-ATGACCCTGACAATCCCCTG-3\'\ 60 5\'-CCCAGATCAGAACACTGCACC-3\' Scleraxis 5\'-GGACCGCAAGCTCTCCAAG-3\'\ 62 5\'-ACCCACCAGCAGCACATTG-3\' Sox9 5\'-GCAGACCAGTACCCGCATCT-3\'\ 62 5\'-CTCGCTCTCGTTCAGCAGC-3\' **Adipocyte-specific** ADD1 5\'-CAGTGACTCTGAGCCCGACA-3\'\ 61 5\'-ATGCCTCGGCTATGTGAAGG-3\' PPARγ 5\'-ATCATCTACACGATGCTGGCC-3\'\ 59 5\'-CTCCCTGGTCATGAATCCTTG-3\' SCD1 5\'-ACACCATGGCGTTCCAAAAT-3\'\ 61 5\'-CGGCGTGTGTTTCTGAGAACT-3\' C/EBPα 5\'-CGCAAGAGCCGAGATAAAGC-3\'\ 60 5\'-GCGGTCATTGTCACTGGTCA-3\' GLUT-4 5\'-ATGGCTGTCGCTGGTTTCTC-3\'\ 59 5\'-ACCCATAGCATCCGCAACAT-3\' AP2 5\'-TGATGCCTTTGTGGGAACCT-3\'\ 58 5\'-GCAAAGCCCACTCCCACTT-3\' acrp30 5\'-AAGAAGGACAAGGCCGTTCTC-3\'\ 60 5\'-GAGGAGCACAGAGCCAGAGG-3\' LPL 5\'-CCAATGGAGGCACTTTCCAG-3\'\ 60 5\'-CCACGTCTCCGAGTCCTCTC-3\' --------------------------------------------------------------------------- Forward primer sequence is depicted from the 5\' to the 3\' end followed by the reverse primer. The appropriate annealing temperature for each set is given as T~m~in °C. ::: Quantification of chondrocyte yield by FACS ------------------------------------------- The GFP expressing plasmid pEGFP-1 (Clontech) under the control of the Aggrecan promotor (kind gift of John R. Matyas) was stably transfected into D3 ES cells using Invitrogen\'s Effectene system followed by neomycin selection. Genomic integration of the reporter was analyzed by RT-PCR with specific GFP primers (data not shown). ES cells were differentiated along the chondrocyte lineage, trypsinized into a single cell suspension and subjected to fluorescence-activated cell sorting on day 32 using a FACS Calibur instrument and the CellQuest software from Becton Dickinson (Germany). Ten thousand events were registered per sample and analysis of whole cells was performed using appropriate scatter gates to avoid cellular debris and aggregates. Oil-Red-O staining ------------------ Cells were washed with PBS and without any fixation directly overlaid with Oil-red-O working solution (0.18% Oil-Red-O dye/60% propanol) After a staining period of 15 minutes, ES cell cultures were rinsed in distilled water until desired colour was achieved. Statistical analysis -------------------- Data analysis was performed with ONE-WAY ANOVA on n = 3 experiments using SigmaStat version 2.03 (SPSS Inc., San Rafael, CA, USA). List of abbreviations ===================== ADD1 Adipocyte determination- and differentiation-dependent factor 1 aP2 Fatty acid-binding protein BMP Bone Morphogenetic Protein Cbfa1 Core binding factor alpha 1 C/EBP CCAAT/enhancer-binding protein Col Collagen DMMB Dimethylmethylenblue ECM extracellular matrix ES embryonic stem EB embryoid body FCS Fetal calf serum FGF Fibroblast growth factor GAPDH Glyceraldehyde-3-phosphate dehydrogenase GLUT4 Glucose transporter 4 IGF Insulin-like growth factor PPAR Peroxisome proliferator-activated receptor SCD Steroyl CoA desaturase Sox Sry-related high mobility group box TGFβ Transforming Growth Factor beta VD~3~Vitamin D~3~ Authors\' contributions ======================= NZN carried out cell culture, biochemical and molecular studies and drafted the manuscript. GK, DER and HJA participated in its design and coordination. All authors read and approved the final manuscript. Acknowledgements ================ This study was funded in part by the German Ministry of Education, Science, Research and Technology (BMBF) grant no. 0312314. NZN is supported by a post-doctoral fellowship from the Alberta Heritage Foundation of Medical Research (AHFMR). DER is an AHFMR Senior Scholar. The authors are thankful to the FACS core facility of the Faculty of Medicine for performing flow cytometry and to Dr. John R. Matyas for providing the Aggrecan-GFP construct. We furthermore appreciate the technical assistance of Lesley Davis.

PubMed Central

2024-06-05T03:55:52.505934

2005-1-26

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548146/", "journal": "BMC Dev Biol. 2005 Jan 26; 5:1", "authors": [ { "first": "Nicole I", "last": "zur Nieden" }, { "first": "Grazyna", "last": "Kempka" }, { "first": "Derrick E", "last": "Rancourt" }, { "first": "Hans-Jürgen", "last": "Ahr" } ] }

PMC548147

Background ========== In recent years, numerous publications -- over 50 pages of them in current listings of the bibliographic search engine Medline -- have detailed changes in biological systems and organisms that appear associated with the climatic changes commonly referred to as global warming (e.g., \[[@B1]-[@B5]\], dealing with observed or impending shifts in the habitats of various organisms). Few studies, however, have documented long-term changes in the *genetic structure*of species populations on a regional scale, necessary for understanding the microevolutionary consequences of global change \[[@B6]\]. In the genus *Drosophila*, comprised of over 1500 species \[[@B7]\], only four species have received such attention. While some long term increases have been documented in the frequencies of chromosomal gene arrangements Standard (*ST*), Tree Line (*TL*), and Pike\'s Peak (*PP*) in *Drosophila pseudoobscura*in the western United States and Canada over ca 40 years \[[@B8]-[@B10]\] and of several arrangements in *Drosophila melanogaster*in Japan \[[@B11]\], little is known of the causes for these changes. Orengo and Prevosti \[[@B12]\] first suggested that climatic warming was responsible for long-term temporal changes in the frequencies of certain gene arrangements in European populations of *D. subobscura*. Later, Rodriguez-Trelles and co-workers \[[@B13]-[@B15]\] and Sole\', [et al]{.underline}. \[[@B16]\] convincingly demonstrated that climatic changes, including global warming, were likely the driving force of microevolutionary changes in these populations. Recently, significant changes have also been documented in the chromosomal variation of *D. robusta*Sturtevant, some of them possibly attributable to global warming \[[@B17]-[@B19]\]. Here we describe additional data that underscore the variety of historical changes being experienced by populations of this species. Clearly, the chromosomal polymorphisms in *D. robusta*are also dynamic, and when compared with the considerable geographical and experimental data available for this species collected over the last 60 years, strongly implicate regional climatic changes as a cause for these temporal frequency shifts. Results ======= Regional patterns of climate change were revealed from ANCOVA analysis of temperature and precipitation data from 1945 -- 2003. No long term tends were detected for precipitation except for a significant increase in Central Park, NY (data available from the authors). Significant long-term temperature changes were apparent at all six 2003 study sites (Fig. [1](#F1){ref-type="fig"}; plus Philadelphia, last studied in 2002) for the three available temperature indicators: average monthly minimum temperature (*MINTMP*), average monthly temperature (*AVETMP*), and average maximum monthly temperature (*MAXTMP*; all 3 ANCOVA models, P \< 0.0001). *AVTEMP*and *MAXTMP*varied significantly from site to site in different years, but *MINTMP*significantly increased over the 58-year period (P \< 0.0001) with no heterogeneity among sites. Thus, the most consistent change in climate across sites in this study was due to significant increases in minimum monthly temperatures. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Map of the eastern United States showing the locations of collecting sites in this study. This modified map is used by permission of the University of Texas Libraries, the University of Texas at Austin. ::: ![](1471-2148-5-4-1) ::: Evidence for site-specific temperature increases from 1945 -- 2003 was evident for these three temperature indicators (Fig. [2](#F2){ref-type="fig"}). Five of the seven sites showed linear or curvilinear increases in *MINTMP*, usually starting in the 1970\'s and extending until 2000. In contrast, both Fayetteville and Englewood, NJ have experienced significant temporal decreases in *MAXTMP*(with a concurrent significant increase in *MINTMP*in Fayetteville). Causes for these decreases are obscure, but in New Jersey may be due to the exaggerated temperature fluctuations in the early 1990\'s (as well as missing data for 1992 and 1993, Fig. [2](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Temperature data for each site in this study plotted from 1945 to 2003. Regression lines and equations are shown only for statistically significant trends in mean maximum monthly temperature (*MAXTMP*), mean monthly temperature (*AVTMP*), and mean minimum monthly temperature (*MINTMP*) at each site. Significance and sign of the regression coefficients are indicated (\*P \< 0.05, \*\* P \< 0.01, \*\*\* P \< 0.001, \*\*\*\* P \< 0.0001). Y axes are in degrees Centigrade. Replicate data plotted for Fayetteville and New Jersey show incomplete secondary weather station data at sites closer to the locations where flies were collected. See text for details. ::: ![](1471-2148-5-4-2) ::: Concurrent with these temperature shifts, chromosome elements in populations of *D. robusta*show systematic temporal frequency changes (all frequency data are available from the authors). X chromosome combinations 1S and S1 show substantial frequency changes with time, with S1 doing so in a site-specific manner (Fig. [3](#F3){ref-type="fig"}). Frequencies of combination 1S have decreased over time, and are negatively correlated with increasing temperatures (Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}). Increases in S1 are only marginally correlated with temperature, but this may be due to other factors, including its low frequency in eastern populations (Fig. [3](#F3){ref-type="fig"}). Gene arrangements 2L-1 and 3R-1 have also increased in frequency since 1945 (Fig. [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}; Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}), and the changes are significantly site-specific as indicated by the significant year × site interactions. While temporal changes in 2L-1 and 3R-1 are correlated (r = 0.419, P = 0.0016, n = 54), only those of 3R-1 are significantly correlated with increasing temperatures (Fig. [5](#F5){ref-type="fig"}; Table [2](#T2){ref-type="table"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Frequencies (in percent) of *D. robusta X*-chromosome arrangement combination *S1*in the seven study localities, including Philadelphia \[18\]. When frequencies across years were statistically homogeneous, they were combined within each locality. Y axes for eastern populations reflect the much lower frequencies for S1 in that part of the species range. ::: ![](1471-2148-5-4-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### ANCOVA results for temporal changes in the frequencies of several X chromosome arrangement combinations and autosomal inversions across the seven collecting sites in this study. r^2^is an estimate of the proportion of the total variance explained by the model used. ::: X chromosome combination 1S ------------ ----------------------------- ------------- --------- ----------- ------- Source df Type III SS F Value Pr \> F r^2^ Model 13 1.151 50.31 \< 0.0001 0.942 Year 1 0.014 5.97 0.019 Site 6 0.207 14.93 \< 0.0001 Year\*Site 6 0.199 14.35 \< 0.0001 X chromosome combination S1 Source df Type III SS F Value Pr \> F r^2^ Model 13 2.589 50.94 \< 0.0001 0.943 Year 1 0.000 0.02 0.894 Site 6 0.206 8.79 \< 0.0001 Year\*Site 6 0.197 8.39 \< 0.0001 Gene arrangement 2L-1 Source df Type III SS F Value Pr \> F r^2^ Model 13 1.317 20.55 \< 0.0001 0.870 Year 1 0.038 7.66 0.0085 Site 6 0.215 7.25 \< 0.0001 Year\*Site 6 0.216 7.29 \< 0.0001 Gene arrangement 3R-1 Source df Type III SS F Value Pr \> F r^2^ Model 13 2.054 54.09 \< 0.0001 0.946 Year 1 0.043 14.76 0.0004 Site 6 0.057 3.28 0.0102 Year\*Site 6 0.060 3.40 0.0084 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Pearson product-moment correlations between average monthly temperature and frequencies of several X chromosome arrangement combinations and autosomal arrangements for all seven localities studied. n = 54 for all tests. ::: 1S S1 2L-1 3R-1 --------------------------------------------- ----------- ----------- ----------- ----------- Average temperature for month of collection \- 0.341 0.250 0.195 0.336 P = 0.012 P = 0.071 P = 0.163 P = 0.014 ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Frequencies (in percent) of *D. robusta*gene arrangement 2L-1 in the seven study localities, including Philadelphia \[18\]. See Fig. 3 for details. ::: ![](1471-2148-5-4-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Frequencies (in percent) of *D. robusta*gene arrangement 3R-1 in the seven study localities, including Philadelphia \[18\]. See Fig. 3 for details. ::: ![](1471-2148-5-4-5) ::: The most consistent historical changes have been gains in the frequency of arrangement 3R-1 with concomitant decreases of its allelic form, 3R (Figure [5](#F5){ref-type="fig"}). Coupled with the clinal tendency of 3R-1, varying between 100% in the southernmost latitudes and zero in the northernmost \[[@B20]\], its similar directional increases of recent years in at least four states, some far apart, and concordance with long term temperature increases indicates that 3R-1 is responding to regional climatic shifts. The frequency of arrangement 2L-1 increased directionally in most of the same localities as 3R-1 (Figure [4](#F4){ref-type="fig"}). The frequency of 2L-2, which is also more common in the south than in the north, has been decreasing steadily, almost to the point of extinction, in the four northern 2003 populations. This is probably not a temperature effect, however, as in the past 2L-2 has been irregularly distributed across the southern states, rarely rising above frequencies of 20 percent, without evidence of a latitudinal cline \[[@B20]-[@B22]\]. Like 2L-1 and 3R-1, the frequency of gene arrangement XL (the sum of data for *SS*, *S1*, and *S2*), tends to be higher in southern populations than in northern ones \[[@B20]\]. Except for a few deviations in small samples, it has increased steadily -- with concomitant decreases in northern arrangement XL-1 -- in all the localities studied except Olivette (where it is already about 95%) and Iowa. At Englewood, for example, it rose from about 40% in 1948 to an average of 78% in 2000--2003, that of northern arrangement XL-1 falling from about 60% to about 22% in the same period. Frequencies of X chromosome combination 1S (XL-1.XR) are clearly consistent with this pattern (Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}). Discussion ========== Studies of *Drosophila*inversion polymorphisms have now provided historical insights into environmental change. On different continents, temporal genetic changes correlated with increasing temperatures in populations of *D. subobscura*\[[@B12]-[@B16]\] and *D. robusta*\[\[[@B17],[@B18]\], this study\] bear the imprint of large scale climate change. For *D. robusta*, not all temporal changes were consistent with this hypothesis. In the course of comparing plant growth in rural and urban areas in relation to ozone exposure, rural areas tended to have lower average temperatures than urban ones \[[@B23]\]; this may explain the smaller (or lack of) change in 3R-1 frequencies in relatively rural Fayetteville and Iowa City as compared to the more urban locales. Also, there are fewer data available for these sites collected over time spans long enough to document temporal trends. Another possibility is that the climatic changes have not been of equal intensity in all localities, as indicated by regional increases and decreases in local temperatures (Fig. [2](#F2){ref-type="fig"}) suggested by the site by year interactions for *AVTMP*and *MAXTMP*in the ANCOVA analyses. Carson \[[@B24]\] noted that the chromosomal variation in populations of *Drosophila robusta*at Olivette, Missouri over a 10-year period was characterized by \"extraordinary stability,\" wherein \"certain frequencies may shift significantly as compared with the previous year, but in every case the observed frequencies approximate some previously observed level.\" This pattern continued until at least 1967 (Fig. [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}). Temperature records from this period seem rather stable before significantly increasing through 2000 (*AVTMP*and *MINTMP*second order regression slopes are both positive; Fig [2](#F2){ref-type="fig"}). Similar stability, frequency changes of less than 10 percent, has also been recorded near Blacksburg, Virginia from 1950 to 1962 (M. Levitan, unpublished data) and in northeastern New Jersey from 1948 until at least 1975 (Fig. [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}). By contrast, every population sampled in 2003 evidenced at least one significant change of chromosomal polymorphism frequency compared to the numbers in the same area ten or more years previously, with indication that the changes in at least three are part of similar, if not identical, directional historical processes. Therefore, documentation of temporal genetic changes in these populations requires at least 10 to 20 years of comparative data. Influences of natural selection due to ambient temperature variation on frequencies of these gene arrangements and X chromosome associations have been demonstrated in laboratory experiments \[[@B24],[@B25]\], and inferences from latitudinal and multiple elevational clines \[[@B26]-[@B31]\]. Frequencies of arrangements XL-1 and 2L-3 are clearly associated with cooler temperatures at higher elevations and latitudes, and 2L-1 increases in frequency in the laboratory under warmer temperatures. Carriers of 2L-3 have shorter egg to adult development times expressed in cooler temperatures, explaining increases in this gene arrangement with elevation and latitude \[[@B26]\]. Evidence of strong natural selection maintaining earlier observed genetic stability has also come from perturbation experiments in the wild \[[@B20]\]. At four time periods, large numbers of flies carrying X-chromosome combinations *S2*and *22*, autosomal combination 2L-1.2R-1 (*11*), and 3R-1 from South Carolina, Alabama, and Mississippi were released in midsummer in the Englewood, NJ woods. In other years the released flies carried XL-1, 2L-3, and 3R from Minnesota and Michigan. Despite evidence of hybridization of the introduced flies with the local population, in each case the following spring saw return to frequencies of the previous year. According to Wright \[[@B32]\], \"The alternative to natural selection in a changing environment is, as noted by Dobzhansky \[[@B33]\], the emergence of superior genetic systems\" in the gene arrangements that have been increasing in frequency. He envisioned this happening in one population by recombination in inversion homozygotes, with the new adaptive gene complex in one or a few flies spreading to other localities by \"occasional very long dispersion by the wind.\" He conceded that such a hypothesis would be most satisfactory for cases where a very rare inversion suddenly started increasing, such as the rises of *TL*and *PP*of *D. pseudoobscura*mentioned above, since high frequencies of inversion homozygotes would undermine the structural stability of the new adaptive complex. The sudden rises of *D. robusta*X-chromosome combination *S1*in New York and New Jersey and of *S2*in Missouri could be cases in point, but it would not explain the near doubling of *S1*in Arkansas from a 35% base between 2001 and 2003 nor the three most consistent directional changes, those of 3R-1, 2L-1, and XL (Figures [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}). Indeed, the nearly simultaneous changes in places as far apart as New York and Allentown, Pennsylvania, let alone New York and Missouri, would be very unlikely to depend on chance wind-blown dispersions. Here, causes for systematic frequency changes in widespread populations can be inferred to be a result of common environmental causes given the large amount of background data available for *D. robusta*. When compared to the inversion frequency data collected over 60 years from more than 150 natural populations \[[@B20],[@B21]\] and results from numerous laboratory experiments, temporal frequency shifts across the range of *D. robusta*strongly suggest temperature variation as a likely mechanism driving microevolutionary change \[[@B17],[@B18]\]. Such regional frequency shifts suggest a common response to temperature fluctuations or causes correlated with them, whether due to regional climatic changes or global warming. Conclusions =========== Although direct evidence for climatic change has been accumulating for many years, its consequences for causing evolutionary changes have only recently been observed. Chromosome polymorphisms in *Drosophila*species have been historically important genetic systems for understanding mechanisms of evolutionary change, and have now been studied long enough to begin revealing widespread, systematic temporal frequency shifts in response to environmental change. These polymorphisms thus represent excellent indicators of future climatic shifts. Methods ======= There are 14 commonly encountered gene arrangements segregating in natural populations of *D. robusta*located on five of the six arms of the 3 major chromosomes. The \"Standard\" arrangements were labeled for the respective chromosome arms: XL, XR, 2L, etc. Others were named in order of their discovery, e.g., XL-1, XL-2, XR-1, 2L-1 \[described and configured in \[[@B20],[@B24]\]\]. The Standard arrangement of each arm was dubbed \"S,\" and the other arrangements are referred to by the Arabic numerals in their names \[[@B34]\]. A fly with karyotype XL/XL-1, XR/XR-2, for example, would be S/1, S/2 in this notation. Depending on the linkage combination of the arrangements, it is also either *SS*/*12*or *S2*/*1S*. Linkage relationships are inferred from karyotypic analyses of adult males and females \[[@B35]\]. Female *D. robusta*, unlike many other drosophilids, quickly deplete stored sperm in the absence of remating so that wild-caught females can be despermed by repeated transfers to fresh food vials and then crossed to homokaryotypic males in a controlled fashion. Karyotypes of at least 6 larvae from these test crosses were prepared in order to infer the linkage combination of X chromosome gene arrangements. Salivary gland smears from larvae derived from matings in the wild, so-called \"egg sample\" data, were included when collected females did not survive the desperming transfers. This report compares data obtained in 2003 to earlier summer collections at a number of geographically isolated populations going back in some cases to 1946: Olivette, a suburb of St. Louis, Missouri; woods alongside Route 4 in Englewood, New Jersey; Trexler Memorial Park on the western outskirts of Allentown, Pennsylvania; the North Woods of Central Park in New York City; Fayetteville, Arkansas; and woods along the Iowa River at Iowa City, Iowa. The significance of year-to-year differences in frequency of each chromosome or chromosome arm obtained prior to 2003 was determined by G-tests \[[@B36]\]. Chronologically contiguous results that proved statistically homogeneous are combined in the figures. Individual X- and second-chromosome arrangements could not be tested in this way due to problems of independence. Analysis of meteorological data =============================== Temperature and precipitation data were obtained online from the National Climatic Data Center <http://www.ncdc.noaa.gov/oa/ncdc.html> for stations with complete records extending from 1945--2003 nearest to each site. Continuous data for Fayetteville and Englewood were not available for the closest weather stations, so records from those stations with complete records were used. Correlations between temperatures from these stations were highly significant: Drake Field and the Agricultural Experiment Station for Fayetteville (r = 0.48 -- 0.90, P \< 0.01), Little Falls and Ridgefield for Englewood (r = 0.84 -- 0.98, P \< 0.0001); see Fig. [2](#F2){ref-type="fig"}. Analysis of covariance in PROC GLM \[[@B36]\] was used to test for overall significance of temperature trends and chromosome frequency changes across sites, and to evaluate regional patterns of temperature and precipitation change. Polynomial regression analyses of temperature and precipitation data with time were performed with PROC REG \[[@B37]\]. In all cases, linear or second order polynomial regression explained the most variation for a particular model. Pearson product-moment correlations between arcsin transformed chromosome frequencies and the average temperature of the month for each collection were calculated with PROC CORR \[[@B37]\]. Non-parametric correlation analyses produced equivalent results. We also assessed correlations with temperatures for the month prior to collection, and the average temperature of the 3 months prior to collection: none were significant. Authors\' contributions ======================= ML collected the Allentown, New York, and New Jersey flies and carried out the chromosomal analyses. WJE contributed the Fayetteville flies. Both authors contributed to planning the study, analyzing the data, and writing and revising the manuscript. Acknowledgments =============== The authors thank the 1996 M. M. Kaplan Family Foundation for grant GCO 00-1223 to ML and NSF grant DEB-0211125 to WJE; A. R. Templeton for the 2002 and 2003 collections in Missouri; B. McAllister for the 2003 collections in Iowa; M. M. Kaplan for advice and comments on the manuscript; S. Beaupré for statistical advice; N. Maj for computer and graphics assistance; and S. Hanada, N. Khanna, S. Gmuca, and K. Chan, sponsored by the New York Academy of Sciences, for assistance in the analysis of the 2003 collections.

PubMed Central

2024-06-05T03:55:52.509995

2005-1-6

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548147/", "journal": "BMC Evol Biol. 2005 Jan 6; 5:4", "authors": [ { "first": "Max", "last": "Levitan" }, { "first": "William J", "last": "Etges" } ] }

PMC548148

Background ========== Positional clustering of co-expressed genes is mainly due to operons in prokaryotes. Genes located within the same operon are transcribed together and thus co-regulated. In general, operons are missing from eukaryotes. Eukaryotic genes appear to be transcribed individually and are thought to be scattered on chromosomes without apparent organization by function or positional expression except for tandem duplication. However, positional clustering has recently been reported in several eukaryotes such as *Saccharomyces cerevisiae*\[[@B1],[@B23]\], *Drosophila melanogaster*\[[@B4],[@B5]\], *Caenorhabditis elegans*\[[@B2],[@B3],[@B7]\], and *Homo sapiens*\[[@B6],[@B8]\]. Cho *et al*. first showed clustering of co-expressed yeast genes on a genome-scale. They found that about 25% of genes with cell-cycle-dependent expression pattern were adjacent to those induced in the same phase of the cell cycle \[[@B23]\]. This fact was also reported by Cohen *et al*. \[[@B1]\]. Cohen *et al*. computationally analyzed the whole-genome gene expression using the same dataset. They consider two ORFs to be adjacent if they occur on the same chromosome and if there are no other ORFs or functional elements such as Ty element, long terminal repeats, tRNAs, snRNAs between them. They observed that adjacent or nearby non-adjacent pairs of genes showed correlated expression independent of their orientation in the yeast genome using chromosome correlation maps that display correlations between the expression patterns of genes on the same chromosome. In addition, they also showed that genes with similar functions tend to occur in adjacent positions along the chromosomes. Spellman *et al*. \[[@B4]\] found that numerous clusters that span 10 to 30 physically adjacent genes share strikingly similar expression profile in *Drosophila melanogaster*and these clustered genes account for over 20% of the total assayed genes. By mapping Expressed Sequence Tags (EST) back to the *Drosophila*genome, Boutanaev *et al*. \[[@B5]\] observed almost one thirds of 1661 testis-specific genes are clustered on chromosomes. Similarly, positional clustering of co-expressed genes was also reported in *Caenorhabditis elegans*. Although operon and tandem duplication are major mechanisms for the observed co-expression gene clusters in the worm, co-expression gene clustering is still evident after exclusion of these two causes \[[@B3],[@B7]\]. Clusters of highly co-expressed genes were also revealed in the human and other mammalian genomes \[[@B6],[@B8],[@B5],[@B12]\]. Most of these studies focused on a set of tissue-specific genes that are highly expressed. For example, by analyzing the expression profiles of genes in carious tissues, Caron *et al*. \[[@B8]\] showed that highly expressed carious genes tend to cluster in large domains, called RIDGEs (*Region of IncreaseD Gene Expression*). However, these RIDGES seem to consist mainly of housekeeping genes \[[@B6]\]. Gabrielsson *et al*. \[[@B9]\] performed a microarray analysis of genes expressed in the human adipose tissue. By mapping these genes back to the human genome, they observed clusters of adipose tissue specific genes. Dempsey *et al*. \[[@B10]\] investigated genes from chromosomes 21 and 22 expressed in the human cardio-vascular system. Using EST (*Expressed Sequence Tag*), they showed positional clustering of these genes. These studies suggest that clusters of tissue-specific genes do exist, and might be more frequent than initially thought. Here we performed a genome-wide analysis of testis-specific genes in the mouse genome using a method similar to one proposed in \[[@B5]\]. We used a publicly available EST (Expressed Sequence Tag) database to identify differentially expressed genes. After mapping testis-expressed ESTs back to the genome, we obtained the testis-expressed gene expression profile by assembling overlapped ESTs. In the same way, we got embryo-expressed gene expression profile. Testis-specific genes were generated by subtracting embryo-expressed gene expression profile from testes-expressed one. From the testis-specific gene expression profile, clustered genes were observed. To further investigate testis-specific gene clusters in the mouse genome, we used the full-length cDNA sequences publicly available in the Fantom 2.1.1 database to identify differently expressed genes. After mapping 60,770 cDNA sequences back to the genome, we obtained the gene expression profile by assembling overlapping cDNA sequences into a single gene model. Testis-specific genes were selected by using only sequences from libraries where the tissue type included the keyword \"testis\" and not the keyword \"embryo\". Again, our statistical analysis shows the existence of testis-specific gene clusters along the mouse chromosomes even after removal of tandem duplicated genes. This suggests that considerable clusters of tissue-specific genes do exist in higher eukaryotes. Our results provide further support for the hypothesis that chromatin domain model may be involved in the regulation of gene expression in higher eukaryotes Results ======= The testis-expressed profile is a global view of gene expression and provides a foundation for understanding how the tissue functions in molecular level. We assume that most of the testis-specific genes are expressed after embryo phases in mouse. Therefore, we selected only testis genes models that exclude any expression in embryo for the construction of the testis-specific gene profile, which reveals positional clustering along chromosomes. Here, we analyzed the cluster distribution on all the chromosomes except for chromosome Y. (In Y chromosome, we only identified 12 testis-specific genes and 7 other genes.) Based on our study, we obtain the following observations. a. Density of testis-specific genes ----------------------------------- The testis-specific gene expression profile along the mouse genome shows that testis-specific genes are distributed on all the chromosomes with different gene density. The ratio (R) of the number of the observed testis-specific genes (O) to the expected number (E) is used to measure the gene density, where E is calculated according to the chromosome\'s size on assumption that the testis-specific genes are uniformly distributed throughout the genome. The lowest gene density appears on chromosomes 1 (R = 0.834), 15 (R = 0.893), 16 (0.855), and 18 (R = 0.767) except for sex chromosomes. But, chromosomes 9 (R = 1.145), 11 (R = 1.374), 14 (R = 1.207) and 19 (R = 1.48) have rather high gene density. Within a chromosome, the distribution of testes-expressed genes also shows diverse density. b. Genomic location is highly correlated with testis-specific gene expression ----------------------------------------------------------------------------- We observed numerous groups of physically neighboring testis-specific genes that shared strikingly similar expression levels. In eukaryotes, it is clear that the activity of a promoter can be modified by transcription factors binding to DNA sequence, which are located from hundreds to thousands of base pairs from the promoter. Recently, increasing evidences show that genes may also be regulated in a group. For example, when transgenes are removed from their local environment and reinserted elsewhere in the same genome, they tend to show variant levels of expression \[[@B13]\]. More commonly, most eukaryotic chromosomes exhibit transcriptionally active (euchromatin) or inactive (heterochromatin) regions. In animals, heterochromatin is typically found near the centromere and other regions of low sequence complexity. Since RIDGEs do exist and neighboring genes show similar expression level, we conclude that genomic location has impact on gene expression. c. A large proportion of testis-specific genes are in adjacent pairs -------------------------------------------------------------------- We consider two genes are adjacent if there are no other genes between them on the same chromosome. We observed that 1170 testis-specific genes appear in adjacent pairs, which account for 31.8% of the testis-specific genes after removal of tandemly duplicated genes. There are 350 adjacent pairs, 86 adjacent triplets, 23 adjacent quadruplets, and 4 quintuplets (see Table [1](#T1){ref-type="table"} for details), significantly more than would be expected by chance. In fact, the number of observed adjacent gene clusters (including singletons) is smaller than the expected number minus the standard deviation for every chromosome other than chromosomes 15 and 19. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Chromosomal distribution of adjacent testis-specific genes after removal of tandemly duplicated genes. ::: Size Cluster distributions of adjacent testis-specific genes on chromosomes ------- ------------------------------------------------------------------------ ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- ---- --- ---- ---- --- --- ---- ----- **2** 26 22 21 26 29 22 17 20 14 24 19 18 18 20 6 15 13 9 9 12 350 **3** 2 5 3 6 6 4 4 4 12 4 8 1 2 5 5 4 3 3 1 4 86 **4** 2 1 1 2 2 3 1 2 1 2 3 2 1 23 **5** 1 1 1 1 4 ::: The genes in the quintuplets are probably worthy to be investigated biologically. There is a quintuplet on each of chromosomes 6, 12, 14 and X. The quintuplet on chromosome 6 is located in a 400 kb segment starting at 144691608 and ending at 145141155; the one on chromosome 12 is located in a 600 K segment starting at 101700908 and ending at 102297618; the one on chromosome 14 is located in a 700 K segment starting at 62802300 and ending at 63500814. However, the one on chromosome X seems less significant, which is located in a 3719 K segment starting at 113253694. d. Testis-specific genes show obvious clustering ------------------------------------------------ We evaluated the positional clustering of testis-specific genes using a model called the neighborhood model. We define clusters using the distance between two neighboring testis-specific genes along the chromosome. Two testis-specific genes are in a cluster if there is a series of testis-specific genes between them such that the distance between two neighboring genes in the series is less than a specified distance (D). We obtained 3679 testis-specific genes on all the chromosomes except for Y after removal of tandemly duplicated genes. We conducted statistical analysis with 4 different values of D: 25 K, 50 K, 75 K, and 100 K (see Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Distribution of testis-specific gene clusters after removal of tandemly duplicated genes. Here only the numbers of clusters having at least 2 genes on all the chromosomes except for chromosome Y are given. ::: Size Cluster distributions on chromosomes (distance = 25 K) ------- --------------------------------------------------------- ------- ------- ------- ------- ------- ------- ------- ------- -------- -------- -------- -------- -------- -------- -------- -------- -------- -------- ------- ------- **2** 18 22 17 22 23 13 17 17 18 22 16 12 15 15 6 8 7 7 7 7 288 **3** 1 1 2 1 2 3 1 2 1 3 1 21 **4** 1 1 2 **5** **6** Size Cluster distributions on chromosomes (distance = 50 K) **1** **2** **3** **4** **5** **6** **7** **8** **9** **10** **11** **12** **13** **14** **15** **16** **17** **18** **19** **X** Total **2** 26 30 22 32 34 20 21 22 19 31 22 16 16 20 15 14 12 8 6 6 392 **3** 2 5 1 2 6 1 8 3 6 3 3 3 4 3 2 1 2 1 56 **4** 1 5 2 1 2 2 1 1 1 1 2 19 **5** 1 1 **6** Size Cluster distributions on chromosomes (distance = 75 K) **1** **2** **3** **4** **5** **6** **7** **8** **9** **10** **11** **12** **13** **14** **15** **16** **17** **18** **19** **X** Total **2** 27 28 23 37 47 29 24 29 17 34 29 17 18 24 16 16 19 11 9 10 454 **3** 1 4 4 5 4 5 9 2 9 2 7 3 3 6 5 4 1 1 3 1 79 **4** 2 6 3 1 3 1 2 4 1 1 1 1 1 1 28 **5** 1 2 1 4 **6** 1 1 Size Cluster distributions on chromosomes (distance = 100 K) **1** **2** **3** **4** **5** **6** **7** **8** **9** **10** **11** **12** **13** **14** **15** **16** **17** **18** **19** **X** Total **2** 32 33 24 31 42 33 28 27 19 39 23 21 21 25 16 15 24 9 9 12 463 **3** 2 6 4 7 8 6 12 8 12 3 11 2 4 7 4 6 2 3 6 1 114 **4** 3 6 5 3 1 1 3 1 3 7 2 2 2 2 1 1 2 1 46 **5** 1 1 1 2 1 1 7 **6** 1 1 2 ::: When D = 25 K, we obtained 288 two-gene, 21 three-gene, and 2 four-gene clusters. In total, 18% of the testis-specific genes are contained in these clusters. By incorporating the variance of gene density in different regions on a chromosome, we conclude that 45 two-gene, 7 three-gene and 2 four-gene clusters are significant with p-value less than 4.9·10^-2^, 3.1·10^-2^and 1.2·10^-2^respectively. The most significant cluster is the four-gene cluster on chromosome 2 (p-value = 1.3·10^-3^). When D = 50 K, we obtained 392 two-gene, 56 three-gene, 19 four-gene and 1 five-gene clusters. In total, 28% of the testis-specific genes are in these clusters. By incorporating the variance of gene density in different regions on a same chromosome, we conclude that 14 two-gene, 6 three-gene and 2 four-gene clusters are significant with p-value less than 4.9·10^-2^, 3.1·10^-2^and 1.2·10^-2^respectively. The most significant one is a two-gene cluster on chromosome 18 (p-value = 5.3·10^-3^). When D = 75 K, we obtained 454 two-gene, 79 three-gene, 28 four-gene, 4 five-gene and 1 six-gene clusters. In total, 35% of the testis-specific genes are in these clusters. By incorporating the variance of gene density in different regions on a chromosome, we conclude that 10 two-gene, 2 three-gene and 3 four-gene clusters are significant with p-value less than 4.1·10^-2^, 3.1·10^-2^and 4.9·10^-2^respectively. The most significant one is a two-gene cluster on chromosome 16 (p-value = 5.2·10^-3^). When D = 100 K, we obtained 463 two-gene, 114 three-gene, 46 four-gene, 7 five-gene and 2 six-gene clusters. In total, 41% of the testis-specific genes are in these clusters. By incorporating the variance of gene density in different regions even on the same chromosome, we conclude that 12 two-gene, 4 three-gene and 1 four-gene clusters are significant with p-value less than 4.9·10^-2^, 4.8·10^-2^and 3.2·10^-2^respectively. The most significant one is a two-gene cluster on chromosome 16 (p-value = 9.2·10^-3^). We also observed that there are more significant gene clusters on chromosomes 5, 7, 12 X than any other chromosomes. Discussion ========== It seems that in most eukaryotes, the transcription factor machinery is sufficient for ensuring co-regulation so that co-localization of genes in a genome is not necessary. However, there could be some degree of selection for keeping co-regulated genes in clusters on a chromosome, for instance, to make them available to transcription more efficient as a group. Gene clusters due to tandem duplication --------------------------------------- Clustering of co-expressed genes could be due to tandem duplication. Tandem duplication and subsequent divergence of amplified copies might result in an array of paralogous genes that may retain common regulatory element. Such a type of clustering is exemplified by the Hox gene family \[[@B14]\]. From the testis-specific gene representation profile, we can observe that besides co-expressed adjacent gene pairs, there are large-scale clusters on chromosomes. To determine to what degree tandem duplication contributes to the clustering of testis-specific co-expressed genes, we analyzed cluster distribution after removal of duplicated genes. To identify duplicated testis-specific genes, we performed an all-against-all BLAST homology search using default BLASTN settings. (See the method part for details.) In total, 240 duplicated genes were identified and removed from the list of the testis-specific genes (see Table [3](#T3){ref-type="table"} for details). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Testis-specific gene distribution before and after removal of tandem duplications. ::: **Chromosome** **Number of genes** **Difference** ---------------- --------------------- ---------------- ----- **1** 249 234 15 **2** 298 280 18 **3** 223 209 14 **4** 230 220 10 **5** 253 232 21 **6** 210 201 9 **7** 229 211 18 **8** 220 205 15 **9** 217 207 10 **10** 223 207 16 **11** 250 239 11 **12** 171 162 9 **13** 179 168 11 **14** 213 199 14 **15** 142 136 6 **16** 128 119 9 **17** 155 145 10 **18** 104 94 10 **19** 109 106 3 **X** 113 105 8 **Y** 12 9 3 **Total** 3928 3688 240 ::: In the most of chromosomes except for chromosomes 2, 5, 7, 10, no more than 15 duplicated genes were identified. On chromosomes 2, 5, 7, 10, we identified 18, 21, 19, 16 duplicated genes respectively. Since, for each of distance threshold value (25 K, 50 K, 75 K and 100 K), we obtained more than 300 gene clusters, we conclude that duplication has no obvious effect on the positional clustering of testis-specific genes in the mouse genome. Non-duplicated clusters of genes -------------------------------- Eukaryotic genes have not only promoters, but also one or more enhancers. An enhancer is a DNA sequence that influences transcription of neighboring genes. Enhancers are different from promoters in their ability to act over thousands of base pairs upstream or downstream of a gene. While a promoter is responsible for initiating low levels of transcription and determining the transcription start site, enhancers are responsible for increasing or \"enhancing\" transcription levels, as well as for regulating cell- or tissue-specific transcription. There are presently two models for non-duplicated gene clustering. One is the incidental expression mode. In this model, co-regulation within an expression neighborhood is due to incidental interactions between transcriptional enhancers and promoters \[[@B16]\]. When transcription factor binds at the appropriate sites and activates nearby genes together with the target gene, a group of nearby genes tend to express together. In this case sites that bind strong transcriptional activators should create new neighboring co-expressed genes. An alternative model of clustering is that gene clusters may correspond to regions of active chromatin. This explanation is called the chromatin domain model. In mammalian genomes, gene clusters are on a much larger scale (Mb rather than 5--20 kb) and may involve long-range chromatin interactions \[[@B18],[@B19]\]. The structure of the chromatin domain model assumes that opening of the chromatin of an entire cluster depends on activation of a target gene within the cluster. Our findings can most be explained as regulation at the level of chromatin structure for the following reasons. First, the regions showing similarities in expression are quite large; each gene presumably has its own core promoter. Second, one or two genes in a group often displays a high level of differential expression. If the chromatin in a region that contains many genes was \'opened\' so that a single target gene could be expressed, it might increase the accessibility of the promoters and enhancers of other genes to transcriptional machinery, leading to corresponding increases in their expression. Such an effect could account for the observations we have made. Although the organization of neighborhoods along a chromosome indicates that there must be cis-acting structures, no known structure correlates with the blocks. The structure basis of positional gene clustering is still under investigation. After removal of tandemly duplicated genes, significant clusters of co-expressed testis-specific genes were still found on all the chromosomes. However, the mechanism responsible for this observation is still unknown. Conclusion ========== We revealed the positional clustering of testis-specific genes in the mouse genome by using EST profiling. Our analysis implies that the mouse genome is enriched with clusters of testis-specific genes. A significant trend for large clusters (at least 3 genes) was discernible. Positional clustering of co-expressed, non-homologous genes has also been reported in nematode \[[@B3]\] and in mammals \[[@B6]\]. This indicates that positional clustering is common in higher eukaryotes. Clustering of tissue-specific genes suggests that there exists some higher order in regulation of gene expression, most probably due to the chromatin domain. From this fascinating starting point we expect further insight into the significance of gene clustering and the mechanisms that generate them as more genomes are sequenced and more expression patterns are studied in the coming years. Methods ======= Testis expressed gene models ---------------------------- A total of 60,770 full-length cDNA sequences are available in the Fantom (Funtional Annotation of Mouse) 2.1.1 database. These sequences were aligned back to the mouse genome using BLAT \[[@B21]\] (a program that specializes in the alignment of EST/cDNA sequences to genomic sequences). Overlapping alignments were assembled to produce a complete gene model. This resulted in 34,402 genes. The 3,928 testis-specific genes were identified on the basis of having the keyword \"testis\" but not the keyword \"embryo\" in the tissue type information associated with the library. Removal of tandem repeats ------------------------- Clusters of co-expressed genes could be due to tandem duplication. To remove effect of local duplication, we did an all-against-all BLAST search on all 3,928 testis-specific genes. As a result, 240 duplicated genes were identified on the basis of an expectancy value less than 10^-50^as proposed by Friedman and Hughes \[[@B20]\]. This results in the final 3,688 testis-specific genes used for our statistical analysis. Statistic analysis ------------------ ### The neighborhood model Recall that we define clusters using the distance between two neighboring testis-specific genes along the chromosome. Two testis-specific genes are in a cluster if and only if there is a series of testis-specific genes between them such that the distance between two consecutive genes in the series is less than a specified threshold (*D*). To incorporate the variance of gene density in different regions on a chromosome, we divide a chromosome into segments of length *L*(2000 Kbp here) so that genes on such a segment are roughly uniformly distributed and analyze the significance of a cluster within the segment according to the number *N*of all the genes (not just testis-specific genes) in the segment and the values of parameters *L*and *D*. Since we assume the (start) position of a gene is uniformly distributed in a segment of length L, it falls in an interval (*x*, *x*+ *D*) in the segment with probability *D*/*L*. Thus, the number of genes that fall in this interval has a binomial distribution with mean *ND*/*L*. In our case, *D*/*L*is smaller than 0.1. This distribution is approximately Poisson with mean *ND*/*L*. Here we use this approximation in our analysis. The Poisson approximation implies that the number of the genes that falls in a randomly chosen interval of length *D*has a Poisson distribution with mean *m*= *ND*/*L*, and so the probability that at least one gene falls in this interval is 1 - *e*^-*m*^. Suppose *g*~1~, *g*~2~, ..., *g*~*n*~form a cluster in the segment. Then, all the distances of two successive genes are less than *D*. Thus, the number of genes in a cluster minus one has a geometric distribution with p = 1 - *e*^-*m*^(see page 10, \[[@B22]\]). This implies that the probability that a cluster has n genes is (1 - *p*) *p*^*n*-1^, *n*= 0, 1, 2, .... Hence, the p-value of a cluster with *n*genes is the probability that a cluster has more than n genes, which is *p*^*n*^. In our analysis, *D*= 25 K, 50, 75 K, 100 K. Actually, the distance between two successive testis-specific genes in the most of observed clusters is much smaller than D in each case. Hence, the real p-value for each cluster could be much smaller than one reported in the result section. ### Adjacent gene clustering To remove the effect of non-uniform distribution of genes on analysis, we adopt an idea from order statistics. Here, we focus on the position rank of a gene rather than its specific position by ordering all the genes according to their positions on a chromosome. We also treat the set of the testis-specific genes and the set of all the other genes as two types of identical objects. In this way, a specific gene distribution on a chromosome is modeled as a 0--1 string in which 0 represents a test-specific gene and 1 a non-testis-specific gene. Assume there are *M*genes in a chromosome and *T*of them are testis-specific genes, where *T*≤ *M*. Then, there are ![](1471-2164-6-7-i1.gif) possible gene arrangements on the chromosome and the number of arrangements with *r*adjacent testis-specific gene clusters is ![](1471-2164-6-7-i2.gif). Thus, the probability that a random arrangement has *r*adjacent testis-specific gene clusters is ![](1471-2164-6-7-i3.gif) Using above formula, we can obtain that the mean number of adjacent testis-specific gene clusters in a random arrangement is ![](1471-2164-6-7-i4.gif) and the standard deviation is ![](1471-2164-6-7-i5.gif). Take chromosome 4 for example. We have *M*= 2060, *T*= 220, we obtain that the mean number is 196.6 and the standard deviation is 4.4. However, we only observed 182 adjacent gene clusters (including singletons) on the chromosome. Authors\' contributions ======================= BL and QL carried out the software development, sequence analysis, and the design of the study. LXZ conceived of the study, performed the statistical analysis, and participated in its coordination. All authors read and approved the final manuscript. Acknowledgement =============== The authors would like to thank the anonymous reviewers for their extremely helpful comments and suggestions particularly on using Fantom database and incorporating gene density variance into statistic analysis. The work was partially supported by Singapore BMRC research grant BMRC01/1/21/19/140.

PubMed Central

2024-06-05T03:55:52.512581

2005-1-19

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548148/", "journal": "BMC Genomics. 2005 Jan 19; 6:7", "authors": [ { "first": "Quan", "last": "Li" }, { "first": "Bernett TK", "last": "Lee" }, { "first": "Louxin", "last": "Zhang" } ] }

PMC548149

Background ========== The nested case-control design employs a case-control approach within an established cohort \[[@B1],[@B2]\] to obtain estimates from a sample of the cohort that are similar to estimates obtained from analysis of the entire cohort \[[@B3],[@B4]\]. The nested case-control design is being increasingly used in large cohorts of patients from prospective studies and randomized clinical trials. This design has become popular because it allows for statistically efficient analysis of data from a cohort with substantial savings in cost and time \[[@B5],[@B6]\]. When studying exposures that vary with time, an additional level of complexity is introduced by the need to account for time-dependent exposure in both the design and analysis \[[@B7],[@B8]\]. This can be accomplished by including time-dependent covariates in a Cox proportional-hazards regression model \[[@B9]\]. Alternatively, a nested case-control approach can be used provided that the exposure and covariate information for controls reflects values corresponding to the time of selection of their respective case. This study compares nested case-control and survival analysis methodologies for evaluating time-dependent exposure. The risk of pacemaker insertion associated with dosage of amiodarone (an anti-arrhythmic medication used for the treatment of atrial fibrillation (AF)), represented by a time-dependent covariate, is evaluated in cohort of patients with AF using both methods for illustrative purposes. The comparability of results is evaluated and differences in computational efficiency are quantified for increasing cohort sizes. Advantages and limitations of the respective methodologies are discussed. Methods ======= Study cohort ------------ A cohort of 11395 elderly (\>65 years of age) Quebec residents with AF and a myocardial infarction (MI) between 1991 and 2000 was created by linking the provincial hospital discharge summary database with the provincial physician and drug claims database, using methods described previously \[[@B10]\]. Approval for the study was obtained from the McGill University Faculty of Medicine Institutional Review Board. In order to evaluate the effect of amiodarone dose on the previously demonstrated association between amiodarone therapy for AF and an increased risk of permanent pacemaker requirement \[[@B10]\], only patients newly started on amiodarone after their diagnosis of AF were included in the study cohort. Amiodarone dose was represented as a binary time-dependent variable comparing daily doses \>200 mg to ≤200 mg. Covariate information included age, sex, calendar year of cohort entry, baseline sinus node or conduction disorder, ventricular arrhythmia, and time-dependent exposure to five categories of medications. The final study cohort included 1340 subjects followed from the date of their first prescription of amiodarone until the first of pacemaker implantation, death, or March 31, 2001. Statistical analysis -------------------- The data for the entire cohort of 1340 subjects including all fixed and time-dependent variables was represented in counting process notation suitable for Cox regression of time-dependent exposure \[[@B11]\]. Multiple records (with consecutive start and end times) were created for each subject to account for every change in exposure to any of the time-dependent variables over the study period. The hazard ratio (HR) of pacemaker insertion associated with amiodarone doses \>200 mg per day was estimated using a Cox proportional-hazards model including all fixed and time-dependent covariates. The timescale used in the model was time since first prescription of amiodarone. Non-significant variables (other than age and sex) were sequentially removed if the resultant model had no significant increase in Akaike Information Criteria (AIC) and no significant change in the HR for amiodarone dose. The nested case-control approach was also used to estimate the HR of pacemaker insertion associated with amiodarone doses \>200 mg per day. Cases of pacemaker insertion were identified and controls were randomly selected from the risk-set of each case (i.e. subjects present in the cohort at the time the case is defined). After selecting all controls and recording their index dates (i.e. the time, in cohort time, at which the respective case is defined) the relevant time-dependent covariate information was retrieved by merging with the database configured in counting process notation. The relevant subject record was selected by requiring that the index date fall within the start and end time of the subject record for each control. The nested case-control approach was repeated using 4, 8, 16, 32, and 64 controls per case. For each number of controls per case, random sampling of controls for all cases and conditional logistic regression analysis was repeated 100 times using the OUTEST option in the PROC PHREG statement to create an output SAS data containing all the parameter estimates \[[@B11]\]. The mean and standard deviation (SD) of the parameter estimates for each number of controls per case was calculated. Computational times for regression models of time-dependent exposures using nested case-control and survival analysis methodologies were compared. The nested case-control samples with 4 and 32 controls per case were analyzed using conditional logistic regression with the PHREG procedure in SAS Release 8.2 \[[@B12]\]. The full cohort was analyzed using Cox regression adapted for analysis of time-dependent covariates with the PHREG procedure in SAS Release 8.2. Ties were handled using the TIES = EFRON option in the PHREG procedure \[[@B11]\]. All analyses were performed using an Intel Pentium 4 computer with a 1.80 GHz central processing unit (CPU) and 256 MB of random access memory (RAM). Relative computational efficiencies were evaluated by comparing the CPU times of the three regression models used to analyze the cohort. Relative increases in computational time as a function of sample size were quantified by repeating the analyses on progressively larger cohorts. This was done by progressive doubling of the original cohort to 2, 4, 8, and 32 times its original size. Given that the objective was to compare computational times, all fixed and time-dependent exposures were included in all models regardless of statistical significance. The computational efficiency of the Cox regression model was also compared to the conditional logistic regression model where the nested case-control sample included all possible controls for each case. This comparison was performed for the original cohort of 1340 subjects with 53 cases (including 2 ties). All analyses were performed using the PHREG procedure. Ties were handled using the TIES = EFRON option in the PHREG procedure, and subsequently using the TIES = DISCRETE option in the PHREG procedure for comparison. Results ======= Comparative risk estimates -------------------------- Pacemaker implantation occurred in 53 of the 1340 subjects during the study period. In the final Cox regression model, amiodarone daily dose (\>200 mg vs. ≤200 mg) was associated with an increased risk of pacemaker insertion (HR: 2.03; 95 percent confidence interval: 1.00, 4.14; *p*= 0.05; Parameter Estimate: 0.71; Standard Error of Parameter Estimate: 0.36), after adjusting for age, sex, as well as baseline sinus node or conduction disorder (the only covariate that was an independent predictor of outcome). The results of 500 nested case-control analyses (i.e. 100 for each of 4, 8, 16, 32, and 64 controls per case) are summarized in Table [1](#T1){ref-type="table"}. When using any number from 4 to 64 controls per case, the mean point estimate (of 100 analyses repeating the random sampling of controls) of the parameter estimate (and HR) was very similar to that obtained using Cox regression on the full cohort (i.e. HR: 2.03; Parameter Estimate: 0.71). The SDs of the parameter estimates decreased with increasing numbers of controls per case (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Nested case-control analyses with repeated sampling for increasing numbers of controls per case: Hazard ratio of pacemaker insertion associated with amiodarone dose\* in 1340 elderly Quebec residents with atrial fibrillation ::: Controls per Case (n) Repeated Sampling (n) Mean HR† SD† of HR Min HR Max HR Mean Parameter Estimate SD of Parameter Estimates ----------------------- ----------------------- ---------- ----------- -------- -------- ------------------------- --------------------------- 4 100 2.14 0.51 1.27 3.59 0.73 0.23 8 100 2.19 0.41 1.39 3.86 0.77 0.18 16 100 2.02 0.22 1.55 2.51 0.70 0.11 32 100 2.07 0.15 1.78 2.55 0.73 0.07 64 100 2.02 0.11 1.82 2.33 0.70 0.05 \* The effect of amiodarone dose, represented by a binary time-dependent covariate (\>200 mg vs. ≤200 mg), was adjusted for age, sex, and baseline sinus node or conduction disorder in all models. † HR, hazard ratio; SD, standard deviation. ::: Comparative computational efficiencies -------------------------------------- The computational time required to analyze the cohort of 1340 subjects with 53 events (cases) in models including four fixed variables and seven time-dependent variables is presented in Table [2](#T2){ref-type="table"}. CPU times are displayed for the Cox regression models and the nested case-control regression models (with 4 or 32 controls per case). For the cohort in its initial size, using 32 rather than 4 controls per case increased CPU time by a factor of 3, whereas using Cox regression increased CPU time by a factor of 42. As the cohort size was increased to 32 times the original (i.e. 42880 subjects), the increase in CPU time was greater for the Cox regression model than the nested case-control models. Figure [1](#F1){ref-type="fig"} displays graphically the increase in CPU time with increasing sample size for nested case-control and Cox regression models. The relative computational efficiency was magnified as the sample size increased, such that the CPU time for Cox regression with a cohort of 42880 subjects was 125 times greater than the nested case-control model with 4 controls per case (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Computational times for nested case-control and survival analyses of time-dependent data for cohorts of increasing sizes: Models\* of the risk of pacemaker insertion in elderly Quebec residents with atrial fibrillation ::: Cohort Size†: \#Subjects (\#Cases) 1340 (53) 2680 (106) 5360 (212) 10720 (424) 21440 (848) 42880 (1696) ------------------------------------ ----------- ------------ ------------ ------------- ------------- -------------- Cohort Size: multiple of original 1 2 4 8 16 32 CPU‡ Time (seconds): Nested: 4 Controls per Case 0.03 0.05 0.07 0.11 0.18 0.4 Nested: 32 Controls per Case 0.08 0.1 0.2 0.41 0.7 1.49 Survival Analysis 1.26 2.51 5.06 9.54 19.53 49.91 CPU Time (multiple of Nested 4): Nested: 4 Controls per Case 1 1 1 1 1 1 Nested: 32 Controls per Case 3 2 3 4 4 4 Survival Analysis 42 50 72 87 109 125 \* All models included four fixed variables (age, sex, calendar year of cohort entry, and baseline sinus node or conduction disorder) and seven time-dependent variables (amiodarone dose, ventricular arrhythmia, and exposure to sotalol, class I antiarrhythmic agents, beta-blockers, calcium channel blockers, and digoxin). † The original cohort of 1340 subjects was increased by progressive duplication to 2, 4, 8, 16, and 32 times its original size. ‡ CPU, central processing unit. ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Increase in computational time with increasing sample size for nested case-control and survival analysis of cohort data with time-dependent covariates ::: ![](1471-2288-5-5-1) ::: The computational time of the Cox regression model was also compared to the nested case-control model including all possible controls for each case. When ties were handled using the TIES = EFRON option, the CPU time for Cox regression was 1.06 times greater than the CPU time for nested case-control model (1.26 vs. 1.19 seconds). When ties were handled using the TIES = DISCRETE option, the CPU time for Cox regression was 3.91 times greater than the CPU time for nested case-control model (14.65 vs. 3.75 seconds). Discussion ========== In this study we illustrate empirically that a nested case-control approach can be used to analyze a cohort with time-dependent covariates, with results that are similar to those obtained by Cox regression. Additionally, given that the nested case-control approach obviates the computationally intensive calculations involved in Cox regression when time-dependent covariates are used, the example also illustrates quantitatively the large reduction in CPU time required for analysis. The similarity between the two methodologies is expected given that conditional logistic regression used to analyze nested case-control studies (as well as other matched case-control studies) is based on inference procedures adapted from Cox regression; i.e. the conditional likelihood used in conditional logistic regression is exactly the same form as the partial likelihood used in Cox regression except that the denominator includes only a selected number of sampled controls as opposed to all subjects available in the risk set \[[@B13]\]. The inclusion of time-dependent covariates adds an additional level of complexity to the analysis but remains based on the same inference procedures. The statistical efficiency of the nested case-control approach for cohort analysis depends on the number of controls per case selected. Our example demonstrates the expected decrease in the SD of the parameter estimates as the number of controls per case increases. This decrease in variance is explained by the fact that as the number of controls per case increases (towards the total number of controls in a case\'s risk-set), the probability of choosing the same controls increases, as does the proportion of available controls selected (i.e. approximating the situation in Cox regression where every case is compared to all controls in its risk-set). In general, the use of 4 controls per case provides a relative statistical efficiency of 0.8 compared to the use of an infinite number of controls \[[@B14]\]. However, the relative efficiency also depends on the probability of exposure among the controls and on the magnitude of the estimated relative risk. Gains in statistical efficiency are possible by using greater than 4 controls per case particularly when the probability of exposure among the controls is \<0.1 \[[@B4],[@B15]\]. In addition to situations where exposure prevalence in controls is low, increasing the number of controls per case is beneficial when the number of case-control sets is small \[[@B16]\]. The major reason for the superior computational efficiency of the conditional logistic regression method for nested-case control analysis of time-dependent covariates is that only a sample of all possible controls are included in the risk set of each case (whereas all are included in Cox regression). As illustrated in Figure [1](#F1){ref-type="fig"} and Table [2](#T2){ref-type="table"}, the impact on computational efficiency of sampling a fixed number of controls per case is greater for larger cohorts because the sample of controls represents a smaller proportion of the all the possible controls for each case. While this effect of sampling controls may be the main reason for the computational efficiency of the nested case-control approach is not the only reason. As demonstrated, even when all possible controls are included in the risk set of each case, the computational time of the conditional logistic regression increases significantly but remains faster than Cox regression. This is because the two analyses process time-dependent covariates differently. In Cox regression, risk sets and time-dependent covariates are calculated at the time of each case failure. In conditional logistic regression, the risk sets and time-dependent covariates are calculated in advance. The relative efficiency also depends on how ties are handled, with Cox regression relatively less efficient when ties are handled using the TIES = DISCRETE option compared to the TIES = EFRON option. The nested case-control approach for cohort analysis offers some advantages over analysis of an entire cohort that may be important regardless of the type of cohort used. A potential advantage with respect to design is the option to match controls to cases on the basis of possible confounding covariates for which estimation of effect is not of interest. Another advantage is that substantial savings in cost and time can be achieved by analyzing the cases and only a sample of the controls (as opposed to the entire cohort), particularly when the collection and/or processing of exposure information is very expensive and/or time consuming \[[@B6]\]. While cost is often a major factor in preferring a nested-case control approach over analyzing an entire cohort, there may be advantages even when differences in cost are not significant. In recent years, large administrative healthcare databases, such as the one from which the example cohort for this study was selected, have become particularly useful in studying outcomes that are very rare because they allow for adequate sample sizes \[[@B17]-[@B20]\]. Once a database with all exposure and outcome information is available, analyzing a sample of rather than the entire cohort does not necessarily decrease costs. However, depending on the size of the cohort (as well as the speed of the processor and amount of memory in the computer), it may not be possible to analyze an entire cohort when complex modeling of time-dependent covariates is needed. Such was the case in another study based on a cohort derived from the Quebec provincial healthcare database, where in one analysis the number of covariates was restricted to four and in another analysis only a sub-cohort (i.e. 15529 of 31062 subjects) could be included because the substantial computational resources required were prohibitive \[[@B21]\]. Depending on the rarity of the outcome under study, it may not be possible to analyze the required sample size when performing Cox regression on the whole cohort, whereas it may be possible to do so using a nested case-control approach. While it is recognized that issues of computational resources are overcome with time as computers and software become more efficient, limitations are likely to remain as the size of databases and complexity of time-dependent analyses will also increase. Both the nested case-control approach described (using conditional logistic regression) and the Cox proportional-hazards model with time-dependent covariates similarly account for the time-dependence of exposure when levels of exposure in subjects vary over time. A different and more complex issue is the possibility that the effect of a given exposure varies over time. This can be addressed by analyzing latency-weighted exposures using either Cox regression or a nested case-control approach, the latter being computationally faster \[[@B22]\]. Alternatively, Cox regression can accommodate changes in the hazard ratio over time with a flexible generalization of the Cox proportional hazards model using a regression spline technique \[[@B23]-[@B25]\]. Conclusions =========== A nested case-control approach is a useful alternative for analysis of a cohort when time-dependent covariates are used. The expectedly similar risk estimates are obtainable with superior computational efficiency. Particularly when studying the effects of time-dependent exposures on rare outcomes in very large databases, study power can be improved by being able to run complex regression models on a larger number of affected subjects. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= All authors participated in the conception and design of the study. VE performed the statistical analysis and drafted the manuscript. All authors contributed to the interpretation of the study and revision of the manuscript. The final manuscript was read and approved by all authors. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2288/5/5/prepub> Acknowledgements ================ This study was funded in part by grant \#53181 from the Canadian Institutes of Health Research (CIHR) and grant \#014100 from the Fonds de la Recherche en Santé du Québec (FRSQ). Dr. Vidal Essebag is the recipient of a Clinician Scientist Award from CIHR. Dr. Platt is a recipient of an Investigator Award from CIHR. Dr. Abrahamowicz is a James McGill Professor at McGill University, Montreal, Canada. Dr. Pilote is a recipient of an Investigator Award from CIHR and the Dawson chair at McGill University, Montreal, Canada.

PubMed Central

2024-06-05T03:55:52.517398

2005-1-25

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548149/", "journal": "BMC Med Res Methodol. 2005 Jan 25; 5:5", "authors": [ { "first": "Vidal", "last": "Essebag" }, { "first": "Robert W", "last": "Platt" }, { "first": "Michal", "last": "Abrahamowicz" }, { "first": "Louise", "last": "Pilote" } ] }

PMC548150

Background ========== Huntington\'s disease (HD) is a severe, dominantly inherited neurodegenerative disorder that typically has its onset in mid-life, though it may occur in the juvenile years or in the elderly, and that produces an inexorable decline to death 10--20 years later \[[@B1]\]. Its cardinal clinical feature is a characteristic motor disturbance involving progressive choreoathetosis, but the disorder also involves psychological changes and cognitive decline. The neuropathological hallmark of HD is the loss of medium spiny striatal projection neurons in a dorso-ventral/medio-lateral gradient that eventually decimates the caudate nucleus, but considerable neuronal loss also occurs in other parts of the basal ganglia and in the cortex \[[@B2]\]. The pathogenic process of HD is initially triggered by an expanded polyglutamine segment near the amino terminus of huntingtin, an \~350 kDa protein whose precise physiological function is uncertain \[[@B3]\]. Huntingtin is required for normal embryonic development and neurogenesis, based on the lethal consequences of mutational inactivation in the mouse \[[@B4]-[@B6]\]. By contrast, the HD mutation itself does not impair this developmental activity but rather produces a \"gain-of-function\" that acts to cause the disorder \[[@B7]\]. Genotype-phenotype studies of HD patients, in comparison with other polyglutamine neurodegenerative disorders, have delineated a number of genetic criteria for the mechanism that triggers HD pathogenesis: 1) a threshold polyglutamine length (within a normal human lifespan); 2) progressive severity with increasing polyglutamine length above the threshold; 3) complete dominance over the wild-type protein; 4) greater dependence on polyglutamine length than on huntingtin concentration (within a physiological range) and 5) striatal selectivity, due to the huntingtin protein context in which the polyglutamine tract is presented \[[@B8],[@B9]\]. The \"gain-of-function\" due to the HD mutation is thought to lie in a novel conformational property conferred on mutant huntingtin by the expanded polyglutamine tract \[[@B10]\]. This has been supported by *in vitro*studies of a small amino-terminal huntingtin fragment, where an expanded polyglutamine tract promotes self-aggregation in a manner that conforms to the first four genetic criteria \[[@B10]-[@B12]\]. The *in vitro*aggregation involves a conformational change of the polyglutamine segment from a random coil to an amyloid structure and is paralleled in cell culture in some ways by the formation of cytoplasmic and nuclear inclusions that also incorporate other proteins \[[@B13]\]. Neuronal inclusions containing amino-terminal fragment have also been detected in HD brain, though their role in pathogenesis remains a matter of debate, as they may occur late in the pathogenic process as a consequence of huntingtin degradation \[[@B14]\]. Precise genetic modeling of HD in the mouse supports the view that *in vivo*, the \"gain-of-function\" property conferred by the expanded polyglutamine acts within full-length huntingtin to cause abnormalities that do not initially involve formation of an insoluble aggregate \[[@B15],[@B16]\]. Knock-in mice in which the HD mutation has been introduced into *Hdh*, the mouse orthologue, display early biochemical and histological phenotypes that are associated with expression of full-length mutant huntingtin at normal physiological levels and in a normal developmental pattern \[[@B7],[@B15]-[@B20]\]. Indeed, the phenotypes associated with expression of full-length mutant huntingtin in these mice, and in neuronal progenitor cells derived from them, also fulfill the genetic criteria for the mechanism triggering HD pathogenesis \[[@B15],[@B20]-[@B22]\] One of the earliest phenotypes is the nuclear localization of full-length mutant huntingtin in the nucleus of striatal neurons \[[@B16]\]. Together, the knock-in mouse data suggest that the process of pathogenesis is triggered by the presence of expanded polyglutamine in full-length huntingtin and leads only after many months to the formation of amino-terminal huntingtin fragment and inclusion formation \[[@B15]\]. We have postulated that the same conformational property that promotes aggregation in the context of a small fragment may also act with the context of full-length huntingtin to trigger pathogenesis, possibly by altering huntingtin\'s interaction with another cellular element. Consequently, we have identified small molecules from the NINDS Custom Collection of bioactive compounds that inhibit *in vitro*aggregation of amino-terminal mutant huntingtin \[[@B23]\]. These have been tested for their ability to reverse the huntingtin localization phenotype associated with full-length mutant huntingtin in cultured striatal progenitor cells from *Hdh*knock-in mice. Our findings indicate that some of these compounds reverse the effects of the expanded polyglutamine in both assays and support the view that some inhibitors of polyglutamine aggregation may lead to viable therapeutics targeted at full-length mutant huntingtin, early in the disease process. Results ======= Screening for inhibitors of aggregation --------------------------------------- We have previously demonstrated that, when released from the protection of a GST fusion protein, the amino terminal fragment 1--171 of mutant huntingtin, forms aggregates in a manner consistent with the genetic criteria for the mechanism of HD pathogenesis \[[@B10]\]. We used a modified version of this assay, implemented using a 96-well format ELIFA dot blot apparatus, to screen the NINDS Custom Collection (NCC) which consists of 1040 small bioactive compounds, both FDA-approved drugs and natural products (Figure [1A](#F1){ref-type="fig"}). The screening was carried out in a blinded fashion as part of the NINDS Neurodegeneration Drug Screening Consortium, with the identities of compounds in the NCC only being made available after completion of the screens \[[@B23]\]. In our primary screen (Figure [1A](#F1){ref-type="fig"}), GST-Q58-Htn (20 μg/ml) was mixed with thrombin (0.5 unit/μg GST-Q58-Htn) and immediately dispensed into a 96-well PCR plate containing compounds diluted to a final concentration of 100 μM. Incubation was continued for 24 hours at room temperature to allow aggregate formation. The aggregation was stopped by 2% SDS/10 mM 2-mercaptoethanol followed by boiling for 5 minutes. The mixture was filtered through a cellulose acetate membrane by using a 96-well ELIFA dot blot apparatus. The aggregates retained on the membrane were detected and quantified by immunoblotting and subsequent image analysis. A typical immunoblot result is shown in Figure [1B](#F1){ref-type="fig"}. Congo Red, a known huntingtin aggregation inhibitor, was used as the positive control \[[@B24]\]. 10 μM Congo Red can completely inhibit the Q58-Htn aggregation. DMSO, used for the negative control, had no impact on Q58-Htn aggregation. Potential inhibitors were distributed evenly cross the whole NCC library (Figure [2](#F2){ref-type="fig"}). Sixty compounds that showed more than 50% inhibitory effect were selected to be retested in a second screen at a lower concentration of 10 μM. The 8 compounds in column 5 of plate 9, were missed in the primary screening at 100 μM, and were therefore also tested in the second screening at 10 μM. In the primary screening, a \"hit\" could have resulted either from direct inhibition of polyglutamine-induced aggregation or indirectly, by inhibition of the thrombin and consequent failure to cleave GST-Q58-Htn, which does not by itself aggregate. Consequently, the second screening at 10 μM was carried out after thrombin digestion, to eliminate thrombin inhibitors. Western blotting showed that more than 95% of GST-Q58-Htn is cleaved by thrombin (at ratio of 0.5 unit/1 μg protein) within 30 minutes (data not shown). Consequently, the mixture of GST-Q58-Htn and thrombin was preincubated for 45 minutes, followed by centrifugation to remove any aggregates already formed, before adding the test compounds. Nineteen of the compounds tested at 10 μM, showed significant direct inhibitory effects on aggregation. The 10 most potent compounds, corresponding to a \'hit\' rate of 1%, are shown in Figure [3](#F3){ref-type="fig"}. Characteristics of aggregation inhibitors ----------------------------------------- To determine the potency of each inhibitor, we performed dose response assays at concentrations ranging from 0.01 μM to 500 μM. Representative curves for the 6 most potent compounds are shown in Figure [4](#F4){ref-type="fig"}. Gambogic acid and celastrol showed strong but incomplete inhibition even at the maximum concentration, permitting approximately 20% residual aggregate to form. The average IC~50~(half-maximal inhibition) values for the most potent 10 compounds, which range from 0.7 to 15 μM, were obtained from at least two independent experiments each (Figure [3](#F3){ref-type="fig"}). The most effective aggregation inhibitor was gossypol-acetic acid complex, followed closely by gambogic acid, and then juglone, celastrol, and sanguinarine nitrate, which all had IC~50~values less than 6 μM. Effect on striatal cells expressing endogenous full-length mutant huntingtin ---------------------------------------------------------------------------- To test the hypothesis that compounds, which inhibit the aggregation-promoting property of amino-terminal mutant huntingtin will also rescue effects of full-length mutant huntingtin, we tested the top six inhibitors in a striatal cell-based assay. Mutant *Hdh*^*Q111/Q111*^and wild-type *Hdh*^*Q7/Q7*^striatal cell lines, ST7/7 and ST111/111, respectively, which have been prepared by transformation with a tsSV40 vector, can be propagated in culture and used for cytological and biochemical comparisons \[[@B25]\]. These cells express full-length mutant or wild-type huntingtin, respectively, with no evidence of truncated amino-terminal fragments, no formation of polyglutamine aggregates and no cell death-producing toxicity. However, like the striatal neurons of *Hdh*^*Q111/Q111*^knock-in mice, the ST111/111 cells show nuclear staining of huntingtin when tested with an amino-terminal huntingtin antibody that is sensitive to the conformation of the full-length protein (Figure [5A](#F5){ref-type="fig"}). By contrast, ST7/7 cells expressing wild type huntingtin show both nuclear and cytoplasmic immunostaining with the same huntingtin antibody (Figure [5A](#F5){ref-type="fig"}). This differential localization phenotype occurs early in the cascade of events detected during the lifespan of *Hdh*knock-in mice, months before the appearance of huntingtin amino-terminal fragment, and fulfills the genetic criteria from genotype-phenotype studies in HD patients, including polyglutamine length progressiveness and striatal specificity, suggesting that it follows from the same property that triggers HD pathogenesis. This huntingtin localization phenotype was used to monitor the effect of inhibitors in the mutant cells (Figure [5B](#F5){ref-type="fig"}), and the results are shown in Table [1](#T1){ref-type="table"}. About 89% of ST7/7 striatal cells showed both nuclear and cytoplasmic immunostaining signal, while ST111/111 striatal cells showed only nuclear signal (99%). Of the six compounds tested, celastrol and juglone both reversed the mutant phenotype in a dose-dependent manner. Juglone showed no evident cell toxicity up to 10 μM, where 68% of mutant cells had reverted to wild-type phenotype. Celastrol reverted up to 81% of the cells but showed toxicity, killing \~4% of cells at 10 μM. Gossypol acetic acid complex was less effective, but showed no toxicity. At 50 μM, only 15% of the mutant cells displayed the wild-type phenotype. Gambogic acid showed a comparable small effect at 10 μM but was very toxic at high concentration, as all cells were killed at 50 μM. Sanguinarine nitrate and anthralin showed little effect on the huntingtin localization phenotype in this assay. Discussion ========== A dramatic marker of pathology in many neurodegenerative disorders is the appearance of intracellular inclusions in some surviving neurons \[[@B13]\]. In HD, these inclusions stain positively for huntingtin, ubiquitin and a number of other proteins, but are thought to be initiated by the aggregation of an amino-terminal fragment of mutant huntingtin, due to its expanded polyglutamine tract \[[@B14],[@B26]-[@B28]\]. A number of model systems have been developed to investigate the polyglutamine-driven aggregation process and its consequences both *in vitro*and *in vivo*, but it remains unclear in HD whether the formation of aggregates plays an essential role in the pathway of pathogenesis or is a downstream by-product of neuronal dysfunction induced by full-length mutant huntingtin \[[@B29]\]. In either event, the search for drugs that alter *in vitro*aggregation of amino-terminal huntingtin fragment is attractive, since the aggregation-promoting physical property exhibits characteristics comparable to the disease-producing property of corresponding human alleles, as defined from genotype-phenotype studies of HD patients. For example, if accumulation of huntingtin inclusions is the proximate cause of neuronal death, compounds that inhibit aggregation would have therapeutic potential. Conversely, if the inclusions are only a downstream marker of the pathogenic process, the same drugs may still have therapeutic potential if they act on the property of full-length mutant huntingtin that triggers pathogenesis. It was with a view to testing whether the *in vitro*aggregation assay could act as a proxy for monitoring the disease-producing property of mutant huntingtin that we undertook this study. A comparable screening assay to the one used here has been employed to screen a large chemical library and has demonstrated the feasibility of identifying small molecule inhibitors of polyglutamine aggregation, including a family of benzothiazole-related compounds \[[@B30]\]. However, the long, arduous and expensive process of developing compounds for use as drugs in humans prompted us to screen a smaller chemical library biased toward drugs already approved by the U.S. Food and Drug Administration. The aggregation-inhibiting compounds that we identified came from a collection of mostly FDA-approved compounds and bioactive natural products that were specifically assembled for a neurodegenerative disease drug screening consortium supported by the National Institute for Neurological Disorders and Stroke and several disease foundations, including the Huntington\'s Disease Society of America \[[@B23]\]. The drugs were distributed to 27 different labs for blinded testing in assays of potential relevance to neurodegenerative disease. Unfortunately, despite the preponderance of FDA approved drugs in the collection, none of our top hits (IC~50~\< 10 μM) is a compound approved for internal use in humans. The most effective inhibitor of aggregation was gossypol-acetic acid complex. Gossypol, a polyphenol found in cottonseed, has been studied extensively as a male contraceptive in China but the World Health Organization has argued against its use because of induced hypokalemia, high toxicity and the risk of permanent sterility \[[@B31]\]. Almost as potent, gambogic acid is a complex ring-structured natural product that is the main active ingredient of gamboge resin from the *Garcinia hanburyi*tree. It has long been used as a pigment for painting and in traditional medicine as a potent purgative. Recently, it was identified in a high-throughput screen as an apoptosis inducer with potential for development as an anti-cancer agent \[[@B32]\]. Juglone is a napthoquinone found in the bark of the black walnut (*Juglans nigra*), which has been used as an herbal medicine for its antihaemorrhagic and antifungal properties \[[@B33]\]. Celastrol is a triterpenoid from the vine *Tripterygium wilfordii*used as an alternative medicine for rheumatoid arthritis that has anti-oxidant, anti-inflammatory activity, immunosuppressive and anti-angiogenic activities. It has been proposed as worthy of exploration as a therapeutic in Alzheimer disease \[[@B34]\]. Sanguinarine, a benzophenanthridine alkaloid from bloodroot (*Sanguinaria canadensis*) has broad antimicrobial and anti-inflammatory, as well as potential anti-angiogenic activity \[[@B35]\]. It has been used as an oral rinse and as a potential antigingivitis/antiplaque agent in toothpaste \[[@B36]\]. Anthralin is a synthetic derivative of chrysarobin, a traditional remedy for various skin ailments from *Andira araroba*, that has been widely used as topical treatment for psoriasis and alopecia areata \[[@B37],[@B38]\]. Although none of these bioactive compounds is a candidate for immediate human trials, they provided a means to test whether compounds that inhibit polyglutamine aggregation might also block the neuronal phenotype caused by an elongated polyglutamine tract in full-length huntingtin. While we do not have a direct physical measure of huntingtin conformation and it remains possible that compounds could reverse the cellular phenotype by different pathways, our finding that juglone and celastrol, two drugs of different structure selected as hits in our primary aggregation assay, are both effective at restoring cytoplasmic huntingtin staining in the striatal cell assay suggests that they both act via a conformational property of mutant huntingtin. That gossypol, gambogic acid, sanguinarine and anthralin were not effective could be due to any of a number of reasons, including cellular uptake, toxicity, interaction with other cellular components, etc. However, it may also indicate that these compounds do not directly modify the conformational property of mutant huntingtin, but instead block a different step in the *in vitro*aggregation process or that they do so by a different physical effect than juglone and celastrol. A detailed analysis of structure-activity relationships using structurally-related compounds and testing *in vivo*in knock-in mice for their ability to reverse the cascade of mutant huntingtin-associated phenotypes will be needed to adequately assess the potential of any of these different types of compounds for testing in HD clinical trials. The remaining compounds identified as weaker aggregation blockers in our primary screen include selamectin, a veterinary anti-parasitic \[[@B39]\], pararosaniline pamoate, a treatment for schistosomiasis \[[@B40]\], tyrothricin, a cyclic peptide antibiotic, and meclocycline, a tetracycline-related antibiotic. The latter is of particular interest since it was the most potent of several tetracycline-related antibiotics present among the NCC compounds, including tetracycline, chlortetracycline, demeclocycline, doxycycline, methacycline, oxytetracycline and notably, minocycline, which has been proposed as a therapeutic in HD and other neurodegenerative disorders. Minocycline is an FDA approved antibiotic used for a variety of infections that has variably been reported to improve symptoms in the R6/2 exon 1 overexpression HD model \[[@B41]-[@B44]\]. It has anti-inflammatory and anti-apoptotic activity that has been proposed to involve several potential mechanisms of action. In our hands, minocycline is a weak inhibitor of polyglutamine aggregation with an IC~50~of 43 μM (unpublished data). This is consistent with the inhibitory effect on huntingtin exon 1 aggregation reported previously at 30 μM in long-term hippocampal slice cultures from the R6/2 mouse \[[@B44]\]. Two safety and tolerability studies of minocycline in human HD are completed \[[@B45],[@B46]\] and can be expected to lead to efficacy trials earlier than any trials for strong aggregation inhibitors. However, it is conceivable that long-term, low level inhibition of mutant huntingtin\'s aggregation-promoting conformational property, independent of minocycline\'s anti-apoptotic activity, may be sufficient to alter detectably the timing of disease onset or early progression. If the hoped for positive results are obtained in minocycline HD trials, this alternative mechanism should be considered since it would have implications for testing of meclocycline and for assessing the potential trade-off between potency and toxicity in choosing other aggregation inhibitors as potential long-term therapeutics. Interestingly, the same set of 1040 NCC compounds were screened for their ability to block toxicity in a PC12 cellular assay where induced expression of huntingtin exon 1 encoding 103 glutamines leads to the accumulation of aggregates and rapid cell death \[[@B47]\]. Although eighteen compounds were found to be completely protective, none was among our hits, suggesting that the mechanism of polyglutamine toxicity in the PC12 cells is fundamentally different than the mechanism(s) involved in the *in vitro*aggregation assay. Among a secondary class of partially protective compounds in the PC12 assay, only celastrol overlapped with our hits. The NCC compounds were also screened in a cellular assay in HEK 293T cells expressing androgen receptor with 112 glutamines \[[@B48]\]. In this model for spinal bulbar and muscular atrophy, accumulation of intracellular inclusions, accompanied by caspase 3 activation, is followed by cell death within 72 hours. Twenty compounds that blocked caspase 3 activation included celastrol, gambogic acid, sanguinarine and tyrothricin, though all but sanguinarine showed toxicity. The major finding from this assay was that several cardiac glycosides were protective, presumably by a different mechanism than our hits. Indeed, although most of the assays in the NINDS consortium involved disorders associated with protein aggregation, including various polyglutamine disorders, amyotrophic lateral sclerosis and Parkinson disease, there was a remarkable lack of overlap in hits suggesting that the individual assays targeted fundamentally different mechanisms. A possible exception was celastrol, which was found as a hit in our aggregation assay, the two assays noted above, and other assays which will be discussed in a summary article describing the consortium. Conclusions =========== The identification and further characterization of chemical inhibitors of *in vitro*aggregation of an amino-terminal fragment of mutant huntingtin offer promise for the development of potentially therapeutic compounds that also target the deleterious conformational property of full-length mutant huntingtin. Methods ======= Chemical library, enzymes and antibodies ---------------------------------------- A library containing 1040 small chemical compounds consisting of FDA-approved drugs and bioactive natural products, the National Institute of Neurodegenerative Diseases and Stroke Custom Collection (NCC), was provided in thirteen 96-well plates by MicroSource Discovery Systems, Inc (Gaylordsville CT). The complete list of NCC compounds is available \[[@B49]\]. All compounds were dissolved in 100% DMSO at a concentration of 10 mM. Thrombin was purchased from Amersham Pharmacia Biotech (Piscataway NJ). Anti-huntingtin antibody HP1, used in the screening assay was described by Persichetti et al. \[[@B50]\]. AP229 used in the cell-based assay was previously described and was a gift of Dr. A.H. Sharp \[[@B25]\]. GST-huntingtin construct and expression --------------------------------------- A recombinant pGEX-2TK expression vector with cDNA fragment encoding amino terminal 171 amino acids of human huntingtin with a polyglutamine tract of 58 was used to prepare the GST-Q58-htn fusion protein. The GST-Q58-htn was overexpressed in BL21 cells and purified by affinity chromatography over glutathione-sepharose 4B beads (Amersham Pharmacia Biotech). The purified proteins were stored at concentration of 2.0 mg/ml at -80°C. Aggregation assay ----------------- For primary screening of the chemical library, 1 μl of 4.0 mM small compound stock, diluted from the original plates, was placed in wells of 96-well plates. The fusion protein, GST-Q58-Htn was mixed with thrombin (0.5 unit/1 μg protein) at a concentration of 20 μg/ml in a buffer of 50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2.5 mM CaCl~2~, 1.0 mM EDTA. The mixture was immediately distributed into the 96-well plates containing diluted compounds at 40 μl/well and mixed well. The final concentration of the small compounds was 100 μM. After 24 hours incubation at room temperature, the reaction was stopped by adding 10 μl of 10% SDS/50 mM 2-mercaptoethanol to each well followed by boiling in a PCR machine for 5 minutes. The mixture from each well was filtered through a cellulose acetate membrane ((0.2 μm, GE Osmonics labstore, Minnetonka MN) by using a 96-well ELIFA (Pierce Biotech). The aggregates retained on the membrane were detected by a specific anti-huntingtin antibody, HP1 (diluted 1:1000), followed by incubation with peroxidase conjugated anti-rabbit antibody (diluted 1:10,000, Sigma). Signals from SDS insoluble aggregates were scanned and quantified by using ImageMaster Totalab image analysis software (Amersham Pharmacia Biotech). In the secondary screening, all steps were the same except the final concentration of compound in each well was reduced to 10 μM and the GST-Q58-Htn/thrombin mixture was preincubated for 45-minutes at room temperature and clarified by centrifugation at 28,000 × g for 30 minutes before being added to the test wells. For IC~50~determinations, the *in vitro*aggregation assay and signal quantification were performed as in the second screening but varying the final concentration of input drug. The data for each inhibitor were obtained from at least two independent experiments in which every sample was analyzed in triplicate with Prism 3.0 software (Graphpad Software, Inc., San Diego, CA). Striatal cell line assay ------------------------ Conditionally immortalized wild-type *Hdh*^*Q7/Q7*^striatal neuronal progenitor cells (ST7/7) expressing endogenous normal huntingtin, and homozygous *Hdh*^*Q111/Q111*^striatal neuronal progenitor cells (ST111/111), expressing endogenous mutant huntingtin with 111-glutamines, have been described previously \[[@B25]\]. The striatal cell lines were grown at 33°C in Dulbecco\'s modified Eagle\'s medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 1% nonessential aminoacids, 2 mM L-glutamine and 400 μg/ml G418 (Geneticin (GIBCO-BRL, Life Technologies, Gaithersburg, MD). In the immunofluorescence and confocal analysis experiment, wild-type ST7/7 and homozygous mutant ST111/111 cells, grown to confluence on glass coverslips, were treated with 6 different drugs at four different concentrations (0.5, 5, 10 or 50 μM) for 30 min at 33°C. After the treatment the drugs were removed and the cells washed twice in PBS. The cells were then fixed by incubation in 4% formaldehyde for 15 min, permeabilized by 0.1% Triton X-100 for 5 min and incubated for 30 min with blocking solution (1% bovine albumin in PBS). Coverslips were then incubated in primary antibody (AP229 1:500 dilution) for 2 h at room temperature and washed three times in PBS before a further 1 h in blocking solution containing the secondary antibody (goat anti-rabbit Cy3, Jackson ImmunoResearch, West Grove, PA. USA). After three washes in PBS coverslips were mounted onto glass slides with Vectashield (Burlingame, CA. USA) and the images were analyzed with a laser confocal microscope (Bio-Rad, Hercules, CA. USA) using 20 × objective. Cell death was quantified by scoring the percentage of cells with apoptotic nuclear morphology, i.e. condensed or fragmented nuclei, under the confocal microscope. In each case five to ten randomly selected fields were counted, comprising at least 200 cells, and each experiment was repeated 3 times. Authors\' contributions ======================= JW carried out the library screen and aggregation assays. SG carried out the cell-based assays. MEM and JFG contributed to the conception of these studies. JW and JFG drafted the manuscript and MEM and SG contributed to its final version. All authors read and approved the final manuscript. Acknowledgements ================ This work was supported by grants from NINDS (NS16367) to JFG and MEM, (NS32765) to MEM and the Huntington\'s Disease Society of America\'s Coalition for the Cure. JW received a fellowship from the Harvard Center for Neurodegeneration and Repair. We thank Jill Heemskerk for organizing the NINDS neurodegenerative disease drug screening consortium and to consortium members for sharing data prior to publication and for helpful discussions. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Schematic diagram of the aggregation screening assay (A) and typical results (B)A**: Scheme of an *in vitro*mutant huntingtin aggregation assay modified for drug screening. In the primary screening, the mixture of fusion protein, GST-Q58-Htn (20 μg/ml) and Thrombin (0.5 unit/μg protein) was immediately distributed into the 96-well plates containing diluted compounds at 40 μl/well. The final concentration of the small compounds is 100 μM. 10 μl of 10% SDS/50 mM 2-mercaptoethanol was added into each well to stop the reaction after 24 hours incubation at room temperature. The aggregates were separated by filtering through a cellulose acetate membrane (0.2 μm). Immunoblotting was done with a specific anti-huntingtin antibody, HP1, followed by incubation with peroxidase conjugated anti-rabbit antibody. The signals of the retained aggregates were scanned and quantified. In the secondary screening, compounds tested positive in were tested at 10 μM and a 45-minute incubation at room temperature was followed after mixing the protein and enzyme. **B**: A typical immunoblot of the huntingtin aggregation inhibitor screening. The aggregates retained on the membrane were visualized by ECL. The intensity of dot reflects the amount of aggregates. The blot shows the positions of each drug in a 96-well plate. Congo Red: positive control (*row*E, *column*12 and *row*F, *column*12). DMSO: negative control (*row*G, *column*12 and *row*H, *column*12). Drugs in wells F2, E4, E9 and D3 are huntingtin aggregation inhibitors. ::: ![](1471-2202-6-1-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Distribution of potential huntingtin aggregation inhibitors in the NCC library**. Most compounds show no or little inhibitory effect on huntingtin aggregation. The potential inhibitors are evenly distributed across the whole chemical library. Due to very high background on particular regions of some dot blotting membranes, some compounds (compounds around 200) show a high apparent promotional effect that is artefactual (as shown in \~-70% inhibition). The 8 compounds located in column 5 of plate 9 were missed and were tested in the second screening at 10 μM. ::: ![](1471-2202-6-1-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Ten most potent aggregation inhibitors from the NCC library ::: ![](1471-2202-6-1-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Dose-response curve of mutant huntingtin aggregation inhibitors**. Compounds were added in 1 μl DMSO to 40 μl 20 μg/ml (0.25 μM) GST-Q58-Htn pre-incubated with thrombin. The reaction was carried out at room temperature for 24 hours. The intensity of each dot of the dot blot was normalized to the DMSO control. Assays were performed twice, in triplicate at each concentration each time (n = 6). ::: ![](1471-2202-6-1-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Localization of huntingtin in wild type and mutant striatal cell from knock-in mice**. Cells were cultured for 48 hours and immunostaining was done with a specific antibody against huntingtin, AP229, followed by Cy3 labeled secondary antibody. The signal was visualized by confocal microscopy (magnification: ×20). A. The localization of full-length huntingtin of striatal cell lines: ST7/7 and ST111/111 with 2.5% DMSO. B. The localization of huntingtin of mutant striatal cell, ST111/111, after treated with drugs at 10 μM. ::: ![](1471-2202-6-1-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Percentage of ST111/111 cells showing cytoplasmic/nuclear huntingtin localization comparable to wild-type ST7/7 cells in response to chemical treatment\* ::: **Compounds** **Concentration (μM)** ---------------------- ------------------------ ------------------- ------------------ -------------- Gossypol-acetic acid 0 4.5 ± 4.3% 3.5 ± 3.5% 15.9 ± 11.1% Gambogic acid 1 ± 2.6% 0 2.1 ± 4.6% Toxic (100%) Juglone 10.4 ± 12.4% 37.9 ± 19.9% 68.8 ± 18.4% N.A. Celastrol 8.7 ± 8.7% 69.2 ± 3.8% (1%) 78.1 ± 3.5% (4%) N.A. Sanguinarine 0 (4.3%) 3.7 ± 5.2% (5.5%) 0 (22.5%) N.A. Anthralin 4.6 ± 7.6% 6.6 ± 9.7% 9.6 ± 12.1% 11.8 ± 14.4% ST111/111 cells with both nuclear and cytoplasmic staining were counted, as well as total cells and dead cells. The effectiveness of the drugs was measured by percentage of cells with nuclear and cytoplasmic staining versus total cells. In untreated cultures, 89% of ST7/7 striatal cells show both nuclear and cytoplasmic immunostaining, while only 1% of ST111/111 striatal cells show this pattern. Entries in the table represent the average values from 5--10 different microcope fields ± standard deviation. Numbers in brackets indicate percentage of dead cells in cases where toxicity was observed. :::

PubMed Central

2024-06-05T03:55:52.520423

2005-1-13

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548150/", "journal": "BMC Neurosci. 2005 Jan 13; 6:1", "authors": [ { "first": "Jin", "last": "Wang" }, { "first": "Silvia", "last": "Gines" }, { "first": "Marcy E", "last": "MacDonald" }, { "first": "James F", "last": "Gusella" } ] }

PMC548151

Background ========== Lung cancer is the leading cause of cancer related deaths all around the world. About 80% of all lung cancers are non-small cell lung cancer (NSCLC) and more than 50% of these patients present with locally advanced or metastatic disease. Meta-analysis of several randomized trials have demonstrated a modest survival advantage for treatment with cisplatin-based regimens in patients with advanced stages of NSCLC \[[@B1],[@B2]\]. Furthermore, chemotherapy (CT) also has been shown to ameliorate symptoms and increase quality of life \[[@B3]\]. Addition of second generation CT regimens with cisplatin or carboplatin plus newer agents, such as taxanes (paclitaxel and docetaxel), gemcitabine, vinorelbine have shown increased response rates and 1-year survival ratios, but overall survivals have not been altered \[[@B4]-[@B6]\]. Being the first of the taxane antimicrotubule agents, paclitaxel (P) demonstrated overall response rates of 21--24% and 1-year survival rates of 37--42% in the phase II trials where it was used as a single agent \[[@B7],[@B8]\]. Antiangiogenic effect of P has also been reported (9). Carboplatin (C) has also demonstrated comparable activity but better toxicity profile than cisplatin in the treatment of advanced NSCLC \[[@B10],[@B11]\]. P and C used in combined chemotherapy regimens have significant activity in NSCLC. PC given every three weeks is considered to be one of the standard regimens being used worldwide \[[@B10]\]. The weekly administration of P is active, dose intense, and has a favorable toxicity profile. To evaluate the efficacy and toxicity of C and day 1 and 8 P in advanced stage NSCLC, we retrospectively reviewed 51 consecutive patients receiving this CT regimen. Methods ======= All patients with stage III or IV NSCLC treated at Medical Oncology Units of Marmara University Hospital, Dr. Lutfi Kirdar Research and Training Hospital, SSK Sureyyapasa Chest and Cardiovascular Diseases Hospital and Gulhane Military Medical Academy Hospital within July 2002 and August 2003 were considered for this protocol. Eligible patients were required to have pathologically proven NSCLC, stage III or IV disease at presentation or progressed after surgery, performance status (PS) ECOG 0--2, objective measurable disease, adequate bone marrow functions (white blood cell count ≥ 3500/mm^3^, hemoglobin ≥ 9 g/dl, and platelet count ≥ 100000/mm^3^), and adequate liver functions (bilirubin ≤ 1.5 mg/dl and alanine aminotransferase ≤ 2 times upper limit of normal and ≤ 5 times upper limit of normal for the patients with liver metastases) and kidney functions (creatinine ≤ 1.5 mg/dl). No prior chemotherapy or radiotherapy (except to bone metastases for palliation) was allowed. Patients presenting with known central nervous system (CNS) disease and uncontrolled cardiac arrhythmia were excluded from this study and they were treated with other chemotherapy regimens (single agent or combination of platinum and vinorelbine, docetaxel or gemcitabine). Patients were treated with P (112.5 mg/m^2^/day) on days 1 and 8, followed by C (AUC 5/6) on day 1, repeated in every three weeks. Both drugs were diluted in 250 ml of normal saline and given intravenously (IV) over 1 hour. No growth factors were administered. Anti-allergic premedication included IV diphenhydramine 50 mg, IV ranitidine 50 mg, and IV dexamethasone 16 mg 1 hour prior to P administration. Toxicity evaluation and routine physical examination were performed in every 3 weeks. Complete blood count (CBC) was done on days 1 and 8 of each cycle, liver and kidney function tests on every 2 cycles. Cranial computed tomography scans (CT), magnetic resonance imaging (MRI) and bone scans were performed as clinically indicated. Side effects of the treatment were graded according to the National Cancer Institute Common Toxicity Criteria (CTC), version 2.0 \[[@B12]\]. Colony stimulating factors were not used. Response was evaluated with CT of chest and/or abdomen on every 3^rd^cycle and standard World Health Organization (WHO) criteria were used to determine response \[[@B13]\]. Independent of the stage at presentation, patients having partial response (PR), stable disease (SD) or progressive disease (PD) during CT were consulted for radiotherapy (RT) for either primary treatment or palliation. The treatment was stopped for patients with PD. Patients with CR, PR or SD after 3 cycles continued their treatment. PC was given for maximum of 6 courses to the patients having PR or SD. Patients were irradiated with CT based treatment planning and multiple fields arrangements with custom blocking to all fields and involved hilar and mediastinal lymph nodes up to 40--41.4 Gy. Boost was given to the primary tumor. Total dose of 60--61.2 Gy was administered in 1.8--2 Gy daily fractions for 5 days a week and completed in 6 weeks. Statistical analysis -------------------- Overall survival (OS) and time to progression (TTP) were assessed from the date of diagnosis to the date of death (any cause) and the date of objective disease progression (death was considered a progression event in patients who died before disease progression), respectively. Survival rates were calculated by using the Kaplan-Meier method \[[@B14]\]. The pre-specified prognostic value of age (\< 60 years vs. ≥ 60 years), gender, PS (0 vs. 1--2), histology (adenocarcinoma vs. squamous cell vs. NSCLC), stage (III vs. IV), smoking history (present vs. absent), and response after third cycle of CT (PR vs. other) were evaluated in univariate and multivariate analyses. Log rank test was used for univariate survival analysis \[[@B15]\]. The multivariate Cox proportional hazard model was applied to identify the variables that can independently influence survival. Results ======= The data of 51 patients receiving PC treatment were collected retrospectively between July 2002 and November 2003. Median follow-up time was 7 months (range, 1--20). Median age was 58 years (range 39--77) and 45% of patients were 60 year-old or above. Eighty percent were male. PS was 0 in 57% of patients and 67% had presented with stage IV disease. Most frequent metastatic sites were the other lung (17), adrenal (10), liver (7) and bone (7). Eighty-two percent of the patients had smoking history, median of which was 40 pack-years (range, 0--135). Patients\' baseline characteristics are presented in Table [1](#T1){ref-type="table"}. The median number of cycles administered was 3 (range, 1--6). Seven patients (14%) did not complete the first 3 cycles either due to death (2), progression (3), grade 3 hypersensitivity reaction to P (1) or lost to follow up (1). Best evaluable response was PR in 45% and SD in 18%. Only 22 (43%) patients continued the treatment after the 3rd course. At the end of treatment of these 22 patients 10 (46%) had PR and 6 (27%) had SD, but the other 6 patients (27%) had PD. No complete remission was seen. Twelve patients (24%) received local RT and 4 of these patients were given low dose gemcitabine (75 mg/m2/week × 5--6 weeks) as radiosensitizing agent. Of these 12 patients 3 presented with stage IIIA and all had PR to PC therapy. But of the 5 patients with IIIB disease who were irradiated only one patient had PR, 3 had SD and another one had PD after the 3 cycles of CT. Four patients with stage IV were offered RT for palliation. Thirteen patients (25%) received 2nd line CT at progression and of those only one patient had PR and another SD to this treatment. For 2nd line CT gemcitabine ± cisplatin or C was used in 10 patients (78%) and the rest received other agents like vinorelbine, C or docetaxel. Details of this data can be seen in Table [2](#T2){ref-type="table"}. At a median follow-up of 7 months 25 (49%) patients died and 35 patients (69%) progressed. Median OS time was 11 ± 2 months (95% CI; 6 to16), 1-year OS ratio was 44% (Figure [1](#F1){ref-type="fig"}). Median TTP was 6 ± 1 months (95% CI; 4 to 8), 1-year progression free survival (PFS) ratio was 20% (Figure [2](#F2){ref-type="fig"}). The most frequent toxicity related symptoms were asthenia (61%), neuropathy (42%) and anorexia (35%). We observed the following grade 3 toxicities: asthenia (10%), neuropathy (4%), anorexia (4%), anemia (4%), hypersensitivity to P (2%), nausea/vomiting (2%), diarrhea (2%) and neutropenia (2%). Two patients (4%) died of febrile neutropenia due to a three day delay in referral to hospital after the onset of fever \> 38°, although they were warned about the side effects of the therapy. Doses of CT was reduced or delayed in 12 patients (24%) (Table [3](#T3){ref-type="table"}). Univariate analysis showed that patients presenting with PS of 0, stage III disease and having PR after the 3rd cycle of PC have statistically higher OS (p = 0.015, p = 0.018 and p = 0.047, respectively)(Table [4](#T4){ref-type="table"}). PS and stage of the disease at presentation and response to the CT after the 3rd cycle were also statistically significant independent prognostic factors influencing the OS in multivariate Cox regression analysis (p = 0.034, p = 0.049 and 0.021, respectively). Discussion ========== Paclitaxel and carboplatin have been shown to be an effective and well tolerated CT regimen in advanced stage NSCLC \[[@B10]\]. PC given once in every three weeks is one of the most widely used standard schedules worldwide based on the spectrum of activity and the ease of administration. This regimen results in an objective response rate of 17--25% with a median survival time of 8 months in stage IIIB and IV NSCLC patients. The major toxicities of this regimen are neuropathy and neutropenia \[[@B10],[@B16]\]. Weekly P is a relatively new strategy for lowering toxicity and increasing dose-intensity and possibly efficacy. Alvarez et al. have used weekly P on patients who progressed or remained stable on P administered in every three weeks and reported that it can induce response in 62.5% of patients with low toxicity \[[@B17]\]. Akerley has also studied weekly P administration on phase I and phase II settings \[[@B18]-[@B20]\]. They started with a P dose of 175 mg/m^2^/week × 6 every 8 weeks in the phase II trial, but they had to reduce the dose up to 50% due to primarily neutropenia and neuropathy with extended therapy. Therefore, they recommended 150 mg/m^2^as the weekly dose of P \[[@B20]\]. Weekly dose of P in combination with cisplatin or C had been administered in NSCLC patients by Belani et al. \[[@B21],[@B22]\]. They used this combination in a multicenter three arm trial in 401 patients with stage IIIB and IV disease \[[@B21]\]. In that trial P was given 100 mg/m^2^/week for 3 weeks out of 4 week cycles in arms I and II, with C either AUC of 6 on day 1 or AUC of 2 on days 1, 8 and 15 of each of four 4-week cycles. Arm III of this trial consisted of P (150 mg/m^2^) and C (AUC = 2) given weekly for 6 out of 8 weeks for a total of two cycles. Greater percentage of the patients on arm I received intended CT (30% of P and 55% of C) compared with the other arms (28--29% of P and 21--22% of C). Patients on arm I received more than half of the planned C dose. The main reasons for discontinuation of therapy were progression of disease (31%) and adverse events (15%). Median time to progression and median survival time were significantly higher for arm I than arm II for patients with stage IIIB disease. Performance status of the patients was also statistically related to the survival times. Patients with PS-0/1 had longer median PFS with treatment arm I than arm II and patients with PS-2 had higher median OS with arm I than arm II. Although arm I was the most easily tolerable schedule between the three arms, grade 3 or 4 neutropenia was observed in 22% of the patients included. In this trial treatment arm I had a response rate of 32%, median TTP of 6.9 months, median OS time of 11.3 months and 1-year survival rate of 47%. In our study, response rate was 45%, median TTP was 6 months, median OS time was 11 months and 1-year survival rate was 44%. The majority of our patients comprised of stage IIIB and IV disease, similar to the patient group in Belani\'s study resulting in similar response rates and survival data \[[@B21]\]. These results also seem more effective than the regimen given once in every three weeks of the same drugs \[[@B10],[@B16]\]. We used the standard dose of P (225 mg/m^2^) given in every three weeks and divided into two consecutive weeks. C dose was calculated according to Calvert formulation with an AUC of 5. This is a lower dose than the dose of C being used in other phase III trials in the literature. In our study only 4 patients (8%) had dose reduction of 10% and 16% of patients had treatment delays of 1 week because of side effects. According to this data, 76% of patients have received the total planned doses of the drugs on scheduled date. Two patients (4%) died of febrile neutropenia due to a three day delay in referral to hospital after the onset of fever \> 38°, although they were warned about the side effects of the therapy. It is worth mentioning that none of our patients received any colony stimulating factors. We have shown that the response to treatment after the third cycles of CT was one of the independent prognostic factors influencing OS. It has already been reported by Socinski et al. that 4 cycles of CT give the maximum benefit which could be obtained from CT in patients with stage IIIB and IV NSCLC \[[@B23]\]. Smith et al. also studied 3 cycles versus 6 cycles of CT in the same group of patients and failed to show any survival advantage for longer treatment durations \[[@B24]\]. In addition, there was an increase in the side effects such as fatigue, nausea and vomiting in the patients receiving six courses. It has been shown that PC combination has relatively mild toxicity profile. Belani et al. observed in their phase I trial that patients who received the PC combination in every three weeks experienced less severe thrombocytopenia than would be expected from C alone. In the view of this finding they suggested that there was a platelet-sparing effect of P on the dose-limiting thrombocytopenia side effect of C \[[@B25]\]. This phenomenon was also shown by Akerley \[[@B18]\] and Kearns \[[@B26]\]. Akerley reported that platelet counts rose by 17000/mL/week with weekly P administration \[[@B18]\]. Belani also speculated on the mechanism for this platelet protective effect and said that it may involve some alteration of megakaryocytopoiesis or thrombocytopoiesis, which could result in increased levels of endogenous thrombopoietin or other cytokines \[[@B27]\]. Kearns et al. suggested that prior exposure to P may suppress the inhibition of platelet formation, which is associated with C \[[@B26]\]. None of our patients experienced thrombocytopenia during our CT treatment with day 1 and 8 P with day 1 C on every three weeks. One of the most frequent side effects during our treatment was neuropathy, but it was usually mild (Grade 1 or 2), only 4% of our patients experiencing grade 3 sensory neuropathy. Grade 3 or 4 neuropathy has been reported to be 10--20% in schedules given every three weeks \[[@B10],[@B16]\]. Belani reported 3--13% of grade 3 or 4 neuropathy, but the incidence was lower for arms 1 (P given weekly and C every four weeks) and 2 (P and C both given weekly), at only 5% and 3%, respectively \[[@B21]\]. This result for arm 1 is similar to the neuropathy rate in our study. Besides the reduced toxicity, weekly administration of P also increases the drugs\' anti-angiogenic and apoptotic effects. The metronomic schedule of P has been studied widely during the last few years. P had been shown to inhibit endothelial cell proliferation, motility, invasiveness, and cord formation both in vitro and in vivo Matrigel assays in a dose dependent manner \[[@B9]\]. Belani et al. randomized the patients having objective response to weekly P and C regimens into two arms (maintenance and observation arms). Patients were either treated with weekly P (70 mg/m^2^/week × 3 weeks out of four weeks cycles) in maintenance arm or observed until disease progression has occurred. They reported that the maintenance arm was compared to the observation arm and had a median PFS of 38 weeks vs. 29 weeks, median OS of 75 weeks vs. 60 weeks, respectively \[[@B21]\]. Although there was not a statistically significant difference between the two arms, authors concluded that this might be a result of low number of patients enrolled in the study (only 65 patients in each arm). It is not yet known whether these responses have an anti-angiogenic basis, or whether such responses will translate into a significant prolongation of survival. Although our study is a retrospective analysis, it is one of the few manuscripts on this PC scheduling in NSCLC in the literature. Conclusions =========== Paclitaxel on day 1 and 8 and carboplatin every three weeks is a practical and fairly well tolerated outpatient regimen. This regimen seems to be comparably active to regimens given every three weeks. This schedule needs to be further evaluated by well planned randomized phase III trials where it could be compared to the standard regimens in patients with advanced stage NSCLC. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= PFY designed the study, followed the patients, collected the data, performed the statistical analysis and drafted the manuscript. NST followed the patients and helped with the manuscript. MG followed the patients and helped with statistical analysis. NFH, OT, AO, TS, MA followed the patients. RA confirmed the diagnosis. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/10/prepub> Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Overall survival Kaplan-Meier curve ::: ![](1471-2407-5-10-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Progression free survival curve ::: ![](1471-2407-5-10-2) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Patient characteristics ::: Characteristics Number of patients Percentage (%) ----------------------------- -------------------- ---------------- Age, years Median 58 Range (39--77) Gender Male 41 80 Female 10 20 Performance Status 0 29 57 1 17 33 2 5 10 Stage IIIA 3 6 IIIB 14 27 IV 34 67 Histology Adenocarcinoma 16 31 Squamous cell carcinoma 18 36 NSCLC 17 33 Sites of metastases Lung 17 50 Adrenal 10 29 Bone 7 21 Liver 7 21 Distant LAP 3 9 Number of metastatic organs 1 24 71 2 9 26 3 1 3 Smoking history Yes 42 82 No 9 18 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Response rates and second line treatments with CT and RT ::: Number of patients Percentage (%) ----------------------- -------------------- ---------------- Number of PC received 1 2 4 2 5 10 3 20 39 4 3 6 5 3 6 6 18 35 Response to PC PR 23 45 SD 9 18 PD 16 31 Not assessed 3 6 Second line CT 13 G ± Cis / C 10 78 Other 3 23 Local RT 12 Stage IIIA 3 25 Stage IIIB 5 42 Stage IV 4 33 CT: Chemotherapy; RT: Radiotherapy; P: Paclitaxel; C: Carboplatin; G: Gemcitabine; Cis: Cisplatin ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Toxicities seen during PC treatment ::: Toxicity Overall (%) Grade 1--2 (%) Grade 3 (%) Grade 4 (%) ----------------------- ------------- ---------------- ------------- ------------- Neutropenia 12 10 2 Febrile neutropenia 4 4 Anemia 22 18 4 Asthenia/Fatigue 61 51 10 Neuropathy 42 38 4 Anorexia 35 31 4 Arthralgia 31 31 Nausea/Vomiting 22 20 2 Diarrhea 8 6 2 Mucositis 6 6 Hypersensitivity to P 2 2 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Prognostic factors in the univariate analyses for overall survival ::: **Variables** Median OS (months) p ------------------------------- -------------------- --------- Age 60 years and over 11 ± 2 0.87 Less than 60 years 12 ± 3 Gender Male 9 ± 3 0.22 Female 15 ± 6 PS 0 15 ± 3 0.015\* 1--2 6 ± 2 Histology Adenocarcinoma 15 ± 5 0.73 Squamous cell 13 ± 7 NSCLC 11 ± 3 Stage at presentation III NR^†^ 0.018\* IV 9 ± 2 Smoking history Yes 9 ± 2 0.20 No NR^†^ Response after 3 cycles of CT PR 13 ± 2 0.047\* Other 6 ± 1 \* Statistically significant † Mean OS is 15 ± 2 months PS: ECOG Performance status; NR: Not reached; CT: Chemotherapy :::

PubMed Central

2024-06-05T03:55:52.523340

2005-1-25

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548151/", "journal": "BMC Cancer. 2005 Jan 25; 5:10", "authors": [ { "first": "Perran F", "last": "Yumuk" }, { "first": "Nazim S", "last": "Turhal" }, { "first": "Mahmut", "last": "Gumus" }, { "first": "Nilgun F", "last": "Hatabay" }, { "first": "Orhan", "last": "Turken" }, { "first": "Alper", "last": "Ozkan" }, { "first": "Taflan", "last": "Salepci" }, { "first": "Mehmet", "last": "Aliustaoglu" }, { "first": "Rengin", "last": "Ahiskali" } ] }

PMC548152

Background ========== The diagnostic stage of colon cancer determines survival. Patients diagnosed with localized tumours have a 75% probability of 5 year survival, whereas patients diagnosed with distant metastases only have a 5--10 % probability of 5 year survival (reviewed in \[[@B1]\]). Recently a number of studies have been published in which Surface Enhanced Laser Desorption/Ionisation-Time Of Flight/Mass Spectrometry (SELDI-TOF/MS) has been applied to biological samples from patients with various forms of cancer \[[@B2]-[@B4]\] leading to the identification of protein markers with clinical potential. Here we present a SELDI-TOF/MS study of colon cancer serum and tumours. We show that the expression of Human Neutrophil Peptides -1, -2 and -3 (HNP 1 -3), also known as alfa-defensin-1, -2 and -3, is upregulated in the tumour microenvironment, as compared to normal colon tissue. This finding is reflected in serum. We find that HNP 1-3 is present in elevated concentrations in serum from patients diagnosed with tumours in the colon, as compared to serum from a healthy control group matched by age and gender. By size-exclusion analysis we add to the existing evidence that HNP 1-3 bind to high mass plasma proteins, explaining the presence of HNP 1-3 in serum. By microflow analysis, we show that HNP 1-3 purified from colon tumours are lethal to mammalian cells. The HNP 1-3 peptides are part of the defensin family of peptides (reviewed in \[[@B5]-[@B7]\]), which are a fundamental component of the immune system and have the capacity to kill / inactivate a broad range of pathogens. Defensins are also known to function as regulators of both the innate and the adaptive immune system. We discuss the possible effects of HNP 1-3 in the tumour microenvironment. Methods ======= Biological samples ------------------ All biological samples were obtained by trained staff at Glostrup Hospital, Denmark. Written consent was obtained from all donators. Permission was obtained from the Danish Scientific Ethical Committee and the Danish Data Protection Agency. Tissue screening ---------------- Normal colon tissue samples and colon tumour samples were obtained from the removed fragment of the patient\'s colon after surgical treatment for colon cancer. Tissue samples were stored at -80°C until use. The protein content was extracted from the tissue: 100 mg tissue sample was thawed on ice and homogenised on a Wheaton Overhead Stirrer for 2 minutes at speed step 2, in 500 ul Lysis buffer (100 mM TRIS-HCl, pH 8.0, 9.5 M UREA, 2% CHAPS). The samples were centrifuged at 14,000 rpm for 10 minutes and the pellet was discarded (repeated twice). The tissue protein extracts were stored at -80°C until use. Pilot studies were performed on different chips (data not shown) and the NP20 (Normal Phase) (Ciphergen) chip was chosen for the tissue screening. NP20 chips were placed in Bioprocessor (Ciphergen) and pre-treated with 50 ul tissue binding buffer (50 mM TRIS-HCl, pH 8.0) for 5 minutes on shaker (250 rpm) (repeated twice). 5 ul tissue protein extract was diluted in 50 ul tissue binding buffer and incubated in Bioprocessor on NP20 chips for 40 minutes at room temperature on shaker (250 rpm). Spots were washed twice in 250 ul tissue washing buffer (50 mM TRIS-HCl, pH 8.0) for 5 minutes. The chips were air dried for 10 minutes, followed by treatment with two times 0.6 ul 100% sinapinic (SPA) matrix solution. SPA was obtained from Ciphergen in 5 mg aliquots and dissolved (150 ul MQ water, 150 ul acetonitrile, 1.5 ul Tri-Fluoro-acetic-Acid (TFA)) immediately before the screenings. Serum screening --------------- Colon cancer serum samples were obtained from patients before surgical treatment. Normal serum was obtained from a group of healthy individuals matched by age and gender to the cancer patients. All serum samples were stored at -80°C until use. Serum pilot studies were performed on different chips to monitor the presence of HNP 1-3 in serum (data not shown). The immobilised metal affinity capture (IMAC30) chip was chosen for the actual screening and was pre-treated with nickel before analysis: 5 ul 100 mM NiSO4 were added to each spot and left on shaker (250 rpm) for 5 minutes (repeated twice). The chips were placed in Bioprocessor and incubated with 100 ul MQ for 5 minutes on shaker (250 rpm). Each spot was treated with 50 ul serum binding buffer (100 mM TRIS-HCl, pH 7.5, 500 mM NaCl, 0,1% Triton X-100) and left on shaker for 5 minutes (250 rpm). Serum samples were thawed on ice and 1 ul serum was diluted in 50 ul serum binding buffer and applied to spots and left on shaker (250 rpm) at room temperature for 40 minutes. The sample solution was removed and the spots were washed twice in 200 ul serum washing buffer (100 mM PBS, pH 7.4, 700 mM NaCl), followed by one wash in 200 ul MQ water. The chips were removed from the Bioprocessor and left to air dry for 20 minutes followed by treatment with two times 0.6 ul SPA. Only freshly made matrix solutions were used and the instrument was calibrated daily. Cancer and normal samples were run side by side. The chips were analysed on a PBS II instrument (Ciphergen). All spectra in each screening were normalised based on total ion current. Purification and identification of HNP 1-3 ------------------------------------------ 100 ul protein extract from colon tumour tissue in tissue lysis buffer was loaded unto a RP-HPLC column (uRPC C2/C18 ST 4.6/100, Pharmacia Biotech, Flow rate: 0.5 ml/min, Fraction size: 0.5 ml) in buffer A (0.065% TFA in MQ-water) and proteins were eluted in a gradient of 0--100% buffer B (0.05% TFA in acetonitrile (ACN)). Elution of peptides was monitored by absorbance at 280 nm. All protein-containing fractions were analysed by Matrix Assisted Laser Desorption/Ionisation-Time of flight (MALDI-TOF) on the PBS II instrument: 1.5 ul fraction was incubated with 0.6 ul SPA on a Gold array (Ciphergen) and left to crystallise, followed by an additional treatment with 0.6 ul SPA and analysed in the PBSII instrument. The HNP 1-3 containing fraction (32% buffer B) was further purified on a peptide gelfiltration column (Superdex Peptide HR 10/30, Pharmacia Biotech, Flow rate 0.9 ml/min, Fraction size: 0.5 ml, Buffer: 50% ACN, 0.1 % TFA). Elution of peptides was monitored by absorbance at 280 nm and protein-containing fractions were again analysed by MALDI-TOF. Purified HNP 1-3 were identified by on-chip trypsin digestion: 10 ul of HNP 1-3 fraction was applied to an NP20 chip and left on shaker (250 rpm) at room temperature for 40 minutes. The solution was removed and the spot was washed twice with 10 ul water. In order to denature peptides prior to digestion, the chip was left on heating block (80°C) for 5 minutes. The chip was cooled on ice for 2 minutes. 10 ul trypsin digestion solution (0.01 ug/ul trypsin in 50 mM NH~4~HCO~3~, pH 8,0) was added, and the chip was left for 10 hours at 40°C in humidity chamber after which the chip was left to air dry for 20 minutes. 1 ul CHCA matrix (prepared as the SPA matrix solution) was added and the peptide map was analysed on the PBS II instrument. Peptide maps of trypsin autodiggest were used as controls. Identification was done with the PepIdent software on the Expasy server. For the reduction experiment, HNP 1-3 were first denaturation by heating (10 minutes at 80°C) followed by treatment with DTT (200 mM, 30 minutes at room temperature) and the peptides were incubated on an NP20 chip and analysed on the PBS II instrument. Size exclusion chromatography of HNP 1-3 ---------------------------------------- 50 ul colon cancer serum was loaded unto a peptide gelfiltration column (Superdex Peptide HR 10/30, Pharmacia Biotech, optimal separation range: 1 to 7 kDa, flow rate: 0.5 ml/min, fraction size: 0.5 ml, buffer: 10 mM Ammonium carbonate, pH: 8.0). Elution of peptides was followed by absorbance at 280 nm. All protein containing fractions were analysed by MALDI-TOF on PBS II (Ciphergen) as described above. Maximum signal intensity of 40 individual peaks was plotted as a function of elution volume and an approximate elution curve was calculated. Study of HNP 1-3 by microflow ----------------------------- For micro flow experiments, canine kidney cells (MDCK cells) were plated onto poly-d-lysine coated cover slips at a concentration 3000 cells/well, grown in Dilbeccoo\'s Modified Eagle Medium (DMEM) with 10% Fetal Bovine Serum (FBS) for five days with the result of confluent islands. Microflow was performed in an Eppendorf micromanipulator 5171 and transjector 5246 system mounted on a Leica DMIRBE inverted research microscope. Micro capillaries (borosilicate with filament, Sutter Instruments Company, Novato, California, USA) were pulled to an outer diameter of 0.85 nm on a Sutter P-97 Micropipette Puller. The dye-loaded cells were visualized by excitation at 470 nm and recorded at 509-nm emission using Haupage version 3.3.18038 software and Kappa CF 15/4 MC-S camera (Leica). The MDCK cells were recorded (in CO2 independent media) on the inverted DMIRBE inverted research microscope. The capillary was placed 20 nmover the confluent cells with a constant flow (1300 hPa). The MDCK cells were exposed to peptide and calcein (20 mM) fractions for 60 minutes. Results ======= HNP 1-3 expression in tissue and serum -------------------------------------- We performed pilot studies of colon tumour and normal colon tissue on a variety of chips with different chemical properties and with different binding and washing conditions. Based on these preliminary studies, we found that the expression of three peptides with mass/charge ratio (m/z) values of 3372, 3443 and 3486 (subsequently identified as HNP 2, 1 and 3, respectively), were upregulated in the tumour samples. The three peptides were visible on different chips and under different binding conditions (data not shown). The strongest signal of HNP 1-3 in tissue extract was obtained on the NP20 (Normal Phase) chip, whereas the strongest signal of HNP 1-3 in serum was observed on the IMAC30 (Ni) (immobilised metal affinity capture chip, activated with nickel), and these conditions were chosen for the actual screenings. We emphasize that in general the protein profiles of serum and tissue were very different when using the same protein chip for serum and tissue extract. However, individual peaks were present on several types of chips, and observed in both serum and in tissue extract, for example the characteristic triplet with m/z 3372, 3443 and 3486. Protein extract from 40 colon tumour and 40 normal colon tissue samples were analysed on NP20 chips and 125 colon cancer serum samples and 100 normal serum samples were analysed on IMAC30 (Ni) chips. All spectres in each screening were pooled and normalised based on overall ion current. Each spectrum produced approximately 40 to 90 protein peaks in the range from 2 to 80 kDa (FIG. [1A--C](#F1){ref-type="fig"}). Statistical analysis of the intensity values of HNP 1-3 in the tissue screening (FIG. [2A](#F2){ref-type="fig"}.) showed that HNP 1-3 were significantly upregulated in tumours (p \< 0.0005) and statistical analysis of HNP 1-3 expression in the serum screening (FIG. [2B](#F2){ref-type="fig"}.) showed that HNP 1-3 were significantly upregulated in cancer serum (p \< 2.2e^-16^). Compared to other peptides in the same range, HNP 1-3 showed average signal intensity in most normal colon tissue extract, whereas the HNP 1-3 signal was extremely high in most tumour samples (in some tumour spectres the HNP 1-3 peaks were the strongest of all peaks). In the normal serum samples the HNP 1-3 signals were weak and only slightly stronger in the cancer serum. Identification of HNP 1-3 ------------------------- The markers were purified by RP-HPLC, peptide gelfiltration and on-chip purification, after which they were identified by peptide mapping as HNP-2 (3372 Da), HNP-1 (3442 Da) and HNP-3 (3486 Da) (Table [1A](#T1){ref-type="table"}). The masses correspond to the peptides in their oxidised states with three disulfide bridges. After reduction with DTT, HNP-1 and HNP-2 increased 6 Daltons in mass, due to reduction of the six cysteines (Table [1B](#T1){ref-type="table"}). We were not able to reduce HNP-3. Size exclusion chromatography of HNP 1-3 ---------------------------------------- 50 ul colon tumour extract in Lysis buffer was applied to a peptide gelfiltration column. Elution of peptides was followed by absorbance at 280 nm. All fractions were analysed by MALDI-TOF on the PBS II instrument (Ciphergen). Maximum signal intensity of 40 individual peaks was plotted as a function of the elution volume and an approximate elution curve was calculated (FIG. [3](#F3){ref-type="fig"}). HNP 1-3 peptides were primarily eluted in early fraction together with high mass proteins above 20 kDa and also, but to a lesser degree, in fractions together with other peptides of similar mass interval (2 to 4 kDa) (FIG. [3](#F3){ref-type="fig"}). Cytoxic assay ------------- The cytotoxicity of HNP 1-3 purified from colon tumours was tested by exposing MDCK cells to different fractions purified from colon tumours. Calcein were added to the fractions and the solutions were left to flow over the cells for one hour. By fluorescence microscopy calcein was observed to accumulate in cells exposed to HNP 1-3/calcein fractions, whereas cells treated with fractions containing other (unidentified) tissue peptides did not uptake calcein (FIG. [4C&D](#F4){ref-type="fig"}). Further, by microscopy we observed that cells exposed to HNP 1-3 appeared more diffuse and had enlarged nuclei, indicating apoptosis (FIG. [4A&B](#F4){ref-type="fig"}). Discussion ========== Elevated concentrations of HNP 1-3 in colon cancer serum -------------------------------------------------------- Abnormal concentration of HNP 1-3 in blood has previously been demonstrated in connection with benign conditions. Elevated concentrations of HNP 1-3 following infection (bacterial- / non-bacterial- infection and pulmonary tuberculosis) has been found in plasma, blood and other body fluids \[[@B8]\], and plasma HNP 1-3 concentrations have been shown to be elevated in patients with septicemia or bacterial meningitis \[[@B9]\]. HNP 1-3 have been found in urine from patients with transitional cell carcinoma of the bladder \[[@B10]\] and HNP-1 has been found in salvia of patients with oral carcinomas \[[@B11]\]. Finally, HNP 1-3 are found in excess amounts in tears after ocular surface surgery \[[@B12]\]. Our study is the first that demonstrate elevated concentrations of HNP 1-3 in blood following tumour growth. Elevated concentrations of HNP 1-3 in colon tumours --------------------------------------------------- HNP expression has previously been linked to different types of tumours and cell lines. HNP-1 has been detected in lung tumours \[[@B13]\] and in the submandibular glands of patients with oral carcinomas \[[@B14]\]. By RT-PCR, mass spectrometry and flow cytometric analysis, HNP 1-3 have been shown to be expressed by cell lines deriving from renal cell carcinomas \[[@B15]\] and the expression of a specific HNP precursor peptide has been shown to be upregulated in human leukemic cells \[[@B16]\]. Our results suggest that HNP 1-3 are extremely abundant in colon tumours. This is in agreement with a study of HNP-1 in lung tumours, where the maximum observed level was 26 nanomoles per gram wet tissue \[[@B13]\]. In order for these excessive amounts of peptide to be detectable in serum, the peptides must be present extracellular in the tumour environment, such as observed in studies of HNP 1-3 expression in kidney \[[@B14]\] and brain \[[@B17]\]. Usually tumour expressed peptides are not easily detected in serum or plasma by SELDI-TOF mass spectrometry techniques; the highly concentrated plasma proteins out compete the signal of low abundance peptides. We suggest that HNP 1-3 are detectable in serum only because they are expressed in exceptionally high amounts in the tumour microenvironment. Previous studies indicate that HNP 1-3 expression in tumours primarily originate from tumour invading eosinophils \[[@B13]\] and neutrophils \[[@B14],[@B18]\]. However, it has been shown that the excess amounts of HNP 1-3 observed in urine from bladder cancer patients were often produced by the actual bladder cancer cells \[[@B10]\], and that highly invasive bladder cancer cells produced more HNP 1-3 than less invasive ones (Holterman DA, in press, personal communication). Since our tissue screening is based on comparison of tissue samples, we cannot say whether the peptides are produced by the colon cancer cells or by tumour infiltrating neutrophils. In the case of active inflammatory bowel disease, it is not clear whether epithelial expression of HNP1-3 is induced by the inflammatory state or the whether the peptides are released by adjacent neutrophils and taken up by the epithelial cells \[[@B6]\]. Here it is believed that the peptides provide protection against microbial invasion when the mucosal barrier is damaged (such as is the case in inflammatory bowel diseases). HNP 1-3 are known to stimulate bronchial epithelial cells to upregulate interleukin-8 production \[[@B19]\], a potent neutrophil chemotactic factor. Thus, the upregulated expression of HNP 1-3 in tumours may primarily originate from invading immune cells, but could be initiated by HNP 1-3 producing cancer cells. Size exclusion chromatography of HNP 1-3 ---------------------------------------- We explain the elevated concentrations of HNP 1-3 in colon cancer serum by unspecific binding between HNP 1-3 and high mass plasma proteins. We suggest that the peptides attach to plasma proteins in the tumour area and are carried into the bloodstream. The HNP 1-3 we observe in high mass fractions from size exclusion, could also be explained by multimerisation: In one study it was demonstrated that defensins form voltage dependent channels in lipid bilayer membranes and further conductance investigations suggested that the channels were formed by multimers containing 2--4 molecules \[[@B20]\] and a crystal structure study \[[@B21]\] of HNP-3 revealed an amphiphilic dimmer. We interpret the size exclusion results as evidence for interaction between HNP 1-3 and unidentified high mass proteins through unspecific interactions; the peptides are eluted in very early fractions which would probably require the presence of multimers of more than five peptides and our study does not reveal the presence of multimers containing four, three or two peptides. Further, our interpretation is in agreement with a number of previous studies that show that HNPs are bound to plasma protein *in vitro*and that high concentrations of HNPs causes precipitation of plasma proteins; specifically 2-macroglubulin and C1 complement \[[@B22],[@B23]\] has been shown to bind defensin. Another study \[[@B24]\] showed that HNP-1 bind to various plasma proteins, notably serum albumin, and it was found that serum, or serum albumin, was able to inhibit the anti-viral activity of HNP-1. This ability to bind to plasma proteins could also explain why HNP 1-3 lysis of mammalian cells is hindered in the presence of serum \[[@B25]\]. Together these observations add to the growing realisation that common plasma proteins may carry disease specific peptides and therefore should not be ignored in biomarker research. Common to beta-defensin 2 (another member of the defensin family) and HNP 1-3 is an uneven distribution of surface charges. Beta-defensin 2 has been shown to bind to a chemokine receptor \[[@B26]\]. It has been suggested that the positively charged cluster found in defensin peptides and chemokines, may play a common role in binding to receptors, but is not important for determining receptor specificity \[[@B27]\]. This surface charge may also explain the binding of HNP 1-3 to plasma proteins. The observation that defensins are localised to lymphocyte nuclei \[[@B28]\], could similarly be explained by unspecific binding to shuttle proteins. The function of HNP 1-3 ----------------------- The exact concentration of HNPs in the tumour microenvironment may decide the *in vivo*function of HNP 1-3. One study showed that HNP 1-3 mediates lysis of tumours in a concentration dependent manner \[[@B25]\]. This is in agreement with another study that show that only relatively high concentrations of HNP-1 (10^-4^M) are cytotoxic for human monocytes, whereas lower concentration of HNP-1 (10^-8^to 10^-9^M) increases TNF-alpha production by monocytes \[[@B29]\]. In a study of renal cell carcinoma lines \[[@B14]\] it was shown that HNP 1-3 were cytotoxic to all tested cell lines when present in high concentrations (above 25 ug/ml), but at lower concentration HNP 1-3 stimulated growth of a subset of tumour cell lines. We add to these results by demonstrating that *in vivo*HNP 1-3 purified from colon tumours are capable of lysing MDCK cells. Our study was based on a 60 minutes microflow study. This screening set-up did not allow us to investigate the minimum concentration of HNP 1-3 necessary for lysis. Conclusions =========== The high concentration of HNP 1-3 observed in tumours and the observation that HNP 1-3 are capable of lysing mammalian cells may lead to the conclusion that the peptides serve to the benefit of the host by killing tumour cells. However, in one study HNP 1-3 were found to bind to HLA-Class II molecules and were capable of reducing the proliferation of a HLA-DR-restricted T-cell line after stimulation \[[@B30]\] and could in this way help the cancer cells avoid local immune recognition. Defensins also regulate the systemic immune response. Through interaction with the chemokine receptor CCR6, beta-defensins recruits dendritic and T cells\[[@B26]\] (discussed in \[[@B31]\]) and HNP 1-3 are capable of recruiting leukocytes to sites of infection in mice \[[@B32]\]. Upregulated immune responses are known to stimulate tumour proliferation; immune cells are actively recruited by tumours to exploit their pro-angiogenic and pro-metastatic effects (reviewed in \[[@B33],[@B34]\]). Whether the high concentrations of HNP 1-3 in the tumour limits the tumour growth or on the contrary stimulate tumour proliferation is not clarified; we emphasise that HNP 1-3 are expressed in inflammatory bowel disease and could be involved in the early stages of carcinogenesis. We suggest that the prominent surface charge on defensins, their unspecific binding to other proteins (such as high mass plasma proteins) and the observed excess amounts of peptides found in tumours, could provide the peptides with broad antagonising effects on numerous receptors in the tumour microenvironment. In this way HNP 1-3 peptides may serve to the benefit of the tumour. Our results add to the evidence that HNP 1-3 bind to high mass plasma proteins. We suggest that the peptides are released in the inflammatory site and passively diffuse into the blood stream. HNP 1-3 have been observed in primary tumours of different tissues and the peptides are known to play a fundamental role in the innate immune system. We suggest that the peptides may serve as markers for colon cancer in combination with established diagnostic tools and as prognostic markers following therapy. Competing interests =================== This study was partly financed by Colotech ltd. and partly by Glostrup Hospital. Jakob Albrethsen, Rikke Bøgebo and Hans Raskov receive salary from Colotech A/S. Steen Gammeltoft, Jesper Olsen and Benny Winther receive salary from Glostrup Hospital. Authors\' contributions ======================= JA performed the SELDI-TOF/MS screenings, the protein identification experiments and the size exclusion study. RB did the statistical analysis, BW did the microflow study, JO obtained the biological samples. HR and SG planned the project. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/8/prepub> Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Protein profiles of serum and tissue.**Representative SELDI-TOF/MS spectra of normal colon tissue (FIG. 1A) on the NP20 chip and normal serum (FIG. 1B) on the IMAC30 (Ni) chip. The two spectra differ significantly and each produce a total of 40 to 60 peaks. The majority of the peaks are present in the specified range from 2 to 10 kDa. FIG. 1C Comparison of a typical colon tumor spectrum (above) and a normal colon spectrum (below) in the range from 3 to 4 kDa. The arrows point to the three differentially expressed peptides, subsequently identified as HNP 1-3. The three peptides are expressed in normal colon tissue and the expression is upregulated in the tumor samples. The three peptides are present in normal serum, but are present in higher concentrations in colon cancer serum. The average peak intensity for the three peptides were significantly lower in serum than in tissue. ::: ![](1471-2407-5-8-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Expression of HNP 1-3 in tumors and serum.**FIG.2A HNP 1-3 profiles of normal and colon tumor tissue. 40 colon tumor and 40 normal colon tissue samples were analysed on NP20 chips. Differences in mean intensities of HNP 1-3 in normal and colon tumor tissue were statistical significant at 5% level (p \< 0.0005). FIG. 2B HNP profiles of normal and colon cancer serum. Serum samples (125 colon cancer and 100 normal) were analyzed on IMAC30 chips. The mean intensities were significantly different at 5% level (p \< 2.2e-16). The reproducibility was significant for all three peptides in both tissue and serum. The standard deviation of HNP 1, 2 and 3 was 70%, 136% and 57% in normal tissue and 11%, 15% and 8% in tumor tissue. The standard deviation of HNP 1, 2 and 3 was 96%, 154% and 81% in normal serum and 234%, 365% and 282% in cancer serum. The boxplot show the 25th quantile, median, 75th quantile, and whiskers extent to min. and max. values. ::: ![](1471-2407-5-8-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Size exclusion study of HNP 1-3.**Protein extract from tumor tissue was separated on a peptide gelfiltration column. The elution volumes of forty (unidentified) peptides was plotted against their respective mass values and an approximate elution curve was calculated. The arrows point to HNP 1-3, which were eluted in two fractions: primarily in the void volume (8 ml) together with high mass proteins (above 20 kDa) and after 14 ml together with peptides of similar mass range (2--4 kDa). We interpret this as evidence for binding between HNP 1-3 and high mass proteins. ::: ![](1471-2407-5-8-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Functional study of HNP 1-3.**Normal microscopy (A&B) and flourescence microscopy (C&D) of MDCK cells. MDCK cells were exposed to calcein with (A&C) and without HNP 1-3 (B&D). By fluorescence microscopy (C&D) the cells were observed to uptake calcein only when treated with fractions containing HNP 1-3/calcein (C). Fractions containing unidentified peptides purified from colon tumors were used as negative controls together with calcein and did not stimulate the cells to uptake calcein (D). Cell islands treated with HNP 1-3 appeared diffuse and showed enlarged nuclei, indicating apoptosis (A). ::: ![](1471-2407-5-8-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Identification of HNP 1-3. Table 1A HNP 1-3 peptides were identified by peptide mapping (on-chip trypsin digest). Three sequence specific fragments were produced (615.8, 930.6, 1061.4 Da.). The peptides were identified as HNP 1-3 using the PepIdent server on the ExPASy homepage. Table 1B The measured masses of the HNP 1-3 peptides were approximately 6 Daltons lower than the expected theoretical masses. This deviation was caused by the *in vivo*formation of three disulfide bridges. By heat denaturation and treatment with DTT we were able to protonate the six cysteines in HNP 1 and 2. (HNP-3 was consistently degraded during the reduction procedure). ::: **A** -------------------------------------------------- ----------------------------------------------------- ------ ---------------------------------------------------------- 615.8 615.7 +0.1 ACYCR 930.6 930.1 +0.5 IPACIAGE 1061.4 1061.2 +0.2 YGTCIYQ **B** **Measured masses of HNP 1-3 peptides (Dalton)** **Theoretical masses of HNP 1-3 peptides (Dalton)** **Measured masses of reduced HNP 1-3 peptides (Dalton)** 3371.2 3377.0 (HNP-2) 3376.2 3442.3 3448.1 (HNP-1) 3448.6 3486.3 3292.1 (HNP-3) \- :::

PubMed Central

2024-06-05T03:55:52.526429

2005-1-19

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548152/", "journal": "BMC Cancer. 2005 Jan 19; 5:8", "authors": [ { "first": "Jakob", "last": "Albrethsen" }, { "first": "Rikke", "last": "Bøgebo" }, { "first": "Steen", "last": "Gammeltoft" }, { "first": "Jesper", "last": "Olsen" }, { "first": "Benny", "last": "Winther" }, { "first": "Hans", "last": "Raskov" } ] }

PMC548262

Background ========== First described in 1997, *Parachlamydia acanthamoebae*is an obligate intracellular bacterium naturally infecting free-living amoebae \[[@B1],[@B2]\]. It was isolated from *Acanthamoeba*spp. recovered from the nasal mucosa of healthy volunteers \[[@B1]\]. Later, additional strains of *Parachlamydiaceae*have been found within about 5% of *Acanthamoeba*spp. and once within *Hartmanella vermiformis*\[[@B2],[@B3]\]. The 16S rRNA sequences of these *Parachlamydiaceae*are about 14% different from those of both genera *Chlamydophila*and *Chlamydia*\[[@B2],[@B3]\]. Since the 16S rRNA sequence difference between *Chlamydophila*sp. and *Chlamydia*sp. is 6% only, it clearly appears that the speciation between the two latter occurred after the divergence between *Parachlamydiaceae*and *Chlamydiaceae*. Like other *Chlamydiales*, *Parachlamydiaceae*can present two developmental stages: the reticulate body, a metabolically active dividing form, and the elementary body, an infective stage; the crescent body is another infective form, not observed in *Chlamydiaceae*\[[@B4]\]. Differentiation of the infective stages in reticulate bodies and multiplication of the latter were recently shown to occur within amoebal vacuoles, that may contain hundreds of bacteria \[[@B4]\]. Depending on the symbiotic/pathogenic relationships prevailing between both organisms, the escape of the bacteria from the amoeba may occur either by the release of secreted vesicles or by the lysis of the host \[[@B4]\]. There is a growing evidence of the human pathogenicity of *Parachlamydiaceae*\[[@B2]\]. For instance, positive *Parachlamydia*serologies were shown to be associated with a febrile epidemic \[[@B5]\], community-acquired pneumonia \[[@B6]\], and inhalation pneumonia \[[@B7]\]. The role of *Parachlamydia*-related bacteria as agents of inhalation pneumonia is further suggested by the temperature-dependent release of the bacteria from their amoebal reservoir \[[@B8]\]. PCR amplification of parachlamydial DNA from monocytes, sputa and bronchoalveolar lavages collected from patients suffering of bronchitis or pneumonia also supports the pathogenic potential of *Parachlamydia*\[[@B9]-[@B12]\]. The survival of these *Chlamydia*-like organisms within human macrophages \[[@B13]\] is an additional hint of parachlamydial pathogenicity. Horn *et al*. \[[@B14]\], by sequencing and annotating the whole genome of the *Parachlamydia*-related UWE25 contributed much to the understanding of the evolution of chlamydiae. Indeed, they demonstrated that major virulence mechanisms of *Chlamydiaceae*such as the Type Three Secretion System (TTSS) and the Chlamydial Protease-like Activity Factor (CPAF) are also encoded by the chromosome of the evolutionary early-branching *Parachlamydiaceae*UWE25. Genome analysis of the parachlamydial endosymbiont also identified Open Reading Frames (ORFs) homologous to Type Four Secretion Systems (TFSS) and characterized by a high G+C content, suggesting that they result from an horizontal transfer. Based on their annotation revealing the apparent absence of genes necessary for DNA transfer, Horn *et al*. \[[@B14]\] proposed that this TFSS was involved in protein export but not in DNA transfer. To date, numerous genomic islands (GIs) were already identified along whole chromosomal sequences of various bacterial species. For instance, 140 GIs are described in the Islander database, including GIs of proteobacteria, firmicutes, actinobacteria and cyanobacteria \[[@B15]\]. Thus, we wondered whether any GIs were located along the bacterial chromosome of the amoebal endosymbiont UWE25. GIs are genetic elements which length vary from 10 to 200 kb and are inserted in a chromosome after a lateral transfer occurring, in some instances, between phylogenetically-distant microorganisms. Their heterologous origins are generally evidenced by a G+C content different from that of the remaining bacterial chromosome and by the presence of various mobility genes (i.e. involved in transposition, transduction or conjugative transfer), that are occasionally source of GI instability \[[@B16],[@B17]\]. They are often flanked by particular DNA sequences, such as direct repeats or insertion sequences. Moreover, tRNA *loci*are generally used as insertion sites by GIs for their chromosomal integration \[[@B16]-[@B18]\]. Since no genetic tools are available for the study of this obligate intracellular bacteria, a bioinformatic approach was chosen to locate putative GIs. Results ======= A genomic island is present in the genome of UWE25 -------------------------------------------------- Using standard G+C content analyses of *Parachlamydia*-related UWE25 chromosome, we observed a G+C-rich region (Figure [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}), similar to that shown by Horn *et al*. \[[@B14]\]. Using the residual cumulative G+C content analysis adapted from the GC profile of Zhang and Zhang \[[@B19]\], we were able to precisely define a 19-kb region (Figure [1C](#F1){ref-type="fig"}). The presence of 17-bp direct repeats flanking a 100-kb chromosome region (1648 to 1748 kb, Table [1](#T1){ref-type="table"}) that encompasses the 19-kb DNA sequence enabled us to define a new region composed of 100 ORFs (See [additional data file 1](#S1){ref-type="supplementary-material"} for the description of these genes and their location on the chromosome of UWE25). Interestingly, this 100-kb region is characterized by a higher level of local gene coorientation (75/100) than that characterizing the remaining of the genome (1015/1931, 52.6%, p \< 0.001) and by a particular signature in the cumulative GC skew analysis. Two identical *gly-tRNA*genes in tandem are located at the proximal end of this 100-kb genetic element (Figure [1A,1C](#F1){ref-type="fig"} and Table [1](#T1){ref-type="table"}). Several mobility genes (eight putative transposases, one recombinase and seven bacteriophage related-proteins) are encoded within the 100-kb region (Figure [1C](#F1){ref-type="fig"}, Table [1](#T1){ref-type="table"}). Thus, this region largely fulfills the accepted criteria of GIs \[[@B16]-[@B18]\]. We termed this newly described GI \"Pam100G\" (*Protochlamydia amoebophila*, 100-kb, Gly-tRNA) according to the nomenclature used in the Islander database \[[@B15]\]. Mosaicism of the 100-kb genomic island -------------------------------------- Interestingly, this GI can be divided into clearly distinct regions, according to their G+C content (Figure [1B](#F1){ref-type="fig"}, Table [2](#T2){ref-type="table"}). The residual cumulative G+C content analysis highlights a modular structure with different slopes, each linear segment indicates that genes of this unit present a rather constant local G+C content (Figure [1C](#F1){ref-type="fig"}). A positive or a negative slope would indicate that each block of genes presents a G+C content higher or lower that of the UWE25 chromosome, respectively. The first module begins with a direct repeat and two identical *gly-tRNAs*in tandem. Composed of 28 ORFs, this unit exhibits a G+C content (36.4%) similar to that of the remaining of the genome of UWE25 (1931 ORFs, 36.1%). Sixteen homologs to these 28 genes (57%) were found in databases, 12 of them (75%) exhibiting a best score in BLAST analyses with a *Chlamydiaceae*ORF (See [additional data file 1](#S1){ref-type="supplementary-material"}). Interestingly, no gene of the other modules of the 100-kb GI exhibited a best hit in similarity analyses with any *Chlamydiaceae*counterpart. Some of the genes present in the first module, such as *sctN*and *sctQ*, are part of a TTSS also present in *Chlamydiaceae*. The other TTSS genes are disseminated along the chromosome of UWE25. The presence of some TTSS genes in the first module and of a gene encoding a putative transposase at the distal end of the first module of this 100-kb GI suggests that this first unit was acquired by chromosomal rearrangements. (See [additional data file 1](#S1){ref-type="supplementary-material"} for the results of BLAST analyses). Characterized by a low G+C content (34.1%), the 2^nd^module encodes 18 ORFs. Only five are similar to known protein sequences (28%), four of them being identified as mobility genes (three bacteriophage-related genes and one putative transposase encoding gene). The 3^rd^module (19 kb), exhibiting the second highest G+C content of the UWE25 genome (40.9%), comprises 21 ORFs. Some of these genes were identified as *tra*genes by Horn *et al*. \[[@B14]\]. Using BLAST analyses and alignment tools, we re-annotated the whole module (see below) and, if we except two transposase genes and one ORF of unknown function, we unveiled that all ORFs of this module belong to a genetic unit similar to the *tra*operons encoding the TFSS previously described in proteobacterial genomes (Figure [2](#F2){ref-type="fig"}, See [additional data file 1](#S1){ref-type="supplementary-material"} for the re-annotations of this module). Presenting a low G+C content (33.4%), the 4^th^module (10 kb) is composed of 13 ORFs. All these ORFs were previously annotated by Horn et al. \[[@B14]\] as encoding hypothetical proteins or without homolog. Our BLAST analysis identified one ORF homologous to genes encoding bacteriophage-related proteins and two genes of proteins involved in DNA metabolism (Table [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}, see also the table in the [additional data file 1](#S1){ref-type="supplementary-material"}). Interestingly, a direct repeat is located between the 9th and 10th genes of the module. This 17-bp direct repeat, that presents 3 mismatches is similar to those present at the proximal and distal ends of the GI, exhibiting the same 14 conserved nucleotides. It may reflect a complex evolutionary history of the GI, possibly enabling it to be mobile as 25-kb, 75-kb or 100-kb DNA segments. A single large protein is encoded along the 5^th^module (6 kb). Its G+C content is one of the highest of the UWE25 chromosome (41.8%). By BLAST analysis, this protein exhibits the strongest similarity with the human Nod3 protein. The 6^th^module (12 kb) is characterized by a low G+C content (33.3%). This unit is composed of 13 ORFs, the first ORF encoding a product similar to the Death on cure (Doc) protein of P1 bacteriophage. Two ORFs code for proteins involved in DNA metabolism and an additional ORF encodes a putative transposase. The 7^th^module is short (2 kb) and present a G+C-rich unit (38.7%). Five of the six ORFs of this unit encode a probable resolvase, three putative transposases and a phage-related Doc protein. The final direct repeat is located at the end of this module. With the only exception of the phage-related protein, all other ORFs of the 7^th^module appear to be similar to gamma-proteobacterial proteins, possibly explaining the observed different signal in the G+C content analysis. Role of the type IV secretion system encoded by the 100-kb genomic island ------------------------------------------------------------------------- The functions of genes encoded by GIs may be related, among others, to pathogenicity such as the ability to exploit the host intracellular environment. Since no genetic system has been described for any obligate intracellular chlamydiae, we investigated the putative functions of this GI by bioinformatics. We focused our analyses on the TFSS, for which a previous annotation of the *tra*genes showed a genetic unit unable to transfer DNA \[[@B14]\]. Using different protein comparison methods described in the [additional data file 1](#S1){ref-type="supplementary-material"}, we identified supplementary *tra*genes, and compared the general organization of this *tra*unit with other genetic elements encoding TFSS genes \[[@B20]\]. The UWE25 *tra*unit displays a striking colinearity with *tra*operons encoding F-like conjugative DNA transfer system, especially to those of the F and pNL1 plasmids of *Escherichia coli*and *Novosphingobium aromaticivorans*, respectively (Figure [2](#F2){ref-type="fig"}). All homologous genes essential for DNA transfer in plasmid F (*traF*, *traG*, *traH*, *traN*, *traU*, *traW*, and *trbC*) and involved in sex pilus retraction and mating pair stabilization \[[@B20]\] are present, strongly suggesting that, similarly to the other F-like TFSSs, the gene products encoded by the UWE25 *tra*unit are devoted to DNA transfer. With the only exception of *traG*, these genes are not present on P-like and I-like plasmids, reinforcing the close relationship prevailing between the UWE25 *tra*unit and their F-like plasmids counterparts. Figure [3](#F3){ref-type="fig"} shows that the UWE25 *tra*unit clusters within F-like TFSSs, confirming that it may function as a F-like conjugative system. Drawn as an UPGMA tree (Figure [3A](#F3){ref-type="fig"}), the comparison of the genetic organization of all *tra*units was performed as a gene order breakpoint analysis developed for the study of the mitochondrial genome evolution \[[@B21]\]. This analysis clearly shows that the closest relatives of the UWE25 *tra*units are the *tra*operons of the F-like conjugative plasmids. The Fitch-Margoliash- and the minimum evolution comparisons performed on the same dataset presented the same tree topologies, confirming the former UPGMA results (data not shown). An *omit*test performed on this tree confirms that the results are robust: with one exception (involving the deep branching of one cluster on one tree), all 11 trees were congruent in all their nodes. Figure [3B](#F3){ref-type="fig"} shows an UPGMA tree comparing the Kimura corrected *p*-distances (the proportion *p*of nucleotide sites at which two sequences are different, taking into account the proportion of transversion- and transition-substitution rates) of nucleotide sequences of the concatenated *traA*, *traK*, *traB*, *traV*, and *traC*genes. A similar topology is observed with (i) neighbor-joining- and minimum evolution trees inferred using the Kimura-corrected *p*-distances and (ii) UPGMA, neighbor-joining- and minimum evolution trees performed on *p*-distance of the whole coding sequences of the concatenated *tra*genes (See [additional data file 2](#S2){ref-type="supplementary-material"} for these trees). Neighbor-joining- and minimum evolution methods comparing Kimura-corrected *p*-distances of the complete coding sequences confirmed that the *tra*unit of UWE25 is phylogenetically closely related to the *tra*operons of the F-like plasmids: the bootstrap values of 94% and 91% respectively, support the node separating the concatenated *tra*genes of the chromosomal UWE25 and the R27 plasmid, a gamma-proteobacterial F-like conjugative plasmid, from those of all other plasmids (See the [additional data file 2](#S2){ref-type="supplementary-material"} for these trees). In neighbor-joining and minimum evolution analyses of *p*-distances, the *tra*unit of UWE25 also clusters with the *tra*operons of gamma-proteobacterial F-like plasmids: the bootstrap of 96% and 92%, respectively, support the node separating the concatenated *tra*genes of UWE25 and RTS1, SXT, R391, three gamma-proteobacterial F-like conjugative plasmid, from their closest relative, R27 plasmid (See [additional data file 2](#S2){ref-type="supplementary-material"} for these trees). Taken together, all these data strongly suggest that the UWE25 *tra*unit is closely related to F-like conjugative *tra*operons. Origin of the genomic island and of its type four secretion system ------------------------------------------------------------------ Our BLAST analyses \[[@B22]\] reveal that a majority (24/43) of genes not presenting a best hit for chlamydial genes but having homologs in other taxa are more related to proteobacterial genes (see Table [2](#T2){ref-type="table"} and the [additional data file 1](#S1){ref-type="supplementary-material"} for similarity analyses indicating for each parachlamydial *tra*gene the most similar gene and its taxonomical background). Moreover, the BLAST analyses of the 21 ORFs of the third module, encoding the *tra*genes, show that most ORFs of this unit (15/21) are of proteobacterial origin. However, since six of them present the highest similarity to alpha-proteobacterial genes and six others to gamma-proteobacterial genes, a more precise origin of the parachlamydial *tra*unit could not be precisely defined by this first approach. The presence of *gly-tRNA*at the proximal end of the GI of UWE25 is consistent with a close relatedness between this GI and proteobacteria: out of 14 GIs described in the *Islander*database of Mantri *et al*. \[[@B15],[@B22]\] inserted along a chromosome by a *gly-tRNA*(14/140), 12 of them (86%) were sequenced in a proteobacterial genome. No GI of Gram-positives described in the Islander database are inserted in a chromosome within a *gly-tRNA*gene. Again, a precise proteobacterial origin could not be proposed, because the distribution of *gly-tRNA*genes in alpha- (4/22) and gamma-proteobacterial (8/72) GIs is not significantly different: by including only the non-redundant GIs, the distribution of *gly-tRNA*genes in alpha and gamma-proteobacterial GIs is 2/20 and 7/71, respectively. Comparison of gene order between all *tra*units also failed in assigning a precise origin to the UWE25 *tra*unit since it branched near the alpha- and gamma-proteobacterial *tra*operons (Figure [3A](#F3){ref-type="fig"}). The only first hint of a possible gamma-proteobacterial origin for the UWE25 *tra*unit was brought by the phylogenetic analyses (Figure [3B](#F3){ref-type="fig"} and additional files [1](#S1){ref-type="supplementary-material"} &[2](#S2){ref-type="supplementary-material"}). Thus, bootstraps values of 94, 91, 96 and 92% supported the node separating the concatenated *tra*genes of UWE25 and several *tra*operons of gamma-proteobacterial F-like plasmids from the F-plasmids of an alpha-proteobacteria and of other gamma-proteobacteria. (See above, and [additional data file 2](#S2){ref-type="supplementary-material"} for these trees). Discussion ========== We showed that the *Parachlamydia*-related endosymbiont UWE25 presents a 100-kb region largely fullfilling the criteria of GIs \[[@B16]-[@B18]\]. Indeed, this DNA region characterized by a high level of gene co-orientation presents a G+C content different from that of the remainder of the genome. The presence of direct repeats flanking this chromosome region enabled us to focus on 100 ORFs. Two identical *gly-tRNA*genes in tandem are present at the proximal end of this genetic element. Moreover, several mobility genes encoding transposases and bacteriophage related-proteins are located within this chromosome region. The cumulative residual G+C content analysis shows that this GI is composed of seven modules. Such a chimeric organization was already described in other GIs \[[@B23],[@B24]\]. The first module contains chlamydiae genes probably brought by chromosome rearrangements. Some of these genes, homologous to TTSS genes of *Chlamydiaceae*, might provide selective advantages to strains that retained the GI. The 2^nd^, 4^th^and 6^th^modules are mainly composed of bacteriophage-related protein genes, that could reflect a putative phage implication in GI formation. The 3^rd^module codes for a TFSS similar to *tra*operons. We propose that this *tra*unit is devoted to DNA transfer, based (i) on similarity analyses demonstrating the presence of all genes encoding proteins used during a DNA transfer, (ii) on phylogenetic analyses of *tra*unit genes and, (iii) on comparison of gene order. These analyses clearly demonstrate that the UWE25 *tra*unit is strongly more related to F-like conjugative system than to P-like and I-like secretion systems. The significant bootstraps of all trees obtained by standard gene phylogeny and their congruent topologies with others obtained by the gene order breakpoint analysis not biased by codon usage homing, strongly support the validity of these analyses confirming the F-like conjugative nature of the parachlamydial *tra*unit. Thus, our model significantly differs from the other proposed by Horn *et al*. \[[@B14]\], who did not identify *traA*, *traL*, *traK*, *traV*, and concluded that the UWE25 *tra*unit is involved in protein export, and not in DNA transfer. The 5^th^module presents a nucleotide composition similar to the *tra*unit and is composed of a single high G+C 6-kb gene, whose product is similar to the human Nod3 protein. The Nod (Nucleotide-binding oligomerization domain) proteins are members of a family that also includes the apoptosis regulator Apaf1 (Apoptotic protease activating factor 1) and plant disease-resistance gene products \[[@B25]\]. The function of the human Nod3 is still unknown. Like Nod1 and Nod2, Nod3 might be involved in the recognition of conserved motifs present at the surface of bacteria, such as peptidoglycan. The nucleotide G+C composition of the 2^nd^, 4^th^, and 6^th^modules are similar, explaining the observed similar negative slope of the residual G+C curves. Moreover, these three modules encode phage-related proteins and proteins involved in DNA metabolism. These modules probably involved in mobility might have a common origin, the ancestral single phage module being currently separated in three pieces by the presence of the *tra*unit and of the Nod3-like protein encoding gene. The positive slope in the G+C analysis of the 7^th^module echoes those of the *tra*unit (3^rd^module) and of the Nod3-like protein (5^th^module). The 7^th^module encodes a transposition resolvase and three transposases similar to gamma-proteobacterial homologs. With the only exception of the phage-related Doc protein, that has an homolog at the beginning of the sixth module, and that might be located there after transposition, the 7^th^module appears thus to have a different origin than the 2^nd^, 4^th^and 6^th^modules, though also encoding mobility genes. The presence of a F-like *tra*unit along the sequences of UWE25 is the first evidence of a putative conjugative system in chlamydiae. If conjugation occurs, it most probably takes place within free-living amoebae, that may contain several hundreds of *Parachlamydia*bacteria tightly packed in their vacuoles \[[@B4]\]. Such a conjugation system would be a mechanism to transfer DNA between internalized bacteria sharing an amoebal vacuole. Moreover, it may provide molecular genetic tools for obligate intracellular bacteria. The presence of *tra*units/operons in the parachlamydial UWE25 and in proteobacteria could be explained by an emergence of this unit in a common ancestor of both clades, and by its subsequent loss in *Chlamydiaceae*. Another evolutionary scenario is that the *tra*unit was acquired from a proteobacteria by a *Parachlamydiaceae*in a common amoebal vacuole. Since the *tra*unit is absent from all sequenced *Chlamydiaceae*genomes, this transfer would have occurred after the divergence of *Parachlamydiaceae*and *Chlamydiaceae*, at a time when *Parachlamydia*was already an intracellular bacteria. An intra-amoebal transfer of this GI is supported by the permissivity of free-living amoebae to proteobacteria \[[@B26]\], and by several hints suggesting its proteobacterial origin. Though phylogenetic analyses suggested a gamma-proteobacterial origin of the F-like parachlamydial *tra*, further analyses have to confirm whether this GI module was acquired from an alpha-, beta-, or gamma-proteobacteria unit. We hypothesize that the F-like parachlamydial *tra*unit has been brought by a lateral transfer from a proteobacterial genome. This hypothesis is strongly supported by the cumulative GC skew analysis \[[@B27]-[@B30]\] producing a signal of the GI differing from that of the remaining of the genome (Figure [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}). The value of nucleotide skew analyses as good taxonomical markers is supported by (i) routine analyses on prokaryotic genome by cumulative TA-skews \[[@B30]\] and (ii) comparison of intragenic nucleotide skews of small subunit ribosomal RNA of the whole living world \[[@B31]\]. The genometric approach appeared to be able to identify GIs of *Chlamydiales*. Sequencing additional genomes of environmental chlamydiae, that present a large biodiversity \[[@B3]\], will provide major insights on bacterial evolution and hopefully a better comprehension of the emergence of this parachlamydial GI. Conclusions =========== We showed that a GI present on the UWE25 chromosome encodes a potentially functional F-like DNA conjugative system. This is the first hint of a putative conjugative system in chlamydiae. Conjugation most probably occurs within free-living amoebae, that may contain hundreds of *Parachlamydiaceae*bacteria tightly packed in vacuoles. Such a conjugative system might be involved in DNA transfer between internalized bacteria. Since this system is absent from the sequenced genomes of *Chlamydiaceae*, we hypothesize that it was acquired after the divergence between *Parachlamydiaceae*and *Chlamydiaceae*, when the *Parachlamydia*-related symbiont was an intracellular bacteria. It suggests that this heterologous DNA was acquired by a *Parachlamydiaceae*from phylogenetically-distant bacteria sharing an amoebal vacuole. Since *Parachlamydiaceae*are emerging agents of pneumonia \[[@B2]\] and since many GIs are also considered as pathogenicity islands \[[@B17]\], the Pam100G GI might be involved in pathogenicity. In future, conjugative systems might be developed as genetic tools for studying *Chlamydiales*. Methods ======= Sequence -------- The genome sequence of UWE25 \[[@B14]\] (Accession number: NC\_005861) is available at the NCBI website \[[@B32],[@B33]\]. In this contribution, the acronym UWE25 refers only to the *Parachlamydia*-related endosymbiont UWE25, and thus not to the *Acanthamoeba*sp. strain UWE25 from which the parachlamydial endosymbiont UWE25 was recovered \[[@B3]\]. Horn *et al*. recently proposed UWE25 as the type strain of a new bacterial species: *Protochlamydia amoebophila*\[[@B34]\]. BLAST analyses -------------- BLAST analyses were performed with BLASTP 2.2.9 \[[@B35]\] available on the NCBI website \[[@B36]\] using the BLOSUM62 matrix, and gap penalties of 11 and 1. Each ORF was compared against all genes of non-redundant databases available at the NCBI website. An e-value of 0.001 was selected as a standard cut-off. To further identify possible homologous ORFs, we also BLASTed each *tra*gene of F plasmid versus all genes of the full genome of *Parachlamydia*and conversely, each ORF of the putative parachlamydial *tra*unit versus counterparts of the different F-like plasmids. CLUSTALW was used to detect the best relatedness of a given parachlamydial Tra protein with its possible homologs encoded by the F and pNL1 plasmids. Residual cumulative GC content ------------------------------ The residual cumulative G+C content, a slightly modified version of the cumulative GC profile defined by Zhang and Zhang \[[@B19]\], was used to reveal local variations of G+C content of a genome, without using sliding windows of arbitrary size. First, a G+C content analysis was performed on 100-bp windows of the selected chromosome sequence, as for a cumulative GC skew analysis. The cumulative G+C content *GC*~*n*~of the *n*^*th*^window is obtained by cumulating the G+C contents from the first to the *n*^*th*^window: ![](1471-2180-4-48-i1.gif) where, in the window *i*, *G*~*i*~and *C*~*i*~are the numbers of Gs and Cs, respectively, and *N*~*i*~is the total number of nucleotides. To visualize genomic regions differing from the average G+C content, a linear regression *y*defined by a slope *k*is performed on the cumulative curve using the least square methods: *y*(*n*) = *kn* where *n*is the position of the center of the *n*^th^window. The residual cumulative G+C content curve *GC*\' can then be drawn as a function of the position of each window center: *GC*\'~*n*~= *GC*~*n*~- *kn* Zhang and Zhang \[[@B19]\] recently demonstrated that, in some instances, abrupt changes in the residual cumulative G+C content curve correspond to genomic islands. Repeats identification ---------------------- The perfect tandem repeats identification was first performed using the EQUICKTANDEM software (Richard Durbin, Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, CB10 1SA, UK) \[[@B37]\] on a 200-kb DNA sequence (UWE25 genome position: 1.6 to 1.8 Mb) encompassing the *tra*genes previously identified by Horn *et al*. \[[@B14]\]. The duplicated genes and the ORFs containing internal repeats were removed. For each pair of direct repeats, potential unperfect matches of flanking nucleotides were scanned using DNA strider 1.2.1 \[[@B38]\], with the following settings: a minimal size of 11 bp and 3 mismatches. Furthermore, sequences similar to direct repeats were searched along the whole chromosome, and sequences also found outside the selected 200-kb region were discarded from our analysis. Finally, the direct repeats positions were compared to the G+C content analysis, the cumulative GC skew curve, and to tRNA genes locations. Phylogenetic analyses --------------------- Since Horn *et al*. \[[@B14]\] did not identified *traA*, *traL*, *traK*, *traV*, re-annotation of the UWE25 *tra*unit was necessary for phylogenetic analyses. We used i) the genes of F-like plasmids encoding the following *tra*genes, i.e. *traA*, *traK*, *traB*, *traV*, *traC*, and ii) the corresponding ORFs of P- and I-like plasmids \[[@B20]\], i.e. *trbC/VirB2*, *trbG/VirB9*, *trbI/VirB10*, *trbH/VirB7*, *trbE/VirB4*of P-plasmids and *traX*, *traN*, *traO*, *traI*, *traU*of I-plasmids, respectively. The genes were concatenated to obtain a single nucleotide sequence and aligned with CLUSTALW (\[[@B39]\] as it was already performed for genes of ribosomal proteins \[[@B40]\]. Using this alignment and the MEGA 2.1 software \[[@B41]\], we inferred phylogenetic relationships by drawing trees using *p*-distances (the proportion *p*of nucleotide sites at which two sequences compared are different) and Kimura corrected *p*-distance (correction for the rates of transition and transversion) with Unweighted Pair Group Method with Arithmetic Mean (UPGMA), neighbor-joining, and minimum evolution methods. To prevent alignment biases, trees were drawn using the complete deletion option implemented on MEGA 2.1. Gene order breakpoint analyses ------------------------------ To quantify the inversion and transposition events leading to the current organization of *tra*operons, the gene order breakpoint analysis developed for small genomes (mitochondria) by Blanchette *et al*. \[[@B21]\] was used to estimate the similarity of gene order existing between the *tra*unit of UWE25 and the *tra*operons reviewed by Lawley *et al*. \[[@B20]\]. The distance calculated for two given operons *O*~*i*~and *O*~*j*~containing homologous genes proposed by Blanchette *et al*. \[[@B21]\] was slightly modified to take into account the variation of gene numbers of *tra*operons: instead of counting the number of minimal breakpoints existing between two *tra*operons, a distance was estimated by measuring the proportion of conserved gene pairs between both genomic entities. Next, a comparison matrix is established by calculating the distance for each pairwise comparison. Finally, a dissimilarity matrix is obtained by subtracting each distance from 1. For instance, if the operon *O*~*i*~encodes sequentially four genes (*a*, *b*, *c*, and *d*) and the operon *O*~*j*~, six genes (*a*, *b*, *e*, *-d*, *-c*, and *-f*; genes labeled by a minus sign are encoded by the complementary strand), the gene order breakpoint analysis reveals that two gene pairs are conserved: *ab*and *cd*. The dissimilarity distances existing between the operons i) *O*~*i*~and *O*~*j*~, and ii) *O*~*j*~and *O*~*i*~would be: 1-(2/3) = 1/3 and 1-(2/5) = 3/5, respectively. From the square dissimilarity matrix, phylogenetic trees were drawn. Three different distance-matrix analyses were used: the UPGMA, the Fitch-Margoliash- and the minimum evolution methods. To assess the robustness of the tree, an *omit*test \[[@B42]\] was performed on 11 UPGMA trees, in each one organism is missing. Authors\' contributions ======================= GG and CAR initiated the project. GG and FC reannotated the parachlamydial *tra*unit and performed all BLAST analyses. FC and LG delimited the GI by direct repeat analyses. GG drew the phylogenetic trees of the concatenated *tra*genes. After developing the residual cumulative G+C content analysis used for a software development, LG performed the G+C content analyses and the gene order comparison of *tra*units by the gene order breakpoint analysis. CAR established the correlation existing between the tRNA genes and the GIs according to taxonomy and coordinated the team work. GG wrote the first draft of the paper. All authors improved the manuscript and approved its final version. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Supplementary table. Results of BLAST \[[@B35],[@B36]\] analyses of 100 ORFs present in the 100-kb region. BLAST analyses were performed using BLOSUM62 matrix and gap penalties of 11 and 1. Chromosome location of each ORF, its G+C content, coding strand, and the presence of at least one homolog in *Chlamydiaceae*are presented. Direct repeats (DR), *gly-tRNA*genes and limits of each modules of the GI are highlighted. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Supplementary figure. Phylogenetic analyses suggest that the UWE25 *tra*unit is phylogenetically closely related to F-like DNA conjugative *tra*operons: (A) UPGMA-, (B) Neighbor-joining-, and (C) minimum evolution-trees comparing p-distances and Kimura corrected *p*-distances of nucleotide sequences of the concatenated *traA*, *traK*, *traB*, *traV*, and *traC*genes (the UPGMA tree comparing the Kimura corrected *p*-distances of *tra*genes, shown in Figure [3B](#F3){ref-type="fig"}, is presented here to facilitate comparison with the other trees). Interestingly, in neighbor-joining and minimum evolution analyses of the *p*-distances, the *tra*unit of UWE25 is clustered with *tra*operons of gamma-proteobacterial F-like plasmids: bootstrap values of 96% and 92%, respectively, support the node separating the concatenated *tra*genes of UWE25 and RTS1, SXT, R391, three gamma-proteobacterial F-like conjugative plasmids, from their closest relative R27 plasmid. Similarly, in neighbor-joining and minimum evolution analyses of the Kimura corrected *p*-distances, bootstrap values of 94% and 91%, respectively, support the node separating the concatenated *tra*genes of the chromosomal UWE25 and the R27 plasmid, another gamma-proteobacterial F-like conjugative plasmid, from those of all other plasmids. ::: ::: {.caption} ###### Click here for file ::: Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The genomic island (GI) present in the chromosome of the endosymbiont UWE25. **(A)**Position of the GI on the UWE25 genome, a 100-kb region (grey area) delimited by two direct repeats (Table 1) at both ends and by two *gly-tRNA*s genes in tandem (all tRNAs genes are represented by \'+\') at its proximal end. A third copy of the direct repeat (Table 1) is indicated by a white line disrupting the grey area. The region is characterized by a different slope in the cumulative GC skew analysis (black curve) and by a higher G+C content (grey curve, windows of 20 kb, 0.1-kb step). The horizontal line indicates the genomic G+C content average. **(B)**Closer view of the 100-kb region (black curve, cumulative GC skew; grey curve, G+C content windows of 5 kb, 0.1-kb step; horizontal line, average genomic G+C content). **(C)**Residual cumulative G+C content (GC\') and genomic features of the 100-kb GI. This region encompasses the region with the highest G+C content in the 20-kb windows analysis of the UWE25 genome. The position of genes is represented by an \'X\' on the upper line if encoded on the positive strand, otherwise by an \'X\' on the bottom line (For details, see Table in Supplementary Material 1). A large majority of genes are co-oriented in the genome region flanked by the direct repeats. The *tra*operon (thick line), present on this GI, exhibits a G+C content (40.0%) clearly higher than that of the whole genome (34.7%). The positions of transposases (open circles) and of phage-related genes (full circles) are indicated. ::: ![](1471-2180-4-48-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Comparison of the *tra*unit of the endosymbiont UWE25 with similar operons of pNL1 (*Novosphingobium aromaticivorans*), F (*Escherichia coli*), R391 (*Providencia rettgeri*) and R27 (*Salmonella*Typhi) plasmids. In the upper part of the figure: the G+C content of the UWE25 chromosome, around the 1.71 Mb location (1-kb sliding window average, 0.1-kb step). The horizontal line represents the genomic G+C content average. Only the ORFs composed of more than hundred amino-acids are presented on genetic maps of *tra*units/operons by arrows according to their transcription direction (adapted from Lawley *et al*. \[20\]). Colors or patterns are used to indicate *tra*gene homologs. White genes represent non-conserved transfer genes. Upper case letters refer to the corresponding *tra*genes, whereas lower case letters f and c stand for *trsF*and *trbC*, respectively. Double slashes indicate non-contiguous regions. Interestingly, the G+C-rich genes encoded by the UWE25 chromosome correspond to the ORFs presenting *tra*homologs. ::: ![](1471-2180-4-48-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Similarity and phylogenetic analyses of *tra*units showing the close relatedness of the UWE25 *tra*unit with the operons involved in the F-like conjugative systems: **(A)**UPGMA tree of gene order analysis and **(B)**UPGMA tree comparing the Kimura corrected *p*-distances of the concatenated *traA*, *traK*, *traB*, *traV*, and *traC*gene present along the UWE25 *tra*unit and the F-like, I-like and P-like plasmids \[20\]. The bar represents estimated evolutionary distance scale. The numbers at each node are the results of a bootstrap analysis; each value is derived from 100 samples. ::: ![](1471-2180-4-48-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Description of main features of the parachlamydial 100-kb genomic island. Chromosome location of direct repeats, tRNA genes, *tra*operon, transposases, bacteriophage-related proteins and proteins involved in DNA metabolism is listed below. ::: Protein number^a^ Position^a^ ----------------------------------------------------------- ------------------- ------------------ Direct repeat \- 1648147--1648157 *gly-tRNA*^a,b^ \- 1648172--1648243 *gly-tRNA*^a,b^ \- 1648332--1648403 Transposase^c^ pc1402 1679924--1680400 Phage-related protein^c^ pc1404 1681569--1682441 Putative transcriptional regulator^c,d^ pc1405 1679924--1680400 Phage-related protein^c^ pc1410 1685329--1686447 Putative transposase^c^ pc1419 1695418--1696245 *tra*operon^e^ pc1420-1441 1696410--1716241 Transposases^e^ pc1426-1427 1700887--1701896 Putative DNA-binding protein^c^ pc1443 1716648--1717004 Phage-related protein^c^ pc1444 1717137--1717400 Direct repeat \- 1723093--1723103 Putative ATPase involved in DNA repair^c^ pc1451 1723169--1723504 Probable Doc (death on cure) protein, bacteriophage P1^a^ pc1456 1732622--1732999 Putative DNA-binding protein pc1461 1735745--1736065 Putative transposase^c^ pc1465 1740371--1741198 Probable DNA double-strand break repair ATPase pc1467 1742079--1744181 Putative transposase pc1468 1744634--1745023 Probable resolvase^a^ pc1469 1745398--1745955 Probable transposases, partial length^a^ pc1470-1471 1745807--1746692 Probable Doc (death on cure) protein, bacteriophage P1^a^ pc1473 1747135--1747512 Phage-related protein^c^ pc1474 1747618--1747809 Direct repeat \- 1747915--1747925 ^a^, according to Horn *et al*. \[14\]; ^b^, positive strand (cooriented as the majority of the genes of the GI); *gly-tRNA*s are separated by 88 nt; ^c^, identified by BLAST \[35; 36\] and CLUSTALW \[39\] by ourselves; ^d^, phage-related protein based on additional BLAST hit; ^e^, partially annotated by Horn *et al*. \[14\] and further characterized by ourselves by BLAST \[35, 36\]. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The genomic island of the UWE25 endosymbiont presents seven different modules. The limit of each module was determined by residual cumulative G+C content analysis. ::: Modules 1^st^mod. 2^nd^mod. 3^rd^mod. 4^th^mod. 5^th^mod. 6^th^mod. 7^th^mod. --------------------------------------- ----------- ----------- ----------- ----------- ----------- ----------- ----------- Length 32 kb 16 kb 19 kb 10 kb 6 kb 12 kb 2 kb Mean G+C content (%) 36.4%^a^ 34.1% 40.9% 33.4% 41.8% 33.3% 38.7% Number of ORFs 28 18 21 13 1 13 6 Number of genes having an homolog 16 (57%) 5 (28%) 16 (85%) 7 (54%) 1 (100%) 5 (38%) 5 (83%) Number of genes having no homolog 12 13 5 6 0 8 1 Best hits with homologs in: \- chlamydiae 12 0 0 0 0 0 0 \- cyanobacteria 2 2 0 3 0 2 0 \- plants^b^ 0 0 0 0 0 0 0 \- α-proteobacteria 0 0 6 0 0 0 0 \- β-proteobacteria 1 0 3 1 0 0 0 \- γ-proteobacteria 0 1 6 2 0 0 4^c^ \- Bacteroidetes group 0 0 1 0 0 2 1 \- others 1 2 0 1 1 1 0 Homologous^d^to: \- phage-related protein 0 3 0 1 0 1 2 \- putative transposase 1 1 2 0 0 2^e^ 2 \- resolvase 0 0 0 0 0 0 1 \- protein involved in DNA metabolism 0 1 0 2 0 2 0 ^a^the G+C content is similar to that of 36.1% of the remaining genome (calculated on 1931 ORFs); ^b^5% of the 2031 ORFs of the genome of UWE25 have products homologous to plant proteins, but no ORFs of the GI were homologous to plant counterparts; ^c^two ORFs which presented best homologs encoded by a plasmid of an uncultured bacteria present in activated sludge have a second best BLAST hit encoded by a gamma-proteobacterial ORFs (*Pseudomonas*sp.); ^d^not only the best BLAST hit is taking into consideration to determine the putative function encoded by the ORF; ^e^one of them has an e-value above 0.001. :::

PubMed Central

2024-06-05T03:55:52.528953

2004-12-22

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548262/", "journal": "BMC Microbiol. 2004 Dec 22; 4:48", "authors": [ { "first": "Gilbert", "last": "Greub" }, { "first": "François", "last": "Collyn" }, { "first": "Lionel", "last": "Guy" }, { "first": "Claude-Alain", "last": "Roten" } ] }

PMC548263

Background ========== Alpha1-adrenergic receptors (α~1~-ARs) are members of the G-protein-coupled receptor superfamily. Both pharmacological and molecular cloning studies have indicated the existence of multiple subtypes of α~1~-ARs including α~1A/C~-AR, α~1B~-AR, and α~1D~-AR \[[@B1]-[@B4]\]. α~1~-ARs play a key role in a variety of physiological processes, such as contraction of vascular and cardiac muscle, contraction of the spleen, liver glycogenesis, or melatonin secretion in the pineal gland \[[@B3],[@B4]\]. Activation of α~1A~-AR promotes hypertrophy of cardiac myocytes \[[@B5],[@B6]\]. Recently it has been shown that all three subtypes of α~1~-AR are also expressed in rat aortic adventitial fibroblasts and vascular smooth muscle cells (SMC) \[[@B7]\] and their activation with norepinephrine stimulates migration, proliferation and protein synthesis \[[@B8],[@B9]\]. However, norepinephrine increased SMC hypertrophy, but not DNA synthesis, through α~1A~-AR stimulation in uninjured aorta whereas norepinephrine stimulated proliferation of adventitial fibroblasts through the α~1B~-AR subtype \[[@B8]\]. Nonvascular fibroblasts, including cardiac fibroblasts \[[@B7],[@B10]\], generally do not express α~1~-AR and have been used for stable transfection of different subtypes of α~1~-AR to study their respective functions. However, a recent study showed the expression of a functional α~1A~-AR in primary tendon fibroblasts \[[@B11]\]. In rat-1 cells, a transformed cell line from embryonic fibroblast, expressing different subtypes of α~1~-AR, phenylephrine (PE), an α~1~-AR agonist, activates phospholipase D and releases arachidonic acid \[[@B12]\]. However, unlike SMC, activation of α~1~-ARs in rat-1 cells also increases cAMP levels and PKA activity \[[@B12]\]. α~1A~-AR is more efficiently coupled to phospholipase D activation, arachidonic acid release and cAMP than α~1B~-AR or α~1D~-AR in these cells \[[@B12]\]. Activation of α~1A~-AR expressed in COS-7 and HeLa cells \[[@B13]\] and α~1B~-AR or α~1D~-AR in COS and CHO cells \[[@B14]\] also increase cAMP levels. In HepG2 and M12 cells expressing α~1B~-AR, PE causes cell scattering and inhibits proliferation through activation of MAP kinases \[[@B15]\]. The family of connective tissue cells includes fibroblasts, cartilage cells, bone cells, fat cells and smooth muscle cells. Fibroblasts seem to be able to transform into any of other members of the family -- in some cases reversibly -- although it is not clear whether this is a property of a single type of fibroblast that is pluripotent or of a mixture of distinct types of fibroblasts with more restricted potentials. These transformations of connective tissue cell type are regulated by the composition of the surrounding extracellular matrix, by cell shape, and by hormones and growth factors \[[@B16]\]. Heterologous expression of α~1A~-ARs in CHO cells inhibits basal and growth factor-stimulated DNA synthesis, in contrast to the α~1D~-AR \[[@B17]\]. A recent study in the same model has reported cAMP as the mediator of the antiproliferative effect of α~1A~-AR stimulation \[[@B18]\]. Therefore, it is possible that activation of α~1A~-AR with PE in rat-1 cells affects their growth and/or differentiation status. To test this hypothesis, we have investigated the effect of PE and cAMP modulators on proliferation, growth and morphology in rat-1 cells expressing α~1A~-ARs. Moreover, we have examined the effect of PE and cAMP modulators on the expression of cell cycle regulators and muscle cell markers, because of the ability of fibroblasts to differentiate into myofibroblasts. Our results show that activation of α~1A~-ARs in rat-1 cells exerts profound effects promoting hypertrophy and expression of specific smooth muscle cell markers. We also show here that α~1A~-AR-induced cessation of DNA synthesis is independent of cAMP and involves the expression of cyclin-dependent kinase (Cdk) inhibitor, p27^kip1^. Results ======= Stimulation of α~1A~-AR inhibits DNA synthesis at the G~1~/S checkpoint of the cell cycle in rat-1 fibroblasts -------------------------------------------------------------------------------------------------------------- Rat-1 fibroblasts stably transfected with α~1A~-AR expressed 288 ± 2 fmol/mg protein of receptors \[[@B12]\]. Cells at 80% confluency were serum-deprived for 48 h in DMEM and then incubated with PE (2, 5 and 10 μM) for different periods prior to the addition of \[^3^H\]thymidine. PE decreased basal \[^3^H\]thymidine incorporation in a concentration-dependent manner with a maximum effect at 10 μM PE (Fig [1A](#F1){ref-type="fig"}). Reduction of basal DNA synthesis was significant after 12 h of treatment with a maximum effect at 18 h of incubation with PE (\> 65% decrease) (Fig [1B](#F1){ref-type="fig"}). Rat-1 fibroblasts stably transfected with α~1A~-AR incorporate \[^3^H\]thymidine in DNA in a time-dependent manner (from 6 to 24 h), even after 48 h of serum-deprivation (Fig [1B](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Effect of PE on \[^3^H\]thymidine incorporation in rat-1 cells. Cells were grown in 24-well plates in DMEM containing 10% FBS at a density of 150,000 cells/well until they reached 80--90% confluency, and then serum-deprived for 48 h in DMEM. A, Cells were treated with different concentrations of PE (2, 5, and 10 μM) for 18 h prior to the addition of \[^3^H\]thymidine (0.50 μCi/ml) for the last 4 h. B, Cells were treated with PE for different incubation periods prior to the addition of \[^3^H\]thymidine (0.50 μCi/ml). Cells were incubated with \[^3^H\]thymidine for the last 4 h and thymidine incorporation was determined as described in Methods. Data are expressed as dpm of \[^3^H\]thymidine incorporated in DNA per well. Values are the mean ± S.E. of six independent experiments performed in quadruplicates on different batches of cells. \* Values significantly different from vehicle, p \< .05. ::: ![](1471-2121-5-47-1) ::: To determine whether PE at different concentrations delays the re-entry of rat-1 cells into the cell cycle, we examined the kinetics of cell cycle re-entry. Flow cytometry studies showed that incubation of the rat-1 cells with 2, 5, and 10 μM PE resulted in inhibition of the re-entry of 83, 81, or 85%, respectively, of the cell population into the S phase of the cell cycle (Table [1](#T1){ref-type="table"}). Therefore, stimulation of α~1A~-AR inhibits DNA synthesis at the G~1~/S checkpoint in rat-1 fibroblasts. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of PE on cell cycle phases. ::: **Treatment** **G~0~/G~1~phase** **S phase** **G~2~/M phase** **% inhibition (S)** --------------- -------------------- -------------- ------------------ ---------------------- vehicle 9269 ± 0.5 319.5 ± 13.5 238.5 ± 23.5 0 % 2 μM PE 9770 ± 9.0 54.0 ± 2.0 125 ± 21.0 83% 5 μM PE 9699 ± 29.0 59.0 ± 4.0 170 ± 11.0 81% 10 μM PE 9761.5 ± 89.5 47.5 ± 13.5 110.5 ± 39.5 85% Cells were subjected to flow cytometric DNA analysis as described in Methods. Different concentrations of PE inhibited the re-entry of the cell population into S phase of the cell cycle. Data show the number of cells in each phase of the cell cycle. Values are the mean ± S.E. of three independent experiments performed in duplicate on different batches of cells (Total events per cycle is 10,000 cells). ::: Stimulation of α~1A~-AR increases protein synthesis and promotes hypertrophy in rat-1 fibroblasts ------------------------------------------------------------------------------------------------- Stimulation of α~1~-ARs in vascular smooth muscle cells \[[@B19]\] and in adult rat ventricular myocytes \[[@B8]\] increases hypertrophy and protein synthesis. However, the α~1~-AR subtype mediating these effects is not known. Exposure to 5 μM PE for 18 h increased \[^3^H\]leucine incorporation by 64% (Fig [2B](#F2){ref-type="fig"}) and decreased basal \[^3^H\]thymidine uptake (Fig [2A](#F2){ref-type="fig"}), as shown earlier in Fig [1](#F1){ref-type="fig"}. These effects were blocked by prazosin (PRZ, 100 nM), an α~1~-AR antagonist. Propranolol (2 μM), a β-AR antagonist (Fig [2A,B](#F2){ref-type="fig"}), or yohimbine (1 μM), an α~2~-AR antagonist (data not shown), had no effect on the decrease in basal \[^3^H\]thymidine or the increase in \[^3^H\]leucine incorporation in rat-1 cells caused by exposure to PE. These results show that PE stimulates protein synthesis and inhibits DNA synthesis in rat-1 cells through activation of α~1A~-ARs. The hypertrophy index, an indicator of cellular hypertrophy, is defined as the ratio of protein synthesis (\[^3^H\]leucine incorporation) to DNA synthesis (\[^3^H\]thymidine incorporation) \[[@B20]\]. At 18 h, this ratio was 4.4 fold as high in PE-treated cells (index = 0.22) than in control cells (index = 0.05, n = 6), indicating that PE promotes rat-1 cell hypertrophy. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Stimulation of α~1A~-AR mediates the effect of PE on DNA and protein synthesis.**A, Effect of prazosin (α~1~-AR antagonist) and propranolol (β-AR antagonist) on PE-induced inhibition of DNA synthesis. Cells were treated with prazosin and/or propranolol for 30 min before the addition of 5 μM PE for 18 h. \[^3^H\]thymidine incorporation was measured as described in Methods. Data are expressed as dpm of \[^3^H\]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in quadruplicates on different batches of cells. \* Value significantly different from the corresponding value obtained without PE treatment, p \< .05. B, Effect of prazosin and propranolol on PE-induced increase in protein synthesis. Experimental conditions were similar as described in A. \[^3^H\]leucine incorporation was measured as described in Methods. Data are expressed as dpm of \[^3^H\]leucine incorporated per well. Values are the mean ± S.E of three independent experiments performed in quadruplicates on different batches of cells. \* Value significantly different from the corresponding value obtained without PE treatment, p \< .05. ::: ![](1471-2121-5-47-2) ::: Protein synthesis elicited by α~1~-AR stimulation was time-dependent and remained significant from 6 to 72 h of exposure with PE (Fig [3A](#F3){ref-type="fig"}). EGF and cAMP have opposite effects on rat-1 fibroblasts proliferation \[[@B21],[@B22]\]. Therefore, we tested the effect of EGF and forskolin (FN), an activator of adenylyl cyclase \[[@B12]\], on protein and DNA synthesis. EGF (30 ng/ml) slightly increased, whereas FN decreased, \[^3^H\]leucine incorporation at 6, 18, 48 and 72 h (Fig [3A](#F3){ref-type="fig"}). Surprisingly, PE was a better activator of protein synthesis than EGF. In contrast, EGF increased \[^3^H\]thymidine incorporation by 272% (Fig [3B](#F3){ref-type="fig"}), an effect blocked by FN and the cAMP analog, 8-cpt-cAMP, as well as by PE. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Effect of PE, cAMP-elevating agents and EGF on protein and DNA synthesis**. A, Cells were treated with 5 μM PE, 1 μM forskolin (FN) and 30 ng/ml EGF for different periods. \[^3^H\]leucine incorporation was measured as described in Methods. Data are expressed as dpm of \[^3^H\]leucine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in sextuplicates. \* Values significantly different from vehicle, p \< .05. B, Effect of FN, 8-cpt-cAMP and PE on EGF-induced \[^3^H\]thymidine incorporation. Cells were preincubated with FN (1 μM), 8-cpt-cAMP (5 μM) and PE (5 μM) for 1 h. Cells were then treated with EGF (30 ng/ml) for 18 h. Incorporation of \[^3^H\]thymidine into rat-1 fibroblasts was determined as described in Methods. Data are expressed as dpm of \[^3^H\]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in duplicate in different batches of cells. \* Value significantly different from vehicle, p \< .05. § Value significantly different from the corresponding value obtained without treatment by FN, cpt-cAMP or PE, p \< .05. ::: ![](1471-2121-5-47-3) ::: PE inhibits DNA synthesis and promotes hypertrophy by a mechanism independent of cAMP ------------------------------------------------------------------------------------- Previously, we have shown that PE induces cAMP accumulation in rat-1 cells \[[@B12]\]. It is also known that cAMP-dependent PKA-mediated inhibition of cell division is due to blockade of growth factor-stimulated cell cycle progression from G~1~to S phase \[[@B23]\]. Therefore, to determine whether cAMP mediates the antiproliferative effect of PE, we examined the effect of FN (0.1--10 μM) and 8-cpt-cAMP (5--20 μM) and of FN and PE on DNA synthesis in the presence and absence of adenylyl cyclase and PKA inhibitors. FN and 8-cpt-cAMP inhibited \[^3^H\]thymidine incorporation to the same extent as PE (Fig [4A](#F4){ref-type="fig"}). The FN but not the PE-induced decrease in \[^3^H\]thymidine incorporation was blunted by the selective inhibitor of adenylyl cyclase SQ 22536 (Fig [4B](#F4){ref-type="fig"}) and by the P-site inhibitors of adenylyl cyclase 2\',5\'dideoxyadenosine, and 2\'-deoxyadenosine 3\'-monophosphate (5 μM each-data not shown). The selectivity of cAMP modulators, FN, 8-cpt-cAMP and SQ 22536 has been tested previously in rat-1 fibroblasts \[[@B12]\]. To further determine the contribution of cAMP pathway, rat-1 cells were transfected with πLXX-PKI \[[@B1]-[@B31]\], a vector that expresses the PKA inhibitor, PKI \[[@B23],[@B24]\]. Transfection with PKI reversed the inhibitory effect of FN, but not that of PE, on \[^3^H\]thymidine incorporation in rat-1 cells (Fig [4C](#F4){ref-type="fig"}). These observations indicate that cAMP and PKA do not mediate the PE-induced inhibition of DNA synthesis. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Effect of PE and cAMP-elevating agents, FN and 8-cpt-cAMP, on DNA synthesis**. Cells were serum-deprived for 2 days. A, Cells were treated with PE (5 μM), FN (0.1, 1, and 10 μM), and 8-cpt-cAMP (5, 10, and 20 μM) for 18 h. Incorporation of \[^3^H\]thymidine into rat-1 fibroblasts was determined as described in Methods. Data are expressed as dpm of \[^3^H\]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed in sextuplicates in different batches of cells. \* Value significantly different from vehicle, p \< .05. B, Effect of inhibitors of adenylyl cyclase inhibition on PE and cAMP-elevating agents. Cells were pre-incubated with SQ22536 (25 μM), an specific adenylyl cyclase inhibitor for 1 h. Cells were then treated with 5 μM PE and/or 1 μM FN for 18 h. \[^3^H\]thymidine incorporation was determined as described in Methods. Values are the mean ± S.E. of three independent experiments performed in quadruplicates on different batches of cells. Data are expressed as dpm of \[^3^H\]thymidine incorporated per well. \* Value significantly different from the corresponding vehicle, p \< .05. § value significantly different from that obtained in the presence of agonist alone, p \< .05. C, Effect of transfection of πLXX-PKI \[1-31\] on PE-induced inhibition of DNA synthesis. Preconfluent cells were transiently transfected with πLXX-PKI \[1-31\], a plasmid encoding for the protein kinase inhibitor, (PKI), using Lipofectamine Plus transfection reagent according to the manufacturer\'s protocol (Life Technologies). Cells were cultured in serum-free DMEM for 24 h and then treated with 5 μM PE and/or 1 μM FN. Cells were assayed for \[^3^H\]thymidine incorporation 48 h post transfection as described in Methods. Data are expressed as dpm of \[^3^H\]thymidine incorporated per well. Values are the mean ± S.E. of three independent experiments performed on different batches of cells. \* Value significantly different from the corresponding vehicle, p \< .05. § value significantly different from that obtained in the presence of agonist alone, p \< .05. ::: ![](1471-2121-5-47-4) ::: The antiproliferative effect of PE, FN and 8-cpt-cAMP could be the result of cell loss due to a cytotoxicity. However, the trypan blue exclusion test and LDH assay showed that more than 90% of cells were viable (Table [2](#T2){ref-type="table"}) and less than 1% of the cells were found in the supernatant, indicating very little cell loss. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Effect of PE and cAMP elevating agents on viability of rat-1 fibroblasts using the hemocytometer trypan blue method. ::: **Treatment** **Total number of cells** **Blue cells** **% of viability** ------------------ --------------------------- ---------------- -------------------- Vehicle 78 8 90% 5 μM PE 142 10 92% 10 μM PE 87 12 86% 10 μM FN 78 8 90% 20 μM 8-cpt-cAMP 84 4 96% 20 μM 8-Br-cAMP 66 2 97% Cells were incubated for 18 h with PE (5 and 10 μM), forskolin (FN), 8-cpt-cAMP and 8-Br-cAMP. The percentage of viable cells was calculated. Values are representative of five different experiments. ::: Morphological change elicited by α~1A~-AR stimulation and by cAMP in rat-1 cells -------------------------------------------------------------------------------- Although both PE and FN inhibited DNA synthesis in rat-1 cells, they produced different effects on cell morphology. PE increased, whereas FN decreased, the apparent size of rat-1 cells as shown in Fig [5](#F5){ref-type="fig"}. Prazosin (100 nM), an α~1~-AR antagonist, reversed the shape change produced by PE. To determine if PE-induced cell cycle arrest and hypertrophy of rat-1 fibroblasts is also independent of cAMP, we preincubated the cells with SQ22536 (25 μM) and then treated them with PE (5 μM) or FN (1 μM) for different time periods. SQ22536 reversed the morphological changes produced by FN and restored the normal fibroblast shape, whereas it enhanced the effect of PE, i.e. the cells became more hypertrophic. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Effect of prazosin and SQ22536 on PE-and FN-induced morphological change in rat-1 cells**. Preconfluent, serum-deprived cells were either stimulated with 5 μM PE and/or 1 μM FN for different periods of time (Fig 5B) and/or pretreated for 30 min with 100 nM prazosin (Fig 5A) or 25 μM SQ22536 (Fig 5C), and then further incubated with 5 μM PE and/or 1 μM FN for 24 h. Morphological studies were performed as described in Methods. The same morphological patterns were obtained in three different experiments. ::: ![](1471-2121-5-47-5) ::: p27^kip1^mediates PE-induced inhibition of DNA synthesis -------------------------------------------------------- Cyclin-dependent kinase (cdk) inhibitors p27^kip1^and p21^cip1/waf1^play an important role in cell-cycle regulation \[[@B25],[@B26]\]. High levels of p27^kip1^and p21^cip1/waf1^in quiescent (G~0~) cells decline upon induction of mitogenesis \[[@B27]\]. This decrease in Cdk inhibitors appears to be critical in enabling cell cycle entry. Western blot analysis showed that α~1A~-AR stimulation increased the expression levels of p27^kip1^(642% of control at 48 h) and p21^cip1/waf1^(935% of control at 48 h) (Fig. [6A](#F6){ref-type="fig"}). The protein levels remained elevated for 48 h, with levels of p27^kip1^reaching 389% of control at 18 h of incubation and remaining elevated for up to 48 h. This increase was concomitant with the inhibitory effect of PE on DNA synthesis, which was also maximal at 18 h of incubation. PE increased the expression of p27^kip1^(552% of control at 24 h) more efficiently than p21^cip1/waf1^(87% of control at 24 h) suggesting a greater contribution of p27^kip1^to PE-induced cell cycle arrest and a delayed induction of p21^cip1/waf1^. Retinoblastoma protein (pRb) hyperphosphorylation and subsequent release of E2F is required for cell cycle progression \[[@B28]\]. However, α-AR stimulation of neonatal myocytes has been shown to induce pRb hyperphosphorylation without induction of cell proliferation \[[@B29]\]. Our results demonstrate that PE promotes an early increase in pRb levels (607% of control at 6 h) in rat-1 cells (Fig [6C](#F6){ref-type="fig"}), associated with a decrease in DNA synthesis (Fig [1B](#F1){ref-type="fig"}). The nuclear envelope proteins lamin A and C, used as control, were not altered by PE treatment (Fig [6D](#F6){ref-type="fig"}). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Effect of PE on the expression of cell cycle regulators, p27^kip1^, p21^ip1/waf1^and pRB**. Preconfluent, serum-deprived cells were incubated with PE (5 μM) or vehicle for up to 48 h. Expression of cell cycle regulators was determined by Western blot analysis of whole cell lysates as described in Methods. The panels are representative Western blots showing the effect of PE on the expression of p27^kip1^(A), p21^cip1/waf1^(B), pRb (C), and lamin A/C (D) protein levels respectively. Percentages representing the quantitation of protein bands by densitometric analysis of blots obtained from three different experiments are indicated on top of each immunoblot. ::: ![](1471-2121-5-47-6) ::: In VSMC, p27^kip1^level is a critical determinant of the cell response, hyperplasia vs. hypertrophy, to angiotensin II and other growth factors \[[@B20]\]. We postulated that the response of rat-1 cells to PE was mediated by p27^kip1^. PE upregulates the expression of p27^kip1^at 18 h of treatment in rat-1 cells at all concentrations (Fig [7A](#F7){ref-type="fig"}). We therefore explored further the role of p27^kip1^in modulating the antiproliferative response of rat-1 cells to PE by transfecting these cells with p27^kip1^antisense and sense ODN. p27^kip1^antisense (102% of control), but not sense ODN (347% of vehicle vs. 338% for PE alone), diminished p27^kip1^level (Fig [7B](#F7){ref-type="fig"}) and blocked the antiproliferative effect of PE in rat-1 cells (Fig [7C](#F7){ref-type="fig"}). These observations suggest that PE-induced inhibition of DNA synthesis is mediated by p27^kip1^. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Effect of PE on p27^kip1^level and effect of p27^kip1^antisense oligonucleotides on PE-induced inhibition of DNA synthesis**. A, Preconfluent rat-1 cells were stimulated with 2, 5, and 10 μM PE for 18 h. Total cell lysates were subjected to Western blot analysis as described in Methods. Percentages representing the quantitation of protein bands by densitometric analysis of blots obtained from three different experiments are indicated on top of each immunoblot. B, Representative Western blot showing the effect of p27^kip1^antisense and sense oligonucleotides treatment on PE-induced increase in p27^kip1^protein level. Cells were transiently transfected with p27^kip^antisense or sense oligonucleotide for 48 h using Oligofectamine™ Reagent according to the manufacturer\'s instruction (Invitrogen). C, Transfected cells as in B were serum-starved for 24 h and then treated with 5 μM PE. Thymidine uptake was measured as described in Methods 48 h post-transfection. Data are expressed as the percent decrease in \[^3^H\]thymidine uptake above the basal value obtained in unstimulated cells. Values are the mean ± S.E of three independent experiments performed in different batches of cells. \* Value significantly different from the corresponding vehicle, p \< .05. ::: ![](1471-2121-5-47-7) ::: PE increases the expression of cyclin D1, proliferating cell nuclear antigen (PCNA), Cdk2 and Cdk4 in rat-1 cells ----------------------------------------------------------------------------------------------------------------- We next investigated the effect of PE on several cell cycle proteins important for G~1~/S phase progression. Fig. [8](#F8){ref-type="fig"} shows that treatment of serum-deprived rat-1 cells with PE increased the levels of cyclin D1 (548% of control at 24 h), PCNA (158% of control at 24 h), and Cdk2 (236% of control at 24 h) in a time-dependent manner. The levels of nuclear envelope proteins lamin A and C, were not altered. These results are unexpected since PE promotes cell cycle arrest at G~1~/S checkpoint (Table [1](#T1){ref-type="table"}). However, the up-regulation of p27^kip1^seems to be sufficient to block cell cycle entry into S phase and DNA synthesis. Surprisingly, we also observed an increase in the protein levels of Cdk4 (280% of control at 24 h), which normally do not change even during mitogenic stimulation. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **Effect of PE on the expression of cyclin D1, PCNA Cdk 2, and Cdk 4**. Preconfluent, serum-deprived cells were stimulated with PE (5 μM) for 6, 12, 24 and 48 h. Cell-cycle regulators expression was determined by Western blot analysis of whole cell lysates as described in Methods. The bar graph represents quantitation of protein bands by densitometric analysis of blots obtained from three different experiments. \* Value significantly different from the corresponding vehicle, p \< .05. ::: ![](1471-2121-5-47-8) ::: PE increases expression of specific smooth muscle differentiation markers as well as MyoD ----------------------------------------------------------------------------------------- Cell cycle arrest is closely coupled to muscle cell differentiation and is required for activation of muscle cell specific gene expression \[[@B16]\]. Since stimulation of α~1A~ARs promoted a change in morphology associated with hypertrophy, we examined the effect of PE on expression of the specific smooth muscle cell markers, α-smooth muscle actin (SMα actin), caldesmon and myosin heavy chain (SM-MHC) \[[@B30]\]. The smooth muscle markers were not expressed in serum-deprived rat-1 cells (Fig [9A,C](#F9){ref-type="fig"}). Stimulation of α~1A~ARs with PE (5 μM) promoted a time-dependent increase in the levels of SMα actin (695% of control at 24 h) (Fig [9A](#F9){ref-type="fig"}), caldesmon (284% of control at 24 h) (Fig [9B](#F9){ref-type="fig"}) and SM-MHC (445% of control at 24 h) (Fig [9C](#F9){ref-type="fig"}) as shown by Western blot analysis We also investigated the expression of MyoD, a helix-loop-helix protein that plays an important role in the regulation of muscle development \[[@B31]\]. Western blot analysis showed that PE also increased the expression of MyoD in a time-dependent manner (457% of control at 24 h), further supporting the evidence that stimulation of α~1A~AR induces differentiation of rat-1 fibroblasts into muscle-like cells, which is closely coordinated with cell cycle arrest (Fig [9D](#F9){ref-type="fig"}). The expression of vimentin, an intermediate filament protein used as a control, was not altered by treatment with PE (Fig [9E](#F9){ref-type="fig"}). The α~1A~AR stimulation of smooth muscle differentiation markers was independent of cAMP because FN did not increase the expression of SMα actin, caldesmon or SM-MHC (Fig [10A,C](#F10){ref-type="fig"}). MyoD expression was not altered by FN (Fig [10D](#F10){ref-type="fig"}). The level of vimentin expression was not altered by PE or FN (Bar Graph). Together, these data demonstrate that stimulation of α~1A~ARs increases expression of smooth muscle markers and MyoD in rat-1 cells, independently of cAMP. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### **Time course of the effect of PE on the expression of the smooth muscle differentiation markers and MyoD**. Preconfluent, serum-deprived fibroblasts were stimulated with PE (5 μM) for up to 48 h. The protein expression of smooth muscle differentiation markers and MyoD was determined by Western blot analysis of whole cell lysates as described in Methods. A, smooth muscle α actin (SMα actin). B, caldesmon. C, smooth muscle myosin heavy chain (SM-MHC). D, MyoD, E, vimentin. VSMC lysate is shown as a positive control. Percentages representing the quantitation of protein bands by densitometric analysis of blots obtained from three different experiments are indicated on top of each immunoblot. ::: ![](1471-2121-5-47-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### **Effect of PE and FN on the expression of smooth muscle differentiation markers and MyoD.**Preconfluent, serum-deprived fibroblasts were stimulated with either PE (5 μM) or FN (1 μM) for up to 48 h. Expression of markers was determined by Western blot analysis of whole cell lysates as described in Methods. A, smooth muscle α actin (SMα actin). B, caldesmon. C, smooth muscle myosin heavy chain (SM-MHC). D, MyoD. VSMC lysate is shown as a positive control in panel A, B, C and D. The bar graph represents quantitation of protein bands by densitometric analysis of blots obtained from three different experiments. \* Value significantly higher than the corresponding vehicle, p \< .05. ::: ![](1471-2121-5-47-10) ::: Discussion ========== This study is the first demonstration of the ability of an α~1A~-AR subtype to promote differentiation of a fibroblast into a SMC/myocyte-like cell. Further, it demonstrates that α~1A~-AR-induced cell cycle arrest and hypertrophy as well as differentiation is mediated through a mechanism dependent upon the increased expression of the critical cell cycle protein p27^kip1^and selective smooth muscle markers. Stimulation of α~1A~-ARs promotes hypertrophy of cardiac myocytes \[[@B6]\]. Norepinephrine increases SMC hypertrophy, but not cell proliferation, through α~1A~-AR stimulation in uninjured aorta \[[@B8]\]. In HepG2 and M12 cells transfected with α~1A~-AR, PE stimulates cell scattering and inhibition of proliferation \[[@B15]\]. Similar observations have been made in CHO cells expressing α~1A~-ARs \[[@B17],[@B18]\]. In the present study, PE inhibited cell proliferation, promoted hypertrophy and differentiation through stimulation of α~1A~-ARs in rat-1 cells expressing this subtype. Analysis of the effect of α~1A~-AR stimulation on cell cycle progression revealed that it inhibits the re-entry of cells from G~0~/G~1~into the S phase of the cell cycle. Next we attempted to define the mechanism mediating α~1A~-AR-induced cell cycle arrest, hypertrophy and differentiation of rat-1 cells. Previous studies have shown that in rat 1-cells expressing α~1A~-ARs, PE promotes increase in cAMP accumulation and PKA activation \[[@B12]\]. Since cAMP has been reported to inhibit growth in various cell types, including cells of fibroblastic origin \[[@B32]\], it seemed possible that cAMP mediates the inhibitory effect of PE on rat-1 cells proliferation. Stable cAMP analogs or adenylyl cyclase activation promoted cell cycle arrest similar to α~1A~-AR stimulation. However, activation of the cAMP/PKA pathway did not promote hypertrophy or differentiation of rat-1 cells. Moreover, inhibition of cAMP/PKA did not reverse the α~1A~-AR-induced inhibition of DNA synthesis. Therefore, α~1A~-AR-induced cell cycle arrest, hypertrophy and differentiation, is independent of the cAMP/PKA pathway in rat-1 cells. Recently, the cAMP pathway has been implicated in α~1A~-AR-induced cell cycle arrest in CHO cells expressing α~1A~-ARs \[[@B18]\]. The difference between these cell lines may be due to the inhibitory effect of PE on ERK in rat-1 cells \[[@B33]\] vs. PE-induced ERK activation in CHO cells \[[@B18]\]. In addition, the potential effect of α~1A~-AR stimulation on CHO cells hypertrophy and differentiation is not known. During our investigation of the possible mechanism by which PE induces cell cycle arrest, we observed that although both PE and FN inhibited DNA synthesis, these two agents produced remarkably different morphological changes in rat-1 cells. FN promoted a change in rat-1 cells to a small, shrinked, round shaped morphology whereas PE shifted the cells to a hypertrophied, enlarged and spindle shaped smooth muscle-like phenotype. This α~1A~-AR-induced morphological change was again independent of cAMP. Differences between PE and cAMP on different cell functions are reported in Table [3](#T3){ref-type="table"}. EGF increases DNA synthesis in rat-1 fibroblasts \[[@B22]\] and slightly increased protein synthesis (our data). However, EGF was a far less potent hypertrophic agent than PE and did not change the morphology of rat-1 cells. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Comparison of the effects of PE and FN on different cell parameters. ::: **Parameter** **PE** **FN** ------------------------- -------------------------- ----------------- cAMP production ↑ ↑ Protein synthesis ↑ ↓ DNA synthesis ↓ ↓ Morphology enlarged; spindle-shaped shrunk; rounded Differentiation markers ↑ ↓ Cdk inhibitors ↑ ↑ The arrows indicate a significant increase or decrease elicited by PE or FN in the corresponding cell parameter studied. These agents\' stimulated cAMP production, decreased basal DNA synthesis, and up-regulated cyclin-dependent kinase inhibitors. PE and cAMP have opposite effects on protein synthesis, cell morphology and smooth muscle differentiation markers. ::: The different effects of α~1A~-AR stimulation and cAMP on rat-1 cell hypertrophy and morphology led us to explore the effects of PE and FN on different cell parameters associated with morphological changes, i.e. cell cycle regulators and smooth muscle cell differentiation markers. Recently, it has been reported that the Cdk inhibitor p27^kip1^mediates angiotensin II-induced cell cycle arrest and hypertrophy in cultured renal tubular cells \[[@B34]\], and vascular smooth muscle cells \[[@B20]\] and induces intestinal epithelial cell differentiation \[[@B35]\]. In the present study, stimulation of α~1A~-ARs increased the expression of p27^kip1^to a much greater extent than p21^cip1/waf1^, suggesting an important role for the former Cdk inhibitor in the action of α~1A~-AR stimulation. Interestingly, the time of upregulation of p27^kip1^by PE correlated closely with its effect on inhibition of DNA synthesis, which was maximal at 18 h. Depletion of p27^kip1^prevented both PE-induced upregulation of p27^kip1^and reversed PE-induced decrease in DNA synthesis. Therefore, p27^kip1^plays a central role as a mediator of α~1A~-AR-induced inhibition of DNA synthesis and probably hypertrophy and differentiation of rat-1 fibroblasts. An important finding in the present study was that PE produced a change in the morphology of rat-1 cells characterized by an increase in cell size. Supporting this phenotypic and hypertrophic change was our demonstration that PE increased global protein synthesis and the expression of markers specific to smooth muscle cells such as smooth muscle actin, caldesmon, and myosin heavy chain. The protein levels of these smooth muscle cell markers were found to remain elevated for up to 48 h, consistent with the PE-induced morphological change that persisted for the same time period. Although stimulation of α~1A~-ARs shifted rat-1 fibroblasts to SMC/myocyte-like phenotype, it also increased the expression of the helix loop helix protein MyoD, a skeletal muscle-specific regulatory transcription factor \[[@B31]\]. Surprisingly, PE also caused an increase in pRb expression to a level similar to p27^kip1^. MyoD has been shown to interact with pRb and to promote muscle gene activation and cell cycle arrest \[[@B28],[@B36]\]. Indeed, pRb has been found to contain a differentiation-promoting activity that is distinct from its cell-cycle progression functions \[[@B37]\]. More recent evidence has indicated an essential role for pRb in promoting functional synergism between MyoD and MEF2 proteins \[[@B38]\]. Although PE increased the expression of MyoD in our study, the rat-1 cells had a smooth muscle/myocyte-like phenotype. It has been reported that vascular smooth muscle cells can spontaneously adopt the skeletal muscle phenotype \[[@B31]\]. Therefore, it is possible that PE increases expression of MyoD in rat-1 cells during differentiation into myocyte-like cells. However, expression of MyoD is not sufficient for the coordinated program of skeletal myogenesis in smooth muscle cells \[[@B31]\]. PE-induced hypertrophy and differentiation of rat-1 cells was also associated with increased levels of cell cycle proteins pRb, cyclin D1, PCNA and Cdk2 that are important for G~1~/S phase progression \[[@B39]\]. These data indicate that stimulation of α~1A~-ARs promotes the transcriptional/translational activation of the machinery required for G~1~/S cell cycle progression. However, a simultaneous increase in Cdk inhibitors such as p27^kip1^prevented DNA synthesis. Elevation of p27^kip1^protein level alone is sufficient for induction of cell cycle arrest, independent of cyclins or Cdk level \[[@B40]\]. Surprisingly, PE also increased the protein level of Cdk4, which is normally not affected by mitogenic stimuli. Although the Cdk inhibitors bind to cyclin/Cdk complexes and reduce their activity, their interaction is probably much more complex \[[@B25],[@B26]\]. Cdk inhibitors may paradoxically activate these kinases, particularly cyclin D/Cdk4, 6 complexes \[[@B26]\]. The mechanism by which stimulation of the α~1A~-AR promotes the up-regulation of Cdk inhibitors (p27^kip1^, p21^cip1/waf1^), smooth muscle cell markers, MyoD, and G~1~/S transition cell cycle proteins (pRb, cyclin D1, PCNA, Cdk2/4) leading to inhibition of proliferation and stimulation of hypertrophy and differentiation is not known. The cAMP/PKA pathway was excluded by our results as well as the ERK pathway \[[@B33]\]. The phosphatidylinositol 3-kinase and Akt/PKB pathway, a pro-survival/mitogenic and hypertrophic pathway is also unlikely to be involved, because PE does not stimulate this pathway in rat-1 cells \[[@B42]\]. Recently, a study on the genetic profiling of rat-1 fibroblasts expressing different subtypes of α-AR has shown that in cells expressing the α~1A~-AR, epinephrine (one hour stimulation) increased the gene expression of IL-6, gp-130 (an IL-6 high affinity receptor and signal transducer) and STAT-3 (an IL-6 activated transcription factor) \[[@B42]\]. Moreover, in cells expressing α~1A~-adrenergic receptor, epinephrine also increased IL-6 secretion and STAT-3 Ser^727^phosphorylation \[[@B42]\]. Therefore, it is possible that IL-6, gp130, and/or STAT-3 contributes to the upregulation of one or more of the cell cycle associated proteins and smooth muscle cell markers responsible for the cells arrest, hypertrophy and/or differentiation caused by α~1A~-AR activation in rat-1 cells. With regard to functional significance *in vivo*, our model uses a transformed embryonic fibroblast cell line that expresses high levels of α~1A~-AR \[[@B12]\]. Therefore, the relevance of these results to fibroblasts in tissues remains to be determined. These features in our model may underlie the ability of PE to cause expression of SMC markers and MyoD through α~1A~-AR. Conclusions =========== This study demonstrates that stimulation of α~1A~-ARs in rat-1 cells promotes cell cycle arrest by increasing levels of Cdk inhibitors and promotes hypertrophy and differentiation into a phenotype having the characteristics of smooth muscle cells by a mechanism independent of cAMP or EGF. Moreover, cell cycle progression was blocked at G~1~/S transition without causing apoptosis, and this cycle arrest was critical for rat-1 cell hypertrophy and differentiation. Reducing p27^kip1^levels reversed α~1A~-AR-promoted inhibition of DNA synthesis. Furthermore, it shows that cell cycle arrest and differentiation are closely coordinated processes but temporally separable. Further studies are underway in our laboratory to characterize the signaling pathway(s) involved in α~1A~-AR-induced differentiation of fibroblasts to smooth muscle cells. Methods ======= Materials --------- \[Methyl-^3^H\]thymidine (20 Ci/mmol) was purchased from NEN Life Science Products, Inc. (Boston, MA). L-\[4,5-^3^H\] leucine from Amercham Pharmacia Biotech. (Piscataway, NJ.). L-phenylephrine hydrochloride, penicillin, streptomycin, prazosin, propranolol, epidermal growth factor (EGF), and propidium iodide were obtained from Sigma (St. Louis, MO). Forskolin (FN), 8-cpt-cAMP and SQ 22536 were purchased from Calbiochem (La Jolla, CA). 2\'-5\'dideoxyadenosine and 2\'-deoxyadenosine 3\'-monophosphates were purchased from Biomol (Playmouth Meeting, PA). G418 sulfate from Invitrogen (Carlsbad, CA). Hanks\' balanced salt solution and fetal bovine serum were from Mediatech, Inc. (Herndon, VA). Dulbecco\'s Modified Eagle\'s Medium (DMEM) and trypsin/EDTA were obtained from Life Technologies Inc. (Grand Island, N.Y). Rat-1 cells and culture conditions ---------------------------------- Rat-1 cells transfected with bovine α~1A~-AR kindly provided to us by Drs. L. Allen, R. J. Lefkowitz, and M. G. Caron (Duke University), were maintained in DMEM supplemented with 10% (v/v) fetal bovine serum, 400 μg/ml G418 sulfate, 50 μg/ml streptomycin and 50 units/ml penicillin at 37°C in an humid atmosphere (5% CO2, 95% air). Cells were serially passaged upon reaching confluence, and all experiments were performed on subculture passages 5--15. Prior to all experiments, preconfluent cell cultures were serum-starved for 48 h. Measurement of DNA synthesis ---------------------------- Cells at a density of 150,000 cells/well were seeded in DMEM containing 10% FBS on 24-well plates until they reached 80--90% confluency and were serum-deprived for 48 h prior to the addition of agonists. Rat-1 cells, even after serum-deprivation for 2 days, exhibit a measurable level of \[^3^H\]thymidine incorporation. Inhibition of \[^3^H\]thymidine incorporation was used as a quantitative measure of reduction in DNA synthesis. Cells were serum-starved in the presence or absence of agonists for the indicated time period, and \[^3^H\]thymidine (0.5 μCi/ml) was added for the last 4 h of the incubation. This time period was chosen because it gave the maximum incorporation of \[^3^H\]thymidine into DNA after addition of agonists. The medium was then removed, and the cells were washed twice with phosphate-buffered saline and three times with 10% ice-cold trichloroacetic acid. Cells were kept in contact with trichloroacetic acid for 10 min during each wash. The precipitated material was dissolved in 1 M NaOH containing 0.1% SDS, and radioactivity was determined by liquid scintillation spectrometry. Measurement of protein synthesis -------------------------------- Rat-1 cells were seeded at a density of 150,000 cells/well in 24-well plates and just prior to confluency were serum-deprived for 48 h. Cells were rinsed with DMEM and 1 μCi/ml \[^3^H\]leucine was added for the last 4 h of each incubation with or without the indicated concentrations of the agonists. Cells were fixed and washed three times with ice-cold trichloroacetic acid (10%). The precipitated material was solubilized with 0.1 M NaOH and the incorporated radioactivity was counted by liquid spectrometry. Flow cytometry -------------- Flow cytometry studies were performed to determine the effect of different concentrations of PE on cell cycle phases. Cells were subjected to flow cytometric DNA analysis as described \[[@B43]\] with some modifications. Briefly, rat-1 cells plated on 100 mm dishes were grown in DMEM containing 10% FBS until they reached 80--90% confluency. Preconfluent cell cultures were serum-starved for 48 h to stop the mitogenic effect of growth factors. The medium was aspirated and cells were incubated for 18 h with different concentrations of PE. Cells were trypsinized in 1 ml trypsin/EDTA for at least 5 min, and then the reaction was stopped by the addition of 1 ml of serum-containing DMEM. The samples were centrifuged at 1000 rpm for 5 min, washed three times in ice-cold PBS containing 1% bovine serum albumin by centrifugation at 1000 rpm for 5 min each, resuspended in 0.5 ml of the same solution, and then fixed with 1 ml of 70% ethanol (-20°C) added dropwise. The fixed cells were stored at 4°C until analyzed. Cells were then washed three times by centrifuging at 1000 rpm for 10 min and resuspended in 3 ml of the BSA buffer. The pellet was then finally resuspended in 1 ml of the BSA buffer to which 100 μg/ml of RNAse A was added to remove interfering RNA, and 5 μg/ml propidium iodide was added to stain DNA. Cells were incubated at 37°C for 10--15 min in the dark to facilitate staining. The cells were analyzed for DNA content using an Epics Profile Analyzer (Coulter Electronics) with an Argon laser emitting at 448 nm. Percentages of cells in various stages of the cell cycles were determined using a multi-cycle program (P. Rabinovitch, Phoenix Flow Systems). At least 10,000 cells per cycle were counted for each treatment. Cell viability -------------- Preconfluent cultures were serum-starved for 48 h. Cells were incubated with different concentrations of PE, FN and 8-cpt-cAMP for 18 h, trypsinized with 1 ml trypsin/EDTA for 5 min and centrifuged for 10 min at room temperature. The pellet was resuspended in 1 ml plain DMEM. 10 μL of the suspension was mixed with 10 μL of 0.4% trypan blue in a 0.5 ml microtube. The total number of cells and the number of blue cells and the percentage of the viable cells in 10 μL of the trypan blue mixture was calculated as follows: % viable cells = \[1-(blue cells/total cells)\] × 100. Morphological study ------------------- Rat-1 cells resuspended in culture medium were seeded on six-well plates (Corning, N.Y.). Preconfluent cells were serum-deprived for 2 days, pre-incubated with inhibitor (s) for 30 min, and then stimulated with 5 μM PE and /or 1 μM FN for the indicated time. Cells were rinsed twice in PBS to remove non-adherent cells and then fixed for 10 min in \[1:1\] methanol-acetone mixture at room temperature. Cells were washed once in distilled water and stained in hematoxylin solution (Sigma, St. Louis, MO) for 15 min at room temperature. Cells were again washed three times in water, air dried and were finally observed under a phase contrast microscope. Images were captured and saved as TIFF files. For each condition, data were collected by random observation. Hypertrophic phenotype was defined as both enlarged, elongated and spindle-shaped whereas rat-1 fibroblasts are round and/or polygonal-shaped cells. Transfection procedures ----------------------- To determine whether protein kinase A (PKA) mediates the antiproliferative effect of PE, rat-1 cells were transiently transfected with πLXX-PKI \[[@B1]-[@B31],[@B24]\], a plasmid encoding for the PKA inhibitor, PKI (A generous gift from Dr. J. Avruch, Massachusetts General Hospital, Boston, MA), using Lipofectamine Plus (Life Technologies, Inc., Grand Island, N.Y.) according to the manufacturer\'s instructions. For p27^kip1^experiments, phosphorothionate oligonucleotides (ODN) (Life Technologies, Grand Island, N.Y.) were used. The antisense ODN sequence used in the experiments was 5\'-CACTCTCACGTTTGACAT-3\' (nuc 1--18 of rat p27 kip1); the sense ODN sequence was 5\'-ATGTCAAACGTGAGAGTG. ODN transfection was performed with oligofectamine in Opti-MEM according to the manufacturer\'s protocol (Invitrogen, Carlsbad, CA). Cells were left in the transfection mixture for 48 h and then incubated for 24 h with or without agonists and harvested for Western blot, or labeled with \[^3^H\]thymidine to determine DNA synthesis. Preparation of cell lysates and western blot analysis ----------------------------------------------------- Cells were rinsed twice with PBS and lysed in ice-cold lysis buffer (1% Igepal CA-630, 25 mM HEPES pH 7.5, 50 mM NaCl, 50 mM NaF, 5 mM EDTA, 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 5 mg/ml pepstatin A, 100 mM PMSF, 100 mM sodium orthovanadate, and 10 μM okadaic acid). Lysates were centrifuged at 4°C for 15 min in a microfuge at maximum speed, and the supernatant was collected for Western blot analysis. Equal amounts of protein were separated on denaturing SDS/polyacrylamide gel and transferred on nitrocellulose blots (Hybond-ECL; Amersham Life Sciences Inc., Arlington Heights, IL) by electrophoresis. Blots were blocked for 1 h in 5% nonfat dry milk in TBST, washed three times 5 min each in TBST and then incubated with the indicated specific primary antibody overnight at 4°C: p27^kip1^, p21^waf1/cip1^, pRb (Biosource International); caldesmon (smooth muscle), myosin (smooth muscle), α-smooth muscle actin, β-actin (Sigma Biosciences); MyoD, cyclin D1, Cdk 2, Cdk 4, PCNA, lamin A/C or vimentin (Santa Cruz Biotechnology). Following incubation, the membranes were washed three times 10 min each in TBST and incubated with a secondary antibody coupled to peroxidase for 1 h at room temperature. Then the membranes were washed three times 10 min each in TBST. Specific proteins were detected by enhanced chemiluminescence (ECL; Amersham Life Sciences Inc.) according to the manufacturer\'s instructions and analyzed with an Alpha Innotech Fluorochem imaging system (Packard Canberra). A lysate from cultured aortic vascular smooth muscle cell (VSMC) was used as a control for the expression of smooth muscle markers. Statistical analysis -------------------- The basal values of incorporation of \[^3^H\]thymidine and/or leucine were variable in different batches of cells. However, the effect of various agents on the incorporation of \[^3^H\]thymidine and \[^3^H\]leucine in rat-1 cells was consistent within each batch of cells. The results are expressed as mean ± SEM. The data were analyzed by one-way analysis of variance; the Newman-Keuls multiple range test was applied to determine the differences among multiple groups, the unpaired Student\'s t-test was applied to determine the difference between two groups. The null hypothesis was rejected at p \< 0.05. The protein level were estimated by densitometric analysis of the Western blots and performed on the indicated number of blots using NIH Image software, and expressed as a percentage of the control, arbitrarily chosen as 100%. Abbreviations ============= AR, adrenergic receptor; Cdk, cyclin-dependent kinase; FN, forskolin; MHC, myosin heavy chain; PCNA, proliferating cell nuclear antigen; PE, phenylephrine; pRb, retinoblastoma protein; SMC, smooth muscle cell Authors contributions ===================== AES performed thymidine/leucine incorporation, morphological studies, cell viability, flow cytometry and Western blot analysis. JHP carried out the antisense design, transfection experiments, statistical analysis and some thymidine/leucine incorporation and Western blot analysis. KUM conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Acknowledgements ================ We would like to dedicate this article to the memory of Dr. Abdelwahab El Saeed, who unexpectedly died in September 2004. He performed this work with great dedication, high enthusiasm and passion and we will miss him. This work was supported by NIH-NHLBI grant 19134. A.E.S was supported by a NIH Minority Postdoctoral Fellowship, supplement to NIH-NHLBI grant 19134-26. J.H.P is the recipient of a Beginning Grant-In-Aid from American Heart Association Southeast Affiliate. We thank Anne Estes for her technical assistance, Felicia Walker (Molecular Resource Center, UTHSC) for performing flow cytometry analysis and Dr. Lauren Cagen for editorial comments. We gratefully acknowledge Dr. J. Avruch (Massachusetts General Hospital, Boston, MA) for generously supplying us with π LXX-PKI \[1-31\].

PubMed Central

2024-06-05T03:55:52.532898

2004-12-16

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548263/", "journal": "BMC Cell Biol. 2004 Dec 16; 5:47", "authors": [ { "first": "Abdelwahab E", "last": "Saeed" }, { "first": "Jean-Hugues", "last": "Parmentier" }, { "first": "Kafait U", "last": "Malik" } ] }

PMC548264

Background ========== An earthquake measuring 6.3 on the Richter scale struck the city of Bam in Iran on the 26th of December 2003 at 5.26 A.M. It was devastating, the city was destroyed, left over 40,000 dead and around 30,000 injured, as well as destroying approximately 20,000 homes, leaving more than 45,000 people homeless \[[@B1]\]. The profound tragedy of thousands killed has caused emotional and psychological trauma for tens of thousands of people who have survived. In general earthquakes are known to be related to increased psychological symptoms among survivors and in particular it has been shown that earthquakes might cause post-traumatic stress disorder (PTSD). Several studies on psychological morbidity have been reported following earthquakes in different parts of the world \[[@B2]-[@B5]\], but there is no report from Iran. Iran is known to be a country of frequent severe earthquakes causing thousands of deaths in recent years. Therefore for the first time a study was conducted to measure psychological distress among the survivors of Bam earthquake in Iran. The objective was to indicate prevalence of psychological distress among survivors and to specify factors associated with psychological symptoms and thus provide information for interventions needed in Bam and in similar conditions in the country. In addition it was thought this might add to a small body of existing literature on the topic from a different population. Methods ======= This was a population-based study of psychological distress among Bam earthquake survivors aged 15 years and over at five months after the event. The pre-earthquake population of Bam was reported to be 97,000 and surprisingly it was increased to around 120,000 at the post-earthquake period (listing from the authorities). This was due to the fact that because of worry many people living in rural areas were moved to the city of Bam after earthquake. At the time of screening about 80% of people stayed inside their homes and the remaining 20% were moved to four specially designated sites with around 4000 tents. However, to select a representative sample of individuals living in Bam a stratified multi-stage area sampling was applied. The stratification was used because people living in the southern region of the city were less affluent and exposed to more damage as compared to those living in the northern part of the city. Every household or tent within the city had the same probability to be sampled. For the purposes of the first stage, units (census sections) were randomly selected after stratifying by region and size of residence. Then, within each census section the homes and tents to be sampled were selected using the procedure of random routes. Finally the last stage sampling units (the individuals) were selected randomly from all persons living in the same home or tent. Random sampling was based on a list of all households (N = 23000) derived from the electricity bills. A sample of 1150 households (5%) was selected as explained. However, we were actually able to screen 1000 households, giving a success rate of 87%. The main reason for the failed attempts for contact was due to the fact that the targeted house was uninhabited because the residents had moved out and there was no possibility to trace the survivors. In addition considering logistic difficulties we did not replace them with other households. A team of trained interviewers collected data in a week and all participants were interviewed in their homes or designated camps. However, the data were collected in a disorganized environment and thus data gathering strategy was based on offering help to survivors. Since there was no validated measure of PTSD in the Iranian language, only psychological distress was measured using the Iranian version of the 12-item General Health Questionnaire, GHQ-12 \[[@B6]\]. The scale examines whether the respondent has experienced a particular symptom or behavior recently. Each item is rated on a four-point scale (less than usual, no more than usual, rather more than usual, or much more than usual). The GHQ-12 is brief, simple, easy to complete, and its application in research settings as a screening tool is well documented, and there is evidence that the GHQ-12 is a consistent and reliable instrument when used in general population samples \[[@B7]\]. The study used the original scoring method. In this method response categories score 0, 0, 1, and 1 respectively. This gives scores ranging from 0 to 12 and higher values indicate more psychological symptoms \[[@B8]\]. In addition, we collected information about demographic and the number of family members that died due to the earthquake. To analyze data Student\'s t-test and one-way analysis of variance were used to find out differences in psychological distress among different sub-groups of the study sample and the logistic regression analysis was performed to investigate factors associated with distress. For the purpose of the logistic regression analysis we used the population mean score on the GHQ-12 as cut-off point as recommended \[[@B9]\]. Results ======= Data were collected from 1000 randomly selected households (600 homes and 400 tents). In total 999 survivors were interviewed, and data for 916 respondents were complete, giving a response rate of 91.7%. The mean age of the respondents was 32.9 years (SD = 12.4), and mostly were male (53%), married (66%) and had secondary school education (50%). The mean score of the survivors on the GHQ-12 was 8.7 (SD = 3.2). The findings showed that 58% of the respondents suffered from severe psychological distress as measured by the GHQ-12. Forty-one percent of the respondents reported that they lost three to five members of their family in the earthquake. The characteristics of the respondents are shown in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The characteristics of the study sample (n = 916) ::: **No.** **%** --------------------------- -------------------- ------------- ------- **Age** ≤ 20 126 14 21--30 352 38 31--40 205 23 41--50 148 16 \> 50 85 9 Mean (SD) 32.9 (12.4) **Gender** Male 486 53 Female 432 47 **Marital status** Single 257 28 Married 600 66 Divorced/widowed 59 6 **Educational level** Illiterate 158 17 Primary 181 20 Secondary 459 50 College/university 118 13 **Employment status** Employed 233 25 Housewife 277 30 Student 97 11 Unemployed 275 30 Retired 34 4 **Number of family loss** None 185 20 \< 3 261 29 3--5 377 41 \> 5 93 10 **GHQ score** Mean (SD) 8.7 (3.2) \< mean score 385 42 \> mean score 531 58 ::: To examine differences in psychological distress among sub-groups of the study sample Student\'s t-test and one-way analysis of variance were performed. The findings showed that there were significant differences between people with different characteristics. People with old age, females, less educated individuals, divorced or widowed and unemployed respondents, and those with loss of more family members in the earthquake reported more severe psychological distress. The results are shown in Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The GHQ-12 score by demographic characteristics (n = 916) ::: **Mean (SD)** **P** --------------------------- -------------------- --------------- ----------- **Age groups** 0.006 ≤ 20 7.8 (3.2) 21--30 8.6 (3.3) 31--40 8.9 (3.0) 41--50 8.9 (3.1) \> 50 9.3 (3.0) **Gender** 0.04 Male 8.5 (3.2) Female 8.9 (3.1) **Marital status** \< 0.0001 Single 8.1 (3.3) Married 8.9 (3.1) Divorced/widowed 9.9 (2.5) **Educational level** \< 0.0001 Illiterate 9.7 (2.7) Primary 8.7 (3.2) Secondary 8.5 (3.2) College/university 8.3 (3.1) **Employment status** \< 0.0001 Employed 8.4 (3.2) Housewife 8.8 (3.4) Student 7.0 (3.2) Unemployed 9.3 (2.9) Retired 9.0 (3.2) **Number of family loss** \< 0.0001 None 7.7 (3.5) \< 3 8.7 (3.2) 3--5 9.1 (2.8) \> 5 9.3 (2.7) ::: To investigate factors associated with severe psychological distress all the significant findings from the univariate analysis were entered into the logistic regression analysis. The analysis indicated that being female (OR = 2.73, 95% CI 1.19--6.26, P = 0.02), illiterate (OR = 3.36, 95% CI 1.11--10.2, P = 0.03), unemployed (OR = 4.39, 95% CI 1.56--12.4, P = 0.005), and the loss of more family members (OR for loss of more than five family members = 2.11, 95% CI 1.22--3.61, P = 0.007) were associated with more severe psychological distress. The results are shown in Table [3](#T3){ref-type="table"}. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### The results of logistic regression analysis ::: **OR (95% CI)** **P** --------------------------- ------------------- ------- **Age** 0.98 (0.95--1.01) 0.25 **Gender** Male 1.0 (ref.) Female 2.73 (1.19--6.26) 0.02 **Marital status** Single 1.0 (ref.) Married 1.56 (0.71--3.41) 0.26 Divorced/widowed 2.17 (0.50--9.37) 0.30 **Educational level** College/university 1.0 (ref.) Secondary 1.09 (0.51--2.31) 0.82 Primary 1.02 (0.43--2.43) 0.96 Illiterate 3.36 (1.11--10.2) 0.03 **Employment status** Housewife 1.0 (ref.) Employed 2.29 (0.85--6.18) 0.10 Student 1.90 (0.57--6.32) 0.29 Unemployed 4.39 (1.56--12.4) 0.005 Retired 1.43 (0.33--6.17) 0.63 **Number of family loss** None 1.0 (ref.) \< 3 1.76 (1.21--2.55) 0.003 3--5 1.98 (1.32--2.98) 0.001 \> 5 2.11 (1.22--3.61) 0.007 ::: Discussion ========== This was a population-based study measuring psychological distress among survivors of the Bam earthquake in Iran. Psychological morbidity was higher among females, less educated respondents and unemployed individuals. Perhaps such observation might relate to the fact that these groups of people were exposed to a relatively homogenous set of psychological stressors, leading to relatively homogenous mental health outcomes \[[@B10],[@B11]\]. In addition although in the univariate analysis there were significant differences among sub-groups of the study sample with regard to their GHQ-12 mean scores, in the logistic regression analysis age and marital status no longer remained significant. This is consistent with other research findings where female gender, lower education, and lower socio-economic status were found to be related to higher PTSD and depression among earthquake survivors \[[@B12],[@B13]\]. However, recent findings on the topic showed a differential predictor pattern for PTSD and depression among earthquake survivors indicating that although certain factors (e.g. grater fear during the earthquake and female gender) relate to PTSD, lower education and loss of family members tend to relate to depression and not to PTSD \[[@B13],[@B14]\]. This suggests that when interpreting the study results one might relate female gender to possible PTSD which we did not measure, and the other variables to depression which was measured using the GHQ-12. Most notably it was found that people who lost family members reported significant severe psychological distress compared to those who did not. Also there was a distinct pattern of psychological distress among those who lost their family members in the earthquake showing a dose-response relationship between loss and the risk of more psychological distress. One might argue that the relationship between loss of relatives and psychological distress may be a reflection of the relationship between severity of trauma exposure and psychopathology. Since no other variables reflecting trauma-exposure (e.g. fear during the earthquake, level of damage to home, injury, etc.) were entered in the regression equation, \'loss of relatives\' was the only variable tapping that, as there is high correlation between loss of first degree relatives (with whom survivors often share the same roof) and exposure to serious damage or collapse of the house. In addition studies have indicated that loss is a strong determinant of PTSD among earthquake survivors and thus it is argued the observation that the risk of PTSD is linked to the amount of loss is an important issue that needs to be incorporated in the development of any effective preventive strategy \[[@B15]\]. As discussed earlier such finding has been challenged and studies have shown that PTSD and depression are distinct issues and loss only relates to depression whereas PTSD is linked to factors relating to exposure to threat or fear for life \[[@B14]\]. However, since in the present study only the GHQ-12 was used, there is no way of knowing which factors related to PTSD and which to depression. The GHQ-12 has items tapping depression but little is known as to how sensitive is to picking up PTSD. Psychological distress among earthquake survivors alongside experience of other problems could be considered a serious issue for people\'s health status living in such difficult conditions. Evidence suggests that severe earthquakes even can cause long-standing morbidity \[[@B12]\]. However, past psychiatric illness also might contribute to this situation \[[@B15],[@B16]\]. Unfortunately one of the shortcomings of the present study was that we did not measure previous psychiatric conditions among survivors and thus it was not possible to comment on this further. It is recommended that the mean GHQ score for the whole population of respondents provide a rough guide to the best cut-off threshold \[[@B9]\]. Thus considering people who scored above the mean, the findings from the present study indicated that 58% of the respondents showed an indication of severe mental health problems. Comparing the figure with the national data this was found to be three times higher than reported mental disorders in the general population \[[@B17]\]. A study using the GHQ-28 measuring prevalence of psychiatric disorder following the China earthquake showed relatively a similar result where the rate of psychological morbidity for earthquake survivors was found to be 51% \[[@B18]\]. Thus it is argued that there is an urgent need to deliver mental health care to disaster victims in local medical settings and health care professionals who work with the earthquake victims need to be promptly and efficiently trained in mental health crisis interventions \[[@B19]\]. The results reported here is an estimate for the overall population experiencing the earthquake. Studies have shown that there is variation in psychological morbidity among earthquake survivors by epicenter proximity and rate of property damage \[[@B12],[@B18]\]. However one should be aware that these variables are not the only information that is needed for studying the relationship between psychological distress and the impact of the earthquake. There is also need to obtain information from earthquake survivors on the extent of damage to their homes, experience of being buried under rubble, participation in rescue operations, and witnessing grotesque sites \[e.g. see \[[@B13],[@B14]\]\]. The present study had certain methodological limitations. Firstly, the results were based on the GHQ-12 scores and we did not measure PTSD symptoms. In this respect also it is important to acknowledge that the GHQ is a general measure of mental health and it is not a measure of diagnostic depression. Thus, since depression is also common in earthquake survivors, our study is limited in not including a validated diagnostic measure of depression. Secondly, no information was obtained on important trauma-exposure variables such as extent of fear or perceived life-threat during the earthquake, rubble experience, disability or injury, et cetera. Thirdly, data on important demographic variables such as past psychiatric illness, and family psychiatric illness were not collected. However in spite of these limitations, the results from this study are useful. This study is the only paper addressing reports on the psychological consequences of the most disastrous earthquakes of the last 50 years. In addition, to our knowledge the international literature does not contain any study on the psychological status of Iranian earthquake survivors, despite the fact that Iran is an earthquake-prone country. It seems that the future research should carefully assess the psychological distress and disruption experiences of the survivors in order to implement necessary interventions. Given that Iran is a country that suffers catastrophic earthquakes relatively frequently, there is need to develop and validate standard instruments such as PTSD measures to include in the future research. Conclusions =========== In conclusion the findings from this study indicated that the survivors of Bam earthquake suffer from psychological distress three times higher than the normal population. In addition female gender, lower education, unemployment, and loss of family members were found to be associated with more severe psychological morbidity among survivors. This suggests that to reduce negative health impacts of the earthquake adequate psychological counseling is needed for those who survived the tragedy. Abbreviations ============= GHQ-12: The 12-item General Health Questionnaire; PTSD: Post-traumatic stress disorder; SD: Standard deviation; OR: Odds ratio, CI: Confidence interval. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= AM was the principle investigator and contributed to the study design, the data analysis and wrote the paper. All other investigators contributed to the study design, the data collection and analysis. All authors reviewed and contributed to the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/4/prepub> Acknowledgements ================ The authors wish to thanks all those who helped to carry this study especially colleagues in the Iranian Academic Centre for Education, Culture and Research-Kerman Province Branch (ACECR). Thanks also go to the Iranian Students\' Polling Agency (ISPA) for helping the authors and to Maria Livanou (Section of Trauma Studies, Division of Psychological Medicine, Institute of Psychiatry, University of London, United Kingdom) for her thoughtful comments to improve the paper.

PubMed Central

2024-06-05T03:55:52.537303

2005-1-11

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548264/", "journal": "BMC Public Health. 2005 Jan 11; 5:4", "authors": [ { "first": "Ali", "last": "Montazeri" }, { "first": "Hamid", "last": "Baradaran" }, { "first": "Sepideh", "last": "Omidvari" }, { "first": "Seyed Ali", "last": "Azin" }, { "first": "Mehdi", "last": "Ebadi" }, { "first": "Gholamreza", "last": "Garmaroudi" }, { "first": "Amir Mahmood", "last": "Harirchi" }, { "first": "Mohammad", "last": "Shariati" } ] }

PMC548265

Background ========== Invasive squamous cell carcinoma(SC) is the most common malignancy of the larynx\[[@B1]\]. Distinguishing between preinvasive squamous neoplasia (high grade squamous cell dysplaisa/ squamous cell carcinoma in-situ, SCIS) and SC may be difficult in small biopsy specimens, particularly when the tissue is superficial and fragmented, a prominent inflammatory infiltrate obscures the epithelial-stromal interface, and/or there is tangential sectioning of the acanthotic neoplastic squamous epithelium. Even in larger resection specimens, the presence of invasion may sometimes be elusive if the invasive element lacks paradoxical maturation characterized by prominent eosinophilic cytoplasm that may undergo either central or individual cell keratinzation, well developed cell borders, and large vesicular nuclei with prominent nucleoli. The existence of an adjunctive feature associated with invasion would be helpful in assessing whether there is any degree of invasion in these challenging cases, or whether such a feature should raise the suspicion that the lesion may harbor an invasive component when it is absent by conventional diagnostic criteria. A moderate to marked stromal eosinophils that may infiltrate into the neoplastic epithelium has occasionally been reported in invasive carcinoma \[[@B2]-[@B4]\]. Spiegel *et al*recently reported that the presence of eosinophils is associated with invasion in the neoplastic squamous lesions in the female genital tract, and proposed that eosinophilia provided as adjunctive morphologic feature in identifying SC in the cervix and vulva\[[@B5],[@B6]\]. One of us (DT) has observed moderate to marked stromal eosinophilia in cases of SC of the larynx, whereas stromal eosinophils were usually either absent or rare in cases of laryngeal SCIS. We speculated that the degree of stromal eosinophilia is a pathologic feature that would provide an adjunctive criterion for distinguishing SC from SCIS in the larynx, and undertook a systematic study to test this hypothesis. In this study, we focused on a single head and neck region, the larynx, to avoid any potential selection bias, since squamous neoplasia and the associated host response and changes in the head and neck are heterogeneous and varied in different anatomic locations\[[@B7]\]. Methods ======= The biopsy and resection specimens with available H&E stained slides of laryngeal SC and SCIS diagnosed at Roswell Park Cancer Institute from 1993 through 2000 were reviewed by two of the authors simultaneously (MZ and DT). Cases with prior radiation and/ or chemotherapy were excluded. All histology specimens at Roswell Park Cancer Institute were fixed in 10% formalin. Paraffin blocks of 5 μm thickness were cut and the sections were stained with conventional hematoxylin and eosin. For each biopsy and resection specimen, the original diagnosis was recorded and compared with the review diagnosis. Cases with any degree of invasion including \"minimal invasion\" or \"microscopic invasion\" in either a biopsy or resection specimen or both were classified as SC. Cases with SCIS only in a biopsy specimen that subsequently had invasion in the resection specimen were classified as SC. On the other hand, a resection specimen lacking invasion was required for a case to be classified as SCIS. For each of specimen, the high-power field(hpf) (Olympus BH2 ×10 ocular and ×40 objective lens) with a maximum number of eosinophils was identified and recorded as eos/hpf. Then, the eosinophils in the adjacent nine contiguous hpf were counted, added to those in the first, and recorded as eos/10hpf. Only nucleated cells with intensely red cytoplasmic granules were accepted as eosinophils, and care was taken to exclude red blood cells with superimposed mononuclear and polymorphonuclear inflammatory cells. And those that were confined to lymphovascular spaces were excluded. During the course of the study, it was noted that frozen section preparation results in the degranulation of eosinophils and leads to difficulty in their being recognized; however, under these conditions, collections extracytoplasmic typical red granules approximately the expected size of an eosinophil allows for their identification. As an internal control, non-neoplastic portions of the specimens, whenever available, were also evaluated. Statistical methods ------------------- Frequency was computed for each eosinophil category in invasive and non-invasive squamous neoplasia specimens obtained from biopsy and excision. Chi-square test was utilized to examine the difference of frequency distribution between each elevated eosinophil category and the referent eosinophil category (0--4 eos/10hpf). This analysis was conducted independently for specimens obtained from biopsy and excision. Sensitivity, specificity, positive predictive value and negative predictive value were computed for eosinophil counts that were exceeding 10 eos/hpf or 20 eos/10hpf to evaluate if these two classifications of eosinophil count would suggest any significance of clinical implication from statistical point of view. P value less than 0.05 was used to determine statistical significance. Elevated eosinophils were defined and categorized as: focally and moderately elevated (5--9 eos/hpf), focally and markedly increased(\>10/hpf), diffusely and moderately elevated(5--19 eos/10hpf), and diffusely and markedly increased (\>20/10hpf), while 0--4 eosinophils/10hpf was used as a baseline. The recorded eosinophil counts were analyzed to determine whether there were thresholds of stromal versus neoplastic squamous eosinophils per 1 hpf and 10 hpf that were significantly associated with invasive tumor in both biopsy and follow-up excisional/resectional specimens. Results ======= A total of 87 cases were evaluated and sixty-eight percent of the cases (n = 59) displayed a chronic inflammation in the stroma. Fifty-seven were biopsy specimens and 30 were ablative resection specimens. The diagnoses of 57 biopsy specimens were 35 invasive carcinoma, 4 minimal invasive carcinoma, 2 suspicious for invasion, and 16 were preinvasive squamous cell neoplasia. 27 of the biopsy specimens (18 invasive carcinoma, 1 minimal invasive carcinoma, 1 suspicious for invasion and six preinvasive squamous cell neoplasm) were followed by ablative resections (7 wide excisional resection, and 20 total laryngectomy). The follow-up specimens confirmed 19 invasive carcinoma and six preinvasive carcinoma, and revealed an invasive carcinoma in the suspicious case. In this case, there were stromal nests of neoplastic squamous cells that were associated with 15 and 34 eosinophils per 1 and 10 hpf, respectively, and clinical image studied revealed an advanced disease (stage IIIa). Additionally, Other 3 ablative resection specimens (all excisional biopsies) were initial operations. There were 2 invasive carcinoma and 1 preinvasive lesion. The distribution of eosinophil counts is summarized in Table [1](#T1){ref-type="table"}. Both diffuse and focal elevated eosinophilic infiltration were noted in invasive tumor and, to much less extend, in the non-invasive counterparts. Typical examples of eosinophilic infiltration in the stroma tissue were illustrated in Figures [1A--1C](#F1){ref-type="fig"}. As shown in Table [2](#T2){ref-type="table"}, thirty-six (57%) of patients with invasive squamous cell carcinoma were found to have diffuse eosinophilic infiltration (\>20/10hpf), whereas elevated eosinophils were not diffusely observed in all non-invasive lesions (p \< 0.05). The same held true for focally marked eosinophilia (\>10/hpf) when compared invasive group with non-invasive group. One exception was observed in a non-invasive neoplasia (Case 37, high grade dysplasia/SCIS) which showed a marked increased eosinophils (10 eosinophils/hpf). Noticeably, this high grade dysplasia with elevated eosinophil infiltrate uncovered an invasive carcinoma in the follow-up resection specimen. These results indicated that a close association exists between the stromal invasion and the presence of elevated tissue eosinophils. Stromal eosinophilia was statistically significantly associated with invasion in squamous cell carcinoma (Table [2](#T2){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Distribution of eosinophils in invasive and non-invasive squamous neoplasia ::: ------------------------ ----------------- ------------------- -------- --------- --------- ----------- ----------- ------------ --------------- Specimen and Diagnosis Eosinophils/hpf Eosinophils/10hpf 0 1--4 5--9 10--20 \>20 0 1--4 5--19 20 or greater Biopsy Invasive CA(41) 1(2%) 5(12%) 8(20%) 15(36%) 12(29%) 1(3%) 5(12%)^1^ 10(24%)^1^ 25(61%)^1^ Non-invasive(16) 13(81%) 1(6%) 1(6%) 1(6%) none 7(44%)^2^ 6(38%)^2^ 3(18%)^2^ none Excision/Resection Invasive CA(22) 1(5%) 4(18%) 8(36%) 6(23%) 3(14%) 1(5%) 3(14%) 7(31%) 11(50%) Non-invasive(8) 6(76) 1(12%) 1(12%) none none 4(50%) 3(38%) 1(12%) none ------------------------ ----------------- ------------------- -------- --------- --------- ----------- ----------- ------------ --------------- ^1^Two invasive carcinoma case less than 10hpf in size ^2^One non-invasive carcinoma case less than 10hpf in size. ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### a\. Absence of eosinophils in normal squamous epithelium. Note that a moderately inflamed submucosal tissue. Arrows point to the inflammatory cells. 200× b. A squamous cell carcinoma in-situ (non-invasive tumor) with no elevated eosinophils in a chronic inflammatory background. Arrows point to the inflammatory cells. 200× c. Markedly increased eosinophils in an invasive squamous cell carcinoma. Note that the eosinophils (arrows) were a major component of the infiltrating nucleated cells. 200× ::: ![](1472-6890-5-1-1) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Significance of eosinophils in invasive and non-invasive squamous neoplasia ::: --------------------------------- -------------------- -------- ------------------- -------- ---------------------- -------- --------------------- --------- Lesion Eosinophils Counts 5--9 eos/hpf ^1^ p \>or = 10 eos/hpf p 5--19 eosin/10hpf^2^ p \>or = 20 eos10/hpf p Invasive CA in biopsy 8/41(20%) \>0.05 27/41(66%) \<0.01 10/41(24%) \>0.05 25(61%) \<0.005 Non invasive lesion in biopsy 1/16(6%) 1/16(6%) 3/16(18%)) 0/16(\--) Invasive CA in Excision 8/22(36%) \>0.05 9/22(41%) \<0.05 7(31%) \>0.05 11((50%) \<0.05 Non-invasive lesion in excision 1/8(12%) 0/8(\--) 1(12%) 0/8(\-\-\--) --------------------------------- -------------------- -------- ------------------- -------- ---------------------- -------- --------------------- --------- ^1^*eos/hpf:*for each specimen, a high power field (hpf) (Olympus BH2 ×10 ocular and ×40 objective lens) with a maximum number of eosinophils in the lesion area ^2^*eos/10hpf*: for each specimen, one high power field (hpf) (Olympus BH2 ×10 ocular and ×40 objective lens) with a maximum number of eosinophils in the lesion area and additional nine hpfs in the contiguous areas ::: There is no association of elevated tissue eosinophils with overall inflammatory response of the stroma in the specimens studied (p \< 0.05). Specifically, among the 37 cases with \> = 10 eosinophils/hpf, 24 cases displayed a non-specific inflammation, while 50 cases with \<10 eosinophils/hpf, 35 displayed a non-specific inflammation. Among the 36 cases with \> = 20 eosinophils/10hpf, 24 cases revealed a non-specific inflammation, while 51 cases with \<20 eosinophils/10hpf, 34 revealed a non-specific inflammation. In fact, some invasive carcinomas (n = 6) virtually contained no chronic inflammatory background, but showed a marked elevated tissue eosinophila (Fig. [2](#F2){ref-type="fig"}). In addition, a number of cases (n = 21) with elevated eosinophila showed a distinct polarization of the infiltrating cells, namely eosinophilic cells accumulating in the tumor invading front (Fig. [2C](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### a\. A low power view of an invasive carcinoma. Note that there was no significant inflammatory background. 40× b. A higher power view of the square area labeled as A in Figure 2a. No eosinophils were present in the stromal tissue between the tumor nests. 200× c. A higher power view of the square area labeled as B in Figure 2a. Elevated eosinophils were present at the invading front of the carcinoma. 200× ::: ![](1472-6890-5-1-2) ::: The predictive values of tissue eosinophils in assessing stromal invasion in squamous neoplastic lesions of larynx are presented in Table [3](#T3){ref-type="table"}. In biopsy specimens, diffusely elevation of eosinophils (\>20/10 hpf) had a sensitivity, specificity and positive predictive value of invasion of 80%, 100% and 100%, respectively. In these specimen, the presence of \>10 eosinophils/hpf predicted invasion in all cases with a sensitivity of 81% and positive predictive value 96%, respectively, while values below this threshold had a predictive value of an absence of invasion 68%. Similarly, the presence of \> 20 eosinophils/10hpf in the excisional specimens had a sensitivity, specificity, and positive predictive value of invasion of 69%, 100% and 100 %, respectively. In these excisional specimens, the presence of \>10 eosinophils/hpf had sensitivity, specificity, and positive predictive values for invasion of 64%, 100% and 100%, respectively. Values below the thresholds of \>10 eosinophils/hpf or 20 eosinophils/10hpf had a predictive value of an absence of invasion of 40% and 42%, respectively. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Predictive value of eosinophils in assessing stromal invasive in squamous neoplasia of larynx ::: Eosinophils in lesion Sensitivity(%) Specificity(%) Positive predictive value(%) Negative predictive value(%) ------------------------ ---------------- ---------------- ------------------------------ ------------------------------ Biopsy specimens \>or = 10 eos/hpf 66% 94% 96% 52% \>or = 20/10hpf 80% 100% 100% 68% Excisonal specimens \>or = 10 eos/hpf 64% 98% 100% 58% \>or = 20 eos/10 hpf 68% 100% 100% 58% Sensitivity, specificity, positive predictive value and negative predictive value were computed for eosinophil counts that were exceeding 10 eos/hpf or 20 eos/10hpf to evaluate if these two classifications of eosinophil count would suggest any significance of clinical implication from statistical point of view ::: Sections with adequate non neoplastic epithelium were available in twelve cases. Eight of them were absent for eosinophils. The highest counts for non-neoplastic epithelium were 4 eosinophils/hpf and 8 eosinophils/10hpf. Although it seems that non-neoplastic epithelium contains less eosinophils than squamous neoplasia, no statistical analyses were performed since the number of available non-neoplastic regions in this series was too small. Discussion ========== For decades, pathologists have used a variety of histologic features, including desmoplastic stromal reaction, intrastromal foreign body reaction to keratin, and the presence of separate minute clusters of intrastromal neoplastic cells, to assess and identify invasion\[[@B8],[@B9]\]. However, when evaluating a small, poorly-oriented, tangentially-cut specimens, one sometimes enters an area replete with uncertainties. The presence of a morphologic feature associated with invasion would be helpful in determining whether any degree of invasion has occurred in the equivocal cases. In practice, we have noticed a frequent presence of eosinophilic infiltration in invasive squamous cell carcinoma of the larynx, which is usually absent in non-invasive neoplastic counterparts. Such a consistent observation has prompted us to carry out the current study. In this series, a systematic study of eosinophils in tissues of squamous neoplasia of larynx suggests that elevated eosinophils are a morphologic marker for assessing tumor invasiveness. We observed that in the invasive squamous carcinomas eosinophils were significantly elevated focally and /or diffusely, statistically more frequent than in non-invasive neoplasia. The increased eosinophil counts (\>10 hpf, and \>20/10 hpf) in laryngeal biopsy and excisional specimens were all statistically significantly associated with stromal invasion. In contrast, values below both of these thresholds had a significant predictive value for the absence of invasion. The slight decrease in the correlation of \>10 eosinophils/hpf with invasion in excisional specimens, relative to that in biopsy counterparts, may be attributed to the increased chance of observing microscopic clusters of eosinophils unrelated to invasion in the larger specimens. It is not surprising to observe inflammation in the specimens examined, likely due to several factors including the specific anatomic location and an overall inflammatory response of the stroma to the tumor, among others\[[@B7],[@B9]\]. However, there is no association of elevated eosinophils with overall inflammatory response of the stroma in the specimens studied. Furthermore, a number of cases with elevated eosinophila showed a distinct polarization of the infiltrating cells, specifically eosinophilic cells accumulating in the tumor invading front (Fig. [2C](#F2){ref-type="fig"}). Cumulatively, our findings strongly indicate that elevated tissue eosinophila is a specific cell response independent of a non-specific inflammatory reaction. Although elevated eosinophil counts are statistically significantlly associated with stromal invasion in squamous cell carcinoma of larynx, occasionally the presence of high number of eosinophils were observed in the non-invasive counterpart tissues (Table [1](#T1){ref-type="table"}). In other words, the presence of eosinophils in squamous neoplasia of larynx is not pathognomonic for stromal invasion and caution must be exerted when evaluating the number of infiltrating eosinophils. However, the quantitation method and thresholds identified in the current experiment may represent an adjunctive feature in assessment of stromal invasion in squamous neoplasia. Specifically, the presence of eosinophils at these thresholds should raise the suspicion that invasive or microinvasive carcinoma is present within the specimen, particularly when \>10 eosinophils/hpf and or 20 eosinophils/10hpf are observed. Since the first observation of malignancy with marked blood eosinophilia described by Rheinbach in 1893, eosinophilia has been described in human cancers from a variety of organs \[[@B10]-[@B12]\]. In head and neck squamous cell carcinoma, it has been reported that the presence of tissue eosinophils ranges between 22 and 89% \[[@B13]-[@B16]\]. Most of these series have focused on whether the presence of a prominent eosinophilic infiltrate has a prognostic value, or is an indicator of response to treatment. Some authors have claimed that the presence of a marked or moderate eosinophilic is associated with a poor prognosis\[[@B12],[@B17]\], while others have found that eosinophilia is a favorable prognostic feature\[[@B13],[@B14]\]. No study has addressed the value of eosinophils in distinguishing invasive from non-invasive squamous neoplasia in the head and neck. The mechanism of eosinophilic accumulation in cases of invasive carcinoma remains largely unknown. It has been suggested that such eosinophilic infiltration may be induced by a tumor-derived eosinophil chemotactic factor\[[@B18],[@B19]\]. A recent study further indicated that stromal eosinophils in squamous cell carcinoma may play a key role in tumor invasion through activation of gelatinase\[[@B20],[@B21]\]. It was found that 92-kd gelatinase, a key member of the matrix metalloproteineaes which are involved in tumor invasion by breaking down the basement membrane and extracellular matrix, is actively expressed by eosinophils. In conclusion, although the etiology of tissue eosinophils in invasive carcinoma is unknown, our study is the first to suggest that an elevated eosinophil count in the squamous neoplasia of larynx may serve as a morphologic feature associated with tumor invasion. The presence of more than individual eosinophils, specifically when the number of infiltrating eosinophils exceeds 10/hpf and or \>20/10 hpf in a biopsy of larynx with squamous neoplasia, represents a histologic marker for the presence of tumor invasion. Similarly, the presence of eosinophils reaching these thresholds in an excisional specimen should prompt a thorough search for invasiveness when evidence of invasion is absent, or when invasion is suspected by conventional criteria in the initial sections. Although the present study assesses a quantitative parameter of tumor invasion, in our daily practice we find it useful that a readily appreciable elevation of tissue eosinophilia alerts us to search for possible invasiveness in tissue biopsy of laryngeal lesions. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= Drs. Said, Speiegel, Tan for study design; Drs. Said, Tan, for pathology evaluation; Drs. Alrwi, Douglas, Hicks, Loree, Riguel, Wiseman for surgical evaluation and clinical follow-up as well as card review. Dr. Yang for statistical analyses. Dr. Cheney for administrative and financial support. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6890/5/1/prepub>

PubMed Central

2024-06-05T03:55:52.539900

2005-1-7

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548265/", "journal": "BMC Clin Pathol. 2005 Jan 7; 5:1", "authors": [ { "first": "Mahmoud", "last": "Said" }, { "first": "Sam", "last": "Wiseman" }, { "first": "Jun", "last": "Yang" }, { "first": "Sadir", "last": "Alrawi" }, { "first": "Wade", "last": "Douglas" }, { "first": "Richard", "last": "Cheney" }, { "first": "Wesley", "last": "Hicks" }, { "first": "Nestor", "last": "Rigual" }, { "first": "Thom", "last": "Loree" }, { "first": "Gregory", "last": "Spiegel" }, { "first": "Dongfeng", "last": "Tan" } ] }

PMC548266

Background ========== In both Canada and the United States efforts are underway to develop systems to assess the quality of health care as a first step to improving services. In the US nursing home sector, the implementation of the Minimum Data Set has been linked to improvements in quality of care \[[@B1]-[@B4]\]. Recently, the Centers for Medicare and Medicaid Services (CMS) has developed web pages to give consumers, and the public at large, further information about the quality of nursing homes and home care services across the country. The \"Nursing Home Compare\" and more recent \"Home Health Compare\" web sites allow individuals to view several quality indicators (QIs) for individual providers\[[@B5],[@B6]\]. When comparing health care providers across a set of QIs there is a concern that they may differentially admit clients with a greater likelihood of triggering on quality issues. Since the indicators are defined as events that are preferable to avoid (e.g., skin ulcers, untreated pain, weight loss), in the absence of risk adjustment, these providers would appear to be delivering poorer quality of care. Risk adjustment attempts to adjust for different populations of clients who may be at greater risk for experiencing quality issues that is a function of their clinical status rather than the quality of care. In the US nursing home sector, the CMS recently funded a large-scale project to review all potential long-term care QIs and the risk adjustment process. The evaluation of risk adjustment included a review of both client-level and agency-level covariates. The research team revised the original set of client covariates to create a set of new models and assessed the effects of using an agency-level covariate, the Facility Admission Profile (FAP). The FAP represents the prevalence of the quality issue for residents newly admitted to the facility and was intended to adjust for potential selection bias (i.e., facilities preferentially admitting clients with a greater likelihood of triggering on the indicator) and ascertainment bias (i.e., ability of providers to detect a quality issue that might increase the QI rate). However, after extensive analyses, the research team did not recommend the use of the FAP, given that the FAP did not appear to be a particularly useful measure of ascertainment bias \[[@B7]\]. Nevertheless, in October 2002, the CMS included the FAP in three quality measures reported on the Nursing Home Compare web page\[[@B8]\]. As part of this same initiative, Kidder et al.\[[@B9]\] examined the Nursing Case Mix Index from the RUG-III grouping system as well as seven of the RUG scales as potential risk adjusters. In their final list of quality measures, 12 indicators for chronic care residents and four indicators for post-acute care residents, included a covariate related to the RUG-III system. Home Care Quality Indicators (HCQIs) ------------------------------------ The current research was part of a project to develop a set of 22 Home Care Quality Indicators (HCQIs) based on items in the Minimum Data Set for Home Care (MDS-HC). Implementation of the MDS-HC is complete or underway in 15 states and in 7 Canadian provinces and territories, where it is being used mainly as a clinical assessment instrument. In Michigan, the MDS-HC is being used as part of the MI-Choice waiver program to reduce nursing home admissions. In Ontario, Community Care Access Centre (CCAC) case managers use the instrument to determine needs, allocate services and make placement decisions. The HCQI derivation was accomplished using data from Ontario and Michigan since these were the regions with the largest scale implementation in Canada and the US, respectively. In their paper describing the HCQI derivation, Hirdes et al.\[[@B10]\] recommended the use of client-level risk adjustment for all but four indicators, and suggested the use of the Agency Intake Profile (AIP) as another method for risk adjustment. These various approaches to risk adjustment are the focus of this study. The set of HCQIs used in this study were developed by members of inter*RAI*, a non-profit multinational organization dedicated to the development and refinement of assessment instruments for older adults and persons with disabilities, and their related applications. The HCQIs represent outcome measures that document an agency\'s rate for triggering on a quality issue. For example, one indicator measures the prevalence of weight loss among individuals who are not considered to be palliative clients. The HCQIs include a mix of prevalence measures (i.e., measured on a cross-section of clients at one point in time) and incidence measures (i.e., failure to improve or incidence of an event measured across two points in time). All HCQIs are defined as events to be avoided such that a higher rate on the indicator is indicative of poorer performance. This paper uses a dataset from two Canadian provinces to further explore these three types of risk adjustment for the inter*RAI*HCQIs. It explores the effects of risk adjustment at both the level of the region and at the level of the individual home care provider. Methods ======= Data ---- The RAI Health Informatics Project was a two and a half year research study begun in 1999 in which fourteen of Ontario\'s 43 CCACs used the MDS-HC as part of usual practice for all adult (18 years and older) home care clients. CCACs provide several different services: information and referral, case management and placement in long-term care facilities. CCACs purchase in-home services from external contracted agencies through a \"request for proposal\" process. Services provided include in-home physiotherapy, occupational therapy, personal support and homemaking, medical supplies and equipment, and care by other professional such as social workers, dietitians and speech-language pathologists. Case managers oversee the assessment process, make referrals to service providers and then monitor the care provided in the home. Funding for CCACs is based on an annual budget provided by the Ontario Ministry of Health and Long-term Care. CCACs are governed by independent, non-profit boards of directors, accountable to the Ministry of Health and Long-term Care. In Ontario, it is now mandatory for all long stay home care clients to be assessed with the MDS-HC. However, at the time of the RAI Health Informatics project, the MDS-HC was not mandated for use. As such, some clients (e.g., those expected to be on service less than two weeks) did not receive an MDS-HC assessment and were not included in the current project. At this same time, Manitoba conducted a pilot implementation of the MDS-HC in the fourteen offices of the Winnipeg Regional Health Authority (WRHA). The WRHA is one of twelve health authorities in the province, and is responsible for providing health services to the approximately 646,000 residents of Winnipeg and the surrounding suburban area. The WRHA receives annual funding from the government of Manitoba. The home care program of the WRHA was established in 1974 with a mandate to provide effective and responsive health care services in the community to support independent living and facilitate admission into LTC facilities when independent community living is no longer an option. Home care services include personal care, nursing, counselling, occupational therapy and physiotherapy assessment, referral to other agencies and coordination of services. In the WRHA region, an MDS-HC assessment was completed by care coordinators only if the client was anticipated to be on service for at least 90 days. As a result, the study sample was focused on a long stay population of home care clients. All participating sites in the WRHA and in Ontario identified a set of case managers who received a two-day training session led by a member of the research team. They then used the MDS-HC instrument as part of their usual in-home assessment. The data used for this study represent a cross-sectional cohort of home care clients assessed between November, 1999 and December, 2002. Two CCACs in Ontario and four WRHA sites, were removed from the database because they submitted fewer than 20 assessments, resulting in a total of ten WHRA and twelve Ontario sites. Of these, three Ontario CCACs and eight offices of the WRHA also submitted client reassessments at approximately 90 days, which allowed for the calculation of failure to improve/incidence HCQIs. The protocol for data collection was reviewed and received ethics clearance through the Office of Research Ethics at the University of Waterloo, Canada. Risk adjustment --------------- Risk adjustment attempts to adjust for differences in client populations that may bias the HCQI rates. Organizations that provide care to more impaired clients will tend to have higher unadjusted rates, regardless of the quality of care they provide. As such, risk adjustment methods are used to maximize the ability to make fair comparisons across providers. With any type of risk adjustment, caution must be exercised to prevent over adjustment (i.e., adjusting away poor practice). The choice of risk adjustment covariates is therefore highly important and should not include variables that would be considered to reflect suboptimal clinical care. There are two generic types of risk adjustment that have been recommended for the HCQIs. The first adjusts for differences in the population at the client-level\[[@B10]\]. Potential adjusters can include both individual assessment items and summary scales embedded within the MDS-HC. The HCQI developers evaluated a large range of potential covariates, considering their distributional properties, strengths of association with the outcomes of interest, consistency of findings across jurisdictions and potential for clinically inappropriate adjustment (e.g., benzodiazepine use was not considered a reasonable adjuster for falls). As a result, from zero to five risk adjusters were recommended for the 22 HCQIs and these client covariates were used in the current project\[[@B11]\]. The second type of risk adjustment was intended to control for two types of bias at the agency level: a) the agency\'s ability to identify differences in clients\' clinical characteristics and b) differences in who an agency selects for admission. These risk adjustments are performed at the agency level after the individual-level risk adjustments are applied. The Agency Intake Profile (AIP) was used following the methodology outlined by Morris et al., for the MDS 2.0 quality indicators\[[@B7]\]. The AIP was calculated for each agency based on clients for whom the MDS-HC assessment was their intake assessment or for clients who had been on service for no more than 30 days. This group of clients was considered to be the intake cohort. More recently, an alternative form of agency-level risk adjustment has been proposed, to control for potential selection and ascertainment bias. This adjustment employs the Case Mix Index (CMI) associated with the RUG-III/HC methodology\[[@B9]\]. In this instance, the CMI for the intake cohort was calculated, and adjustments to the HCQIs were performed similar to those used for the AIP. The process followed for adjusting HCQI rates was the same as that used by Morris et al. in adjusting the US nursing home QI rates\[[@B7]\]. Three sources of information are required: the agency-level observed rate, the agency-level expected rate and the grand mean across all agencies. The first data element was simply the raw observed HCQI rate for a given home care agency. The next data element involved the creation of an expected rate for each client within a given agency, based on output from a logistic regression model. In this model, each HCQI acts as a dichotomous dependent variable (i.e., triggering on the HCQI or not) and the client-level and/or agency-level covariates are entered simultaneously as independent variables. The expected value for each client is then pooled to create an expected value for the agency. Three separate risk adjusted HCQI rates are thus computed, based on which method is used for adjustment: the expected rate based on the client covariates only (CC), on client covariates plus AIP (+AIP) and on client covariates plus CMI (+CMI). The final adjusted value can be thought of as an estimate of an agency\'s HCQI rate if the agency had clients with an average level of risk\[[@B7]\]. This risk adjustment method is similar to the concept of indirect standardization, in which the ratio of the observed to expected events is calculated then multiplied by the crude rate in the standard population\[[@B12]\]. In the current project, the standard population used was the combined set of agencies from both the Winnipeg Regional Health Authority (WRHA) and from Ontario. HCQI rates ---------- For each participating agency, the unadjusted HCQI rates were calculated for all 22 indicators. These rates represent the average rate across all eligible clients for a given agency. For prevalence indicators, the rates were calculated only for clients who had been on service for at least 30 days to avoid penalizing an organization for quality issues that were newly recognized. Several methods were utilized to assess the impact of risk adjustment, both at the regional and at the agency-level. The unadjusted and three adjusted rates were compared between Ontario and the WRHA, to assess the effects at the regional level. At the level of the individual agency, ranks were calculated for each HCQI and a count was created to examine how often a given agency was among those with the four highest rates. Since higher rates on each HCQI were indicative of higher prevalence or incidence of undesirable outcomes, agencies ranked within the four highest rates were considered to be among the \"worst performers.\" The range between agencies with the highest and fourth highest rates was also examined for both unadjusted and adjusted rates to assess the degree of variation among the group of worst performers. Results ======= The two regions were very similar on average age and sex. The WRHA clients were significantly less likely to have some level of cognitive impairment, as measured by the Cognitive Performance Scale (CPS)\[[@B13]\], compared to Ontario clients, although the actual difference was only 2.6% (WRHA: 37.0% vs. Ontario: 39.6%; p \< 0.0001). They were also significantly less likely than clients in Ontario to require some assistance with ADLs (22.0% vs. 27.1%, respectively; p \< 0.0001), as measured by the ADL Self-performance Hierarchy Scale\[[@B14]\]. Severe daily pain was experienced by 17.2% of the Ontario clients compared to 14.2% of the WRHA clients (p \< 0.0001). Although the differences were statistically significant, with Ontario having significantly lower rates of both arthritis and hypertension, the absolute difference between the regions on the most common diagnoses was less than 5% (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of home care clients in the WRHA and Ontario\* ::: **WRHA (n = 6704)** **ON (n = 5063)** **p value** ------------------------------------------------- --------------------- ------------------- ------------- **%** **%** **Age** Mean (95% CI\*\*) 76.0 (74.9, 77.0) 75.6 (75.2, 76.0) 0.49 18--64 13.4 16.5 \<0.0001 65--74 16.5 19.2 75--84 39.9 40.7 85 and older 30.0 23.6 **Sex** Female 69.2 70.5 0.14 **Marital status** Never married 11.5 8.7 \<0.0001 Married/widowed 79.4 82.7 Separated/divorced 8.2 7.9 Other 0.8 0.7 **Primary language** English 85.6 85.1 0.63 French 3.5 3.8 Other 10.9 11.1 **Aboriginal status** Origin is Inuit, Metis or North American Indian 3.2 1.6 \<0.0001 **Cognitive Performance Scale** Mean (95% CI) 0.8 (0.8, 0.8) 0.8 (0.8, 0.9) 0.07 0 -- intact 63.0 60.4 \<0.0001 1 -- borderline intact 15.0 18.4 1 -- borderline intact 15.0 18.4 2 -- mild impairment 8.4 7.3 3 -- moderate impairment 10.9 10.1 4 -- moderate to severe impairment 0.8 1.0 5 -- severe impairment 1.5 2.5 6 -- very severe impairment 0.5 0.4 **ADL Self-Performance Hierarchy Scale** Mean (95% CI) 0.5 (0.5, 0.6) 0.7 (0.6, 0.7) \<0.0001 0 -- independent 78.0 72.9 \<0.0001 1 -- supervision required 5.2 7.9 2 -- limited impairment 8.9 8.5 3 -- extensive assistance required (level I) 4.7 5.4 4 -- extensive assistance required (level II) 1.6 2.6 5 -- dependent 1.1 2.0 6 -- total dependence 0.5 0.7 **Pain Scale** Mean (95% CI) 1.2 (1.2, 1.2) 1.3 (1.3, 1.4) \<0.0001 0 -- no pain 39.8 34.9 \<0.0001 1 -- less than daily pain 14.4 14.1 2 -- daily pain but not severe 31.6 33.8 3 -- severe daily pain 14.2 17.2 **Top 3 medical diagnoses\*\*\*** Arthritis 49.4 44.8 \<0.0001 Hypertension 42.2 37.4 \<0.0001 Diabetes 19.1 20.1 0.24 \* client characteristics and scale values based on first submitted MDS-HC assessment \*\* CI = confidence interval \*\*\* disease was present and was or was not being treated or monitored by a home care professional ::: Unadjusted rates ---------------- When comparing the two regions, there were statistically significant differences for five of the 22 unadjusted HCQIs (Table [2](#T2){ref-type="table"}). In only one of these five cases (ADL rehabilitation potential and no therapies) was the rate in the WRHA significantly higher than the rate in Ontario. However, the actual size of the absolute difference between regions was small (0.3% on average). Among the prevalence HCQIs, the largest unadjusted difference was for disruptive/intense daily pain, which was 9.1% higher in Ontario. There were no statistically significant differences between the regions for any of the incidence HCQIs. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Unadjusted HCQI rates comparing the WRHA and Ontario\* ::: **WRHA Offices** **Ontario CCACs** **p value\*\*** ----------------------------------------------------------------- ------------------ ------------------- ----------------- ------------ -------- **n = 10** **n = 12** ***Prevalence HCQIs*** **Mean (sd)** **95% CI** **Mean (sd)** **95% CI** Inadequate meals 2.7 (1.2) 1.9, 3.6 4.4 (1.9) 2.1, 7.1 0.02 Weight loss 5.7 (3.4) 3.2, 8.1 7.6 (2.2) 5.1, 10.3 0.13 Dehydration 0.6(0.7) 0.08, 1.1 2.4 (1.6) 1.0, 5.0 0.003 Not receiving a medication review by a physician 3.8 (1.8) 2.5, 5.0 10.3 (4.3) 6.6, 17.1 0.0002 No assistive device among clients with difficulty in locomotion 13.8 (16.4) 2.1, 25.5 10.2 (5.2) 4.2, 15.6 0.52 ADL/rehabilitation potential and no therapies 84.0 (7.4) 78.7, 89.3 73.7 (5.5) 69.6, 78.5 0.001 Falls 20.8 (7.8) 15.2, 26.3 24.4 (4.5) 19.7, 30.3 0.18 Social isolation 21.6 (4.5) 18.4, 24.8 24.3 (5.9) 17.6, 32.4 0.24 Delirium 5.1 (2.3) 3.4, 6.7 5.9 (2.6) 3.4, 9.0 0.45 Negative mood 7.9 (3.2) 5.6, 10.1 11.6 (6.2) 5.2, 18.4 0.10 Disruptive or intense daily pain 30.5 (4.0) 27.6, 33.3 39.6 (6.3) 33.6, 44.3 0.0008 Inadequate pain control among those with pain 26.6 (5.3) 22.8, 30.4 29.7 (7.4) 22.3, 35.8 0.27 Neglect/abuse 3.0 (2.6) 1.2, 4.9 2.5 (2.1) 0.8, 5.2 0.58 Injuries 13.6 (6.4) 9.0, 18.2 11.0 (3.7) 8.6, 14.3 0.26 No influenza vaccine 28.4 (7.1) 23.2, 33.5 30.3 (9.8) 21.1, 44.0 0.61 Hospitalization 32.0 (7.2) 26.8, 37.2 36.4 (4.9) 31.1, 40.4 0.11 ***Incidence HCQIs*** **n = 8** **n = 3** Failure to improve/incidence of bladder incontinence 29.5 (9.7) 21.4, 37.5 23.1 (7.4) 4.8, 41.4 0.33 Failure to improve/incidence of skin ulcers 3.4 (4.54) 0.0, 7.1 3.4 (3.2) -5.0, 11.3 0.96 Failure to improve/incidence of decline on ADL long form 41.5 (12.5) 31.1, 51.9 30.3 (4.4) 19.3, 41.3 0.17 Failure to improve/incidence of impaired locomotion in the home 11.0 (9.0) 3.5, 18.5 16.5 (9.2) -6.5, 38.4 0.44 Failure to improve/incidence of cognitive decline 44.6 (13.7) 33.1, 56.0 36.1 (3.9) 26.3, 45.9 0.33 Failure to improve/incidence of difficulty in communication 13.7 (8.1) 6.9, 20.4 14.4 (3.4) 6.0, 22.9 0.88 \* in the event that the lower value of the 95% confidence interval was less than zero, the value was set to zero. \*\* based on a t-test of independent means. ::: Agency-level covariates ----------------------- The AIP was calculated for each home care agency for each of the 19 HCQIs for which client-level risk adjustment was recommended. The AIP values shown represent the HCQI rate among an admission cohort within each region and were used in the risk adjustment models that included client-level covariates together with the AIP covariate. Ontario CCACs had a significantly higher AIP value for eight indicators, demonstrating that they admit or at least assess individuals as more likely to have these conditions on admission (Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### AIP values for the WRHA and Ontario for each of the 19 risk-adjusted HCQIs\* ::: **WRHA n = 10** **Ontario n = 12** **p value** ----------------------------------------------------------------- ------------------- -------------------- ------------- ***Prevalence HCQIs*** **mean (95% CI)** Inadequate meals 2.9 (1.7, 4.1) 6.8 (5.3, 8.2) 0.0003 Weight loss 8.2 (4.7, 11.7) 12.8 (10.7, 14.8) 0.02 Dehydration 0.6 (0.2, 1.1) 4.0 (2.5, 5.5) 0.0005 No assistive device among clients with difficulty in locomotion 13.4 (10.8, 16.0) 19.1 (12.7, 25.4) 0.09 Falls 24.4 (20.6, 28.2) 31.4 (27.5, 35.3) 0.01 Social isolation 22.4 (19.4, 25.3) 25.3 (21.2, 29.4) 0.23 Delirium 5.2 (4.0, 6.4) 10.2 (7.8, 12.6) 0.0008 Negative mood 7.7 (5.5, 9.9) 12.8 (8.7, 17.0) 0.03 Disruptive or intense daily pain 31.5 (28.1, 34.9) 42.3 (37.5, 47.0) 0.0008 Inadequate pain control among those with pain 30.6 (26.6, 34.6) 31.4 (26.5, 36.4) 0.79 Neglect/abuse 2.5 (1.5, 3.5) 2.9 (1.3, 4.6) 0.65 Injuries 11.5 (8.5, 14.5) 12.8 (9.1, 16.5) 0.55 Hospitalization 35.3 (31.6, 39.1) 52.0 (47.4, 56.6) \<0.0001 ***Incidence HCQIs*** **n = 8** **n = 3** Failure to improve/incidence of bladder incontinence 0.9 (0.8, 1.0) 0.9 (0.5, 1.2) 0.81 Failure to improve/incidence of skin ulcers 6.3 (5.6, 7.0) 7.6 (4.8, 10.3) 0.07 Failure to improve/incidence of decline on ADL long form 1.9 (1.6, 2.3) 2.8 (0.0, 6.9) 0.45 Failure to improve/incidence of impaired locomotion in the home 0.3 (0.2, 0.4) 0.6 (0.0, 1.6) 0.36 Failure to improve/incidence of cognitive decline 0.8 (0.7, 0.9) 1.0 (0.2, 1.7) 0.21 Failure to improve/incidence of difficulty in communication 0.5 (0.4, 0.6) 0.6 (0.0, 1.6) 0.65 \* for prevalence HCQIs and failure to improve/incidence of skin ulcers, the AIP value represents the mean rate (expressed as a percent) of the HCQI on an intake cohort of clients and for the remaining incidence HCQIs, the AIP value represents the mean value for the MDS item(s) involved in calculating the HCQI. ::: Among new home care clients in Ontario, 52% of clients triggered on the HCQI for the prevalence of hospitalization versus 35% in the WRHA (difference of 17%). Ontario also had a significantly (p \< 0.0001) higher Case Mix Index (CMI) on intake (i.e., the CMI covariate), at 0.84 (95% CI: 0.81, 0.86), compared with the WRHA at 0.70 (CI: 0.68, 0.71). Risk adjustment at the regional level ------------------------------------- In general, the risk adjustment process minimized the differences between the two regions compared with the unadjusted rates. For example, the unadjusted difference between regions for the prevalence of disruptive/intense daily pain was 9.1%, however, the difference was reduced to 8.0% with the CC adjustment, 5.7% with the +CMI adjustment and 2.8% with the +AIP adjustment (Table [4](#T4){ref-type="table"}). The +AIP adjusted rates showed the most variability, so that for five HCQIs, the direction of the difference was reversed. For example, the unadjusted rate of falls was 3.6% ***higher***in Ontario than in the WRHA. Following the +AIP adjustment, Ontario had a rate that was 1.4% *lower*than in the WRHA. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Difference between Ontario and WRHA HCQI unadjusted and adjusted rates\* ::: **Unadjusted** **Client covariates only** **AIP + client covariates** **CMI+ client Covariates** ----------------------------------------------------------------- ------------------------------------------- ---------------------------- ----------------------------- ---------------------------- **Difference (ON-WRHA) expressed as a %** ***Prevalence HCQIs*** Inadequate meals 1.7 1.7 0.2 1.5 Weight loss 1.9 1.9 0.2 1.5 Dehydration 1.8 1.9 1.1 1.7 No assistive device among clients with difficulty in locomotion -3.6 -1.9 -2.8 -3.3 Falls 3.6 1.7 -1.4 0.7 Social isolation 2.7 0.8 1.1 0.6 Delirium 0.8 1.1 -0.4 0.8 Negative mood 3.7 3.1 -1.0 1.8 Disruptive or intense daily pain 9.1 8.0 2.8 5.7 Inadequate pain control among those with pain 3.1 2.8 1.7 3.3 Neglect/abuse -0.5 -0.4 -0.5 -1.0 Injuries -2.6 -3.0 -3.3 -1.8 Hospitalization 4.3 2.8 -1.4 2.1 ***Incidence HCQIs*** Failure to improve/incidence of bladder incontinence -6.4 -5.7 -5.6 -2.3 Failure to improve/incidence of skin ulcers 0.0 0.0 -1.2 -0.8 Failure to improve/incidence of decline on ADL long form -11.2 -11.0 -10.7 -6.6 Failure to improve/incidence of impaired locomotion in the home 5.0 5.3 -0.1 4.2 Failure to improve/incidence of cognitive decline -8.5 -6.1 -7.7 -4.1 Failure to improve/incidence of difficulty in communication 0.7 1.3 0.0 1.7 ***Mean*** ***0.3*** ***0.2*** ***-1.5*** ***0.3*** \* differences were always calculated as Ontario rate minus WRHA rate ::: Agencies with the highest rates ------------------------------- A summary measure was created to count the frequency with which an agency was ranked among the worst performers. Overall, only three of the ten offices within the WRHA did not change their ranking when rates were adjusted (Table [5](#T5){ref-type="table"}). For example, Office 6 was ranked within the four highest (i.e., worst) agencies for three out of the 19 HCQIs both for unadjusted and for the three adjusted rates. In another six offices, the effect of risk adjustment was a decline in their standing (i.e., poorer quality) so that after at least one type of risk adjustment they were ranked among the worst performers. For example, in Offices 2, 3 and 7, the number of times they were ranked within the four highest rates increased as a result of the +AIP adjustment. The most dramatic effect was evident in Office 7 such that the unadjusted rates ranked this group among the worst performers only once, but the +AIP adjustment increased this frequency to five. There was no instance of an office in the WRHA consistently benefiting from risk adjustment. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Number of times a given agency was ranked within the four highest rates ::: **Number of times agency ranked within the four highest rates** ------------- ----------------------------------------------------------------- ----------------------- ----------------------------- ----------------------------- **Unadjusted** **Client covariates** **AIP + client covariates** **CMI + client covariates** **WRHA** 1 8 9 10 9 2 1 1 4 3 3 2 2 5 3 4\* 0 0 0 0 5 3 5 4 5 6 3 3 3 3 7 1 2 5 2 8 0 0 2 0 9 7 6 8 6 10\* 4 4 4 4 **Ontario** 100\* 3 3 2 4 200 5 5 4 4 300\* 1 1 2 2 400\* 5 5 6 4 500\* 1 2 0 2 600\* 4 3 3 1 700\* 1 1 2 1 800\* 5 4 2 4 900\* 6 6 6 6 1000\* 1 1 0 1 1100 10 9 3 8 1200 6 4 1 4 \* indicates agencies for which the count was based on 13 HCQIs since incidence HCQIs could not be calculated ::: In Ontario, only CCAC 900 experienced no change in their ranking following risk adjustment (Table [5](#T5){ref-type="table"}). In another nine cases, at least one type of adjustment resulted in an improved standing, with the agency ranking among the four highest rates less often. For example, CCAC 1100 ranked among the worst performers ten times across the 19 HCQIs prior to risk adjustment, but the +AIP adjustment reduced this value to three. A similar effect was observed for CCAC 1200 which was ranked in the worst four performers six times across the unadjusted rates, but only once following +AIP adjustment. Examining only the ranking of agencies may be misleading if very small changes in the actual rate resulted in an increase or decrease in the rank for a given agency. For example, the change in the rate for a given agency could be very small, say 5%, but could result in the organization moving from the fifth highest rate (i.e., fifth worst performer) to the third highest rate. Examining only the ranking tells one little about the actual magnitude of change among the worst performers. Therefore, it is also important to explore the range in the rates across the four highest agencies. The unadjusted and CC adjusted rates had the largest degree of variation between the highest and fourth highest agency, with a mean of 11% across the 19 HCQIs. The +CMI adjustment had the next largest amount of variation at 10% and the +AIP adjustment at 8% (Table [6](#T6){ref-type="table"}). These results further reinforce the finding that the +AIP method of adjustment consistently had the largest impact compared with the other types of risk adjustment. ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Range in the HCQI rates comparing agencies with the highest rates ::: **Unadjusted** **Client covariates** **AIP + client covariates** **CMI + client covariates** ----------------------------------------------------------------- ------------------------------------------------ --------------------------------------------- ------------------------------------------------ --------------------------------------------- ------------------------------------------------ --------------------------------------------- ------------------------------------------------ --------------------------------------------- **Range between 1^st^and 4^th^highest agency** **Difference between 1^st^and 4^th^agency** **Range between 1^st^and 4^th^highest agency** **Difference between 1^st^and 4^th^agency** **Range between 1^st^and 4^th^highest agency** **Difference between 1^st^and 4^th^agency** **Range between 1^st^and 4^th^highest agency** **Difference between 1^st^and 4^th^agency** **%** **%** **%** **%** ***Prevalence HCQIs*** Inadequate meals 7.4 -- 4.9 2.5 8.3 -- 5.4 2.9 6.4 -- 4.6 1.8 8.2 -- 5.4 2.9 Weight loss 13.3 -- 9.3 4.0 13.3 -- 9.2 4.1 10.7 -- 9.2 1.5 13.6 -- 9.8 3.8 Dehydration 6.1 -- 2.6 3.5 6.6 -- 3.0 3.6 3.8 -- 2.3 1.5 6.5 -- 2.8 3.7 No assistive device among clients with difficulty in locomotion 60.0 -- 15.3 44.7 50.4 -- 14.8 35.6 53.8 -- 15.6 38.2 52.0 -- 14.3 37.7 Falls 38.4 -- 28.8 9.6 38.4 -- 28.8 9.6 36.5 -- 28.4 8.1 39.1 -- 29.1 10.0 Social isolation 33.1 -- 28.6 4.5 31.8 -- 30.2 1.6 31.5 -- 30.3 1.2 31.9 -- 30.2 1.7 Delirium 11.0 -- 8.1 2.9 14.3 -- 12.4 1.9 12.8 -- 11.9 0.9 14.2 -- 12.9 1.3 Negative mood 26.2 -- 13.4 12.8 30.4 -- 16.1 14.3 18.0 -- 15.0 3.0 23.4 -- 15.9 7.5 Disruptive or intense daily pain 54.1 -- 42.5 11.6 51.6 -- 40.8 10.8 43.7 -- 39.4 4.3 44.5 -- 41.2 3.3 Inadequate pain control among those with pain 46.5 -- 32.6 13.9 47.6 -- 33.8 13.8 36.6 -- 33.0 3.6 47.4 -- 34.2 13.2 Neglect/abuse 9.7 -- 3.9 5.8 9.5 -- 4.0 5.5 6.7 -- 3.7 3.0 9.8 -- 4.4 5.4 Injuries 22.4 -- 18.9 3.5 22.8 -- 18.3 4.5 21.9 -- 16.1 5.8 22.0 -- 19.0 3.0 Hospitalization 46.8 -- 40.1 6.7 44.6 -- 40.8 3.8 47.7 -- 38.4 9.3 46.2 -- 41.2 5.0 ***Incidence HCQIs*** Failure to improve/incidence of bladder incontinence 48.0 -- 29.0 19.0 48.4 -- 28.4 20.0 44.5 -- 31.9 12.6 47.3 -- 27.9 19.4 Failure to improve/incidence of skin ulcers 12.9 -- 4.2 8.7 13.2 -- 4.3 8.9 11.1 -- 5.5 5.6 14.1 -- 4.9 9.2 Failure to improve/incidence of decline on ADL long form 56.0 -- 42.9 13.1 55.1 -- 43.4 11.7 54.9 -- 43.3 11.6 53.9 -- 41.2 12.7 Failure to improve/incidence of impaired locomotion in the home 29.0 -- 14.1 14.9 33.4 -- 19.1 14.3 36.6 -- 22.2 14.4 34.1 -- 18.4 15.7 Failure to improve/incidence of cognitive decline 72.0 -- 48.4 23.6 70.1 -- 46.7 23.4 69.5 -- 46.3 23.2 69.6 -- 46.0 23.6 Failure to improve/incidence of difficulty in communication 23.8 -- 18.3 5.5 30.7 -- 22.3 8.4 29.3 -- 22.4 6.9 30.6 -- 22.4 8.2 **Mean difference** **11.1** **10.5** **8.2** **9.9** ::: Discussion ========== Ontario CCACs exhibited several key differences when compared to the WRHA. Ontario sites had significantly higher unadjusted rates across four HCQIs. However, the degree of variation between the sites was small, with a mean difference across indicators of less than 1%. Ontario as a region had significantly higher AIP values for eight HCQIs, indicating a greater propensity in Ontario to admit clients exhibiting quality issues, and they also had a higher mean Case Mix Index. Overall, the change in the actual rates at the regional level was small following risk adjustment. When risk adjustment was applied, for the client covariates alone or the client covariates with the CMI covariate, there was little effect on the mean difference between the two regions. In each case, the mean difference was positive (i.e., Ontario had a higher rate on average) and less than 1%. The +AIP adjustment, however, resulted in a negative mean difference (i.e., WRHA higher than Ontario on average) across the set of risk-adjusted HCQIs. At the agency level, there was a greater influence of risk adjustment, as assessed by agency rankings across the set of indicators. In general, sites in Ontario benefited from risk adjustment and were less likely to be ranked among the worst performers. The opposite was true within the WRHA. Again, the +AIP adjustment had the largest influence when compared to the other two types of risk adjustment. It is possible that the level of variation between agencies on the AIP covariate was greater than the level of variation between clients on the individual-level covariates. Although a detailed analysis is beyond the scope of this paper, use of a single HCQI example may prove beneficial. When examining the prevalence of falls (an HCQI that showed the largest changes in the agency rankings following risk adjustment), the coefficient of variation (CV) for the AIP value was 23.0. The CV is a relative measure of dispersion about the mean and is calculated by dividing the standard deviation by the mean and multiplying by 100. This CV value was much lower than any of the corresponding CV values for the various MDS outcome scales that reflect differences at the level of the individual client. For example, the CV for the Cognitive Performance Scale was 158.5 and for the ADL Self-performance Hierarchy Scale, the CV was 195.6 (data not shown). Clearly, the +AIP adjustment had the effect of minimizing differences between the individual providers and it also resulted in rates that had less variability, as demonstrated by the drop in the CV value when comparing the unadjusted and +AIP adjusted rates for the prevalence of falls indicator (CV of 27.7 and 19.1, respectively; data not shown). However, this effect cannot be explained by differences in the variances of the adjusters since the AIP had a lower level of variance than the individual covariates. It is also possible that the AIP covariate serves as a proxy for regional differences in practice patterns. If the Ontario agencies are grouped into four main geographic regions, the AIP value for the falls HCQI ranged from 24.1% to 34.0%. The degree of variation appears modest, but cannot be discounted as one factor in the ability of the AIP adjustment to minimize differences between providers and between regions. Although there continues to be support for the conceptual notion of risk adjustment, the potential to over adjust remains a concern. In a recent publication, Mor et al\[[@B15]\] recognize the possibility for over adjustment and conclude that in the absence of a simple solution to this problem, researchers must carefully consider each performance measure on an individual basis in an attempt to minimize this issue. In the current project, the AIP covariate represents a continuous, numeric value that can range from zero to one. Thus, it does not simply serve as an agency identifier, but represents the rate among an intake cohort. Furthermore, in the US, Morris et al. determined that the Facility Admission Profile, the conceptual cousin to the AIP, did not substantially improve the risk adjustment models and they did not recommend its use. This decision was not based on a fear of over adjustment, but rather a concern that the FAP added increased complexity without significantly improving the risk adjustment process\[[@B7]\]. It is also important that to develop a clearer understanding of what is meant by \"over adjustment\". For example, it would seem reasonable to differentiate between instances of truly *inappropriate*risk adjustment where variance is due to poor practices (e.g., adjusted for benzodiazepine use for a falls indicator) and \"over adjustment\" due to the excessive use of spurious risk adjusters resulting in suppression of variance. Another possible example of over adjustment is the use of individual level covariates that are too closely related to the quality indicator. For example, one might argue that using dressing of the upper body as a risk adjuster for a QI on dressing the lower body is a form of over adjustment because the dependent variable is represented on both sides of the regression equation. This area of research continues to present many challenges. Given the current results and those from the long-term care sector, it appears advantageous to undertake some form of risk adjustment, even though the definitive method may not yet be best understood. Given that these HCQIs are new and so little is known about the risk adjustment process, it seems appropriate that all three type of risk adjustment be considered by researchers and policy makers. The +AIP adjustment represents the most conservative approach and may therefore be most appropriate for public reporting of HCQI results. The assessment of quality of care, and ongoing refinement of risk adjustment methods, is ultimately intended to provide information to different audiences to assist in continuously improving the quality of care. To date, many initiatives in the US have taken place to provide additional information on the quality of LTC and home health services\[[@B6]\]. Similar efforts have not begun in the Canadian home care sector. Several important issues should be addressed prior to moving towards more public reporting of these types of quality data. For example, there needs to be a discussion of the relative importance of the HCQIs. The current project made no attempt to prioritize the indicators, although clearly some would have higher clinical priority than others. To some degree, individual agencies must determine their own priorities. However, a simple system based on prevalence, severity and modifiability may be useful in determining relative importance. For example, pain is a prevalent condition in this population, can be severely debilitating for clients and is clearly modifiable. One might argue that it is of higher importance to address than declining cognitive performance, which is less prevalent and in many cases not modifiable. On the other hand, cognitive decline can have extremely serious consequences for the individual In addition, a decision is needed regarding the calculation of the HCQIs, in particular, which pairs of assessment are to be used in the calculation of failure to improve/incidence HCQIs and what time period in between assessments is acceptable (e.g., a minimum of 90 or 120 days). There will need to be further analysis to explore the relationship between the length of stay and triggering on the HCQIs. It is anticipated that clients who have been on service longer will show important clinical differences from short-stay clients and would therefore be expected to have differential HCQI rates. Tracking of the HCQI rates over time will be essential to determine the level of stability in the indicators. It would be useful to measure the amount of variation and to assess methods to summarize the rates over time in order to maximize their stability. For example, it may be important to report annual HCQI rates for a given organization or province if in fact the rates are found to be highly variable over shorter periods of observation. A shorter time frame may also lead to unstable rates if the number of observations is small. In this paper, a minimum of 20 observations was required and a decision would also be needed for the minimum sample size for public reporting of HCQIs. Finally, it will be essential to assess possible methods to create summary measures across the set of HCQIs. This is not a simple task and one for which little research has been conducted to date. Previous research has pointed to the lack of correlation between measures of quality, but has also suggested that summary measures would be useful for consumers who would likely prefer less complicated information\[[@B15]\]. Knowledge about the home care sector in Canada remains rudimentary, and our knowledge and understanding of quality assessment in this sector is still in its infancy. The results from the current project serve to enhance the overall understanding of the issues involved in quality assessment and our ability, through the risk adjustment process, to create fair comparisons across providers. Conclusions =========== Risk adjustment of quality indicators is important to enable fair comparisons across geographic regions or across home care providers. To date, little research has examined the quality of home care services. This project, using a set of HCQIs developed by inter*RAI*, provides an important first step in assessing quality and the variable effects of different types of risk adjustment. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= DD participated in the coordination of the study, assisted in the conceptual design of the study, carried out the data analysis and took the lead in developing the manuscript. JH designed the original study, oversaw the development of the indicators, gave input into the data analysis and development of the draft manuscript. BF was the Co-Principal Investigator in the development of the indicators and provided feedback in terms of data analysis and choice of statistical methods. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/5/7/prepub> Acknowledgements ================ The authors would like to acknowledge John Morris, Naoki Ikegami, David Zimmerman and Richard Jones as well as Pablo Aliaga and Suzanne Hammer who were instrumental in the development of the home care quality indicators. The authors are grateful for contributions to the development effort by Mary James, Nancy Curtin-Telegdi, Jeff Poss, Colleen Maxwell, Lori Mitchell, Trevor Smith, Paula Fletcher, Gary Teare, Michael Stones, Kim Voelker, and Judy Bowyer. The authors also wish to thank Roy Cameron, John Goyder and Margaret Denton for helpful comments on an earlier report on which this manuscript is based. The authors wish to thank the following agencies for financial support of this research: Agency for Health Care Policy Research (Grant 5 U18 HS09455), the Health Transition Fund -- Health Canada (ON 421), inter*RAI*, the State of Michigan and the Robert Wood Johnson Foundation.

PubMed Central

2024-06-05T03:55:52.542305

2005-1-18

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548266/", "journal": "BMC Health Serv Res. 2005 Jan 18; 5:7", "authors": [ { "first": "Dawn M", "last": "Dalby" }, { "first": "John P", "last": "Hirdes" }, { "first": "Brant E", "last": "Fries" } ] }

PMC548267

Background ========== Alzheimer\'s disease (AD) is the most common form of dementia affecting elderly people in the United States. Prevalence is 1% to 2% at age 65 years, but increases markedly to 35% or greater by age 85. Because of a demographic shift toward a more aged population, the percentage of affected individuals is rapidly increasing. This trend is expected to continue for the foreseeable future. Therefore, accurate and timely diagnosis and effective treatments are critical to optimal outcomes over the 8- to 10-year course of the illness \[[@B1]\]. Traditionally, a probable diagnosis of AD was accomplished by history, clinical examination, neuroimaging, and neuropsychological and laboratory testing to rule out treatable causes for the patient\'s symptoms and to differentiate AD from other possible causes of dementia \[[@B2],[@B3]\]. Much effort has gone into defining risk factors for the development and progression of Alzheimer\'s dementia, as well as to identify biological markers for the disease. Clinical-demographic variables that are consistently associated with AD in prior studies include family history of AD, age, and Down\'s syndrome \[[@B1],[@B3]\]. None of these variables has been demonstrated to affect the rate of disease progression or show any utility in defining subgroups that may be more amenable to therapy. Currently, predominant symptoms of dementia are treated primarily with second-generation cholinesterase (ChE) inhibitors. These drugs have demonstrated efficacy, as measured by cognitive, behavioral, and functional outcomes, in randomized, placebo-controlled clinical trials, the majority of which have been of 6 months\' duration \[[@B4]-[@B6]\]. In an open-label extension study of the cholinesterase inhibitor donepezil, Doody et al \[[@B7]\] concluded that donepezil was safe and effective for treating the symptoms of mild to moderate AD for up to 2 1/2 years. Cognitive, behavioral, and functional outcomes in patients treated with ChE inhibitors over the longer term are of great interest given the substantial social and economic implications of AD, which has a course that averages 8 to 10 years. Due to their relatively recent approval, however, longer-term data on the clinical benefits and/or limitations of ChE inhibitor therapy in AD patients is virtually nonexistent \[[@B8]\]. Rivastigmine\'s approval by the FDA in 2000 was supported by several pivotal trials, including a randomized US trial (ENA 713 B352)\[[@B5]\]. In this pivotal trial, 699 patients with mild to moderately severe AD were randomized to high dose rivastigmine (6--12 mg/day), low dose (1--4 mg/day) or placebo with a 7 week fixed dose-titration phase followed by a flexible dosing phase during weeks 8--26. Results of the 26-week open-label extension of this study found that at 52 weeks, patients originally treated with 6--12 mg/day rivastigmine had significantly better cognitive function than patients originally treated with placebo \[[@B9]\]. In this paper the authors present descriptive findings for a cohort of 37 patients who participated in the long-term open-label extension of the ENA713B352 rivastigmine trial. Much work remains to be done to more definitively answer questions about when to start therapy, which patients are most likely to benefit, what constitutes clinically relevant beneficial effects over the longer term, and when these drugs are no longer clinically effective. Consideration should also be given to withdrawal of therapy. Findings presented in this article will add to the current limited dataset for long-term efficacy and outcomes with cholinesterase inhibitor therapy for persons with probable AD. This report describes our experience in following the cohort of patients at our center with AD treated with the ChE inhibitor rivastigmine (a medication that inhibits both butyl- and acetylcholinesterase) as part of the ENA 713 B352 pivotal trial for a period up to 5 years. Methods ======= Data in this analysis came from a subgroup of 37 patients with originally mild- to moderate-stage (defined by a Mini-Mental State Examination \[MMSE\] score of 10 to 26) AD followed at a large Mid-Western university site in a 26-week, prospective, randomized, double-blind, placebo-controlled, parallel-group study of rivastigmine as therapy for AD conducted at 22 research sites across the United States (ENA713, B352 Study Group, 1998) \[[@B5]\]. Patients were enrolled according to previously described inclusion criteria \[[@B5]\]. Of note, this study allowed rather broad inclusion of AD patients with other comorbid illnesses; presumably this would allow this cohort to more closely mirror real-world populations. The study was conducted in accordance with ethical standards of the institutional committee on human experimentation and with the Helsinki Declaration of 1975, revised 1983. The initial study protocol was therefore reviewed in our center by the institutional review board and all patients or caregivers consented to inclusion based on appropriate informed consent. Additional consent was obtained for the open-label extension of the study. Cholinesterase inhibitor treatment ---------------------------------- Immediately following the double-blind phase of the study, open-label rivastigmine was flexibly titrated over a 12-week time period to a maximum tolerated dosage of up to 6 mg BID. By the end of the 12 week titration 25 participants were on 4--6 mg. of rivastigmine and 11 participants had dropped from the study. One participant remained on a 2 mg. dose and dropped from the study between weeks 52 and 78. Assessment of treatment response -------------------------------- Outcomes measures included the Alzheimer\'s Disease Assessment Scale-cognitive subscale (ADAS-cog) and the Clinician\'s Interview-Based Impression of Change, with caregiver input (CIBIC-Plus). Ability to carry out activities of daily living (ADL) was assessed by the Progressive Deterioration Scale (PDS). Disease-staging measures included the Geriatric Deterioration Scale (GDS) and the MMSE. In the open-label phase of the study, efficacy evaluations were performed every 6 to 8 weeks for titration and early maintenance, and every 26 weeks for the next 5 to 6 years. Statistical analysis -------------------- These data are predominantly descriptive, with analyses including Kaplan- Meier survival plots when appropriate. All statistical analyses on our single center extension were performed at our center using SPSS. Subgroup analyses by initial treatment randomization were also performed. Results ======= Demographics and population --------------------------- Twenty-one Caucasian women and 11 Caucasian men participated in the open label extension of this pivotal study. Twenty-two participants reported a family history of AD. Selected demographic and baseline characteristics of the subsample are presented in Table [I](#T1){ref-type="table"}. Of the 32 patients, 11 were originally randomized to the high dose (6--12 mg/day) group, 10 to the low dose group, and 11 to placebo. Five of the patients in the cohort chose not to participate in the extension. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Selected baseline and demographic characteristics (N = 32). ::: Characteristic Mean (SD) --------------------------- ------------- Age (y) 71.8 (7.5) Length of symptoms (mo)\* 29.6 (18.3) Baseline MMSE 21.3 (4.1) Baseline GDS 3.7 (0.78) MMSE = Mini-Mental Status Examination; GDS = Geriatric Deterioration Scale. \*Length of symptoms prior to enrollment in the double-blind study. ::: A total of 25 patients during the 5-year term eventually withdrew from the study. Reasons for termination, stratified by original group during the double-blind phase, are summarized in Figure [1](#F1){ref-type="fig"}. The most frequent reason for termination of participants initially randomized to the high-dose group was the lack of availability of free rivastigmine following FDA approval, which had been provided at pre-launch at no charge as part of the clinical study. It is of interest that no deaths occurred in the group initially randomized to high-dose rivastigmine during the initial double-blind, placebo-controlled trial. Furthermore, disease severity, as measured by the GDS, was greater in the original high-dose group (mean = 4.0; median = 4.0) as compared with the low-dose (mean = 3.7; median = 3.5) and placebo groups (mean = 3.4, median = 3.0). A total of 8 terminations were due to withdrawal of consent (n = 3) or caregiver discontinuation (n = 5). Five of the 8 were in the original placebo group. Seven patients withdrew because of adverse events; 2 each in the original low-dose and placebo groups (57%) and 3 in the original high-dose group (43%). Treatment failure was cited as the reason for termination of one 69-year-old man in the original placebo group. His baseline MMSE score was 21, and his final MMSE score at week 26 was 17. No other patients withdrew as a result of treatment failure. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Reason for termination stratified by original group. ::: ![](1471-2318-5-3-1) ::: Of the 4 patients who withdrew from the study due to disease progression, 3 were men between the ages of 57 and 64 years. The 57-year-old man was in the original high-dose group, with a baseline MMSE score of 16 and an MMSE score of 0 at the 234-week data collection. The other two male participants were aged 62 and 64 years with baseline MMSE scores of 22 and 20, respectively. The 62-year-old man in the original low-dose group withdrew at week 104 with an MMSE score of 9; the 64-year old man in the original high-dose group withdrew at week 156 with an MMSE score of 11. Interestingly, the 76-year-old woman in the original low-dose group, with a baseline MMSE score of 22, withdrew after the 26-week data collection (MMSE = 21). Quantitative (objective) analysis --------------------------------- The Kaplan-Meier survival analysis, shown in Figure [2](#F2){ref-type="fig"}, illustrates time to dropout from week 26 to week 234. The survival curve reveals a relatively steep decline in participation in the first 9 months of open-label extension, mostly related to adverse events (AEs), since almost all AEs causing discontinuation occurred during this phase, followed by a \"flattening\" of the curve. Fourteen participants were still taking high-dose rivastigmine at 2 years, 12 participants at 3 years, and 10 participants at 4 years. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Kaplan-Meier analysis of time to dropout from week 26 to week 234 ::: ![](1471-2318-5-3-2) ::: Of the subjects starting open-label rivastigmine, 25% were still participating at week 234. Of the 8 subjects still participating, 5 were female and only 1 reported a family history of AD. Interestingly, this group was characterized by a broad age range (57--85 years), a broad range of baseline MMSE scores (15--26), and by the fact that 5 of the 8 remaining participants at week 234 were in the original high-dose rivastigmine group. For the end point defined as a 5-point drop from the baseline MMSE score, the group mean was 94 weeks. However, an age-related observation was noted such that the mean time to a 5-point drop from baseline MMSE for the ≤ 70 age group was 67 weeks as compared with 122 weeks for the \>70 years age group. A similar trend was observed for scores on the ADAS-cog. For the group as a whole, mean time to a 4-point deterioration on the ADAS-cog was 84 weeks; with a mean of 70 weeks for the younger group and 104 weeks for the older group. \[In the original study, mean age across the 22 research sites was 74.5 years; mean age for participants in this report was 71.8 years\] Figures [4](#F4){ref-type="fig"} through 8 summarize mean scores for the ADAS-cog, CIBIC, GDS, PDS, and MMSE from week 26 through week 234. As expected, mean values for cognitive and functional status decline over time; however, significant within subject variability is present. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Mean scores on the Alzheimer\'s Disease Assessment Scale-cognitive subscale (ADAS-cog) for weeks 26--234. ::: ![](1471-2318-5-3-3) ::: Qualitative analysis -------------------- When evaluating qualitative data, it is important to examine particular patients in terms of the data collected and treatment response at end of study. Data for the youngest man and the elderly woman, considered side-by-side, seem counterintuitive. Part of the explanation, however, may rest with the caregivers\' experiences with and beliefs about the patients. This information can be accessed by reviewing the \"symptoms most troubling\" item from the CIBIC-Plus. This item asks, \"With respect to the above symptoms, which are the biggest problems for you and/or other caregivers?\" The spouse of the 57-year-old man reported that the \"symptoms most troubling\" for her were the patient\'s \"selective difficulties and his attitude problem\" (week 104, MMSE = 11) and that the patient \"doesn\'t try\" (week 130, MMSE = 7). These comments suggest a lack of acceptance of the patient\'s diagnosis and a lack of belief regarding the patient\'s documented decline that could explain continued clinic visits until, at week 234, the patient\'s MMSE score was 0. In contrast, the caregiver for the 76-year-old woman cited the fact that the patient \"doesn\'t want to leave the house\" (week 12) and the patient\'s anxiety about coming to clinic appointments (week 26) as the \"most troubling\" symptoms. These comments suggest that the caregiver was encountering patient resistance and observing patient distress, which were exacerbated by efforts to participate in the study. Therefore, further clinic visits were declined. Retrospectively, it is impossible to determine how the caregiver\'s beliefs and experience with the patient affect perceptions about disease status, treatment response, and decisions to continue or terminate treatment. These are interesting hypothesis-generating observations that should be explored in future investigations. Discussion ========== Limited data are available on the tolerability and effectiveness of cholinesterase inhibitor therapy for periods up to 3 years \[[@B4]-[@B7]\], but nothing has been reported in the literature concerning the percentages of patients remaining on therapy and its effect beyond this time point. This study adds to the limited long term data on patients treated with high-dose rivastigmine therapy by reporting descriptive data for up to 5 years. Findings presented in this report indicate that, in this sample of patients, high-dose rivastigmine therapy was well tolerated over a 5-year period. Of note, approximately two thirds of the participants still enrolled at week 234 were in the original high-dose rivastigmine group during the double-blind phase; this finding suggests that early therapy with rivastigmine may confer some benefit in delaying long-term progression of symptoms, as has been previously suggested by analysis of the combined 26 weeks of double-blind and first 26 weeks of open-label data from the B352 US trial \[[@B9]\]. Throughout the initial 26-week double-blind portion, patients receiving placebo steadily deteriorated, while those treated with high-dose rivastigmine were able to maintain their baseline level of performance on the ADAS-Cog \[[@B5]\]. This approximated a delayed-start design for the open-label portion, which demonstrated that patients who started rivastigmine late never \"caught up\" with patients who had been on high-dose rivastigmine from the beginning of the trial. This suggests a disease-progression-delaying effect of the drug, which may allow this population to maintain their autonomy for a longer period of time. However, it is important to emphasize the limitations of this data; the analysis was retrospective, the sample was small, there were significant numbers of drop-outs, and the availability of free rivastigmine ceased with FDA approval which occurred near to the end of the study Conclusions =========== In summary, long-term therapy with the ChE inhibitor rivastigmine was well tolerated with no dropouts due to adverse effects past the first 9 months of the open-label extension. In contrast to the generally reported community experience of relatively brief duration of therapy with ChE inhibitors in AD, 25% of patients in this study were still taking rivastigmine by the end of 5 years. Of interest is the multifactorial nature of reasons for treatment discontinuation. Only 1 of these patients withdrew from the study because of perceived treatment failure over the 5-year period. Disease progression accounted for the withdrawal of 4 patients. These results are informative both for the duration of treatment, and because the majority of the patients who continued treatment during this 5 year period were from the group originally titrated up to a high dose during the initial double-blind phase. Given that the sample size prohibits significance testing, these data suggest that rivastigmine therapy may be sustained over long periods of time in a significant percentage of patients with AD, and that high-dose therapy, particularly when begun early, may have an advantage in delaying the progression of the illness. Competing interests =================== MRF has received grant support and has served as consultant and received honoraria from Novartis Pharmaceuticals Corporation. MLL has no financial interest to declare. Authors\' contributions ======================= MF carried out the original clinical trial, conceived of the design for the observational study, and participated in the drafting and revisions of the manuscript. ML participated in the design of the observational study, performed statistical analysis, and participated in the drafting and revisions of the manuscript. Both authors read and approved the final manuscript. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Change in Clinician\'s Interview-Based Impression of Change for weeks 26--56. ::: ![](1471-2318-5-3-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Mean scores on Geriatric Deterioration Scale for weeks 26--234. ::: ![](1471-2318-5-3-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Mean scores on Progressive Deterioration Scale for weeks 26--156. ::: ![](1471-2318-5-3-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Mean scores on Mini-Mental Status Examination for weeks 26--234. ::: ![](1471-2318-5-3-7) ::: Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2318/5/3/prepub> Acknowledgments =============== \*The principal investigators for the ENA713 B352 Study Group are listed in the Acknowledgments. This study was supported by Novartis Pharmaceuticals Corporation, East Hanover, NJ. The participants in the ENA 713 B352 Study Group were as follows: Marc Bauder, MD, and Troy Willaims, MD, Clinical Studies, Peoria, AZ; Louis Cohen, MD, Clinical Studies, Sarasota, FL; Martin Farlow, MD, Indiana University Alzheimer\'s Clinic, University Hospital, Indianapolis; Michael Gold, MD, Behavioral Neurology Memory Disorders Clinic, University of South Florida, Tampa; John Feighner, MD, Feighner Research Institute, San Diego, CA; Donald Hay, MD, St Louis University Medical Center, St Louis, MO; Ranga Krishnan, MD, Duke University Medical Center, Durham, NC; Vinod Bhatnagar, MD, Center for Clinical Trials & Research Inc, Venice, FL; David Margolin, MD, Fresno, CA; Peter Ripley, MD, Clinical Studies, Cape Cod, S Yarmouth, MA; Nunzio Pomara, MD, Nathan S Kline Institute for Psychiatric Research, Orangeburg, New York and Brookdale Hospital Medical Center Neurology Department, Brooklyn, NY; Benjamin Seltzer, MD, Tulane University Medical Center, New Orleans, LA; James Webster, Jr, MD, McGaw Medical Center, Northwestern University, Chicago, IL; Lorna Charles, MD, Southern New Jersey Medical Institute, Stratford, NJ; George Hanna, MD, Neurology Suite, Fontaine Research Park, Charlottesville, VA; Walter Brown, MD, Clinical Studies, Ltd, Providence, RI; Robert Linden, MD, Pharmacology Research Institute, Long Beach, CA; Barry Gordon, MD, Cognitive Neurology, Johns Hopkins University School of Medicine, Baltimore, MD; Stuart Stark, MD, The Neurology Center, Alexandria, VA; Patricia Walicke, MD, and Michael Jann, PharmD, Buckhead Neurology, PC, Atlanta, GA and Center for Clinical Research, Mercer University Southern School of Pharmacy, Atlanta, GA; Jody Corey-Bloom, MD, PhD, Department of Neuroscience, UCSD, La Jolla, CA; Charles Echols, Jr, MD, Phoenix, AZ. Also, Richard D Hartman, PhD, Stephen M Graham, PhD, Peggy E Hayes, PharmD, John C Messina, Jr, PharmD, Peter Irwin, Novartis Pharmaceuticals Corporation, East Hanover, NJ. This study was monitored by Quintiles Pacific, Inc, San Diego, CA. The authors would also like to thank Amy Rothman Schonfeld, PhD, Robert Gilbert, STL, Mary Ellen S Congleton, and Pietro Mastrangeli for their editorial and artistic assistance.

PubMed Central

2024-06-05T03:55:52.548307

2005-1-19

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548267/", "journal": "BMC Geriatr. 2005 Jan 19; 5:3", "authors": [ { "first": "Martin R", "last": "Farlow" }, { "first": "Mary L", "last": "Lilly" } ] }

PMC548268

Background ========== Suction drainage in the management of mastectomy patients was used for the first time in 1947 \[[@B1]\] and has been found in various studies superior to other methods of fluid evacuation to minimize the dead space. The mechanism proposed is that the suction helps skin flaps to adhere to the chest wall and axilla sealing off all the leaking lymphatics\[[@B2],[@B3]\]. This reduces the incidence of post-operative seromas, hematoma formation and flap necrosis, which are, recognized complications of modified radical mastectomy \[[@B2],[@B3]\]. When no postoperative suction drains were used the incidence of seromas was found to be unacceptably high in various studies \[[@B4]\]. Prolonged drainage on the other hand, may increase the hospital stay and increase the risk of infection by allowing retrograde migration of bacteria \[[@B4]\]. Indiscriminate or premature withdrawl of postoperative drains irrespective of the amount of fluid drained may be accompanied by an increase in the incidence of axillary seromas \[[@B4]-[@B6]\]. If kept for longer periods it has been observed that drain itself might contribute to increased drainage and the risk of infection in addition to the increased hospital stay resulting in to wasteful utilization of the hospital resources. The amount of postoperative drainage is influenced by various factors like the clinical profile of the patient including the body mass index, extent of axillary lymph node dissection, number of lymph nodes dissected, use of elctrocautery, co morbid conditions and also the *negative pressure*on the suction drain \[[@B4]-[@B10]\]. The amount of postoperative fluid drained has been found to be significantly influenced by the negative pressure on the suction drainage. While the negative suction drain is logically expected to drain the fluid, a high negative suction drain may prevent the leaking lymphatics from sealing off thus leading to prolonged drainage leading to increased hospital stay \[[@B4]\]. The present prospective randomized clinical trial compared the postoperative wound drainage in patients with full suction drain (high) and those with half vacuum drainage system (low). The study also compared the drain volume, average hospital stay and postoperative morbidity between full vacuum and half vacuum suction groups. Methods ======= The study was conducted in one surgical unit of a tertiary care center over a period of two years.85, FNAC (fine needle aspiration cytology) proven cases of locally advanced breast cancer were randomized (using randomly ordered sealed envelops, which were opened immediately before the closure of the wound) into full vacuum suction (pressure = 700 g/m2) group -- (A) and 35 cases into half vacuum suction (pressure = 350 g/m2) group -- (B). The two groups were comparable in respect of age, weight and type of operation i.e. modified radical mastectomy (MRM). Following complete routine and metastatic work up, all patients received three cycles of Neoadjuvant chemotherapy (NACT) using CAF regime (Cyclophosphamide, Adriamycin, 5-Fluorouracil) and underwent Patey\'s modified radical mastectomy after three weeks of the last cycle. Surgery was performed by the same surgical team comprising of five surgeons (two senior and three resident surgeons) using a standardized technique with electro cautery. Axillary dissection was done up to level- III in all the cases. The boundaries of axillary dissection were defined by superior limit as the posterolateral border of the Pectoralis major muscle and axillary vein, medial limit being clavipectoral fascia or Hallstead\'s ligament, lateral limit as the anterior border of lattismus dorsi and the inferior limit being the angular vein joining the thoracodorsal vein. The long thoracic and thoracodorsal nerves were identified, dissected and preserved. Two silicone tube drains (12Fr) (one axillary and pectoral) were inserted in all the patients. All resected specimens were examined and the lymph nodes dissected, counted and assessed histo-pathologically for metastases. No patient received intra-operative blood transfusion. Both the drains were connected to a single 600 ml suction bottle (Romovac -Romson). In-group A (n = 50), drainage was performed using complete vacuum negative suction (700 g/m2) and in-group B (n = 35) with half vacuum suction drainage (350 g/m2). The pressure was also measured by attaching a manometer to the exit opening of the drainage bottle. The two groups were comparable with respect to age, weight (body mass index), type of operation indicating the success of randomization (Table. [1](#T1){ref-type="table"}). The drain was emptied every 24 hours to reset suction at the respective pressures and to measure the daily drain out put. External compression dressing was provided over the axilla for first 48 hrs and following that the patients were encouraged to do active and passive shoulder exercises. The outcomes measured were morbidity and the length of hospital stay. The total drain output was measured and recorded daily in both the groups, the drains were removed once the output was less than 30 ml in 24 hrs and the patients were discharged on the same day. The mean total drain output was measured in each group and compared. The mean hospital stay in both the groups was calculated and compared. The associated morbidity in the form of seroma formation, flap necrosis and wound infection during the postoperative period was recorded and compared in both the groups Statistical methods used ------------------------ Descriptive studies were performed with SPSS version 10 and group characteristics were compared using student t-test (Tables. [2](#T2){ref-type="table"}&[3](#T3){ref-type="table"}). Results ======= 1\. Half vaccum suction drains were removed earlier than full vacuum suction drains without any significant addition to the postoperative morbidity. 2\. The use of half vacuum drains after modified radical mastectomy reduced the hospital stay significantly without any increase in the postoperative morbidity. 3\. The high negative suction is an important contributory factor to the amount of drainage following breast surgery along with axillary dissection. While negative suction helps in prevention of postoperative seroma formation, a high negative suction may affect adversely by increasing the amount and duration of drainage. This probably is on account of persistent drainage from the lymphatics, which do not close due to high negative suction. Discussion ========== Seroma formation is the most frequently observed early complication after breast and axillary surgery. The use of closed suction drainage is a common practice that has been shown to reduce the incidence of seroma formation \[[@B1]-[@B6]\]. These drains are generally removed once the lymph production falls to less than 35--50 ml/24 hours, a level generally reached between 3--17 days after surgery \[[@B1]\]. The length of postoperative axillary drainage is a major cause of morbidity after axillary dissection as the patients are usually discharged once the drains are removed. The patients with suction drains in situ are normally managed in the hospital (although some authors advocate discharge with the drains in situ)\[[@B13]\]. Migration of bacteria along these drains has also observed to increase the risk of infection if the drains stay in situ for a long time \[[@B7]\]. Early or premature removal however has been found to be associated with an unacceptably high incidence of seroma formation and its continuation until fluid discharge is acceptably low leads to a prolonged stay in the hospital, which has a bearing on the cost of surgical management of breast cancer \[[@B1],[@B11]-[@B13]\]. Shortening the hospital stay has been shown to be an effective way of reducing the costs in the case of surgery for breast cancer and axillary drains are the main obstacles in achieving it \[[@B1],[@B8]-[@B10]\]. To reduce the hospital stay after MRM, early discharge with the drains in situ has been reported but discharging patients with drains in situ has an inherent difficulty faced by the patients in management of drains besides higher incidence of wound infection \[[@B13],[@B14]\]. The other disadvantages are discomfort for the patients, with difficulties undressing or using the toilet. It may be feasible with patients of higher cultural and social standing, but not all the patients have the required background. In a third world country where the patients are poor, uneducated coming from far and remote areas with limited medical facilities, there is an added difficulty in management of the drains away from the hospital. As most of our patients come from far flung rural areas with limited education, poor medical and communication facilities they were managed indoors until the drains were removed. There are other solutions proposed for prevention or reduction of fluid accumulation and early discharge after axillary dissection e.g by Patrek et al \[[@B15],[@B16]\] where several parallel drains were used. Suture obliteration of axillary space under skin flaps with sutures to the chest wall, approximation of the pectoralis major and the latissimus dorsi muscle in the form of axillary padding has been suggested by some authors \[[@B9],[@B17]\]. The incidence of seroma formation had reduced but the length of drainage was not specified in these studies. Further more suture approximation of the muscles may limit movement of the arm leading to shoulder dysfunction. Harada et al \[[@B14]\] used fibrin glue in rats to occlude transected lymph channels and obliterate the subcutaneous cavity. The association of seroma formation with large amounts of drainage before removal of the drain has already been established \[[@B18]-[@B20]\]. In one study it was observed that when the amount of fluid drained before removal of the catheter was less than 250 ml in three days no seromas developed and they concluded that it is safe to remove drains if the total amount of fluid drained during the first postoperative days is low. Yii et al \[[@B20]\] reported that removal of drains after 48 hours did not result result in seroma formation if the total amount of fluid drained before removal was less than 150 ml Proposed factors contributing to the increased drainage and seroma formation \[[@B2],[@B3],[@B8]-[@B15]\] --------------------------------------------------------------------------------------------------------- Patrek et al \[[@B15]\] examined 13 factors influencing fluid drainage. Only two (a large number of positive lymph nodes and previous biopsy) predicted greater drainage. 1.**Body mass index**. A significant linear relation exists between BMI and increased seroma formation was reported by (Boonman et al) 2\. **Technique**: Use of elctrocautery has been reported to be associated with increased incidence of seroma formation as compared to cold knife. It has also been reported that tissue ligation around the axillary vein rather than mere transection with knife or diathermy may reduce the amount of postoperative discharge, the technique was followed in the presented study \[[@B8]\]. 3 **Drains themselves**encourage drainage by stimulating tissue reactions or by suction \[[@B20]\] 4.**Early shoulder exercises**have been implicated but were not observed to be a factor in the present study \[[@B10],[@B11]\] Although early mobilization of the shoulder did not increase fluid discharge in various studies but it was reported to be an additional factor leading to increased drainage after axillary dissection \[[@B10],[@B11]\]. 5 **The negative suction**applied may prevent the lymphatics from closing leading to continuous leakage and discharge \[[@B18]\]. 6.**Extent of axillary dissection**. More seromas were seen when more lymph nodes were dissected from the axilla. The higher lymph node yield may well be an indirect measure of more extensive dissection performed. \[[@B3]\]. The drainage may also reflect the damage to the lymph vessels and therefore the number of lymph nodes dissected may have a bearing on the amount of drainage. In our study also, it was observed that patients with higher lymph node yield had a higher volume and duration of drainage although it was not found to be significantly different in both the groups because they were matched in all respects except the negative suction pressure of the drainage. Negative suction and the drainage --------------------------------- It is an accepted fact that negative suction prevents seroma collection and helps in the adherence of the walls of the axilla thus reducing the dead space and allowing the lymphatics to close. High negative suction pressure generated by the drain can maintain lymph drainage by a negative pressure gradient \[[@B18]\]. It is also reported that the high negative suction pressure does not allow the lymphatic channels to close leading to continuous drainage and a higher incidence of seroma formation \[[@B18]-[@B20]\]. There are studies to suggest that high negative suction may be beneficial in the sense that the amount of drainage would be more thus allowing an early adherence of walls of the axilla to the chest wall and reduction in the seroma formation \[[@B18]\]. However in the present study it was observed that high suction caused prolonged drainage, which can possibly be explained by the hypothesis that high negative suction may not allow, leaking lymphatics to close. Therefore no suction or high suction drainage both may contribute to the same result that is higher incidence of seroma formation and longer hospital stay. To strike a balance between not having suction at all and having a very high or full negative suction, half negative suction drainage was used in the present study to achieve a shorter hospital stay without any increase in the rate of post operative seroma formation. The external compression dressings in the first forty-eight hours perhaps helped in the adherence of the flaps and reduction of dead space without compromising on the shoulder mobility. This was found to effectively reduce the Hospital stay and also did not increase the postoperative morbidity as compared to high (full) negative suction group. Conclusions =========== Reducing the negative suction pressure applied to the drain (making it half suction) along with external compression dressings applied for first 48 hours can significantly reduce drainage from the axilla following modified radical mastectomy without increasing the incidence of seroma formation as was observed in this randomized prospective clinical study. The hospital stay was reduced considerably compared to a matched group with full suction drain (p \< 0.001). Half suction drain following axillary dissection in patients with carcinoma breast may thus be recommended as an effective approach to reducing the hospital stay and the cost of treatment without adding to the morbidity. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= CM was the surgeon in charge of the project and prepared the study design. VS was the first surgical assistant and did the data processing and statistical analysis. JP, the second surgical assistant assisted in the preparation of the manuscript. SS the Director of ICMR and AB the research officer, Tumor Biology Lab ICMR were responsible for the histopathological analysis and contributed to the preparation of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/11/prepub> Figures and Tables ================== ::: {#T1 .table-wrap} Table 1 Full vaccum group n = 50 Half vaccum group n = 35 p- value ------------------------------------ -------------------------- -------------------------- ---------- Mean Age (years) 49.54 (S.D = 9.821) 46 (S.D = 7.580) \- Mean Weight (kg) 54 (S.D = 6.543) 55 (S.D = 5.285) \- No. Of lymph nodes resected (mean) 12.5 (S.D = 1.619) 12 (S.D = 1.328) \- \% With positive lymph nodes 28% 30% \- NACT regime CAF CAF \- Surgery MRM MRM \- Mean volume drained (ml) 525 (S.D = 66.282) 325 (S.D = 39.612) \<0.001 Mean hospital stay (days) 10.8 (S.D = 1.603) 6 (S.D = 1.414) \<0.001 BMI 21.6 (S.D = 2.347) 21.7 (S.D = 1.800) \- Morbidity Flap necrosis 1 1 NS Wound infection 4 3 NS Seroma formation 2 1 NS Seroma aspirations required None None \- ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Non parametric test ::: Group n Mean Rank ----------- ------- ---- ----------- Drain vol 1.00 50 60.49 2.00 35 18.01 Total 85 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Mann Whitney U test ::: Drain vol ------------- ----------- Chi square 61.044 df 1 Asymp. Sig. \<0.05 :::

PubMed Central

2024-06-05T03:55:52.550300

2005-1-27

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548268/", "journal": "BMC Cancer. 2005 Jan 27; 5:11", "authors": [ { "first": "Vinay", "last": "Singhal" }, { "first": "JP", "last": "Singh" }, { "first": "Anju", "last": "Bansal" }, { "first": "Sunita", "last": "Saxena" } ] }

PMC548269

Background ========== Insulin-like growth factor binding protein (IGFBP)-3 is a multifunctional protein ferrying insulin-like growth factors (IGFs) in circulation and mediating growth suppression signals in cells. Serum IGFBP-3 protein (\< 5000 ng/ml) complexes with IGFs and an acid labile subunit (ALS), to extend the half lives and modulate the bio-availability of IGFs \[[@B1]\]. While a precise mechanism of action is not clear, the growth suppressive activity of IGFBP-3 depends on its nuclear translocation \[[@B2]\]. Other growth suppressors such as p53, retinoic acids, transforming growth factor (TGF)-β, and tumor necrosis factor (TNF)-α induce IGFBP-3 as a mediator of growth suppression \[[@B3]-[@B6]\]. The growth suppression by IGFBP-3 is independent from the modulation of IGFs action \[[@B7]-[@B9]\]. The functional importance of the IGFBP-3 in the growth suppression is noteworthy. IGFBP-3 is produced in most tissues, but the main site of production is liver. It is produced by non-parenchymal cells (endothelial and Kupffer cells) while parenchymal cells (hepatocytes) do not produce it under normal condition \[[@B10]\]. We postulate that IGFBP-3 is a gene induced by growth suppression signals such as p53 in hepatocytes. While the growth suppression imported by IGFBP-3 suggests the potential for tumor suppression, polymorphisms, but no significant mutations were observed in a survey of several tumors \[[@B11]\]. As gene silencing may occur without mutations, we recently investigated IGFBP-3 promoter hypermethylation in human hepatocellular carcinoma \[[@B12]\]. These promoter hypermethylations were subsequently reported in other tumors systems \[[@B13],[@B14]\]. Promoter analysis of IGFBP-3 indicated that the NaB-RE sequence is essential for the sodium butyrate (NaB) induced IGFBP-3 expression \[[@B15]\], but the importance of the eleven upstream p53 binding sites reported by Bourdon *et al*. were not confirmed until now \[[@B16]\]. The methylation hot spot we identified exactly matched the putative p53 binding sites that Bourdon *et al*. indicated. Thus, we postulated that these sites are important for the expression of IGFBP-3 induced by p53. Moreover, we hypothesized that the suppression of apoptosis mediated by IGFBP-3 due to the promoter hypermethylation will be a possible pathway of hepatocarcinogenesis. To explore this possibility, the functions of the promoter upstream binding sites of p53 were examined precisely in this study. Methods ======= Cell culture ------------ HepG2 cells were obtained from Japanese Cancer Research Resources Bank (Tokyo, Japan), and maintained in D-MEM supplemented with 10 % FCS (Life Technologies, Tokyo, Japan), antibiotic-antimycotics at 37°C in a humidified atmosphere of 95 % air and 5 % CO~2~. Plasmids -------- pGL2-IGFBP-3, kindly provided by Dr. Youngman Oh (Oregon Health Sciences University, Portland, OR), carries a 1.9 kb IGFBP-3 promoter (-1805/+69) in pGL2-Basic (Promega Corp., Madison, WI). A series of deletion mutant constructs, pGL2-270, pGL2-240, pGL2-210, pGL2-180, pGL2-150, pGL2-120, pGL2-90, pGL2-60, pGL2-30, and pGL2-1 containing the indicated fragments upstream of the transcription start site and 60 bp of fragments downstream of the transcription start site, were generated by PCR amplification of the promoter fragment and subsequent subcloning of the *Mlu*I-*Bgl*II fragment to pGL2-Basic (Table [1](#T1){ref-type="table"}, Fig. [2](#F2){ref-type="fig"}). The transcription start site of the IGFBP-3 promoter, +1, is based on the sequence determined by Cubbage *et al*. \[[@B17]\]. The plasmid containing site-directed mutations in putative p53 binding sites was generated by replacing the wild type *Mlu*I-*Xho*I fragment of pGL2-210 with a mutant fragment synthesized artificially (pGL2-210B, Table [1](#T1){ref-type="table"}, Fig. [3](#F3){ref-type="fig"}). A fusion gene with site-directed methylation was constructed by methylating the *Mlu*I-*Xho*I fragment (-210/-174) of pGL2-210 *in vitro*with *Sss*I methylase (New England Biolabs, Inc., Beverly, MA), and reconstituting the fragment and unmethylated vector (Fig. [4](#F4){ref-type="fig"}). pCMV-p53 and pCMV-p53mt135 were obtained from BD-Biosciences (East Meadow Circle, Palo Alto, CA). Transient transfection ---------------------- HepG2 cells were transiently transfected using the FuGENE 6 transfection reagent according to the manufacturer\'s instructions (Roche Molecular Biochemicals, Indianapolis IN). Cells were seeded at a density of 5 × 10^4^cells/well in 24-well plates. After 24 hours, cells were transfected with 0.25 μg/well of reporter plasmid DNA in serum-containing medium. Forty-eight hours post transfection, cells were washed twice with PBS and collected for luciferase assays. Transfections were performed in quadruplicate and experiments were performed at least two times. Luciferase assay ---------------- Luciferase activities of cell lysates were measured according to the manufacturer\'s instructions (Promega Corp. Madison, WI) using a liquid scintillation counter (Aloka, LSC-700, Tokyo, Japan). Luciferase activities were normalized for total protein determined using the Bradford Assay (Bio-Rad Laboratories, In., Hercules, CA). EMSA (electrophoresis mobility shift assay) ------------------------------------------- 278 bp of the *Mlu*I-*Bgl*II fragment of pGL-210 were labelled using \[α-^32^P\]dCTP by end filling with Klenow fragment, and used for EMSA. Oligonucleotide DNAs (BP3WPSF: 5\'-GGCTGCAGCG GGCGTGCGCA CGAGGAGCAG GTGCCCGGGC GAGTCTCGAG CTGCACGCCC CCGAGCTCGG-3\', BP3WPSR: 5\'-CCGAGCTCGG GGGCGTGCAG CTCGAGACTC GCCCGGGCAC CTGCTCCTCG TGCGCACGCC CGCTGCAGCC-3\'), comprising the promoter sequence of -210/-149, were custom-made (Sigma-genosys Japan, Ishikari, Japan), annealed one hour at room temperature, and used as cold competitor in the assay. EMSA was performed in a 20 μl reaction containing 10 mM Tris (pH 7.5), 2.5% Glycerol, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, and in the presence of 50 ng/μl of double-stranded poly \[d(I-C)\], 5000 cpm (2 ng) of ^32^P-labeled probe DNA, and 2.5 μg of H~2~O~2~treated MCF7 nuclear extract (Active motif LLC, Palomar, CA). The reaction mixture was incubated at 14°C for 20 min. 20 μl of each reaction mixture was then loaded onto a native 4 % polyacrylamide gel containing 0.5 × Tris-Glycine buffer (25 mM Tris, 190 mM Glycine, 1 mM EDTA pH 8.3), and electrophoresed at 14°C, 100 V for 1 hr. For the supershift assay, a p53 antibody (Ab-2, Oncogene Research Products, San Diego, CA) was used. Results ======= Deletion analysis ----------------- We identified a hot spot of promoter hypermethylation in human hepatocellular carcinoma and its cell lines in the promoter upstream region of IGFBP-3 (Fig. [1](#F1){ref-type="fig"}). As methylation sites were identified to the cluster of putative p53 binding sites, we examined the role of these sites in IGFBP-3 expression. First, we constructed promoter deletion mutants of IGFBP-3 and examined the expression of the reporter gene in the absence (Fig. [2A](#F2){ref-type="fig"}) and presence (Fig. [2B](#F2){ref-type="fig"}) of p53 expression by the co-transfection of a p53 expression plasmid (pCMV-p53). As the transfection efficiency was not fully controlled, and p53 overexpression may to cause massive changes in cellular conditions, we did not compare results between the presence and absence of p53. Even though, we can obtain clear results about the effect of p53 to IGFBP-3 expression. The pattern of expression of deletion mutants was clearly different in the absence and presence of p53. In the absence of p53 overexpression, we identified the NaB-RE sequence as an essential site for expression, and deletion of p53 binding site enhanced the gene expression (Fig. [2A](#F2){ref-type="fig"}). These observations were similar to those of Walker *et al*. \[[@B15]\], but differed as HepG2 cell does not require NaB or Tricostatin A (TSA) for its activation. In contrast, in the presence of p53 over-expression, we identified that four p53 binding sites between -210 to -150 (relative transcription start site as +1) were essential for IGFBP-3 expression (Fig. [2B](#F2){ref-type="fig"}). When the deletion constructs were co-transfected with pCMV-p53mt135, the expression of the IGFBP-3 promoter was suppressed to a background level (almost same as blank constructs) in all constructs (data not shown). This indicates that IGFBP-3 expression we observe is tightly regulated by p53. Site-directed mutagenesis ------------------------- To confirm the importance of p53 binding sites between -210 to -150, we abrogated one of p53 binding sites by site-directed mutagenesis (C to T at -179 and G to C at -176, Fig. [3A](#F3){ref-type="fig"}). In the absence of p53 overexpression, there exist little differences in expression between the wild type and mutant construct (74 % relative to the wild type) (Fig. [3B](#F3){ref-type="fig"}). However, in the presence of p53 overexpression, IGFBP-3 expression was strongly decreased in the mutant construct (3.4 %) relative to wild type (Fig. [3B](#F3){ref-type="fig"}). For reasons already mentioned, we did not compare the result between in presence and absence of p53. Site-directed methylation ------------------------- Next, we constructed *in vitro*methylated promoter constructs to evaluate the effect of methylation (Fig. [4A](#F4){ref-type="fig"}). For methylation, the *Mlu*I-*Xho*I fragment of pGL2-210 was methylated with *Sss*I methylase and reconstituted with unmethylated reporter vector fragment. As we used linear constructs for the transfection, the expression of luciferase was strongly suppressed compared to circular plasmids (0.7 %). But similar patterns compared to site directed mutation were observed. Although the expression of IGFBP-3 was slightly enhanced in the absence of p53 overexpression in the methylated construct, it was decreased in the presence of p53 overexpression in the methylated construct (Fig. [4B](#F4){ref-type="fig"}). In this experiment, at a glance, we observed induction of IGFBP-3 expression by p53, but the transfection efficiency of linear plasmids is low, while the relative levels of p53 and the availability of putative negative regulators are extremely different from other experiments. Thus, we cannot conclude in this case, whether or not there is induction by p53. EMSA ---- EMSA was performed to determine whether p53 protein binding was disturbed by methylation. In this assay, the wild type and methylated promoter fragments (278 bp, -210/+60) were incubated with H~2~O~2~-treated MCF7 nuclear extract that express high level of p53. In the wild type promoter probe, we observed supershifted complex (Fig. [5](#F5){ref-type="fig"}, lane 4, indicated by arrows) when p53 antibody (Ab-2) is added to the reaction mixture. A 100-fold molar excess of a cold 70 bp sequence containing a p53 binding site (-210/-149), competed the probe (Fig. [5](#F5){ref-type="fig"}, lane 5). In methylated probe, we observed no supershifted complex (Fig. [5](#F5){ref-type="fig"}, lane 9). As we used probes that contain a Sp1/GC box and TATA box, we observed the shift and supershift bands in the methylated probe as well as unmethylated probe. These bands were postulated to be due to p53 binding to the nuclear factor such as p300. These observations indicate that p53 binding to the *IGFBP3*promoter sequence is blocked or at least attenuated by hypermethylation. Discussion ========== We have indicated that the p53 binding sites upstream of IGFBP-3 promoter are essential for its induction by p53, and that the induction can be suppressed by promoter hypermethylation in the human hepatoblastoma cell line HepG2. A working model of the p53 action in IGFBP-3 promoter upstream binding sites is summarized in Fig. [6](#F6){ref-type="fig"}. In this model, in normal cells, IGFBP-3 is induced by factors such as growth hormone (GH) and IGFs, and steady levels of expression are observed in some cells. That the deletion of p53 binding sites enhances the expression of IGFBP-3, suggests existence of negative regulators (Fig. [6A](#F6){ref-type="fig"}). In apoptotic cells, p53 tetramers at high levels of expression bind to upstream binding sites. These tetramers recruit the p300 complex to its binding site, thereby a p53-dependent high level of expression (Fig. [6B](#F6){ref-type="fig"}). In tumor cells, deletions, mutations, methylations of p53 binding sites, or mutations of p53 such as p53mt135, disturb the binding of p53 tetramers to their binding sites, and this prevents the binding of the p300 complex to IGFBP-3 promoter. As a result, the expression of IGFBP-3 is suppressed in tumor cells (Fig.[6C](#F6){ref-type="fig"}). In this model, promoter hypermethylation has the same effect as promoter mutation in determining IGFBP-3 expression. Our observations of promoter hypermethylation in human hepatocellular carcinomas and derivative cell lines (12), and the observations in this report strongly support the notion that *IGFBP3*is a true tumor suppressor gene. *IGFBP3*is a gene that is silenced by biallelic hypermethylation or hypermethylation and loss of heterogeneity (LOH) in human hepatocellular carcinoma. As reported recently \[[@B14]\], we have also observed the reduced expression of IGFBP-3 in several tumors such as, breast (9/41), uterus (11/42), ovary (6/16), kidney (6/20), and prostate (1/4) using the cancer profiling array (BD bioscience, data not shown). We therefore postulate that the tumor suppressor role of IGFBP-3 will not be limited to HCCs. In addition, there are also many reports of IGFBP-3 overexpression in tumors from breast \[[@B18]\], prostate \[[@B19]\], kidneys \[[@B20]\], and lung squamous cells \[[@B21]\], so on. We thus anticipate the existence of additional defects, such as papilloma virus infections that inactivate IGFBP-3 \[[@B22]\], TGF-β / Rb signalling abnormalities that often coincide with IGFBP-3 overexpression \[[@B23]-[@B25]\], or as yet unknown defects in IGFBP-3 receptor function leading to IGFBP-3, for these overexpression in tumors. IGFBP-3 is a ubiquitous, multifunctional protein, whose importance as a carrier of IGFs is evident. The absence of gross loss-of-function mutations of IGFBP-3 observed to date likely underscores its functional importance. We hypothesize that IGFBP-3 is a gene whose basal level of expression is essential for cell survival, but upon induction by p53, high levels of expression of IGFBP-3 induces apoptosis. Alternatively, IGFBP-3 may be a gene that is essential for cell survival when induced by growth hormones or IGFs, but functions as an apoptotic mediator when induced by p53. We observed slight base changes within p53 binding sites strongly influenced the induction of IGFBP-3 by p53. As SNPs that change the expression level of IGFBP-3 were within p53 binding sites \[[@B26]\], and it was reported that the IGFBP-3 is differentially activated by p53 mutants \[[@B27],[@B28]\], we postulate that the expression and functions of IGFBP-3 is controlled in some way by p53 binding sites in the promoter of IGFBP-3. This may include the intronic p53 binding sites as well as the upstream sites explored here. IGFBP-3 may, therefore, have an important function in tumor development through p53 control. We identified the MyoD (-195/-186) and WT1 (-164/-156) binding sites as well as p53 binding sites at the hypermethylation hot spot in HCC by promoter analysis using TRANSFAC (v 4.0). As MyoD is a transcription factor that can induce apoptosis, and WT1 is a tumor suppressor gene, the existence of a binding site for these putative regulator genes in the hot spot of the promoter of IGFBP-3 suggest the possibility that these genes also use IGFBP-3 as a mediator of their actions. Conclusions =========== We conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells. As IGFBP-3 functions downstream of many growth suppressors and its growth suppression effects are drastic, and as it is a small-sized secreted protein, the use of IGFBP-3 in tumor therapy will be a promising option. Abbreviations ============= ALS; acid labile subunit, D-MEM; Dulbecco\'s modification of Eagle\'s medium DTT; dithiothreitol, EDTA; ethylenediaminetetra-acetic acid, EMSA; electrophoresis mobility shift assay, FCS; fetal calf serum, HCC; hepatocellular carcinoma, IGF; Insulin-like growth factor, IGFBP-3; Insulin-like growth factor binding protein-3, LOH; loss of heterogeneity, NaB; sodium butyrate, NaB-RE (sodium butyrate-responsive region), PBS; phosphate buffered saline, PCR; polymerase chain reaction, TGF-β; transforming growth factor-β, TNF-α; tumor necrosis factor-α, SEM; standard error of mean, SNP; single nucleotide polymorphism. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= TH carried out these studies and manuscript preparation, KN and YI participated plasmid construction and reporter assay, TS and HS participated the EMSA, EY helped the array study, TO participated in the design of the study and performed the statistical analysis. NK conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/9/prepub> Acknowledgements ================ We Thank Dr. Y. Oh (Oregon Health Sciences University, Portland, OR) for providing pGL2-IGFBP-3 plasmid and Dr. R. Chaparro (Albert Einstein College of Medicine, New York, NY) for critical reading of this manuscript. This work was supported in part by grants-in aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (\#11770273). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Schematic genomic structure of the *IGFBP3*gene**. There exist 11 p53 consensus sequences upstream of the promoter \[16\] and two binding sites in introns \[3\]. Polymorphisms and hypermethylations are identified in the promoter upstream p53 binding sites \[26, 12\]. ::: ![](1471-2407-5-9-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Deletion analysis of IGFBP-3 promoter for the induction by p53**. HepG2 cells were transiently transfected with the panel of IGFBP-3 promoter-luciferase reporter constructs with (B) or without (A) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown. ::: ![](1471-2407-5-9-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Mutant analysis of IGFBP-3 promoter for the induction by p53**. A, Schematic of four wild type p53 consensus sequence of IGFBP-3 (-159/-209) of pGL2-210 and mutant sequence carrying point mutations in the core consensus sequence (-179 C to T, -176 G to C) of pGL2-210B. B, HepG2 cells were transiently transfected with wild type IGFBP-3 promoter-luciferase reporter constructs pGL2-210 or mutant IGFBP-3 promoter-luciferase reporter constructs pGL2-210B with (filled box) or without (open box) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown. ::: ![](1471-2407-5-9-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Methylation analysis of IGFBP-3 promoter for induction by p53**. A, Schematic of four wild type p53 consensus sequence of IGFBP-3 (-159/-209) of pGL2-210 and a sequence of the methylated construct carrying the CpG methylation in the core consensus sequence (underlined) of pGL2-210. B, HepG2 cells were transiently transfected with wild type IGFBP-3 promoter-luciferase reporter construct or methylated IGFBP-3 promoter-luciferase reporter construct with (filled box) or without (open box) co-transfection by pCMV-p53, and luciferase activity was measured after 48 h. Mean count of luciferase activity +/- SEM is shown. ::: ![](1471-2407-5-9-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Specific binding of p53 to IGFBP-3 promoter and its inhibition by methylation**. 2 ng of end-labelled 278 bp of *Mlu*I-*Bgl*II fragment of pGL-210 were used for EMSA. To construct the methylated probe, a labelled fragment was methylated *in vitro*with *Sss*I methylase. Each reaction contained the components listed in Materials and Methods. 2.5 μg of H~2~O~2~-treated MCF7 with an induced p53 expression was used as nuclear extract. Supershift was obtained using 0.1 μg of anti-p53 (Ab-2). As competitor, 200 ng of the fragment that carries the core p53 binding site (-210/-149) were used. The mixture was incubated at room temperature for 20 min and analyzed on 4 % nondenaturating polyacrylamide gel in 0.5 × Tris-Glycine buffer. Supershift bands observed in lane 4 were indicated by arrows. ::: ![](1471-2407-5-9-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Working model of p53 action to IGFBP-3 promoter**. In normal cells, IGFBP-3 is induced by such as growth hormone and IGFs, and steady level of expression is observed in some cells. As the deletion of p53 binding sites enhances the expression of IGFBP-3, the binding of negative regulators is expected (Fig. 6A). In apoptotic cells, p53 tetramers at high level of expression bind to upstream binding sites. These tetramers recruit the p300 complex to its binding site, and a p53 dependent high level of expression is observed (Fig. 6B). In tumor cells, deletions, mutations, methylations of p53 binding sites, or mutations of p53 such as p53mt135, disturb the binding of p53 tetramers to its binding sites, and this prevents the binding of p300. As a result, the expression of IGFBP-3 is suppressed in tumor cells. ::: ![](1471-2407-5-9-6) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Oligonucleotide DNAs used for construction of IGFBP-3 promoter mutants. ::: ------------------------------------------------------------------------------------------------------------------- **Plasmid** **Cloned fragment\*** **sense primer\*\***(5\' to 3\') **antisense primer\*\***(5\' to 3\') ----------------- ----------------------- ---------------------------------- -------------------------------------- pGL2-270 -270/+60 ggacgcgtctggagtgccggggtggc ggagatctgctgtggaatccaggcag pGL2-240 -240/+60 ggacgcgtggttcttgtagacgacaa ggagatctgctgtggaatccaggcag pGL2-210 -210/+60 ggacgcgtcgggcgtgagcacgagga ggagatctgctgtggaatccaggcag pGL2-180 -180/+60 ggacgcgtgcgagtctcgagctgcac ggagatctgctgtggaatccaggcag pGL2-150 -150/+60 ggacgcgtggccccggctgctcaggg ggagatctgctgtggaatccaggcag pGL2-120 -120/+60 ggacgcgtcccgcagccgtgcctgcg ggagatctgctgtggaatccaggcag pGL2-90 -90/+60 ggacgcgtcctcccaacccccactcc ggagatctgctgtggaatccaggcag pGL2-60 -60/+60 ggacgcgtccggggcgtgtcctgggc ggagatctgctgtggaatccaggcag pGL2-30 -30/+60 ggacgcgttatatacgggccggcgcg ggagatctgctgtggaatccaggcag pGL2-1 +1/+60 ggacgcgtagatgcgagcactgcggc ggagatctgctgtggaatccaggcag pGL2-210B\*\*\* -210/+60 ggacgcgtctgcagcgggcgtgagcacga\ agctcgagagtcacccgggcacctgctcc\ ggagcaggtgcccgggtgactctcgagct tcgtgctcacgcccgctgcagacgcgtcc ------------------------------------------------------------------------------------------------------------------- \*; The number is indicated by transcription start site as +1. \*\*; *Mlu*I restriction site is included in sense primers and *Bgl*II restriction site is included in antisense primers. \*\*\*; pGL-210B was made by replacing *Mlu*I-*Xho*I fragment of pGL2-210 with the oligonucleotide DNA indicated. :::

PubMed Central

2024-06-05T03:55:52.552213

2005-1-20

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548269/", "journal": "BMC Cancer. 2005 Jan 20; 5:9", "authors": [ { "first": "Tadashi", "last": "Hanafusa" }, { "first": "Toshiyuki", "last": "Shinji" }, { "first": "Hidenori", "last": "Shiraha" }, { "first": "Kazuhiro", "last": "Nouso" }, { "first": "Yoshiaki", "last": "Iwasaki" }, { "first": "Eichiro", "last": "Yumoto" }, { "first": "Toshiro", "last": "Ono" }, { "first": "Norio", "last": "Koide" } ] }

PMC548270

Background ========== Type II restriction endonucleases (REases) comprise one of the major families of endonucleases and one of the largest groups of experimentally characterized enzymes (comprehensively reviewed in: \[[@B1]\]). The \"orthodox\" Type IIP REases are dimeric, they recognize a short (4--8 bp) palindromic sequence of double-stranded DNA and in the presence of Mg^2+^, catalyze the hydrolysis of phosphodiester bonds at precise positions within or close to this sequence, leaving \"blunt\" or \"sticky\" ends (with a 5\' or 3\' overhangs). The enzymes that do not fit this definition or exhibit certain structural and functional peculiarities, have been classified into several subtypes (review: \[[@B2]\]). REases coupled with DNA methyltransferases (MTases) of similar specificity form restriction-modification (RM) systems, which are ubiquitous among Bacteria and Archaea \[[@B3]\]. While cleavage at specific sequences provides efficient means of destroying foreign DNA, methylation of these sequences in the prokaryotic chromosome renders them resistant to REase and thereby protects the own DNA from cleavage. Because cleavage of the chromosomal DNA in unmodified sequences would be deletorious for the cell, the REases must maintain extremely high specificities, tightly coupled with that of the methyltransferase. A change in just one base pair of the \"cognate\" site can reduce the ratio *k*~cat~/*K*~m~for DNA cleavage by a factor ≥10^6^\[[@B4]\]. To date, only crystal structures of 15 REases have been solved, compared to over 3000 biochemically characterized enzymes (review: \[[@B1]\]; see also \[[@B3]\] for updates). It was found that they share a characteristic structural core and a very weakly conserved catalytic motif (P)[D]{.underline}-X~n~-([D/E]{.underline})-X-[K]{.underline} (where X is any amino acid), together with a number of non-specific and structure-specific nucleases, suggesting that these proteins are evolutionarily related despite the absence of overall sequence similarity (review: \[[@B5]\]). The comparison of crystal structures of members of this so-called \"PD-DxK\" superfamily suggested that the catalytic and DNA-binding regions are major determinants of structural stability of these proteins \[[@B6]\]. Structural comparisons revealed also two major branches or classes, α and β, whose archetypal members were the enzymes that cleave DNA to generate 4 nt long 5\' \"sticky ends\", for instance EcoRI (α-class), and those that generate \"blunt\" ends after the cleavage, for instance EcoRV (β-class) \[[@B7]-[@B9]\]. Using the experimentally solved structures as templates, bioinformatics methods such as iterative sequence database searches and protein fold-recognition have been used to predict the active site in some REases \[[@B10]-[@B13]\]. From these analyses it was learned that certain functional peculiarities, like the requirement of a binding of an uncleaved effector site for cleavage of another site characteristic for Type IIE enzymes, evolved independently in the α and β branches of the PD-DxK superfamily \[[@B11],[@B13]-[@B15]\]. It was also found that other REases belong to completely unrelated superfamilies, with different three-dimensional folds and catalytic sites (review: \[[@B9]\]): BfiI is a member of the phospholipase D (PLD) superfamily \[[@B16]\], Eco29kI belongs to the GIY-YIG superfamily \[[@B17]\], and KpnI and a few other REases belong to the HNH superfamily \[[@B17]-[@B19]\]. While the structural information is essential to infer the molecular basis of sequence specificity in REases, the lack of overall sequence conservation in these enzymes, the absence of invariable residues even in the active site and the presence of several alternative folds make structure prediction and classification extremely difficult. SfiI is a REase isolated from *Streptomyces fimbriatus*. It recognizes the interrupted palindromic sequence 5\'GGCCNNNN\^NGGCC3\', where N denotes any base, and cleaves it as indicated by \"\^\", leaving 3\' extensions 3 nt long \[[@B20]\]. SfiI is a prototype of Type IIF enzymes, which function as tetramers that bind simultaneously to two recognition sites and cleave both sites concertedly \[[@B21]\]. However, a structural model of SfiI, which could be used as a platform to study its sequence-function relationships, is not yet available. Thus, despite the availability of a large body of biochemical data on how SfiI interacts with the DNA substrate (mainly on the kinetics of protein-DNA interactions with different substrates and the geometry of DNA looping \[[@B22]-[@B25]\], but not on the \"residue-level\" details thereof), the identity of amino acid residues important for dimerization, tetramerization, DNA binding and cleavage remains completely unknown. We have therefore carried out bioinformatics analyses of SfiI that allowed to identify its closest relative amongst REases with known structure and use this information to construct a tertiary model of SfiI in complex with its target DNA. Results and discussion ====================== In the absence of experimentally determined protein structures, homology-based models may serve as working models for the investigation of sequence-structure-function relationships between diverged enzymes \[[@B26]\]. Homology-modeled structures may be of too low resolution to characterize the protein-protein or protein-DNA contacts at the atomic level, but they can suggest which sequence regions or individual amino-acids are essential components of the binding surfaces. In particular, identification of amino acids potentially involved in protein-DNA contacts may guide mutagenesis experiments aimed at the engineering protein variants with novel specificities. However, homology modeling requires a homologous template structure to be identified and the sequence of the protein of interest (a target) to be correctly aligned to the template. Identification of the three-dimensional fold of SfiI ---------------------------------------------------- The sequence of SfiI showed no significant similarity to any other protein sequences. Also among the proteins reported by BLAST with sub-optimal scores, there were no proteins of known structure and no nucleases (data not shown) that could hint at potential relationships of SfiI to any previously characterized protein superfamily. Thus, in order to identify a template structure for modeling of SfI we used the threading approach, which allows to assess the compatibility of the target sequence with the available protein folds based not only on the sequence similarity but also on the structural considerations (match of secondary structure elements, compatibility of residue-residue contacts, etc.) (reviews: \[[@B27],[@B28]\]). The SfiI sequence was therefore submitted to the GeneSilico protein fold-recognition metaserver \[[@B29]\]. As expected, fold-recognition methods that rely only on sequence similarity (PDB-BLAST, and FFAS) failed to identify any significant matches between SfiI and proteins with known structrues. However, several threading methods that explicitly use the structural information from the templates reported a match between SfiI and the structure of a Type II REase BglI \[[@B30]\], a member of the PD-DxK superfamily of nucleases (FUGUE \[[@B31]\]: 4.25, INBGU \[[@B32]\]: 3.8, SAM-T02 \[[@B33]\]: 0.13, 3DPSSM \[[@B34]\]: 4.4; note that these scores are not normalized as each server uses a different evaluation system; see the individual references for details). Additionally, FUGUE reported a match (low score 3.17) between SfiI and the structure of another REase, EcoRV \[[@B35]\]. Despite the scores reported by the individual threading methods (except FUGUE for BglI) were hardly significant, the consensus server Pcons5 \[[@B36]\] assigned a significant score (1.35) to the BglI structure as a potential modeling template. Homology modeling of the SfiI monomer ------------------------------------- A homology model of SfiI was constructed based on the alignments reported by threading methods, using the \"FRankenstein\'s Monster\" approach \[[@B37]\] (see Methods). Since the PD-DxK nuclease fold was selected by Pcons as the only reasonable template and no other nuclease folds were identified by the FR methods, only alignments between SfiI and the PD-DxK superfamily members BglI and EcoRV were used. The final model was constructed by iterating the homology modeling procedure (initially based on the raw FR alignments), evaluation of the sequence-structure fit by VERIFY3D, merging of fragments with best scores, and local realignment in poorly scored regions. Local realignments were constrained to maintain the overlap between the secondary structure elements found in the bglI structure used as the modeling template, and predicted for SfiI. This procedure was stopped when all regions in the protein core obtained acceptable VERIFY3D score (\>0.3) or their score could not be improved by any manipulations, while the average VERIFY3D score for the whole model could not be improved. The final model, comprising residues 13--240 obtained the average VERIFY3D score of 2.6. The alignment between SfiI and BglI is shown in Figure [1](#F1){ref-type="fig"}, the corresponding final model of the monomer is shown in Figure [2](#F2){ref-type="fig"}. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Alignment between SfiI and structurally characterized REases.**A) Fold-recognition alignment between full-length sequences of SfiI and BglI. Amino acids are colored according to the physico-chemical properties of their side-chains (negatively charged: red, positively charged: blue, polar: magenta, hydrophobic: green. Pairs of residues conserved between SfiI and BglI are highlighted. Putative catalytic residues are indicated by \"\#\", putative DNA-binding residues are indicated by \"\*\". Secondary structure elements of BglI are shown below the alignment. Numbers of amino acid residues at the N-terminus of each panel are shown. B) Structure-based sequence alignment of the conserved core, corresponding to the PD-(D/E)XK motif, including SfiI, BglI, and other selected REases from the β-class. Conserved residues of the active site are highlighted. ::: ![](1472-6807-5-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Homology model of the SfiI monomer.**A) Superposition of the BglI template structure (red) and the SfiI model (blue). The CCGG half-site of the DNA target is shown in green. The Ca^2+^ions from the BglI structure are shown as white dots. B) SfiI model colored according to the sequence conservation: residues identical between SfiI and BglI are shown in blue, residues with physico-chemically similar side chains are in green, dissimilar residues are in yellow and red. The putative conserved active site is shown in the wireframe representation. ::: ![](1472-6807-5-2-2) ::: Modeling of the SfiI dimer in complex with the DNA -------------------------------------------------- BglI belongs to the \"EcoRV-like\" β-class of PD-DxK nucleases. The most typical features of REases from this class are: antiparallel orientation of the 5^th^strand of the common β-sheet and recognition of the DNA by an additional β-sheet formed by extended loops between the common secondary structure elements \[[@B5],[@B7],[@B9]\]. Most of β-class PD-DxK REases (including EcoRV) exhibit a similar mode of dimerization, which results in positioning of the two active sites as to cut the pair of the opposite phosphodiester bonds in the middle of the recognition sequence and thereby produce the \"blunt\" ends. BglI is exceptional in that its mode of dimerization is completely different, which leads to a different arrangement of the active sites and the sequence-recognition loops, resulting in the recognition of an interrupted sequence 5\'GCCNNNN\^NGGC3\' and cleavage in the position indicated by \"\^\" that yields 3\' ends 3 nt long. SfiI also recognizes an interrupted sequence 5\'GGCCNNNN\^NGGCC3\' and cleaves it in the same manner and therefore can be regarded as a more specific variant of BglI. These striking functional similarities, together with the results of the threading analysis, suggest that SfiI is indeed closely related to BglI and that both enzymes interact with their substrate DNA in a similar manner. Thus, we modeled the structure of the SfiI dimer in complex with the DNA based on the available crystal structure of BglI \[[@B30]\]. Briefly, the SfiI monomer model was duplicated and each of the copies was superimposed onto the corresponding monomer in the BglI dimer. A few minor steric clashes between the side-chains of residues at the protein-protein interface were removed by choosing alternative rotamers for the respective amino acids. The DNA duplex (sequence 5\'ATCGCCTAATAGGCGAT3\') was copied from the BglI co-crystal structure (1 dmu) \[[@B30]\] and \"mutated\" to 5\'AT[G]{.underline}GCCTAATAGGC[C]{.underline}AT3\' using HyperChem 7.1 (Hypercube, Inc.), followed by local geometry optimization. One of the adenine residues in the mismatched A/A base pair in the middle of the DNA molecule was \"mutated\" to T/A. The curvature of the DNA remained unchanged. Essentially, the global structure of the protein-DNA complex for SfiI remains exactly as in the BglI structure, as reliable modeling of macromolecular interactions remains beyond the capabilities of the existing methods. The model of SfiI dimer is shown in Figure [3](#F3){ref-type="fig"} and is available for download from <ftp://genesilico.pl/iamb/models/R.SfiI/>. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Model of the SfiI dimer.**Individual subunits are shown in yellow and blue. The modeled DNA sequence is shown in green, the specifically recognized CCGG half-sites are in red. ::: ![](1472-6807-5-2-3) ::: Model-based identification of amino acid residues important for catalysis, DNA-binding and dimerization of SfiI --------------------------------------------------------------------------------------------------------------- In the proposed model of SfiI, the spatial configuration of the catalytic residues is typical for an active site architecture conserved among PD-DxK nucleases. We predict that the active site of SfiI comprises residues: E55, D79, D100 and K102, which superimpose well on the catalytic residues of BglI: E87, D116, D142 and K144, respectively (Figure [4](#F4){ref-type="fig"}). We predict that the DNA-binding mode of SfiI will be very similar to that of BglI, with the side chains of residues S210 and R218 (homologs of D268 and R277 in BglI) involved in the recognition of the inner C/G base pair (Figure [5a](#F5){ref-type="fig"}), backbone oxygen and the side chain of K208 (a homolog of K266 in BglI) recognizing the middle C/G base pair (Figure [5b](#F5){ref-type="fig"}), and the side chain of R220 (a homolog of R279) recognizing the G of the middle G/C base pair (Figure [5c](#F5){ref-type="fig"}). The specificity of SfiI towards the outer G/C base pair, not discriminated by BglI, can be explained by the development of new contacts made by residues from a divergent loop adjacent to the REase active site and comprising residues 104--110 of SfiI and 146--155 of BglI. In BglI, D150 makes specific contacts to the middle C/G base pair and the G/C base pair \[[@B30]\], however this residue is not conserved in SfiI (Figure [1](#F1){ref-type="fig"}). Instead, we predict that the changes of the loop length and the amino acid substitutions lead to a different conformation of the corresponding loop in SfiI, which allows R109 (not present in BglI) to make a specific contact to the G of the outer G/C base pair (Figure [5d](#F5){ref-type="fig"}). Other residues from the same loop, such as K107 may also contribute to the specific sequence recognition by SfiI by making contacts to either of the two G/C base pairs. It is noteworthy that according to our model of SfiI, the majority of specific contacts are achieved by three Arg residues (R109, R218, and R220). These predictions can be tested by site-directed mutagenesis of the respective residues to Ala and testing whether the mutant proteins are proficient in DNA cleavage and/or binding. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Superposition of SfiI and BglI structures.**The predicted active site of SfiI (in blue) superimposed onto the BglI structure (red) Individual subunits are shown in yellow and blue. The Ca^2+^ions from the BglI structure are shown as cyan spheres. Only the two nucleotides adjacent to the scissile phosphodiester bond are shown. ::: ![](1472-6807-5-2-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Predicted specific protein-DNA contacts.**A) Recognition of the inner C/G pair B) Recognition of the middle C/G pair. C) Recognition of the middle G/C pair. D) Recognition of the outer G/C pair. ::: ![](1472-6807-5-2-5) ::: Interestingly, our model suggests that SfiI lacks the counterpart of a loop corresponding to aa 63--80 in BglI used by this enzyme to interact with the target site from the minor groove side. Thus, SfiI appears to recognize its target solely from the major groove side and to use fewer specific contacts than BglI to recognize its cognate site. This suggests that SfiI may be an easier target for the engineering of REases with new sequence specificities. The dimerization interface of SfiI is comparable to that of BglI. We predict that the following residues may be important for monomer-monomer interactions: Q59, Y60, E63, E66, R73, F74, G76 and that mutating them to change the volume of the side chain (for instance G76R) or introducing (or reversing) the charge (E63R, E66R, R73D, F74R) could disrupt the formation of the SfiI dimer and destroy the REase activity. Prediction of the dimer-dimer interaction surface in the SfiI tetramer ---------------------------------------------------------------------- SfiI is a Type IIF enzyme, i.e. a tetramer that binds simultaneously to two recognition sites and cleaves both sites concertedly \[[@B21]\], while BglI is an orthodox IIP enzyme, i.e. a dimer that acts on single sites \[[@B38]\]. Therefore, the crystal structure of BglI cannot be used to model the tetrameric structure of SfiI. To date, the only Type IIF enzymes, for which crystal structures have been solved, are NgoMIV \[[@B39]\], Cfr10I \[[@B40]\] and Bse634I \[[@B41]\], which are all relatively closely related to each other and exhibit similar mode of interactions between two dimers within the tetramer. These enzymes, however, even at the level of a dimer exhibit a completely different arrangement of monomers, compatible with the generation of 5\' overhangs 4 nt long (compared to 3\' overhangs 3 nt long in the case of SfiI and BglI). Therefore, it is impossible to obtain a meaningful superposition of the BglI or SfiI dimer onto any pair of subunits in the NgoMIV, Cfr10I or Bse634I tetramer. However, it is tempting to speculate that SfiI may tetramerize in a similar manner to these enzymes, i.e. to use surface regions on the opposite sites of the molecule to the protein-DNA and protein-protein binding. A highly speculative model of SfiI tetramer obtained by manual docking of two dimers is shown in Figure [6](#F6){ref-type="fig"}. Based on this model, we predict that the dimer-dimer interface will be composed mostly of hydrophobic and polar residues (and very few charged ones), involve the following segments of the amino-acid sequence, corresponding to loops on the surface of the dimer: 26--34, 67--69, and 86--93. The putative dimer-dimer interactions involve contacts between hydrophobic regions (aa 86--93 from different subunits) as well as hydrophilic ones (aa 26--34). It is possible that the C-terminal region of the SfiI sequence, which could not be modeled (aa 241--269) may also participates in tetramerization. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Putative structure of the SfiI tetramer.**Individual subunits are shown in yellow, green, red, and magenta. The two DNA substrates are shown in white. ::: ![](1472-6807-5-2-6) ::: Conclusions =========== Implications for the evolutionary history of different (sub) Types of REases ---------------------------------------------------------------------------- Comparative analysis of nucleases from the PD-DxK superfamily suggests that they can be classified into two remotely related lineages: α (EcoRI-like) and β (EcoRV-like) \[[@B7]-[@B9]\]. It was proposed that extant REases evolved independently from non-specific or structure-specific nucleases from both lineages (review: \[[@B5]\]). Interestingly, the phylogenetic tree of the PD-DxK superfamily revealed intriguing cases of convergent evolution. So far, it was found that Type IIE enzymes that bind two copies of the recognition site (the actual target of cleavage and the non-cleaved allosteric effector), evolved independently at least three times: EcoRII is an α-lineage member that apparently evolved from IIP enzymes similar to SsoII or PspGI by acquisition of an N-terminal effector-binding domain \[[@B12],[@B42]\]. NaeI is a β-lineage member remotely related to IIP enzymes EcoRV and HincII that acquired a C-terminal effector-binding domain unrelated to that of EcoRII \[[@B7]\]. Finally, Sau3AI is a β-lineage member that apparently evolved by a duplication of a catalytic domain closely related to a DNA repair enzyme MutH, followed by the loss of catalytic residues in the C-terminal domain, thereby adapted to function as an effector-binding domain \[[@B14],[@B15]\]. Another type of evidence for convergent evolution is provided by the finding that the specificity for the GATC sequence appeared independently in the α-lineage (MboI and its close homologs \[[@B5],[@B13]\]) and in the β-lineage (Sau3AI and its close homologs \[[@B14],[@B15]\]). Our results strongly suggest that the archetypal Type IIF enzyme SfiI is closely related to a β-lineage member, an \"orthodox\" Type IIP REase BglI. Another well-characterized group of Type IIF REases comprises α-lineage members for which crystal structures were solved (NgoMIV, Cfr10I, Bse643I) \[[@B39]-[@B41]\]. Thus, our analysis provides evidence that the ability of Type IIF REases to tetramerize and cut two target sites in a concerted manner was developed independently at least two times in the evolution of the PD-DxK superfamily. Different Type IIF REases appear to have evolved independently from the simplest, \"orthodox\" Type IIP enzymes, like previously found for Type IIE REases. It was previously demonstrated that deletion of the effector-binding domain converts the Type IIE REase EcoRII to function as a Type IIP enzyme \[[@B43]\]. However, tetramerization seems to be important for the catalytic activity of the Type IIF enzyme Cfr10I, since the DNA cleavage activity of the dimeric W220A mutant of this REase is \<0.1% of that of the wild-type enzyme \[[@B44]\]. In the absence of a high resolution co-crystal structure of SfiI in complex with DNA, our model will serve as a convenient platform to study sequence-structure-function relationships in this enzyme. In particular, it will facilitate the mutagenesis of residues potentially involved in tetramerization, dimerization, DNA-binding and catalysis. Methods ======= Structure prediction -------------------- Sequence searches of the non-redundant (nr) database and of the putative translations from finished and unfinished microbial genomes were carried out at the NCBI using PSI-BLAST \[[@B45]\]. Secondary structure prediction and tertiary fold-recognition was carried out via the GeneSilico meta-server gateway \[[@B29]\]. Secondary structure prediction was predicted using PSIPRED \[[@B46]\], PROFsec \[[@B47]\], PROF \[[@B48]\], SABLE \[[@B49]\], JNET \[[@B50]\], JUFO \[[@B51]\], and SAM-T02 \[[@B33]\]. Solvent accessibility for the individual residues was predicted with SABLE \[[@B49]\] and JPRED \[[@B52]\]. The fold-recognition analysis (attempt to match the query sequence to known protein structures) was carried out using FFAS03 \[[@B53]\], SAM-T02 \[[@B33]\], 3DPSSM \[[@B34]\], INBGU \[[@B32]\], FUGUE \[[@B31]\], mGENTHREADER \[[@B54]\], and SPARKS \[[@B55]\]. Fold-recognition alignments reported by these methods were compared, evaluated, and ranked by the Pcons server \[[@B36]\]. Homology modeling ----------------- The alignments between the sequence of SfiI and the structures of selected templates (members of the fold identified by Pcons) were used as a starting point for modeling of the SfiI tertiary structure using the \"FRankenstein\'s Monster\" approach \[[@B37]\], comprising cycles of model building by MODELLER \[[@B56]\], evaluation by VERIFY3D \[[@B57]\] via the COLORADO3D server \[[@B58]\], realignment in poorly scored regions and merging of best scoring fragments. The positions of predicted catalytic residues and secondary structure elements were used as spatial restraints. This strategy has previously helped us to build accurate, experimentally validated models of other REases, such as SsoII \[[@B11]\], PspGI \[[@B12]\], MboI \[[@B13]\], and KpnI \[[@B19]\]. List of abbreviations ===================== aa, amino acid(s); bp, base pair(s); nt, nucleotide; e, expectation; REase, restriction endonuclease; MTase, methyltransferase; ORF, product of an open reading frame, RM, restriction-modification; Authors\' contributions ======================= JMB carried out the fold-recognition analysis for SfiI, built the preliminary models, drafted the manuscript and coordinated the whole study. AC built the final models and identified functionally important residues. KS participated in interpretation of the data and writing the manuscript. All authors have read and accepted the final version of the manuscript. Acknowledgements ================ This analysis was funded by KBN (grant 3P04A01124 to JMB). The work of KJS and AAC was supported by the NIH (Fogarty International Center grant R03 TW007163-01). JMB was supported by the EMBO/HHMI Young Investigator Award and by the Fellowship for Young Scientists from the Foundation for Polish Science. We would like to thank Alfred Pingoud and Virgis Siksnys for stimulating discussions concerning the evolution of sequence-structure-function relationships in REases of various subTypes.

PubMed Central

2024-06-05T03:55:52.554423

2005-1-24

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548270/", "journal": "BMC Struct Biol. 2005 Jan 24; 5:2", "authors": [ { "first": "Agnieszka A", "last": "Chmiel" }, { "first": "Janusz M", "last": "Bujnicki" }, { "first": "Krzysztof J", "last": "Skowronek" } ] }

PMC548271

Background ========== There is a long tradition of studying at population level the quality of medical care provided to patients who died from conditions amenable to medical intervention. This type of study (so called \'in-depth\' or \'audit\' study), aims to identify deficiencies in medical care that may have contributed to death. It was first systematically carried out on maternal death, and later on other causes of avoidable death \[[@B1]-[@B4]\]. This method can be applied to other potentially avoidable conditions, e.g. those that could be avoided by appropriate preventive care. The general approach is to document in detail the process of care provided to a single patient preceding the occurrence of an adverse event, followed by an assessment of the quality of care by an expert panel, either with or without the use of explicit criteria \[[@B5]\]. An important limitation of this type of study, without control subjects, is its inability to fully establish a causal relationship between identified deficiencies in care and the adverse outcome, and to determine to what extent identified deficiencies are associated to the occurrence of such an event. Identified deficiencies in care are expected to indicate only to a certain extent an increase in risk of an adverse health outcome, while the probability of having an adverse outcome can be calculated only if we compare the care provided to patients who suffered an adverse outcome with that of patients who did not suffer such an event. For this reason it has been proposed to perform a case-control study with patients with an adverse event as \'cases\' and a comparable group of patients without an adverse outcome as \'controls\' \[[@B6]\]. We performed a case-control study with the aim to assess the effect of guideline adherence for stroke prevention on the occurrence of stroke in general practice. Unfortunately, we encountered various obstacles in the design and conduct of this study, in particular related to the recruitment of cases and controls, in availability of information on the care delivery process in the GP\'s data registration system, and in controlling for differences other than differences in the quality of care. The aim of this paper is to highlight the problems related to a variant of confounding by indication, that may be common in quality of care studies. Observational studies of intended treatment effects are particularly prone to \'confounding by indication\', and can produce misleading estimates on either, or both, the size and direction of treatment effects \[[@B7],[@B8]\]. Confounding by indication refers to an extraneous determinant of the outcome parameter that is present if a perceived high risk or poor prognosis is an indication for intervention. This means that differences in care, for example, between cases and controls may partly originate from differences in indication for medical intervention such as the presence of risk factors for particular health problems. The latter has frequently been reported in studies evaluating the efficacy of pharmaceutical interventions \[[@B9],[@B10]\], screening tests \[[@B11]\], and vaccines \[[@B12]\]. We hypothesise that this may not only apply to indications for medical intervention but also for guideline adherence and quality of care. In comparing retrospectively the quality of care between patients with and without a stroke, stroke patients may have received more preventive care because more indications for preventive interventions were present. Because differences in indications for preventive intervention correspond with the probability of an adverse outcome (more indications will be associated with a higher risk of an adverse outcome), when comparing care between cases and controls it is necessary to control for these differences. If one omits to control for confounding by indication, it is expected that more and probably better care, correlates with a higher risk of stroke. In quality of care research, there is, as yet, little information regarding the role of confounding by indication in studies that investigate the effect of quality of care on disease outcomes. Methods ======= Sample ------ From the Dutch national GP register, a random sample was taken of 58 GPs working in Rotterdam and the surrounding region. The study was restricted to patients with a first-ever stroke meeting the following criteria for inclusion: (a) diagnosis of intracerebral haemorrhage or infarction according to the World Health Organization (WHO) definition of stroke \[[@B13]\], (b) age between 39--80 year, (c) occurrence of stroke in the period 1996--1997, (d) stroke caused by cardiovascular disease (CVD) and not by trauma, infection or malignancy, (e) presence of hypertension, (f) GP of the patient practising in the southern part of Rotterdam or surrounding region, (g) patient registered with local GP for not less than two years, and (h) patient not living in a nursing home during the two years period prior to stroke. Cases and controls were selected from the GPs\' patient register, using health outcome (stroke) and risk factor (e.g. hypertension) entries. For each case, two controls were randomly selected and matched with the cases in terms of overall distribution on sex, age, and hypertension (most important risk factor for stroke). Cases and controls were not matched on the same GP. Data collection --------------- In a pilot study among 32 GPs, the quality of care measurement instruments (audit procedure and questionnaire) were tested. GPs participating in the pilot study did not participate in this study. Data on the process of care, two years prior to the occurrence of stroke (for controls from January 1995 to January 1997), were collected by means of structured face-to-face interviews with the GP, using separate questionnaires for each stroke patient. GPs were interviewed between March and October 1999. At the time of interview, GPs used either hand-written or electronic patient records to retrieve patient information. In case information was not available in the patient\'s record, information was drawn from the GP\'s memory. For each question, the type of data source was registered. The questionnaire comprised questions related to patient characteristics and family and medical history of CVD and risk factors, and the detection and treatment of cardiovascular risk factors such as hypertension, diabetes mellitus, transient ischemic attack (TIA) and cardiac failure. Similarly, data were collected on lifestyle-related risk factors such as smoking status, overweight, and excessive alcohol intake. Expert panel and assessment method ---------------------------------- The quality of preventive care and its potential to prevent stroke was assessed and valued by a six-member panel of experts. The panellists (three neurologists and three GPs) were selected on the basis of their clinical expertise with respect to stroke prevention, experience in quality of care evaluation, academic or non-academic background and professional discipline. Six practice guidelines relevant to stroke prevention (hypertension, diabetes mellitus, TIA, peripheral vascular disease, cardiac failure and angina pectoris) were selected by the panel \[[@B14]\]. These guidelines, based on scientific evidence, broad consensus, and clinical evidence, are developed and implemented by the Dutch College of General Practitioners as part of a national guideline program operational since 1987 \[[@B15]\]. From each guideline, the panellists identified specific elements of care and systematically converted these into review criteria (n = 65), allowing detailed measurement of GP\'s adherence \[[@B16]\]. All these criteria were all used to construct the patient questionnaire. In a two-round evaluation, with a final plenary round, cases were assessed by the panellists (panellists were divided in sub-panels). Each sub-panel assessed a specific number of cases. Based on identified elements of sub-optimal care and seriousness of shortcoming in terms of \'minor\' and \'major\', the panellists allocated grades on a scale of 0 to 3 (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Grades of (sub)optimal care given by the expert panel (in both groups allpatients are hypertensive) ::: *Cases* *Controls* ------------------ --- ------------------------------------------------------------------------------------------------------------------------ --------- ------------ ---- ----- Grading: 0 No sub-optimal factors have been identified 12 43 18 31 1 Sub-optimal factor(s) have been identified, but are unlikely to be related to the occurrence of stroke in this patient 8 29 18 31 2 Sub-optimal factor(s) have been identified, and possibly have failed to prevent the stroke in this patient 4 14 18 31 3 Sub-optimal factor(s) have been identified, and are likely to have failed to prevent the stroke in this patient 4 14 4 7 Sub-optimal care Grading 1, 2, 3 16 57 40 69 Total Grading 0, 1, 2, 3 28 100 58 100 ::: The two-round process was focussed on detecting consensus among the panellists (providing the same grade), and no attempt was made to force the panellists to consensus. The intersubpanel agreement was k = 0.63 (overall agreement on assigned grades between sub-panels was 74%). A detailed description of the assessment method is provided elsewhere \[[@B17]\]. Analysis -------- Analysis of the data was done by using simple cross-tabulations, and by using logistic regression analysis to model the chance of getting a stroke as a function of the presence of sub-optimal care (as ascertained by the panel), age and sex, and risk factors for stroke. Results ======= GP Participation and recruitment of cases / controls ---------------------------------------------------- The rate of participation was 62% (36 GPs). The main reason for GPs not to participate in the study was lack of time and interest (68%). Participating and non-participating GPs did not differ significantly in age, practice type, and date of qualification. Ninety-two percent of the GPs used electronic GP information systems. Among cases and controls there was a nonsignificant difference in mean age, however, cases were slightly older than controls (67 versus 65 years). Initially, before we excluded patients \'without\' hypertension, GPs identified and selected 50 cases and 58 controls (1.4 case and 1.6 control per GP). Expected number of cases was 2.5 stroke patients per GP per year \[[@B18]\]. After excluding patients without hypertension, 28 cases and 58 controls with hypertension entered the study. Availability of data -------------------- Overall, data for verification of the initial diagnosis of stroke, assessment of GPs\' guideline adherence, and judgement of the causality of the relationship between non-adherence and the occurrence of stroke could be collected from the patient records. However, information on risk factors such as family history of CVD, body weight (overweight), excessive alcohol intake, and smoking was less easily obtained. Depending on the type of risk factor, in 8--56% of all subjects, information on risk factors was unknown to the GP (8% in patients with overweight, 11% in patients smoking cigarettes, 17% in patients with excessive alcohol consumption, and 56% in patients with a family history of CVD). In 41--58% information was taken from the GP\'s memory, instead of the patient register. Indications for confounding by indication ----------------------------------------- In 43% of the cases and 31% of the controls, no sub-optimal care could be identified (grade 0), whereas in 57% and 69%, respectively, sub-optimal care was identified (grade 1, 2 or 3). Thus the Odds Ratio for a case to receive sub-optimal care was 0.60 (95%CI 0.24 -- 1.53) compared to a control (Table [1](#T1){ref-type="table"}). Compared with controls receiving sub-optimal care, the number of shortcomings in care per case receiving sub-optimal care was higher (28/16 = 1.7 versus 41/40 = 1.0) (Table [2](#T2){ref-type="table"}). The percentage of shortcomings in hypertensive care, however, was considerably higher among controls (90% versus 57%, respectively). The latter, apparently, correlates with the fact that controls less often have risk factors other than hypertension (next paragraph). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Guideline-derived elements of care used to indicate shortcomings in care among stroke patients and controls ::: *Practice guideline* *Elements of care* *Cases* *Controls* -------------------------------------------------------------- ------------------------------------------------ --------- ------------ Arguments derived from practice guideline: Hypertension \- Detection of hypertension 1 2 \- Confirmation diagnosis hypertension 2 1 \- Pharmacologic therapy (anti-hypert. med) 2 1 \- Follow-up (quarterly) 8 17 \- Follow-up (annually) 3 16 Arguments derived from practice guideline: Diabetes mellitus \- Follow-up (quarterly) 4 3 \- Laboratory evaluation 1 0 \- Referral to eye specialist 1 0 Arguments derived from practice guideline: TIA \- Treatment (therapy and follow-up after TIA) 1 1 Arguments derived from more than one practice Guideline \- Advice to quit smoking 2 0 \- Dietary advice (overweight) 1 0 \- Evaluation of cardiovascular risk profile 2 0 Total number of shortcomings 28 41 Total number of patients with shortcomings 16 40 TIA, Transient Ischemic Attack Note: each patient could have more than one element of sub-optimal care ::: The mean number of risk factors among cases (6.1 per patient) was higher than among controls (4.4 per patient) (Figure [1](#F1){ref-type="fig"}). Multivariate logistic regression indicates that cases receiving sub-optimal care (grade 1, 2, or 3) have a lower risk of stroke (crude OR 0.60) (Table [3](#T3){ref-type="table"}). If adjusted for sex and age distribution, the odds ratio does not change significantly (adjusted OR 0.64). Subsequently, in an attempt to investigate the possible role of confounding by indication we adjusted for risk factor prevalence. Indeed, with an adjusted OR of 0.82 (95% CI 0.29--2.30), it seems that risk factor prevalence to some extent explains why patients receiving sub-optimal care have a lower risk of stroke. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Risk factor distribution. Prevalence (%) of risk factors for stroke among stroke patients (n = 28) and controls (n = 58). Total number of risk factors among stroke patients is 172, and among controls 277. Mean number of risk factors per case is 6.1, and for controls 4.4. This relationship is statistically borderline significant (p = 0.096), and could be an explanation for the somehow surprising result found earlier, that is, that cases receive sub-optimal care less often than controls. ::: ![](1472-6963-5-10-1) ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Relationship between quality of care and the occurrence of stroke(Odds Ratio and 95% CI) ::: MODEL 1 MODEL 2 MODEL 3 --------------- ------------------- ------------------- ------------------- Care: Optimal 1.00 (ref.) 1.00 (ref.) 1.00 (ref.) Sub-optimal 0.60 (0.24--1.53) 0.64 (0.25--1.65) 0.82 (0.29--2.30) Sex: Male 1.00 (ref.) 1.00 (ref.) Female 0.90 (0.36--2.30) 0.61 (0.22--1.72) Age: 1.03 (0.98--1.08) 1.03 (0.98--1.08) Risk factors: 0.76 (0.61--0.94) Note: to control for risk factors, the number of risk factors per patient were included in the regression model. ::: Patients with a higher number of risk factors for stroke, indeed have a lower risk of sub-optimal care (OR for the number of risk factors present 0.76; 95% CI 0.61--0.94). As expected, higher numbers of risk factors per patient also increases the risk of stroke (OR for the number of risk factors present 1.34; 95% CI 1.10--1.62). Discussion ========== This study demonstrated confounding by indication in a case-control study analysing the association between guideline adherence and the occurrence of stroke in general practice. It also provided insight into the possibilities for controlling for this confounding bias. We learned that, at present, difficulties in patient recruitment and data retrieval seriously limit the potential use of a case-control method to assess the relationship between guideline adherence for stroke prevention and stroke in general practice. We found that in specific domains data were incomplete and not readily available in the patient records. As a consequence, in many cases GPs were unable to identify stroke patients from their patient register, which most likely introduced under-reporting of stroke patients. As compared to national frequencies (2.5 stroke patients per GP per year) \[[@B18]\], GPs participating in our study identified less stroke patients, 1.7 stroke patient per GP. The same applies to information on patients\' family history of CVD and lifestyle-related risk factors, which was inaccurate and in many cases not available in the patient\'s register. The latter finding is consistent with previous work on the accuracy of information on CVD risk factors in GPs\' patient records \[[@B19],[@B20]\], indicating that data from GP\'s record on lifestyle-related risk factors of CVD are frequently incomplete or absent. Incomplete information on risk factors for stroke is a serious threat to the validity of the results of case-control studies investigating the relationship between process of care and health care outcome. It complicates evaluation of GP\'s adherence to recommended guidelines, and makes it difficult, if not impossible, to control for confounding by indication. Apart from that, information on risk factors that was available in the patient records is presumably not 100% valid. Strong indications for the existence of confounding by indication were found, albeit different from how it is usually described in literature. Confounding by indication, which is conceived as a substantial problem in observational studies of treatment efficacy, usually refers to a situation in which patients who are more in need both receive more care have a higher risk of adverse health outcome \[[@B8]\]. In our study, we show that confounding by indication can also cause patients with an adverse health outcome (stroke) to appear to receive better quality of care. A more detailed analysis showed, similar to results found in previous studies, that this result partly emanates from a higher prevalence of risk factors for stroke among patients suffering stroke at a later stage in life, which not only increases the risk of stroke but also GPs\' compliance to guidelines. We hypothesize that, on average, patients with more risk factors for stroke receive more attention or visit their GP more frequently, which in turn facilitates guideline adherence (e.g. compliance to quarterly follow-up of treated hypertensive patients) and at the same time results in better quality of care. Controlling for (recorded) risk factors reduced the counter-intuitive result by approximately one half, and we hypothesise that incomplete registration of risk factors for stroke explains why the risk of stroke in stroke-prone or high-risk patients associated with sub-optimal care remained below 1.00, even after controlling for risk factors. We hope that our paper draws the attention of quality of care researchers to this variant of confounding by indication, that may lead to biased associations between process measures of quality of care and care outcomes. Conclusions =========== This study shows that, at present, difficulties in patient recruitment and data retrieval seriously limit the potential use of a case-control method to assess the relationship between guideline adherence for stroke prevention and stroke in general practice. It demonstrates the role of confounding by indication, causing patients with an adverse health outcome to appear to receive better quality of care. Competing interests =================== The author(s) declare that they have no competing interests Authors\' contributions ======================= JK, NK, PK, AP and JM conceived and designed the study. Analyses were performed by JK and GB. All authors contributed to this article and earlier drafts of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/5/10/prepub> Acknowledgements ================ The audit was carried out by a panel of experts. The members were: *Chairman*: Jan W. Van Ree, MD. *Members*: Diederik W.J. Dippel, MD (neurologist); Gabriël J.E. Rinkel, MD (neurologist); Jan Heeringa, MD (general practitioner); R.P. Kleyweg, MD (neurologist); Arie Knuistingh-Neven, MD (general practitioner); Jan W. Van Ree, MD (general practitioner).

PubMed Central

2024-06-05T03:55:52.557201

2005-1-27

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548271/", "journal": "BMC Health Serv Res. 2005 Jan 27; 5:10", "authors": [ { "first": "Johan S", "last": "de Koning" }, { "first": "Niek S", "last": "Klazinga" }, { "first": "Peter J", "last": "Koudstaal" }, { "first": "Ad", "last": "Prins" }, { "first": "Gerard JJM", "last": "Borsboom" }, { "first": "Johan P", "last": "Mackenbach" } ] }

PMC548272

Background ========== Priority setting, also known as rationing or resource allocation, occurs at all levels of every health care system, including governments, funded provincial/territorial agencies, pharmaceutical benefit-management organizations, hospitals and clinical programs \[[@B1]\]. Countries with very different health care systems and levels of health care are all grappling with the problem of how to reconcile growing demands and constrained resources \[[@B2]\]. Hospitals, in particular, are struggling to meet growing demands, affordably, without compromising delivery of services \[[@B3],[@B4]\]. Daniels and Sabin have proposed a framework for priority setting in health care institutions called \'accountability for reasonableness\' \[[@B5]-[@B7]\], which links priority setting to theories of democratic deliberation, operationalizing the ethical concept of fairness. Fairness is a key goal of priority setting. According to \'accountability for reasonableness\', health care institutions engaged in priority setting have a claim to fairness if they satisfy four conditions of relevance, publicity, appeals/revision, and enforcement (described in Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The four conditions of \'accountability for reasonableness\' ::: ------------------ -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Relevance Rationales rest on evidence, reasons, and principles that fair-minded parties can agree are relevant to deciding how to meet the diverse needs of a covered population under necessary resource constraints. Publicity Limit-setting decisions and their rationales must be publicly accessible. Appeals/Revision There is a mechanism for challenge and dispute resolution regarding limit-setting decisions, including the opportunity for revising decisions in light of further evidence or arguments. Enforcement There is either a voluntary or public regulation of the process to ensure that the first three conditions are met. ------------------ -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: \'Accountability for reasonableness\' may be an effective framework for describing the components of priority setting, evaluating the fairness of priority setting in hospitals and identifying \'good\' practices and opportunities for improvement. It can help hospitals improve their priority setting practices and can be an effective driver of health care reforms \[[@B8]-[@B10]\]. \'Accountability for reasonableness\' has been used to evaluate priority setting in health systems \[[@B11]\]. However, only a limited number of studies have addressed how priority setting occurs in hospitals \[[@B12]-[@B17]\], and no study has attempted to survey the views of hospital decision makers throughout an entire health system about the fairness of priority setting in their institutions. The purpose of this study is to elicit hospital decision-makers\' self-report of the fairness of priority setting in their hospitals using an explicit conceptual framework, \'accountability for reasonableness\'. Methods ======= Design ------ We conducted a survey of Chief Executive Officers, or their designates, of Ontario hospitals. The survey questionnaire covered 102 items, including hospital profile information (e.g. hospital name, number of beds, operating budgets). In this paper we focus on the results of 9 questions concerning priority setting, fairness, and the four conditions of \'accountability for reasonableness\' (refer to Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Survey Questions -- Rating\* and Open Scale ::: --------------------------------------------------------------------------------------------------------------------------- Overall, how fair is priority setting at your hospital? (Rating Scale). Please explain your response (Open-ended). How well does your hospital meet its priority setting goal(s)? (Rating Scale). Please explain your response (Open-ended). What do you see as the goal(s) of priority setting in your hospital? (Open-ended) How well is the relevance condition met at your hospital? (Rating Scale) How well is the publicity condition met at your hospital? (Rating Scale) How well is the appeals condition met at your hospital? (Rating Scale) How well is the enforcement condition met at your hospital? (Rating Scale) --------------------------------------------------------------------------------------------------------------------------- \* Ratings were on a five-point scale, from 1 (not well) to 5 (very well) or from 1 (not fair) to 5 (very fair). ::: Setting and sample scope ------------------------ With 12 million people, Ontario is the largest province in Canada. Like the rest of the country, it is a single payor, predominately publicly-funded health care system with some privately funded services and products (e.g. dental services, drugs). There are 160 hospitals in Ontario and all were invited to participate in this study. Participants ------------ 160 Ontario hospital Chief Executive Officers, or their designates, were asked to participate. 86 Ontario hospitals completed this survey, for a response rate of 54%. The average bed count of the responding hospitals was 250.4, with a range of 18 to 1265 beds. The average operating budget of the hospital sample was \$75.8 million, ranging from \$3.3 million to \$733 million. The sampled hospitals represented a blend of teaching, small, community and specialized service facilities across the province. Data collection --------------- The survey was pre-tested and mailed to Chief Executive Officers of all Ontario hospitals in January 2001. Data from returned surveys were entered into an electronic database for further analysis. Of the 9 questions analyzed in this study, 6 asked for a rating on a 5-point scale ranging from \'Not Well\' or \'1\' to \'Very Well\' or \'5\', and 3 were open-ended. Data analysis ------------- Close-ended ratings were analyzed statistically \[[@B18]\], with ratings equal to and below the mid-point combined to identify proportions of respondents suggesting improvement, using a conservative bivariate cut-off point to discriminate among reported responses. P values for all hypothesis tests were two tailed. Open-ended responses were analyzed using a modified thematic analysis involving open and axial coding techniques \[[@B19],[@B20]\]. In open coding, data was segmented and coded with a label identifying parts of text relating to a concept or idea. For axial coding, concepts were organized into overarching themes, and compared, both within and between questions, in search of patterns in responses and to ensure consistency. Results ======= Overall, 60.7% of respondents indicated their hospitals\' priority setting was fair, while 79% stated their hospitals met their priority setting goals. With respect to the \'accountability for reasonableness\' conditions, respondents indicated their hospitals performed best for the relevance (75.0%) condition, followed by appeals/revision (56.6%), publicity (56.0%), and enforcement (39.5%). (Refer to Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Summary of Decision Maker Responses ^a,b^ ::: How well does your hospital meet its priority setting goal(s)? Overall, how fair is priority setting at your hospital? How well is the relevance condition met at your hospital? How well is the publicity condition met at your hospital? How well is the appeals condition met at your hospital? How well is the enforcement condition met at your hospital? ----------------- ---------------------------------------------------------------- --------------------------------------------------------- ----------------------------------------------------------- ----------------------------------------------------------- --------------------------------------------------------- ------------------------------------------------------------- \# Respondents 81 84 84 84 83 81 5 \'Very Well\' 15 (18.5%) 15 (17.9%) 21 (25.0%) 14 (16.7%) 12 (14.4%) 9 (11.1%) 4 49 (60.5%) 36 (42.8%) 42 (50.0%) 33 (39.3%) 35 (42.2%) 23 (28.4%) 3 12 (14.8%) 28 (33.3%) 17 (20.2%) 26 (30.9%) 23 (27.7%) 34 (41.9%) 2 3 (3.7%) 4 (4.8%) 3 (3.6%) 9 (10.7%) 10 (12.0%) 9 (11.1%) 1 \'Not Well\' 2 (2.5%) 1 (1.2%) 1 (1.2%) 2 (2.4%) 3 (3.6%) 6 (7.4%) (SD) 3.89 (0.84) 3.71 (0.86) 3.94 (0.84) 3.57 (0.97) 3.52 (1.00) 3.25 (1.04) a Due to rounding, frequencies may not add up to 100% b Based on survey scale from 1 to 5 ::: Respondents rated the relevance (![](1472-6963-5-8-i1.gif) = 3.94) condition significantly higher than the publicity (![](1472-6963-5-8-i1.gif) = 3.57), appeals (![](1472-6963-5-8-i1.gif) = 3.52), and enforcement (![](1472-6963-5-8-i1.gif) = 3.25) conditions (p \< .05) (paired-samples t test). While publicity and appeals conditions were not significantly different, respondents rated these conditions significantly higher than the enforcement condition (p \< .05). The distribution of ratings suggested that there was ample opportunity for improvement, with most room for improvement in meeting the enforcement condition. Priority setting and fairness ratings were positively correlated (r = .51, p \< .01). Fairness ratings were also positively correlated with each of the 4 \'accountability for reasonableness\' conditions (p \< .01), as were meeting priority setting goals (p \< .05). The bivariate correlation between each of the accountability variables of relevance, publicity, appeals/revision and enforcement was strongly positive (p \< .01). The Pearson correlation coefficients among the 4 conditions ranged from 0.41 to 0.57. The internal consistency (Cronbach\'s α) of all of the \'accountability for reasonableness\' conditions was very good (α = .78). Bed size (r = -.24, p \<.05) and operating budget (r = -.32, p \< .01) were negatively correlated with the rating of fairness in priority setting. Analysis of respondent comments ------------------------------- In this section we describe participants\' responses to the open-ended questions organized according to themes. We have included verbatim responses to help illustrate the themes. What do you see as the goals of priority setting in your hospital? ------------------------------------------------------------------ Decision makers emphasized four priority setting goals which were complex, interdependent, and required balancing. Some respondents said that (1) meeting needs existed in relation to delivering (2) quality services, or was constrained by resource availability. Examples of these goals include \"quality care within available resources\" and \"access to high quality service in areas of greatest need\". While (3) meeting budgets was identified as a goal by some decision makers, it did not exist in isolation from other goals such as meeting strategic directions or needs. One decision maker said his goal was \"ensuring a balanced situation at year end while meeting the direction set out in our strategic plan\". Achieving (4) organizational goals was expressed as a limiting, or organizational focusing process, which involved alignment or balancing of multiple goals and values. How well does your hospital meet its priority setting goals? Please explain your response ----------------------------------------------------------------------------------------- Decision makers described a deliberative process by which their priority setting goals were operationalized, providing examples of good practices and challenges or barriers for achievement. Overall, decision makers appeared confident that their priority setting goals were the correct ones, in no case did they comment that expressed goals were unrealistic, so contributing to lack of achievement. Decision makers pointed to five factors of importance in meeting their priority setting goals. Review processes (1) were described in which different review processes are brought to bear pointing to areas requiring additional resources. For example, a decision maker said \"annually we review and ensure that resources are being allocated to priority programs and services\". Leadership (2) was implicated as an important factor in meeting priority-setting goals. For example, a decision maker said \"We do not set goals that are pie in the sky, they must be achievable\". With respect to (3) stakeholder consultation, some respondents pointed to the need to involve the wider community in decision making. Decision-makers felt that improvements in priority setting were contingent upon (4) access to relevant information. Finally, some decision-makers emphasized the importance of (5) decision making tools, or benchmarking, to improve decision making. Overall, how fair is priority setting at your hospital? Please explain your response ------------------------------------------------------------------------------------ Respondents gave information to support their self-evaluation, and provided examples to demonstrate the fairness of priority setting processes from their perspective. In evaluating the fairness of priority setting, decision makers emphasized five themes. Stakeholder consultation raises the level of (1) inclusivity, bringing different points of views and interests to bear in solving common problems. For example, a decision maker said \"we have a service providers network in our community that has direct input into resource allocation and program planning\". Respondents described review processes (2) involving the deliberation of concerned parties, using relevant data, based on need, cost and other values (e.g. evidence). Reporting systems (3) provide an institutional feedback loop, pointing to areas where limits may be fairly set. With respect to (4) revision/appeals, revising arguments or values based on iterative processes involving various stakeholders is a feature of fairness, and helps to improve buy-in, but may increase organizational tensions. Finally, (5) governance may also be involved in developing decision-making models, and in ensuring the conditions of relevancy, appeals/revision, and publicity are met. For example, a decision maker said \"The Board and senior management have developed decision-making models for key program/service changes\...our appeals process is informal, but open to access including the Board\". Discussion ========== To our knowledge, this is the first published survey of hospital decision makers covering an entire health system, using Daniels and Sabin\'s framework of \'accountability for reasonableness\'. It provides data to understand how fair priority setting processes are, what decision makers\' priority setting goals are, and how well these goals are met. Several studies, using either survey or qualitative methodologies sometimes in combination with hypothetical \"tradeoffs\", have examined priority setting from theoretical perspectives aimed at capturing the public or professional\'s preferences or values in priority setting \[[@B21]-[@B24]\]. Relatively few studies have described what is happening in real-world contexts with a view of evaluating and improving priority setting activities \[[@B25]-[@B31]\]. These have described actual priority setting in individual hospitals, focusing on strategic planning \[[@B12]\], surgery \[[@B13]\], critical care \[[@B14]\], new technologies \[[@B15]\], and the rationing of new drugs \[[@B17]\], with no study reviewing priority setting in hospitals across the health system. This study makes five contributions to knowledge on priority setting. First, there is ample room for improvement in fair practices within Ontario hospitals as described by their Chief Executive Officers. Although many hospital CEOs felt that their priority setting was fair, ample room for improvement was noted, especially for the enforcement condition, followed by publicity, appeals/revision and relevance. While 79% of decision makers felt their hospitals met their priority setting goals, only 60.7% felt their processes were fair. According to decision makers, the perceived \'gap\' was greater in meeting fairness than in priority setting goals. Second, it is feasible for decision makers to assess the fairness of priority setting in their institutions on a quantitative basis according to the \'accountability for reasonableness\' framework, with greater than half of respondents saying their processes are fair. There is a high degree of association between \'fairness\' and the internal components of \'accountability for reasonableness\', lending support that fairness can be operationalized through \'accountability for reasonableness\'. As well, the internal conditions of \'accountability for reasonableness\' are highly related, and positively correlated, suggesting relevance, publicity, appeals/revision, and enforcement are measuring comparable aspects of fairness. Consistent with this, the high Cronbach\'s α finding is promising for the development of an \'accountability for reasonableness\' scale of perceived fairness of priority setting in health care institutions. Measuring decision makers\' self-report of their perception in meeting the various conditions of \'accountability for reasonableness\', the scale would provide a practical tool for decision makers to assess self-reported views over time, noting progress in meeting conditions, while providing a reference point for needed improvement (i.e. enhance meeting \'publicity\' condition). Finally, the finding of a negative relationship between number of hospital beds or budgets and self-report on fairness is consistent with the view that smaller hospitals may be perceived to have fairer processes, with greater involvement of their local communities in decision making and emphasis on transparency and \"trust\". Third, decision makers\' views were surveyed at a health system level to determine what their actual priority setting practices were. Four complex, interrelated priority setting goals were described, suggesting a balance of need, quality, budget and organizational goals in priority determination. In addition, the study points to a close alignment of factors required in the meeting of priority setting goals and in the elements of fairness. In providing decision maker perspectives on characteristics of fairness, this study also adds to previous empirical work suggesting fair priority setting depends on a fair priority setting process \[[@B31]\]. Additional research is required to understand the capacity of organizations to improve such practices. Four, this study has shown, from a blend of qualitative and quantitative approaches, that it is feasible to operationalize the \'accountability for reasonableness\' framework, through the design of survey instruments to facilitate self-evaluation, and identification of good practices. This data can be shared with decisions makers to improve the fairness of their priority setting processes in meeting the four conditions of \'accountability for reasonableness\'. Finally, this study expands on the likely relationship between leadership and priority setting in which leadership was found to contribute to perceptions of fairness in two committees engaged in priority setting for new health care technologies. Study results indicate that greatest room for improvement exists in meeting the \'enforcement\' condition, with decision-makers describing critical leadership success factors, including: the role of governance in establishing policy and in meeting this condition, the need to set achievable corporate goals, and the significance of a lobbying funding function. Further study is needed to clarify the nature of this leadership contribution. Limitations ----------- The study is limited, first, in the response rate from hospitals. Only 54% of Ontario\'s 160 hospital CEO\'s responded to the survey. It is possible that hospitals responding to this survey were those doing, or perceived doing, better on self-report than others. However, there is no evidence to suggest that this is the case, and the sample did include small, medium and larger teaching hospitals, as well as specialized facilities to mitigate against representative selection bias. Second, social desirability bias was possible in that the views of decision makers expressed in the survey may not have corresponded to what they actually believed, or did. However, unpublished data \[[@B32]\] based on in-depth interviews with Ontario hospital decision makers suggest self-reported views in relation to fairness were similar in both survey respondents and non-respondents, and that reasons for non-response were related to convenience, workload and priority, and not social desirability. Third, corroborative evidence of \'fairness\', obtained through in-depth interviews with staff or community stakeholders, or through review of other relevant information (e.g. meeting minutes, publication of rationales on web sites) was not done. The study design was a survey, focusing on the self-report of fairness from the perspective of Chief Executive Officers, or delegates. In-depth interviews with Chief Executive Officers, or additional individual hospital case studies would be helpful in understanding actual priority setting practices, building on these survey results. Conclusions =========== In this first survey of Chief Executive Officers within a health region reporting on their assessment of the fairness of priority setting practices in their institutions according to \'accountability for reasonableness\', ample room for improvement was observed, especially for the enforcement condition. Additional study is required to understand how application of \'accountability for reasonableness\' will vary within and across institutions, and is shaped or influenced by various parameters, including the nature of corporate leadership. These evaluations can help to identify good practice opportunities for improvement that can be shared between local institutions or used within government to drive health care reforms. Competing interests =================== The author(s) declare they have no competing interests. Authors\' contributions ======================= DR was the primary analyst and principal author of the manuscript. DKM, CK and PAS were involved in study conception, analysis and drafting the manuscript. CK and DKM were involved in data collection. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/5/8/prepub> Acknowledgements ================ Funding for study provided by a grant from The Change Foundation and by an Interdisciplinary Capacity Enhancement grant from the Canadian Institutes of Health Research. DKM is supported by an Ontario Ministry of Health and Long-Term Care Career Scientist Award. PAS is supported by a Canadian Institutes of Health Research Distinguished Scientist Award.

PubMed Central

2024-06-05T03:55:52.560423

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548272/", "journal": "BMC Health Serv Res. 2005 Jan 21; 5:8", "authors": [ { "first": "David", "last": "Reeleder" }, { "first": "Douglas K", "last": "Martin" }, { "first": "Christian", "last": "Keresztes" }, { "first": "Peter A", "last": "Singer" } ] }

PMC548273

Background ========== The glucose-6-phosphatase system consists of the glucose-6-phosphate catalytic subunit (EC 3.1.3.9), embedded in the membrane of the endoplasmic reticulum (ER) via nine transmembrane domains, and the membrane spanning translocases, responsible in carrying either the substrate into the ER or the product from the ER \[[@B1]\]. Transport of substrate and product is necessary due to the orientation of the active site of the glucose-6-phosphatase enzyme towards the luminal side of the ER. Glucose-6-phosphate, the end product of both gluconeogenesis and glycogenolysis in the liver, is hydrolyzed by the glucose-6-phosphatase system allowing the liberation of glucose into the circulation. Thus, glucose-6-phosphatase plays a key role in the homeostasis of blood glucose. Mutations in the gene encoding the catalytic subunit of glucose-6-phosphatase are responsible for the development of glycogen storage disease type 1, also known as Gierke disease \[[@B2]\]. In animal models of diabetes mellitus, glucose-6-phosphatase activity is increased along with mRNA and protein levels \[[@B3]\]. Accordingly, inhibitors of glucose-6-phosphatase or the G6PT transporter are used for the treatment of type 2 diabetes. The glucose-6-phosphatase encoding gene is regulated by a variety of extracellular signaling molecules, including glucose, insulin, glucocorticoids, and cAMP. Insulin decreases the level of glucose-6-phosphatase mRNA in the liver and in hepatoma cells and three functionally distinct insulin response elements have been identified \[[@B4]\]. Glucocorticoids elevate glucose-6-phosphatase promoter activity mediated by a glucocorticoid response element and hepatocyte nuclear factor 1 \[[@B5]\]. Conflicting results were published concerning the stimulation of glucose-6-phosphatase promoter activity by elevated intracellular cAMP concentrations. While dibutyryl cAMP alone did not significantly stimulate transcription of a luciferase reporter gene under control of 1.2 kb of the human glucose-6-phosphatase promoter in H4IIIE hepatoma cells \[[@B6]\], a later report by the same group described a ≈ 2-fold stimulation of glucose-6-phosphatase promoter activity with dibutyryl cAMP \[[@B7]\]. The sequence from -161 to -152 of the human glucose-6-phosphatase promoter, including the motif 5\'-TTTACGTAA-3\', was proposed to mediate the effect of dibutyryl cAMP on reporter gene transcription \[[@B7]\]. In HepG2 cells a glucose-6-phosphatase promoter/chloramphenicol acetyltransferase reporter gene showed a 2 to 3-fold enhancement in transcription following stimulation of the cells with dibutyryl cAMP \[[@B8]\]. Here, the sequence from -136 to -129 of the human glucose-6-phosphatase gene, including the motif 5\'-TTGCATCAA-3\', was proposed to be essential to couple dibutyryl cAMP stimulation with enhanced reporter gene transcription \[[@B8]\]. The most important and best characterized protein that connects an elevation of intracellular cAMP concentrations with enhanced transcription of selected genes is the CRE binding protein CREB, a basic region leucine zipper protein. CREB plays an essential role in the regulation of glucose-6-phosphatase gene transcription in the liver, as exemplified by the fact that transgenic mice expressing a dominant-negative CREB mutant in the liver show reduced mRNA levels of glucose-6-phosphatase \[[@B9]\]. CREB not only mediates stimulus-transcription coupling of the cAMP signaling pathway but functions as a point of convergence of many other signaling molecules involving calcium, neurotrophins, tumor promoters, and growth factors \[[@B10]\]. CREB is inactive in the dephosphorylated state and turns into an activator upon phosphorylation. The key enzyme leading to CREB activation is the cAMP-dependent protein kinase (PKA), but CREB serves also as a substrate for calcium/calmodulin-dependent protein kinase IV and the mitogen-and stress-activated kinases MSK1 and 2 \[[@B11],[@B12]\]. To study CREB regulated gene transcription, a signaling cascade needs to be activated that leads to phosphorylation and activation of CREB. This can be accomplished by adding cAMP analogues such as dibutyryl cAMP that passes the plasma membrane or via a direct activation of adenylate cyclase by forskolin. This experimental setting is problematic in that high levels of phosphodiesterase enzymes in the cells may immediately hydrolyze cAMP, with the result that no cAMP-mediated gene transcription can be monitored \[[@B13]\]. Moreover, elevated levels of cAMP may additionally activate the EPAC/Rap1 pathway, making it somewhat difficult to attribute the effect of cAMP solely to the cAMP/PKA signaling pathway. Many functions of cAMP previously attributed to PKA may be in fact the result of EPAC activation \[[@B14]\]. Instead of increasing the intracellular levels of cAMP some investigators employed an overexpression strategy using the catalytic subunit of cAMP-dependent protein kinase to analyze cAMP regulated genes. This approach has the advantage that by excluding a parallel signaling cascade via the cAMP-inducible nucleotide exchange factor EPAC, only the biological outcome of cAMP-dependent protein kinase activation is studied. However, the translocation of the catalytic subunit of cAMP-dependent protein kinase into the nucleus is not very efficient and may rely solely on diffusion \[[@B15]\]. Thus high amounts of expression vector in the range of 1 to 5 μg/plate are often transfected \[[@B16]-[@B18]\] in order to observe an effect on gene transcription. Naturally, these high amounts of expressed catalytic subunit are far away from the physiological concentrations within the cells. Here, we report on the regulation of glucose-6-phosphatase gene transcription by CREB and PKA. Using a constitutively active CREB2/CREB fusion protein, that does not require phosphorylation for activation, and a nuclear-targeted mutant of the catalytic subunit of PKA, we show that CREB activates transcription of glucose-6-phosphatase promoter/luciferase reporter genes via two distinct genetic elements. In contrast, the bZIP protein ATF2, known to compete with CREB for binding to the canonical CRE, did not transactivate these reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Results ======= The proximal region of the human glucose-6-phosphatase gene contains two cyclic AMP response element like motifs ---------------------------------------------------------------------------------------------------------------- Fig. [1A](#F1){ref-type="fig"} shows part of the proximal region of the human glucose-6-phosphatase gene. As indicated, two CRE-like sequences are present, resembling the canonical CRE sequence 5\'-TGACGTCA-3\'. The distal CRE-like site (CRE1) and the proximal CRE-like site (CRE2) differ on two or three positions, respectively, in comparison to the canonical CRE. To investigate the biological function of these elements we constructed a battery of reporter plasmids containing both CRE-like elements or only one of them (Fig. [1B,C](#F1){ref-type="fig"}). Mutations were introduced into CRE1 and CRE2 sites to prevent CREB binding \[[@B19]\]. As a control, we generated a reporter plasmid containing two copies of a CRE/AP1-like element derived from the human tumor necrosis factor (TNF) α gene promoter (Fig. [1D](#F1){ref-type="fig"}). The TNFα gene belongs to the target genes of CREB \[[@B10]\]. Additionally, the reporter genes contained a TATA box derived from the HIV long terminal repeat, an initiator element from the adenovirus major late promoter and the luciferase open reading frame. A constitutively active CREB2/CREB fusion protein transactivates reporter genes with either CRE1 or CRE2 of the glucose-6-phosphatase gene in their regulatory regions ---------------------------------------------------------------------------------------------------------------------------------------------------------------------- The fact that unphosphorylated CREB is transcriptionally silent excludes a simple overexpression strategy to analyze the biological impact of the CRE-like sequences within the glucose-6-phosphatase gene. The use of natural inducers of CREB such as glucagon or epinephrine, that all elevate the cAMP concentration in the cells, also triggers pleiotropic responses by activating the cAMP-dependent protein kinase and the EPAC/Rap1 pathway, so that the biological outcome cannot be attributed solely to CREB activation. We therefore designed a constitutively active CREB2/CREB fusion protein to uncouple the investigation of glucose-6-phosphatase gene transcription from signaling pathways in the cell. Fig. [2A](#F2){ref-type="fig"} shows the structural domains of the basic region leucine zipper (bZIP) transcription factors CREB and CREB2. Both proteins contain a bZIP domain on their C-termini responsible for dimerization and DNA-binding. The N-termini of CREB and CREB2 contain activation domains. While the activation domain of CREB2 is constitutively active and transferable to heterologous DNA-binding domains \[[@B20]\], the major activation domain of CREB is controlled by phosphorylation. We expressed the bZIP domain of CREB, which is responsible for DNA binding and dimerization, as a fusion protein with the constitutively active transcriptional activation domain of CREB2, generating the chimeric transcription factor C2/CREB (Fig. [2A](#F2){ref-type="fig"}). The C2/CREB fusion protein contains additionally an immunological tag used for the detection of the protein. Proteins derived from nuclear extracts of HepG2 human hepatoma cells (mock) or HepG2 cells transfected with an expression vector encoding C2/CREB were fractionated by SDS-PAGE. The fusion protein was identified by Western Blot analysis using antibodies targeting the FLAG epitope. Fig. [2B](#F2){ref-type="fig"} shows that the CREB2/CREB fusion protein was synthesized as expected. To study the regulation of the glucose-6-phosphatase promoter/luciferase reporter genes by C2/CREB, we decided to use the human hepatoma cell line HepG2, as it has been reported that activation of the cAMP signaling pathway stimulates glucose-6-phosphatase gene transcription in these cells \[[@B17],[@B21],[@B22]\]. HepG2 cells were transfected with one of the luciferase reporter plasmids pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc, pG6PCRE1/CRE2mutluc, pG6PCRE1luc or pG6PCRE2luc together with either the \"empty\" expression vector pCMV5 (denoted \"-\") or an expression vector encoding C2/CREB (plasmid pCMV-FLAG-C2/CREB) (denoted \"+\"). As a control, we transfected the pTNFα(CRE/AP1)^2^luc reporter plasmid that contains two copies of the composite CRE/AP1 element of the TNFα promoter. We transfected additionally plasmid pRSVβ, encoding β-galactosidase under the control of the Rous sarcoma virus long-terminal repeat, to correct for variations in transfection efficiencies. Luciferase activities were normalized for transfection efficiency by dividing luciferase light units by β-galactosidase activities. The results of the transfection experiments are depicted in Fig. [3](#F3){ref-type="fig"}. Transcription of the pG6PCRE1/CRE2luc reporter gene, that contains both CRE-like sequences in its regulatory region, was strongly induced following expression of C2/CREB (Fig. [3A](#F3){ref-type="fig"}, upper panel). Mutation of CRE1 or CRE2 did not abolish the transactivation potential of C2/CREB (Fig. [3A](#F3){ref-type="fig"}, middle, bottom panel), indicating that both CRE-like sequences function independently as *bona fide*CREs. This conclusion was confirmed by transfection experiments of reporter plasmids having only one of the CRE-like sequences in the regulatory regions. Fig. [3B](#F3){ref-type="fig"} shows that both reporter genes pG6PCRE1luc and pG6PCRE2luc were transactivated by C2/CREB. Finally, C2/CREB also transactivated the pTNFα(CRE/AP1)^2^luc reporter gene via the CRE/AP1 element (Fig. [3C](#F3){ref-type="fig"}). We conclude that both CRE1 and CRE2 motifs of the glucose-6-phosphatase gene promoter function as *bona fide*CREs. Expression of a nuclear-targeted catalytic subunit of cAMP-dependent protein kinase ----------------------------------------------------------------------------------- The structural domains of the catalytic subunit of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) are depicted in Fig. [4A](#F4){ref-type="fig"}. The protein is myristylated on the N-terminus, but myristylation is nonessential for enzyme activation \[[@B23]\]. We modified the N- terminal region of the protein by adding a nuclear targeting signal derived from the SV40 large T antigen (NLS) and a FLAG epitope to facilitate immunological detection. This mutant termed NLSCα should be sorted to the nuclear compartment, due to the presence of the NLS. HepG2 cells were transfected with an expression vector encoding NLSCα. Nuclear extracts of these cells were fractionated by SDS-PAGE and analyzed by Western blotting. Fig. [4B](#F4){ref-type="fig"} shows that the modified catalytic subunit of cAMP-dependent protein kinase was synthesized as expected. To test the biological activity of NLSCα, in comparison to the wild-type form of the catalytic subunit (Cα), we measured the activity of the phosphorylation-regulated transcription factor CREB using a fusion protein consisting of the GAL4 DNA-binding domain fused to the kinase inducible activation domain of CREB. The modular structure of the GAL4-CREB fusion protein is depicted in Fig. [4C](#F4){ref-type="fig"}. Transcriptional activation was monitored by co-transfection of the reporter plasmid pUAS^5^luc that contains five copies of the GAL4 binding site termed \"upstream activating sequence\" (UAS) upstream of a luciferase reporter gene (Fig. [4C](#F4){ref-type="fig"}). Since mammalian cells do not express transcription factors that bind to the UAS, this system directly measures the effect of NLSCα or Cα on the transcriptional activation potential of CREB. The reporter plasmid pUAS^5^luc and the expression plasmid that encodes GAL4-CREB were transfected into HepG2 cells together with either an \"empty\" expression vector (denoted \"-\") or expression vectors encoding Cα or NLSCα. Transfection efficiency was monitored by co-transfecting pRSVβ. As a control, plasmid pM1 encoding only the DNA binding domain of GAL4 (GAL4~DBD~) was transfected. Cell extracts were prepared forty-eight hours later and β-galactosidase and luciferase activities were determined. Fig. [4C](#F4){ref-type="fig"} shows that the modified form of PKA catalytic subunit (NLSCα) was highly potent in stimulating the transcriptional activation potential of CREB. The activation was on the order of 60-fold. In contrast, no transcriptional activation was observed following overexpression of the wild-type form of the catalytic subunit. Next, we tested the biological activity of NLSCα with regard to transcription of the glucose-6-phosphatase promoter/luciferase reporter genes. In the experiments, we additionally overexpressed wild-type CREB, the CREBS133A containing a serine to alanine mutation at position 133, and K-CREB, a CREB mutant containing the point mutation R286L within the basic DNA-binding domain, impairing DNA-binding \[[@B24]\]. Transfection of expression vectors encoding the wild-type form of CREB, CREBS133A, or K-CREB did not significantly change basal transcription of the reporter genes. However, transfection of an NLSCα expression vector strongly stimulated transcription of the glucose-6-phosphatase promoter/luciferase reporter genes, containing both or one of the CRE-like sequences of the glucose-6-phosphatase promoter (Fig. [5A,B](#F5){ref-type="fig"}). These results confirm the previous observations, obtained via overexpression of C2/CREB, that both CRE motifs of the glucose-6-phosphatase gene function as independent genetic elements responsive to couple elevated cAMP and PKA levels with enhanced glucose-6-phosphatase gene transcription. Similarly, coexpression of wild-type CREB and NLSCα stimulated reporter gene transcription mediated by the CRE/AP1 element derived from the TNFα gene (Fig. [5C](#F5){ref-type="fig"}). Surprisingly, we still detected reporter gene activation following coexpression of NLSCα with CREBS133A, lacking the major PKA phosphorylation site. Compared to the coexpression experiments of NLSCα with the wild-type form of CREB, the reduced activation of reporter gene transcription in coexpression experiments of NLSCα with CREBS133A indicates that NLSCα catalyzed phosphorylation of serine residue 133 of CREB is important for reporter gene transcription. The fact that CREBS133A is still able to transactivate the reporter genes following expression of NLSCα suggests that NLSCα triggers further phosphorylation reactions leading to enhanced transcription via the CRE-like sequences within the glucose-6-phosphatase gene. In contrast, expression of K-CREB is transcriptionally inactive, in the presence or absence of NLSCα, indicating that DNA-binding is a prerequisite for CREB and CREBS133A to transactivate the reporter genes. Biological activity of a constitutively active CREB2/ATF2 fusion protein on glucose-6-phosphatase promoter/luciferase reporter genes ------------------------------------------------------------------------------------------------------------------------------------ The transcription factor ATF2, a substrate of stress-activated protein kinases, has been reported to bind to the classical cAMP responsive element (CRE) 5\'-TGACGTCA-3\' \[[@B25]\]. Recently, we confirmed this observation showing that ATF2 and CREB compete for binding to the CRE of the secretogranin II gene \[[@B26]\]. ATF2 was proposed to also bind with high affinity to the related DNA target sequence 5\'-TTACGTAA-3\' \[[@B19]\], which is identical to the CRE1 of the glucose-6-phosphatase gene promoter. We tested the biological activity of ATF2 on reporter genes containing sequences of the glucose-6-phosphatase promoter region. As a control, we used a reporter gene containing two copies of the CRE/AP-1 element of the TNFα gene. ATF2 has been shown to stimulate TNFα promoter activity \[[@B27]\]. The unphosphorylated form of ATF2 is transcriptionally inactive, due to an inhibitory intramolecular interaction between the activation domain of ATF2 and the bZIP domain of ATF2 \[[@B28]\]. We therefore expressed a constitutively active CREB2/ATF2 fusion protein to investigate whether ATF2 functions as a transactivator for reporter genes containing the CRE1 and/or CRE2 sequences of the glucose-6-phosphatase gene in the regulatory region. The domain structure of this C2/ATF2 fusion protein is depicted in Fig. [6A](#F6){ref-type="fig"}. HepG2 cells were transfected with one of the reporter plasmids (pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc, pG6PCRE1/CRE2mutluc, pG6PCRE1luc, and pG6PCRE2luc), the internal standard plasmid pRSVβ, and an expression vector encoding C2/ATF2. As a control, we analyzed the reporter plasmid pTNFα(CRE/AP1)^2^luc. The results show that C2/ATF2 was unable to transactivate the reporter genes containing CRE1, CRE2, or both CRE-like sequences derived from the glucose-6-phosphatase gene in the regulatory region, indicating that glucose-6-phosphatase gene transcription is not regulated by ATF2 via the two CRE-like sequences. In contrast, C2/ATF2 strongly activated transcription of the pTNFα(CRE/AP1)^2^luc reporter gene, confirming that TNFα gene transcription is regulated by ATF2. Discussion ========== The gene encoding glucose-6-phosphatase is regulated by increased levels of intracellular cAMP, but the regulatory sites responsible for cAMP-induced gene transcription are still a matter of controversy. The objective of this study was to characterize the genetic elements that function as *cis*-acting sites for transactivation by CREB and that also respond to activated PKA in the nucleus. Two distinct genetic elements had been suggested to couple enhanced levels of cAMP and elevated PKA activity with increased glucose-6-phosphatase gene transcription \[[@B8],[@B17]\] and we intended to clarify which of these elements is required for transactivation of the glucose-6-phosphatase gene by CREB. The reason for the differences in the published reports concerning cAMP-regulated glucose-6-phosphatase gene transcription can be explained by the technical problems that occurs following elevation of the intracellular cAMP concentration, as outlined in the introduction section. To solve these problems, we prefered to measure \"transcriptional activation\" instead of \"transcription factor/DNA-binding\" because although DNA-binding is required for a subsequent transcriptional activation by CREB, an enhanced binding activity of a transcription factor to DNA, monitored by an *in vitro*binding assay, does not necessarily prove an enhanced transcriptional activation potential of this protein \[[@B29]\]. In addition, we expressed a constitutively active CREB2/CREB fusion protein that was highly active in transactivating CREB-responsive target genes. Using this strategy we avoided the use of dibutyryl cAMP or forskolin, that may trigger other biological responses. Furthermore, only nanomolar levels of the expression vector encoding C2/CREB were required to show that both CRE-like sequences (CRE1 and CRE2) within the glucose-6-phosphatase gene function as target sites for an active CREB. Thus, both the reported genetic elements, the sequence from -161 to -152 \[[@B7]\] and the sequence from -136 to -129 \[[@B8]\] of the human glucose-6-phosphatase promoter, mediated reporter gene transactivation by CREB. The fact that the glucose-6-phosphatase gene contains two distinct genetic elements for the regulation by CREB is not surprising as multiple CREs have been found in other genes encoding for instance ICER, MKP-1, Nur77 or Egr-4 \[[@B30]\]. Using an overexpression strategy for the catalytic subunit of PKA, it has been reported that PKA directly stimulates reporter gene transcription under control of CRE1, whereas CRE2 was unable to connect elevated PKA activity with enhanced CRE2-controlled reporter gene transcription \[[@B17]\]. The authors concluded that only the CRE1, but not the CRE2 functions as *bona fide*CRE \[[@B17]\]. In this study a concentration of 5 μg of expression vector encoding the catalytic subunit of PKA was used. In the past, we also employed this overexpression strategy for the catalytic subunit of PKA in the laboratory \[[@B29],[@B31]\] and observed that high levels of expression vectors are required to monitor an effect on gene transcription. We also observed that transcription of the reference gene encoding β-galactosidase under control of the Rous sarcoma virus long terminal repeat was changed following overexpression of the catalytic subunit, although no CRE has been mapped within this promoter/enhancer region. This fact indicates that transfection of micromolar levels of expression vectors encoding the catalytic subunit of PKA disturbs the machinery of transcription and the data obtained by this approach may not always depict the real picture. Likewise, it has been known for many years that expression of the strong transcriptional activator VP16 induces transcriptional repression within the cells, due to squelching. In the study reported here, expression of a modified catalytic subunit that had a nuclear targeting signal, activated reporter gene transcription very efficiently following transfection of nanomolar amounts of the expression vector. In fact, we used a 50-fold lower amount of expression vector compared to the study by Streeper et al. \[[@B17]\]. Transfection of nanomolar concentrations of an expression plasmid encoding the wild-type catalytic subunit of PKA did not show any effect on reporter gene transcription. These experiments, involving an overexpression of NLSCα, showed an enhanced transcription of reporter genes having either an intact CRE1 or CRE2 in its regulatory region, indicating that both CRE1 and CRE2 functioned as *bona fide*CRE. We also observed an activation of glucose-6-phosphatase promoter/luciferase reporter gene transcription following coexpression of CREBS133A with NLSCα. Phosphorylation of the serine 133 residue of CREB is the predominant mechanism to enhance the transcriptional activation potential of CREB via recruitment of the coactivator CREB binding protein (CBP) and its paralogue p300 to the promoter. Accordingly, we observed a clear reduction of reporter gene transcription in coexpression studies of NLSCα together with CREBS133A instead of wild-type CREB. However, expression of CREBS133A still contributed to reporter gene transcription in the presence of NLSCα, suggesting that either other residues of CREBS133A or other promoter-bound proteins involved in the regulation of CREB-mediated transcription are phosphorylated. PKA can phosphorylate CBP directly or other components of the transcriptional machinery downstream from CREB \[[@B32],[@B33]\]. Nonetheless, the experiments confirmed that both CRE-like sequences within the glucose-6-phosphatase gene function as genetic elements to mediate transactivation via CREB. In contrast to the expression experiments involving C2/CREB, we did not detect an effect of C2/ATF2, a constitutively active CREB2/ATF2 fusion protein, on glucose-6-phosphatase promoter/luciferase reporter gene transcription, despite the fact that the sequence 5\'-TTACGTAA-3\', which is identical to the CRE1 site of the glucose-6-phosphatase promoter, has been shown to function as a high affinity binding site for ATF2 in vitro \[[@B19]\]. This discrepancy supports the view that transcriptional bioassays and not in vitro DNA-protein binding experiments describe the biological activities of transcription factors. The CRE-like sequences CRE1 and CRE2 of the glucose-6-phosphatase gene are therefore strikingly different from the CRE/AP1 site of the TNFα gene, that is strongly activated by C2/ATF2. Recently, we showed that ATF2 is able to specifically transactivate CRE-containing genes and we identified the secretogranin II gene as a target gene for ATF2 \[[@B26]\]. We also showed that C2/ATF2 only marginally enhanced transcription of a reporter gene carrying four copies of the c-Fos CRE in its regulatory region, while transcription regulated by the tyrosine hydroxylase promoter was not upregulated at all. Thus, ATF2 distinguishs between different CRE-containing genes. The biological activity of ATF2 is regulated by stress-activated protein kinases and ATF2 is thought to play an important role in the cellular stress response. Our data sheds light on the fact that expression of glucose-6-phosphatase is not connected -- via ATF2 -- to the cellular stress response, in contrast to the TNFα gene. Conclusions =========== We have shown that there are two distinct CREB-responsive sites in the glucose-6-phosphatase gene promoter that are responding to either a constitutively active CREB or elevated concentrations of the catalytic subunit of cAMP-dependent protein kinase in the nucleus. This study further shows that the expression vectors encoding C2/CREB and NLSCα are valuable tools for the investigation of CREB-mediated gene transcription and the biological functions of CREB. CREB is a key molecule in neuronal survival, as demonstrated by the fact that a dominant-negative A-CREB induced apoptosis in sympathetic neurons grown in NGF \[[@B34]\]. The C2/CREB fusion protein, that directs CREB-mediated gene transcription in the absence of PKA activation, can be used in gain-of-function experiments to investigate the cytoprotective activity of CREB on the molecular level. Most importantly, the C2/CREB protein can be used to identify anti-apoptotic genes that are controlled by CREB. Likewise, expression of NLSCα will permit the analysis of PKA-regulated gene transcription, without influencing other functions of PKA in the cytosol. Methods ======= Reporter constructs ------------------- The glucose-6-phosphatase promoter/luciferase reporter genes pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc, and pG6PCRE1/CRE2mutluc were constructed by the insertion of the annealed oligonucleotides depicted in Fig. [1B](#F1){ref-type="fig"} with KpnI/XhoI cohesive ends into the KpnI/XhoI sites of plasmid pHIVTATAluc \[[@B35]\]. The glucose-6-phosphatase promoter/luciferase reporter genes pG6PCRE1luc and pG6PCRE2luc that contain either the CRE1 or the CRE2 sequence derived from the human glucose-6-phosphatase gene were constructed by the insertion of the annealed oligonucleotides depicted in Fig. [1C](#F1){ref-type="fig"} with XhoI/SalI cohesive ends into the XhoI/SalI sites of plasmid pHIVTATA-CAT \[[@B36]\]. These plasmids termed pG6PCRE1CAT and pG6PCRE2CAT were digested with XbaI, filled in with the Klenow fragment of DNA polymerase I, recut with XhoI and cloned into plasmid pGL3-Basic (Promega). The reporter gene pTNFα(CRE/AP-1)^2^luc, that contains two copies of the CRE/AP1 element of the human tumor necrosis factor α gene, was generated in a similar way with annealed oligonucleotides encompassing the sequence depicted in Fig. [1D](#F1){ref-type="fig"}. Plasmid pUAS^5^luc containing the luciferase reporter gene, a TATA box derived from the HIV long terminal repeat, an initiator element from the adenovirus major late promoter, and five binding sites for GAL4 (termed \'upstream activating sequence\', UAS) has been described \[[@B35]\]. Expression vectors ------------------ The expression vector pCMV-FLAG-C2/CREB, encoding a constitutively active CREB2/CREB chimera, has been described \[[@B26]\]. The CREB2/CREB fusion protein consists of the amino-terminal 187 amino acids from CREB2, encompassing the phosphorylation-independent transcriptional activation domain, and amino acids 182 to 326 of CREB, including the bZIP domain. Expression vectors encoding CREB, CREBS133A, and K-CREB were kind gifts of Wilhart Knepel and Elke Oetjen, Department of Molecular Pharmacology, University of Göttingen, Germany. The expression vector encoding a constitutively active CREB2/ATF2 fusion protein termed C2/ATF2 has been described \[[@B27]\]. The GAL4 expression plasmid pFA2CREB was purchased from Stratagene. The fusion protein encodes the transcriptional activation sequence of CREB (amino acids 1--281) fused to the DNA-binding domain of GAL4. An expression vector encoding the catalytic subunit of PKA (pCMVCα) was a kind gift of Michael Uhler from the University of Michigan, Ann Arbor \[[@B37]\]. The construction of the NLSCα encoding expression vector has been described \[[@B26]\]. NLSCα has a triple FLAG-tag and a nuclear localization signal on the N-terminus, followed by amino acids 19 to 351 of Cα. The expression vector pRSVβ, encoding β-galactosidase under the control of the Rous sarcoma virus long terminal repeat, has been described \[[@B29]\]. Cell culture, transfections, and reporter gene assays ----------------------------------------------------- Human HepG2 hepatoma cells were cultured and transfected as described \[[@B38]\]. The amounts of expression vectors transfected are indicated in the figure legends. The luciferase reporter plasmids (1 μg) and the internal reference plasmid pRSVβ were transfected into cells grown on 60 mm plates. Lysates were prepared forty-eight hours later using cell culture lysis buffer (Promega) and β-galactosidase and luciferase activities were measured as described \[[@B35]\]. Western Blots ------------- Nuclear extracts were prepared as described \[[@B39]\]. 20 μg of nuclear proteins were separated by SDS-PAGE and the blot was incubated with the M2 monoclonal antibody directed against the FLAG epitope (Sigma, \# F3165). Blots were developed with a horseradish peroxidase conjugated anti-mouse secondary antibody and ECL (Amersham, Freiburg, Germany). Abbreviations ============= ATF activating transcription factor bZIP basic region leucine zipper CRE cAMP response element CREB cAMP response element binding protein G6P glucose-6-phosphatase PKA protein kinase A TNF tumor necrosis factor Authors\' contributions ======================= GT designed the study, generated the reporter plasmids and the expression vectors encoding C2/CREB, C2/ATF2, and NLSCα, performed part of the transfection experiments and drafted the manuscript. JAS performed the transfection experiments depicted in Figs. [3](#F3){ref-type="fig"}, [4C](#F4){ref-type="fig"}, and [5](#F5){ref-type="fig"}. LS performed the C2/ATF2 transfection experiments depicted in Figs. [6](#F6){ref-type="fig"}. All authors read and approved the manuscript. Acknowledgement =============== We thank Karl Bach for excellent technical help, Wilhart Knepel, Elke Oetjen and Michael Uhler for plasmids, and Libby Guethlein and Oliver Rössler for critical reading of the manuscript. This work was supported by the Medical Faculty of the University of Saarland via HOMFOR and by fellowships from the \"Deutscher Akademischer Austauschdienst\" (DAAD) to Jude Al Sarraj and from the European Graduate School of Neuroscience (EURON) to Luisa Stefano. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **The human glucose-6-phosphatase gene promoter**. (A) Sequence of a portion of the human glucose-6-phosphatase gene promoter including the CRE-like sequences CRE1 and CRE2. (B) The reporter plasmid pG6PCRE1/CRE2luc contains a minimal promoter consisting of the human immunodeficiency virus TATA box, the adenovirus major late promoter initiator element, the luciferase open reading frame, and glucose-6-phosphatase promoter sequences encompassing both CRE-like sequences CRE1 and CRE2. The reporter plasmids pG6PCRE1mut/CRE2luc and pG6PCRE1/CRE2mutluc carry mutations in either the CRE1 or the CRE2, to inactivate these sites. (C) Reporter plasmids pG6PCRE1luc and pG6PCRE2luc contain glucose-6-phosphatase promoter sequences encompassing either the CRE1 or the CRE2. (D) The reporter plasmid pTNFα(CRE/AP1)^2^luc contains two identical copies of the composite CRE/AP1 sequence derived from the human TNFα gene, upstream of a minimal promoter. The sequence of one of these motifs is depicted. ::: ![](1471-2199-6-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Schematic representation and expression of the constitutively active CREB2/CREB fusion protein C2/CREB**. (A) Schematic representation of the modular structure of CREB, CREB2, and C2/CREB. The basic region leucine zipper domain (bZIP) is indicated. The chimeric bZIP protein C2/CREB consists of the constitutively active transcriptional activation domain of CREB2 and the bZIP domain of CREB, responsible for dimerization and DNA-binding. (B) Western Blot analysis of HepG2 cells transfected with an expression vector encoding C2/CREB. As a control, extracts from mock transfected HepG2 cells were analyzed. Western blots were probed with an antibody against the FLAG-tag. ::: ![](1471-2199-6-2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Biological activity of C2/CREB, a constitutively active CREB2/CREB fusion protein**. One of the reporter plasmids pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc pG6PCRE1/CRE2mutluc (A), pG6PCRE1luc, pG6PCRE2luc (B), or pTNFα(CRE/AP1)^2^luc (C) (1 μg/plate) was transfected into HepG2 cells together with the pRSVβ internal standard plasmid (2 μg/plate), encoding β-galactosidase under the control of the Rous sarcoma virus long terminal repeat, and either the \"empty\" expression vector pCMV5 or an expression vector encoding C2/CREB (20 ng plasmid/plate). The data are presented as the ratio of luciferase activity (light units) to β-galactosidase units (OD units) measured in the cell extracts. The mean +/- SD is depicted. ::: ![](1471-2199-6-2-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Schematic representation and biological activity of the nuclear targeted mutant of the catalytic subunit of cAMP-dependent protein kinase**. (A) Schematic representation of the modular structure of the catalytic subunit of cAMP-dependent protein kinase (Cα) and the mutant NLSCα. The wild-type enzyme is myristylated as indicated (Myr). The sequence of the nuclear localization signal derived from the SV40 large T antigen and the triple FLAG epitope in NLSCα are shown. (B) Western Blot analysis of HepG2 cells transfected with an expression vector encoding NLSCα. As a control, extracts from mock transfected HepG2 cells were analyzed. Western blots were probed with an antibody against the FLAG-tag. (C) Modular structure of the GAL4-CREB fusion protein. The protein consists of the DNA-binding domain of GAL4 (amino acids 1--147) and the activation domain of CREB (amino acids 1--281). The reporter plasmid pUAS^5^luc contains a transcription unit encompassing the luciferase open reading frame and a minimal promoter that consists of five copies of the upstream activating sequence (UAS), a TATA box derived from the HIV long terminal repeat and the initiator element from the adenovirus major late promoter. The reporter plasmid pUAS^5^luc (1 μg/plate) was transfected into HepG2 cells together with the pRSVβ internal standard plasmid (2 μg/plate) and either an expression vector encoding the DNA binding domain of GAL4 (plasmid pM1) or the GAL4-CREB fusion protein (1 μg plasmid/plate). In addition, cells were transfected with an expression vector encoding either the wild-type (Cα) or mutated (NLSCα) form of cAMP-dependent protein kinase (100 ng/plate). Forty-eight hours post-transfection cell extracts were prepared and the β-galactosidase and luciferase activities of these extracts were determined. ::: ![](1471-2199-6-2-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Transcriptional activity of CREB and CREB mutants in the presence of a nuclear targeted mutant of the catalytic subunit of cAMP-dependent protein kinase**. One of the reporter plasmids pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc pG6PCRE1/CRE2mutluc (A), pG6PCRE1luc, pG6PCRE2luc (B), or pTNFα(CRE/AP1)^2^luc (C) (1 μg/plate) was transfected into HepG2 cells together with the pRSVβ internal standard plasmid (2 μg/plate) and the \"empty\" expression vector pCMV5 or an expression vector encoding either CREB, CREBS133A, or K-CREB (20 ng plasmid/plate). In addition, an expression vector encoding NLSCα (100 ng/plate) was transfected. Forty-eight hours post-transfection cell extracts were prepared and the β-galactosidase and luciferase activities of these extracts were determined. The data are presented as the ratio of luciferase activity (light units) to β-galactosidase units (OD units) measured in the cell extracts. The mean +/- SD is depicted. ::: ![](1471-2199-6-2-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Biological activity of constitutively active ATF2 fusion protein towards glucose-6-phosphatase promoter-containing reporter genes**. (A) Schematic representation of the modular structure of C2/ATF2. This chimeric bZIP protein consists of the constitutively active transcriptional activation domain of CREB2 and the bZIP domain of ATF2, responsible for dimerization and DNA-binding. (B, C, D) HepG2 cells were transfected with one of the reporter plasmids pG6PCRE1/CRE2luc, pG6PCRE1mut/CRE2luc pG6PCRE1/CRE2mutluc (B), pG6PCRE1luc, pG6PCRE2luc (C), or pTNFα(CRE/AP1)^2^luc (D), the pRSVβ internal reference plasmid, and either the \"empty\" expression vector pCMV5 or an expression vector encoding C2/ATF2 (100 ng expression plasmid/plate). Lysates were prepared forty-eight hours post-transfection and β-galactosidase and luciferase activities were measured. The mean +/- SD is depicted. ::: ![](1471-2199-6-2-6) :::

PubMed Central

2024-06-05T03:55:52.563018

2005-1-19

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548273/", "journal": "BMC Mol Biol. 2005 Jan 19; 6:2", "authors": [ { "first": "Gerald", "last": "Thiel" }, { "first": "Jude", "last": "Al Sarraj" }, { "first": "Luisa", "last": "Stefano" } ] }

PMC548274

Background ========== Among many trophic factors that act on sensory neurons, three have been studied extensively: nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). During development, NGF, GDNF and BDNF, along with neurotrophin-3 (NT3), support the survival of subpopulations of sensory neurons through their cognate trk receptors \[[@B1]-[@B3]\]. In the adult, uninjured rat, receptors for NGF, GDNF and BDNF are found in partially distinct subpopulations of sensory neurons. NGF-responsive, trkA-containing neurons are mostly small diameter, contain the neuropeptide calcitonin gene-related peptide (CGRP), and are generally thought to have nociceptive properties \[[@B4],[@B5]\]. The BDNF-responsive, trkB-containing population of sensory neurons is predominantly larger diameter and mechanosensitive \[[@B6]\], although there is considerable overlap with the trkA population \[[@B7]\]. GDNF receptors are more widespread, with as many as 60% of dorsal root ganglion (DRG) neurons containing c-ret, while GFR components are found throughout the c-ret population, as well as in c-ret negative neurons \[[@B8],[@B9]\]. Additionally, sensory neurons that are postnatally dependent on GDNF for survival bind the isolectin B~4~(IB~4~, \[[@B2]\]). NGF is thought to play a primary role in the development and maintenance of several pro-algesic states. NGF produces sensitization of nociceptive responses in vivo and increases responsiveness to chemical stimuli associated with nociceptive neurotransmission in vitro \[[@B10]-[@B12]\]. NGF increases the expression of the pro-inflammatory neuropeptide CGRP \[[@B13]\] and increases substance P (SP) and CGRP content in sensory neurons \[[@B14]-[@B17]\]. NGF also promotes the development and maintenance of hyperalgesia following chronic constriction injury \[[@B18],[@B19]\]. Additionally, NGF increases capsaicin (CAP) sensitivity both in vivo and in vitro \[[@B20]-[@B24]\]. In accordance with this increased CAP sensitivity, NGF increases the expression of the CAP- and noxious heat-sensitive ion channel vanilloid receptor type 1 (TRPV1) through the ras/p38 MAP kinase signaling pathway, thereby promoting thermal hyperalgesia \[[@B25],[@B26]\]. Additionally, NGF has recently been shown to modulate TRPV1 by releasing the receptor from phosphotidylinositol-4,5-bisphosphate-mediated inhibition \[[@B27]\]. GDNF also plays a role in modulating nociceptive processing, but with contrasting in vitro and in vivo effects. In cultured DRG neurons, GDNF increases CAP sensitivity and TRPV1 expression \[[@B16],[@B26]\] and increases neuropeptide content \[[@B16]\]. On the other hand, intrathecal injection of GDNF has no effect on C-fiber evoked outflow of SP and does not induce thermal hyperalgesia, while NGF does both \[[@B15]\]. This, coupled with the observation that GDNF-overexpressing mice do not develop thermal or mechanical hypersensitivity \[[@B28]\], suggests that GDNF might not lead to nociceptor sensitization in vivo. In fact, in nerve injured rats, GDNF has antihyperalgesic effects \[[@B29]\] and promotes the functional regeneration of DRG-spinal cord connections \[[@B30]\]. It is not understood how this dissociation of the in vitro and in vivo effects of GDNF is manifested. Inflammation and nerve injury both increase BDNF content in DRG neurons and in the spinal cord, and this increase in BDNF is associated with the maintenance of a hyperalgesic state \[[@B31]-[@B36]\]. Furthermore, BDNF is released in the spinal cord upon noxious afferent stimulation \[[@B37],[@B38]\], and peripheral CAP application increases BDNF release at central terminals of sensory neurons \[[@B39]\]. Hence, BDNF might act as a neurotransmitter in the pain pathway in adult animals \[[@B40]\]. The trophic properties of BDNF on adult sensory neurons, particularly nociceptors, are poorly understood. In addition to CAP, a number of other TRPV1 agonists have been described. Among these are compounds that are also cannabinoid receptor agonists, including the endogenous cannabinoid anandamide (AEA, \[[@B41],[@B42]\] and the synthetic AEA analogue arachidonyl-2-chloroethylamide (ACEA, \[[@B43]\]). AEA causes antinociception in vivo through central \[[@B44]\] and peripheral mechanisms \[[@B45],[@B46]\]. It is not known, though, whether the endogenous production of AEA can reach sufficient concentrations under pathophysiological states to act as an endogenous activator of TRPV1. One potential mechanism whereby growth factors could contribute to the development of nociceptor sensitization is through the modulation of the efficacy or potency of AEA at TRPV1.Thus, growth factors might unmask conditions under which AEA could be capable of functioning as an endogenously produced TRPV1 agonist, potentially leading to neurogenic inflammation and thermal hyperalgesia. The aim of the present work was to gain a more precise understanding of NGF-, GDNF- and BDNF-dependent alterations of cultured TG sensory neuron survival and phenotype, with attention to markers and functions that are related to nociception. Furthermore, by relating these findings to neurosecretion associated with pharmacological stimulation of TRPV1, we hope to present a rationale for differences in how these neurotrophins might contribute to altered nociception at the level of the TG sensory neuron. Moreover, there is a gap in knowledge concerning neurotrophic factor influence over TG neurons, as the vast majority of our understanding of how neurotrophins alter sensory neurons stems from studies of the DRG. The recent advances of CGRP receptor antagonists for the treatment of migraine \[[@B47],[@B48]\] raises the possibility that manipulations which influence CGRP expression and/or secretion might be beneficial in the treatment of migraine or conditions related to the cerebral vasculature. A fuller understanding of the impact of NGF, GDNF and BDNF on TG neurochemical properties has the potential to lead to novel therapeutic strategies concerning disease states involving neuropeptide secretion in the trigeminal system, such as migraine. Results ======= Effects of NGF, GDNF or BDNF treatment on neuronal survival in vitro -------------------------------------------------------------------- TG neuronal cultures were plated at equal densities (\~5000 neurons / well) on 8-well culture slides (experimental design shown in Figure [1](#F1){ref-type="fig"}). After five days of growth factor treatment, immunocytochemistry (ICC) for 200 kD neurofilament (NF-H) present in all sensory neurons \[[@B49]\] and not other ganglion cells (i.e., glia), was performed to determine the number of neurons in each well. Representative photomicrographs are shown in Figure [2](#F2){ref-type="fig"}. NGF did not significantly influence the number of neurons in culture following five days of treatment (Fig [3](#F3){ref-type="fig"}). On the other hand, GDNF significantly enhanced the number of neurons per well versus no growth factor treated cultures at both 1 and 100 ng/ml (Fig. [3](#F3){ref-type="fig"}). The enhanced neuronal survival seen with GDNF was biphasic, as 10 ng/ml GDNF did not significantly enhance neuronal survival. It should be noted that in GDNF-treated cultures we observed a robust sprouting of TG neurons, appearing far more robust than in NGF- or BDNF-treated cultures (although sprouting was still evident in these cultures). Effects of NGF, GDNF or BDNF treatment on TRPV1 mRNA and protein levels ----------------------------------------------------------------------- To directly assess changes in TRPV1 mRNA levels with growth factor treatment, quantitative, realtime PCR was conducted. NGF, GDNF or BDNF (100 ng/ml) did not significantly alter TRPV1 mRNA levels after 5 days of treatment (Fig. [4](#F4){ref-type="fig"}). To evaluate whether growth factor treatment might exert post-transcriptional regulation of TRPV1 gene expression, we next examined their effects on TRPV1 protein levels by Western blot. As shown in Fig. [5](#F5){ref-type="fig"}, a major band was detected with the TRPV1 antibody at \~130 kD, consistent with the glycosylated form of TRPV1 \[[@B25]\]. NGF and GDNF (100 ng/ml) each was demonstrated to have increased TRPV1 protein levels by approximately 50% compared with no growth factor-treated cultures (Fig [5](#F5){ref-type="fig"}). BDNF effects on TRPV1 protein levels were not assessed, because preliminary studies indicated that BDNF did not influence CAP-evoked CGRP release. Effects of NGF, GDNF or BDNF treatment on K^+^-evoked CGRP release and CGRP content ----------------------------------------------------------------------------------- We next examined the effect of growth factor treatment on K^+^-evoked CGRP release and total CGRP content in TG neuronal cultures. NGF treatment significantly and concentration-dependently increased 50 mM K^+^-evoked CGRP release at every concentration, peaking (10.5-fold increase vs. control) at 100 ng/ml (Fig. [6A](#F6){ref-type="fig"}). Similarly, GDNF significantly and concentration-dependently increased K^+^- evoked release at every concentration, again peaking (9.7-fold increase vs. control) at 100 ng/ml. On the other hand, BDNF did not alter K -evoked CGRP release. Because there were differences in neuron numbers between growth factor conditions, we examined under each condition the proportion of neurons that were positive for CGRP, TRPV1 or IB~4~. Representative photomicrographs for each of these conditions are shown in Figure [7](#F7){ref-type="fig"}. The proportion of neurons positive for CGRP, TRPV1 or IB~4~are shown in Table [1](#T1){ref-type="table"}. In no cases were CGRP- or TRPV1-immunoreactive or IB~4~-binding neurons not likewise immunoreactive for NF-H. We then utilized the proportion of CGRP-expressing neurons under NGF, GDNF or BDNF treatment conditions to normalize K^+^- evoked CGRP release to the number of CGRP-immunoreactive neurons under each condition, respectively (see *Methods*for normalization calculation). Following this transformation, NGF treatment still augmented K^+^-evoked CGRP release at every concentration tested, with the magnitude of the effect reaching 18 times that of control (Fig [6B](#F6){ref-type="fig"}). While GDNF treatment again enhanced K^+^-evoked CGRP release at all concentrations following normalization, its peak effect was relatively lower. BDNF treatment continued not to have an effect following normalization to the number of CGRP-immunoreactive neurons. Much like K^+^-evoked CGRP release, total CGRP content was increased significantly by NGF and GDNF treatment, while BDNF had no effect (Fig. [6C](#F6){ref-type="fig"}). Both NGF and GDNF increased CGRP content concentration-dependently at every concentration (p \< 0.001), with peaks at 10 ng/ml (3.2-fold increase) for NGF and 100 ng/ml (3.0-fold increase) for GDNF (Fig. [6C](#F6){ref-type="fig"}). Again, we normalized these data to CGRP-immunoreactive neurons to assess changes in relation to alterations in CGRP-immunoreactive neurons between the growth factor conditions. Interestingly, the GDNF augmentation of CGRP release appeared to be due primarily to the large increases in neuron survival, because only the 10 ng/ml GDNF condition was significantly greater than control (p \< 0.001), but still substantial lower compared with the untransformed lysis figures (Fig. [6D](#F6){ref-type="fig"}). On the other hand, normalization led to a further augmention in the effect of NGF on total CGRP content, up to a 5.8-fold increase. BDNF treatment did not alter the normalized CGRP content. Effects of NGF, GDNF or BDNF treatment on CAP-evoked CGRP release ----------------------------------------------------------------- To assess the effect of growth factor supplementation on the neurosecretory function of nociceptors in culture, we evaluated CAP-evoked CGRP release. Treatment with 100 nM CAP for 10 min led to a significant enhancement of CGRP release over baseline under all conditions. Treatment of cultures with NGF or GDNF resulted in a concentration-dependent enhancement of this effect, with significant increases in CAP-evoked CGRP release at 1, 10 and 100 ng/ml. BDNF also enhanced the ability of CAP to evoke CGRP release; however, the effect was only seen at the 100 ng/ml concentration. In every case, GDNF and NGF (except 1 ng/ml NGF) supplementation significantly enhanced CAP-evoked CGRP release over the same concentration of BDNF (Fig [8A](#F8){ref-type="fig"}). To determine to what extent the enhancement of CAP-evoked CGRP release in response to growth factor treatment might derive from increased neuronal survival and/or up-regulation in the proportion of neurons expressing TRPV1, release data were normalized to the number of TRPV1-immunoreactive neurons (Fig. [8B](#F8){ref-type="fig"}; TRPV1 neuron proportions shown in Table [1](#T1){ref-type="table"}). In this case, NGF treatment still significantly increased CAP-evoked CGRP release over no growth factor treatment at 1, 10 and 100 ng/ml. However, this normalization procedure led to a relative reduction in the GDNF effect on CAP-evoked CGRP release, such that only the 10 ng/ml condition reached a significant increase in CGRP outflow. After normalization, BDNF treatment had no significant effect on CAP-evoked CGRP release at 1 or 100 ng/ml and actually reduced the amount of release at 10 ng/ml. Moreover, following normalization, NGF-treated neurons displayed a significantly higher CGRP release than either GDNF- or BDNF-treated neurons at every concentration of growth factor tested (Fig [8B](#F8){ref-type="fig"}). Because treatment with either GDNF or NGF induced a large increase in 100 nM CAP-evoked CGRP release, we assessed the effect of supplementation with these growth factors on the receptor pharmacodynamics of this response compared with no growth factor supplementation. GDNF or NGF treatment significantly increased the Hill slope of the concentration-response function from 1.12 ± 0.47 in the no growth factor condition to 3.760 ± 0.057 for 100 ng/ml NGF and to 4.48 ± 0.90 for 100 ng/ml GDNF (Fig [9A](#F9){ref-type="fig"}) but had no effect on the EC~50~(41 nM for GDNF or NGF and 47 nM for no growth factor). GDNF (100 ng/ml) or NGF (100 ng/ml) treatment caused a significant, five-fold increase in the E~max~of the CAP concentration-response curve (Fig. [9B](#F9){ref-type="fig"}). A common feature of CAP concentration-response functions is that they frequently exhibit an inverted U-shape, likely attributable to TRPV1 desensitization at higher concentrations. Thus, whereas the concentration-response function in the absence of growth factor displayed desensitization only at 1 uM, that for either GDNF- and NGF-treated TG cultures showed desensitization of the CAP-evoked CGRP response at concentrations above 100 nM (Fig. [9B](#F9){ref-type="fig"}). Effects of NGF, GDNF or BDNF treatment on AEA- and ACEA-evoked CGRP release --------------------------------------------------------------------------- To test whether growth factor-mediated enhancement of neuropeptide release might be generalizable to other TRPV1 secretagogues, we evaluated the dual cannabinoid-vanilloid agonists AEA and ACEA. As in the case with CAP, the effects of AEA and ACEA on evoked CGRP release were augmented by supplementation of TG cultures with NGF (Fig. [10A](#F10){ref-type="fig"}) or GDNF (Fig. [10B](#F10){ref-type="fig"}). While TG neurons not treated with growth factors were largely unresponsive to AEA or ACEA, in terms of CGRP release, treatment of cultures with either NGF or GDNF for 5 days at 1, 10 or 100 ng/ml caused a concentration-dependent increase in CGRP release evoked by 30 μM AEA or ACEA. Furthermore, the estimated EC~50~values for each of the individual growth factors to increase evoked CGRP release were equivalent for CAP, AEA and ACEA (NGF = 3.5 ng/ml; GDNF = 1.3 ng/ml). In contrast, BDNF supplementation did not augment AEA- or ACEA-evoked CGRP release, while a small, yet significant, increase in CAP-evoked CGRP release was observed with increasing BDNF concentration (Fig. [10C](#F10){ref-type="fig"}). When TG neurons were supplemented with either 100 ng/ml NGF or GDNF, the E~max~and EC~50~values for AEA did not change (F = 2.082~(4,85)~) ; however, when compared with non-growth factor-treated TG neurons, the E~max~was significantly augmented (Fig. [10D](#F10){ref-type="fig"}). Discussion ========== This study demonstrates that NGF, GDNF and BDNF differentially influence neuronal survival, neuropeptide content and stimulated secretion of TG neurons in culture. GDNF significantly augments TG neuronal survival, while both NGF and GDNF modulate CAP sensitivity, and alter the pharmacodynamics of the concentration-response function for CAP-evoked CGRP release. Furthermore, NGF and GDNF increased the releasable pool and total content of CGRP while increasing TRPV1 protein, without increasing its mRNA. These data support the hypothesis that GDNF and NGF, but not BDNF, alter CAP sensitivity in cultured TG neurons, and taken together, suggest that the differential effects of NGF and GDNF in vitro may reflect their differential effects in vivo, particularly with regard to TRPV1-mediated nociception. GDNF, but not NGF or BDNF significantly increased the number of neurons present in TG culture 5 days post-plating. The finding that this effect of GDNF was biphasic, as the 10 ng/ml concentration did not enhance the number of neurons in TG culture, suggests the possible involvement of different receptors with different concentrations of GDNF. Multiple receptors exist in the GDNF receptor family, and they function in concert with the protein c-ret. GDNF binds most readily to GFR alpha-1, but also binds to GFR alpha-2 / c-ret heterodimers \[[@B50]\]. GFR alpha-1 and -2 are expressed in sensory ganglia and are found mostly in IB~4~-binding neurons \[[@B51]\] that also contain c-ret \[[@B8]\]. Notably, it has been suggested that GFR alpha-1 and GFR alpha-2-containing neurons in the adult rat make up two distinct populations in the DRG within the IB~4~-binding class \[[@B8]\]; hence, the effects observed here may be due to GDNF acting through its high affinity GFR alpha-1 site at the 1 ng/ml concentration and through either or both GFR alpha-1 and GFR alpha-2 / c-ret, for which it has a lower affinity, at the 100 ng/ml concentration. While the observation here that GDNF promotes survival is a novel finding for rat TG sensory neurons in primary culture, the neuroprotective effects of GDNF are well documented. GDNF is protective against the loss of dopaminergic neurons in animal models of Parkinson\'s disease \[[@B52],[@B53]\], and GDNF reduces the number of apoptotic bodies in DRG explants from adult mice \[[@B54]\]. GDNF also rescues the reduction of P2X3 expression that occurs in the DRG following axotomy \[[@B55]\]. Although BDNF and NGF did not significantly increase the number of TG neurons in culture, we did observe a significant, linear trend for increased neuron survival as a function of increasing BDNF concentrations, an observation not present in NGF-treated TG cultures. Hence, BDNF and GDNF both promoted the survival of TG neurons in vitro. Primary cultures generated here were grown in the presence of mitotic inhibitors, which greatly reduce the supportive glial cells and the trophic factors they normally provide TG neurons in the native ganglia (although some of these cells are still present). Removal of astroglial support promotes apoptosis in cerebral neuronal cultures, an effect which is reversed by addition of GDNF or BDNF, but not NGF, to the culture medium \[[@B56]\]. The addition of GDNF or BDNF to the culture medium here may have re-supplied, at least partially, the withdrawn glial-supplied trophic factors that maintain sensory neurons in vivo, thereby increasing the number of neurons in culture at 5 days. It should be noted, however, that despite the significant effects of the growth factors observed in this study on neuronal survival, from an original density of nearly 5,000 neurons / well in the original culture homogenate, only about 10% of neurons could be counted at 5 days post-platting, in the control condition. A maximum of just over 20% were counted in the 100 ng/ml GDNF treated TG cultures. Therefore, it stands to reason that GDNF and BDNF are supporting the survival of certain classes of sensory neurons that are not supported either without growth factors or with NGF alone. The proportions of TG neurons in culture that expressed CGRP- or TRPV1-immunoreactivity or IB~4~-binding sites were assessed with attention to how these proportions changed in the presence of NGF, GDNF and BDNF. Notably, the proportion of sensory neurons in vitro that expressed CGRP (\~65%) was much larger than the known proportion of CGRP-containing neurons in native TG (\~35%, \[[@B57]\]). This likely indicates that the culturing process conditions are either selectively preserving peptidergic neurons or that normally non-peptidergic neurons novelly express CGRP in culture. On the other hand, discrepancies on reports of the percentage of IB~4~-binding neurons in native TG, ranging from \~35--60% \[[@B58],[@B59]\] make it difficult to assess whether there is an increased proportion of IB~4~-binding neurons in culture; however, our data indicate that this proportion is at least in the upper range, if not greater than native TG. Furthermore, a probable significant overlap exists between CGRP-immunoreactive and IB~4~-binding neuronal populations observed here, as both were found in the majority of TG neurons in culture, in agreement with the demonstration that these populations overlap significantly in native, adult rat TG and DRG (Price and Flores, unpublished observations). We observed that a greater proportion of TRPV1-immunoreactive neurons were present with GDNF supplementation (nearly 30% increase), again indicating either that GDNF preferentially supports the survival of TRPV1-expressing sensory neurons or that neurons that do not normally express TRPV1 begin expressing TRPV1 when GDNF is included in the culture medium. The latter proposition is supported by the finding that peripheral treatment with anti-GDNF antibodies suppresses the novel expression of TRPV1 in IB~4~-binding neurons following peripheral inflammation \[[@B60]\]. The percentage of TRPV1-expressing neurons in TG cultures not treated with growth factors was essentially equivalent to the percentage in native TG \[[@B57]\]. This finding indicates that, not only does GDNF promote survival of TG neurons in culture, but also enriches for TRPV1-expressing neurons, suggesting that GDNF might be preferentially neuroprotective for sensory neurons in adult animals that express TRPV1. We have also illustrated that chronic application of NGF or GDNF, but not BDNF (except at high concentrations, and to a much lesser degree), increases CAP-evoked CGRP release from TG neurons in vitro. NGF and GDNF each increased TRPV1 protein, and both upregulated the CGRP content of TG neurons. Both the NGF- and GDNF-induced upregulation of TRPV1 appears to be translationally regulated, as neither of these growth factors altered TRPV1 mRNA levels. NGF, in the setting of inflammation, is known to increase TRPV1 protein, but not mRNA, through activation of the p38/MAP kinase pathway \[[@B25],[@B26]\], and NGF-mediated upregulation of TRPV1 is blocked by over-expression of dominant-negative ras \[[@B26]\]. Interestingly, the study by Bron et al. (2003) indicated that GDNF is also able to upregulate TRPV1 expression; although this conclusion was based on immunofluorescence and cobalt uptake assays, it is consistent with our direct demonstration of GDNF-induced upregulation of TRPV1 protein by Western blot. After normalization to the number of neurons that express TRPV1 and CGRP, we found that NGF had a much greater effect on CAP-evoked CGRP release and neuropeptide content, on a per cell basis, than did GDNF. On the other hand, GDNF increased the proportion of neurons in culture that express TRPV1, such that GDNF-maintained cultures contained a higher number of TRPV1-expressing neurons compared with NGF-maintained cultures. Hence, our findings suggest that NGF increases CAP responsiveness in individual cultured TG neurons. On the other hand, GDNF appears to increase the responsiveness of the in vitro population by altering the proportion of TRPV1 neurons and upregulating the releasable pool of CGRP, as evidenced by the persistent increase in 50 mM K^+^-evoked CGRP release after normalization. This difference in the ability of GDNF and NGF to enhance individual neuronal neuropeptide content and CAP responsiveness could partially explain why NGF induces hyperalgesia \[[@B10],[@B12],[@B19],[@B61]\] while GDNF does not \[[@B15],[@B28],[@B29]\]. Our findings suggest that while GDNF might play a role in maintaining CAP sensitivity in vitro, its effects in vivo might be of a preservative nature that prevents the development of pain exacerbation following experimental manipulation. Furthermore, NGF and GDNF might differentially/predominantly subserve two of the main physiologic processes following injury: hyperalgesia to protect the organism from further injury (NGF) and regeneration/repair to restore function (GDNF). While it has been shown, using a variety of dependent measures, that both NGF and GDNF increase CAP responsiveness \[[@B16],[@B17]\], this is the first demonstration that these growth factors qualitatively alter the pharmacodynamics of the neuronal response to CAP in TG neurons. The increase in the Hill slope of the CAP response following exposure to NGF or GDNF indicates that these growth factors induce positive cooperativity, possible at the level of TRPV1. While the mechanism underlying this effect is not known, it could involve an alteration in post-translational modifications and/or protein interactions of TRPV1 in response to NGF or GDNF. NGF or GDNF also decreased the concentration of CAP necessary to induce tachyphylaxsis compared with control cultures. These findings indicate that TRPV1-mediated sensory neuron desensitization might be a more efficacious therapeutic strategy in pathologies known to be associated with increased levels of NGF and/or GDNF. While we are unaware of any data linking GDNF with migraine, increased cerebrospinal fluid levels of NGF are associated with chronic headache \[[@B62]\]. Interestingly, a TRPV1 targeted approach has been utilized in a clinical trial for migraine treatment in which intranasal civamide (a TRPV1 agonist) was shown to be effective for acute treatment of migraine \[63\], indicating that TRPV1 agonists might be employed in this condition, possibly to desensitize CGRP-containing TG nerve endings. Similarly to CAP-evoked CGRP release, the ability of AEA and ACEA to evoke release was concentration-dependently augmented by NGF or GDNF supplementation but was unaltered by BDNF. Moreover, the respective potencies of NGF and GDNF to enhance the CGRP release in response to CAP, AEA and ACEA were equivalent. This suggests that the pharmacology of CAP, AEA and ACEA at TRPV1, at least with respect to neuropeptide secretion, is similarly regulated in the presence of either of these growth factors. Insofar as NGF and GDNF have been implicated in the development of nociceptor sensitization in a number of pathological states, it should be considered that endocanninoids are apt to have enhanced TRPV1-mediated peripheral neuromodulatory effects under these conditions. Conclusions =========== Taken together, our results illustrate that NGF, GDNF and BDNF differentially alter sensory neuron survival, neurochemical properties and TRPV1-mediated neuropeptide release of TG neurons in culture. GDNF and, to a lesser extent, BDNF promote survival of TG neurons and GDNF enhances the proportion of neurons that exhibit TRPV1-immunoreactivity. GDNF or NGF enhanced CAP-, AEA- and ACEA-evoked CGRP release from TG neurons in vitro and increased TRPV1 protein, likely through translational regulation, and overall CGRP content. On the other hand, our findings suggest that GDNF and NGF differentially modulate TRPV1-mediated neuropeptide secretion sensitivity, with NGF having a much greater effect on a per neuron basis, providing a possible explanation for why NGF promotes thermal hypersensitivity in vivo while GDNF apparently does not. Although the present studies were conducted on cultured neurons under artificially controlled conditions, these findings contribute to a growing body of work concerning neurotrophin modulation of the properties of nociceptors. Thus the results described here have implications for advancing our understanding of how this system might be advantageously manipulated in a therapeutic setting especially as it concerns the TG system. Methods ======= Experimental chemicals ---------------------- Capsaicin (CAP, 8-methyl-N-vanillyl-trans-6-nonenamide) was from Fluka-Aldrich (St Louis, MO). AEA (*N*-(2-hydroxyethyl)-5Z,8Z,11Z,14Z-eicosatetraenamide, in water soluble emulsion), and ACEA (*N*-(2-chloroethyl)-5Z,11Z,14Z)-eicosatetraenamide were from Tocris (Ellisville, MO). Recombinant rat GDNF and recombinant human (100% homologous to rat) BDNF were from Sigma (St Louis, MO). Rat NGF was from Harlan (Indianapolis, IN). All other chemicals were from Sigma, unless otherwise stated. TG culture ---------- Adult, male Sprague-Dawley rats weighing 250--300 g were used in this study. All procedures utilizing animals were approved by the Institutional Animal Care and Use Committee of The University of Texas Health Science Center at San Antonio and were conducted in accordance with policies for the ethical treatment of animals established by the National Institutes of Health. Animals were euthanized by decapitation and their TGs were rapidly dissected (\~30 s) and placed in ice-cold Ca^++^- and Mg^++^- free Hank\'s balanced salt solution (HBSS, Gibco, Carlsbad, CA). TGs were enzymaticaly digested for 30 min with 5 mg/ml collagenase followed by 25 min with 0.1% trypsin type IX supplemented for the last 10 min with 10 units of DNase I (Roche, Indianapolis, IN). TG cell suspensions were then centrifuged at 2000 RPM for 2 min, vortexed briefly and centrifuged again. They were then resuspended in basal culture medium containing high glucose Dulbeco\'s Modified Eagle\'s Medium (DMEM, Gibco), 1X pen-strep (Gibco), 1X glutamine (Gibco) and 3 μg/mL 5-FDU and 7 μg/mL uridine as mitotic inhibitors. TG cell suspensions were gently triturated with a Pasteur pipette followed by successive triturations through 19- and 23-gauge needles. TG cell suspensions were then transferred to a separate container and adjusted to the total volume needed for plating at a density of \~5000 neurons / well. The appropriate growth factors were added to this suspension prior to plating. Immunocytochemistry (ICC) and Image Aquisition ---------------------------------------------- TG cultures were first washed in PBS and fixed for 1 hr in 3.7% formaldehyde in PBS. Next, TG cultures were washed 3 times in PBS and permeabilized in PBS containing 10% normal goat serum (NGS, Gibco) and 0.2% Triton X-100 (Sigma) for 1 hr. Finally, TG cultures were blocked 3 × 10 min in PBS containing 10% NGS and then exposed to NF-H mouse-monoclonal antibody (1:300; Sigma) overnight at 4°C. Primary antisera were then washed off and goat anti-mouse Alexa-Fluor-488 (1:300, Molecular Probes, Eugene, OR), or goat anti-mouse Alexa-Fluor-594 (1:300) was applied for 1 hr at room temperature. For double-labeling either CGRP rabbit polyclonal (1:750, Peninsula Labs) or TRPV1 guinea pig polyclonal (1:3000, Neuromics) or IB~4~-conjugated to Alexa-Fluor-488 (1:1000, Molecular Probes) was then added overnight at 4°C. CGRP or TRPV1 antisera were followed by goat anti-rabbit Alexa-Fluor-594 (1:300) for 1 hr at room temperature. All images were acquired using a Nikon E600 microscope (Melville, NY) equipped with a Photometrics SenSys digital CCD camera (Roper Scientific, Tucson, AZ) connected to a computer equipped with Metamorph V4.1 image analysis software (Universal Image Corporation, Downingtown, PA). Twenty 20X images were taken of each well to capture the complete cellular area for neuron counts. For these experiments, all neurons displaying fluorescent signal above background were counted as positive for the specific marker. This was defined by scaling the image, using Metamorph\'s built-in scaling feature, to an average pixel value for negative neurons in the case of TRPV1, CGRP and IB~4~(no scaling was performed for NF-H ICC) and establishing all neurons as positive that were above that threshold. For double labeling experiments, the thresholded images were then overlaid and analyzed for the presence of both signals to assess colocalization. Neuronal Counting (Survival) ---------------------------- TG cell suspensions (\~5000 neurons/well) were added to 8-well poly-D-lysine Lab-Tek II chamber slides (Nalge/Nunc, Naperville, IL). Neuron density for plating was determined by counting neurons with a hemacytometer with Neubauer rulings and the cultures for each slide were generated independently. Neurons were easily distinguished from other cell types for plating density measurements due to their large size and opaqueness. Medium was changed after 24 hr and 72 hr, and on day 5, ICC was performed (as described above). In total, 12 chamber slides were utilized. The experimental design is shown in figure [1](#F1){ref-type="fig"} with 4 slides each for NGF, GDNF and BDNF. Neurons on the NF-H alone slides were not counted as these slides were used only for images shown in figure [2](#F2){ref-type="fig"}. To assess neuronal survival, all NF-H- immunoreactive neurons were counted for each growth factor concentration and are presented as mean ± SEM. The 2 matching wells for each slide were averaged and this average was used for 1 observation (as described in Fig [1](#F1){ref-type="fig"}). For colocalization studies, the previously generated NF-H-immunoreactive neuron counts for each well were followed by counting of CGRP- or TRPV1-immunoreactive or IB~4~-binding neurons in the same wells to generate the proportion of neurons expressing these markers. The total for the 2 matching wells were summed to yield the final proportion. At least 500 NF-H-immunoreactive neurons were counted under every growth factor condition to calculate proportions of neurons expressing the three markers examined. In no cases were CGRP-, TRPV1- or IB~4~-binding neurons not likewise positive for NF-H. Colocalization is presented only as an overall percentage. Realtime PCR ------------ TG cultures were prepared on 48-well poly-D-lysine pre-coated plates (Becton Dickinson, Franklin Lakes, NJ) at an initial density of \~5000 neurons/well. For each 48 well plate 12 wells received no growth factor, 12 received 100 ng/ml NGF, 12 received 100 ng/ml GDNF and the remaining 12 received 100 ng/ml BDNF. A total of 3 plates were utilized and each culture plate was generated independently. Following 5 days of culture, RNA was extracted from TG cultures using a ToTALLY RNA (Ambion, Austin, TX) total RNA extraction kit and each of the 12 wells per condition were pooled together to yield sufficient RNA. RNA samples were subsequently treated with DNA-free DNAse (Ambion) for removal of trace amounts of DNA. RNA concentrations were determined by UV absorbance, and RNA samples were then diluted to equal concentrations in TE buffer. For realtime PCR assessment of TRPV1 mRNA levels, samples were loaded in triplicate in 96-well reaction plates with each sample containing 150 ng RNA, 2X RT-PCR Taqman Master Mix, 40X Multiscribe and RNase Inhibitor solution, forward primer (tcc agt caa gcc cca cat c), reverse primer (tcc gag tca ccc ttc cca) and Taqman probe (6FAM tca cta cca gga gtc gta ccc ggc ttt TAMRA) (all 300 nM, all reagents Applied Biosystems, Foster City, CA) in a final reaction volume of 50 μL. Controls were run concomitantly using the same reaction recipe with a rodent GAPDH control kit (Applied Biosystems), primers and probe at 50 nM. Reactions were run on an ABI Prism 7700 (Applied Biosystems) with an initial RT step of 48°C for 30 min followed by a 95°C 10 min denaturation step and then a repeating denaturation, extension cycle of 95°C for 15 s and 60°C for 1 min for 55 cycles in order to reach a full plateau for all samples. All data were normalized to GAPDH mRNA levels to account for any variation in RNA concentrations between samples. Western blotting ---------------- TG cultures were prepared on 48-well poly-D-lysine pre-coated plates at an initial density of \~5000 neurons/well. For each 48 well plate 12 wells received no growth factor, 12 received 100 ng/ml NGF, 12 received 100 ng/ml GDNF and the remaining 12 received 100 ng/ml BDNF. A total of 3 plates were utilized and each culture was generated independently. After five days, total protein was extracted by first lysing cells with lysis buffer (1 mM Na pyrophosphate, 50 mM HEPES, 1% Triton X-100, 50 mM NaCl, 50 mM NaF, 5 mM EDTA and 1 mM Na orthovanadate) supplemented with 1% protease inhibitor cocktail and then homogenizing the combined lysate from the 12 wells per condition by pumping it through a 25 gauge needle 20 times. Extracted proteins were cleared of nuclei and cellular debris by spinning the homogenate at 1000 × g for 5 min at 4°C. Protein levels were then measured by the Bradford method. Proteins were run at a concentration of 20 μg/lane on a 12.5% SDS-PAGE gel and transferred to Immobilon -- P membranes (Millipore). Membranes were blocked in 5% dry milk for 1 hr and then exposed to rabbit anti-TRPV1 antibody (Neuromics, Minneapolis MN), at a concentration of 1:1000, overnight at 4°C. Membranes were then incubated with donkey anti-rabbit horseradish peroxidase-linked secondary antibody (Amersham, 1:5000) for 1 hr followed by ECL Western blotting detection for 1 min (Amersham). Blots were next exposed to film and subsequently scanned on a flatbed scanner. To control for protein loading, membranes were then stripped and reblotted for β-actin. Images were assessed for changes in TRPV1 protein using NIH image and normalized to β-actin protein levels. CGRP release and CGRP content ----------------------------- All experiments were performed in 48-well poly-D-lysine pre-coated plates. Data shown are representative of at least 3 independently conducted CGRP release experiments with consistent results and neurons were plated at the same density as indicated for survival, ICC, realtime PCR and Western blotting experiments (\~5000 neurons/well). Culture medium was changed at 24 and 72 hr, and all CGRP assays were performed on day 5. TG cultures were washed free of culture medium by 2 successive washes with release buffer (Hank\'s balanced salt solution (Gibco) supplemented with 10.9 mM HEPES, 4.2 mM sodium bicarbonate, 10 mM dextrose and 0.1% bovine serum albumin (BSA), pH 7.4). Growth factors were not included in the release buffer. Following washing, TG cultures were exposed for 10 min to the indicated concentrations of CAP, AEA or ACEA or to 50 mM K^+^buffer (containing 2.5 mM CaCl~2~, 50 mM KCl, 1.2 mM MgCl~2~, 90 mM NaCl, 25 mM NaHCO~3~, 1 mM NaH~2~PO~4~, 10 mM dextrose, 15 mM Hepes, 16 uM thiophan and 0.1% BSA at pH = 7.4), after which the CGRP-containing supernatant was removed and transferred to glass culture tubes (Fisher). Content was assessed by hypotonic lysis with deionized H~2~O supplemented with 1% protease inhibitor cocktail (Sigma) for 30 min. CGRP release or content for each well was subsequently measured by radioimmunoassay. CGRP radioimmunoassay --------------------- Following culture release assays, individual aliquots of the superfusate (0.5 ml) were incubated with a C-terminally directed anti-CGRP antiserum (kindly donated by Dr Michael Iadarola, NIDCR, NIH, Bethesda, MD, USA). After 24 h, 100 μL of \[125^I^\]-CGRP~28--37~(approximately 20000--25000 cpm) and 50 μL of goat anti-rabbit antibody conjugated to ferric beads were added. Following another 24 h, bound peptide was separated from free peptide via immunomagnetic separation (PerSeptive Biosystems, Framingham, MA, USA). All incubations were carried out at 4°C. The minimum detection limit for this assay is approximately 1--2 fmol per tube, with 50% displacement occurring at 20--40 fmol per tube. To account for the possibility of any nonspecific effects on the RIA, all drugs used in the release experiments were included in separate standard curves for the purposes of data analysis. We did not observe any alterations in the standard curve for any of the compounds utilized in these studies Data analysis and statistics ---------------------------- All data are presented as mean ± SEM unless otherwise stated. When normalizations for CGRP release or content to neuron numbers for either CGRP or TRPV1-immunoreactive neurons were made the equation shown in figure [11](#F11){ref-type="fig"} was used. All data were analyzed using GraphPad Prism for Mac OSX (GraphPad, San Diego, CA). To assess statistical differences, data were analyzed by one-way ANOVA followed by Tukey\'s post-test, for multiple comparisons, or Dunnett\'s post-test to compare all groups to the control group. For differences between growth factors at the same concentrations differences were assessed by two-way ANOVA with Bonferroni post-test for between group comparisons. All nonlinear regressions were fit to a sigmoidal curve with variable slope. Abbreviations ============= ACEA: arachidonyl-2-chloroethylamide, AEA: anandamide, BDNF: brain-dervied neurotrphic factor, CAP: capsaicin, CGRP: calcitonin gene-related peptide, DRG: dorsal root ganglion, GDNF: glial cell-line derived neurotrphic factor, IB~4~: isolectin B~4~, ICC: immunocytochemistry, NGF: nerve growth factor, SP: substance P, TG: trigeminal ganglion, TRPV1, transient receptor potential receptor vanilloid type 1 Author\'s Contributions ======================= TJP performed and conceived (or participated in their conception) experiments in each section, analyzed all data and authored the manuscript. MDL performed immunocytochemistry and neuron counts. DCS conducted the intitial CAP-evoked CGRP release study. GOD assisted in conceiving the experiments and performing the pilot studies. NAJ conducted the Western blots. AP assisted in conducting the CGRP release experiments. AD assisted in real-time PCR experiments. AAT assisted in generating cultures and in conducting pilot studies for CAP-evoked CGRP release. KMH assisted in conceiving the experiments and gave critical readings of the manuscript. CMF supervised all studies and conceived the original experimental designs as well as critically editing the manuscript. Acknowledgments =============== This work was supported by National Institute of Drug Abuse grants DA06085 and DA11959. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *Experimental design for neuronal counting*. 12 slides were utilized in these experiments, with 4 slides for each growth factor. The antibodies used on each slide are shown to the right of the slide along with the wavelength for the corresponding secondary antibody. For the 2 matching wells for each slide (growth factor concentration), the neuron numbers were averaged to give one observation (since the wells were not derived from independent cultures). Each slide contained a no growth factor control (blue), hence n = 9 for this condition. All other growth factors at 1 (i.e. NGF, red), 10 (i.e. GDNF, green) or 100 ng/ml (i.e. BDNF, yellow) are n = 3. The same slides were then utilized to ascertain the proportions of neurons expressing CGRP- or TRPV1-immunoreactivity or IB~4~-binding, as described in Methods. This figure refers to Figures 2 and 3 and Table 1. ::: ![](1471-2202-6-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### *Representative 20X photomicrographs of growth factor-treated TG neurons*. TG cultures were fixed and labeled for NF-H-immunoreactivity (green), and the 20X photomicrographs depicted here are representative of the 20 images taken per slide for each condition for neuron counting. The upper frame shows the no growth factor treated control with corresponding growth factor treatments shown for BDNF, GDNF and NGF at 1, 10 and 100 ng/ml concentrations. ::: ![](1471-2202-6-4-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### *NGF, GDNF and BDNF and TG neuronal surival*. All NF-H-immunoreactive neurons were counted for each growth factor-treated TG culture (n = 9 no growth factor; n = 3 all other conditions) and compared with the control (no growth factor-treated) TG cultures to assess neuronal survival at day 5 post-plating (\* p \< 0.05, \*\*\* p \< 0.001). ::: ![](1471-2202-6-4-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### *Assessment of TRPV1 mRNA levels by Realtime PCR*. Levels of TRPV1 mRNA are depicted as fold change compared with no growth factor-treated cultures for each growth factor condition normalized to its corresponding GAPDH mRNA levels (n = 3). ::: ![](1471-2202-6-4-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### *Assessment of TRPV1 protein by Western Blot*. Panel A, 20 μg total protein was electrophoretically seperated per lane and transferred to membranes that were subsequently probed with an anti-TRPV1 antibody and then reprobed for standardization with β-actin (X = no growth factor; N 100 = NGF 100 ng/ml; G 100 = GDNF 100 ng/ml). Image is of a representative Western blot. Immunoreactivity to a protein corresponding to the size of the glycosyated form of TRPV1 was detected at \~130 kD. Panel B depicts alterations in TRPV1 protein levels standardized to β-actin (\* p \< 0.05, n = 3) ::: ![](1471-2202-6-4-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### *50 mM K+ -evoked CGRP release and total CGRP content in TG cultures*. Panel A illustrates the effect of growth factor treatment on 50 mM K+ -evoked CGRP release, while panel B shows the data standardized to the number of CGRP-positive neurons per condition as a proportion of the no growth factor-treated cultures. Panel C shows total CGRP content by growth factor treatment, and again, panel D shows this data standardized to CGRP-positive neurons, as stated above (\*\*\* p \< 0.001, n = 6). ::: ![](1471-2202-6-4-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### *Representative 20X photomicropgraphs of colocalization of sensory neurons markers with NF-H in growth factor-treated TG neurons*. Immunoreactivity for CGRP and TRPV1 and staining for IB~4~-binding sites (red) was assessed following immunocytochemistry for NF-H (green) to assess the proportion of neurons expressing these population markers. Representative 20X photomicrographs of each growth factor at 100 ng/ml for CGRP (left), TRPV1 (middle) and IB~4~(right) are shown as well as control (no growth factor-treated) TG cultures (top panels). All cell bodies containing both NF-H-immunoreactivity and CGRP- or TRPV1-immunoreactivity or IB~4~-binding appear yellow from the overlay of the red with green. In no cases were neurons observed that contained CGRP- or TRPV1-immunoreactivity or IB~4~-binding without the co-presence of NF-H-immunoreactivity. ::: ![](1471-2202-6-4-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### *Growth factor treatment increases CAP (100 nM)-evoked CGRP release in TG cultures*. Panel A illustrates the total amount of CGRP released by 10 min treatment with capsaicin for each growth factor. Panel B illustrates the same data standardized to the number of TRPV1-positive neurons under each growth factor condition (\*\* p \< 0.01, \*\*\* p \< 0.01 for comparisons to no growth factor treated controls; \#\# p \< 0.01, \#\#\# p \< 0.001 for comparisons to NGF at the equivalent growth factor concentration by two-way ANOVA with Bonferroni post-test; n = 6). ::: ![](1471-2202-6-4-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### *NGF and GDNF alter capsaicin-evoked CGRP release*. Concentration-response functions (molar, log units) are shown for capsaicin-evoked CGRP release in TG cultures maintained in 100 ng/ml NGF, 100 ng/ml GNDF or no growth factor conditions (n = 6). Panel A illustrates the maximum relative effect of capsaicin under each condition matched to the maximum evoked-CGRP release for that given condition. Panel B depicts the CAP-evoked CGRP release data in terms of CGRP released in fmoles in the presence or absence of NGF or GDNF to illustrate the magnitude of increases in E~max~and the biphasic nature of the capsaicin-evoked CGRP concentration-response function. ::: ![](1471-2202-6-4-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### *The effect of growth factor treatment on AEA- and ACEA-evoked CGRP release*. TG neurons were grown in the presence of the indicated concentrations of NGF (panel A), GDNF (panel B) or BDNF (panel C) for five days and then exposed to 100 nM CAP, 30 μM AEA or 30 μM ACEA for 10 min, and the released CGRP was quantified (n = 12). Panel D, TG neurons were grown in the absence of growth factors or in the presence of 100 ng/ml NGF or GDNF for 5 days and exposed to the indicated concentrations of AEA (molar, log units) to assess CGRP release (\#\#\# p \< 0.001; GDNF and NGF vs. no growth factor; two-way ANOVA, Bonferroni post-test, n = 6). ::: ![](1471-2202-6-4-10) ::: ::: {#F11 .fig} Figure 11 ::: {.caption} ###### *Equation used to normalize CGRP release*. \"No GF\" refers to no growth factor controls and \"GF condition\" refers to any given growth factor treated condition ::: ![](1471-2202-6-4-11) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Percentage of neurons expressing markers. ::: **X** **N1** **N10** **N100** **G1** **G10** **G100** **B1** **B10** **B100** ----------- ------- -------- --------- ---------- -------- --------- ---------- -------- --------- ---------- **TRPV1** 37.9 37.5 37.4 40.0 48.4 57.5 66.6 50.1 50.9 47.8 **CGRP** 67.1 61.4 61.0 54.3 64.6 74.4 76.0 65.0 68.7 63.5 **IB~4~** 56.5 61.3 73.1 62.0 63.3 65.0 63.9 59.6 61.8 47.2 The percentage of total NF-H-positive neurons expressing TRPV1, CGRP or IB~4~is shown for each growth factor condition (X: no growth factor, N: NGF, G: GDNF, B: BDNF). :::

PubMed Central

2024-06-05T03:55:52.566587

2005-1-24

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548274/", "journal": "BMC Neurosci. 2005 Jan 24; 6:4", "authors": [ { "first": "Theodore J", "last": "Price" }, { "first": "Michael D", "last": "Louria" }, { "first": "Damaries", "last": "Candelario-Soto" }, { "first": "Gregory O", "last": "Dussor" }, { "first": "Nathanial A", "last": "Jeske" }, { "first": "Amol M", "last": "Patwardhan" }, { "first": "Anibal", "last": "Diogenes" }, { "first": "Amanda A", "last": "Trott" }, { "first": "Kenneth M", "last": "Hargreaves" }, { "first": "Christopher M", "last": "Flores" } ] }

PMC548275

Background ========== Over recent years increased emphasis has been given to performance monitoring of NHS hospitals and various quality indicators based on analysis of routine administrative data have been devised\[[@B1]\]. The 2003 dataset of the Commission of Health Improvement indicators includes 28-day hospital readmission rates following any hospital admission, and also readmission rates following admission for stroke and hip fracture\[[@B2]\]. Although indicators are currently standardised for age and sex^1^, they are not adjusted for potential case-mix differentials in disease severity, co-morbidity and patient deprivation status. Unplanned hospital re-admissions may represent adverse events and could therefore indicate poor quality of care\[[@B3],[@B4]\]. The interpretation of variation in readmission rates between healthcare organisations is nevertheless complicated\[[@B5],[@B6]\]. Broadly a readmission could be due to healthcare factors (e.g. sub-optimal health and social care, either at hospital or within primary/social care structures), patient factors (e.g. poor treatment adherence), disease factors (e.g. natural disease progression), or a combination of all the above. Readmissions due to healthcare and patient factors could be assumed to be potentially avoidable. There is insufficient evidence about the proportion of hospital readmissions that could be judged to be due to healthcare factors, as estimates of the proportion of medical readmissions that are due to healthcare factors vary widely between 9 and 48%\[[@B4]\]. Although current NHS performance indicators use readmission rates at 28-days\[[@B1],[@B2]\], there is lack of consensus in the literature about the choice of optimal time interval, with different studies choosing different intervals, ranging from one week to one year\[[@B4]\]. Intuitively shorter time intervals are more likely to represent \"avoidable\" readmissions due to poor quality care, nevertheless this may be more applicable in the case of elective surgical admissions rather than emergency medical ones. Moreover, for chronic medical conditions (such as diabetes, chronic obstructive pulmonary disease and heart failure), even a \"delayed\" readmission may represent a failure of disease management due to deficiencies in healthcare quality\[[@B4]\]. It can be hypothesised that factors such as patient sex, length of stay, number of coded co-morbidities, method of admission (e.g. presentation to the Accidents and Emergency department or GP referral), as well as patient deprivation, independently influence the probability of hospital readmission. Lower socioeconomic status in particular is independently associated with increased risk for many adverse health outcomes\[[@B7]\], including hospital readmission due to conditions such as heart failure\[[@B8]\]. The increasing demand for emergency medical admissions\[[@B9]\] makes epidemiological studies of medical readmissions a priority. Additionally, emergency medical admissions constitute a sizeable proportion of all hospital admissions, and can therefore play an important role in the overall performance of NHS hospitals under the current set of indicators\[[@B1],[@B2]\]. We therefore conducted a study to examine the effect of several patient and disease factors on the risk of emergency medical readmission at various time intervals following an index emergency medical admission. Methods ======= Context ------- This work was carried out as part of the routine function of Stockport NHS Trust Clinical Effectiveness Unit, and in relation to work commissioned by the then \"Emergency Demand Management Group\" of the Stockport Primary Care Trust. The objective was to accurately describe the epidemiology of emergency medical readmissions so that demand management strategies (e.g. appropriate targeting of resources to patients with certain conditions, or certain types of presentation) could be informed. Setting ------- Stockport NHS Trust is a district general hospital in Greater Manchester, serving a reference population of about 300,000. About 85% of all patients emergency medical admissions are from Stockport, a population with a slightly lower, compared to the England and Wales average, Standardised Mortality Ratio from all causes (all ages) of 96 (95% CI 94--98)\[[@B10]\]. Data source, population and follow-up period -------------------------------------------- Hospital Episodes Statistics Data from April 1997 to September 2001 for Stockport NHS Trust were analysed and all emergency medical admissions in Stockport residents leading to live discharge were identified. An emergency medical admission was defined as an emergency hospital admission to any medical specialty in person over 18 years of age. Some persons had more than one emergency medical admission during the study period, and emergency medical admissions other than the index admission (defined as the chronologically first admission during the 4.5-year study period) were excluded. This was because not restricting analysis to index admissions would have meant that any deprivation gradients in readmission rates would have been confounded by deprivation gradients in index admissions, as previously described\[[@B11]\]. This is an important difference of the methodology used in this study in relation to the way the relevant performance indicators are presently calculated, including \"all\" (as opposed to index/first only) admissions in the denominator\[[@B12]\]. An emergency medical re-admission was defined as the first subsequent emergency medical admission during a follow-up period of either 28-days, or 3, 6 and 12 months respectively, following a first (index) emergency medical admission that led to a live discharge, and through the use of a single patient identifier. Observations were censored at the end of the chosen follow-up periods (as above) or at the time of intervening death unrelated to readmission to the study hospital. The latter was ascertained by data-linking to the Stockport Health Authority Public Health Mortality File produced by the Office for National Statistics. Measurement and definitions --------------------------- Index admission data were originally available on: sex; age; length of stay of index admission; International Classification of Diseases (ICD)-10 coded primary diagnosis; number (up to four) of coded co-morbidities; patient post code and admission method (referral by Accidents and Emergency Department, General Practitioner or other). Information on primary diagnosis was aggregated into five categorical groups comprising chronic obstructive pulmonary disease / asthma (ICD codes J44.0--45.9 respectively), heart failure (I50.0--50.9), acute coronary syndrome (I20.0 \[unstable angina\] and I21.0--9 \[acute MI\]), stroke (I60.0--I67.0) and all other conditions (all other codes). Length of stay was divided into quartiles (\<2, 2--5, 6--11 and \>11 days). Deprivation status was subsequently ascribed with an area-based measure, using the 1991 Census enumeration district (ED) of patient\'s post-code, and by the use of Townsend multiple deprivation index score. Four deprivation groups were defined, using quartiles of the range of the Townsend scores between Stockport EDs. Analysis -------- Kaplan-Meier readmission-free curves at 28 days, and 3, 6 and 12 months were constructed for each of the following variables: *sex*, *age group*, *diagnostic group*(defined as above), *admission method*, *number of coded co-morbidities*(0--4), *length of stay group*(quartile) and *deprivation group*(quartile). Statistical significance for each of the above variables was assessed by the log rank test. A series of proportional hazards models with follow-up at 28 days, 3, 6 and 12 months were subsequently constructed to examine the adjusted hazard (risk) ratio of emergency medical readmission. Each model included all variables found to be significant in the uni-variable analysis at the 0.05 probability level. The proportional hazards assumption was tested using the Schoenfeld residuals as per the *stphtest*command in STATA. This showed that a time varying co-variate should be included in all four models, in relation to the *length of stay*variable (i.e. that an interaction term between *length of stay*and time of follow-up should be included), and this was hence included in the models. Additionally, for the number of coded co-morbidities and deprivation group variables, a test for trend was performed, entering the actual values as continuous variables. In this context, the test for trend value indicates the proportional change in the risk of readmission associated with one unit change in the exposure variable (i.e. number of coded co-morbidities, Townsend deprivation score). Results ======= There were 21,118 index emergency medical admissions corresponding to an equal number of patients leading to a live discharge during the study period, but primary diagnosis information was only available for 20,209 index emergency medical admissions (Table [1](#T1){ref-type="table"}). Cases without diagnostic information were excluded from further analysis. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Basic characteristics of index admissions in study participants (n = 20,209) ::: **Variable** **Category** **n** **%** -------------------------- ------------------------- ------- ------- **Sex** Male 9397 46.5 Female 10812 53.5 **Age group** \<60 8094 40.1 60--74 5526 27.3 75+ 6589 32.6 **Diagnostic group** Acute coronary syndrome 4283 21.2 COPD/asthma 1594 7.9 Heart failure 587 2.9 Stroke 575 2.8 All other diagnoses 13170 65.2 **Length of Stay** \<2 5666 28.0 2--5 5184 25.7 6--11 5174 25.6 \>11 4185 20.7 **Deprivation Group** Affluent 5057 25.0 2 5003 24.8 3 5113 25.3 Deprived 5015 24.8 Unknown 21 0.1 **Admission method** A&E 12604 62.4 GP referral 7113 35.2 Other 492 2.4 **No of co-morbidities** 0 2963 14.7 1 3796 18.8 2 3790 18.8 3 8801 43.5 4 859 4.3 ::: Uni-variable analysis --------------------- The proportion of patients readmitted at 28 days and 3, 6, 12 months progressively increased from 7.2% at 28-days to 23.3% at 12 months respectively (Table [2](#T2){ref-type="table"}). Male sex, older age group, length of stay, higher number of coded co-morbidities and any primary diagnosis other than the \"all other diagnoses\" category were significantly associated with higher readmission rates independently of duration of follow-up (Table [2](#T2){ref-type="table"}). Higher deprivation status was significantly associated with increased readmission rates in follow-up periods longer than three months, but not at 28 days (Table [2](#T2){ref-type="table"} and Figure [1](#F1){ref-type="fig"}). Admission method was not significantly associated with deprivation risk. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Proportion of patients readmitted (%) by patient subgroup and different periods of follow-up (log rank test p values from relevant Kaplan-Meier readmission-free curves) ::: **28 days** **p\*** **3 months** **p\*** **6 months** **p\*** **12 months** **p\*** ------------------------------------ --------------- ------------- --------- -------------- --------- -------------- --------- --------------- --------- **Sex** Men 7.7 0.017 13.5 0.009 18.3 0.003 24.1 0.01 Women 6.8 12.4 16.8 22.7 **Age group** \<60 5.4 \<0.001 8.8 \<0.001 11.8 \<0.001 15.8 \<0.001 60--74 8.2 15.1 19.7 26.4 \>75 8.5 15.8 22.3 29.6 **Admission method** A&E 6.9 0.07 12.7 0.2 17.4 0.79 23.3 0.93 GP referral 7.7 13.4 17.7 23.4 Other 7.8 11.6 17.6 23.9 **Length of Stay** \<2 5.9 \<0.001 8.6 \<0.001 11.2 \<0.001 14.7 \<0.001 2--5 6.5 10.9 15.5 21.4 6--11 8.0 15.6 21.2 28.0 \>11 8.8 17.4 22.3 31.0 **Number of coded co-morbidities** None 6.1 \<0.001 9.6 \<0.001 13.2 \<0.001 17.3 \<0.001 1 6.0 9.8 13.3 17.5 2 7.0 12.3 16.4 22.2 3 8.3 15.8 21.6 28.9 4 7.3 14.3 18.7 24.4 **Diagnosis** ACS 7.2 \<0.001 13.0 \<0.001 17.2 \<0.001 22.3 \<0.001 COPD/Asthma 8.7 15.7 22.1 30.1 Heart Failure 11.1 22.7 31.3 37.5 Stroke 4.5 9.6 14.6 23.0 All other 7.0 12.3 16.6 22.3 **Deprivation** Affluent 7.1 0.44 11.8 0.002 15.8 \<0.001 21.0 \<0.001 2 7.2 12.5 17.0 22.2 3 6.9 13.0 18.1 24.2 Deprived 7.7 14.3 19.2 25.9 ***Total*** ***7.2*** ***12.9*** ***17.5*** ***23.3*** \*Log Rank test A&E: Accident and Emergency, GP: General Practitioner, ACS: Acute Coronary Syndrome, COPD: Chronic Obstructive Pulmonary Disease ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Kaplan-Meier readmission-free curves by deprivation group. ::: ![](1471-227X-5-1-1) ::: Multi-variable analysis ----------------------- Male sex, older age group, and primary diagnosis of heart failure and chronic obstructive pulmonary disease/asthma were significantly associated with increased readmission risk, independently of length of follow-up (Table [3](#T3){ref-type="table"}). With the \"all other diagnoses\" as the reference category, primary diagnosis of acute coronary syndrome was associated with a significantly increased risk of readmission at 3 and 6 months, but not at 28 days or 12 months. Independently of length of follow-up and all other variables, primary diagnosis of stroke was significantly associated with reduced readmissions risk compared with the \"all other diagnoses\" category as reference. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Hazard ratios (HR) by variable and follow-up length, with associated 95% confidence intervals ::: **Variables in the Equation** **28 days HR (95% CI)** **3 months HR (95% CI)** **6 months HR (95% CI)** **12 months HR (95% CI)** ---------------------------------------- ------------------------- -------------------------- -------------------------- --------------------------- ------------ ------------------ ------------- ------------------ **Female** \- \- \- \- Male 1.17\*\* *(1.06 -- 1.30)* 1.14\*\*\* *(1.06 -- 1.23)* 1.15\*\*\* *(1.08 -- 1.23)* 1.13\*\*\* *(1.07 -- 1.20)* **Age \<60** \- \- \- \- Age 60--74 1.41\*\*\* *(1.23 -- 1.62)* 1.47\*\*\* *(1.33 -- 1.64)* 1.44\*\*\* *(1.32 -- 1.58)* 1.46\*\*\* *(1.35 -- 1.58)* Age \>75 1.45\*\*\* *(1.26 -- 1.67)* 1.45\*\*\* *(1.30 -- 1.62)* 1.56\*\*\* *(1.42 -- 1.71)* 1.57\*\*\* *(1.45 -- 1.70)* **Affluent** \- \- \- \- 2 1.01 *(0.87 -- 1.17)* 1.05 *(0.94 -- 1.17)* 1.07 *(0.97 -- 1.18)* 1.07 *(0.98 -- 1.16)* 3 0.98 *(0.84 -- 1.13)* 1.09 *(0.98 -- 1.22)* 1.15\*\* *(1.05 -- 1.27)* 1.17\*\*\* *(1.08 -- 1.27)* Deprived 1.09 *(0.94 -- 1.26)* 1.21\*\* *(1.08 -- 1.35)* 1.21\*\*\* *(1.10 -- 1.33)* 1.25\*\*\* *(1.16 -- 1.36)* *Test for trend (Deprivation Index)*\^ 1.02 *(0.98 -- 1.07)* 1.06\*\*\* *(1.03 -- 1.10)* 1.07\*\*\* *(1.04 -- 1.10)* 1.08\*\*\* *(1.05 -- 1.11)* **All other diagnoses** \- \- \- \- Heart Failure 1.32\* *(1.02 -- 1.70)* 1.43\*\*\* *(1.19 -- 1.71)* 1.47\*\*\* *(1.26 -- 1.71)* 1.33\*\*\* *(1.16 -- 1.53)* COPD/asthma 1.25\* *(1.04 -- 1.50)* 1.23\*\* *(1.08 -- 1.41)* 1.29\*\*\* *(1.15 -- 1.45)* 1.34\*\*\* *(1.21 -- 1.48)* ACS 1.12 *(0.97 -- 1.28)* 1.15\*\* *(1.04 -- 1.27)* 1.12\* *(1.02 -- 1.22)* 1.07 *(0.99 -- 1.15)* Stroke 0.54\*\* *(0.37 -- 0.81)* 0.57\*\*\* *(0.44 -- 0.75)* 0.65\*\*\* *(0.52 -- 0.81)* 0.76\*\* *(0.63 -- 0.90)* **Without co-morbidity** \- \- \- \- 1 co-morbidity 1.02 *(0.83 -- 1.25)* 1.04 *(0.89 -- 1.23)* 1.05 *(0.91 -- 1.20)* 1.10 *(0.97 -- 1.24)* 2 co-morbidities 1.13 *(0.93 -- 1.38)* 1.21\* *(1.03 -- 1.41)* 1.19\* *(1.04 -- 1.36)* 1.29\*\*\* *(1.15 -- 1.45)* 3 co-morbidities 1.25\* *(1.04 -- 1.49)* 1.39\*\*\* *(1.21 -- 1.60)* 1.41\*\*\* *(1.25 -- 1.59)* 1.54\*\*\* *(1.38 -- 1.71)* 4 co-morbidities 1.26 *(0.93 -- 1.69)* 1.46\*\* *(1.17 -- 1.82)* 1.42\*\*\* *(1.18 -- 1.73)* 1.49\*\*\* *(1.26 -- 1.76)* *Test for trend (number of com.)*\^ 1.08\*\* *(1.03 -- 1.14)* 1.11\*\*\* *(1.08 -- 1.17)* 1.13\*\*\* *(1.09 -- 1.17)* 1.15\*\*\* *(1.12 -- 1.18)* **A&E referral** \- \- \- \- GP referral 1.09 *(0.98 -- 1.22)* 1.00 *(0.92 -- 1.09)* 0.96 *(0.89 -- 1.03)* 0.93\* *(0.88 -- 0.99)* Other referral 1.17 *(0.85 -- 1.60)* 0.84 *(0.64 -- 1.09)* 0.91 *(0.74 -- 1.13)* 0.94 *(0.78 -- 1.13)* **\<2 days LoS** \- \- \- \- 2--5 days LoS 0.54\*\*\* *(0.42 -- 0.69)* 0.82\* *(0.69 -- 0.99)* 0.92 *(0.79 -- 1.08)* 1.06 *(0.93 -- 1.21)* 6--11 days LoS 0.52\*\*\* *(0.41 -- 0.66)* 0.89 *(0.74 -- 1.05)* 1.14 *(0.98 -- 1.32)* 1.31\*\*\* *(1.15 -- 1.49)* \>11 days LoS 0.54\*\*\* *(0.41 -- 0.70)* 0.95 *(0.79 -- 1.14)* 1.23\*\* *(1.06 -- 1.44)* 1.42\*\*\* *(1.25 -- 1.62)* **\<2 days LoS \* Time\^\^** \- \- \- \- 2--5 days LoS \* Time\^\^ 1.07\*\*\* (1.05 -- 1.09 1.01\*\*\* (1.01 -- 1.02) 1.01\*\*\* (1.00 -- 1.01) 1.002\*\*\* (1.001 -- 1.003) 6--11 days LoS \* Time\^\^ 1.08\*\*\* (1.06 -- 1.11 1.02\*\*\* (1.01 -- 1.02) 1.01\*\*\* (1.00 -- 1.01) 1.002\*\*\* (1.001 -- 1.003) \>11 days LoS \* Time\^\^ 1.09\*\*\* (1.06 -- 1.11 1.02\*\*\* (1.02 -- 1.02) 1.01\*\*\* (1.00 -- 1.01) 1.002\*\* (1.001 -- 1.002) \*: p \< 0.05, \*\*: p \< 0.01, \*\*\*: p \< 0.001 \^ : Denotes the proportion change in probability of outcome associated with one unit change in continuous variable (e.g. Townsend deprivation score index, number of co-morbidities) \^\^ In days HR: Hazard Ratio, COPD: Chronic obstructive airways disease, ACS: Acute coronary syndrome, LoS: Length of stay, A&E: Accident and Emergency, GP: General Practitioner. ::: More than two coded co-morbidities were associated with higher readmission risk only in follow-up periods of more than three months duration. However test for trend indicated a strong and significant positive effect of the number of coded co-morbidities independently of follow-up length. Higher deprivation status was independently associated with higher readmission risk at 3, 6 and 12 months, but did not significantly influence readmission risk short term (at 28 days). Test for trend confirmed the strong and statistically significant effect of deprivation at 3--12 months but there was no effect at 28 days. Admission method via a GP referral was significantly associated with a lower readmission risk at one year, but not at any other time intervals. Length of stay of the index admission influences readmission risk differently, depending on the length of follow-up as there was a highly significant interaction between length of stay group and time of follow-up (see [Additional file 1](#S1){ref-type="supplementary-material"}). Taking into account the relevant time varying co-variate, shorter length of stay is associated with higher readmission risk at discharge and immediately afterwards, but with lower readmission risk thereafter. Discussion ========== The findings indicate that in the study hospital about a quarter of patients with an index emergency medical admission will be readmitted in the same hospital during the subsequent year. Male sex and older age were strongly and independently associated with higher readmission risk, along with diagnosis of heart failure and chronic obstructive pulmonary disease or asthma. Effective measures to reduce readmission rates for patients suffering from these two conditions in particular are available\[[@B13],[@B14]\] but not always used\[[@B13],[@B15],[@B16]\]. Improving the availability of effective treatments for these two conditions could contribute greatly to the management of demand for emergency care in general. The present study, at the local health economy level, has helped support the decision making process that allocated increased resources to the management of these two conditions by the expansion or creation of relevant specialist services. As originally hypothesised, patient deprivation status exerted a significant independent effect on the risk of emergency medical readmission at 3--12 months of follow-up, with more deprived patients having had a higher readmission risk. There are several theoretical reasons why deprived patients may be at higher readmission risk, including: disease factors, such as greater disease severity in deprived patients\[[@B17]\]; patient factors, such as poor adherence to treatment and advice because of educational or behavioural reasons; and health and social care factors, such as differentials in the type and quality of primary care in particular, in a way analogous to the \"inverse care law\", originally describing differentials in access, rather than quality, of care\[[@B18]\]. A clearer understanding of the exact mechanisms responsible for deprivation group gradients through further research is necessary for future policy measures aiming at reducing such gradients. Although this is a single-centre study, the results may also have implications for the way current and future NHS performance indicators relating to readmission rates are both constructed and interpreted. NHS hospitals serving pre-dominantly deprived populations might in principle be disadvantaged if indicators are not adjusted for the impact of deprivation on case-mix. Although this study showed no significant effect of deprivation status on readmission risk at 28-days, which is the follow-up period currently used by the performance indicators\[[@B1],[@B2]\], care should be taken when interpreting this \"negative\" finding. Firstly, this analysis included in the denominator only index (as opposed to \"all\") admissions, in contrast to the technical specification of the performance indicators\[[@B12]\]. Because more deprived patients have higher rates of index emergency medical admissions\[[@B11]\], including all index admission in the calculation will accentuate any deprivation differences in readmission risk, and the performance indicators as they are currently calculated may for this reason be misleading. Previous analysis of the same dataset including \"all\" admissions provides empirical evidence that this is true\[[@B19]\]. Secondly, it is possible that a true effect of deprivation status on index readmission rates at 28-days also exists but it was not detected by our study due to its single-centre nature, or insufficient sample size. A larger study, ideally using data from more than one hospital may be warranted. The Department of Health includes performance indicators in the calculations of award of \"three-star\" status, which in turn is the \"gateway\" to \"Foundation\" status\"\[[@B20]\]. Standardising, or otherwise adjusting, for patient deprivation is feasible using the HES data, as this study indicates. Standardisation of readmission indicators for patient deprivation status would be prudent. This would ensure that NHS organisations serving deprived communities would not be unfairly \"punished\" for poor performance because of factors outside their control. It will also increase the perception of validity of the indicators. Unlike information on disease severity, which is difficult to measure accurately for most medical conditions, information about patient socioeconomic status using area-based (ecological) deprivation measures is relatively easy to obtain, using patient postcodes, routinely included in the Hospital Episodes Statistics dataset. All studies using administrative data are sensitive to the quality of routine data collection. The validity of HES data in relation to age, sex, length of stay, admission method, and area of residence is generally good, but misclassification errors may occur in relation to diagnostic codes and the extent to which co-morbidities are recorded and coded\[[@B21]\]. Currently the degree of miscoding in our data is uncertain, but an audit of 200 cases in the study hospital has shown diagnostic inaccuracy to be in the order of 7.5%, comparable with levels quoted in the published literature\[[@B22]\]. Misclassification of primary diagnosis might be assumed to have occurred non-differentially between patients of different deprivation groups, and is so it would have diminished rather than exaggerated any association observed in this study, including the observed effect of deprivation. Misclassification error may have also resulted by the use of ecological measures of socioeconomic status (ecological fallacy). Again, this would reduce the effect size, if one exists. Therefore the effect of deprivation status on readmissions risk reported in our study may be an under-estimate of a true association. A limitation of the study is that, besides the very large sample size, the findings are based on one single hospital in an urban English setting, and in principle the results are not generalisable. Similarly, the study was not population-based, so readmissions that may have occurred to other hospitals (either because of where patients happened to be taken if fallen acutely ill, or due to migration) were not ascertained. In theory such readmissions may have occurred at a differential rate between different deprivation groups. However this factor is unlikely to have biased the results in any considerable way for two reasons. First, the emergency (as opposed to elective) nature of the studied condition (emergency medical admission) makes it unlikely that either patients or doctors exercise an important degree of choice on which hospital a patient is admitted or readmitted, independently of patient deprivation status. Second, due to local geography and service configuration, 85% of the total medical admissions in Stockport residents occur at Stockport NHS (unpublished data). Similarly, by the nature of the hospital-centred nature of the study, admissions to private hospitals could not have been accounted. However, most admissions to private hospitals are for elective surgical procedures (rather than emergency medical reasons) for which we believe this is unlikely to have introduced considerable degree of bias. Lastly it is worth remembering that current NHS performance indicators for hospital Trusts are not population-based. Conclusions =========== Our study suggests that there is an important potential for both managing emergency demand and improving individual patient experience by focusing on the effective management of heart failure and chronic obstructive pulmonary disease. Although there is a similar potential by reducing differentials in readmission risk between deprivation groups, more research is required in order to understand reasons for such differentials in order to inform relevant policy measures. In the mean time, standardisation or other adjustment of hospital readmission indicators for patient socio-economic status in the future would be prudent. Failure to do so may disadvantage hospitals serving primarily deprived communities. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= The study was conceived and designed by GL and GC. GL, DH and IG analysed data. All authors contributed in the interpretation of findings and in the writing of the paper. GL and GC are guarantors. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-227X/5/1/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Length of stay group and readmission free Kaplan-Meier curves (0--28 days). This file demonstrates that during follow-up of 0--28 days, length of stay is not proportional to readmission risk, reason for which a time varying co-variate was included in the Cox regression model (see main article Text). ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We are grateful to Professor RF Heller for useful comments on the manuscript.

PubMed Central

2024-06-05T03:55:52.571760

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548275/", "journal": "BMC Emerg Med. 2005 Jan 21; 5:1", "authors": [ { "first": "Georgios", "last": "Lyratzopoulos" }, { "first": "Daniel", "last": "Havely" }, { "first": "Islay", "last": "Gemmell" }, { "first": "Gary A", "last": "Cook" } ] }

PMC548276

Background ========== Case-control research on the association between surgery and sporadic Creutzfeldt-Jakob Disease (CJD) encompasses six studies and one meta-analysis \[[@B1]-[@B8]\], which frequently yielded diverging, partly inconsistent, positive \[[@B5],[@B6]\] or negative \[[@B7],[@B8]\] results, potentially attributable to methodological difficulties. This paper constitutes the first part of methods development for a case-control study summarily described in the Methods/Design section. \"Development\" in this sense refers to theoretical development built on scientific evidence and will focus on classification of surgical procedures, using technical and biological criteria appropriate for identifying characteristics of the intervention reflecting the putative risk level of sporadic CJD transmission. Methods ======= In this section, we summarily describe the on-going study, review reports that possibly shed light on the biological plausibility of surgery-related CJD transmission, and propose principles for re-classification of surgical procedures. Study design ------------ The on-going case-control study, entitled \"Surgery and risk of Creutzfeldt-Jakob Disease\" (EUROSURGYCJD), constitutes a Concerted Action funded by the EU Research Commission, contract QLG3-CT-2002-81223. The main objective of this study is to quantify a putative excess risk of CJD associated with surgery. A secondary objective is to establish a basis for the design of preventive strategies. The most relevant methodological characteristics are: case-control design; exposure measurement prior to disease onset, registered as codes for surgical procedures; matched 5:1, randomly chosen population controls and random sample of population controls. The population base is the resident population in Denmark, Finland and Sweden, covered by the respective hospital in-patient registers. Cases are individuals with diagnoses corresponding to ICD-9 codes 046.1 and 331.5 and ICD-10 code A81.0 at death or at hospital discharge for the period 1987--2002, identified from the respective national hospital in-patient registers and corresponding national surveillance units. A questionnaire will be mailed to the heads of the registered hospital department or surveillance unit, and a copy of the medical record will be obtained for diagnosis validation. Approximately 300 patients will fulfil criteria for definite or probable sporadic CJD, and constitute the study cases. Population controls are: 1) 5 × 1 controls (approximately 1500), matched to the corresponding case by age, sex, county of residence of the case (at first discharge from hospital with CJD diagnosis or death if never hospitalised with CJD diagnosis); and 2) a 20/million sample of the 1987--2002 resident population aged \>40 years, randomly selected from the corresponding national population registers. Individual person-numbers will be used for each resident case or control. Diagnoses and surgical procedures at hospital discharge of the corresponding CJD case at any registered time before date of death will be obtained from the three national hospital discharge registers in Sweden, Denmark and Finland. Perusal of surgical records might be undertaken for selected associations in order to understand transmission mechanisms and interpret results. Open-care surgery or dentistry will not be studied. Surgical procedures coded in registers as per national or NOMESCO classifications will be re-classified in accordance with putative levels of transmission risk based on scientific evidence/plausibility. The quality of CJD diagnoses will be assessed by a review of medical records. The accuracy of surgical history given by the registers will be assessed by comparison with that of a sample of controls and surrogate respondents obtained by interview. Centralised data analyses will be conducted by the Spanish team. In specific instances, risk due to blood transfusion, whether or not performed during surgical procedures, might also be studied. Review of reports and proposal ------------------------------ Surgery may be a pathway for patient-to-patient transmission of sporadic Creutzfeldt-Jakob Disease (CJD). In many invasive surgical procedures, non-disposable surgical instruments come in contact with tissues that are known to be infective in CJD patients. These same instruments may retain a considerable level of infectivity after routine sterilisation, and in successive patients can come into contact with tissues that may act as entry sites for CJD transmission. Among the almost 300 recorded cases of iatrogenic transmission of CJD, 5 cases have been attributed to surgical instruments employed in neurosurgical procedures, whilst 2 additional cases were caused by the use of a contaminated intracerebral EEG electrode \[[@B9]\]. To date, proven surgical transmission of CJD has only been shown to have taken place through instruments contaminated with high-infectivity tissues (brain). However, stainless steel instruments exposed to infective tissue can acquire a maximum load of infectivity in a considerably short period of time (5 minutes) and are highly efficient in transmitting disease even after thorough washing \[[@B10]\]. The possibility of prion transmission through surgical interventions involving nervous or peripheral tissue has raised concern about decontaminating procedures, particularly after the emergence of variant CJD in the United Kingdom and several other countries \[[@B11]\]. In the above-mentioned studies \[[@B1]-[@B7]\], surgical procedures have been grouped and analysed according to gross anatomical regions (e.g., thyroid, gallbladder, prostate, etc.), which limits an interpretation of results based on biological inference. For a surgical instrument to act as a vehicle of prion transmission, it should come into contact with infective tissue during surgery of the \"donor\" (contaminating procedure), should maintain any adhered infectivity after being washed and sterilised, and, finally, should make contact with receptive tissues in the \"recipient\" patient (transmitting procedure). Different surgical interventions on the same organ may result in direct exposure of different tissues to surgical instruments, and may consequently involve a different risk of prion transmission. Within the context of a case-control study designed to address surgical risk in sporadic CJD in Nordic European countries (EUROSURGYCJD Project), we adopted the strategy of categorising all reported surgical procedures (putative transmitting procedures) in terms of potential risk of CJD acquisition. For this purpose, a classification of exposed tissues and anatomic structures has been drawn up on the basis of their specific putative role as entry site for prion transmission through surgical instruments. This classification can serve, both in our study and in further epidemiological studies, as a reference for a categorisation of surgical procedures in terms of risk of CJD acquisition. According to the \"protein only\" hypothesis \[[@B12]\], pathogenic prion protein (PrP^Sc^) is a conformational isoform of PrP^C^, a normal host protein present in neurons and other cell types. In sporadic and familial transmissible spongiform encephalopathies (TSEs), \"spontaneous\" conversion of PrP^C^into PrP^Sc^is the key pathogenic event, followed by the accumulation, deposition and further conversion of PrP^Sc^in tissues, together with its propagation along specific neural pathways. In the case of transmitted TSEs -when these are not due to direct inoculation into the CNS- a peripheral phase of neuroinvasion by PrP^Sc^is followed by a subsequent phase of prion replication and propagation along the peripheral nervous system, with final access to the central nervous system \[[@B13]\]. In scrapie, bovine spongiform encephalopathy (BSE) and variant CJD, neuroinvasion follows widespread deposition of PrP^Sc^in mucosae-associated lymphoid tissue. For the purpose of classifying tissues susceptible to prion inoculation, the following considerations can be derived from this pathogenic model: i) a tissue can act as entry site for prion transmission if it normally expresses PrP^C^; ii) the level of PrP^Sc^expression of an infected tissue correlates positively with the risk of prion acquisition by that tissue; and, iii) all tissues involved in the propagation chain of infection from peripheral tissues to the central nervous system can act as entry sites for prion transmission. The recently published WHO classification of tissue infectivity in TSEs \[[@B14]\], though aimed at public health issues radically different from those addressed in our study, may nonetheless serve as a conceptual framework for a tissue classification in terms of risk level of prion acquisition. This approach is based on our above-mentioned assumption (ii). The WHO classification groups tissues in three levels (high, lower and no detected infectivity) on the basis of bioassay infectivity data and/or detection of PrP^Sc^by Western blot. This three-level classification correlates quite closely with the distribution and levels of PrP^C^expression in normal nervous and non-nervous tissues in mammals.\[[@B15]\] The WHO tissue classification presents data on vCJD, other human TSEs, BSE and scrapie. Since no vCJD cases have been registered in Nordic countries, our epidemiological study must be limited to sporadic CJD. Consequently, our working classification excludes all tissues where positive data on infectivity or PrP^Sc^detection have been obtained exclusively in animal TSEs and/or vCJD. This is the case of the small bowel, large bowel (including enteric nerve plexuses), adrenal tissue, pancreas and bone marrow. Further relevant data for tissue classification derive from iatrogenic CJD cases and from experimental transmission of prion diseases to animals. In roughly half of iCJD cases the entry site has been the CNS or the eye (dura mater transplants, neurosurgical instruments or devices, corneal transplants), whilst in the other half, injection of pituitary hormones means that a peripheral route of entry has to be assumed \[[@B9]\]. Experimental efficiency of prion disease transmission to animals depends on various factors, such as the inoculum dose, the species barrier between the species of origin of the inoculum and the host, and the route of administration, among others. Under the same experimental conditions, different routes of administration show different efficacy of disease transmission, in terms of length of incubation period and percentage of infected animals \[[@B16],[@B17]\]. While the most efficient route of transmission is intracerebral administration, other routes, such as intraperitoneal, intraneural, intraocular, intravenous, subcutaneous and intramuscular administration, have been used successfully in bioassays and other experimental models \[[@B18]\]. Still other routes, such as oral administration and conjunctival instillation \[[@B19]\], have shown a lower efficiency of transmission. Accordingly, clinical and experimental evidence includes several routes of prion transmission that cannot be easily reduced to a simple tissue classification involving tissues of known infectivity in CJD and/or expression of PrP^C^under normal conditions. This is the case of anterior ophthalmic tissues, skeletal muscle, peritoneum, and subcutaneous tissue rich in sensitive nerve fibres. These anatomical structures have therefore been independently added to our classification as putative routes of entry, with a lower level of risk compared to the high level represented by the central nervous system, sensitive ganglia and posterior eye tissues. The fact that PrP^Sc^has been recently found in 1/4 skeletal muscle samples of sCJD cases\[[@B20]\] prompted us to classify it as a tissue for potential entry rather than a route. A final classification of entry sites for putative surgical transmission of CJD contains tissues, including all those showing positive results for sporadic and familial CJD in the WHO classification \[[@B14]\], with minor additions (tonsil and thymus), and maintains the three risk levels of the original classification along with several putative routes of entry, based on clinical and experimental evidence (see Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Proposed classification of entry sites for putative surgical transmission of CJD by risk level. ::: ------------------------------------------------------------------------------------------------------ **Risk level** **Tissues** **Anatomical structures / routes** ---------------- ----------------------- ------------------------------------------------------------- **High** Brain\ Spinal cord\ Retina, optic nerve\ Spinal ganglia\ Trigeminal ganglia\ Pituitary gland\ Dura mater^a^ **Lower** Peripheral nerves^b^\ Anterior ophthalmic\ Spleen\ Peritoneum\ Lymph nodes^c^\ Subcutaneous (high density of sensitive nerve terminals)^f^ Tonsil^d^\ Thymus^d^\ Placenta\ Lung\ Liver\ Kidney\ Blood vessels^e^\ Olfactory mucosa\ CSF\ Skeletal muscle **Lowest** Other Other ------------------------------------------------------------------------------------------------------ ^a^Dura mater does not contain pathological PrP in CJD patients and its infectivity has not been tested. It is included among high-infectivity tissues in the WHO classification because of evidence of iatrogenic transmission through dura mater grafts.^11^The same rationale has been applied to its inclusion in the present table. ^b^Only surgical procedures on peripheral nerves (e.g., amputation, vagotomy, etc.) will be classified according to this tissue. ^c^Surgical procedures that include this tissue as putative risk are those involving direct manipulation of lymph node chains, e.g., lymph node excision, oncological lymphadenectomy, and intra-abdominal procedures with extensive section of lymph node chains, such as cholecystectomy, gastrectomy and diverse types of bowel resection. ^d^Although tonsil and thymic tissue have yielded negative results for infectivity and presence of pathological PrP in sporadic CJD tissue (tonsillar tissue has not yet been tested for infectivity), they are included in the table in the lower level group, together with the spleen and lymph nodes for biological reasons, in order to assess the role of peripheral lymphoid tissue in surgical transmission. ^e^Only procedures involving direct surgery on blood vessels will be classified according to this tissue. ^f^Hand and facial subcutaneous tissue will be included under this heading. ::: Discussion ========== We propose a list intended to be used to generate attributes of single, well-defined surgical procedures and criteria for their classification in terms of putative risk level of transmission. The two main attributes will be: 1) use of non-disposable surgical instruments; and, 2) exposure during surgery of tissues included in the preceding list. High- and lower-risk surgical procedures will be defined by exposure of tissue corresponding to the respective risk level during surgery. In addition, the lowest risk level is represented by those surgical procedures where disposable surgical instruments are not employed or where no listed tissue or anatomical structure is exposed to surgical instruments. A categorisation of surgical procedures based on the above attributes is inevitably prone to some degree of subjectivity, something that should be minimised by adequate methodological assessment. However, this drawback is more than offset by the possible benefits of identifying specific surgical procedures that pose a significant risk of CJD transmission, in terms of increased study power and control of misclassification of exposure by the removal of surgical procedures, which are probably irrelevant for CJD transmission, from gross anatomical classifications of surgery. We are also aware of the fact that whereas the same surgical instruments are commonly employed in an homogeneous group of procedures in some countries, as is the case of Nordic countries, the same instruments may circulate through a much wider range of procedures and putative risk levels in other countries. Final risk for disease transmission in each surgical procedure combines postulated risk related to tissues exposed in that procedure with the highest risk level of tissues to which instruments have been previously exposed. Accordingly, results should always be interpreted in the light of a known or assumed pattern of instrument circulation between surgical procedure groups. Finally, it is worth stressing that the aim of the approach presented here is exclusively to produce a useful tool for epidemiological research in CJD transmission. Under the present state of knowledge, no consequences for the possible adoption of any practical recommendation concerning surgery or further preventive measures are to be drawn from this approach. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= AR assumed the basic task of reviewing literature, proposing tissues and structures, and drafting the first manuscript version. JPC indicated the subject domain, suggested differences between disease transmission and disease acquisition as seen from experimental and observational epidemiological research, generated first paragraphs relating to epidemiological aspects. KM suggested changes in epidemiological aspects. ÅS provided some criticisms. MC gave diverse comments, particularly on biological plausibility. HL clarified the need for methodological refinement in future work. All authors read and accepted the final version. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/9/prepub> Acknowledgements ================ The EUROSURGYCJD group members are grateful to Prof. Maurizio Pocchiari, Italy, for the substantial contribution to this proposal as regards his stance on the biological plausibility of surgical transmission of prion disorders, and to Maria José Bleda and Margarita Ramírez, Spain, for editorial help. **EUROSURGYCJD group members.**Spanish team: Javier ALMAZÁN, María J. BLEDA, Miguel CALERO, Pablo MARTÍNEZ-MARTÍN, Jesús de PEDRO-CUESTA (P. Leader), Alberto RÁBANO. Danish team: Kåre M∅LBACK, Henning LAURSEN. Finnish team: Jussi KOVANEN. Swedish team: Åke SIDEN, Inger NENNESMO. **Consultant experts**: Dr. Annick ALPEROVITCH, Prof. Paul BROWN, Prof. James IRONSIDE, Prof. Maurizio POCCHIARI, Prof. Robert G. WILL

PubMed Central

2024-06-05T03:55:52.576382

2005-1-24

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548276/", "journal": "BMC Public Health. 2005 Jan 24; 5:9", "authors": [ { "first": "Alberto", "last": "Rábano" }, { "first": "Jesús", "last": "de Pedro-Cuesta" }, { "first": "Kåre", "last": "Mølbak" }, { "first": "Åke", "last": "Siden" }, { "first": "Miguel", "last": "Calero" }, { "first": "Henning", "last": "Laursen" } ] }

PMC548277

Background ========== Reflection is considered by many \[[@B1]-[@B3]\] in the Western world to be an important part of clinical practice and clinical decision-making. In order to learn from experience, health care professionals must reflect on the events and determine where the information \'fits\' in relation to their assumptions, previous experience and knowledge. Reflection is the process where individuals think about and evaluate their experience in order to come to new understandings and appreciations \[[@B2]-[@B4]\]. All promoters of reflection refer to it as a process of thinking about experiences. The process leads to a change in perspective or understanding. Schön \[[@B2],[@B3]\] suggested that reflective practitioners are better able to manage the uncertainty and complexity of clinical practice, and therefore asserted that a major part of preparation of professionals should be centred on enhancing their ability to reflect. Journal writing is a method that has been used to promote reflective thinking in health science students and professionals \[[@B5],[@B6]\], to facilitate the link between academic learning and clinical practice \[[@B7]\], and to assist students to explore and change their attitudes toward patients \[[@B7],[@B8]\]. When writing a reflective journal, students are asked to describe a learning event or situation, explain how the experience led to new understandings and appreciations, and consider how they might act differently in the future. Students have indicated that the writing of the journal provides a framework for the reflective process).)\[[@B9],[@B10]\]. Shepard and Jensen \[[@B11]\] specifically referred to the need for physiotherapists to have \'reflective\' knowledge in order to deal with uncertainties and value conflicts. Two recent Canadian studies \[[@B12],[@B13]\]. have described the reflections of physiotherapy students during clinical placement. The students wrote weekly in journals, and reflected on a number of topics that were classified into six major themes \[[@B12]\]. These included: 1) the process of making clinical decisions; 2) the complexity and richness of interactions with clients; 3) the influence of the practice environment on learning and patient care; 4) acquisition of clinical and administrative skills; 5) the value of clinical experiences in validating and integrating previous learning; and 6) acknowledgment and evaluation of different learning methods. Students frequently commented on their own values and beliefs and whether these were in harmony or conflict with the beliefs and actions of other health professionals or clients \[[@B13]\]. They discussed professional behavior, professional collegiality, respect for others, advocacy and informed consent. In considering the student reflections, the investigators wondered about the potential impact of culture and working environment on the content of the reflective journals. Would students from a non-Western culture and a different health care system be concerned about the same issues? Would they feel comfortable with the reflective process, particularly documenting their thoughts in a journal? How might the hospital/institutional infrastructure and norms affect their learning? We had a unique opportunity to answer some of these questions by comparing the themes from reflective journals of physiotherapy students in the United Arab Emirates (UAE) with those taken from the above mentioned studies \[[@B12],[@B13]\]. conducted at McMaster University in Canada. The physiotherapy program at the University of Sharjah in the UAE was developed with a philosophy similar to that of the program at McMaster, and the Chair of the program was a McMaster faculty member. Students in both programs have worked in small groups, studied similar content, and considered the same professional and attitudinal issues such as moral reasoning, client-centred care, professional behavior and professional code of ethics. Although both the UAE and Canada have many of the conveniences of modern society, there are major societal and cultural differences. The students in both countries have similar standards of living and have ready access to education and health services. Many of the health problems of modern society -- diabetes, heart disease, arthritis, cancer, and trauma from traffic accidents -- are common in Canada and the UAE. However, Canada is a democracy with a predominantly Christian and European population, whereas the UAE is a system of emirates with a population comprising mainly Arabic and Eastern cultures and where the Muslim religion predominates. The students in the two countries might be expected to have different values and beliefs, and different societal norms that could affect their reflections. The purpose of this study was to describe the reflections of physiotherapy students from the University of Sharjah during their clinical placements. A second objective was to compare the themes generated from the Sharjah data to the themes from the previous two studies on reflections of McMaster students during their clinical placements. Because the students in the two programs have similar socioeconomic status and similar curricula, it was anticipated that differences in reflections would be due to differences in culture and life experiences related to the cultures. Methods ======= This qualitative study was conducted during the students\'clinical placements in a summer semester of the Physiotherapy Program at the University of Sharjah, United Arab Emirates, a program developed in affiliation with McMaster University, Hamilton, Ontario, Canada. Curriculum and semester description ----------------------------------- The baccalaureate physiotherapy program in the UAE comprises one pre-professional year (year 1) and three professional years (years 2--4) of study. To be admitted to the program, students must have completed the UAE Secondary School Scientific Certificate (equivalent to grade 12), or equivalent program accredited by the Ministry of Education. They must have also scored at least 500 on the Test of English as a Foreign Language (TOEFL). While in the physiotherapy program, students need to complete university required and elective courses. Basic science courses are offered in years 1 and 2. Physiotherapy courses in years 2--4 include small-group, problem-based learning tutorials and clinical skills laboratories based on problems/ case scenarios enhanced with resource sessions. The male and female students are taught separately in discrete areas of the campus. Students -------- Six senior physiotherapy students (3 females and 3 males) with a mean age of 22.6 years (range 20.5 to 23.1) and 15 junior, female students with a mean age of 20.1 years (range 19.9 to 22.8) formed the subject group of this study. These students were respectively from the first and second cohorts of students admitted to the new physiotherapy program at the University of Sharjah. All students spoke Arabic and English. The senior students had completed the academic component of year 3 and were in their third clinical placement, which provided them with six weeks experience primarily with clients with cardio-respiratory conditions. The junior students had just completed two academic semesters of year 2, and were in their first clinical placement -- four weeks with clients with peripheral musculoskeletal conditions. Students practiced in five different clinical sites in the UAE, under the supervision of clinical preceptors and university instructors/coordinators. The study was approved by the Ethics Committee of the College of Health Sciences, and all participating students signed an informed consent. Evaluators ---------- Two instructors were involved in reading, coding and identifying the major themes that emerged from the students\' reflective journals. One instructor (HL) from the University of Sharjah, Department of Physiotherapy, had taught one course to the junior group and two courses to the senior group. The second instructor (JW) from McMaster University Physiotherapy Program, had taught one course to the senior group as a consultant to the program. The two instructors were not involved with the students during their summer clinical placements. A third evaluator (AAS) independently read all the journals. She was an instructor in the Sharjah program, a UAE national, and fluent in both Arabic and English. She had taught two courses to the junior students. Her analysis was used to confirm the interpretation of the entries made by the other two evaluators who were both from Western cultures. Procedure --------- The students were asked to write a reflective journal, a new exercise for all students. This assignment was one of the requirements for a Pass mark in their summer clinical placement. The students were required to make one entry per week for the duration of their clinical placement (four weeks for junior students and six weeks for senior students), and to submit the journal weekly to the university instructor involved in their clinical supervision. However, the instructor did not provide any feedback to the student at that time. The students were instructed to include the following in their reflective journals: *Reflective journals should include observations, impressions, and reactions to what you have learned in the academic portion of the semester and how you are applying it to clinical practice. How does the clinical experience change what you thought, felt, or did in the past, and how you may respond in the future? You are expected to write at least one journal entry per week during your clinical placement*. 1\. Describe the learning event, issue or situation. Describe prior knowledge, feelings or attitudes with new knowledge, feelings or attitudes. What happened? 2\. Analyse the learning event, issue or situation in relation to prior knowledge, feelings or attitudes. What was your reaction to the learning event, issue or situation? Your response may include cognitive and emotional reactions. Why did it happen? 3\. Verify the learning event, issue or situation in relation to prior knowledge, feelings or attitudes. What is the value of the learning event, issue or situation that has occurred? Is the new knowledge, feeling or attitude about the learning event, issue or situation correct? 4\. Gain a new understanding of the learning event, issue or situation. What is your new understanding of the learning event, issue or situation? 5\. Indicate how the new learning event, issue or situation will affect future behavior. Determine the clarification of an issue, the development of a skill or the resolution of a problem. How will you approach the same or similar event, issue or situation in the future? Data analysis ------------- The method described by Coffey and Atkinson \[[@B14]\] was used to analyse and interpret the data in the students\' journals. The analysis was conducted in two phases after the students had completed their clinical placements and submitted their entire journal. In Phase 1, one evaluator (HL) selected five journal entries based on their readability level and diverse content and forwarded them to the second evaluator (JW). The two evaluators read and coded the five journals independently. Then they met to discuss and establish agreement on the coding. In Phase 2, the two evaluators independently read and coded all remaining journals. They met again to 1) determine agreement on the content of each entry; 2) group the codes into categories; and 3) determine major themes. When coding discrepancy occurred, the evaluators discussed the discrepancy and came to a consensus. The following two methods were used to further validate the analysis and interpretation of the data. 1) The third evaluator independently read and labelled all journal entries to confirm the interpretation of the content. 2) The themes established by the evaluators were presented to two students who had completed the journals, and who volunteered to provide feedback on the authors\' interpretation of their reflections. Themes for the present study were developed without direct reference to the themes found in the Canadian studies \[[@B12],[@B13]\]. The Emirati and Canadian themes were then compared by examining their descriptions and content. Results ======= The two initial evaluators (HL, JW) came to a consensus on four themes concerning the clinical learning experience of the students. These themes did not change as a result of the coding of the third reader (AAS) or the feedback from the students. However, these additional data occasionally resulted in a change in emphasis of some of the content within a theme. Whenever this happened, the differences are described. All students outlined the positive impact and value of their learning from the clinical placement. They discussed events and issues that fit into the following four themes: 1\. Professional behaviors: skills and attitudes, scope of practice, time management, professional boundaries, and respect for clients and colleagues. 2\. Awareness of learning: clinical versus academic learning, gaining of important new knowledge, self-directed and life-long learning, and client as a source of learning. 3\. Self-development and shift to a more client-oriented focus over the time of the clinical placement. 4\. Identification and analysis of ethical issues. Each of these themes is discussed below with quotes provided for illustrative purposes. Professional behaviors ---------------------- The students appeared very aware of their own behavior and that of other health professionals and students. They reflected on how they should behave as professionals, but this also included how they should behave in general. The events that precipitated discussion on this theme included actions of themselves and other health professionals and included what they regarded as both positive and negative behaviors. They referred to physiotherapists and other health professionals who helped the students and who modelled good professional behavior. They also described some situations where health professionals did not show sufficient respect for their clients or their colleagues, or did not provide \'best practice\'. They commented on professional boundaries, being concerned about the client-therapist relationship becoming too personal and limits to the scope of physiotherapy practice. In addition, the students noted that self-directed learning and life-long learning were professional responsibilities. This topic is discussed under the next theme, Awareness of Learning. The students providing feedback emphasized that professional behavior (both negative and positive) was also learned from patients who might show their appreciation, be patient or uncooperative, or provide specific feedback regarding the student\'s communication or skills. The students also provided examples of \"crossing boundaries\" in terms of professional competencies and relationships with their patients and supervising therapists. *One patient asked me angrily where \[were the physiotherapists\]? I responded politely to wait until they finish their work with the patients\... I calmed her down and asked her to be patient\...I really felt that the \[physiotherapist\] and I should maintain and manage our time\...It is really a big responsibility we have*. \[student 11, week 3\] *The child came crying and refusing to look at her forearm. We should respect the emotional status of the child and try to understand it because the child has been through a lot. The accident\..., surgery,\...and functional limitations\...The stress should be controlled before giving physiotherapy\...I should be patient and try to give the most enjoyable set of exercises to gain the child\'s trust*. \[student 10, week 3\] *Sometimes patients are shy to tell that there is no benefit from the treatment programme. So I asked the patient if he feels any improvements and if not to inform me in order to change the plan of treatment. The patient responded very well to that*. \[student 5, week 3\] Awareness of learning --------------------- Although the entire reflective journal assignment was designed to have students reflect on their learning, this theme was included to capture the many components of learning discussed by the students. They gained knowledge, but also commented on the unique aspects of clinical learning. Clinical experiences provided an opportunity to apply and enhance their academic learning. Junior students particularly noted how observing surgery helped them understand their anatomy, or how working with \"real patients\' was not as straight forward as their academic learning. The senior students realized the need to be life-long and self-directed learners because they were always encountering new conditions/situations and they wanted to provide their clients with \'best practice\'. Students acknowledged the clients as a source of knowledge, and noted that clinical situations provided a stimulus to learn. In the feedback session, students particularly emphasized how the clinical experience had enhanced their learning \"a lot!\" They also indicated that clinicians reinforced the need for life-long learning because of the constant changes in science. Some therapists welcomed new knowledge from students, for example, learning about outcome measures, while others indicated they did not have the time. *It was really good to have a mix of people \[clinicians\] and to learn from their experience in the work field\...Each one of them has given us a new way to deal with patients and\... also a new experience to add to our future knowledge\... The learning is not finished\...We have to keep our hands on the best information available and the best experience*. \[student 4, week 6\] *\[I\] try to apply the theoretical bases learned at the university. \[I\] interact with patients and take their histories, try to know their goals and what they need from physiotherapy. I try to invent new materials for the exercises. In my opinion, when we apply what we learned in the theory, the information sticks in our minds and is hardly forgotten*. \[student 21, week 2\] Self-development and orientation shift -------------------------------------- The theme of self-development was derived from the content of individual student entries, but was also seen in the change in the reflections as students proceeded through their placements. Often at the beginning of the clinical encounter, the students were unsure of themselves, but they gained confidence as they worked with the client and/or learned from their colleagues. The change across the journal entries was even more noticeable. In many of the early entries, students described their own emotional response to a client or a situation. They were concerned about their own performance, and about what they were learning from the placement. They indicated how an event had an impact on them. The later entries were more client-centred. The focus was more on how the client responded to an event, or how helpful a physiotherapist was. Students also referred to an increased confidence, the more experiences they had. It became evident from the third reader\'s summary that students were aware of how their professional behavior, outside direct patient care, could ultimately have an impact on the patient. They referred specifically to the maintenance of knowledge and skills and to relationships with other health professionals. Students in the feedback session referred to their initial lack of confidence which improved during the clinical placement. They added, however, that it was difficult to demonstrate the \"correct confidence level\" to their instructor/evaluator, particularly when students were occasionally told they were \"over-confident\". They felt that self-development was affected by behavior of other physiotherapy students, method of feedback from instructors and whether their clients improved or not. The quote immediately below illustrates how a student confronted his/her uncertainties during one client encounter. The next quote concerns a student\'s acknowledgement of the change in his/her confidence with increasing experience. *First patient with tetraplegia\...What if I do something wrong?\...First, worried\... and unable to do anything, I observed other colleagues\... I could do it too!*\[student 6, week 2\] *A one-month old infant was in the ICU. I thought I would not be able to touch her as usual, but things are now different. I did all the treatment\... with my hands with no hesitation and I was not scared\... My reaction was totally changed and it is becoming more regular\... I am happy\... I trust myself better and it is increasing with time\...and will increase the more cases I face*. \[student 3, week 6\] In the quotes below, the student initially talks about his/her own emotional response to a patient, but later in the placement discusses the need to adapt to patients\' situations and provide them with the necessary treatment. In both journal entries, the student discusses a client with a novel and somewhat \"unpleasant\" condition. However, the student reacts differently in week 5 than week 1. *At that moment I was shocked because I was not familiar with the neurology cases. When the physiotherapist asked me to help\...I hesitated in the beginning. \...This situation had a bad effect on me \... I was depressed and I couldn\'t do any single movement*\[student 4, week 1\] *I have seen a patient who was always under sedation due to his pain and his anger which was there almost all the time. \... I was always confused \...not knowing what to do for him and what is the best for him. \... Such a case made me think \[very\] much before any and each simple movement. \... So this made me prevent \[other\] problems such as ulcers, and invent any simple movement just to change his lying position*\[student 4, week 5\] As the quotes illustrate, the student was concerned about what to do in both cases, but by week 5 was able to overcome the emotional response, problem solve about the management of the client, and take action. Ethics ------ Students commented on what they believed to be ethical or unethical behavior of the health professionals they were in contact with. They noted when informed consent was not obtained from clients and they discussed ways to get consent from uncooperative clients. They talked about having respect for their clients, and maintaining confidentiality. They noted \"ethical\" and \"unethical\" behavior in the same professionals under different circumstances. *The staff is cooperative and helpful, respect each other, but there is no confidentiality or respect of time with patients*. \[student 20, week 1\] They were concerned when individuals (clients or professionals) were being treated unfairly or differently because of their health condition or socio-economic status. In some cases they thought that the client might be harmed by the practice. One student was concerned about how to \'tell the truth\' to a client who had a poor surgical result possibly due to the questionable practice of the surgeon. One student described an event where a therapist refused to see a patient when she changed her appointment time, but treated another patient who did not have an appointment. The student felt that the latter patient was treated because she was a \"VIP (very important person)\". *I felt sad for the \[person who did not receive treatment\], and I said to myself, where are the ethics? I learned before \... that all patients should be at the same level without any bias. \...all persons who work in health care fields specially the professional therapists must \[follow\] the code of ethics because they have humans in their hands. \... I will delete the word VIP from my life work*. \[student 12, week 2\] In the feedback session, the students also indicated how clients could be discriminated against because of their health condition and the therapist\'s level of confidence. Therapists might provide less treatment to a child if they felt uncomfortable treating children. They might leave a student to treat an older person if they felt the management of older persons was less important or less effective. Feedback from students ---------------------- The students agreed with the four themes and did not think that any major concept was omitted in this analysis. However, they added some insight to the content and interpretation of each theme. The students emphasized that many people affect their learning -- university instructors, therapists, other health professionals, students and patients/clients. They felt that the information derived from the analysis of the reflective journals was very important. They strongly recommended that the results be disseminated to clinicians, instructors and future students to increase their awareness of the learning process and help the students to improve. Comparisons of themes of students from McMaster University and the University of Sharjah ---------------------------------------------------------------------------------------- The content of the journals indicated that students from both universities were comfortable with the reflective process. They frequently shared quite personal information, including their positive and negative emotions, concerns about their own behavior, and a discussion of their own beliefs. Some students in both countries, simply described an event and provided minimal personal reflection, but these students were in the minority. Table [1](#T1){ref-type="table"} summarizes the comparable themes of the Emirati and Canadian students. Themes from both countries related to professional behaviors, learning and ethical issues. Self-development was not identified per se as a theme in the Canadian studies, but change and growth were mentioned under the other themes. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Themes from Emirati and Canadian students during clinical placements ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------ UAE Canada ----------------------------------------------- ------------------------------------------------------------------------------------------------------------------ Professional behaviors † Process of making clinical decisions † Complexity and richness of interactions with patients ‡ Respect: for uniqueness of individual ‡ Professionalism: responsibility and behavior as a member of a profession ‡ Professional collegiality: interaction with and respect for health professionals including physical therapists Awareness of learning † Effects of practice environment on learning and patient care † Acknowledgment and evaluation of different learning methods † Value of clinical experiences in validating and integrating previous learning † Acquisition of knowledge and clinical and administrative skills Identification and analysis of ethical issues ‡ Allocation of resources\ ‡ Advocacy: for clients, society or health policy\ ‡ Informed consent: right of person to decide what will happen to him/her ------------------------------------------------------------------------------------------------------------------------------------------------------------------ † Themes from Williams et al \[12\] ‡ Themes from Geddes et al \[13\] ::: In the area of professional behavior, students in both countries commented on role models (both positive and negative) in the clinical environment, as well as on their own behavior or response to clients. Both groups demonstrated a respect for their clients and saw their role as going beyond physical treatment. Canadian students mentioned advocacy in the community while students in the UAE discussed supporting their clients within the hospital milieu. Both groups talked about the uniqueness of learning in clinical placements. The clinical experience enhanced and confirmed their academic learning, and stimulated them to learn more. They enjoyed learning new information and using their knowledge to help others. They were anxious when they \"didn\'t know what to do\", but expressed increased confidence with each experience. They appreciated constructive feedback on their performance from clinical supervisors, health professionals and clients. Both groups acquired new skills related to direct client care (therapeutic techniques and communication) and administrative activities (time management, organizational skills and charting). Only Canadian students referred to writing reports and billing, tasks that students in the UAE were not expected to do in their clinical placements. Both groups, however, broadened their view of the scope of practice of physiotherapy. Students from both countries noted ethical issues pertaining to inequalities in their respective health care systems. Canadian students were concerned about the shortfall of resources (time, personnel, funding), and about differences in health care provided to private clients or those funded through provincial health insurance. Emirati students noted that the health or societal status of a client could affect their treatment within the health system. Discussion ========== Physiotherapy students from Sharjah demonstrated the ability to reflect on their clinical practice at the highest level (discussion of how experience will impact on future behavior). They described not only the learning event, but also their emotional and cognitive reactions to it. They analysed the situations to arrive at new understandings and considerations of how they would behave differently in the future. Like the students in the Canadian studies, the subjects in the present study described three types of learning that are considered essential for clinical decision-making. These are propositional (academic learning -- facts, concepts), professional craft (learning from experience), and personal knowledge (knowledge of self and unique frame of reference) \[[@B15]\]. The students commented on the knowledge that they gained (e.g., anatomy, treatment techniques, medications), and the unique learning opportunities of fieldwork (e.g., application and enhancement of academic learning, learning new skills, response to novel situations). They acknowledged that their personal frame of reference affected their clinical interactions, but was also modified by their clinical experience. Although the students did not categorize their learning in the three areas, they were able to describe the acquisition of knowledge and skills required for making clinical decisions. The reflection on one\'s own development is a common theme in the reflections of health professionals. Jensen and Denton\[[@B7]\] referred to themes of student self-adequacy and inadequacy in their study of 23 physiotherapy students completing journals in their first clinical placement. Tryssenaar and Perkins\[[@B16]\] demonstrated that reflection on personal growth was still evident at the end of the educational programs and the beginning of careers for physical and occupational therapists. Subjects in their study described initial anxiety and then increasing confidence about their ability as graduate therapists. They became more patient-centred with experience. Jensen and Paschal\[[@B17]\] suggest that the shift to a client-centred approach is part of the transition that students go through from their first to later clinical placements. Because we did not follow one group of students over several placements, we cannot comment on the change in the focus of students with each additional clinical experience. However, both junior and senior students described their personal reactions more frequently at the beginning of the placement, and the patient\'s unique situation more frequently in the later weeks. Perhaps with each new experience (e.g., start of a clinical placement) students will be temporarily more self-centred as they deal with their anxieties and uncertainties. As they become more comfortable with their own abilities, they can focus more on the client. Such a pattern is in agreement with the finding mentioned above, that therapists become more client-centred with experience. \[[@B16]\] One other study \[[@B18]\] examined the reflections of non-Western physiotherapy students during their clinical placements. Although the students were practicing in an English, Western environment, rather than in their own culture and language, the students and their supervisors did raise some issues that may have relevance to the present study. The Chinese students and their Australian supervisors noted that the students were reluctant to self-evaluate, to express their opinions, to admit they did not understand or to take active steps to acquire needed information. In the present study, the students certainly voiced their opinions, admitted their perceived inadequacies, and expressed pride in their achievements in the reflective journals. However, some may have been less forthcoming with their clinical preceptors or university instructors/coordinators, particularly when they did not agree with them. In the sample quotes in the Results section, the students did not mention discussing their concerns and feelings with their supervisors. On the other hand, the students in the present study were working in their own language and culture, and would be better able to express their views, and to do it in a \"politically correct\" manner. Unlike the Chinese students, the students in the UAE discussed the need for self-direction in their learning. This difference could be cultural and/or due to the self-directed nature of the problem-based learning method used in the physiotherapy program at the University of Sharjah. Language might have been a factor in the reflections of the students from the UAE, however. Although the students worked in Arabic in the clinical environment, they conducted their physiotherapy education and wrote their journals in English. It is possible that some students had difficulty accurately expressing their reflections in English, and that there was some misinterpretation by the evaluators. However, two of the evaluators (HL, AAS) knew the students well and were used to their writing. One of them (AAS) was a UAE national and fluent in both English and Arabic. When the two initial evaluators (HL, JW) worked together in Phase 1 to determine coding methods, part of the discussion was on how to interpret the \"second-language writing\" of the journals. In spite of these precautions, it is still possible that the themes may have varied somewhat if the journals had been written in Arabic and interpreted by Arabic-speaking evaluators. Conclusions =========== Physiotherapy students from a Middle East culture consider many of the same issues as students from a Western culture when asked to reflect on their clinical experience. They reflect on their personal growth, on how they learn in a clinical setting, and on the ethical and professional behaviors of themselves and others. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= HL and JW were involved in the conception and design of the study, in the qualitative analysis, and in the writing of the manuscript. HL coordinated the data collection. AAS assisted with data analysis. All authors were involved in the preparation of the manuscript, and read and approved the final version. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6920/5/3/prepub> Acknowledgements ================ We thank all the students who participated in the study and provided feedback on the analysis. This study was supported by an Education Grant from the School of Rehabilitation Science, McMaster University.

PubMed Central

2024-06-05T03:55:52.578117

2005-1-20

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548277/", "journal": "BMC Med Educ. 2005 Jan 20; 5:3", "authors": [ { "first": "Hélène", "last": "Larin" }, { "first": "Jean", "last": "Wessel" }, { "first": "Amal", "last": "Al-Shamlan" } ] }

PMC548278

Background ========== Single nucleotide polymorphisms (SNPs) are the most common form of genetic variation within species. As a result, SNPs are now becoming the most popular type of marker in genetic association and mapping studies. SNPs are also most likely to be the molecular basis for the majority of phenotypic variation in (outbred) populations. In particular, SNPs in regulatory and protein-coding regions can have an effect on gene expression levels and protein activity, respectively. The phenotypic differences observed between selected (sub) strains in model organisms may be the result of specific (combinations of) natural occurring polymorphisms. Hence, a comprehensive inventory of SNPs, including extensive annotation will be extremely valuable in the search for functional polymorphisms. There is often a vast unexplored potential in large sequence datasets that have been collected for other purposes, for example, EST and whole genome sequencing (WGS) projects. In an effort to address these two issues, we have developed an *in silico*candidate SNP mining pipeline that uses all publicly available sequence data for a specific organism, and designed a database, CASCAD (CAscad SNP CAndidates Database), that allows storage of a wide variety of primary source data, cross-annotation to other databases, and analysis parameters for SNPs associated with expressed sequences. Construction and content ======================== We applied the SNP discovery pipeline to both rat \[[@B1]\] and zebrafish (unpublished results) and identified about 33,000 and 52,000 high-quality candidates, respectively, that were extensively annotated and stored in the CASCAD database (Table [1](#T1){ref-type="table"}). The database includes detailed primary information on which the discovery of the candidate SNPs was based. This information, including sequence quality information (Phred score), the number of supporting reads for every nucleotide observed at the SNP position, and expected alignment lengths, was found to be very valuable for filtering for predicted variants that have the highest likelihood to be experimentally confirmed. Two verification experiments for the rat resulted in confirmation rate estimates of 59% (68 candidate SNPs in 10 different laboratory rat strains) and 50.3 % (340 candidates in 5 laboratory and 2 wild rat isolates) \[[@B1]\]. A set of 139 CASCAD entries tested in 7 zebrafish isolates confirmed 67.6% of them as true polymorphisms (unpublished data). The success rate values obtained are likely to be underestimates since only limited number of isolates/samples were used and we were unable to include exactly the same isolates that were used for generating the primary data (e.g. EST sequencing). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Input data (number of sequence reads) for the CASCAD pipeline and number of predicted candidate SNPs. ::: ***Rattus norvegicus*** ***Danio rerio*** -------------------------- ------------------------- ------------------- Input data mRNA 25, 634 3, 366 EST 244, 518 283, 572 WGS 19, 813, 313 11, 588, 394 Candidate SNPs predicted 33, 305 51, 769 synonymous 3, 842 9, 111 non-synonymous 3, 708 6, 217 nonsense 162 158 ::: We designed a web-based interface with Perl scripts communicating to a MySQL database, and displaying HTML pages through Apache server running on SuSE Linux. The interface provides simple, advanced (Figure [1](#F1){ref-type="fig"}), and sequence-based search forms. Parameters that can be used in a search include strain information, different formats of sequence identifiers (e.g. GenBank, UniGene, LocusLink accessions or gene symbol), map positions (genetic and physical), and a wide variety of SNP characteristics, such as experimental evidence, a likelihood score for verification as deduced from extensive verification experiments \[[@B1]\], and information regarding restriction sites that have been affected, facilitating the design of RFLP based assays. To this end CASCAD is tightly linked to primer design \[[@B2]\] and local genome assembly \[[@B3]\] interfaces, enabling a fast, reliable, and universal primer design for a chosen SNP candidate even when there is no assembled genome sequence available. To facilitate the retrieval of candidate SNPs with higher verification rates, represented by more reliable and common SNP candidates, we implemented the possibility to restrict searches to entries characterized by location at hypervariable CpG site, by minimal basecalling quality (Phred score), sequence match size, and/or minimal number of supporting reads for either allele. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### CASCAD advanced search form. ::: ![](1471-2164-6-10-1) ::: In addition to primary sequence data analysis, the effect of all SNPs on protein coding capacity was evaluated and non-synonymous SNPs were categorized in classes reflecting the severity of the polymorphism using a BLOSUM-based score. The predicted missense SNPs were analyzed by SIFT \[[@B4]\] and Polyphen \[[@B5]\] programs that utilize not only substitution information but also phylogenetic conservation and structural protein information to predict a potential effect of the polymorphism on protein function. Query results are summarized on the SNP details page (Figure [2](#F2){ref-type="fig"}), listing the SNP characteristics and including active links to other databases and resources, such as dbSNP, Ensembl, UniGene, and LocusLink. More detailed information regarding raw data underlying the candidate SNP (links to the original sequence files and a full nucleotide and protein alignment) can be obtained by clicking on the observed nearly exact hits between nucleotide sequences. Moreover, statistics on the data that support the SNP (number of occurrences for every nucleotide at SNP position, range of Phred basecalling quality scores) are provided. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### SNP details page ::: ![](1471-2164-6-10-2) ::: Utility and discussion ====================== For many applications, it is important to be able to distinguish between SNP candidates by their characteristics, as they may be predictive for verification success rate or carry biologically relevant information. Non-confirmed candidate polymorphisms may represent variants uncommon for a given population, but also sequencing errors (all types of sequences), RNA editing events and reverse transcriptase errors (EST reads). In order to minimize the contribution of false positives, one can exclude polymorphisms based on a single read for either allele, as is common for many *in silico*discovery pipelines \[[@B6]\]. Although this is a valid approach when selecting SNPs for population or association genetics, one could inadvertently discard many rare variants that may be associated with phenotypic variation, for example by affecting protein structure or function. Information on such polymorphisms can be very useful when mapping disease or QTL alleles. We have developed our database to fulfill the needs of any particular SNP application by providing control over every parameter we used in the polymorphism discovery step. Applications of the CASCAD database include queries for potentially deleterious SNPs in a specific genomic region of interest, for example a QTL interval, design of SNP-based mapping panels using either RFLP or any other technology, and identification of informative SNPs for fine-mapping. Custom sequences can be provided to search for known SNPs in any sequence of interest. In addition, the CASCAD pipeline \[[@B1]\] can be used to build a candidate SNP database for any model organism of interest for which sufficient sequencing data is available. Conclusions =========== The main purpose of CASCAD database is to provide flexible access to candidate single nucleotide polymorphisms, which were predicted using a computational approach from publicly available sequence data of the rat and zebrafish. The resulting database is crosslinked to most common public databases and can be queried for SNPs using accession numbers, sequence context, SNP characteristics, but also using parameters specific to the SNP discovery process, allowing stringent or relaxed conditions suitable for different types of applications. Availability and requirements ============================= The database is freely accessible through the website <http://cascad.niob.knaw.nl>. Programs, scripts, MySQL database dumps, and instructions for setting up a species-specific SNP database can be obtained from the authors upon request. Authors\' contributions ======================= VG designed and implemented the CASCAD database. EB tested database and interface. EC provided supervision and guidance for the project. Acknowledgements ================ This work was supported by the Dutch Ministry of Economic Affairs through the Innovation Oriented Research Program on Genomics, grant \#IGE010017.

PubMed Central

2024-06-05T03:55:52.582399

2005-1-27

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548278/", "journal": "BMC Genomics. 2005 Jan 27; 6:10", "authors": [ { "first": "Victor", "last": "Guryev" }, { "first": "Eugene", "last": "Berezikov" }, { "first": "Edwin", "last": "Cuppen" } ] }

PMC548279

Background ========== In many developing countries, social marketing program have become an essential component, if not the main component, of the national family planning and HIV prevention strategy. Social marketing programs in reproductive health all aim to improve reproductive health of the target population by using commercial approaches to promote healthy behaviors and/or to expand access to essential products and services. While improving reproductive health of the target population is the ultimate aim, donors recognize the potential of social marketing programs to build the sustainable delivery of reproductive health and family planning products and services in developing countries. Different types of social marketing models have been used to achieve these objectives. Social marketing programs have often been classified as either using the *non-governmental organization (NGO) model (*sometimes also referred to as the *social marketing organization model*) or the *manufacturer\'s model*. \[[@B1]-[@B3]\]. The NGO model typically focuses on achieving the largest possible health impact among the target population. Hence, the NGO model typically heavily subsidizes products. By contrast, the manufacturer\'s model was specifically developed in response to the need not only to improve reproductive health, but also to do so in a financially sustainable manner. Programs under the manufacturer\'s model are treated as temporary interventions with a realistic exit strategy. Such programs typically use experts to provide temporary technical assistance to existing private sector companies, which eventually are expected to continue the program without subsidies. Although the two models are defined largely by the type of management and financial structure, they often also differ in terms of branding, pricing, distribution, and other factors. Moreover, context-specific variations on these two models are common, and some programs use hybrid approaches that integrate features from each of the two broader models \[[@B4]\]. While many social marketing variants have proven successful, it is generally assumed that the manufacturer\'s model is more feasible in middle-income countries with a fairly well developed commercial infrastructure, while NGO models are more appropriate in lower-income countries with less less-developed commercial infrastructure \[[@B4]\]. Although scattered case studies support these assumptions, as yet there has not been any comprehensive empirical assessment of the socio-economic context in which each model works best. This paper analyzes a comprehensive database of annual social marketing statistics. The database is unique in that it includes annual data on social marketing programs in reproductive health in over 70 countries, which cumulatively represent 555 years of experience with social marketing programs. We analyze these data to provide empirical information on the extent to which different social marketing models have been used in different socio-economic contexts, and to identify the socio-economic context in which each model is most effective. Our analysis is restricted to programs that social market condoms or oral contraceptives. As only limited data on the features of social marketing programs are routinely collected, we focus on differences in management structure. In many countries, social marketing programs are an essential component of the national family planning and HIV prevention programs. At present, large-scale social marketing programs for reproductive health are in operation in over 60 countries worldwide. A growing body of evidence shows that social marketing programs can make an important contribution to the reproductive health of the target population \[[@B5]-[@B19]\]. In 2002, social marketing programs in 69 countries sold almost 1.6 billion condoms \[[@B20]\] accounting for more than half of all condoms available from public sources. Total sales of condoms, pills, vaginal foaming tablets and IUDs amounted to over 28 million couple-years of protection (a measure of the number of couples that could be protected from pregnancy for one year). In these countries (excluding China and Russia) social marketing sales of temporary methods of contraception excluding condoms accounted for almost 10 percent of all use \[[@B21]\]. Social marketing tends to be a cost-effective approach to achieving widespread access to health products and services \[[@B22],[@B23]\]. It uses private sector incentives and organizations to achieve efficiency and revenues from sales offset some of the program costs. The total cost of \$6 per couple-year of protection compares favorably with costs to implement public sector family planning programs, which are around \$15--20 per couple-year of protection \[[@B24]-[@B27]\]. Some programs may eventually become self-sustaining and graduate from donor assistance altogether. Some social marketing programs in low-income countries are now operated by independent local social marketing foundations (e.g., the Ghana Social Marketing Foundation (GSMF) and ADEMAS in Senegal) and in some middle-income countries social marketing sales are continuing well after donor assistance has ended (e.g., condoms in Turkey, oral contraceptives in Morocco, and injectables in Brazil) \[[@B28]-[@B30]\]. Three different management structures are common in social marketing programs: 1) management by an affiliate of an international NGO, 2) management by local clinic-based or non-clinic-based organizations, and 3) partnerships with a commercial organization. The most commonly used management structure in health-oriented social marketing programs is management by an NGO. Typically this implies management by an affiliate of an international social marketing organization that provides an expatriate resident advisor. Several social marketing programs are managed by local organizations. For example, some social marketing programs are managed by a specially developed new organization that is independent of international affiliation. These are often called *social marketing foundations*. This organization may receive technical assistance from an international social marketing organization but it operates as an independent entity. Implementation may also be by an existing organization, such as a family planning association (FPA) or another type of clinic-based organization. In such case an international NGO provides technical assistance to the local organization to develop and operate the social marketing program. Social marketing programs may also be implemented through a partnership between an international NGO and an existing commercial organization. In this case, the international NGO often helps to develop the market for the product through research and promotion, while the commercial partner handles all aspects of packaging, sales and distribution. Not all management approaches are feasible in every context. For example, in countries with relatively high-income levels and a strong commercial sector there may be many opportunities for commercial partnerships, while in other countries there may be few such opportunities, if any \[[@B4]\]. Similarly, opportunities for management through existing local organizations do not exist in all countries. Furthermore, the context in which social marketing programs are implemented has a crucial impact on the types of programs that can be successful. For example, in a poor country with a rudimentary commercial sector, it is unlikely that commercial partnerships will be successful. On the other hand, in a middle-income country with a well-developed commercial sector and a rapidly developing middle class, it may be counterproductive to use an NGO affiliate model with a highly subsidized product that competes with existing brands. Methods ======= This section analyses empirical data to study differences in social marketing models and their impact. Since data about program characteristics are scarce, we focus on differences in management structure. Specifically, our objectives are to 1) describe to what extent the type of social marketing management structure actually varies according to the socio-economic context in which the programs are implemented, and 2) to examine the effectiveness of various management structures in increasing socially marketed product sales in different contexts. Data ---- For the purpose of this study, we compiled a database that comprises data on most of the major social marketing programs that were in operation at any time during the period between 1988 and 2001. Many, but not all, of these programs were still in operation in 2001. The database contains annual data on several program characteristics and on indicators of the local socio-economic context. The main sources of information for data on program characteristics were DKT International\'s \"Contraceptive Social Marketing Statistics,\" Population Services International\'s (PSI) MIS database, and published data on Futures Group International (TFGI) programs \[[@B26]\]. These three programs are the dominant implementing organization in contraceptive social marketing \[[@B1]\]. Data on the local context were obtained from the \"World Development Indicators 2003 CD-ROM\" \[[@B31]\]. For each social marketing program, the database contains one case for each year of operation within the 1988--2001 study period. In other words, our unit of analysis is the \"program-year.\" Using \"program-years\" as our unit of analysis has the advantage that all indicators, including program characteristics, can change over time. In addition, it implies that in the analyses programs that have been in operation for a longer period of time are given more weight than newer programs. For example, a social marketing program that has been operating since 1988 will contribute 14 program-years to the database (one case for each year of operation); a program that started in 1995 and was terminated in 1997 will contribute three program-years, and a program that started in 2001 will contribute only one program-year. After restricting the database to those social marketing programs that have a reproductive health component, the database contains information on social marketing programs in over 70 countries, which represent a total of 555 years of program experience. This includes 508 years of experience with condom social marketing and 208 years of experience with OC social marketing. ### Indicators Our measures of market potential are based on the indicators used by Michigan State University\'s Center for International Business Education and Research \[[@B32]\]. Specifically, we use the following indicators of market size, market intensity, and commercial infrastructure: ### Market size • Population size • Percentage of population living in urban areas ### Market intensity • Per capita GNI in current international dollars. This indicator measures the GNI converted in international dollars using purchasing power parity. An international dollar has the same purchasing power as a dollar in the U.S. \[World Bank 2003\] ### Commercial infrastructure • Telephone mainlines per 1,000 inhabitants • Television sets per 1,000 inhabitants The database also includes selected indicators of the characteristics of the social marketing programs, including: • Management structure (NGO affiliate, local clinic-based and non-clinic-based organizations, and commercial partnerships) • Program maturity/years of operation (less than 3 years, 4--6 years, and 7+ years) • Number of reproductive health products social marketed (one vs. two or more). Only condoms, oral contraceptives, injectables and IUDs are considered. In addition to these indicators of the socio-economic context and of the characteristics of the programs, information is available on the following outcome measures: • Per capita condom sales • Per capita OC sales While some social marketing programs distribute and/or promote injectables or IUDs, their number is not sufficiently large to allow multivariate analyses. Hence, data on sales of injectables and IUDs are not examined in this study. Other indicators of program effectiveness, such as user-prevalence rates are not collected on an annual basis. Ideally, one would also want to examine the cost-efficiency of implementing different social marketing models. A commonly used approach to estimate cost-efficiency is to conduct a cost-effectiveness analysis. By definition, any cost-effectiveness analysis requires obtaining data on both the effectiveness of the program, i.e. the impact of the program, and on the cost of implementing the program \[[@B33],[@B34]\]. Unfortunately, only limited cost information on social marketing programs is available, and the cost data that are routinely collected by the main social marketing organizations are not comparable. Hence, we are unable to assess the cost-efficiency of the programs in our database. Methods ------- In the next sections, we use cross-tabulations to examine to what extent management structure varies by socio-economic context. Since our unit of analysis consists of program-years of operation, these tabulations show the percentage of program-years that had each management type. We also test the extent to which different indicators of socio-economic context affect effectiveness of social marketing programs with different management structures. For this purpose, we use multiple regression analyses to assess the effect of indicators of socio-economic context on per capita condom sales for different management structures. Because each social marketing program has multiple entries in the data set (one for each year of operation), the standard errors in ordinary linear regression are incorrect. We use random-effects regression to solve the problem that observations for a given country-program are not independent \[[@B35]\]. For each type of management structure, we assess the net effect of socio-economic context on per capita condom sales, after controlling for the number of reproductive health products marketed, program maturity, time period (1995--2001 vs. earlier). Similar analyses are conducted for per capita OC sales. Results ======= Variations in management structure by socio-economic context ------------------------------------------------------------ We now analyze our database to illustrate to what extent different management structures have been used in social marketing programs (see Table [1](#T1){ref-type="table"}). Overall, data on the management structure of the social marketing programs were available for 555 program years. Of these 55 program years, 72% were managed through an NGO affiliate, 16% by local organizations, and 12% through a commercial partnership. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Distribution of Years of Social Marketing Program Experience by Management Structure and by Socio-Economic Context ::: Distribution of Program Years --------------------------- ------------------------------- ------- ------- ----- Population Size \<10 Million 76.3% 17.1% 6.6% 211 10--25 Million 62.7% 24.0% 13.3% 150 25+ Million 72.5% 9.0% 18.5% 189 Urban Population \<25% 86.1% 13.9% 0.0% 101 25--49% 80.8% 7.7% 11.5% 260 50+% 47.0% 31.0% 22.0% 168 GNI per capita \<\$1000 97.0% 3.0% 0.0% 132 \$1000--3000 68.2% 18.6% 13.2% 258 \$3000+ 52.3% 24.5% 23.2% 151 Phone mainlines per 1,000 \<5 87.1% 11.8% 1.1% 178 5--30 73.4% 9.2% 17.4% 184 30+ 53.8% 27.4% 18.8% 186 Television sets per 1,000 \<20 88.2% 10.1% 1.8% 169 20--100 70.9% 18.7% 10.4% 182 100+ 48.5% 23.3% 28.2% 163 Time Period 1986--93 56.4% 25.4% 18.3% 126 1994--97 66.2% 18.5% 15.3% 216 1998--01 85.9% 8.0% 6.1% 213 Region Eastern Europe 100.0% 0.0% 0.0% 16 Africa 89.7% 7.7% 2.6% 273 Asia 64.2% 17.5% 18.3% 137 Latin America 42.9% 41.9% 15.2% 105 Mid. East/N. Afr. 13.6% 0.0% 86.4% 22 Total 71.5% 16.0% 12.4% 555 ::: Social marketing programs in countries with populations smaller than 10 million have predominantly been managed by NGO affiliates (76% of program-years). In larger countries, other management structures have been somewhat more common. Breakdown by level of urbanization confirms that the range of management strategies used increased with socio-economic status. The percentage of social marketing program-years managed by NGO affiliates ranged from 86% when urbanization was low to 47% when urbanization was high. In highly urbanized populations, 31% of program-years were managed by existing local organizations and 22% by commercial partnerships. Social marketing programs in countries with a low per capita GNI (\<\$1,000) have been managed predominantly by NGO affiliates (97% of program-years), with local organizations being a distant second (3%). In countries with higher GNI levels, a wider range of management strategies has been used. When the per capita GNI levels are medium-low, management through NGO affiliates was still the most common (68% of all program-years). However, in medium-low GNI countries local organizations accounted for 18% of program years and commercial partnerships for 13%. Finally, when the GNI exceeded \$3,000 per capita, NGO affiliates accounted for only 52% of program-years, followed by local organizations (25%) and commercial partnerships (23%). Our indicators of the level of development of the commercial infrastructure show that the NGO model has dominated in settings with a relatively poorly developed commercial infrastructure. For example, NGO affiliates accounted for roughly 87% of program years in countries with fewer than 5 phone mainlines per 1,000 inhabitants, and for 88% of program years in countries with fewer than 20 television sets per 1,000 in habitants. Management through existing local organizations and commercial partnerships is most common in settings with over 30 phone mainlines and over 100 television sets per 1,000 inhabitants. Management structures have also changed over time. For example, the percentage of program-years managed through NGO affiliates has steadily increased from 56% in 1986--93 to 86% in 1998--2001. Simultaneously, management through existing local organizations and commercial partnerships has gradually declined. The last panel of Table [1](#T1){ref-type="table"} shows that management through commercial partnerships has been most common in the Middle East and Northern Africa (87% of program-years), while management through existing organizations has been most common in Latin America (86% of program-years). In Eastern Europe and Africa management through NGO affiliates has dominated (90% and over). In Latin America, management through NGO affiliates and through local organizations have both been common (43% and 42%, respectively). The effect of socio-economic context on the effectiveness of different social marketing management structures ------------------------------------------------------------------------------------------------------------- We now use random-effect GLS regression analyses to examine to what extent market size, market intensity, commercial infrastructure, and program features (number of products social marketed and program maturity) affect per capita condom sales and per capita OC sales. The variable \"number of television sets per 1,000 inhabitants\" was removed from the multivariate analyses as it correlated too much with some of the other predictor variables. We conduct separate analyses for social marketing programs managed by NGO affiliates, commercial partnerships, and local organizations. Due to the small number of cases, we are unable to differentiate between existing organizations and new organizations. ### Management by NGO affiliates The second column in Table [2](#T2){ref-type="table"} shows the predictors of per capita condoms sales in social marketing programs managed by NGO affiliates. The results indicate that among social marketing programs managed through NGO affiliates per capita condoms sales increase significantly with the number of years of program maturity (β = .181 for programs with 4--6 years of maturity; β = .348 for programs with 7 or more years of maturity). Programs that social marketed two or more products have higher per capita condom sales than programs that just market condoms (β = .159). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Effect of Socio-Economic Context on Condom Sales and CYP Among Social Marketing Programs Managed Through an NGO Affiliate (Random-Effects Regression GLS Coefficients) ::: Per Capita Condom Sales OC Sales ---------------------------------- ------------------------- ----------- Markets Two or More Products .159\*\*\* .017 Program Maturity 1--3 (ref) 4--6 .181\*\*\* .001 7+ .348\*\*\* .003 Time Period \<1994 (ref) 1995--2001 .143\*\* .004 Population (in 10 millions) -.006\*\*\* -.000\* Percent Urban -.002 -.000 Per Capita Gross National Income -.003 .007 Phone mainlines per 1,000 -.001 -.000 Constant .260\*\*\* (dropped) R Square .236 .082 Number of Program-Years 374 90 Number of Countries Included 58 26 \*\*\* p \< .01; \*\* p \< .05; \* p \< .10 ::: As expected, social marketing programs managed through an NGO affiliate tend to be more effective, as measured by per capita condom sales, in countries with smaller populations (β = -.006). Per capita condom sales do not vary with the level of urbanization, per capita Gross National Income (an indicator of market intensity), nor with the number of phone mainlines (an indicator of the strength of commercial infrastructure). The third column in Table [2](#T2){ref-type="table"} shows the predictors of per capita OC sales. The results indicate that there is no evidence that our indicators of market size, market intensity, and commercial infrastructure have any significant impact on per capita OC sales. ### Management by commercial partnerships Table [3](#T3){ref-type="table"} shows the predictors of per capita condom sales in social marketing programs managed using the manufacturer\'s model. The results shown in Table [3](#T3){ref-type="table"} indicate that among social marketing programs managed through commercial partnerships, per capita condom sales tend to be higher in countries with smaller populations (β = -.012), and with a lower per capita gross national income (β = -.111). The level of commercial infrastructure, as measured by the number of telephone mainlines per 1,000 inhabitants, has a small but significant positive effect on per capita condom sales (β = .001). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Effect of Socio-Economic Context on Condom Sales and CYP Among Social Marketing Programs Managed Through the Manufacturer\'s Model (Random-Effects Regression GLS Coefficients) ::: Per Capita Condom Sales OC Sales ---------------------------------- ------------------------- ---------- Markets Two or More Products .078 -.015\* Program Maturity 1--3 (ref) 4--6 -.018 .003 7+ -.069 .007 Time Period \<1994 (ref) 1995--2001 .099 .007 Population (in 10 millions) -.012\*\*\* -.002 Percent Urban .001 -.000 Per Capita Gross National Income -.111\*\* .015\*\* Phone mainlines per 1,000 .001\*\* -.000 Constant .278\*\* .030 R Square .384 .185 Number of Program-Years 54 55 Number of Countries Included 9 12 \*\*\* p \< .01; \*\* p \< .05; \* p \< .10 ::: The third column in Table [3](#T3){ref-type="table"} reveals a different pattern for factors affecting per capita OC sales. Among social marketing programs managed through commercial partnerships, per capita OC sales appear to be lower for those programs that also market other products (β = -.015; p \< .010). Our indicator of market intensity, per capita gross national income, has a significant positive effect on per capita OC sales (β = .015). However, population size and urbanization do not have any significant effect. ### Management by local clinic-based and non-clinic-based organizations Table [4](#T4){ref-type="table"} shows similar analyses for programs managed by local organizations (including both clinic-based and non-clinic-based organizations). The results show that programs in countries with larger populations have significantly higher per capita condom sales (β = .036). However, program in countries with higher levels of urbanization have lower per capita condom sales (β = -.012). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Effect of Socio-Economic Context on Condom Sales and CYP Among Social Marketing Programs Managed Through Local Organizations (Random-Effects Regression GLS Coefficients) ::: Per Capita Condom Sales OC Sales ---------------------------------- ------------------------- ------------- Markets Two or More Products .075 -.012 Program Maturity 1--3 (ref) 4--6 .158 .022\* 7+ .181 .028\*\* Time Period \<1994 (ref) 1995--2002 .138 -.010 Population (in millions) .036\*\* .008\*\* Percent Urban -.012\*\*\* .001 Per Capita Gross National Income .006 .028\*\*\* Phone mainlines per 1,000 .001 -.001\*\*\* Constant .436\*\* -.021 R Square .309 .401 Number of Program-Years 80 63 Number of Countries Included 17 12 \*\*\* p \< .01; \*\* p \< .05; \* p \< .10 ::: The third column in Table [4](#T4){ref-type="table"} shows that among social marketing programs implemented by local organization, OC sales are significantly higher for those programs that have been in operation for seven or more years (β = .031). For these types of social marketing programs per capita OC sales increase with population size (β = .008) and with level of urbanization (β = .028). The level of commercial infrastructure, as measure through the number of telephone mainlines per 1,000 inhabitants, has a small negative effect on per capita OC sales. Discussion ========== Several studies have commented on the differences between social marketing programs that use the non-governmental organization model and those that use the manufacturer\'s model \[[@B1]-[@B3]\]. Although it is increasingly recognized that context-specific variations on these models are common, and that hybrid models exist \[[@B4]\], it is generally assumed that social marketing programs that use the manufacturer\'s model are most feasible in middle-income countries with a fairly well-developed commercial infrastructure, while NGO managed models work best in lower-income countries with less-developed commercial infrastructure. As yet, however, there has been little empirical evidence to verify these assumptions. This paper has used a database of annual data on social marketing programs in over 70 countries to verify the extent to which the different social marketing models that have been used vary across socio-economic contexts, and to test in which socio-economic context each of the models is most successful at increasing use of socially marketed products. A rigorous analysis of the relative effectiveness of different social marketing models would require detailed data about the characteristics of the program, including data about financial structures, branding, pricing (and level of subsidies), distribution, and promotion. Because data on the features of social marketing programs are limited, we focused on differences in the programs\' management structure. Ideally, we would also like to examine are range of outcome measures, including per capita sales, the user-prevalence, and the unit-cost for each method. Since such prevalence and unit-cost data are unavailable, we have focused on per capita condom and OC sales. We have used empirical analyses to obtain a better understanding of the variations that exist in social marketing programs. Specifically, we have analyzed a database of annual statistics on social marketing programs in over 70 countries, accounting for a total of 555 years of program experience. The database contains annual data on the management structure, program maturity, the number of reproductive health products marketed, and on product sales. The database also contains indicators of the local socio-economic context (market size, market intensity, and commercial infrastructure), including the population size, level of urbanization, per capita GNI, number of telephone mainlines and television sets per 1,000 inhabitants. Our analyses have shown that of the 555 years of social marketing program experience covered by the database, over 70% has used the NGO management structure. Nevertheless, there are considerable variations in management structure. The NGO affiliate management structure has been the dominant model when the per capita GNI is below \$3,000 and the level of urbanization below 50%. The data also show that use of the NGO management structure has increase dramatically over the course of our study period. The findings support the theory that there may be more opportunities for successful partnerships with the private sector in countries with a high per capita GNI. The highest percentage of such partnerships (23%) was found in countries with a GNI exceeding \$3,000 per capita. This percentage decreases to 13% in countries with a per capita GNI between \$1,000 and \$3,000. None of the commercial partnerships in our database were in countries with a per capita GNI below \$1,000. One of the reasons for this pattern may be that there is more commercial activity and a wider range of possible partners in countries with a higher per capita income. Users in those countries are also more likely to be able to afford the commercially sustainable brands that are marketed under this approach. Although the program characteristics usually appear to be appropriate for the local socio-economic context, it is questionable whether this has always been the case. For example, over 50% of program-years in countries with a medium-high GNI (over \$3,000) used the NGO model, even though countries with such income levels typically provide opportunities for commercial partnerships. Our multivariate analyses identified the factors that affect per capita condom and OC sales for each of the four social marketing models. The reader is cautioned, however, that condom and OC sales may also be influenced by other factors that were not measured in our data base, such as HIV prevalence and overall levels of contraceptive prevalence. Bearing these cautions in mind, the analyses yielded the following key findings: Programs managed by NGO affiliates ---------------------------------- • Per capita condom sales are higher in countries with smaller populations. This may be influenced by the difficulty of achieving high distribution coverage in large, mostly rural countries (e.g., India, Indonesia, Vietnam, Nigeria) and/or the time it takes to large such large populations with social marketing campaigns. Also, social marketing programs in countries with a larger population may have more competition (e.g., India, South Africa). By contrast, several small countries have a high HIV/AIDS prevalence and/or limited method choice (e.g., sub-Saharan Africa); • Per capita condom sales are higher for more mature programs and those that market more than one product. This probably reflects improvements in distribution capability over time, as well as the cumulative effect of behavior change programs that stimulate demand. More developed programs with stronger distribution are also more likely to market several products; • Per capita condom sales do not vary with the level of urbanization, per capita income, or commercial infrastructure. These variables are more likely to affect access-related factors, such as product access and ability to pay. Other factors to look for that may affect sales are related to demand: level of effort of the social marketing program (which reflects funding levels), health context (HIV/AIDS and contraceptive prevalence) and competition from the public sector and other NGOs that may cancel out the higher market potential in the more developed countries; • None of the factors examined had any significant influence on the per capita OC sales of social marketing programs managed through an NGO affiliate. Programs managed by commercial partnerships ------------------------------------------- • Per capita condom sales tend to be higher in countries with smaller populations and with a lower per capita growth. At first glance, this finding is counter-intuitive. That is, initially one would expect sales for commercial partnerships to increase with population and per capita GNI, because commercial program need both a high demand and ability to pay. However, as population size and income increase sales may decline due to increased competition from commercial and other suppliers. Several countries with middle-high incomes (Turkey, the Philippines, and Jordan) or large populations (Turkey, the Philippines, and Indonesia) have strong public sector programs that offer free contraceptives, as well as many commercial suppliers. Also, demand for condoms in these countries has been very low, given that HIV prevalence is low that other contraceptive methods are available. By contrast, smaller countries such as Haiti and Zimbabwe have high HIV rates, less choice of methods, and probably less competition from other suppliers. Programs managed by local clinic-based and non clinic-based organizations ------------------------------------------------------------------------- • Programs in countries with larger populations have significantly higher per capita condom sales. One likely explanation for this pattern is that local organizations tend to have good distribution networks, which pays off in countries with a larger population. Some of the larger countries (e.g., Bangladesh and Colombia) have well-established organizations with a big market share. For example, in Bangladesh SMC provides almost all condoms and the demand is very high; • OC sales are significantly higher for programs that have been in operation for seven or more years. The longer a program operates, the more developed its distribution is likely to be. Social marketing communication efforts are also more likely to pay off over the long run; • Programs in countries with a higher level of urbanization have lower per capita condom sales. This was anticipated, given that NGOs and family planning associations have a strong track record of serving users in peri-urban and rural areas. In addition, when urbanization increases, access to commercial products sold in pharmacies and other private sector outlets increases as well, which allows commercial suppliers to grow their market share and compete with social marketing and public sector products; • The level of commercial infrastructure, as measured through the number of telephone mainlines per 1,000 inhabitants has a small negative effect on per capita OC sales. This finding may reflect that a better commercial infrastructure implies more competition from commercial suppliers. Conclusions =========== Our analyses of records on 555 years of experience with social marketing programs have illustrated the tendency to design social marketing programs with a management structure suitable for the local context. However, the evidence also shows examples where this has not been the case. While socio-economic context clearly influences the effectiveness of some of the social marketing models, program maturity and the size of the target population appear equally important. Consequently, it is crucial that social marketing programs are designed using the model or approach that is most suitable for the local context. Competing interests =================== Dr. Meekers is the former Research Director of PSI (1996--2001), and has been a paid consultant for other PSI research projects. Author\'s contributions ======================= Dr. Meekers developed the study design, assisted with the analysis, and contributed to the writing of the final manuscript. Mr. Rahaim collected data, conducted literature reviews, and contributed to the editing of the paper. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/10/prepub> Acknowledgements ================ This study was funded by the United States Agency for International Development (USAID) through the Commercial Market Strategies (CMS) Project. The authors are grateful to Ruth Berg and Françoise Armand for comments and suggestions on earlier drafts of this report.

PubMed Central

2024-06-05T03:55:52.583437

2005-1-27

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548279/", "journal": "BMC Public Health. 2005 Jan 27; 5:10", "authors": [ { "first": "Dominique", "last": "Meekers" }, { "first": "Stephen", "last": "Rahaim" } ] }

PMC548280

Background ========== Phenotypic robustness refers to the invariance of the specified target phenotype given the genetic makeup and environmental conditions. Whereas the presence of naturally occurring phenotypic variation is at the core of evolutionary biology, developmental geneticists have traditionally considered it as a nuisance. Instead, they have relied on the study of single or multiple mutant combinations to reveal the generation of phenotypic patterns (e.g. \[[@B1]\]). A resurgence of interest in the issue of phenotypic robustness has emerged in recent years, partly due to experimental results showing that many knock-out mutations have little effect on phenotype (\[[@B2]\]; although Papp\'s et al. \[[@B3]\] metabolic network analysis found that the majority of genes that looked dispensable turn out to be such only under laboratory conditions), and that developmental systems show a high degree of stability with respect to perturbations \[[@B4],[@B5]\]. Three major processes are involved in the control of phenotypic variability (the potential or propensity to vary, in the terminology of Wagner and Altenberg \[[@B6]\]): canalization, developmental stability (DS), and plasticity \[[@B7]\]. As first defined by Waddington \[[@B8]\] the term canalization could be understood as a morphogenetic constrain \[[@B9]\], where development appears to be buffered so that slight abnormalities of genotype or slight perturbations in the environment do not lead to the production of abnormal phenotypes. However, evolutionary geneticists define canalization as the tendency of traits to evolve a reduction in variability \[[@B4],[@B10]\]. DS can be defined as the ability of organisms to buffer against the random noise that arises spontaneously as a consequence of stochastic variation in the cellular processes that are involved in the development of morphological structures \[[@B11]\]. Therefore, canalization and DS are subcategories of developmental buffering: the first can be appraised by estimating interindividual variance whereas the most commonly used estimate of DS in bilaterally symmetrical organisms is fluctuating asymmetry (FA); i.e. the intraindividual variation due to random differences between left and right sides. The question of whether or not canalization and DS are different buffering mechanisms has been a constant source of debate. Two recent reviews implicitly \[[@B4]\] or explicitly \[[@B10]\] assume that DS is a special case of canalization, a viewpoint also embraced by several authors (e.g. \[[@B12]-[@B14]\]). Thus, by using geometric morphometrics Klingenberg and McIntyre \[[@B13]\] found that the vectors describing inter- and intraindividual variation of landmark position for fly vein traits were highly concordant. On the other hand, Debat et al. \[[@B15]\] came to the opposite conclusion applying the same methods to cranial landmarks in the house mouse -- although Klingenberg\'s et al. \[[@B16]\] work with mouse mandibles found patterns of intra- and interindividual variation that were only partly consistent --. At first glance, the different results may suggest that the mechanisms that affect canalization and DS are related in some developmental contexts but not in others. The problem is, however, that according to the causes of phenotypic variation a distinction between genetic and environmental canalization is necessary \[[@B17],[@B18]\]. Selection for environmental canalization may produce genetic canalization as a by-product \[[@B4],[@B10]\], but this may not always be the case. The better way to address these contentious issues is to rely on quantitative genetic analyses devised to partition phenotypic variation into genetic and environmental components \[[@B19]\]. Environmental variation can be further partitioned into general (![](1471-2148-5-7-i1.gif)) and special (micro) environmental effects (![](1471-2148-5-7-i2.gif)): the first refer to influential factors (e.g. temperature) that are shared by groups of individuals, whereas the latter are residual deviations from the phenotype that would be specified on the basis of genotype and general environmental effects. Such deviations are unique to individuals and are largely unpredictable. The variance associated with special environmental effects can be estimated when experiments are performed on completely inbred lines (i.e., there is no genetic variance). In bilaterally symmetrical organisms it is also feasible to estimate the two sources that contribute to those special environmental effects: among-individual (![](1471-2148-5-7-i3.gif)) and within-individual variance (![](1471-2148-5-7-i4.gif)). If the only real cause of asymmetry is variation due to stochasticity in development, then FA can be taken as an estimated of ![](1471-2148-5-7-i4.gif). Therefore, FA is only one source of the phenotypic variation within environments (excluding environmentally induced asymmetry), contrarily to the arguments in Nijhout and Davidovitz \[[@B20]\]. The other source is ![](1471-2148-5-7-i3.gif). The third process involved in the control of phenotypic variability is plasticity, which can be defined as the ability of an individual to express one phenotype under one set of environmental circumstances and another phenotype under another set. The expressed phenotypes can be discontinuous thus eliciting discrete morphs (i.e., polyphenism), or there can be a continuous range of potential phenotypes (i.e., reaction norm). The reaction norm is thus a property of the genome: genetic canalization and phenotypic plasticity are not mutually exclusive and can combine to form canalized reaction norms \[[@B7],[@B17]\]. Plasticity is thus an alternative to genetic change allowing populations to adapt to changing environmental conditions. To summarize, phenotypic plasticity increases the variance among groups of individuals that produce different phenotypes in different environments, canalization decreases the within-group interindividual variance around the target phenotype by reducing the sensitivity to genetic and environmental conditions, and DS buffers against random perturbations in development (i.e., decreases FA). Because the left and right body sides share the same genome (barring unusual somatic mutation or somatic recombination) and in most organisms also very nearly the same environment, FA provides an intrinsic control for genetic and environmental effects and the important question is to what extent these two sources of variation share underlying regulatory mechanisms. Within the framework of recently developed geometrically based methods for the statistical analysis of size and shape variation (collectively referred to as geometric morphometrics \[[@B21],[@B22]\]), the wing vein network of *Drosophila*is regarded as an excellent model system to investigate those problems \[[@B23],[@B24]\]. Wing development in *Drosophila*is well understood \[[@B25]\], and the vein pattern is highly conserved across species (e.g. \[[@B26]\]). When flies are reared at low temperatures it is well known that the final wing size increases because of an increase in adult cell size \[[@B27]\]. This plastic response is parallel to what has been commonly observed in laboratory experiments on thermal evolution, where adaptation to lower temperature resulted in increased wing size (a proxy for body size) entirely as a consequence of cell size divergence \[[@B28]\]. However, there is circumstantial evidence suggesting that developmental and evolutionary temperature-related cell size divergence have contrasting effects on wing shape. Thus, Birdsall et al. \[[@B29]\] concluded that wing shape in *Drosophila melanogaster*is quite resistant to developmental temperature. Conversely, in *D. subobscura*there are changes in wing proportions along a latitudinal size cline mediated by cell area \[[@B30],[@B31]\]. These populations exhibit, in addition, prominent latitudinal clines for chromosomal inversion polymorphisms, and there is compelling evidence showing that the inversion clines underlie the latitudinal changes in wing proportions \[[@B32],[@B33]\]. Here we report on the effects of clinal genetic variation (inversion polymorphism) on wing form (size and shape) and bilateral asymmetry using isochromosomal lines of *D. subobscura*. We consider the consequences of inbreeding and temperature on the two components of developmental homeostasis (canalization and DS), and the relationship between them. The remainder of the paper is planned as follows. First, we provide a short account of the inversion polymorphism in *D. subobscura*and the experimental settings. Then, based on the well balanced data set rendered by the experimental design we used the standard least-squares (ANOVA) method to decompose sources of variation for wing size and shape into causal components at the core of further analyses. Furthermore, because the underlying assumption to use FA as a measure of DS is that left -- right-side variation has not heritable basis, the genetic and environmental components of bilateral asymmetry were partitioned. As a result, our approach is unusual in studies of DS in providing estimates of the two components of special environmental effects (co-) variance under different genetic backgrounds and general environmental settings. We also present some evidence for the presence of genetic variation in directional asymmetry (DA) but not in FA. Next, we test whether or not the vectors describing variation of landmark position for fly vein traits are concordant, and finally we discuss the main findings in relation with the evolution of buffering mechanisms and the putative adaptive value of natural wing shape variation in *D subobscura*. Experimental settings ===================== *D. subobscura*is a particularly inversion-rich species, with up to 38 natural chromosomal arrangements already reported for the largest chromosome O (homologous to arm 3R in *D. melanogaster*\[[@B34]\]) for which a balancer stock is available. In colonizing populations of the New World only six gene arrangements are segregating for that chromosome: O~st~, O~3+4~, O~3+4+2~, O~3+4+7~, O~3+4+8~and O~5~(arrangement O~7~is also present at very low frequency but it is probably the result of a recombination event in the O~st~/O~3+4+7~heterokaryotype \[[@B35]\]). In native Palearctic populations arrangements O~3+4+2~and O~3+4+8~are restricted to the Mediterranean region (the likely area from which the original American colonists derived \[[@B36]\]) and are not involved in latitudinal clines \[[@B35]\]. On the other hand, arrangement O~st~shows a world-wide positive correlation with latitude, while arrangements O~3+4~and O~3+4+7~show a contrasting pattern \[[@B35]\]. Therefore, six independent isochromosomal lines for each of these three chromosome arrangements (i.e., ![](1471-2148-5-7-i5.gif), \..., ![](1471-2148-5-7-i6.gif); *j*= st, 3+4, 3+4+7) were used in the present experiments. The experimental flies were obtained from 54 crosses, which will be referred to as inbred (isogenic; i.e., ![](1471-2148-5-7-i7.gif)) with 18 crosses in total, or outbred (including both structural homo- and heterokaryotypes) with 36 (18 + 18) crosses in total. The six lines with a given gene arrangement were crossed to produce the three different outbred homokaryotypes (i.e., ![](1471-2148-5-7-i8.gif)). The three kinds of heterokaryotypic flies were similarly obtained but using lines with different gene arrangements (i.e., ![](1471-2148-5-7-i9.gif)). Since all isochromosomal lines were homogeneous for the same genetic background (except for the male sex chromosome), maternal effects were not considered to be critically important. Anyhow, experimental flies were randomly derived form reciprocal crosses for all outbred combinations. Two developmental temperatures were used in the experiment: optimal (18°C) and warm (23°C). Results and discussion ====================== Variation and asymmetry in size ------------------------------- ### a) Basic statistics Signed left-right (![](1471-2148-5-7-i10.gif)) differences of centroid size did not significantly departure from normality in any case (*D*~max~ranging from 0.032 for inbred females at 18°C to 0.073 for inbred males at 23°C; *P*\> 0.05). In addition, none of the regressions of centroid size FA on average wing size was statistically significant (ranging from *β*= -0.045 (95% C.I.: -0.091, 0.001) for inbred females at 18°C to *β*= 0.030 (-0.005, 0.064) for inbred females at 23°C), thus suggesting independence between size and size FA. ### b) Causal components of variation For each sex two-way mixed ANOVAs were separately performed for inbred and outbred crosses at each experimental temperature (Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}). Size variation (CS: centroid size) among individuals comprised the largest part (\> 90%) of the variation. The fraction of the total phenotypic variance in wing size associated to genetic differences among karyotypes and/or lines (i.e., ![](1471-2148-5-7-i11.gif)) ranged from 0.235 (inbred males at 18°C) to 0.602 (inbred females at 23°C). (Bear in mind that there is nothing in the ANOVA method of estimation that will prevent a negative variance estimate \[[@B37]\].) ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Asymmetry of overall wing size for females raised at 18°C *Drosophila subobscura*flies raised from inbred (isogenic) and outbred crosses reared at 18°C. Centroid size (CS, estimated in a normalized form \[22\]) is the dependent variable (values in pixels^2^: 1 mm = 144 pixels). The ANOVAs assess measurement error, directional asymmetry (Sides effect), fluctuating asymmetry (Individuals × Sides interaction effect), and genetic components of the trait () and DA of the trait ((DA)). (CS) and (DA~CS~) provide here unbiased estimates of the among-fly (i.e. ) and within-fly ( or FA) special environmental effects. (⊂ means \'nested in\'.) ::: Inbred Outbred --------------------- -------------------- -------- --------------- -------------------- ------ --------------- -------------------- Source of variation Variance component d.f. Mean Square Estimated variance d.f. Mean Square Estimated variance Individuals (I) 107 39.747\*\*\* 9.6175 215 45.773\*\*\* 11.2830 Karyotypes (K) (CS) 2 114.593^n.s.^ 0.1389 5 214.204^n.s.^ 0.7014 Cross ⊂ K (CS) 15 94.589\*\*\* 2.7352 30 113.199\*\*\* 3.4726 Among flies (CS) 90 28.944\*\*\* 6.9166 180 29.857\*\*\* 7.3040 Sides (S) 1 15.982\*\*\* 1 18.549\*\*\* I × S (CS) 107 1.278\*\*\* 0.5467 215 0.641\*\*\* 0.2225 Karyotypes (K) (DA~CS~) 2 0.067^n.s.^ -0.0520 5 0.457^n.s.^ -0.0123 Cross ⊂ K (DA~CS~) 15 1.938^¶^ 0.1239 30 0.899^¶^ 0.0492 Within flies (DA~CS~) 90 1.194\*\*\* 0.5051 180 0.603\*\*\* 0.2036 Measurement error (CS) 216 0.184 0.1841 432 0.196 0.1962 Average CS for left (*L*) and right (*R*) wings: inbred females = 0.9918 mm, = 0.9891 ; outbred females = 1.0022, = 1.0002. ^n.s.^*P*\> 0.10; ^¶^0.10 \>*P*\> 0.05; \*\*\* *P*\< 0.001. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Asymmetry of overall wing size for males raised at 18°C Same as in Table 1. ::: Inbred Outbred --------------------- -------------------- -------- -------------- -------------------- ------ --------------- -------------------- Source of variation Variance component d.f. Mean Square Estimated variance d.f. Mean Square Estimated variance Individuals (I) 107 43.303\*\*\* 10.4842 215 38.600\*\*\* 9.4200 Karyotypes (K) (CS) 2 232.359^¶^ 1.1331 5 115.718^n.s.^ 0.1908 Cross ⊂ K (CS) 15 69.186\* 1.4332 30 88.246\*\*\* 2.5026 Among flies (CS) 90 34.788\*\*\* 8.3554 180 28.184\*\*\* 6.8159 Sides (S) 1 1.140^n.s.^ 1 22.492\*\*\* I × S (CS) 107 1.366\*\*\* 0.5045 215 0.920\*\*\* 0.3297 Karyotypes (K) (DA~CS~) 2 0.385^n.s.^ -0.0176 5 1.156^n.s.^ 0.0035 Cross ⊂ K (DA~CS~) 15 1.017^n.s.^ -0.0715 30 1.031^n.s.^ 0.0226 Within flies (DA~CS~) 90 1.446\*\*\* 0.5444 180 0.895\*\*\* 0.3172 Measurement error (CS) 216 0.357 0.3574 432 0.261 0.2609 Average CS for left (*L*) and right (*R*) wings: inbred males = 0.8942, = 0.8935; outbred males = 0.9003, = 0.8980. ^n.s.^*P*\> 0.10; ^¶^0.10 \>*P*\> 0.05; \* *P*\< 0.05; \*\*\* *P*\< 0.001. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Asymmetry of overall wing size for females raised at 23°C Same as in Table 1 for *Drosophila subobscura*flies reared at 23°C ::: Inbred Outbred --------------------- -------------------- -------- --------------- -------------------- ------ --------------- -------------------- Source of variation Variance component d.f. Mean Square Estimated variance d.f. Mean Square Estimated variance Individuals (I) 107 64.857\*\*\* 15.8796 215 49.100\*\*\* 11.9347 Karyotypes (K) (CS) 2 27.808^n.s.^ -1.8893 5 457.293\*\*\* 2.6178 Cross ⊂ K (CS) 15 299.873\*\*\* 11.3901 30 80.332\*\*\* 1.9907 Among flies (CS) 90 26.511\*\*\* 6.2931 180 32.556\*\*\* 7.7987 Sides (S) 1 33.413\*\*\* 1 16.825\*\*\* I × S (CS) 107 1.339\*\*\* 0.5446 215 1.361\*\*\* 0.5916 Karyotypes (K) (DA~CS~) 2 0.681^n.s.^ -0.0125 5 4.333\* 0.0781 Cross ⊂ K (DA~CS~) 15 1.132^n.s.^ -0.0427 30 1.520^n.s.^ 0.0447 Within flies (DA~CS~) 90 1.388\*\*\* 0.5692 180 1.252\*\*\* 0.5371 Measurement error (CS) 216 0.250 0.2496 432 0.178 0.1778 Average CS for left (*L*) and right (*R*) wings: inbred females = 0.8999 mm, = 0.8960; outbred females = 0.9203, = 0.9184. ^n.s.^*P*\> 0.10; \* *P*\< 0.05; \*\*\* *P*\< 0.001. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Asymmetry of overall wing size for males raised at 23°C Same as in Table 1 for *Drosophila subobscura*flies reared at 23°C ::: Inbred Outbred --------------------- -------------------- -------- --------------- -------------------- ------ --------------- -------------------- Source of variation Variance component d.f Mean Square Estimated variance d.f. Mean Square Estimated variance Individuals (I) 107 44.690\*\*\* 10.9045 215 28.772\*\*\* 6.8138 Karyotypes (K) (CS) 2 41.926^n.s.^ -0.6021 5 112.284^n.s.^ 0.3480 Cross ⊂ K (CS) 15 128.628\*\*\* 4.0778 30 62.165\*\*\* 1.7199 Among flies (CS) 90 30.762\*\*\* 7.4224 180 20.887\*\*\* 4.8425 Sides (S) 1 36.691\*\*\* 1 13.586\*\* I × S (CS) 107 1.072\*\*\* 0.3553 215 1.517\*\*\* 0.6465 Karyotypes (K) (CS) 2 2.596^n.s.^ 0.0366 5 0.862^n.s.^ -0.0110 Cross ⊂ K (DA~CS~) 15 1.277^n.s.^ 0.0454 30 1.259^n.s.^ -0.0532 Within flies (DA~CS~) 90 1.004\*\*\* 0.3213 180 1.578\*\*\* 0.6771 Measurement error (CS) 216 0.361 0.3615 432 0.224 0.2240 Average CS for left (*L*) and right (*R*) wings: inbred males = 0.8112, = 0.8072 ; outbred males = 0.8277, = 0.8260. ^n.s.^*P*\> 0.10; \*\* *P*\< 0.01; \*\*\* *P*\< 0.001. ::: No significant size differences were generally detected among karyotypes for average CS, although O~3+4~flies were always the biggest within inbred lines (Fig. [1](#F1){ref-type="fig"}). On the other hand, in outbred crosses heterokaryotypes were bigger than homokaryotypes (females: 18°C *F*~(1,195)~= 9.78, *P*= 0.002; 23°C *F*~(1,195)~= 9.19, *P*= 0.003; males: 18°C *F*~(1,195)~= 1.84, *P*= 0.176; 23°C *F*~(1,195)~= 4.23, *P*= 0.041), but interactions of dominance effects were observed in all samples with discernible heterosis in O~st~/O~3+4~lines when compared to their homokaryotypic counterparts. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Inbreeding and temperature effects on size**Homokaryotipic averages for centroid size and centroid size FA (index FA1 in \[39\]) in inbred (black symbols) and outbred (open symbols) crosses. Small symbols give the average values for each of the three different homokaryotypes to appreciate the dispersion from the corresponding grand average (large symbols connected by lines). Squares give the values at 23°C and circles at 18°C. ::: ![](1471-2148-5-7-1) ::: In concert with some independent preliminary results using a set of O~st~isochromosomal lines \[[@B38]\] a quite remarkable finding here was that left wings were consistently bigger than the right ones, thus causing a generally highly significant DA (i.e., \"sides\" effect in Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}) of overall wing size even though DA was fairly subtle (see bottom statistics in Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}). Each *Drosophila*wing vein has dorsal and ventral components that come together after the apposition of the dorsal and ventral surfaces, but each vein protrudes only in one wing surface (\"corrugation\") \[[@B25]\]. When wings were mounted no attempt was made to standardize the surface position: in females 394 (60.8%) left and 387 (59.7%) right wings were mounted on the slides with the dorsal side up (*χ*^2^= 0.16, 1 df, *P*= 0.691); in males the corresponding figures were 383 (59.1%) and 401 (61.9%), respectively (*χ*^2^= 1.05, 1 df, *P*= 0.306). Potential biasing effects when measuring wings; namely, dorsal or ventral Bitmap images or possible differences between left and right wings when Bitmap images are captured from the top or bottom of the microscope slide, were checked from a subset of 75 females and 75 males. An additional set of two images for each wing were taken in the same session from the top and bottom of the slide and digitized once. The centroid size differences between the averages of both measurements was apparently random with respect to digitizing procedure and always lower than 0.07%, whereas left wings were 0.26% bigger than the right ones in females and 0.34% in males. We are, therefore, quite confident that the fairly subtle DA for wing CS is not an experimental artifact but a real phenomenon. In addition to DA, there was subtle but significant FA in all crosses (i.e., \"individuals × sides\" interaction effect in Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}) together with a small amount of genetic variation for DA in some of them. This last finding could hardly be attributable to a type I error because similar results had been previously obtained \[\[[@B38]\]; see below\]. Conversely, two-level nested ANOVAs to test for genetic components of overall size FA (using index FA1 in Palmer \[[@B39]\]) failed to show any statistically significant effects whatsoever (variance components ranging from -0.0047 to 0.0071 for karyotypes, and from -0.0343 to 0.0406 for crosses within karyotypes; values in pixels^2^). ### c) Consanguinity and temperature effects Inbreeding and environmental effects were simultaneously analyzed by contrasting isogenic *vs*. outbred homokaryotypic flies reared at both experimental temperatures (Fig. [1](#F1){ref-type="fig"}). Flies were obviously bigger when raised at the lowest temperature, and three-way factorial ANOVAs performed separately for each sex using CS (as log~*e*~(pixels), but results were qualitatively identical without a log-transformation) as the dependent variable, with karyotype, temperature and inbreeding as fixed effects, and crosses nested within karyotypes, clearly indicated inbreeding depression together with temperature by inbreeding interaction (i.e., inbreeding was most noticeable at the sub-optimal temperature of 23°C), but no karyotype by temperature interaction was detected. These results confirm that wing size is not a purely additive trait in *D. subobscura*, in agreement with the previous observation that heterokaryotypes were bigger than homokaryotypes in outbred crosses (see also \[[@B40]\]). Both inbreeding and (sub-optimal) temperature effects were also apparent in females when overall size FA (index FA1) was used as the dependent variable in three-way factorial ANOVAs, with no differences among karyotypes. On the other hand, no statistically significant effects were detected for males, basically because inbred crosses performed approximately equal at both temperatures (Fig. [1](#F1){ref-type="fig"}). However, overall asymmetry augmented in inbred crosses because DA largely increased (mainly in males) at the highest temperature (\"temperature × inbreeding\" interaction: *F*~(1,856)~= 9.46, *P*= 0.002). It is worth mentioning here that in outbred crosses overall size FA was about the same for homokaryotypes and heterokaryotypes: the only significant effect was again an increase in FA at the sub-optimal temperature (more than two-fold; c.f. ![](1471-2148-5-7-i13.gif)(DA~CS~) values in Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}). Finally, inbreeding appears to have affected among-fly variation only in males as suggested by the consistently lower ![](1471-2148-5-7-i13.gif)(CS) estimates in outbred crosses within rearing temperature. In conclusion, overall size DS was positively correlated with levels of heterozygosity (i.e., inbred vs. outbred homokaryotypes) and development at the optimal temperature of 18°C. However, no positive association was found between DS and chromosomal heterozygosity in outbred crosses. Variation and asymmetry in shape -------------------------------- ### a) Sources of variation Two-way MANOVA analyses to quantify inter- and intra-individual variation in wing shape are shown in Tables [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}, [7](#T7){ref-type="table"}, [8](#T8){ref-type="table"}. For the present study of 13 landmarks, with 2 coordinates each, the shape dimension is 22. Sums of squares and cross-products (SSCP) matrices are therefore not full-ranked, and we retained 22 PC (principal components \[[@B41]\]) scores for outbred crosses and only 15 PC scores -- which accounted for more than 98% of the total shape variance -- for inbred crosses to be capable of testing for genetic components. The degrees of freedom in Tables [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}, [7](#T7){ref-type="table"}, [8](#T8){ref-type="table"} (columns \"df 1\") are simply the corresponding degrees of freedom in the ANOVAs for centroid size (Tables [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}, [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"}) times the number of PC scores retained in each sample. Likewise, the overall covariation in wing shape (\"individuals\" effect) was decomposed into causal components (karyotypes, crosses in karyotypes, and among flies); and the overall covariation in wing shape FA (\"individuals × sides\" interaction effect) was decomposed into causal components attributable to wing shape DA (karyotypes, crosses in karyotypes, and within flies). ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Asymmetry of overall wing shape for females raised at 18°C Flies raised from inbred (isogenic) and outbred crosses of *Drosophila subobscura*reared at 18°C. For the inbred crosses 15 PC scores were retained for analyses (proportion of total shape variance accounted is given in parenthesis). For the outbred crosses 22 PC scores were retained. (⊂ means \'nested in\'.) ::: Inbred (98.6%) Outbred --------------------- ---------------- --------- ------ --------- ---------------- ------ ------ --------- Source of variation Wilks\' lambda df 1 df 2 *P* Wilks\' lambda df 1 df 2 *P* Individuals (I) 1.13 × 10^-11^ 1605 1464 \<0.001 5.14 × 10^-15^ 4730 4442 \<0.001 Karyotypes (K) 0.002 30 2 0.505 7.44 × 10^-5^ 110 48 \<0.001 Cross ⊂ K 1.51 × 10^-4^ 225 843 \<0.001 3.68 × 10^-4^ 660 2932 \<0.001 Among flies 2.23 × 10^-8^ 1350 1457 \<0.001 7.55 × 10^-12^ 3960 4430 \<0.001 Sides (S) 0.597 15 93 \<0.001 0.563 22 194 \<0.001 I × S 1.58 × 10^-9^ 1605 3083 \<0.001 6.46 × 10^-11^ 4730 9192 \<0.001 Karyotypes (K) 0.003 30 2 0.546 0.002 110 48 0.301 Cross ⊂ K 0.074 225 843 0.362 0.018 660 2932 0.023 Within flies 9.69 × 10^-9^ 1350 3070 \<0.001 7.91 × 10^-10^ 3960 9169 \<0.001 ::: ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Asymmetry of overall wing shape for males raised at 18°C Same as in Table 5. ::: Inbred (98.5%) Outbred --------------------- ---------------- --------- ------ --------- ---------------- ------ ------ --------- Source of variation Wilks\' lambda df 1 df 2 *P* Wilks\' lambda df 1 df 2 *P* Individuals (I) 7.18 × 10^-12^ 1605 1464 \<0.001 3.51 × 10^-14^ 4730 4442 \<0.001 Karyotypes (K) 0.004 30 2 0.633 2.49 × 10^-4^ 110 48 0.006 Cross ⊂ K 1.61 × 10^-4^ 225 843 \<0.001 2.98 × 10^-4^ 660 2932 \<0.001 Among flies 1.47 × 10^-8^ 1350 1457 \<0.001 3.62 × 10^-11^ 3960 4430 \<0.001 Sides (S) 0.658 15 93 \<0.001 0.569 22 194 \<0.001 I × S 5.58 × 10^-8^ 1605 3083 \<0.001 1.28 × 10^-10^ 4730 9192 \<0.001 Karyotypes (K) 0.004 30 2 0.605 0.003 110 48 0.449 Cross ⊂ K 0.068 225 843 0.236 0.019 660 2932 0.036 Within flies 3.05 × 10^-7^ 1350 3070 \<0.001 1.73 × 10^-9^ 3960 9169 \<0.001 ::: ::: {#T7 .table-wrap} Table 7 ::: {.caption} ###### Asymmetry of overall wing shape for females raised at 23°C Same as in Table 5 for *Drosophila subobscura*flies reared at 23°C. ::: Inbred (98.3%) Outbred --------------------- ---------------- --------- ------ --------- ---------------- ------ ------ --------- Source of variation Wilks\' lambda df 1 df 2 *P* Wilks\' lambda df 1 df 2 *P* Individuals (I) 1.07 × 10^-12^ 1605 1464 \<0.001 1.08 × 10^-13^ 4730 4442 \<0.001 Karyotypes (K) 2.18 × 10^-4^ 30 2 0.200 0.001 110 48 0.146 Cross ⊂ K 3.31 × 10^-4^ 225 843 \<0.001 1.81 × 10^-4^ 660 2932 \<0.001 Among flies 2.32 × 10^-9^ 1350 1457 \<0.001 1.54 × 10^-10^ 3960 4430 \<0.001 Sides (S) 0.450 15 93 \<0.001 0.585 22 194 \<0.001 I × S 2.57 × 10^-9^ 1605 3083 \<0.001 3.21 × 10^-13^ 4730 9192 \<0.001 Karyotypes (K) 0.007 30 2 0.725 0.006 110 48 0.842 Cross ⊂ K 0.055 225 843 0.062 0.034 660 2932 0.889 Within flies 1.95 × 10^-8^ 1350 3070 \<0.001 3.54 × 10^-12^ 3960 9169 \<0.001 ::: ::: {#T8 .table-wrap} Table 8 ::: {.caption} ###### Asymmetry of overall wing shape for males raised at 23°C Same as in Table 5 for *Drosophila subobscura*flies reared at 23°C. ::: Inbred (98.3%) Outbred --------------------- ---------------- --------- ------ --------- ---------------- ------ ------ --------- Source of variation Wilks\' lambda df 1 df 2 *P* Wilks\' lambda df 1 df 2 *P* Individuals (I) 5.39 × 10^-12^ 1605 1464 \<0.001 6.41 × 10^-14^ 4730 4442 \<0.001 Karyotypes (K) 8.81 × 10^-4^ 30 2 0.364 1.18 × 10^-4^ 110 48 \<0.001 Cross ⊂ K 2.75 × 10^-4^ 225 843 \<0.001 1.96 × 10^-4^ 660 2932 \<0.001 Among flies 8.92 × 10^-9^ 1350 1457 \<0.001 7.94 × 10^-11^ 3960 4430 \<0.001 Sides (S) 0.642 15 93 \<0.001 0.540 22 194 \<0.001 I × S 8.58 × 10^-9^ 1605 3083 \<0.001 9.84 × 10^-12^ 4730 9192 \<0.001 Karyotypes (K) 5.13 × 10^-5^ 30 2 0.102 6.40 × 10^-4^ 110 48 0.052 Cross ⊂ K 0.060 225 843 0.111 0.024 660 2932 0.250 Within flies 5.79 × 10^-8^ 1350 3070 \<0.001 1.26 × 10^-10^ 3960 9169 \<0.001 ::: Similarly to what had been found for CS, differences between left and right wings were also highly significant (\"sides\" effect), thus indicating that DA was present for overall wing shape. This finding is contrary to our previous claim from a subset of O~st~isochromosomal lines, where DA for some landmarks (e.g. those defining the position of the anterior crossvein) but not for overall wing shape was detected \[[@B38]\]. After plotting the Procrustes grand mean shapes of both wings it also became apparent here that the location of the anterior crossvein was indeed slightly more distal in the right wings. Furthermore, the individuals × sides interaction effects were highly significant in all cases and, hence, wing shape FA greatly exceeded measurement error. ### b) Causal components of variation As has been forcefully stressed \[[@B42]\] shape is an inherently multidimensional concept and cannot be easily reduced to a scalar index without severe loss of information. Therefore, for a quantitative genetic analysis of shape data a multivariate approach is required \[[@B43]\]. For overall wing shape, genetic differences among karyotypes were mostly detected for outbred crosses (Tables [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}, [7](#T7){ref-type="table"}, [8](#T8){ref-type="table"}), and we have estimated the covariance matrices **P**= **K**+ **C**+ **E**as a simple multivariate extension of the two-level nested ANOVAs, where **P**is the phenotypic covariance matrix and **K**, **C**, and **E**are, respectively, the covariance matrices for karyotypes, crosses within karyotypes, and the residuals. Fig. [2](#F2){ref-type="fig"} shows the amount of variation associated with the different dimensions in shape space. Much of the variation was concentrated in the first few PCs, but the **K**matrices showed the clearest trend to quickly decrease after the first PC. Permutation tests indicated that matrix correlations (MCs) between **K**and **C**matrices were generally higher at 18°C (females MC = 0.258, *P*= 0.1908 ; males MC = 0.305, *P*= 0.1963) than at 23°C (females MC = 0.157, *P*= 0.3356 ; males MC = 0.250, *P*= 0.2665), but none of the MCs was statistically significant. On the other hand, VCV matrices were correlated across rearing temperatures (females: MC **K**= 0.716, *P*= 0.0163; MC **C**= 0.818, *P*= 0.0001; males: MC **K**= 0.706, *P*= 0.0160 ; MC **C**= 0.587, *P*= 0.0399 ; this last correlation was no longer significant after the Bonferroni procedure \[[@B44]\]). A close inspection to Fig. [2](#F2){ref-type="fig"} reveals an increase in the genetic components of overall wing shape at 23°C, which agrees with our preliminary findings \[[@B38]\]. Thus, the ratio between the total variance of genetic (**G**= **K**+ **C**) covariance matrix onto the total variance of the phenotypic covariance matrix was lower at 18°C in both sexes (females: 0.1312 *vs*. 0.5450; males: 0.2365 *vs*. 0.2522). A caveat: these ratios cannot be interpreted as estimates of shape heritability \[[@B43]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Eigenvalues of causal covariance matrices for wing shape**First 15 eigenvalues of the phenotypic (black bars), karyotype (hatched) and crosses (open) covariance matrices from outbred crosses. ::: ![](1471-2148-5-7-2) ::: MANOVA results in Tables [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}, [7](#T7){ref-type="table"}, [8](#T8){ref-type="table"} also point to the presence of genetic variation for overall shape DA, mainly at 18°C (i.e., the \"crosses in karyotypes\" component from the decomposition of the I × S interaction effect). As far as we are aware, these are the first experiments that found detectable genetic variation in DA for wing traits. The uncovering of DA (i.e., \"side\" effect) for fly wings is quite general when quantitative analyses of form are carried out using the powerful methods of geometric morphometrics to reveal even small morphological variation that otherwise would remain hidden with less effective techniques \[[@B13],[@B45]\]. This has raised concerns against the conventional wisdom that left and right are not distinguished in *Drosophila*development \[[@B46]\] because it provides compelling evidence that DA in fly wings may signal the presence of genetic variation in a phylogenetic conserved left-right developmental axis (i.e., an imaginary plane between the two lateral sides of the body), as discussed by Klingenberg et al. \[[@B45]\]. Actually, modern treatises in developmental biology (e.g. \[[@B9]\]) distinguish the left-right axis besides the customary anterior-posterior and dorsal-ventral axes, and several asymmetrically expressed genes (e.g. *sonic hedgehog*) have recently been discovered. In *Drosophila*, Ligoxygakis et al. \[[@B47]\] were the first (and to our knowledge the only ones) who showed a developmental mechanism for the developmental asymmetry. It seems, therefore, that the detection of genetic variation for DA in this genus appears to be basically a methodological problem, including statistical power and the environmental conditions where the experiments are performed. The mechanisms that constitute the genetic basis of morphological asymmetry in *Drosophila*obviously require further study. ### c) Genetic components of wing shape FA Following \[[@B13]\] a multivariate equivalent of FA1 (i.e., the \"unsigned\" left-right differences) was defined by changing the signs of all coordinate differences (from left-right to right-left) whenever the inner product (also referred to as the dot product) of a left-right difference vector with the vector of mean left-right difference was negative. For the univariate case (CS) this procedure would render here the absolute (![](1471-2148-5-7-i10.gif)) differences, but notice that for the multivariate case it is not equivalent to calculate the absolute (![](1471-2148-5-7-i10.gif)) differences of all Procrustes coordinates. MANOVA analyses of these \"unsigned\" shape asymmetries in outbred crosses did not detect any genetic variation for shape FA at 18°C or 23°C (Tables [9](#T9){ref-type="table"}, [10](#T10){ref-type="table"}). However, the approach used to define the multivariate equivalent of FA1 might be influenced by the arbitrary choice of the plane (i.e., the mean left-right differences) to subdivide the shape space into \"positive\" and \"negative\" halves (Christian P Klingenberg, pers. comm. 2004). A modified Procrustes shape distance for non-isotropic variation (i.e., landmarks usually differ in their amounts of variation) has been recently developed by Klingenberg and Monteiro \[[@B48]\], and can be used here as a scalar measure of the amount of shape asymmetry because FA is random in origin (i.e., only the magnitude and not the direction may usually be the interesting component of FA shape variation). When this scalar was used in our data set the same conclusion was obtained; namely, there was no detectable genetic variation for wing shape FA in any case (results not shown). ::: {#T9 .table-wrap} Table 9 ::: {.caption} ###### MANOVAs for female wing shape fluctuating asymmetry A multivariate equivalent of FA1 (i.e., the \"unsigned\" left-right differences) was defined as explained in the text. Flies raised from outbred crosses of *Drosophila subobscura*(⊂ means \'nested in\'). ::: 18°C 23°C --------------------- ---------------- ------ ------ ------- ---------------- ------ ------ ------- Source of variation Wilks\' lambda df 1 df 2 *P* Wilks\' lambda df 1 df 2 *P* Karyotypes (K) 0.008 110 48 0.908 0.007 110 48 0.856 Cross ⊂ K 0.022 660 2932 0.169 0.029 660 2932 0.604 ::: ::: {#T10 .table-wrap} Table 10 ::: {.caption} ###### MANOVAs for male wing shape fluctuating asymmetry Same as in Table 9. ::: 18°C 23°C --------------------- ---------------- ------ ------ ------- ---------------- ------ ------ ------- Source of variation Wilks\' lambda df 1 df 2 *P* Wilks\' lambda df 1 df 2 *P* Karyotypes (K) 0.004 110 48 0.627 0.009 110 48 0.938 Cross ⊂ K 0.024 660 2932 0.243 0.042 660 2932 0.988 ::: ### d) Consanguinity and temperature effects on wing shape To investigate allometric and nonallometric temperature effects on overall wing shape we performed a multivariate analyses of covariance (MANCOVA) of the Procrustes coordinates (after averaging both sides and the two replicated measurements per side) considering temperature and inbreeding (i.e., isogenic *vs*. outbred homokaryotypic flies) as the categorical predictors and CS (as log~*e*~(pixels)) as the covariate. Temperature effects were only significant in males, but inbreeding and temperature × inbreeding interaction effects were highly significant in both sexes (results not shown), which suggests a strong effect of the categorical predictors on the nonallometric component of shape. Size effects were also found to be significant (females: Wilks\' *λ*= 0.881, *F*~(22,405)~= 2.496, *P*\< 0.001; males: Wilks\' *λ*= 0.915, *F*~(22,405)~= 1.715, *P*= 0.024), but the allometric effect on shape remained relatively consistent at both temperatures in females (size × temperature interaction: Wilks\' *λ*= 0.930, *F*~(22,405)~= 1.395, *P*= 0.111) but not in males (Wilks\' *λ*= 0.853, *F*~(22,405)~= 3.165, *P*\<0.001). The association between size and temperature (Fig. [1](#F1){ref-type="fig"}), measured by the variance inflaction factor (*VIF*\< 5; \[[@B49]\]), was found to be lower than the suggested guideline for serious collinearity (i.e. *VIF*≥ 10), which indicates that the effects of temperature and size on wing shape could be effectively separated. The conclusion is, therefore, that *Drosophila*wing shape does not seem to be as resistant to environmental temperature as previously claimed from the analysis of 12 highly inbred *D. melanogaster*lines \[[@B29]\]. Inbreeding effects (isogenic *vs*. outbred homokaryotypic flies) on wing shape FA were tested from the ratio between the traces of the corresponding \"individual × side\" VCV matrices. Notice that the traces of these interaction matrices are equal to the respective mean squares of the Procrustes ANOVA as implemented by Klingenberg and McIntyre \[[@B13]\], and are simply the sum of ![](1471-2148-5-7-i20.gif) (index FA4 in \[[@B39]\]) for each *x*and *y*coordinates of the corresponding aligned configurations divided by the shape dimension. We performed 10,000 randomization runs for each test. Inbreeding effects were detected at 18°C but only in females (18°C: female *F*= 1.694, *P*= 0.0003 ; male *F*= 0.963, *P*= 0.6037 ; 23°C: female *F*= 0.834, *P*= 0.9231; male *F*= 0.984, *P*= 0.5541). Patterns of wing shape variation -------------------------------- ### a) Fluctuating asymmetry Principal component analyses were only implemented for the outbred crosses since they are more representative of the natural situation. The percentages of total shape variation, together with the features of variation associated with the dominant PCs, are graphically plotted in Figs. [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}, [6](#F6){ref-type="fig"}. For the individual variation several PCs accounted for relatively large amounts of variability. On the contrary, for FA and measurement error PC1 explained almost all total variance (\>80%). For all levels in the analysis (i.e. individuals, FA and measurement error) the dominant PCs were connected to the relatively large variability of landmarks 3, 6, 7 and, to a lesser extent, landmark 2. However, the disproportionate amount of variation associated with these landmarks did not spread to all sources of causal variation because their coefficients were relatively small for the PC1 of karyotype variation (which explained \~60% of the total variance; see below). Furthermore, for the individual variation the first two PCs were also linked to the shift of the anterior (landmarks 11 and 12) and posterior (landmarks 7 and 13) crossveins along the adjoining longitudinal veins. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Vectors of the landmarks displacements**First two axes of wing shape variation for each effect in the two-way mixed MANOVA (individuals, individuals × sides interaction, and measurement error) for females from outbred crosses reared at 18°C. Also plotted are the percentages of total wing shape variation explained by the principal components for the corresponding covariance matrices. ::: ![](1471-2148-5-7-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Vectors of the landmarks displacements**Same as Fig. 3 for males from outbred crosses reared at 18°C. ::: ![](1471-2148-5-7-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Vectors of the landmarks displacements**Same as Fig. 3 for females from outbred crosses reared at 23°C. ::: ![](1471-2148-5-7-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Vectors of the landmarks displacements**Same as Fig. 3 for males from outbred crosses reared at 23°C. ::: ![](1471-2148-5-7-6) ::: Permutation tests indicated that VCV matrices were mostly correlated for FA and measurement error effects within samples (MCs \> 0.95, *P*\< 0.01; Table [11](#T11){ref-type="table"}). The individual VCV matrix was significantly correlated with the FA and measurement error matrices only for females at 18°C. Between temperatures the VCV matrices were highly correlated for FA and measurement error (results not shown), but loosely correlated for the individual variation (females MC = 0.668, *P*= 0.0355 ; males MC = 0.494, *P*= 0.1066 ; statistical significance vanishes after the Bonferroni procedure). ::: {#T11 .table-wrap} Table 11 ::: {.caption} ###### Correlations between VCV matrices of landmarks displacements within groups Results of the permutation tests used for the analyses within sexes and temperatures. ::: Group Effects Correlation *P*(permutation) *P*(Bonferroni) -------------- -------------------- ------------- ------------------ ----------------- Females 18°C Individual / FA 0.7699 0.0001 \*\* Karyotype / FA -0.1691 0.7583 n.s. Cross / FA 0.5773 0.0871 n.s. Between-fly / FA 0.7517 0.0001 \*\* Individual / error 0.7550 0.0001 \*\* FA / error 0.9953 0.0001 \*\* Males 18°C Individual / FA -0.3998 0.8694 n.s. Karyotype / FA 0.0067 0.4393 n.s. Cross / FA -0.0706 0.6202 n.s. Between-fly / FA 0.2060 0.3296 n.s. Individual / error -0.4280 0.9436 n.s. FA / error 0.9964 0.0001 \*\* Females 23°C Individual / FA 0.1233 0.2881 n.s. Karyotype / FA -0.0151 0.4771 n.s. Cross / FA 0.6516 0.0264 n.s. Between-fly / FA 0.5764 0.0744 n.s. Individual / error 0.1093 0.3141 n.s. FA / error 0.9959 0.0001 \*\* Males 23°C Individual / FA 0.5278 0.0523 n.s. Karyotype / FA -0.1469 0.7545 n.s. Cross / FA 0.2752 0.1817 n.s. Between-fly / FA 0.4033 0.1241 n.s. Individual / error 0.5165 0.0519 n.s. FA / error 0.9922 0.0001 \*\* n.s. = *P*\> 0.05; \*\* = *P*\< 0.01. ::: The angles between the PC1s for FA and measurement error were very much alike (ranging from angle *α*= 2.1° to *α*= 3.4° ; recall that the 0.1% quantile of the resulting distribution between pairs of random vectors in 22-dimensional space was 50.3°), which reflects the similarity due to landmarks 3, 6 and 7. However, the first three PCs for interindividual variation were generally distinct to those of FA: the only clear correspondences were between the PC1s for females at 18°C (*α*= 21.5°), and the PC2 of interindividual variation with the PC1 of FA for males at 18°C (*α*= 11.8°). (The correspondences were qualitatively the same for interindividual variation and measurement error; results not shown.) Overall, these results seem to suggest that canalization and DS do not generally share the same underlying regulatory mechanisms (but see below). A potentially important problem with the foregoing approach to compare the patterns of intra- and interindividual variation is to rely on the interaction VCV matrix as the source of variation due to FA. As has been previously argued the uncovering of DA is almost ubiquitous for shape data when using the methods of geometry morphometrics, and there was evidence here for statistically significant genetic variation of overall shape DA at 18°C (Tables [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}). Therefore, the VCV matrix from the \"individuals × sides\" interaction effect gives a biased estimate of developmental stability and cannot be taken as the covariance matrix for FA. In other words, this VCV matrix also includes all causal components due to genetic variation for DA, and the corresponding unbiased VCV matrix for FA is that for the within-fly component of the interaction effect (i.e., after removing the genetic variation for DA \[[@B50],[@B51]\]). In any case, all results were qualitatively similar and, hence, the conclusion that canalization and DS seem to be different mechanisms remains unchanged. However, it is difficult to appraise how this potential problem could have affected the previously published conclusions when comparing interindividual variation and \"FA\" in fly wings and mouse skulls (see Background section). Between rearing temperatures the congruence of PC1 eigenvectors was also very high for FA (females *α*= 4.0°; males *α*= 3.5°) and measurement error (females *α*= 3.1°; males *α*= 4.1°). For the interindividual variation the correlations between PC1s were significant only in males (females *α*= 74.3°; males *α*= 19.3°); however, the PC1 vector describing the joint interindividual variation of landmark position in females at 18°C matched the PC2 of the interindividual covariance matrix at 23°C (*α*= 49.6°; recall that the direction of PCs is arbitrary and all the movements in Figs. [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}, [6](#F6){ref-type="fig"} can be simultaneously reversed by 180°) and *vice versa*(i.e., PC1 at 23°C *vs*PC2 at 18°C: *α*= 26.4°). ### b) Causal components Besides the interindividual variation in the two-way MANOVAs (which comprises genetic plus environmental covariances due to special environmental effects) it is important here to asses the patterns of joint displacements of landmarks for each of the causal components of wing shape variation (Figs. [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}, [9](#F9){ref-type="fig"}, [10](#F10){ref-type="fig"}). For karyotype variation PC1 accounted for \~60% of the total variance and was linked to a great extent with equivalent movements of those landmarks defining the location of the crossveins, which shifted in the same direction. Landmarks 4 and 5 tended to move away each other, stretching the wing margin between longitudinal veins III and IV. Landmark 9 budged in the opposite direction to crossveins shifts, thus shaping the relationship between L1 to the total length of longitudinal vein IV (i.e. shape index L1 WL ; Fig. [12](#F12){ref-type="fig"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Vectors of the landmarks displacements**First two axes of wing shape variation in the two-level nested MANOVA (karyotypes, crosses nested in karyotypes, and within crosses) for each causal component effect pertaining to the inter-individual variation in females from outbred crosses reared at 18°C. Also plotted are the percentages of total wing shape variation explained by the principal components for the corresponding covariance matrices. ::: ![](1471-2148-5-7-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### **Vectors of the landmarks displacements**Same as Fig. 7 for males from outbred crosses reared at 18°C. ::: ![](1471-2148-5-7-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### **Vectors of the landmarks displacements**Same as Fig. 7 for females from outbred crosses reared at 23°C. ::: ![](1471-2148-5-7-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### **Vectors of the landmarks displacements**Same as Fig. 7 for males from outbred crosses reared at 23°C. ::: ![](1471-2148-5-7-10) ::: ::: {#F12 .fig} Figure 12 ::: {.caption} ###### **Left wing of *Drosophila subobscura***The image shows the thirteen landmarks (1 -- 13) used in this work. I -- VI longitudinal veins; cv-a and cv-p anterior and posterior crossveins; Co costal or marginal veins; L1 and L2 lengths of the proximal (Euclidian distance between landmarks 9 and 13) and distal (Euclidian distance between landmarks 13 and 5) segments of longitudinal vein IV, respectively. Wing shape index ![](1471-2148-5-7-i21.gif) has been previously used to study shape clines in this species \[30\]. ::: ![](1471-2148-5-7-12) ::: A relative shortening of the basal length of longitudinal vein IV relative to the total wing length with an increasing dose of standard gene arrangements in all five major chromosomes of *D. subobscura*had been previously identified in an outbred stock \[[@B32],[@B33]\]. A similar pattern regarding O~st~dose is also clear here when considering the six karyotypes (Fig. [11](#F11){ref-type="fig"}), but rearing temperature quantitatively modified the shape index (L1/WL was lower at the highest temperature). However, there was no statistically significant karyotype × temperature interaction. The wing shape index appears to be a purely additive trait since heterokaryotypes were always intermediate to their corresponding homokaryotypes (Fig. [11](#F11){ref-type="fig"}). Actually, none of 12 within- group (i.e., sex and temperature) possible contrasts comparing all three heterokaryotypes with the average of the corresponding homokaryotypes was statistically significant (the mean square for \"crosses\" was used as the error term; see legend in Fig. [11](#F11){ref-type="fig"}). ::: {#F11 .fig} Figure 11 ::: {.caption} ###### **Wing shape index**Averages of the relative length (with 95% confidence intervals) of the basal portion of longitudinal vein IV (L1) to the total wing length (WL = L1 + L2) versus karyotype for outbred crosses at the two rearing temperatures. A two-way factorial ANOVA using the shape index as ![](1471-2148-5-7-i22.gif), with karyotype and temperature as fixed effects, and crosses nested within karyotypes, detected statistically significant differences for the main effects (karyotype: female *F*~5,30~= 12.625, *P*\< 0.001; male *F*~5,30~= 9.785, *P*\< 0.001. Temperature: female *F*~1,390~= 30.219, *P*\< 0.001; male *F*~1,390~= 61.835, *P*\< 0.001) but no karyotype × temperature interaction (females: *F*~5,390~= 1.570, *P*= 0.168; males: *F*~5,390~= 1.111, *P*= 0.354). ::: ![](1471-2148-5-7-11) ::: PC2 for karyotypes was also connected to the variability of landmarks 3, 6 and 7. For the crosses component, several PCs explained relatively large amounts of variation, and shifts of crossveins now seem to be independent of each other at 18°C but not at 23°C. Finally, for the within-fly variation several PCs accounted for relatively large amounts of variability. PC1s were again connected to the variability of landmarks 3, 6 and 7; and PC2s to shifts in the anterior crossvein. The large amount of variation of the anterior and posterior crossveins for karyotypes and crosses can be interpreted in terms of developmental processes. The crossveins are determined after the longitudinal veins, and mutations that eliminate crossveins (e.g. *crossveinless*) do not affect the longitudinal veins; however, some mutants that affect the longitudinal veins also influence the crossveins (e.g. the *vn*group in \[[@B1]\]). Intra- and interespecific studies in several *Drosophila*species have found displacements of one or both crossveins along their longitudinal veins, and such shifts also occur in a number of mutants (see \[[@B23]\]). However, these shifts do not occur in isolation an also include other landmarks as well (e.g., landmarks 9 and 5 on L4; landmarks 1 and 2 on L1; Figs. [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}, [9](#F9){ref-type="fig"}, [10](#F10){ref-type="fig"}). The matrix permutation tests (Table [11](#T11){ref-type="table"}) indicated that the VCV matrices of karyotypes and crosses were never significantly correlated with the VCV matrices of FA and measurement error. The high correlation between the VCV matrices of the interindividual and FA effects for females at 18°C was basically due to the (micro-) environmental component. Also notice that all correlations between the VCV matrices of karyotype and FA effects were close to zero or even negative, which clearly suggests that this genetic component of canalization is unrelated to DS. In addition, the PC1s of karyotypes and FA were nearly at right angles (18°C: females *α*= 85.8°, males *α*= 77.3°; 23°C: females *α*= 75.6°, males *α*= 78.5°). The only matches were between PC2 of karyotypes and PC1 of FA for females at 18°C (*α*= 13.0°) and males at 23°C (*α*= 31.2°). The PC1s of crosses and FA were also poorly correlated; the only exception being females a 23°C (*α*= 43.3°). These results clearly support the hypothesis that genetic canalization and DS are not functionally the same mechanism. On the other hand, all observed angles involving PC1s between \"replicated genotypes\" (i.e. the between-fly component) and FA were relatively small and highly significant (18°C: females *α*= 20.1°, males *α*= 15.9° ; 23°C: females *α*= 22.7°, males *α*= 36.7°). (Results were qualitatively the same for all observed angles involving PC1s of the between-fly and measurement error covariance matrices; results not shown.) Together with the overall comparisons of the covariance matrices (Table [11](#T11){ref-type="table"}), these results indicate that (micro-) environmental canalization and DS share underlying regulatory mechanisms but are not identical. There was not a complete congruence as PC1 of FA accounted for most part of the variation, while PC1 of between-fly variation usually explained less than 50% of the total variance (Figs. [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}, [9](#F9){ref-type="fig"}, [10](#F10){ref-type="fig"}). To conclude, the theoretical lower limit for (micro-) environmental canalization (i.e., the environmental variance among genetically identical individuals) would be FA because the two sides share the same genome (barring unusual somatic mutation or somatic recombination) and nearly the same environment, so differences between sides are likely to be small. Under stabilizing selection this lower limit is obviously associated with higher fitness. However, this \"canalization limit\" would hardly ever be observed because of unavoidable additional sources of environmental variance (e.g. variation between vials, the position of the pupae in a vial, etc.). A similar logic than the one used in this work has been applied to distinguish between intrinsic and extrinsic stochastic variation in gene expression: intrinsic noise can be separated by contrasting the levels of gene expression in a construct with two identically regulated but fluorescently distinguishable *gpf*genes in the *Escherichia coli*chromosome, whereas extrinsic noise is inferred by the correlated variation between the two copies in the same environment \[[@B52],[@B53]\]. Conclusions =========== This study applied the methods of geometric morphometrics in the context of quantitative genetics of wing form variation using isochromosomal lines of *D. subobscura*. The main findings can be summarized as follows: (i) for the analysis of overall size, DS was positively correlated with levels of heterozygosity (i.e., inbred *vs*. outbred homokaryotypes) and development at the optimal temperature; however, no positive association was found between DS and chromosomal heterozygosity in outbred crosses; (ii) there was detectable genetic variation (mainly for overall shape) for the directional component of morphological asymmetry (i.e., DA) but not for FA, which likely reflects variation due to stochasticity in development; (iii) for analyses of shape, the patterns of covariation for FA and measurement error were highly concordant in all samples, which also provides strong reasons to conclude that FA is generated by random perturbations of developmental processes (obviously, this does not imply that DS is independent of the genetic background: wing shape FA was found to be higher in inbred females at 18°C when compared to their outbred homokaryotypic counterparts); (iv) the inter- and intraindividual variation patterns were generally poorly correlated, which supports the hypothesis that canalization and DS are distinct mechanisms; however, (v) the patterns of variation due to the (micro-) environmental component of canalization (i.e., the among-fly special environmental effects covariances) were quite similar to those observed for FA; (vi) the lack of a significant within-group correlation between the VCV matrices associated with the interindividual genetic components of canalization and FA, as well as the low similarity between the corresponding vectors describing variation of landmark position, strongly suggest that genetic and environmental canalization are not similar mechanisms. In addition, (vii) a discrepancy between sexes was observed in some situations; e.g. overall size FA increased with inbreeding and (sub-optimal) temperature effects mainly in females, and the allometric effect on wing shape at both experimental temperatures was similar in females but not in males. It is also interesting to note here that wing size (measured as WL; Fig. [12](#F12){ref-type="fig"}) clines for *D. subobscura*developed in North America after \~20 years since colonization, but males were clearly lagging behind females \[[@B54]\]. What is not obvious, however, is why there is a difference between the sexes. It has been suggested that a relationship between canalization and DS could only reflect a common underlying association between character and fitness \[[@B55],[@B56]\]. Those traits under strong stabilizing selection may not be genetically canalized and the major source of selective pressure for canalization can result from the benefits gained by buffering the effects of environmental perturbations \[[@B4],[@B10]\]. The strongest evidence in favor of this hypothesis comes from the well-known genotype-phenotype mapping of RNA folding. Conservation of RNA secondary structure is under strong selection, and low structural plasticity is achieved through increasing the thermodynamic independence of any one structural component from the remaining structure \[[@B57]\]. Likewise, the flux summation theorem developed in the field of metabolic control analysis implies, if true, that phenotypic robustness is an inevitable outcome of the underlying metabolism and not a result of evolution (see \[[@B58]\]). However, it is still an open question whether or not natural wing shape changes in *Drosophila*are adaptive. There are no consistent patterns between latitude and wing shape (e.g. \[[@B30]\]), contrarily to what happens for size-related traits where world-wide latitudinal clines are found with genetically larger individuals derived from higher latitudes (e.g. \[[@B30],[@B59]\]). Many genes with small additive effects on features of wing shape are dispersed along the *Drosophila*genome (e.g. \[[@B60],[@B61]\]), and we have shown here that the wing shape index L1/WL appears to be a purely additive trait since heterokaryotypes were always intermediate to their homokaryotypic counterparts. The wing shape cline in North America colonizing populations of *D. subobscura*\[[@B30]\] can be largely accounted for parallel latitudinal clines in chromosomal gene arrangements \[[@B32],[@B33]\], and the small shifts of (e.g.) the anterior and posterior crossveins in relation to karyotype variation (Figs. [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}, [9](#F9){ref-type="fig"}, [10](#F10){ref-type="fig"}; notice that the plotted joint variation in landmark positions is an exaggeration of the actual variation in the data set) are difficult to link with any adaptive response to a better flight capacity. Actually, we lack even hypothetical functional explanations for subtle shape variation: Gilchrist et al. \[[@B54]\] speculated that wing shape variation in *D. subobscura*may simply represent drift around an optimum. Our present results (points (v) and (vi) above) give some credence to that conjecture. Genetic canalization on wing shape does not seem to arise as a by-product of environmental canalization and, therefore, canalization is not a single mechanism to buffer any source of variation as has been suggested \[[@B10]\]. According to Graham et al. \[[@B62]\] the classical linear theory of DS can successfully account for both normally distributed error distributions and leptokurtic distributions caused by the admixture of individuals having different levels of DS, but cannot account for transitions between FA and DA. We have previously suggested, however, that a transition from \"ideal\" FA (i.e., a normal distribution of left -- right-side scores whose mean is zero) to a distribution showing DA could be made entirely compatible with what it is already known from classical quantitative genetics \[[@B38]\]. Shifts between asymmetry types (FA, DA and antisymmetry) have been reported to happen along a species distribution range \[[@B63]\], but unless the genetic component can be partitioned out the variation in left-right differences cannot be assumed to describe DS. From the results of outbred crosses reared at 18°C (Table [5](#T5){ref-type="table"}, [6](#T6){ref-type="table"}) it is possible to test here for the congruence between patterns of morphological variation with respect to the variation attributable to FA (i.e. the within-fly environmental component of the interaction term) and that attributable to genetic variation for DA (the within-fly genetic component due to crosses in karyotypes of the interaction term). The corresponding VCV matrices were highly correlated for females (MC = 0.914, *P*~(permutation)~= 0.0001) but not for males (MC = 0.064, *P*~(permutation)~= 0.485). The angles between the PC1s also reflect this discrepancy between samples (females *α*= 8.6° ; males *α*= 45.9°). When considered together, these results clearly suggest that FA and genetic variation for DA may or may not be functionally linked. Methods ======= Extraction of O chromosomes and fly handling -------------------------------------------- A large number of *D. subobscura*isochromosomal lines for the O chromosome in an otherwise homogeneous genetic background were derived from an outbred stock collected at Puerto Montt (Chile; 41° 28\' S) in November 1999 as previously indicated \[[@B38]\]. Briefly, wild-type males were individually crossed to three or four virgin females from the highly homogeneous *ch-cu*marker strain, which is homozygous for the morphological recessive markers on the O chromosome *cherry*eyes (*ch*) and *curled*wings (*cu*) and fixed for the gene arrangement O~3+4~. A single ![](1471-2148-5-7-i23.gif) / O~+\ *cu*+\ *ch*~male from the offspring was backcrossed to *ch-cu*females, and its arrangement on the wild-type chromosome was identified after four generations of backcrosses. Followed by at least another backcross to *ch-cu*females, a single male from each line carrying the wild chromosome was crossed to two virgin females from the *Va/Ba*balanced marker stock. This strain was derived from the *ch-cu*stock and carries the dominant lethal genes *Varicose*(*Va*) and *Bare*(*Ba*) on the O chromosome. The isochromosomal lines were established from the final crosses ♀♀ O~*Va\ ch*+\ *cu*~/ ![](1471-2148-5-7-i23.gif) × ♂♂ O~*Va\ ch*+\ *cu*~/ ![](1471-2148-5-7-i23.gif). All lines used here had a quasi-normal viability according to the recorded proportions of wild-type flies raised in the final crosses to obtain the isochromosomal lines \[[@B38]\]. The lines were kept at 18°C (12:12 light/dark cycle) in 130-mL bottles with low adult density to standardize the rearing conditions before egg collections. As previously indicated the experimental flies were obtained from 54 crosses. Reciprocal crosses were made for all outbred combinations by mating one-week virgin females and males. After three days the males were discarded and equal numbers of females from each reciprocal cross were placed together in a plastic chamber with a spoon containing non-nutritive agar with a generous smear of live yeast for egg collections. To standardize the experimental conditions, eggs from the inbred (isogenic) crosses were also obtained in a similar way; namely, after mating the flies in bottles and transferring the females to plastic chambers. Eggs were placed in six 2 × 8 cm vials with 6 mL of food (26 eggs/vial); three vials were kept at 18°C (optimal temperature) and the other three at 23°C (sub-optimal temperature). Within each experimental temperature the vials were randomly placed on the same incubator shelf. As a result, the total experiment consisted of 324 vials (162 vials at each experimental temperature), and all eggs were sampled on the same day. Emerging flies (not less than 2 or 3 days old) were stored in Eppendorf tubes with a 3:1 mixture of alcohol and glycerol at 4°C before wing measurements. All fly handling was done at room temperature using CO~2~anesthesia on flies not less than 6 h after eclosion. Wing measurements ----------------- Two randomly sampled females and males emerged from each vial were used for morphometric analyses. Both wings were removed from each fly and fixed in DPX under coverslips on microscope slides. Bitmap images were captured with a video camera (Sony CCD-Iris, Tokyo, Japan) connected to a PC computer with MGI VideoWave software and mounted on a compound microscope (Zeiss Axioskop, Jena, Germany), using a 2.5 × objective. Calibration of the optical system was checked at each session. The images were stored on a Dell Workstation PWS350. To quantify and minimize measurement error all wings were digitized two times at different sessions as follows: images of both the left and right wings were captured during a given session and after an entire round on all individuals the same process was repeated again. A similar procedure was also used to record the *x*and *y*coordinates of 13 morphological landmarks (i.e., labeled geometric points located at the intersections of wing veins or at sites where veins reach the wing margin; Fig. [12](#F12){ref-type="fig"}) by using the Scion Image for Windows software \[[@B64]\]. Therefore, the process we used guaranteed that the observer was blind with respect to the results from previous measurements. Analysis of wing size and shape ------------------------------- Geometric morphometrics precisely separates morphological variation (i.e., variation in form) into size and shape components \[[@B21],[@B22]\]. Size is a one-dimensional trait and the measure most widely used in geometric morphometrics is centroid size (CS), computed here in a normalized form as the square root of the sum of squared Euclidian distances between each landmark to the centroid (center of gravity) of all landmarks divided by the square root of the number of landmarks. Individual size is therefore represented by four scalars, one for each side and session. The shape of an original configuration of landmarks is the geometrical information that is invariant to uniform scaling (variation in size), translation (differences in position), and rotation (differences in orientation). In contrast to size, shape is an inherently multidimensional space and we used Procrustes superimposition to characterize shape variation. This method allows comparing configurations of landmarks by optimally superimposing (according to a least-squares criterion) homologous landmarks in two or more specimens to achieve an overall best fit \[[@B65]\].Because the data set included both left and right wings (i.e., we are dealing with \"matching symmetry\" \[[@B66],[@B67]\]) our analyses also removed differences due to reflection by changing the sign of the *x*coordinate of every landmark for configurations from the right side. The reflection, scaling, and superposition steps were performed for all wings within each cross and temperature simultaneously, which allows contrasting wing shapes between different lines or crosses. The final iteration to minimize the sum of the squared distances between the landmarks of all wings in the sample was done without additional scaling and, consequently, we performed a partial Procrustes fit according to Dryden and Mardia \[[@B22]\]. Given the small amounts of shape variation in this analysis rescaling the coordinates of each configuration by the scaling option 1/cos(*ρ*) \[[@B65]\] would have negligible effects on the results. The landmark coordinates after Procrustes superimposition are amenable to standard multivariate analyses. However, it is important to remember that the removal of size, position (in two dimensions), and orientation reduces the dimensional space to 2*p*-- 4, where *p*is the number of landmarks \[[@B22]\]. Thus, for the present study of 13 landmarks, with 2 coordinates each, the shape dimension is 22. Sums of squares and cross-products (SSCP) matrices are therefore not full-ranked, and the degrees of freedom need to be adjusted. There are three alternative ways of avoiding these difficulties \[[@B22],[@B67]\]: (i) to omit, after Procrustes superimposition of the complete configurations, the coordinates of any two landmarks; (ii) to retain 22 PC scores from the covariance matrix of the data set; (iii) to slightly modify the multivariate statistics (see below) by using the Moore-Penrose generalized inverse of the SSCP matrices so they can tolerate singular matrices, and compute the product of nonzero eigenvalues instead of the determinant of SSCP matrices. We have used here the second scheme. Experimental design and asymmetry analysis ------------------------------------------ Quantitative genetic studies of directional and fluctuating asymmetry obviously require measures from individuals that can be grouped into families or independent lines. Our final data set was a fully balanced design, comprising 54 crosses × 3 vials per cross × 2 females per vial × 2 males per vial × 2 sides per fly × 2 measurements per wing × 2 temperatures = 5,184 wing landmark configurations in total. Within each sex and temperature, least-squares (ANOVA) estimates of variance components (i.e. CS) can be easily obtained from the linear model: *Y*~*ijkl*~= *μ*+ *κ*~*i*~+ *l*~*j*(*i*)~+ *ν*~*k*(*ji*)~+ *ε*~*l*(*kji*)~, where *μ*is the overall grand mean, *κ*~*i*~is the effect of the *i*th karyotype, *l*~*j*(*i*)~is the random effect of the *j*th cross within karyotype *i*, *ν*~*k*(*ji*)~is the random effect of the *k*th vial within cross *j*and karyotype *i*, and *ε*~*l*(*kji*)~is the residual error associated with the trait (i.e. the individual means computed from both sides and the two replicated measurements per side) of the *l*th individual within vial *k*, cross *j*, and karyotype *i*. Since there was no genetic variation within crosses, the residual error provides an estimate of the total special environmental effects variance (i.e. ![](1471-2148-5-7-i2.gif)). Variation among the three replicated vials was generally negligible (results not shown) and, therefore, we have conveniently reduced the previous model to a two-level nested ANOVA after grouping flies across vials. To first partition the total phenotypic variation into interindividual, intraindividual and measurement error components, we used the conventional mixed model, two-way ANOVA (or its MANOVA generalization; see below) for the study of left-right asymmetries \[[@B39]\]. In this ANOVA the main random effect of individuals stands for phenotypic variation in the trait (i.e. CS), the main fixed effect of body side is for directional asymmetry (DA) and tests whether or no the signed differences between the left and right wings \[designated as (![](1471-2148-5-7-i10.gif))\] have a mean of zero, the interaction term is a measure of fluctuating asymmetry (the variation in left-right differences among individuals) provided that there is no genetic variation for DA \[[@B51]\], and the error term gives an estimate of the measurement error. The two-level nested ANOVA can be straightforwardly subsumed within the two-way ANOVA. We now digress slightly to point out some inconsistencies in the literature on what is the appropriate error term to test for the \"interindividual\" effect in the mixed model, two-way ANOVA (either the individual × side interaction effect or the measurement error \[[@B13],[@B15],[@B68],[@B69]\]). Interindividual variation, even if of no general interest in most studies of asymmetry, comprises here genetic components (\"karyotype\" plus \"crosses within karyotypes\") and special environmental effects variance (![](1471-2148-5-7-i2.gif); there is no genetic variance within crosses). An estimated of the among-fly special environmental effects variance (i.e. ![](1471-2148-5-7-i3.gif)) is therefore obtained by subtracting the individual × side interaction effect (which includes ![](1471-2148-5-7-i4.gif) plus measurement error) as the appropriate error term. However, when genetic variation for DA is present the unbiased within-fly special environmental effects variance (i.e. FA) is estimated after partitioning the individual × side interaction effect into its causal components \[[@B51]\]. As pointed out by Klingenberg et al. \[[@B67]\] it is fairly straightforward to extent the preceding ANOVA approach to a full two-factor MANOVA to analyze wing shape asymmetry since all effects are computed from averages or contrasts in the same shape space. Recall that the traces of the corresponding SSCP matrices are just the sum of squares in the Procrustes ANOVA as implemented by Klingenberg and McIntyre \[[@B13]\], but this ANOVA is based on an isotropic model (i.e., it assumes that there is an equal amount of non-directional variation at each landmark \[[@B70]\]) that is not generally correct for any real data. Covariance (VCV) matrices for each effect in the MANOVA were calculated as a simple multivariate extension of the two-way ANOVA. Thus, the SSCP matrices were divided by the appropriate degrees of freedom, and effects were separated according to the expected mean squares in the ANOVA by subtracting the interaction covariance (VCV) matrix from the interindividual VCV matrix, and the error VCV matrix from the interaction one. Therefore, for (e.g.) outbred crosses the interindividual covariance components were calculated as ![](1471-2148-5-7-i24.gif) and the covariance components of FA as ![](1471-2148-5-7-i25.gif), were SSCP~I~is the interindividual SSCP matrix, SSCP~IS~is the interaction SSCP matrix, and SSCP~ME~is the measurement error SSCP matrix. The SSCP~I~matrix was further partitioned into among-karyotype SSCP~K~matrix, among-cross within karyotype SSCP~C⊂K~matrix, and the residual SSCP~*e*~matrix corresponding to the special environmental effects. As a result, genetic effects for overall wing shape were separated from special environmental effects according to the expected means squares in the two-level nested ANOVA. Therefore, for (e.g.) outbred crosses the karyotype covariance components were calculated as ![](1471-2148-5-7-i26.gif) (remind that the entries in the SSCP~I~matrix are equal to those computed from individual means times twice the number of independent measurements per wing), the cross covariance components as ![](1471-2148-5-7-i27.gif), and the among-fly special environmental effects covariance components as ![](1471-2148-5-7-i28.gif). Similarly, genetic effects for DA can be investigated after partitioning the SSCP~IS~matrix into their causal components \[[@B51]\]. Morphological patterns of variation ----------------------------------- Within each sex and temperature, principal component analyses \[[@B41]\] of the VCV matrices were performed for each source of variation with the purpose of describing the landmark displacements corresponding to each emerging principal component (PC), and also to test for the congruence of these displacements between effects. This technique extracts new shape variables (PCs) which successively account for the maximal amount of shape variation and contain information on how the variables relate to each other. The PCs form an orthonormal set of vectors (i.e., the inner product ![](1471-2148-5-7-i29.gif) for i ≠ j, ![](1471-2148-5-7-i30.gif) for i = j; superscript \'denotes transposition) in an n-dimensional space. Correlations between corresponding VCV matrices were computed from the upper triangular part (diagonal entries were included) since covariance matrices are symmetrical, and statistical significance was assessed using permutation tests designed to maintain the associations between pairs of *x*- and *y*-coordinates (i.e., by permuting pairs of rows and columns \[[@B13],[@B15]\]); otherwise the null hypothesis would imply the complete absence of all geometric structure. The permutation procedure was carried out 10,000 times. Correlative patterns of whole shape variation are difficult to interpret: a significant correlation would suggest a real congruence, but a weak congruence does not imply a significant correlation. A second test examined the congruence of the landmark displacements corresponding to each emergent PC for the different effects within groups. Because the PCs correspond to directions in the multivariate shape space, correlations can be obtained by angular comparisons of component vectors. Statistical significance of these correlations was then assessed by comparing those observed values to a null distribution of absolute angles between 100,000 pairs of 22-dimensional random vectors \[[@B71]\]. The 0.1% and 0.001% quantiles of the resulting distribution were 50.3° and 41.6°, respectively. Antisymmetry and allometric effects ----------------------------------- The occurrence of antisymmetry (AS: a bimodal distribution of signed (![](1471-2148-5-7-i10.gif)) \[[@B39]\]) for centroid size was investigated within each sample using the Lilliefors (Kolmogorov-Smirnov) test for the composite hypothesis of normality \[[@B69]\]. The independence between size and size FA within each sample was assessed by a linear regression of unsigned (![](1471-2148-5-7-i31.gif)) against mean centroid size ![](1471-2148-5-7-i32.gif). Scatter plots of left-right differences for each landmark after Procrustes superimposition were visually checked to see whether or not there was evidence for clustering of these vectors that would have argue for the occurrence of AS \[[@B13],[@B15]\]. No indication of AS was detected. Finally, to test for size effects on shape asymmetry within each sample we used multivariate regression of vectors of both signed and \"unsigned\" shape asymmetries onto mean centroid size \[[@B13]\]. Shape asymmetries were not related to size (*P*-values \> 0.10) and, therefore, no size corrections were necessary. Computer software for statistical analysis ------------------------------------------ The computer programs used for statistical data analyses were MATLAB (V.6. \[[@B72]\]) together with the collection of tools supplied by the Statistics Toolbox (V.3. \[[@B73]\]). Some helpful functions in morphometrics from the MATLAB toolboxes Res5 and Res6 developed by R. E. Strauss \[[@B74]\] were also used. Results (e.g., derivation of SSCP matrices) were checked with the statistical software packages STATISTICA V.6 \[[@B75]\] and SPSS V.11 \[[@B76]\]. Authors\' contributions ======================= MS conceived the study, carried out extraction of O chromosomes, experimental crosses, egg collections, statistical analyses, and drafted the final manuscript. PFI carried out extraction of O chromosomes, experimental crosses, egg collections, wing measurements, and preliminary statistical analyses and drafts of results. WC read all salivary gland squashes for gene arrangement identification and mounted the wings on microscope slides. All authors read and approved the final manuscript. Acknowledgments =============== We thank Chris Klingenberg for illuminating discussions during the preparation of the manuscript, for providing insightful comments on an earlier draft, and for sharing with us unpublished manuscripts. We are also grateful to Andrew Pomiankowski and two anonymous reviewers for their thoughtful and highly constructive criticisms that substantially improved the manuscript. PFI was supported by a postdoctoral fellowship (SB2000-0370) from the Secretaría de Estado de Educación y Universidades del Ministerio de Educación, Cultura y Deporte (Spain). WC is supported by a postgraduate fellowship (FP2000-7001) from the Ministerio de Ciencia y Tecnología (Spain). This work was supported by grants BOS2000-0295-C02 and BOS2003-05904-C02 from the Ministerio de Ciencia y Tecnología (Spain), 2001SGR-00207 from the Direcció General de Recerca (Generalitat de Catalunya) to the GBE, and by Fundación Ramón Areces (Spain).

PubMed Central

2024-06-05T03:55:52.587143

2005-1-22

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548280/", "journal": "BMC Evol Biol. 2005 Jan 22; 5:7", "authors": [ { "first": "Mauro", "last": "Santos" }, { "first": "Pedro Fernández", "last": "Iriarte" }, { "first": "Walkiria", "last": "Céspedes" } ] }

PMC548281

Background ========== Ribosomal protein (RP) S6 is a phosphorylated protein that resides on the small subunit of eukaryotic ribosomes. Phosphorylation occurs on a cluster of five serine residues near the C-terminal end of the protein. Although details remain unclear, the phosphorylation state of RPS6 is believed to influence translational efficiency of some mRNAs \[[@B1]\], possibly mediated by direct contact between RPS6 and the 28S rRNA in the large subunit. RPS6 has also been implicated in ribosome biogenesis, and is thought to play a conserved role in the initiation of protein synthesis \[[@B2]\]. In *Aedes aegypti*and *Aedes albopictus*mosquitoes, the RPS6 protein is \~17 kDa larger than its *Drosophila*homolog, and on polyacrylamide gels, it migrates as the largest protein from the small ribosomal subunit. *Ae. aegypti*and *Ae. albopictus*RPS6 cDNAs encode an approximately 100 amino acid extension at the C-terminal end of the protein. The extension is particularly rich in lysine, alanine and glutamic acid, and most closely resembles the sequence of histone H1 proteins from diverse sources \[[@B3]\]. Because RPS6 is thought to have regulatory function(s) in a variety of cell signaling pathways \[[@B2]\], we were surprised to uncover this difference between mosquito and *Drosophila*RPS6 proteins. We have recently shown that RPS6 protein isolated from ribosomal subunits retains its histone H1-like tail \[[@B4]\]. Thus, unlike the case with the ubiquitinated ribosomal protein S27a in the rat \[[@B5]\], the histone tail is not removed from the mosquito ribosomal protein prior to ribosome assembly. *RpS6*cDNA from an *Anopheles stephensi*cell line encodes an approximately 170 amino acid histone H1-like C-terminal extension, and in silico analysis reveals a similar modification encoded by the *rpS6*gene in *Anopheles gambiae*. In both *Aedes*and *Anopheles*mosquitoes, the C-terminal extension was completely encoded in Exon 3, directly contiguous with upstream open reading frame encoding the series of serines that may be phosphorylated \[[@B4]\]. Anopheline mosquitoes are believed to be ancestral to the Culicidae, which includes the genera *Aedes*and *Culex*\[[@B6]\]. Thus, to a first approximation, we infer that the longer tail in *Anopheles*mosquitoes represents the ancestral state, and that the RPS6 tail has been lost in the higher Diptera, which include *D. melanogaster*. Although mosquito RPS6 tails in general resemble histone H1 proteins, their divergence between *Aedes*and *Anopheles*mosquitoes was high, relative to the conventional portion of the RPS6 coding sequence. Because histone H1 is the most variable of the histone proteins, and functions as a linker, rather than as a component of the histone octamer, we set out to clone a cDNA encoding a bona fide histone H1 protein from an *An. stephensi*cell line. In a phylogenetic comparison, the *An. stephensi*histone H1 protein clusters with homologs from *Drosophila*and *Chironomus*, rather than with RPS6 histone H1-like tails from mosquitoes. These results indicate that the histone H1-like tails on mosquito RPS6 proteins are evolving independently of conspecific histone H1 proteins. Results ======= Design of PCR primers --------------------- The gene encoding *Drosophila melanogaster*histone H1 spans 1204 nucleotides, and encodes a 256 amino acid protein in a single exon \[[@B7]\]. There is a single recorded *His1*allele in *Drosophila*\[[@B8]\], while multiple histone H1 variants have been described in Chironomid flies \[[@B9]-[@B11]\]. When the deduced sequence of the *Drosophila*histone H1 protein (Accession NM\_058232) was compared to the *Anopheles gambiae*genome using the program BLAST \[[@B12]\] on the NCBI website (National Center for Biotechnology Information; <http://www.ncbi.nlm.nih.gov/>), we obtained 5 accessions with E values ranging from 3e-35 to 8e-43, distributed on mosquito chromosomes 2 and 3. Upon further examination, we noted that XP\_314184 and XP\_314186 (chromosome 2) corresponded to the same protein. Two additional histone H1 candidates (XP\_311486 and XP\_309451) were encoded on chromosome 3. These three conceptual *Anopheles*proteins shared 70--80% identity to one another, and about 50% identity to the *Drosophila*H1 protein sequence. In the EST-other database, we found a single uninformative match to an unidentified *An. gambiae*entry (dbEST id = 11236311), with the relatively modest E value of 0.055. Histone H1 sequences from *Aedes*mosquitoes are not yet in existing databases. The 50% identity between *Drosophila*and *Anopheles*histone H1 proteins was relatively low, compared to approximately 80% amino acid identity between *Drosophila*and *Anopheles*RPS6, exclusive of the histone-H1-like tail in the mosquito protein. The *Drosophila*H1 histone was also \~50% identical to that from *Chironomus thummi*, a fly closely related to mosquitoes in the infraorder/superfamily Culicomorpha \[[@B13]\]. To design primers that would amplify a histone H1 gene, and not the histone H1-like tail in mosquito *rpS6*, we aligned one of the *An. gambiae*H1 candidate proteins (XP\_311486) to a histone H1 protein from *C. thummi*, and examined the alignment for precise matches (Fig. [1A](#F1){ref-type="fig"}) that did not match well in a separate alignment of the *An. gambiae*histone H1 protein with the *An. gambiae*RPS6 tail (Fig. [1B](#F1){ref-type="fig"}). The forward primer (F1) corresponded to amino acids PKKPKKP in *An. gambiae*, and a reverse primer (R1) corresponded to residues AAKKPKA (Fig. [2](#F2){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Primer design. To design primers, we aligned an *An. gambiae*putative histone H1 candidate XP\_311486 (Panel A, top) with a histone H1 protein (Q07134; Panel A, bottom) from *C. thummi*. Boxed residues were chosen for design of primers, according to the *An. gambiae*nucleotide sequence. Panel B shows these primer residues aligned between the *An. stephensi*RPS6 tail (top), and the putative *Anopheles gambiae*histone H1 (bottom). Vertical bars designate identities. ::: ![](1471-2164-6-8-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Sequence of *An. stephensi*histone H1 gene. The positions of internal primers F1 and R1, and primers F2 and R2 are designated by arrows. The ATG start codon and TAA stop codon are boxed. ::: ![](1471-2164-6-8-2) ::: Recovery of *An. stephensi*histone H1 gene ------------------------------------------ We used F1 and R1 primers with *Hin*dIII-digested genomic DNA from *An. stephensi*cells to obtain an approximately 450 bp PCR product, which was sequenced and verified to encode a histone H1 protein. The 5-end of the gene, which extended 81 nucleotides upstream of the ATG start codon, was obtained using primer R1 with the GeneRacer kit (Invitrogen, Carlsbad, CA), with total RNA as the template. The absence of a poly(A) tail on histone mRNAs required an unconventional strategy to obtain the 3\'-end of the coding sequence. First, we used *Hin*dIII-digested genomic DNA template, with a primer based entirely on the 3\'-UTR of *An. gambiae*XP\_314184, without success. When we designed a second primer (R2, in Fig. [2](#F2){ref-type="fig"}) extending from the 3\'-UTR through the TAA stop codon and into the coding region, we obtained the 3\'-end of the coding sequence. Finally, primers F2 and R2 (Fig. [2](#F2){ref-type="fig"}) were used to verify the complete nucleotide sequence. Southern blots with *An. stephensi*genomic DNA ---------------------------------------------- The likelihood that the mosquito genome contains multiple histone H1 gene variants is consistent with the multiple H1 variants that have been described in *Chironomus*\[[@B9]-[@B11]\] and eight histone H1 subtypes that have been described in mammals \[[@B14],[@B15]\]. When we used the *An. stephensi*cDNA to probe Southern blots of genomic DNA digested with various restriction enzymes with 6 bp recognition sites, most enzymes gave multiple bands, with the notable exception of *Bam*HI, which hybridized to a single band longer than 10 kb (Fig. [3](#F3){ref-type="fig"}). Based on the observation that *D. melanogaster*H1, H2A, H2B, H3 and H4 histone genes are organized in approximately one hundred 5 kb repeats per haploid genome \[[@B16]\], the large *Bam*HI fragment from *An. stephensi*may be a starting point for recovery of a complete cluster of the *An. stephensi*histone gene family. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Southern blot of *An. stephensi*genomic DNA hybridized to the *An. stephensi*histone H1 probe. DNA was digested with *Bam*HI (B), *Eco*RI (E), *Hin*dIII (H) and *Pvu*I (P). Positions of size markers are shown at right. ::: ![](1471-2164-6-8-3) ::: The *An. stephensi*nucleotide sequence (GenBank accession \# AY672907) matched *An. gambiae*histone H1 candidates on chromosomes 2 and 3 with an E value of 0.0. In addition, 6 unmapped sites also had E values of 0.0. A final two sites had E values of 4e-170 and 3e-127. The deduced *An. stephensi*protein sequence was 92% identical to *An. gambiae*protein XP\_314184 on chromosome 2 (Fig. [4A](#F4){ref-type="fig"}). A similar level of identity was obtained with *An. gambiae*XP\_309451 on chromosome 3, but the alignment required introduction of a 58 amino acid gap in the shorter (190 residue) deduced *Anopheles gambiae*protein (not shown). Identity with *An. gambiae*XP-311486 was 79%. Based on these criteria, we have cloned the *An. stephensi*homolog of *An. gambiae*XP\_314184. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Comparison of mosquito histone H1 proteins and RPS6 histone H1-like tails. Panel A shows the alignment of the experimentally-determined *An. stephensi*histone H1 amino acid sequence, compared to *An. gambiae*conceptual protein XP\_314184. Panel B shows a phylogram produced in PAUP\* by neighbor joining, with the nematode *C. elegans*histone H1-like protein 2 (AAM44399) designated as the outgroup. The alignment includes histone H1 proteins from various Diptera, and the known histone H1-like tails on mosquito RPS6. Values on the horizontal lines indicate branch lengths, defined as the fraction of substitutions between the nodes that define the branch. Bootstrap values based on 1000 replicates are shown within circles. A single tree with identical topology was obtained with the optimality criterion set to parsimony. ::: ![](1471-2164-6-8-4) ::: Comparisons of histone H1 proteins with mosquito RPS6 C-terminal extensions --------------------------------------------------------------------------- The identity between *Drosophila*and *Anopheles*(or *Drosophila*and *Chironomus*) histone H1 proteins was only 50%. This divergence undoubtedly reflects the \~250 million years \[[@B6]\] separating Nematoceran from Cyclorrhaphan diptera. In this study, we were interested in comparing mosquito histone H1 proteins to the histone H1-like tails of mosquito RPS6. Fig. [4B](#F4){ref-type="fig"} shows a neighbor-joining analysis in which we compared protein sequences from *Aedes*and *Anopheles*RPS6 histone H1-like tails, exclusive of the conventional RPS6 protein sequence, with histone H1 proteins from the nematode *Caenorhabditis elegans*(AAM44399), the closely-related flies *Chironomus thummi*(Q07134) and *Chironomus tentans*(AAB62239), *Drosophila*, and the *Anopheles gambiae*and *Anopheles stephensi*homologs (Fig. [4A](#F4){ref-type="fig"}). With the *C. elegans*sequence designated as the outgroup, the phylogram shows that the RPS6 tails cluster into a distinct group relative to the Dipteran histone H1 proteins. Circled values indicate bootstrap values based on 1000 replicates. When the analysis was repeated with the optimality criterion set to parsimony, we obtained a tree with the same topology, with the 77% value shown in Fig. [4B](#F4){ref-type="fig"} reduced to 59%, and the 97% value reduced to 94%. The 100% values remained unchanged. In an alignment of mosquito RPS6 tails with the *Anopheles*H1 histones (Fig. [5](#F5){ref-type="fig"}), we note that while some degree of identity covers the entire histone H1 protein, the C-terminal half of the H1 histone has a higher proportion of identities to the RPS6 tail, as indicated by the distribution of consensus residues. Within the RPS6 tails, however, the boxed motifs:VAKK(D/E)A, KKEVKK, AAPA, KKEAPKRKPE, KG(D/E)ASAAK(E/D) are shared by all four mosquitoes. In contrast, the additional amino acids in the *Anopheles*RPS6 tails, which are represented by gaps in the *Aedes*sequences (Fig. [5](#F5){ref-type="fig"}), did not show regions of homology with *Anopheles*histone H1. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Alignment of mosquito RPS6 tails with mosquito histone H1 proteins. Angam (CAD89874), *An. gambiae*; Anstep (AY237124), *An. stephensi*; Aealbo (Q9U762), *Ae. albopictus*; Aeaegy (Q9U761), *Ae. aegypti*. The alignment was produced with ClustalX (version 1.83), using default settings. Indicators of consensus residues are shown below the alignment. Boxes in the top four entries indicate identities (aside from D, E substitutions) shared by the mosquito RPS6 tails. ::: ![](1471-2164-6-8-5) ::: Discussion ========== An important rationale for cloning an *An. stephensi*histone H1 was to compare its sequence to the histone H1-like tails on mosquito RPS6 ribosomal proteins. Our choice of an *Anopheles*histone H1 was based on the existing database for *An. gambiae*, the observation that the tail in *Anopheles*RPS6 is nearly twice as long as that in *Aedes*RPS6 proteins \[[@B4]\], and evidence that the genus *Anopheles*is ancestral to *Aedes*\[[@B6]\]. Because putative homologies to *Drosophila*histone H1 protein could be recovered as conceptual translation products from the *An. gambiae*database, we used these sequences to design primers that would discriminate between an *An. stephensi*histone H1 gene, and the histone H1-like extension in *An. stephensi*RPS6. Because the *Drosophila*gene was encoded in a single exon, and the histone message was unlikely to be polyadenylated \[[@B14]\], we used genomic DNA from *An. stephensi*as a template for our PCR reaction. The gene we recovered had more than 90% identity to XP\_314184 in *An. gambiae*. The proteins differed in length by a single amino acid residue, and showed 92 % identity. When we analyzed RPS6 tails and histone H1 genes, we found that the Dipteran histone H1 proteins and the RPS6 tails each fell into distinct groups, suggesting that in present-day mosquitoes, these proteins are evolving independently. Although these data are consistent with the possibility that present-day histone H1 proteins and the histone H1-like tails on mosquito RPS6 protein share a common ancestral gene, the histone tails seem to be evolving independently in the two mosquito genera, and have changed more rapidly than the conventional portion of mosquito RPS6 proteins. Because RPS6 is considered an important functional component of the ribosome, it seems surprising that a histone H1-like tail occurs at the C-terminal end of this particular protein. However, histone H1-like tails have been reported at the N-terminus of *Drosophila melanogaster*ribosomal proteins L22 and L23a \[[@B17]\]. The *An. gambiae*homolog of *D. melanogaster*L23a also contains an N-terminal histone-like extension. The N-terminal tails of *Drosophila*L22 and L23a were found in an effort to identify proteins that interact with poly (ADP-ribose) polymerase (PARP). In future studies, we plan to explore whether the histone H1-like tail undergoes posttranslational modification, and whether it plays a functional role in ribosome biogenesis, perhaps through the activity of PARP. Experimental procedures ----------------------- ### Mosquito cells and culture conditions We used the ASE-IV *Anopheles stephensi*mosquito cell line \[[@B18]\], which was adapted to Eagle\'s minimal medium, supplemented with non-essential amino acids, glutamine and 5% heat-inactivated fetal bovine serum \[[@B19]\]. This formulation is called E-5 medium. ### Genomic DNA preparation Cells grown as suspended vesicles for 4 to 5 days in twenty 60 mm plates were collected by centrifugation, and the cell pellet was washed twice with phosphate-buffered saline (PBS; \[[@B20]\]). The cell pellet was resuspended in 20 ml lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM EDTA, 200 μg/ml proteinase K), and SDS was added to a final concentration of 0.5%. The lysate was incubated at 37°C overnight. NaCl was added to a final concentration of 0.4 M, and the DNA was extracted once with 20 ml phenol, twice with an equal volume of phenol:chloroform (1:1), and twice with an equal volume of chloroform. Two volumes of ethanol were added, and DNA was spooled onto a clean glass rod. The DNA was dried, and dissolved in 10 ml of TE (10 mM Tris-HCl, pH 8.0, containing 1 mM EDTA) at 37°C. RNase A was added to a final concentration of 200 μg/ml and incubated at 37°C for 4 hours. DNA was phenol extracted, ethanol precipitated and dissolved in TE as described above. ### DNA amplification by PCR Genomic DNA (0.4 mg) was digested with *Hin*dIII (Promega) at 37°C overnight. Enzyme was removed by phenol:choloroform extraction, and the DNA was recovered by precipitation with ethanol and dissolved in TE. Digested DNA (100 ng) was used as template for the PCR reaction, which contained 1X PCR buffer, 1.5 mM MgCl~2~, 0.2 mM of each of the four dNTPs, 0.4 μM of primer F1 (5\'CCG AAG AAG CCG AAG AAG CCC) and R1 (5\'TGC TTT CGG CTT CTT GGC AGC) and 2.5 units of Taq DNA polymerase (Promega, Madison, WI). PCR was performed with an initial denaturation at 94°C for 2 minutes. The next 35 cycles included 94°C denaturation for 45 sec, 55°C annealing for 1 minute, and 72°C extension for 1 minute. The reaction was terminated by a final elongation cycle at 72°C for 2 minutes. The PCR product was recovered from a 0.9% agarose gel, purified using Ultra-Clean 15 (MO Bio Laboratories Inc., Solana Beach, CA) and cloned into PGEM T-Easy vector (Promega). The 3\'-end of the gene was obtained in a similar manner, using primers R2 (Fig. [2](#F2){ref-type="fig"}) and F1. ### Amplifying the 5\'-end of the cDNA Total RNA was recovered from ASE-IV cells by guanidine isothiocyanate extraction and cesium chloride centrifugation as described by Davis et al. \[[@B21]\]. The final RNA pellet was dissolved in DEPC-treated water and stored at -70°C. RNA (1 μg) was used with the GeneRacer kit (Invitrogen) to obtain the 5\' end of the mRNA, using primer R1 as the reverse primer. ### Programs and accession numbers The analysis in Fig. [4A](#F4){ref-type="fig"} was produced using the Genetics Computer Group (GCG; Madison, WI) program \"gap\". The tree in Fig. [4B](#F4){ref-type="fig"} and the alignment in Fig. [5](#F5){ref-type="fig"} were produced by an alignment of amino acid residues using default parameters of Clustal X (version 1.83) \[[@B22]\]. The tree was created in PAUP\* \[[@B23]\], with the *C. elegans*H1 protein designated as an outgroup. The *An. stephensi*histone H1 sequence has GenBank accession \# AY672907. Authors\' contributions ======================= YZ did the experimental work, AMF helped with experimental design and manuscript preparation. Both authors read and approved the final manuscript. Acknowledgments =============== We acknowledge support from the National Institutes of Health (AI 20385) and from the University of Minnesota Agricultural Experiment Station, St. Paul, MN.

PubMed Central

2024-06-05T03:55:52.595713

2005-1-24

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548281/", "journal": "BMC Genomics. 2005 Jan 24; 6:8", "authors": [ { "first": "Yongjiao", "last": "Zhai" }, { "first": "Ann M", "last": "Fallon" } ] }

PMC548282

Background ========== Relatively little documentation supports a scientific basis for alternative healing therapies involving the manipulation of a purported healing energy associated with the body. Despite this paucity of evidence, such *energy healing*modalities are becoming increasingly popular. Recent surveys indicate the majority of the United States public has used alternative medical therapies, and that energy healing therapies are among the fastest growing complementary and alternative medicine treatments \[[@B1],[@B2]\]. Johrei is one such energy healing practice with origins in Japan. Johrei was founded by Mokichi Okada in the 1920\'s and is now practiced worldwide with significant numbers of adherents in Asia, Europe and the Americas. Practitioners of Johrei believe it possible to improve the health of others by directing a universal healing energy toward them. The Johrei philosophy, as it relates to healing energy, emphasizes what can be described as \"spiritual purification.\" Other aspects of the Johrei philosophy include fostering an appreciation of beauty, and a form of organic farming. Two scientific studies evaluating Johrei healing practices have found provocative results. In one, Johrei was shown to have a beneficial affect on the mood of practitioners \[[@B3]\]. Results from a second study suggested that those practicing Johrei may display an immune profile consistent with stress reduction \[[@B4]\]. Clinical studies evaluating potential efficacy of Johrei are ongoing \[[@B5],[@B6]\]. An in vitro study revealed an apparent influence of Johrei treatment on the germination rate of irradiated seeds but the study did not follow standard scientific methods \[[@B7]\]. To investigate whether Johrei can have a direct effect on human cells, we exposed cultured cancer cells to Johrei treatment from a short distance. Johrei treatments were maintained continuously for four hours by teams of Johrei practitioners. Experiments were conducted using strict blinding procedures, exceeding standards commonly used to evaluate conventional therapeutics. Methods ======= Overall study design -------------------- For each session Johrei practitioners were asked to direct healing intention toward cell cultures in a temperature, CO~2~, and humidity controlled time-lapse microscope incubator chamber. Johrei practitioners participated in teams of two, alternating every half hour such that a total of 4 hours of Johrei treatment was delivered. A total of eight control and eight Johrei intervention experiments were accomplished. Johrei treatments were initiated after 4 hours of baseline data had been collected and the observation period extended for a total of 22 hours. We quantified tumor cell death and proliferation throughout the observation period. We also quantified the rate of cellular emigration (i.e. how many tracked cells left the microscopic field per hour) in order to accurately assess cell population dynamics (i.e. cell death and proliferation rates). Blinding procedures ------------------- Experiments were conducted with blinding applied to each of the scientists based on previously reported methods \[[@B8]\]. The experimental protocol was divided among scientists such that those responsible for preparation of the cell cultures, data acquisition, and data analysis were blind to each other\'s activities and results until data analysis was complete. Cell culture ------------ As the target of Johrei healing intentionality in these studies we used a human cell line derived from a brain tumor biopsy specimen from a patient with glioblastoma multiforme (SF188 GBM). This cell culture model is used widely and can show responsiveness to conventional therapeutic agents including ionizing radiation and chemotherapeutics. SF188 GBM cells were grown in RPMI media supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate, and 0.25 μg/ml amphotericin. A fresh aliquot of cryogenically preserved cells was thawed at the start of each experiment to ensure uniformity in the genetic profile of the target cells throughout the experiments. Cells were plated at a density of 5,000 cells per well in six-well culture plates and allowed to grow uninterrupted for 20 hours in a humidified incubator maintained at 37°C and 5% CO~2~before beginning each time-lapse experiment. Time-lapse microscopy --------------------- For each experiment cell cultures were transferred from the incubator to a time-lapse microscope in our laboratory equipped with a heated stage, CO~2~chamber, and a plexi-glass environmental chamber (Axiovert 200; Zeiss, Gottingen, Germany). Cell cultures were maintained at routine incubation settings (37°C, 5% CO~2~) and optimum humidity. Temperature and CO~2~concentration were independently maintained using digital controlling units (Zeiss, Gottingen, Germany). Two sets of phase contrast images (100 X magnification) from each well were taken in 300 second intervals using a Cohu 2600 Series compact monochrome interline transfer CCD camera. Images were acquired during a four-hour baseline period and then continuously for another 18 hours. An Openlab software automation (Improvision, Lexington, MA) drove the camera and stage movements, and compiled the acquired phase images. Images were subsequently processed as Quicktime movies using Openlab. Johrei intervention ------------------- Johrei practitioners were selected based on experience and willingness to participate by the Center for the Science of Life, a Johrei organizational body in the United States. Five Johrei practitioners with a minimum of 17 years experience participated in the experiments in teams of two. Johrei treatment was administered by the practitioner teams for a total of four hours, beginning after the four-hour baseline period. This exceptionally long treatment period (4 hours) was chosen as the highest \"dose\" that was practical within the experimental model. Each practitioner treated the cells for a total of 2 hours per experiment, switching every half hour with the team member. Treatment began with one of the two practitioners being seated in front of the time-lapse microscope and raising one hand toward the cellular target. One hand remained raised toward the cell cultures for the duration of any given Johrei treatment. Johrei treatments were delivered from a distance of 6 -- 9 cm, from outside the plexiglass environmental chamber. In collaboration with The Center for the Science of Life, we developed a set of standard mental procedures for practitioners to follow to minimize variability in the mental states among practitioners. These mental cues were used by all practitioners in all experiment. The mental cues can be summarized briefly as: 1) establishing a connection to the divine, 2) consciously relaxing body and mind, 3) visualizing healing energy penetrating the cellular target, 4) taking enjoyment in participating in the experiment, 5) maintaining a feeling of gratitude. Time-lapse microscopy data analysis ----------------------------------- Every cell in the initial microscopic field was identified and numbered. For each experiment, we counted the initial number of cells in each of 12 microscopic fields. All numbered cells and their progeny were then tracked for the duration of their onscreen viability using compiled Quicktime movies. Each cell\'s life events were identified using a modified version of a previously described cell pedigree system \[[@B9]\]. Cells identified as dead at the start of the video or that entered the microscopic field after the initial frame were not included in this analysis. Cataloged data was entered into a Microsoft Excel spreadsheet (using blinding codes) for further analysis. Cell deaths and divisions occurring per half hour were recorded. Statistical analysis -------------------- Statistical analysis was based on a model which categorizes a cell as engaged in any one of four activities at any time: 1) division, resulting in an additional cell (division), 2) death, resulting in the loss of a cell (death), 3) movement out of the microscopic field (emigration), or 4) the cell can remain unchanged \[[@B10]\]. Under this model three transition probabilities plus the total number of cells at a previous time determine the number of cells at a future time. The expected number of cells at time t, N(t), is given by the equation N(t) = N(t-1) exp(λ(t) - μ(t) - ν(t))\], where λ(t), μ(t) and ν(t) are the transition probabilities for division, death and emigration, respectively, at time t. We estimated the transition probabilities in one-hour time blocks. For example, the estimate for λ(t) is λ(t) = \[ln(N(t) + div(t)) - ln(N(t-1))\], where div(t) is the number of divisions during (t-1, t). A similar equation was used for estimating death and emigration transition probabilities at each hour. Statistical analyses focused on two types of comparisons: comparisons within the Johrei treatment experiments, where division and death rates (transition probabilities) pre-treatment were compared with those during and post-treatment; and comparisons between Johrei and control experiments at similar times. Data from each experiment was pooled at 30 minute time intervals. Each of the cellular events (division, death or emigration) occurs infrequently to each cell, so it is necessary to pool results from many cells in order to have nonzero data. Additionally, in instances where few events were recorded due to relatively few cells being observed it was necessary to pool over several time intervals in order to have enough data to perform statistical tests. Results ======= Overall we documented the behavior of 336 cells in the eight control experiments and 351 cells in the eight Johrei intervention experiments. Initial numbers of cells per microscopic field ranged from 5 to 10; the average cell count per field was 7.0 for controls and 7.3 for Johrei. The numbers of cells per experiment ranged from 40 to 48 with averages of 42.0 and 43.9 for controls and Johrei. Observation over 22 hours showed 312 divisions, 15 deaths and 17 emigrations in the control experiments and 316 divisions, 21 deaths and 7 emigrations in the Johrei experiments. Differences in number of divisions or deaths is not significant (p = 0.19 for divisions and p = 0.37 for deaths based on chi-square test comparing frequencies). While the difference in emigrations is statistically significant (p = 0.03, chi-square test comparing frequencies), this was largely affected by a difference present in the 0--4 hour baseline period making it unlikely that the observed effect is due to Johrei treatment. Experimental treatments (control or Johrei) were confined to a four-hour period starting after collection of four hours of baseline data. During the four-hour treatment period the numbers of divisions were 84 (control), 59 (Johrei); deaths were 4 (control), 5 (Johrei); and emigrations were 0 (control) and 2 (Johrei). Taking into account the numbers of cells at the start of the treatment period, only the difference in divisions is statistically significant (p = 0.018 based on comparing the division rates during the treatment period). This result is based on pooling observations from all cells over the four-hour period and the p-value is based on an assumption that counts observed follow a Poisson distribution. It is possible that counts do not follow a Poisson distribution and that their variability is greater than that expected for Poisson counts. To examine this we calculated division rates separately for each of the eight control experiments and eight Johrei experiments and then compared these rates using the nonparametric Mann-Whitney rank sum test. This analysis confirmed the result based on the total counts (p = 0.016 for Mann-Whitney test). We also tried fitting parametric models to the data in an effort to gain degrees of freedom for performing statistical tests. For example, we tried fitting low order polynomials to division rates over time, but the fits were unsatisfactory, as judged by assuming counts were Poisson distributed with rates predicted from the polynomial fit. The fits were particularly deficient in following the rapid initial rise in division rates over the first four hours of baseline data (Figure [1](#F1){ref-type="fig"}). We obtained similar results attempting to fit a sum of exponentials model (Figure [2](#F2){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Rates of cell division are shown as plotted against time in hours. The plot depicts a decrease in divisions in Johrei treated cells (pink line) during the 4.0--8.0 treatment period which is statistically offset by a similar dip in the control period. Control and pooled data are depicted by green and blue lines respectively. The solid black line depicts the fit of a 4^th^degree polynomial to half-hourly cell division rates for all experiments. ::: ![](1472-6882-5-2-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The fit of the sum of two exponentials to pooled division rate data for all experiments is depicted. The maroon line is pooled data and the blue line is fit. ::: ![](1472-6882-5-2-2) ::: The division rates for control and Johrei experiments plotted in Figure [1](#F1){ref-type="fig"} suggest another method of comparison. The experimental conditions should be identical during the 0--4 hour baseline period, yet we see wide variation in division rates for the two conditions. We can measure the variance between control and Johrei experiments at each half-hour time point and use the pooled value, over the first eight time points, as a baseline standard. We computed the pooled variances for the 4--8 hour time period (treatment) and compared the two using an F-test for equality of variances. The pooled variance for the 0--4 hours period was 5.8 × 10^-5^compared to 6.8 × 10^-5^for the 4--8 hours period. The ratio of these is 1.17 which is not statistically significant (p = 0.41 based on F distribution with numerator and denominator df = 8). This suggests that the discrepancies between the rates during the 4--8 hour period are statistically no larger than those observed during the 0--4 hour period when there were no treatment differences. This comparison requires no assumption of Poisson variability of counts and takes advantage of a priori knowledge that treatment conditions were identical in the first four hours of the experiments. The only assumption is that the variances arise from normally distributed data, so this requirement was tested. A Mann-Whitney comparison of the ranks of the absolute differences in control and Johrei rate during 0--4 hours vs. 4--8 hours give a p-value of 0.92. Thus, there appears to be no evidence that differences in rates of division during 4--8 hours for control and Johrei were any different from those during 0--4 ours. A plot of these absolute differences is shown in Figure [3](#F3){ref-type="fig"}. The original data are provided as supplementary material \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Absolute differences in half-hourly rates of cell division for all experiments are depicted. The plot suggests that differences between control and Johrei experiments were largest during the first 8 hours and that the differences during Johrei treatment (4--8 hrs) were not statistically larger than those during the previous baseline time period, when the treatment conditions were identical. ::: ![](1472-6882-5-2-3) ::: Discussion ========== Our initial examination of the data suggested that Johrei may have affected the rate of division of cultured human brain tumor cells. This analysis, however, was based on the assumption that the baseline period in both the control and Johrei treated cultures were statistically identical with respect to cell divisions and cell deaths. Subsequent analysis revealed that a significant difference existed in the baseline period between control and Johrei treated samples, with Johrei cultures exhibiting fewer divisions. Taking this into account it appears that there was no observable effect of Johrei on these cell behaviors. The failure to observe evidence of a reproducible cellular response to Johrei treatment is consistent with prior studies in our laboratory evaluating another popular energy medicine modality, external Qigong. Like Johrei, practitioners of external Qigong generally claim the ability to emit or direct healing energy to treat patients. Qigong practices originate from China and are based on the manipulation of a purported healing energy called \"Qi.\" Our prior study investigated the ability of experienced Qigong practitioners to enhance the growth of normal human brain cells in culture as measured by a colony-forming efficiency assay. Following a rigorously designed protocol with randomization, blinding and controls for variability, we did not observe reproducible effects of external Qigong treatment on the growth of these cells \[[@B11]\]. Such \"negative\" data do not negate the possible therapeutic effects of such practices. Cultured cells offer an incomplete system that may not be sensitive to treatments of this kind. More complete models (e.g., a clinical research model) may be more useful to evaluate Johrei treatment for a variety of reasons. Conclusion ========== Cell death and proliferation rates of cultured human cancer cells do not appear responsive to Johrei treatment from a short distance. Competing interests =================== The Center for the Science of Life, a Johrei organizational body in the United States, has provided funding for this work. Authors\' contributions ======================= RT and GY conceived of the study and participated in its design, coordination and implementation. DM performed statistical analysis. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6882/5/2/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Johrei Experimental Data. The supplementary file includes the raw data for all experiments, including number of events for cell death, division, and emigration by half-hourly intervals and cumulative totals. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This work was supported by grants from the Samueli Institute for Information Biology, the Rockefeller-Samueli Center for Research in Mind-Body Energy and the Center for the Science of Life. The authors thank Tri Luu and Aly Pennucci for preparation of samples and performing data analysis.

PubMed Central

2024-06-05T03:55:52.597687

2005-1-24

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548282/", "journal": "BMC Complement Altern Med. 2005 Jan 24; 5:2", "authors": [ { "first": "Ryan", "last": "Taft" }, { "first": "Dan", "last": "Moore" }, { "first": "Garret", "last": "Yount" } ] }

PMC548283

Background ========== In the Netherlands, little information is available about prevalence, incidence, etiology and prognosis of pregnancy-related pelvic girdle pain (PPGP). It is hypothesized that during pregnancy many women (about 80%) experience some degree of pain in the pelvic region and/or the low back and that in some of these patients pain becomes chronic or recurrent. Often, symptoms impact on activities of daily life, hobbies, participation in society, planning of next pregnancies and sometimes lead to a chronic disabling condition with considerable work absenteeism in the future \[[@B1]\]. Treatment and indirect costs of these chronic or recurrent patients constitute a considerable burden on health care services, health care insurers and other parties. The total health care expenditures incurred by patients with back pain in the United States in 1998 were approximately \$91 billion, accounting for about 1 % of the Gross National Product \[[@B2]\]. Van Tulder et al. estimated the costs of back pain for Dutch society in 1991 at 1.7% of the Gross National Product \[[@B3]\]. These costs consist almost totally of indirect costs (97%) such as absenteeism and disablement; for that reason chronic back pain can be considered a major economical problem. It is therefore important that PPGP can be diagnosed and treated before PPGP becomes chronic. Consequently, tracking risk factors and characteristics influencing etiology and prognosis of pregnancy-related pelvic girdle pain is important. Absence of knowledge of risk and prognostic factors and the absence of evidence-based treatment strategies about PPGP has prompted the start of a large cohort study in the Netherlands. In January 2000, the Maastricht PPGP cohort study started. It was established (I) to examine the prevalence and incidence of pregnancy-related pelvic girdle pain (PPGP) during and after pregnancy, (II) to identify risk factors involved in the onset of PPGP and to identify which factors can play an important role in the early detection of PPGP and finally (III) to determine the prognosis of PPGP and to identify prognostic factors. Furthermore, a clinical trial is embedded in this cohort, aimed at studying the effectiveness of a tailor-made treatment program in PPGP after delivery \[Bastiaenen et al., treatment, submitted\]. Methods/design ============== Design and study population --------------------------- In an observational prospective cohort study, etiology and prognosis of PPGP will be studied in 7526 pregnant women. The source population for the study comprises of pregnant women from the southeastern area of the Netherlands. Both midwives and gynecologists recruit women for the study when pregnant between 10 and 14 weeks. Women are considered eligible if they meet the following inclusion criteria: women are well versed in the Dutch language and at least 18 years. At inclusion, women receive a leaflet containing information about the research project, an informed consent form and a baseline questionnaire. After providing informed consent and filling out the first questionnaire, the women receive a second questionnaire at 30 weeks of pregnancy, a third one at 2 weeks after delivery and a fourth and fifth at 6 months and 12 months postpartum, respectively. In this study, extensive questionnaires are used with questions ranging from physical complaints (past and present), limitations in activities, restriction in participation, work situation, demographics, lifestyle, pregnancy-related factors and psychosocial factors. With the help of these questionnaires the prevalence and incidence of PPGP in the Netherlands will be described. In addition, possible risk factors and prognostic factors of PPGP will be examined. Exposure variables ------------------ In the Maastricht PPGP cohort study several domains of exposure were measured, including individual characteristics, lifestyle, work situation, pregnancy-related factors and psychosocial factors. The majority of factors were assessed with existing, validated questionnaires. The Dutch translation of the Quebec Back Pain Disability Scale (QBPDS) \[[@B4]\] will measure low back functional status \[[@B5]\]. The QBPDS is a 20-item 6-point scale describing activities commonly affected by back pain. This questionnaire is not developed to study a pregnant population and some activities were unsuitable for women who were pregnant or just gave birth. We therefore added a 7^th^option to the questionnaire, namely \"not applicable\". We also changed the phrase \"because of my back \" into \"because of my back and/or pelvic pain\" in questionnaires. Fear of movement is measured by means of the Dutch translation of the Tampa Scale for Kinesiophobia (TSK) \[[@B6],[@B7]\]. The TSK consists of 17 items; each rated on a 4-point Likert scale. Pain catastrophizing is measured by the Pain Catastrophizing Scale (PCS) \[[@B8]\]. The PCS is a 13-item 5-point scale. A woman is said to catastrophize pain, when she views pain as extremely threatening. To measure the experience of negative affect and positive affect we used the 14-item Negative Emotionality Scale (NEM) and the 11-item Positive Emotionality Scale (PEM) \[[@B9]\]. Current mental health was measured by the General Health questionnaire (GHQ). The questionnaire was originally designed as a 60-item instrument, but we used the shortened version GHQ-12 \[[@B10]\]. The perceived stress scale (PSS) was used to measure to assess stress. The PSS is a 14-item instrument with a 5-point scale \[[@B11]\]. Outcome measurements -------------------- Pregnancy-related pelvic girdle pain is currently not an entity that can be clearly diagnosed and described. Therefore, Bastiaenen et al. studied separate diagnostic strategies of four international authors in the field of PPGP \[Bastiaenen et al., submitted\]. They concluded that there was no similarity in the selection of patients with PPGP between the authors. Most of these classification-strategies of PPGP are based on expert-opinions. Therefore, a possible reason for the lack of similarity in the selection of patients can be that they all select different small parts of the same large patient-group. Because of the relatively unknown etiology of pregnancy-related pelvic girdle pain and the lack of an all-embracing definition, we will use an extensive description of PPGP. We expect that during pregnancy almost all women experience some form of pain in the lower back, the buttocks, the symphyses, the groins and/or radiation into the legs. This pain is probably caused by hormonal and physiological changes which are considered normal during pregnancy. However, some women experience pain in a very early stage of pregnancy while others only experience pain in the final stages of pregnancy. In addition, some women are more limited in their activities (due to pain) than others. This suggests that other factors might influence the hormonal or physiological changes during pregnancy \[[@B12]\]. Most women who had developed PPGP during pregnancy quickly recover after delivery \[[@B13]\]. In this study, pain during or after pregnancy is measured by using patients\' self-reports. Women with pain can be identified by the question whether they experienced pain in the lower back, the buttocks, the symphyses, the groins or radiation into the legs during or after this pregnancy. To study etiology of PPGP, women who gave a positive answer to this question during this pregnancy were selected. For the prognosis of PPGP it is important that women have pain that started during pregnancy and persisted after delivery. At several moments during and after pregnancy, experienced pain in the lower back, the buttocks, symphyses, groins or radiation into the legs was measured. The answers to this question were assimilated into a flowchart (Fig. [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Flowchart of pain in the lower back, the buttocks, the symphyses, groins and/or radiation into the legs: description of cases. ::: ![](1471-2458-5-1-1) ::: Based on their self-reports, the women are stratified into a case group without a history of LBP/PPGP or a case group with a history of LBP/PPGP. Some women (N = 246) were not classified into groups for the following reasons; specific disorders of the spine, rheumatism, neurological disorders and cancer. The first group consists of women without a history of LBP/PPGP, but they experience pain during pregnancy and this pain does not disappear until (at least) 2 weeks after delivery. The second group experiences pain during and after pregnancy, but they also have a history of LBP/PPGP. Both groups of cases will be analysed to study the prognosis of PPGP. Women who experience recurrent pain episodes during pregnancy, that resolve within 2 weeks after delivery, will not be considered cases in the analyses for the prognosis of PPGP. They form a miscellaneous group. However, this miscellaneous group and women who experience no pain after delivery were not excluded from follow-up. Data analyses ------------- Participation rates and descriptions of baseline characteristics of the Maastricht PPGP cohort study will be presented. We calculated prevalence rates for PPGP by dividing the numbers of prevalent cases at several moments during pregnancy by the total number of subjects. Characteristics of the study population at baseline --------------------------------------------------- Recruitment of women into the study began in November 2000 and ended in November 2002. Approximately 10,850 women were asked to participate in the study by midwives and gynecologist in the southeast of the Netherlands. The locations of the participating midwives and gynecologists are shown in Fig. [2](#F2){ref-type="fig"}. At the end of the recruitment period 7,526 pregnant women (73.4%) were willing to participate and were included in the cohort. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### The location of participating midwives and gynaecologists in the Netherlands. ::: ![](1471-2458-5-1-2) ::: In Table [1](#T1){ref-type="table"} a number of selected characteristics of the study population at baseline are presented, including details of their age, education, BMI, smoking, work and reproductive history. Mean age of the study population is 31.5 years. The educational level of the participants is very high. Approximately 38 % of the participants have had higher vocational or academic education. The use of alcohol during pregnancy is limited. While 44.8% of the study population did not use alcohol before pregnancy, 91.2% did not use alcohol during pregnancy. With exception of the country of birth, the study population is heterogeneous with respect to demographics, work status and pregnancy-related factors. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of the study population (N = 7526) at baseline (14 wks pregnancy) ::: **Aspect** \% ----------------------------------------------- --------------------------------------------------------------------------------------- -------------------- ------- ------ Age (yrs) Mean 31.54 N = 7523 \< = 20 0.4 21--25 5.3 26--30 32.4 31--35 47.8 36--40 12.8 \>40 1.2 Country of origin Netherlands 96.5 N = 7516 Belgium, Germany, France, Austria, Switzerland, Luxemburg, Ireland and United Kingdom 1.9 Other countries 1.6 Education Primary school 0.6 N = 7498 Preparatory vocational education 7.2 Lower general secondary education 6.6 Vocational education 31.9 Higher general secondary education 8.5 Pre-university education 2.3 Higher vocational education 27.7 Academic education 10.3 Different 4.9 BMI before pregnancy \< 18.5 Underweight 3,1 N = 7437 18.5--24.9 Normal weight 68,1 25.0--29.9 Overweight 20,5 \> = 30 Extreme overweight 8,3 Smoking habits at 14 wks pregnancy Never 59.7 N = 7488 Ex 29.8 Current during pregnancy 10.5 Alcohol-usage (glasses/week) before pregnancy 0 44.8 N = 7482 1--10 53.5 11--20 1.6 \>20 0.2 Alcohol-usage (glasses/week) during pregnancy 0 91.2 N = 7210 1--10 8.8 11--20 0.0 \>20 0.0 Work (hours/week) No job 13.9 N = 7428 1--10 3.8 11--20 22.9 21--30 21.1 31--40 36.9 \>40 1.5 Number of pregnancies 1 42.3 N = 7519 2 36.6 3 14.2 4 4.6 \>4 2.3 ::: To examine whether the response in our study affected the determinant distributions (e.g. did primarily women in their first pregnancy respond?), a comparison of response rates was carried out. We performed a pilot-study in which data was recorded from April 2001 until November 2001 of every pregnant woman who attended one of the cooperating practices. Information about parity, PPGP during pregnancy, delivery and PPGP after delivery (until 6 weeks) was collected of 283 women (170 participants and 113 non-participants). Results of this pilot-study showed that primipara compared to multiparous women were more willing to participate in the cohort study. Furthermore, data from the responders in the cohort was compared to available data on pregnant Women in The Netherlands from the \"Centraal Bureau voor Statistiek\". The results of this comparison are shown in table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### A comparison between the total Dutch female population and the study population ::: ------------------------------------------------------------------------------------------------------------------------------------------------ **Total Dutch population** **Study population** --------------------------------- ---------------------------- ---------------------- ------------- -------------- -------------- -------------- Period 2001 Period 2002 Period 2003 Period 2001\ Period 2002\ Period 2003\ (N = 2892) (N = 3567) (N = 1061) BMI \%\* \%\* \%\* \% \% \% - Underweight (\<18.5) 5.3 4.9 3.0 2,9 3,2 3,5 - Normal weight (18.5--24.9) 70.3 71.9 71.2 66,8 68,5 70,4 - Overweight (25.0--29.9) 18.5 17.8 18.0 21,3 20,3 18,9 - Extreme overweight (\> = 30) 5.9 5.4 7.8 9,1 8,1 7,2 Smoking \%\* \%\* \%\* \% \% \% - Current/before pregnancy 35.7 34.0 33.3 29.7 29.3 31.7 **Pregnancy-related factors** Mean age mother - Total pregnancies 30.8 30.9 31.0 31.1 31.7 32.4 - First pregnancies 29.2 29.2 29.3 29.6 30.2 30.9 Child \% \% \% \% \% \% - Boy 51.2 51.3 51.4 51.0 52.2 50.5 - Girl 48.8 48.7 48.6 49.0 47.8 49.5 Number of pregnancies: \% \% \% \% \% \% - First 46.3 45.8 45.5 44,6 41,0 41.0 - Second 36.3 36.7 36.9 35,6 37,2 37.3 - Third 12.3 12.5 12.6 13,6 14,7 13.9 - Fourth or more 5.1 5.0 5.0 6,2 7,1 7.8 Multiple pregnancy \% \% \% \% \% \% - Twin 1.9 1.9 1.8 1.0 0.9 1.1 ------------------------------------------------------------------------------------------------------------------------------------------------ \* Age-group comparable to study-population ::: Data are presented in 3 separate years to show possible fluctuations. It is noticeable that the mean age of pregnant women is increasing during the 3-year period in the study population and in the total Dutch population. In the study population, pregnant women are slightly older and heavier compared to the total Dutch population. Approximately 34% of all Dutch women (age 18--45 years), compared to 30% in the study population, are smoking (see table [2](#T2){ref-type="table"}). During pregnancy, 10.5% of the study population will continue smoking (see table [1](#T1){ref-type="table"}). In general, data of the study population correspond with data of the total Dutch population. Prevalence of pregnancy-related pelvic girdle pain during pregnancy ------------------------------------------------------------------- To determine the prevalence of PPGP during pregnancy, data at baseline (14 wks), 30 weeks of pregnancy and 2 weeks after delivery (information about 34--40 weeks of pregnancy) were used. Almost every woman develops pain in the lower back, the buttocks, the symphyses, the groins or radiation into the legs at some time in their pregnancy. Of the 7527 women, 84% reported pain in any or all of these areas during pregnancy. Women with a history of LBP/PPGP are more likely to develop PPGP during pregnancy then women without a history of LBP/PPGP (see fig. [3](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The point prevalence of PPGP during pregnancy for women with (■) and without (▲) a history of LBP/PPGP. ::: ![](1471-2458-5-1-3) ::: Discussion ========== In this article, we describe the main characteristics of the Maastricht PPGP cohort study in terms of study design, study population, exposure variables and outcome measures. This design, in which both risk factors and outcomes are frequently measured between 14 weeks of pregnancy and 1 year after delivery, enables us to examine the etiology and prognosis of PPGP. Advantages and disadvantages of the study ----------------------------------------- Although a cohort design (even incorporating a randomized controlled trial) has advantages over other epidemiological designs, it poses also a burden on the project in terms of logistics and recruitment. First of all, we are totally dependent on the cooperation and recruiting power of midwives and gynecologists. The number of pregnant women is about evenly divided between the two professions. Although professional workload for midwives in the Netherlands is extremely high, 62 % of the midwife practices took part in this study, recruiting about 90% of the patients. Whereas 58% of the hospitals in the recruitment area participate, they only recruit about 10% of the women. Coordination of the recruitment and frequent staff changes in the hospitals themselves seems to be a key problem. In etiological research there should be sufficient contrast in exposure. In this study, the study population at baseline is heterogeneous with respect to demographic variables, lifestyle characteristics and work-related factors. However, due to logistic constraints we have restricted the recruiting area to the southeast of the Netherlands, posing questions about the representation of our sample for the whole Dutch population. For instance the number of immigrants is significantly less in the southeast of the Netherlands. However, for future genetically oriented studies, this could be a major advantage. We have shown previously that our sample is slightly different from the national population of pregnant women with regards to several determinants (e.g. age, BMI). At baseline, 7526 pregnant women participated in our study. This is a response rate of 73.4%. Non-response and loss to follow-up might introduce selection bias in prospective studies. Loss to follow-up, especially in time series designs with repeated measurements, pose threats to representation of the sample. Especially when losses-to-follow-up are connected with negative pregnancy outcomes (miscarriage, birth defects), co-morbidity of the mother or factors predicting for PPGP. To evaluate whether differential loss to follow-up occurs, we will compare the profile of those lost to follow-up with other participants. In this study we were able to evaluate if there are significant differences between participants and non-participants. This evaluations shows that primiparous women are more willing to participate in the study compared to multiparous women. We expect that multiparity plays an important role in the etiology and prognosis of PPGP. Therefore, an underestimation of the prevalence of PPGP reported in this study cannot be ruled out. However, the collected sample should be able to provide reliable preliminary insights in incidence and prevalence of PPGP and factors related to etiology and prognosis of PPGP in the Netherlands. PPGP is a complex syndrome and for a greater understanding of pregnancy-related pelvic girdle pain, future studies should further disentangle the multifactorial etiology and prognosis of PPGP. Future research within the framework of the Maastricht PPGP cohort study will focus on disentanglement of the complex syndrome. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= All authors participated in the design of the study. JMB drafted the manuscript with input from the other authors. All authors read, revised and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/1/prepub> Acknowledgements ================ The Maastricht PPGP cohort study was funded by the Dutch board of health insurance companies. The authors would like to thank the participating midwives and gynecologists as well as all the women who completed the questionnaires. We also would like to thank Conny de Zwart for the logistic assistance.

PubMed Central

2024-06-05T03:55:52.599351

2005-1-3

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548283/", "journal": "BMC Public Health. 2005 Jan 3; 5:1", "authors": [ { "first": "Janneke M", "last": "Bastiaanssen" }, { "first": "Rob A", "last": "de Bie" }, { "first": "Caroline HG", "last": "Bastiaenen" }, { "first": "Annie", "last": "Heuts" }, { "first": "Mariëlle EAL", "last": "Kroese" }, { "first": "Gerard GM", "last": "Essed" }, { "first": "Piet A", "last": "van den Brandt" } ] }

PMC548284

Introduction ============ Fetal development and morphogenesis of various tissues and organs such as hair follicles, teeth, salivary glands, mammary glands, kidneys, liver, pancreas, and lungs depend on epithelium-mesenchyme interactions \[[@B1],[@B2]\]. Such interactions are believed to be important for the tissue regeneration necessary for wound healing in adults \[[@B3]\]. Pulmonary fibrosis is thought to be a result of wound healing or regeneration after lung injury \[[@B4]-[@B11]\]. Lung injury causes the epithelial basement membrane to be destroyed, which enables migration of interstitial cells into intra-alveolar spaces where they produce and deposit extracellular matrix (ECM). Regenerated epithelial cells then cover the surface of the intra-alveolar fibrotic area. If the injury is mild and focal and re-epithelialization has occurred, these epithelial cells appear widespread over early intraluminal fibrotic lesions and form intra-alveolar buds, which later become small collagen globules and do not contribute to alveolar structural remodeling \[[@B5]-[@B7],[@B9]-[@B13]\]. For example, intra-alveolar buds, in a so-called organizing pneumonia pattern, are frequently observed in nonspecific interstitial pneumonia (NSIP); NSIP usually has a better prognosis than does usual interstitial pneumonia (UIP), which has fibroblastic foci without good re-epithelialization \[[@B8]-[@B10],[@B13],[@B14]\] as early fibrotic lesions. The re-epithelialization around early fibrotic lesions is similar to the process of fetal epithelial development \[[@B4]-[@B7]\], which suggests that epithelium-mesenchyme interactions may play a key role in repair of pulmonary fibrosis. Epimorphin is a mesenchymal cell surface-associated protein that modulates epithelial morphogenesis in embryonic mouse organs including lungs and skin \[[@B15]\]. Epimorphin expression was found in fetal lungs and skin rudimentary cells, at the mesenchyme-epithelium interface. Epimorphin also directs epithelial morphogenetic processes in other gastrointestinal organs and mammary glands \[[@B15]-[@B21]\]. For example, primary cultured rat hepatocytes (an epithelial cell) were used to show that epimorphin induces formation of hepatocyte spheroids with a bile canaliculi-like structure, which maintained albumin production even without growth factors \[[@B16]\]. Epimorphin mediated mammary luminal morphogenesis by controlling expression of CCAAT/enhancer binding protein Î² \[[@B22]\], which is essential for proper mammary morphogenesis and for determination of the fate of mammary epithelial cells \[[@B23]\]. In cultured mammary epithelial cells, epimorphin augmented expression of matrix metalloproteinase 2 (MMP-2), an important proteinase in matrix degradation, and both epimorphin and MMP-2 were required for mammary gland morphogenesis \[[@B24]\]. During fetal rabbit lung development, MMP-2 and MT1-MMP (an activator of MMP-2) were detected in epithelial cells, and expression of active MMP-2 and MT1-MMP increased dramatically; MMP-2 and MT1-MMP were especially predominant during late development, in which there was an extremely wide alveolar surface, which indicated an important role for MMP-2 in alveoli formation \[[@B25]\]. MMP-2 was also up-regulated and activated in regenerated alveolar epithelial cells, which may lead to elongation and migration of these cells for repair of pulmonary fibrosis \[[@B26],[@B27]\]. The epimorphin gene is highly conserved among mouse, rat, and human \[[@B15],[@B28]\]: the 289 amino acid sequence of rat epimorphin/syntaxin 2 exhibits 86% homology to human epimorphin \[[@B29]\]. However, the distribution and function of epimorphin in human lungs, including during fibrosis, are unknown. To clarify the role of epimorphin in human lungs, especially in fibrosis, we evaluated epimorphin expression and localization in normal control lungs and in lungs affected by NSIP or UIP, especially in early intra-alveolar fibrotic lesions. We also used cultured human alveolar epithelial cells to assess the effect of recombinant epimorphin on MMP-2 expression. Materials and Methods ===================== Patients -------- All histologic slides from open or thoracoscopic lung biopsies in the files of the Department of Pathology, Kumamoto University Hospital, from 2000 to 2003, were evaluated. From among these samples, 17 patients were selected, with 8 fulfilling the histologic and clinical criteria for NSIP and 9 showing UIP. To confirm the histologic diagnosis, two pulmonary pathologists reviewed the slides; they were informed only of the age and sex of each patient and the presence of bilateral pulmonary disease determined by chest posteroanterior radiography and high-resolution computed tomography. All patients had had no steroid therapy before the biopsy. Normal control lung tissues were obtained from the normal areas of lungs that had been surgically removed from eight patients because of cancer. Table 1 shows the characteristics of the nine patients with UIP, eight patients with NSIP, and eight normal control subjects. In addition to the histologic diagnosis, we confirmed that the clinical data, pulmonary function test results, bronchoalveolar lavage findings, chest radiographic findings, and follow-up information obtained from the patients were consistent with the diagnoses. Diagnostic criteria of the American Thoracic Society/European Respiratory Society consensus classification system were applied \[[@B30]\]. We also verified that none of the patients with UIP had a connective tissue disease. In the group of patients with NSIP, two with SjÃ¶gren\'s syndrome and one with rheumatoid arthritis were included. In all other cases, no etiologic agent was found. These tissue samples were used for microscopic immunohistochemistry, Western blotting, and Northern blotting assays. The procedures used in this study were in accordance with those recommended by the regional ethical committee on human experimentation. Northern Blotting Studies ------------------------- Human epimorphin DNA fragments were isolated by reverse transcriptase-polymerase chain reaction. Strand cDNA was synthesized with random primers from human lung total RNA. Polymerase chain reaction assays were carried out at 95Â°C for 1 minute, 64Â°C for 1 minute, and 72Â°C for 1 minute for 35 cycles. The following primer pairs were used: human epimorphin forward 5\'-GGA ACC GGA CTT CAG TGG ATC-3\' and reverse 5\'-CAGC CAA TGA TTA GAG CCA GGA-3\'. Polymerase chain reaction products were subcloned into the *Nco*I and *Spe*I sites of the pGEM-T Vector (Promega, Madison, WI), and authenticity was confirmed by sequencing. Total cellular RNA was isolated from lung tissues from each case by using the acid guanidinium-isothiocyanate-phenol-chloroform method. The RNA samples (10 Î¼g/lane) were fractionated by electrophoresis on 1% agarose-formaldehyde gels under denaturing conditions and were transferred to Nytran by capillary action. The blots were then probed with the 339-bp *Nco*I/*Spe*I fragment of the human epimorphin cDNA labeled with \[^32^P\]dCTP (3000 Ci/mmol) by means of the Nick Translation System kit (GIBCO/BRL, Grand Island, NY). After hybridization, blots were washed and were processed by autoradiography. Washed blots were analyzed by using a Fujix BAS2000 Bio-Imaging analyzer system (BASTM, Fuji Photo Film Co., Ltd., Tokyo, Japan) and were visualized on Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY). Signal intensity was quantified in digital images via BASTM analysis (FUJIX BAStation, Fuji Photo Film Co., Ltd.). To control for differences in gel loading, for each sample the RNA hybridized with the epimorphin probe was normalized to the expression of Î²-actin mRNA in the same sample. Western Blotting Studies ------------------------ Although the monoclonal antibody to mouse epimorphin (MC-1) has been reported to cross-react with human epimorphin \[[@B31],[@B32]\], we performed Western blotting to confirm whether the anti-epimorphin antibody (MC-1, a gift from Dr. Hirai, Osaka R&D Laboratory (Yokohama-lab), Sumitomo Electric Industries, Yokohama, Japan) cross-reacted with the human epimorphin in our lung samples and to make comparisons of epimorphin peptide expression between normal and fibrotic groups. For immunoblotting, lung tissues from each case were dissolved in sample buffer containing 2% sodium dodecyl sulfate in 8 M urea and were kept at 4Â°C overnight; the same protein concentration (30 Î¼g) in samples from each lung extract was ensured by using Protein Assay (Bio-Rad Laboratories, Hercules, CA). Lung tissues from normal adult mice were also dissolved in sample buffer as above and served as positive controls. Those samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to the method of Laemmli (1970), and proteins in the gels were transferred onto nitrocellulose filters and were incubated sequentially with 5% skim milk in 0.5% Tween 20 overnight. Blots were then incubated at 4Â°C overnight with 10 Î¼g/ml rat anti-mouse epimorphin primary monoclonal antibody MC-1 used at a dilution of 1:1000 in 1% bovine serum albumin. Samples were then stained by incubation with horseradish peroxidase-conjugated anti-rat goat IgG F(ab\')~2~antibody (Biosource International Inc., Camarillo, CA) used at a dilution of 1:1000 in Tris-buffered saline with 0.05% Tween 20 for 2 hours at room temperature. The bound antibody was detected by using the enhanced chemiluminescence method (Amersham Pharmacia Biotech UK Limited, Little Chalfont, Buckinghamshire, England) according to the manufacturer\'s protocol. Light Microscopic Immunohistochemistry -------------------------------------- A portion of each lung specimen was fixed immediately in a solution of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), after which samples were sequentially washed for 4 hours in 10% sucrose in 0.01 M phosphate-buffered saline (pH 7.4, 4Â°C), 4 hours in 20% sucrose in phosphate-buffered saline, and overnight in 30% sucrose in phosphate-buffered saline. They were then snap-frozen in OCT embedding medium and stored at -80Â°C. Frozen tissues were cut into 4-Î¼m sections, which were incubated for 30 minutes with a biotin blocking system (Dako Corporation, Carpinteria, CA) and for 30 minutes with 0.3% hydrogen peroxide in methanol to eliminate endogenous peroxidase activity. After treatment with normal goat serum, tissues were incubated overnight at 4Â°C with 10 Î¼g/ml rat anti-mouse monoclonal epimorphin antibody and were then incubated for 1.5 hours at 37Â°C with 1 Î¼g/ml biotinylated goat anti-rat IgG (ZYMED, San Francisco, CA) and streptavidin-biotin-horseradish peroxidase complex (Dakopatts, Glostrup, Denmark). Bound antibody was visualized after incubation for 10 minutes in a Coplin jar with 100 ml of Tris-HCl buffer (pH 7.6) containing 20 mg of diaminobenzidine and 17 ml of 30% H~2~O~2~. Counterstaining was with Mayer\'s hematoxylin. Lung tissue of normal adult mice was stained as a positive control. Nonspecific labeling of primary antibody was evaluated with normal rat serum. Each tissue sample was also stained for keratin (rabbit anti-bovine antibody raised against the 58-, 56-, and 52-kd subunits of muzzle epidermal keratin; Dakopatts, Santa Barbara, CA), Î±-smooth muscle actin (mouse anti-human Î±-smooth muscle actin monoclonal antibody; Dakopatts, Santa Barbara, CA), and MMP-2 (mouse anti-human MMP-2 monoclonal antibody; clone 42-5D11, IgG1 isotype, purified antibody; Daiichi Fine Chemical Co., Ltd., Takaoka, Japan). Serial sections were also stained with hematoxylin-eosin (H&E) and Alcian blue-periodic acid-Schiff (AB-PAS). Confocal Microscopy ------------------- To localize epimorphin as precisely as possible, epithelial and stromal cells were double labeled by means of immunofluorescent probes, and the distribution of epimorphin was compared with that of keratin and vimentin (mouse monoclonal anti-vimentin \[V9\] antibody; Dakopatts, Glostrup, Denmark). Briefly, after sections were exposed to primary antibodies, they were exposed to secondary antibodies: for the first analysis: fluorescein isothiocyanate-conjugated goat anti-rat IgG (American Qualex, San Clemente, CA) or Texas Red-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., Eugene, OR); for the second analysis: fluorescein isothiocyanate-conjugated goat anti-rat IgG (American Qualex) or Texas Red-conjugated goat anti-mouse IgG (Molecular Probes). The nuclei were counterstained with 4,6-diamidino-2-phenylindole dihydrochloride (Vector Laboratories, Inc., Burlingame, CA). Specimens were examined under a confocal laser scanning microscope (TCS-SP; Leica Lasertechnik, Heidelberg, Germany), based on an upright microscope (DMRB, Leica Lasertechnik) equipped with a krypton/argon laser \[[@B33]\]. The excitation wavelengths for fluorescein isothiocyanate and Texas Red were 498 nm and 568 nm, respectively. Green fluorescein isothiocyanate emission was selected and recorded by using a 500- to 550-nm bandpass filter; red Texas Red emission was selected and recorded by using a 581- to 631-nm bandpass filter. In addition, 4,6-diamidino-2-phenylindole dihydrochloride was excited at 350-nm by using a UV laser, and its blue emission was recorded via a 401- to 551-nm bandpass filter. Localization of MMP-2 and Epimorphin in Lungs with NSIP ------------------------------------------------------- To compare the precise localization of MMP-2 and epimorphin in NSIP, a confocal microscope was used for immunohistochemical study of lung tissues from patients with NSIP, by means of the same technical immunohistochemical staining and analysis method as that described previously for epimorphin and keratin detection \[[@B34]\]. Mouse anti-human MMP-2 monoclonal antibody and rat anti-mouse monoclonal epimorphin antibody served as primary antibodies. Secondary antibodies for double-labeling immunohistochemistry studies via confocal microscopy were goat anti-mouse IgG antibody (Alexa Fluor 546; Molecular Probes) and goat anti-rat IgG antibody (Alexa Fluor 488; Molecular Probes). Cell Culture and Treatment Conditions ------------------------------------- Human lung-derived alveolar epithelial cells (A549), an epithelial cell line derived from lung adenocarcinoma (Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University), were cultured in RPMI (GIBCO/BRL) supplemented with 10% fetal bovine serum, 2 mM l-glutamine, 50 U/ml penicillin, and 50 Î¼g/ml streptomycin. All cells were maintained at 37Â°C in a humidified incubator containing 5% CO~2~and 95% air. Detection of MMP-2 Expression in A549 Cells ------------------------------------------- Four-well chamber slides (Lab-Tek II, Nalge Nunc International, Naperville, IL) were coated with human recombinant epimorphin fragment (H12; 20 Î¼g/well, dissolved in 1.5 mM HCl; a gift from Dr. Hirai), negative control solution prepared from non-transfected *Escherichia coli*strain BL21 (BL; 20 Î¼g/well, dissolved in 1.5 mM HCl; a gift from Dr. Hirai), or Type IV collagen from human placenta collagen (20 Î¼g/well, dissolved in 0.4% glacial acetic acid; Sigma Chemical Co., St. Louis, MO), after which samples were dried at room temperature. After A549 cells were washed two times with phosphate-buffered saline, the cells (5 Ã--- 10^5^) were suspended in RPMI medium with 2% fetal calf serum and were incubated at 37Â°C on the chamber slides coated with recombinant epimorphin, BL, or Type IV collagen. After incubation for 9 h, culture dishes were placed on ice, medium from each dish was collected, and cells were washed twice with phosphate-buffered saline. Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline for 5 minutes at -20Â°C and then were washed in phosphate-buffered saline and permeabilized in acetone for 5 minutes at -20Â°C. Cells were again washed in phosphate-buffered saline and incubated with a blocking solution containing 1% bovine serum albumin/phosphate-buffered saline for 1 hour at room temperature. Immunohistochemistry was performed via the same technical method described above for epimorphin detection. The primary antibody was 5 Î¼g/ml mouse anti-human MMP-2 monoclonal antibody, and the secondary antibody was horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Amersham Pharmacia Biotech UK Limited). The A549 culture supernatant collected after incubation for 9 hours on the chamber slides coated with recombinant epimorphin, BL, and Type IV collagen was used to determine the amount of MMP-2 activity by means of the MMP-2 Biotrack Activity Assay System (Amersham Pharmacia Biotech UK Limited), according to the manufacturer\'s instructions. All samples were assayed in triplicate, and assays were repeated three times. Statistical Analysis -------------------- Densitometric analysis of the bands of Northern and Western blotting was performed with Macintosh G4 computer (Apple Japan, Inc., Tokyo, Japan) using high-resolution scanner and NIH image software (version 1.62, National Institutes of Health, Bethesda, Maryland). The data are expressed as a ratio of a standard normal control subject to each case\'s band density units (arbitrary units) and reported as mean Â± standard error of the mean (SEM). Statistical significance was established using the one-way analysis of variance test followed by Tukey-Kramer multiple intergroup comparison test. Probabilities less than 0.05 were considered significant. Results ======= Expression of Epimorphin mRNA in Normal Human Lungs and Lungs of Patients with NSIP or UIP ------------------------------------------------------------------------------------------ Expression of 3.2-kb epimorphin mRNA was revealed in all lung samples from patients with NSIP, UIP and normal control subjects by Northern blotting (Figure [1A](#F1){ref-type="fig"}). To confirm that the blots reflected samples of equal size, they were reprobed for expression of Î²-actin mRNA. Compared with corresponding densities of Î²-actin, the densities of epimorphin expression in NSIP samples were significantly higher than those in control lungs and lungs with UIP, as determined by NIH Image analysis (*P*\< 0.05) (Figure [1B](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### \(A) Representative Northern blots of epimorphin mRNA and Î²-actin mRNA. Epimorphin mRNA (3.2-kb) was expressed in samples of normal lung (Control: *lanes 1â€"3*) and lungs from patients with UIP (*lanes 4â€"6*) and patients with NSIP (lanes 7â€"9) (*upper blot*). To confirm that the blots reflected samples of equal size, they were reprobed for expression of Î²-actin mRNA (*lower blot*). (B) The data are expressed as a ratio of epimorphin to Î²-actin band density units (arbitrary units) and reported as mean Â± SEM measured by means of NIH Image analysis. In NSIP samples, the density of epimorphin expression was significantly higher than that in control and UIP samples (\**P*\< 0.05). ::: ![](1465-9921-6-6-1) ::: Tissue Localization of Epimorphin in Normal Human Lungs ------------------------------------------------------- Western blotting showed that rat anti-mouse epimorphin monoclonal antibody (MC-1) recognized the 150-kd bands in the extracted samples from both mouse lung and normal human lung (Figure [2A](#F2){ref-type="fig"}), similar to results reported elsewhere \[[@B31],[@B32]\]. We thus confirmed that anti-mouse epimorphin antibody demonstrated specificity and cross-reactivity with the human epimorphin of our lung samples. Normal lung tissues of the human samples had normally opened alveoli with infiltration of a few inflammatory cells. In immunohistochemical studies of the human samples, as with normal mouse lung specimens \[[@B34]\], epimorphin was weakly stained in vascular and alveolar walls (Figure [2B](#F2){ref-type="fig"}). Negative control staining with normal rat serum showed no positive findings in the serial section (data not shown). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Representative Western blots for epimorphin in normal mouse and human lung samples (A), and representative epimorphin immunohistochemistry (B) in normal mouse lung. (A) Recognition of the 150-kd bands by rat anti-mouse epimorphin monoclonal antibody (MC-1) in extracted lung samples. (B) Weak epimorphin staining in vascular walls (*arrowheads*) and alveolar walls (*inset*). Scale bar = 50 Î¼m. ::: ![](1465-9921-6-6-2) ::: Amount of Epimorphin in Normal Human Lungs and Lungs of Patients with NSIP or UIP --------------------------------------------------------------------------------- In the lung tissue homogenates of patients with NSIP, UIP and of control subjects, Western blotting with antibody for epimorphin also recognized the 150-kd bands (Figure [3A](#F3){ref-type="fig"}) and the densities of the bands of patients with NSIP are relatively higher than those of UIP and control subjects by methods of NIH image analysis (p \< 0.05) (Figure [3B](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### \(A) Representative Western blots for epimorphin in normal control subjects and fibrotic groups. Epimorphin protein (150-kd) was expressed in samples of normal lung (Control: *lanes 1â€"3*) and lungs from patients with UIP (*lanes 4â€"7*) and patients with NSIP (*lanes 8â€"10*). (B) The data are expressed as a ratio of a standard normal control subject to each case\'s band density units (arbitrary units) and reported as mean Â± SEM measured by means of NIH Image analysis. In NSIP samples, the density of epimorphin protein expression was significantly higher than that in control and UIP samples (\**P*\< 0.05) ::: ![](1465-9921-6-6-3) ::: Tissue Localization of Epimorphin in NSIP and UIP by Means of Immunohistochemical Analyses ------------------------------------------------------------------------------------------ In lungs of all patients with NSIP, alveolar walls were thickened with edema, fibrosis, and inflammatory cell infiltration; the appearance was typically uniform (Figure [4A](#F4){ref-type="fig"}). There was slight or no dense fibrosis; intra-alveolar organizing fibrosis was seen as pale eosinophilic fibrosis with H&E staining and as Alcian blue positive area with AB-PAS staining as early stage of fibrosis (Figure [4A](#F4){ref-type="fig"} and [4B](#F4){ref-type="fig"}). Epimorphin immunostaining was strong in the early intra-alveolar fibrotic areas with a covering of regenerated alveolar epithelial cells (Figure [4C](#F4){ref-type="fig"} and [4E](#F4){ref-type="fig"}), which contained a few Î±-smooth muscle actin-positive cells (Figure [4F](#F4){ref-type="fig"}), in addition to immunostaining seen in vascular and alveolar walls. No positive findings was shown using normal rat serum as fist antibody for negative control (Figure [4D](#F4){ref-type="fig"}). The epimorphin immunohistochemical staining patterns for all eight patients with NSIP were quite similar. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Representative histology stained with hematoxylin-eosin (H&E)(A) and Alcian blue-periodic acid-Schiff (AB-PAS)(B) and immunohistochemistry for epimorphin (C), rat normal serum as negative control for epimorphin (D), keratin (E), and Î±-smooth muscle actin (F) for all patients with NSIP. (A) Thickened alveolar walls showed edema, fibrosis, and inflammatory cell infiltration. Dense fibrosis was inconspicuous or absent. (A and B) Intra-alveolar organizing fibrotic areas were seen as pale eosinophilic fibrosis with H&E staining and as Alcian blue positive area with AB-PAS staining as early stage of fibrosis (*asterisks*). (C) Positive epimorphin immunostaining appeared in areas of early fibrosis (*inset*in C shows a close-up view; asterisks indicates early intra-alveolar fibrotic area) covered with regenerated alveolar epithelial cells (*arrowhead*in E), in addition to the alveolar walls. A few Î±-smooth muscle actin-positive cells were noted in the fibrotic areas (*arrowhead*in F). Scale bars = 60 Î¼m. ::: ![](1465-9921-6-6-4) ::: In lungs of all patients with UIP, the fibrotic zones showed temporal heterogeneity, with dense acellular collagen and scattered fibroblastic foci with intervening nearly normal alveoli. Most fibrotic zones had honeycombing with complete destruction of the architecture (Figure [5A](#F5){ref-type="fig"}). Though clear immunostaining in the vascular walls, only weak epimorphin immunostaining occurred in dense fibrotic lesions and scattered fibroblastic foci, as in areas of early fibrosis (Figure [5B](#F5){ref-type="fig"} and [5F](#F5){ref-type="fig"}) with Alcian blue positive (Figure [5E](#F5){ref-type="fig"}). The scattered fibroblastic foci contained many Î±-smooth muscle actin-positive cells (Figure [5D](#F5){ref-type="fig"}) with the desquamative regenerated alveolar epithelial cells overlying these fibroblastic foci (Figure [5C](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Representative histology (A, C \[close-up view\]; H&E staining) and immunohistochemistry for epimorphin (B, F \[close-up view\]) and Î±-smooth muscle actin (D \[close-up view\]) of scattered fibroblastic foci in early fibrotic areas as Alcian blue positive area (E; AB-PAS staining) for patients with UIP. (A and C) Honeycombing with complete architectural destruction occurred in fibrotic zones. (C and E) Arrowheads indicate scattered fibroblastic foci. In addition to clear immunostaining in the vascular and alveolar walls (*arrows*), only weak epimorphin immunostaining was seen in scattered fibroblastic foci (*arrowheads*) (B and F); many Î±-smooth muscle actin-positive cells (D) and desquamative regenerated alveolar epithelial cells (C) were found. Scale bars = A, B: 200 Î¼m; Câ€"E: 80 Î¼m. ::: ![](1465-9921-6-6-5) ::: Tissue Localization of Epimorphin in NSIP by Means of Double-labeled Immunohistochemical Analyses ------------------------------------------------------------------------------------------------- Double-labeled confocal fluorescent images confirmed the presence of epimorphin in early fibrotic lesions and keratin-positive epithelial cells overlying the lesions (Figure [6A](#F6){ref-type="fig"} and [6B](#F6){ref-type="fig"}). Double-labeled confocal fluorescent images also verified the localization of epimorphin in vimentin-positive stromal cells and in surrounding ECM (Figure [6C, D](#F6){ref-type="fig"}, and [6E](#F6){ref-type="fig"}) in early fibrotic lesions. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Representative double-labeled confocal fluorescent images of staining for epimorphin plus keratin (A), epimorphin (C), vimentin (D), and epimorphin plus vimentin (E) in an early fibrotic lesion from the lung of a patient with NSIP. (A) The presence of epimorphin (green) in areas of early fibrosis and the presence of keratin (red) in epithelial cells overlying the lesions were confirmed. (B) The same image shown in part A but with Nomarski optics used. (Câ€"E) Epimorphin (C, green) localized in vimentin-positive (D, red) stromal cells (yellow-orange) and in surrounding ECM in early fibrotic areas (E). Nuclei were 4,6-diamidino-2-phenylindole dihydrochloride-positive (blue). Asterisks indicate early fibrotic areas. Scale bars = B: 20 Î¼m, E: 5 Î¼m. ::: ![](1465-9921-6-6-6) ::: Localization of MMP-2 and Its Relation to Epimorphin in NSIP ------------------------------------------------------------ Areas of early fibrosis in lung tissues from patients with NSIP showed epimorphin immunoreactivity (Figure [7A](#F7){ref-type="fig"}), and regenerating epithelial cells overlying these lesions demonstrated MMP-2 labeling (Figure [7B](#F7){ref-type="fig"}). Furthermore, in tissues from patients with NSIP, double-labeled confocal fluorescent images confirmed the expression of MMP-2 (Figure [7C](#F7){ref-type="fig"}) in re-epithelialized cells overlying fibrotic lesions in which epimorphin was also clearly expressed. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Representative images of immunostaining for epimorphin (A) and MMP-2 (B) in NSIP; the double-labeled confocal fluorescent image of in an early fibrotic lesion from the lung of a patient with NSIP was stained for epimorphin plus MMP-2 (C). (A) Epimorphin immunoreactivity was observed in early fibrotic areas, with (B) MMP-2 labeled in regenerated epithelial cells overlying the fibrotic lesion. Asterisks indicate early fibrotic areas. (C) MMP-2 (red) was strongly expressed in re-epithelialized cells overlying early fibrotic lesions with clear epimorphin expression (green). Nuclei were 4,6-diamidino-2-phenylindole dihydrochloride-positive (blue). Asterisks indicate early fibrotic areas. Scale bars = 20 Î¼m. ::: ![](1465-9921-6-6-7) ::: Increased Expression of MMP-2 Induced in A549 Cells by Epimorphin ----------------------------------------------------------------- Nine hours after plating of BL or Type IV collagen-coated chamber slides with cells of the human lung-derived alveolar epithelial cell line A549, the cells had spread and formed a loose sheet (Figure [8A](#F8){ref-type="fig"}, left panel). However, during the same time period, in recombinant epimorphin-coated chamber slides, the A549 cells formed some monolayer cell islands in addition to the loose cellular sheet, in about 50% of the chamber slide area (Figure [8A](#F8){ref-type="fig"}, right panel). Moreover, the immunoperoxidase method revealed strong MMP-2 immunostaining in cells of these cell islands in epimorphin-coated slides after 9 hours of incubation (Figure [8A](#F8){ref-type="fig"}, right panel). The MMP-2 Biotrack Activity Assay System revealed significantly higher MMP-2 activity levels in the supernatants of cultures of A549 cells with recombinant epimorphin compared with those for cultures with BL or Type IV collagen after 9 hours of incubation (Figure [8B](#F8){ref-type="fig"}). ::: {#F8 .fig} Figure 8 ::: {.caption} ###### \(A) Representative images of MMP-2 immunostaining in A549 cells cultured with recombinant epimorphin (*right panel*) or with BL (*left panel*) after 9 hours of incubation. In the epimorphin-coated slides, plated cells formed some monolayer cell islands in addition to loose sheets of cells, and cells in these cell islands showed strong MMP-2 immunostaining (by the immunoperoxidase method). Scale bars = 30 Î¼m. (B) Via the MMP-2 Biotrack Activity Assay System, MMP-2 activity in the supernatants of A549 cells cultured with recombinant epimorphin was higher than in supernatants of cells cultured with BL or Type IV collagen (*P*= 0.01). ::: ![](1465-9921-6-6-8) ::: Discussion ========== This is the first study to examine in detail the distribution and magnitude of epimorphin expression in normal human lungs and lungs from patients with NSIP or UIP. We found clear expression of epimorphin mRNA and protein in lung samples from normal control subjects and patients with NSIP or UIP. Expression of Epimorphin in Normal Human Lungs ---------------------------------------------- Consistent with previous reports \[[@B32]\], epimorphin was expressed in connective tissue components of walls of the alveoli and blood vessels in normal lungs, as determined by immunohistochemistry. Epimorphin expression was also confirmed at the mRNA and protein levels by Northern and Western blotting assays, similar to results found previously for mice \[[@B15],[@B32]\]. Although the specific function of epimorphin in normal lungs is not yet known, it may serve as an epithelial and endothelial cell morphogen to maintain the cell turnover necessary for normal structure and function. Alveolar epithelial cells are replaced at regular intervals under physiologic conditions. For example, the estimated turnover time for normal mouse alveolar epithelial cells ranges from 28 to 35 days \[[@B35]\], and daily endothelial cell turnover is reported to be about 1% \[[@B36]\]. Also, in the normal adult human kidney, similar to results for normal adult mouse kidney, low levels of epimorphin mRNA and protein were detected in the glomerular mesangium and peritubular interstitium (interstitial fibroblast-like cells), as revealed by reverse transcriptase -polymerase chain reaction and immunohistochemistry \[[@B37]\]. These findings are consistent with our result that epimorphin is expressed in the interstitium in human control lungs, as in normal adult mouse lungs \[[@B34]\]. Expression of Epimorphin in Lungs of Patients with NSIP or UIP -------------------------------------------------------------- We found, by means of Northern blotting assays, distinct expression of epimorphin mRNA and protein in normal lungs and lung samples of patients with NSIP or UIP, with significantly higher epimorphin expression in lungs of patients with NSIP than in other lung samples. Also, serial sections and double-labeled immunohistochemistry analyses of NSIP samples demonstrated strong expression of epimorphin in mesenchymal cells situated within active intra-alveolar fibrotic lesions and the presence of epimorphin in the ECM in the vicinity of these cells. These findings are consistent with our previous report that epimorphin was synthesized by mesenchymal cells and localized to these cells and the ECM of early intra-alveolar fibrotic lesions in a murine bleomycin-induced pulmonary fibrosis model \[[@B34]\]. They are also consistent with a report that epimorphin was synthesized by normal human skin fibroblasts \[[@B38]\]. It is well known that regeneration of alveolar and bronchiolar epithelial cells is crucial for repair of pulmonary fibrosis, and the ECM in early fibrotic lesions provides fibronectin and other adhesion molecules as ligands for regenerating epithelial cells to ensure successful repair \[[@B3],[@B4],[@B39]-[@B41]\]. In the murine bleomycin-induced pulmonary fibrosis model, we suggested that epimorphin in the early fibrotic lesions participated in adhesion of the regenerating alveolar epithelial cells. Involvement of epimorphin in the repair process was also reported for other organs including the gut during tissue repair in an isograft in rats \[[@B20]\] and the liver during regeneration after partial hepatectomy in mice \[[@B17]\]. Increased expression of epimorphin during epithelial cell regeneration was also reported in human skin ulcers and active ulcerative colitis \[[@B32],[@B42]\]. We observed strong epimorphin immunostaining in early fibrotic areas with a few Î±-smooth muscle actin-positive cells in NSIP, as well as weak epimorphin immunostaining in fibroblastic foci with many Î±-smooth muscle actin-positive cells and desquamative regenerated alveolar epithelial cells in UIP. It is believed that in UIP, a failure of re-epithelialization of fibroblastic foci maintains fibroblast/myofibroblast activity and ECM synthesis \[[@B43],[@B44]\]. We suggest that highly expressed epimorphin in the lungs of patients with NSIP may be necessary during wound healing, especially for regeneration of alveolar epithelial cells in early fibrotic lesions, and we also suggest that poorly expressed epimorphin in the lungs of patients with UIP may lead to failure of re-epithelialization of fibroblastic foci and the contraction of fibrotic lungs during the end stage of UIP. Role of Epimorphin Associated with MMP-2 in Lung Repair ------------------------------------------------------- To understand the role of epimorphin in the repair process in the lungs of patients with NSIP, we evaluated whether epimorphin enhanced expression of MMP-2 (gelatinase A). Recombinant epimorphin induced formation of some monolayer cell islands in addition to a loose cellular sheet and augmented expression of MMP-2 in cultured human alveolar epithelial cells, as demonstrated by immunohistochemistry and the MMP-2 Activity Assay System. We suggest that epimorphin-induced monolayer cell islands are a type of formation of differentiated alveolar epithelial cells, like the epimorphin-induced spheroid formation of rat hepatocytes, and suggest that the augmented expression of MMP-2 may be similar to the increased albumin production of rat hepatocytes. We also demonstrated expression of MMP-2 in re-epithelialized cells overlying epimorphin-positive early fibrotic areas by means of double-labeled confocal fluorescent images. MMP-2, which is an important proteinase needed for matrix degradation, is secreted by type II alveolar epithelial cells *in vitro*\[[@B45]\]. Fukuda et al. \[[@B25]\] observed the localization of MMP-2 in fetal rabbit alveolar epithelial cells and gelatinolytic activities of MMP-2 in fetal rabbit lung, which indicated an important role for MMP-2 in formation of alveoli. These authors also observed the localization of MMP-2 in regenerating alveolar epithelial cells covering intra-alveolar fibrotic areas in a study of bronchiolitis obliterans organizing pneumonia, which is a reversible fibrotic human lung disease \[[@B13]\]. Yaguchi et al. \[[@B26]\] reported that MMP-2 was found in regenerating alveolar epithelial cells and that gelatinolytic activities of the active forms of MMP-2 increased in later repair stages in bleomycin-induced pulmonary fibrosis, during reconstruction of alveoli. Moreover, Fukuda et al. \[[@B46]\] reported that in bleomycin-induced pulmonary fibrosis, re-epithelialization on the surface of early intra-alveolar fibrotic lesions in gelatinase A^-/-^mice was markedly reduced compared with that of gelatinase A^+/+^controls. Thus, MMP-2 was up-regulated and activated in regenerating alveolar epithelial cells, which may allow elongation and migration of these cells for successful repair of pulmonary fibrotic lesions \[\[[@B26],[@B27]\], 47\]. We therefore suggest that epimorphin expression in early fibrotic lesions in the lungs of patients with NSIP may contribute to the repair process in pulmonary fibrosis, in part by inducing MMP-2 expression. What may follow this MMP-2 expression may be elongation and migration of regenerating epithelial cells and degradation of ECM in alveolar spaces. Thus, epimorphin, as a component of proteolytic systems, may contribute to cell migration and tissue remodeling during repair of human lung fibrosis. Although epimorphin receptors have not yet been identified, binding ECM molecules has been suggested as involved in establishing epithelial polarity, thus helping form an organized basement membrane and cell-ECM junctional complexes \[[@B21]\]. Our results are consistent with the idea \[[@B22],[@B34]\] that epimorphin has a high affinity for ECM molecules and could modify functions of adhesion and migration of regenerating alveolar epithelial cells by altering signaling through MMP-2. In conclusion, epimorphin expressed in lungs may have important roles as a morphogen not only in mice but also in humans, and epimorphin may contribute to the repair process in human pulmonary fibrosis via epithelium-mesenchyme interactions. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Table 1. Characteristics of subjects, including pulmonary function test results ::: ::: {.caption} ###### Click here for file ::: Acknowledgments =============== The authors thank Dr. Yohei Hirai, Osaka R&D Laboratory (Yokohama-lab), Sumitomo Electric Industries, Yokohama, for generous gifts of anti-epimorphin antibody and human recombinant epimorphin; Mr. Osamu Nakamura, Mr. Takenobu Nakagawa, Mrs. Emi Miyata, and Ms. Makiko Tanaka for their skillful technical assistance; and Ms. Judith B. Gandy for editing the manuscript. This work is supported by a Grant-in-Aid for Young Scientists from the Ministry of Education, Science, Sports, Science and Technology of Japan.

PubMed Central

2024-06-05T03:55:52.602942

2005-1-16

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548284/", "journal": "Respir Res. 2005 Jan 16; 6(1):6", "authors": [ { "first": "Yasuhiro", "last": "Terasaki" }, { "first": "Yuh", "last": "Fukuda" }, { "first": "Moritaka", "last": "Suga" }, { "first": "Naoki", "last": "Ikeguchi" }, { "first": "Motohiro", "last": "Takeya" } ] }

PMC548285

Background ========== The current generation of adolescents -- over one billion -- is the largest in history \[[@B1]\] and, far from representing a picture of health \[[@B2]\], many will suffer untimely disease and death. HIV and malaria are responsible for much of the disease burden affecting female adolescents, who suffer disproportionately from these combined infections relative to other age groups. This is due to a high HIV incidence during the period when many adolescents become pregnant for the first time -- an event which greatly increases their susceptibility to *Plasmodium falciparum*malaria \[[@B3]\]. Biological interactions between malaria and HIV in pregnancy complicate therapy, which can also be compromised by inappropriate adolescent health-seeking behaviour. The public health importance of HIV and malaria synergism is only now emerging. It has led to a recommendation by the World Health Organization (WHO) that the two disease programs should collaborate to ensure integrated service delivery, especially within the framework of reproductive health services \[[@B4]\]. A case has also been made for linking disease control programs as a more efficient and effective way for lowering the malaria and HIV disease burden \[[@B5]\]. Nevertheless, it is difficult to seen how these goals will be attained unless adolescents are accorded a much higher priority by disease control programs. In this paper we consider the justification, as well as the challenges, of using common approaches to improve delivery of HIV and malaria interventions to female adolescents. HIV and malaria burden in pregnant and non-pregnant adolescents --------------------------------------------------------------- Prevalence estimates for HIV infection \[[@B1]\] for 35 sub-Saharan African countries, for young males and females (15--24 years), are shown in Figure [1](#F1){ref-type="fig"}. Estimations are often derived from sentinel surveillance of women attending antenatal care (ANC) and tend to overestimate prevalence in adolescents due to the selection of sexually active \[[@B6]\] and less-educated groups \[[@B7]\]. Accepting this bias, in every country, listed HIV prevalence is two to three times higher among females than males. Having an older male partner is associated with a modest increased HIV risk \[[@B8]\] but probably does not explain a sex difference that occurs uniformly across all countries and cultures and at all levels of HIV endemicity. In Rakai, Uganda, only 12.4% of HIV cases in 15--19 year olds was attributed to relationships with men 10 years older \[[@B9]\]. Other sexually transmitted infections (STIs) increase susceptibility to HIV but do not account for the sex difference \[[@B10]\]. Host biological factors may be critical \[[@B11]\]. Sexual maturation takes several years to complete, but menarche is delayed amongst socio-disadvantaged girls, and many will be sexually active before biological maturation is complete. Interventions that delay the onset of sexual activity could reduce exposure at a time of peak biological susceptibility to HIV and highest risk of malaria in pregnancy. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Female and male HIV prevalence among young people 15--24 years of age in sub-Saharan Africa. Compiled from UNFPA^1^ ::: ![](1475-2875-4-2-1) ::: Malaria is an important cause of adolescent hospital admissions in many sub-Saharan African countries with stable malaria transmission \[[@B12]\]. Malaria mortality in African adolescents (both sexes) aged 10--14 years has been estimated at over 45,000 deaths per annum \[[@B13]\]. The highest prevalence of *P. falciparum*infection after childhood is also in young, mostly adolescent, women. Data from Malawi (Table [1](#T1){ref-type="table"}) shows that non-pregnant and pregnant adolescents had significantly higher parasite rates than women above 19 years. Primigravidae also have a markedly increased susceptibility to malaria \[[@B3]\]. Young age and nulliparity in the same individual lead to greatly increased malaria risk. This partly relates to reduced acquisition of acquired malaria immunity in young individuals \[[@B14]\] as well as the lack of parity-specific malaria immunity acquired during the first pregnancy \[[@B15]\]. Risk factors for symptomatic malaria in adolescents have not been studied, but both symptomatic and asymptomatic malaria infection will contribute to the development of adolescent and newborn anaemia \[[@B16]\]. HIV infection increases *P. falciparum*prevalence during pregnancy and this is marked during the first pregnancy \[[@B17],[@B18]\], which in Africa, is nearly always in adolescence. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Malaria prevalence in non-pregnant and pregnant adolescents and adults in the Shire Valley, Malawi ::: **Adolescent** **Adult** ------------------ ----------------- ------------------ ---------------- **Non-Pregnant** **Pregnant \*** **Non-Pregnant** **Pregnant\*** **% (n)** **% (n)** **% (n)** **% (n)** 41.4(29)† 45.9 (122)‡ 20.3 (74) 35.4 (345) \* At delivery. Includes women who had received anti-malarials during pregnancy † Difference with non-pregnant adults χ^2^= 4.8; p = 0.028 ‡ Difference with pregnant adults χ^2^= 4.3; p = 0.04 ::: HIV and malaria interventions during adolescence ------------------------------------------------ For both HIV and malaria there are effective interventions for disease control. HIV and malaria frequently occur in the same African populations and the same individuals, yet control activities are directed to different target groups, using different channels. Adolescent HIV prevention, with its focus on behavioural change, has taken place largely outside conventional health settings. Few HIV strategies have been directed at pregnant adolescents and/or their partners -- an omission noted by a WHO Technical Consultation on married adolescents \[[@B19]\]. In some African countries, by the age of 19 years, half of all female adolescents were in marital or permanent consensual unions \[[@B9]\] that will almost certainly lead to pregnancy. In contrast, malaria control efforts for women have focused on pregnancy, but not on adolescents and certainly not on adolescents before they become pregnant. As a result, in many sub-Saharan African settings, adolescents are often parasitaemic and anaemic when they first become pregnant. The problem is that currently no overall strategy for adolescent health has been approved for most African countries. The burden of infection and disease among adolescents is of sufficient magnitude that a way must be found to provide services to this population. The existence of an appropriate strategy would allow both HIV and malaria interventions to be delivered within a common framework. In traditional cultures, menarche often marks the transition from childhood to adulthood because it signifies reproductive potential. Nevertheless, adolescence is a physical process that takes a number of years and is usually completed by the late teens. This developmental process should be encompassed within health care provision. Pregnancy may punctuate that process, but during it, the adolescent continues to grow physically and mentally \[[@B20]\]. Strategies to reduce adolescent malaria or HIV should, therefore, provide a continuum of care -- before conception, during and after pregnancy -- encompassing appropriate information, preventive and curative services. For prevention, adolescents need access to condoms and bednets, information on how to use them and, when and where to go to deal with infection. Services must be available for infection monitoring and treatment, including HIV testing and drug therapy for malaria, anaemia and HIV-related opportunistic infections. System improvements may not be strictly about adolescent health services, but about support for ANC. Adolescents need good access to pregnancy care and this includes attendance for early pregnancy assessment and screening for HIV and other sexually transmitted infections (STIs). ANC presents an opportunity for HIV prevention, support for discordant couples and care and referral for HIV-infected adolescents. Early assessment is important as the HIV positive individual needs to plan for anti-retroviral treatment (ART) at delivery to prevent mother-to-child transmission. It is also important for malaria control in pregnancy. Intermittent preventive antimalarial treatment (IPT) is the key malaria prevention policy that improves birthweight outcomes and reduces pregnancy anaemia \[[@B21]\]. Its effectiveness depends on the prevalence of HIV infection \[[@B22]\]. It also depends on adherence to the treatment regimen. Coverage with the standard two dose intermittent sulphadoxine-pyrimethamine preventive malaria treatment was lower in younger adolescents in the Shire Valley, Malawi ≤ 17 years, 35.1% versus 18--19 years, 53.7%; p = 0.028, Verhoeff, personal communication), and more than half of all pregnant adolescents received inadequate antimalarial treatment. This demonstrates the importance of pre-conceptual malaria health education for girls. Adolescent mothers need instruction on how to recognize and respond to infant malaria infection and encouragement for using bednets for themselves and their babies. A major challenge for malaria control is to improve adolescent health prior to the first pregnancy. For non-pregnant adolescents there are several conundrums. On account of drug resistance, malaria combination therapy with artemisinin derivatives is being introduced into an increasing number of sub-Saharan African countries. Artemisinin may be foeto-toxic and should be avoided, except for symptomatic infections with multi-drug resistant malaria, in the first trimester of pregnancy \[[@B23]\]. Inadvertent use or self-treatment by adolescent girls could frequently occur in early pregnancy, before they recognise their pregnancy. The issue of pregnancy testing adolescent girls before treatment with potentially foeto-toxic drugs will need to be considered. A further issue is that the benefits of IPT extend to pregnant but not nulliparous adolescents who may be anaemic or parasitaemic at the start of their pregnancy. The use of permethrin-treated bednets would protect against anaemia and parasitaemia during the first trimester, but adolescent pregnant girls and their newborns are the least likely to be net users \[[@B24]\]. Reaching the target population ------------------------------ The growing literature on adolescent-friendly health services shows that adolescents have substantial concerns about the delivery of interventions through health services \[[@B25]\]. Almost universally, adolescents complain of lack of information, confidentiality issues and judgmental attitudes of service providers. There may be barriers of physical inaccessibility, service fees and non-supportive community attitudes. A WHO consultation in Africa concluded that adolescents had \"a right to access health services that can protect them from HIV/AIDS and from other threats to their health and well-being, and that these services should be made adolescent friendly\" \[[@B26]\]. A WHO Global Consultation on Adolescent Friendly Health Services stated that government ministries should take appropriate action to ensure service provision for adolescents, taking into account cost, epidemiological factors and national health priorities \[[@B27]\]. The role of non-governmental organizations and the private sector in spearheading adolescent projects was acknowledged, but alone, these organizations cannot obtain sufficient coverage of the adolescent population to substantially reduce the HIV and malaria burden. Governments and disease control programs must also consider training health care providers and modifying service delivery, so as to reduce barriers to adolescent uptake. The only country in Africa that has scaled up adolescent health services is South Africa \[[@B28]\]. The South African National Adolescent-Friendly Clinic Initiative has strengthened the public health sector\'s ability to respond to adolescent health needs by building capacity and establishing good clinical services which, eventually, will be maintained by district and provincial health authorities. It has demonstrated that an adolescent strategy is achievable if there is political will. The South African approach may not be suitable or affordable for lower income countries. Well-focused services, training of key staff at selected sites, outreach preventive services and good quality, adolescent-oriented ANC are potential service reconfigurations that could be used to extend coverage and increase uptake of both HIV and malaria interventions without creating a separate adolescent service. What about young men? Available evidence suggests that young men are not attracted to clinic services which are perceived as \"female \"spaces \[[@B29]\]. In Ghana, for example, 76--89% of all adolescent clinic users were female \[[@B30]\]. Malaria is a reproductive health issue for females, but not for males, and the disease burden of both malaria and HIV infections is much lower in young males. Adolescent females require more health-service based care than males, and this should be reflected in service configuration. Creating an enabling environment to promote adolescent access to HIV and malaria interventions ---------------------------------------------------------------------------------------------- Behavioural change, including appropriate health-seeking practices, is essential for disease reduction. A decline in HIV prevalence amongst adolescents has been reported from Zambia \[[@B7]\], Uganda \[[@B31],[@B32]\] and Tanzania \[[@B33]\] and is attributed to changes in sexual behaviour. In Zambia, between 1996 and 1999, urban adolescent males reported less frequent sexual activity, fewer partners and were more likely to have used a condom at the last sexual encounter. Changes in these indices were less marked, or even deteriorated, among rural female adolescents \[[@B7]\]. Whether behaviour change is the result of HIV programme interventions or other factors is unclear. Recent surveys in sub-Saharan Africa reported that higher female education was associated with lower HIV prevalence and lack of schooling with higher HIV incidence \[[@B6]\] and lower condom use \[[@B34]\]. In western Uganda the proportion of illiterate women fell from 41.6% to 24.6% over six years and may have facilitated behavioural changes, but this link has been little investigated. Illiterate females are likely to be young and married, and to be in a situation where they cannot reduce frequency of intercourse, negotiate condom use or delay having children \[[@B19]\]. In a large pregnancy study in southern Malawi, 73% of 469 nulliparous adolescents were illiterate \[[@B35]\]. They made significantly fewer ANC visits, were less likely to have a supervised delivery and had a higher risk of low birthweight. Reducing illiteracy and increasing educational and vocational opportunities for female adolescents may be critical enabling actions to promote risk reduction, to enhance their social status and to decrease female vulnerability. \[[@B36]\] Developing policies to support HIV and malaria control for female adolescents ----------------------------------------------------------------------------- Few developing countries have well-defined policies for delivering care to the non-pregnant or pregnant adolescent, especially for sexual health. Existing malaria guidelines also do not mention adolescents specifically \[[@B12]\]. An important aspect is the issue of the adolescent\'s right to sexual and reproductive information and services vis à vis the legitimate rights of parents to act in the best interests of a child, and the health care provider\'s right to work within the law. These are issues of consent and confidentiality for adolescents considered \"minors\" and under parental control, or wives under their husband\'s or husband\'s family\'s jurisdiction. When these issues are ignored, adolescents cannot be assured of confidentiality, and health workers are neither bound, nor protected by, the legal system. Rights established in law, or by signed Conventions (eg Convention of the Rights of the Child), may not be implemented. Health policy falls down because it is not explicit about the vulnerability of female adolescents and does not challenge cultural and social norms that can perpetuate the disease burden. Similarly, encouragement for improving female literacy and education may be lacking, reducing the supportive environment for promoting uptake of adolescent health services and safer sex. Advocacy is essential to co-ordinate law, custom and health practice. At another level, it is important to acknowledge that interventions for female adolescents raise ethical issues that are avoided when they are excluded from care. The policy of avoiding treatment of adolescent girls (and adult women) because they might be in the first trimester of pregnancy, raises issues of gender discrimination and has been a serious issue for parasitic disease control programs \[[@B37]\]. Reducing the necessity for first trimester treatments for malaria can be achieved by increasing efforts to redress the malaria burden prior to pregnancy, but this requires reconfiguring service delivery. HIV-positive adolescents who are at the start of their sexual and reproductive lives might be prioritized to receive anti-retroviral therapy, but their relative poverty and lack of social status may lead to their exclusion from access to this intervention. Conclusions =========== For both HIV and malaria, although interventions are available to reduce the disease burden, adolescents are not prioritized. Effective policies and services are needed to reduce the substantial disease burden they experience from these infections. To secure this goal, concerted action across HIV and malaria disease control programs is required. This will reinforce support for adolescent programs rather than weakening them by multiple separate activities, which can confuse implementation. A schedule of activities is required that is reinforced by other systems (e.g. education) to facilitate embedding it into a way of life. These issues need to be addressed head-on by the adolescent health community to achieve the focus needed, and to establish the realistic steps forward. In the short term, developing services to deliver HIV and malaria interventions can act as a catalyst for addressing broader adolescent health service needs. In the medium-term adolescent-friendly health services provide the required platform for the evaluation and delivery of future vaccines and drug therapies for HIV, malaria \[[@B38]\] and other diseases. In the longer term an inter-sectoral adolescent strategy provides a basis for breaking the cycle of maternal-child ill health. Authors\' Contributions ======================= Both authors made substantial contributions to the analysis, interpretation, drafting and revision of this article. Both have agreed to authorship. Acknowledgements ================ L. Brabin is funded by the Max Elstein Trust. Dr. F. Verhoeff provided unpublished data. B. Brabin has contributed to this review as a member of the PREMA-EU Concerted Action on Malaria and Anaemia Control in Pregnancy (European Commission Research Directorate General, Fifth Framework. Contract: PREMA-EU-ICAL-CT-111012).

PubMed Central

2024-06-05T03:55:52.605506

2005-1-10

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548285/", "journal": "Malar J. 2005 Jan 10; 4:2", "authors": [ { "first": "Loretta", "last": "Brabin" }, { "first": "Bernard John", "last": "Brabin" } ] }

PMC548286

Background ========== *Leishmania*is an intracellular pathogen that causes Leishmaniasis, which remain an important medical problem in several countries. Protozoan parasites of the genus *Leishmania*result in a spectrum of human disease that range from self-healing cutaneous ulcers to potentially fatal visceral infection, depending primarily upon the species of parasite involved \[[@B1],[@B2]\]. *Leishmania*live as either extracellular flagellated promastigotes in the digestive tracts of their sand fly vectors or as nonflagellated amastigotes within macrophages where they survive and replicate within phagolysosome. *Leishmania*are known to export large range of proteins and glycoconjugates including lipophosphoglycan \[[@B3]\]. In a previous paper we have characterized a *Leishmania major*gene encoding a protein carrying extensive homology to SIR2 of yeast that we consequently termed LmSIR2rp \[[@B4]\]. It belongs to a large family of closely related NAD-dependent deacetylase named Hst proteins (Homologous of Sir two) or sirTuins, present in both prokaryotic and eukaryotic species \[[@B5]\]. Post-translational modifications of histones, like acetylation by histone acetylase (HAT) and deacetylation by histone deacetylase (HDAC), within the context of chromatin has been shown to regulate gene expression \[[@B6],[@B7]\]. To date, three classes of HDACs are characterized in eukaryotes, the class I and II HDACs, and the class III defined by the sir2 family. In eukaryotic species some members of the Sir2 family are much more closely related to the core domain of the yHst2p protein than to the core domain of ySir2p itself \[[@B8]\]. These are: the SIR2 family members from *Schizosaccharomyces pombe*, *C albicans*, *Leishmania*, chicken, human (hSirT2p, hSirT3p) and the closely related mouse protein (MmSirT2p and MmSirT3p) \[[@B8]\]. This group has been designated Hst2-like, as it forms an independent branch within the Sir2 family tree. Five members of this group (LmSIR2rp), like it yeast (yHst2) human (SIR2L) and mouse (MmSIR2L2 and MmSIR2L3), are cytoplasmic \[[@B4],[@B9],[@B8],[@B11]\]. The implication of SIR2 proteins bearing nuclear localization, in aging process has been extensively studied. In yeast, the deletion of SIR2 shortens lifespan and over expression extends lifespan \[[@B12]\]. This phenotype of life extension has been also observed in *Caenorhabditis elegans*carrying extra copies of the SIR2 orthologue, Sir-2.1 \[review in \[[@B13]\]\]. In mammalian cell, SIRT1 promote cell survival \[[@B14]\]. SIRT1 is able to deacetylate FOXO3 and/or FOXO4, thus attenuating FOXO-induced apoptosis and potentiating FOXO-induced cell-cycle arrest. Thus SIRT1 might increase longevity by shifting FOXO dependent responses away from cell death and toward cell survival \[[@B15]\]. Also, the unusual NAD-requirement for the Sir2 deacetylase may link metabolic rate to silencing and life span \[[@B16]\]. We have shown that the cytosolic LmSIR2rp protein, when over expressed in *Leishmania*amastigotes, was able to promote parasite survival in *in vitro*culture systems \[[@B9]\]. Further, we also observed that LmSIR2rp can be detected in the excreted/secreted material of *Leishmania*parasites when using radiolabelled immunoprecipitation technique, suggesting therefore that fraction of LmSIR2 is actively excreted by parasite (personal observations). We then initially surmised that this protein could also be involved in some aspect of host cell-parasite interplay, particularly in pathways that could contribute to cell survival. To test this hypothesis, the *leishmania*LmSIR2 gene was cloned in a mammalian shuttle vector and the expression of the protein was performed in fibroblasts. Unexpectedly, expression of the *Leishmania*Sir2 protein leads to a cell growth arrest phenotypes, which correlates, with the expression by the cells of an acidic (pH 6.0) β-galactosidase activity, known to be a senescence biomarker. Furthermore, fibroblasts expressing LmSIR2 were more permissive towards *Leishmania*infection than control ones. Altogether our results may suggest that the excreted forms of LmSIR2 could participate in the perturbation observed during chronic disease. Materials and Methods ===================== Molecular construct and transfection procedure ---------------------------------------------- *LmSIR2*was amplified using sense GAA TTC GAT ATG ACA GGG TCT CCG and antisense CTC GAG CAG TCA CCA TGT TGG CAG primers. The 1119 bp PCR amplified genomic fragment subcloned into pCR2.1 was digested with *Eco*RI and inserted into the *Eco*RI digested and dephosphorylated pcDNA3.1 shuttle vector (Amersham). Restriction analysis of the recombinant plasmids using *Eco*RI and *Xho*I allowed the identification of recombinant vector carrying the *LmSIR2*gene in sense orientation. Large-scale preparations of pcDNA-*LmSIR2*and pcDNA empty vectors were performed using the QIAGEN plasmid maxi kit. Transfected cells were selected in RPMI 1640 medium containing 10% FBS and 400 μg/ml G418. Each G418-resistant clone, isolated by limiting dilution, were propagated and screened by immunofluorescence. Four clones were isolated and named Cl2, Cl9, Cl10 and Cl11. Production of monoclonal antibodies (mAb) against the fusion protein -------------------------------------------------------------------- LmSIR2 recombinant protein containing 6 X histidine residues at its N-terminal (hisLmSIR2) was previously obtained after having subcloned the encoding gene in the pQE-expression vector \[[@B17]\]. Mice of BALB/c strain were injected intraperitoneally with 50 mg of hisLmSIR2 in saline solution emulsified with 0.1 ml of Freud\'s Complete Adjuvant. After 2 weeks, a booster injection of the same preparation was given. The antibody response of the animals was tested 1 month later. The same dose of antigen was injected intravenously 3 days before the hybridization experiment. The fusion procedure and the screening of mAb were carried out as previously described \[[@B18]\]. The class of mAb was determined by agarose double diffusion of supernatants against antisera specific for immunoglobulin subclasses obtained from cliniscience (Montrouge, France). Several hybridoma clones producing mAb reacting in an ELISA test with LmSIR2 protein were obtained; only one mAb of the IgG1 isotype (IIIG4) reacted in Western blot against the fusion as well as the native LmSIR2 protein. Growth kinetic and ^3^H-thymidine incorporation ----------------------------------------------- L929 cells were inoculated at 400 cells/cm^2^, after various period of incubation they were collected by using Trypsin-EDTA (0.025%). The mean number of viable cells was determined at 100-× magnification after staining with trypan blue. In order to evaluate the rate of ^3^H-thymidine incorporation, cells were seeded in 96 wells plates at 1000 cells/cm2, after various periods of incubation 1 μCi of ^3^H-thymidine was added to each well. Cells were further incubated for 4 hours before reading the level of incorporated radioactivity. Giemsa staining and Immunofluorescence analysis ----------------------------------------------- L929 cells were seeded in 16-well Labteck chambers for 48 hrs. Cells were then washed with PBS and fixed for 30 minutes with 4% paraformaldehyde at 4°C. After two washes with PBS, cells were permeabilized with 0.01 M PBS containing 0.5% TritonX-100 and 2% BSA for 30 min at 4°C. Slides were then washed and incubated for 1 hour with mAb IIIG4 in 0.01 M PBS containing 2% BSA. After two washes, slides were incubated for 45 min with fluoresceine-conjugated rabbit anti-mouse IgG (Diagnostic Pasteur, Marnes la Coquette, France) diluted 1:100 in PBS containing Evan\'s blue (final dilution in PBS: 1: 10.000) and mounted in glycerol PBS (1:1). Giemsa staining of the cell culture was performed as follows: cells were inoculated at 400 cells/cm^2^in 25 cm^2^flasks, after 7 days of growth they were fixed with methanol and stained with Giemsa and examined observed on inverted microscope (40× magnification) after adding a solution of PBS 0,01 M-Glycerol (1/1). RT-PCR analysis --------------- Total RNA was isolated from wild type and transformed cells using the Rneasy Mini Kit following the manufacturer\'s instructions. One microgram of RNA was reverse transcribed to cDNA with an oligonucleotide \[poly (dT)~12--18~\] using the Superscript II RNase H-reverse transcriptase. PCR amplifications were performed using 1--2 μl of reverse-transcription of each product and 20 pmol of each primer, using Taq DNA polymerase. Each PCR cycle consisted of a denaturation step (94°C, 1 min), an annealing step (55°C, 1 min) and an elongation step (72°C 1 min). For the last cycle, the elongation step was extended to 10 min at 72°C. Reactions were carried out for 25--35 cycles in a thermocycler (PTC-100™, MJ Research, Inc.). PCR products were analyzed on 1.5% agarose gel and visualized with ethidium bromide. As an internal control, a housekeeping gene, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript, was amplified. *LmSIR2*primers were: Sense GAA TTC GAT ATG ACA GGG TCT CCG and antisense CTC GAG CAG TCA CCA TGT TGG CAG. GAPDH primers were: Sense GTC TTC ACC ACC ATG GAG and antisense CCA AAG TTG TCA TGG ATG ACC. β-galactosidase pH6 analysis ---------------------------- Acidic (pH 6.0) β-galactosidase was assayed according to Dimri et al. \[[@B19]\]. Briefly, stationary-phase cells were washed three times in phosphate buffered saline (0.1 M PBS, pH 7.4), fixed in 2% formaldehyde/0,2% glutaraldehyde for 5 min at room temperature, washed three times with PBS, then incubated at 37°C without CO~2~for 12 h with freshly prepared β-Gal staining solution pH 6.0, containing the 5-bromo-4-chloro-3-indoyl-b-D-galactosidase (X-Gal) chromogenic substrate which produces blue 5-bromo-4-chloro-3-indole upon hydrolysis. Cell preparation was stored at 4°C in 70% glycerol upon analysis. Results and Discussion ====================== Expression of LmSIR2 by L929 fibroblasts ---------------------------------------- The Level of the LmSIR2 gene transcription was evaluated by RT-PCR analysis. As shown in Figure [1](#F1){ref-type="fig"}, an amplification product of around 1.2 Kb was observed only in recombinant L929 cells transformed with the pcDNA carrying LmSIR2 gene (Fig [1](#F1){ref-type="fig"} lane 2), No amplification could be detected in Wild-type fibroblasts or in fibroblasts carrying the empty pcDNA vector (Lane 1 and 3 respectively). Accordingly, Western blot analysis of cellular extracts derived from the clones 2 and 11 reacted with with the mAb IIIG4 showed a polypeptide of 50 kDa (Fig [2](#F2){ref-type="fig"} lanes 3 and 5), which is slightly higher than the molecular size of the pcDNA encoded protein (≈ 43 kDa). However, it is noteworthy that this molecular size is similar to that of the parasite native protein shown in previous studies \[[@B9]\]. It is therefore likely that the transfected gene product is submitted to posttranslational modifications that occur during its processing. No cross reactivity was observed on cell extract derived from Wild-type cell or fibroblast carrying the empty vector (Fig [2](#F2){ref-type="fig"} Lane 1 and 2). Thus, these results confirm that mouse L929 fibroblast cell line properly expressed the *LmSIR2*gene. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Expression of LmSIR2 gene in L-929 cells.**RT-PCR analysis of total RNA from various L-929 clones was carried out using LmSIR2 or GAPDH primers. The lanes correspond to the following samples: standard size marker (PM); WT L929 cells (1); L929 clone 2 (Cl2) (2); L929 carrying an empty pcDNA plasmid (3). ::: ![](1475-9292-4-1-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Detection of the LmSIR2 protein in L929 cell extracts.**The lanes correspond to the following samples: WT L929 cells (1); L929 carrying an empty pcDNA plasmid (2), L929 Cl2 (3); Molecular weight markers (4); L-929 Clone 9 (5). ::: ![](1475-9292-4-1-2) ::: The *LmSIR2*gene was originally isolated via immunoscreening of a cDNA library, using polyclonal antibodies raised against excreted factors which could bind to glutathione \[[@B4]\]. The presence of such protein in the *leishmania*\'s excreted material as previously demonstrated raised the question of its function during the infection process. Thus, heterologous expression in eukaryotic cells could represent a useful approach to give some insight into the biological activity of secreted parasite material. Immunolocalization of LmSIR2 shows that cells expressing LmSIR2 (Figure [3B](#F3){ref-type="fig"}) react with the monoclonal antibody as compared to cells carrying the empty pcDNA vector (Figure [3A](#F3){ref-type="fig"}). Interestingly, high number of cells positive for LmSIR2 bears abnormal morphology i.e. giant multinucleated cells (Figure [3B](#F3){ref-type="fig"}). In these cells, the protein is localized both in the cytosol and the nucleus. Functional studies of several mammalian SIR2 related proteins have been carried out including those who are mainly localized in the cytosol. Nevertheless, similar cellular localization pattern has never been reported nor such modification of cell physiology. Our observations suggest that LmSIR2 possesses unrelated specificity that can strongly affect the biological properties of the host cell. Interestingly, LmSIR2 protein presents a NES (the nuclear export signal or NES)-like motif LX~3~L~X~LX~3~L at its carboxy-terminal region, where L represents Leucine and X any amino acid \[[@B20]\]. The intracellular localization of key regulatory proteins tagged with a short Leucine-rich motif (nuclear export signal) is controlled by CRM1/exportin1, which is involved in the export of these proteins from the nucleus \[reviewed in \[[@B21]\]\]. However, the ability of the *Leishmania*SIR2 to shuttle between the cytoplasm and nucleus of the host cell through the NES putative sequence, await further investigations. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Indirect immunofluorescence analysis and morphology of transgenic cells**. Localisation of LmSIR2 using immunofluorescence analysis (A) L929 cells carrying an empty pcDNA vector (B) L929 (Cl2) expressing LmSIR2 protein. Morphology of cells in culture: (C) L929 cells carrying an empty pcDNA vector and (D) L929 Cl2. Note the reduced cell density and the presence of cells bearing abnormal morphology (arrow). ::: ![](1475-9292-4-1-3) ::: When L929 cell culture, of either clone 1, clone 2, clone 9 or clone 10, were stained with Giemsa, a high rate of cells with unusual shape and size associated with the presence of multiple nuclei was revealed (Figure [3 D](#F3){ref-type="fig"}) as compared to cells carrying the empty pcDNA vector (Figure [3C](#F3){ref-type="fig"}). The rate of multinucleated cells was determined by counting the number of cells bearing more than 2 nucleus per 10 champs at 10-× magnification. In fact all the clone cultures present a 10 to 15-fold increase in giant multinucleated cells as compared to cultures of L-929 fibroblast transformed with the empty vector (Figure [3 C](#F3){ref-type="fig"}). Moreover, majority of the cells if not all, present an enlarged and flattened morphology (Figure [3 D](#F3){ref-type="fig"}). Growth kinetic parameters of L929 cell expressing LmSIR2 -------------------------------------------------------- In order to investigate the potential functions of LmSIR2 protein, when delivered intracellularly in L929 fibroblasts, we have determined the growth kinetic parameters of cells via two different methodologies. Using a counting method, we observed a high reduction in the saturation density of cells expressing LmSIR2 (Figure [4A](#F4){ref-type="fig"}). The reduction observed in this experiment was as high as 40% as compared to cells carrying the empty pcDNA vector. These preliminary observations were further confirmed using ^3^H thymidine. Indeed, as shown in figure ([4B](#F4){ref-type="fig"}), expression of LmSIR2 strongly reduced the thymidine incorporation over the time course of the experiment. The most dramatic reduction of 3H-thymidine incorporation was observed a day 7 which correspond to the maximal cell density observed for both Wild type control and the four clones studied (reduction in ^3^H-thymidine incorporation of about 61% for clone 2, 60% for clone 9, 52% for clone 10 and 42% for clone 11). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Growth kinetics analysis.**Cell proliferation was determined using two methods: cell counting (A), the results are given as a mean value of a triplicate experiment ; and ^3^H-thymidine incorporation (B), the results are given as mean value of a sextuplate experiment ::: ![](1475-9292-4-1-4) ::: Expression of senescence biomarker ---------------------------------- Parameters of senescence include increased cell size, reduced saturation density as well as altered expression of some gene products that have been proposed to serve as biomarkers of aging. Reduction in the final cell yield or saturation density is known to occur in human diploid fibroblast senescence and is considered as a criterion of the onset of senescence \[[@B22]\]. With respect to biomarkers, pH 6 β-galactosidase is particularly useful, since this activity has been found to distinguish senescent from quiescent cells both *in vivo*and *in vitro*\[[@B19]\]. We found that L929 cells expressing LmSIR2 exhibit reduced saturation densities (40% to 60%) and altered cell morphology. We have thus evaluated the occurrence of the pH 6 β-glactosidase activity. As shown on Figure [5](#F5){ref-type="fig"} (B and C), a strong pH-6 β-glactosidase was detected in L929 cells expressing the *LmSIR2*gene but was not detected in wild-type cells or cells transformed with the empty pcDNA vector (Figure [5A](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Expression of a senescence biomarker.**(pH6.0) β-galactosidase activity detected in L929 cells expressing LmSIR2 (Cl2) (B and C) but not in L929 carrying an empty pcDNA plasmid (A). Cells were seeded at 40 cells/mm^2^in 25 cm^2^flasks and after 13 days of growth, the presence of pH6-galactosidase activity (Arrow) was determined as described in the Materials and Methods section. N nucleus, note the perinuclear galactosidase activity. ::: ![](1475-9292-4-1-5) ::: Permissivity of fibroblast expressing LmSIR2 towards *Leishmania amazoensis* ---------------------------------------------------------------------------- Fibroblasts at the lymph are used as safe target for long lasting residual parasite population \[[@B23]\]. However, only few reports deal with *in vitro*infection of fibroblast. In 1978, Chang \[[@B24]\] demonstrated that promastigotes of *L. amazonsensis*but not *L. donovani*were able to infect human skin fibroblasts. However, once differentiated into amastigotes the parasites were unable to multiply inside the cell. Dedet and co-workers further confirmed these observations \[[@B25]\]. Thus, we decided to test the capacity of *L. amazonensis*to infect L929 cell lines. When *L. amazonensis*promastigotes were incubated with Wild-type fibroblasts the proportion of infected cells reached a peak of about 50% after 24 hours of contact, after what the proportion of infected fibroblasts decreased quickly. On day 2, only few parasites could be seen and after 3 days of incubation less that 1% of fibroblasts remain infected (data not shown). As shown in Figure [6](#F6){ref-type="fig"}, fibroblasts expressing LmSIR2 were more permissive toward *Leishmania*infection since the mean percentage of infected fibroblast reach 70% after 24 hours of contact. However, once inside fibroblasts no differences in the capacity of parasites to survive and/or multiply was observed and the intracellular amastigote population was eliminated after three days of contact (data not shown). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Permissivity of L929 cells toward *Leishmania amazonensis*infection.**L929 cells were seeded in 25 cm2 flasks and treated with mitomycin. They were then infected, at different parasite/fibroblast ratio, with freshly differentiated stationary phase *L. amazonensis*promastigotes. After 24 hours of contact cells were washed and the percentage of fibroblats bearing attached or internalized parasites was determined. Results are expressed as a mean value of a triplicate experiment. ::: ![](1475-9292-4-1-6) ::: Conclusions =========== Our study demonstrates that LmSIR2 is able to down regulate the proliferation of fibroblasts and to induce a senescence-like state in L929 fibroblast cell line. The most interesting finding is that a parasitic protein is able to modify the physiology of the host cell making them more permissive toward *Leishmania*infection. These results suggest that LmSIR2 has dual function according to the cell type in which it acts: in *Leishmania*parasites, LmSIR2 is able to promote cell survival while in the host fibroblast it induces a senescence phenotype. This effect could be beneficial for *Leishmania*since senescent cells are no longer capable of dividing yet remaining metabolically active. They could thus represent safe target for parasite. It is documented that senescent fibroblasts present an inherent higher capacity to resist to apoptotic death \[[@B26]\]. In this view, it will be of interest to determine if fibroblasts that express LmSIR2 were more resistant to apoptotic stimuli. It is known that infected macrophages are usually more resistant to apoptosis, however it is not know if such mechanism operate in infected fibroblast. Thus, the implication of LmSIR2 in a general mechanism leading to apoptosis resistance of both macrophages and fibroblasts await further investigations. The *in vivo*relevance of our observation is rather difficult to appreciate because; (1) the level of the protein produced in fibroblasts transformed with the pcDNA is certainly far higher than the level achievable *in vivo*, and (2) the presence of excreted parasite material inside the fibroblast cytosol has yet not been reported. However, an action of the excreted form of LmSIR2 on he host cell could no be ruled out. Two series of observations support this possibility: (1) it has been shown that the *Leishmania*EF1-a could be translocated from the parasitophorous vesicle into the macrophage cytosol \[[@B27]\]; (2) the ultrastructural examinations of skin biopsies obtained from patients with cutaneous leishmaniasis showed that intracellular amastigotes could be frequently identified in vacuoles and in the cytosol of the host cell \[[@B28]\]. In conclusion, although the effect of the protein, when delivered extracellularly, on the host cells have not being examined in the present study, our data support the notion that the excreted form of LmSIR2 is able to interfere with the host cell physiology, when delivered intracellularly. Unexpectedly the expression of the protein is associated with the appearance of a senescence biomarker. Relevance of such finding during the *in vivo*infection process awaits further investigations Acknowledgement =============== This work was supported in part by a grant from IRD and INSERM. AMA was supported by a DSF IRD (France) grant. BV was supported by an ANRS (France) grant.

PubMed Central

2024-06-05T03:55:52.607218

2005-1-24

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548286/", "journal": "Kinetoplastid Biol Dis. 2005 Jan 24; 4:1", "authors": [ { "first": "Denis", "last": "Sereno" }, { "first": "Laurent", "last": "Vanhille" }, { "first": "Baptiste", "last": "Vergnes" }, { "first": "Adriano", "last": "Monte-Allegre" }, { "first": "Ali", "last": "Ouaissi" } ] }

PMC548287

Review ====== Assessments of patient-focused health status and health-related quality of life (HRQL) are being recognized increasingly by clinicians, patient advocates, regulatory authorities, administrators and policy makers as primary measures of the need, efficacy, effectiveness and efficiency associated with health care services. These types of measures are commonly reported in the published literature of many health care disciplines although there are few published reports concerning orthopedic surgery. However, the orthopedic community is becoming interested in using evidence reported by patients and patients\' family members to determine what is best for patients. Patient-focused evidence (including patient-reported outcomes, PROs) refers to results from functional health status (FHS) and HRQL studies of orthopedic outcomes with measurements obtained from the perspective of patients or their non-clinician caregivers (e.g., parents of patients too young to self-report). The field of orthopedics is concerned with all musculo-skeletal problems from the top to the bottom of the human body. This broad anatomical range is associated with a wide range of functional problems. Some orthopedic problems are highly focused in regards to anatomy and associated with highly effective therapies. However, other orthopedic problems are of a diverse nature, the etiology is often poorly understood, and effective treatment strategies are sometimes elusive. The breadth and depth of some particularly challenging orthopedic problems has stimulated an interest in patient-based perspectives of their own FHS and HRQL. These types of measures will be referred to as \"health measures\" for the purposes of this paper. Health measures can be used to assess the burden of morbidity associated with orthopedic diagnoses, and to assess the effectiveness and efficiency of therapies. The diversity of issues also led the American Academy of Orthopedic Surgeons and the Pediatric Orthopedic Society of North America to develop the Pediatric Outcomes Data Collection Instrument (PODCI), and led other researchers to develop function-specific instruments such as the Activities Scale for Kids (ASK). However, there are a large variety of potentially appropriate health measures and there are limits to the number of these measures that can be used in any given study. The most appropriate set of health measures should be selected on the basis of underlying study objectives and design criteria \[[@B1]\]. Objectives and designs vary greatly across orthopedic studies. In general, orthopedic procedures and programs are undertaken to improve the health of patients. The health of patients can be measured in different ways. Conventional clinical or physiological measures are generally very useful for diagnostic and therapeutic purposes. Conventional clinical measures may, however, provide an incomplete assessment of health. For example, clinical examination and gait measures have been used to describe the quality and quantity of limitations in walking ability associated with hip flexion contracture but these types of assessments provide little information about the importance of this contracture relative to that of other serious health problems. In addition, various measures may provide conflicting results: some indicating improvement while others suggest decline in patients\' health. Therefore, in the interest of scientific rigor, protocols to assess the overall effectiveness of therapy should include *a priori*specification of the comprehensive measure of health and a credible assessment viewpoint for the purposes of primary analysis of study results. Secondary study objectives may involve use of data collected from other health measures or from other assessment perspectives. FHS and HRQL measures are important for a variety of reasons that complement conventional clinical measures \[[@B2]\]. FHS measures provide descriptive information and HRQL measures add some type of valuation about the desirability of overall health status. Valuation may be based on preference-based scores or by including only items identified as important to patients. HRQL is a more comprehensive concept than FHS as noted in this leading definition. \"Health-related quality of life is the value assigned to duration of life as modified by the impairments, functional states, perceptions, and social opportunities that are influenced by disease, injury, treatment or policy\" \[[@B3]\]. It is generally accepted that a goal of therapy is to make patients feel better \[[@B4]\] and this is supported by statements of leading policy analysts such as \"The goal of health care is to protect, promote, and maintain the health status of people\" \[[@B5]\] and \"Since the ultimate goal of all health care is to improve, restore, and preserve HRQL\" \[[@B6]\]. However, physiological measures may change without people feeling better and people may feel better without measurable change in physiological function. Furthermore there is often a need for trade-offs between treatment-related benefits and adverse side effects. There is an increasing awareness also that various stakeholders in clinical decisions often have differing opinions about disability and that the opinions of patients should count. Numerous valid and reliable questionnaires are now available to collect FHS and HRQL measurements from patients and their representatives. The measures may be used for discriminative (comparing groups at a point in time such as assessing the burden of illness), evaluative (assessing within-person change over time as in clinical trials), and predictive purposes (providing prognostic information) \[[@B7]\]. A series of studies from a randomized controlled clinical trial (RCT) and prospective cohort study of elective total hip arthroplasty are illustrative of these purposes. These examples, while not from a pediatric setting, are orthopedic and demonstrate how data can be used for a variety of purposes. In the RCT, Rorabeck and colleagues \[[@B8]\] undertook a discriminative analysis of a set of FHS and HRQL measures, and reported there were no differences in outcomes between cemented and non-cemented protheses. Evaluative analyses of data from the same trial have documented improvements according to six HRQL measures and the six-minute-walk test \[[@B9]\], and have shown important differences among results from four major generic HRQL measures \[[@B10]\]. In the prospective cohort study by Mahon et al \[[@B11]\], waiting time for surgery was inversely related to baseline WOMAC (Western Ontario and McMaster University Osteoarthritis Index) scores for patients at time of referral to an orthopedic surgeon. There are many factors to consider when designing studies using patient-focused health measures for orthopedic patients. Among the most important factors are the study objectives, types of patients, the dimensions and FHS constructs associated with the health problem under study, the appropriateness of the questionnaire for the age range of the patients, the viewpoint of the assessors answering the questionnaire, the method of collecting questionnaire information (self-completion or interviewer-administration), the period of time respondents are asked to consider when answering the questionnaire (i.e., assessment recall period), and measurement properties of assessment tools. Measurement properties include content and construct validity, test-retest and inter-rater reliability, responsiveness, and practical limitations of data collection. It takes many years, and often decades, for credible and relevant evidence to be accumulated about the measurement properties of instruments. Instruments with well-established measurement properties should be used whenever possible. Two prime issues for assessments of children are the developmental stage of the subjects and the most appropriate respondent for questionnaires. The methods section of the paper presents a framework of important factors for consideration in designing studies to assess FHS and HRQL for orthopedic patients. The results were generated by applying the framework in the context of developing a proposal for a comprehensive international study of patients with neurofibromatosis type 1 (NF1) and congenital tibial dysplasia (CTD). The NF1-CTD protocol provides a rich illustration because it encompasses great variability in the instrument selection factors, and these patients with their supportive caregivers face numerous complex health status challenges with long-term implications for HRQL. Furthermore, there is little information in the published literature about the comprehensive health status and HRQL of patients with NF1-CTD. The rationale and approach to studying FHS and HRQL from a patient perspective have relevance to many other areas of orthopedics. Methods: a framework for orthopedic applications ================================================ This section identifies specific study design and measurement instrument factors that should be considered in selecting measures for use in studies, and presents some background information for illustrating the application of these factors. Study design factors -------------------- Study design factors are specified in detailed protocols that clearly define the study objectives and conditions. Well-defined study objectives identify the study subjects, and type and number of measurements required. Study conditions describe practical issues including data collection sites, and budgets for time and resources. Variability in study objectives ------------------------------- A highly focused, single objective study may require relatively few instruments while a broad-based, multi-faceted study will require numerous measures. For example, a RCT of efficacy for an experimental technique to achieve union of bone compared to a conventional technique might specify patients\' reports from a single well-established walking ability scale as the primary outcome measure. However, an economic evaluation of the cost-effectiveness and cost-utility of the experimental technique would require at least two instruments: the single well-established walking ability scale to estimate effectiveness for the numerator in the cost-effectiveness ratio; and a utility-based scale of overall HRQL to estimate quality-adjusted life years for the numerator in the cost-utility ratio. Age range of subjects --------------------- The age of study subjects affects the choice of measurement instruments and the method of collecting data. Each instrument is valid for a specific age range and the age range is quite small for many pediatric instruments. For example, developmental changes during childhood make it difficult for single instruments to be valid from infancy through adolescence. Many instruments are valid for one or more of the following age categories: infants, pre-school children, primary-school children, adolescents, young / middle-age adults, seniors and elderly. Inability to read well (e.g., young children or populations with high illiteracy rates), or see well (e.g., many elderly populations), or concentrate well (e.g., people on certain strong medications) inhibits use of self-complete questions. Well-designed interviewer-administered questionnaires pose clear, short, well-focused questions with readily understood and easily remembered response options (e.g., yes or no response options). Assessment perspective ---------------------- It is becoming widely accepted in many disciplines that studies of health care programs and technologies should include measures of patients\' perceptions of their FHS and HRQL \[[@B12]\]. Measurements of these types have been shown to vary by assessment viewpoint and, although many different viewpoints are valid, the patient perspective is considered to be one of the most important. It has been shown that children as young as 7 years can reliably complete interviewer-administered disease-specific and generic questionnaires about their own health \[[@B13]\]. Type of data collection sites ----------------------------- The locations and cultural characteristics of the study population will determine the language and sometimes the choice of assessors. Relatively few instruments have been carefully translated and culturally-adapted to facilitate use in a large variety of communities. Some cultures accept a variety of assessment viewpoints (e.g., patient, family, nurse, physician, and other allied health professionals) while other cultures recognize only a physician viewpoint for health assessments. Mode of data collection ----------------------- The literature suggests that there may be important effects due to mode of data collection \[[@B14]\] and, therefore, mode of administration should be standardized across subjects, assessors, and assessment points. In general, self-complete questionnaires tend to elicit reports of greater morbidity than interviewer-administered questionnaires. It is hypothesized that subjects may be less inhibited to report disabilities on self-complete questionnaires than to report disabilities to interviewers. The mode of data collection for a study may be determined by numerous factors other than the characteristics of study subjects mentioned above. A low budget study may rely on a mail survey, or distribution of questionnaires in a busy clinic setting, and use self-complete questionnaires. Alternatively, a study involving serial assessments may require follow-up data to be collected by telephone and therefore require use of questionnaires designed for interviewer-administration. Assessment recall period ------------------------ FHS questionnaires should have well-specified assessment periods to help ensure that the subjects and researchers know the period of time covered by the responses. Assessment recall period refers to the period of time the assessor is asked to consider when answering the questionnaire. The period should match the objectives of the study. For instance, if a new surgical technique is thought to reduce the peri-operative burden of morbidity then frequent assessments with a brief recall period (24 hours) might be suitable. Standard assessment periods include the past 24 hours, the past 1 week, the past 2 weeks and the past 4 weeks. Short assessment periods should be used in studies of patients whose FHS varies over time and in studies involving serial assessments to ensure that there is no overlap in assessment times. Relatively long recall assessment durations may be used when it can be assumed patients\' health status is fairly stable. For example, a four-week recall assessment period was used to measure the HRQL of patients in a randomized clinical trial and economic evaluation of two alternative treatment strategies for patients with knee osteoarthritis \[[@B15],[@B16]\]. Other factors ------------- There are limits to the number and type of measures that respondents can be expected to complete without getting tired or frustrated, and/or that the study budget can afford in regards to both data collection and analysis. The evidence about the limits of respondent burden is sparse. All else being equal, studies involving serial assessments should expect to collect fewer measurements per assessment than single-assessment cross-sectional surveys. To maximize efficiency, instruments should be selected to provide complementary rather than overlapping information. Instrument factors ------------------ ### Types of measures There are numerous definitions of FHS and HRQL. For the purposes of the paper, a FHS measure is defined as being descriptive in terms of functional ability and HRQL is defined as involving some form of valuation of that health status. One published taxonomy \[[@B7]\] suggests that measures may be classified as specific or generic. Specific measures are focused on a specified health problem, disease, or age group of subjects. An example is the Western Ontario McMaster Osteoarthritis Index \[[@B17]\]. Specific measures designed for evaluative purposes are often able to detect small but clinically important differences among subjects and be responsive to small but clinically important intra-subject changes over time. Generic measures are applicable to a broad range of subjects, including a wide variety of clinical groups and general populations. There are 2 types of generic measures: generic health profile instruments such as the Rand-36 \[[@B18]\] and SF-36 \[[@B19]\]; and generic preference-based instruments. There are 2 types of generic preference-based instruments: direct measurement instruments, such as standard gamble \[[@B20]\]; and multi-attribute classification systems with preference-based scoring functions \[[@B21]\], such as the Health Utilities Index \[[@B22],[@B23]\] and the Quality of Well-Being Scale \[[@B24],[@B25]\]. A detailed description of direct measurement techniques is beyond the scope of this paper but provided in a recent review paper by Torrance and colleagues \[[@B20]\]. Direct preference measurement is generally not practical for application in most clinical studies, especially those involving very young children. Customized instrumentation, usually relying on administration by highly skilled interviewers, must be developed for each study. Further, measurement questions are cognitively demanding. Direct preference measurement will, therefore, not be considered further in this paper. Each multi-attribute system includes a descriptive classification scheme to describe and assess health status, and a preference-based valuation system. HRQL scores for health states defined by multi-attribute systems are calculated from models fitted from directly measured preference measurements (see below). Multi-attribute systems provide descriptive information about comprehensive health status, and interval-scale preference scores of overall HRQL from a community perspective on a scale where 0.00 is the score for being dead and 1.00 is the score for being in perfect health. Several multi-attribute systems define negative scores of overall HRQL to represent preferences for states considered worse than being dead. A few systems include single-attribute preference-based scales of morbidity. Single-attribute morbidity scales are defined such that the least desirable level within an attribute (dimension of health status such as vision) has a score of 0.00 (blind) and the most desirable level has a score of 1.00. The community perspective is most widely recommended for technology assessment and reference case economic evaluation analyses \[[@B26]-[@B29]\]. Interval-scale properties, and a score of 0.00 for being dead, are important features of HRQL scales for integrating the effects of morbidity and mortality in descriptive studies and in cost-utility economic evaluations. Interval-scale preference scores of HRQL may be either utilities (e.g., Health Utilities Index Mark 3) or values (e.g., EQ-5D). Utility preference measures are based on von Neumann-Morgenstern utility theory, include an element of risk attitude, and are therefore appropriate for decision problems with uncertainty. Value scores are preferences measured under certainty. Details about differences between utilities and values, and about direct preference measurement, appear in recent papers by Torrance et al \[[@B20],[@B30]\]. Uncertainty is an important factor in many orthopedic procedures and therefore utility scores are more appropriate than value scores in this context. Evidence of measurement properties: validity, reliability, and responsiveness ----------------------------------------------------------------------------- A valid measure is \"sound and sufficient\" \[[@B31]\]. There are many ways to assess validity of measures. Assessments of FHS and HRQL measures should consider at least six types of validity: face validity, content validity, construct validity, convergent validity, discriminative validity, and predictive validity \[[@B32]\]. Face validity requires that a measure appear on the surface to make sense in regards to being relevant and useful. Content validity requires that the measure include all important and relevant domains or dimensions of health status. Construct validity describes the extent to which a measure corresponds to theoretical concepts and convergent validity describes the association between related variables. Discriminant validity is a lack of correlation between dissimilar variables or groups. Predictive validity, one type of criterion validity, describes the relationship between current and future measurements. A measure is reliable if it is sound and dependable \[[@B31]\]. Reliability is assessed by tests of repeatability or reproducibility. Reliability is often assessed in terms of agreement between intra-subject test-retest measurements and inter-assessor measurements \[[@B33]\]. Responsiveness is also referred to as sensitivity to change. It is an important feature for determining a measure\'s ability to detect effects of treatments or natural changes over time (e.g., due to the aging process). Husted and colleagues reviewed the literature and defined two major types of responsiveness: internal and external responsiveness \[[@B34]\]. Internal responsiveness describes the ability of a measure (instrument) to change and has been assessed using a variety of techniques including the magnitude of statistical significance tests (e.g., p \< 0.05 versus p \< 0.001), the mean change score divided by the standard deviation of scores at baseline (effect size), and a sensitivity coefficient calculated as the proportion of the variance in change scores due to treatment \[[@B32]\]. It has also been assessed as the ratio of the mean change in patients\' scores and the pooled standard deviation of the mean change scores \[[@B35]\], and as the mean change score among those who changed divided by the standard deviation of change scores among stable patients \[[@B33]\]. External responsiveness is concerned with the relationship between change in a measurement and change in a reference measurement of health status. External responsiveness has been assessed using the receiver operating characteristic method, correlations (e.g., Pearson product moment correlation), and regression models. The minimum important difference (MID) is the smallest size of difference that is important from patients\' or clinicians\' perspectives. The MID between two measurements is a concept closely related to responsiveness when assessing change over time \[[@B36]\]. Ceiling and floor effects are undesirable properties that reduce the validity, reliability, and responsiveness of measures. A ceiling effect may occur when a large proportion of measurement observations are close to the upper bound of the measurement scale. A ceiling effect results in a positively skewed distribution of measurements, limited ability of the measure to discriminate among subjects at the upper end of the scale, and attenuated responsiveness to improvements in health in longitudinal studies. A floor effect may occur when a large proportion of measurement observations are close to the lower bound of the measurement scale. Floor effects create a negatively skewed distribution of measurements, limited ability of the measure to discriminate among subjects at the lower end of the scale, and decreased responsiveness to decrements in health in longitudinal studies. Many generic and specific measures of HRQL may be subject to ceiling effect problems in that they may not be able to describe patients or subjects with above average (supra-normal) health. Some measures are subject to floor effect problems. Some are subject to both. Typically floor effect problems are more serious in clinical studies (which often involve patients with disabilities) and ceiling effect problems may be more problematic in general population studies. Limits of respondent burden --------------------------- The limits of respondent burden depend upon many factors including the number of questions presented, how the questions are presented, the complexity of questions, the sophistication of respondents, and the respondents\' interest in the questions. In general, the allowable length of questionnaire is shorter for mail and phone administration than for face-to-face interviewer administration \[[@B37]\]. One set of guidelines specifies the following maximum lengths: 20 questions for phone surveys; 60 questions for mailed surveys; and 80 questions for face-to-face interview surveys \[[@B38]\]. Another guideline recommends that telephone interviews not exceed 5 to 10 minutes \[[@B39]\]. These guidelines are in general agreement with maximum recommended number of pages for self-administered questionnaires: 2 to 4 page upper limit for topics not especially salient \[[@B40]\]; 12 page upper limit for self-administered questionnaires \[[@B41]\]; and 4 to 6 page upper limit for mailed surveys \[[@B42]\]. For mail-out surveys, the evidence suggests no effect of length on response rates for questionnaires varying from 3 to 9 pages \[[@B43],[@B44]\] but reduced response rates with questionnaires greater than 12 pages \[[@B41]\]. Availability of support services -------------------------------- Applications of FHS and HRQL measures are greatly facilitated by expert advice, detailed instructions and other services designed to support users of a measure. Supporting documentation is usually protected by copyright and should not be used without written permission of the original developers. Documentation obtained from third-party sources should be considered suspect because it is frequently invalid. Licensing fees are used to fund high quality, readily accessible service centers. Permission to use copyright materials is typically granted one study at a time. Support services may also include consultation about the most appropriate versions of questionnaires for use in a specific study. Application packages may include data collection instruments such as questionnaires, procedure manuals, coding algorithms and scoring systems, as well as background information about the conceptual and measurement properties of the instrument. NF1-CTD: A case study --------------------- Recently there has been interest in using measures of FHS and HRQL to evaluate treatment alternatives for NF1-CTD. NF1 is one of the most common genetic disorders in childhood \[[@B45]\]. It is estimated that at least 1 million people throughout the world have NF1 \[[@B46]\]. NF1 has a wide range of clinical manifestations including abnormalities of the skin, nervous system, bones and soft tissues \[[@B46]\]. Other conditions experienced by children with NF1 include short stature and neurologic problems such as learning disabilities or unspecified school performance problems (36%), frequent headaches (28%), mental retardation (6%), and reduced reproductive potential \[[@B46]-[@B49]\]. CTD is rare in the general population, approximately 1 per 140,000 \[[@B46]\]. It has been estimated that approximately 1% of people with NF1 have CTD \[[@B46]\]. CTD is diagnosed usually during the first year of life and fractures often occur before 3 years of age. Frequently, initial presentation is tibial bowing followed by subsequent fracture and pseudoarthrosis \[[@B45]\]. There is no generally accepted standard for management of CTD although most surgeons would suggest initial treatment of either intramedullary fixation with bone grafting or resection and bone transplant. Surgical procedures for the treatment of CTD are fraught with complications and failure of union. For the treatment of CTD, pre-fracture bracing until skeletal maturity may be a better alternative than surgery. CTD is associated with severe complications due to nonunion or pseudoarthrosis after osteotomy and amputation may be required. Conventional clinical measures of CTD include the Crawford classification system \[[@B46]\]. These measures provide clinicians with important information used in diagnosis and management of well-established symptoms. A list of important concerns could be prepared by interviewing patients and members of their families. Standardized comprehensive tools that integrate multi-dimensional effects would also be useful in quantifying the number and extent of problems experienced by NF1-CTD patients, and other pediatric orthopedic patients with complex issues. The published literature on NF1 and NF1-CTD contains virtually no information based on FHS or HRQL measurements. The exception is a recent paper by Wolkenstein and colleagues \[[@B50]\] who reported results from 128 adult patients in France using the generic health profile SF-36 and a skin-disease-specific measure, Skindex-France. Surveys of the published literature, experts in the fields, web sites and other sources of information were conducted to determine the dimensions of health that are affected by NF1-CTD, the types of FHS and HRQL measures that have been used, which measures should be considered as potentially useful for studies of NF1-CTD, the measurement properties of potentially useful measures, and the relative merits of various measures. A review of the on-line Quality of Life Instruments Database (QOLID) developed by Dr. Marcello Tamburini and the MAPI Research Institute \[[@B51]\], and correspondence with instrument developers, identified a short list of potentially useful measurement tools in each of the following categories: generic preference-based HRQL systems, major pediatric and other generic health profiles, and disease or function specific measures. Selected measures should have demonstrated properties in accordance with currently accepted criteria \[[@B12],[@B52],[@B53]\] and should provide commensurate measurements for patients across a wide age range. Problems with mobility, cognition, pain, emotion (including impacts of problems with self-image), self-care, vision, and fertility are aspects of health reported in the published literature to be compromised in NF1 patients. Illustrative study design criteria ---------------------------------- There are five important research objectives of an NF1-CTD study that provide a context for applying the framework described in the Methods section: 1\) to document long-term health outcomes associated with the disease and its treatment; 2\) to measure the burden of disease and treatment during active therapy; 3\) to investigate the hypothesis that improved HRQL is associated with initial amputation compared with multiple limb-saving procedures; 4\) to determine relationships of FHS and HRQL with conventional clinical variables used in diagnosis and management; and 5\) to assess the measurement properties (e.g., construct validity, patient versus parent inter-rater reliability, and responsiveness to change) of selected FHS and HRQL measures in NF1-CTD patients. These detailed objectives require the identification and assessment of leading FHS and HRQL measures for use in both cross-sectional and prospective longitudinal surveys. The prevalence of NF1-CTD is relatively low. Patients will need to be recruited from numerous clinical centers in North America to generate precise estimates of FHS and HRQL. Questionnaires should be available in at least 3 major languages: English, French and Spanish. The survey population ranges in age from newborn into adulthood and linking results across the study objectives requires that at least some of the assessment tools be in common across the age range of study patients. To avoid potential confounding effects, data collection techniques should be consistent across subjects and measures. The patient-focus will be represented by collecting measurements from all patients old enough to provide self-assessments, and from parents acting as proxy assessors for all children and adolescents. Self-complete questionnaires requiring minimal supervision should be used to eliminate the need for interviewers at each clinical center, to facilitate use of mail-out surveys, and to avoid potential \"interviewer\" effects. The number and type of measures per assessment, and the number of serial assessments per patient, should be sufficient to address all the study objectives within the limits of study resources and assessor burden. Measures of morbidity associated with NF1-CTD should be comparable with data on norms from surveys of general populations and other patient groups, and be useful for assessing the effectiveness and efficiency of health care services. Existing patient-focused health measures ---------------------------------------- The HRQL measure should be comprehensive and preference-based, to facilitate a broad variety of comparisons. A pediatric health profile measure and other specific measures will be selected to complement the selected preference-based HRQL measure. FHS measures may be focused on one or more of the following: the population of interest (e.g., pediatrics); the major underlying disease (e.g., NF1); the major human function of most interest (e.g., walking ability); the medically-defined health problem of most interest (e.g., tibial dysplasia); the medical speciality most involved with treatment of the health problem (e.g., pediatric orthopedics). There are six major generic preference-based HRQL utility systems \[[@B21]\], presented here in chronological order of development: QWB \[[@B25]\], 15D \[[@B54]\], HUI \[[@B23]\], EQ-5D \[[@B55]\], AQOL \[[@B56]\] and SF-6D \[[@B57],[@B58]\]. HRQL scores from these systems represent mean community preference scores. The 15D and AQOL have not been widely used outside of Finland and Australia respectively and, therefore, will not be described further in this paper. The SF-6D has been developed only recently so there is as yet little evidence to report. The major features of QWB, HUI, EQ-5D, and SF-6D systems are summarized in Table [1](#T1){ref-type="table"}\[[@B21],[@B25],[@B57],[@B59],[@B60]\]. The major characteristics vary greatly among the systems. For example, linear additive scoring models do not include effects of preference interactions among attributes or domains but multiplicative scoring functions include these effects. The QWB is available in both self-complete and interviewer-administered formats \[[@B61]\]. The symptoms attribute is a dominant feature of the QWB health status classification system. This emphasis is reflected in the population-derived preference weights. HUI health status classification systems cover more than 10 attributes. There is evidence that HUI scores agree well with mean directly measured standard gamble utility scores from a representative sample of the general population \[[@B59],[@B60],[@B62],[@B63]\]. Numerous versions of HUI questionnaires are available and HUI has a service center \[[@B64]-[@B66]\]. It is available in numerous languages. A closely-related comprehensive health status classification system for pre-school children (CHSCS-PS) has been developed recently \[[@B67]-[@B69]\] for children age 2 through 5 years of age. EQ-5D is very simple and concise. It consists of 5 attributes with 3 levels per attribute, assesses \"current\" health status, has been used in a large number of studies, and is available in numerous languages. Information, including a long list of references, about EQ-5D is available on the EuroQol Group web site \[[@B70]\]. SF-6D is a multi-attribute health status classification system based on the SF-36 \[[@B19],[@B71],[@B72]\]. The SF-36 was not designed to be commensurate with the fitting of a multi-attribute utility function. The SF-6D health status classification system is a sub-set of the attributes defined in the SF-36 health status classification system \[[@B57]\]. SF-6D utility scores may be useful in retrospective studies analyzing previously collected SF-36 data. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Major Characteristics of Five Generic Preference-Based Multi-Attribute Systems ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Instrument** **QWB** **HUI** **EQ-5D** **SF-6D** --------------------------------------------- -------------------------------------------------------------------- ------------------------------------------- --------------------------------------------------------------------------- --------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------- **Developed** 1970s 1980s 1990s 1990s 1990s **\# Attributes (\# levels per attribute)** 4 (3 to 27) 7 (3 to 5) 8 (5 or 6) 5 (3) 6\* (4 to 6) **\# of unique health states** 1215 24000 972000 243 18000 **Attributes** Mobility, Physical activity, Social activity, Symptoms / problems. Sensation, Mobility, Emotion, Cognition,\ Vision, Hearing, Speech, Ambulation, Dexterity, Emotion, Cognition, Pain. Mobility, Self-care, Usual activities, Pain / discomfort, Anxiety / depression. Physical functioning, Role limitations, Social functioning, Pain, Mental health, Vitality. Self-care,\ Pain, Fertility. **Types of HRQL scores and measurement** Values; VAS Utilities; VAS and SG combination Utilities; VAS and SG combination Values; TTO Utilities; SG **HRQL scoring model form** Linear additive Multiplicative Multiplicative Modified linear additive Modified linear additive **HRQL scale interval** 0.00 to 1.00 -0.03 to 1.00 -0.36 to 1.00 -0.59 to 1.00 0.00 to 1.00 **Questionnaire formats (languages)** 2 (numerous) 16 (numerous) 1 (numerous) 2 SF-36 versions (numerous) ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- SG -- standard gamble; TTO -- time trade-off; VAS -- visual analog scale. \* -- 6 attributes but role limitations attribute above includes emotional and physical so encompasses 7 of 8 SF-36 domains. ::: The major population-specific health profiles include the Child Health Questionnaire (CHQ), Pediatric Inventory of Quality of Life (PedsQL), Pediatric Evaluation of Disability Inventory (PEI) and TNO-AZL Pre-School Children Quality of Life questionnaire (TAPQOL). The PEI is limited to children age 0.5 -- 7 years of age and requires a structured parent interview or clinician observation \[[@B73]\]. TAPQOL \[[@B74],[@B51]\] is limited to children 0.5 to 5 years of age. Therefore, PEI and TAPQOL will not be discussed further. The major pediatric disease-/function-/specialty-specific instruments include the PODCI (also referred to as the POSNA or Pediatric Orthopedic Society of North America instrument), ASK, Gillette Functional Assessment Questionnaire Walking Scale (FAQ walking scale), and Wee-FIM. Wee-FIM \[[@B75]\], a popular measure of functional independence, is not being considered because it involves clinician assessments rather than assessments from a patient or parent perspective. In general, disease-specific scales in orthopedics focus on pain and physical function because these factors are major areas of concern for orthopedic patients and no generic health measures have been developed specifically for orthopedic application \[[@B73]\]. No relevant disease-specific measures or disease-specific preference-based tools were identified. A summary of the major pediatric generic health profiles appears in Tables [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}. The CHQ \[[@B76]\] covers relevant physical domains and provides detail on emotion/psychological health. The PedsQL \[[@B77]\] assesses physical, emotional, social and school functioning. It has demonstrated a return to health 3 months after acute limb fractures \[[@B78]\] and has been used in large general population surveys \[[@B79]\]. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Major Characteristics of Two Pediatric Health Profile Systems ::: ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Instrument** **CHQ** **PedsQL 4.0** ------------------------------- -------------------------------------------------------- ----------------------------------------------- ------------------------------ ------------------------------------------------------ ---------------------- **Full Name** Child Health Questionnaire Pediatric Inventory of Quality of Life **Origin** 1996 1998 **Version** Parent Forms (PF) Parent Form for Infants & Toddlers Youth- Completion Form Child Self-Report Forms\ Parent Report Forms\ 1) ages 5--7 years\ 1) ages 2--4 years\ 2) ages 8--12 years\ 2) ages 5--7 years\ 3) ages 13--18 years 3) ages 8--12 years\ 4) ages 13--18 years **Measurement Constructs** Measures of physical and psycho-social health concepts Measures of core dimensions delineated by WHO **Age Bounds (years)** 5 to 17 2 months to 5 years 11 to 17 5 to 18 2 to 18 **\# Items** 98 (PF98) or 50 (PF50) or 28 (PF28) short form 102 87 (working on shorter form) 23 **\# Domains** 10 child & 4 parent concepts 8 infant & 5 parent concepts 10 child concepts 4 \"Generic Core\" **Overall Score** 2 Global Physical & Global Psycho-Social 1 overall, for primary analyses **Sub-scale Scores** 14 13 10 4 sub-scales, for secondary and descriptive analyses **Completion Time (minutes)** 10 to 20 for PF50;\ 20 to 30 15 to 30 n/a \< 4 7 to 15 for PF28 **Spanish Language** Yes Yes **Assessment Perspective** Parents\' Parents\' Patients\' Patients\' Parents\' ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Legend: WHO -- World Health Organization. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Domains and Constructs of Forms for Two Pediatric Health Profile Systems ::: ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **Instrument** **CHQ: Child Health Questionnaire** **PedsQL 4.0** ------------------ ----------------------------------------------------------------------------------------------------------------------- ------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- --------------------------------------------------- ---------------------------------------------------------- **Version** Parent Forms (PF) Youth-Completion Form Parent Form for Infants & Toddlers Child Forms (3 versions: 5--7, 8--12, 13--18 yrs) Parent Forms (4 versions: 2--4, 5--7, 8--12, 13--18 yrs) **Domain Names** 1\. Physical functioning -- 6 items.\ 1\. Physical Functioning.\ 1.. Physical Functioning -- 8 items.\ - e.g., Child is greatly limited in performing all physical activities, including self-care, due to physical health.\ 2. Behavior (perception).\ - hard to walk more than one block, hard to run, hard to do sports or exercises, hard to lift something heavy, hard to take a bath, hard to do chores around the house, hurt or ache, low energy.\ 2. Role (school and activities) limitations due to emotional.\*\ 3. Growth & Development.\ 2. Emotional Functioning -- 5 items.\ 3. Role limitations due to behavioral problems\* -- 3 items.\ 4. Pain.\ - feel afraid or scared, feel sad or blue, feel angry, trouble sleeping, worry about what will happen.\ - e.g., Child is limited a lot in school work or activities with friends due to behavioral problems.\ 5. Behavior (getting along).\ 3. Social Functioning -- 5 items.\ 4. Role limitations due to physical problems -- 2 items.\ 6. Temperament & Mood.\ - trouble getting around, other kids not wanting to be friends, teased, doing things other peers do, hard to keep up when playing with others.\ - e.g., Child is greatly limited in school work or activities with friends due to physical health.\ 7. General Health.\ 4. School Functioning -- 5 items.\ 5. Bodily pain -- 2 items.\ 8. Change in Health. - hard to concentrate, forget things, trouble keeping up with schoolwork, miss school because not well, miss school for doctor appointments. - e.g., Child has extremely severe, frequent and limiting body pain.\ 6. General behavior -- 6 items.\ - e.g., Child very often exhibits aggressive, immature, delinquent behavior.\ 7. Mental health -- 5 items.\ - e.g., Child has feelings of anxiety and depression all the time.\ 8. Self-esteem -- 6 items.\ - e.g., Child is very dissatisfied with abilities, looks, family/peer relationships and life overall.\ 9. General health perceptions -- 6 items.\ - e.g., Parent believes child\'s health is poor and likely to get worse.\ 10.Change in health -- 1 item.\ - e.g., Child\'s health is much worse now than 1 year ago. ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- \*Role Emotional and Role Behavioral are separate scales in PF98 but combined into one in the PF50 and PF28. ::: Table [4](#T4){ref-type="table"} summarizes the major characteristics of four orthopedic-specific measures. The PODCI \[[@B80]\] was designed specifically as a very comprehensive measure of musclo-skeletal outcomes associated with pediatric orthopedic problems. ASK was designed to measure children\'s activities in terms of both capacity and performance \[[@B81]\], and it assesses domains not covered in detail by other instruments \[[@B82]\]. The FAQ walking scale provides the most complete measure of walking abilities. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Major Characteristics of Orthopedic-Specific Systems ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ **Instrument** **PODCI** **ASK** **FAQ walking scale** ------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------ ------------------------------------------------ **Full Name** Pediatric Outcomes\ Activities Scale for Kids Gillette Functional\ Data Collection\ Assessment Questionnaire:\ Instrument Functional Walking Scale **Origin** 1997 1996 1993 **Measurement constructs** Musculoskeletal outcomes Physical disability in terms of capacity or performance dimensions Complete range of functional walking abilities **Age range (years)** 2 -- 18 2 -- 18 2 + **\# Items** 108 30 1 **\# Domains; and Domains** 8\ 9\ 1\ Comorbidity, Upper Extremity & Physical Function, Transfers & Basic Mobility, Sports & Physical Function, Pain, Treatment Expectations, Happiness, Satisfaction with Symptoms. Personal Care, Dressing, Eating & Drinking, Miscellaneous, Play, Locomotion, Standing Skills, Stairs, Transfers. Walking. **Overall Score** Yes, Global Function, mean of sub-scales for domains 1 to 4 above Yes, additive and not weighted Yes **Sub-scale Scores** Yes, additive No No **Completion Time (minutes)** 10 -- 20 5 -- 12 \< 2 **Spanish Language version** No No No **Assessment Perspective** Patient &/or Parent (Diagnostic, complications and aims section by clinician) Patient Parent ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ::: The choice of existing measures is based on a process of elimination considering the relative strengths of each instrument and the complementarities among measures. Neither the SF-36 nor the EQ-5D is valid for use in adolescent patients with orthopedic problems \[[@B83]\]. A review of measurement of HRQL in children by Eiser & Morse \[\[[@B84]\]; see also \[[@B85],[@B86]\]\] identified HUI and CHQ and PedsQL as the only 3 generic measures that fulfill all specified review criteria: established reliability and validity; suitable for self- and proxy-report; and brief (\<30 items). PODCI outperformed CHQ physical functioning scale for orthopedic patients \[[@B87]\]. However, PODCI has considerable problems with missing data, especially in upper extremity function and physical function and sports scales for children ages 2 to 5 years, associated with the use of \"too young\" response options \[[@B88]\]. ASK is reported to be more sensitive to change in disability levels than HUI \[Young N, personal email communication to W Furlong 2002-02-18\]. The FAQ walking scale provides the most complete assessment of functional walking abilities, especially at the upper end of the scale \[[@B89]\]. In summary, there are few measures available for assessing subjects less than 5 years of age and even fewer for subjects less than 2 years of age. Most relevant measures are available in self-complete format only. Preliminary recommendations for the NF1-CTD study were that PedsQL be used as the generic health profile, HUI be the multi-attribute preference-based measure of HRQL utility scores for children age 5 years and older and that CHSCS-PS be the measure for children age 2 through 4 years, ASK be the measure of activity limitation, FAQ walking scale be the measure of walking ability, and that a small feasibility study of these instruments be completed with a convenience sample. Feasibility study ----------------- A pilot feasibility study surveyed 8 NF1 patients using HUI and FAQ walking scale measures. Questionnaires were completed by 6 NF1 patients and 3 parents. The combined HUI and FAQ walking scale questions took respondents an average of 13 minutes (range, 9--20 minutes) to complete. The patients were 11 to 50+ years old and had health problems ranging from mild to severe. One patient with tibial dysplasia and 2 patients with scoliosis were included. HUI data were collected from 5 patients, 2 parents and both the patient and parents in one case. Health problems were reported in 7 of the 8 HUI3 attributes (vision, speech, ambulation, dexterity, emotion, cognition, and pain; no problems with hearing were reported). The attributes associated with the most morbidity, as assessed using HUI3 single-attribute utility scores \[[@B60]\], were pain (mean score = 0.81), speech (0.94), cognition (0.94), and emotion (0.94). For the 7 patients having complete data, 5 had two or more HUI2 and HUI3 attributes at less than full function. On the conventional utility scale in which being dead = 0.00 and in perfect health = 1.00, the HUI3 scores ranged from 0.45 to 1.00. The mean HUI3 score, 0.73, is similar to the mean score of 0.77 for adults with arthritis \[[@B90]\]. FAQ walking scale data were collected from 4 patients (1 of the 5 survey patients did not answer the question), 2 parents and both the patient and parents in one case. Five patients were reported to be at Level 10 (walks, runs, and climbs on level and uneven terrain and does stairs without difficulty or assistance), one patient to be at Level 8 (walks outside the home for community distances, is able to get around on curbs and uneven terrain in addition to level surfaces, but usually requires minimal assistance or supervision for safety), and one patient at Level 6 (walks more than 15--50 ft. outside the home but usually uses a wheelchair or stroller for community distances or in congested areas). In summary, the feasibility study showed that the HUI and FAQ walking scale questions were acceptable to patients\' families and that results, especially for HUI, reflected the large variability in HRQL of the sample of patients. Choice of measures for illustrative study ----------------------------------------- No single measure will provide sufficient data to address all the important study objectives. A set of measures is required. The set of measures should provide complementary data of health status and preference-based scores of HRQL. Redundancy in measurement is reduced, and efficiency of measurement is increased, by selecting the most comprehensive generic measures and then supplementing these measures with the most appropriate set of specific measures. It is recommended that HUI be selected as the comprehensive generic measure for ten reasons: a\) it includes both generic health profile and preference-based scoring systems; b\) the preference-based scoring systems are well-validated; c\) it is the most comprehensive, compact and efficient of these types of systems; d\) it includes many of the most important domains in the context of NF1-CTD; e\) it is applicable for all people age 5 years and older; f\) well-developed data collection questionnaires are available to match the study design criteria; g\) HUI results facilitate integrating effects of morbidity and mortality, and cost-utility economic evaluations; h\) it has been used successfully in a variety of studies of musculoskeletal problems; i\) population norm data are available; and j\) a closely-related health status system, the CHSCS-PS, is available to assess children 2 through 4 years of age. The HUI will provide a broad set of measures for comparisons with other populations and for estimating HRQL on a general scale such that dead = 0.00 and perfect health = 1.00. As a generic measure, HUI also has the ability to capture side effects and the effects of co-morbidities. However, these broad measures may not be responsive to small but important changes in health status. Therefore, HUI should be complemented by a set of instruments focused on pediatric, orthopedic and walking issues. The PedsQL 4.0, a pediatric generic health profile, should also be included in the set of measures because: a\) it includes domains, social and school function, which complement HUI and CHSCS-PS domains; b\) it is appropriate for children ages 2 through 18 years; c\) it is not overly burdensome in terms of data collection; d\) patient and parent assessment questionnaires are available; and e\) it can be interviewer-administered to facilitate data collection by telephone, if necessary. Two specific measures should also be part of the set of instrumentation: the ASK and the FAQ walking scale. ASK is an orthopedic-specific instrument which has been shown to cover the most important domains in the context of musculoskeletal disorders, including the impact of limb lengthening surgery experienced by many children with tibial dysplasia. ASK is also attractive because it provides overall summary scores for both performance and capability measures, and is only moderately burdensome to complete. Walking ability is one of the most important aspects of health that is frequently compromised in NF1-CTD patients, and the FAQ walking scale is the most complete scale of functional walking ability currently available and it only requires asking one question. CHQ is not recommended because it does not add much to the set of recommended measures and it is burdensome to complete. PODCI is not recommended because it is very burdensome to complete, it has been reported to have major problems with \"missing data\", and the system for collapsing questionnaire responses into summary scores is not well validated. Children ages 2 to 5 years of age should be assessed by their parents using three questionnaires: the CHSCS-PS (12 questions), the PedsQL (23 questions); and the FAQ walking scale (1 question). It is expected that all three of these questionnaires can be completed in an average of 15 minutes. Children and adolescents ages 5 to 17 years of age should be assessed by their parents using four questionnaires: the HUI (15 questions), the PedsQL (23 questions), the FAQ walking scale (1 question), and the ASK (30 questions). These four questionnaires are expected to be completed in an average of 20 to 30 minutes. Children and adolescents older than 11 years should provide self-assessments using four questionnaires: the HUI (15 questions), the PedsQL (23 questions), the FAQ walking scale (1 question), and the ASK (30 questions). On average, it is expected that respondents will complete all four questionnaires in 20 to 30 minutes. Conclusions =========== This paper highlights reasons why patient-focused measures of FHS and HRQL should be considered important tools in the field of orthopedic surgery. It has also noted that there is increasing competition for scarce health-care resources, that allocation decisions about these resources are being informed by evidence based on patient-focused health measures, and that these measures are being under-utilized by the orthopedic surgery community. The orthopedic community faces numerous obstacles in utilizing FHS and HRQL measures. One major obstacle is that the multitude of existing measures makes it difficult to decide which measures may be appropriate for a specific application. A second obstacle is that most of the information about FHS and HRQL measures is not reported in the orthopedic literature. A third obstacle is that usually no one measure can capture all the important aspects associated with a specific orthopedic issue. The framework outlined in the paper provides guidance for selecting appropriate FHS and HRQL measures. The framework guides orthopedic investigators to combine their basic study criteria, including objectives and clinical context, with key criteria for FHS and HRQL measures from the published literature. The results in this paper identify some major sources of information about health measures, identify some of the most widely used measures of FHS and HRQL, and provide summaries of key characteristics for selected measures in three major taxonomical classes: generic preference-based multi-attribute systems; generic pediatric health profile systems; and orthopedic-specific systems. It is clear that there are many important differences among measures both within and across taxonomical classes. All measures are not equal. There are sound factors for making judgements about which measures are most appropriate for a given application. A process of appraisal and elimination was used to select one measure from each taxonomical class for inclusion in the NF1-CTD study illustrative example, and a pilot study of the most readily available selected measures confirmed the feasibility of their use in a small sample of NF1-CTD patients. The paper shows that a set of relevant, valid, reliable, responsive and practical patient-focused health measures for use in an orthopedic study can be readily identified and selected from the published literature and information available on the worldwide web. We encourage orthopedic researchers to use the framework to identify and select appropriate patient-focused health measures in their future studies. Conflict of Interest ==================== W. Furlong and D. Feeny have a proprietary interest in Health Utilities Inc. which distributes copyright Health Utilities Index (HUI^®^) instrumentation and provides methodological advice on the use of HUI. Authors\' contributions ======================= All authors were involved in critical review of drafts for intellectual content and have given final approval to the version submitted for publication. W Furlong was responsible for much of the overall design, acquisition of data, and initial drafting. R Barr and D Feeny made important contributions to the analysis and interpretation of results. S Yandow conceived the idea of presenting the information in a published manuscript. Acknowledgments =============== We are pleased to acknowledge funding from a Shriner\'s Foundation Planning Grant and support of the following members of the Planning Grant team: Dr. John Carey in the Department of Pediatrics at the University of Utah School of Medicine and member of the medical staff at Intermountain Shriners Hospital for Children for his role as Principal Investigator of the Shriner Foundation Planning Grant; and Dr. Jan Friedman and Patricia Birch in the Department of Medical Genetics at the University of British Columbia for providing the HUI and FAQ walking scale feasibility study data reported in this paper. The granting agency played no role in the design, interpretation, or analysis of the work reported here and have not reviewed or approved of this manuscript. Dr. James Wright\'s review comments identified some important points that we used to improve the paper.

PubMed Central

2024-06-05T03:55:52.609552

2005-1-12

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548287/", "journal": "Health Qual Life Outcomes. 2005 Jan 12; 3:3", "authors": [ { "first": "William", "last": "Furlong" }, { "first": "Ronald D", "last": "Barr" }, { "first": "David", "last": "Feeny" }, { "first": "Suzanne", "last": "Yandow" } ] }

PMC548288

Because bone marrow and adipocytes are derived from the same stem cells, it is not surprising to find so many inflammatory peptides expressed in fat tissue. Many of these are classical inflammatory peptides derived from components of the adipose tissue and all are known also as fat derived peptides (FDP). With aging, there is a linear accumulation of adipose cells and percent of body fat increases. This increased body fat is characterized by increased visceral adiposity \[[@B1]\] and occurs despite the decreased subcutaneous fat and progressive sarcopenia typical of aging \[[@B2]\]. Visceral adiposity has been associated with greater risks for age-related diseases \[[@B3]\]. In addition, fat infiltration typical of aging occurs in many organs including liver and bone marrow. As adipose tissue accumulates throughout the body and in other organs, it is possible that this hyperplastic adipose tissue over expresses FDP. The metabolic syndrome is a common disorder consisting of a cluster of abnormalities including insulin resistance, dyslipidemia, and hypercoagulability and is associated with increased risk for cancer, Alzheimer\'s disease, type-II diabetes and atherosclerosis \[[@B4]\]. It is also associated with increased fat mass and increased inflammatory peptides. The obesity epidemic of the rapidly growing aging population makes understanding underlying relationship between adiposity, chronic inflammation and the metabolic syndrome essential. Increased inflammatory peptides are being studied as possible modifiable markers of the increased risk predictors of disease and possibly the underlying link between obesity and the poor clinical outcomes seen with the metabolic syndrome. More specifically, C-reactive protein (CRP) is the most well established inflammatory cytokine in the clinical setting but there are other inflammatory cytokines including IL-6, leptin, TNF-*α*, and other (non-cytokine) FDP, such as PAI-1, adiponectin and resistin, which may play a role in the pathogenesis, and/or serve as markers of risk in the metabolic syndrome. The fact that many of these peptides are derived from adipose tissue leads us to the question of whether adipose tissue itself is the underlying pathophysiological link between obesity and the poor clinical outcomes associated with the metabolic syndrome. We will provide a brief overview of some of the peptides associated with the metabolic syndrome. Cytokines with a potential role in the metabolic syndrome ========================================================= *TNFα*, leptin, and IL-6 are examples for cytokines that may have a role in the metabolic syndrome. TNF*α*, previously known as lymphotoxin and cachetin, is believed to be involved in the wasting that occurs during acute and chronic illness and malignancy. In the basal state TNF*α*is directly proportional to fat mass and has been shown to be involved in the development of insulin resistance \[[@B5]\]. In-vitro studies have demonstrated that TNF*α*decreases the insulin receptor tyrosine phosphorylation, and down regulates several steps in the insulin signaling pathway \[[@B6]-[@B9]\] while neutralizing agents for TNF*α*have been shown to improve insulin resistance. \[[@B10]\] Thus, *TNFα is not only a classical cytokine but may be causal in the insulin resistance of the metabolic syndrome of aging*. *Leptin*is a peptide derived from adipose tissue and like other cytokines acts through a cytokine receptor. It is expressed and secreted in direct proportion to fat mass. Leptin exerts is effect predominantly through receptors in the hypothalamus but it may also have peripheral actions \[[@B5]\]. Leptin serves as a marker of energy sufficiency by rapidly decreasing during starvation and weight loss. \[[@B11]\] With obesity, leptin levels are increased in proportion to fat mass, but its activity to decrease appetite seems reduced. *Leptin appears to have an important role in energy regulation but no apparent role in development of inflammation*. *IL-6*is another cytokine derived from adipose tissue. Its expression and circulating levels correlate directly with obesity, and weight loss will lower circulating levels. Elevation of circulating IL6 is a predictor of the development of cardiovascular disease and diabetes \[[@B12]\]. Infusion of IL6 results in hyperlipidemia, hyperglycemia and insulin resistance in experimental models. \[[@B13]\] Additionally, IL6 decreases the expression adiponectin, an \'anti-diabetic\' cytokine. \[[@B14]\]*IL-6 plays a role in the development of insulin resistance and may directly cause induction of CRP*. Other inflammatory cytokines such as *IL-1*, IL-8, IL18, Serum Amyloid A, have been shown to be increased with obesity and may have a yet undetermined role in the syndrome. These cytokines are other examples of inflammatory markers which do not have a clear role in the causation of systemic inflammation. Non-cytokine Fat Derived Peptides with a role in the metabolic syndrome ======================================================================= *Adiponectin*is highly expressed in adipose tissue, and is the one non-cytokine FDP that is protective from inflammation. Unlike most FDP, circulating levels are inversely proportional to obesity and therefore tend to be low in obesity. Adiponectin levels increase with weight loss and with use of insulin sensitizing drugs. \[[@B15]\] Adiponectin administration has been shown to improve insulin sensitivity. \[[@B16]\] Low levels of adiponectin have been linked to inflammatory arthrosclerosis in humans.\[[@B17]\] Animal models have shown that low adiponectin levels increase smooth muscle proliferation in response to injury, increase free fatty acids levels and cause insulin resistance.\[[@B18]\] The pro-diabetic and pro-atherogenic effects of low adiponectin levels seen in the metabolic syndrome provide a link between inflammation and obesity. *Plasminogen activator inhibitor type-1 (PAI-1)*is the primary inhibitor of fibrinolysis and is highly expressed in adipose tissue. Levels of PAI-1 are elevated in acute conditions such as deep venous thrombosis, and chronic conditions such as obesity, the metabolic syndrome of aging and diabetes. PAI-1 levels are correlated with adiposity and significantly overexpressed in the adipose tissue of obese compared to lean animals. \[[@B19]\] Levels are decreased by weight loss and drugs that improve insulin sensitivity \[[@B20]\]. The relationship of PAI-1 to obesity provides a potential link between the metabolic syndrome and hypercoagulabilty. *Angiotesinogen (AGT)*is a peptide that is produced in the liver and in adipose tissue. The strong correlation between obesity and hypertension implies that adipose tissue may play a role in blood pressure regulation and in fact there is a correlation between circulating AGT levels and obesity/hypertension \[[@B21]\]. Animal studies have shown that overexpression of AGT results in hypertension while under expression of AGT results in decreased blood pressures \[[@B22]\]. *Resistin*is a peptide which is elevated in obesity and appears to play a role in glucose homeostasis in rodents. In experimental models, resistin induces hepatic insulin resistance while anti-resistin antibodies have the opposite effect \[[@B23]\]. In humans, the role of resistin is less clear and it is not known what role it has glucose homeostasis or whether it directly relates to adipose tissue mass. The role of resistin in pathogenesis of inflammation is also unclear. Markers of inflammation or markers of obesity? ============================================== Low grade inflammation is a predominant feature in the metabolic syndrome of aging and seems to be linked to the development of diabetes and poor vascular outcomes. We have briefly named several cytokines and other FDP that are generally increased with fat mass. Although many of these FDP have a role in metabolic homeostasis, many seem to lack distinct role in inflammatory pathogenesis. While many FDP have roles in *vivo*metabolism, we suggest that some levels of cytokines are increased because of the hyperplastic characteristic of adipose tissue, and their levels are better serve as marker of adipose tissue hypertrophy, rather than having a causal role in aging. Thus, whether aging is inflammatory state or whether it is a state associated with increased inflammatory marker is subject for further studies.

PubMed Central

2024-06-05T03:55:52.615828

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548288/", "journal": "Immun Ageing. 2005 Jan 21; 2:1", "authors": [ { "first": "Eric", "last": "Rudin" }, { "first": "Nir", "last": "Barzilai" } ] }

PMC548289

Introduction ============ Celso-Ramon Garcia (Figure [1](#F1){ref-type="fig"}), who departed this world in 2004 at the age of 82, was a remarkable human being who did much to shape the specialty of reproductive medicine as it is known today. His research yielded important discoveries that continue to influence the daily lives of millions of women and men around the globe. Reviewing his career and his accomplishments is like looking through a kaleidoscope -- elements of greatness and the purest embodiment of the medical arts are brought together and with a small twist transformed into a new vision. Hard work and determination brought him from a relatively modest but academically-oriented Spanish immigrant family to medical training in community hospitals in Brooklyn, and then into the elite circles of East Coast academic medicine. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Celso-Ramon Garcia, M.D. (1922--2004) ::: ![](1743-1050-2-2-1) ::: Background ========== Garcia was raised speaking Spanish, but his English diction and elocution were so honed by his teachers in grammar and high school that hardly a trace of accent was left to be detected by the Boston Brahmans he would later encounter. Along the way his interests took seemingly improbable turns, yet there was a clear rationale that was beyond the imagination of the average physician. Indeed, his work presaged several important developments in the field of reproductive medicine. Garcia began his career with an interest in obstetrics, but ended up conducting clinical research on contraception that on more than one occasion was nominated for the Nobel Prize in Medicine or Physiology. He was revered as a meticulous abdominal infertility surgeon, yet he embraced the fledgling field of endoscopy and endoscopic surgery. While being celebrated for his surgical technique, he promoted research into the psychological aspects of infertility. Towards the end of his career he became interested in sexuality in the menopause, bringing his career through the full cycle from obstetrics, to family planning, to infertility to reproductive aging. His accomplishments in each of these spheres were well recognized by his peers who chose him President of the American Association of Planned Parenthood Physicians, Chairman of the Board of Directors of Planned Parenthood of America, Founding President of the Society of Reproductive Surgeons, and President of the American Society of Reproductive Medicine and bestowed upon him the Scientific Leadership Award from the Global Alliance for Women\'s Health, a non-governmental organization of the United Nations. Early years =========== Garcia\'s course to greatness was based on a diverse set of skills that he used creatively throughout his career. He majored in chemistry at Queens College where he revealed a remarkable aptitude for analysis of organic compounds. During his undergraduate studies, Garcia served as a teaching fellow in chemistry at New York University and authored two papers on qualitative and quantitative analysis. These are the earliest public manifestations of his passion for rigor and precision, which subsequently guided his clinical practice and his scholarship. After receiving his baccalaureate degree he entered medical school at the Long Island College of Medicine (now SUNY-Downstate) and graduated in 1945. He completed a rotating internship at the Norwegian Hospital in Brooklyn and then served two years in the U.S. Army Medical Corps in Fairbanks Alaska and then the Phoenixville General Army Hospital outside of Philadelphia. Garcia started a residency in pathology at Cumberland Hospital in Brooklyn where he was introduced to Sam Lubin, who became his friend and mentor. Lubin\'s work focused on uterine pathology and contractility and this sparked Garcia\'s interest in obstetrics and gynecology. With Lubin, Garcia would later publish papers on the effects of meperidine on labor. Further studies and research ============================ After a year of pathology residency and a year as a research fellow, Garcia transferred to the obstetrics and gynecology program at Cumberland Hospital. With a pathologist\'s keen eye and an appreciation of tissue biology/healing, Garcia was well-equipped to excel in the art of reproductive surgery. In the operating rooms at Cumberland Hospital Garcia met Shirley Stoddard, the newly appointed Director of Surgical Nursing; she was to become his wife and lifelong companion. After completing residency, Garcia explored openings in academia but few opportunities were available. One exception was an advertisement for assistant professor of obstetrics and gynecology at the newly established medical school at University of Puerto Rico, for which Garcia applied and was accepted in 1953. Garcia\'s research during residency, mentored by Lubin, had focused on obstetrical issues -- something that he could have easily developed as a junior faculty member at the University of Puerto Rico. However, Garcia was about to embark on the first of several sharp turns that would lead him into seemingly improbable arenas. His arrival in Puerto Rico fortuitously coincided with the time for the first large scale clinical trials of the oral contraceptive pill. Within a year of Garcia\'s arrival, Gregory Pincus (director of the Worcester Foundation for Experimental Biology in Shrewsbury, Massachusetts) was in Puerto Rico looking for sites to conduct these trials. It was there that Pincus was first introduced to Garcia. Pincus had an uncanny ability to sense talent; he always surrounded himself with the brightest people. Pincus, undoubtedly impressed by Garcia\'s fund of knowledge in organic chemistry, training in pathology, and of course, the fact that he was bilingual in addition to his training in obstetrics and gynecology, promptly recruited Garcia to oversee his important research program in Puerto Rico. Pincus would subsequently introduce Garcia to John Rock, who arranged for Garcia to be named Sydney Graves Fellow in infertility and gynecology at the Free Hospital for Women in Brookline, Massachusetts. There, Garcia continued to work on oral contraceptives but also pursued clinical work in infertility. Although the Graves fellowship no longer exists, contemporary reproductive medicine was developed by its incumbents. Rock had an interesting manner of selecting these fellows. He usually interviewed candidates over breakfast in his hotel room at the annual meeting of the American College of Obstetricians and Gynecologists. He could identify the smart ones within a minute, and had little patience for individuals who were not well-spoken. The diction and elocution drilling Garcia had endured and his insistence on precision brought him through with flying colors. Garcia quickly became indispensable to Rock. Garcia moved to Boston, where he held assistant and instructor faculty posts at Harvard Medical School. During this time, Garcia also commuted to Puerto Rico to manage the oral contraceptive trials. Rock had a thriving infertility practice and Garcia did much of the surgery. Garcia\'s contributions to Rock\'s clinical practice were, however, underappreciated and when it came time for Garcia to be proposed for a full-time position at the Free Hospital for Women, the job did not materialize. This scenario is not unfamiliar to practitioners in our field, and it elicited the obvious reaction: Rock and Garcia pulled up stakes and took their hugely lucrative practice to the Faulkner Hospital in Boston. But after a short interval, the Rock/Garcia partnership was persuaded to return to the Free Hospital where an appropriate appointment was arranged for Garcia. Mrs. Stanley McCormick, a benefactor of remarkable vision who had provided initial funds to Pincus and colleagues at the Worcester Foundation to begin studies on oral contraceptive steroids, helped establish the Rock Reproduction Study Center at the Free Hospital campus, greatly facilitating research and training. The Worcester Program and Massachusetts General Hospital years ============================================================== By 1960, Pincus had recruited Garcia to the Worcester Foundation as a senior scientist and director of the training program in Physiology of Reproduction. Sponsored by grants from the Ford Foundation and National Institutes of Health, this was the first training program of its kind. The discipline of reproductive biology as we know it today had its roots in the Worcester program. Although Garcia was appointed chief of the infertility clinic at Massachusetts General Hospital in 1962, he continued to visit Puerto Rico to perform follow-up examinations on women who had participated in the oral contraceptive trials in gratitude for their role and with genuine concern for potential untoward long-term effects. That was a personal mission, not a mandated component of the study protocol. This post-trial surveillance helped identify rare side effects that ultimately led to product reformulation and lowering of the steroid content of oral contraceptives. Garcia subsequently carried out groundbreaking studies on patient acceptance of oral contraceptives, recognizing that future developments in family planning must address the social and demographic aspects, including cultural differences in preferences for methods. The technologies that gave men and women the ability to control their own reproduction through contraception and infertility treatments are arguably among the most important medical advances of the 20^th^century. This is in good part Garcia\'s legacy \[[@B1]-[@B3]\]. While his career embraced the full spectrum of reproductive medicine and surgery, undoubtedly Garcia\'s most significant role was as co-developer of the first oral contraceptive approved by the U.S. Food and Drug Administration. Important medical advances like \"the pill\" or IVF do not come without controversy, societal baggage, and rhetoric. The pill\'s creators fully understood these implications, and knew such peripheral distractions would take away from the significance of their work. Consequently, there was no phone call from Sweden despite several nominations to the Nobel committee. But it is best to look back on the history of the development of the oral contraceptive with awe and wonder. Indeed, the commercial availability of oral contraceptives was also important because it established a formal approval and regulatory framework for pharmacologic agents of an entirely new class: drugs designed to be used by healthy people for long periods of time. It provided a clear understanding of how post-marketing surveillance can lead to product reformulations that reduce side effects without reducing efficacy. It yielded an understanding of how sociology and demography must be incorporated into the development of pharmaceuticals and the practice of medicine. Garcia the peerless surgeon =========================== Garcia maintained his connections with Rock and Rock\'s family of protégés, one of whom (L.M.) later lured him and other Rock/Pincus trainees, Edward Wallach and John V. Kelly, to the Department of Obstetrics and Gynecology at the University of Pennsylvania. Garcia spent the rest of his career at Penn developing the specialty of reproductive surgery, particularly microsurgical approaches. Here again came the totally un-expected. Garcia was at his best when confronted with the most challenging open abdominal procedures, which always ended with the pelvis in a pristine state, not a drop of blood, all surfaces meticulously re-peritonealized, no risk of inflammation or adhesion. Garcia\'s novel regimen of intraperitoneal corticoids and antihistamines (an early anti-adhesion therapy) was merely icing on the cake. The two-stage tuboplasty procedure was celebrated both locally and internationally. Yet this master of the open abdomen quickly recognized the potential of minimally invasive surgery, establishing one of the first endoscopic surgery programs in USA after a brief sabbatical with his colleague, Professor Kurt Semm in Kiel, Germany. During his career, Garcia authored numerous textbook chapters on surgery of the ovaries, tubes and uterus that guided a generation of gynecologists in reconstructive surgery. He was also an early advocate of laser and electrosurgical methods, as well as microsurgical techniques designed to minimize tissue trauma. When Garcia\'s residents and fellows under-performed, he could not mask his displeasure, yet when they excelled he could not hide his joy. His concern for the outcomes of a rapidly evolving medical specialty led him to develop one of the first electronic infertility data registries \[[@B4]\]. In 1968, Garcia made yet another change in tack, developing an interest in psychological aspects of infertility. While this may have puzzled colleagues who knew Garcia only by reputation as a surgeon, those of us who worked with him knew well his holistic commitment to medicine and his devotion -- unprecedented even in the current era -- to his patients and their families. He had a profound understanding of the human frailties that emerge during the rigors of infertility treatment. One of us (J.F.S.) assisted Garcia in the care of an attorney\'s wife who underwent a two-stage tuboplasty. The husband demanded to be present during all medical examinations of his wife, he tape recorded all sessions, and refused to pay for Garcia\'s services, despite the fact that she conceived within three months after the hoods were removed from her reconstructed tubes. Garcia did not press the attorney for payment -- he knew that the treatment course was exceptionally arduous with considerable post-surgery discomfort. When this same lawyer called eight months later asking if Garcia would serve as an expert witness in a plaintiff\'s medical malpractice case, Garcia thanked him for his call and politely said his schedule would not permit it, never mentioning the unpaid bill. He forgave, but he did not forget. Final years and retirement ========================== Later in his career, Garcia and his colleagues undertook studies on the menopause that presaged the current interest in female sexual dysfunction. In retrospect, this was a logical extension of his long-standing interest in the social and psychological aspects first of contraception and family planning, and later of infertility. Garcia published on perimenopausal sexuality, and reported the first studies the relationship between sex steroid levels and sexual behavior. Garcia also recognized the need for multidisciplinary collaboration in the care of women, particularly in the post-reproductive years, and co-authored an early self-help manual for menopausal women \[[@B5]\]. The Women\'s Wellness Program he established at the Hospital of the University of Pennsylvania opened years ahead of similar facilities elsewhere. Garcia gracefully retired from practice to devote time to his wife and family. This life transition, seamlessly executed, surprised many who were familiar with Garcia\'s intensity. But there should have been no surprise. Garcia demanded the highest technical standards from residents and fellows in the operating room, the same standards that he held for himself. He would not carry on if he could not perform to those standards, and he would not neglect his wife who needed increasing medical attention. Epilogue ======== Throughout his professional life, Celso-Ramon Garcia was a major force driving innovation in clinical care, and ultimately the evolution of the field of reproductive medicine. Let all who follow in the arena of reproductive endocrinology honor his contributions, appreciate the unique attributes that made him a great physician and educator, and gain insight from a truly remarkable career. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JLS and LM contributed equally to this work and its revisions.

PubMed Central

2024-06-05T03:55:52.616593

2005-1-26

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548289/", "journal": "J Exp Clin Assist Reprod. 2005 Jan 26; 2:2", "authors": [ { "first": "Jerome F", "last": "Strauss" }, { "first": "Luigi", "last": "Mastroianni" } ] }

PMC548290

Introduction ============ It is estimated that more than 110 million Africans live in areas prone to epidemics of malaria. Populations in these areas are infrequently challenged by malaria and, therefore, do not fully develop acquired immunity. As a result, the disease remains life threatening to all age groups. The impact of malaria epidemics could be greatly reduced by timely detection or, ideally, by prediction and prevention through vector control and deployment of appropriate drugs \[[@B1]\]. Rainfall is recognized as one of the major factors influencing variability in malaria transmission in warm semi-arid and desert-fringe areas of the Sahel, the Greater Horn and Southern Africa. Explosive epidemics may occur in these regions after excessive rains, usually with a lag-time of several weeks during which time mosquito vector populations and malaria infections increase rapidly. Epidemics can be especially severe among communities stressed by recent periods of drought and poor food security. Recent research has found that rainfall estimates would be able to provide useful epidemic early warning information, even in highland-fringe settings, such as those in Kenya and Ethiopia, where temperature is also an important limiting factor for the development of the malaria parasite \[[@B2]-[@B4]\]. Consequently, rainfall monitoring forms one of the essential elements for the development of integrated Malaria Early Warning Systems for sub-Saharan Africa, as outlined by the World Health Organization \[[@B5],[@B6]\]. During a meeting of the Roll Back Malaria Technical Resource Network on Prevention and Control of Epidemics (RBM-TSN) it was decided that immediate benefit could be realized from the routine availability of a simple indicator of changes in epidemic risk in these regions of marginal transmission \[[@B7]\]. The indicator was to be based on the difference between current rainfall (derived from meteorological satellite estimates) and the expected (average) for the particular time of the year; results were to be made available on the internet in a frequently updated format. The purpose was to provide timely alerts to malaria control programme staff and RBM partners working in areas of increased epidemic risk \[[@B8]\]. The following article provides an update on existing online rainfall monitoring resources and a description of a new tool that has been developed to address remaining needs of the malaria control community Discussion ========== Following discussion among members of the RBM-TSN a consensus map of epidemic risk zones was produced \[[@B7]\]. The map, shown in Figure [1](#F1){ref-type="fig"}, was used as a mask to exclude areas where malaria transmission is considered absent or endemic, as opposed to epidemic. This mask is based purely on climatic constraints to malaria transmission, and does not yet account for areas in the northern and southern margins of the continent where control has eliminated malaria risk. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Epidemic risk zones in Africa (adapted from \[1\]). ::: ![](1475-2875-4-6-1) ::: The epidemic risk map was then combined with rainfall anomaly data (i.e., the difference between observed rainfall and the expected, i.e. average, rainfall for a particular time of the year) to provide a simple indicator of changes in risk in epidemic prone areas. Figure [2](#F2){ref-type="fig"} illustrates the resulting dekadal (i.e., \~10-daily) rainfall anomaly maps, which are updated approximately every 10 days, and have been available in experimental form through the Africa Data Dissemination Service (ADDS) since June 2002. The ADDS is an operational part of the Famine Early Warning Systems Network (FEWS NET) which is maintained by the United States Geological Survey (USGS) and supported by the U. S. Agency for International Development (USAID). The maps can be accessed at: <http://igskmncnwb015.cr.usgs.gov/adds/> under \"RFE Anomaly -- Malaria\", where the anomaly map for the most recent dekad is displayed along with documentation on how the map is produced. The existence of this online monitoring resource was publicized and their use and validation by control services and researchers was encouraged \[[@B8]\]. The rainfall estimate data underlying these maps was tested against laboratory-confirmed malaria incidence figures for selected districts in Southern Africa, where they showed a good association \[[@B9]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Rainfall Anomalies in Zones with Malaria Epidemic Potential: 21--31 December 2004 ::: ![](1475-2875-4-6-2) ::: In the year following the launch of the ADDS dekadal rainfall anomaly maps, WHO commissioned field visits to a number of epidemic prone countries to evaluate whether the National Malaria Control Programmes were aware of this resource and how useful they considered it may be for their efforts. Sudan, Uganda, Niger, Mali and Burkina Faso received field visits. In general, all of the control programmes had been aware of the rainfall anomaly maps, but only those in Uganda and Sudan had monitored them regularly during the previous year. The control programmes in the Sahelian countries did not agree with the epidemic risk zone used in the mask because their recent experience was that epidemic outbreaks had occurred beyond the northern boundary of the epidemic risk zone. This was also partly true in Sudan where epidemic outbreaks have been known to occur along the Nile River margins in the northern half of the country. Uganda\'s malaria control programme, however, had found the maps to be reasonably accurate and a useful monitoring resource. Further dialogue with malaria control programmes in West Africa and Southern Africa also raised the point that a single dekadal rainfall anomaly map could raise an alert; when in fact the rainfall levels were not abnormally high -- but just 10 days earlier than \'normal\'. This suggested that additional information about the temporal distribution of rainfall was necessary. In order to respond to these issues, USGS and WHO-HealthMapper agreed to collaborate on the development of the dekadal anomaly maps in a format which could be downloaded, viewed and archived by surveillance staff directly in HealthMapper, a basic mapping and surveillance software developed by WHO\'s Communicable Disease Surveillance and Response Department <http://www.who.int/csr/mapping/tools/healthmapper/healthmapper/en/>. In addition to the most recent map, it is possible to download the dekadal maps for the previous six months and begin to construct a seasonal time series. The integration of the rainfall anomalies maps within HealthMapper also allowed the users to improve their analyses by combining ancillary data related to malaria directly on top of the rainfall anomalies maps for their country. Figure [3](#F3){ref-type="fig"} provides an example for Niger. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### 10 daily rainfall anomaly map for Niger viewed in the HealthMapper ::: ![](1475-2875-4-6-3) ::: Staff working at the International Research Institute for Climate Prediction (IRI) have since developed a web-based Malaria Early Warning System (MEWS) interface that enables the user to gain a broader contextual perspective of the current rainfall season by comparing it to previous seasons and climatological averages. The interface is in the IRI Data Library and takes the form of an online \'clickable map\' of Africa: <http://iridl.ldeo.columbia.edu/maproom/.Regional/.Africa/.MEWS/>. It displays the most recent dekadal rainfall map (Figure [4](#F4){ref-type="fig"}) over which national and district administrative boundaries and the epidemic risk zone can be overlaid (in this case as a guide rather than an absolute mask which may have excluded districts of local interest). These visual features can be toggled on or off and the user can zoom in to any region for more clarity. The user can \'zoom\' into a more localized region of interest. Dekadal rainfall can be spatially-averaged over a variety of user-selected areas, including administrative districts and 11 × 11 km, 33 × 33 km, 55 × 55 km and 111 × 111 km boxes. Upon the selection of this sampling area and a specific location of interest (by a click on map at the location of interest), four time-series graphs are generated (Figure [5](#F5){ref-type="fig"}). These time-series provide an analysis of recent rainfall with respect to that of recent seasons and the overall climatology. A description of the time-series figures, the data used and its source is also provided. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### MEWS \'Clickable Map\' for Rainfall Monitoring: 21--31 December 2004 ::: ![](1475-2875-4-6-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Summary information on current rainfall/seasonal development and expected rainfall for location of interest. ::: ![](1475-2875-4-6-5) ::: Conclusions =========== Access to frequently-updated rainfall information is an important requirement for the development of integrated early warning systems for malaria and other climate sensitive diseases \[[@B6],[@B10]\]. These operational rainfall monitoring tools have been developed primarily for application in warm semi-arid regions where rainfall anomalies are the main determinant of epidemic outbreaks, however they may also be an important information source for highland-fringe epidemic settings. They are offered as an experimental resource for testing within MEWS applications in Africa -- and may be modified in future in response to user feedback and further evaluation. While it is recognized that much of Africa does not (yet) have easy access to the internet, email is becoming more prevalent and it is now relatively easy for regional support centres, such as the WHO-Intercountry Programme for Malaria Control in Southern Africa, to prepare bulletins with malaria relevant climate data for distribution by email and courier to district health teams in epidemic prone areas as part of an overall MEWS process \[[@B11]\]. Acknowledgements ================ We would like to thank Charles Delacollette and other members of the Roll Back Malaria Technical Support Network on Epidemic Prevention and Control for their inputs during the development of these monitoring tools. We would also like to acknowledge Madeleine Thomson (IRI) and Jean Pierre Meert (WHO) for the field evaluations in Mali, Niger and Burkina Faso. The technical assistance provided by John del Corral (IRI) is also greatly appreciated.

PubMed Central

2024-06-05T03:55:52.618675

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548290/", "journal": "Malar J. 2005 Jan 21; 4:6", "authors": [ { "first": "Emily", "last": "Grover-Kopec" }, { "first": "Mika", "last": "Kawano" }, { "first": "Robert W", "last": "Klaver" }, { "first": "Benno", "last": "Blumenthal" }, { "first": "Pietro", "last": "Ceccato" }, { "first": "Stephen J", "last": "Connor" } ] }

PMC548291

Background ========== HCMV is a member of the betaherpesvirus family \[[@B42],[@B48]\]. Other members of this family include human herpesvirus 6 (HHV-6), and human herpesvirus 7 (HHV-7), and all are widely distributed in human populations. During productive replication, the 230 kilobase pair (kbp) viral genome replicates by a rolling circle mechanism, which generates long head-to-tail concatemers that are cleaved to unit length and packaged in capsids. The state of the HCMV genome during latency remains unidentified and is likely to be circular and extrachromosomal \[[@B6]\]. The HCMV genome has been detected in cells within the hematopoietic lineage as early as CD34+ progenitors and up through differentiated macrophages \[[@B23],[@B29],[@B38],[@B54]\]. Defective HSV viruses created by high multiplicity serial passage of virus stocks have been described on numerous occasions and have been characterized in detail at the molecular level \[[@B13],[@B18],[@B31],[@B43],[@B52],[@B67]\]. Naturally occurring defective HSV viruses and laboratory derived HSV amplicon vectors are composed of head-to-tail tandem reiterations of subgenomic regions containing a functional origin of DNA replication (Ori~S~or Ori~L~) and a DNA cleavage/packaging signal \[[@B3],[@B4],[@B30],[@B57],[@B60]-[@B62]\]. These two *cis*-acting functions can be relatively small ranging from *ca*. 90--150 base pairs (bp) for the ori and *ca*. 250--300 bp for the *a*sequence. The functional HCMV oriLyt is much more complex than either of the HSV oris; the HCMV oriLyt consists of multiple direct and inverted repeats and extends over at least 1500 bp \[[@B1],[@B2],[@B24],[@B37]\]. HCMV is unique among the herpesviruses in not having an origin binding protein homolog that is required for DNA replication \[[@B45]\]. The HCMV *a*sequence varies in size from *ca*. 550 bp to 762 bp, however, the conserved pac-1 and pac-2 *cis*-elements which determine the sites for cleavage of replicated viral DNA are present \[[@B15],[@B28],[@B58],[@B64],[@B65]\]. In contrast to HSV, HCMV does not efficiently produce defective virus genomes, this difference may be related to the distinct biology of the two viruses \[[@B45]\]. However two reports described the identification of what may potentially be HCMV defective viruses created by serial high multiplicity passage \[[@B47],[@B59]\]. These reports characterized HCMV defectives as very large subgenomic DNA molecules, in some cases extending over two thirds of the genome. In addition to these replication defective HCMV viruses, a recent report by Borst *et al*. 2003 \[[@B7]\], described the construction of an HCMV amplicon. In this report we further utilized the HCMV amplicon for gene delivery to human CD34+ cells. HCMV infects cells of the hematopoietic lineage \[[@B34],[@B38],[@B39],[@B55],[@B68]\]. Viral genomes can be found in CD34+ cells from seropositive individuals and granulocyte-macrophage progenitors and differentiated macrophages can be infected experimentally \[[@B56]\]. We were interested in determining whether the tropism of HCMV can be exploited to construct defective HCMV virus vectors (amplicons) targeted to hematopoietic stem cells. The general feasibility of such an approach for other cell types has been shown using other herpesviruses, e.g. HSV, EBV, and HHV-7 \[[@B20],[@B25],[@B26],[@B30],[@B35],[@B49],[@B70],[@B71]\]. Methods ======= Cells and virus --------------- HCMV Toledo (passage 8, from Dr. S. Plotkin, Aventis Pasteur, Doylestown, PA), and HCMV Towne*var*RIT (passage 134, from Dr. Plotkin via Dr. Ed Mocarski, Stanford University), were propagated in human fibroblasts (HF) cultured in Dulbecco\'s modified Eagle\'s medium supplemented with 10% fetal calf serum (JRH Biosciences, Lenexa, Kans.). Recombinant HCMV, RC2.7EGFP, expressing enhanced green fluorescent protein (EGFP) (Clonetech, Palo Alto, CA), under the control of the major early 2.7 promoter, was constructed by cotransfection of plasmid pEAG2.7EGFP with a set of overlapping cosmid clones derived from HCMV Toledo (G.M. Duke, unpublished data). Plasmid constructions --------------------- Plasmid pON205 (Spaete and Mocarski, 1985), contains the Towne strain *a*sequence, was obtained from Ed Mocarski (Stanford University). pEAG2.7EGFP was derived by cloning the EGFP gene from plasmid pEGFP-N2 (Clonetech, Palo Alto, CA) between the *Eag*I and *Sma*I site of the β2.7 gene taken from Toledo (G.M. Duke, unpublished data). HCMV amplicon plasmid Tn9-8 was derived by inserting the 6 kpb *Dra*I fragment of Towne*var*RIT (corresponding to nucleotides 91,166 -- 95,909 relative to AD169) (Figure [1B](#F1){ref-type="fig"}), spanning the HCMV oriLyt into the *Eco*RI site of pON205. Tn9-8 was partially sequenced by single-cycle and multicycle dideoxy-nucleotide chain termination method of Sanger *et al*., \[[@B51]\]. The plasmids designated Tn9-8GF5 and Tn9-8GF7 incorporating the EGFP gene with the HCMV Major Immediate Early (MIE) promoter and SV40 poly A sequence was isolated as a 2,334 bp *Nsi*I fragment from plasmid pEGFP-N2 (Clonetech, Palo Alto, CA), and cloned into the HCMV amplicon Tn9-8 at the *Pst*I site in both orientations. The *gpt*gene in Tn9-8-*gpt*was derived by cloning a PCR fragment from *Escherichia coli*DH5α using the primer pairs 5\'CTGCAGCTAGTCTAGACTGGGACACTTCACATGAGC3\'and 5\'CTGCAGCTATGTATCTAGAGCCAGGCGTTGAAAAGATTA3\'. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Schematic representation of amplified region near *ori*Lyt of the HCMV genome. **A**. Restriction enzyme map of minimal *ori*Lyt and adjacent region of heterogeneity (block). **B**. Region of heterogeneity shown as a dimer. Arrow indicates junction of the repeat segment. The number 91,166 to the left of the restriction map corresponds to the nucleotide position of the AD169 genome (EMBL accession number X17403). **C**. Autoradiograph of Southern blot utilizing a minimal *ori*Lyt probe, pON2623 (Kemble et al., 1996). Monomers and dimers are depicted with one and two arrows, respectively. Trimers and tetramers can be seen in the Towne*var*RIT viral stock. ::: ![](1479-0556-3-1-1) ::: Generation of viral stocks containing amplicons ----------------------------------------------- Plasmid DNA was transfected by CaPO~4~precipitation of approximately 4 μg of Tn9-8 amplicon DNA. The Tn9-8 DNA was transfected into approximately 1 × 10^6^passage 16 human fibroblast (HF) cells. At 24 hours post transfection, the cells were infected with CMV Towne at a multiplicity of infection (MOI) of 5 plaque forming units (PFU) per cell. Fresh medium was added to cells four days after infection and cells were harvested at 6 to 7 days post infection as described previously (Spaete and Frenkel, 1982). Virus stocks are prepared by three freeze-thaw cycles. Serial passages of amplicon-containing viral stocks on fresh HF cells were superinfected with CMV Towne as a helper virus at a MOI of 1. Southern blot analysis ---------------------- Viral DNAs were digested with restriction enzyme, electrophoresed in 0.8% agarose gels, transferred to Hybond-N+ nylon membranes (Amersham Corp.), (Maniatis et al., 1989), and immobilized with a UV Crosslinker 1000 (Hoefer Scientific Instruments, San Francisco, CA). DNA on the membrane was probed with fluorescein-labeled pUC9 DNA using conditions previously described (Spaete and Mocarski, 1985). Isolation of CD34^+^cells and infection with CMV ------------------------------------------------ The isolation of cord blood CD34+ stem cells was carried out by All Cells Inc. (Berkeley, CA) using CD34 Progenitor Cell Isolation Kit (Miltenyi Biotech, Auburn, CA). The positive selection of the CD34+ cells was carried out using hapten-conjugated antibody to CD34+ followed by anti-hapten antibody coupled to MACS Microbeads. The magnetically labeled cells are enriched on positive selection columns in the magnetic field. The purity of the CD34+ population was \>95% as analyzed by flow cytometry. The purified CD34+ cells were suspended in Iscove\'s modified Dulbecco\'s Minimal Essential Medium containing 5% fetal bovine serum. 2 × 10^5^CD34+ cells were used for each infection with TN9-8GF5 amplicon containing stocks, RC2.7EGFP virus, CMV Towne virus, or uninfected cell control. The cells mixed with virus were centrifuged at 500 × g for 10 mins at room temperature and were then placed in 37°C water bath for one hour. Following this the cells were cultured in 6-well cell culture plates (Costar) for 18--72 hours. At the end of the incubation the cells were harvested for CD34 staining. EGFP expression and immunostaining for flow cytometric analysis --------------------------------------------------------------- Amplicon containing viral stocks prepared from passage 1 were used to infect HF or human CD34+ cells maintained in 12 well culture plates. At 24 hour intervals post infection, the wells were observed for EGFP expression with a Nikon TE2000 microscope under UV illumination. Immunostaining for CD34+ cells was done using Phycoerythrin (PE)-conjugated anti-CD34 antibody (Becton Dickinson, San Jose, CA). Infected or control cells were incubated with 20 μl of PE-labeled anti-CD34 antibody for 45 minutes at room temperature and subsequently were washed twice with PBS containing 0.1% BSA. The cells were directly analyzed for EGFP and CD34+ staining on a FACSCalibur instrument (Becton Dickinson, San Jose, CA), at 18 and 36 hours post infection. Results ======= In order to exploit the natural tropism of HCMV for cells of the hematopoietic lineage, in a nonlytic manner, an HCMV amplicon i.e. a plasmid containing the HCMV oriLyt and *a*sequences was constructed. Theoretically, due to the large size of the HCMV genome, an amplicon derived from this virus should be able to carry the large DNA inserts and be capable of efficient introduction into hematopoietic cells by infection. Heterogeneity at oriLyt ----------------------- During analysis of cosmid clones of HCMV strain Towne, sequence heterogeneity was observed in the *Eco*RI E fragment of Towne that was not present in the Toledo strain \[[@B27]\]. The *Eco*RI E region spans in part the complex oriLyt region \[[@B1],[@B2],[@B24],[@B37]\]. Sequences in a 1.2 kbp repeat fragment were shown to give rise to the heterogeneity observed at this locus in the Towne genome (Figure [1](#F1){ref-type="fig"}). The coordinates of a single repeat unit starts at nucleotide 94,561 relative to the AD169 sequence and end at nucleotide 95,807 \[[@B10]\]. This segment can repeat at least three times in Towne strains from different passage histories (Fig. [1](#F1){ref-type="fig"}). This heterogeneity is different from the 189 bp repeat region previously described for the Towne strain oriLyt which occurs near the *Bam*HI sites in Figure [1](#F1){ref-type="fig"} (nt 93337--93525 relative to AD169), \[[@B11],[@B12]\]. Since Towne replicates to relatively high titers in cell culture, it was deemed advantageous to incorporate this heterogeneity in the origin containing sequences to be used in the amplicon construct. Construction of the HCMV amplicon --------------------------------- As a test of the feasibility of the system, an HCMV amplicon was constructed which incorporated the two *cis*-acting functions required for the propagation of the defective virus genomes in the presence of helper virus (Figure [2](#F2){ref-type="fig"}). HCMV amplicon plasmid Tn9-8 was derived by inserting the 6 kpb *Dra*I fragment of Towne (corresponding to nucleotides 91,166--95,909 relative to AD169) \[[@B10]\] (Figure [1B](#F1){ref-type="fig"}), spanning the HCMV oriLyt into the *Eco*RI site of pON205. The resulting amplicon was designated Tn9-8 (Figure [2](#F2){ref-type="fig"}), and was partially sequenced to verify its structure. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Schematic representation of the HCMV amplicon plasmids Tn9-8*gpt*, Tn9-8GF7 and Tn9-8GF5. The EGFP expression cassette was cloned in two orientations in the unique *Pst*I site in Tn9-8. The *gpt*expression cassette was cloned between the unique *Pst*I and *Hind*III sites. ::: ![](1479-0556-3-1-2) ::: Generation of viral stocks containing amplicons ----------------------------------------------- As a test of the ability of this construct to function as an amplicon, plasmid Tn9-8 was transfected in human fibroblast (HF) cells, and subsequently infected with HCMV Towne strain at an MOI of 5 to provide helper virus replication functions. Seven days later, infected cells were harvested, sonicated, and viral stocks were prepared for passage to fresh HF cells. Fresh HF cells were infected with the progeny of the transfection/infection and incubated for 7 days. The DNA from these infected cells was harvested (designated passage 1), restricted with *Hind*III and *Dpn*I, and Southern blotted \[[@B36]\]. Southern blot analyses of DNA demonstrated that Tn9-8 was susceptible to digestion with *Dpn*I, consistent with replication of the plasmid in bacteria (Figure [3](#F3){ref-type="fig"}, lane 2). In contrast, Tn9-8 in infected cells was resistant to *Dpn*I demonstrating that it had replicated in eucaryotic cells (Figure [3](#F3){ref-type="fig"}, lanes 3--5). This observation is consistent with replication and packaging of Tn9-8 into infectious virions. These results demonstrate that foreign DNA sequences, exemplified by the plasmid pUC9, can be introduced into defective genomes that are packaged and propagated in serially passaged virus stocks. To examine whether these results were the consequence of amplicon replication and packaging or integration of the amplicon plasmid into the helper virus, DNA prepared from passage 1 infected cells was digested with *Cla*I, *Xba*I, *Afl*II and *Dpn*I. These enzymes digested the Towne helper virus DNA to fragments no larger than 13.6 kbp but do not cut within Tn9-8. The amplicon DNA was significantly larger than 23 kbp consistent with the amplicon being replicated as a concatamer (Figure [4](#F4){ref-type="fig"}). This result indicated that the high molecular weight DNA containing plasmid sequences was packaged independently and was not integrated into helper virus. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Southern blot analysis of passage 1 of HCMV amplicon DNA probed with plasmid pUC9. Lane 1. Plasmid Tn9-8 linearized with *Hind*III serves as a marker for correct migration of monomeric repeats. Lane 2. Plasmid Tn9-8 restricted with *Hind*III and *Dpn*I as a control for non-replicating DNA. Lanes 3--5. Infected cell DNAs restricted with *Hind*III and *Dpn*I. The signal in lanes 3--5 (arrow) demonstrates authentic replication and packaging of amplicon Tn9-8 in eucaryotic cells. ::: ![](1479-0556-3-1-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Southern blot analysis of high molecular weight HCMV amplicon DNA at passage 1 probed with plasmid pUC9. DNA prepared from passage 1 infected cells was digested with *Cla*I, *Xba*I, *Afl*II and *Dpn*I, Southern blotted and probed with plasmid pUC9. The samples are from those shown in Fig. 3, lanes 3 and 5. The high molecular weight DNA containing plasmid sequences (arrow) demonstrates the major hybridizing species migrating slower than the 23 kbp lambda DNA *Hind*III digest indicated as the marker on the left of the autoradiograph. ::: ![](1479-0556-3-1-4) ::: Packaged defective viral genomes derived from Tn9-8 or a derivative containing a selectable marker (Tn9-8-*gpt*), were serially passaged in HF cells. Defective viruses could be detected at passage 3 when probed with plasmid pUC9; however, the copy number appeared to diminish upon serial passage (not shown). Selection with mycophenolic acid on Tn9-8-*gpt*amplicons did not enhance recovery. Rescue of monomeric repeat units in bacteria -------------------------------------------- Concatemeric DNA was prepared from passage 2 and 3 virus stocks containing the defective virus genomes (Tn9-8-*gpt*), digested with *Pst*I and *Hind*III, respectively, in order to analyze monomeric repeat units. The *Hind*III-digested DNA was circularized by ligation and used to transform *E. coli*bacteria to analyze structure and to demonstrate shuttle vector capability between eucaryotic and bacterial hosts. A number of plasmids prepared from the rescue attempt had a restriction enzyme pattern indistinguishable from the input (Fig. [5A](#F5){ref-type="fig"}, lanes 1 and 2). Other plasmids however exhibited the expected restriction pattern consistent with a head-to-tail amplification of the *a*sequence (lanes 3 and 4). Digestion with *Nae*I produced a fragment of the predicted size of a unit length *a*sequence (762 bp), and this product hybridized with an *a*sequence specific probe (*Pst*I-*Sgr*AI fragment from Tn9-8), (Figs. [2](#F2){ref-type="fig"} and [5B](#F5){ref-type="fig"}, lanes 3 and 4). This type of amplification has been readily seen in restriction enzyme digested DNA preparations of parental genomes of both HSV and HCMV \[[@B31],[@B40],[@B41],[@B58],[@B64],[@B69]\] and has also been observed in HSV amplicons using Southern blot hybridizations \[[@B15]\]. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Tn9-8-*gpt*was rescued from concatamers following serial passage in HF cells. (A) Tn9-8-*gpt*was passaged in HF cells and monomer units were recovered by linearizing concatemeric DNA from serial passage 3 with *Hind*III and cloning in bacteria (lanes 2 and 4) and also by linearizing passage 2 DNA with *Pst*I and cloning in bacteria (lane 3). Lane 1 represents the unpassaged clone for comparison. Following rescue in bacteria, DNA was prepared and cut with *Nco*I. Fragments were separated on an agarose gel and visualized with ethidium bromide staining (left panel). The gel was transferred to a nylon membrane and probed with an *a*sequence specific probe (right panel). (B) Fragments from an *Nae*I digest were also separated on an agarose gel and visualized as in panel A. The gel was transferred to a nylon membrane and probed with an *a*sequence specific probe. Lane 5 shows a 100 bp ladder. Lanes 3 and 4 show a *ca*. 800 bp fragment that hybridizes to *a*sequences. ::: ![](1479-0556-3-1-5) ::: Expression of heterologous genes in an HCMV amplicon ---------------------------------------------------- To demonstrate that the HCMV amplicon could be used as a vector system to support the expression of a foreign gene, EGFP under the transcriptional control of the HCMV major immediate early (MIE) promoter was used as test reporter gene. Two resulting amplicon plasmids designated Tn9-8GF5 and Tn9-8GF7 both expressed EGFP following transfection of HF cells in the absence of helper virus, as expected (not shown). Packaged amplicons were generated by introduction of Tn9-8GF5 into cells and infecting with HCMV 24 hours later at an MOI of 5. Transfection-derived viral stocks were passaged onto fresh HF cells supplemented with Towne helper virus at an MOI of 1. Viral stocks prepared from passage 1 were used to infect HF cells and grown on 12-well tissue culture plates. A limited number (ca. 0. 1%) of brightly fluorescing cells could be seen by microscopic examination at 24, 72 and 96 hours post-infection (Figure [6](#F6){ref-type="fig"}). This demonstrates that a foreign gene can be expressed in the context of a HCMV amplicon viral stock in infected HF cells. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Fluorescent Microscopic Analysis of TN9-8GF5 amplicon infected cells. Human fibroblast cells (HF) or human cord blood CD34^+^cells were infected with TN9-8GF5 amplicon-containing stocks, or mock infected. Cells were observed at different time-points 24, 72 and 96 hrs post infection with TN9-8GF5 amplicon under the fluorescent microscope (Nikon TE2000 microscope). EGFP expressing fluorescent cells were observed in the TN9-8GF5 amplicon infected human fibroblast cells or human CD34+ cells at different time-points. Control uninfected cells were negative (not shown). ::: ![](1479-0556-3-1-6) ::: To test the utility of the HCMV amplicon in gene therapy or gene delivery, we used packaged amplicons in viral stocks to infect and deliver an expressed gene into human CD34+ progenitor cells. Viral stocks containing amplicons carrying EGFP under the transcriptional control of the HCMV major immediate early (MIE) promoter prepared from passage 0 and passage 1 were used to infect CD34+ cells derived from cord blood. Starting at 24 h after infection, CD34+ cells were examined for EGFP expression by fluorescent microscopy. EGFP expression was observed in TN9-8GF5 amplicon-infected CD34+ cells starting at 24 h post-infection. The cells remained positive for EGFP expression for more than 96 hrs, at which point the cells were terminated (Figure [6](#F6){ref-type="fig"}). In a separate experiment, at 36 hours post infection, the cells were stained with PE-labeled anti-CD34 and analyzed for the CD34 marker and EGFP expression. EGFP expression was observed in the CD34+ population in the TN9-8GF5 amplicon (0.3%, 0.1%) (Figure [7a](#F7){ref-type="fig"}, &[7b](#F7){ref-type="fig"}) or the CMV-EGFP virus (0.6%) (Figure [7c](#F7){ref-type="fig"}) at 36 hours post infection. The CMV Towne control-virus infected cells or uninfected CD34+ cell control were negative for EGFP expression (Figure [7d](#F7){ref-type="fig"}, &[7e](#F7){ref-type="fig"}). A small population (7--12%) of the cells lost expression of the CD34+ marker upon *in vitro*culture. EGFP expression was also observed in a CD34(-) population infected with either TN9-8GF5 amplicon containing viral stocks (0.8%, 0.1%) or with the CMV-EGFP virus, RC2.7EGFP (3.6%) (Figure [7a, 7b](#F7){ref-type="fig"} &[7c](#F7){ref-type="fig"}). The CMV Towne infected cells or uninfected control cell also had a significant CD34 negative population but were negative for EGFP expression (Figure [7d](#F7){ref-type="fig"}, and [7e](#F7){ref-type="fig"}). These results clearly demonstrate that CD34+ cells can be infected with replication competent or incompetent CMV vectors expressing a foreign gene. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Flow cytometry analysis of human cord blood CD34^+^cells infected with CMV amplicon containing stocks, virus, or uninfected cell control. TN9-8GF5 amplicon (*a,b*), CMV-EGFP (RC2.7EGFP) virus (*c*), CMV (Towne) infected (*d*), or control uninfected human cord blood CD34 cells (*e,f*), were stained 36 hours post-infection with PE-antiCD34 antibody (*a-e*), or were left unstained (*f*), and were analyzed for two-color cytometry analysis using a FACS Calibur instrument. The dot-plots are generated using Cell Quest software and reveal the EGFP^+^cells populations. Numbers in the upper right and lower right quadrants indicate percentage of the EGFP^+^CD34^+^and EGFP^+^CD34^-^cells respectively. A frequency lower than 0.01% is considered negative. ::: ![](1479-0556-3-1-7) ::: Conclusions =========== We have shown that a replication-defective virus vector system that is derived from HCMV is capable of delivering and expressing foreign genes in infected primary cells including progenitor stem cells such as human CD34+ cells. Further improvement and optimization of the system offers the potential to deliver gene-based therapies to multipotent cells. Advantages for use of the HCMV amplicon --------------------------------------- Foremost among the advantages of the vector system we have described is the potential ability to efficiently infect and deliver genetic information to hematopoietic stem cells (CD34+) and other dividing and non-dividing cell types which may support HCMV infection \[[@B34],[@B38],[@B39],[@B55],[@B68]\]. Genetic hematological disorders such as thalassemias and sickle-cell anemia and other hemaglobinopathies could therefore be targeted for therapy with this strategy. Another potential advantage for the system is that vector DNA could possibly be maintained as an episome with minimal concern for the potential consequences of random integration of vector DNA (i.e. activation of oncogenes or inactivation of tumor suppressor genes). In order to insure efficient segregation as an episome, the EBV latent replication origin, oriP, and the transactivator, EBNA-1, could be added as was previously shown for another hybrid herpesvirus vector \[[@B71]\]. However such a modification may not be necessary because HCMV genomes appear to be carried continuously in cells of hematopoietic origin in infected individuals. Yet another potential advantage as with other herpesviral vectors, is that the HCMV vector system should have the capacity for very large inserts. Infection of CD34+ cells with HCMV ---------------------------------- The infectivity of CD34+ cells from seropositive and seronegative subjects with HCMV has been tested both *in vivo*and *in vitro*\[[@B53]\]. Furthermore, hematopoietic stem cells are also reported as a site for HCMV latency. Efficient transduction of human CD34+ cells with retroviral and non-viral vectors has been unsatisfactory due to the lack of maintenance of high levels of expression of the transgene following engraftment of the engineered cells \[[@B16]\]. The HCMV MIE promoter may not be the right promoter for optimal expression in a CD34+ cells, since it has been shown that in the context of a lentiviral-based gene transfer system this promoter appeared to function less efficiently due to a cell-type specific expression defect \[[@B16]\]. The approaches to improving the efficiency of gene transfer into human cells have focused on improving gene delivery vectors and optimizing *ex vivo*culture conditions, which preserve the developmental properties of the stem cells \[[@B14],[@B22]\]. Umbilical cord blood is recognized as a rich source of hematopoietic CD34+ stem cells \[[@B33]\]. In our experiments we used cord blood derived CD34+ cells for infection with HCMV amplicon containing stocks or HCMV-EGFP virus. However, bone marrow derived CD34+ cells have also been shown to be infectable *in vitro*with HCMV \[[@B34]\]. Gentry & Smith \[[@B21]\], reported a progressive loss of primitive cell properties including a reduction of CD34 expression upon *in vitro*culture of cord blood derived CD34+ cells. In a separate study, cord blood derived CD34+ cells cultured with IL-3 *in vitro*showed a progressive decline of the CD34+ population and more differentiated cells originating in the CD34(-) population \[[@B9]\]. In our experiments with \>95% pure cord blood derived CD34+ cell population, a loss of CD34 expression in a small percent population (9--12%) of stem cells upon *in vitro*culture has been observed. EGFP expression was also seen in the CD34(-) population (Fig [7a, 7b](#F7){ref-type="fig"}, &[7c](#F7){ref-type="fig"}). It is possible that HCMV infection of CD34 cells could induce cell differentiation and loss of primitive properties including reduction of CD34 expression. Further studies of HCMV infection in CD34 cells will help in defining whether CD34+ infected cells undergo cell differentiation by increased expression of other markers such as CD33, CD38, HLA-DR or cytokines. It is also relevant to note here that HCMV virus carries homolog sequences for HLA-related and cytokine-related molecules and infection can induce cellular cytokines \[[@B5],[@B8],[@B19],[@B32],[@B44],[@B46],[@B50],[@B66]\]. The HCMV amplicons contain only the *cis*-acting ori and packaging sequences, and have no structural gene sequences. However, amplicon containing viral stocks are a mixture with HCMV replication competent helper virus. HCMV induced cell-differentiating effect, if any, might be minimized using a helper virus-free amplicon system. In this regard, it should be possible to test a number of strategies to prepare helper virus-free stocks \[[@B17],[@B63]\]. These preparations would be useful for therapeutic applications in immuno-compromised patients. Competing Interests =================== The authors of this publication are supported financially by salary and shares of MedImmune Inc., during the completion of this work. A patent application has been filed with the United States Patent Office relating to the content of this manuscript. The authors have assigned all rights of ownership to MedImmune Inc. The authors declare that they have no other competing interest. Authors\' Contributions ======================= KM carried out the expression analysis in human fibroblasts and CD34 cells. MNP & GMD generated amplicon stocks and MNP did the \'*a*\' sequence analysis. GWK provided critical intellectual input. RRS provided the original idea and experimental design as well as cloning of the amplicon, generation of amplicon stocks and Southern analysis.

PubMed Central

2024-06-05T03:55:52.620132

2005-1-26

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548291/", "journal": "Genet Vaccines Ther. 2005 Jan 26; 3:1", "authors": [ { "first": "Kutubuddin", "last": "Mahmood" }, { "first": "Mark N", "last": "Prichard" }, { "first": "Gregory M", "last": "Duke" }, { "first": "George W", "last": "Kemble" }, { "first": "Richard R", "last": "Spaete" } ] }

PMC548292

Background ========== As health care professionals begin to understand the importance of quality of life (QL) and emotional and social well-being in the treatment and progression of cancer, it has become standard practice, and in fact has been described as a bioethical imperative, to include QL assessment in all oncology clinical trials \[[@B1]-[@B7]\]. This is particularly the case in Phase I trials for novel therapeutics, where in many cases the patients admitted have exhausted other treatment options and likely face death within months under best supportive care \[[@B8]\]. A Phase I trial is the first test of a new therapeutic in humans and aims to establish a maximum tolerated dose, to evaluate dose limiting toxicity, and to examine the drug\'s pharmacology. There is generally little chance of clinical disease response and relatively high potential risk of toxicity in this type of trial. For patients in this situation, quality of life remains perhaps the most important variable to consider in the evaluation of the new treatment \[[@B9],[@B10]\]. We report here on the initial QL of patients enrolled in the Reolysin Phase I trial, the first clinical usage of the reovirus in humans with cancer. The reovirus as a possible treatment for cancer attracted a great deal of media attention when the journal Science, in 1998, published very encouraging results showing tumor regression in animal models \[[@B11]\]. Further work has established the reovirus as a potential therapeutic in the treatment of brain, colorectal, ovarian and breast cancer cell lines \[[@B12],[@B13]\]. A small sample of patients with different types of primary cancers that had exhausted other treatment options, all of whom had subcutaneous tumors that were easily palpable and injectable with Reolysin, were enrolled in the current trial. Due to the nature of the population under study, it seemed especially important to use instrumentation that was flexible enough to allow these very ill patients to identify what was important to each of them as individuals, as well as to collect data from standardized instruments that would allow direct comparisons with other patient populations. Thus, a battery of tests to measure both individualized and standardized QL, distress levels, and spirituality was selected. Important aspects of QL were measured, including physical, psychological, emotional, social, and spiritual functioning, with emphasis on mood states and psychopathology. Expectations and hopes regarding participation in the trial were also elicited during a semi-structured interview. The specific instruments used included an interview-based subjective measure of QL, the Schedule for the Evaluation of Individual Quality of Life -- Direct Weighting (SEIQoL -- DW)\[[@B14]\]. The SEIQoL-DW is a relatively new measure and unique in QL measurement in that it elicits from patients their own self-generated list of the \"five most important domains of QL\" for them, rather than asking about pre-set areas. After patients identify their most important domains, they rate how well each domain is for them currently, and how important overall in their lives each domain is. This instrument and its predecessor, the SEIQoL \[[@B15]\], have been used in oncology in several published studies \[[@B16]-[@B18]\]. The SEIQoL differs from the SEIQoL-DW in that a procedure called judgement analysis is used in the SEIQoL to arrive at the relative importance of each of the domains for each patient. This process is much more time consuming, abstract and complicated than the direct weighting procedure used in the SEIQoL-DW. While the SEIQoL procedure has been deemed overly burdensome for some patients with both early and advanced cancer \[[@B16],[@B17]\], primarily due to the judgement analysis portion of the procedure, the SEIQoL-DW has been found to be acceptable and practical to use in a validation study of patients on Phase I clinical trials \[[@B18]\]. Another study of the SEIQoL-DW found that patients with advanced cancer were good judges of their own QL, and able to complete the interview with little difficulty \[[@B17]\]. In past studies, the most important areas of QL that patients on Phase I clinical trials identified were equally health and family \[[@B18]\]. However, in a sample of advanced cancer patients family concerns were consistently identified as more important than health issues \[[@B17]\]. The patient population in the Campbell & White study \[[@B18]\] was not defined except by their participation in Phase I trials, whereas the Waldron & O\'Boyle sample \[[@B17]\] all had advanced incurable cancer. It may be the case that as illness severity progresses, concerns are directed toward domains that offer more hope. Other studies that have used the SEIQoL found family, health and finances to be the top three areas in men with early stage prostate cancer \[[@B16]\], and similarly family, health, marriage and leisure/hobbies were most important to a group of cardiac patients \[[@B19]\]. Thus, although there does seem to be some consistency in the areas nominated by diverse patient groups, differences in the relative importance of the areas are commonly found. The other QL questionnaire used in this study is the very widely used European Organization of Research and Treatment of Cancer (EORTC) Quality of life Questionnaire (QLQ C-30), a 30 item standardized self-administered questionnaire that taps into important domains of QL, including physical, psychological, emotional and social functioning. This was included to allow comparisons with a well-known and validated standardized quality of life questionnaire with set domains and subscale scores. Another study at our Centre directly compared the SEIQoL to the EORTC QLQ C-30 in a sample of early stage prostate cancer patients \[[@B16]\]. The authors compared the domains nominated by the patients to those included on the EORTC QLQ C-30, and concluded that although there was substantial overlap on some items, many items identified by patients were not included on the standardized questionnaire. The area of spirituality was also of interest in this population of very ill people, as issues of death and dying are often accompanied by questioning in the realm of spirituality. The Spiritual Health Inventory \[[@B20]\] was used for this purpose, as it measures self-acceptance, relationships with others, and hope, which may be an important factor as patients participate in the trial. In terms of depression, anxiety and other psychiatric symptoms that are frequent in cancer patients, the Brief Symptom Inventory (BSI)\[[@B21]\] was used to broadly assess many areas of psychopathology, and the Beck Depression Inventory (BDI)\[[@B22]\] to focus in more detail on depressive symptomatology. Both of these instruments are widely used in the oncology literature and thus there are many published reports with values that can be used for comparison purposes. Previous work with patients in Phase I trials has found that patients\' expectations going into trials are generally more optimistic than oncologists\'. Patients with incurable malignancy in a Phase I trial estimated a greater potential for benefit from the experimental therapy than did oncologists \[[@B10]\]. They also estimated that the experimental therapy had less potential for toxicity than the standard treatment. The oncologists estimated the potential for toxicity on both treatments to be about equal, and lower, than did patients. Patients in Phase I trials for new drugs, when asked why they had agreed to try the new treatment, cited the potential for helping their disease to be the number one reason for participation \[[@B9]\]. Indeed, despite cautious words from the medical staff, it is not surprising that patients with a disease resistant to all other treatments might hope for at least a slowing of their disease progression with an experimental treatment. Thus, the preliminary QL of such patients, as measured in the current study, may be inflated by these hopes. Other studies looking at the effects of participation in Phase I trials on QL have found either no detrimental effects of participation \[[@B8]\], or enhancement of QL over the course of the trial compared to a control group that received supportive care \[[@B23]\]. The purpose of the current study was to investigate individualized QL in a group of patients with metastatic incurable cancer participating in a Phase I trial of a highly media lauded new therapeutic, and investigate their expectations regarding the trial. Areas of importance and SEIQoL Index scores for these patients will be compared with those of patients in other studies, and compared to their own scores on the standardized QL and psychological measures assessed. Relationships between the different measures will also be explored. Methods ======= Subjects -------- Patients were recruited as specified in the protocol: \"A Phase I Clinical Trial to Evaluate Dose Limiting Toxicity and Maximum Tolerated Dose of Intralesional Administration of REOLYSIN for the Treatment of Histologically Confirmed Malignancies\". All patients had histologically confirmed evaluable palpable tumors of any histological type that had failed to improve on existing standard therapy. The injectable lesion was required to be between 1 and 10 cm^2^, and accessible and measurable for delivery of an intralesional injection. Patients were required to have a life expectancy of at least 12 weeks and a ECOG performance status of ≤ 3 and must not have received active cancer treatment for least 21 days prior to entrance onto the trial. Adequate organ reserve in terms of bone marrow, hepatic, renal and cardiac functions was required. Patients on immunosuppressive therapy or alternative/complimentary/unproven systemic or local therapies were ineligible. Procedures ---------- After patients had been referred to the above mentioned trial, but prior to being definitively accepted (pending complete assessment of inclusion/exclusion criteria), patients met with a psychologist (LC) for the assessment protocol. At that time they were interviewed concerning their expectations about their health, both without any further conventional treatment and with the potential experimental treatment. They then completed the interview-based individualized QL interview, followed by the quantitative questionnaires as detailed below. All 16 patients followed the same procedures. Instruments ----------- ### Demographics Form Demographic information including age, education, marital status, occupation and current employment status was obtained on a form created for this study. Medical history including type of illness, dates of first diagnosis and subsequent relapses and site of metastases were collected, and later verified from patient charts. ### Expectations Interview Patients were asked three questions in a semi-structured interview: How do you feel about potentially being a part of this trial? How do you see your disease progressing without any further conventional treatment? Once on the trial, how do you see your disease progressing? Short answers to these questions were recorded verbatim at the time of the interview. Quality of Life --------------- ### Schedule for the Evaluation of Individual Quality of Life -- Direct Weighting (SEIQoL -- DW) \[[@B14]\] This schedule takes the form of a semi-structured interview, in which the investigator first describes quality of life as an individually defined construct, then elicits from the patient their own five most important domains of QL, rather than asking them about pre-set areas. Patients are asked to think of what areas of life determine their own happiness, or quality of life. After patients identify their most important domains (\"cues\"), they rate the quality of each domain currently in their lives by drawing a bar graph on a 100 mm scale from worst possible to best possible. This is called the \"level\" of the cue. Finally, they rate how important each domain is overall in their lives using a direct weighting disk. This disk consists of five overlapping different colored laminated disks that can be rotated around a central point to form a pie chart. Each piece represents one of the five chosen domains. The patient manipulates the disk until the proportion of each piece making up the pie represents the relative importance of that domain in their lives (\"weight\"). The weight value of each cue is calculated by determining the percentage of the overall pie that each piece covers by reading off a larger backing disk that is labeled with a 0--100 scale around the pie. Then by multiplying the level of each domain by its weight and summing the product for all five items, a summary score representing overall subjective quality of life can be calculated. This is called the SEIQoL Index Score. More detailed descriptions of the procedures are available in other publications \[[@B14],[@B18],[@B24]\]. ### The European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30 Quality of Life Questionnaire \[[@B25]\] This 30-item questionnaire includes five functional domains of quality of life: physical function (5 items), emotional function (4 items), cognitive function (2 items), social function (2 items) and role function (2 items). There are also several symptom scales: fatigue (3 items), pain (2 items), nausea and vomiting (2 items), and one item each for dyspnea, sleep disturbance, appetite, constipation, diarrhea and financial difficulties. Finally, two items assess global quality of life. The questionnaire shows high internal consistency, and overall reliability and validity of the survey has been demonstrated in international clinical trials with cancer patients of heterogeneous diagnoses including lung cancer \[[@B26]\]. Distress -------- ### Beck Depression Inventory (BDI) \[[@B22]\] This 21-item questionnaire gives a global score on depressive symptoms, and norms are available for many different populations, including cancer patients. Higher scores represent more depressive symptoms. ### Brief Symptom Inventory (BSI) \[[@B21]\] A general mental health measure of 58 questions which provides scores on nine dimensions of psychopathology or psychological distress: somatization, obsessive-compulsive, interpersonal sensitivity, depression, anxiety, hostility, phobic anxiety, paranoid ideation, and psychoticism. Three global scores can be calculated: the Global Severity Index (GSI), the Positive Symptom Total (PST) and the Positive Symptom Distress Index (PSDI). The GSI was used as the global score in this study. Spirituality ------------ ### Spiritual Health Inventory (SHI) \[[@B20]\] This instruments defines spiritual health as the capacity to transcend oneself and meet three basic needs; the need for self-acceptance, the need for relationships with others and/or a supreme being, and the need for hope. These three factors accounted for 71% of the variance in validity studies. A single total score is calculated by summing all items. The possible range of scores is 31--155, with higher scores indicating higher levels of spiritual health. The 31-item questionnaire takes very little time to complete. Results ======= Subjects -------- Demographic characteristics and disease variables of participants are presented in Table [1](#T1){ref-type="table"}. Patient \#10 was registered in the trial but too ill to complete any of the questionnaires or the interview. Therefore no data for this patient is included in the study. The remaining 16 patients who provided data all had metastatic disease that was considered incurable, 6 men and 10 women. The largest patient group consisted of five women who had metastatic breast cancer, followed by three patients with malignant melanoma. Patients ranged in age from 32 to almost 76 years old, with a median age of 53 years. They had been diagnosed with cancer for a median of 3.3 years (range 0.5--26.9 years) before entrance to the study. They had on average 16 years of education (range 12--25), and therefore represented a highly educated group. At the time of analysis, nine of the patients had died, at a median of 136 days from the time of the interview (range 21--664 days). The remaining seven were still alive, a median of 242 days from the time of the interview (range 207--709 days). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographic and Disease Characteristics ::: Patient Number Gender Age (Years) Cancer diagnosis Years since first diagnosis Location of metastases Days from interview to death ---------------- -------- ------------- ------------------------------------------ ----------------------------- ---------------------------------------------------- ------------------------------ 1 Female 45 Mucoepidermoid carcinoma (head and neck) 14.7 Lymph nodes 28 2 Male 50 Squamous carcinoma (head and neck) 0.7 Lymph nodes Alive-709 3 Female 54 Anaplastic thyroid carconoma 10.8 Lungs/liver/bone 21 4 Female 47 Malignant Melanoma 12.7 Eye/breast/liver/chest wall/lung 664 5 Female 47 Metastatic Breast carcinoma 6.6 Chest wall/bone 241 6 Female 60 Metastatic Breast carcinoma 2.7 Chest wall /lungs /retroperitoneum Alive-529 7 Male 32 Soft tissue sarcoma 0.5 Right lower extremity 96 8 Male 56 Neuroendocrine islet cell tumor 2.4 Liver/face/neck/ Scalp 44 9 Female 56 Metastatic Breast carcinoma 2.7 Chest wall/ left supraclavicular skin/ intrabdomen 142 11 Male 42 Malignant melanoma 6.4 Liver/lung/spleen Alive-368 12 Male 64 Klatskin\'s tumor 1.4 Abdomen 171 13 Female 76 Metastatic Breast carcinoma 26.9 Lung/bone/liver 136 14 Female 55 Malignant melanoma 1.8 Axilla /lung /liver Alive-242 15 Female 48 Metastatic breast carcinoma 2.8 Neck /Chest Wall /Brain Alive-242 16 Female 46 Soft tissue sarcoma 3.9 Lung/skin/breast/ retroperitoneal Alive-227 17 Male 70 Squamous carcinoma (head and neck) 9.7 Head/neck Alive-207 ::: Expectations Interview ---------------------- Interviews were conducted with all of the 16 patients. To the question \"How do you see your disease progressing without further conventional treatment?\", nine of the patients indicated they felt it would get worse and they would eventually die of their disease. This was stated in different ways: Nothing else left\...getting gradually worse\...terminal -- could be months, could be years\...wouldn\'t go very well. The other seven patients offered more hopeful or neutral prognoses: don\'t think about it -- stay positive\...still hopeful and optimistic\...other things available still\...would still take chemo, hope it would work\...wouldn\'t ever give up on hope\...faith\...not sure, unknown. When asked how they felt about being in the trial, most patients indicated feeling excited, fortunate, grateful and hopeful. One indicated that they felt scared as well as hopeful, not knowing what to expect, and one said they felt like a guinea pig. To the question \"Once on the trial, how do you see your disease progressing?\", ten of the patients mentioned hoping for the tumor to shrink, for a remission, or for some extension of life. Five patients mentioned hoping for a cure, to be cancer free. One patient just mentioned hoping to help others, and four others said that although they were hoping for personal benefit, if it didn\'t help them it might help others in the future. In general, patients were hopeful yet philosophical about the trial. The 54-year old woman with melanoma captured these sentiments with her comments: It feels hopeful. Maybe it won\'t help me -- I won\'t be disappointed. It might help others down the road. It\'s on the frontier -- exciting. If it works it\'s a bonus. I don\'t totally expect anything. It may extend life. Just day by day carry on. EORTC QLQ C-30 -------------- Quality of life scores on the EORTC QLQ C-30 are presented in Table [2](#T2){ref-type="table"}. All 16 patients completed the questionnaire. On the functional scales, where higher scores indicated better functioning, scores ranged from a low of 55 on social functioning, to a high of 76 for cognitive functioning, on a scale of 1--100. The overall global QL rating was 57. On the symptom scales, where higher scores indicate more symptomatology, scores ranged from a low of 14 (nausea and diarrhea) to a high of 42 on pain and fatigue. The next most prevalent symptoms were sleep problems and appetite loss. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### EORTC Scores ::: Functional Scales (Higher scores = higher function) Mean SD ----------------------------------------------------- --------------------- ----------------------------------- Physical Function 63.75 32.02 Role Function 62.50 38.73 Emotional Function 75.52 18.12 Cognitive Function 76.04 24.32 Social Function 55.21 32.04 Global Quality of Life 57.22 21.79 Symptom Scales (Higher scores = more symptomatic) Fatigue 41.67 25.82 Nausea 14.58 14.75 Pain 41.67 25.82 Dyspnea 22.92 26.44 Sleep 35.42 30.96 Appetite 33.33 32.20 Constipation 25.00 28.55 Diarrhea 14.58 17.78 Finances 29.17 26.87 ::: Correlations between subscales are presented in Table 6 (additional file [1](#S1){ref-type="supplementary-material"}). The subscales of role function, cognitive function, global QL, fatigue and appetite loss were significantly related to seven other subscales each. Social functioning was associated with scores on six other subscales. Finances, diarrhea, and sleep were unassociated with any other subscales, and pain was associated only with dyspnea. All significant correlations were in the expected directions. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### SEIQoL Items ::: Patient Number Item 1 Item 2 Item 3 Item 4 Item 5 ---------------- -------------- --------------- -------------------- -------------------- ------------ 1 Children Spouse Religion Physical Fitness Finances 2 Family Exercise Nature Computer Work 3 Spouse Children Friends Activities Father 4 Family Friends Dog Gardening Fun 5 Pain Control Finances Health Energy Activities 6 Travel Health Family and Friends Spouse Activities 7 Children Family Mobility Hope Work 8 Work Family Finances Health Activities 9 Family Grandchildren Friends Travel with Spouse Finances 11 Family Friends Active at Home Work Finances 12 Spouse Friends Family Belief Art 13 Activity Grandchildren Sewing Gardening Travel 14 Family Faith Positivity Activities Friends 15 Work Recreation Mobility Family Friends 16 Spouse Children Family Faith Exercise 17 Family Spouse Work Family tree Religion Mean Level 70.9 68.4 64.6 56.9 62.5 SD 31.3 26.0 32.0 32.1 27.1 Mean Weight 0.25 0.23 0.19 0.20 0.16 SD 0.08 0.08 0.06 0.08 0.09 ::: Psychological Scores -------------------- Scores on the BDI, BSI and SHI are presented in Table [3](#T3){ref-type="table"}. Fifteen of the 16 patients completed the questionnaires. Scores on the BDI averaged 11, in the moderate range of depressive symptomatology. On the BSI, scores ranged from a low of 0.17 on paranoid ideation, to a high of 0.91 on the obsessive-compulsive subscale. The overall global severity index was 0.55. These scores are higher than those of the general population, but quite a bit lower than those of psychiatric outpatients \[[@B21]\]. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Psychological Scores ::: Mean SD ---------------------------------------- ---------------------- ----------------------------------- BSI Somatization 0.74 0.56 BSI Obsessive Compulsive 0.91 0.63 BSI Interpersonal Sensitivity 0.38 0.36 BSI Depression 0.76 0.62 BSI Anxiety 0.50 0.40 BSI Hostility 0.21 0.27 BSI Paranoid Ideation 0.17 0.17 BSI Psychoticism 0.16 0.17 BSI Global Severity Index 0.55 0.36 Beck Depression Inventory Total Score 11.40 9.46 Spiritual Health Inventory Total Score 118.33 16.02 ::: Patients scored an average of 118 on the SHI. The most highly endorsed items on the scale of 1--5, where 1 corresponds with the heading \"not at all\", and 5 with \"very much\", were the following: \"I believe other people accept me even with my faults\" (4.5); \"I actively participate in decisions concerning my health care\" (4.5); \"I believe my nurses and doctors care about me\" (4.3); \"My life has a purpose\" (4.2); \"I feel accepted and forgiven despite some past actions\" (4.0). The lowest scores were on the following items: \"I wonder if God is angry with me\" (1.1); \"I feel angry with others\" (1.3); \"I feel a need to be forgiven for some of my thoughts and feelings\" (1.7); \"I worry about life after death\" (1.9); \"I feel angry with myself\" (1.9); and \"I feel out of touch with my own feelings and with others\" (1.9). SEIQoL ------ All 16 patients completed the SEIQoL. The average time taken was 13.5 minutes, range 5--30 minutes. Areas identified by patients as the most important in determining their overall quality of life are presented in Table [4](#T4){ref-type="table"} by patient, along with average levels and weights associated with each cue by order of identification. As can bee seen, most patients identified family, children, or spouse as the single most important factor in determining their current quality of life. The average level of each of the five cues ranged from 57--71 on a scale of 0--100, where 100 was the best possible state for that cue. The weights assigned to the cues varied from 16% to 25%, a fairly narrow range, with those cues identified earlier in the process generally being assigned higher importance. The overall index scores, which take into account both the level of the cue and its weight, were an average of 69, SD 20.5 and ranged from 27--100. The frequency of nomination of different cues as any of the five domains is presented in Table [5](#T5){ref-type="table"}. All but one patient mentioned some family relationship as one of the five domains, while some patients nominated several different specific family relationships. This was followed by the general ability to participate in chosen activities (e.g. exercise, recreation, travel, gardening, sewing). Seventy-five percent of the patients nominated some type of activity in their top five. The next most frequent category was friends, endorsed by 44% of the patients. This was followed equally by health (mobility, fitness, energy), faith (religion, belief, hope), and work, with 38% of the patients nominating each category. Finances were nominated by 31% of the patients, followed by several items that were mentioned by only one person each and warranted separate categories. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Frequency of Cue Nomination ::: Cue N (out of 16) \% --------------------------------------------------------------- ----------------------------- ---------------------------------- Family (Children, Spouse, Grandchildren, Parent, Family Tree) 15 93.8 Activities (exercise, gardening, sewing, recreation, travel) 12 75 Friends 7 43.8 Health (mobility, physical fitness, energy) 6 37.5 Faith (religion, belief, hope) 6 37.5 Work 6 37.5 Finances 5 31.3 Pet 1 6.3 Computer 1 6.3 Pain Control 1 6.3 Art 1 6.3 Fun 1 6.3 Positivity 1 6.3 Nature 1 6.3 ::: The internal validity of each of the cues was assessed by performing regressions of the combination of each cue level and its weight onto the total index scores. The resulting R^2^values ranged from .19--.76, median .47, mean .50. The highest R^2^value was for the first cue generated, and the lowest value was associated with the fourth cue. This is much lower than in previous reports \[[@B16],[@B17]\] and may constitute reason to pause before attributing high levels of credence to the validity of all of the cues in influencing overall QL. Two examples of cues, cue levels and cue weights are illustrated in Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}. Figure [1](#F1){ref-type="fig"} illustrates the responses of patient \#1, a 45 year old woman with mucoepidermoid cancer of her head and neck region who died four weeks following the interview. The most important areas to her were health, followed by children, spouse, religion and finally finances. This profile is unusual for this group in that most patients, if they nominated health as a cue at all, did so later in the process. She indicated that the areas that were going the best were finances and religion, followed by children, spouse and health. This combination of things not rated as going very well in some important areas resulted in an index score of 59 on the SEIQoL This is quite a bit higher than her global QL score on the EORTC of just 25. Another example is patient \#2 (figure [2](#F2){ref-type="fig"}), a 50 year-old male with head and neck cancer who, as of this writing, has been alive for 709 days following the interview. For him, things were going well in the areas most important to him; family, work and computers. This resulted in an index score of 81, consistent with his global QL score on the EORTC of 75. These examples illustrate that the SEIQoL scores are related to overall QL scores, and suggest that they may be related to health status as well. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Patient \#1: Cues, Levels and Weights ::: ![](1477-7525-3-7-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Patient \#2: Cues, Levels and Weights ::: ![](1477-7525-3-7-2) ::: Correlations between measures ----------------------------- Correlations between the SEIQoL index and scores on the other measures are presented in Table 6 (see additional file [1](#S1){ref-type="supplementary-material"} -- Carlson Table 6.doc). The index score was significantly positively correlated with the global QL score (r = .53, p \< .05), and negatively associated with the symptoms of nausea (r = -.58, p \< .05), pain (r = -.53, p \< .05) and appetite loss (r = -.59, p \< .05) on the EORTC QLQ C-30. Scores on the BDI were positively associated with appetite loss (r = 0.56, p \< .05) and fatigue (r = 0.56, p \< .05), not surprising since these are both symptoms of depression assessed by the BDI. Depression scores were also negatively related to social functioning (r = -.79, p \< .01) and global quality of life (r = -.69, p \< .01), indicating that people who endorsed more depressive symptoms also tended to report lower social functioning and lower overall QL. Higher scores on the Global Severity Index of the BSI were associated with worse emotional (r = -.55, p \< .05), cognitive (r = -.58, p \< .05) and social (r = -.69, p \< .01) functioning, as well as worse global QL (r = -.70, p \< .01) and more sleep disturbance on the EORTC. Higher scores on the spiritual health inventory were associated with lower scores on the global severity index of the GSI (r = -.54, p \< .05) and less nausea (r = -.63, p \< .05). Significant correlations between days to death and psychological scores were found on two instruments in the nine patients who had passed away at the time of writing. The BDI total score was negatively correlated (r = -.75, p \< .05), and the EORTC global QL score was positively correlated (r = .77, p \< .05) with time to death. This indicates an association between higher levels of depression and lower global QL at the time of the interview, and fewer days to death. Discussion ========== This study is the first to use the SEIQoL-DW instrument to assess individualized QL in a population of patients with advanced cancer participating in a Phase I clinical trial. Of note is that the instrument was easy to use in this population and acceptable even to those patients who were quite ill. Replication of the wide range of individual differences in the cues chosen and the weights associated with each cue was seen in this group. The areas of QL that were nominated by patients as the most important factors in the determination of their overall QL were primarily family relationships, the ability to participate in pleasurable activities, and friendships. Only 38% of this sample mentioned health or health-related domains such as mobility, fitness and energy as one of the five cues. This is in contrast to other samples of cancer patients where over 70% of patients nominated health as an important domain. For example, health was nominated by 73% of cancer patients in phase I trials (not necessarily advanced cancer) \[[@B18]\], 87% of men with early stage prostate cancer \[[@B16]\], and 70% of patients with advanced incurable cancer (these patients were not on trials) \[[@B17]\]. The commonality between these studies is that family was consistently nominated as the most frequent domain. In terms of the index scores, our average of 69 was higher than that of the group of advance incurable cancer patients (mean = 58) and the patients participating in Phase I trials (mean = 61). It was comparable to the men with early stage prostate cancer (mean = 71), but the relatively small range of mean scores among these studies is notable. Construct validity is supported in that those patients whom one might expect to have higher QL (i.e. less ill patients), indeed reported higher QL. That the scores in the current sample were more comparable to the early stage prostate cancer patients than the incurable cancer patients may speak to the hope patients were feeling regarding the potential of the reovirus treatment. In terms of the index scores for other illness populations, patients prior to hip arthroplasty scored 59, after the surgery scores increased to 69 \[[@B27]\], on par with the cancer patients in this study (mean 69). Another study of hip replacement patients found scores of 62 prior to surgery, with improvements to 71 following surgery \[[@B28]\], a similar improvement pre- to post-surgery. Severely disabled multiple sclerosis patients scored 61 \[[@B29]\], patients with ALS had a higher mean index of 76 \[[@B30]\], and cardiac patients scored a high index of 82 after myocardial infarction or coronary artery bypass graft surgery and prior to beginning cardiac rehab \[[@B31]\];. Interestingly, only 24% of the ALS patients nominated health (disease progression) as a cue. Thus, although the areas of importance varied by individual in all these studies, the resultant index scores seem to demonstrate some consistency across similar populations. Another indication of the construct validity of the SEIQoL-DW is its high correlation to global QL scores on the EORTC QLQ C-30. This speaks to the validity of the self-generated items, as the sum of the products of their importance and current status was associated with the overall global assessment of QL on standardized domains. Predictably, our patients had a global QL on the EORTC of 57, much lower than the 77 reported in a large normative community sample \[[@B32]\]. Scores on all five functional scales and symptom scores were all also worse in this population, not surprising considering the extent of their disease status. However, they did have the same global QL scores compared to a large sample of patients with advanced malignancy from 12 institutions in 10 countries (both 57), but scored higher on most of the other functional scales than this group (Physical function: 64 vs. 60; Role function: 63 vs. 50; Emotional function: 76 vs. 50; Cognitive function: 76 vs. 63; Social Function: 55 vs. 50) \[[@B33]\]. The greatest differences in favor of the patients in this trial were seen on emotional, cognitive and role function. Interestingly, lower global QL scores on the EORTC and higher depression scores on the BDI were associated with a shorter time to death in those patients who had already passed away, an association that has been reported in other studies \[[@B33]-[@B35]\]. In fact, in an international sample of 411 patients with advanced malignancy, similar to the current group, the single-item global QL scale remained independently prognostic of death in a proportional hazards model stratified on diagnostic category, after allowing for performance status and age, and, among solid tumor patients, metastatic site \[[@B33]\]. Patients who were in terminal care from a large sample from 12 oncology outpatients departments around the United States scored an average BSI Global severity index score of .93. Those who were under symptom control scored .80, whereas lower scores were seen for patients in active therapy (.59), adjuvant therapy (.60) or no current therapy (.65)\[[@B36]\]. Our sample mean of .55 is quite low in comparison and most comparable to patients in active therapy. An even larger sample of over 4000 patients of all disease sites and stages of illness from Johns Hopkins Oncology Centre reported an average global severity index very similar to our patients, at .54 \[37\]. This seems to indicate that our sample is reporting less psychopathology than would be expected of patients in similar disease states, but comparable levels to cancer patients in general. They also scored an average of 11 on the BDI, which indicates mild depressive symptomatology. The spirituality scores of this group average 118. This is very similar to a group of lung cancer patients who had a mean of 120 \[[@B20]\]. Analysis of the individual items showed that these patients endorsed feeling well supported by the medical team and quite peaceful and accepting of themselves, and well accepted by others. They reported not feeling angry at themselves or others, or worried about life after death. The spirituality scores were correlated with the global severity index of the BSI, indicating that those who felt more spiritually at ease also endorsed fewer symptoms of psychopathology. In summary, these patients in a Phase I trial of a promising novel therapeutic were easily able to complete the SEIQoL-DW interview as well as a battery of other psychological questionnaires. They reported feeling excited and hopeful about the trial, with about two-thirds hoping for disease regression, and another third optimistically hoping for a cure. However, most acknowledged that although they hoped for the best they were realistic in their expectations. The individuality of QL as defined by each person was reinforced in this group, as many different cues were nominated as important and variable weights were assigned to the same cues. Consistent with reports from other seriously ill groups, health status received less focus than other aspects of life, primarily family relationships and activities. Overall QL on standardized measures and psychological status was generally better than other seriously ill patient groups, but comparable to cancer patients in general. QL and depression scores were related to time until death in those patients who had passed away. Authors\' contributions ======================= LC wrote the research proposal and ethics applications, conducted the interviews with the patients, designed the database, conducted the statistical analysis, and wrote the first draft of the paper. BB is the department head for Psychosocial Resources, and in conjunction with LC conceived of and designed the study. He also made the linkages with medical oncology to facilitate the study. DM is the oncologist and principal investigator of the Phase I Reovirus trial, and recruited all the patients. All authors reviewed and edited the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Table 6: Correlations between scores on the SEIQoL, BDI, BSI, SHI and EORTC QLQ C-30 ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ Dr. Linda Carlson was a Terry Fox Postdoctoral Research Fellow of the National Cancer Institute of Canada during the time the study was run. She is currently a Canadian Institutes of Health Research New Investigator. Special thanks to our research nurse Ms. Sally Lim for her tireless efforts in assuring the smooth running of the study, and to Ms. Jodi Cullum for data management.

PubMed Central

2024-06-05T03:55:52.622805

2005-1-27

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548292/", "journal": "Health Qual Life Outcomes. 2005 Jan 27; 3:7", "authors": [ { "first": "Linda E", "last": "Carlson" }, { "first": "Barry D", "last": "Bultz" }, { "first": "Donald G", "last": "Morris" } ] }

PMC548293

Background ========== In a recent study, University of Toronto researchers identified the \"Top 10 Biotechnologies to Improve Health in Developing Countries\" \[[@B1]\]. The study underscores the importance of harnessing new technologies to improve global health and development, a belief that is gaining widespread acceptance. For instance, the overall goal of the United Nations Millennium Project\'s Science and Technology Task Force is to address how science and technology can be leveraged to help countries achieve the Millennium Development Goals (MDGs) \[[@B2]\]. Its mission is guided by the understanding that most of the MDGs cannot be reached without a strong contribution from science and technology. The potential contribution of genomics and biotechnology to these goals has also been demonstrated \[[@B3]\]. However, these technologies are most beneficial to countries that have the scientific capacity to absorb and use them. The aim of the Inter-Academy Council on Science and Technology Capacity (IAC) was to develop a global strategy for promoting capacities in science and technology and the first report of the Council was recently presented to UN Secretary General Kofi Annan \[[@B4]\]. There is a wide range of scientific capacity and health system development across the Eastern Mediterranean Region (EMR) \[[@B5]\], which consists of 21 countries. Some countries in the region have taken the initiative in biotechnology by establishing regulations and encouraging private sector involvement. In other countries of the region, there is a serious lack of scientific capacity not only to conduct research and development in biotechnology but even to absorb the benefits of biotechnology and apply them to help meet the health and socio-economic needs of the population \[[@B6]\]. According to UNDP\'s Arab Human Development Report 2003, research and development in the Arab world represents less than 0.2% of Gross National Product (GNP). Fewer than one in 20 Arab university students were pursuing scientific disciplines, compared, for instance, to one in five in South Korea \[[@B7]\]. There is a risk that, as the genomics revolution gathers momentum, the imminent genomics divide between developed and developing countries will increase unless urgent action is taken to reverse the trend \[[@B8]\]. In order to assess the potential of genomics to address health needs in the Region, the World Health Organization-Eastern Mediterranean Regional Office (WHO-EMRO) and the University of Toronto Joint Centre for Bioethics jointly organized a Genomics and Public Health Policy Executive Course, held September 20^th^--23^rd^, 2003, in Muscat, Oman. This 4-day course/workshop was sponsored by WHO-EMRO with additional support from the Canadian Program in Genomics and Global Health. The overall objective of the Genomics Policy Executive Course was to familiarize participants with the potential of genomics and related biotechnologies to address health needs and to collectively address the question of how best to harness genomics to improve health in the region (table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Objectives of the Genomics Policy Executive Course ::: -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- To familiarize participants with the current status and implications of health genomics/biotechnology, and to provide information relevant to public policy on health genomics/biotechnology To provide frameworks for analyzing and debating the policy issues and related ethical questions in health genomics/biotechnology, and to help understand, anticipate and possibly influence the legal and regulatory frameworks under which health biotechnology industries will operate, both nationally and internationally To begin developing an opinion-leaders network across different sectors (industry, academic, government, NGOs) by sharing perspectives and building relationship To formulate recommendations for future policy and strategic directions at the regional, national and individual levels. -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: Methods ======= We based the program and designed the sessions and lecture topics of this course on prior courses held in Nairobi, Kenya in March 2002; in Toronto, Canada in May 2002; and in Kumarakom, India in January 2003. The sessions and presenters of the course are shown in table [2](#T2){ref-type="table"}. The course participants and facilitators for each session were identified through a combination of recommendations from field experts in the region and from WHO\'s network of experts. People identified to participate in the course included scientists from academic institutions and industry, industry executives, regulatory officials, representatives from the legal sector, and media. The participants were carefully chosen in an attempt to represent a wide range of interests relevant to the emerging area of genomics and to have geographical, discipline and gender balance. They included scientists from academic institutions and industry, industry executives, legal and regulatory officials, WHO representatives and the media. In total there were 51 participants, from 13 countries of EMR. We drew as many of the faculty as possible from the region. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Program ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- **[Saturday, 20 September 2003]{.underline}** ----------------------------------------------- ------------------------------------------------------------------------------------------------------------------------- **08:00 -- 08:30** Registration **08:30 -- 10:30** Session I:\ Opening Address\ *H.E. Dr Ali Bin Moosa, Minister of Health, Oman*\ *Dr H. A. Gezairy, Regional Director, EMRO*\ Introduction and Course Overview\ *Team from University of Toronto* Session Chair: *Dr Ali Jaffer Suleiman, Ministry of Health, Oman* **10:30 -- 11:00** Coffee break **11:00 -- 12:30** Genomics: Scientific Developments\ Professor Riad Bayoumi, Sultan Qaboos University **12:30 -- 14:00** Lunch Break **14:00 -- 15:30** Session III:\ WHO Report on Genomics and World Health\ *Professor Alexander Capron, Director, Department of Ethics, Trade and Human Rights, WHO/HQ* **15:30 -- 16:00** Coffee break **15:45 -- 16:30** Session IV:\ Top 10 Biotechnologies for Improving Health in Developing Countries *Professor Abdallah S. Daar, University of Toronto* **[Sunday, 21 September 2003]{.underline}** **08:30 -- 09:00** Golden Nuggets (previous day\'s summary)\ *Dr Peter A. Singer, University of Toronto* **09:00 -- 10:30** Islamic Perspective on Stem Cells, Cloning, Genetic Engineering, ..etc\ *Dr Mohammed Al Bar, King Fahd Medical Research Centre, Saudi Arabia* **10:30 -- 11:00** Coffee break **01:00 -- 12:30** Intellectual Property Rights\ Professor Richard Gold, McGill University, Centre for Intellectual Property Policy **12:30: -- 14:00** Lunch Break **14:00 -- 15:30** Business Models\ *Mr Khalil Ahmed, Managing Director, Shantha Biotech, India*\ *Dr Peter A. Singer, University of Toronto* **15:30 -- 16:00** Coffee break **16:00 -- 17:30** Group Work **[Monday, 22 September 2003]{.underline}** **08:30 -- 09:00** Golden Nuggets (previous day\'s summary)\ *Dr Peter A. Singer, University of Toronto* **09:00 -- 10:30** Innovation Systems\ *Dr Peter A. Singer, University of Toronto* **10:30 -- 11:00** Coffee break **11:00 -- 12:30** Regulatory Systems and Related Issues\ *Dr D.C. Jayasuriya, Director, UNAIDS, Pakistan*\ *Dr Anwar Nasim, Chairman, National Council of Biotechnology, Pakistan* **12:30 -- 13:00** TRIPS and Pharmaceutical Issues in Public Health.\ *Dr. Abdel Aziz Saleh Special Advisor to the Regional Director for Medicine, WHO/EMRO* **13:00 -- 14:00** Lunch Break **14:30 -- 15:30** Public Engagement\ *Mr Ehsan Masood, Scidev.net, United Kingdom* **15:30 -- 16:00** Coffee Break **16:00 -- 17:30** Group Work **[Tuesday, 23 September 2003]{.underline}** **08:30 -- 09:00** Golden Nuggets (previous day\'s summary)\ *Dr Peter A. Singer, University of Toronto* **09:00 -- 10:00** Opinion Leaders Network\ *Dr Peter A. Singer, University of Toronto, Dr Tara Acharya, University of Toronto* **10:00 -- 10:30** Break **10:30 -- 12:30** Group Work **12:30 -- 14:00** Lunch break **14:00 -- 15:30** Group Presentations and Discussion **15:30 -- 16:00** Coffee Break **16:00 -- 17:30** Recommendations, Concluding Remarks and Closure of the Workshop\ *Dr Peter Singer, University of Toronto* ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: The sessions dealt with a wide range of relevant topics, starting with recent scientific advances in genomics and stem cell research, followed by discussions on business models in genomics and biotechnology, intellectual property rights and regulatory frameworks, public engagement and an internet-based opinion leaders\' network. The presentations were designed to be interactive and foster active discussion from, and among, the participants, and each presentation was followed by a moderated discussion period. Early in the course, the attendees were placed into one of five study groups -- these groups were carefully designed to capitalize on the diverse backgrounds of the participants. Each group was assigned the task to on the deliberate the key question **\"How best to harness genomics and biotechnology to improve the health of the people in the Eastern Mediterranean Region?\"**The groups met frequently to discuss the presentations, and each participant was also provided a course reader with additional literature on the lecture topics (table 3). The overall task of these study groups was to draw upon the course material and their own experiences and propose a set of recommendations for genomics and biotechnology policy in the region. On the last day of the meeting each group presented these recommendations. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Course Readings ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Bhutta ZA (2002) Ethics in international health research: a perspective from the developing world.\ *Bull World Health Organ*.;80(2):114--20. Review. Bloom BR & Trach D (2001) Genetics and Developing Countries. *BMJ*;322:1006--7. Capron AM (2001) Stem Cells: Ethics, Law and Politics. Biotech Law Report;5:678--699. Collins FS, Green ED, Guttmacher AE, Guyer MS; US National Human Genome Research Institute. (2003) A vision for the future of genomics research.\ Nature.24;422(6934):835--47. Daar AS, Thorsteinsdóttir H, Martin DK, Smith AC, Nast S, Singer PA. (2002) Top ten biotechnologies for improving health in developing countries. *Nat Genet*.;32(2):229--32. Gold ER (2003) SARS genome patent: symptom or disease? *Lancet*.;361(9374):2002--3. Juma C & Konde V (2002). The New Bioeconomy: Industrial and Environmental Biotechnology in Developing Countries. Geneva, Switzerland: United Nations, July 2002. Lundvall B, Johnson B, Andersen EA, Dalum B (2002) National systems of production, innovation and competence building. *Research Policy*;31:213--231 Nasim A (2000) Ethical Issues of the Human Genome Project: An Islamic Perspective *in*Bioethics in Asia. Eubios Ethics Institute Eds: N Fujiki and DRJ Macer p. 209--214 Pang T & Weatherall D (2002) Genomics and Global Health. *BMJ*;324:p.1051--52 Singer PA, Daar AS (2001) Harnessing genomics and biotechnology to improve global health equity. *Science*;294(5540):87--9. Sulston J (2003) Beyond release: the equitable use of genomic information.\ *Lancet*.;362(9381):400--2. Thorsteinsdóttir H, Daar AS, Smith RD, Singer PA (2003) Genomics -- a global public good?\ *Lancet*.;361(9361):891--2. Time to Unite Islam and Science. *Nature*. 2003 Mar 13;422(6928):99. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: In order to assess the level of interest in the formation of an email-based opinion leaders\' network to continue discussion among the participants following the course, a brief survey was conducted on the participants\' internet access and their expectations of the network. Results ======= Dr Peter A. Singer, Director of the University of Toronto Joint Centre for Bioethics, described the overall aim of the course \"How to best harness genomics to improve health in the region\", as well as the 4-day program. Dr Ali Jaffer Suleiman of the Ministry of Health in Oman acted as chairperson of the meeting. Professor Riad Bayoumi of Sultan Qaboos University highlighted new scientific developments that have resulted from the genomics revolution, such as proteomics; mapping of single nucleotide polymorphisms (SNPs) to understand human genetic variation and its relationship with disease; gene expression chips to monitor differential gene expression and identify drug targets; and bioinformatics as a new field that combines biology, mathematics, statistics and computer programming to mine large-scale biological data. Professor Alexander Capron, Director, Department of Ethics, Trade and Human Rights at WHO, described the 2002 report of the World Health Organization \"Genomics and World Health\" \[[@B9]\]. He called attention to the recommendations from the report -- these include improving technical cooperation between WHO and its member states (e.g. assessing the health impacts of genomics research to support informed priority setting; capacity building for genomics research and biotechnology in developing countries; development of ethical review structures and bioethics capacity). Professor Abdallah Daar described a recent study, conducted by the University of Toronto Joint Centre for Bioethics\' Canadian Program on Genomics and Global Health, to identify the ten most promising biotechnologies for improving health in developing countries in the next five to ten years. These technologies offer guidance to those who can influence the direction of R&D in developing countries and challenge common assumptions about the relevance of biotechnology for these countries, as shown by the mapping of the Top 10 Biotechnologies onto the UN Millennium Development Goals \[[@B10]\]. However, to foster biotechnology in developing countries it is essential to build capacity (among researchers, politicians, legislators, entrepreneurs, etc). Professor Al Bar of King Fahad University, Saudi Arabia, illustrated Islamic perspectives on genetic testing, cloning, recombinant DNA technology and other genomics-related technologies. He showed that while Islam and science have always been aligned through history, today\'s religious leaders in the region must take the initiative to develop and formulate recommendations for genomics. Dr. Peter Singer described the concept of innovation systems and presented some observations of other countries\' innovation systems and their successes. One definition of a national system of innovation (NSI) is the \"network of institutions in the public and private sector whose activities initiate, import, modify and diffuse new technologies\" \[[@B11]-[@B13]\]. The application of NSI to developing countries is a fairly recent concept. The Canadian Program on Genomics and Global Health is currently conducting studies of the NSI of 7 developing countries: Brazil, China, Cuba, Egypt, India, South Africa and South Korea \[[@B14]\]. Despite the identification of factors that foster innovation systems, there is no one model that guarantees success -- each country follows its own unique path. Mr. Khalil Ahmed, Managing Director of Shantha Biotechnics, described the successes of this biotechnology company based in Hyderabad, India. Shantha was established in 1993, in part with seed money from the Government of Oman. Today, it is the first Indian company to receive WHO certification for a recombinant hepatitis-B vaccine Shanvac-B™, paving the way for UNICEF to buy 8.5 million doses for distribution globally. Hepatitis B vaccine is currently priced internationally as high as \$8--10 per dose, while Shantha is selling it at \$2 per dose \[[@B15],[@B16]\]. Professor Richard Gold of McGill University\'s Centre for Intellectual Property Policy described the basics of intellectual property rights, patents and copyright issues, as well as policy options for developing countries in the international context. He outlined the characteristics of international agreements, which offer considerable flexibility to countries on how to apply patent laws to genomics and biotechnology. Developing countries have a number of options, such as compulsory licensing and research exemptions, to deal with property rights. Dr Jayasuriya, Director of UNAIDS in Pakistan described ways in which legal systems can facilitate best use of biotechnology. It is essential that legal reform keep up with the rapidly evolving science of genomics. Health law can facilitate best use of biotechnology -- by providing for fast-track approval of biotechnology products; reducing import duties on health interventions; and allowing multiple channels of procurement and distribution to improve access and optimize prices. Dr Anwar Nasim, Chairman of Pakistan\'s National Council of Biotechnology, described the role of regulations both to promote useful biotechnologies and limit their risks for human health and the environment. A national-level regulatory body could provide guidelines for the use and release of biotechnology products, conduct biosafety reviews and risk assessments and formulate feedback mechanisms to improve the system through experience. The National Commission of Biotechnology of Pakistan, established in November 2001, focuses on biotechnology regulatory issues in health, agriculture, environment and industry. Similar commissions could be set up in other countries of the region and their interaction could further regional cooperation in biotechnology. Mr. Ehsan Masood of Scidev.net pointed out that active public engagement based upon knowledge can stimulate action to improve public health. Public engagement is far greater in today\'s world than it has ever been in the past. This is because of several factors including; a) the growing implications of research on public health, b) the increased awareness in civil societies to invest in health care and research, and to influence policy change and action, c) development and access of information technologies. On the last day of the course the five participant groups, who had been assigned the task to on the deliberate the key question **\"How best to harness genomics and biotechnology to improve the health of the people in the Eastern Mediterranean Region?\"**, were invited to present their findings. The main points of these group presentations are summarized in the recommendations that emerged from the course. Discussion ========== The session discussions and group presentations underscored the urgent need for action to create the enabling environments (at regional, national and individual levels) for research and development in genomics and biotechnology. The issue of awareness of biotechnology among political leaders to garner support at the highest level was raised early on in the course, and was reinforced throughout the course discussions. The participants felt strongly that political commitment is crucial to the advancement of biotechnology in the region. Political leadership is a critical factor in raising the profile of science in developing countries. A prominent example in the Eastern-Mediterranean Region is that of the Sultan Bin Mohammed Al-Qassimi, Shaikh of the Arab emirate of Sharjah. He has made a dedicated effort to change the face of science in the Gulf \[[@B17]\]. For example, in an attempt to attract Arab scientists working abroad to return to work in the Gulf, and more specifically to Sharjah, he has built two universities, six museums and established a science foundation in just a decade. He is also actively engaged in creating an environment of regional cooperation. The course attendees recommended that WHO-EMRO should take the responsibility to engage political leaders in the region\'s national governments. The participants shared experiences of the importance of government support for biotechnology -- in Egypt there appears to be relatively strong support for biotechnology by the government, with biotechnology activity in academic and other research institutions as well as in the private sector. In Iran, considerable advances have been made in private and academic research centres, leading to a number of biotechnology products that are soon to reach the market, as well as well-respected scientific journals such as the Iranian Journal of Biotechnology \[[@B18]\]. Other countries such as Pakistan, Tunisia, Lebanon, and Morocco are making steady gains in biotechnology, some in conjunction with their powerful public health sectors and others as a result of their strong scientific bases. It will also be crucial to involve, inform and engage religious leaders in the region in order to promote genomics and biotechnology for improving public health. In doing so, it is worthwhile to note that Islam and science have always been aligned through history and it is well-established that Islam has historically contributed tremendous achievements to the advancement of science. \[[@B19]\] A recent article argues that the Muslim world has neglected to pay attention to contemporary ethical issues in science and technology \[[@B20]\]. The author calls for the establishment of an independent Islamic bioethics panel to advise Islamic governments and communities. Other measures, such as training Muslim bioethicists, incorporating biomedical issues into school curricula and educating the community about such issues are also recommended. The active participation and involvement of organizations like COMSTECH was recommended to give support and guidance to WHO-EMRO\'s efforts. COMSTECH was established by the Islamic Summit in 1981 and includes among its objectives the building of indigenous capabilities in the fields of science and technology, promotion and continuing cooperation and coordination in scientific and technological areas of is member states and creation of effective institutional structure for planning research, development and monitoring of scientific and technological activities. COMSTECH has already launched networks across the region for exchange of information, and has valuable experience in the promotion of cooperation and coordination amongst the member states in science and technology activities in high technology areas \[[@B21]\]. A major discussion point at the workshop was the effective assessment of existing capacity in the region. Several participants shared disappointment over the relative lack of participation of the region in global science and technology advances, citing, for instance, poor representation of the region at international scientific conferences. Many felt the need to evaluate and assess the level of scientific activity and capacity in individual countries in the region, in order to identify, among other things, strengths and weaknesses, entry points for countries, opportunities for regional collaboration, and areas for improvement. The workshop attendees felt it important to carry out this type of survey of each country\'s innovation system as a prerequisite to revising and revamping national and regional genomics policy. The factors identified by the participants as important for assessment in this survey -- scientific capacity within public and private sector, private enterprise, religious and political leadership and intellectual property rights -- can be considered to be part of the National System of Innovation (NSI). This proposed survey of NSI in the region can help to identify factors for successful growth of biotechnology sectors. Similarly, the recently completed study by the Canadian Program on Genomics and Global Health of the health biotechnology innovation systems of Brazil, China, Cuba, Egypt, India, South Africa and South Korea may also help to identify some of these factors \[[@B22]\]. Workshop participants from Egypt, Iran, Lebanon, Pakistan, and Tunisia contributed to the discussion of NSI with descriptions of components of their countries\' biotechnology innovation systems. In Egypt, there is increased recognition of the importance of intellectual property rights in fostering innovation. The Minister of Health has taken a keen interest in improving linkages between sectors to enhance inter-sectoral communication. The president himself was instrumental in the establishment of the Mubarak City for Scientific Research and Technology (MCSRT), which is currently engaging in collaborative projects with the United States and with China. Private sector development is of high priority, as indicated by government support of companies such as Vacsera, which is now making recombinant insulin in Egypt \[[@B23]\]. One successful story from Egypt is the development by scientists at the Agricultural Genetic Engineering Research Institute in Giza of a powerful bio-pesticide based on a new strain of *Bacillus thuringiensis*\[[@B24]\]. In Iran, the trade embargo has led to a shortage of funds, but has in some ways helped to strengthen the innovation system through the need for self-reliance. Iran and Cuba have reached an agreement for cooperation and transfer of technology to produce hepatitis-B vaccine, interferon-α, streptokinase, and erythropoietin. Iran has developed several products based on recombinant DNA technology, and the country is also actively building research collaboration networks. Pakistan has had some successes in agricultural biotechnology and made some advances in bioethics. Although there are powerful institutes in place, the country needs to continue to strengthen facilities, resources and human capital. There is a need to bring products to the public. Lebanon has a well-developed healthcare industry, and has developed expertise in genetic testing, bioethics as well as intellectual property rights. Tunisia has strong research infrastructure and has made advances in genetic counseling, cytogenetics, and diagnosis of genetic diseases, along with regulation and legislation. However the pharmaceutical industry is not able to optimize the potential of public sector research, for which regional cooperation would be valuable. The participants identified strengthening the linkages between academia and the private sector as key to strengthening capacity in genomics. One example of a concerted effort to improve academic-private sector links is Jeddah BioCity, a research facility with close ties to the King Faisal Specialist Hospital and Research Center. Founded by Sultan Bahabri, head of King Faisal Specialist Hospital and Research Center in Jeddah and other Saudi scientists, this private venture plans the construction of state of the art biotechnology laboratories and companies and is intended to make Saudi Arabia a world leader in biotechnology. As highlighted by the 2004 report of the UN Commission on Private Sector and Development report \[[@B25]\], the process of commercialization for development involves the dissemination and facilitation of knowledge flows between public and private sectors of both developed and developing markets. The report recommends action in both the public and private spheres, and emphasizes the linkages between these spheres, recognizing the importance of cooperation and partnerships to achieve goals. With stronger public-private linkages, the private sector in developing countries will be able to help provide new genomics-based technologies at affordable prices. Given that over the last few decades, market forces have driven the R&D agenda of pharmaceutical companies based in the North to de-emphasize the health concerns of developing countries, it is becoming increasingly important to develop indigenous capacity in private enterprise. The successes of biotechnology firms in developing countries such as India and Egypt were seen as a source of inspiration by the course attendees. The attendees observed that the key to effective participation in the genomics revolution is building scientific capacity, and expressed serious concern over the region\'s limited capacity to absorb and utilize genomics and biotechnology. Critical to capacity-building is access not just to technology but, more importantly, to scientific knowledge. One point-of-entry that was greeted with enthusiasm by the participants was bioinformatics, which is typically less-resource intensive compared with other genomics-related sciences, such as sequencing and proteomics. According to the report on the \"Top 10 technologies to improve health in developing countries\" countries of EMR can take advantage of genomic data and apply the power of bioinformatics to local health problems without having to invest heavily in the technologies used to produce them, A dedicated Genomics and Health Research Fund for the region may help to break down financial barriers to encourage scientific research and development in the region. The participants expressed enthusiasm for setting up National Biotechnology Commissions to address genomics policies at the national level and to contribute to the development of the above-mentioned national biotechnology strategy. A National Commission on Biotechnology (NCB) has been set up in Pakistan. The overall goal would be to help devise regulations to facilitate innovation in biotechnology. The NCB could help coordinate national biotechnology activities and policy, with activities including evaluation of biotechnology capacity (see above) to priority setting and public engagement. Accordingly, the NCB should have broad cross-sectoral representation in order to minimize the negative effects of inter-institutional rivalry. One of the main goals of the NCB (or equivalent national agency), following the above-mentioned national biotechnology survey, will be to help develop and adopt a national biotechnology strategy, perhaps as part of a long-term science and technology policy. Other important objectives are discussed below. At the outset of the conference, participants voiced their concern about the low level of awareness (which goes beyond just *public*awareness) of biotechnology and genomics in the region, and that this lack of awareness may be the biggest barrier to advances in genomics. Public awareness and engagement can help change the pace of research, leading to increased opportunities for greater societal involvement for improved health care. Media can be used as a catalyst to raise community awareness for social beneficence, equity and justice in health care. Recently, in May 2004, science journalists from across the region met in Cairo to discuss the hurdles they face in science reporting. The hurdles identified at this meeting included bureaucracy and poor access to scientific research taking place in the region \[[@B26]\]. The meeting concluded with the creation of a provisional network of Arab science journalists that will aim to provide its members with training, skills and contacts, as well as promote the coverage of scientific issues from a development perspective. Similarly, public health specialists and scientists should also be encouraged to engage actively disseminate evidence based and correct information in disease prevention and control through media. While it was agreed that science should be permitted to march forward, the participants emphasized that ethics, regulations and laws must keep up with the science \[[@B27]\]. For example, an important focus area of the NCB is that of intellectual property rights. The participants agreed that developing countries must build capacity and knowledge to choose the best policy options to benefit from the international patent regime. Compulsory licensing under specific circumstances may be a good option for developing countries and national laws should permit compulsory licensing \[[@B28]\]. The forces of globalization must balance forces of protectionism, especially by the developed world, and both national and regional regimes should respect international patent law to take advantage of globalization. If developing countries are involved in international research collaborations, they should ensure that they obtain patents. In view of this, it is essential for developing countries to build capacity and training in patent law and learn how to formulate effective patents, and the NCB could play a key role in this effort. Regional cooperation in science was given a boost this year, with the establishment of a network of science academies of the Organisation of Islamic Conference \[[@B29]\] at a meeting organized by the Third World Academy of Sciences. This formal network aims to provide the partner states with mutual support and supports discussion of the scientific aspects of common problems. It could help to build a unified approach to capacity building in science and technology within member states. Health advances in developing countries have lagged behind those in the developed world. The rapid advance in genomics research in developed countries compared with the relatively slow progress of genomics R&D in developing countries threatens to create a North-South genomics divide in the coming years, which may enhance existing health inequities. With the appropriate emphasis on its health needs, incentives for public-private R&D partnerships, and a sound set of regulatory policies, the Eastern Mediterranean Region may well reap the benefits of genomics and biotechnology. The overall goal of the Genome Policy Executive Course, a WHO-University of Toronto initiative, was to help provide the impetus for cross-sectoral dialogue on genomics and health policy in the region. The internet-based opinion leaders\' network is expected to foster dialogue to help achieve the objectives outlined by the participants of the course. The participants and the organizers of the course felt strongly that the recommendations formulated at the course must be shepherded by individuals. People felt that progress could be achieved if individuals make a significant and concerted effort to ensure that these recommendations are fulfilled. One way to spur action and maintain the momentum generated by this course is by coordinating the participants into a network within which they can continue to interact and share information and experiences. This internet-based network will be moderated in order to streamline discussions. A number of short-term projects are envisioned that could be coordinated by various expert members of the network. The results of the survey (table [4](#T4){ref-type="table"}) administered to the participants during the course suggested that 80% of them had reliable access to internet and would be willing to spend 1--2 hours a week taking part in the discussion. The main objectives of the network, as identified by the participants in the survey, would be dissemination of information, exchange of ideas, maintaining inter-connectivity, consensus building through wide participation, and influencing policy and media. The network is now established and is being used by the participants to exchange information. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Opinion leaders\' network survey results in brief ::: Goals of network • Dissemination of information ---------------------------- ----------------------------------------------------------------------------------------------------------------------------- Access • 41 (79%) no access issues -- reliable connectivity from work • 3 needed some assistance with email access (internet connection at work; compensation for access; help to post responses) Obstacles to participation • 47 (92%) identified lack of time due to professional responsibilities • 98% of those with connectivity willing to dedicate 1 hour a week to the network • 3 people identified lack of connectivity as a barrier ::: Conclusions =========== The meeting concluded with a set of recommendations for the EMRO and Member States (table [5](#T5){ref-type="table"}) \[[@B30]\]. The recommendations were developed through consensus among the participants. The process of consensus development involved the following steps: (i) the recommendations were drafted based on the group presentations, (ii) any recommendations which the participants did not support were deleted (iii) recommendations that were missing but deemed to be important were added (iv) the final list was scrutinized to sharpen language and consolidate points where possible. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Recommendations ::: --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- [Recommendations for the Eastern Mediterranean Regional Office]{.underline} The workshop recommends that the Regional Director EMRO may be requested to address the governments at the highest level for actively considering the proposals of this workshop and for giving priority attention to genomics for health and health biotechnology. The political leadership may be provided effective advocacy material, with special reference to its link with poverty alleviation, public health objectives, and need for transfer (and internalization) of technology. EMRO and Organization of the Islamic Conference Standing Committee for Science and Technology (COMSTECH), and possibly other groups should provide coordination and networking among national biotechnology bodies (see below) and coordinators to exchange information, expertise, training, and Regional cooperation in production and utilization of health biotechnology. EMRO, in collaboration with member states and their national biotechnology bodies, should coordinate a national survey/inventory/situation analysis/needs assessment of health biotechnology innovation systems, including scientific and management capacity, government policies, legislation and regulations, intellectual property policies, private sector activity, and strengths/weaknesses, opportunities and threats. EMRO, in collaboration with COMSTECH and member states, should develop a proposal/feasibility study for a Regional Genomics and Health Research Fund emphasizing both peer-reviewed research and capacity strengthening. [Recommendations for Member States]{.underline} Each member state should create an effective National Commission on Genomics, Biotechnology and Health, if this function has not otherwise been established, including a coordinator who will serve as the focal point for this activity. The membership should be multisectoral and include youth, women, and civil society. The focus should include ethical issues. Based on evidence from the national survey described above, governments of member states should develop and adopt, at the highest level, a national biotechnology strategy. The National Commission on Biotechnology should develop programs of public awareness and engagement. Important \"publics\" here include media and religious leaders as well as the public at large. The discussion should include ethical issues. The National Commission on Biotechnology should encourage academic institutions including schools and universities, to include health biotechnology topics within their curricula and create specialized programs and degrees where appropriate. There should be particular emphasis on ICT and bioinformatics. The National Commission on Biotechnology, in collaboration with the relevant ministries, should develop a plan to integrate genetic and genomics products (including diagnostics, vaccines, therapies, and other genomic priorities), within the health system and public health programs. The emphasis should be on accessibility and equity to improve the health of the poor. [Recommendations for Individuals]{.underline} There is a need for strong personal commitment to strengthen the initiative on genomics and biotechnology to improve health and well-being of people in the EMRO Region. Workshop participants, as well as other concerned individuals, should are therefore encouraged to actively engage in the implementation of these recommendations. --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: The participants felt the need for EMRO to: a\) Request regional governments and policy makers at highest level to give priority to genomics for health and health biotechnology b\) Develop linkages with Organization of Islamic Conference Standing Committee for Science and Technology (COMSTECH) and other international partners to build Regional networking and cooperation for developing and utilizing health biotechnology. c\) In collaboration with Member States undertake a national survey/situation analysis/needs assessment of health biotechnology innovation systems including resource capacities, government policies (legislation, regulations intellectual property policies and private sector activity. d\) In collaboration with COMSTECH initiate a research grant for applied (health) genomics and biotechnology The participants agreed that each member state would benefit from creating effective National Commissions on Biotechnology (NCB) to develop national biotechnology strategies. NCB should; a) develop national priorities, programmes and guidelines aimed at raising public education and awareness and b) collaborate with civil sectors to develop plans to integrate genetic and genomic products (including diagnostics, vaccines, therapies and other genomic priorities within the health systems and public health programmes and c) build capacities for utilization and access of health biotechnology to the needy and e) ensure ethical safeguards against unwanted harm and exploitation and improve equity to improve health of the poor. The participants also felt that there was a need for strong personal commitment at individual level by experts and key actors to engage actively in the implementation of the recommendations to strengthen the initiative on genomics and biotechnology to improve the health and well-being of people in EMR. One concrete outcome of the workshop that may contribute to strengthening capacity in the region is the joint agreement between WHO/EMRO and the University of Toronto Joint Centre for Bioethics to provide scholarships to the Master of Health Sciences Program at the JCB. Furthermore, there seems to be growing need to establish a Regional Health Biotechnology Network for EMR, and WHO-EMRO is now planning to hold a Regional meeting to discuss this in Iran in July 2004. WHO-EMRO has initiated follow-up of these recommendations. Ministers of Health from the region, at the 50^th^Regional Committee Meeting held in October 2003, urged Member States to establish national bodies for genomics and biotechnology to formulate strategic vision for creating public awareness and for developing biotechnology for equitable health care in the region. Recently, a paper on harnessing genomics and biotechnology for public health was presented at the EM 28th Regional Consultative Committee Meeting (RCC) held in Cairo in April of this year. The recommendations in the paper were derived from those developed at the workshop. The paper will be presented at this year\'s Regional Committee Meeting to be held in October 2004. The methods and recommendations outlined in this paper demonstrate progress in bringing genomics and biotechnology to the forefront of science policy in developing countries. Our procedure has now led to the formation of three regional networks -- the two previous ones being the African Genome Policy Forum, which encompasses participants from 10 African nations, the Indian Genome Policy Forum. We have also now held a course for Latin America and the Caribbean in association with PAHO and the UN University in Venezuela May 23--26 2004, and a similar network is being created of participants of that course. The next one will be held in the Southeast Asian region, most likely based in Hong Kong. These regional genome policy networks will provide models to establish a Global Genome Policy Forum. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= TA drafted the manuscript; PAS and ASD conceived of the course, and all authors participated in its design and coordination. All authors revised the manuscript for critical content and approved the final draft. Acknowledgements ================ We would like to thank the course participants and members of the EMRO Genome Policy Forum for their insightful comments and suggestions. Funding was obtained from WHO-EMRO and the Canadian Program on Genomics and Global Health (CPGGH). CPGGH funds for this course derived primarily from Genome Canada and the International Development Research Centre (Canada). A full list of CPGGH funders is available at <http://www.geneticsethics.net>. ASD receives support from the McLaughlin Centre for Molecular Medicine and PAS is supported by a Distinguished Investigator award from the Canadian Institutes of Health Research.

PubMed Central

2024-06-05T03:55:52.627325

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548293/", "journal": "Health Res Policy Syst. 2005 Jan 21; 3:1", "authors": [ { "first": "Tara", "last": "Acharya" }, { "first": "Mohammed Abdur", "last": "Rab" }, { "first": "Peter A", "last": "Singer" }, { "first": "Abdallah S", "last": "Daar" } ] }

PMC548294

Background ========== Medawar et al. \[[@B1]\] showed almost half a century ago that rodents injected at birth with splenocytes from genetically different donors could accept transplants from that donor as an adult. These milestone experiments guided the notion that the introduction of antigens during neonatal life leads to tolerance and that the immune system functions by making a distinction between self and nonself. For some years, Matzinger et al. have persevered on the hypothesis that tolerance is not an intrinsic property of the newborn immune system \[[@B2],[@B3]\]. For example, many studies have shown that neonatal exposure to antigen may prime T cells and induce both Th1 and Th2 cells \[[@B4]-[@B7]\]. Moreover, Adkins et al. have demonstrated that although neonates develop compartmentally distinct primary responses to antigen immunization (mixed Th1/Th2 in lymph nodes and Th2 in spleen), after rechallenge the elicited secondary response is always of the Th2 type \[[@B7],[@B8]\]. They have also proved that even in the lymph nodes, the Th2 function persists for a prolonged period after a single immunization, and that animals initially immunized as neonates are impaired in their capacity to develop the expected Th1 memory effector function observed in adults \[[@B9]\]. The biased immunogenic neonatal immunity may be attributable to factors associated with antigen presentation such as type of antigen-presenting cell, accompanying adjuvant and the nature, concentration and in vivo availability of the antigen \[[@B5],[@B10]-[@B13]\]. Resting T cells need two signals to be activated; signal 1 from TCR binding to MHC/peptide and signal 2 (co-stimulation) from a professional phagocyte, such as a dendritic cell or a macrophage. Tolerance is associated to a lack of co-stimulation that usually occurs when antigen is encounter by a non-professional phagocyte, or by professional phagocytes in a non-APC tissue (lymphoid tissue, skin, etc)\[[@B14]\]. In this study, we have evaluated the effect of adoptive transfer of DCs from adult and neonatal mice infected with *L. (L.) mexicana*, and from healthy adult and neonatal mice. As in the *L. major*mouse model, we have shown that infection with *L. (L.) mexicana*strain MHOM/BZ/82/BEL21, generates a Th1 response associated to protective immunity in C57BL/6 mice, and a Th2 response related to non-healing disease in BALB/6 mice \[[@B15]\]. Leishmaniasis is an excellent model to study the extremes of host/parasite relationships, particularly the diversity of the immune response associated to the genetic background of the host. In addition, mice can reproduce the distinct clinical forms observed in humans \[[@B16],[@B17]\]. These models have been particularly important to show that skin-derived DCs including Langerhans cells play an important role in cutaneous leishmaniasis, where they can transport *Leishmania*antigens to the lymph nodes and induce specific immune responses \[[@B18]-[@B24]\]. Moll et al. have also shown that Langerhans cells may act as reservoirs sustaining parasite-specific stimulation of T memory cells, thus protecting animals from reinfection \[[@B25]\]. Results and Discussion ====================== Establishment of a *L. (L.) mexicana*infection model in neonatal BALB/c mice ---------------------------------------------------------------------------- The progress of *L. (L.) mexicana*infection in neonatal BALB/c mice, after the inoculation with 5 × 10^4^, 1 × 10^5^, 2 × 10^5^or 5 × 10^5^promastigotes was determined by measuring the footpad thickness during 6 weeks. All 4 experimental groups developed lesions. Mice that received 1 × 10^5^, 2 × 10^5^and 5 × 10^5^promastigotes respectively, showed a significant increase (p ≤ 0.05) on footpad thickness starting from the second week, reaching a maximal value on the sixth week of evaluation (Fig. [1A](#F1){ref-type="fig"}). This increase in footpad thickness was much greater (p ≤ 0.05) in the group inoculated with 5 × 10^5^promastigotes, with lesions appearing from the first week (Fig. [1A](#F1){ref-type="fig"}). Moreover, this experimental group presented a similar evolution to that observed in *L. (L.) mexicana*-infected adult BALB/c mice inoculated with 1 × 10^6^promastigotes (Fig. [1B](#F1){ref-type="fig"}). The statistical analysis using a Wilcoxon matched-pairs signed-ranks test of the percentage increase from the starting footpad thickness in both neonatal and adult BALB/c mice infected with 5 × 10^5^and 1 × 10^6^promastigotes, respectively, showed a significant (p ≤ 0.05) two-tailed value and a very significant Spearman correlation (r = 1.000, p = 0.0014). The starting footpad thickness in neonatal and adult BALB/c mice was 1.67 mm and 1.85 mm, respectively. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Progression of *L. (L.) mexicana*infection in neonatal BALB/c mice. A. Footpad thickness of adult mice infected with 1 × 10^6^promastigotes (■), non-infected mice (○), neonatal mice infected with 5 × 10^4^promastigotes (△), neonatal mice infected with 1 × 10^5^promastigotes (▲), neonatal mice infected with 2 × 10^5^promastigotes (□) and neonatal mice infected with 5 × 10^5^promastigotes (●). B. Percentage increase from the starting footpad thickness in both neonatal (□) and (■) adult BALB/c mice infected with 5 × 10^5^and 1 × 10^6^promastigotes, respectively. ::: ![](1475-9292-4-2-1) ::: We used 5 × 10^5^promastigotes as the optimal concentration for *L. (L.) mexicana*infection in all the subsequent experiments including the infection control group. This neonatal murine model of *L. (L.) mexicana*infection used half the numbers of promastigotes previously described to infect adult BALB/c mice \[[@B16]\]. A significant Spearman correlation attested that our neonatal model was comparable to the adult model of infection. Although infected neonatal mice have a statistically similar clinical outcome that infected adult mice, we ignore whether these mice have similar level of infection and therefore similar concentrations of antigens carried over by the transferred DCs, however, looking at the present results one can speculate that DCs from mice infected during neonatal life induced tolerance probably due to a high parasite burden, and not a lack of adjuvancity since DCs from healthy neonatal mice were able to partially protect against *Leishmania*infection. Other studies have shown a similar pattern of Th2-biased immune response in other models of neonatal infection \[[@B7],[@B11]\]. We also observed that even after the inoculation of considerable numbers of parasites, neonatal mice differed significantly from adult mice in their percentage increment from the starting footpad thickness, suggesting a functional impairment of the primary immune response. This may be explained, first by the fact that in BALB/c mice carry a point mutation in the Nramp1 (natural-resistance-associated macrophage protein) gene that allows the mRNA degradation of macrophage activation genes, increasing susceptibility to *Leishmania*infection \[[@B26]\]. Susceptibility associated with the dominant expression of the costimulatory molecule CD86 (B7-2) and the subsequent generation of the Th2-mediated response \[[@B27]-[@B31]\]. Second, the proof that murine naïve neonatal T cells, unlike adult T cells, express a Th2 phenotype and are highly deficient in Th1 functions \[[@B32],[@B33]\]. Morphological and immunophenotypic characterization of murine splenic dendritic cells ------------------------------------------------------------------------------------- DCs obtained by our purification method showed characteristic dendritic cell morphology, and a 97% purity as determined by CD11c immunostaining and flow cytometry. A minor fraction of about 3.5 % expressed CD3 and NK1.1 (Fig. [2](#F2){ref-type="fig"}). The expression of CD11c, MHC-II and CD86 molecules was detected by immunocytochemistry, thus demonstrating that these cells showed characteristics of functionally mature DCs. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Frequency distributions of purified dendritic cells labelled CD11c-FITC showing 97.17% purity (right), and FITC-isotype control (IgG1) (left). The information shown is from a single cell isolation procedure, representative of various separate experiments. ::: ![](1475-9292-4-2-2) ::: Splenic DCs were isolated for our adoptive transfer experiments since they are mobile antigen-presenting cells that migrate to peripheral lymph organs where they stimulate naive T cells, thus initiating primary T cell responses \[[@B34]-[@B36]\]. Further, splenic DCs have been isolated by standardized procedures based on the high expression of CD11c and the lack of CD205 \[[@B37]\]. Progression of the infection in neonatal recipient BALB/c mice after adoptive transfer of dendritic cells from the distinct experimental groups ----------------------------------------------------------------------------------------------------------------------------------------------- The adoptive transfer of 1 × 10^3^, 1 × 10^5^or 1 × 10^6^DCs from adult BALB/c mice infected during adulthood with *L. (L) mexicana*on neonatal recipient mice modified the course of infection, showing a delayed lesion growth from the second week onward (Fig. [3](#F3){ref-type="fig"}) as compared with the infection control group. This reduction in footpad thickness was dependent of DC numbers, since at the highest concentration of 1 × 10^6^, lesions were smaller than those observed with 1 × 10^3^and 1 × 10^5^DCs from the fifth week onward (Fig. [3](#F3){ref-type="fig"}). At the seventh week of infection, lesion size decreased by 40% after the adoptive transfer of 1 × 10^6^DCs, whereas in animals inoculated with 1 × 10^5^and 1 × 10^3^DCs the decrease was of 33% and 22%, respectively. In contrast, the adoptive transfer of 1 × 10^5^or 1 × 10^6^DCs from adult BALB/c mice infected during neonatal life with *L. (L) mexicana*fail to modify the course of infection of neonatal recipient BALB/c mice as compared with infection control animals (Fig. [4](#F4){ref-type="fig"}). However, those mice receiving 1 × 10^6^DCs showed a significant reduction in lesion growth (p ≤ 0.05) on weeks 2, 3 and 4. This effect disappeared from the fifth week onwards (Fig. [4](#F4){ref-type="fig"}). Moreover, the adoptive transfer of 1 × 10^5^or 1 × 10^6^DCs from healthy adult BALB/c mice modified the course of infection in neonatal recipient mice, showing a delayed and significant decrease (p ≤ 0.05) in lesion growth from the second week of infection (Fig. [5](#F5){ref-type="fig"}). This reduction in footpad thickness was dependent on DC numbers, since at 1 × 10^6^lesions were significantly (p ≤ 0.05) smaller than those observed in mice transferred with 1 × 10^5^DCs, which also initiated their lesions on the third week (Fig. [5](#F5){ref-type="fig"}). At the seventh week of infection, lesion size decreased by 30% after the adoptive transfer of 1 × 10^6^DCs and by 10% in mice receiving 1 × 10^5^DCs. similarly, the adoptive transfer of 1 × 10^3^or 1 × 10^5^DCs from healthy neonatal BALB/c mice modified the course of infection of neonatal recipient mice, showing a delayed and significant decrease (p ≤ 0.05) in lesion growth from the second week of infection. This reduction in footpad thickness was very similar in both tested concentrations (Fig. [6](#F6){ref-type="fig"}). At the seventh week of infection, lesion size decreased by 35% in both groups. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Progression of infection in neonatal recipient BALB/c mice after adoptive transfer of DCs from adult BALB/c mice infected during adulthood with *L. (L) mexicana*. Footpad thickness of neonatal mice infected with 5 × 10^5^promastigotes (■); neonatal mice transferred with 1 × 10^6^(○;), 1 × 10^5^(△) and 1 × 10^3^(□) DCs and subsequently infected with 5 × 10^5^promastigotes. ::: ![](1475-9292-4-2-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Progression of the infection in neonatal recipient BALB/c mice after adoptive transfer of DCs from adult BALB/c mice infected during neonatal life with *L. (L) mexicana*. Footpad thickness of neonatal mice infected with 5 × 10^5^promastigotes (■); neonatal mice transferred with 1 × 10^6^(○) and 1 × 10^5^(△) DCs and subsequently infected with 5 × 10^5^promastigotes. ::: ![](1475-9292-4-2-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Progression of the infection in neonatal recipient BALB/c mice after adoptive transfer of DCs from healthy adult BALB/c mice. Footpad thickness of neonatal mice infected with 5 × 10^5^promastigotes (■); neonatal mice transferred with 1 × 10^5^(○) and 1 × 10^6^(△) DCs and subsequently infected with 5 × 10^5^promastigotes. ::: ![](1475-9292-4-2-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Progression of the infection in neonatal recipient BALB/c mice after adoptive transfer of DCs from healthy neonatal BALB/c mice. Footpad thickness of neonatal mice infected with 5 × 10^5^promastigotes (■); neonatal mice transferred with 1 × 10^5^(△) and 1 × 10^3^(◇) DCs and subsequently infected with 5 × 10^5^promastigotes. ::: ![](1475-9292-4-2-6) ::: Disease progression was substantially decreased after transferring cells from adult BALB/c mice infected during adulthood with *L. (L) mexicana*, healthy adult BALB/c mice and healthy neonatal BALB/c mice. The reduction in these 3 groups was statistically significant (p ≤ 0.05) as compared with the infection control group. This reduction in footpad thickness was absent or considerably diminished in mice receiving DCs from adult BALB/c mice infected during neonatal life with *L. (L) mexicana*(Fig. [7](#F7){ref-type="fig"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Progression of the infection in neonatal recipient BALB/c mice after the adoptive transfer of DCs from the different experimental groups. Footpad thickness of neonatal mice infected with 5 × 10^5^promastigotes (■); neonatal mice transferred with 1 × 10^6^DCs from adult BALB/c mice infected during adulthood (◇), adult BALB/c mice infected during neonatal life (○), healthy adult BALB/c mice (●) and 1 × 10^5^CDs from healthy neonatal BALB/c mice (△) and subsequently infected with 5 × 10^5^promastigotes. ::: ![](1475-9292-4-2-7) ::: Our results showed that the preceding intraperitoneal adoptive transfer of DCs diminished the progression of *L. (L.) mexicana*infection in neonatal BALB/c recipient mice. These results contrast with those of Moll and Berberich \[[@B38]\] showing that only intravenous administration of antigen-pulsed Langerhans cells, but not intradermal or intraperitoneal inoculation, induced resistance against *Leishmania*infection. In this study, the observed protection depends on the quantity and provenance of the transferred DCs, since the effect was more evident with high cellular numbers of DCs from adult BALB/c mice infected during adulthood and healthy neonatal mice, where lesions were about 40% smaller than in the infection control group. DCs from these two groups have the intrinsic capacity to induce protective or resistant immune responses very early in life. That neonatal DCs appear to be more protective, on a per cell basis, than adults DCs is a very striking result since only Dadaglio et al.\[[@B39]\] have shown that neonatal DCs are as effective as adult DCs in expressing MHC and costimulatory molecules; taking-up, processing and presenting antigens to T cells inducing CTL responses in vivo. Others have shown that neonatal DCs are not fully functional \[[@B40],[@B41]\]. Also, animals receiving DCs from healthy adult mice showed a slightly but significantly reduced protection from that observed with DCs from adult mice infected during adulthood and healthy neonatal mice. Various studies have shown that epidermal DCs in aged skin are reduced significantly compared with young skin in mice and humans \[[@B42]-[@B48]\]. This cellular reduction may be the consequence of a decreased production in the bone marrow of DC progenitors or alternatively, these stem cells may be less responsive to cytokine and chemokine signals required for their homing to the skin \[[@B49]-[@B51]\]. Our results favor the latter hypothesis, since the same numbers of transferred DCs from healthy neonatal or adult mice induced a somewhat different disease outcome. More notable was the observed absence of a protective effect in mice receiving DCs from adult BALB/c mice infected with *L. (L) mexicana*during neonatal life. This result confirmed recent studies by Adkins et al. showing that animals initially immunized as neonates are unable to develop the expected Th1 memory effector function observed in adults \[[@B9]\]. These investigators proposed that in neonates, the spleen is the primary site of tolerance induction to self-antigens whereas the lymph nodes are the sites of immune responsiveness to foreign antigens. The initial and transitory protection observed at the greatest concentration of DCs from adult mice infected during neonatal period, suggests impairment in their accessory functions specifically in those associated with signal 2 and signal 3. Signal 2 comprises co-stimulatory factors essential for the clonal expansion of T cells and signal 3 involves in situ properties of DCs such as tissue interaction and migration where cytokines, chemokines and extracellular matrix components are crucial \[[@B36]\]. Conclusions =========== Our results show that tolerizing DCs from animals initially immunized as neonates play a key role in the attenuation of Th1 responses. The present results may have a considerable epidemiological impact on leishmaniasis, where infection at early stages of life may impose a tolerogenic state that favors the development of visceral or diffuse cutaneous leishmaniasis, both characterized by Th2-type responses. In this study, we have shown that intraperitoneal adoptive transfer of splenic DCs is able to surpass the genetic bias of the mice, allowing the development of an immune response that modifies the progression of *L. (L.) mexicana*infection. Methods ======= Animals ------- Adult (6 weeks) and neonatal (about 24 hour newborn) female BALB/c mice (Taconic, Germantown, NY, U.S.A.) were raised in the Animal House of the Instituto de Biomedicina, under appropriate conditions of temperature, water and feeding. Specific Antibodies ------------------- The following rat monoclonal antibodies were used to isolate and characterize dendritic cells: CD19 (B cells, clone IBL-2), MOMA-2 (Macrophages/Monocytes), CD45R (B and NK cells; clone RA3-6B2), CD3 (T cells; clone KT3), CD11c (dendritic cells and other leukocytes, clone N418), CD19 (clone 6D5) conjugated to phycoeritrine (PE), NK1.1 (clone PK136) conjugated to PE, Macrophages-Monocytes (MOMA-2) conjugated to fluorescein isothiocyanate (FITC), CD3 (clone KT3) conjugated to FITC. All were purchased from Serotec Ltd. (Oxford, United Kingdom) except CD205 (dendritic cells, clone NLDC-145, DEC205) donated by Georg Kraal, Vrije Universiteit, Amsterdam, The Netherlands; I-A^d^(MHC-II, clone AMS-32.1) and CD86 (B7.2, clone GL1) purchased from BD Pharmigen (San Diego, USA). Parasite culture and isolation of *L. (L.) mexicana*promastigotes ----------------------------------------------------------------- Amastigotes of *Leishmania (Leishmania) mexicana*(MHOM/BZ/82/BEL21) were extracted from footpad nodules of hamsters infected a month earlier with 1 × 10^6^amastigotes. The nodules were aseptically dissected out and washed in phosphate-buffered saline (PBS, pH 7.4) with 100 U/ml penicillin and 100 μg/ml streptomycin, and finely cut and ground in a Petri dish containing cold PBS. The suspension was filtered through a sterile sieve to remove large debris. These parasites were cultured on blood agar base (Sigma-Aldrich, St. Louis, U.S.A.) at room temperature for 7 days (the stationary growth phase) to obtain infective promastigotes. For an enriched population of parasites, free of erythrocytes and cellular debris, 100 μl of that sample were cultured in 2 ml Schneider\'s insect cell culture medium (Sigma-Aldrich, St. Louis, U.S.A.) for one week at room temperature. Promastigotes were isolated after 3 washes with sterile PBS and centrifugation at 1000 g at 4°C for 15 min. Pellets were resuspended in 1 ml of sterile PBS. Viable parasites were counted by trypan blue exclusion. Parasite concentration was adjusted to 5 × 10^4^, 1 × 10^5^, 2 × 10^5^and 5 × 10^5^per μl to be used in the different experimental groups. Experimental infection with promastigotes of *L. (L.) mexicana* --------------------------------------------------------------- A similar pattern of *L. (L.) mexicana*infection to that established in adult mice \[[@B52]\] was determined in neonatal BALB/c mice. Neonatal BALB/c mice (n = 12) were inoculated subcutaneouslly into the left hind footpad with 5 × 10^4^, 1 × 10^5^, 2 × 10^5^, or 5 × 10^5^promastigotes suspended in 10 μl sterile PBS, applied with a tuberculin syringe (29-gauge needle) connected to a stepper repetitive pipette (Tridak, Danbury, U.S.A.). For comparison, adult BALB/c mice were infected the standardized optimal parasite load of 1 × 10^6^promastigotes of *L. (L.) mexicana*\[[@B52]\]. The course of infection was evaluated weekly for 6 weeks, measuring the experimental left footpad using a dial gauge caliper (Mituyoto N° 7300, U.S.A.). Isolation and purification of dendritic cells --------------------------------------------- DCs from adult and neonatal BALB/c mice were isolated from the spleen. Under sterile conditions, spleens were minced on a metallic mesh with RPMI-1640 (Life Technologies, Rockville, U.S.A.) supplemented with 10% of decomplemented fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol and 100 U/ml penicillin (complete RPMI-10). The cell suspension was filtered on a nylon sieve and transferred to 15 ml centrifuge tubes (Corning Life Sciences, Acton, U.S.A.) and spun at 250 g at 4°C for 10 min. Viable cells were counted by trypan blue exclusion. Cell concentration was adjusted to 1 × 10^7^cells/ml in complete RPMI-10 and 8 ml plated in tissue culture flasks. The flaks were incubated for 2 hr at 37°C in a 5% CO~2~incubator (NuAire, Inc., Plymouth, U.S.A.), allowing DCs to adhere. Non adherent cells were carefully removed and placed in sterile 50 ml centrifuge tubes and spun at 250 g, 4°C for 10 min. Adherent cells were covered with 10 ml complete RPMI-10 and incubated as before for 16--18 hours, allowing DCs to detach. After gently washing the surface of the flasks with a plugged Pasteur pipette and complete RPMI-10, pools of the eluted cells were placed in sterile 15 ml centrifuge tubes and spun at 250 g, 4°C for 10 min. For each tube, the cell pellet was resuspended in 6 ml complete RPMI-10. This volume was carefully layered over a 3 ml NycoPrep™ density gradient (Nycomed Pharma AS, Torshov, Norway) and centrifuged at 600 g, 20°C for 20 min. Mononuclear cells were removed from the interface ring using a Pasteur pipette, transferred to a sterile 15 ml centrifuge tube and spun down in 10 ml complete RPMI-10 at 400 g, 20°C for 15 min three times. The final pellet was resuspended in 1 ml of cold (4°C) Hanks balanced salt solution supplemented with 10% decomplemented FBS and 2 mM HEPES. Cells were quantified and viability assessed by trypan blue exclusion. The final purification stage consisted of an immunomagnetic negative selection of DCs. The cell suspension obtained above was incubated under continuous agitation at 4°C for 1 hour, with primary rat anti-mouse monoclonal antibodies recognizing B and T lymphocytes, NK cells and monocytes/macrophages (1.5 μg/ml antibody per 1 × 10^6^cells). After incubation, cells were washed three times in Hanks centrifuging at 250 g, 4°C for 10 min. The pellet was resuspended in 1 ml cold Hanks in a sterile 15 ml centrifuge tube and incubated under continuous agitation at 4°C for 1 hour with a secondary sheep anti-rat IgG polyclonal antibody coupled to magnetic microspheres (Dynabeads^®^M-450, Dynal Biotech Inc., Lake Success, U.S.A.) at a 7:1 sphere/target ratio. Non-dendritic magnetic-coated cells were removed by positive selection in three sequential depletions using a magnetic gadget (Dynal MPC^®^Dynal Biotech Inc., Lake Success, U.S.A.) at 4°C for 6 min. Characterization of dendritic cells ----------------------------------- DC purity was determined by flow cytometry and immunocytochemistry. For flow cytometry, 1 × 10^5^cells were suspended in PBS (1% FBS) and incubated with 10 μl primary monoclonal antibodies directly coupled to PE or FITC recognizing T and B lymphocytes, NK cells and monocytes/macrophages. DCs were characterized by an indirect method using primary monoclonal antibodies to CD11c and a secondary antibody, hamster anti-rat IgG1conjugated to FITC (clone MARG1-2, Serotec Ltd., Oxford, United Kingdom). The incubations were carried out in the dark at 4°C for 45 min, followed by 3 washes and centrifugation at 250 g, 4°C for 10 min. The cell pellet was resuspended in 500 μl PBS and the percentage of labeled cells determined in a flow cytometer (FACScan, Becton Dickinson, Franklin Lakes, U.S.A.). The control consisted of an antibody of irrelevant specificity conjugated to FITC. For immunocytochemistry, 1 × 10^5^cells were suspended in PBS (1% FBS) and spun down at 50 g in a Cytospin (Shandon Inc., Pittsburg, U.S.A.). Sample slides were hydrated in PBS, fixed in fresh acetone for 5 min. and sequentially incubated for 90 min with primary rat monoclonal antibodies to CD11c and CD205, biotinylated goat anti-rat IgG (50 μg/ml) (Vector Laboratories, Burlingame, U.S.A.) for 45 min., and Vectastain^®^Elite ABC kit (Vector Laboratories, Burlingame, U.S.A.) at 1:100, 30 min. Five-minute washes with PBS were done between incubations. The reactions were developed for 3 minutes in Vector^®^NovaRed™ substrate. The slides were then washed and counterstained with methyl green. Omissions of the primary antibody and incubation with an antibody of irrelevant specificity at the same protein concentration were used as controls. Adoptive transfer of dendritic cells ------------------------------------ DCs were isolated from the spleens of the following donor groups: a) Adult BALB/c mice infected during adulthood with *L. (L.). mexicana*(n = 4); b) Adult BALB/c mice infected during neonatal life with *L. (L.). mexicana*(n = 4); c) Healthy neonatal BALB/c mice (n = 4); d) Healthy adult BALB/c mice (n = 4). The infection control group consisted of neonatal BALB/c mice infected with 5 × 10^5^promastigotes of *L. (L) mexicana*. DCs from the 4 experimental groups were adjusted to 1 × 10^3^, 1 × 10^5^, or 1 × 10^6^cells/ml in sterile PBS for intraperitoneal transfer to neonatal recipient BALB/c mice. Cells, at the mentioned concentrations, were injected in 20 μl volumes using a tuberculin syringe (29-gauge needle) connected to a stepper repetitive pipette (Tridak, Danbury, U.S.A.). After sixteen hours of adoptive transfer, neonatal recipient BALB/c mice were infected with 5 × 10^5^promastigotes of *L. (L) mexicana*. Isolation of DCs and adoptive transfer experiments were done in duplicates. Statistical analysis -------------------- The results were expressed as mean ± standard error of the mean (SEM). Each experimental group consisted of 4--5 individuals. Comparisons between groups were made with Student t test and Welch t test for unpaired samples. Any value of p ≤ 0.05 was considered significant. All tests were performed using GraphPad InStat 3.02 (GraphPad Software, San Diego California USA, <http://www.graphpad.com>). List of abbreviations ===================== APCs: antigen-presenting cells DCs: dendritic cells TCR: T-cell receptor Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= LVP carried out most of the experimental work and drafted the manuscript. JC developed the experimental design, carried out part of the experimental work and drafted the manuscript. NLD participated in the in experimental design and evaluated the progression of infection in the mice. FJT conceived the study, participated in the experimental design and coordinated the work. All authors read and approved the final manuscript. Acknowledgements ================ We are grateful to Becky Adkins for insightful discussions and critical appraisal of the manuscript; Marian Ulrich for reading and commenting on the manuscript; Martin A. Sanchez for excellent scientific consultation on the flow cytometry information. We would like to acknowledge the personnel from the Animal House of the Instituto de Biomedicina and the Flow Cytometry Facility of Banco Municipal de Sangre in Caracas. This work was supported by Fondo Nacional de Ciencia, Tecnología e Innovación (FONACIT) Proyecto S1- 98000041 and Millennium Scientific Initiative Grant 4572VE, UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, European Commission INCO Programme, and CDCH- Universidad Central de Venezuela.

PubMed Central

2024-06-05T03:55:52.631328

2005-1-25

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548294/", "journal": "Kinetoplastid Biol Dis. 2005 Jan 25; 4:2", "authors": [ { "first": "Loida V", "last": "Ponce" }, { "first": "José", "last": "Corado" }, { "first": "Nilka L", "last": "Díaz" }, { "first": "Felix J", "last": "Tapia" } ] }

PMC548295

Background ========== Peritoneal adhesions resulting from surgical injury are often associated with pelvic pain, bowel obstruction and infertility \[[@B1]\]. Epidemiological studies conclude that 30 to 35% of all hospital readmissions are associated with adhesion associated complications, of which 4.5 to 5.1% are directly related to adhesions \[[@B2]\]. Mechanisms of adhesion formations are not completely known. It is also not clear why adhesion form in some patients and not in others. Therefore, deciphering genetic components that signal adhesion formation may help diagnose adhesion-prone patients prior to surgery. Needless to mention that such information will facilitate finding ways to prevent post-surgical adhesion formation. Parietal and visceral peritoneum that surfaces the intraperitoneal organs is covered by a layer of squamous epithelial cells, the mesothelium. The submesothelial layer consists of fibroblasts, macrophages and blood vessels. Surgical abrasion to the peritoneum releases mesothelial cells, macrophages, fibroblasts, and blood containing cytokines and several cell types at the site of injury. Coagulation of blood creates a fibrinous mass between injured surfaces. In some patients fibrinolysis of clot followed by proliferation of mesothelial cells covers the wound. In others, failure of fibrinolysis followed by proliferation and migration of fibroblasts into the proteinous mass generates fibrous tissues of adhesion. Consequently, the process of adhesion formation include inflammatory response, fibrin deposition, cell-proliferation, -differentiation, -migration, -death, angiogenesis, extra cellular matrix (ECM) turnover regulated by cytokines, hypoxia, genetic and epigenetic factors \[[@B3]\]. Recent studies illustrate roles of peritoneal fibroblasts in adhesion development \[[@B4]-[@B10]\]. It is also proposed that fibroblasts from the chronic wounds migrate into the fibrin deposit; secrete ECM proteins causing wound contraction and scar formation \[[@B11]\]. The migration of fibroblasts may be coordinated by TGF-β1 mediated interactions of integrin receptors \[[@B10]\] with the RGD sequence of the fibrin, fibrinogen and fibronectin at the fibrin clot \[[@B12]\]. Additional cytokines and the hypoxic condition at the site of injury may also influence peritoneal fibroblasts to attain a phenotype supporting formation of adhesion tissue. This change in the phenotype of fibroblasts may be induced by changes in expression pattern of several genes during the process of adhesion development. Therefore, identifying differences in the global gene expression pattern between normal and adhesion fibroblasts may provide additional clues to the mechanisms by which normal fibroblasts attain the adhesive, proliferating and migratory phenotype required for the formation of fibrous tissues of adhesions. In the present study, we compared gene expression patterns between adhesion and normal peritoneal fibroblasts using GF211 gene filters (Research genetics) containing 4325 randomly selected known genes. Furthermore, we confirmed the expression pattern of genes of interest by a semiquantitative RT-PCR method and examined possible contribution/s of TGF-β1 and hypoxia in the transformation of normal peritoneal fibroblasts into an adhesion phenotype. Methods ======= Peritoneal-tissue collection, fibroblast-isolation and culture -------------------------------------------------------------- Tissues were collected at the initiation of surgery and after the entry into the abdominal cavity of female patients (25--50 years) undergoing laparatomy for pelvic pain as described earlier \[[@B4]\]. All patients gave informed written consent for the tissue collection, which was conducted under a protocol approved by the Wayne State University Institutional Review Board. Normal parietal peritoneal tissues were collected from these patients from the anterior abdominal wall, approximately midway between the umbilicus and symphyses pubis, and lateral to the midline incision. Peritoneal tissues from adhesions, that were at least 3 inches away from the site of normal tissue collections, were also collected from the same patient. The peritoneal fibroblasts were isolated and separated from mesothelial cells by a differential centrifugation procedure that is briefly described earlier \[[@B4]\]. The isolation of fibroblasts from mesothelial cells were also verified by the RT-PCR detection of Collagen type I, Matrix metalloproteinase-2 (MMP-2) and Transforming growth factor-β3 (TGF-β3) \[[@B13]-[@B15]\]. The primary cultures were maintained in a humidified incubator (37°C, 5% CO~2~) for 3 days in DMEM (Life Tech.) supplemented with 10% fetal bovine serum (Life Tech.) and antibiotics (Penicillin and Streptomycin 50 U/ml; Life Tech.). The monolayer of cells were passaged in trypsin-EDTA solution (Life Tech.). Cells at 3--5 passages were cultured in serum free medium in 75 cm^2^flasks (Fisher Scientific, Pittsburgh, PA) to 75% confluency prior to studies. Gene expression pattern in the fibroblasts from adhesion and normal peritoneum ------------------------------------------------------------------------------ Total RNA was isolated from monolayer of fibroblasts at 12 h of culture in serum free medium using Trizol reagent (Invitrogen Inc.). Human Gene Filters (GF211; Research Genetics, Inc., Huntsville, AL) containing 4325 known human cDNA spots were used for the identification of differentially expressed genes between adhesion and normal fibroblasts from human peritoneum. Method suggested by the manufacturer was strictly followed. In brief, 10 μg of total RNA from monolayer cultures of fibroblasts were subjected to cDNA synthesis in presence of 10 μl ^33^dCTP (10 mCi/ml; ICN Radiochemicals, Irvin, CA). Radiolabeled cDNA was separated from the free nucleotides using Bio-Spin 6 chromatography column (Bio-Rad Laboratories). Gene filters were labeled as adhesion or normal fibroblasts and washed with 0.5% sodium dodecyl sulfate (SDS) prior to prehybridization. Individual membrane was transferred to separate roller tubes of the hybridization oven (Fisher Scientific, Inc., Pittsburg, PA), each containing MicroHyb hybridization solution (Research Genetics) supplemented with Human Cot-1 DNA (Life Technology) and Poly dA (Research Genetics). The membranes were rotated at 10 RPM and at 42°C for 2 h. Radio-labeled cDNA prepared from adhesion and normal fibroblasts total RNA was denatured by heating in a boiling water bath for 3 min. The denatured probes were then injected into the prehybridization solution containing respective membrane. The membranes were hybridized with respective probe for 18 h at 42°C. The hybridization solution was then replaced with washing solution (2 × SSC containing 1% SDS). The temperature of the oven was raised to 50°C and RPM of rotors was increased to 15. Membranes were washed for 20 minutes when washing solution was replaced with a batch of fresh and prewarmed (50°C) washing solution. Washing was continued for additional 20 min. A third wash was performed with 0.5 × SSC solution containing 1% SDS at 55°C for 15 minutes. Membranes with cDNA spots facing up were covered with Saran wrap and exposed to phosphor screen (Kodak) for overnight. The screen was scanned with a Phosphor Imager (Storm System; Amersham Biosciences Corp., Piscataway, NJ). After acquisition of signal intensities from the normal and adhesion fibroblasts of one patient, filters were stripped according to protocol and subjected to gene filter experiments with the RNA samples from a second patient and images were scanned. Tiff images obtained from normal and adhesion fibroblasts of two patients were analyzed using Pathway 4 software (Research Genetics) for the identification of differentially expressed genes between the normal and adhesion fibroblasts of each patient. Relative abundance of selected genes in the fibroblasts from adhesion and normal peritoneum ------------------------------------------------------------------------------------------- Steady-state levels of mRNA of selected genes that are known to have a role in cellular adhesion, proliferation, migration, apoptosis and demonstrating different expression levels between adhesion and normal fibroblasts in the gene filter experiments were verified further by a previously described semiquantitative RT-PCR method \[[@B16]\]. Total RNA (1 μg) from the monolayer culture of adhesion or normal fibroblasts was subjected to reverse transcription as described earlier. Complementary DNA (100 ng) was subjected to PCR amplification of the cDNA of interests in a 25 μl reaction mixture containing 50 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl~2~, 0.2 mM dNTP, 0.5 U Taq Polymerase (all from Life Technology, BRL) and 1 μM each of sense and antisense primers. Primer sequences were determined using Primer3 software from the Internet <http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi>. The control primers (*sense*5\'-ggaggttcgaagacgatcag-3\' and *antisense*5\'-cgctgagccagtcagtgtag-3\') were expected to provide an amplicon of 509 bp from human 18S ribosomal subunit cDNA (gi: 337376). Accession numbers of genes of interests are provided in Table [2](#T2){ref-type="table"} and nucleotide sequences of primers and expected size amplicons are provided in the Table 3. Each PCR cycle consisted of a hot start at 95°C for 1 min, followed by melting at 95°C for 30 sec, annealing at 58°C for 1 min and extension at 72°C for 1 min. At the end an extension reaction at 72°C for 10 min was performed. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Genes differentially expressed in the adhesion fibroblasts and known to have roles in cell-adhesion, -proliferation, -migration, -differentiation and -death. ::: **Accession Number** **Definition** **Fold Change** **Functions** ------------------------------ ---------------------------------------------------- ----------------- --------------- ----------------------------------------------------------------------------------------------------------------- ***Increased in Intensity*** gi:17986276 Collagen, type IV alpha 2 2.4 2.7 See Discussion gi:4506760 S100 calcium-binding protein A10 2.3 2.7 See Discussion gi:6679055 Nidogen 2 6.4 7.1 See Discussion gi:14250074 Transmembrane 4 superfamily member 1 3.7 2.6 See Discussion gi:4758081 Chondroitin sulfate proteoglycan 2 3.4 3.2 See Discussion gi:187538 Metallothionein 1E 4.3 4.0 See Discussion gi:4336324 Small membrane protein 1 2.4 2.6 Cell viability \[54\] gi:17738299 Cyclin-dependent kinase inhibitor 2A 2.0 2.0 Cell proliferation \[55\] gi:16359382 Nuclear receptor subfamily 4, group A 1.6 2.1 Antagonizes TNF-α induced apoptosis \[56\] gi:40353726 Synaptopodin 2.9 2.4 Actin cytoskeleton dynamics \[57\] gi:23398519 Vasodilator-stimulated phosphoprotein 1.5 2.1 Enhances actin based cell motility, Cytoskeltal dynamics \[58\] gi:28329 α-Smooth muscle actin 3.0 3.2 Myofibroblast transformation \[44\] gi:14574570 Bcl-2 related gene bfl-1 1.6 1.4 Anti apoptotic; inhibitor of p53 induced apoptosis gi:796812 p53 tumor suppressor 1.5 1.6 Cell cycle arrest and apoptosis \[52\] ***Decreased in Intensity*** gi:184522 Insulin-like growth factor binding protein 3 3.2 2.3 See Discussion gi:4504618 Insulin-like growth factor binding protein 7 2.3 2.0 Growth suppressing factor \[59\] gi:28610153 Interleukin 8 3.2 2.6 Inhibits fibroblast migration, delays wound healing, reduces wound contraction \[60\] gi:4504982 Lectin, galactoside-binding, soluble 3 \[galectin) 3.0 3.0 Tumor-suppressive and pro apoptotic \[61\] gi:12803916 Gap junction protein, beta 1, \[Connexin 32) 1.8 2.2 Tumor suppressive and Proapoptotic \[62\] gi:14589894 Cadherin 5, type 2, VE-cadherin \[vascular\] 2.3 1.7 Down regulation associates with tumor metastasis, Initiates endothelial-mesenchymal transdifferentiation \[63\] gi: 16198356 Lactotransferrin 2.2 2.1 Inhibits growth of malignant tumors. Elevated by high level of estrogen \[64\] gi:21619838 Lipocalin 2, Oncogene 24p3 3.3 2.5 Proapoptotic \[65\] gi: 23273645 Calponin 1, basic, Smooth muscle cell 1.7 2.5 Inhibits smooth muscle cell contraction and Tumor Suppressive \[66\] gi:40225461 RAP1A, member of RAS oncogene family 1.8 1.6 Inhibits cell proliferation \[67\] gi:4507112 Synuclein-gamma 1.5 1.3 Expression reduced in carcinoma \[68\] \* Adhesion/Normal peritoneal fibroblasts values of gene expression intensity from patient 1 (P1) and 2 (P2). ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### PCR primers, amplicon size and expression ratios of genes between adhesion and normal peritoneal fibroblasts ::: Transcripts Primer Sequence (5\' to 3\') Amplicon size (base pairs) Adhesion/Normal Ratio Gene Filter\* Adhesion/Normal Ratio (RT-PCR)\*\* ----------------------------------------------------------------- ------------------------------ ---------------------------- ------------------------------------- ------------------------------------ ------ 18S Ribosomal Subunit Sense ggaggttcgaagacgatcag 509 (No spot) 0.9 Antisense cgctgagccagtcagtgtag Collagen type IV alpha 2 chain(COL4A2) Sense caccatgcccttcctgtact 351 2.6 2.3 Antisense ttgcattcgatgaatggtgt S100 Calcium binding protein A10 (S100A10) Sense cacaccaaaatgccatctca 389 2.5 2.1 Antisense cttctatgggggaagctgtg Nidogen 2 (NID2) Sense gcttacgaggaggtcaaacg 500 6.8 2.9 Antisense ttcacccggaaggtattcag Transmembrane 4 superfamily member 1 (TM4SF1) Sense tcgcggctaatattttgctt 500 3.2 1.9 Antisense gcctccaagcactccattta Chondoitin sulfate proteoglycan 2 (CSPG2) Sense gaaccaaattatggggcaga 400 3.3 3.0 Antisense ctcccaatccttcgtcgata Insulin-like growth factor binding protein 3 precursor (IGFBP3) Sense gggtaggcacgttgtaggaa 603 -2.8 -2.8 Antisese gtgaggctggctaagaatgc Metallothionine (hMT-Ie) Sense cagagggtctctgggtttca 400 4.2 3.3 Antisense gccccatgtcctctcactaa \* Average intensity of Adhesion/Normal peritoneal fibroblast gene expression data from patient 1(P1) and 2 (P2) presented in Table 2. Minus (-) sign indicate fold decrease in intensity in the adhesion fibroblasts. \*\* Ratios of Adhesion/Normal mean values from 4 patients. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Expression profiles of genes in the adhesion vs. normal peritoneal fibroblasts and the effects of TGF-β1 or hypoxia on the expression level of genes in the normal peritoneal fibroblasts ::: **Transcripts** **Adhesion/Normal fibroblasts**(Gene Filter & RT-PCR) **TGF-β1 Effects**(RT-PCR) **Hypoxia Effects**(RT-PCR) ----------------- ------------------------------------------------------- ---------------------------- ----------------------------- COL4A2 ↑ ↑ ↑ S100A10 ↑ ↑ ↑ NID2 ↑ **---** **ND** TM4SF1 ↑ **---** **ND** CSPG2 ↑ ↑ **---** IGFBP3 ↓ **---** **ND** hMT-Ie ↑ ↑ ↑ ↑ = Up regulation (p \< 0.05); ↓ = Down Regulation (p \< 0.05); **---**= No Change **ND**= Not determined ::: Initially cDNA of interests were amplified from normal peritoneal fibroblasts at different (25 to 35) PCR cycles. PCR products were subjected to agarose gel electrophoresis. Molecular weight marker (100 bp DNA ladder; Life Technology) were also loaded in adjacent lanes. DNA in the gel were stained with 1:10,000 dilution of SYBR Green I dye (Molecular Probes, Inc., Eugene, OR) and photographed using a DC 120 Kodak digital camera (Eastman Kodak, Rochester, NY) for the verification of size and analysis of band intensity using Image J software <http://rsb.info.nih.gov/ij/>. Band intensities were plotted to determine the linearity of PCR reactions for the amplification of target transcripts. Target cDNA were amplified by PCR from normal and peritoneal fibroblasts at specific PCR cycle within its linear range of amplification. Total RNA samples from normal and adhesion fibroblasts of 4 patients (included RNA from normal and adhesion fibroblasts of two patients used for the gene filter experiments) were used for the RT-PCR experiments. Optical densities obtained from amplicons of 4 patients (1 normal and 1 adhesion fibroblast RNA sample per patient) were used to derive mean ± standard error of mean values representing relative levels of each mRNA species in normal and adhesion fibroblasts. Effects of TGF-β~1~or hypoxia on gene expression pattern -------------------------------------------------------- Effects of TGF-β1 or hypoxic conditions on the steady state levels of specific gene transcripts in the normal peritoneal fibroblasts were also studied to examine the possibility of adhesion causing factors potentiating the gene expression pattern in the normal fibroblasts similar to adhesion fibroblasts. Normal peritoneal fibroblasts were cultured in six well culture plates (FALCON). When confluent, monolayer of cells in culture were exposed to 1 ng/ml TGF-β~1~(Sigma Chemical Company, St. Louis, MO) or hypoxia (2% Oxygen) for 24 h. Control plates were cultured for the same duration in absence of TGF-β1 or hypoxia. Total RNA was isolated from the control, TGF-β1 and hypoxia treated cells and subjected to RT-PCR reactions as described above to determine relative levels of 18S ribosomal subunit and gene specific transcripts in the control and treated cells. RT-PCR experiments were conducted twice with the normal peritoneal fibroblasts isolated from 3 patients to have six control, six TGF-β1 treated and six hypoxia exposed amplicons. This included normal fibroblasts from one new patient and two patients that were used exclusively for RT-PCR experiments for the confirmation of gene array data. Optical densities of amplicons from six control or treated cells per mRNA species were used to derive the mean ± standard error of mean values for comparison. Statistical analysis -------------------- Band intensity value of each RT-PCR experiment (normal, adhesion or treated fibroblasts) was used to derive Mean ± Standard error of Mean using Statview 4.5 software (Abacus Concepts, Berkley, CA). Differences between Means were tested for significance by one-way analysis of variance with the specific post hoc test using the same software to compare differences in the steady state levels of different mRNA species. Results ======= Expression pattern of genes between adhesion and normal peritoneal fibroblasts ------------------------------------------------------------------------------ Hybridization intensities of radio labeled cDNA from normal or adhesion fibroblasts from both the patients were different when analyzed using Pathway software. Comparison of hybridization intensities from individual gene spots between normal and adhesion fibroblast RNA (Figure [1](#F1){ref-type="fig"}) demonstrated that the expression levels of \~4% genes were \>1.5 fold different. BLAST search of the accession number of genes from the list provided by the manufacturer showed that genes with altered expression level between normal and adhesion fibroblasts are reported to be involved in cell adhesion and migration; transformation, transcription, translation and growth factors as well as cytokines and signaling molecules. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Images depicting radioactive signals from GF211 filters hybridized with radiolabeled cDNA.**Gene filters were hybridized with ^33^P labeled cDNA from normal peritoneal fibroblasts or fibroblasts from adhesion tissue. Unbound signals were washed and relative radioactive signal intensities were detected using a Phosphoroimager as described in the Methods. **A.**Tiff images of radioactive signals from individual gene spots of filters hybridized with normal (above) and adhesion fibroblasts, both isolated from Patient 1. **B.**Scatter plot showing signal intensities from normal peritoneal (Intensity I) and adhesion (Intensity II) fibroblasts. Dotted lines indicate a two fold changes in hybridization intensities from the median (solid line). ::: ![](1477-7827-3-1-1) ::: Gene filter data from two patients showed similar expression pattern of collagen type 1 (alpha 2), Collagen type III (alpha 1), fibronectin 1, Matrix metalloproteinase-1 (MMP-1), Transforming Growth Factor beta-1 (TGF-β1), TGF-β2 and tissue plasminogen activator as reported earlier using multiplex PCR technique (Table-[1](#T1){ref-type="table"}). Signal intensities representing TGF-β3 (gi:22531293), TGF-β III Receptor (gi:26251745), VEGF-A (gi:2565322), VEGF-B (gi:39725673) and VEGF-C (gi:19924300) expression levels were respectively 1.6, 1.5, 1.9, 1.3 and 1.3 fold (average values from two patients) lower in the adhesion compared to normal fibroblasts. No spots for antiapoptotic bcl-2 and proapoptotic bax were present in GF211 filters. Signal intensities representing anti apoptotic molecule bcl-2 related gene bfl-1 (gi: 14574570) and pro-apoptotic molecule p53 (gi:796812) were higher (Table [2](#T2){ref-type="table"}) in adhesion compared to normal fibroblasts. Expression levels of proapoptotic molecule bad (gi: 14670387) and bak1 (gi: 33457353) were not different between normal and adhesion fibroblasts. A list of additional genes that are differentially expressed between normal and adhesion fibroblasts and known to be involved in apoptosis as well as cell adhesion, proliferation and migration are listed in Table [2](#T2){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Ratios of signal intensities from adhesion and normal peritoneal fibroblasts detected from gene filters representing relative expression level of genes in patient 1 (P1) and 2 (P2). ::: **Gene** **Accession Number** **P1 (A/N)** **P2 (A/N)** **Reference\*** ----------------------------- ---------------------- -------------- -------------- ----------------- Collagen Type I (alpha 2) gi:48762933 1.4 1.5 \[4,15,53\] Collagen Type III (alpha 1) gi:15149480 2.0 1.7 \[15\] Fibronectin 1 gi:53791222 1.5 1.2 \[4,15\] MMP-1 gi:13027798 1.6 1.4 \[4\] TGF-β1 gi:10863872 1.4 1.7 \[4,15\] TGF-β2 gi:339549 1.5 1.3 \[4\] tPA gi:2161467 -1.5 -2.0 \[8\] Minus (-) sign represents lower signal intensity in adhesion (A) compared to normal (N) fibroblasts (gene filter data) \* Citations reporting expression levels of respective genes in fibroblasts from normal human peritoneum and adhesion using multiplex PCR technique ::: Semiquantitative RT-PCR experiments (Figure [2](#F2){ref-type="fig"}) conducted to verify expression pattern of specific genes from the gene filter experiments that were not studied earlier in the peritoneal fibroblasts confirmed higher expression (p \< 0.05) of Collagen type IV (alpha 2) chain (COL4A2), S100 Calcium binding protein A10 (S100A10), Nidogen 2 (NID2), Transmembrane 4 superfamily member 1 (TM4SF1), Chondroitin sulfate proteoglycan 2 (CSPG2) and Metallothioneine (hMT-Ie) in adhesion compared to normal fibroblasts. The semiquantitative RT-PCR experiments also confirmed lower expression levels of Insulin-like growth factor binding protein 3 precursor (IGFBP3) mRNA in the adhesion compared to normal peritoneal fibroblasts. Transcript levels of 18S ribosomal subunit estimated by RT-PCR method was not significantly different between fibroblasts isolated from normal and adhesion peritoneum (Figure [2](#F2){ref-type="fig"} and Table [3](#T3){ref-type="table"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Relative abundance of specific mRNA species in the normal and adhesion fibroblasts.**Genes differentially expressed between the normal and adhesion fibroblasts, as identified by gene filter experiments, were amplified by the RT-PCR technique at 26 PCR cycle. PCR products (20 μl) were subjected to electrophoresis, stained with fluorescent dye, photographed and optical density determined as described in Methods. **A**: Representative gel showing amplicons from normal (odd lane numbers) and adhesion (even lane numbers) fibroblasts. Lanes **1**&**2, 3**&**4; 5**&**6; 7**&**8; 9**&**10**and **11**&**12**show RT-PCR products from COL4A2; NID2; CSPG2; S100A10; 18S ribosomal subunit and TM4SF1 mRNA respectively. Lanes **13**&**14; 15**&**16**and **17**&**18**show RT-PCR products from 18S ribosomal subunit, IGFBP3 precursor and MET-1e mRNA respectively. **L:**Lanes loaded each with 7 μl of 100 bp DNA ladder. The 600 bp band of the ladder is shown by arrow head. **B**. Histogram showing mean and standard error of mean values of optical densities derived from amplicons of specific genes (x axis) from normal (empty bars) and adhesion (filled bars) fibroblasts isolated from 4 patients as described in Methods. \*Significantly different (*p*\< 0.05) between normal and adhesion fibroblasts. ::: ![](1477-7827-3-1-2) ::: Effects of TGF-β1 or hypoxia on the expression levels of specific genes in the normal peritoneal fibroblasts ------------------------------------------------------------------------------------------------------------ Exposure to TGF-β~1~or hypoxic conditions for 24 h altered expression levels of specific genes in the normal peritoneal fibroblasts as evidenced by semiquantitative RT-PCR. Transcript levels of COL4A2, S100A10, CSPG2 and hMT-Ie were up regulated by TGF-β1 in the normal peritoneal fibroblasts (Figure [3](#F3){ref-type="fig"}), whereas transcript levels of NID2, TM4SF1, and IGFBP3 were not altered by TGF-β1 treatment. Hypoxic conditions elevated expression levels of COL4A2, S100A10 and hMT-Ie transcripts in the normal peritoneal fibroblasts (Figure [4](#F4){ref-type="fig"}). Transcript levels of CSPG2 were not significantly altered by hypoxia. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Effects of TGF-β1 on the steady state levels of specific mRNA species in normal peritoneal fibroblasts.**Normal peritoneal fibroblasts were cultured for 24 h in absence or presence of TGF-β1 and total RNA from cells were examined for the steady-state levels of different mRNA species by semiquantitative RT-PCR technique as described in Methods. **A**. Representative gels showing amplicons generated by RT-PCR from specific gene transcripts (denoted on the left of the panel) from control (lanes 1, 2 and 3) and TGF-β1 (lanes 4, 5 and 6) treated cells. Complementary DNA for all genes except IGFBP3 precursor was amplified at 26 PCR cycles. IGFBP3 precursor transcripts were amplified at 25 cycles. **L**Lane loaded with 100 bp DNA ladder. **B**Histogram showing mean and standard errors of mean of optical densities from amplicons representing specific mRNA species (x axis). The RT-PCR experiments were conducted twice from normal and peritoneal isolated from 3 patients to obtain OD values of six amplicons from control (empty bars) or treated (shaded bars) fibroblasts statistical analysis. \* Significantly different from control conditions at *p*\< 0.05. ::: ![](1477-7827-3-1-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Effects of hypoxia on the steady state levels of specific genes in normal peritoneal fibroblasts.**Normal peritoneal fibroblasts were cultured for 24 h in normoxic and hypoxic conditions and total RNA from cells were examined to determine the steady state levels of specific transcripts as described in Methods. Complementary DNA for all genes was amplified at 26 PCR cycles. Histogram showing mean and standard errors of mean of optical densities of amplicons representing specific mRNA species (x axis) from control (empty bars) or hypoxia exposed cells (shaded bars) from 3 patients. The RT-PCR experiments were conducted twice to obtain OD values of six amplicons from normoxic or hypoxic fibroblasts for statistical analysis. Images of gels with amplicons from cells treated with hypoxia are not shown. \* Significantly different from control conditions at *p*\< 0.05. ::: ![](1477-7827-3-1-4) ::: Discussion ========== We present evidence that the expression pattern of large number of genes differ between the fibroblasts isolated from adhesion tissues and normal human peritoneal supporting the notion that adhesion fibroblasts may attain a different phenotype following peritoneal injury. Genes that displayed altered expression levels in this transition included those involved in cell proliferation, differentiation, signaling molecules, transcription and translation factors, proteolysis and cytokines. Results indicate that fibroblasts from adhesion tissue may perceive and respond to external and internal cues differently than those residing in normal human peritoneum. We attempted to decipher the functional consequences of altered gene expression pattern in the adhesion fibroblast to further elucidate the mechanism of adhesion formation and point to additional ways adhesion development may be restrained. Expression pattern of genes in the fibroblasts from normal and pathological sites are shown to be different also in earlier studies \[[@B17]\]. More relevant to the present study are the reports \[[@B4],[@B8]\] on the mRNA levels of human type I collagen (alpha 2), fibronectin 1, MMP-1, TIMP-1, TGF-β1, TGF-β2, IL-10, PAI-1, tPA and COX-2 in adhesion and normal peritoneal fibroblasts from humans estimated by multiplex PCR technique. Gene filter data from two patients also showed similar pattern of collagen, type 1 (alpha 2), fibronectin 1, MMP-1, TGF-β1, TGF-β2 and tPA mRNA levels in the normal and adhesion fibroblasts (Table [1](#T1){ref-type="table"}). Expression pattern of TIMP-1, IL-10, PAI-1, COX-2 in the adhesion and normal peritoneal fibroblasts as reported earlier \[[@B4],[@B8],[@B9]\] could not be verified by gene filter experiments because GF211 filters do not have spots representing these genes. Even so, similarities in the expression pattern of many genes between two patients (Tables 1--3) and those reported earlier using multiplex PCR technique \[[@B4],[@B8]\] validate our findings. The semiquantitative RT-PCR experiments conducted to verify expression pattern of specific genes recorded from gene filter experiments show that mRNA levels of COL4A2, S100A10, nidogen-2, TM4SF1, CSPG2, MT-1e and IGFBP3 precursor indeed differ between normal and adhesion fibroblasts. Even though expression levels of these transcripts were significantly different between normal and adhesion fibroblasts, only minor variations in the optical densities of amplicons were recorded within normal or adhesion tissues of patients of different age groups. This indicate that age dependent differences in the expression levels of genes in the fibroblasts from normal or adhesion tissues may tend to attain a relatively similar expression levels when in culture. Despite the fact that our study focused on the steady state levels of mRNA species and not on translation or posttranslational events, analysis of the functional consequences of altered expression of encoded proteins from the literature as referred below indicated that changes in the pool of these mRNA species may lead to the transformation of normal peritoneal fibroblasts into a specialized phenotype during the healing process. COL4A2 is a major structure-defining component in all basement membranes \[[@B18]\] and forms a framework for the ordered aggregation of additional molecules like laminin, heparin sulphate proteoglycans, and nidogen \[[@B19]\]. Relatively higher levels of COL4A2 observed in the adhesion fibroblasts may enhance synthesis of basement membrane in the tissues of adhesions. As COL4A2 gene is up regulated during malignant transformation and tumor vessel proliferation \[[@B20]\], it is anticipated that up regulated levels of COL4A2 in the adhesion fibroblasts may aid to the formation of adhesion tissue by increasing proliferation of adhesion fibroblasts and supporting new vessel formation for the nourishments of growing tissue. S100A10 proteins interact with Annexin A2 forming a heterotetrameric structure AIIt; that dock into the cell membrane promoting F-actin reorganization and cell migration \[[@B21]\]. AIIt also colocalizes with uPAR and plasminogen in the cells \[[@B22]\]. Heightened levels of S100A10 may enhance migration of adhesion fibroblasts by changing F-actin dynamics and influencing Cathepsin B and plasminogen machinery \[[@B23]\]. S100A10 also interacts with cytosolic phospholipase A2, inhibits its activity and decreases synthesis of archidonic acid \[[@B24]\]. Therefore, increase in S100A10 levels in the adhesion fibroblasts may deplete intracellular levels of archidonic acid and Prostaglandin E2 (PGE2) that are known to inhibit cell proliferation, collagen I synthesis, contraction of ECM and fibroblast migration \[[@B25]\]. Nidogen-2 (entactin-2) interacts with laminin1 P1, collagen I, collagen IV, perlecan and fibulin-2 in the extracellular space and stabilizes the basement membrane. It also interacts with α6β1 and α3β1 integrin receptors on cells \[[@B26]\]. Relatively higher levels of nidogen-2 secreted by adhesion fibroblasts in the extracellular space may strengthen the basement membrane and enhance integrin mediated adhesion and migration of fibroblasts into the growing tissue of adhesion. TM4SF molecules (tetraspanins) play important roles in cell migration and in the generation of complexes with integrins functionally relevant for cell motility, tumor progression and wound healing \[[@B27]\]. It is proposed that tetraspanins can influence cell migration by (i) modulating integrin signaling and integrin-mediated reorganization of the cortical actin cytoskeleton; (ii) regulating compartmentalization of integrins on the cell surface or (iii) directing intracellular trafficking and recycling of integrins \[[@B27]\]. Therefore, heightened intercalation of TM4SF1 in the cell surface of adhesion fibroblasts may facilitate their integrin-mediated migration into the developing tissues of adhesion. Versicans (CSPG2) are also known to influence α4β1 and α2β1 integrin mediated invasion of melanoma cells \[[@B28]\]. Higher CSPG2 in the fibroblasts of adhesion tissues may assist in the integrin-CSPG2 mediated migration of peritoneal fibroblasts to the site of injury and increase the number of fibroblasts by enhancing proliferation and decreasing apoptosis as evidenced in other cell types \[[@B28],[@B29]\]. Veriscan interacts with hyaluronan and CD44 and increase the viscoelastic nature of the pericellular matrix, creating a highly malleable extracellular environment that supports a cell-shape change necessary for cell proliferation and migration \[[@B30]\]. Because MT-1e transcripts are detected in cell types that have undergone myoepithelial differentiation \[[@B31]\], significant differences in the MT-1e mRNA levels between adhesion and normal peritoneal fibroblasts indicate that fibroblasts in the adhesions are at different state of differentiation compared to normal peritoneum. Molecules including IL-1; IL-6, TNF-α, EGF, bFGF, glucocorticoids, LPS, and estrogen that promote post surgical adhesion formation \[[@B32]-[@B34]\] directly or indirectly increase MT-1 transcripts and proteins in several tissues and cell types \[[@B35]\]. Therefore, it is likely that these molecules may increase adhesion formation by augmenting MT-IE levels which in turn may increase proliferation, reduce cell death and confer invasiveness of adhesion fibroblasts \[[@B36]\]. Contrary to increase in the above mentioned mRNA species in the adhesion fibroblasts, steady state levels of IGFBP3 precursor transcript were found to be lower. Because IGFBP-3 is known to inhibit cell growth by sequestering IGF, its decreased level may enhance proliferation of adhesion fibroblasts \[[@B37]\]. Reduced levels of IGFBP3 mRNA are reported in the tumorigenic cells \[[@B38]\]. Therefore, reported lower incidence of pelvic adhesion formation in the primates on anti-estrogenic therapy \[[@B32]\] could be due to the antiproliferative effects of anti-estrogens mediated in part, by IGFBP-3 \[[@B39]\]. IGFBP-3 also induces growth inhibition and apoptosis \[[@B40]\]. Decrease in the levels of IGFBP-3 in the adhesion fibroblasts may promote adhesion development both by increasing proliferation and reducing apoptosis at the site of injury. Our attempts to examine the regulatory roles of TGF-β1 and hypoxia, factors known to promote adhesion development \[[@B3]\], on the expression pattern of specific genes show that not all changes in the gene expression pattern between the normal and adhesion fibroblast are the function of these factors (Figure [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}; Table [4](#T4){ref-type="table"}). Our data show that while mRNA levels of COL4A2, S100A10 and MT-1e are elevated by both TGF-β1 and hypoxia in the human peritoneal fibroblasts, the mRNA levels of only CSPG2 is influenced by TGF-β1. Moreover, transcript levels of nidogen-2, TM4SF1 and IGFBP3 mRNA were not influenced by TGF-β1. Based on these results we hypothesize that genes that are not influenced by TGF-β1 and hypoxia in the peritoneal fibroblasts may be influenced by factors such as interleukins and TNF-α that are also known to play role in adhesion formation. Alternately, TGF-β1 and/or hypoxia may influence actions of these genes at the post transcriptional level without altering transcript levels. TGF-β1 induced up regulation of integrin α5, αv and α6 subunits in the normal human peritoneal fibroblasts without altering mRNA levels \[[@B10]\] is consistent with this possibility. It is also possible that TGF-β1 and hypoxia may alter expression of these genes in mesothelial and other cell types following peritoneal injury. Likewise lower levels of VEGF transcripts in adhesion fibroblasts may be compensated by its higher levels in other cell type required for angiogenesis during adhesion formation \[[@B3]\]. Detected lower intensity of VEGF-A isoform in the adhesion fibroblasts may also be due to the fact that spots representing this isoform do not distinguish different VEGF-A splice variants that are known to be up or down regulated during adhesion formation \[[@B16]\]. It is known that a new phenotype of fibroblasts is induced during wound healing. These fibroblasts, termed- myofibroblasts, express higher levels of α-smooth muscle actin and vinculin-containing fibronexus adhesion complexes \[[@B41]\]. Fibroblasts isolated from adhesion tissues express higher levels of α-smooth muscle actin transcripts compared to normal peritoneal fibroblast (Table-[2](#T2){ref-type="table"}) \[[@B42]\] and TGF-β1 induces formation of adhesion complex in these cells \[[@B10]\]. These observations in addition to the known roles of TGF-β in the development of post surgical adhesions \[[@B43]\] and transformation of fibroblasts into smooth muscle α-actin expressing myofibroblasts \[[@B44]\] imply that this cytokine may influence transformation of normal fibroblasts into a phenotype similar to myofibroblasts in the developing tissues of adhesion. Therefore, hindering this transformation may reduce adhesion formation. For instance, augmenting E prostanoid 2 (EP2) receptor pathways may be a way to reduce the incidence of adhesion formation because prostaglandin E~2~(PGE~2~) is shown to inhibit TGF-β1 induced expression of α-SMA, production of Collagen I and the transformation of fibroblasts to myofibroblasts via EP2 signaling \[[@B45]\]. Additionally, adhesion formation may be reduced by *P311*(*PTZ17*) and Interferon γ treatments, which inhibits TGF-β1 induced myofibroblast transformation \[[@B46],[@B47]\]. During the course of normal wound healing, myofibroblasts disappear, possibly by apoptosis \[[@B48]\]. In contrast, when there is abnormal wound healing, myofibroblasts persist \[[@B49]\]. Data obtained in our study also indicate that adhesion fibroblasts may resist apoptosis due to anti apoptotic effects mediated by increased hMet1-e and CSPG2 levels and down regulation of IGFBP3. They may also attain a high proliferating nature due to up regulation of S100A10 and CSPG2 genes, and down regulation of IGFBP3 (Table [2](#T2){ref-type="table"}). Higher proliferating and reduced apoptotic nature of adhesion fibroblasts derived from altered ratio of bcl-2 and bax expression is suggested in an earlier study \[[@B5]\]. It is apparent now that higher proliferative and reduced apoptotic nature of adhesion fibroblasts in human as reported earlier \[[@B5]\] could also derive from altered expression of hMet1-e, CSPG2, S100A10, CSPG2, IGFBP3, and the Bfl-1 that inhibits p53-induced apoptosis and is induced by cytokines TNF-α and IL-1β \[[@B50]\]. This altered phenotype of adhesion fibroblasts, acquired during the healing process, may lead to the accumulation of extra number of cells at the site of peritoneal injury resulting fibrosis and scar formation. Of note, one of the pivotal differences between wounds that proceed to normal scar compared with those that develop hypertrophic scars or fibrosis may be a lack of or reduced cell death \[[@B51]\]. Therefore, excess fibroblasts at the site of peritoneal wound healing may divert the normal process of healing towards fibrosis and adhesion. The elevated levels of p53 in the adhesion fibroblasts during this disarray, as evident from the gene filter data (Table [2](#T2){ref-type="table"}), may guard against its transition towards malignancy \[[@B52]\]. Conclusions =========== It is evident from our study that steady state levels of several genes are different between adhesion and normal peritoneal fibroblasts in human and that adhesion development may be a function of several genes. Changes in the functional interdependence of these genes at the site of injury may transform normal peritoneal fibroblast into cell type/s with altered phenotype. These cells- designated as adhesion fibroblasts may mimic previously known myofibroblasts and are highly proliferative. These cells resist apoptosis and secrete ECM molecules to renovate basement membrane. With changed expression pattern of cell surface molecules these cells may respond to intracellular signaling for migration over the fibrin clot. This altered nature of adhesion fibroblasts therefore may play a major role in the formation of the fibrous mass of adhesion-tissue that bridges adjacent and injured peritoneum. Blocking changes in the expression or function of genes necessary for this transformation of normal peritoneal fibroblasts may curtail adhesion formation. This could be achieved by the application of PGE~2~, EP~2~blockers, interferon γ, *P311*and applying measures to induce apoptosis in the peritoneal fibroblast at the site of injury. The obvious question -- \"how to maintain apoptosis at a desired level for normal peritoneal healing?\" however, remains to be answered. List of Abbreviations ===================== Collagen type IV (alpha 2) chain (COL4A2), Nidogen 2 (NID2), Chondroitin sulfate proteoglycan 2 (CSPG2), S100 Calcium binding protein A10 (S100A10), Transmembrane 4 superfamily member 1 (TM4SF1), Metallothioneine (hMT-Ie), Insulin-like growth factor binding protein 3 precursor (IGFBP3), Transforming growth factor (TGF), Prostaglandin E2 (PGE2), Urokinase Plasminogen activator receptor (uPAR), Annexin 2 and S100A10 complex (AIIt), tissue Plasminogen Activator (tPA), Plasminogen Activator Inhibitor (PAI), Cyclooxigenase (COX), Matrix metalloproteinase (MMP), Tissue Inhibitor of Metalloproteinase (TIMP), Interferon γ (IFN-γ), IL (Interleukin). Authors\' Contributions ======================= GMS and MPD were responsible for the isolation of peritoneal fibroblasts from normal peritoneum and adhesion tissues as well as establishing hypoxia chambers. MPD provided patient information and valuable suggestions during writing the manuscript. UKR performed microarray and semiquantitative RT-PCR experiments, analyzed the data and wrote the manuscript. Acknowledgment ============== U. K. Rout thanks Research Genetics (Invitrogen Corporation) for the license to use Pathway Universal Software and acknowledges a Department of Obstetrics and Gynecology, WSU Grant support for this study.

PubMed Central

2024-06-05T03:55:52.633596

2005-1-10

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548295/", "journal": "Reprod Biol Endocrinol. 2005 Jan 10; 3:1", "authors": [ { "first": "Ujjwal K", "last": "Rout" }, { "first": "Ghassan M", "last": "Saed" }, { "first": "Michael P", "last": "Diamond" } ] }

PMC548296

Review ====== This paper reviews the growth of the Pharmaceutical Benefits Scheme (PBS) during 2002--03; concerns about the sustainability of the Scheme, the government\'s response, a potential new threat that emerged and issues that remain to be tackled. The growth and sustainability of the PBS ---------------------------------------- From March 2003 to March 2004, a total of \$5.8 billion was spent on prescription medicines subsidised under the PBS. Of this, \$4.89 billion (84%) was paid by the Commonwealth, the remaining \$0.91 billion through patient co-payments \[[@B1]\]. In comparison, in 2002--03, the Commonwealth spent \$7.24 billion on public hospital services \[[@B2]\] and \$8.17 billion on medical and diagnostic services (through Medicare benefits) \[[@B3]\]. Although the PBS is the smallest of these components of Commonwealth expenditure, it has the highest average annual growth rate over the last decade (around 12% pa), compared to 6% pa for public hospital services, and 5% pa for medical services. At these rates, by 2011 the Commonwealth would be spending more on subsidised pharmaceuticals than it would spend on either public hospital or medical services, and by 2022, more on pharmaceuticals than both public hospital and medical services together. Such projections make the sustainability of the PBS a major concern, especially given an aging population and the continued introduction of useful (but increasingly expensive) new medicines \[[@B4]\]. While the growth rate of the PBS has slowed over the last two years (10% during March 2002--03 and 8% from March 2003--04) the past history of PBS expenditure shows considerable fluctuations over the years. These fluctuations are caused by expensive but valuable new drugs coming onto the Scheme, more cost-effective generic drugs replacing older drugs whose patent has expired and administrative changes, such as increased patient co-payments, transiently reducing usage. The government\'s response -------------------------- In 2003, as in 2002, the government proposed a 27% increase in PBS patient co-payments and safety-net thresholds in order to transfer more of the cost of the PBS from the government to consumers. Once again this measure was rejected by Labor and other opposition parties in the Senate because of concern that such increases would impact on equitable access to necessary medicines \[[@B5]\]. Regardless, the government continued to argue that without increased patient contributions (and patient restraint) the PBS would become unsustainable. By mid 2004, the Labor party was faced with an impending Federal election and had serious trouble costing its tax and spending promises. As a consequence, Labor abandoned their previous principled stand in the Senate of blocking the government\'s proposed increase in PBS copayments and safety-net thresholds \[[@B6]\] arguing that they needed the additional \$1.1 billion to spend on election promises \[[@B7]\]. They also stated that, if returned to government, a substantial proportion of the \$1.1 billion might be achieved through administrative reforms to the PBS and savings achieved by the use of cheaper generic drugs as expensive drugs moving off patent. Not surprisingly, consumer and public health groups were appalled with this Labor \"back-flip\" while the Greens and Democrats said the decision was a disgrace \[[@B8]\]. Labor said the decision was difficult but necessary. Several other measures were introduced by the government in order to improve the community\'s understanding of PBS processes and costs. From June 2003, all recommendations of the Pharmaceutical Benefits Advisory Committee (PBAC) to list, not list or defer a decision to list a medicine on the PBS were made publicly available on the PBS website \[[@B9]\]. Unfortunately, only summary information was provided; commercial-in-confidence concerns of pharmaceutical manufacturers precluded making more detailed information available, such as cost-effectiveness data, on which PBAC based its decision. From 1 August 2003 the full cost of PBS medicines appeared on medicine labels if the price was greater than the co-payment. The full cost included what the consumer has paid and the amount that is paid through the PBS. The aim was to help people understand what medicines really cost and how the PBS helps make medicines affordable for all. In addition, the government commissioned a \$24 million advertising campaign that emphasised that patient responsibility was, \"the prescription for a healthy PBS\". Critics noted that by neglecting to inform the public that pharmaceutical marketing and inappropriate prescribing habits of doctors also produced pressures on the PBS, the campaign missed an opportunity to initiate a more balanced and constructive debate about the viability of the PBS \[[@B10]\]. During the year under review, pharmaceutical reforms designed to stop PBS cost-shifting in Victorian public hospitals were evaluated \[[@B11]\]. The reforms were a joint initiative of Victorian Department of Human Services (DHS) and the Australian Government Department of Health and Ageing (DoHA). Since the early 1990s there had been increasing cost pressures on State and Territory funded public hospitals. Their response included restricting drug supplies to discharged patients, often to only two or three days of treatment. Patients then needed to see their GP to obtain a PBS prescription to cover their needs. The effect was to \"cost shift\" pharmaceutical supplies from the State and Territories to the Australian Government. The reforms trialed in Victoria allowed public hospital doctors to write PBS prescriptions for both outpatients and discharged inpatients. They also allowed PBS access to a group of cancer chemotherapy drugs for use by day-admitted patients and outpatients. The qualitative evaluation undertaken was generally positive although it noted the reforms had increased the administrative work of both doctors and pharmacists. There was also concern that PBS rules (designed for general practice) were not always appropriate for specialised public hospitals. The Society of Hospital Pharmacists of Australia supported the need to modify PBS procedures to take into account public hospital expertise but also noted the need for more integration of medicines funding \[[@B12],[@B13]\]. Subsequently, the Victorian reforms are being implemented in other States and Territories. Programs promoting the quality use of medicines (QUM) were further developed throughout 2002--03. Specific programs were coordinated by the National Prescribing Service (NPS), Australian Divisions of General Practice and the Pharmacy Guild of Australia. The NPS was set up in 1999 with government funding but with an independent Board of Directors in order to provide unbiased educative activities to assist health practitioners (and more recently consumers) to use medicines wisely. Evaluation of NPS activities has consistently shown that spending money on targeted QUM interventions can save considerably more money on the PBS by reducing inappropriate prescribing. It was estimated that NPS activities during the period 1 July 2000 to 30 June 2002 generated PBS savings in the range of \$55.6 million to \$83.9 million through the following prescribing intervention programs: antibiotics in primary care; peptic ulcer management; management of dyspepsia; COX-2 selective NSAIDs; managing hypertension; and managing dyslipidaemia \[[@B14]\]. In 2003, the NPS received an additional allocation of government money to provide educational material about drugs newly listed on the PBS (the RADAR project). The latter was in response to considerable evidence that intensive pharmaceutical promotion at the time of PBS listing was associated with drugs being prescribed for broader indications than those indicated in the PBS listing (causing so-called PBS \"leakage\" or \"blow-outs\") \[[@B15]\]. However, the 2003 NPS educational budget of \$12.5 million needs to be compared with the estimated \$1.0 billion promotional budget of the Australian pharmaceutical industry \[[@B16]\]. The Enhanced Divisional Quality Use of Medicines (EDQUM) program was a 1999--2000 Federal Budget initiative, originally announced as the Incentives for Quality Prescribing (IQP) program. The program offered Divisions of General Practice (Divisions) a proportion of monies saved if Divisional QUM activities improved prescribing and lowered PBS costs. The Divisions were allowed to use any savings made for a range of primary health care activities. The program evolved significantly due to feedback from the medical profession. It was implemented on a pilot basis in thirteen Divisions on 1 July 2002. Under the program, Divisions were encouraged to invest their own resources in a range of drug utilisation data collection and/or education related activities. Activities were implemented in close consultation with the NPS and focused on one or more of the following target drug groups: antibiotics, peptic ulcer drugs and cardiovascular drugs. An evaluation of the pilot EDQUM project was undertaken in early 2004 \[[@B17]\]. Barriers to implementation included perceptions that the program was primarily focused on reducing pharmaceutical costs to government; limited capacity of existing prescribing software systems to extract drug utilisation data; and the need for Divisions to take a commercial risk in developing their EDQUM initiatives (due to the absence of up-front funding) which limited their capacity to systematically implement a comprehensive range of strategies. Program achievements included the development of a wide range of shared resource material \[[@B18]\] and the creation (by some Divisions in association with software vendors) of data extraction tools. The latter have allowed a small number of practices to gain access to comprehensive information from which further initiatives to improve quality or change practices can evolve. The evaluation report recommended that standards should be established for prescribing software so that comparable data could be extracted from different systems to facilitate comparison of individual prescribing practice with evidence based guidelines. It also noted the difficulties of attributing any cost-savings in the PBS to Divisional activities. Following this report, the Government supported a four year extension to the EDQUM program in the 2003--2004 Budget. The Third Community Pharmacy Agreement between the Government and the Pharmacy Guild of Australia (1 July 2000 to 30 June 2005) also provided a range of QUM activities over the year in question including medication reviews of problem patients (conducted at the request of GPs), quality care pharmacy programs and the provision of consumer medicine information \[[@B19]\]. A potential new threat that emerged ----------------------------------- In 2003--2004 the PBS became caught up in negotiations concerning the Australia-United States Free Trade Agreement (AUSFTA). This saga has been extensively reported elsewhere \[[@B20],[@B21]\]. The government remained adamant that the AUSFTA provisions concerning the PBS were benign and would also increase the transparency of PBAC decision-making. Others were concerned that the AUSFTA contained major concessions to the US pharmaceutical industry that undermined the egalitarian principles and operation of the PBS and had the potential to increase the costs of medicinal drugs to Australian consumers. Time will tell who is right. Issues still to be tackled -------------------------- The EDQUM project highlighted the needs for software standards in order to extract comparable drug utilisation data from different prescribing systems. The need for prescribing software standards has also been raised in connection with three other issues of relevance to the PBS: pharmaceutical promotion, independent therapeutic information and drug-drug interaction checking. The uptake of computers by Australian general practitioners (GPs) was stimulated by the Australian government in 1999. A one-off grant of around \$10,000 was offered to those practices that purchased a computer, acquired internet connectivity (an E-mail address) and promised to use computer prescribing software to write the majority of their prescriptions. This increased the numbers of GPs writing prescriptions with the aid of a computer from around 50% in 1999 to more than 90% in 2004 \[[@B22]\]. Legible, printed prescriptions have been one of a number of positive outcomes of this initiative. However, new problems emerged. One software vendor (Health Communication Network Ltd.) became the dominant market leader because its business model relied on pharmaceutical promotion to heavily subsidise the cost of GPs purchasing and updating its prescribing software (Medical Director™). This business model facilitated software uptake but also resulted in advertisements for the latest and most expensive drugs appearing on the computer screen at the time of prescribing (and elsewhere). GPs using this software package were shown to prescribe more antibiotics per patient than those who wrote \'scripts manually. It was suggested that this may have been due to default settings in the software automatically writing in the maximum number of repeat prescriptions allowed.\[[@B23]\] Another default option in this software was the automatic production of a, \"Do not substitute generic drugs\" message on the prescription. The latter was eventually changed by the government amending regulation 19(5) of the National Health (Pharmaceutical Benefits) Regulations 1960 \[[@B24]\]. However, the issue of pharmaceutical promotion in prescribing software has yet to be tackled. Pharmaceutical promotion has never been allowed on government supplied \'script pads. It is hard to understand why it was allowed on the computerised equivalent. Pharmaceutical promotion distorts the information flow to physicians by selectively promoting the benefits of the latest and most expensive drugs. It provides minimal information about drug side-effects, contra-indications and opportunity costs. Cost-effective generic drugs are rarely promoted and non-drug solutions usually not at all. Pharmaceutical promotion has clearly been shown to influence physician\'s prescribing \[[@B25]\] and has resulted in cost-blow outs on the PBS due to \"leakage\" of prescribing away from cost-effective indications approved by PBAC \[[@B26]\]. In addition, pharmaceutical promotion in prescribing software, occurring at the time of physician decision making, is likely to be much more influential than promotion in medical journals, gimmicks and give-ways. As a consequence, several medical and consumer organisations have advocated further amendment of the National Health (Pharmaceutical Benefits) Regulations 1960, Part V, Regulation 19, to prohibit prescribing software from displaying pharmaceutical advertisements. Government intervention is also required to ensure that key national resources of objective therapeutic information, such as the Australian Medicines Handbook and Therapeutic Guidelines, are incorporated in prescribing software. The provision of objective therapeutic information is an important strategy of the QUM component of Australian National Medicinal Drug Policy \[[@B27]\]. Ironically, while both the Australian Medicines Handbook and Therapeutic Guidelines have been converted into electronic formats they are not yet included in computerised prescribing software. The problems have included arguments between software vendors and guideline producers over who should pay for the integration and a lack of defined standards for electronic information representation and interfacing. More recently, the NPS RADAR project has shown the way forward. RADAR provides independent information to health professionals about medicines that have a new or a changed listing on the PBS. RADAR drug monographs have recently been incorporated in four leading GP prescribing packages using an open standard interface. This project has moved ahead because the Australian government provided financial support to both the NPS and software vendors to enable the RADAR integration to take place. Following a workshop on electronic decision support, HL7 Australia has presented a work plan to the Australian Health Information Council Electronic Decision Support Steering Committee that would build on the RADAR project by incorporating the Australian Medicines Handbook and Therapeutic Guidelines into clinical software in a standard manner \[[@B28]\]. However, this plan has yet to proceed because of a current review of E-Health policy and reorganisation of its governance \[[@B29]\]. The third area of prescribing software requiring government intervention is standards for drug-drug interaction checking. The NPS tested four popular GP software packages by entering a common set of elderly patients on multiple medications \[[@B30]\]. This revealed very different behaviour by different software packages; some missed serious drug-drug interactions, others produced numerous trivial and clinical unimportant alerts. GPs noted that the latter behaviour caused them to turn off all alerts \[[@B31]\]. There is an urgent need for standards concerning acceptable drug-drug interaction detection &/or external assessment of prescribing software, another item on the HL7 Australia work plan. Conclusions =========== The PBS remained in the media and policy spotlight during 2003--04. While the growth rate of the PBS has slowed during the year under review the sustainability of the Scheme remains an ongoing concern. One strategy adopted by the government was to transfer more of the cost of medicines to consumers through higher PBS co-payments and increased safety-net thresholds. However, such measures can result in higher costs elsewhere if poorer patients forgo necessary medicines and end up being hospitalised with uncontrolled disease. Cost-shifting (and patient inconvenience) was reduced by allowing State and Territory public hospitals limited access to the PBS but these reforms also showed the need for changes in the PBS to make it more suitable for hospital practice and the desirability of further integrating health funding systems. Educational strategies focusing on the quality use of PBS medicines were successfully pursued but would benefit from increased funding. In addition, there was an exploratory attempt to focus the attention of Divisions on PBS costs by rewarding them with a moiety of any money saved by their members through more cost-effective prescribing. However, the difficulties experienced by the EDQUM project in extracting useful drug utilisation data from computerised prescribing systems highlighted the need for prescribing software standards as did other problems with such software. Information communication technology and information management (ICT/IM) has the potential to allow individual health practitioners, Divisions and governments to compare what is being done with what is recommended best-practice, highlight major discrepancies, and provide targeted education and appropriate incentives to reduce the gap. However, as the events of 2003--04 show, this potential is unlikely to be realised if the development of clinical computer systems is left solely to market forces. Competing interests =================== Dr. Harvey is a past Board member of Therapeutic Guidelines Limited, Co-Chair of HL7 Australia\'s Decision Support Technical Committee, a Councillor of the Australia Consumer\'s Association and a member of the Australian Labor Party.

PubMed Central

2024-06-05T03:55:52.638067

2005-1-12

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548296/", "journal": "Aust New Zealand Health Policy. 2005 Jan 12; 2:2", "authors": [ { "first": "Ken J", "last": "Harvey" } ] }

PMC548297

Background ========== Ovaries are the primary source of estrogen. In ovariectomized rats, the production of estrogen is shifted from ovary to a number of extragonadal sites \[[@B1],[@B2]\]. Simpson developed the intriguing concept of extragonadal aromatization, i.e. androgens, particularly androstenedione produced primarily in the adrenal glands, can be converted (aromatized) into estrogens at extraglandular sites, including the mesenchymal cells in adipose tissue and skin, osteoblasts and chondrocytes in bone, vascular endothelial, aortic smooth muscle cells as well as numerous sites in brain \[[@B2],[@B3]\]. Estrogen synthesised within these sites is biologically active in a paracrine or intracrine fashion, although it may escape the local metabolism and enter the circulation \[[@B2],[@B3]\]. Estrogen production in extragonadal sites is dependent on an external source of C19 androgenic precursors \[[@B4]\]. Circulating levels of testosterone (T) and androstenedione as well as dehydroepiandrosterone (DHEA) and DHEA sulphate (DHEAS) become extremely important in terms of providing adequate substrate for estrogen biosynthesis in extragonadal sites. It is now recognized that much of the physiology of androgens is consistent with the concept that T functions as a circulating pro-hormone. In the postmenopausal woman, circulating T levels are an order of magnitude greater than circulating estrogen levels, which by itself suggests the importance of T for maintaining local estrogen levels in extragonadal sites \[[@B2]\]. 65--75% of the circulating T is formed peripherally from androstenedione, DHEA and DHEAS secreted by the adrenals, which form a large precursor reservoir that is available for conversion to T and finally to estrogen \[[@B2]\]. Thus in the species of rat, it is still unknown that whether it is the same situation of T in extragonadal aromatization. We have observed that the release of corticotrophin-releasing hormone (CRH) in the hypothalamic paraventricular nucleus of the ovariectomized rats increased significantly compared with the intact rats \[[@B5]\]. And more interestingly, the hypothalamic CRH expressions showed a gradual elevation along with time after ovariectomy (submitted data), which suggested that the activity of hypothalamus-pituitary-adrenal axis (HPAA) might promote. These observations lead us to hypothesize that more androgens from adrenal cortex might gradually be converted into estrogens by extragonadal aromatase in the rats along with time after ovariectomy. To test the hypothesis, we measured the blood concentrations of estradiol (E~2~) and T as well as the aromatase expressions in adipose and liver tissues in the ovariectomized rats. Methods ======= Animals ------- Female Sprague-Dawley rats (180--200 g), with regular 4-day estrus cycles were purchased from Medical Experimental Animals Centre of Fudan University (Shanghai, China). The animals were housed under laminar flow in an isolated room with controlled temperature and at a 12 /12 (light /dark) schedule. Forty-eight of them underwent ovariectomy with ether anaesthesia, which were then divided randomly into six groups: 1 month after ovariectomy (OVX1M), OVX2M, OVX3M, OVX4M, OVX5M, OVX6M. The corresponding control groups were: intact 1 month (INT1M), INT2M, INT3M, INT4M, INT5M, INT6M. All experimental procedures involving the use of animals were conducted in accordance with NIH Guidelines and were reviewed and approved by the Animal Use and Care Committee for the Fudan University. Tissue collection and preparation --------------------------------- At the time of sacrifice, the vaginal cytology of each rat was first examined. The tissues of the rats were collected respectively, and those of the intact control animals, during the period of diestrous. All the operations were carried out at 4°C. The liver tissues, subcutaneous abdominal (SA) adipose tissues and the adrenals were excised, and then snap-frozen in liquid nitrogen, and stored at -80°C. The preparation of the microsomal pellet was accordance with the report by Hiroshi \[[@B6]\]. Total tissue RNA was extracted using \'TRIzol Regent\' (Biobasic Inc, Canada), and the purity and integrity of the RNA were checked spectroscopically and by gel electrophoresis before analytical procedures. Semiquantitative RNA analysis ----------------------------- To compare the level of adrenal cytochrome P450 17α hydroxylase/lyase (P450c17), liver and SA adipose P450arom expressions after different treatments, PCR methologies were adapted to provide a semiquantitative measure of mRNA levels. Primers were synthesized based on published reports \[[@B7],[@B8]\]. Table [1](#T1){ref-type="table"} summarizes the anticipated size of PCR products, sense and antisense sequences, and locations of primers. Total RNA (2 μg) was transcribed in reverse, in a final volume of 20 μl, using 200IU M-MLV reverse transcriptase in the presence of 25 pmol downstream primer (Sangon Inc), 0.5 mM deoxy-NTP and 20IU Rnasin (from Promega) for 60 min at 42°C before heat denatured for 5 min at 95°C. The cDNAs obtained were further amplified by PCR using 25 pmol of upstream primer (Sangon Inc). We first determined the linear range of amplification of cDNA using each of the primer sets, and then chose an appropriate amplification cycle within this range for each cDNA species. For P450arom and P450c17, we used 35 PCR amplification cycles, and 20 cycles for β-actin gene expression. Each PCR reaction underwent an amplification regimen characterized by (P450arom: 1 min at 94°C, 1 min at 60°C, 2 min at 72°C; P450c17: 1.5 min at 72°C, 2 min at 56°C, 4 min at 72°C) with Taq DNA polymerase (3U per tube) and 2.2 mM magnesium chloride (from Promega) in a final volume of 50 μl. To check the presence of DNA contamination, RT-PCR was performed on 2 μg of total RNA without M-MLV reverse transcriptase (negative control). An internal control (water instead of RNA) for each RT-PCR was performed to investigate RNA contamination of the mixture. For each sample 5 μl of the PCR amplification products were analysed on 2% agarose gels and stained with ethidium bromide. The intensities of the bands were evaluated using the Image Master Software (SYDR-1990, SYNGENE, U.S.A.). The RT-PCR products were extracted and purified from agarose gel by Golden Beads Gel Extraction kit (Sangon Inc., China) and sequenced using radioactive dideoxychain terminating method (Sangon Inc., China). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Properties of oligonucleotide primers used for PCR. ::: ---------------------------------------------------------------------------------------------------------- Target mRNA Size of PCR products (bp) Sense (S) or antisense (AS) Primer sequence (location) ------------- --------------------------- ----------------------------- ---------------------------------- P450c17 299 S\ 5\'-TCATCAAGAAGGGAAAAGAA-3\'\ AS 5\'-TGAAGCAGATAGCACAGATG-3\' P450arom 289 S\ 5\'-GCTTCTCATCGCAGAGTATCCGG-3\'\ AS 5\'-CAAGGGTAAATTCATTGGGCTTGG-3\' β-actin 550 S\ 5\'-AAGCAGGAGTATGACGAGTCCG-3\'\ AS 5\'-GCCTTCATACATCTCAAG TTGG-3\' ---------------------------------------------------------------------------------------------------------- ::: Western blotting ---------------- A 50 μg sample of the microsomal protein was loaded into each lane along with a prestained protein size marker (Bio-Rad Laboratories, Inc., Hercules, CA), electrophoresed on a 10% SDS-polyacrylamide gel at 18 V/cm, and electroblotted onto a polyvinylidene difluoride membrane (Micron Separations, Westboro, MA) using a wet electroblotter. After blocking in fat-free milk, incubation was conducted with the antiaromatase antibody (1:200; Boster Biological Technology LTD., China) and β-actin antibody (1:3000) at room temperature in 18°C for 4 h in TBS-T solution (20 mM Tris, 137 mM NaCl, and 0.1% Tween-20, pH 7.6). After extensive washing, blots were incubated with AP-labelled goat antirabbit antiserum (Sino-American Biotechnology Co., China) for 60 min at room temperature in 18°C and developed using NBT/BCIP detection system (Amersham Pharmacia Biotech). The intensities of the bands were evaluated using the Image Master Software (SYDR-1990, SYNGENE, U.S.A.), and values were normalized to β-actin immunoreactivity in each sample and expressed as percent of the control. Specificity of the aromatase immuno-staining was determined by preincubation of antiserum for 24 h at 4°C with varying concentrations of aromatase, with the primary antibody omitted to identify non-specific staining as well. RIA of blood estrogen and testosterone concentrations ----------------------------------------------------- At the time of sacrifice, the blood samples (0.8 ml) of the rats were collected from tail veins respectively, and the corresponding intact controlling animals, during the period of diestrous. The plasma was separated by centrifugation and stored at -70°C until assayed. Concentration of blood hormones were determined by double-antibody RIA kits purchased from the National Atomic Energy Research Institute (Beijing, China.). The samples were assayed in duplicate, and all the subjects\' samples were assayed together. The sensitivity of the kit was 0.8 pg/ml (testosterone) and 1.4 pg/ml (estrogen), the intra- and interassay coefficients of variation, 3.7--8.0% and 4.74--7.7%. Statistical analysis -------------------- All data are presented as means ± S.E.M. Statistical analysis was performed on raw data using two-way analysis of variance (ANOVA), with the significance set at p \< 0.05 and p \< 0.01 in two-tailed testing chosen. Results ======= Vaginal cytology of the animals ------------------------------- The epithelial cells were stained by haematoxylin-eosin (HE). The intact rats (INT1M, 2M, 3M, 4M, 5M, 6M) showed regular 4-day estrus cycle change. The cyclic change disappeared in the ovariectomized (OVX1M, 2M, 3M, 4M, 5M, 6M) rats. A few of mature vaginal epithelia were observed in the smears of the OVX5M and OVX6M rats, and the percent of mature epithelia increased significantly in the OVX6M rats (p \< 0.05) (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The percent of mature vaginal epithelia and the blood concentrations of E~2~and T of the rats ::: Groups The percent of mature vaginal epithelia Blood E~2~level (pg/ml) Blood T level (pg/ml) -------- ----------------------------------------- ------------------------- ----------------------- ---------------- 1M INT (15.6 ± 0.88)% 56.60 ± 14.13 27.92 ± 1.74 OVX (0.19 ± 0.019)%\* 4.06 ± 1.36\* 18.91 ± 2.53\* 2M INT (15.8 ± 0.91)% 59.12 ± 11.23 26.39 ± 2.04 OVX (0.17 ± 0.015)%\* 8.94 ± 1.78\* 18.03 ± 2.20\* 3M INT (14.8 ± 0.90)% 58.25 ± 13.93 28.86 ± 2.19 OVX (0.20 ± 0.025)%\* 16.72 ± 3.71\* 20.21 ± 1.96\* 4M INT (15.1 ± 0.86)% 59.35 ± 10.35 26.99 ± 2.14 OVX (0.19 ± 0.020)%\* 28.10 ± 8.88\*^\#^ 20.89 ± 2.69\* 5M INT (16.4 ± 1.01)% 63.34 ± 15.85 27.03 ± 1.93 OVX (0.26 ± 0.034)%\* ^\#^ 28.97 ± 6.61\*^\#^ 21.35 ± 2.33\* 6M INT (16.8 ± 1.06)% 59.15 ± 13.34 29.36 ± 2.56 OVX (0.29 ± 0.027)%\* ^\#^ 31.05 ± 6.61\*^\#^ 21.54 ± 2.09\* \*p \< 0.05 vs corresponding intact; \# p \< 0.05 vs OVX1M ::: Blood concentrations of estrogen and testosterone ------------------------------------------------- The blood E~2~concentrations decreased significantly in the OVX1M, 2M and 3M (p \< 0.01) compared with those in the INT1M, 2M and 3M. The concentrations in OVX4M, 5M and 6M groups increased significantly (p \< 0.05) compared with OVX1M, though still lower than INT4M, 5M and 6M. There were no disparities between the INT1M, 2M, 3M, 4M, 5M and 6M groups (Table [2](#T2){ref-type="table"}). The blood T concentrations decreased significantly in the OVX groups compared with corresponding intact controls (p \< 0.05). Though there were slightly increases in the OVX3M, 4M, 5M and 6M compared with OVX1M, no statistical significances were detected (Table [2](#T2){ref-type="table"}). RT-PCR analysis: Effects of ovariectomy on adrenal P450c17, liver and SA adipose P450arom mRNA levels ----------------------------------------------------------------------------------------------------- Comparison of the amplified PCR fragments with rat P450c17 and ovary aromatase cDNA sequences revealed 100% homology (data not shown). Densitometric analysis of the mRNA concentration using target product/β-actin was expressed as the mean with SEM. The ratios of liver P450arom to β-actin in the OVX1M, 2M, 3M, 4M and 5M groups were lower than the corresponding intact controls (p \< 0.05). The ratio increased significantly in the OVX6M compared with OVX1M (p \< 0.05), and no difference was detected between OVX6M and INT6M (Fig [1](#F1){ref-type="fig"}). The ratios of SA adipose P450arom to β-actin in the OVX1M, 2M, 3M, 4M and 5M groups appeared no significant changes compared with those in the corresponding intact control, and the ratio in the OVX6M was higher than that in all other groups (p \< 0.05) (Fig [2](#F2){ref-type="fig"}). The ratio of adrenal P450c17 to β-actin in the OVX1M decreased significantly compared with that in the INT1M (p \< 0.05), and those in the OVX4M and OVX5M increased slightly, with statistical disparities between OVX4M and INT4M, or OVX5M and INT5M. In the OVX6M, the ratio increased markedly, and was higher than OVX1M (p \< 0.05) (Fig [3](#F3){ref-type="fig"}). However, no changes occurred on the ratios of adrenal P450c, liver and SA P450arom among INT1M, 2M, 3M, 4M, 5M and 6M(Fig [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"},[3](#F3){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **RT-PCR analysis of liver P450arom mRNA expressions of the rats.**The upper picture shows the gel electrophoresis of the RT-PCR products for the liver P450arom. Densitometric analysis of the mRNA concentration using PCR product/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. \* p \< 0.05 vs corresponding intact controls, \# p \< 0.05 vs OVX1M. ::: ![](1477-7827-3-6-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **RT-PCR analysis of SA adipose P450arom mRNA expressions of the rats.**The upper picture shows the gel electrophoresis of the RT-PCR products for the SA adipose P450arom. Densitometric analysis of the mRNA concentration using PCR product/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. \* p \< 0.05 vs corresponding intact controls, \# p \< 0.05 vs OVX1M. ::: ![](1477-7827-3-6-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **RT-PCR analysis of adrenal P450c17 mRNA expressions of the rats.**The upper picture shows the gel electrophoresis of the RT-PCR products for the adrenal P450c17. Densitometric analysis of the mRNA concentration using PCR product/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. \* p \< 0.05 vs corresponding intact controls, \# p \< 0.05 vs OVX1M. ::: ![](1477-7827-3-6-3) ::: Western blot analysis: Effects of ovariectomy on liver and SA adipose P450arom protein levels --------------------------------------------------------------------------------------------- Densitometric analysis of the protein concentration using aromatase/β-actin was expressed as the mean with SEM. The ratios of liver P450arom to β-actin in the OVX groups were lower than the corresponding intact controls (p \< 0.05). But the ratio increased significantly in the OVX6M compared with OVX1M (p \< 0.05) (Fig [4](#F4){ref-type="fig"}). The ratios of SA adipose P450arom to β-actin in the OVX1M and OVX6M increased significantly compared with those in the INT1M and INT6M (p \< 0.05). And the ratios in the OVX2M, 3M, 4M and 5M produced no disparities compared with corresponding intact controls (Fig [5](#F5){ref-type="fig"}). No disparities were detected among INT1M, 2M, 3M, 4M, 5M and 6M (Fig [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}). No immunoreactive bands detected in the samples when using antiserum after preabsorption with excessive antigens and omission of the primary antibody. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Western blot analysis of liver P450arom expressions of the rats.**The upper picture shows the Western blot analysis of the liver aromatase P450. Densitometric analysis of the protein concentration using aromatase/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. \* p \< 0.05 vs corresponding intact controls, \# p \< 0.05 vs OVX1M. ::: ![](1477-7827-3-6-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Western blot analysis of SA adipose P450arom expressions of the rats.**The upper picture shows the Western blot analysis of the SA adipose aromatase P450. Densitometric analysis of the protein concentration using aromatase/β-actin expressed as the mean with SEM bar in each column indicated in the lower panel. \* p \< 0.05 vs corresponding intact controls, \# p \< 0.05 vs OVX1M. ::: ![](1477-7827-3-6-5) ::: Discussion ========== The most interesting findings in the present study were that the circulating E~2~levels increased gradually along with time after ovariectomy in the rats. If ovary is not the source of estrogen in ovariectomized rats, then the question arises as to the origin of the estrogens found in the circulation. Extragonadal aromatization has been in a general sense recognized, although its significance is only becoming to be appreciated, as will be explained further. It has been reported that aromatization in the adipose tissue is not negligible under normal and pathological conditions \[[@B9]\]. Due to the highest conversion of C19 precursor such as androstenedione to estrogen was observed in adipose tissue obtained from the subcutaneous abdominal wall but not from intraperitoneal cavity \[[@B10]\], our research focused on the aromatase activity of the SA adipose tissues. In the present results, increased aromatase protein and mRNA expressions were observed in the OVX6M rats\' SA adipose tissue. Hemsell and co-workers \[[@B11]\] first addressed the significance of adipose tissue as a major source of estrogen production, i.e. there is a progressive increase in the conversion efficiency with advancing age, and the increase of estrogen production as a function of obesity \[[@B12]-[@B14]\]. The body fat of ovariectomized rats increases significantly \[[@B15]\], and the mesenchymal cells from the SA adipose tissue could be active, which might be closely related to the higher aromatase expression in the OVX6M. Yet, only in the OVX6M was there an associated significant increase of the aromatase expression in the SA adipose tissue, but not in other OVX groups, which by itself suggested that the elevated expression of aromatase in the OVX6M was not closely implicated in the obesity of the rats. We have also observed that the content of hypothalamic CRH increased along with time after ovariectomy in the rats, with a significant elevation in the OVX6M (submitted data). It has been reported that CRH may regulate the expressions of extragonadal aromatase via CRH receptor-II \[[@B16]\], which may help us understand the involved mechanism of the enhanced SA adipose aromatization and the eventual increased E~2~in the OVX6M rats, but further studies are earnestly needed. However, in the present results, it\'s confusing that the protein but not mRNA expression of SA adipose aromatase in the OVX1M showed an elevation compared with that in the INT1M. This might provide evidence indicating the inconsistency on the levels between transcription and translation of the aromatase gene. The factors possibly involved in the regulation of aromatase expression are still poorly understood \[[@B17]\]. Many studies have been performed to assess the possible dependence of the enzyme on androgens, though the data available are conflicting \[[@B17],[@B18]\]. The situation is complicated by the fact that, in postmenopausal women, only about 25% of circulating T is derived by direct secretion from the ovaries \[[@B2]\]. The rest is formed largely from circulating precursors derived from the adrenal cortex. In the present study, the blood concentrations of T in the rats were detected. In the OVX groups there were decreases of the T level compared with corresponding intact ones, and no significant changes occurred along with the time after ovariectomy. These might be implicated in the less importance of T in the extragonadal aromatisation in rats than in women. Nevertheless, it\'s the first time to present the dynamic changes of T levels in the rats after ovariectomy. But further explorations are still needed on the way by which the aromatase enzyme expression is activated in ovariectomized rats. The microsomal enzyme cytochrome P450c17 is an important regulator of steroidogenesis. The enzyme has two functions: 17alpha-hydroxylase and 17,20-lyase activities. These functions determine the ability of adrenal glands and gonads to synthesize sex steroids (17,20-lyase activity) \[[@B19]\]. Its activity is abundant in testis, and lesser in ovary, and low levels of P450c17 activity in adrenals \[[@B20]\]. It is well known that adrenal is the principle organ to secrete sexual hormones except ovarian in females \[[@B21]\]. In the present study, due to the T levels did not show significant change along with the time after ovariectomy, the mRNA expressions of P450c17 were semi-quantified by RT-PCR to indirectly report the androgen synthesis in the adrenals. Excitedly, the ratio of adrenal P450c17 to β-actin in the OVX4M and OVX5M increased slightly, and in the OVX6M, the ratio increased markedly, which was higher than OVX1M. These suggested that the androgen synthesis activity of adrenal might be enhanced, and the more androgens might secrete from adrenal cortex. Though it has been reported that the splanchnic tissue is a minor site for extragonadal aromatization of androgens \[[@B22]\], there is a significant conversion of androstenedione to estrone by liver tissues \[[@B15]\]. In adult liver homogenates, C19 norsteroid (19-nortestosterone; NT) is readily aromatized to estrogens \[[@B23],[@B24]\]. The present results showed that the aromatase expressions (mRNA and protein) of the liver tissue in the OVX1M, 2M, 3M, 4M and 5M groups were lower than the corresponding intact controls, and in the OVX6M the ratio increased significantly compared with that in the OVX1M. On one hand, after ovariectomy, the diminution of C19 precursor from ovaries for aromatization may induce the decreased expressions of aromatase in the OVX rats compared with the gonad-intact rats in our results. On the other hand, the results suggested that aromatization of liver tissue enhanced six months after ovariectomy. However, in the present study, the aromatase expressions in the OVX rats did not show consistent changes between the SA adipose and liver tissues, which may suggest that the role of SA adipose and splanchnic tissues in the extraglandular aromatisation might be different. Nonetheless, in the final analysis, our results suggested that both the SA adipose and liver tissues contributed to the extragonadal aromatisation to promote the circulation estrogen concentrations. Conclusions =========== Both the subcutaneous abdominal adipose tissues and the liver tissues contributed to the extragonadal aromatization to promote the circulating E~2~levels in the rats along with time after ovariectomy; the adrenal compensation might also be activated naturally. Authors\' contributions ======================= Hong Zhao and Zhanzhuang Tian designed the study, performed the studies and the statistical analysis, and drafted the manuscript. Junwei Hao performed the animal experiment. Boying Chen conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript. Acknowledgements ================ This project was financed by the Shanghai Acupuncture Research Centre (No. 03DZ19554-3) and Shanghai Medical Health Bureau (the Key Project 20003 on Traditional Chinese Medicine).

PubMed Central

2024-06-05T03:55:52.640138

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548297/", "journal": "Reprod Biol Endocrinol. 2005 Jan 21; 3:6", "authors": [ { "first": "Hong", "last": "Zhao" }, { "first": "Zhanzhuang", "last": "Tian" }, { "first": "Junwei", "last": "Hao" }, { "first": "Boying", "last": "Chen" } ] }

PMC548298

Background ========== World Health Organization published the Global Tuberculosis Control Report (2003) on \'World Tuberculosis Day\' where India is ranked number one in the world for high incidence of smear positive cases of pulmonary (about 0.9 million) and extrapulmonary TB (about 0.2 million) every year \[[@B1]\]. Undoubtedly, TB itself has reemerged in India as a serious problem since 1985 with the advent of HIV/AIDS \[[@B2]\]. However, cutaneous TB is still rare in countries like India (0.10%), Hong Kong (0.07%) and Madrid (0.14%) \[[@B2]\] and it manifests either as a true bacterial invasion or as a tuberculid (hypersensitivity reaction) with primary focus elsewhere. Evidences of bacterial invasion are found in lupus vulgaris, the most common manifestation (55%) in patients with cutaneous TB (1975--95) in northern India \[[@B2]\], primary chancre, tuberculous verrucosa cutis, scrofuloderma, tuberculous cutis orificialis and tuberculous cutis miliaris disseminates \[[@B3]\] whereas erythema induratum (Bazin disease) and lichen scrofulosorum are tuberculid lesions \[[@B2]\]. Moreover, atypical mycobacteria such as *M. kansasii*, *M. scrofulaceum*, rather than *M. tuberculosis*are the most common etiological agents for cutaneous TB in HIV/AIDS and other immunocompromised patients. WHO defines acquired drug resistance as the isolation of drug-resistant *M. tuberculosis*from a patient who has been treated for TB for 1 month or longer \[[@B4]\] and primary drug resistance as the isolation of drug-resistant strain from a patient without a history of previous treatment. The selection of *M. tuberculosis*with mutations conferring resistance to antitubercular drugs may result from poor management including the prescription of incorrect regimens and non-compliance with treatment. We report a rare case of reinfection with same but MDR *M. tuberculosis*in a polymyositis patient on immunosuppressive therapy resulting in multiple cutaneous abscesses and miliary TB. Case Presentation ================= A 23-year-old polymyositis patient with suggestive clinical symptoms, muscle biopsy, enzyme assay, needle EMG and NCV test was on immunosuppressive drugs such as corticosteroids at 1 mg/kg/day for 3 to 4 weeks, tapered gradually over a period of 10 weeks to 1 mg/kg every alternate days and methotrexate at 7.5 mg weekly with gradual increase to 25 mg weekly for a period of 1 year when he experienced slightly improved muscle power. He had single cutaneous abscess 6 months later on the right side of the neck with no sinus formation and cervical and axillary lymphadenopathy. He was HIV-negative with normal CXR and there was no AFB in three consecutive (spot-early morning-spot) sputum samples (induced in two occasions). However, the aspirated pus from cutaneous abscess and biopsy of the axillary lymph node showed plenty of *M. tuberculosis*as culture was confirmed by niacin and nitrate reduction tests, rapid bacteriophage assay (FAST *Plaque*Tuberculosis kit, BIOTEC Laboratories Ltd, UK) and RFLP analysis (figure [1](#F1){ref-type="fig"}) using species-specific probes for *devR*(Differentially expressed virulent gene) \[[@B5]\]. In RFLP analysis, the *devR*gene (Gene bank nucleotide sequence accession no. U22037) encodes a response regulator that is part of a two-component signal transduction system of *M. tuberculosis*. The authenticity of the amplified product was established by hybridization of immobilized PCR products to an internal oligonucleotide, *devR1*, mapping within the *devR*gene. The chromosomal DNA was prepared by chloroform-isoamyl alcohol DNA extraction and 4.5 μg of DNA from the isolate was restricted with *Pvu*II. Separation of *Pvu*II-restricted DNA by electrophoresis, Southern blot hybridization with a 513-bp PCR probes for *devR*(*devRf*, 5\' GGTGAGGCGGGTTCGGTCGC 3\'; *devRr*, 5\' CGCGGCTTGCGTCCGACGTTC 3\') and chemiluminescence detection were done according to the standard method recommended for the DNA fingerprinting of *M. tuberculosis*\[[@B6]\]. Histopathologically there were nonspecific necrosis with disintegrating polymorphs, very few granulomatous cells and no epithelioid cells in the biopsy specimen. USG of kidney, liver or spleen revealed no abscess. The strain was susceptible to the 4 first line drugs \[isoniazide (H), rifampicin (R), ethambutol (E) and pyrazinamide (Z)\] by BACTEC 460TB system. Treatment with 4-drug regimen (H = 300 mg/d, R = 450 mg/d, E = 800 mg/d and Z = 1.5 g/d) for 8 months (2EHRZ/6HR) \[[@B7]\] with first 2 months supervised showed clinical improvement. However, the patient returned 6 months later with multiple cutaneous abscesses, mainly on his back, left thigh and left arm and miliary mottling in CXR. He denied any irregularity in antituberculosis therapy. All immunosuppressive drugs (corticosteroids and methotrexate) were discontinued as he was readmitted. Isolates from 2 of 3 consecutive sputum samples and aspirated pus samples from all 5 completely drained abscesses were reconfirmed as the identical *M. tuberculosis*strain by phage assay and RFLP analysis (figure [1](#F1){ref-type="fig"}). The isolate was resistant to 4 first-line drugs by BACTEC 460TB system and to 9 drugs, tested into Loewenstein Jensen media (Hi-media, Mumbai, India): H (1 μg/ml), H (10 μg/ml), R (20 μg/ml), R (50 μg/ml), E (2 μg/ml), E (10 μg/ml), Z (50 μg/ml, at pH 5.5), streptomycin (5 μg/ml), streptomycin (50 μg/ml), paraaminosalicylate (2.5 μg/ml), cycloserine (30 μg/ml), amikacin (700 μg/ml) and ciprofloxacin (12.5 μg/ml). Treatment was continued with H (5 mg/kg/d), R (10 mg/kg/d), Z (30 mg/kg/d), E (15 mg/kg/d), kanamycin (15 mg/kg IM 5 times weekly) and sparfloxacin (500 mg/d) for 18 months with first 2 months under supervision. A slow but complete clinical, microbiological and radiological cure after 1.5 year was followed up for another 16 months. Discussion ========== India is an endemic country for TB with an estimated 20,000 infectious cases and 1--3.3% of new cases of MDR TB every year \[[@B8]\]. Infections are common with more than one strain of *M. tuberculosis*during the same episode (multiple infection), in different lesions (multiple infection) or during successive episodes (reinfection) in HIV-positive as well as -negative individuals \[[@B9]\]. In this study identical strains of *M. tuberculosis*were isolated at 6 months interval, but contrary to the first, the strain in the second episode was resistant to all the first line and most of the second line antitubercular drugs. As far as could be ascertained, this is the first case of isolation of MDR strain of *M. tuberculosis*from a HIV-negative but immunosuppressed patient who had an infection with the same, but susceptible strain 6 months before. As evident from the study, the chance of exogenous reinfection with the same but drug-resistant strain from some undetected source within the endemic community is the most likely explanation. This explains the acquisition of resistance against all those drugs, to which the patient was not exposed earlier. Reinfection though occurs usually after first two to five years in immunocompetent hosts, may progress to active disease at any time after treatment has been discontinued and even during treatment for active tuberculosis \[[@B10]\]. Moreover, an ongoing tuberculous infection and simultaneous immunosuppressive therapy might significantly divert the immune response, thereby increasing the overall susceptibility to \'superinfection\' \[[@B10]\] with the same but MDR strain. The chances of \'simultaneous infection\' \[[@B11]\] with both the strains (susceptible and MDR) could be another possibility. Drug-susceptibility testing could discriminate simultaneous infection with different susceptibility profiles if individual colonies from the isolate were tested whereas in our case the sensitivity pattern of the susceptible strain might have been obtained at first attempt. Even RFLP and phage typing were not enough to determine simultaneous infections or reinfection (superinfection). Endogenous reactivation which is higher than the rate of exogenous reinfection in endemic countries \[[@B12],[@B13]\] with multi- \"drug resistance in previously treated case\"\[[@B14]\] might be a remote possibility. The history of regular medication itself might be notoriously misleading in patients with multidrug resistance \[[@B14]\]. However, it is difficult still to explain the acquisition of multidrug resistance of the strain in endogenous reactivation in such a short period against those drugs, to which the patient was not exposed. Any switch in specimens or cross-contamination of cultures in laboratories were unlikely since no other sample to switch or contaminate was identified. Niacin test (95% positive \[[@B15]\]) and nitrate reduction test (97% positive \[[@B15]\]) differentiated *M. tuberculosis*from other mycobacteria in *M. tuberculosis*complex whereas RFLP and phage typing excluded atypical mycobacteria, like *M. kansasii*and *M. scrofulaceum*. The overall acuracy of the drug susceptibility test ranges between 84--100%, since mycobacteria often clump \[[@B16]\]. We tried thorough vortexing with glass beads to obtain homogenous inoculum suspensions. It was a rare case of miliary TB of non-reactive type \[[@B17]\] with MDR *M. tuberculosis*invasion of the skin without any muscle involvement and sinus formation as the biopsy of the axillary lymph node showed non-specific necrosis containing disintegrating polymorphonuclear leukocytes and enormous number of AFB. This type is more often seen with severe HIV infection than in patients with immunosuppressive therapy \[[@B18],[@B19]\], where the liver and the spleen are most commonly involved followed by the lung, the bone marrow and the kidney \[[@B17]\], granulomas and epithelioid cells are lacking and CXR shows inconspicuous diffuse mottling. In our case, there was no other organ involvement. Moreover, the rapid skin and lung involvement despite adequate antituberculosis therapy suggests that the tubercle bacilli were somehow protected from the drugs in the cutaneous and subcutaneous tissue (\'paradoxical expansion of disease during therapy\' \[[@B20]\]) either due to polymyositis-associated subcutaneous calcification or due to obstruction in the small arteries with the large mass of bacilli leading to necrosis and abscess formation in the surrounding areas. This is probably the first reported case of cure where all first and most of the second line drugs (a total of 9 antitubercular drugs) were found to be resistant. However, the 6-drug antituberculosis therapy might not be considered as the only reason for cure, since 4 first line drugs showed high-level resistance and chances of cross-resistance between closely-related aminoglycosides, amikacin and kanamycin and quinolones, sparfloxacin and ciprofloxacin could not be ignored \[[@B21],[@B22]\]. Discontinuation of immunosuppressive therapy with a reconstitution of the immune system and complete surgical drainage of the abscesses might have proved to be an important adjunct to the treatment. Conclusions =========== Severe immunosuppression may lead to disseminated TB such as miliary TB or other rare types of extra-pulmonary TB such as cutaneous abscesses. Follow-up of patients is important, and if response to treatment is poor, adherence to treatment, drug resistance and other possible reasons such as continuation of immunosuppressive therapy should be considered. In this case intervention by drainage of abscesses, discontinuation of immunosuppressive treatment and possibly long-term treatment with additional second line antituberculosis drugs eventually lead to cure. Abbreviations ============= AFB -- Acid fast bacilli AIDS -- Acquired immunodeficiency disease syndrome CXR -- Chest radiograph DNA -- Deoxyribonucleic acid EMG -- Electromyography HIV -- Human immunodeficiency virus MDR -- Multidrug resistant *M. tuberculosis*- *Mycobacterium tuberculosis* NCV -- Nerve conduction velocity PCR -- Polymerase Chain Reaction RFLP -- Restriction-fragment length polymorphism TB -- Tuberculosis UK -- United Kingdom USG -- Ultrasonography WHO -- World Health Organisation Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= CM conceived the study, carried out the case study and follow-up, in the clinical as well as microbiological aspects, formatted the study design, performed sensitivity of the organism, participated in the FAST *plaque*assay and molecular identification method and drafted the manuscript. AG carried out FAST *plaque*assay and molecular identification method. AA participated in the design of the study and acted as an overall supervisor. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/4/63/prepub> Acknowledgements ================ We acknowledge the cooperation on behalf of the patient and his relatives and the helpful attitude on behalf of our clinician friends during the study. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The PCR amplified product for *devR*gene (513 bps) of *M. tuberculosis*. (From left to right) Lane M; 100 bp ladder Banglore Genei (India), Lane 1; Negative control, Lane 2; *M. tuberculosis*H37Rv, Lane 3; Patient\'s isolate of sensitive *M. tuberculosis*strain (on first admission), Lane 4; Patient\'s isolate of MDR *M. tuberculosis*strain (on readmission), Lane 5; *M. kansasii*, Lane 6; *M. scrofulaceum*. ::: ![](1471-2334-4-63-1) :::

PubMed Central

2024-06-05T03:55:52.642409

2004-12-23

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548298/", "journal": "BMC Infect Dis. 2004 Dec 23; 4:63", "authors": [ { "first": "Chiranjoy", "last": "Mukhopadhyay" }, { "first": "Ankita", "last": "Garg" }, { "first": "Archana", "last": "Ayyagari" } ] }

PMC548299

Background ========== Lipoprotein lipase (LPL) hydrolyzes triglycerides in very low-density lipoproteins and chylomicrons \[[@B1]\]. Tissue-specific regulation of LPL activity is a major mechanism to distribute lipids among tissues according to the physiological needs \[[@B2]\]. Current information indicates that in adipose tissue, the regulation is post-translational and occurs by a shift of the lipase protein towards an inactive form under the influence of another gene with short-lived message and product \[[@B3]\]. This information derives from *in vivo*experiments. To study the underlying mechanism an *in vitro*model is urgently needed. Experiments with isolated adipocytes do not seem to bring out the mechanism and the *in vivo*experiments indicate that it is the extracellular LPL that is the target for the regulation \[[@B4]\]. We have therefore explored the possibility to use tissue explants and report our experiences in this paper. The results support the view that it is the extracellular enzyme that is being regulated. Unfortunately the preparation of tissue explants seems to trigger a proteolytic response in the tissue. Results ======= LPL activity and mass decreased when explants of rat adipose tissue were incubated ---------------------------------------------------------------------------------- In the first set of experiments we incubated explants of rat adipose tissue and followed LPL mass and activity (Figure [1](#F1){ref-type="fig"}). LPL mass decreased by more than 50% in three hours. The decrease then continued so that after six hours only around 20% of the original mass remained. With tissues from fed rats, in which most of the LPL protein is in the catalytically active form, the LPL activity decreased in parallel to LPL mass. In tissues from fasted rats, in which most of the LPL protein is in the catalytically inactive form, the decrease was less steep for LPL activity than for LPL mass. The inset in Figure [1](#F1){ref-type="fig"} shows that there was a delay of about two hours before the decrease of LPL mass accelerated. There was no difference between tissue from fed and fasted rats. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Changes in LPL mass and activity during incubation of adipose tissue explants.**Epididymal adipose tissue was dissected from fed and 24 h-fasted rats, cut into small pieces and rinsed in cold PBS. About 100 mg tissue was then incubated at 37°C for the indicated times as described in the Methods section. LPL mass (triangles, panel A) and activity (circles, panel B) in tissue explants from fed (filled symbols, ● and ▼) or fasted (open symbols, ○ and ▽) rats. Values at the start of the incubations were for LPL mass: 17.4 ± 1.1 and 17.1 ± 2.5 ng/μg DNA and for LPL activity: 6.3 ± 1.8 and 3.5 ± 0.7 mU/μg DNA, in tissues from fed and fasted rats, respectively. The inset shows a separate experiment where incubations were stopped at shorter times. Only LPL mass was followed in that experiment. Some of the data points for fed and fasted rats fall on top of each other. Values are mean ± SEM for five parallel incubations. ::: ![](1471-2121-6-4-1) ::: We tried variations in technique and several different incubation media in experiments such as those in Figure [1](#F1){ref-type="fig"}. There was some variation in the absolute values but the results were in principle the same, a rapid decline of LPL mass and activity. The rate at which LPL mass decreased was similar with tissue explants from fed and fasted rats, and LPL activity roughly followed LPL mass in tissues from fed rats. One possible explanation could be that LPL was released from the tissue into the medium. The amounts of LPL mass or activity that appeared in the medium were, however, small (Table [1](#T1){ref-type="table"}). To test whether lipase might have adsorbed to the plastic dishes these were rinsed out with warm SDS solution, but only small amounts of LPL protein were recovered (Table [1](#T1){ref-type="table"}). Hence it is clear that there was a loss of LPL mass from the system. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Changes in LPL mass during incubation. Explants of rat adipose tissue were incubated for three hours as in Figure 1. The tissue explants and the medium were recovered and then the vessel was rinsed out with warm (\~80°C) SDS solution. This was then suitably diluted with Triton X-100 to match the composition of the medium used for the ELISA. Mean ± SEM of five parallel incubations. ::: LPL mass (ng/g tissue) ------------------ ------------------------ ------------------ Before incubation After incubation Adipose tissue 1882 ± 13 665 ± 11\* Culture medium 26 ± 6 Washing solution 12 ± 3 Total 1882 ± 13 702 ± 8 \* \* P \< 0.001 compared with before incubation ::: Another possibility was that the cells lost their ability to produce LPL. Isolated adipocytes, incubated under the same conditions as the tissue explants, released LPL activity (Figure [2](#F2){ref-type="fig"}) and mass (data not shown) to the medium, while cellular activity (Figure [2](#F2){ref-type="fig"}) and mass (data not shown) increased slightly. Total LPL activity in the system increased by about 60% during four hours of incubation. When cycloheximide was added, the release of LPL to the medium was virtually abolished and cellular LPL activity decreased with time. Total LPL activity in the system decreased by almost 70%. This demonstrates that the cells depend on synthesis of new LPL protein to sustain LPL activity and secretion to the medium. Adipocytes isolated from tissue explants that had been incubated for three or six hours as in Figure [1](#F1){ref-type="fig"} retained the ability to release LPL to the medium (not shown). Hence, the loss of LPL activity that occurred when tissue explants were incubated was not due to a loss of LPL production within adipocytes. In these experiments we also noted that the release of LPL from adipocytes was similar whether the cells were isolated from fed or fasted rats (not shown). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **LPL activity in cells and in medium during incubation of isolated adipocytes, and the effect of heparin.**Adipocytes (from 180 -- 200 g rats) were incubated under the same conditions as used for the tissue explants in Figure 1 without (filled symbols) or with (open symbols) 0.1 mg/ml cycloheximide. ●, ○ -- adipocytes; ▼, ▽ -- medium. Mean ± SEM for five wells at each time. ::: ![](1471-2121-6-4-2) ::: Is the loss of LPL mass an intra- or extracellular event? --------------------------------------------------------- Heparin release is often used to assess the LPL activity of tissues and releases mainly extracellular LPL \[[@B4]\]. Figure [3](#F3){ref-type="fig"} shows that in fresh explants of adipose tissue a substantial fraction of tissue LPL could be released by heparin. More LPL was released from tissues of fed rats (Figure [3](#F3){ref-type="fig"}). When the tissue explants were incubated for three hours before the heparin challenge, much less LPL was released and after six hours virtually no LPL was released (Figure [3](#F3){ref-type="fig"}). This suggested that the decrease of LPL affected mainly the extracellular enzyme. To test this hypothesis we isolated adipocytes from tissue explants after three or six hours of incubation (Figure [4](#F4){ref-type="fig"}). The LPL activity and mass in the adipocytes was the same when the cells were isolated from tissue explants that had been incubated for three or six hours as when they were isolated from fresh tissue explants. Hence, it was extracellular LPL (calculated as the difference between tissue total and adipocytes) that accounted for the rapid decrease of tissue LPL during incubation. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Changes in the amount of heparin-releasable LPL mass during the incubation.**Conditions as in Figure 1 but at the designated times, the explants of adipose tissue were transferred to new medium containing heparin and incubated for a further 45 min. Values are means ± SEM for five parallel incubations. Black bars represent explants from fed rats; grey bars represent explants from fasted rats. ::: ![](1471-2121-6-4-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **LPL activity and mass within and outside the adipocytes.**Conditions as in Figure 1 but adipose tissue explants from 180--200 g rats were used to get enough material to isolate adipocytes. At the end of the incubation some of the tissue explants were incubated with collagenase and adipocytes were isolated as described in the methods section. Filled symbols -- fed rats; open symbols -- fasted rats. Panel A shows LPL mass. ▲, △ -- tissue, ▼, ▽ -- adipocytes. Panel B shows LPL activity. ■, □ -- tissue, ●, ○ -- adipocytes. Values are means ± SEM for five parallel incubations. Some of the data points for fed and fasted rats fall on top of each other. ::: ![](1471-2121-6-4-4) ::: Is the LPL protein degraded? ---------------------------- These results indicated that the decline of LPL mass occurred through proteolytic cleavage of the extracellular enzyme. To test this hypothesis, we included a cocktail of protease inhibitors in the medium used for incubation of tissue explants. This slowed down the decrease of LPL mass (Figure [5A](#F5){ref-type="fig"}). Chloroquine had no effect (Figure [5B](#F5){ref-type="fig"}), indicating that the degradation did not occur in lysosomes. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Effect of protease inhibitors on the decrease of LPL mass during incubation of tissue explants.**Conditions as in Figure 1. A cocktail of protease inhibitors (panel A) or chloroquine (final concentration 150 mM, panel B) were included in the medium of some of the incubations. The tissue was from fed rats. Similar results (not shown) were obtained with tissue from fasted rats. ○ -- without protease inhibitor, ● -- with protease inhibitor. Mean ± SEM for five parallel incubations. ::: ![](1471-2121-6-4-5) ::: To further study the proteolytic process, ^125^I-LPL was added to the incubations. TCA soluble material appeared in the medium demonstrating that proteolytic degradation took place (Figure [6](#F6){ref-type="fig"}). We noted that some of the TCA precipitable material became associated with the tissue explants suggesting, binding and/or uptake of the lipase. To explore if the degradation required that the lipase was taken up into cells in the tissue we incubated the labelled lipase in conditioned medium. Analysis by SDS-PAGE showed that several fragments were formed (Figure [7](#F7){ref-type="fig"}). Addition of either of two non-specific MMP inhibitors, Captopril or GM6001, prevented the degradation almost completely. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Fate of ^125^I-LPL added to the incubation.**Conditions as in Figure 1 but ^125^I-LPL was added to the medium. ● -- TCA precipitable radioactivity in medium, ▲ -- TCA precipitable radioactivity in the tissue explants, ■ -- TCA soluble radioactivity in medium. Mean ± SEM for five parallel incubations. ::: ![](1471-2121-6-4-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Analysis by SDS-PAGE of the cleavage of ^125^I-LPL in conditioned medium and the effect of an MMP inhibitor**Explants of adipose tissue from fed rats were incubated as in Figure 1 for four hours and the medium was collected. ^125^I-LPL was then incubated at 4°C (to minimize the risk of conformational changes) in this medium with or without the MMP inhibitor GM6001 (5 μg/ml). Lane 1 -- fresh medium, lane 2 -- conditioned medium, lane 3 -- conditioned medium + inhibitor. The lower band in lane 1 is a proteolytic fragment of LPL that is always present in preparations of the enzyme from bovine milk \[23\]. ::: ![](1471-2121-6-4-7) ::: Discussion ========== The objective for this study was to find an *in vitro*system to study the mechanism for down-regulation of LPL activity in rat adipose tissue that occurs on food deprivation. Isolated adipocytes have been tried in several laboratories \[[@B5]-[@B9]\], but the differences with nutritional state are rather small. This is true whether one measures LPL within the cells or the rates at which the cells secrete LPL to the medium. These observations are repeated here. The lack of difference within adipocytes indicates that other cell types are needed and may in fact be responsible for the pronounced down-regulation of extracellular LPL activity that occurs on food deprivation \[[@B3],[@B4]\]. We therefore tried to use tissue explants, which have proved valuable in other studies of adipose tissue \[[@B10]\]. Our results show that degradation of the enzyme was a major process when explants were incubated. The degradation occurred extracellularly; LPL mass and activity in the adipocytes did not change during incubation for up to six hours. Added ^125^I-LPL was degraded and this was prevented by addition of MMP inhibitors to the medium. There must be damage of cells when the tissue is cut into small pieces, and there is probably some degree of hypoxia during incubation of the pieces. Cultured explants have, however, been widely and successfully used to explore various aspects of adipose tissue biology (\[[@B10]\] and references therein). In preliminary experiments we found that the explants retained their ability to take up glucose, to synthesize proteins and to secrete leptin. Adipocytes isolated from the explants after several hours of incubation had the same LPL activity as adipocytes from fresh tissue (Figure [4](#F4){ref-type="fig"}). This indicates that the cells produced LPL at an essentially unchanged rate, since the LPL activity in adipocytes decreased rapidly when protein synthesis was inhibited by cycloheximide (Figure [2](#F2){ref-type="fig"}). Hence, the rapid decrease of LPL when explants were incubated was not due to a general loss of functionality, but reflected specific processes leading to degradation of extracellular LPL. Our results are in line with observations made already in the sixties. There was evidence from chromatographic separations for at least two different forms of LPL in rat adipose tissue \[[@B11]\]. Cunnigham and Robinson found that incubation of fat pads from fed rats resulted in a rapid loss of LPL activity until a low activity, stable to prolonged incubation, was attained \[[@B12]\]. In contrast, the LPL activity of isolated fat cells was stable to prolonged incubation. The concept of stable and unstable forms of the lipase can now be interpreted as a reflection of extracellular lipase that is exposed to proteolysis, and intracellular lipase that is protected from the extracellular proteases. The degradation of LPL in conditioned medium was almost completely abolished by the two non-specific MMP inhibitors that we tested. We have not characterized the proteolytic activity further but note that adipose tissue produces at least two MMPs, 2 and 9 \[[@B13]\]. The loss of LPL mass during incubation of tissue explants was relatively slow during the first two hours and then accelerated. It is likely that the tissue trauma and/or the loss of blood circulation triggered an activation of the MMP system. It has been shown that primary culture of human adipose tissue explants dramatically alters adipocyte gene expression \[[@B14]\]. It is of interest to note that LPL activity does not decrease during perfusion of fat pads \[[@B15]\], whereas it does decrease when whole fat pads are cut out and incubated *in vitro*\[[@B12]\]. Two pathways for turnover of adipose tissue LPL have been demonstrated so far. One is dissociation of the lipase, perhaps after loss of catalytic activity, into the blood and degradation in the liver \[[@B16]\]. Release of LPL into blood from adipose tissue has been directly demonstrated by measurement of arterio-venous difference in man \[[@B17]\]. This pathway can, however, not operate in tissue explants. Another pathway, demonstrated with cultured fat cells, is endocytosis and degradation in lysosomes \[[@B18]\]. This pathway did not seem to contribute significantly in the present system since the rate at which LPL mass decreased was not affected by chloroquine. The present findings suggest a third pathway, extracellular proteolysis in the tissue. Conclusions =========== The rapid decrease of LPL that occurs when adipose tissue explants are incubated engages only the extracellular enzyme. The adipocytes continue to produce and secrete the enzyme and intracellular LPL remains essentially constant for at least six hours. The decrease in extracellular LPL is due to proteolytic cleavage/degradation of both active and inactive forms of the enzyme. The effects of inhibitors indicate, but do not prove, that the degradation is mediated by MMPs. It appears that this process is accelerated in the tissue fragments compared to intact tissue. Methods ======= Animals ------- Male Sprague-Dawley rats were from Möllegaard Breeding Center (Ejby, Denmark). Unless otherwise stated, the rats were 23 days old and weighed around 60 g. After transport to Umeå they were allowed to acclimatize for seven to ten days by which time they had reached a weight of approximately 120 g. The rats were kept in a well ventilated, temperature (21°C) and humidity (40--45%) controlled room with free access to a standard laboratory chow (Laktamin AB, Stockholm) and tap water. The light in the room was on between 6 a.m. and 6 p.m. In experiments where the rats were to be fasted, food was withdrawn from the cages at 6 a.m. and a grid was placed at the bottom of the cages to prevent coprophagia. The adipose depot used in all experiments was the periepididymal one. The rats were killed by decapitation. Animal experiments were approved by the animal ethics committee in Umeå. Materials --------- Cycloheximide, bovine serum albumin (BSA), the MMP inhibitors GM6001 (Galardin) and Captopril, chloroquine and collagenase were from SIGMA (St. Louis, MO, USA). Protease inhibitor cocktail tablets \"Complete Mini\" were from Roche Diagnostics, Mannheim, Germany. Heparin was from Lövens (Malmö, Sweden). Substrate for the LPL activity assay was ^3^H-labelled triolein in Intralipid (10%) kindly prepared by Pharmacia-UpJohn (Stockholm, Sweden). Parker medium (Parker 199) was from SBL (Stockholm, Sweden). ^125^I-LPL was prepared as before \[[@B19]\]. All other reagents were of the highest commercial grade possible. Assays ------ LPL was extracted from tissues by homogenization in a Tris-HCL buffer (pH 8.2) containing detergents and protease inhibitors as described \[[@B20]\]. The homogenate was centrifuged for 15 min at 3000 rpm after which the intermediate phase (between the floating fat droplets and the pellet) was used for assay of LPL activity and mass. In most cases the extract was kept on ice and assayed within a few hours. Under these conditions LPL activity is stable. In some cases the extracts were frozen and kept at -70°C for later assay. LPL activity was measured as described previously \[[@B20]\]. Briefly, two μl of tissue homogenate (triplicate samples) was incubated for 60 min at 25°C with substrate in the presence of ten μl heat-inactivated serum from fasted rats (as source of apolipoprotein CII) and 6% BSA. The total volume was 200 μl. After termination of lipolysis by addition of organic solvents, the fatty acids were extracted and counted for radioactivity. One mU of lipase activity represents one nmol of fatty acids released per minute. LPL mass was measured with an ELISA as described \[[@B20]\]. The chicken antibodies used recognize both active and inactive forms of the lipase \[[@B21]\]. Briefly, three different dilutions of tissue homogenate were incubated in microtiter plate wells previously coated with affinity-purified chicken anti-LPL IgG. Detection was mediated via the 5D2 monoclonal antibody (a kind gift by Dr John Brunzell, University of Washington, Seattle) followed by a peroxidase conjugated anti-mouse IgG antibody. Absorbance at 490 nm was measured in a Spectramax microplate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). DNA content was assayed using Labarca\'s method \[[@B22]\]. *In vitro*incubation of adipose tissue -------------------------------------- Epididymal adipose tissue was dissected out from fed or 24 h fasted rats. The tissue was cut into small pieces (5 mg or less). A total of about maximal 100 mg tissue pieces were immediately put into culture plates. Each well contained 15 ml of Parker Medium 199 supplemented with 2% BSA, 0.5% casein hydrolysate, 10 mM glucose and adjusted to pH 7.4. Incubations were at 37°C and 5 % CO~2~: 95 % O~2~with continuous gentle shaking motion in a Cellstar Incubator (Queue Systems, Asheville, Canada). After incubation, the tissues were prepared for measurement of LPL activity and mass as described above. In some experiments samples of the medium were taken for assay of LPL activity and/or mass. In some experiments adipocytes were prepared after collagenase treatment of the tissue pieces as described \[[@B4]\]. To measure heparin releasable LPL (HR-LPL), adipose tissue explants were incubated with heparin (final concentration was 50 IU/ml) for 45 min at 37°C. Statistics ---------- Student\'s t-test was used for analysis of the data. Authors\' contributions ======================= GW carried out the experiments and participated in their design and in writing of the manuscript, TO participated in the design of the study and drafted the manuscript. GO conceived of the study and coordinated the work. All authors read and approved the final manuscript. Acknowledgements ================ This study was funded by the Swedish Medical Research Council Projects no. 727, 12203 and 12663.

PubMed Central

2024-06-05T03:55:52.643966

2005-1-25

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548299/", "journal": "BMC Cell Biol. 2005 Jan 25; 6:4", "authors": [ { "first": "Gengshu", "last": "Wu" }, { "first": "Gunilla", "last": "Olivecrona" }, { "first": "Thomas", "last": "Olivecrona" } ] }

PMC548300

Background ========== The coalescent is a very versatile stochastic model of the genetic variation in a set of sequences sampled from a population. It allows to accommodate a wide range of assumptions about rates and modes of evolution, and of population history \[[@B1]-[@B5]\]. As the observed sequence data are positively correlated due to common ancestry, coalescent theory also provides a framework for understanding the relationship between a population\'s history and its genealogy. For instance, it has long been noted that genealogies of samples taken from exponentially growing populations tend to be \"star-like\" with short branch lengths near the root of the tree. In contrast, the inter-node distances in genealogies from constant-size populations typically are much more evenly spaced. Thus, coalescent theory quantifies the imprint that demographic development of a population leaves in the data. While the original theory was outlined for constant population size \[[@B1],[@B2]\], Slatkin and Hudson \[[@B6]\] soon developed a coalescent model for the case of an exponentially growing population. Subsequently, a general approach allowing arbitrary population size variation through time was presented by Griffith and Tavaré \[[@B7]\]. Therefore at least in principle the coalescent model provides a basis for *statistically inferring the demographic history*as a function of time from the sampled sequences \[[@B3],[@B8]-[@B12]\] or, alternatively, from the corresponding inferred genealogies \[[@B13]-[@B15]\]. In practice, however, application of coalescent theory to this problem has been restricted to very simple demographic scenarios such as constant size, exponential or logistic growth. Only recently methods have emerged that attempt the completely non-parametric estimation of the demographic function from the data. Polanski et al. proposed an approach based on pairwise distances \[[@B16]\], hence generalizing the method by Slatkin and Hudson \[[@B6]\]. Pybus et al. \[[@B14]\] presented the \"skyline plot\" method that uses a step-function to approximate the population history obtained from an estimated genealogy. This method was subsequently refined to the \"generalized skyline plot\" \[[@B17]\] which is essentially a regularized version of the classic skyline plot. If the population size is truly constant through time the generalized skyline plot estimate of population size collapses to the phylogenetic coalescent estimator proposed by Felsenstein \[[@B13]\]. The advantage of the skyline plot over the method suggested by Polanski et al. \[[@B16]\] is that it takes into account the genealogical relationship among the sequences. This helps to decrease bias and improves the efficiency of the resulting estimator compared to methods based on summary statistics and pairwise distances \[[@B13]\]. Unfortunately, the skyline plot approach also has several deficiencies. First, it is unclear how to extend the approach to allow multiple genealogies as input. This is important in order to accommodate phylogenetic error, and to allow non-parametric inference of population history in coalescent approaches that take all possible genealogies into account \[[@B7]-[@B10]\]. Second, and perhaps more critical, the (generalized) skyline plot only provides a population size trend rather than a realistic estimate of population size changes, as by construction the population function is modeled by a step function. Moreover, the change-points of this function are fixed at the inter-nodes of the underlying tree. In this paper we propose a novel framework to non-parametric estimation of the demographic history. This approach relies on Bayesian reversible-jump MCMC inference \[[@B18]\] to obtain a smooth population size function from a given set of genealogies. The new method not only renders many deficiencies of the classic and generalized skyline plot obsolete but it is also computationally efficient, with running times of the algorithm for typical data in the order of minutes on standard PC hardware. The framework has been implemented in the computer language R \[[@B19]\] and incorporated in the R package APE \[[@B20]\]. The remainder of the paper is organized as follows. In the next section we describe the mathematical and statistical theory of the new framework. Subsequently, we apply the method to simulated and biological sequence data and discuss the results. In the last section we briefly outline possible further extensions and related directions of research. Results ======= Background in coalescent theory ------------------------------- ### Basic model In a pan-mictic population with constant effective population size *N*~*e*~, where every individual has a single parent, the waiting time *w*~*n*~until any two of *n*sampled lineages coalesce is exponentially distributed with rate ![](1471-2148-5-6-i1.gif)\[[@B1],[@B2]\]. For *n*sequences there are therefore *n*- 1 intervals *I*~*n*~, *I*~*n*-1~,\..., *I*~2~with rates *r*~*n*~, *r*~*n*-1~,\..., *r*~2~and interval lengths *w*~*n*~, *w*~*n*-1~,\..., *w*~2~. With ![](1471-2148-5-6-i2.gif) we denote the time until all samples have reached the most recent common ancestor. The coalescent model implies that the waiting time to the next coalescent event follows an inhomogeneous Poisson-process with a hazard rate *r*~*n*~that varies in time *t*because of the change in the number of lineages. Thus, it is straightforward to also include variable population size in the coalescent simply by using the hazard rate ![](1471-2148-5-6-i3.gif). From standard theory in survival analysis \[[@B21]\] it follows that the corresponding density for the waiting times is given by ![](1471-2148-5-6-i4.gif) where *τ*~*i*~is the time at the beginning of the interval *I*~*i*~. This is exactly the distribution from the variable population size coalescent ![](1471-2148-5-6-i5.gif) as developed in \[[@B7]\]. The coalescent model can be further expanded to diploid populations \[[@B22]\] or to include other effects like selection, recombination or geographical structures \[[@B4]\]. In this paper, however, we focus solely on the coalescent/survival model given by Eq. 2. ### Estimation of population size If the waiting times *w*~*i*~are known Eq. 2 can be used directly to estimate *N*~*e*~(*t*). This is typically done by maximizing the likelihood ![](1471-2148-5-6-i6.gif) assuming a simple parametric model for the population size change. For constant population size this has been done in \[[@B13]\], for more complicated scenarios such as logistic growth see, e.g., \[[@B14]\]. In a typical setting, however, the waiting times are themselves estimated from sequence data. In this case the total likelihood function will be a weighted sum of the likelihoods for all possible waiting times, so that in effect the *w*~*i*~are marginalized out in favor of the actually observed data. In practice exact calculation of this sum is prohibitive, hence one relies on approximating MCMC methods \[[@B8]-[@B10]\]. As a shortcut to avoid these computationally very expensive procedures one may also substitute the \"true\" waiting times by those obtained from inter-node distances of a single estimated gene tree (see, e.g., \[[@B23]\] for an overview of relevant likelihood-based tree inference methods) and proceed as above. Note that the resulting plug-in approximation ignores the uncertainty from estimating the *w*~*i*~in the inference of demographic parameters. However, this is justifiable if the phylogenetic error is much smaller than the error introduced by the coalescent. This will be the case if sequences are sufficiently long and the substitution rate is comparatively high (a typical example would be virus data). For non-parametric estimation of population size, Pybus et al. suggested the \"skyline plot\" \[[@B14]\]. This method assumes a piece-wise constant function for the population size *N*~*e*~(*t*) and allows population size changes only at the beginning and end of an interval *I*~*i*~. The estimated effective population size ![](1471-2148-5-6-i7.gif) in interval *I*~*i*~according to the skyline plot is given by the simple relation ![](1471-2148-5-6-i8.gif) This is the maximum likelihood estimate under the assumed model of fixed change-points. The \"generalized skyline plot\" subsequently introduced by Strimmer and Pybus \[[@B17]\] reduces the over-fitting present in the classic skyline plot by applying a simple form of regularization: adjacent intervals that alone are likely to have high stochastic noise are pooled together (cf. Fig. [2b](#F2){ref-type="fig"} and [2d](#F2){ref-type="fig"}). Choice of an optimal grouping of intervals (i.e. model selection) is performed by employing a second-order variant of the Akaike criterion \[[@B24]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Comparison of prior and posterior demographic functions***Top row*: Bayesian inference using a prior demographic function with constant mean and constant variance (a 95% confidence band is indicated by showing the 2.5% and 97.5% quantiles). *Bottom row*: Bayesian inference using the \"skyline plot\" prior function. ::: ![](1471-2148-5-6-2) ::: A Bayesian non-parametric approach to estimating demographic history -------------------------------------------------------------------- ### Outline In this paper we present a non-parametric approach to infer population size changes in time that overcomes the limitations of previous approaches. More specifically, we develop a non-parametric Bayesian estimator for the function *N*~*e*~(*t*) conditioned on observed or sampled inter-node distances *w*~*n*~, *w*~*n*-1~,\..., *w*~2~by determining the posterior distribution P(*N*~*e*~(*t*)\|*w*~*n*~, *w*~*n*-1~,\..., *w*~2~). In order to sample the non-parametric demographic function from this posterior we use the reversible jump Markov chain Monte Carlo (rjMCMC) algorithm \[[@B18]\]. As a result, we obtain for any given time *t*both a point estimate ![](1471-2148-5-6-i9.gif) -- here we choose the posterior median -- as well as the associated credible interval (e.g., the lower and upper 2.5% quantiles). If the considered inter-node distances *w*~*n*~, *w*~*n*-1~,\..., *w*~2~are fixed and obtained from a single estimated tree, the resulting method is already directly applicable to phylogenetically informative data such as viral sequences (this is the focus of this paper). However, sampling of non-parametric demographic functions can also be combined in a conceptually straightforward fashion with sampling of trees, as outlined below. ### Bayesian inference using reversible jump MCMC In a nutshell, Bayesian inference of a parameter *x*consists of updating its prior distribution P(*x*) to a posterior distribution P(*x*\|*D*) that takes account of the information in the observed data *D*. The relative evidence of the data for different values of *x*is summarized in the likelihood *L*= P(*D*\|*x*) that accordingly plays a central role in the computation of the posterior via Bayes\' theorem ![](1471-2148-5-6-i10.gif) For most realistic problems the posterior distribution cannot be computed analytically, in particular if *x*is a high-dimensional vector. Instead, one utilizes computational procedures to efficiently draw random samples from the posterior. This in turn allows computation of summary statistics such as the median or the upper and lower 2.5% quantiles. Markov chain Monte Carlo (MCMC) is one particularly useful sampling algorithm as it doesn\'t require calculation of the sum (or integral) in the nominator of Eq. 4. Briefly, sampling via MCMC is done by constructing a Markov chain with the possible combinations of parameter values as \"states\", and the desired posterior as its stationary distribution. These properties can be guaranteed by following certain rules for accepting or rejecting proposed new parameter values. Here we use the Metropolis-Hastings-Green method, i.e. the reversible jump MCMC algorithm \[[@B18]\], that has the advantage of not only allowing changes in the parameters values but also in the dimension of the parameter vector itself. Specifically, if *x*is the initial state, and ![](1471-2148-5-6-i11.gif) a proposed new state with proposal density ![](1471-2148-5-6-i12.gif), then the acceptance probability according to Green \[[@B18]\] is ![](1471-2148-5-6-i13.gif) where ![](1471-2148-5-6-i14.gif) is the likelihood ratio P(*D*\|![](1471-2148-5-6-i11.gif))/P(*D*\|*x*), ![](1471-2148-5-6-i15.gif) is the prior ratio P(![](1471-2148-5-6-i11.gif))/P(*x*), ![](1471-2148-5-6-i16.gif) is the proposal ratio *q*(![](1471-2148-5-6-i11.gif))/*q*(*x*), and ![](1471-2148-5-6-i17.gif) is the determinant of the Jacobian resulting from the potential change of dimension of the parameter vector. Accordingly, for the application of MCMC to infer the functional form of demographic history a variety of components need to be specified: • a suitable *parameterization*of the estimated function *N*~*e*~(*t*) • the *likelihood function*, • a *prior distribution*for each considered variable, and, • rules to construct the *Markov chain*(i.e. acceptance probabilities). In the following sections we now describe each of these elements in detail. For further general information on the statistical and mathematical background of the MCMC algorithm we refer to the many excellent monographs on this topic (e.g., \[[@B25]\]). ### Parameterization of *N*~*e*~(*t*) In our suggested procedure we approximate the sampled demographic history *N*~*e*~(*t*) by a piecewise linear function. This spline of first order degree consists of a first node at position *a*~0~= 0 and height *h*~0~, followed by *k*internal supporting nodes at (*a*~1~; *h*~1~), (*a*~2~;*h*~2~),\..., (*a*~*k*~; *h*~*k*~), and a terminal node at ![](1471-2148-5-6-i18.gif) with height *h*~*k*+1~Hence, the spline is defined for all *t*∈ \[0, *T*\], and for any given *k*the it contains *k*free position parameters and *k*+ 2 free height parameters. Note that, unlike in the skyline plot, we do not constrain the change-points *a*~1~,\..., *a*~*k*~to lie on the grid points defined by the inter-node distances *w*~*i*~. Moreover, we also allow that the number of internal nodes *k*changes during sampling of the population function from the posterior. Hence, *k*is technically a hyper-parameter that controls the roughness of the resulting spline. As will be clear from the outline of the MCMC algorithm below, note that the final point estimate ![](1471-2148-5-6-i9.gif) obtained from posterior sampling will be a mixture of linear splines (i.e. a smooth and possibly nonlinear function) rather than a single spline. ### Likelihood function The likelihood *L*employed in our procedure is the product of the densities of the waiting times between subsequent coalescence events, i.e. ![](1471-2148-5-6-i19.gif). This function depends via Eq. 2 on the effective population size *N*~*e*~(*t*), and hence indirectly on the spline parameters *a*~*i*~, *h*~*i*~and *k*. Because *N*~*e*~(*t*) is represented by a linear spline, calculation of the likelihood can be done in a computationally efficient fashion. ### Prior distributions #### Number of change-points Following \[[@B18]\] we employ a truncated Poisson-distribution as the prior distribution for *k*, i.e. ![](1471-2148-5-6-i20.gif) where *c*is a normalizing constant to ensure that P(*k*) is a proper distribution. For the hard upper limit of the number of change-points we use *k*~max~= 30. The parameter *λ*acts as a smoothing parameter, set in a typical analysis to about *λ*= 0.1 - 1.0. As an alternative to using a fixed *λ*we also suggest a hierarchical Bayes approach where *λ*is drawn from a Gamma distribution ![](1471-2148-5-6-i21.gif) with some shape parameter *a*and scale parameter *b*(for instance, *a*≈ 0.5 and *b*≈ 2 so that E(*λ*) = *ab*≈ 1 and Var(*λ*) = *ab*^2^≈ 2). #### Positions We assume that the internal nodes of the spline are *a priori*uniformly distributed in the interval \[0, *T*\]. As a simple trick to avoid very small inter-node distance we generate *2k*+ 1 random variables, and set the change-points *a*~*j*~= *z*~\[2*j*\]~for *j*= 1,\..., *k*. The corresponding joint density is ![](1471-2148-5-6-i22.gif) with *a*~0~= 0 and *a*~*k*+1~= *T*. #### Heights As prior distributions for the heights *h*~*i*~we assume a Gamma distribution Gamma(*h*~*i*~\|*α*~*i*~, *β*~*i*~) (9) which ensures that sampled heights are always positive. The parameters *α*~*i*~and *β*~*i*~determine the *a priori*mean and variance of height *h*~*i*~. More generally, one can also allow fully time-dependent prior parameters *α*(*t*) and *β*(*t*). This is particularly advisable if the population size is known in advance to be subject to large changes in time. In a strict Bayesian approach, the choice of the prior distribution for the heights is completely external to the observed data. One simple possibility would, e.g., be to assume an arbitrary constant for the mean and variance. However, we recommend to follow a more pragmatic \"empirical Bayes\" route and to use the data at hand (or some other related data set) to obtain an informed guess about the prior heights. For example, an assumed constant population size as prior mean could be estimated using the method by Felsenstein \[[@B13]\]. Another possibility is to employ the skyline plot as a prior mean estimate (this is the default in our program). However, note that in practice the actual choice of prior height distribution seems to matter only little for estimating the posterior demographic function (see Figure [2](#F2){ref-type="fig"} and the section on simulated data below). Only when there are few coalescent events per unit of time will the posterior estimate of the demographic function be dominated by the prior. ### Construction of the Markov chain There are four different possibilities to change the state defined by the parameters *c*~*i*~, *h*~*i*~, and *k*of the spline describing the effective population size *N*~*e*~(*t*): 1\. varying the position of a change-point (i.e. internal node), 2\. changing the height at a certain change-point, 3\. generating a new change-point (\"birth\" step), and 4\. deleting an existent change-point (\"death\" step). Let *η*~*k*~, *π*~*k*~, *b*~*k*~, and *d*~*k*~the probabilities of the four moves given *k*, with *η*~*k*~+ *π*~*k*~+ *b*~*k*~+ *d*~*k*~= 1. In order to satisfy the requirement of detailed balance in the corresponding Markov chain the probabilities of birth and death steps (*b*~*k*~and *d*~*k*~) need to be synchronized \[[@B18]\]. This can be achieved, e.g., by setting ![](1471-2148-5-6-i23.gif) and ![](1471-2148-5-6-i24.gif) where *c*is chosen so that *b*~*k*~+ *d*~*k*~\< 0.9 for all *k*. Next, we describe the individual procedures to propose and accept one of the above four moves as implemented in our program. #### Height change First, a height *h*~*j*~is selected out of the *k*+ 2 existing heights with probability ![](1471-2148-5-6-i25.gif). Second, a new height ![](1471-2148-5-6-i26.gif) is generated by ![](1471-2148-5-6-i26.gif) = *h*~*j*~exp(*z*), where *z*is a uniformly distributed random variable on ![](1471-2148-5-6-i27.gif). Third, the new height is accepted with probability ![](1471-2148-5-6-i28.gif) where *α*and *β*are from the prior distribution and ![](1471-2148-5-6-i14.gif) denotes the ratio of the likelihood of the new state ![](1471-2148-5-6-i11.gif) (with modified height) and the likelihood of the current state *x*. #### Position move First, a change-point *a*~*j*~is chosen randomly with probability ![](1471-2148-5-6-i29.gif). Second, its new position ![](1471-2148-5-6-i30.gif) within \[*a*~*j*-1~, *a*~*j*+1~\] is determined by drawing from the corresponding uniform distribution. Third, ![](1471-2148-5-6-i30.gif) is accepted with probability ![](1471-2148-5-6-i31.gif) #### Birth step First, the position *a*\* of the new change-point is found by uniformly drawing from (0, *L*), and let the neighboring nodes left and right of *a*\* have positions *a*~*j*~and *a*~*j*+1~. Second, the corresponding new height *h*\* is generated by randomly disturbing the current height *N*~*e*~(*a*\*) on the position *a*\* according to *N*~*e*~(*a*\*) + *zN*~*e*~(*a*\*) where *z*is a uniformly distributed random variable on the interval ![](1471-2148-5-6-i27.gif). Note that the birth step increases the dimension of the parameter vector from 2*k*+ 2 to 2*k*+ 4 as a new change-point and a new height are generated. The corresponding acceptance probability of the birth step is computed according to Eq. 5 with likelihood and prior ratios as above, and with proposal ratio ![](1471-2148-5-6-i32.gif) and Jacobi determinant ![](1471-2148-5-6-i33.gif) #### Death step This is the inversion of the birth step and consists of removing a change-point. First, *a*\* chosen from *a*~1~,\..., *a*~*k*~with probability ![](1471-2148-5-6-i29.gif). Second, the corresponding height *h*\* is also removed from the vector of spline parameters. The acceptance probability for the death step is ![](1471-2148-5-6-i34.gif) where the proposal ratio and the Jacobi determinant is the same as for the birth step. ### Computation of estimated *N*~*e*~(*t*) and associated confidence intervals In order to obtain an estimate of the effective population size in time we now proceed as follows. First, the Markov chain is started with an initial state that corresponds to a completely flat demographic function, i.e. *N*~*e*~(*t*) = *c*, where c is some rough estimate of population size, and *k*= 0. Second, 100,000 repeats of the MCMC algorithm are performed, of which the first 5,000 are ignored to allow for a \"burn-in\" period. Third, the remaining samples are thinned out by a factor of 1:50 to remove auto-correlation. As a result, 1900 independent samples from the joint posterior of the spline parameters *a*~*i*~, *h*~*i*~and *k*are obtained. Subsequently, in order to obtain a point estimate ![](1471-2148-5-6-i9.gif) and associated confidence bands we compute the distribution of the effective population size at a number of fixed equidistant time points *t*~1~, *t*~2~,\..., *t*~1000~∈ \[0, *T*\]. Finally, we report as summary statistics the corresponding median and the lower and upper 2.5% quantiles. ### Extension to multiple genealogies In this paper we have introduced non-parametric sampling of demographic histories assuming a fixed underlying genealogical tree (or equivalently, a fixed set of inter-node distances *w*~*n*~, *w*~*n*-1~,\..., *w*~2~.) However, in our approach -- unlike previous non-parametric methods such as the skyline plot -- it is also conceptually straightforward to incorporate phylogenetic error. This can be done by joint sampling of trees and demographies according to the following simple algorithm: 1\. Given sequence data *D*, sample a tree *G*\* with clock-like branch lengths (see, e.g, refs. \[[@B8],[@B9],[@B11],[@B12],[@B26]\] for suitable methods). 2\. Use the method described in this paper to sample the demographic function conditioned on the inter-node distances ![](1471-2148-5-6-i35.gif) from *G*\*. 3\. Repeat steps 1 and 2 to obtain the posterior distribution for the population size function, now conditioned on *D*rather than on some given *w*~*n*~, *w*~*n*-1~,\..., *w*~2~. Note that each sampled tree may have a different depth ![](1471-2148-5-6-i36.gif). This means that the interval \[0, *T*\] for the prior (and posterior) height distribution has to be set in advance (and independent of the *T*\*). For the case of 0 \<*t*\<*T*\* sampling of heights then proceeds as described above, while for *T*\* \<*t*\<*T*-- the region with no data from a given sampled tree -- the heights are simply drawn from the respective prior distribution. Discussion ========== In order to test the potential of the proposed reversible jump MCMC algorithm we first applied it to synthetic data simulated according to various demographic scenarios. Subsequently, we reanalyzed two viral data sets from Central Africa and Egypt. Computer program ---------------- The proposed framework has been implemented by us for the case of a single underlying genealogy. The program is written in the statistical computer language R \[[@B19]\] and is incorporated in recent versions of the R package APE \[[@B20]\]. To install the APE package, simply run the R program, and enter at the R prompt install.packages(\"ape\") This downloads the APE package from the Internet. The proposed reversible jump MCMC approach is implemented in the function \"mcmc.popsize\" of which an extensive description along with examples can be obtained online by typing library(\"ape\") help(mcmc.popsize) into the R command window. The APE package also includes routines for plotting the inferred population function (e.g., all figures in this paper were prepared with APE). Note that the use of this R program is only valid if the phylogenetic error is low -- this is typically the case when the evolutionary rate is high and the available sequences are long (e.g. viral data). If the phylogenetic error is not negligible compared to the coalescent error, please use software such as BEAST \[[@B27]\]. Simulated data -------------- In the simulation setup we followed Pybus et al. \[[@B14]\] and Strimmer and Pybus \[[@B17]\]. Specifically, we performed simulations assuming constant population size (*N*~*e*~(*t*) = 100) as well as exponential population growth (*N*~*e*~(*t*) = l000*e*^-*t*^), using 25 and 100 sampled lineages, respectively. To estimate the population size function we employed the proposed MCMC algorithm and the classic and generalized skyline plot. In the former the smoothing parameter *λ*was drawn from the hierarchical model with default parameters (*a*= 0.5 and *b*= 2). Figure [1](#F1){ref-type="fig"} shows the results from a typical run of the simulations. The top row illustrates the case of constant population size, whereas the bottom row demonstrates exponential growth. On the left in Figure [1](#F1){ref-type="fig"}, top row, the true underlying constant population size is shown (the thick dashed line), together with the estimate provided by the classic skyline plot. On the right, this is contrasted with the estimate obtained by using our reversible jump MCMC algorithm. Clearly, the median of the posterior distribution of *N*~*e*~(*t*) is a very good point estimator of the true demographic history. In addition, the 95% confidence band is also automatically obtained by the MCMC method. Interestingly, it can be immediately seen that the uncertainty in *N*~*e*~(*t*) increases with a growing distance from the present. This simply reflects the fact that near the root of the tree for constant population size there are only few coalescent events. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Simulated data***Top row*: Example of a simulation with constant population size: (left) true demographic history (dashed line) and estimate obtained with the classic skyline plot; (right) point estimate obtained with rjMCMC and 95% confidence band. *Bottom row*: Example with exponential population growth: (left) true population growth and classic skyline plot; (right) results from rjMCMC approach. ::: ![](1471-2148-5-6-1) ::: In Figure [1](#F1){ref-type="fig"}, bottom row, an example for a simulation with an exponentially growing population is shown. As for the constant population, the rjMCMC algorithm is capable of recovering the original population size function (shown as thick dashed line) complete with confidence bands, whereas the skyline plot contains a large amount of stochastic noise, and only provides a rough exploratory picture of the population size changes. In Figure [2](#F2){ref-type="fig"} the influence of the choice of prior demographic function on the final posterior estimate is investigated using further simulations of an exponentially growing population. The left column depicts the prior distributions (specifically the 2.5%, 50% and 97.5% quantiles for each time point) for two typical cases: a constant prior function (= constant population size with constant variance), and the \"skyline plot\" prior function (= time dependent piecewise- constant population size and variance). The right column of figure [2](#F2){ref-type="fig"} presents the corresponding posterior distributions as obtained with the present rjMCMC approach. The results for both cases are very similar. This indicates that there is sufficient signal in the data to make the posterior demographic function (almost) independent from the choice of prior distribution. Note that only near the left and right end of the investigated time intervals there are some slight differences. These can be explained by the lack of data points near the borders. HIV-1 in Central Africa ----------------------- Next, we applied our method to infer the demographic history from a set of HIV-1 sequences from Central Africa. These data was originally used by Vidal et al. \[[@B28]\] who examined the genetic diversity of HIV-1 type M in this region. Further detailed analysis can be found in Rambaut et al. \[[@B29]\] and Yusim et al. \[[@B30]\]. Here we use the reconstructed phylogeny of Yusim et al. with which Strimmer and Pybus also estimated the demographic history by means of the generalized skyline plot \[[@B17]\]. Figure [3](#F3){ref-type="fig"} shows the result of the analysis with the reversible jump MCMC algorithm compared with the predictions from the classic and generalized skyline plots. As in Yusim et al. \[[@B30]\] an evolutionary rate of 0.0023 substitutions per year was assumed to convert the time axis into units of years. The first row of Figure [3](#F3){ref-type="fig"} displays the tree of Yusim et al. \[[@B30]\] and the corresponding classic skyline plot. The latter exhibits a large amount of noise, nevertheless the main demographic signal is clearly visible in the graph. In contrast, in Figure [3c](#F3){ref-type="fig"} (second row) the effective population size as estimated by the rjMCMC algorithm is displayed. The thick line shows the median and the thin lines the 95% confidence interval. Especially in the middle part of the figure, where most of the data is located, the confidence interval is very narrow, indicating a stable estimation of the demographic history. Also note that for this data the average number of change-points in the MCMC run was *k*= 9.25, i.e. the estimated effective degree of freedom is much less than that implicitly assumed in the classic skyline plot. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **HIV-1 in Central Africa***Top row*: a) underlying genealogy; b) classic skyline plot. *Bottom row*: c) population size function estimated with rjMCMC and corresponding 95% confidence band; d) comparison rjMCMC versus generalized skyline plot. ::: ![](1471-2148-5-6-3) ::: A comparison with the generalized skyline plot \[[@B17]\] is shown in Figure [3d](#F3){ref-type="fig"}. This demonstrates that the generalized skyline plot, in contrast to its classic cousin, provides a very good noise-reduced approximation to the demographic history as estimated by the reversible jump MCMC approach. However, especially near the present the step function employed in the generalized skyline plot leads to unrealistic jumps in the population size that are not present in the smooth estimate provided by the proposed MCMC method. HCV in Egypt ------------ In Egypt 10%-20% of the general population are infected with the Hepatitis C virus (HCV) \[[@B31]\]. This endemicity seems mainly to be caused by percutaneous medical procedures such as needle injections that took place during a countrywide health campaign between 1964 and 1982 against schistosomiasis. In order to investigate this phenomenon blood samples were obtained from various regions of Egypt and used to study the epidemic history of Hepatitis C. For instance, Tanaka et al. \[[@B32]\] analyzed the molecular evolution of HCV genotype 4a. Specifically, they utilized 47 sequences (AF217800-AF217812 \[[@B31]\] and AB103424-AB103457 \[[@B32]\]) from the NS5B region of the HCV subtype 4a to reconstruct the respective phylogeny, and subsequently applied the skyline plot method to infer the demographic history. We repeated their analysis with the reversible jump MCMC approach developed in this paper. We downloaded the sequence data from the HCV sequence database \[[@B33]\] and inferred the corresponding maximum-likelihood genealogy using the TREEFINDER program \[[@B34]\]. This tree is depicted in Figure [4a](#F4){ref-type="fig"}, next to the demographic history estimated from it by the classic skyline plot (Figure [4b](#F4){ref-type="fig"}). In the bottom of the figure we show the estimated population size function and its 95% confidence bands as obtained by our rjMCMC method (Figure [4c](#F4){ref-type="fig"}) and we also compare our results with those of the generalized skyline plot (Figure [4d](#F4){ref-type="fig"}). For the generating the time axis in these plots we assumed an evolutionary rate of 0.00045 substitutions per year. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **HCV in Egypt***Top row*: a) underlying reconstructed genealogy; b) classic skyline plot. *Bottom row*: c) population size function estimated with rjMCMC and corresponding 95% confidence band; d) comparison rjMCMC versus generalized skyline plot. ::: ![](1471-2148-5-6-4) ::: Generally, the star-like shape of the inferred tree already is indicative of exponential growth. This is confirmed by both the skyline plot as well as by our analysis (Figure [4d](#F4){ref-type="fig"}). Moreover, it can be seen that around 1940 the growth rate increased (i.e. the slope of *N*~*e*~(*t*) in the log-plot changes). Near the present, the rate decreased again. Also note the broadening of the confidence interval since 1940 which reflects the sparsity of available observations. This implies that the claim of Tanaka et al. \[[@B32]\] that the demographic history recently changed back to constant population size after an exponential growth is not firmly backed by the data. For further biological analysis of the HCV data we refer to Pybus et al. \[[@B35]\]. Conclusions =========== We have presented a new approach to non-parametric inference of demographic history from an inferred genealogy. This method is based on reversible jump MCMC sampling of the population size function *N*~*e*~(*t*). Unlike its predecessors, the classic and generalized skyline plots, it returns a smooth and realistic estimate of the demographic history and thus overcomes the constraints due to assuming a step function. Moreover, it automatically provides confidence limits. Nevertheless, the procedure is still computationally fast and can be run on any standard PC hardware. In our examples we demonstrated the advantage of non-parametric estimation of demographic history. Parametric estimation always assumes a certain functional form of population growth which may lead to problematic statements (cf. the HCV data set), in particular if the confidence bands of the estimated function *N*~*e*~(*t*) are not taken into account. From the methodological point of view, model selection via rjMCMC has the advantage that the effective dimension, i.e. the degree of smoothing, is automatically chosen in a data-driven manner. There is only one parameter (*λ*) that controls the *a priori*degree of smoothing, and this is adjusted accordingly by the investigated data. In addition, a further advantage of our MCMC approach is that -- in contrast to the skyline plot -- at least in principle it is straightforward to incorporate it in more general MCMC sampling schemes that also take account of the uncertainty in the genealogy. During the referee process we have learned that the authors of the software package BEAST \[[@B27]\] have developed a similar non-parametric method to Bayesian coalescent inference of population history (A.J. Drummond et al., in preparation). We plan to work with Dr. Drummond to make available in BEAST joint sampling of sampling of demographic histories and of trees. This would combine the present rjMCMC approach and the method developed by Drummond and colleagues. Authors\' contributions ======================= This paper summarizes the main results from a master\'s thesis of R.O. supervised by K.S. and L.F. Accordingly, K.S. and L.F. jointly devised the project and R.O. carried out all analyses and simulations. All authors participated in the development of methodology. R.O. and K.S. wrote the manuscript. All authors approved of the final version. Acknowledgements ================ We are grateful for financial support from the Deutsche Forschungsgemeinschaft (DFG) in the Emmy Noether program (R.O. and K.S.) and from the SFB 386 (L.F). We thank G??nter Ra??er and Leonhard Held for valuable comments and discussion and Juliane Sch??fer for critical reading of the manuscript.

PubMed Central

2024-06-05T03:55:52.646601

2005-1-21

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548300/", "journal": "BMC Evol Biol. 2005 Jan 21; 5:6", "authors": [ { "first": "Rainer", "last": "Opgen-Rhein" }, { "first": "Ludwig", "last": "Fahrmeir" }, { "first": "Korbinian", "last": "Strimmer" } ] }

PMC548301

Background ========== The mammalian myocardium contains several cell types, of which the cardiac myocytes (CM) make up most of the heart\'s mass. Although a small proportion of CM in the adult myocardium remains mitotic, most CM lose the capacity to undergo cell division shortly after birth \[[@B1]\]. In the adult heart, approximately 70% of the cells is represented by non-myocytes, most of which belong to the fibroblast compartment. In this study, we investigate how the stress response of cardiac cells to increased mechanical stress (MS) can be compared to genotoxic stresses induced by two DNA damaging agents, ionising radiation (IR) and ultraviolet radiation (UV). In cardiac cells, MS is increased during excessive pressure or volume load of the heart, as seen in hypertensive and valvular heart disease. This results in an adaptive growth response leading to structural and functional cardiac changes, including CM hypertrophy and hyperplasia of fibroblasts, to compensate for the increased workload \[[@B2]\]. In vitro cyclic stretch of rat cardiac cells has been shown to be an appropriate model for cellular changes that occur during overload of cardiac muscle in vivo \[[@B3]\]. Mechanical signals may be transferred to the nucleus of cells through integrin receptors, cytoskeletal filaments and nuclear scaffolds \[[@B4]\] and through ion channels, ion exchangers and hormone receptors \[[@B5]\]. Radiation induced heart disease (RIHD) has been recognised as a late adverse effect of thoracic radiotherapy if the heart was situated in the radiation field \[[@B6]\]. IR induces the formation of reactive oxygen species that react with different components of the cell, thereby inducing macromolecular lesions. IR can activate several signal transduction pathways, involving growth factor receptors, death receptors and DNA damage sensing proteins \[[@B7]\]. Primary fibroblasts in culture are known to go into senescence shortly after IR \[[@B8]\], after which terminal differentiation of these cells is induced \[[@B9]\]. Cultures of CM do not demonstrate cell death, nor a loss of function upon a single dose of 10,000 rad (\~100 Gy) \[[@B10],[@B11]\]. The density of DNA damaging events after UVC, which include helix-distorting photolesions, is three orders of magnitude higher than the density of DNA damage that occurs after IR \[[@B12]\] and sufficient to block cellular DNA replication and transcription. Signal transduction after UV is mediated via components of the cellular membrane, involving growth factor receptors, and via DNA damage sensing proteins \[[@B7]\]. UV is known to induce cell cycle arrest and apoptosis in several cell types \[[@B13],[@B14]\]. The aim of the study was to identify and compare differentially expressed genes (up-regulated or down-regulated when compared with untreated controls) in cardiac cells in response to MS, IR or UV. To this purpose, cultures enriched for ventricular CM or fibroblasts were exposed to one of the three stressors. Differentially expressed genes were identified using Affymetrix GeneChips. Several statistical methods have been developed to analyse Affymetrix gene expression microarrays. We used a method based on Linear Modelling on Probe Level \[[@B15]\] to describe the signal of every perfect match (PM) probe. Because overall changes in the expression of functionally related genes are more informative than the expression pattern of single genes, genes in microarray studies can be assigned to functional groups \[[@B16]\]. In the present study, such an approach was used to classify genes, based on biological function or on the role of a gene product in common intracellular pathways. Results ======= Accuracy of the linear model ---------------------------- As an example for the accuracy of the linear model that was used to describe the PM probe signals, Figure [1A](#F1){ref-type="fig"} shows a dot plot of all PM probe signals as calculated by the linear model, against the actual PM probe signals determined from fibroblasts after UV. These data were used to calculate correlation coefficients (R^2^) between the signals calculated by the model and the actual PM signals obtained. Figure [1B](#F1){ref-type="fig"} shows the distribution of R^2^values for fibroblasts after UV. The majority of probe-sets have a correlation coefficient ≥ 0.90, indicating that the model used fitted the data accurately. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Accuracy of the linear model**. Dot plot of all PM probe signals as calculated by the linear model, against actual PM probe signals determined from fibroblasts after UV (A). The data presented in Figure 1A were used to calculate correlation coefficients (R^2^) between the signals calculated by the linear model and the actual PM signals of fibroblasts after UV (B). ::: ![](1471-2164-6-6-1) ::: Numbers of differentially expressed genes ----------------------------------------- A Series entry (accession number GSE2032) at Gene Expression Omnibus (GEO), a public gene expression database of NCBI \[[@B17]\], gives access to all microarray data generated in this study. Figure [2](#F2){ref-type="fig"} represents the numbers of genes with a unique LocusLink ID that were up-regulated or down-regulated (q \< 0.005) in CM-enriched cultures/fractions and cultures of fibroblasts after one of the three stressors. When using these criteria, the numbers of differentially expressed genes (up-regulated or down-regulated) after UV were considerably higher than after IR or MS. After each of the three stressors more genes were up-regulated in CM-enriched cultures/fractions than in cultures of fibroblasts. Conversely, higher numbers of down-regulated genes were determined in fibroblasts. These differences were most pronounced after MS (Figure [2A](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Numbers of differentially expressed genes**. Numbers of differentially expressed genes (q \< 0.005) with a unique LocusLink ID in cultures of fibroblasts (dotted) and CM-enriched cultures after MS (A), in cultures of fibroblasts (dotted) and CM-enriched fractions after IR (B), or in cultures of fibroblasts (dotted) and CM-enriched fractions after UV (C). Overlapping parts of the circles represent genes that show differential expression both in CM-enriched cultures/fractions and cultures of fibroblasts. ::: ![](1471-2164-6-6-2) ::: Based on information available at NettAffx™ \[[@B18]\], extended with standard textbooks and recent literature, genes with a unique LocusLink ID were assigned to several functional groups. Subsequently, the status of each gene within a functional group was determined in both cell populations (CM-enriched cultures/fractions and cultures of fibroblasts). Individual probe-sets within these functional groups and their q-value after the three stressors are listed in table 1 (see [additional file 1](#S1){ref-type="supplementary-material"}). In this table, down-regulated genes are distinguished from up-regulated genes by a minus-sign in front of their q-value. Figure [3](#F3){ref-type="fig"} shows the percentage of genes in a functional group that are differentially expressed after MS, IR or UV. In accordance with Figure [2](#F2){ref-type="fig"}, the highest percentages of genes were differentially expressed after UV (on average 39.4% for both cell populations), followed by IR (13.0%). In general, the percentages of differentially expressed genes were lowest after MS (8.3%). Several functional groups, including heat shock proteins and genes involved in cholesterol biosynthesis, showed high proportions of differentially expressed genes with a hypergeometric probability P \< 0.005. These functional groups were considered to have significantly high percentages of differentially expressed genes. On the other hand, in the group of genes encoding for ion channels and exchangers, low percentages of differentially expressed genes with a hypergeometric probability P \< 0.005 were determined, both after IR and UV. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Percentages of differentially expressed genes**. Percentage of total number of genes within functional groups that are differentially expressed in CM-enriched cultures/fractions and cultures of fibroblasts after MS, IR or UV. Numbers between brackets represent total numbers of genes within a functional group. For example, of the 22 MAPkinases and phosphatases found to be represented by the array, 27% were differentially expressed in CM-enriched fractions after UV. AA: amino acid. \*Hypergeometric probability P \< 0.005 ::: ![](1471-2164-6-6-3) ::: Percentages of up-regulated genes --------------------------------- Figure [4](#F4){ref-type="fig"} shows the percentages of differentially expressed genes that were up-regulated after IR or after UV. In several functional groups, including p53 target genes and genes involved in mitosis, a significant percentage of differentially expressed genes was up-regulated (hypergeometric probability P \< 0.005). Other functional groups, including cytoskeletal components and genes involved in cholesterol biosynthesis mainly had down-regulated genes. In all 25 functional groups, the hypergeometric probability P of the percentage of up-regulated genes after MS was not below the pre-set threshold of 0.005. Therefore, no significant percentages of up-regulated genes were determined in any of the functional groups after MS (data not shown). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Percentages of up-regulated genes**. Percentage of changed genes that were up-regulated per functional group after IR or UV. Numbers between brackets represent numbers of differentially expressed genes in CM-enriched fractions and cultures of fibroblasts, respectively. For example, of the 8 MAPkinases and phosphatases that were differentially expressed in CM-enriched fractions after UV, 63% were up-regulated. \*Hypergeometric probability P \< 0.005 ::: ![](1471-2164-6-6-4) ::: General stress response genes ----------------------------- Probe-sets that showed an up-regulation or down-regulation after more than one stressor are listed in table 2 (see [additional file 2](#S2){ref-type="supplementary-material"}). The overlap in responsive genes between IR and UV was larger than the overlap between MS and one of the radiation types. Both after IR and UV, several genes that are known to play a central role in the radiation response of cells, including p21, GADD153 and mdm2, were up-regulated. These genes were not up-regulated after MS. A striking large proportion of genes encoding cytoskeletal components were down-regulated both after IR and UV. Validation of microarray results by semi-quantitative PCR --------------------------------------------------------- Gene expression changes detected using microarrays were validated by semi-quantitative PCR, using RNA from CM-enriched cultures at 24 hours after MS. The increased gene expression of Tenascin C and biglycan were confirmed with PCR in two independent experiments. In these two experiments, Tenascin C gene expression increased 1.59 and 1.64 times, respectively. Biglycan gene expression increased 1.42 and 1.25 times, respectively. Figure [5](#F5){ref-type="fig"} shows a representative result of the Tenascin C PCR. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Representative result of the Tenascin C PCR**Total RNA was isolated from CM-enriched cultures at 24 hours after MS or control treatment. After cDNA synthesis, semi-quantitative PCR was used to determine Tenascin C gene expression. Lane 1: smart ladder; lane 2: negative control; lane 3: positive control; lanes 4 and 5: CM-enriched after control treatment; lanes 6 and 7: CM-enriched after MS. ::: ![](1471-2164-6-6-5) ::: Discussion ========== In this study, a linear model was used to describe GeneChip PM probe signals and to determine effects of three types of stressors on gene expression in two neonatal rat heart cell populations, consisting of CM that are known to be terminally differentiated cells and fibroblasts that still have the capacity to undergo mitosis. Several studies have shown a good correlation between Affymetrix GeneChip data and RT-PCR \[[@B16]\] or Northern-blot \[[@B19],[@B20]\]. In a previous study neonatal rat CM-enriched cultures and cultures of neonatal rat cardiac fibroblasts were irradiated with a single dose of 8.5 Gy and some mRNA transcripts were quantified by competitive PCR \[[@B21]\]. In accordance with the present study, no significant changes in gene expression of transforming growth factor-β1 (TGF-β1), fibroblast growth factor-2 (FGF-2) and collagen type I were determined at 24 h after IR in cultures of cardiac fibroblasts. Moreover, no significant IR-induced changes were determined in gene expression of atrial natriuretic peptide (ANP) in CM-enriched cell populations in both the latter and the present study. On the other hand, CM-enriched cultures showed a reduced TGF-β1 expression (by PCR) at 24 h after 8.5 Gy, which was not observed in the present study. This might be due to differences in experimental design between the two studies, as in the former study CM-enriched cultures were obtained by incubation with bromodeoxyuridine to prevent fibroblast proliferation. Also, during and after irradiation, the cells were incubated in higher serum concentration than used in the present study. Here, differences between the two cell populations are observed in the predominant type of gene expression response, i.e. up-regulation versus down-regulation. After all three stressors, differentially expressed genes were mostly up-regulated (q \< 0.005) in CM-enriched fractions, while in cultures of fibroblasts the majority of changed genes were down-regulated. After MS, these differences were most pronounced. Paracrine signalling is involved in the response of CM and cardiac fibroblasts in co-cultures subjected to MS \[[@B22]\]. In the CM-enriched cultures used in this study, remaining fibroblasts might stimulate gene transcription of the CM, resulting in higher numbers of up-regulated genes. It cannot be excluded that differences in level of toxicity between the three stressors applied in this study caused differences in gene expression levels. Of the three stressors examined in this study, both IR and UV are known to induce oxidative stress. Accordingly, both stressors induced high numbers of up-regulated genes involved in anti-oxidative processes. The high percentages of up-regulated genes encoding heat shock proteins in both cell populations after IR and UV might indicate oxidative stress induced protein damage in these cells. In comparison, after MS only few genes encoding heat shock proteins were up-regulated in either cell population. Heinloth et al. (2003) proposed a model for the regulation of several gene expression patterns after IR and UV in human dermal fibroblasts, based on microarray analysis. The general view that p53 plays a central role in signal transduction after IR and UV was confirmed in their study. Moreover, IR down-regulated the expression of genes involved in mitosis. UV, on the other hand, induced the expression of genes involved in protein degradation and the MAPK pathway \[[@B23]\]. The latter data are in accordance with the data of the present study, showing that UV did affect genes participating in the MAPK pathway (although not significantly) in both cell populations, but IR did not. Moreover, in CM-enriched fractions, IR resulted in a down-regulation of a large percentage of genes involved in mitosis. Several genes, involved in cell cycle regulation and mitosis are expressed in foetal cardiac myocytes and down-regulated in adult myocytes, in accordance with their terminal differentiation \[[@B24],[@B25]\]. Due to the close relation between foetal and neonatal cells, an expression of mitotic genes in the neonatal CM used in this study can be expected. The high numbers of up-regulated genes involved in ubiquitination and protein degradation in both cell populations after UV are also in accordance with the study of Heinloth et al. (2003) and with other studies on cultured cells after UV \[[@B20]\]. These results might reflect the need of cells to replace molecules that were damaged by UV irradiation, although the proteasome is also proposed to play a role in several cellular processes, including DNA-repair, cell cycle regulation and cell survival after irradiation \[[@B26]\]. In CM-enriched cultures, MS led to a relatively high proportion of up-regulated genes that are involved in cholesterol biosynthesis. Among these genes, expression of 3-hydroxy-3-methylglutaryl-Coenzyme A reductase, which forms the starting enzyme of the mevalonate pathway and is considered to be the key enzyme in cholesterol biosynthesis, was up-regulated. This suggests that in neonatal rat cardiac myocytes cholesterol biosynthesis is stimulated after MS. Neonatal rat heart myocytes that undergo hypertrophy in culture show an increased biosynthesis and intracellular accumulation of cholesterol \[[@B27]\]. Moreover, the mevalonate pathway has been proposed to play a role in Ras activation in neonatal rat cardiac myocytes subjected to MS in culture, leading to hypertrophy \[[@B28]\]. *In vivo*hypertrophy of the heart is also associated with elevated myocardial cholesterol contents \[[@B29]\]. An increased cholesterol biosynthesis in cardiac myocytes that undergo MS might therefore accompany a hypertrophic response of these cells. However, of the three foetal genes that are known to be re-expressed in hypertrophic myocytes, i.e. smooth muscle alpha-actin, ANP and beta-myosin heavy chain, only the last gene was up-regulated at the time point of investigation. In contrast to MS, IR and UV led to a down-regulation of genes involved in cholesterol biosynthesis in CM-enriched fractions. In previous studies, alterations in cholesterol contents of cardiac myocytes and fibroblasts were associated with alterations in protein to DNA ratios, levels of several enzymes including ATPases and phosphatases, and diffusion rates of membrane proteins \[[@B30]-[@B32]\]. Moreover, a decrease in beating rate observed in aging cultures of CM was opposed by increased membrane amounts of cholesterol \[[@B33]\]. The role of cholesterol biosynthesis in the cellular functions of cardiac cells after MS, IR or UV needs further investigation. Higher numbers of differentially expressed genes involved in ECM formation, including genes encoding collagens, fibronectin and laminin, were determined in CM-enriched cultures after MS than in cultures of fibroblasts. As mentioned before, paracrine signalling between CM and remaining fibroblasts might play a role in cellular responses in these CM-enriched cultures. Alterations in expression of genes involved in ECM formation might originate from remaining fibroblasts after stimulation by CM. Interestingly, MS does not affect the expression of these genes in cultures of fibroblasts, which suggests that paracrine signalling from CM is necessary for alterations in expression profiles of genes involved in ECM formation in fibroblasts. The down-regulated genes encoding cytoskeletal components in cultures of fibroblasts after IR and UV, including myosin and troponin, are mostly CM-specific. Therefore, these genes are likely to originate from remaining CM in the fibroblast cultures, although their numbers were low (3--5%). The extremely low numbers of differentially expressed genes encoding for ion channels and exchangers might be explained by a low number of cardiac cell type-specific genes within this functional group. Conclusions =========== MS, IR and UV mainly induce stress-specific and cell-type specific gene expression profiles in neonatal rat CM-enriched cultures and cultures of neonatal rat heart fibroblasts. Functional groups that show significant percentages of differentially expressed genes suggest that certain cellular pathways are activated after one or more stresses. Methods ======= Cell culturing -------------- The experiments were performed with permission of the local committee on animal experiments, installed by the University of Leiden according to the Dutch law. Cardiac cells were isolated from ventricles of neonatal rat hearts, and cultured as described before \[[@B14],[@B34]\]. By applying a pre-plating method, cultures enriched for CM and cultures of fibroblasts were obtained. After 2 days of culturing with 5% horse serum (HS), culture media of CM-enriched cultures were replaced by media containing 2.5% HS. One day later, these cultures were subjected to UV, IR or MS. Three days after isolation, cultures of fibroblasts were subcultured for 6 days in medium containing 10% foetal bovine serum (FBS). Then, culture medium was replaced by medium containing 2.5% FBS. One day later, confluent cell cultures were subjected to UV, IR or MS. Irradiation and cyclic stretch ------------------------------ Cells were subjected to a single dose of 8.5 Gy of X-rays at room temperature, using a 6 MV accelerator (SL 75-5 Philips), operated at a dose rate of 8 Gy min^-1^. During irradiation, sham-irradiated control cells were kept at room temperature. After irradiation, cell cultures were maintained at 37°C for 24 h. In another experiment cells were irradiated with 10 J/m^2^UVC at room temperature at a dose rate of 0.2 J/m^2^per s. Before irradiation, medium was collected and cells were rinsed with PBS. Following irradiation, growth medium was returned to the cultures and the cells were incubated for an additional 24 h. Control cells received a similar treatment except for UV irradiation. To subject cells to cyclic stretch, CM-enriched cells (54 ± 5% CM) and fibroblasts (95 ± 2% non-myocytes) were cultured in 6-well Flex I culture plates (Flexcell, Hillsborough, NC, USA), coated with collagen I. Medium was changed to medium containing 2.5% serum and after 24 h plates were placed in the Flexercell Strain Unit FX-2000^®^(Flexcell) in which the frequency and magnitude of stretch were regulated by a computer-controlled vacuum pump. The apparatus applied an equiaxial cyclic stretch of 20% elongation to the wells at a frequency of 60 cycles/min (1 Hz). Stretch was applied for 24 h. Control cells were grown in identical culture plates and incubated in the same incubator as the stretched cultures, but were not mounted in the Flexercell Strain Unit. Cell harvesting after irradiation --------------------------------- Twenty-four hours after IR or UV, non-irradiated and irradiated CM-enriched cultures were trypsinised and subjected to centrifugal elutriation \[[@B35]\]. The proportion of CM in each elutriation fraction was determined by flow cytometric analysis of myosin expression, as described before \[[@B35]\]. After elutriation of IR-exposed cultures, CM-enriched fractions contained 74 ± 3% CM and after elutriation of UV-exposed cultures, CM-enriched fractions contained 85 ± 3% CM. Because of the high purity of the fibroblast cultures (95 ± 2% non-myocytes), no centrifugal elutriation was applied on these cells. CM-enriched fractions and cultures of fibroblasts were used for RNA isolation. Cell harvesting after MS ------------------------ After undergoing MS or control treatment for 24 h, cells were collected from CM-enriched cultures and from cultures of fibroblasts by trypsinisation. The proportion of CM in the cell cultures was determined by flow cytometric analysis of myosin expression as described before \[[@B35]\]. CM-enriched cell cultures, containing 54 ± 5% CM, and fibroblast cultures, containing 95 ± 2% non-myocytes, were used for RNA isolation. RNA isolation and labelling --------------------------- RNA was isolated applying an RNeasy^®^kit (Qiagen GmbH, Hilden, Germany), according to the manufacturer\'s instructions. Ten to 14 μg of total RNA from CM-enriched fractions and 7.5 to 14 μg of total RNA from fibroblast cultures was used for labelling. Per experiment, the quantities of total RNA from irradiated and control cells were equal. cDNA was synthesised using the Gibco BRL Superscript system (Invitrogen, Carlsbad CA, USA). Briefly, single stranded cDNA was synthesised using Superscript II reverse transcriptase and T7-oligo(dT)24 primers at 42°C for 1 h. Double stranded cDNA was obtained by using DNA ligase, DNA polymerase I and RNAse H at 16°C for 2 h, followed by T4 DNA polymerase at 16°C for 5 min. After clean up with a phase lock gel (Qiagen), ds-cDNA was used for *in vitro*transcription (IVT). cDNA was transcribed using the BioArray HighYield^®^RNA transcript labelling kit (Enzo Lifesciences, Pharmingdale NY, USA) in the presence of biotin-labelled ribonucleotides, or using the MEGAScript T7^®^kit (Ambion, Austin TX, USA), in the presence of biotin-labelled CTP and UTP. In each experiment, cDNAs from control cells and stressed cells were labelled simultaneously using the same labelling kit. After clean up with a RNeasy kit (Qiagen), the biotin-labelled IVT-RNA was fragmented in a buffer containing 40 mM Tris-acetate (pH 8.1), 100 mM potassium-acetate and 30 mM magnesium-acetate, at 94°C for 35 min. Hybridisation of IVT-RNA ------------------------ RG-U34A arrays (Affymetrix, Santa Clara CA, USA) were used, representing \~7000 transcripts, of which 6399 had a unique LocusLink identification number (ID) at the time of data analysis. Biotin-labelled IVT-RNA was hybridised to the arrays at 45°C for 16 h according to the manufacturer\'s instructions. After hybridisation, the arrays were washed in a GeneChip Fluidics Station 400 with a non-stringent wash buffer at 25°C followed by a stringent wash buffer at 50°C. After washing, the arrays were stained with a streptavidin-phycoerythrin complex. After staining, intensities were determined with a GeneChip scanner, controlled by GeneChip software (Affymetrix). The intensities were background corrected using gcrma \[[@B36]\] and normalized at the probe level by VSN \[[@B37]\]. Data analysis ------------- For each stressor, three independent experiments were performed. To describe the signal of every PM probe, the following linear model was used: signal \~P + T + E + TE, based on a method described before \[[@B15]\]. In our model, the symbols P, T, E and TE represent the effects of probe, treatment, experiment per stressor (1, 2, or 3) and the interaction between treatment and experiment, respectively. Subsequently, analysis of variance was applied on the treatment effect to determine the p-value for each probe-set. The p-values were corrected for multiple testing using the Benjamini Hochberg step-up procedure \[[@B38]\] which yielded q-values for the false discovery rates (FDR). The FDR level of control was set to 0.005, equivalent to selecting genes with a q-value\<0.005 (keeping up- and down-regulated genes separate by the sign of the treatment coefficient). Some genes are represented by more than one probe-set. A gene was determined to be differentially expressed when at least 50% of the probe-sets representing this gene showed significant up-regulation or down-regulation. To determine significant proportions of differentially expressed genes within functional groups, the hypergeometric probability P was calculated. P \< 0.005 was considered significant. To determine the accuracy of the linear model that was used to describe PM signals, R^2^was calculated for every probe-set, using the following standard formula: R^2^= 1-ΣR~i~^2^/Σ(signal~i~-mean signal)^2^, where R~i~= observed-fitted value for signal~i~and mean signal = the mean of observed signal~i~. To compare data on differentially expressed genes with microarray data in literature on species other than rat, RESOURCERER of the Institute of Genomic Research \[[@B39]\] was used. RNA isolation and semi-quantitative PCR --------------------------------------- Total RNA isolation, cDNA synthesis and semi-quantitative PCR were performed as described before \[[@B40]\]. In short, total RNA was isolated using an RNeasy^®^kit (Qiagen). Following DNAse (Amersham Pharmacia Biotech, Uppsala, Sweden) treatment, cDNA was synthesised using M-MLV reverse transcriptase (Life Technologies, Rockville, MD, USA) and oligo(dT) primers (Amersham). Semi-quantitative PCR was performed, using the following primers and annealing temperatures: Tenascin C sense: CGA CAG TTT TGT TAT CAG GAT CAG, Tenascin C antisense: GGC ACA TAA GTA ATC CGG AAA T, 60°C; Biglycan sense: CAA CAA CCC TGT GCC CTA CT, Biglycan antisense: GGT GT GCT TCT TTG CTT CC, 65°C. A competitive PCR was performed on GAPDH to correct for differences in cDNA concentrations. After agarose gel electrophoresis, intensities of PCR product bands were determined by Scion image analysis software (Scion Corporation, Frederick, MD, USA). Authors\' contributions ======================= MB performed experiments, developed and analyzed functional groups of genes and prepared the manuscript. CGCW performed experiments, developed and analyzed functional groups of genes and participated in discussions. HV and AL participated in discussions and gave textual advice. PS performed microarray normalization and data analysis. JW and LHFM participated in discussions. AAZ participated in discussions, suggested the assignment of genes to functional groups and gave textual advice. All authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 **q-values of genes within functional groups**Table 1. Excel-file containing the 25 functional groups of genes used in this study. Of every gene, Affymetrix probe-set IDs and q-values after the three types of stress are listed. Down-regulated genes are represented by a minus-sign in front of their q-value. q-values \< 0.005 are marked red (in case of up-regulation) and green (in case of down-regulation). ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 **General stress response genes**Table 2. Excel file containing Affymetrix probe-set IDs, LocusLink ID and gene description of all probe-sets that showed an up-regulation or down-regulation after two or more stresses. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This study was financially supported by the Netherlands Heart Foundation (grant no. 97.176), the Interuniversity Research Institute of Radiopathology and Radiation Protection (grant no. 9.0.13) and the European Union. The authors would like to thank Rebecca Esveldt-Van Lange, Cindy Schutte-Bart and Minka Bax for skilful technical support.

PubMed Central

2024-06-05T03:55:52.649799

2005-1-18

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548301/", "journal": "BMC Genomics. 2005 Jan 18; 6:6", "authors": [ { "first": "Marjan", "last": "Boerma" }, { "first": "Caroline GC", "last": "van der Wees" }, { "first": "Harry", "last": "Vrieling" }, { "first": "J Peter", "last": "Svensson" }, { "first": "Jan", "last": "Wondergem" }, { "first": "Arnoud", "last": "van der Laarse" }, { "first": "Leon HF", "last": "Mullenders" }, { "first": "Albert A", "last": "van Zeeland" } ] }

PMC548302

Background ========== Bacterial strains belonging to the *Enterobacteriaceae*species *Escherichia coli*are common as commensals in the intestinal flora of humans and warm-blooded animals \[[@B1]\]. Typing systems for identification of related *E. coli*strains were developed in the early 1940ies when it became evident that certain *E. coli*strains were agents of infantile gastroenteritis \[[@B2]\]. In 1944, Kauffmann established the method of serological typing for *E. coli*O- (lipopolysaccharide) and H-(flagellar) antigens which allowed the grouping of *E. coli*strains according to their O:H-types (serotypes) \[[@B3]\]. Serotyping proved to be widely useful for identification of enteropathogenic *E. coli*(EPEC) strains from stools of diarrhoeic infants \[[@B4],[@B5]\] and is successfully employed for characterization of pathogenic *E. coli*strains from both humans and animals \[[@B2],[@B3]\]. The genetic analysis of *E. coli*populations by multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST) allowed the detection of clonal types of strains which carry specific virulence markers and are associated with disease in humans \[[@B6],[@B7]\]. It was shown that the O:H serotype is a good marker for identification of strains belonging to clonal types of pathogenic *E. coli*\[[@B6],[@B7]\]. At present, 176 O- and 53 H-antigens are described for *E. coli*which can occur in different combinations in wildtype isolates of strains \[[@B2],[@B3],[@B5],[@B8]\]. However, the large number of O- and H-antisera which are needed for *E. coli*serotyping and the laborious typing procedure restricts its usage to a few reference laboratories. Therefore, alternative typing methods were developed including molecular characterization of genes coding for the O- and H-antigens in *E. coli*\[[@B9]-[@B12]\]. Typing of *fliC*genes by PCR was successfully employed for characterization of human pathogenic O157:H7 and O26:H11 strains \[[@B13],[@B14]\]. Analysis of the nucleotide sequence of *fliC*genes coding for flagellar antigens H7 and H6 revealed large sequence similarities between strains sharing the same H-type but different O-types \[[@B15],[@B16]\]. Moreover, molecular typing of the *fliC*gene allows H-typing of non-flagellated (non-motile) *E. coli*isolates which cannot be analyzed for their flagella with H-specific antisera \[[@B14],[@B15],[@B17]\]. The flagellar type H4 is frequently occurring in *E. coli*belonging to many different O-groups including strains of shiga toxin-producing *E. coli*(STEC) and extraintestinal pathogenic serotypes \[[@B18]-[@B20]\], (K.A. Bettelheim, The VTEC table, May 2003, <http://www.microbionet.com.au/vtectable.htm>). Moreover, a cryptic *fliC*-H4 gene was described to be present and to be spontaneously expressed in *E. coli*strain P12b (O15:H17) which carries another type of flagella called H17 which is not encoded by the *fliC*gene \[[@B21],[@B22]\]. Therefore, we became interested in the genetic variability of flagellar H4 genes in *E. coli*strains belonging to different O-serogroups and pathotypes. We have used the published nucleotide sequence of the *fliC*gene present in the *E. coli*H4 reference strain U9-41 (accession AB028472) to develop a PCR which allows discrimination between the *fliC*-H4 gene variants. Nucleotide sequence analysis of the *fliC*gene was performed on other *E. coli*H4 strains that either showed deviations in the PCR analysis or were reported to harbour allelic types of the H4-*fliC*gene \[[@B9]\] or revealed differences in the agglutination reaction compared to the reference strains. To study the expression of different flagellar H4 types we have cloned their corresponding *fliC*genes and have introduced them into the laboratory *E. coli*K-12 strain JM109 \[[@B23]\]. Expression of recombinant flagella was demonstrated by serotyping and by immuno electron microscopy. Results ======= Serological detection of the flagellar H4 antigen in different *E. coli*wildtype host strains --------------------------------------------------------------------------------------------- *E. coli*reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) \[[@B3]\] and the *E. coli*K-12 strain JM109 (O-rough:H48) (this study), which was used for expression studies were investigated for inhibition of motility in swarm-agar containing 1:600 dilutions of either H4, or H48-antiserum (see Methods). U9-41 and P12b were fully inhibited for their motility in the presence of H4 antiserum derived from strains U9-41 or from P12b but not in the presence of H48 specific antiserum. The *E. coli*K-12 strain JM109 was not inhibited for motility by H4 antisera but by H48 antiserum. These findings indicate that H4 antisera specifically inhibited the motility of *E. coli*H4 strains and that the antigenically different flagellar type H17 was not expressed or lost in our P12b isolate, similar as previously described with spontaneously arising variants of P12b \[[@B24]\]. We became interested if other *E. coli*H17 strains would also carry the genes for expression of H4 type flagella as it was described for P12b \[[@B21],[@B22],[@B24]\]. For this, we have investigated five additional *E. coli*H17 strains (872-69, 107-74, 305-78, 870-69 and 327-01) for their serological reaction with different H4 specific antisera and all strains showed specific positive reactions (Table [1](#T1){ref-type="table"}). H-serotyping performed with strains from the collection of the Robert Koch-Institute revealed 88 *E. coli*strains which agglutinated with H4 antisera and the results obtained with 10 representative strains are shown in Table [1](#T1){ref-type="table"}. All strains agglutinated with both, H4~U9-41~and H4~P12b~antiserum, but were not agglutinating with antisera made against other H-antigens (data not shown). Differences in agglutinating titers between H4~U9-41~and H4~P12b~antisera were not more than twofold with either strain indicating that both sera were similar for their specificity (Table [1](#T1){ref-type="table"}). The strains P7d, E1541-68, 107-74 and 305-78 showed significantly lower agglutination titers with H4 antisera than did the reference strains U9-41 and P12b, which had been used for production of H4 typing sera, respectively (Table [1](#T1){ref-type="table"}). These findings prompted us to compare all the H4 strains with all other *E. coli*H-types for polymorphisms in the *fliC*gene by HhaI digestion of *fliC*-PCR products as described in the Method section. HhaI-RFLP typing of *fliC*genes in *E. coli* -------------------------------------------- PCR products obtained with primers fliC-1 and fliC-2 were digested with HhaI to obtain H-serotype specific RFLP patterns from reference strains for the 53 different *E. coli*H-serotypes \[[@B3],[@B9]\]. HhaI-RFLP typing of *E. coli fliC*genes coding for flagellar types H1 to H56 revealed individual patterns corresponding to the different H-serotypes (Fig. [1](#F1){ref-type="fig"}). Flagellar antigens H3, H17, H35, H36, H44, H47, H53, H54 and H55 were reported to be not encoded by *fliC*but by other genes (*flkA*, *fllA, flmA*and others) in the corresponding *E. coli*strains and the *fliC*HhaI patterns obtained from these strains do therefore not correspond to their H-serotypes \[[@B21],[@B25]-[@B27]\]. HhaI-RFLP patterns were found conserved among strains sharing the same H-serotype independent of their O-antigen as previously described \[[@B9]\] (data not shown). An exception was found for H-serotypes 2, 8, 18, 19, 21, 33 and 47 in which single strains possessed different HhaI-RFLP patterns when compared with the corresponding H-type reference strain (data not shown). The HhaI-RFLP patterns of different H-types were distinguishable from each other (Fig. [1](#F1){ref-type="fig"}). The results from HhaI-RFLP typing corresponded with the earlier reports showing that the *fliC*gene in strain P12b codes for flagella of serotype H4. The relationship between *fliC*genes present in the different *E. coli*strains was further investigated by nucleotide sequencing. Nucleotide sequence analysis of the *fliC*genes present in representative *E. coli*H4 strains --------------------------------------------------------------------------------------------- To obtain the entire coding region of the *fliC*gene in *E. coli*strains oligonucleotide primers fliC-5 and fliC-6 were deduced from the *fliC*-H4 chromosomal region and applied to amplify the corresponding chromosomal regions from *E. coli*strains U9-41 (O2:K1:H4), P12b (O15:H17), U1-41 (O5:K4:H4), P7d (O68:H4), C107-74 (O15:H17) and E1541-68 (O154:H4) as listed in Table [1](#T1){ref-type="table"}. These strains were taken as flagellar type H4 representatives according to previously published results \[[@B3],[@B9]\] and according to the H-serotyping performed in this study. The analysis of the coding sequences of the *fliC*genes present in these strains revealed in all cases a length of 1050 bp. As a control, the *fliC*gene of the strain U9-41 was sequenced and found to be identical to the previously deposited *fliC*sequence (accession AB028472). The identity of the *fliC*-H4 sequences ranges from 97,6 % to 100 %. The greatest sequence difference to the *fliC*gene coding sequence of strain U9-41 was found in the *fliC*gene of strain P7d (exchange of 20 nucleotides), whereas the *fliC*sequences of strains U1-41 and U9-41 were identical to each other. All deduced flagellins have a length of 349 amino acids. The greatest deviation in the primary structure to the FliC protein of the reference strain U9-41 was again observed for the FliC protein of strain P7d (exchange of 9 aa in a stretch of 349 aa). Fig. [2](#F2){ref-type="fig"} shows the alignment of the deduced FliC proteins of all investigated strains. The FliC protein of strain U1-41 is 100% identical to that of strain U9-41 and therefore not shown. Similarities of the deduced amino acid sequences of the FliC proteins (flagellins) are summarized in Table [2](#T2){ref-type="table"}. PCR based detection of *fliC*-H4 specific DNA sequences ------------------------------------------------------- Amplification of the *fliC*genes present in *E. coli*strains U9-41 and P12b with primers fliC-1 and fliC-2 resulted in the generation of a 953 bp internal PCR product with both strains (Fig. [3](#F3){ref-type="fig"}, lanes 4+5). The nucleotide sequences of this stretch of DNA of strains U9-41 and P12b were compared for restriction enzymes which cut at different sites in the *fliC*-H4~U9-41~and *fliC*-H4~P12b~sequence. HhaI was found to cut at identical sites confirming the results from HhaI RFLP typing (Table [3](#T3){ref-type="table"}). In contrast, enzymes HpaII and MboI were found to generate each different restriction fragments from PCR products of the *fliC*-H4~U9-41~and *fliC*-H4~P12b~gene, respectively. Both enzymes were taken for RFLP typing of amplified *fliC*genes (primers fliC-1 and fliC-2) from 88 *E. coli*strains which showed agglutination reactions with H4 antisera (Table [3](#T3){ref-type="table"}). Eighty-six of the 88 strains showed HpaII and *Mbo*I restriction profiles which corresponded to the patterns obtained with strain U9-41 (O2:K1:H4) (Table [3](#T3){ref-type="table"} and Fig. [3](#F3){ref-type="fig"}). Exceptions were made by strains P7d (O68:H4) and P12b (O15:H17) which showed individual restriction patterns which differed from all other H4 strains investigated in this study. (Table [3](#T3){ref-type="table"}). Cloning and expression of *fliC*-H4 genes in the *E. coli*K-12 strain JM109 --------------------------------------------------------------------------- In order to study the functional expression of the *fliC*-H4 genes in a different genetic background we cloned the corresponding PCR products of strains U9-41 and P12b into the vector pLITMUS38 as described in the Methods section. The *fliC*coding regions were inserted downstream of the *lacZ*promoter of pLITMUS38 and the *fliC*recombinant plasmids were transformed into the laboratory *E. coli*K-12 strain JM109 \[[@B23]\]. JM109 was serotyped as O-rough:H48, and it showed the same HhaI-RFLP *fliC*-pattern as the *E. coli*reference strain P4 (O16:H48) \[[@B3]\] (Fig. [1](#F1){ref-type="fig"}). The functional expression of the cloned *fliC*-H4 genes in the JM109 derivative strains TPE1976 (*fliC*-H4~U9-41~clone) and TPE1978 (*fliC*-H4~P12b~clone) was analyzed by tube agglutination with H4 and H48 antisera, respectively. The strains TPE1976 and TPE1978 showed agglutination with H48 and H4 sera whereas the parental JM109 strain reacted only with H48 serum (Table [4](#T4){ref-type="table"}). To find out if the flagellins of the *fliC*recombinant plasmid carrying JM109 strains were co-assembled in all flagella or assembled separately we studied the parental strain JM109 and its *fliC-*H4 derivative TPE1978 by IEM (Fig. [4B--G](#F4){ref-type="fig"}). Both strains were found to express 2-4 flagella per cell which appeared morphologically typical as long helical filaments (diameter 18 nm, length up to 20 μm) (Fig. [4A](#F4){ref-type="fig"}), \[[@B29]\]. The reaction of H48 antiserum with bacteria followed by detection of adsorbed antibodies with anti-rabbit-IgG coupled with 10 nm immuno-gold particles showed a specific and homogeneous labelling of flagella present on the surfaces of JM109 (Fig. [4C](#F4){ref-type="fig"}) and of TPE1978 (Fig. [4B](#F4){ref-type="fig"}). When H4 serum was used, only flagella of strain TPE1978 became immuno-labelled (5 nm gold particles) (Fig. [4D](#F4){ref-type="fig"}) and no labeling was observed with JM109 (Fig. [4E](#F4){ref-type="fig"}), confirming H-serotyping results. Sequential double labeling experiments with H48 and H4 antibodies resulted in staining of all flagella on the surfaces of JM109 (Fig. [4G](#F4){ref-type="fig"}) and TPE1978 (Fig. [4F](#F4){ref-type="fig"}). However, both 5 nm (H4 label) and 10 nm (H48 label) gold particles were only bound to flagella of TPE1978 (Fig. [4F](#F4){ref-type="fig"}) whereas the flagella of JM109 were exclusively labelled with 10 nm gold particles which indicates the H48 antigen (Fig. [4G](#F4){ref-type="fig"}). These results demonstrate that both H48 and H4 flagellins are co-assembled in the flagella made by the *fliC*-H48/H4 genes in strain TPE1978. Discussion ========== The *fliC*genes of representative *E. coli*strains for the 53 different H-types were recently investigated and compared for their nucleotide sequences \[[@B21]\]. Among the H-type reference strains which were not analyzed for their complete *fliC*gene sequences, were the reference strains for the *E. coli*H4 (U9-41) and H17 (P12b) flagellar antigen \[[@B21],[@B22]\]. In this work, we performed a complete characterization of the *fliC-*H4 genes from different *E. coli*strains with primers which were generated on the basis of the previously published *fliC*sequence (accession AB028472) of the *E. coli*H4 reference strain U9-41. The comparison of the *fliC*gene sequence encoding H4 flagella in strain P12b with the *fliC*sequences of five other *E. coli*H4 or H17 strains revealed a high similarity on the DNA and on the amino acid level (Fig. [2](#F2){ref-type="fig"} and Table [2](#T2){ref-type="table"}). We established a PCR/RFLP typing assay for genotypic investigation of clinical *E. coli*isolates which reacted with H4 antisera. Genotyping of 88 *E. coli*strains comprising 20 different O-serogroups (Table [3](#T3){ref-type="table"}) revealed that 86 of the strains gave RFLP patterns with HhaI, MboI and HpaII which were indistiguishable from the prototype *fli*C-H4 gene and only two strains showed alternative patterns. The detection of some genetic variants in the *fliC*-H4 gene of *E. coli*strains studied here points to a sequence diversity similar as described previously for the *fliC*genes of *E. coli*H6 and H7 strains \[[@B15],[@B16]\]. These results correspond to previous findings indicating that the H4 antigens in different *E. coli*typing strains are not fully identical \[[@B20],[@B24],[@B28]\]. It was suggested earlier that the strain P12b encodes two flagellins, H4 and H17, which are subject to phase variation for their expression \[[@B22]\] and mutants of P12b could be isolated which expressed only the H4 antigen \[[@B24]\]. Recent studies have demonstrated that the *fliC*gene of strain P12b encodes flagellar type H4 and it was suggested that the gene for the H17 flagellin is encoded by a locus outside *fliC*, however the gene responsible for the flagellar type H17 was not identified in the study \[[@B21]\]. In our study, cultures of strain P12b were fully inhibited for motility and swarming in the presence of H4 antiserum but not in the presence of H48 antiserum which was used a non-specific control. This result indicates that H17 type flagella were not expressed or lost from our P12b isolate, similar as previously described with mutant strains of P12b \[[@B24]\]. The functional expression of the cloned *fliC*-H4~U9-41~and *fliC*-H4~P12b~genes in the genetic background of *E. coli*K-12 strain JM109 which shows flagellar serotype H48 confirmed their coding capacity. Since we found coexpression of cloned flagellins with the parental H48 flagellin, we became interested in the composition of flagella made by the *fliC*recombinant plasmid carrying JM109 strains. Electron microscopy of flagella from JM109 and from the *fliC*-H4~P12b~recombinant plasmid carrying strain revealed no differences between JM109 and TPE1978 showing both a typical helical organization of normal sized flagella on their surface (Fig. [4](#F4){ref-type="fig"}). Immuno-gold staining of bacteria which were prior incubated with H48 and H4 antisera revealed high specificity of the rabbit antisera for the flagellar structure (Fig. [4B--G](#F4){ref-type="fig"}). Single (Fig. [4 B+D](#F4){ref-type="fig"}) and double labelling (Fig. [4 F](#F4){ref-type="fig"}) experiments with different sized gold markers demonstrated that all flagella present on the surface of TPE1978 were labelled after incubation with H48 and H4 antiserum. Our results indicate that both H48 and H4 flagellins are coassembled in the flagella made by the *fliC*-H4~P12b~recombinant plasmid carrying strain. This finding is surprising in view of the molecular weight size differences found between H48 and H4 flagellin. To our knowledge, the assembling of two different flagellins in the same filament has not yet been demonstrated before. The H48 flagellin of *E. coli*K-12 (accession AE000285) is a 51.3 kDa protein consisting of 498 amino acids and thus much larger than the 36.3 kDa H4 flagellin which is composed of 349 amino acids. However, both flagellins share conserved N- and C-terminal sequences which are known to be involved in the structural assembly of flagella \[[@B29]\]. H48 and H4 flagellins differ largely for their central regions which are not involved in flagellar assembly and function but which contain flagellar antigenic epitopes \[[@B29]\]. We were able to show that the introduction of an isolated *fliC*gene in *E. coli*can change the antigenic properties of the flagella made by this strain. Horizontal transfer of *fliC*genes may contribute to the diversity of flagellar serotypes by recombination within *E. coli*recipient strains. The mammalian host immune system is the driving force for continuous selection of new flagellar antigens in *E. coli*. Published data indicate that both mutation and recombination events in the *fliC*gene have taken place in the evolution of *E. coli*flagellar antigens \[[@B15],[@B16]\]. Conclusions =========== Our *fliC*sequence data have shown that the flagellar type H4 which is present in *E. coli*strains of clinical importance covers several genetic variants which are closely related to each other. We have shown for the first time that flagellins of different molecular size are expressed and coassembled into functional flagella in a laboratory *E. coli*K-12 strain. Methods ======= Bacterial strains ----------------- The reference strains used for production of antisera for O- and H-typing of *E. coli*were obtained from the International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen, Denmark and are described elsewhere \[[@B3]\]. Origin and serotype data on five additional strains with flagellar type H17 are listed in Table [1](#T1){ref-type="table"}. A laboratory collection of 88 *E. coli*isolates originating from humans and animals was investigated by PCR/RFLP typing for *fliC*-H4 genes. These strains were previously investigated for the O-types and for production of Shiga-toxins (Stx) and were isolated in different countries between 1941 to 2002 \[[@B3],[@B19],[@B30]\] (Table [3](#T3){ref-type="table"}). The *E. coli*K-12 strain JM109 is described elsewhere \[[@B23]\]. Production of *E. coli*O and H-specific antisera ------------------------------------------------ Rabbit antisera against the different O- and H-antigens of *E. coli*were prepared according to ∅rskov and ∅rskov \[[@B3]\]. Antisera for typing of flagellar antigens H4 were produced with reference strains U9-41 (O2:K1:H4) and P12b (O15:H17) \[[@B3]\]. Our strain P12b was found to express its *fliC*encoded H4 antigen and produced flagellar type H4 (this work). Motility inhibition test ------------------------ Expression of flagella and swarming of *E. coli*strains was tested by inoculating bacteria in tubes containing 10 ml portions of swarm-agar (L-broth + 0.3% agar) as described \[[@B3]\]. Inhibition of motility of *E. coli*strains in the presence of flagellar-specific antiserum (H4 and H48) was tested in swarm-agar containing a 1:600 dilution of the respective antiserum. Cultures which were inhibited for motility were observed over two weeks for possible switch to motility by phase variation. Serological typing of H-antigens of *E. coli* --------------------------------------------- H-serotyping was performed as described \[[@B3]\]. In brief, bacteria were grown in tubes containing 10 ml 0.3% semi-solid LB-agar \[[@B23]\] for two to three passages. Highly motile bacteria were transferred in LB-medium, incubated 6h at 37°C and inactivated by addition of 0.5% formaldehyde in solution. Agglutination reactions were performed in two fold dilutions of 0.5 ml portions of serum in phosphate-buffered saline pH 7.4 (PBS) \[[@B22]\] with 0.5 ml formalized bacteria in glass tubes which were incubated for 2h at 50°C. Agglutination tests were read by eye immediately after incubation as described \[[@B3]\]. PCR-typing of *fliC* genes -------------------------- The oligonucleotide primers fliC-1 (5\' CAA GTC ATT AAT AC(A/C) AAC AGC C 3\') and fliC-2 (5\' GAC AT(A/G) TT(A/G) GA(G/A/C) ACT TC(G/C) GT 3\') were used for amplification of internal parts of *fliC*genes present in the *E. coli*reference strains as described \[[@B9]\]. The PCR was performed for 25 cycles at 94°C for 60 sec, 55°C for 60 sec and 72°C for 120 sec \[[@B9]\]. PCR products of sizes varying between 950 to 2500 bp were obtained with *E. coli*reference strains for 53 different H-types \[[@B3]\] (Figure [1](#F1){ref-type="fig"}). Amplified DNA was digested with HhaI and the resulting restriction fragments were compared on 2% agarose gels. Restriction enzymes HpaII and MboI were used for characterization of fliC-H4 specific PCR products. Gel images were stored digitally and analyzed with BioNumerics software, version 2.5 (Applied Maths, Kortrijk, Belgium) for similarity (Dice, complete linkage) (Fig. [1](#F1){ref-type="fig"}). Nucleotide sequence analysis of *fliC*genes ------------------------------------------- Two primers deduced from the published *fliC*-H4 sequence (AB028472) were used for the amplification of the entire *fliC*-H4 coding regions. The PCR was performed for 30 cycles: 30 sec at 94°C, 60 sec at 58.1°C and 90 sec at 72°C with primers fliC-5 (5\'-TGA GTG ACC AGA CGA TAA CAG GG-3\') and fliC-6 (5\'-GGA CGA TTA GTG GGT GAA ATG AGG-3\') and yielded a 1243 bp product. PCR products were purified with the QIAquick™ PCR Purification Kit (Qiagen, Hilden, Germany) and used for sequencing. Sequencing reactions were carried out using the dye terminator chemistry (PE Applied Biosystems, Darmstadt, Germany) and separated on an automated DNA sequencer (ABI PRISM^®^3100 Genetic Analyzer). The sequences were analysed using the Lasergene software (DNASTAR, Madison,WI, USA) and the Mac Vector software (Oxford Molecular Group, Campell, CA, USA) to assemblings and alignings. Nucleotide sequence accession numbers ------------------------------------- The nucleotide sequence of the genomic region of *E. coli*strain P12b (O15:H17) with a size of 1234 bp containing the *fliC*gene for flagellin has been submitted to EMBL data library under accession number AJ515904. The coding sequences of the different *fliC*genes from the following strains have been assigned the following accession numbers: AJ605764 for strain U1-41 (O5:K4:H4), AJ605765 for strain P7d (O68:H4), AJ605766 for strain C107-74 (O15:H17) and AJ536600 for strain E1541-68 (O154:H4). The origin of the strains is listed in Table [1](#T1){ref-type="table"}. Molecular cloning of *fliC*gene PCR products -------------------------------------------- PCR products encompassing the complete coding sequences of the *fliC*-H4 genes were obtained from genomic DNA prepared as described \[[@B9]\] of *E. coli*strains U9-41 and P12b using primers fliC-5 and fliC-6. The amplification products were inserted into the vector pLITMUS38 (New England Biolabs, Beverly, MA, USA) digested with EcoRV. The orientation of the the insert PCR products was determined by using commercially available sequencing primers LITMUS forward 28/38 and LITMUS Reverse 28/38 (New England Biolabs). Immuno electron microscopy (IEM) of *E. coli*flagellar antigens --------------------------------------------------------------- Motile cultures of *E. coli*strains were produced by repeated passage on semi-solid agar followed by growth in L-Broth as described above. Cultures carrying recombinant pLITMUS38 plasmids were grown in the presence of 100 μg/ml ampicillin. IEM was performed using fresh, non-formalized cultures of motile bacteria. For IEM, aliquots of respective bacterial cultures were diluted 1:2 in PBS pH 7.2 and adsorbed onto glow-discharge treated 400 mesh grids coated with Pioloform and carbon (Wacker Chemie, Munich, Germany) \[[@B31]\]. Grids with adsorbed bacteria were preincubated for 30 min at room-temperature with blocking buffer (0.1 % bovine serum albumine (Sigma, Deisenhofen, Germany) in PBS). Rabbit anti-H48- and anti-H4 hyperimmune sera were diluted 1:1000 in blocking buffer. After conditioning, specimens were incubated for 30 min at room temperature on droplets of the specific, unlabelled antibodies. Non-bound antibody was removed by washing the grids twice for 10 min on blocking buffer. Immuno-specifically bound primary antibodies were detected using anti-rabbit-IgG-gold 5 or -gold 10 nm conjugates (British Bio Cell International Ltd, Cardiff, UK). The conjugates were diluted 1:20 in blocking buffer and reacted for 30 min at room temperature. Unbound conjugate was removed by a sequence of washing steps (two times with blocking buffer for 5 min each; once with PBS for 3 min and a final wash with double destilled water for 3 min) at room temperature. Before negative staining with 1 % uranyl acetate (pH 4.0--4.5), the grids were washed rapidly on 4 droplets of double destilled water. The preparations were analyzed with an EM 10 electron microscope (Zeiss-LEO, Oberkochen, Germany) at an accelerating voltage of 80 kV. To look for different antigenic determinants expressed on the flagella of strains JM109 or TPE1978, double immuno-labelling was performed using H48 and H4 antisera sequentially and two anti-rabbit-IgG-gold conjugates with different sized markers for the detection of the bound primary unlabelled rabbit antibody. Both *E. coli*strains were incubated with rabbit anti-H4 (1:1000) followed by anti-rabbit-IgG-5 nm gold and two washing steps on droplets of blocking buffer for 10 min. The samples were subsequently incubated with anti H48 antibody (1:1000) followed by incubation with anti-rabbit-IgG- 10 nm gold. Removal of surplus conjugate and negative staining were performed as detailed above. Author\'s contribution ====================== LB conceived of the study and carried out PCR genotyping and coexpression studies. ES carried out sequence determination and alignments and construction of recombinant plasmids. SZ and SK performed serological assays and PCR genotyping. CS performed analysis of *fliC*recombinant plasmid carrying strains. AM and HG developed the IEM methodology and HG contributed to the data analysis. All authors participated in review and preparation of the final manuscript. Acknowledgements ================ We are grateful to Ida and Frits ∅rskov and to Flemming Scheutz (International Escherichia and Klebsiella Centre, Statens Seruminstitut, Copenhagen, Denmark) for donating *E. coli*serotype reference strains. We thank Bärbel Jungnickel for excellent processing of the electron micrographs. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### HhaI digested *fliC*PCR products obtained with primers fliC-1 and fliC-2 from *E. coli*reference strains for 53 different expressed H-types as indicated at the right side. Similarity of restriction fragment patterns was calculated with BioNumerics software and is indicated by the dendrogram on the left side. The flagellar antigens of strains encoding H-types H3, H17, H35, H36, H44, H47, H53, H54 and H55 are not encoded by *fliC*but by other genes (*flkA*, *fllA, flmA*and others) in the corresponding *E. coli*strains and the *fliC*HhaI patterns obtained from these strains do therefore not correspond to their H-serotypes \[21, 25, 26, 27\]. ::: ![](1471-2180-5-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Alignment of the deduced FliC (flagellin) sequences from *E. coli*strains representing the flagellar antigen H4 and its genetic variants: U9-41 (accession BAA85081); C107-54 (accession CAE53943), E1541-68 (accession CAD60547); P12b (accession CAD56695); and P7d (accession CAE53942). ::: ![](1471-2180-5-4-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Electrophoretic separation of restriction enzyme digested *fliC*PCR products (primers fliC-1 and fliC-2) of *E. coli*strains U9-41 and P12b on 2% agarose: Lanes: 1+8= molecular weight standard; 2 = U9-41 (HpaII); 3 = P12b (HpaII); 4 = U9-41 (undigested); 5 = P12b (undigested); 6 = U9-41 (MboI); 7 = P12b (MboI). Sizes of restriction fragments are listed in Table 2. ::: ![](1471-2180-5-4-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### \(A) Low power micrograph of *E. coli*strain TPE1978 cells showing the density of the bacterial samples used for indirect IEM and the presentation of flagella (bar length = 1 μm) (B) IEM of strain TPE1978 flagella after incubation with rabbit flagellar H48 antiserum (1:1000) and detection of bound antibody by anti-rabbit-IgG- 10 nm gold (1:20), bar length = 100 nm. (C) Strain JM109 flagella after incubation with rabbit flagellar H48 antiserum and detection of bound antibody by anti-rabbit-IgG- 10 nm gold. (D) Strain TPE1978 flagella after incubation with rabbit flagellar H4 antiserum (1:1000) and detection of bound antibody by anti-rabbit-IgG- 5 nm gold. (E) Strain JM109 flagella after incubation with rabbit flagellar H4 antiserum and detection of bound antibody by anti-rabbit-IgG- 5 nm gold. (F) Double-labeling IEM of strain TPE1978 after sequential incubations with rabbit flagellar H4 antiserum and anti-rabbit-IgG 5 nm gold, followed by rabbit flagellar H48 antiserum detected by anti-rabbit-IgG- 10 nm gold. Both, 5 nm and 10 nm gold markers are bound at comparable amounts over all flagella present on the bacteria. (G) Double-labeling IEM of strain JM109 after sequential incubations with rabbit flagellar H4 antiserum and anti-rabbit-IgG- 5 nm gold followed by rabbit flagellar H48 antiserum and anti-rabbit-IgG- 10 nm gold. Only H48 specific (10 nm) gold particles are bound to the flagella of JM109. ::: ![](1471-2180-5-4-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Agglutination titers with H4 antisera derived from strains U9-41 and P12b ::: Strain reported serotype agglutination with antisera^a^ ---------- ------------------- -------------------------------- ------- U9-41 O2:K1:H4^b^ 12800 12800 U1-41 O5:K4:H4^b^ 25600 25600 P7d O68:H4^b^ 1600 3200 E1541-68 O154:H4^b^ 3200 1600 P12b O15:H17^c,e^ 6400 12800 872-69 O20:H17^d,e^ 12800 12800 107-74 O15:H17^d,e^ 3200 3200 305-78 O15:H17^d,e^ 3200 3200 870-69 O20:H17^d,e^ 6400 12800 327-01 O77:H17^d,e^ 12800 12800 a\) reciprocal value of agglutination titers with antisera (the same results were obtained in two separate experiments). H4~U9-41~and H4~P12b~indicates the H4 antisera produced with strains U9-41 and P12b, respectively. b\) *E. coli*standard test strain \[3-5\] c\) *E. coli*standard test H-test strain \[3-5\], able to produce spontaneously a variant expressing flagellar antigen H4 and containing a *fliC*-H4 gene \[21, 22\] d\) strains and serotype data provided from Flemming Scheutz, Statens Seruminstitut, Copenhagen, Denmark e\) all H17 strains were shown to carry a *fliC*-H4 genotype and expressed flagellar H4 antigens (this work). ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Amino acid identitity/divergence between the deduced FliC proteins of the six investigated *E. coli*strains (aligned length 349 aa, no gaps). ::: percent identity -------------------- ---------- ------------------ -------- --------- ---------- -------- -------- U9-41 U1-41 C107-74 E1541-68 P12b P 7d Percent divergence U9-41 \*\*\* 100.0 99.7 99.4 98.6 97.4 U1-41 0.0 \*\*\* 99.7 99.4 98.6 97.4 C107-74 0.3 0.3 \*\*\* 99.1 98.3 97.1 E1541-68 0.6 0.6 0.9 \*\*\* 98.0 98.0 P12b 1.4 1.4 1.7 2.0 \*\*\* 96.6 P 7d 2.6 2.6 2.9 2.0 3.5 \*\*\* ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### PCR/RFLP typing of *fliC*genes in *E. coli*H4 strains with restriction enzymes HhaI, HpaII and MboI ::: DNA fragments (bp) obtained by enzymatic digestion of the 953 bp PCR product obtained with primers fliC-1 and fliC-2 ------------------------------------ ---------------------------------------------------------------------------------------------------------------------- --------------------------- ---------------------- *fliC*-genotype (prototype strain) HhaI HpaII MboI *fliC*-H4 (U9-41)^a^ 362, 304, 104, 66, 50, 29^d^, 20^d^, 16^d^2^d^ 296, 240, 225, 159, 33^d^ 429, 313, 182, 29^d^ *fliC*-H4 (P7d)^b^ 362, 304, 104, 66, 50, 29^d^, 20^d^, 16^d^,2^d^ 296, 273, 225, 159 429, 313, 182, 29^d^ *fliC*-H4 (P12b)^c^ 362, 304, 104, 66, 50, 29^d^, 20^d^, 16^d^,2^d^ 296, 273, 225, 159 495, 458 a\) PCR/RFLP patterns found in strains U9-41, U1-41, E1541-68, C107-74 and in 82 *E. coli*strains which reacted with H4 antisera. The serotypes of all 86 strains are: O2:K1 (4 strains), O5 (2), O7 (1), O8 (1), O13 (1), O15 (2), O20 (2), O50 (1), O60 (1), O77 (1), O78 (1), O99 (3), O111 (9), O113 (32), O114 (5), O117 (1), O119 (1), O141 (3), O154 (1), O176 (1), O181 (1), O-untypable (9), O-rough (3). Forty-two of the strains belonging to O-groups O60, O113, O114, O141 as well as O-rough and O-untypable strains produced Shiga-toxins. b\) RFLP patterns found with strain P7d (O68:H4) c\) RFLP-patterns found with strain P12b (O15:H17) d\) fragments smaller than 50 bp are not detectable on 2% agarose gels (Fig. 3). Exact fragment sizes were calculated on the basis of nucleotide sequence analysis for each of the restriction enzymes used ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### H-Agglutination reaction of *fliC*-H4 recombinant plasmid carrying *E. coli*K-12 strains ::: Strain serotype or *fliC*recombinant JM109 derivative agglutination with H-specific antisera^a^ ---------- ------------------------------------------------ ------------------------------------------- ---------- ---------- U9-41 O2:K1:H4 \<200^b^ 12800 12800 P12b O15:H17 \<200^b^ 6400 12800 JM109^c^ O-rough:H48 12800 \<200^b^ \<200^b^ TPE1976 JM109 (pLITMUS38-*fliC*-H4~U9-41~)^d^ 12800 12800 12800 TPE1978 JM109 (pLITMUS38-*fliC*-H4~P12b~)^e^ 12800 6400 12800 a\) reciprocal value agglutination titers with antisera (the same results were obtained in two separate experiments). H4~U9-41~and H4~P12b~indicates the H4 antisera produced with strains U9-41 and P12b, respectively. b\) no agglutination with start serum dilution 1:200 c\) *E. coli*K-12 \[23\]. d) *fliC*-H4 gene cloned from strain U9-41 e\) *fliC*-H4 gene cloned from strain P12b :::

PubMed Central

2024-06-05T03:55:52.652675

2005-1-24

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548302/", "journal": "BMC Microbiol. 2005 Jan 24; 5:4", "authors": [ { "first": "Lothar", "last": "Beutin" }, { "first": "Eckhard", "last": "Strauch" }, { "first": "Sonja", "last": "Zimmermann" }, { "first": "Stefan", "last": "Kaulfuss" }, { "first": "Christoph", "last": "Schaudinn" }, { "first": "Andrea", "last": "Männel" }, { "first": "Hans R", "last": "Gelderblom" } ] }

PMC548303

Background ========== Persistent atrial fibrillation (AF) is difficult to treat. In the absence of class I or III antiarrhythmic drugs sinus rhythm is maintained in only 30--50% of patients during the first year after Direct Current electrical cardioversion (ECV)\[[@B1],[@B2]\]. Furthermore, even following an aggressive approach with repeated ECVs and use of prophylactic drugs, arrhythmia-free outcome is still poor: only 39% of patients maintain sinus rhythm during two years of follow-up\[[@B1],[@B2]\]. Notwithstanding the recent results of AFFIRM and RACE showing no beneficial effect of rhythm control over rate control a rhythm control strategy may be indicated in severely symptomatic patients and those with a tachycardiomyopathy\[[@B3]\]. In recent years research has focused on the atrial remodeling processes that are induced by AF itself and that trigger the arrhythmia to become sustained: \"AF begets AF\"\[[@B4]\]. One of the remodeling processes induced by AF is fibrosis. Fibrosis causes dispersion of conduction, which, in its turn is related to inducibility of AF\[[@B5]\]. The renin-angiotensin system seems to play an important role in the development of fibrosis in heart failure. It was shown that pre-treatment with enalapril may attenuate atrial fibrosis and conduction abnormalities in a canine model of heart failure, and the occurrence of AF in patients with left ventricular dysfunction \[[@B5]-[@B7]\]. A recent experimental study showed that angiotensin II blockers may prevent electrical remodeling when started before start of AF\[[@B8]\]. In the present study we report on the effects of ACE inhibition on the outcome of ECV and the prevention of early recurrences after ECV of persistent AF. Methods ======= One hundred-seven consecutive patients with persistent AF, defined as the presence of AF for at least 24 hours were included in this study\[[@B9]\]. ECV was performed according to a previously described step up protocol\[[@B10]\]. Successful ECV was defined as the presence of sinus rhythm for at least 4 hours after ECV. No difference was made between unsuccessful ECV due to shock failure or due to an immediate recurrence of AF (within 2 minutes after successful ECV). ACE pre-treatment was defined as use of ACE inhibitors before *onset*of the current AF episode. Most patients on ACE inhibitors used these drugs for hypertension or congestive heart failure. To make sure that all patients were not completely remodeled at the very moment of start of the current episode of AF (and thereby verifying the fact that they were treated with ACE inhibitors before the process of electrical remodeling started), only patients with at least 1 month sinus rhythm before the current episode of AF were included in this study. Duration of AF was determined as precisely as possible by previous electrocardiograms, 24-hours Holter registrations, and by the patient\'s history. None of the patients were on class I or III antiarrhythmic drugs neither at the moment of ECV nor during follow-up. Statistical analysis -------------------- Quantitative variables were compared between groups using a two-tailed t-test for normally distributed variables or a Wilcoxon two-sample test for skewed distributed variables. For qualitative variables (categorical or ordered), group differences were evaluated using a Fisher\'s exact test or a Chi-square test. Accordingly, baseline characteristics are given in mean ± SD, median and range (min-max) or percentages. To determine the predictive factors for successful ECV, an univariate logistic regression analysis was performed using the relevant baseline predictors. Variables with a p-value \< 0.20 were selected for the multiple logistic regression analysis to derive a model with statistically significant predictors, by using a backward selection method. All p-values are two-sided and a p-value of \< 0.05 was considered statistically significant. SAS version 6.12 (Cary, NC) was used for all statistical evaluations. Results ======= Baseline characteristics are listed in table [1](#T1){ref-type="table"}. Twenty-eight patients (26%) were treated with ACE inhibitors *before*start of the current episode of AF (pre-treated patients). In the latter group, ECV was successful in 96% while in the group of patients not pre-treated with ACE inhibitors before start of the current AF episode only 80% had a successful ECV (p = 0.04). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Baseline characteristics ::: All Successful ECV Unsuccessful ECV P ----------------------------------- --------------- ---------------- ------------------ ------ N = 107 N = 90 N = 17 Duration in days, median (range) current episode 115(71--175) 113 (61--175) 126 (81--189) ns total AF duration 144 (95--232) 142 (86--213) 160 (102--340) Ns Number of previous ECVs, N(range) 1 (0--5) 1 (0--5) 1(0--3) Ns Underlying heart disease \* (%) Left ventricular dysfunction 21% 25% 0% 0.02 Hypertension 30% 32% 17% ns Valve disease 19% 19% 22% ns Coronary artery disease 22% 21% 28% ns Lone AF 16% 16% 15% ns Echocardiography (mm ± SD) LA parasternal long axis 47 ± 8 47 ± 9 47 ± 8 ns LA 4 chamber view long axis 67 ± 9 67 ± 10 67 ± 6 ns LA 4 chamber view short axis 46 ± 8 46 ± 9 43 ± 5 ns RA 4 chamber view long axis 62 ± 8 62 ± 8 63 ± 7 ns LVEDD 50 ± 8 50 ± 8 50 ± 7 ns LVESD 35 ± 10 35 ± 10 33 ± 9 ns Medication Beta blocker 49% 50% 48% ns Calcium channel blocker 36% 36% 37% ns Digoxin 47% 46% 49% ns ACE pretreatment 26% 30% 6% 0.04 Diuretics 31% 31% 29% ns Angiotensin receptor blocker 1% 1% 0% ns \* more then 1 entity per patient AF = atrial fibrillation, ECV = electrical cardioversion, LA = left atrium, RA = right atrium, LVEDD = left ventricular end diastolic diameter, LVESD = left ventricular end systolic diameter ::: In general, patients treated with ACE inhibitors before start of the current episode of AF had more evidence for heart disease than those who were not pre-treated. Prevalence of hypertension (46% versus 24%, respectively, p = 0.03), and left ventricular dysfunction (40% versus 14%, respectively, p = 0.01) was significantly higher in the pre-treated group in comparison to those who were not pre-treated with ACE inhibitors. (Table [2](#T2){ref-type="table"}) Furthermore, there was a trend towards a higher prevalence of coronary artery disease in the pre-treated group compared to the not pre-treated group (36% versus 18%, respectively p = 0.07). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Baseline characteristics divided in ACE pre-treatment and no ACE pre-treatment. ::: No ACE pre-treatment ACE-pre-treatment P ----------------------------------- ---------------------- ------------------- ------ N = 79 N = 28 Duration in days, median (range) current episode 121 (74--178) 96 (59--135) ns total AF duration 161 (105--247) 117 (63--160) ns Number of previous ECVs, N(range) 1 (0--5) 1(0--3) ns Underlying heart disease \* (%) Left ventricular dysfunction 14% 40% 0.01 Hypertension 24% 46% 0.03 Valve disease 19% 21% ns Coronary artery disease 18% 36% 0.07 Lone AF 17% 14% ns Echocardiography (mm ± SD) LA parasternal long axis 46 ± 7 50 ± 10 0.03 LA 4 chamber view long axis 66 ± 9 67 ± 12 ns LA 4 chamber view short axis 45 ± 8 49 ± 10 ns RA 4 chamber view long axis 61 ± 8 64 ± 7 ns LVEDD 49 ± 7 54 ± 8 0.01 LVESD 34 ± 9 38 ± 12 ns Medication Beta blocker 51% 47% ns Calcium channel blocker 35% 39% ns Digoxin 45% 51% ns Diuretics 29% 34% ns Angiotensin receptor blocker 1% 0% ns \* more then 1 entity per patient AF = atrial fibrillation, ECV = electrical cardioversion, LA = left atrium, RA = right atrium, LVEDD = left ventricular end diastolic diameter, LVESD = left ventricular end systolic diameter ::: An additional 21 patients received ACE inhibitors *after*start of the current episode of AF because of either insufficiently treated hypertension or left ventricular dysfunction newly documented with echocardiography. Success of ECV in these patients was comparable to patients who were not treated with ACE inhibitors at all (80% and 82%, respectively). Use of beta adrenergic receptor blockers, calcium channel blockers or digoxin, started before the present episode of AF or not, did not influence success of ECV. After 1 month of follow-up 49% of the pre-treated patients compared to 50% of those who were not pre-treated with ACE inhibition were still in sinus rhythm (Table [3](#T3){ref-type="table"}, Figure [1](#F1){ref-type="fig"}). ::: {#T3 .table-wrap} Table 3 All No ACE-pretreatment ACE-pretreatment P ----------------------------- ----- --------------------- ------------------ ------ Successful ECV (%) 84% 80% 96% 0.04 SR at 1 month follow-up (%) 50% 50% 49% ns ECV = electrical cardioversion, SR = sinus rhythm ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Effect of pretreatment with ACE-inhibitors before start of AF. ::: ![](1471-2261-5-3-1) ::: Multivariate analysis showed that pre-treatment with ACE inhibitors and a smaller left atrial size were independent predictors of successful ECV (OR = 5.8, C.I. 1.3--26.1 and OR = 5.6, C.I. 1.2--25.3, respectively). Discussion ========== Main results ------------ This post-hoc retrospective analysis shows that use of ACE inhibitors before the onset of AF enhances acute ECV outcome but it does not improve maintenance of sinus rhythm. Furthermore, when ACE inhibitiors are instituted later, i.e. after the start of AF, it does no longer improve success of ECV. Effect of ACE-inhibition on structural remodeling ------------------------------------------------- ACE inhibitors reduce the incidence of AF in patients with left ventricular dysfunction\[[@B7],[@B6]\]. This may be related to the protective effects of ACE inhibitors, which help to maintain atrial integrity and attenuate fibrosis\[[@B5]\]. In the present study pre-treatment with an oweveHowACE inhibitor (i.e. use of ACE inhibitors before onset of the arrhythmia) improved acute outcome of ECV. In view of the above this suggests that ACE inhibitors may prevent or diminish AF induced structural remodeling. These clinical findings are compatible with experimental findings showing that ACE-inhibition could attenuate heart failure induced atrial functional remodeling and fibrosis in dogs\[[@B11]\]. In atrial tissue from AF patients an ACE-dependent increase of activated extracellular signal-regulated kinase (Erk) type 1 and 2 was found, which may contribute to the development of atrial fibrosis during AF\[[@B12]\]. Effect of ACE-inhibition on electrical remodeling ------------------------------------------------- In 1995 it was shown that AF induces several electrophysiological changes, called electrical remodeling\[[@B4]\]. Experimental data show that ACE inhibition prevents short-term (\< 2 hours) tachycardia-induced atrial electrical remodeling\[[@B8],[@B13]\]. However, enalapril could not attenuate or prevent the long-term (7 days) effects of tachycardia on remodeling\[[@B8]\]. Several studies have investigated the role of calcium channel blockers on electrical remodeling. In these studies it was also shown that although there was a short-term prevention of electrical remodeling, calcium channel blockers did not have a long-term protective effect on electrical remodeling \[[@B14]-[@B16]\]. Effect on AF recurrences ------------------------ Madrid et al. investigated the role of the angiotensin II type 1 receptor antagonist irbesartan in amiodarone treated patients\[[@B17]\]. Persistent AF patients were randomized to either treatment with amiodarone alone or treatment with amiodarone in combination with irbesartan. Drug treatment was started at least 3 weeks before cardioversion but *after*the start of AF. No differences were found in electrical cardioversion outcome between the two treatment groups which is in contrast to the present study. This may relate to the fact that all patients were in AF at the very moment of start of irbesartan. Two months after cardioversion it appeared that patients treated with irbesartan and amiodarone had a significantly lower AF recurrence rate compared to amiodarone alone treated patients, 15% versus 37%, respectively (p = 0.007). This difference was maintained during a median follow-up of 254 days. According to their figure 2, the benefit of irbesartan (in combination with amiodarone) was mainly achieved by reduction of recurrences after the first two weeks after cardioversion. An earlier study on the role of ACE inhibitors in patients with heart failure and AF showed a trend towards more patients maintaining sinus rhythm after ECV when instituted on lisinopril in comparison to patients not treated with lisinopril\[[@B18]\]. In the present study no difference in recurrence rate could be found between patients with and without ACE-pretreatment. Limitations of the study ======================== This was a non-randomized and post-hoc analysis. This implies that all findings can only be used to generate hypotheses. However, this is the first clinical study showing that ACE-inhibition initiated before start of AF enhances direct ECV outcome. Only a very small amount of patients was on angiotensin receptor blockers. Whether the effect of pretreatment with angiotensin receptor blockers on cardioversion outcome would be the same as the effect of pretreatment with ACE inhibitors could not be investigated in this study. However, in this context the results of the study of Madrid et al. are encouraging \[[@B17]\]. Conclusions =========== Pretreatment with ACE-inhibitors significantly improves acute outcome of ECV when initiated before the onset of AF but it does not lead to better maintenance of sinus rhythm. When ACE inhibitiors are, however, instituted later, i.e. after the start of AF, it does no longer improve success of ECV. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= TVN carried out the study and drafted the manuscript, HC designed the study, and interpreted the data, MB participated in the draft of the manuscript and interpreted the data, DVV participated in the draft of the manuscript and interpreted the data, IVG participated in the design of the study and interpreted the data. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2261/5/3/prepub>

PubMed Central

2024-06-05T03:55:52.656246

2005-1-24

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548303/", "journal": "BMC Cardiovasc Disord. 2005 Jan 24; 5:3", "authors": [ { "first": "Trudeke", "last": "Van Noord" }, { "first": "Harry JGM", "last": "Crijns" }, { "first": "Maarten P", "last": "van den Berg" }, { "first": "Dirk J", "last": "Van Veldhuisen" }, { "first": "Isabelle C", "last": "Van Gelder" } ] }

PMC548304

Background ========== *Clostridium difficile*infection may be a compounding factor in ulcerative colitis (UC) with data suggestive of a role in disease exacerbation or initiation by the organism in a subset of patients \[[@B1]\]. Adjunctive antibiotic therapy, either broad-spectrum or gram positive-specific, has shown limited efficacy in UC \[[@B2],[@B3]\]. However, sporadic reports in which significant therapeutic potential was achieved \[[@B4]-[@B6]\] suggest that a distinct subclass of patients with an infectious-associated phenotype may exist. Although there have been an increasing number of case reports on ulcerative colitis complicated by cytomegalovirus infection \[[@B7]-[@B9]\], there are few with conclusive evidence of infection with *Clostridium difficile*. Herein, we report a case of steroid-resistant UC with profound rheumatologic involvement immediately preceded by *C. difficile*infection in which remission of UC symptoms was twice achieved in response to adjunct metronidazole and vancomycin therapy. The patient remained symptom-free without supportive therapy for a four-year period. Case presentation ================= A 32-year-old white male presented with nausea, vomiting, crampy lower abdominal pain and diarrhea. His initial lab values were hemoglobin 15.6 g/dl (14--18 g/dl), hematocrit 45.8% (40--54%), sodium 131 mEq/l (135--146 mEq/l), potassium 3.9 mEq/l (3.5--5.5 mEq/l), bicarbonate 24 mEq/l (22--24 mEq/l), chloride 91 mEq/l (95--112 mEq/l), BUN 37 mg/dl (7--25 mg/dl), serum creatinine 1.6 mg/dl (0.7--1.4 mg/dl), and normal liver function tests. He was treated with intravenous fluids, Ciprofloxacin and anti-emetics, to which the diarrhea resolved in 2--3 days. After one month he had recurrent watery diarrhea accompanied with rectal pain, bleeding and vomiting. He also developed fever of 101.6 F. The physical exam was remarkable for decreased bowel sounds and moderate lower abdominal tenderness. There were no masses or organomegalies palpable. The rectal exam was remarkable for tenderness and heme-positive mucus. The remainder of the physical exam was uneventful. Stool samples from the initial visit were positive for *Clostridium difficile*cytotoxin, and depicted many WBCs (neutrophils 99% and eosinophils 1%), suggesting an infectious etiology. Stool sample was negative for giardia lamblia, salmonella, shigella, campylobacter, aeromonas and plesiomonas. There was no predominance of staphylococcus, yeast, or pseudomonas. The patient was initially started on Ciprofloxacin (500 mg bid.) for 3 days without symptom resolution. Metronidazole was added (250 mg tid.), but intolerance of the patient to metronidazole with exacerbations of nausea prompted the replacement to Vancomycin (250 mg qid) in conjunction with Ciprofloxacin for 7 days. During this initial treatment period, the patient characteristically developed migratory polyarticular joint pain and swelling involving the shoulders, elbows, fingers, hips and knees. Lack of response to antibiotics suggested an autoimmune disease as a contributing factor. At this time a sigmoidoscopy and pinch biopsy revealed diffuse acute and chronic inflammation with cryptitis, mucin depletion, and glandular foreshortening and branching suggestive of UC (Fig. [1](#F1){ref-type="fig"}). The patient was treated with Asacol (400 mg bid.) in conjunction with metronidazole (250 mg qid). After 7 days of combined therapy with continued weight loss and no improvement, prednisone (60 mg qid.) was added, resulting in cessation of GI symptoms and moderate improvement in joint pain and swelling. After 7 days of continued improvement, the antibiotic and Asacol therapy were stopped. The patient was maintained on high dose steroids for 14 days. However, fever, gastrointestinal, and joint symptoms recurred upon tapering of prednisone to 20 mg a day. When symptoms recurred, a stool culture was negative for infectious agents including *Clostridium difficile*antigen, confirming an autoimmune component to disease pathology in this patient. Prednisone was increased to 30 mg per day at which point gastrointestinal symptoms resolved but joint swelling and pain continued. Over the course of 3 months prednisone tapering was attempted three additional times, with each resulting in symptom recurrence. After 3 months of treatment, the patient experienced an acute dental abscess and was prescribed a combination vancomycin (250 mg tid) and metronidazole (250 mg tid). During this period, the patient was able to gradually reduce prednisone dosage without symptom recurrence. Antibiotics were continued for 30 days during which time prednisone was tapered completely. Symptoms did not recur and the patient remained symptom-free without further therapeutic intervention for 4.5 years. After this prolonged period of remission of UC symptoms, a second episode similar to the first occurred. The patient once again presented with bloody diarrhea, nausea, vomiting, and joint pain. Stool cultures were *Clostridium difficile*positive by culture and cytotoxin. Once again the patient was treated with 60 mg daily of prednisone and metronidazole (250 mg tid). Symptoms did not resolve, and vancomycin (125 mg tid) was added, resulting in a significant improvement in symptoms. After 2 weeks of combined therapy metronidazole treatment was stopped and symptoms recurred within a week. Metronidazole was once again added, leading to resolution of symptoms. Combined antibiotic and steroid therapy continued for 2 months during which time prednisone was tapered completely and a second remission of UC symptoms was induced. Discussion ========== The patient was remarkable in several regards. The onset of UC and spondyloarthropathy coincided with *C. difficile*infection. While various therapeutic regimens of prednisone, vancomycin and metronidazole preceded a negative *C. difficile*toxin test, remission of UC symptoms could only be induced by the combination of these three medications. *C. difficile*toxin tests have shown varying efficacy in diagnosing pseudomembranous colitis. It has been demonstrated that toxin-negative patients with *C. difficile*positive stool culture may still have symptomatic pseudomembranous colitis \[[@B10]\]. Lynch et al showed that 46% of stool specimens from patients test negative by cytotoxin but positive by culture \[[@B11]\]. Likewise, others have shown that some cases of *C. difficile*infection cannot be detected by the cytotoxin assay and suggested that the organism has the ability to vary toxin production \[[@B12]-[@B16]\]. The bacterium also tends to persist as an antibiotic-resistant vegetative spore, which explains the high frequency at which the symptoms recur following treatment. Since toxin-negative patients have presented with *C. difficile*related diarrhea, it is reasonable to assume that the organism may exist undetectably in a symptomatic patient, but especially in a patient with inflammatory bowel disease (IBD). The elusiveness of the bacterium in diagnosis of *C. difficile*infected IBD patients was confirmed in 2001 by Markowitz et al. They demonstrated that single-toxin assays failed to identify *C. difficile*infection in approximately 40% of pediatric IBD patients and in a case report where a toxin A positive, toxin B negative variant was detected \[[@B14],[@B17]\]. It has also been shown that IBD patients have a higher incidence of *C. difficile*infection than in healthy controls \[[@B18]\]. When considered together with the case reported here, this information leads us to the hypothesis that a small subclass of UC pathology may actually have an infectious etiology resulting from chronic *C. difficile*infection. IBD has been shown to be more prevalent in industrialized nations, where antibiotic treatment has its longest history \[[@B19]\]. The increase in IBD prevalence parallels rising antibiotic use over the last 50 years \[[@B20]\]. If an undetected antibiotic-resistant enteric pathogen is responsible for inducing or exacerbating IBD, then widespread antibiotic use may have contributed to the increased prevalence of IBD in these industrialized nations. This case is especially interesting considering *H. pylori*and its recently discovered tendency to adhere to glycoconjugates expressed in inflamed gastric mucosa \[[@B21]\]. In light of this new information, it is tempting to speculate that *C. difficile*could similarly bind to inflamed colonic tissue in this subclass of UC patients. Further research in this area could yield valuable new data. The potential utility of combined therapy is further suggested by the fact that antimicrobial resistance among C. difficile strains to metronidazole and with intermediate resistance to vancomycin is emerging in countries like Hong Kong (where one of 100 C. difficile isolates was resistant to metronidazole) and Spain (where 9% of 469 clinically significant C. difficile isolates were resistant to metronidazole, particularly in isolates recovered from HIV-positive patients and few patients also had intermediate resistance to vancomycin) \[[@B22]-[@B24]\] It is interesting to note that no resistance was found to metronidazole or vancomycin among C. difficile strains from isolates in patients from UK, Germany, Brazil, Poland or Kuwait \[[@B25]-[@B29]\]. The Public Health Laboratory Service (PHLS) Anaerobe Reference Unit (ARU) has also not been successful in detection of metronidazole resistance in any of their over 1000 C. difficile isolates tested \[[@B24]\]. The impact of drug resistance should be considered if long-term treatment is utilized in patients. However, a complicating factor in this context is that for metronidazole and vancomycin, minimum inhibitory concentration susceptibility breakpoints are usually set for isolates causing systemic infections that are based upon antimicrobial levels in blood (serum) and not on the levels in the intraluminal area, where higher drug concentrations can be achieved \[[@B30]\]. Conclusions =========== Recurrent relapses could only be suppressed in this patient by the combination therapy of corticosteroids, metronidazole and vancomycin. Our observations suggest that this combination therapy may be effective to confront and induce remissions of UC symptoms in patients with *C. difficile*toxin-positive refractory autoimmune symptomatologic UC as opposed to the use of a single agent. An opportunistic *C. difficile*infection commonly results from the use of broad-spectrum antibiotics like quinolones. The frequent use of these antibiotics in treating IBD suggests that these patients could develop *C. difficile*infection during treatment, thereby exacerbating symptoms and preventing remission of UC symptoms. It may therefore be practical to probe for *C. difficile*infections more meticulously in patients with IBD. Competing interests =================== The author(s) declare they have no competing interests. Authors\' contributions ======================= All authors contributed equally to this work. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-230X/5/3/prepub> Acknowledgements ================ Written consent was obtained from the patient for the publication of patient\'s details. This work was supported by grants NIH 1 P20 RR15577, and NIH 1 P20 RR16478 from the National Institutes of Health, and a grant from FAIR, the Fund for Arthritis and Inflammatory Research Inc. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Colonic mucosal inflammation in ulcerative colitis with loss of goblet cells and neutrophilic infiltrate (magnification ×100) ::: ![](1471-230X-5-3-1) :::

PubMed Central

2024-06-05T03:55:52.658539

2005-1-24

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548304/", "journal": "BMC Gastroenterol. 2005 Jan 24; 5:3", "authors": [ { "first": "Jonathan", "last": "Miner" }, { "first": "M Monem", "last": "Gillan" }, { "first": "Philip", "last": "Alex" }, { "first": "Michael", "last": "Centola" } ] }

PMC548305

Background ========== In 1943 Kanner coined \"infantile autism\" (autism derived from Greek *autos*, or self) after observing 11 children, mostly boys, on the basis of their social isolation. Autism, often referred to as autistic disorder or infantile autism, is a complex behavioral disorder, which, by definition, develops prior to age three. Autism is defined completely on the basis of impairments in social interaction and communication, repetitive and stereotypic behaviors. Recent research has examined autistic traits in a population of twins and found that social impairment actually follows a unimodal distribution without a clear demarcation to separate cases of the disorder \[[@B1]\]. For this reason, future discussion of autism may be referred to as autism spectrum disorder (ASD) \[[@B2]\]. For most children, the onset of autism is gradual; however, about 30% have a \"regressive\" onset. Fifty to seventy percent of children with autism are defined as mentally retarded by nonverbal IQ testing. Seizures develop in about 25% of children with autism. About 25% of children who fit the diagnostic criteria for autism at age two or three years subsequently begin to talk and communicate, and by six or seven years blend to varying degrees into the regular school population. The remaining 75% continue to have a life-long disability requiring intensive parental, school, and societal support. There are no biologic markers, therefore the standard criteria, compiled by the American Psychiatric Association Manual of Psychiatric Diseases, 4th edition (DSM-IV), is the primary diagnostic reference used in the United States for autism. The causes of autism can be divided into \"idiopathic,\" which comprises the majority of cases, and \"secondary,\" in which an environmental agent, chromosome abnormality, or single gene disorder can be identified. About 5--10% of individuals with autism can be diagnosed with secondary autism; the remaining 90--95% have idiopathic autism. About 30% of children with idiopathic autism have complex autism, defined by the presence of dysmorphic features or microcephaly or a structural brain malformation \[[@B3]\]. About 70% of children with idiopathic autism have essential autism, defined as the absence of physical abnormalities. A recent CDC case-finding study in Brick Township, New Jersey reported prevalence at 40 per 10,000 for autism \[[@B4]\]. While the latest epidemiologic study from the United Kingdom utilizing specialized visiting nurses who monitored child health and development at seven months, 18 to 24 months, and three years of age reported a prevalence rate of 16.8 per 10,000 for autism \[[@B5]\]. The sex ratio for autism has been estimated at 4 males:1 female \[[@B6],[@B7]\]. Social cognition and communication in autism may be related to dysfunction in the amygdala, hippocampus, and related limbic and cortical structures. The cerebellum may also form part of a distributed neuronal network responsible for social cognition and communication. Serotonin is the neurotransmitter implicated in autism \[[@B8]\] The single gene disorders in which secondary autism is observed include fragile X syndrome, tuberous sclerosis, phenylketonuria, Rett syndrome \[[@B3],[@B9]\], Sotos, Neurofibromatosis I, Joubert syndrome \[[@B10]\], and Smith-Lemli-Opitz syndrome \[[@B11]\]. Risk to sibs of idiopathic cases is 75 times greater than the prevalence in the general population \[[@B7]\] and higher concordance for autism among monozygotic (60--90%) than dizygotic (0--10%) twins \[[@B12]\] argue for a genetic predisposition to idiopathic autism. Multiple independent whole genome scans and chromosomal abnormalities studies have pointed out several candidate regions on chromosomes 2q, 3q, 7q, 6, 13q, 15q, 16p, 17q and sex chromosomes. These regions possess candidate genes that have been screened for mutations or association with autism \[[@B13]\]. However, a clear involvement of a major susceptibility gene (or genes) in autism remains far from clear. The results from linkage studies and the drop in the concordance rates between monozygotic and dizygotic twins suggests that the genetic etiology of autism is certainly heterogeneous (different genes in different families), polygenic (more than one affected gene per individual) with epigenetic influences and allelic heterogeneity (different variants in the same gene may lead to different patterns of genetic disease) \[[@B13]-[@B15]\]. To gain further insight into secondary autism we have compiled data on patients with autistic traits tested for fragile-X syndrome using molecular methods and chromosome abnormalities using cytogenetic analysis and FISH. Methods ======= A search was initiated using the key word autism in the indication field of the Genzyme Genetics, Orange laboratory database. For each case the electronic data and/or files were reviewed. The referral center, age, sex, karyotype, fluorescence in situ hybridization (FISH) and fragile X results were extracted and tabulated. G-banded metaphases were prepared using standard procedures and FISH was performed using the protocol provided by the manufacturer of the commercial probes (Vysis Inc., Illinois, MI). Fragile X test: Isolated DNA was tested by both Southern blot analysis and Polymerase chain reaction (PCR) for the size and methylation status of the CGG repeat expansion within the FMR-1 gene. Southern blot analysis was performed with the probe StB12.3 on EcoR1 and Eag1 digested DNA. PCR products were separated by acrylamide gel electrophoresis and detected with a CGG repeat probe. Results ======= Patients with autistic traits were referred by physicians to our laboratory for genetic testing. The clinical diagnostic criteria applied were not specified on the test request forms. A total of 433 patients with an indication of autism were sent to our laboratory for genetic diagnosis. The median age was 4 years. Sex ratio was 4.5 males to 1 female \[354:79\] (Table [1](#T1){ref-type="table"}). A chromosome abnormality was found in 14/421 \[3.33 %\] cases. A Fragile-Xq27.3 was diagnosed in 7/316 \[2.2 %\]. Chromosome abnormalities are summarized in Table [2](#T2){ref-type="table"}: The aberrations were: 4/14 \[28%\] supernumerary markers from cs 15 \[[@B3]\] and cs 2 \[[@B1]\] (Fig [1a](#F1){ref-type="fig"}); 4/14 \[28%\] deletions of 2q37.3, 3p25, 12q21.2q23.3 and 13q13.2q14.1(Fig [1b](#F1){ref-type="fig"}); 1/14 \[7%\] duplication of 15q11.2q13; 3/14 \[21%\] inversions of 10p11.2q21.2, 17q23q25 (de novo) and 14q11.2q33 (mosaic) (Fig [1c](#F1){ref-type="fig"}); 2/14 \[14%\] translocations, one balanced t(1;14) and one unbalanced der(14;18) (Fig [1d](#F1){ref-type="fig"}). The 3/4 marker/ring had a 15 centromere signal by FISH and had either nil, one or two signals for D15S11, that demarcates the involvement of the critical region of Prader Willi/Angelman syndrome (Table [2](#T2){ref-type="table"}). Fluorescence in situ hybridization was performed on 23 of 433 patients for reasons other than to characterize a cytogenetic abnormality, 3 to rule out Williams-Beuren Syndrome, 5 for DiGeorge syndrome, 7 for proximal duplication of D15S11, 6 for subtelomere rearrangements, 1-for subtelomere rearrangement & proximal duplication D15S11 and 1 for duplication D15S11, Smith-Magenis Syndome, and DiGeorge Syndrome. All 23 FISH tests were negative (Table [3](#T3){ref-type="table"}). Fragile X (Table [4](#T4){ref-type="table"}): The mutations detected were: A full mutation (fM) and abnormal methylation in 3 \[43 %\] and mosaic mutations with partial methylation of variable clinical significance in 3 \[43%\]. The mosaic size mutations were: an fM \[200--900 repeats(r)\] / deletion mutation \[30 r\] with partial methylation. The faint bands for the full mutation and deletion mutation may be a reflection of the sample quality (Fig [2](#F2){ref-type="fig"}, Lane 5). Two size mosaics had permutation (pM) \[150 r\]/fM \[400 r\] (Fig [2](#F2){ref-type="fig"}), and pM \[155 r\]/fM \[800 r\] with normal and abnormal methylation. A premutation mosaic female carrier with an atypical EcoR1 and Eag1 pattern and a typical BssH1 pattern gave 2.8, 3.0, 5.2--5.4 Kb bands, the Eag1 pattern suggested a DNA sequence change \[14%\]. PCR gave reproducible bands corresponding to 29, 65, 80 repeats and a faint band for 39 repeats. Intermediate mutation (45--54 r) was found in 5 males and 2 females. Discussion ========== Chromosomal causes of secondary Autism Spectrum Disorder (ASD) -------------------------------------------------------------- About 1.7 % to 4.8 % of individuals with ASD have chromosome abnormalities, including unbalanced translocations, inversions, rings, and interstitial deletions and duplications (Table [5](#T5){ref-type="table"}). The chromosome abnormalities that have been reported on more than one occasion are duplication of 15q, deletions of 18q, Xp, 2q and the sex chromosome aneuploidies 47,XYY and 45,X \[[@B16]\]. A recent FISH subtelomere study found one out of ten unselected patients with ASD had a subtelomeric 2qter deletion \[[@B17]\]. In our experience 7/7 ASD patients were negative for subtelomeric rearrangements. Children with Down syndrome have autism more commonly than expected. The incidence was at least 7 % in one study \[[@B18]\]. This finding suggests that chromosome abnormalities may lower the threshold for the expression of autism. In our study (4/14 = 28 %) and in other surveys, the common recurring chromosomal abnormality was duplication of the proximal 15q region (Table [5](#T5){ref-type="table"}). About 1% of individuals with ASD have a chromosomal duplication in the Prader-Willi/Angelman region of proximal 15q \[\[[@B19],[@B20]\], present study 4/421\[0.95%\]\]. The duplicated region q11.2q13 is on the maternal chromosome 15 in autistic patients \[[@B21]-[@B23]\]. Most commonly, this is a supernumerary isodicentric 15q chromosome detectable by routine cytogenetic studies or, less commonly, an interstitial duplication of the region detected by FISH analysis for the *SNRPN*gene region. These two chromosome abnormalities have only subtle effects on the physical phenotype. Supernumerary isodicentric 15q chromosomes are *de novo*occurrences. Duplication of proximal 15q may result from segregation of a parental chromosome translocation or an interstitial 15q duplication. An abnormal gene dosage within 15q11.2-q13 might cause susceptibility to autism. The 15q11-q13 region is shown schematically in Figures [3](#F3){ref-type="fig"} &[4](#F4){ref-type="fig"}. Chromosome 15q11.2-q13 has gained support as an autism candidate region on the basis of the association of maternally derived chromosomal duplications of this region with an autistic phenotype \[[@B20],[@B24]-[@B27]\] and genetic evidence for linkage and allelic association in the same interval in chromosomally normal autism families \[[@B28]-[@B34]\]. The maternal specificity of chromosome 15 duplications in autism suggests a genomic imprinting effect. There are multiple imprinted genes in 15q11.2-q13, and two neurodevelopmental disorders exhibiting opposite patterns of genomic imprinting have been mapped to this region \[[@B35]-[@B37]\]. Interstitial deletion of 15q11.2-q13 specific for the paternal chromosome is the most frequent cause of Prader-Willi syndrome (PWS; MIM 176270), whereas maternal-specific deletion of the same common interval results in Angelman syndrome (AS; MIM 105830). The converse of Angelman Syndrome is observed in autism, that is a maternal duplication. The four causes of Angelman syndrome are 1) maternal deletion of 15q11.2q13, 2) paternal UPD15 3) mutations in UBE3A 4) mutations leading to imprinting errors of this region. A population-based study showed a high rate of ASD in AS \[[@B38]\]. But, a mutation was not identified in the UBE3A putative promoter or coding region in 10 idiopathic ASD patients \[[@B39]\]. Lack of expression of the maternally expressed UBE3A gene in the brain is thought to be the cause of AS. Since patients with deletions compared to other types of AS mutations have a more severe phenotype, suggests the involvement of additional gene losses, such as GABAA receptor gene cluster \[[@B40]\]. Transcripts increased in patients with duplications 15q11.2q13 are maternal UBE3A \[[@B41]\], maternal ATP10C \[[@B40],[@B42]\] and other transcripts including antisense transcripts that could regulate gene expression \[[@B43]\] and may contribute to the duplication phenotype. Therefore, over expression of genes in 15q11-q13 probably confers ASD risk. Region proximal to D15S11 is considered to have no phenotypic effect. However, one of our patients had a marker 15 negative for D15S11, therefore, some duplications of 15, proximal to D15S11 and the autism candidate region may also influence susceptibility to autistic traits. Initial studies to characterize the phenotype of 15q11.2q13 duplication patients have found variation among affected people including mental retardation, motor coordination problems, seizure disorder, and impairments in attention, communication, and social function (some but not all with ASD or attention deficit hyperactivity disorder (ADHD)) \[[@B44],[@B45]\]. It appears there may be a parent-of-origin effect on the linkage and association signals in this region of UBE3A and ATP10C \[[@B46],[@B32],[@B33]\]. Further studies across data sets, and rigorous evaluation of potential functional effects of associated alleles, and a thorough assessment of haplotype transmission within ATP10C and neighboring genes would be conclusive. The majority of linkage and association data point to the *GABRB3*gene, which is one of a cluster of γ-aminobutyric acid (GABA) receptor subunits that map to the distal, apparently nonimprinted segment of the duplicated region (Fig. [3](#F3){ref-type="fig"}). Although a number of groups have detected genetic effects at *GABRB3*in independent autism populations \[[@B29]-[@B31]\], other studies have failed to replicate these observations \[[@B47]-[@B49]\]. Yardin et al 2002 \[[@B50]\] recommended a systematic screening by FISH, of chromosome region 15q11.2q13 in cases with autistic-like syndrome A deletion of 2q37.3 was identified by FISH in a patient with autism and macrocephaly in our study. Wolff et al 2002 \[[@B51]\] also reported an autistic patient with a 2q37.3 deletion detected using subtelomere probes. Whole genome screens have suggested several chromosomal regions that are potentially associated with a susceptibility gene for autism. \[[@B52]-[@B57]\]. Three studies revealed positive linkage to 2q \[[@B52],[@B53],[@B57]\] and a third study demonstrated linkage to distal 2q in a subset of patients with autism and delayed onset of phrase speech \[[@B53]\]. Genomic scans are limited by the number of loci that are assessed; therefore, not all areas may be equally represented. It is important to note that telomeric regions may have increased meiotic recombination and may be under-represented in these types of analyses. Thus, the FISH approach is an important correlative study in the search for susceptibility genes. Macrocephaly in ASD: most children with autism are born with normal head circumference and about 20% meet the criteria for macrocephaly \[[@B58]\]. The increased rate of growth in head circumference appears to be most dramatic in the first year of life and corresponds to increased growth of the cerebral cortex as measured by MRI \[[@B59]\]. A de novo deletion of chromosome 3, del(3)(p25), was found in one case with ASD and development delay in our patient pool. A deletion of 3q region was found by Konstantareas & Homatidis 1999 \[[@B60]\]. Genome wide scan found a major susceptibility locus at 3q25-27 and there was also allelic association in families with autism spectrum disorder originating from a subisolate of Finland \[[@B61],[@B62]\]. Animal models and linkage data from genome screens implicate the oxytocin receptor at 3p25-p26 \[[@B61],[@B62]\]. To date, there have been no reports of 12q deletions in patients with ASD, to the best of our knowledge. For the first time we report an interstitial deletion 46,XY,del(12)(q21.2q23.3) in a patient with ASD, development delay, mental retardation, multiple congenital abnormality, and family history of Down syndrome. 46,XY,del(13)(q13.2q14.1)de novo was found in one of our patients\' with ASD. Hyperserotonemia in autism is one of the longest-standing biochemical findings. The serotonin 2A receptor gene (HTR2A) on chromosome 13q14q21 is a primary candidate gene in autism. Converging data from recent genome screens also implicates the genomic region containing HTR2A \[[@B63]-[@B66]\]. Correlation of HTR2A disruption or deletion in our case with a 13q13.2q14.1 deletion and other 13q deletion in ASD patients would complement genome screen data. The recent physical mapping of the serotonin 5-HT(7) receptor gene (HTR7) to 10q23 \[[@B67]\] raises the question if the inversion inv(10)(p11.2q21.2) present in our patient, which is considered a normal familial variant could have long range influence on HTR7 and susceptibility to autism in some cases. So far there has been no observed association or link between chromosome 14 and ASD. Also, the mosaic inversion inv(14)(q11.2q33) \[3/20\] found in one of our patients is considered a cultural artifact when seen in 1or 2 cells. In this study, a patient with inv(17)(q23q25)de novo, had ASD, hypotonia and developmental delay. Chromosome 17q shows association with autism by genome wide scans and in linkage studies \[[@B57],[@B66],[@B68]\]. There is also interest in 17q since serotonin transporter gene (SERT) has been mapped to17q11-q12 \[[@B69]\]. We report a patient with 18p deletion due to an unbalanced translocation between 14 and 18. Majority of the reported cases with autism involve deletion of 18q \[[@B70]-[@B72]\]. A deletion of the 18p-arm (at band 11.3) in about 50% cells and 50% of the cells with a duplication of the long arm in peripheral blood was described in a mildly obese girl with DSM-III-R autistic disorder and moderate mental retardation \[[@B73]\]. Another preschool girl with selective autism and a deletion of Chromosome 18p11.l has also been described \[[@B74]\]. She had communication problems consistent with a diagnosis of autism. However, in the area of reciprocal social interaction she was a little less deviant than most children and had no major behavior problems typical of autistic disorder. Linkage and association studies have suggested at least 2 candidate loci, one on the short and the other on the long arms of chromosome 18 \[[@B75]\]. One of the breakpoints in the balanced translocation, 46,XX,t(1;14)(q25;q31.2) was 1q25, in the present study. A recent report, links D1S1675 that maps to chromosome 1q24 with autism \[[@B76]\] using obsessive-compulsive behaviors as a restricting criterion for the analysis. The proximity of our breakpoint and D1S1675 may be coincidental or causal. Although no cases with X- rearrangements were identified, in this study, the literature on abnormal X chromosome and autism is discussed as it has been cited in multiple cases. An autistic (ICD-10) woman had a translocation, t(X;8)(p22.13;q22.1) \[[@B77]\], a boy with \"autistic disorder\" had duplication of Xp22 \[[@B78]\], and a de novo Xp22.3 deletion was observed in 3 autistic females \[[@B79]\]. Further, mutations in cell adhesion genes NLGN4 on Xp22.3 and NLGN3 on Xq13 are reported in patients with autism \[[@B80]\]. Therefore, subtle Xp rearrangements have to be considered in the cytogenetic assessment of ASD patients Fragile X syndrome ------------------ The typical clinical picture in FRAXA includes mental retardation, macro-orchidism, large ears, and prominent jaw. Within neurons, the FMR protein (FMRP) interacts with mRNA and ribosomes, suggesting a role in regulating protein synthesis \[[@B81]\]. FMRP is heavily synthesized in dendritic spines in response to synaptic activity, and abnormal dendritic spine size and shape have been noted in FRAXA patients and fmr1 knockout mice \[[@B82]\]. These abnormalities may correspond to an abnormal postsynaptic response that weakens synaptic connections \[[@B83]\]. A multicenter study in Sweden \[[@B84]\] found fragile X in 13 of 83 boys (16%) with infantile autism but in none of 19 girls with infantile autism. Klauck et al. (1997) \[[@B85]\] concluded from molecular genetic studies of 141 patients from 105 simplex and 18 multiplex families that an association of autism with fragile X is nonexistent and that the Xq27.3 region is not a candidate for autism. Stoll (2001) \[[@B86]\] presented 11 children under the age of 8 years and the difficulties in diagnosis of fragile X syndrome at this age. The author concluded on the importance of fragile X DNA test for all children with mental retardation, autism, or significant developmental delay without a clear etiology. Whereas only a few percent of children with autism have fragile X syndrome, at least half of children with fragile X syndrome have autistic behaviors, including avoidance of eye contact, language delays, repetitive behaviors, sleep disturbances, tantrums, self-injurious behaviors, hyperactivity, impulsiveness, inattention, and sound sensitivities. The frequency of the fragile X syndrome among individuals with autism was ascertained up to 1993 using cytogenetic method. The incidence ranged from 12.7%--1.6%. However, these studies had different criteria for classifying positive cases. The differences were: the number of metaphases analyzed ranged from 20 to 100 and the cut-off range from 1% to 4% metaphases with a fragile X \[[@B84],[@B87]-[@B94]\]. Using molecular analysis the incidence of fragile X syndrome was 5% (1/20) \[[@B95]\], 3.3%(1/30) \[[@B96]\], 12% (3/25) \[[@B97]\] and the present study 2.2 % (7/316). The difference in incidence between the present (2.2%) and previous studies (3.3% -- 12%) may be due to small sample size in the other studies or clinical criteria for selection of patients or both. ### Mosaicism for FRAX mutation: Forty-three percent of FRAX patients had a mosaic mutation in our study. The prevalence of males who carry a full mutation and a permutation is 15--20% \[[@B98]-[@B102]\] among the affected individuals. Nolin et al 1994 \[[@B103]\] analyzed a group of affected fragile X males by Southern blotting and found 41% (61/148) to be mosaic. This observation of 41% is significantly higher than previous reports 15--20%. The difference could be technical modifications, which permitted the identification of faint premutation bands in some patients. The higher percentage (43 %) of affected males with mosaicism in our study suggests that the occurrence of such individuals may be frequent in patients with ASD. The degree of mental retardation seemed not to be influenced by the presence of premutation alleles in some of the cells and a full mutation in the rest of the cells \[[@B104]\]. While Merenstein et al 1996 \[[@B105]\] study suggests there may be some variation of clinical expression in fragile X males with a full mutation and permutation. It has been hypothesized that these mosaic cases should show higher levels of functioning than those who have only the inactive full mutation gene, but previous studies have provided negative or equivocal results. In one study, the cross-sectional development of communication, self-care, socialization, and motor skills was studied in 46 males with fragile X syndrome under age 20 years as a function of two variables: age and the presence or absence of mosaicism. The rate of adaptive skills development was 2--4 times greater in mosaic cases versus full mutation cases. FMR1 protein (FMRP) levels was shown to correlate with IQ, even in mosaic males for 38% of the IQ variance \[[@B106],[@B107]\]. There was also a trend for cases with autism to be more prevalent in the full-mutation group \[[@B108]\]. However, we found a high incidence (43 %) of mosaic FRAX mutation in autistic patients, which requires confirmation in other FRAX studies of large cohorts of ASD patients. ### Deletion mutation The molecular mechanism of the FRAX is based on the expansion of a CGG repeat in the 5\' UTR of the FMR1 gene in the majority of fragile X patients. The instability of this CGG repeats containing region is not restricted to the CGG repeat itself but expands to the flanking region as well. de Graaff et al 1996 \[[@B109]\] described four unrelated fragile X patients mosaic for both a full mutation and a small deletion in the CGG repeat containing region. Sequence analysis of the regions surrounding the deletions showed that both the (CGG)n repeat and some flanking sequences were missing in all four patients. The 5\' breakpoints of the deletions were found to be located between 75--53 bp proximal to the CGG repeat. This suggests the presence of a hot spots for deletions in the CGG repeat region of the FMR1 gene and emphasizes the instability of this region in the presence of an expanded CGG repeat \[[@B110]-[@B113]\]. All reported cases had fragile X phenotype (which may be an ascertainment bias), and the deletion was usually a faint band suggesting a recent mutation in a small population of cells. Our case had an unusual Southern band pattern, consistent with the presence of both a full mutation (3.7 and 5.8--7.9 kb; 200--900 r)and a deleted (2.8 kb; 30 r) mutation with partial methylation. Since immunohistochemical staining or Western blot analysis was not performed to assess the FMRP production it precludes clinical correlation. A 6-year old mosaic permutation female carrier had 29,65,80,39 repeats. The autism spectrum disorder in our patient may be due to diminished translational efficiency in FMRP production. Tassone et al (2000) \[[@B114]\] \[have shown FMRP in 61% and 70% lymphocytes in two females 91/2 years and 33 years of age with 103/33, 180/30 repeats and IQ of 49 and 90, respectively. The first patient has physical, cognitive, and behavioral features of the fragile X phenotype but FMRP was in the normal range, while the second patient also had FMRP level in the normal range was treated for depression and has a history of ovarian cyst, premature menopause at 27 years, and a hysterectomy at 31 years. She also experienced social anxiety, panic attacks, mood swings, and mild obsessive compulsive behaviors. Johnston et al. (2001) \[115\] recently observed that emotional problems, including depression and interpersonal sensitivity, were more likely to occur in carrier females with \>100 repeats. Austism spectrum disorder has been observed mostly in males with permutations \[[@B114],[@B116],[@B117]\]. The mosaic permutation in our female patient was 80 repeats as the largest expansion which is still below 100 repeats found in the affected cases in literature. Clinical correlation in our permutation mosaic case is hindered by the lack of FMRP studies and even so, the tissue distribution of mosaicism and FMRP profile would be the necessary phenotype determinant. The significance of intermediate alleles found in 7/316 (Average age 4.36 years) of our patients requires further exploration on larger independent samples as they may raise the threshold for important developmental disabilities and/or physical features \[[@B116]\]. The limitations of our retrospective study were a lack of uniform clinical criterion for inclusion and limited behavioral diagnostic information for purposes of dissecting the phenotype. However, for autism diagnosis it may be reliable, as shown in a recent study \[[@B118]\] which examined the UK General Practitioner Research Database (GPRD) and found the diagnosis of autism among general practitioners in the UK had a high positive predictive value. Conclusions =========== In our experience, the incidence of chromosome (3.33%) and fragile-X (2.2%) abnormalities totals to 5.53% in a population of patients with an indication of autism sent for genetic testing. Since, 28% percent of chromosome abnormalities detected in our study were subtle; a high resolution cytogenetic study for ASD patients with a scrutiny of 15q11.2q13, 2q37 and Xp23.3 region should be standard practice. The higher incidence of mosaic fragile-X mutations with partial methylation in our ASD population vs incidence of mosaicism in reported populations with fragile X syndrome \[50% vs 15--40%\], suggests that faint bands and variations in the Southern band pattern may occur rather frequently in fragile X positive autistic patients. The mosaic FRAXA mutation with normal and abnormal or partial methylation may also enhance the threshold for autism spectrum disorder. Careful analysis and high quality gels are required to rule out the type of mutation in patients with autistic traits. FRMP estimates in positive cases would be an extremely useful adjunct especially in mosaic cases. Future studies are necessary to corroborate the high incidence of mosaicism and their role in ASD. Competing interests =================== The author(s) declare that they have no competing interests. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2350/6/3/prepub> Acknowledgements ================ I thank Bernice Allitto, Deborah Anaya for their help and everyone who contributed to this database. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Chromosome abnormalities in patients with autistic traits (A) 4 markers: \[multiple copies\] derived from chromosome 2 \[1\], 15 \[3\] with nomenclature (B) 1 duplication of chromosome 15 with arrow denoting the region involved (C) 3 partial deletions (right homolog)(multiple copies) of 3p25, 12q21.2q23.3 & 13q13.2q14.1 with the ideogram, the arrows denote the deleted region. (D) 3 inversions (the right homolog) (multiple copies), inv(10)(p11.2q21.2), inv(14)(q11.2q32), inv(17)(q24.2q25.3) with arrows on the ideogram showing the inverted region (E) 2 translocations (partials in 2 copies, chromosomes involved (right) and their normal homolog (left)) one apparently balanced t(1;14)(q25;q31.2) and one unbalanced der(14;18)(q10;q10). The ideogram with arrows show the breakpoints ::: ![](1471-2350-6-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Mosaic fragile X mutations: Southern blot using probe StB12.3 on EcoR1 and Eag1 (methylation-sensitive enzyme) digested DNA. Lane 1 -- permutation carrier, Lane 2 & 3 -- normal female, Lane 4 -- normal male, Lane 5 -- an ASD male mosaic with fm (3.7 and 5.8--7.9 kb, 200--900 r)(arrows)/a deletion mutation (2.8 kb faint band, 30 r)(arrow). By PCR a 30 repeats and \>200 repeats were amplified. Lane 6 -- an ASD male mosaic fm (6.4 kb, 400 r)/pm (3.3 kb, 150 r). ::: ![](1471-2350-6-3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Chromosome 15q11-q13 region showing the autism candidate region. A schematic representation of the 15q11-q13 interval duplicated in autism cases and deleted in Prader-Willi/Angelman syndrome is shown. IC denotes the position of the 15q imprinting center. Loci corresponding to previous reports of linkage and association are indicated by dark and light arrowheads, respectively, below the map. (Adapted with permission from Sutcliffe J et al article \"Dense linkage disequilibrium mapping in the 15q11-q13 maternal expression domain yields evidence for association in autism\' in Molecular Psychiatry (2003) 8, 624--634) ::: ![](1471-2350-6-3-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### The map location of D15S11 probe used to characterize the markers ::: ![](1471-2350-6-3-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographics of the ASD patients tested ::: Demographics ------------------------------------------------------ Patients with ASD referred for genetic testing = 433 Median age = 4 years Sex ratio = 4.5 males to 1 female ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The chromosome abnormality found in 3.33 % (14/421) patients with an indication of autism ::: **Sl \#** **Age** **Sex** **Clinical Indication** **Chromosome Result** **FISH Result** **Fragile X Result** ----------- --------- --------- --------------------------------------------------------- ----------------------------------------------- --------------------------------------------------- ---------------------- **Marker 4/14 = 28%** 1 6.1 M Autism 47,XY,+mar\[27\]/46,XY\[3\] .ish der(2)(D2Z+) NRM 2 5 F Autism 47,XX,+mar .ish der(15)(D15Z+,D15S11++, GABRB3+) NT 3 3 M Autism 47,XY,+mar de novo .ish i(15)(q11.2)(rRNA++,D15Z+,D15S11-) NT 4 5.1 F Autism, MR mos47,XY,+r\[15\]/46,XX\[15\] .ish der(15)(rRNA+,D15Z+,D15S11+,GABRB3+). NT **Duplication 1/14 = 7%** 5 16.75 F Autism 46,XX,dup(15)(q11.2q13)de novo .ish dup(15)(q12)(D15S11++,GABRB3++)de novo NT **Deletion 4/14 = 28%** 6 6.5 F Autism, DD, MR, macrocephaly .ish del(2) (q37.3)(D2S447-) NT 7 6.6 F Autism, DD 46,XX,del(3)(p25) de novo NT 8 11.7 M Autism, DD, MR, multiple congenital abnormality, h/o DS 46,XY,del(12)(q21.2q23.3) .ish 12(wcp12x2) NRM 9 3.6 M Autism 46,XY,del(13)(q13.2q14.1)de novo .ish 13q13(D13S6x2),13q14(RBx2) NT **Inversion 3/14 = 21%** 10 2.8 F Autism 46,XX,inv(10)(p11.2q21.2)\* NT NRM 11 3.5 M Autism Mos46,XY,inv(14)(q11.2q33)\[3\]/46,XY\[17\]\* NT NRM 12 3.25 M Autism, hypotonia, DD 46,XY,add(17)(q23) or inv(17)(q23q25)de novo .ish inv(17)(q24.2q25.3)(wcp17x2,MPOx2,D17S928x2) NT **Translocation 2/14 = 14%** 13 2.7 F Autism 46,XX,t(1;14)(q25;q31.2) NT NT 14 3.3 M Autism 46,XY,der(14;18)(q10;q10) NT NRM DD = developmental delay, MR = mental retardation, DS = Down syndrome, NT = not tested NRM = normal \*inv(10) is a normal familial variant and inv(14) is a frequently observed artifact of culture ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Summary of FISH cases ::: **Sl \#** **Age in years** **Sex** **Clinical Indication** **Karyotype** **FISH** **FRAX** ----------- ------------------ --------- ---------------------------------------------- --------------- -------------------------------------------------------------------- ---------- 1 5.25 M Autism 46,XY Normal ish 7q11.23(ELNX2) 2 7 M Autism, FTT, MR, DD 46,XY Normal ish 7q11.23(ELNX2) NRM 3 M Autism 46,XY Normal ish 7q11.23(ELNX2) NRM 4 3.6 M Autism, DD, hypocalcemia, cardiac defect, MR 46,XY Normal ish 22q11.2(TUPLEx2) 5 6.3 M Autism, DD, hypotonia 46,XY Normal ish 22q11.2(TUPLEx2) 6 3.5 M Autism not ordered Normal-ish 22q11.2(TUPLEx2) 7 M Autism 46,XY Normal-ish 22q11.2(TUPLEx2) 8 M Autism 46,XY Normal-ish 22q11.2(TUPLEx2) NRM 9 6.5 M Autism, DD, MR 46,XY Normal, ish 15q12(D15S11x2) NRM 10 11.6 M Autism, DD 46,XY Normal, ish 15q12(D15S11x2) NRM 11 3.5 M Autism 46,XY Normal, ish 15q12(D15S11x2) NRM 12 3 M Autism 46,XY Normal, ish 15q12(D15S11x2) 13 2 M Autism, DD,MR 46,XY Normal-ish 15q12(D15S11x2) NRM 14 2.5 F Autism, DD,MR 46,XX Normal ish 15q12(D15S11) NRM 15 3.6 M Autism, DD, patch hyperpigmentation 46,XY Normal, ish 15q12(D15S11x2) NRM 16 3.5 M Autism, DD, seizures 46,XY Normal- subtelomere panel NRM 17 3.5 F Autism, DD 46,XX Normal-subtelomeres NRM 18 4.6 M Autism, DD 46,XY Normal-subtelomeres NRM 19 8.6 M Autism, DD 46,XY Normal-subtelomeres NRM 20 M Autism 46,XY Normal-subtelomeres NRM 21 2.7 M Autism 46,XY Normal-subtelomeres ABN 22 16 M Autism, DD, dysmorphic features, MR 46,XY Normal, subtelomeres Normal, ish 15q12(D15S11x2) 23 17.5 F Autism 46,XX Normal ish 15q11.2q13(D15S11x2), 17p11.2(SMSx2), 22q11.2(TUPLE1X2) M = male, F = female, NRM = normal, ABN = abnormal, DD = development delay, MR = mental retardation, FTT = failure to thrive. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### The details of Fragile X mutations found in 2.2 % (7/316) patients with a clinical indication of autism ::: **Sl \#** **Age In years** **Sex** **Clinical indication** **FRAXA Result** **Methylation** **Chrom Result** **FISH Result** ----------- ------------------ --------- ------------------------- ---------------------------------------------------------------------- ------------------------------- ------------------ ----------------- **Full mutation 3/7= 43%** 1 4.5 M Autism ABN-Full mutation (400,533,667repeats \[r\]) abnormal methylation 46,XY NT 2 5 M Autism ABN-Full mutation (600--1100 r) abnormal methylation 46,XY NT 3 4.5 M Autism, DD ABN- Full mutation (267--933 r), abnormal methylation 46,XY NT **Mosaic mutation 3/7 = 43%** 4 2.3 M Autism ABN-size mosaic Full mutation \[800 r\]/ premutation \[155 r\] Normal & abnormal methylation 46,XY NT 5 1.7 M Autism ABN-size mosaic Full mutation \[400 r\]/ premutation \[150 r\] Normal & abnormal methylation 46,XY Normal-sub tel 6 8.5 M Autism, DD ABN-mosaic Full mutation \[200--900 r\]/ deletion mutation \[30 r\], partial methylation 46,XY NT **Mosaic Premutation carrier 1/7 = 14%** 7 6 F Autism ABN- premutation mosaic \[29,65,80,39(light band)r\] 46,XX NT Av 4.36 years 2F, 5M **Intermediate mutation **7 cases with 45--54r DD = developmental delay, NT = not tested ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Summary of reported population studies to assess the frequency of chromosome abnormalities (excluding fragile sites, polymorphisms and single cell abnormalities) ::: Study Subjects karyotyped Chromosome abnormality Clinical diagnosis ---------------------------------------- --------------------- --------------------------------------------- -------------------------------------------------------------------------------------------------- Konstantareas & Homatidis 1999 \[123\] 127 6 \[4.7%\] Diagnosed using the clinical criteria of autistic disorder by DSM-III (1983--1989) 46,XY,inv(2)(p11q13)pat,3q+ 47,XY,+mar 47,XY,+mar 47,XX,+13 47,XX,+inv dup(15)(pter→ q13::q13→ pter) 47,XY,+der(15)(pter→ q15::p11→ pter)de novo Gillberg & Wahlstrom 1985 \[124\] 46 2 \[4.3%\] Diagnosed using the American Psychiatric Association (1980) criteria DSM-III 47,XY,+21 47,XYY Lauritsen 1999 \[122\] 145 4 \[2.8%\] Cases with psychotic symptoms before 2--3 years or beginning at 2--3 or later between 1969--1993 47,XX,+mar,?t(13;22) 46,XX,t(9;10)(p23;q23.1) 46,XY,inv(10)(p11.21;q21.2)mat 46,XY,t(7;12)(q21.4;q15)de novo Li 1993 \[121\] 104 5 \[4.8%\] Diagnosed using the American Psychiatric Association (1980 & 1987) guidelines, DSM-III & III-R 47,XY,+21 46,XY/47,XY,+21 \[12/88\] 46,XY,t(5;6)(q13;p23)de novo 46,X,inv(Y)(p11q11) 46,fra(X)(q27.3),inv(Y)(p11q11) Weidmer-Mikhail 1998 \[120\] 59 1 \[1.69 %\] DSM-III-R (1991--1995) Tetrasomy 15 Ritvo 1990 \[6\] 233 9 \[3.9%\] DSM-III (1984--1988) 6-trisomy 21 Partial trisomy 8 Deletion 9p 46,XX,t(5q;11q)pat Wassink 2001 \[119\] 278 13 \[4.7 %\] DSM-III,-III-R & -IV (1980--1999) 46,XX,del(8)(p23) MR, diaphragmatic hernia, hemivertebra, 2-vessel umbilicus, strabismus 47,XX,der(14)t(14;?)(q22;?) MR, abnormal facies & palate, failure to thrive 46,XX,dup(15)(q11.2;q13) MR, abnormal EEG, precocious puberty 2--46,XY,del(15)(q11.2q13) Mild MR Moderate MR & Seizures mat47,XX,+mar de novo 47,XX,+mar McCune-Albright syndrome Microcephaly, abnormal facies 47,XY,+del(15)(q22) MR 46,XY,del(16)(q13q22) MR, Seizures, failure to thrive, abnormal facies, webbed neck macrocephaly, syndactyly 46,XY,add(17)(q23) MR & abnormal facies 2--47,XX,+21 MR, heart murmur, esotropia, recurrent pneumonia. MR1VSD, pneumonia and seizures 46,XY,add(22)(q13) Macrocephaly and failure to thrive Present 421 14 \[3.3%\] Physician referrals to a genetic lab. 1995--2003 March :::

PubMed Central

2024-06-05T03:55:52.659769

2005-1-18

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548305/", "journal": "BMC Med Genet. 2005 Jan 18; 6:3", "authors": [ { "first": "Kavita S", "last": "Reddy" } ] }

PMC548306

Background ========== In the past ten years there has been a growing interest in engineering Gram-positive bacteria for biotechnological applications, including vaccine delivery. \[[@B1]-[@B4]\], and *in situ*production of anti-infective protectants \[[@B5]\] and microbicides \[[@B6]\]. A common approach to genetic manipulation of bacteria is based on the use of plasmid expression vectors since these recombinant molecules can be introduced into bacterial cells by a variety of genetic techniques such as natural transformation, artificial transformation, transduction, conjugative mobilization, and electroporation \[[@B7]-[@B9]\]. However, the major limitation of this approach is due to the fact that recombinant plasmids are often lost from the bacterial culture upon removal of antibiotic selection. Certainly, this has consequences when using recombinant bacteria *in vivo*where their replication occurs in the absence of selection. An alternative approach is to integrate recombinant DNA molecules into the bacterial chromosome since this method allows increased *in vivo*stability of the genetic constructs. Therefore a lot of efforts have focused on the development of efficient expression systems based on chromosomal integration of expression cassettes \[[@B10],[@B11]\]. Naturally transformable bacteria represent a convenient model, since heterologous DNA can be easily integrated into their chromosomes, whereas genetic manipulation of non-transformable bacteria is more difficult and relies mainly on electroporation and conjugative mobilization of foreign DNA molecules. We have previously described a genetic system based on conjugative transposons allowing stable integration of recombinant DNA into the chromosome of transformable and non-transformable streptococci \[[@B12],[@B13]\]. A series of transposon insertion vectors containing two regions of homology with Tn*916*\[[@B14]\] have been created in order to manipulate both naturally transformable and non-transformable Gram-positive bacteria carrying Tn*916*\[[@B12]\]. The aim of this work was to select a strong promoter to improve this genetic system making it suitable for expression of single-copy recombinant genes in a broad spectrum of Gram-positive bacteria. Results and discussion ====================== Promoter selection by chromosomal integration --------------------------------------------- To select resident promoters from the genome of *Streptococcus gordonii*, we performed a random ligation of streptococcal DNA to a promoterless *cat*gene, conferring resistance to chloramphenicol (Cm). The ligation mixture was used to transform the naturally transformable *S. gordonii*«Challis» strain V288 and transformants were selected for Cm resistance. Chromosomal DNA flanking the promoterless *cat*gene provided the homology for the random integration of *cat*into the chromosome during transformation (Fig. [1](#F1){ref-type="fig"}). 71 Cm-resistant (Cm^R^) transformants were isolated, presumably as a result of transcriptional fusions of streptococcal promoters to the promoterless *cat*gene. Eighteen Cm^R^transformants were selected for further characterization. The strategy commonly used to select promoters is based on cloning random chromosomal DNA fragments in a promoter probe vector upstream of a promoterless reporter gene. However, integrating the promoterless reporter gene (*cat*) directly into the streptococcal chromosome, allowed us to select resident chromosomal promoters expressing *cat*after *in vivo*transcriptional fusion at a single locus on the chromosome. This is preferable when looking for promoters to express heterologous genes integrated into the chromosome in a single copy. *In vivo*analysis of promoter strength was determined in the eighteen selected transformants by measuring the minimum inhibitory concentration (MIC) of Cm. Fifteen transformants exhibited a MIC of 16 μg/ml, whereas the remaining three transformants exhibited a MIC of 8 μg/ml. The fifteen strains with higher MIC were tested for the stability of the chloramphenicol-resistance phenotype: after 50 generations of growth without selection, bacterial cultures were plated on non-selective medium and at least 200 colonies were picked and tested for Cm resistance. The stability of the resistance phenotype varied considerably among the different transformants (data not shown). Three strains (GP214, GP215 and GP216) showed a 100% stability and were chosen for further analysis. Cloning of a promoter from *S. gordonii* ---------------------------------------- The structure of the integrated *cat*gene in GP214, GP215 and GP216 was analyzed by Southern blot. Only GP215 showed to have a single *cat*copy integrated into the chromosome, whereas in GP214 and GP216 integration occurred at multiple sites (data not shown). In order to clone the regions flanking *cat*integration site in GP215, the chromosome of this strain was cut with *Taq*I whose recognition sequence is absent inside the *cat*gene. The derivative fragments were ligated to pBLUESCRIPT, and the ligation mixture was used to transform *Escherichia coli*cells; transformants were then selected for Cm^R^. All transformants analyzed for plasmid content showed to carry a plasmid of the same size. One of these transformants (GP334) was selected for further analysis and the transforming plasmid was named pVMB5. By restriction analysis we showed that pVMB5 contains a 2.2 kb *Taq*I insert where a 600 base pairs (bp) streptococcal DNA fragment was cloned upstream of the *cat*gene. This DNA fragment was stably maintained in *E. coli*, where it retained its promoter activity, conferring Cm^R^to *E. coli*. This is of considerable interest since it is known that very often cloning streptococcal promoters in a high copy number plasmid results in the failure of that plasmid to be established \[[@B15]\]. Sequence analysis ----------------- The streptococcal DNA upstream of the *cat*gene in pVMB5 was sequenced (GenBank accession number: U74080). Analysis of the sequence revealed the presence of an open reading frame (ORF1), preceded by a typical ribosome binding site (RBS) (Fig. [2](#F2){ref-type="fig"}). A BLAST search with the partial sequenced genome of *S. gordonii*<http://www.tigr.org/index.shtml> showed that ORF1 represents the 5\'-end of a gene encoding the first 48 amino acids (aa) of an uncharacterized protein conserved in bacteria and whose function is unknown. As indicated in figure [2](#F2){ref-type="fig"}, this truncated streptococcal protein is translationally fused to the N-terminus of CAT. Sequence analysis also revealed that the first 122 bp of the cloned streptococcal fragment belong to the 3\'-end of a gene encoding a putative acetyltransferase (ORF2). To obtain information about the chromosomal region containing the 600 bp streptococcal DNA fragment cloned in pVMB5, we looked throughout the whole contig sequence and we found out that upstream ORF2, and partially overlapping with it, there is an ORF encoding a DltD horthologue (*dltD*), a protein involved in D-alanine incorporation into lipoteichoic acid (LTA) \[[@B16]\], whereas downstream ORF1, there is a gene encoding a Tmp7 transmembrane protein (Fig. [2](#F2){ref-type="fig"}). Identification of the transcriptional start site ------------------------------------------------ To identify the sequences responsible for the observed promoter activity, the 5\'-end of the *cat-*specific mRNA was mapped by primer extension with a specific primer. Total RNA was isolated from *E. coli*GP334 (containing pVMB5) and *S. gordonii*GP215. In both strains, the position of the 5\'-end of the mRNA was located at the same purine residue at position 423 of the 600 bp region of streptococcal DNA, 26 nucleotides upstream of the ORF1 translational start site (Fig. [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}). Putative -35 and -10 sequences closely resembling the consensus *E. coli*σ^70^and *Bacillus subtilis*σ^43^\[[@B17]\] recognition sequences TTGACA (-35) and TATAAT (-10) could be identified. The -35 region was TTGCAA, and the -10 region was TAGAAT. The spacing between the -35 and -10 region was 17 bp and was thus similar to the spacing in *B. subtilis*(17 to 19 bp) and *E. coli*(16 to 18 bp) promoters. Moreover, a TG nucleotide pair was found 1 bp upstream of the -10 region; such a structure is typical of Gram-positive bacteria promoters \[[@B18]\]. Based on this information, we concluded that indeed we isolated a streptococcal promoter which was designated PP. The spacing between the 5\'-end of the mRNA and the -10 hexanucleotide was only 2 bp, which is unusually short. A second putative -10 region could be identified in the TATGAT hexanucleotide (Fig. [2](#F2){ref-type="fig"}), whose distance from the 5\' end of the mRNA is 7 bp. However, since this sequence is not preceded by the TG nucleotide pair typical of Gram-positive promoters, and is separated from the -35 region only by 12 bp, we suppose that this -10 region of PP is probably not active. Insertion vectors to express heterologous proteins -------------------------------------------------- We have previously developed a genetic system based on the use of *Streptococcus pyogenes*surface fibrillar M6 protein, as a partner for the construction of translational fusions to deliver foreign proteins on the surface of *S. gordonii*\[[@B10]\]. To determine the possibility of using PP for chromosomal single copy heterologous gene expression in Gram-positive bacteria, we first generated a transcriptional fusion of PP with a promoterless gene encoding the M6 protein (*emm6*) in pSMB47, a suicide vector capable of integrating heterologous DNA into the conjugative transposon Tn*916*via homologous recombinantion \[[@B12]\]. The recombinant plasmid was named pSMB139 (see Methods) (Fig. [4](#F4){ref-type="fig"}). To construct a Tn*916*insertion vector that could be used to express translational fusions between M6 and any heterologous proteins under the PP promoter control, a 900 bp *Avr*II-*Hind*III fragment internal to *emm6*in pSMB139 was replaced by a 390 bp *Avr*II-*Hind*III fragment of pSMB55 containing a multiple cloning site \[[@B10]\] (Fig. [4](#F4){ref-type="fig"}). The resulting vector, named pSMB148 (Fig. [4](#F4){ref-type="fig"}), allows to create translational fusions of heterologous proteins between the first 122 N-terminal aa and the last 140 aa of the M6 protein, which provides sequences necessary for cell wall anchoring. pSMB148 was used to create two derivative vectors in which the *emm6*gene was fused respectively with a DNA sequence encoding 339 aa of the chicken ovalbumin (OVA) (pSMB156), and a DNA sequence encoding 458 aa of the tetanus toxin fragment C (TTFC) (pSMB288) (see Methods). A schematic representation of the recombinant proteins is shown in Fig. [5A](#F5){ref-type="fig"}. Expression of recombinant proteins in Gram-positive bacteria ------------------------------------------------------------ *S. gordonii*, *B. subtilis*, and *Enterococcus faecalis*were three Gram-positive hosts used to analyze the capability of PP to direct transcription of the heterologous genes expressing M6, M6/TTFC and M6/OVA recombinant proteins. Expression of M6 in *S. gordonii* --------------------------------- pSMB139, bearing a transcriptional fusion of PP with *emm6*, was introduced by natural transformation in *S. gordonii*GP201, a strain with a single copy of Tn*916*integrated into the chromosome. One of the transformants (GP1241), in which the integrative suicide vector drove the integration of the PP-*emm6*fusion into Tn*916*, was isolated and analyzed for M6 protein expression. Envelope fractions (containing surface associated proteins) prepared from equal amounts of cells grown to mid-log, early and late stationary phase, were analyzed by Western-blotting with an anti-M6 monoclonal antibody. Multiple bands could be detected in fractions of cells grown to mid-log and early stationary phase, whereas no band was detected in the fraction of cultures grown to late stationary phase (Fig. [6A](#F6){ref-type="fig"}). The intensity of the signal was higher during the mid-log growth phase suggesting that either PP is more active during exponential growth or that the M6 protein is being degraded during stationary phase. The presence of multiple reactive bands of molecular masses close to the hypothetical size of M6 (predicted molecular weight, 49 kDa) is probably due to the fact that coiled-coil proteins like M6 run at aberrant sizes on denaturing gels \[[@B19]\]. Expression of M6/TTFC in *B. subtilis* -------------------------------------- Competent cells of *B. subtilis*GP800.2, containing one copy of Tn*916*integrated into the chromosome, were transformed with the insertion vector pSMB288, bearing the transcriptional fusion PP-*emm6/ttfc*. One transformant, GP848, was chosen for further studies. A culture of GP848 was grown to mid-exponential phase and analyzed by Western-blotting for the presence of recombinant M6/TTFC. As shown in Fig. [6B](#F6){ref-type="fig"}, two reactive bands could be detected in the envelope fraction. The lower band, indicated by an arrow, corresponds to the mature protein (predicted molecular weight, 82 kDa), while the upper band probably represents an unprocessed form (predicted molecular weight, 86.4 kDa). Expression of M6/OVA in *E. faecalis* ------------------------------------- Using a previously described genetic system \[[@B12]\], we constructed a derivative of the *E. faecalis*strain OG1SS \[[@B20]\] expressing the recombinant M6/OVA protein under PP control. pSMB156, bearing the PP-*emm6/ova*fusion, was first introduced in the Tn*916*containing *B. subtilis*GP800.2 by natural transformation, to obtain a recombinant conjugative transposon containing the transcriptional fusion. The recombinant transposon was then transferred by conjugation into *E. faecalis*OG1SS. Transconjugants were detected at a frequency of 4 × 10^-10^transconjugants/recipient. One of them (GP431) was analyzed for cell-surface expression of M6/OVA by flow-cytometric analysis using an anti-ovalbumin polyclonal antibody. The presence of recombinant M6/OVA on the surface of GP431 was clearly demonstrated by the increase of the fluorescence intensity in this strain, as compared to the parental control OG1SS (Fig. [7](#F7){ref-type="fig"}). Conclusions =========== We have isolated and characterized a promoter from the chromosome of *S. gordonii*, and demonstrated that it can be used to direct expression of heterologous genes in different Gram-positive bacteria when integrated in a single copy into the chromosome. This promoter, together with the genetic system based on suicide vectors able to integrate into conjugative transposons, represents a useful tool for the stable manipulation of a broad spectrum of Gram-positive bacteria. Methods ======= Bacterial strains, plasmids and growth conditions ------------------------------------------------- All strains and plasmids used in this work are listed in Table [1](#T1){ref-type="table"}. *E. coli*strains DH5α and HB101 were cultured in Luria-Bertani (LB) broth. For maintenance of plasmids, ampicillin (100 μg/ml), chloramphenicol (20 μg/ml) or erythromycin (100 μg/ml) was added to the growth medium. Streptococcal strains were cultured in Brain Heart Infusion medium (BHI, Difco) or Tryptic Soy Broth (TSB, Difco) in the presence of chloramphenicol (5 μg/ml), erythromicin (100 μg/ml) or streptomycin (500 μg/ml) whenever required. Transformation of naturally competent cells of *S. gordonii*V288 and GP201, scoring and genetic analysis of transformants was carried out as already described \[[@B21],[@B22]\]. *B. subtilis*strains were grown in LB broth with erythromicin (3 μg/ml) when it was required. Competent cells of *B. subtilis*GP800.2 were prepared and transformed according to described procedures \[[@B23]\]. Agar (1.5%) was added to LB, BHI or TSB to obtain solid media. All cultures were incubated at 37°C. DNA manipulation ---------------- Total DNA preparation of *S. gordonii*was performed as previously described \[[@B21]\]. Plasmid DNA was prepared using the Qiagen Plasmid Kit (Qiagen) according to the manufacturer\'s instructions. All recombinant techniques were performed following standard procedures \[[@B24]\], using *E. coli*HB101 or DH5α as a host. DNA restriction enzymes were obtained from Roche and used according to the manufacturer\'s instructions. Promoter selection by chromosomal integration --------------------------------------------- A 1.6 kb *Hind*III-*Bam*HI fragment of plasmid pKT \[[@B25]\], containing a *cat*gene, was ligated with random chromosomal DNA fragments of *S. gordonii*V288 previously cut with both *Hind*III and *Bam*HI. The initiation codon of *cat*was 34 bp downstream of the *Hind*III site, therefore *Hind*III cleavage would leave *cat*promoterless and preceded by an intact ribosome binding site. The ligation mixture was introduced in *S. gordonii*V288 and transformants were selected for Cm^R^. MIC determination ----------------- The MIC of chloramphenicol for *S. gordonii*was determined following standard procedures \[[@B26]\]. Construction of recombinant vectors ----------------------------------- A 1648 bp fragment containing the *emm6*gene (encoding M6, a fibrillar surface protein of *S. pyogenes*) was amplified by PCR from plasmid pVMB20 \[[@B22]\] using the oligonucleotides 5\'-AT[GGATCC]{.underline}AT[CATATG]{.underline}GCTAAAAATAACACGAAT-3\' (upstream primer, containing a *Bam*HI site and introducing a *Nde*I site at the ATG translation initiation codon) and 5\'-GCAT[GTCGAC]{.underline}CATAATCATTAAATGTATCTCAT-3\' (downstream primers containing a *Sal*I site). This 1648 bp PCR fragment was digested with *Bam*HI and *Sal*I and cloned in pVA891 \[[@B27]\] previously digested with *Bam*HIand *Sal*I, resulting in plasmid pSMB89. A 451 bp region containing the streptococcal promoter PP was amplified from pVMB5 using the following primers: 5\'-CGA[GGATCC]{.underline}TTTA[ATCGAT]{.underline}ACTCATG-3\' (upstream primer, containing a *Bam*HI and a *Cla*I site) and 5\'-CCG[CATATG]{.underline}GTTCTCCTTTTTATTTGT-3\' (downstream primers containing a *Nde*I site). After digestion with *Bam*HI and *Nde*I, this PCR product was inserted between the *Bam*HI and *Nde*I site of pSMB89 to obtain a transcriptional fusion of PP with *emm6*. The resulting plasmid was named pSMB128. This plasmid was first cut with *Bam*HI, treated with Klenow enzyme to generate blunt ends, and finally cut with *Sal*I to obtain a 2.0 kbfragment containing PP-*emm6*fusion. This fragment was gel-purified and ligated to the suicide integrative plasmid pSMB47 \[[@B12]\] previously cut with *Hind*III, treated with Klenow enzyme to generate blunt ends, and finally cut with *Sal*I. The resulting plasmid was named pSMB139 (Fig. [4](#F4){ref-type="fig"}). To introduce a multiple cloning site in the *emm6*gene contained in pSMB139, the 900 bp *Avr*II-*Hind*III fragment internal to *emm6*was replaced with the 390 bp *Avr*II-*Hind*III fragment of *emm6*from pSMB55 \[[@B10]\] (Fig. [4](#F4){ref-type="fig"}). The resulting plasmid was named pSMB148. A 1016 bp DNA region encoding 339 aa of the chicken ovalbumin (OVA) (Gene Bank accession number: V00383) was amplified with the following primers: 5\'-CT[AGATCT]{.underline}GACAGCA CCAGGACAC-3\' (upstream primer containing a *Bgl*II site) and 5\'-TA[AAGCTT]{.underline}TAGGGG AAACACATCTG-3\' (downstream primer containing a *Hind*III site). After digestion with *Bgl*II and *Hind*III, this segment was introduced in pSMB148 previously digested with *Bgl*II and *Hind*III. The resulting plasmid, named pSMB156, contained a translational fusion of M6 with OVA. To create a translational fusion of M6 with the tetanus toxin fragment C (TTFC) a 1374 bp *Bgl*II-*Hind*III fragment encoding 458 aa of TTFC was isolated from pSMB158 \[[@B28]\] and cloned between the *Bgl*II and *Hind*III sites of pSMB148. The resulting plasmid was named pSMB288. Western-blot analysis --------------------- Preparation of *S. gordonii*and *B. subtilis*cell envelope fractions (representing the protoplast surface containing the cell membrane together with cell wall fragments associated to the protoplasts) was performed as already described \[[@B29],[@B30]\]. The monoclonal antibody 10B6 \[[@B31]\] diluted 1:1000 was used to detect the presence of M6 protein. M6/TTFC fusion protein was visualized with an anti-TTFC rabbit serum (Calbiochem-Novabiochem Corporation) diluted 1:1000. Flow-cytometric analysis ------------------------ Flow-cytometric analysis of *E. faecalis*was performed as already described \[[@B28],[@B32]\] using an anti-OVA rabbit serum diluted 1:300 \[[@B29]\]. DNA sequence determination -------------------------- The promoter containing fragment cloned in pVMB5 was sequenced by dideoxy chain termination method \[[@B33]\] as already described \[[@B34]\]. Denatured plasmid DNA was used as template. RNA isolation ------------- Total RNA was isolated from a 50 ml cell culture of *S. gordonii*and *E. coli*grown to late exponential phase (OD~590~≌ 0.5). Cells were harvested by centrifugation at 6000 × *g*at 4°C and lysed according to the following procedures. *E. coli*cells were first resuspended in hot (100°C) lysis buffer (50 mM Tris/HCl pH8, 1 mM EDTA, 1% SDS) and lysed by boiling the suspension for 5 minutes. *S. gordonii*cells were resuspended in lysozyme buffer (25 mM Tris/HCl (pH8), 10 mM EDTA, 50 mM glucose) and subjected to three cycles of freezing in liquid nitrogen and thawing at 52°C. After incubating with 0.2 mg/ml of lysozyme for 30 min at 37°C, one volume of hot (100°C) lysis buffer (100 mM Tris/HCl (pH8), 2 mM EDTA, 2% SDS) was added, and complete lysis was obtained by boiling cells for 5 minutes. After boiling, all lysates were cooled on ice for 5 min and total RNA was purified using the SV Total RNA Isolation System (Promega). Primer-extention analysis ------------------------- Primer extention analysis was performed with a synthetic oligonucleotide 5\'-GTTCTTTACGATGCC-3\' (position 47 to 61 relative to the initiation of the *cat*gene). Two pmol of the oligonucleotide, labeled with \[γ-^32^P\] ATP (3000 Ci/mmol, Amersham) using T~4~polynucleotide kinase (New England Biolabs), were precipitated with 10 μg of RNA and the pellet was resuspended in 8 μl of Moloney Murine Leukemia Virus (M-MuLV) Reverse Transcriptase buffer (50 mM Tris/HCl (pH8.3), 8 mM MgCl~2~, 10 mM DTT). The mixture was heated at 65°C for 3 min, cooled rapidly at -80°C for 1 min and then transfered on ice until it was completely thawed. 1 μl of a 3.75 mM deoxynucleoside triphosphate solution and 10 U of M-MuLV Reverse Transcriptase (New England Biolabs) were added to the RNA-primer hybrid. The reaction mixture was incubated at 48°C for 30 min and terminated with 10 ml of stop solution (95% formamide, 20 mM EDTA pH8.0, 0.05% bromophenol blue, and 0.05% xylene cyanol FF). The reverse transcriptase reactions were analyzed by electrophoresis on a 6% polyacrylamide-7 M urea gel with sequencing reaction obtained with the same primer used as size standards. Conjugation ----------- Conjugation experiments were performed on solid media as previously described \[[@B12]\]. Authors\' contributions ======================= RP, characterization of promoter, engineering of *S. gordonii*, writing of manuscript. TM, engineering of *B. subtilis*and *E. faecalis* MRO, participation in experimental work, data evaluation RM, participation in experimental work, data evaluation, writing of manuscript GP, design and coordination of the study, data evaluation, direct supervision of experimental work, writing of manuscript All authors read and approved the final manuscript. Acknowledgements ================ The work was supported in part by the European Commission grant QLK2-CT2000-00543 to GP, a grant from MIUR (COFIN 2002) to GP and from MIUR (FIRB RBAU01X9TB) to MRO. The authors wish to thank Claudio Gualerzi for help and advice with the primer extention analysis. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### ***In vivo*transcriptional fusion by integration of a promoterless *cat*gene into the chromosome of a naturally transformable streptococcus**. A promoterless *cat*gene is ligated *in vitro*to random chromosomal fragments, using a restriction site a few base pairs upstream of its translation initiation codon (a). The ligation mixture is then used to transform the recipient strain (b), and the homologous sequences allow chromosomal integration of the promoterless *cat*gene (c). By this process, the *cat*gene is integrated into the chromosome between two direct repeats. Since some of the chromosomal fragments contain promoters (P), it is possible to obtain expression of the promoterless *cat*gene after *in vivo*transcriptional fusion with resident chromosomal promoters. ::: ![](1472-6750-5-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Schematic representation of the *S. gordonii*locus containing PP promoter**. DltD orthologue, an ORF encoding a putative acetyltransferase (ORF2), ORF1, the gene encoding a Tmp7 transmembrane protein, and the *cat*gene are indicated by arrows. Dashed arrows designate that the ORF is only partially represented in the scheme. The 600 bp *Taq*I-*Hind*III fragment including PP promoter and cloned upstream of the *cat*gene in pVMB5 is indicated by a dotted box. Nucleotide sequence of the PP promoter region (332 bp) is reported inside the box. The transcriptional start site determined by primer extention analysis is marked with an asterisk. Proposed -35 and -10 regions are underlined. A second putative -10 sequence is overlined. ORF1 putative ribosome binding site (RBS) is boxed. ATG initiation codon of *cat*is in bold characters and the sequence of *Hind*III site is in italic letters. The complete sequence of the 600 bp cloned fragment is available on GenBank (Accession number: U74080). T, *Taq*I site; B, *Bam*HI site; H, *Hind*III site ; loop, putative transcriptional terminator. ::: ![](1472-6750-5-3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Primer extension analysis of the PP promoter**. Localization by primer extension of the transcriptional start site of the *cat*mRNA specified by the PP promoter in *E. coli*GP334 (lanes 1--2), and *S. gordonii*GP215 (lanes 3--4). The sequence of the region upstream of the *cat*gene in pVMB5 was used as standard. The A residue, complementary to the T at position 423, indicated by an arrow, represents the transcriptional start site used in both strains. ::: ![](1472-6750-5-3-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Construction of the insertion plasmid pSMB148**. pSMB139 was constructed by introducing a 2.0 kb fragment, containing a transcriptional fusion of PP promoter with *emm6*, in the Tn*916*insertion vector pSMB47 (see Methods). pSMB148 is a derivative of pSMB139 in which a 900 bp *Avr*II-*Hind*III fragment was replaced with a 390 bp *Avr*II-*Hind*III fragment from pSMB55 containing a multiple cloning site \[10\]. ::: ![](1472-6750-5-3-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Schematic representation of recombinant M6, M6/TTFC and M6/OVA expressed on the surface of *S. gordonii*, *B. subtilis*and *E. faecalis***. The 458 aa protein TTFC (white bar) and the 339 aa protein OVA (light gray bar) were fused with the first 122 N-terminal aminoacids and the last 140 C-terminal aminoacids of M6 (dark gray bar). The predicted molecular weight of M6, M6/TTFC and M6/OVA is 49 kDa, 82 kDa and 66.9 kDa respectively. ::: ![](1472-6750-5-3-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Western blot analysis of recombinant *S. gordonii*and *B. subtilis*strains expressing M6 protein and M6/TTFC fusion protein**. (A) *S. gordonii*envelope fractions. Lane 1 through 3, GP1241 expressing M6 under the control of PP promoter. Lane 1, GP1241 harvested after overnight growth. Lane 2, GP1241 harvested after early stationary phase. Lane 3, GP1241 harvested after exponential phase. Lane 4, recipient strain GP201 (negative control). Lane 5, GP231 (positive control). Blot was developed with anti-M6 monoclonal antibody 10B6. (B) *S. gordonii*and *B. subtilis*envelope fractions. Lane 1, *S. gordonii*GP204 (negative control). Lane 2, *S. gordonii*GP1253 expressing M6/TTFC (positive control). Lane 3, *B. subtilis*recipient strain GP800.2 (negative control). Lane 4, *B. subtilis*GP848 expressing M6/TTFC under the control of PP promoter. Blot was developed with anti TTFC rabbit serum. Molecular weight markers are shown in the right side of panels. ::: ![](1472-6750-5-3-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Flow-cytometric analysis of *E. faecalis*expressing M6/OVA**. (A) OG1SS, recipient strain not expressing M6/OVA. (B) GP431, recombinant strain expressing M6/OVA on the surface. Bacterial cells were treated with anti-OVA rabbit serum and than with FITC-conjugated goat anti-rabbit IgG. *x*axis, arbitrary units (a.u.) of fluorescence intensity (log~10~); *y*axis, relative cell number. ::: ![](1472-6750-5-3-7) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Strains and plasmids used in this work ::: **Strain** **Relevant genotype**^a^ **Source/reference** --------------- ------------------------------------------------------------------------ ---------------------- *S. gordonii* V288 Recipient in transformation \[35\] GP201 ΩTn*5253*, Sm^R^, Cm^R^, Tc^R^ \[13\] GP204 *str-204*, Sm^R^ \[13\] GP214 pKT random in V288, Cm^R^ This work GP215 pKT random in V288, Cm^R^ This work GP216 pKT random in V288, Cm^R^ This work GP231 *emm6*, Er^R^, Sm^R^ \[21\] GP1241 ΩTn*5253*(Δ*tetM*::pSMB139) Er^R^, Sm^R^, Cm^R^, Tc^S^ This work GP1253 *emm6.1*::TTFC \[28\] *E. faecalis* OG1SS Recipient in conjugation, Sm^R^, Sp^R^ \[20\] GP431 ΩTn*916*, Er^R^, Tc^S^(Δ *tetM*::pSMB156) This work *B. subtilis* GP800.2 ΩTn*916*, Tc^R^ \[12\] GP847 ΩTn*916*, Er^R^, Tc^S^(Δ *tetM*::pSMB156) This work GP848 ΩTn*916*, Er^R^, Tc^S^(Δ *tetM*::pSMB288) This work **Plasmid** **Description** **Source/reference** pKT Ap^R^, Cm^R^ \[25\] pBluescript Ap^R^ Stratagene pVMB5 pBluescript::PP-*cat*, Ap^R^, Cm^R^ This work pVMB20 pBluescript::*emm6.1*::*ermC*, Ap^R^, Er^R^ \[21\] pVA891 Cm^R^, Er^R^ \[27\] pSMB47 pVA891 derivative containing DNA sequences from Tn*5253*, Cm^R^, Er^R^ \[12\] pSMB89 pVA891::*emm6*, Cm^R^, Er^R^ This work pSMB128 pVA891::PP-*emm6*, Cm^R^, Er^R^ This work pSMB139 pSMB47::PP-*emm6*, Cm^R^, Er^R^ This work pSMB148 pSMB47::PP-*emm6/*55, Cm^R^, Er^R^ This work pSMB156 pSMB47::PP-*emm6/*55::*ova*, Cm^R^, Er^R^ This work ^**a**^Sm, streptomycin; Cm, chloramphenicol; Tc, tetracyclin; Er, erythromycin; spectinomycin; Ap, ampicillin. :::

PubMed Central

2024-06-05T03:55:52.665499

2005-1-14

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548306/", "journal": "BMC Biotechnol. 2005 Jan 14; 5:3", "authors": [ { "first": "Roberta", "last": "Provvedi" }, { "first": "Tiziana", "last": "Maggi" }, { "first": "Marco R", "last": "Oggioni" }, { "first": "Riccardo", "last": "Manganelli" }, { "first": "Gianni", "last": "Pozzi" } ] }

PMC548319

Background ========== Because of its intense oxidative metabolism, the brain consumes a high fraction of total oxygen generating large amounts of reactive oxygen species \[[@B1],[@B2]\]. Although brain antioxidant defenses function properly during most of human life, a number of neurodegenerative processes which involve redox-active iron accumulation become evident with age \[[@B3]-[@B5]\]. Iron is a pro-oxidant that in the reductive intracellular environment catalyses hydroxyl radical formation through the Fenton reaction \[[@B6]\]. At present, the crucial components of the iron homeostasis machinery have been identified. Thus, current efforts should be directed to the understanding of the mechanisms that regulate cellular iron levels and antioxidant defenses. This is of primary importance for the development of strategies to ameliorate iron accumulation and oxidative damage in neurons. In vertebrates, cellular iron levels are post-transcriptionally controlled by the activity of iron regulatory proteins (IRP1 and IRP2), cytosolic proteins that bind to structural elements called iron-responsive elements (IREs). IREs are found in the untranslated region of the mRNAs of the major proteins that regulate cellular iron homeostasis: the transferrin receptor, involved in plasma-to-cell iron transport, and the iron-storage protein ferritin. IRP2-/- mice are born normal but in adulthood develop a movement disorder characterized by ataxia, bradykinesia and tremor \[[@B7]\]. IRP1-/- mice are normal with slight misregulation of iron metabolism in the kidney and brown fat \[[@B8]\]. Thus, IRP2 seems to dominate the physiological regulation of iron metabolism whereas IRP1 seems to predominate in pathophysiological conditions. Iron is internalized into cells by the import transporter DMT1. Four DMT1 isoforms have been identified that differ in both the N-and the C-termini \[[@B9]\]. Two of the isoforms have a 3\' iron responsive element (IRE) in their mRNA. Additional variation is given by exons 1A and 1B in the 5\' end. Expression of DMT1 in response to iron availability follows a pattern similar to transferrin receptor \[[@B10]\], but its control by the IRE/IRP system is not clear \[for review see \[[@B11]\]\]. A new iron transporter, IREG1, also known as ferroportin or MTP1, was recently described \[[@B12],[@B13]\]. The protein is expressed mainly in enterocytes and macrophages \[reviewed in \[[@B14]\]\]. In enterocytes IREG1 is responsible for iron efflux during the process of intestinal iron absorption, while in Kupffer cells IREG1 mediates iron export for reutilization by the bone marrow \[[@B15]\]. The presence of both DMT1 and IREG1 has been described in neurons, glioma cells and astrocytes \[[@B16]-[@B18]\]. The presence of IREG1 in neurons opens the possibility that they may be able to down-regulate intracellular iron concentration through its expression. In this study we examined iron homeostasis in SH-SY5Y neuroblastoma cells and hippocampal neurons. We found that iron accumulation killed a large proportion of cells, but a sub-population became resistant to iron accumulation developing an adaptative mechanism intended to decrease intracellular iron content. Results ======= Iron accumulation and cell death -------------------------------- Iron accumulation was determined in SH-SY5Y cells grown to confluence and then cultured for two days in media containing from 1.5 to 80 μM iron (Figure [1A](#F1){ref-type="fig"}). Total cell iron increased with increasing extracellular iron, reaching a plateau at 40--80 μM Fe (Figure [1B](#F1){ref-type="fig"}). The observed increase in cell iron was accompanied by increases in the labile iron pool (Figure [1C](#F1){ref-type="fig"}). Iron accumulation indeed caused loss of cell viability, with hippocampal neurons demonstrating higher sensitivity than SH-SY5Y cells to iron treatment (Figure [2](#F2){ref-type="fig"}). Nevertheless, a sub-population of cells survived to high iron concentrations. It was of interest to inquire into the processes underlying this adaptation, since they could help to understand iron accumulation observed in a number of neurodegenerative diseases. Consequently, we characterized the components of the iron homeostasis machine during the process of iron accumulation. Ferritin and DMT1 regulation ---------------------------- Ferritin, the main iron-storage protein in mammalian cells, is considered the first line of defense against iron overload. Increasing iron from 1.5 to 5 μM produced a robust 4-fold increase in cell ferritin content (Figure [3A](#F3){ref-type="fig"}). Further increases in iron induced additional increases in ferritin. At 80 μM extracellular iron, ferritin increased 11-fold compared to the basal 1.5 μM iron condition. In molar base, ferritin increased more than iron. The iron to ferritin mol : mol ratio decreased from 1500 at 1.5 μM Fe to 400 at 10 μM Fe to 200 at 80 μM Fe (Figure [3B](#F3){ref-type="fig"}). We further characterized iron homeostasis in SH-SY5Y cells by examining the expression of the iron importer DMT1 (Figure [4](#F4){ref-type="fig"}). A 3.5-fold decrease in DMT1 protein expression was observed when iron increased from 1.5 to 80 μM. The presence of DMT1 even at high iron concentration explains the sustained iron uptake observed at 80 μM Fe \[[@B19]\]. Thus, DMT1 activity persisted even under conditions of iron accumulation that preceded cell death. IREG1 expression and functionality ---------------------------------- Given that the presence of IREG1 in the central nervous system has been reported \[\[[@B16]\]\], it was of interest to examine if it participates in neuronal iron homeostasis. Western blot analysis revealed that SH-SY5Y cells expressed anti-IREG1 reactive bands of 65.3 and 122.1 KDa molecular weight (Figure [5A](#F5){ref-type="fig"}). Densitometric analysis revealed a 10-fold increase in the 122.1 KDa band in the 1.5 to 80 mM Fe range while the 65.3 band had a minor increase (Figure [5A](#F5){ref-type="fig"}). Both bands were eliminated if the antibody was incubated with the immunogenic peptide before the assay (Figure [5B](#F5){ref-type="fig"}). A similar pattern was obtained with an independent anti-IREG1 antibody (the kind gift of Dr. David Haile). Thus, it is most likely that the 65.3 and 122.1 KDa bands correspond to the monomer and dimer of IREG1. The stability of the 122.1 KDa band was dependent of the concentration of b-mercaptoethanol in the sample buffer since increasing b-mercaptoethanol produced a shift in the 122.1 KDa /65.3 KDa band ratios (Figure [5C](#F5){ref-type="fig"}). It is possible that in neuronal cells Ireg1 tends to form S-S bridged dimers resistant to the electrophoresis conditions. The presence of IREG1 in SH-SY5Y neuroblastoma cells and hippocampal neurons was further documented by immunocytochemistry. IREG1 was detected in both types of cells, with a predominantly cytosolic distribution pattern (Figure [6](#F6){ref-type="fig"}). The levels of IREG1 expression were directly proportional to the amount of iron in the culture. Thus, it was determined by two independent methods that IREG1 expression in neuronal cells increased with cell iron content. Efflux of iron from neurons has never been reported. In view of the presence of IREG1, we tested whether SH-SY5Y cells actually had an iron efflux function. To that end, iron efflux from cells pre-cultured for 2 days with varied iron concentrations was determined by atomic absorption spectrometry. This method was preferred to the use of radioisotopic iron since the latter could underestimate a putative iron efflux because of isotope dilution with the pre-existing iron pool (see Figure [1B](#F1){ref-type="fig"}). SH-SY5Y cells had discrete but measurable iron efflux activity (Figure [7A](#F7){ref-type="fig"}). The iron efflux rate increased markedly in the 20--80 μM Fe range (Figure [7B](#F7){ref-type="fig"}). Interestingly, the efflux rate correlated closely with the presence of the 122.1 KDa band, while the correlation between efflux activity and the 62.5 KDa band was weaker (Figure [7C](#F7){ref-type="fig"}). Discussion ========== The number of neurological diseases associated with iron accumulation in the brain underlines the need for increased knowledge of the mechanisms of brain iron homeostasis. In this study we show that iron accumulation by SH-SY5Y neuroblastoma cells and hippocampal neurons resulted in cell death of part of the population, while another fraction survived by adapting the expression of iron homeostasis proteins. Iron content increased significantly as a function of Fe in the culture up to 20--40 μM Fe, increasing very little thereafter up to 80 μM Fe. Cell iron increase was accompanied by increased ferritin content. The increase in ferritin more than compensated for the increase in iron. Iron to ferritin mol ratios of 1500, 260 and 190 were obtained for 1.5, 20 and 80 μM Fe in the culture media. Thus, the IRE/IRP system of SH-SY5Y cells over-responded to iron accumulation in terms of ferritin expression. Despite the increase in ferritin, the LIP increased between 1.5 and 80 μM Fe. This finding clearly indicates that in SH-SY5Y cells the level of labile iron is a function of total iron, even in the presence of ample ferritin supply. It is possible that ferritin-stored iron contributes to the LIP each time that ferritin undergo lisosomal degradation. Iron accumulation was accompanied by a marked decrease in DMT1 expression. Nevertheless, some DMT1 persisted even at 40--80 μM iron. The persistence of DMT1 at high iron concentrations could underline the continuous iron uptake observed under these conditions \[[@B19]\]. This is curious because at 40--80 μM Fe cells were dying. Sustained DMT1 expression points to the inability of neuronal cells to shut-off iron uptake and the need for additional defense mechanisms to prevent iron-mediated cell death. The discovery of increased IREG1 expression in response to cell iron accumulation is a major break-through in the understanding of cell survival under conditions of iron accumulation. Total IREG1, and especially a putative IREG1 dimer, increased markedly in the 20--80 μM Fe range. Thus, in SH-SY5Y cells IREG1 is up-regulated by increased cell iron. Expressed IREG1 was functional since it associated with increased iron efflux activity. Iron efflux activity in astrocytes \[[@B18]\] and neurons (this work) indicate that iron efflux from brain cells is a dynamic process, and highlights the importance of iron transporters as determinants of iron accumulation. The regulation of IREG1 expression is unknown but seems to be cell-specific. In enterocytes, IREG1 expression is induced by iron deficiency \[[@B13]\] while in macrophages iron increases IREG1 expression \[[@B20]\]. The findings reported here indicate that in neuronal cells IREG1 has a macrophage-like regulation. This is certainly the case for cells in the 40--80 μM range that survived to iron accumulation. IREG1\'s predominantly cytosolic distribution pattern is similar to that of Kupffer cells \[[@B12]\]. Again, this distribution points to macrophage-like behavior of neuronal IREG1. In examining brain biopsies from Alzheimer\'s patients an intriguing question arises: Why do some neurons die or present evident signs of degeneration while others in the vicinity show a normal phenotype? Extrapolating on the data presented here, it is tempting to hypothesize that surviving neurons induce IREG1 expression while sick neurons do not. Nevertheless, at present we cannot exclude that other regulatory molecules may play a pivotal role under these conditions. Conclusions =========== Hippocampal neurons and SH-SY5Y cells displayed an active system to regulate iron content. Nevertheless, this system was unable to block iron accumulation which resulted in death of part of the cell population. Another fraction of the cell population developed an adaptative mechanism that includes decreased expression of the import transporter DMT1 and increased expression of ferritin and the efflux transporter IREG1. The finding that neurons regulate the expression of functional IREG1 opens new avenues for the understanding and possible treatment of iron-related neurodegenerative processes. Methods ======= Antibodies and immunodetection ------------------------------ Antibody D-1, prepared against the C-terminal end of the IRE-containing isoform of DMT1 was used as described previously \[[@B10]\]. Additionally, a rabbit polyclonal antibody against peptide CGPDEKEVTKENQPNTSVV, corresponding to the consensus sequence of human, rat and mouse carboxyl-terminal sequence of IREG1, was obtained from BioSonda, Chile <http://www.biosonda.cl>. Western analysis ---------------- Cell extracts, cells were prepared treating cells with lysis buffer (50 μl per 1 × 106 cells of 10 mM MOPS, pH 7.5, 3 mM MgCl2, 40 mM KCl, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 0.5 μg/ml aprotinin, 0.7 μg/ml pepstatin A, 5% glycerol, 1 mM dithiothreitol, 0.1% Triton X-100). The mixture was incubated for 15 min on ice and centrifuged for 10 min at 5,000 × g. Protein concentrations were determined using the bicinchoninic acid (BCA) protein assay. The supernatant was stored at -70°C. For Western analysis, 30 micrograms of protein from each sample were boiled in Laemmli sample buffer for 5 min and subjected to SDS-PAGE on a 7.5% acrylamide gel. Proteins were transferred to nitrocellulose membrane and blocked for 1 hr at 25°C with 5% nonfat dry milk in blocking saline (20 mM Tris, 0.5 M NaCl, 0.05% Tween-20). Membranes were incubated with primary antibody overnight at 4°C, rinsed with blocking saline and incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody for 1 hr at 25°C. Transferred proteins were detected with a peroxidase-based chemiluminiscence assay kit (SuperSignal, Pierce Chem. Co., Rockford, IL). Chemiluminiscence was detected using a Molecular Imager FX device (Bio-Rad, Hercules, CA). The bands were quantified by densitometry using the Quantity One (Bio-Rad) software. Cell culture and iron challenge ------------------------------- Human neuroblastoma SH-SY5Y cells (CRL-2266, American Type Culture Collection Rockville, MD), were seeded at 1 × 105 cells in 2-cm^2^plastic wells and cultured in a 5 % CO~2~incubator in MEM/F12 medium supplemented with 10 % fetal bovine serum and 5 mM glutamine. The medium was replaced every two days. Under these conditions, doubling time was about 48 hours. After 8 days in culture, the culture reached a steady-state number of cells. At this time, cells were challenged with iron for the next two days as described \[[@B19]\]. In brief, low-iron culture media was supplemented with either 1, 5, 10, 20, 40 or 80 μM Fe^3+^as the complex FeCl~3~-sodium nitrilotriacetate. Cell viability was quantified by the MTT assay (Molecular Probes, OR) following the manufacturer\'s instructions. This model of iron loading attempts to replicate neuronal iron accumulation that occurs during life \[[@B4]\]. Hippocampal neurons were prepared from E18.5 rat embryos \[[@B21]\]. Neurons were plated over poly-L-lysine coated cover slips at 100,000 cells/cm^2^. Cultures were maintained in 10% bovine serum until 3 hours after plating, when the culture medium was replaced with medium containing B27 supplement \[[@B22]\]. After 3 days in culture, the cells were challenged with iron as described above. Labile iron pool ---------------- The intracellular labile or reactive iron pool of neuroblastoma cells was determined as described \[[@B23],[@B24]\]. The increase in fluorescence after the addition of SIH chelator is directly proportional to the iron labile pool, i.e., iron in complexes with affinity constant \< 10^6^. Immunocytochemistry ------------------- Cells grown in cover slips were sequentially fixed with 2% and 4% parafolmaldehyde (PFA) in Eagles\' MEM, and then washed three times with phosphate-buffered saline (PBS). The fixed cells were permeabilized with Triton-X-100 (0.2%) in PBS at room temperature for 3 min and blocked with defatted milk (10%) in PBS for more than 1 h. The cells were incubated with anti-IREG1 antibody (1:500) overnight at 4°C, washed with PBS and then incubated with Alexa-546-conjugated goat anti-rabbit IgG. The labeled cells were observed with a Zeiss LSM 510 Meta confocal laser scanning microscope. Data analysis ------------- Variables were tested in triplicates, and experiments were repeated at least twice. Variability among experiments was \<20%. One-way ANOVA was used to test differences in mean values, and Turkey\'s post-hoc test was used for comparisons (In Stat program from GraphPad Prism). Differences were considered significant if P \< 0.05. Authors\' contributions ======================= MTN conceived of the study, participated in its design and coordination and drafted the manuscript. PA performed the experiments with hippocampal neurons, did the ferritin assays and participated in the analysis and interpretation of data. MN optimized the immunocytochemistry detection of IREG1, performed the confocal microscope observations and participated in the analysis and interpretation of data. VT did the Western blot, labile iron pool and viability assays and contributed to the discussion of the results. MA set up the method to determine total Fe concentration, did the sample and control measures of iron and participated in the analysis and interpretation of data. All coauthors participated in refining the text. Acknowledgements ================ We are indebted to Dr. David Haile for supplying anti-IREG1 antibody. This work was supported by project P99-031 of the Millennium Institute for Advanced Studies in Cell Biology and Biotechnology, Santiago, Chile. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Total and labile iron in the progressive iron accumulation model*A:***SH-SY5Y cells were grown for 8 days and then challenged for 2 days with different concentrations of iron (range 1.5--80 μM). ***B:***total iron content determined by mass spectrometry. Increased level of iron in the culture media produced increased levels of intracellular iron. Data is mean ± SD of 4 independent determinations. **C:**The calcein-sensitive iron pool increased at 80 μM Fe compared with 7 μM Fe. Representative tracings are shown. ::: ![](1471-2202-6-3-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Cell viability upon iron load.**SH-SY5Y cells and hippocampal neurons were subjected to the progressive iron accumulation protocol described in Figure 1A. Viability in the cell cultures was determined by the MTT method. Representative curves from 4 (SH-SY5Y cells) or 3 (hippocampal cells) independent experiments are shown. ::: ![](1471-2202-6-3-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **Ferritin content*A:***ferritin was determined in extracts from cells cultured for 2 days with different iron concentrations as described in Figure 1A. Data is mean ± SD from 4 independent determinations. ***B:***iron : ferritin molar ratio. Total iron and ferritin total data were from Figure 1B and 3A, respectively. Ferritin synthesis response was larger that iron accumulation, an indication of a very active IRE/IRP system. ::: ![](1471-2202-6-3-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Changes in DMT1 expression*Upper panel:***DMT1 from cells cultured for 2 days with different iron concentrations was determined by Western blot. A continuous decrease in DMT1 expression was observed in the 1.5--80 μM Fe range. Shown is one of three similar experiments. ***Lower panel:***densitometric analysis of the DMT1 bands shown in Figure 4A. ::: ![](1471-2202-6-3-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### IREG1 expression increases upon increasing iron exposure. IREG1 from cells cultured for 2 days with different iron concentrations was determined by Western blot. A shows results of one of two similar experiments. Bands of 65.3 and 122.1 KDa were evident. The side panel shows the densitometric analysis of the bands. The 122.1 KDa band increased in the 1.5 to 40 mM Fe range. B: Both the upper, 122.1 KDa, band and the lower, 65.3 KDa, band diminished when the antibody was pre-incubated with peptide CGPDEKEVTKENQPNTSVV, prior to Western blot of 30, 20 and 10 mg of cell extract. C: treatment of 80 mM Fe extracts with increasing b-mercaptoethanol. A decrease in the 122.1 KDa band with increased b-mercaptoethanol was apparent. ::: ![](1471-2202-6-3-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Immunocytochemistry determination of IREG1.**Hippocampal neurons and SH-SY5Y cells, labeled with rabbit anti-IREG1 antibody and with Alexa-546-conjugated goat anti-rabbit IgG, were imaged in a confocal microscope. Shown are representative fields of cells cultured at different iron concentrations. Note the preferentially cytosolic distribution of IREG1. ::: ![](1471-2202-6-3-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### **Kinetics of iron efflux from SH-SY5Y cells.**SH-SY5Y cells were cultured for 2 days with varied amounts of iron as described in Figure 1A. The culture medium was changed to fresh DMEM and the kinetic of iron exit from the cells was followed by determining the concentration of iron in the medium by absorption spectrometry (***A***). Data is means ± SD from 3 experiments. The efflux rate, obtained from the slope of the curves in Figure 7A, was plotted against iron concentration during the 2-day culture period (***B***). In ***C***, the efflux rate was plotted against the density of the 65.3 KDa or 122.1 KDa bands. To that end, the 65.3 KDa or 122.1 KDa bands shown in Figure 5 were quantified by densitometry and paired with the efflux rates determined in Figure 7A at the same iron concentrations. A good correlation between efflux rate and the presence of the 122.1 KDa band was observed. ::: ![](1471-2202-6-3-7) :::

PubMed Central

2024-06-05T03:55:52.668422

2005-1-24

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548319/", "journal": "BMC Neurosci. 2005 Jan 24; 6:3", "authors": [ { "first": "Pabla", "last": "Aguirre" }, { "first": "Natalia", "last": "Mena" }, { "first": "Victoria", "last": "Tapia" }, { "first": "Miguel", "last": "Arredondo" }, { "first": "Marco T", "last": "Núñez" } ] }

PMC548382

Background ========== A common idea in animal ecology is that adverse or stressful environmental conditions facilitate the evolution of social behaviour \[[@B1]\]. The formation of groups across a huge number of animal taxa is thus considered to have broad implications for the benefit of individuals, including mate finding, the efficient location and use of resources, thermoregulation, energetic benefits and defence against natural enemies or competitors \[[@B2],[@B3]\]. Basic proximate prerequisites for communal behaviour are cues indicating the location of conspecifics and the ability to receive and process information regarding these cues, which in turn induce inter-individual attraction \[[@B3]\]. Because the costs and benefits of communal behaviour typically vary with environmental conditions, the degree to which individuals are mutually attracted is regulated by signals indicating the presences of predators, food availability, etc. \[[@B4]\]. In the vast group of insects that depend on ephemeral resources, such as decaying plant tissues, dung and carrion, aggregation in the immature stages across resource patches is the result of the choice of a female to lay batches of eggs and/or to aggregate with conspecifics \[[@B5]-[@B8]\]. In studies of *Drosophila*as an ecological model system, one benefit that females flies seem to achieve by this spatial aggregation is that larval survival probability to the adult stage is highest at intermediate densities \[[@B9],[@B10]\], indicating the existence of so-called Allee effects \[[@B11]\]. Competing filamentous fungi co-occurring with *Drosophila*larvae on the same patches have been demonstrated to cause high rates of mortality when larvae feed solitarily or in small groups, whereas larger groups are able to hamper mould growth \[[@B12]\] (Fig. [1](#F1){ref-type="fig"}), which in turn increases larval survival \[[@B9],[@B13],[@B14]\]. Although the mechanisms leading to mould suppression are not fully understood, physical damage of the fungal tissue from the feeding (shovelling food with the mouth hooks) and locomotor (crawling and digging) behaviour of the fly larvae \[[@B15]\] seems to be the major cause of the repression of mould growth \[[@B12],[@B14]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **The effect of larval density on mould growth.**The effect of *Drosophila*larval density (a. one larva, b. 5 larvae, c. 10 larvae) on the growth of *Aspergillus niger*. Patches (2.5 cm diameter) contained standard *Drosophila*rearing medium. Photographs were taken 10 days after infection with fungal spores. Spores and fly larvae were simultaneously transferred to the patches. Whereas one larvae did not significantly hamper mould development (a), five and ten larvae caused a substantial reduction in fungal growth (b) or even entirely suppressed fungal development (c). (unpublished study) ::: ![](1742-9994-2-2-1) ::: Given the relationship between spatial oviposition patterns, Allee effects and the suppression of mould, spatial aggregation in *Drosophila*can be interpreted as an adaptive behaviour against competing fungi on larval feeding sites in order to enhance offspring survival. These ecological interrelationships might set conditions for facilitating social behaviour in the fly larvae because, at the level of larval behaviour, a more efficient strategy that might control the rapid establishment of noxious fungi would be to exert physical stress directly on fungal colonies. Thus, larvae should display an assortative behaviour on the site on which fungi are growing, rather than moving randomly and independently of each other across a resource patch, by which the fungal tissues might only incidentally be destroyed. In the present study, I have provided groups of *Drosophila melanogaster*Meigen (Diptera, Drosophilidae) larvae with fungal-infected (2-day-old colonies of *Aspergillus niger*van Tieghem) and uninfected (control patches) (F-C treatment) food patches and examined whether the distribution of larvae across the patches is driven by fungal infection. In comparison with this naturally occurring heterogeneity in patch quality, I have also studied the distribution of fly larvae when they were offered only infected (F-F treatment) or uninfected (C-C treatment) food patches in order to test for the existence of grouping behaviour in two types of homogenous larval environment. If grouping is irrelevant under the given experimental setting, no deviation from the regular larval distribution across the food patches would be expected, i.e larvae should distribute themselves across patches in order to minimise larval competition for food \[[@B16]\]. Although *Drosophila*is a thoroughly studied model organism in foraging biology \[[@B17],[@B18]\], knowledge about social interactions between the insect larvae is surprisingly limited. This is intriguing because drosophilids are also model organisms in spatial ecology in which *Drosophila*communities are characterised by strong intraspecific aggregation across patchily distributed substrates (e.g. decaying plant tissues) \[[@B19]-[@B21]\]. The lack of knowledge concerning social interactions among larvae and its possible role in competition with filamentous fungi have provided the specific impetus of the present study. Results ======= Larval aggregation in the F-C treatment (Δ*pl*) ----------------------------------------------- The proportion of larvae on fungal-infected patches minus the proportion on uninfected patches, Δ*pl*, was used as a measure of the way in which *Drosophila*larvae distributed themselves between the two types of food patches in the F-C treatment (see method section for details). The number of larvae in both food patches (LARVAE) and the experimental day (DAY) did not influence Δ*pl*(Table [1](#T1){ref-type="table"}). The estimated intercept for Δ*pl*was significantly different from zero (GLM *d.f.*= 1, *mean square*= 4.475, *F*= 20.13, *P*\< 0.0001, *N*= 35), the positive value for Δ*pl*(Fig. [2a](#F2){ref-type="fig"}; *intercept estimate*: 0.3576 ± 0.0797, *t*= 4.49, *P*\< 0.001) indicating the aggregation of larvae on fungal-infected sites (see method section). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of LARVAE and DAY on larval aggregation in the F-C treatment. Analysis of variance for the effect of the number of larvae in both food patches (LARVAE) and experimental day (DAY) on *Drosophila*larval distribution between fungal-infected and uninfected food patches (F-C treatment). ::: **Explanatory variable** ***d.f.*** **Mean square** ***F*-value** ***P*** -------------------------- ------------ ----------------- --------------- --------- LARVAE 1 0.0361 0.15 0.7042 DAY 3 0.0470 0.19 0.9018 Error 30 0.2462 ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Larval aggregation in the heterogeneous (F-C) and two types of homogeneous (F-F and C-C) larval environment.**(a) Δ*pl*(where Δ*pl*= proportion of larvae from the fungal-infected patch -- proportion of larvae from the uninfected patch) as a measure of larval aggregation in the F-C treatment (Δ*pl*= 0: no effect of fungal-infected patches on larval distribution behaviour; Δ*pl*\> 0: aggregation of larvae on fungal infected patches; Δ*pl*\< 0: larvae avoid fungal colonies). (b) \|Δ*pl*\| as a measure of the general tendency of *Drosophila*larvae to aggregate with conspecifics in the heterogeneous environment (F-C) and two types of homogeneous environment (F-F and C-C). Because larval aggregation in the F-C treatment was measured independently of the patch type (see Methods), \|Δ*pl*\| is larger than Δ*pl*(2a). (F: fungal-infected patches, C: uninfected control patches) ::: ![](1742-9994-2-2-2) ::: Comparison of larval aggregation in the F-C, F-F and C-C treatment (\|Δ*pl*\|) ------------------------------------------------------------------------------ \|Δ*pl*\|, the absolute value of Δ*pl*, was used as a measure of the general tendency of *Drosophila*larvae to aggregate with conspecifics in the heterogeneous environment (F-C) and the two types of homogenous environment (F-F or C-C). By using \|Δ*pl*\|, aggregation in the F-C treatment was quantified independently of whether a food patch was infected with fungi or not. With regard to all three larval environments, the estimated intercepts for \|Δ*pl*\| were significantly different from zero, and hence indicate larval aggregation (Table [2](#T2){ref-type="table"}). Within each treatment LARVAE and DAY had no effect on \|Δ*pl*\| (Table [3](#T3){ref-type="table"}). In comparison with the homogenous environments (F-F and C-C treatment), the F-C treatment induced stronger larval aggregation (Fig. [2b](#F2){ref-type="fig"}, Table [4](#T4){ref-type="table"}). Moreover, there is a statistical trend of LARVAE influencing fly larval aggregation (Table [4](#T4){ref-type="table"}). This was due to differences in LARVAE as a function of TREATMENT (GLM *d.f.*= 2, *mean square*= 0.0134, *F*= 3.34, *P*= 0.0393, *N*= 105). Significantly fewer larvae were found to be feeding in both patches in the C-C treatment (8.89 ± 1.64 SE) than in the F-C (9.43 ± 0.95 SE) or the F-F treatment (9.46 ± 0.61 SE). However, LARVAE within one type of environment had no effect on larval aggregation (Table [3](#T3){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The general tendency to aggregate with conspecifics (\|Δ*pl*\|) in the heterogeneous (F-C) and two types of homogeneous (F-F and C-C) larval environment. Test of the effect of intercept as the only explanatory variable for the general tendency of *Drosophila*larvae to aggregate with conspecifics (measured as \|Δ*pl*\|, see text for details) in three types of larval environment (F-C, F-F or C-C). Whereas \|Δ*pl*\| = 0 and no explanatory power of intercept would indicate a regular distribution of larvae across the food patches, \|Δ*pl*\| \> 0 and a significant effect of intercept indicates larval aggregation in one of the experimental food patches (see also Fig. 2b). Note that, in contrast to Δ*pl*(Fig. 2a), \|Δ*pl*\| measures larval aggregation in the F-C treatment independently of whether a food patches was fungal-infected or not. For each type of larval environment an individual test was performed, with *N*= 35 for each treatment. ::: ***Parameter estimate*** ------------------------ -------------------------- --------------- ---------- **Larval environment** **Intercept ± SE** ***t*-value** ***P*** F-C 0.51 ± 0.05 10.55 \<0.0001 F-F 0.37 ± 0.05 8.13 \<0.0001 C-C 0.35 ± 0.04 9.24 \<0.0001 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### The effect of LARVAE and DAY on the general tendency of *Drosophila*larval aggregation (\|Δ*pl*\|) under three environmental conditions. Analysis of variance for the effect of LARVAE and DAY on *Drosophila*larval distribution between food patches in three different larval environments (F-C, F-F or C-C). ::: **Larval environment** **Explanatory variable** ***d.f.*** **Mean square** ***F*-value** ***P*** ------------------------ -------------------------- ------------ ----------------- --------------- --------- F-C LARVAE 1 0.2120 2.63 0.1154 DAY 3 0.0931 1.15 0.3433 Error 30 0.0807 F-F LARVAE 1 0.0682 1.03 0.3177 DAY 3 0.1132 1.71 0.1855 Error 30 0.0661 C-C LARVAE 1 0.0329 0.66 0.4236 DAY 3 0.2090 4.18 0.4998 Error 30 0.0400 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### The effect of the larval environment on the general tendency of *Drosophila*larvae to aggregate with conspecifics (\|Δ*pl*\|). Mixed model analysis of variance for the tendency to aggregate with conspecific larvae in *D. melanogaster*in three types of larval environment (F-C, F-F or C-C). Larval aggregation was measured as \|Δ*pl*\|, the absolute value of Δ*pl*(see Methods). TREATMENT (F-C, F-F or C-C) and LARVAE were fixed main effects, whereas experimental day (DAY) was a random factor. DAY is nested within TREATMENT and was used as the error term in testing the effect of TREATMENT. ::: **Explanatory variable** ***d.f.*** **Mean square** ***F*-value** ***P*** -------------------------- ------------ ----------------- --------------- --------- TREATMENT 2 0.4168 5.08 0.0302 LARVAE 1 0.2237 3.44 0.0670 TREATMENT (DAY) 9 0.0831 1.28 0.2603 Error 92 0.0651 ::: Discussion ========== On the background of ecological interactions between insects and filamentous fungi on ephemeral resources, the experiment presented in this study was designed to test for social attraction in *Drosophila*larvae, an attraction that I hypothesised to be advantageous when larvae are confronted with noxious moulds. The results demonstrate that the fly larvae significantly aggregated on food patches on which young fungal colonies were growing (F-C treatment, Fig. [2a](#F2){ref-type="fig"}). Moreover, when provided with a homogeneous environment (F-F or C-C treatment), larvae displayed significant aggregation across the two food patches (Fig. [2b](#F2){ref-type="fig"}). In comparison, however, aggregation was significantly enhanced when larvae had the choice between a mould-free and a mould-infected site (Fig. [2b](#F2){ref-type="fig"}). Thus, the results suggest that grouping behaviour in *Drosophila*larvae involves both mutual attraction between group members \[[@B2]\] and the attraction of individuals to the same environmental stimulus \[[@B22]\], i.e. cues emitted by the fungi. Group formation in eusocial insects and those living in groups for part or most of their lives is often mediated by pheromones, e.g. cuticular hydrocarbons that induce attraction between individuals \[[@B22],[@B23]\]. Chemical communication is also widespread in drosophilid behaviour, including those associated with spatial aggregation in adult flies \[[@B24]-[@B26]\]. Because several receptors on the cephalic lobe of *Drosophila*larvae have gustatory, mechanosensory and olfactory functions \[[@B27]\], both chemical and physical cues (e.g. substrate vibrations caused by larval movements) might be involved in mutual attraction. However, the mechanisms leading to grouping and group cohesion in *Drosophila*larvae are unknown. Interestingly, strong social attraction (communal digging) between *Drosophila*larvae is present in third-instar larvae, a behaviour regulated by a peptide neuromodulator (*Drosophila*neuropeptide F, dNPF) \[[@B28]\]; this shows striking similarities to the correlation of social feeding and the expression of a neuropeptide Y receptor homologue (NRP-1) in *Caenorhabditis elegans*\[[@B29]\]. Moreover, neurons that detect aversive environmental stimuli have been demonstrated to induce social feeding in *C. elegans*\[[@B30]\], thus providing support for the proposed relationship between environmental stress and social behaviour \[[@B31]\]. In contrast to *C. elegans*, the intimate communal digging behaviour in old third-instar *Drosophila*larvae does not occur in the context of food foraging behaviour but is part of the post-feeding phase prior to pupation \[[@B28]\]. Whereas downregulation of dNPF expression coincides with social behaviour in old and non-feeding *Drosophila*larvae, higher levels of dNPF expression in younger larvae seem to suppress strong larval aggregation and communal digging behaviour \[[@B28]\]. Therefore, it remains to be seen whether similar neural regulatory mechanisms are involved in earlier developmental stages of *Drosophila*larvae with respect to social affinity related to foraging for food and attraction to fungal competitors. With regard to the proximate causes of attraction to mould-infected sites, fungal-borne volatiles such as CO~2~or ethylene \[[@B32]\] might be perceived by fly larvae and might guide them to young mould colonies. A general reason for the formation of social groups seems to be an adaptive response to stressful environmental conditions \[[@B1],[@B31]\]. As outlined in the introduction, filamentous fungi co-occurring with drosophilids on larval feeding sites can impede fly larval development; indeed, larval aggregations have been shown successfully to suppress mould growth \[[@B12]\] (Fig. [1](#F1){ref-type="fig"}). Consequently, the presence of fungi may indicate stressful ecological conditions that initiate attraction towards fungal patches and enhance the mutual attraction of *Drosophila*larvae (Fig. [2](#F2){ref-type="fig"}). In connection with the benefits that accrue from mould suppression, the present study demonstrates that the larval-driven inhibition of fungal development is not a mere by-product attributable to the maternal decision to aggregate eggs across patches but is the consequence of a positive response of individuals to conspecifics. Because adult density-dependent oviposition choices influence a larva\'s food quality and its susceptibility to natural enemies \[[@B33]\] or abiotic stress, as well as its probability of coming into contact with intra- and interspecific competitors, the present study demonstrates the possibility of adaptive behavioural relationships between the well-known adult gregariousness in *Drosophila*and communal behaviour in the immature stages. Further investigating this kind of behavioural adult-offspring correlations would strongly contribute to our understanding of the evolutionary costs and benefits of spatial aggregation in insect communities \[[@B34]\]. Conclusion ========== The study presented here demonstrates that fungal-infected food patches (1) attract first-instar *Drosophila*larvae and (2) enhance group foraging behaviour. Because the larval-driven reduction in mould growth \[[@B12]\] has been shown to improve the chance of larval survival to the adult stage \[[@B9],[@B13],[@B14]\], social attraction to fungal-infected sites may reflect an adaptive behavioural response. In connection with the maternal behaviour of aggregating eggs across substrate patches, the condition-dependent mutual attraction of larvae can be discussed as a communal defence behaviour against competing mould. Given that filamentous fungi seriously deteriorate the developmental conditions for insect larvae, mould may constitute one important ecological factor that has, at least in the larval stages, facilitated social attraction in *Drosophila*. Thus, this study highlights the largely unappreciated role of inter-kingdom competition as a potentially important driving force in the evolution of insect behavioural traits. In general, the group formation of *Drosophila*larvae in response to well-defined ecological conditions might be an interesting model system for the study of proximate and ultimate aspects of social biology. Methods ======= Experimental set-up ------------------- I experimentally analysed the grouping behaviour of *Drosophila*larvae using a *D. melanogaster*strain that originated from wild animals caught in 2003 near Kiel, northern Germany (approx. 54° N, 10° E). Flies had been reared for 18 generations on standard *Drosophila*medium (30 gram corn meal, 30 gram sugar, 30 gram brewer\'s yeast extract (Leiber, Germany), and Nipagin) under constant environmental conditions (photoperiod of 16 hours and a temperature of 22°C). In order to obtain first instar larvae, a population of approx. 300 individuals (five to seven days old) were offered a 10 cm Petri dish containing a hard Agar medium (22 gram Agar, 90 cm^3^sugar beet syrup and 9.5 cm^3^Nipagin (10% in 95% ethanol) per 500 cm^3^water), on which they were allowed to oviposit for a period of 16 to 18 hours, including a period of darkness. Subsequently, flies were removed and the eggs were incubated at 22°C over a 16-hour photoperiod. After 24 hours, almost all larvae had hatched from the eggs and were then isolated from the medium by washing them off the Agar plate onto fine-meshed gauze with water. These larvae were used in the experiments. I used the same medium as for fly rearing (without adding the antifungal agent Nipagin) to simulate an uninfected or a fungal-infected larval environment as follows: aliquots of 3.5 cm^3^hot medium were transferred to each of two small pots (10 mm diameter, 5 mm high) that were glued to the bottom of a Petri dish (45 mm diameter, 13 mm high). Within one Petri dish, the larval food patches were placed at a distance of approx. 10 mm and surrounded by an Agar layer (5 mm high), so that the surfaces of the Agar and the food patches were at the same level. After the food patches had cooled down, one patch was provided with 1 μl of water containing approx. 800 conidiospores of the fungus *A. niger*(F treatment) and, as a control, the second patch was provided with spore-free water (C treatment). In addition to this F-C treatment, I simultaneously prepared arenas in which both patches were infected with spores (F-F) or both patches remained uninfected (C-C). The arenas were immediately sealed with lids and incubated under the aforementioned conditions. Two days later, the fungal spores had germinated and tiny translucent hyphal colonies were visible. A group of ten *Drosophila*larvae were transferred, with a fine brush, to each arena at a distance of approx. 10 mm from each of the two food patches. Subsequently, the arenas were sealed with lids and stored at 22°C in an illuminated incubator. In order to avoid any systematic effects on larval distribution behaviour that might be caused by the position of the light tubes, the arenas of all three treatments were randomly arranged in the incubator. After six hours, the number of larvae in each food patch was recorded. Preliminary experiments had shown that this time period was sufficient to obtain final larval distribution patterns across the two patches, which remained nearly constant until the next day. Five to ten replicates for each treatment were simultaneously prepared at four different days. Statistical analysis -------------------- Based on the number of larvae that were found to be feeding in both patches, I calculated the proportion of larvae in each patch. Larvae that were not found on any of the food patches were ignored. However, I tested for the effect of the number of larvae in both patches (LARVAE) on the degree to which larvae aggregated across the food patches (see below). To obtain a measure of the degree of larval aggregation across the two food patches in the F-C treatment, the proportion of larvae on the fungal-infected patch in each arena was reduced by the proportion of larvae on the uninfected patch, yielding Δ*pl*. Δ*pl*= 0 would indicate no effect of the presence of fungal-infected and uninfected food sites on larval distribution patterns. Whereas Δ*pl*\> 0 would indicate aggregation on fungal-infected sites and thus larval attraction to fungal colonies, Δ*pl*\< 0 is expected if larvae avoid fungal colonies and aggregate on uninfected patches. Subsequently, I used the absolute values of Δ*pl*, \|Δ*pl*\|, that were obtained in all three types of treatment (F-C, F-F and C-C) in order to compare the tendency to aggregate with conspecific larvae in the heterogeneous larval environment (F-C) with larval aggregation in two types of homogeneous environment (F-F or C-C). Note that, because the absolute value of Δ*pl*can only be equal to or larger than zero, \|Δ*pl*\| measures larval aggregation in the F-C treatment independently of whether a food patch was fungal-infected or not. I applied the GLM procedure provided by SAS version 8.2 to test if *Drosophila*larvae aggregated on fungal infected food patches, i.e. if Δ*pl*is significantly larger than 0 (see above). For this only the intercept was tested as an effect in the statistical model \[[@B35]\]. The result of the parameter estimate for the intercept are given. Before this test, I verified that LARVAE and experimental DAY did not affect Δ*pl*(Table [1](#T1){ref-type="table"}), which justifies the removal of these variables from the full model (backward elimination of non-significant variables) \[[@B36]\]. The same procedure was applied to test for the general tendency of larval aggregation (measured as \|Δ*pl\|*) under different environmental conditions (see Table [2](#T2){ref-type="table"} to [4](#T4){ref-type="table"}). To analyse the effect of TREATMENT (F-C, F-F or C-C) and LARVAE on the general propensity of *Drosophila*larvae to aggregate across the experimental food patches (\|Δ*pl*\|), I used the aforementioned GLM procedure with a RANDOM statement to account for possible effects of experimental DAY on larval distribution patterns. In this model, TREATMENT and LARVAE were fixed main effects. Since five to ten replicates for each treatment were prepared at four different days, DAY was considered as a categorical random factor. DAY is nested within TREATMENT and was used as the error term in testing for the effect of TREATMENT \[[@B37]\]. The results of the tests of hypotheses for mixed model analysis of variance are shown in Table [4](#T4){ref-type="table"}. Acknowledgments =============== Frank Kempken and Hanna Schmidt provided spores of *A. niger*. Denise Olbrich, Katherina Habekost and Saskia Heppner are acknowledged for help with data collection and preparation of experimental arenas. I thank Jacqui Shykoff and two anonymous reviewers for their valuable comments on an earlier version of this paper.

PubMed Central

2024-06-05T03:55:52.670567

2005-1-27

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548382/", "journal": "Front Zool. 2005 Jan 27; 2:2", "authors": [ { "first": "Marko", "last": "Rohlfs" } ] }

PMC548501

Background ========== Prostate cancer is the most common cancer and the second leading cause of cancer related deaths in men in the United States with an estimated 230,110 new cases and 29,500 deaths in 2004 \[[@B1]\]. Initial treatment for prostate cancer is usually androgen-ablative therapy, radiotherapy or radical prostatectomy and the patients respond to androgen-ablative therapy in the beginning of treatment. However, many patients eventually fail this therapy and die of recurrent androgen-independent prostate cancer and metastasis \[[@B2]\]. Up to 30% of men undergoing radical prostatectomy will also relapse, often as a result of micrometastatic cancer present at the time of surgery \[[@B3]\]. The efficacy of cytotoxic chemotherapy for treatment of hormone-refractory prostate cancer has been tested in clinical trials. In general, response rates of \<10% were observed in single-agent studies \[[@B2]\]. Thus, there is a tremendous need for the development of mechanism-based targeted strategies for treatment of prostate cancer. Taxotere, a member of taxane family, is semi-synthesized from an inactive taxoid precursor extracted from the needles of the European yew, *Taxus baccata*. Its known basic mechanism of action is that it binds to tubulin and deranges the equilibrium between microtubule assembly and disassembly during mitosis \[[@B4]\]. Stabilization of microtubules by Taxotere impairs mitosis and exerts an anticancer effect in tumors \[[@B4]\]. Taxotere has shown clinical activity in wide spectrum of solid tumors including breast, lung, ovarian, prostate cancers, etc \[[@B5],[@B6]\]. In metastatic breast, lung, and ovarian cancer, randomized trials have shown that Taxotere-containing therapies are superior to or as effective as established standard chemotherapeutic regimens and are often associated with an improved safety profile \[[@B6]\]. Clinical trials have also found that weekly Taxotere in patients with metastatic hormone-refractory prostate cancer is associated with improvements in clinical benefit response and quality of life \[[@B7],[@B8]\]. Thus, Taxotere is currently considered to be among the most important anticancer drugs in cancer chemotherapy. In addition to its effects on microtubules, Taxotere also induces apoptosis with down-regulation of bcl~XL~and bcl-2, and upregulation of p21^WAF1^and p53 \[[@B9],[@B10]\]. We have previously reported that Taxotere down-regulates some genes for cell proliferation, mitotic spindle formation, transcription factors, and oncogenesis, and up-regulates some genes related to induction of apoptosis and cell cycle arrest in prostate cancer cells, suggesting the pleiotropic effects of Taxotere on prostate cancer cells \[[@B11]\]. Capecitabine is an orally administered systemic prodrug of 5\'-deoxy-5-fluorouridine (5-DFUR or Furtulon) which is converted to 5-fluorourasil (5-FU) \[[@B12]\]. Capecitabine is readily absorbed from the gastrointestinal tract. In human and animals, carboxylesterase hydrolyzes much of Capecitabine to 5\'-deoxy-5-flurocytidine (5-DFCR). Cytidine deaminase, an enzyme found in most tissues including tumors, subsequently converts 5-DFCR to 5-DFUR. The enzyme, thymidine phosphorylase (dThdPase), then hydrolyzes 5-DFUR to the active drug 5-FU both *in vivo*and *in vitro*. After being converted to 5-FU, Capecitabine inhibits essential cellular biosynthetic processes and is incorporated into DNA to inhibit normal bioprocess function of the cell \[[@B13]\]. Capecitabine has shown anti-tumor activity in various cancers including prostate cancer \[[@B14]-[@B16]\]. 5-FU-based chemotherapy improves overall and disease-free survival of patients with cancer. However, response rates for 5-FU-based chemotherapy are low and a large number of tumors eventually becomes resistant to 5-FU \[[@B13],[@B17]\]. Clinical trials showed that chemotherapeutic combination with Taxotere and Capecitabine resulted in improved objective response rate and overall survival without a significant increase in the treatment related adverse effects in metastatic breast cancer and advanced non-small cell lung carcinoma \[[@B18],[@B19]\]. However, the molecular mechanism(s) of action of Taxotere and Capecitabine have not been fully elucidated. In this study, we utilized high-throughput gene chip, which contains 22,215 known genes, to determine the alternation of gene expression profiles of hormone insensitive (PC3) and sensitive (LNCaP) prostate cancer cells exposed to Taxotere and Furtulon. The purpose of this study was: 1) to identify novel genes that have key roles in cancer cell killing and resistance induced by Taxotere and/or Furtulon; 2) to test whether similar genes are altered by Taxotere and Furtulon; 3) to test whether combination therapy alters genes that may reflect better treatment outcome or may provide information whether combination therapy could be antagonistic; 4) finally to provide molecular information for further mechanistic investigation and future clinical application. Methods ======= Cell culture and growth inhibition ---------------------------------- PC3 (ATCC, Manassas, VA) and LNCaP (ATCC) human prostate cancer cells were cultured in RPMI-1640 media (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum and 1% penicillin and streptomycin in a 5% CO~2~atmosphere at 37°C. Taxotere (Aventis Pharmaceuticals, Bridgewater, NJ) was dissolved in DMSO to make 4 μM stock solution. Furtulon (Roche, Palo Alto, CA) was dissolved in PBS to make 100 mM stock solution. For growth inhibition, PC3 and LNCaP cells were treated with Taxotere (1, 2, and 4 nM), Furtulon (50, 100, and 200 μM), or 1 nM Taxotere plus 50 μM Furtulon for one to three days. Control PC3 and LNCaP cells received 0.01% DMSO or 0.1% PBS for same time points. After treatment, PC3 and LNCaP cells were incubated with MTT (0.5 mg/ml, Sigma, St. Louis, MO) at 37°C for two hours and then with isopropyl alcohol at room temperature for one hour. The spectrophotometric absorbance of the samples was determined by using ULTRA Multifunctional Microplate Reader (TECAN, Durham, NC) at 595 nm. The concentrations of Taxotere and Furtulon used for our *in vitro*studies are easily achievable in humans, suggesting that our experimental results are relevant for human applications. The experiment was repeated three times and a *t*test was performed to verify the significance of cell growth inhibition after treatment. Microarray analysis for gene expression profiles ------------------------------------------------ PC3 and LNCaP cells were treated with 2 nM Taxotere, 110 μM Furtulon, or 1 nM Taxotere plus 50 μM Furtulon for 6, 36, and 72 h. Total RNA from each sample was isolated by Trizol (Invitrogen, Carlsbad, CA) and purified by RNeasy Mini Kit and RNase-free DNase Set (QIAGEN, Valencia, CA) according to the manufacturer\'s protocols. cDNA for each sample was synthesized by Superscript cDNA Synthesis Kit (Invitrogen, Carlsbad, CA) using the T7-(dT)~24~primer instead of the oligo(dT) provided in the kit. Then, the biotin-labeled cRNA was transcripted *in vitro*from cDNA by using BioArray HighYield RNA Transcript Labeling Kit (ENZO Biochem, New York, NY), and purified by RNeasy Mini Kit. The purified cRNA was fragmented by incubation in fragmentation buffer (40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc) at 95°C for 35 min and chilled on ice. The fragmented labeled cRNA was applied to Human Genome U133A Array (Affymetrix, Santa Clara, CA), which contains 22,215 human gene probes, and hybridized to the probes in the array. After washing and staining, the arrays were scanned. Two independent experiments were performed to verify the reproducibility of results. Microarray data normalization and analysis ------------------------------------------ The gene expression levels of samples were normalized and analyzed by using Microarray Suite, MicroDB™, and Data Mining Tool software (Affymetrix, Santa Clara, CA). The absolute call (present, marginal, absent) and average difference of 22,215 gene expressions in a sample, and the absolute call difference, fold change, average difference of gene expressions between two or several samples were normalized and identified using these software. Statistical analysis of the mean expression average difference of genes, which show \>2 fold change, was performed using a *t*test between treated and untreated samples. Clustering and annotation of the gene expression were analyzed by using Cluster and TreeView \[[@B20]\], Onto-Express \[[@B21]\], and GenMAPP \[[@B22]\]. Genes that were not annotated or not easily classified were excluded from the functional clustering analysis. Real-time RT-PCR analysis for gene expression --------------------------------------------- To verify the alterations of gene expression at the mRNA level, which appeared on the microarray, we chose representative genes (Table [1](#T1){ref-type="table"}) with varying expression profiles for real-time RT-PCR analysis. Two micrograms of total RNA from each sample were subjected to reverse transcription using the Superscript first strand cDNA synthesis kit (Invitrogen, Carlsbad, CA) according to the manufacturer\'s protocol. Real-time PCR reactions were then carried out in a total of 25 μL reaction mixture (2 μl of cDNA, 12.5 μl of 2X SYBR Green PCR Master Mix, 1.5 μl of each 5 μM forward and reverse primers, and 7.5 μl of H~2~O) in SmartCycler II (Cepheid, Sunnyvale, CA). The PCR program was initiated by 10 min at 95°C before 40 thermal cycles, each of 15 s at 95°C and 1 min at 60°C. Data were analyzed according to the comparative Ct method and were normalized by actin expression in each sample. Melting curves for each PCR reaction were generated to ensure the purity of the amplification product. Western blot analysis --------------------- We also conducted Western Blot analysis to verify the alterations of genes at the level of translation for selected genes with varying expression profiles. The PC3 and LNCaP cells were treated with 1 and 2 nM Taxotere or 50 and 110 μM Furtulon for 24, 48, and 72 hours. After treatment, the cells were lysed in 62.5 mM Tris-HCl and 2% SDS, and protein concentration was measured using BCA protein assay (PIERCE, Rockford, IL). The proteins were subjected to 10% or 14% SDS-PAGE, and electrophoretically transferred to nitrocellulose membrane. The membranes were incubated with anti-cathepsin C (1:200, Santa Cruz, Santa Cruz, CA), anti-p16 (1:200, Santa Cruz, Santa Cruz, CA), anti-IKKα (1:100, Santa Cruz, Santa Cruz, CA), anti-p21^WAF1^(1:500, Upstate, Lake Placid, NY), anti-Bax (1:10000, Trevigen, Gaithersburg, MD), anti-survivin (1:200, Alpha Diagnostic, San Antonio, TX), anti-CDC2 (1:200, Santa Cruz, Santa Cruz, CA), anti-cyclin A (1:250, NeoMarkers, Union City, CA), anti-cyclin B (1:200, Santa Cruz, Santa Cruz, CA), anti-cyclin E (1:250, NeoMarkers), and anti-β-actin (1:10000, Sigma, MO) primary antibodies, and subsequently incubated with secondary antibody conjugated with fluorescence dye. The signal was then detected and quantified by using Odyssey infrared imaging system (LI-COR, Lincoln, NE). Results ======= Cell growth inhibition ---------------------- MTT assay showed that the treatment of PC3 and LNCaP prostate cancer cells with Taxotere, Furtulon, or lower concentration of Taxotere plus Furtulon resulted in dose and time-dependent inhibition of cell proliferation (Figure [1](#F1){ref-type="fig"}), demonstrating the inhibitory effect of Taxotere and Furtulon on the growth of PC3 and LNCaP prostate cancer cells. Regulation of mRNA expression by Taxotere and Furtulon treatment ---------------------------------------------------------------- Microarray analysis showed that the alterations of gene expression were occurred as early as 6 hours of Taxotere and/or Furtulon treatment, and were more evident with longer treatment (Table [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). Clustering analysis based on gene function showed down-regulation of some genes for cell proliferation and cell cycle progression (cyclin A, cyclin F, CDC2, CDK2, etc), transcription factors (transcription factor A, ATF5, TAF1131L, FOXM1, etc), and oncogenesis (GRO oncogene, BRCA1 associated RING domain, tumor-associated nuclear protein p120, etc) in Taxotere and/or Furtulon treated prostate cancer cells (Table [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). In contrast, Taxotere and/or Furtulon up-regulated some genes that are related to the induction of apoptosis (GADD45A, GADD45B, etc), cell cycle arrest (p21^CIP1^, VDUP1, BTG, etc), and tumor suppression (Table [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). Combination treatment with Taxotere and Furtulon also altered expression of some genes (CDC27, CDK9, p18, IKKα, etc) that showed no change in mono-treatment (Table [4](#T4){ref-type="table"} and [5](#T5){ref-type="table"}), suggesting the synergic effects of combination treatment on some genes. Taxotere and Furtulon also up-regulated some genes (S-100P, ALDH1A3, casein kinase, annexin, etc) responsible for chemotherapeutic resistance, suggesting the induction of cancer cell resistance to these agents (Table [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). Taxotere and Furtulon also showed differential effects on PC3 cells with alteration of metastasis-related genes and on LNCaP cells with down-regulation of survivin, cyclin B & E, CDC2, CDC25, and specifically AR by Furtulon, suggesting their effects mediated by both AR-independent and dependent pathways (Table [2](#T2){ref-type="table"} and [3](#T3){ref-type="table"}). Target verification by real-time RT-PCR and western blot -------------------------------------------------------- To verify the alterations of gene expression at the mRNA level, which appeared on the microarray, we chose representative genes with varying expression profiles for real-time RT-PCR and Western Blot analysis. The results of real-time RT-PCR for these selected genes were in direct agreement with the microarray data (Figure [2](#F2){ref-type="fig"}). The same alternations of gene expression were observed by real-time RT-PCR analysis, although the fold change in the expression level was not exactly same between these two different analytical methods. The results of Western Blot analysis were also in direct agreement with the microarray and real-time RT-PCR data (Figure [3](#F3){ref-type="fig"} and our earlier report \[[@B11]\]). These results support the findings obtained from microarray experiments. Discussion ========== It has been known that Taxotere binds to microtubules while Capecitabine is incorporated into DNA, inhibiting the bioprocess in cancer cells \[[@B4],[@B13]\]. However, the precise molecular mechanisms for inhibiting cancer cell growth by Taxotere and/or Capecitabine have not been fully elucidated. From gene expression profiles of Taxotere and/or Capecitabine treated prostate cancer cells, we found that these chemotherapeutic agents caused alterations in the expression of many genes related to the control of cell proliferation, apoptosis, transcription, translation, cell signaling, oncogenesis, and angiogenesis (Figure [4](#F4){ref-type="fig"}), although the cellular target of Taxotere or Capecitabine appears to be different. It has been well known that CDCs regulate the molecules related to the cell cycle initiation and progression and that cyclins associate with cyclin-dependent protein kinases (CDKs) and CDCs to control the process of cell cycle \[[@B23],[@B24]\]. The CDK inhibitors including p21^WAF1^, p16^INK4A^, and p18^INK4C^have been demonstrated to arrest the cell cycle and inhibit the growth of cancer cells \[[@B23],[@B24]\]. Our results showed that Cyclins (cyclin A2, cyclin E2, cyclin F, cyclin B1), CDK2, CDC2, and other cell growth promotion genes (pescadillo, spermidine synthase, mitotin) \[[@B25]-[@B27]\] were down-regulated in Taxotere and/or Furtulon treated prostate cancer cells, while CDK inhibitor p21^WAF1^and other growth inhibitor genes (BTG2, VDUP1, anti-proliferative B-cell translocation gene 1) \[[@B28],[@B29]\] were up-regulated, suggesting that Taxotere and/or Furtulon inhibited the growth of prostate cancer cells through the arrest of cell cycle and the inhibition of cell proliferation (Figure [4](#F4){ref-type="fig"}). The down-regulation of CDC27, CDK9, EGF, and FGF12B, and up-regulation of p16^INK4A^and p18^INK4C^were also observed in combination treatment but not in mono-treatment, suggesting the synergic effect of combination treatment. These observations are novel in Taxotere and/or Furtulon treated prostate cancer cells. Induction of apoptosis by chemotherapeutic agents also leads to the inhibition of cancer cell growth. It has been reported that Taxotere is able to induce apoptosis by caspase-3 dependent or independent cell death mechanism \[[@B30]\]. Capecitabine may induce apoptosis through Fas/FasL or Bax/Bcl-2 pathway \[[@B31],[@B32]\]. From gene expression profile, we found that Taxotere and/or Furtulon increased level of growth arrest and DNA-damage-inducible alpha (GADD45A), GADD45B, p53 regulated PA26 nuclear protein (PA26), and p53-induced protein 11 (PIG11), all of which are related to the induction of apoptotic processes. GADD45A and GADD45B have been known to promote apoptosis and regulate G2/M arrest \[[@B33]\]. PA26 is a target of the p53 tumor suppressor and a member of the GADD family with the properties of inducing apoptosis \[[@B34]\]. PIG11 as a downstream target of p53 is also involved in the apoptotic processes \[[@B35]\]. The combination treatment also showed down-regulation of negative regulator of programmed cell death ICH-1S and Bcl-2-associated transcription factor, which was not occurred in mono-treatment. The induction of apoptosis mediated by GADD45A, GADD45B, PA25, and PIG11 could be another molecular mechanism by which Taxotere and/or Furtulon inhibit the growth of prostate cancer cells. We also found that Taxotere and/or Furtulon inhibited the expression of transcription factors (FOXM1, ATF5, TFAM, TAFII31L), translation factors (EIF1A, EIF5A), oncogene (GRO1, GRO3, BRCA1-associated protein, tumor-associated nuclear protein p120), and heat shock protein, and up-regulated the genes for differentiation (prostate differentiation factor). These results are novel, and suggest the beneficial effects of Taxotere and/or Furtulon on the inhibition of cancer cell growth and oncogenesis. It is important to note that Taxotere and/or Furtulon also up-regulated the expression of some genes which are known to induce cell resistance to chemotherapeutic agents and to favor cell survival. Among these genes, calcium-binding protein S100P has been found to be highly expressed in cells which develop acquired resistance to anti-tumor agents \[[@B36]\]. The overexpression of aldehyde dehydrogenase 1 (ALDH1) has also been detected solely in classical multidrug resistance cancer cells \[[@B37],[@B38]\]. It has been reported that Annexin-I, casein kinase 1, and cisplatin-resistance associated protein expressions modulate drug resistance in tumor cells \[[@B39],[@B40]\]. The up-regulation of these molecules by Taxotere and/or Furtulon could induce cell resistance to chemotherapeutic agents. Also, Taxotere and/or Furtulon were found to up-regulate the expression of Notch 3, angiopoietin, activating transcription factor 3, which could favor cell survival \[[@B41]-[@B43]\]. Further in depth mechanistic studies are needed to address these issues. The investigation on overcoming these unbeneficial effects with other agents must be devised, which is ongoing in our laboratory. Taxotere showed no effect on AR expression while Furtulon down-regulated AR expression in LNCaP cells, suggesting that the combination could be superior in AR-positive cells. The genes altered by Taxotere and/or Furtulon with respect to the control of cell growth, apoptosis, transcription, oncogenesis, and metastasis in androgen insensitive PC3 cells are different from that in androgen sensitive LNCaP cells, suggesting that the effects of Taxotere and Furtulon may be mediated by both AR-dependent and independent signaling pathways. We observed up-regulation of tissue inhibitor of metalloproteinase 1 (TIMP1), TIMP2, and protease inhibitor 3 in Taxotere and/or Furtulon treated PC3 cells, suggesting that Taxotere and/or Furtulon may exert anti-metastatic effect. However, we also observed increase in the expression of MMP1, MMP9, cathepsin B, uPA, and tPA in Taxotere and Furtulon treated PC3 cells, therefore, more experimental studies are needed to reveal the overall effect of Taxotere and Furtulon on metastatic processes. These results were not observed in androgen sensitive LNCaP cells, suggesting difference in effects that could be mediated through different cell signal transduction pathways. Conclusions =========== In conclusion, Taxotere and/or Furtulon directly and indirectly caused changes in the expression of many genes that are critically involved in the control of cell proliferation, apoptosis, transcription, translation, oncogenesis, angiogenesis, metastasis, and drug resistance (Figure [4](#F4){ref-type="fig"}). These findings could provide molecular information for further investigation on the mechanisms by which Taxotere and Furtulon exerts their pleiotropic effects on prostate cancer cells. These results could also be important in devising mechanism-based targeted therapeutic strategies for prostate cancer, especially in devising combination therapy for drug resistant prostate cancers. However, further in-depth investigations are needed in order to establish cause and effect relationships between these altered genes and therapeutic response in prostate cancer cells. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= FHS designed the study and prepared the manuscript. YL carried out cell growth inhibition, microarray and Western Blot analysis and drafted the manuscript. MH participated in the design of the study. RL and SHS carried out real-time PCR. JE prepared Furtulon reagent. All authors read and approved the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/5/7/prepub> Acknowledgements ================ This work was partly funded by a grant from Aventis Pharmaceuticals (awarded to F.H.S.). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Effects of Taxotere and/or Furtulon on the growth of PC3 and LNCaP Cells. PC3 and LNCaP prostate cancer cells treated with Taxotere and/or Furtulon resulted in a dose and time-dependent inhibition of cell proliferation (\* : *p*\< 0.05, n = 3) ::: ![](1471-2407-5-7-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Real-time RT-PCR showing the altered expression of specific genes in Taxotere and Furtulon treated PC3 and LNCaP cells. (C: control; T+F: Taxotere and Furtulon combination treatment.) ::: ![](1471-2407-5-7-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Western Blot analysis showing the altered expression of specific genes in Taxotere and Furtulon treated PC-3 or LNCaP cells. (C: control; T: Taxotere treatment; F: Furtulon treatment; T+F: Taxotere and Furtulon combination treatment.) ::: ![](1471-2407-5-7-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effects of Taxotere and Furtulon on cell cycle, apoptosis, and other pathway related gene expression analyzed and visualized by GenMAPP software integrated with cDNA microarray data. A: PC3 cells. B: LNCaP cells. (positive value: increase in fold change; negative value: decrease in fold change; A: PC3 cells; B: LNCaP cells) ::: ![](1471-2407-5-7-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The primers used for real-time RT-PCR analysis ::: ------------------------------------------------------------------ **Genes** **Primer Sequence** **PCR Product** ----------- ------------------------------------ ----------------- ATF5 ctc ctc ctt ctc cac ctc aa\ 103 bp gcc gac ttg ttc tgg tct ct Cyclin A2 aat cag ttt ctt acc caa tac\ 127 bp ctg atg gca aat act tga Fas/Apo-1 caa aag tgt taa tgc cca agt\ 187 bp gca gtc tgg ttc atc cc FOXM1 gcc aca ctt agc gag acc c\ 189 bp atc aca agc att tcc gag aca GADD45 cgc ctg tga gtg agt gc\ 154 bp ctt atc cat cct ttc ggt ctt IGFBP2 atg ggc gag ggc act t\ 189 bp cag ctc ctt cat acc cga ctt uPA ggg agc aga gac act aac gac t\ 108 bp ctc aca gcc cac aca aca ag Ki-67 ccg ggc tcc atc atc t\ 148 bp ctc cag acg cca aaa taa gac p21^WAF1^ ctg gag act ctc agg gtc gaa\ 98 bp gga tta ggg ctt cct ctt gga p27^KIP1^ cgc tcg cca gtc cat t\ 187 bp aca aaa ccg aac aaa aca aag PIR cac tag ccc tcc atc ctc tac\ 151 bp ggg tct gcc aat gct tct MMP1 gct ttc ctc cac tgc tgc t\ 146 bp aac ttg cct ccc atc att ctt STK6 tca gcg ggt ctt gtg t\ 162 bp ctc ttt tgg gtg tta ttc agt Survivin cca ctg ccc cac tga gaa c\ 118 bp acc gga cga atg ctt ttt atg TRIP13 tct ggc agt gga caa gca gtt\ 136 bp tgg gag acg gct gtg tgg GAPDH ctg cac cac caa ctg ctt ag\ 222 bp ttc agc tca ggg atg acc tt β-actin cca cac tgt gcc cat cta cg\ 99 bp agg atc ttc atg agg tag tca gtc ag ------------------------------------------------------------------ ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Fold change of genes in PC3 cells exposed to combination treatment or mono-treatment with Taxotere or Furtulon ::: **Gene** **Taxotere** **Furtulon** **Taxotere+Furtulon** ----------------------------------------------------------------------- -------------- -------------- ----------------------- ------ ------ ------ ------ ------ ------- ***Cell cycle and apoptosis*** NM\_001237.1 cyclin A2 (CCNA2) NC -1.1 -2.0 -1.1 -1.7 -3.0 -1.1 -1.4 -2.5 BE407516 cyclin B1 -1.1 -1.1 -2.3 NC -3.0 -6.1 -1.1 -3.0 -5.3 NM\_001761.1 cyclin F (CCNF) -1.3 -1.3 -2.5 -1.5 -2.5 -2.6 -1.6 -1.6 -2.5 NM\_014303.1 pescadillo homolog 1 NC -1.5 -2.3 1.3 -1.9 -2.6 -1.1 -2.1 -3.5 NM\_003132.1 spermidine synthase (SRM) NC -1.5 -2.5 -1.1 -1.3 -2.5 -1.1 -1.1 -2.3 NM\_000389.1 cyclin-dependent kinase inhibitor 1A (p21, Cip1) 1.7 1.0 2.6 1.9 2.6 3.5 NC 1.7 3.5 NM\_003914.1 cyclin A1 (CCNA1) 1.3 2.3 2.8 NC 1.5 2.5 NC NC 2.0 L49506.1 cyclin G2 -2.1 2.0 2.6 -2.8 2.3 2.3 NC 3.7 2.5 NM\_004354.1 cyclin G2 (CCNG2) -1.9 3.0 3.2 NC 2.5 2.3 1.0 4.6 3.5 AL535380 B-cell translocation gene 1, anti-proliferative 1.1 1.7 3.5 NC 1.4 2.8 -1.5 1.6 2.6 NM\_006472.1 upregulated by 1,25-dihydroxyvitamin D-3 (VDUP1) 1.3 2.1 -2 1.0 2.1 2.3 1.0 2.3 NC NM\_015675.1 growth arrest and DNA-damage-inducible, beta (GADD45B) 1.2 2.5 6.5 1.6 1.5 2.6 1.0 NC 3.5 AF087853.1 growth arrest and DNA damage inducible protein beta NC 2.0 4.6 1.3 1.2 2.3 NC NC 3.2 AF078077.1 growth arrest and DNA-damage-inducible protein GADD45beta 1.5 2.0 5.3 1.1 1.2 2.1 NC 1.4 2.8 ***Transcription and translation*** NM\_007111.1 transcription factor Dp-1 (TFDP1) NC -1.6 -3.2 -1.3 -1.1 -2.0 NC -1.1 -2.0 NM\_012068.2 activating transcription factor 5 (ATF5) NC -2.1 -3.5 NC -1.5 -2.1 -1.3 -1.1 -2.6 NM\_012251.1 transcription factor A, mitochondrial (TFAM) 1.3 -1.5 -2.5 NC -1.6 -2.0 -1.1 -2.0 -3.7 AF220509.1 transcription associated factor TAFII31L NC -2.3 -3.2 -1.3 -1.3 -2.3 NC -1.2 -5.7 AA393940 eukaryotic translation initiation factor 5A -1.3 -1.5 -3.0 -1.6 -2.0 -3.5 -1.1 -1.1 -2.0 NM\_001674.1 activating transcription factor 3 (ATF3) 1.4 1.9 14.9 1.5 2.1 3.5 1.1 2.6 4.9 ***Oncogenesis and other*** NM\_001511.1 GRO1 oncogene (GRO1) 2 -1.5 -3.0 1.7 1.2 -2.0 NC NC -4.0 NM\_002090.1 GRO3 oncogene (GRO3) 2.8 -3.0 -4.0 2.5 -1.4 -6.5 NC -2.8 -13.9 NM\_005754.1 Ras-GTPase-activating protein SH3-domain-binding protein NC -1.7 -2.8 NC -2.0 -2.5 NC -2.6 -4.6 NM\_000026.1 adenylosuccinate lyase (ADSL) NC -1.3 -2.5 NC -1.4 -2.6 NC -1.6 -3.0 M80261.1 apurinic endonuclease (APE) -1.1 -1.4 -3.0 -1.1 -1.9 -2.6 NC -2.1 -3.7 D13413.1 tumor-associated 120 kDa nuclear protein p120 -1.1 -2.0 -3.2 -1.7 -1.7 -2.5 -2.3 -1.1 -3.0 AI743685 methionine aminopeptidase; eIF-2-associated p67 NC -2.6 -2.0 1.5 -1.5 -3.2 NC -2.3 -2.8 NM\_002634.2 prohibitin (PHB) NC -1.4 -3.0 NC -1.9 -2.1 NC -1.5 -2.6 NM\_002546.1 osteoprotegerin 1.5 -1.3 -3.0 1.2 -2.1 -3.0 NC -2.8 -5.3 AF003934.1 prostate differentiation factor mRNA -1.5 4.3 26.0 -1.7 4.9 16.0 NC 4.3 12.1 NM\_000177.1 gelsolin (amyloidosis, Finnish type) (GSN) NC 1.5 2.8 NC 2.0 3.5 NC 1.9 2.3 ***Invasion and metastasis*** NM\_003254.1 tissue inhibitor of metalloproteinase 1 (TIMP1) NC 2.0 2.5 NC 2.6 3.7 NC 2.6 3.7 NM\_003255.2 tissue inhibitor of metalloproteina(TIMP2) NC 1.6 2.5 NC 2.3 3.5 NC 1.6 3.5 NM\_002638.1 protease inhibitor 3 (PI3) 1.2 2.0 2.0 1.1 3.7 5.7 1.0 3.0 4.6 NM\_005562.1 laminin, gamma 2 (nicein (100 kD) 1.3 1.5 2.0 1.1 2.5 3.7 NC 2.3 3.7 NM\_001908.1 cathepsin B (CTSB) NC 1.7 3.0 NC 1.7 2.8 1.1 1.3 2.5 NM\_002658.1 plasminogen activator, urokinase (PLAU) NC 2.3 3.5 NC 2.8 2.3 1.0 2.1 1.9 NM\_000930.1 plasminogen activator, tissue (PLAT) 1.1 2.5 4.6 NC 5.7 9.8 NC 3.2 4.9 NM\_002421.2 matrix metalloproteinase 1 (MMP1) NC 1.4 4.3 NC 4.9 18.4 NC 5.7 13.0 NM\_004994.1 matrix metalloproteinase 9 (MMP9) 1.1 1.7 2.0 1.0 1.9 2.0 NC 2.5 2.1 NM\_000435.1 Notch homolog 3 (NOTCH3) NC 2.0 2.6 -2 2.3 3.2 NC 3.5 3.5 ***Resistance to chemotherapeutic agents*** NM\_000499.2 cytochrome P450, subfamily I (CYP1A1) 1.4 3.0 4.0 NC 3.5 5.3 1.2 9.2 10.6 NM\_006697.1 cisplatin resistance associated (CRA) 1.0 1.7 2.0 -1.3 2.0 2.6 NC NC 2.6 NM\_005980.1 S100 calcium-binding protein P (S100P) -1.2 4.9 24.3 1.1 10.6 27.9 NC 6.5 19.7 NM\_002961.2 S100 calcium-binding protein A4 (S100A4) 3.0 3.7 5.3 1.0 5.7 11.3 2.8 6.5 10.6 NM\_020672.1 S100-type calcium binding protein A14 (LOC57402) NC 1.5 2.0 NC 2.1 2.6 1.1 1.6 2.1 NM\_001894.1 casein kinase 1, epsilon (CSNK1E) NC 1.7 3.2 NC 2.0 2.6 1.1 1.7 3.2 NM\_000700.1 annexin A1 (ANXA1) NC 1.9 2.3 NC 2.0 2.3 NC 1.6 2.1 The genes in this list showed a \>2 fold change in expression in at least one time point in both mono and combination treatment. NC: No change; Negative value: Decrease; Positive value: Increase. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Fold change of genes in LNCaP cells exposed to combination treatment or mono-treatment with Taxotere or Furtulon ::: **Gene** **Taxotere** **Furtulon** **Taxotere+Furtulon** -------------------------------------------------------------------- -------------- -------------- ----------------------- ------ ------ ------ ------ ------ ------- ***Cell cycle and apoptosis*** NM\_001786.1 cell division cycle 2 (CDC2) -1.2 -7.5 -12.1 -1.1 -2.8 -1.6 NC -4.0 -14.9 D88357.1 mRNA for CDC2 delta T -1.1 -6.1 -14.9 NC -2.8 -1.6 -1.1 -4.3 -13.0 NM\_001237.1 cyclin A2 (CCNA2) -1.2 -5.7 -12.1 NC NC -2.1 -1.1 -8.6 -13.9 NM\_004702.1 cyclin E2 (CCNE2) 1.6 -5.3 -3.2 NC -2.3 -1.7 -1.7 -4.9 -8.0 AF112857.1 cyclin E2 splice variant 1 mRNA 1.2 -3.5 -3.2 NC -2.0 -1.6 -1.4 -7.0 -8.6 NM\_001761.1 cyclin F (CCNF) -1.5 -2.5 -3.7 -1.1 -1.1 -2.0 -1.4 -2.3 -2.8 AB012305.1 cyclin-dependent kinase 2 -1.7 -3.5 -5.3 -2.0 -2.1 -2.1 NC -2.8 -3.5 U30872.1 mitosin mRNA -1.2 -4.6 -6.1 NC -2.1 -1.3 NC -3.2 -7.0 NM\_000389.1 cyclin-dependent kinase inhibitor 1A (p21, Cip1) 1.3 9.8 8.6 1.0 2.0 2.0 1.0 8.0 7.5 NM\_006763.1 BTG family, member 2 (BTG2) 1.5 6.1 6.5 1.1 1.7 2.1 1.4 5.3 3.7 NM\_006472.1 upregulated by 1,25-dihydroxyvitamin D-3 (VDUP1) 1.0 3.5 3.5 1.0 1.5 2.6 1.0 2.3 3.0 NM\_001924.2 growth arrest and DNA-damage-inducible, alpha 1.4 7.5 7.5 NC 1.9 2.6 1.1 7.0 7.5 BC003637.1 Similar to DNA-damage-inducible transcript 3 1.0 2.6 2.6 1.1 2.1 2.5 NC 2.6 2.8 NM\_014454.1 p53 regulated PA26 nuclear protein (PA26) 1.1 4.0 3.0 1.1 1.4 2.6 1.1 3.7 3.7 NM\_004701.2 cyclin B2 (CCNB2) -1.1 -8 -13.9 NC 1.3 -1.3 -1 -4.9 -19.7 NM\_001255.1 CDC20 (cell division cycle 20) -1.9 -90.5 -147 NC 1.6 -1.9 -1.6 -13 -157 NM\_021873.1 cell division cycle 25B (CDC25B) -1.1 -1.6 -2.5 NC NC -1.4 -1 -2.3 -3.25 NM\_001827.1 CDC28 protein kinase 2 (CKS2) -1.1 -4.6 -6.5 NC 1.6 -1.6 -1.2 -3.5 -5.7 NM\_001168.1 survivin (BIRC5) -1.1 -27.9 -294 NC NC -1.6 NC -6.9 -181 ***Transcription, translation, oncogenesis, angiogenesis, other*** NM\_021953.1 forkhead box M1 (FOXM1) -1.2 -13.9 -42.2 -1.1 -1.2 -2.0 NC -2.8 -45.3 NM\_000465.1 BRCA1 associated RING domain 1 (BARD1) -1.3 -1.9 -2.0 NC -1.1 -2.1 -1.2 -2.6 -4.9 NM\_003368.1 ubiquitin specific protease 1 (USP1) NC -2.6 -1.6 NC -2.0 -1.4 NC -2.1 -1.9 NM\_006716.1 activator of S phase kinase (ASK) -1.2 -6.5 -5.3 -1.2 -1.3 -2.0 -1.1 -4.6 -11.3 NM\_003246.1 thrombospondin 1 (THBS1) NC -2.0 -3.5 -1.9 -1.4 -2.6 -1.1 -1.6 -4.6 NM\_001147.1 angiopoietin 2 (ANGPT2) 3.5 3.5 8.0 1.7 5.3 8.0 -0.5 4.9 2.8 AF187858.1 angiopoietin-2 isoform-1 NC 2.6 4.3 NC 1.5 3.5 NC 2.0 2.0 NM\_000435.1 Notch (Drosophila) homolog 3 (NOTCH3) 1.3 3.0 2.8 1.0 2.3 2.1 NC 2.1 3.0 NM\_001674.1 activating transcription factor 3 (ATF3) NC 1.5 2.5 NC 1.7 2.5 NC 2.6 4.3 NM\_003158.1 serinethreonine kinase 6 (STK6) -1.7 -11.3 -9.8 1.1 1.2 -1.7 -1.3 -8.6 -26 NM\_003600.1 serinethreonine kinase 15 (STK15) -1.2 -3.7 -8.6 NC NC -1.6 -1.3 -4 -6.1 AF162704.1 androgen receptor mRNA NC 1.3 -1.6 1.2 1.3 -2.1 -1.1 -1.6 -2 ***Resistance to chemotherapeutic agents*** NM\_000693.1 aldehyde dehydrogenase 1A3 (ALDH1A3) NC 4.0 3.2 1.0 1.1 2.1 1.2 4.9 4.3 NM\_005980.1 S100 calcium-binding protein P (S100P) 1.0 5.7 4.0 1.5 2.6 3.0 NC 2.6 2.6 The genes in this list showed a \>2 fold change in expression in at least one time point in both mono and combination treatment. NC: No change; Negative value: Decrease; Positive value: Increase. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Comparison of difference in gene expression between combination treatment and mono-treatment in PC3 cells ::: **Gene** **Taxotere+Furtulon** ----------------------------------------------------------------------------------- ----------------------- ------ ------ ***Decrease in combination treatment, No change or increase in mono-treatment.*** NM\_001261.1 cyclin-dependent kinase 9 (CDC2-related kinase) (CDK9) -1.3 -1.2 -2.3 NM\_016507.1 CDC2-related protein kinase 7 (CrkRS) NC -1.6 -2.3 AF080157.1 IkB kinase-a (IKK-alpha) NC -2.0 -1.6 U62296.1 transcription factor NF-YC -1.1 -1.7 -2.3 BC005003.1 nuclear transcription factor Y, gamma -1.1 -2.0 -2.1 BC001771.1 transcription factor IIF NC -1.6 -2.3 AI434345 activating transcription factor 1 NC -2.5 -2.6 BC001173.1 eukaryotic translation initiation factor 3 NC -1.3 -2.0 U78525.1 eukaryotic translation initiation factor (eIF3) NC -1.4 -2.0 NM\_001814.1 cathepsin C (CTSC) NC -1.3 -2.0 NM\_003377.1 vascular endothelial growth factor B (VEGFB) -2.1 NC NC AF035620.1 BRCA1-associated protein 2 (BRAP2) -2.3 NC NC AF035620.1 BRCA1-associated protein 2 (BRAP2) -2.3 -2.0 -1.4 NM\_005346.2 heat shock 70 kD protein 1B (HSPA1B) -1.1 -2.1 -1.1 BC000478.1 heat shock 70 kD protein 9B NC -1.7 -2.3 NM\_014278.1 heat shock protein (hsp110 family) (APG-1) -1.2 -1.3 -2.0 BC002526.1 Similar to heat shock protein, 110 kDa -1.1 -1.4 -2.1 ***Increase in combination treatment, No change or decrease in mono-treatment.*** NM\_000077.1 cyclin-dependent kinase inhibitor 2A (p16, inhibits CDK4) NC 1.7 2.1 NM\_001262.1 cyclin-dependent kinase inhibitor 2C (p18, inhibits CDK4) NC 2.1 2.0 J03202.1 laminin B2 chain mRNA NC 1.3 2.0 BG164365 microtubule-associated protein 1B NC 2.3 2.0 NM\_000594.1 tumor necrosis factor, member 2 NC 2.5 1.6 NM\_001065.1 tumor necrosis factor, member 1A NC 2.0 1.9 BC000125.1 Similar to transforming growth factor, beta 1 -1.2 1.9 2.1 NM\_005649.1 transcription factor 17 (TCF17) NC 1.1 2.3 NM\_005923.2 mitogen-activated protein kinase kinase kinase 5 (MAP3K5) NC 1.6 2.1 NM\_005204.1 mitogen-activated protein kinase kinase kinase 8 (MAP3K8) NC 2.3 4.3 NM\_000785.1 cytochrome P450, subfamily XXVIIB NC 2.1 1.5 NM\_002960.1 S100 calcium-binding protein A3 (S100A3) NC 2.5 2.5 NM\_002962.1 S100 calcium-binding protein A5 NC 8.6 10.6 NC: No change; Negative value: Decrease; Positive value: Increase. The genes in this list showed a \>2 fold change in expression in at least one time point in combination treatment. ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Comparison of difference in gene expression between combination treatment and mono-treatment in LNCaP cells ::: **Gene** **Taxotere+Furtulon** ----------------------------------------------------------------------------------- ----------------------- ------ ------- ***Decrease in combination treatment, No change or increase in mono-treatment.*** NM\_001256.1 cell division cycle 27 (CDC27) NC -1.6 -2.0 NM\_001963.2 epidermal growth factor (beta-urogastrone) (EGF) NC -3.5 -1.9 NM\_004113.2 fibroblast growth factor 12B (FGF12B) NC -4.3 -1.2 U13022 negative regulator of programmed cell death ICH-1S (Ich-1) NC -2.3 -2.0 AF249273.1 Bcl-2-associated transcription factor short form mRNA NC -1.7 -2.3 NM\_001071.1 thymidylate synthetase (TYMS) NC -1.4 -2.6 AF005068.1 breast and ovarian cancer susceptibility protein (BRCA1) NC -4.6 -17.1 NM\_012068.2 activating transcription factor 5 (ATF5) NC -2.0 -2.8 NM\_021809.1 TGF(beta)-induced transcription factor 2 (TGIF2) NC -1.6 -2.1 NM\_001412.1 eukaryotic translation initiation factor 1A (EIF1A) NC -1.2 -2.3 NM\_002758.1 mitogen-activated protein kinase kinase 6 (MAP2K6) NC -1.7 -2.1 ***Increase in combination treatment, No change or decrease in mono-treatment.*** NM\_006034.1 p53-induced protein (PIG11) NC 6.1 7.5 NM\_000227.1 laminin, alpha 3 NC 1.7 2.0 NM\_000094.1 collagen, type VII, alpha 1 (COL7A1) NC 1.4 6.5 NM\_016437.1 tubulin, gamma 2 (TUBG2) NC 1.4 2.1 NM\_000853.1 glutathione S-transferase theta 1 (GSTT1) NC 2.0 2.1 NC: No change; Negative value: Decrease; Positive value: Increase. The genes in this list showed a \>2 fold change in expression in at least one time point in combination treatment. :::

PubMed Central

2024-06-05T03:55:52.673268

2005-1-18

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548501/", "journal": "BMC Cancer. 2005 Jan 18; 5:7", "authors": [ { "first": "Yiwei", "last": "Li" }, { "first": "Maha", "last": "Hussain" }, { "first": "Sarah H", "last": "Sarkar" }, { "first": "James", "last": "Eliason" }, { "first": "Ran", "last": "Li" }, { "first": "Fazlul H", "last": "Sarkar" } ] }

PMC548502

Background ========== Many chromosomal regions have been shown to be linked or associated to asthma and asthma-associated traits in humans \[[@B1]\]. More recently, asthma genes have been identified on chromosomes 2 \[[@B2]\], 13 \[[@B3]\], 14 \[[@B4]\], and 20 \[[@B5]\]. The investigation of the genetic aetiology aims at the improvement of preventive strategies, diagnostic tools and therapeutic alternatives \[[@B6]\]. These final steps have not yet been reached or are even in sight, while the reasons for this delay are unclear. The mainstay of all genetic studies has been genome-wide linkage scans in families with at least two asthma-affected siblings. Based on a previous analysis of a genome-wide scan of asthma \[[@B7],[@B8]\] with inconsistent chromosomal findings to earlier studies, we decided to expand the initial sample with additional families by the same core protocol for clinical examination and using the same set of microsatellite markers \[[@B7],[@B8]\]. The increased the number of identically pheno- and genotyped families could be used to define sub-phenotypes, which may be a promising strategy to explain the aetiological heterogeneity observed so far. Relevant clinical subsets may be defined by different age of onset, different disease course by degree of severity, extrinsic (allergic sensitization detectable) and intrinsic (no allergic sensitization detectable, symptoms often during infections of the upper respiratory tract) asthma type, and house dust mite allergy (HDM), as well as genetic background as judged by geographical origin of parents (table [1](#T1){ref-type="table"}). We hypothesized that the restriction to a smaller well-defined sample would reduce heterogeneity and improve the power of detecting linkage. Linkage regions should show higher lod scores than in the total sample and lead from phenotype subgroups to a genotypic dissection. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Phenotypes of the 201 families included. ::: **Phenotype** **Definition** **No. of families** ---------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------- --------------------- **Early onset asthma** occurrence of asthmatic symptoms for one child before the age of 2 years and for his or her sib pair at least before the age of 4 years 67 **Extrinsic asthma** all asthmatic children are positive for at least one SPT or specific IgE 134 **HDM SPT positive** at least 3 family members with a positive skin prick test (SPT) for HDM 33 **HDM RAST positive** at least 3 family members with a positive result of serum specific IgE for HDM 42 **Severe asthma** asthma severity index (see table 3) for one asthmatic child is 4 or 5 and for his or her sib pair 3, 4 or 5 56 **Seasonal variation:**\"Winter-Type\" one affected child suffers from asthma attacks in the winter half-year and his or her sib pair at least not solely in the summer half-year 39 **Seasonal variation:**\"Summer-Type\" one affected child suffers from asthma attacks in the winter half-year and his or her sib pair at least not solely in the summer half-year 35 **German nationality** both parents are of German descent 170 ::: Methods ======= Clinical evaluation ------------------- 97 families consisting of at least two children with confirmed clinical asthma were collected during the first stage of the German genome scan \[[@B7]\]. We now recruited another set of families during a period of 18 months. Trained staff from 3 university hospitals as well as 6 pediatric pulmonary practices carried out an identical phenotyping procedure as described previously \[[@B7]\]. This procedure contained detailed interviews of every family member, skin prick tests (SPT) of frequent allergens, blood samples (for IgE and allergen-specific IgE (RAST) measurements, eosinophil count), peak flow tests for a period of 10 days and dust collection at patients\' homes. SPT and RAST assays included several pollens, animal furs, mould, and house dust mite allergens (ALK-SCHERAX, Hamburg, Germany). The ethics commission of \"Nordrhein-Westfalen\" approved all study methods and informed consent was obtained from all parents and children. Children with premature birth, low birth weight and any severe pulmonary disease other than asthma were excluded as prematurity (and or associated low birth weight) may be a major non-genetic risk factor for the development of pulmonary symptoms. Another 4 families were excluded after genotyping because of Mendelian errors. The additional 104 families comprised 452 individuals (table [2](#T2){ref-type="table"}). Their 244 children had a mean age of 10.8 years. 75 families contributed 2 children, 22 families 3 children and 7 families 4 children). In total, the 201 families had 465 children; 255 were male, 210 female, and 413 had physician-diagnosed asthma. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Clinical characteristics of the 201 families (867 participants) that also include non-affected siblings of the core families. Descriptors of categorical variables include n/total sample in the first row and percent in the second row. Descriptors of continuous variables include mean values in the first row and standard deviation in the second row. ::: -------------------------------------------------------------------------------------------- **Parents** **Children** ---------------------------------- -------------- -------------- ------------- ------------- **Stage 1** **Stage 2** **Stage 1** **Stage 2** **sex females** 97/194\ 104/208\ 96/221\ 114/244\ 50.0% 50.0% 43.4% 46.7% **Asthma diagnosis** 33/194\ 60/208\ 200/221\ 213/244\ 17.0% 28.8% 90.5% 87.3% **D.pter skin \> = 3 mm** 68/193\ 58/206\ 111/217\ 107/242\ 35.2% 28.2% 51.2% 44.2% **D.far skin \> = 3 mm** 57/193\ 48/207\ 101/217\ 88/242\ 29.5% 23.2% 46.5% 36.4% **D.pter. CAP class \>1** 51/183\ 43/147\ 124/217\ 102/198\ 27.9% 29.3% 57.1% 51.5% **D.far. CAP class \>1** 46/183\ 39/147\ 122/217\ 100/198\ 25.1% 26.5% 56.2% 50.5% **age (years)** 39.8\ 40.9\ 11.0\ 10.8\ +/-5.7 +/-5.5 +/-4.2 +/-3.4 **onset asthma (years)** not reliable not reliable 3.8\ 4.3\ +/-2.8 +/-3.1 **height (cm)** 172.3\ 172.3\ 146.9\ 156.3\ +/-9.0 +/- 9.9 +/-19.2 +/-18.1 **weight (kg)** 76.0\ 78.0\ 41.5\ 47.4\ +/-14.9 +/-16.6 +/-17.0 +/-16.6 **ln(Ige) (kU/L)** 4.1\ 4.1\ 5.0\ 5.0\ +/-1.7 +/-1.6 +/-2.0 +/-1.8 **ln(eosinophil) (count / mm3)** 0.3\ 0.7\ 1.1\ 1.1\ +/-1.7 +/-1.5 +/-1.6 +/-1.6 **FEV1 (ml)** 3.594\ 3.464\ 2.312\ 2.861\ +/-804 +/- 957 +/-883 +/-1.061 -------------------------------------------------------------------------------------------- ::: The trait \"asthma\" was based on the clinical diagnosis as described earlier \[[@B7]\]. \"lnIgE\" was used for analysis as a quantitative variable and categorized by cut-off point of 100 kU/l. Furthermore, the following phenotype subsets (see also table [1](#T1){ref-type="table"}) were selected from the total study sample: ### Early onset asthma We created a subset of 67 families with one child having asthma symptoms before the age of 2 years and another child having asthma symptoms before the age of 4 years. In about half of the families within this subtype, both affected children had asthmatic symptoms before the age of 2 years. We were of course dependent on the recollection of the parents -- but keeping in mind that the state of health of the children is of high priority in those families who joined our study and the period of time is quite clear, the data seemed valuable to us. ### Extrinsic/intrinsic asthma sample The group of families with atopic (\"extrinsic\") asthma was large, consisting of 134 families all with at least one positive skin prick test reaction or one increased specific IgE. ### Positive Dermatophagoides farinae skin prick test and increased specific IgE in serum Almost all family members participated in a skin prick test (SPT). We selected an \"HDM SPT positive\" group of 33 families with at least 3 family members showing a skin prick test reaction ≥ 3 mm. House dust mite allergy is quite common in asthmatic patients, and a known trigger factor for asthma attacks. Some studies have successfully used the combination with asthma as lead trait \[[@B9],[@B10]\]. As the functional relevance might be slightly different in study participants with serum antibodies, another subgroup was based on the measurement of HDM-positive specific IgE at concentrations ≥ 0.35 kU/l, which resulted in a group of 42 families. ### Asthma severity Several interview questions related to asthma symptoms, diagnostic findings and therapy. We used information about asthma attack frequency, actual medication and emergency hospital visits for a severity index with 5 levels. This index is based on subjective patient information, current/previous therapy and influence on quality of life (table [3](#T3){ref-type="table"}). \"Severe Asthma\" was seen in 56 families where one sib had at least grade 4 and another grade 3, 4 or 5. \"Moderate Asthma\" with one sib of maximum grade 2 and another with maximum grade 3 was defined in the same way. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Asthma severity grade definition. ::: **Severity grade** **Frequency and percentage in asthmatic children** **Clinical criteria** -------------------- ---------------------------------------------------- ----------------------- ------ ------ **1** 29 (7.2%) none none none **2** 118 (29.3%) none none yes none yes none **3** 164 (40.8%) none yes yes once/month yes none **4** 72 (17.9%) once/month yes yes at least once/month yes none **5** 19 (4.7%) at least once/month yes yes ::: ### Seasonal variation of asthma attacks All participants were asked to specify the months when asthma attacks occurred. 39 \"winter-type\" families had one affected child with symptoms only in the cold months and his or her sibs not a fully \"summer-type\". In contrast 35 \"summer-type\" families were found with one affected child with summer attacks and his or her sibs not purely \"winter-type\". Attacks year-round usually were seen with severe disease only. Sporadic attacks usually did not have any seasonal preferences while both the intrinsic, usually infection-related \"winter-type\", as well as the extrinsic, hay fever related \"summer-type\", appeared to be concordant within families. This observation may already support the hypothesis of different genes in different subtypes. ### German sample In 170 of 201 families, both parents were of German descent (\"German\"). In this subgroup, we tried to reduce the genetic heterogeneity by leaving out the remaining 31 families, of which 5 were from Sweden and 5 from Turkey. DNA analysis ------------ DNA was isolated from peripheral white blood cells using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) according to the manufacturer\'s recommendation. For genotyping we used almost the same microsatellite marker as in the first scan \[[@B7],[@B8]\]. The final analysis included 408 markers of which 364 were autosomal and 7 were based on the X-chromosome, all included in the previous scan as well as a set of 37 previous fine mapping markers. For the baseline marker set, the mean distance was 10 cM, with an average marker-information content of 0.87 and a mean heterozygosity of 0.79. Marker amplification was performed in microtiter plates, either in 96- (Peltier Thermal Cycler PT-225, MJ Research, Waltham, MA) or in 384-well format (GeneAmp PCR System 9700, Applied Biosystems, Foster City, CA). Fragment analysis of PCR pools was conducted on an ABI 3700 DNA sequencer and genotypes were scored using GENESCAN and GENOTYPER (ABI) software. In the second scan, 98% of all possible genotypes could be unequivocally determined. Statistical analysis -------------------- Genotyping data were transferred to a SQL 2000 database, pre-checked with previously developed routines and exported in ASCII format for multipoint linkage analysis with MERLIN 0.9.3 \[[@B11]\]. Allele frequencies were estimated from the (unrelated) parental alleles. As different primers were used in some assays between the first scan in 1997 and the second scan in 2002, we adjusted the original allele size before replacing the allele size with its respective order. This procedure led to comparable allele frequency distributions in both scans. For ordering of all markers we used the Marshfield comprehensive human genetic linkage map that was slightly modified according to the marker order given by Golden Path 13 and expressed intermarker distances to Haldane cM. Error detection as implemented in MERLIN was then applied to discard unlikely genotypes from the analysis. Finally, P-values for qualitative traits were derived from the Kong and Cox method based on the score statistic S\_all \[[@B12]\]. For the quantitative trait ln(IgE) a variance component was applied including only age and sex as covariates. Results ======= Figure [1](#F1){ref-type="fig"} shows the linkage results for asthma, total IgE and all other subgroups. Chromosomes are arranged by number from p-ter to q-ter with distance in centimorgans on a linear scale. Table [4](#T4){ref-type="table"} reports all loci with p-values below 0.01 in at least two adjacent markers for one phenotypic subgroup. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Multipoint linkage results for all traits. Chromosomes are arranged with increasing numbers with orientation from p-ter to q-ter. Distance is given in centimorgans (cM) on a linear scale. A = asthma all, B = asthma German families, C = extrinsic asthma, D = HDM RAST positive, E = HDM SPT positive, F = severe asthma, G = early onset asthma, H = winter-type, I = summer-type, J = LnIgE continuous, K = LnLgE categorical. ::: ![](1471-2466-5-1-1) ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Linkage results in 201 core families with at least 2 children having asthma. Weak linkage (p \< 0.01) and suggestive linkage (p = 0.0007) is indicated in bold. ::: GDB PIC Chr. cM (Marshfield) Asthma All Asthma German Extrinsic Asthma HDM RAST +ve HDM SPT +ve Severe Asthma Early Onset Winter-Type Summer-Type Ln(IgE) continuous Ln(IgE) categorical ---------- -------- ------ ----------------- ------------ --------------- ------------------ -------------- ------------- --------------- ------------- ------------- ------------- -------------------- --------------------- D1S478 0.8404 1 48.53 0.03 0.03 0.04 0.5 0.9 0.12 0.3 0.13 0.8 **0.002** **0.0011** D1S234 0.8559 1 55.1 0.07 0.08 0.04 0.3 0.9 0.12 0.2 0.2 0.8 **0.0002** **0.00009** D1S255 0.7710 1 65.47 **0.005** 0.04 **0.0015** 0.04 0.4 0.03 0.15 0.7 0.2 **0.002** **0.005** D1S197 0.8285 1 76.27 **0.007** 0.06 **0.002** 0.12 0.4 0.09 0.07 0.5 0.11 0.08 0.2 D1S484 0.8050 1 169.68 0.08 0.12 0.012 **0.004** **0.003** 0.3 0.3 0.08 0.3 **0.007** 0.013 D1S431 0.9008 1 182.35 0.12 0.13 0.02 0.03 0.04 0.5 0.3 0.3 0.2 **0.005** **0.005** D1S2815 0.9201 1 188.85 0.06 0.09 0.012 0.06 0.07 0.3 0.3 0.2 0.03 **0.0008** **0.0007** D1S238 0.8722 1 202.73 0.4 0.7 0.4 0.2 0.1 0.3 0.8 0.2 0.6 **0.004** **0.003** D1S2655 0.8976 1 216.82 0.7 0.9 0.6 0.2 0.2 0.2 0.9 0.7 0.7 0.05 **0.007** D2S2374 0.9039 2 54.96 0.03 0.07 0.04 0.011 **0.008** 0.04 0.02 0.8 0.4 0.3 0.5 D2S2328 0.9545 2 61 0.02 0.04 0.014 **0.007** **0.007** **0.008** 0.04 0.8 0.06 0.14 0.4 D2S2294 0.9617 2 64.84 0.02 0.03 0.02 **0.003** 0.02 **0.003** 0.5 0.8 0.02 0.13 0.4 D2S2298 0.9585 2 65.94 0.02 0.02 0.02 **0.003** 0.02 **0.002** 0.06 0.8 0.014 0.11 0.3 D2S2113 0.9331 2 88.15 0.3 0.5 0.3 0.2 0.4 **0.008** 0.13 0.7 0.3 0.5 0.5 D3S1597 0.8199 3 29.92 0.7 0.7 0.4 0.6 0.9 0.4 0.4 0.6 0.8 **0.009** **0.006** D3S1286 0.9021 3 41.56 0.4 0.3 0.3 0.2 0.3 0.13 0.2 1 0.4 0.02 **0.007** D3S1300 0.8748 3 80.32 0.12 0.3 0.2 0.09 **0.0014** 0.3 0.5 0.3 0.8 0.4 0.09 D3S1285 0.7673 3 91.18 0.07 0.08 0.2 0.07 **0.0008** 0.2 0.2 0.13 0.6 0.5 0.5 D4S1607 0.8843 4 183.63 0.3 0.3 0.3 0.07 0.05 0.2 **0.005** 0.07 0.6 0.4 0.3 D4S1535 0.8938 4 195.06 0.05 0.07 0.013 **0.009** 0.03 0.03 **0.006** **0.007** 0.11 0.07 0.09 D4S2924 0.8934 4 199.93 0.06 0.09 **0.007** **0.002** 0.03 0.02 **0.009** 0.02 0.2 0.08 0.15 D5S426 0.8990 5 51.99 **0.006** **0.006** 0.03 **0.008** 0.14 0.04 **0.002** **0.007** 0.2 0.09 0.4 D5S418 0.9101 5 58.55 0.015 0.03 0.06 **0.004** 0.2 0.012 **0.007** 0.012 0.4 0.02 0.07 D7S484 0.9204 7 53.5 0.5 0.7 0.2 0.4 0.5 0.5 0.4 0.5 0.9 0.02 **0.009** D7S528 0.9481 7 57.79 0.2 0.4 0.014 0.14 0.3 0.3 0.5 0.5 0.6 **0.0012** **0.0007** D7S510 0.9783 7 59.93 0.14 0.4 0.02 0.3 0.3 0.2 0.6 0.6 0.4 **0.0009** **0.0013** D7S485 0.9688 7 60.68 0.1 0.3 0.01 0.3 0.3 0.13 0.6 0.7 0.4 **0.0007** **0.0004** D7S2548 0.9596 7 62.28 0.2 0.4 0.05 0.4 0.5 0.12 0.7 0.7 0.3 **0.003** **0.002** D7S691 0.9403 7 63.67 0.08 0.2 0.02 0.4 0.5 0.08 0.6 0.6 0.3 **0.003** **0.007** D7S2506 0.9313 7 69.56 0.2 0.4 0.13 0.9 0.8 0.14 0.7 0.7 0.3 **0.007** **0.011** D7S663 0.9049 7 78.65 0.06 0.05 0.08 0.3 0.4 0.2 0.5 0.3 0.14 **0.0007** **0.001** D7S669 0.8351 7 90.42 0.08 0.05 0.04 0.4 0.2 0.13 0.5 0.5 0.02 **0.0014** **0.0012** D9S257 0.9630 9 91.87 0.09 0.06 0.3 0.3 0.4 0.4 0.4 0.4 **0.004** 0.5 0.5 D9S283 0.9681 9 94.85 0.015 0.01 0.1 0.2 0.3 0.2 0.11 0.1 **0.002** 0.2 0.3 D9S1796 0.9745 9 97.53 0.02 0.014 0.07 0.3 0.2 0.05 0.07 0.14 **0.006** 0.07 0.13 D9S1781 0.9565 9 99.4 0.015 **0.007** 0.05 0.14 0.2 0.11 0.03 0.03 **0.003** 0.07 0.15 D9S1851 0.9708 9 103.42 0.02 **0.008** 0.04 0.06 0.2 0.2 0.05 0.02 **0.003** 0.3 0.5 D9S1786 0.9825 9 104.48 0.013 **0.006** 0.03 0.04 0.11 0.13 0.05 0.02 **0.002** 0.4 0.5 D9S176 0.9798 9 105.02 0.02 **0.009** 0.05 0.09 0.1 0.2 0.14 0.03 **0.005** 0.5 0.5 D9S1690 0.9709 9 106.63 0.06 0.04 0.2 0.2 0.14 0.4 0.4 0.13 **0.007** 0.5 0.5 D9S1784 0.9667 9 111.99 0.11 0.04 0.2 0.07 0.2 0.3 0.6 0.4 **0.008** 0.5 0.5 D10S547 0.8313 10 29.15 0.14 0.2 0.4 0.8 0.8 0.6 0.3 0.2 0.9 **0.007** **0.005** D10S191 0.8726 10 37.9 0.4 0.5 0.2 0.3 0.5 0.8 0.5 0.3 0.8 **0.008** 0.012 D11S922 0.9045 11 2.11 0.2 0.4 0.4 0.3 0.02 0.13 0.3 0.9 0.06 **0.0005** **0.0006** D11S902 0.8133 11 21.47 0.3 0.3 0.5 0.9 0.3 0.3 0.5 0.6 0.5 **0.006** **0.003** D11S935 0.7997 11 45.94 0.5 0.4 0.4 0.4 0.4 0.4 0.6 0.9 0.05 **0.003** 0.04 D11S968 0.8094 11 147.77 **0.003** **0.007** 0.05 0.11 0.3 0.04 0.12 0.6 0.2 **0.0005** **0.0002** D12S355 0.8343 12 74.58 0.07 0.08 0.06 **0.003** 0.04 0.4 0.2 0.012 0.6 0.3 0.4 D12S1684 0.9339 12 86.4 0.02 0.015 0.05 **0.002** 0.05 0.9 0.15 0.2 0.4 0.08 0.4 D12S1667 0.9727 12 92.89 0.05 0.08 0.2 **0.003** 0.04 0.8 0.15 0.08 0.4 0.03 0.2 D12S81 0.9707 12 94.44 0.08 0.12 0.2 **0.005** 0.02 0.8 0.3 0.09 0.3 0.013 0.2 D12S351 0.9822 12 95.56 0.02 0.013 0.05 **0.002** **0.0003** 0.5 0.3 0.05 0.3 **0.0009** 0.02 D12S95 0.9785 12 96.09 0.012 **0.007** 0.04 **0.002** **0.0002** 0.5 0.2 0.06 0.4 **0.0012** 0.02 D12S327 0.9757 12 97.78 0.02 **0.008** 0.07 **0.008** **0.0009** 0.4 0.05 0.13 0.2 0.04 0.2 D12S1716 0.9590 12 101.45 0.04 **0.005** 0.2 0.02 **0.006** 0.4 0.06 0.2 0.2 0.011 0.06 D12S1706 0.9696 12 104.12 0.12 0.015 0.3 0.07 0.06 0.6 0.3 0.4 0.2 **0.007** 0.2 ::: On [chromosome 1]{.underline}, two linkage areas for total IgE could be found at about 55 cM (p = 0.00009) and at 188 cM (p = 0.0007), which reached the threshold for suggestive linkage (p = 0.0007) \[[@B13]\]. On [chromosome 2]{.underline} at about 64 cM (p = 0.002) evidence was found for a locus for severe asthma and for house dust mite sensitive asthma. Early onset, together with HDM, is represented both on [chromosome 4]{.underline} at about 195 cM (p = 0.002) and on [chromosome 5]{.underline} at about 55 cM (p = 0.002). Three loci for total IgE regulation were found on [chromosome 7]{.underline} (65 cM, p = 0.0004) and chromosome 11 (2 cM, p = 0.0005; 147 cM, p = 0.0002), all with suggestive linkage according Kruglyak-Lander. On [chromosome 9]{.underline} at about 104 cM (p = 0.002) there is evidence for a locus linked to allergic rhinitis plus asthma (\"summer-type\"), having an effect mainly in families of German descent. Lastly, a locus showing suggestive linkage could be identified on [chromosome 12]{.underline} (95 cM, p= 0.0002) for house dust mite sensitised asthmatic patients. Discussion ========== Several linkage regions in asthma families could be found in this extended sample, although again no linkage with P \< 0.003 could be found with any marker for asthma. Asthma is apparently of such a heterogeneity that even investigating affected sib pairs from more than 200 families failed to yield significant results. Nevertheless, the quantitative trait IgE in these families, both as a discrete and continuous variable, led to three suggestive linkage findings on chromosomes 1, 7, and 11, which have all been discussed in prior publications (table [5](#T5){ref-type="table"}). ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Comparison of asthma loci ::: Chr. Position This study (2004) Bradley (2002) \[14\] Cookson (2001)\[15\] Daniels (1996) \[16\] Dizier (2000) \[17\] Laitinen (2001) \[18\] Malerba (1999) \[19\] Ober (1998. 1999, 2000) \[20-22\] Xu J (2000, 2001) \[23, 24\] Xu X (2001) \[25\] Yokouchi (2000) \[9\] CSGA (1997) \[26\] --------------- -------------------- ----------------------- ---------------------- ------------------------- ---------------------- ------------------------ ----------------------- ----------------------------------- ------------------------------ -------------------- ----------------------- -------------------- **1**55cM IgE Asthma Asthma, Atopy Asthma Hispanic population **1**188cM IgE AD Slope IgE **2**64cM severe asthma HDM **3**50cM IgE HDM AD Loose Asthma **4**195cM early onset HDM Slope HDM and Asthma **5**55cM early onset HDM Slope BHR Asthma **7**65cM IgE IgE, Slope, Eosinophils SPT Asthma, IgE **9**104cM German summer type Asthma symptoms, Atopy **11**2cM IgE IgE SPT Asthma Hispanic population Asthma **11**147cM IgE IgE **12**95cM HDM Eosino-phils, IgE Asthma Asthma IgE, Asthma HDM and Asthma Asthma ::: Phenotypic dissection --------------------- Moreover, the strategy of phenotypic dissection proved to be quite successful, though the sample size of subsets seems to be very small. The size of the sample in a linkage study is known to be one of the most important factors for a significant finding \[[@B1]\], making the artificial limitation of subsets a double-edged decision. Clearly, there is a remarkable phenotypic heterogeneity of asthmatic diseases but -- as these families represent different genetic influences-limitation of the sample size did not affect statistical properties as significance values were smaller compared to the whole study sample. In particular, the subgroups segregated by high disease severity, sensitisation by HDM, early onset and population origin attained better lower significance levels than the main trait (table [4](#T4){ref-type="table"}). So far, results were not adjusted with the Bonferroni correction for multiple testing as this would have been too conservative for this explorative investigation. Subgroups are often closely related and highly overlapping. Nevertheless, there might be a problem with multiple testing and the criteria for suggestive linkage in a genome scan on a single phenotype \[[@B13]\]. Sample recruitment with restricted phenotypic requirements proved to be successful for some other traits as well. Early onset definitions have been used for cancer studies and metabolic disorders to increase the underlying genetic component \[[@B27],[@B28]\]. Separation in sub-samples of ethnically diverse populations has also been conducted \[[@B26]\], but higher disease activity scores \[[@B29]\] seemed to give an advantage in other fields also. Other Asthma Genome Scans ------------------------- Recently, evidence was shown for asthma genes on chromosomes 2 \[[@B2]\], 13 \[[@B3]\], 14 \[[@B4]\], and 20 \[[@B5]\]. Our study does not support linkage loci in those regions. As the region on chromosome 13 was responsible for only 10% of the observed IgE serum level variability, this small effect may be easily overlooked in another study. A lack of replication does not necessarily mean an error in the primary study. Most likely, genetic heterogeneity is the cause, where the proportion of patients with a particular gene variant is different between studies. A polygenic model of the inheritance mode of asthma with minor effects by single genes could explain the highly diverse association and linkage results thus far. Frequent replication, however, may point toward lead genes. Most regions listed in table [4](#T4){ref-type="table"} have already been described and some of the traits are supported by the phenotypes proposed in our analysis. Table [5](#T5){ref-type="table"} summarizes previously published asthma/atopy loci in approx. 20 cM distance that would be consistent with our findings. Both loci on chromosomes 4 and 5 are supported by the phenotypes of early onset, HDM and \"winter-type\", which might relate to the clinical picture of chronic obstructive bronchitis. Phenotypes described by other studies include lung function variation and HDM, which is supported by our data. The \"atopy\" locus (defined by SPT and RAST) on chromosome 3 confirms linkage studies of atopic dermatitis (AD) \[[@B14]\] and might be the genetic link between both diseases. The locus on chromosome 12 with suggestive linkage for HDM in our study is known from many other reports. Also our first scan showed weak evidence for linkage in this region at D12S351 (p = 0.01) \[[@B7]\], whereas the best result for the HDM subset lies now about 0.5 cM apart at D12S95 with p = 0.0002. These consistent findings are probably based on frequent alleles playing an important role in extrinsic asthma. Linkage results for SPT and RAST for HDM were quite similar, but the HDM SPT positive group -- which is a smaller group-, reached the suggestive linkage level on chromosome 12. SPT, even more than RAST, might represent the individual\'s allergic disposition. On chromosome 2, we found a locus in families with severe asthma that has not yet been described in other studies. A p-value of 0.007 has already been found in our first genome-wide scan \[[@B7]\] for D2S2298 and our main trait asthma. The same marker for severe asthma in both samples improved to a p-value of 0.002. Severe asthma is rather rare, and a disease-aggravating allele on chromosome 2 could be easily overlooked in a heterogeneous asthma sample. Severe asthma may be a lethal disease, while a severe-asthma gene would be of high importance for intensified treatment. The German \"summer-type\" locus on chromosome 9 is also not frequent in other studies. This might be due to the fact that many, but not all, genes are necessary for a common trait. Our first asthma scan has already shown linkage in this region with a p-value of 0.007 for D9S1784 \[[@B7]\], which now improves to p = 0.002 for the \"summer-type\" subset at D9S1786, about 7.5 cM apart. Conclusions =========== We conclude that this phenotypic dissection is a useful tool to detect linkage in a heterogeneous disease like asthma because some of the sub phenotypes reached even better significance values than the main trait. We show that the precision of the phenotype can be more effective than expanding the sample size only. Unfortunately, large sample sizes are needed to assure at least moderate sample size in subsets. Grouping by early onset or disease severity could be applied to almost every complex disease, but for a more specific dissection, clinical expert knowledge is required. A prior analysis of clinical data can help to identify symptom clustering, which -if consistent within families- can reduce genetic heterogeneity. Competing interests =================== The author(s) delcare that they have no competing interests. Authors\' contributions ======================= MW, JA, and CS were involved in the study design, JA organised the sample collection, conducted the genetic analysis, and prepared the manuscript, MW organized funding and supervised the study. YAL and PN organised and helped with genotyping and revised the article critically for important intellectual content. SL, FR and MW carried out the statistical analysis and made substantial contributions to its conception and design, CS, DB, FF, HJ, JK, AK, AS, MS, WW, PW assisted in the recruitment of families and examined patients and therefore made substantial contributions to the acquisition of data, asthma severity grades are part of the thesis of CS. GS was responsible for the IgE analysis. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2466/5/1/prepub> Acknowledgements ================ We thank all participating families for their help as well as the members of the clinical centres for their work. We wish to thank Guido Fischer for data handling, Liane Thaller for secretarial assistance, Margret Bahnweg, Bettina Wunderlich, Regina Pospiech and Inka Szangolies for excellent laboratory work; Michelle Emfinger and Leah Bajc for proof-reading the manuscript. The project was funded by the Deutsche Forschungsgemeinschaft DFG WI621/5-1, DFG FR1526/1, DFG KN378/1-1, GSF FE 73922, GSF FE 73915, BMBF 07ALE087 and the National Genome Network 01GS0122.

PubMed Central

2024-06-05T03:55:52.681341

2005-1-5

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548502/", "journal": "BMC Pulm Med. 2005 Jan 5; 5:1", "authors": [ { "first": "Janine", "last": "Altmüller" }, { "first": "Corinna", "last": "Seidel" }, { "first": "Young-Ae", "last": "Lee" }, { "first": "Sabine", "last": "Loesgen" }, { "first": "Dieter", "last": "Bulle" }, { "first": "Frank", "last": "Friedrichs" }, { "first": "Heidemarie", "last": "Jellouschek" }, { "first": "Julika", "last": "Kelber" }, { "first": "Angela", "last": "Keller" }, { "first": "Antje", "last": "Schuster" }, { "first": "Michael", "last": "Silbermann" }, { "first": "Wolfgang", "last": "Wahlen" }, { "first": "Peter", "last": "Wolff" }, { "first": "Gerhard", "last": "Schlenvoigt" }, { "first": "Franz", "last": "Rüschendorf" }, { "first": "Peter", "last": "Nürnberg" }, { "first": "Matthias", "last": "Wjst" } ] }

PMC548503

Introduction ============ Health reforms that aim at increasing efficiency, quality and users\' satisfaction need to take into consideration human resource issues, because the health sector is labor-intensive and the performance of health systems depends on qualified and motivated workers \[[@B1]-[@B4]\]. At the same time, the support of the workforce is crucial to ensure successful implementation of reforms. In Latin America, the need to improve the performance of the workforce had been pointed out in many health sector assessments conducted in the 1970s and 1980s by the United States Agency for International Development (USAID), the World Bank (WB), other agencies and independent researchers. (See for Argentina \[[@B5]-[@B7]\], for Bolivia \[[@B8]-[@B10]\], for Brazil \[[@B11]\], for Chile \[[@B7],[@B12]\], for Colombia \[[@B12]-[@B14]\], for Costa Rica \[[@B15]\], for the Dominican Republic \[[@B16]-[@B18]\], for Ecuador \[[@B19],[@B20]\], for El Salvador \[[@B21]\], for Guatemala \[[@B22],[@B23]\], for Mexico \[[@B12],[@B24]-[@B26]\], for Nicaragua \[[@B27]\], for Panama \[[@B28],[@B29]\], and for Uruguay \[[@B7]\].) From these reports and studies, and notwithstanding the differences among the countries in the region, we can summarize the problems present during the 1970s and 1980s as follows: • The skill mix of health personnel was often inadequate to meet the needs of the communities, and highly qualified staff often performed tasks that could be conducted by less-trained providers. The health systems of the region were characterized by an excess number of medical specialists and insufficient numbers of other professionals such as primary care providers, nurses, pharmacists, public health specialists, epidemiologists, health economists, accountants, social workers, administrators, communication experts, planners, health educators, nutritionists, physical therapists and sanitary engineers; • There was an over-concentration of qualified health personnel in hospitals and urban centers, coupled with shortages in poor neighborhoods and rural areas; • A large majority of physicians held at least two jobs, one in the government and one in the private sector. In countries with fragmented health systems, physicians could even have three jobs: they worked part time for the social security institute, they worked for the ministry of health, and also held a private practice. Dual or triple employment generated conflicts of interests; physicians used the public sector to draw patients for their private practice, and their productivity in the public post was low and absenteeism high; • Human resources management systems were weak, largely due to dispersal of accountability: in many countries the terms and conditions of employment were under the control of the public service commission or the ministry of finance, and the education of human resources was under the control of the ministry of education or the private sector. Ministries of health did not have any input in determining the types and number of persons to be trained, and their involvement in hiring and managing the health workforce was limited. Health managers handled relations with the labor unions, had some limited supervisory roles, ensured organizational adherence to recruitment policies, and were responsible for some training. • Salary increases were generally based on years of service. In the majority of countries, central labor unions negotiated working conditions directly with governments and signed collective agreements that left administrators with little room to compensate workers according to performance; • Personnel decisions (hiring and promotion) were too often guided by favoritism, political dictates, and nepotism; • Health professionals were insufficiently committed to the public system due to the conflict of interests mentioned above, poor personnel management systems and the perception that wages were low; • The medical profession strongly dominated the definition of health sector policies and the regulation of the conditions of practice of all health professions; • Communication between providers and patients was poor, and providers and service users had very different social and cultural backgrounds. In countries with Amerindian-speaking populations, providers did not speak their languages; • The regulation of training institutions and conditions of practice was weak; • The training of health promoters and other auxiliary personnel such as dental assistants, midwives, laboratory technicians, equipment maintenance and repair technicians, and pharmacy clerks was poor or non-existent, thus their performance was poor. According to the literature reviewed, these conditions led to low productivity and efficiency; inadequate equipment; shortages of supplies and drugs; unmotivated and inadequately trained staff; questionable quality of care; and low users\' satisfaction. By the mid-1970s, the need to reform the human component of the health services was very urgent, and the urgency increased with the severe economic downturn that countries of the region suffered during in the early 1980s. The size of the Latin America health labor force (about nine million \[[@B30]\]) implied that reformers attempting to resolve the human resources problems mentioned above needed to dedicate a large amount of time and resources to it. This paper reviews the impact of the health reforms carried out under the leadership of the World Bank. Data come from a review of the literature including the leading Latin American and non-Latin American journals, monographs, documents found in ministries and reform offices, technical reports, papers presented at conferences and fieldwork carried out by the authors between 1980 and the present in Bolivia, Colombia, Costa Rica, Dominican Republic, Ecuador, El Salvador, Honduras, Mexico and Peru. The World Bank\'s neoliberal health sector reforms ================================================== In the early 1980s, with a few exceptions, countries in the region entered a severe economic downturn. The International Monetary Fund and the World Bank required -- as a condition for new lending and for refinancing the debts -- reduction of public spending. The impact of structural adjustment programs on health services was severe and compromised the ability of governments to maintain the physical infrastructure of the facilities, provide the necessary supplies and equipment, and maintain competitive salaries for the workforce. The World Bank took advantage of this situation to provide loans to the ministries of health and social security funds. Together with the loans, the Bank offered guidelines for the reorganization of the health services according to the ideological economic principles held by the institution. The World Bank had started its activities in the health sector in the early 1970s, and by the early 1990s it was the world\'s largest health sector lender. In 1999, the total accumulated value of health, nutrition, and population loans worldwide amounted to USD 16.8 billion (in 1996 dollars) \[[@B31]\]. By the late 1980s, the Bank placed health financing at the center of its health policy dialogue with borrowers. In 1987 the Bank proposed four changes \[[@B32]\]: to impose user fees at government facilities; introduce insurance or other risk coverage systems; use nongovernmental resources more effectively; and decentralize planning, budgeting and purchasing for government health services \[[@B32]\] A few years later, it looked at the roles of the government and the market in the health sector, and described the main components that have guided most World Bank-led health reform efforts \[[@B33]\]. These principles were reiterated in 1997 \[[@B34]\]. The underlying principles of the Bank-led reforms include the belief that the private sector is more efficient than the public sector, and that decentralized administrative units are better-equipped to respond to the needs of the population than centralized governments. The Bank also proposes to limit government financing to a basic package of services, to be determined for each country by cost-effectiveness studies and the country\'s ability to pay. Services included in the basic package are made available at no cost to the indigent population; governments and the rest of the population subsidize them. In the Bank\'s health reform model, the role of the state is limited to that of a regulator of the health care market. The United States health system is closer to the World Bank model than the national health services or social security funds of other industrial nations. The World Bank-led reforms did not include strategies to correct the human resources problems presented earlier, even if some of them had been identified by Bank-sponsored studies; the World Bank reformers believed that market forces would resolve them. As we will see, the contrary occurred and privatization and decentralization had some negative consequences for the workforce. The reliance on the market also hid the structural problems that needed to be taken into account at the time reformers designed human resource policies. The design and implementation process which were characterized by secrecy, generated dissatisfaction among health workers. We have organized our analysis of human resources during the reforms under five categories: resistance of the workforce to the implementation of a market-oriented health care model; faulty implementation processes; inadequately trained personnel in managerial positions and in service delivery; institutional and legal dimensions including insufficient and faulty information, lack of financial resources, and civil service statutes; and weak regulatory legislation to ensure the quality of professionals and the performance of the sector. Resistance to the implementation of a market-oriented health care model ======================================================================= The neoliberal health reforms intended to change the values that had inspired the Latin American systems and the relations between the government and the health workforce. Thus, health would no longer be considered a right; only the insured would be entitled to receive a broad array of services. Health workers would lose the protection they had enjoyed as public servants and become part of a flexible workforce (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Resistance of the workforce to a market-oriented health care model ::: ---------------------------------------------------------------------------------------------------------------------------------- a\. Health is not a right, and the reformed system is no longer based on the principles of solidarity and access to health care. b\. Health workers become part of a flexible workforce and are encouraged to compete, instead of collaborate, among themselves. c\. Worker unions lose power to influence the system and negotiate work conditions of behalf of affiliates. d\. The reform is an abdication of the responsibilities of the government to protect the population. e\. Physicians fear losing their professional autonomy. ---------------------------------------------------------------------------------------------------------------------------------- ::: Humans tend to resist change, but opposition to health reform was compounded by conflicts with the value system that inspired it. Most Latin American constitutions recognize health as a right and most governments had adopted Alma Ata\'s primary health care principles as their strategy to promote Health for All. Health policies were based on the principle of solidarity and fostered cooperation among health workers and also between them and other workers in related sectors such as education and agriculture \[[@B35]\]. It was assumed that health workers would do the best for their clients out of a sense of personal duty \[[@B4]\]. Human resource changes were needed to implement Alma Ata, but the budget reductions required by the World Bank caused a significant deterioration of working conditions. As their purchasing power worsened, health workers intensified undesirable behaviors to increase their income. These included levying illegal fees, diverting patients from the public sector to their private clinics \[[@B15]\], using public supplies and equipment for personal profit \[[@B36]\], and reducing their productivity in the public sector \[[@B15]\]. As indicated, Latin American physicians have supplemented their income from the public system by means of their private practice. Physicians value the stability and fringe benefits of a public post, but as the public delivery system deteriorated in many countries, private clients became their main source of revenue. By the late 1980s in most Latin American countries (with the exception of Argentina, Costa Rica, Cuba, Dominican Republic, Guatemala and Panama), more than half of the health expenditures, including the cost of medicines, occurred in the private sector \[[@B37]\], and in most countries of the region, personnel expenditures accounted for about 60% to 70% of the public health budget \[[@B38]\]. One of the objectives of the neoliberal reforms was to create a more flexible labor force by decreasing the number of tenured public employees and increasing the number of temporary personnel. This change threatened: the job security of the civil service, a very important dimension in countries with little political stability and where workers are frequently exposed to arbitrary political removals; the providers\' income, which now would depend on their ability to compete for contracts and clients; and that which workers expected from their employer (recognition, opportunities for self-actualization and promotion). The promoters of health reforms failed to acknowledge that in a politically unstable region, civil service tenure was necessary to maintain an efficient, productive, and loyal labor force. Predictably, these threats triggered the opposition of professional associations and unions, led to strikes, and lowered productivity during the reform process. Health workers of most countries of the region are unionized \[[@B39]\]. The unions protect the workers from the politicization of the appointments and promotions, and their leaders regularly engage in collective bargaining with the managers of the public sector. The stability provided by the civil service status facilitates the formation of strong union leadership: union leaders remain in office longer than policymakers. Labor unions anticipated that the decentralization and privatization of the sector would have a negative impact on their membership and their bargaining power. Labor unions in Bolivia, Ecuador, El Salvador, Mexico, Nicaragua, Peru and Venezuela expressed their opposition to the reforms on the grounds that they were an excuse for governments to relinquish their constitutional responsibility to ensure access to care and would lead to dramatic changes in work conditions \[[@B39]\]. In some countries, such as El Salvador and Mexico, unionized workers successfully stalled or delayed the implementation of the reform. Analysts of the health reform policies agree with the workers\' concerns. Segall \[[@B4]\] asserts that a market system does not nurture the service ethic that should characterize the workforce and leads providers to adopt self-seeking behaviors instead of working in the interests of the patient or the community. Others worry that the uncertainties generated by the reform, the stress and demands added to the workforce and the misalignment between the values of the workers and those of the reformed system are very detrimental to the workers\' motivation \[[@B1],[@B40]\]. According to Rigoli and Dussault \[[@B41]\] the unions\' fears were justified; union membership appears to have decreased in recent years. In addition, health professionals strongly opposed the idea of having their professional autonomy limited by professional administrators who would force them to adhere to diagnostic and treatment protocols based on economic principles rather than on technical criteria. The cool reception that Mexican professional associations offered to foreign insurance companies and health maintenance organizations is a good example of this concern \[[@B42]\]. Institutional and legal constraints =================================== The large majority of countries in Latin America do not have accurate information on the numbers and distribution of the health workforce. There is no centralized entity responsible for gathering information on the health personnel practicing in the country or in the different geographical regions, and even when only public sector workers are examined, the numbers collected at different administrative levels differ (people may move to a different location, and regional or local governments hire additional staff without reporting to the central authorities). The reforms worsened the possibility of having accurate information. If the system is decentralized and the majority of providers are in the private sector, the sources of information from which human resources data will need to be collated multiplies. The challenge may be even greater if each agency gathers the information differently, and without this basic information it is very difficult to engage in human resources development planning (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Institutional and legal dimensions ::: ------------------------------------------------------------------------------------------------------------------------- a\. Lack of accurate information on the availability of human resources and their distribution. b\. Civil service status limits the capacity of managers to change the working conditions of personnel. c\. Decentralizing human resources is expensive: homologation of salaries and benefits; hiring of additional personnel. d\. Decentralized governments have limited ability to manage personnel to respond to the needs of the population. ------------------------------------------------------------------------------------------------------------------------- ::: Civil service laws and negotiated agreements with worker unions have limited the freedom of public sector managers to reorganize the workforce; managers had to \"administer the rules\" \[[@B43]\] issued elsewhere. For instance, managers could not change the status of public sector employees to flexible contract workers, dismiss employees, increase workloads or change the working schedule. They may also have had difficulties rewarding their employees because the salaries and the conditions of employment were set outside the health sector by the public employment commission, the ministry of labor, or the ministry of finance or their corresponding decentralized entities. Reformers did not foresee these limitations. Managers overcame these constraints by hiring additional personnel under flexible contracts. In Brazil there are now about 15 different types of contracts for public sector employees; a significant expansion of the use of temporary workers and contracts without fringe benefits has been reported in Argentina \[[@B44]\], Colombia \[[@B45]\], Ecuador \[[@B2]\], El Salvador \[[@B30]\], Panama and Peru \[[@B2]\]. Funds for the temporary positions have different origins, including extrabudgetary government allocations, revenues generated through user fees, or savings from positions left empty due to retirements or administrative leaves. Transferring human resources to other politico-administrative levels was more difficult than anticipated. Kolehmainen-Aitken \[[@B46]\] and Homedes and Ugalde \[[@B47]\] have suggested that this is the most complex part of the decentralization process. The fear of transfers caused discontent and anxiety. On the other hand, managers did not want to absorb everybody who was transferred \[[@B46]\]. The decentralized personnel felt insecure about the new reporting mechanisms and the new managers\' expectations, and vulnerable to political crossfire. Decentralizing human resources is expensive. Prior to decentralization -- in countries such as Bolivia, Colombia and Mexico -- local or regional governments frequently hired additional personnel under different pay scales and benefit packages than those used by the central government. After decentralization, each decentralized administrative unit had to set pay scales, fringe benefits, performance assessments and reward systems for its workers. Because it was not possible to lower the salaries and reduce the benefits of workers with higher salaries and benefits -- generally federal/national workers -- it was necessary to bring the salaries and benefits of all the workers to the level of those who had the most generous package. Higher salaries and benefits represent an increase in the fixed costs of the system for an indefinite period. Moreover, the creation and/or strengthening of the decentralized administrative structures are often not accompanied by a decrease in the number of federal employees, further raising the costs of personnel to the system \[[@B48]\]. In Mexico, from 1985 to 1987 the cost of transferring federal employees to 14 states was 140,000 million pesos (approximately 452 million US dollars) \[[@B49]\], and in Colombia the World Bank indicated that the cost of transferring state and departmental health personnel to Cali\'s Municipal Health Secretariat was prohibitive \[[@B50]\]. Decentralization has been promoted under the assumption that the proximity of decision makers to the communities facilitates providing services more in accordance with the needs of the community. But the decentralized structures\' ability to respond to local needs has been constrained by: the civil servants they inherited, who are often inadequate in terms of skill mix and geographical distribution; the conditions of employment imposed by other government sectors and the unions; and the local elite, who do not have in mind the health needs of the communities and who lobby to have relatives and friends hired by local health authorities. In several countries of the region, decentralization has broadened the urban-rural inequities in the distribution of personnel and in the quality of services \[[@B51]-[@B54]\]. Rich decentralized units are able to offer better working conditions and attract qualified personnel from poorer municipalities, a sort of brain drain. Similarly, the development of the private sector also tends to drain qualified resources from the public sector \[[@B43]\]. Inadequately trained personnel ============================== Countries considered the human resource implications of reform only when they faced resistance from the unions or realized they did not have the financial and human resources required to implement the reform \[[@B55]\]. The health reform plan in Costa Rica recognized the ambiguity of the policies and procedures related to human resources but did not modify them. As planned, incentives were established to increase workers\' productivity and the use of short-term contracts. These two strategies ended up triggering greater grievances on the part of public sector employees \[[@B43]\]. The human resources units of the health ministries and the regional and local administrations were inadequately prepared for the reform (Table [3](#T3){ref-type="table"}). Traditionally they had had a narrow scope of responsibilities, often limited to managing the relationship with the unionized workforce, ensuring compliance with national/provincial policies on recruitment and deployment of personnel, and organizing continuing education activities \[[@B56]\]. The personnel attached to these departments generally had limited or no human resources development training \[[@B30],[@B57]\]. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Inadequately trained personnel ::: ----------------------------------------------------------------------------------------------------- a\. Human resources units are not adequately staffed, especially to manage change. b\. Lack of management experts, especially experts in insurance systems and contract managers. c\. Insufficient numbers of people trained in primary health care and public health related fields. d\. Training centers unable to produce personnel to operate the reformed health system. ----------------------------------------------------------------------------------------------------- ::: The tasks required by the reform overstretched the capacity of these units. Included among these tasks were: the development of new organizational structures, defining new job descriptions, reassigning responsibilities and designing new reporting systems, establishing performance evaluation methods and assisting all decentralized units to carry out similar duties in their jurisdictions. The decentralization of personnel often unveils rivalries and discontent among personnel who feel unfairly treated. All these issues need to be addressed, negotiated and resolved; and most human resources units were ill-prepared to lead the process \[[@B56]\]. For example, when the health system in Bolivia was decentralized, the salaries for several workers in Santa Cruz were delayed for months. Costa Rica introduced performance-based contracts between the Social Security Fund (Caja Costarricense de Seguro Social) and the health units, but according to a senior executive of the Ministry of Health, the targets were set at minimum levels and some units decreased their level of production \[personal communication with the authors\]. The mismatch between the abilities of the workforce and the needs of the system documented in the 1980s still prevails. The lack of coordination between training institutions and employers is at the root of the problem, along with weaknesses in the regulation of health professions and the dominance of physician\'s groups on the policy making process. According to Bach \[[@B56]\], shortages of personnel trained in disciplines such as primary health care, health economics, public health, health communication, health education, nutrition, and environmental engineering continue to severely limit the possibilities for improving the quality and efficiency of the health care system. Only in very few instances has reform included the human resources development activities to address these issues. For example, Costa Rica trained multifunctional teams for their lowest-level facilities and promoted the training of general doctors instead of specialists \[[@B58]\]. The National Autonomous University of Mexico modified its curriculum to promote family medicine; it introduced public health concepts and interdisciplinary experiences around issues related to promoting the health and wellbeing of the elderly and protecting workers from occupational hazards \[[@B59]\]. In most countries, managerial positions were traditionally given to physicians with little or no management training \[[@B55]\]. The neoliberal reforms require managers and staff with experience in specific dominions such as insurance, capacity to write contracts and enforce them, ability to monitor performance, knowledge of performance-based reimbursement systems, and expertise in health services research to evaluate progress. Decentralization to the state and municipal levels generates the need for even more managers, and countries that have given autonomy to hospital executives to manage their human and financial resources face additional challenges. With the exception of Chile, countries in the region had limited expertise in private health insurance \[[@B60]\]; Costa Rica, the Dominican Republic, Mexico and Venezuela engaged expensive foreign technical assistance to develop performance-based contracts and management information systems. The promoters of the reforms recognized the need for good managers but, because they had heavily criticized the public sector and questioned its role, it was difficult to recruit qualified staff into managerial positions and, in turn, it was difficult for those recruited to motivate and retain qualified staff \[[@B56],[@B57]\]. Most health reform projects included management training. The Pan American Health Organization \[[@B61]\] evaluated 15 such training programs and concluded that the training did not change the performance of the systems and that for only two projects was management capacity improved. The reasons for the failure included: difficulties in recruiting trainers; inability of the local universities to respond to the needs of the projects; inappropriate selection of training participants; conflict between project units and the ministries of health; political interference; and the absence of a human resource development plan. Theoretically the deficiencies in formal training can be corrected with supervision and continuing education activities, but this did not occur in Latin America. The authors of the report emphasize the need for countries to develop comprehensive human resources development plans to ensure the efficacy and sustainability of the training programs. After having spent millions of dollars in training and infrastructure, the region\'s capacity to manage contracts is still limited \[[@B62]\], and when contracts are in place, they are expensive to administer and the legal system is insufficiently developed to enforce them. In Colombia, most public hospitals were unable to compete with the private sector and are now bankrupt \[[@B63]\]. Poor management in decentralized entities has been considered one of the main reasons for the decentralization failure \[[@B64],[@B65]\]. Faulty reform implementation process ==================================== Countries in the region used a top-down approach to define and implement reform. The implementation was often led by a handful of top health executives, newly created reform offices, the political elite and international agencies, which in turn contracted for the technical assistance of international consultants and universities closely aligned with the neoliberal ideology of the World Bank. In general, there was little interest in involving professional associations, unions or even local universities in this process \[[@B2],[@B56]\] (Table [4](#T4){ref-type="table"}). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Faulty reform implementation process ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------ a\. Lack of involvement of professional associations and labor unions in the definition of the reform. b\. Secrecy surrounding definition of the reform raises suspicions among those responsible for implementing it and predisposes them to resist the changes. c\. Lack of transition plan. ------------------------------------------------------------------------------------------------------------------------------------------------------------ ::: In Costa Rica the labor unions were involved initially in the reform, but their influence was undermined by the World Bank and the Inter-American Development Bank, which adopted a more closed, centralized decision-making style \[[@B58]\]. According to a top executive of the Ministry of Health in El Salvador, the group preparing the reform operated in secret, and in his view, the secretive process was desirable because if health workers had participated they would have created obstacles to its implementation \[[@B66]\]. Indeed, it appears likely that the labor unions and professional associations in this country and many others would not have approved neoliberal reforms. Some authors have offered a different interpretation of the exclusion of the workforce and assert that it was the complexity of the human resources issues and the need to involve many players (the ministries of education, labor, finance and health, and professional associations and unions) in their solutions that led reform promoters to ignore the labor force and other stakeholders \[[@B30]\]. Regardless of the underlying motivation, the secrecy that surrounded the process of defining and implementing the reforms produced rumors, confusion and hostility against the reform among civil servants and professional groups \[[@B30],[@B39],[@B67],[@B68]\]. The objectives and processes by which reforms were introduced were never made clear; and often the reforms were perceived as responding more to ideological concerns of international organizations, in particular the International Monetary Fund and the World Bank, than to the needs, resources and sociopolitical realities of the countries \[[@B57]\]. The president of the Medical Association of El Salvador said that his association had tried to obtain information about the reform for over a year and that all his knowledge was based on \"rumors and guesswork that led nowhere\" \[[@B66]\]. In addition, health reformers did not consider the strategies and resources needed during the transition and early stages of implementation; such as allocating new financial resources and establishing clear communication channels. In addition, as discussed in the previous section, the reforms failed to adequately train managers who could lead the transition to a new management system \[[@B40]\]. Inadequate regulatory system to ensure high-quality training and health providers ================================================================================= The need for regulation increases in health systems where the private sector plays a prominent role. In Latin America it was not until the early 1990s, largely as a consequence of the health reforms, that health policy makers became aware of the urgency to regulate the health system. A regulatory system includes adequate regulations, the institutions to enforce them, and a judicial system that ensures a timely response in the event of conflict. Enforcing regulations is important to guarantee that trained personnel provide safe and adequate services (Table [5](#T5){ref-type="table"}). ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Inadequate regulatory framework to ensure the quality of professionals and the performance of the sector ::: --------------------------------------------------------------------------------------------------------------------------------------------------------------- a\. Limited quality controls in training institutions. b\. Physician-dominated field that precludes other professional groups from being recognized as health care providers within the official health care system. c\. Limited accreditation of health care professionals. --------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: Prior to the reforms, the regulatory systems were poorly developed and, when they existed, they were not tailored to the needs of consumers and enforcement was very limited \[[@B69]\]. For instance, the licensing of providers was a purely bureaucratic formality with no assessment of qualifications. The patronage and bossism observed in many countries were further expressions of regulatory deficiencies \[[@B70]\]. The number of medical schools has grown spectacularly in the last two decades and, in most countries, there were no mechanisms to ensure the quality of the training institutions or to test the abilities of the graduates. The number of medical schools in Chile grew by 68% between 1992 and 2000, in Peru the growth was also by 68%, in Argentina by 61% and in Brazil by 21%. The growth occurred mainly in the private sector \[[@B71]\]. Costa Rica had no private universities until the 1970s; now there are 70, several of which train health professionals \[[@B72]\]. By its very nature the regulation of the health profession relies heavily on the opinions of professionals and especially on physicians, who in turn place a great value on their autonomy and have had little interest in responding to social and political demands \[[@B55]\]. Medical associations have traditionally opposed health reforms and have had a very strong influence on health policy-making \[[@B29],[@B66],[@B73],[@B74]\]. The dominance of physicians has alienated other professions such as therapists, nurses, pharmacists, optometrists and psychologists \[[@B75]\]. For example, in Chile the government proposed to train and use more optometric technicians, but medically trained ophthalmologists opposed the proposal. After a long negotiation process involving ophthalmologists, optometric technicians, insurance companies, universities, parliamentary representatives and consumers, an agreement was reached. The four-year trial period allows optometric technicians to expand their scope of practice while medical schools take in more ophthalmologists for training \[[@B58]\]. The regulation of the health professions has a long way to go in Latin America, and it is probably impossible to establish sustainable regulatory mechanisms in the absence of political and judicial reforms; for the system to work, it needs to free itself from political interference \[[@B70]\]. Some argue that the separation between professional associations and licensing bodies \[[@B76]\] must be increased, and there is general agreement that the perspective of the general public \[[@B75]\] must be included. Consequences of the health reform on human resources ==================================================== Bach \[[@B56]\], Brito et al. \[[@B77]\], Dussault and Dubois \[[@B78]\], Rigoli and Dussault \[[@B41]\] have identified human resources issues as the main obstacle for the success of the reforms. The neoliberal health reforms did not solve the workforce problems that had previously been identified, and created additional ones that have had a negative impact on the health systems (see Table [6](#T6){ref-type="table"}). ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Consequences of the health reform on human resources ::: ----------------------------------------------------------------------------------------------------------------- a\. Working conditions have worsened, and talented workers migrate to the private sector or to other countries. b\. The motivation of workers has deteriorated. c\. Productivity and quality may have deteriorated. d\. The uneven ratio of specialists to primary physicians has not changed. e\. The uneven distribution of personnel (hospital and urban bias) persists. f\. Corruption has not decreased. ----------------------------------------------------------------------------------------------------------------- ::: The implementation of the reforms has been uneven in the Latin American region. Technical, logistical, political and financial problems have surfaced everywhere. While most countries decentralized, a few -- such as Colombia and Chile -- managed to significantly expand private insurance and, with the exception of Brazil, very few have engaged in large contracts with private sector providers. The most salient feature has been significant changes in hiring modalities. In Brazil there are 15 different types of contract arrangements \[[@B30]\] and in Peru the need to expand service coverage led to hiring 10 000 health professionals (physicians, nurses and technicians) between 1992 and 1996 under temporary contracts without social security; by the late 1990s about 12% of the health workers did not have social security \[[@B77]\]. Health workers in Ecuador have suffered wage reductions, in Mexico with decentralization the states have increasingly hired temporary workers \[[@B47]\], and in Argentina there has been a rise in precarious contracts, even fraudulent ones, such as full-time jobs under the label \"autonomous professional\" \[[@B30]\]. Another important result of the reform is the surge of multiple jobs, particularly in Argentina, Brazil, El Salvador, Panama, Peru, Uruguay, and to a lesser degree Chile \[[@B30]\]; that has caused stress and dissatisfaction among physicians. A survey of nurses conducted in Argentina, Brazil, Colombia and Mexico \[[@B44]\] revealed that the reforms increased stressful conditions at work, job dissatisfaction, insecurity from flexible contracts, malpractice concerns, inter-institutional migration, and new bureaucratic tasks for which nurses were not trained. The nurses specifically mentioned that they needed to do more work in less time with fewer staff; and complained about excessive paperwork, including billing, and about having less time for direct patient care than before the reform. One of the nurses who participated in the study said \"Patients may feel that we really don\'t care that much about them, because we just don\'t have enough time to spend with them and really know what is going on \[[@B44]\].\" In a different survey, nurses who had gone through reform restructuring held more negative perceptions of patient care than those who had not; and they also expressed a higher desire to unionize \[[@B41]\]. Furthermore, according to the Tavistock Group \[[@B79]\] \"cooperation throughout a health care system can produce better outcomes and much greater value for individuals and for society. Such cooperation requires agreement across disciplinary, professional and organizational lines about the fundamental ethical principles that should guide all decisions in a truly integrated system of health care delivery.\" If this statement is correct, by fragmenting the system through privatization and decentralization, and by introducing competition among health professionals, the neoliberal health reforms have compromised the quality of care. Similarly, one of the basic principles of neoliberal reforms -- that the efficiency of the system will increase by using flexible contracts and rewarding productivity -- is not supported by the data. The World Bank conducted an evaluation of civil service reforms in 15 countries and concluded: \"None of the cases reviewed so far have revealed any empirical evidence that the Civil Service Reform and related Technical Assistance Loans have succeeded in fostering the needed change in work attitudes, ethics and organizational culture that could lead to greater efficiency/productivity in the civil service\" \[[@B1]\]. Research has also uncovered problems with using performance-based payment schemes. For example, in health it is often difficult to establish who is responsible for the outcome. Costa Rica implemented a pilot project in Barva de Heredia in which physicians received an incentive based on productivity, but this was not extended to the nurses and the other clinic staff \[[@B80]\]. The project increased the costs to the system without increasing the productivity of physicians or the quality of care, and as a result the government halted its plans to extend the model to other health facilities. Health providers can also manipulate the information to maximize their benefits rather than the well-being of the patients; and substandard working conditions rather than workers\' action may be responsible for a poor outcome \[[@B81]\]. Mexican providers opposed a malpractice evaluation system because they did not want to be held liable for errors due to equipment deficiencies and lack of supplies \[[@B42]\]. Health workers in Costa Rica feared that the focus on productivity compromises the commitment to patients \[[@B58]\] and discourages the provision of services that require extra time, such as health education \[[@B2],[@B55]\] and mental health counseling. Establishing a valid and reliable merit-pay system is extremely complicated; placing too much emphasis on material rewards may displace more intrinsic motivators such as the pleasure of doing good, or caring for the patient. Bennet and Franco \[[@B1]\] even suggest that loyalty to the organization may decrease as the worker becomes aware of more lucrative opportunities with other employers. This could have serious consequences. Attracted by NGOs and the higher salaries of private hospitals, the most talented public servants could leave the public sector. Others raised questions about the sustainability of these strategies. In Brazil, productivity-based payment systems resulted in increased productivity, but the increase was not sustained over time and created competition among workers who were expected to collaborate \[[@B82]\]. For an interesting discussion on incentive systems and motivation in a different context, see Le Grand \[[@B83]\]. There is a belief among neoliberal economists that private sector workers are more productive and less corrupt than public employees. Recent hospital studies confirm that because of fear of termination, absenteeism is less frequent among non-tenured physicians hired through short-term contracts than among civil servants, but short-term contracts have not increased commitment to the institution \[[@B84]\]. Corruption continues to be pervasive in both private and public hospitals, and productivity differences between the private and public hospitals have not been documented \[[@B85]\]. Costa Rica has attempted to reduce the waiting lists by contracting for the provision of services with private groups, under the condition that the recipient of the contract is someone not working for the clinic that makes the referral, a condition that is often violated \[[@B72]\]. Decentralization can also be seen as a transfer of financial responsibilities from central government to local authorities, which has the potential to affect wages and job stability \[[@B39]\] and increase inequity. Poor local authorities cannot compete with the conditions of employment offered by wealthier municipalities and have difficulties in attracting personnel. In a decentralized system it is more difficult to structure career ladders, especially for workers who choose to locate in rural areas \[[@B81]\]; and decentralization can also exacerbate forms of patronage and political domination. The experience in many countries has proven that it is more difficult to resist the politicization of decision-making when health managers interact with local political leaders without central controls \[[@B46],[@B57],[@B70]\]. In decentralized health systems, particular attention needs to be paid to establishing good coordination among those responsible for the vertical program at the central level, the decentralized administrative units, and the clinical staff. The absence of good coordination may result in health workers\' reporting to two supervisors: the person responsible for the vertical program and the supervisor of the health facility or region, as is the case in Mexico \[[@B47]\]. In sum, in the majority of Latin American countries, the neoliberal reforms have not made the health delivery model more responsive to the needs of the community; have not increased the productivity of health workers; have had a negative impact on working conditions and staff motivation; appear to have further compromised the quality of care; and have had a limited impact on the capacity to regulate the health professions and training institutions. Discussion ========== More recent studies suggest that many of the old workforce problems remain unresolved \[[@B56],[@B70],[@B86],[@B87]\]. Even the World Bank, which promoted the reforms, has finally recognized \[[@B88]\] that the neoliberal strategies are not having the desired impact. The Bank questions the performance of the private sector and highlights the need to find the institutional arrangements and policies that best respond to local conditions and resources. Health reform provided a perfect opportunity to promote and encourage workforce improvement. Prerequisites for the progress of such processes are political will, effective relationships between the educational and service-providing institutions, and the open collaboration of professional groups. However, the reforms had the opposite effect. The neoliberal orientation challenged the use of conventional regulation strategies because, by encouraging professionals to seek their own interest instead of the interests of society as a whole, it questioned whether society and regulatory bodies could continue to trust and have faith in the criteria expressed by health professionals. Human resources account for the lion\'s share of health budgets, and poor performance had been identified as the main constraint to efficiency, quality of services and user\'s satisfaction. The values that guided the neoliberal reform and the privatization and decentralization initiatives worsened the problems affecting the Latin American workforce and added new challenges. The reform implementation process was also responsible for the failures. The promoters of the neoliberal health reforms underestimated the importance of involving professional organizations and unions in the planning and implementation of the reform efforts, raising suspicion and resistance among organized health workers. In addition, the reforms were designed in secret and implemented using a top-down approach. The only significant advance in human resource development in the last 10 years is the increased interest in strengthening the capacity to regulate training institutions and practitioners. Solving the problems that affect human resources is no easy task, and probably the solutions are different for each country; ignoring the problems and hoping that the market will resolve them is a recipe for failure. Managing change is very complex; embarking on reform without having secured the collaboration of the workforce and ensuring the availability of sufficient qualified staff is irresponsible. The exclusion of the organized labor force from reform discussions is indefensible; the collaboration and motivation of the health workers is essential to the reform, and the leaders of the organized groups can assist in informing and aligning the workforce \[[@B40],[@B89]\]. Policy changes requiring a different skill mix of health personnel require careful planning because there is a significant time lag between deciding that there is a need to train additional professionals and having them available. For example, a 10% rise in the number of students in a medical school produces only a 2% increase in the supply of doctors 10 years later \[[@B78]\]. Most countries did include a training component, but as mentioned earlier it was insufficient, and part of the problem was that the reform implementation was rushed, without the benefit of field-testing the underlying theories, gathering evidence on the appropriateness of the strategies, or learning from the reform experience. One thing that reforms could have done was to support interventions that, independently from the reform, some countries had designed to overcome workforce weaknesses. Some interventions were national programs, others were pilot projects, and there were also experimental projects. The following are examples of these autochthonous interventions. To solve the urban-rural gap and improve equity, most ministries of health in the region created the obligatory rural health service known as pasantía or servicio social that requires physicians to spend one year in a rural health center prior to graduation or immediately after receiving their degree \[[@B35]\]. A few social security institutes have also found ways to reduce health inequities; such is the case of the Mexican and Ecuadorian Institutes of Social Security (IMSS and IESS). IMSS organized COPLAMAR, an extensive primary and secondary care program for poor rural populations, which brought general practitioners and specialists to rural areas. The Ecuadorian program known as Seguro Social Campesino offered primary care services for rural dwellers and, when needed, hospitalization at the Ecuadorian Social Security Institute; this program aimed at reducing the rural-urban gap in a country of which 70% of the population then lived in the rural areas \[[@B90]\]. Mexico created a training program for traditional midwives who worked in dispersed rural populations; the objective of the program was to enhance the quality of their services and reduce maternal and infant mortality \[[@B91]\]. The Ministry of Health of the Dominican Republic, in an effort to increase health equity, trained and deployed more than 5 000 health promoters in rural centers. The promoters periodically visited every rural household to monitor infant growth, promote nutrition and sanitation, and assist in immunization campaigns \[[@B35]\]. Costa Rica attracted the world\'s attention with the Open Walls Hospital, a program that required the specialists of a regional hospital to schedule -- when needed -- weekly visits to dispersed rural populations. The program also intended to convey the message to specialists that they were not different from other health workers and had an obligation to serve poor rural dwellers even when doing so would involve personal inconvenience \[[@B80]\]. To enhance the professional status of primary care practitioners, reduce referrals, and improve quality of care, IMSS created many positions in the specialty of family physician, forcing the medical profession to recognize the status of the new specialty. Colombia\'s Ministry of Health sent nurses to a graduate health education program taking advantage of a US fellowship program in order to diversify the human resource composition of the Ministry. Some of these projects were successful; this was the case of family physicians and COPLAMAR in Mexico. However, the first 14 Mexican states that decentralized in the 1980s dismantled the program, transferring it to the states\' departments of health to create the state health system, and the quality of rural health deteriorated rapidly \[[@B92]\]. Others function poorly, as is the case of the compulsory year of social service in all the countries where it was established. Similarly, the health promoters program in the Dominican Republic suffered from insufficient training, lack of continuous education, absence of efficient supervision, and poor remuneration, comments that can be extended to all health promoter programs of the region. The Seguro Social Campesino has suffered from inadequate financing, and plans to extend it to the entire rural population have been placed on permanent hold. Other programs were discontinued because of indifference and lack of support from policy makers. Thus, the Hospital without Walls ceased after all Ministry of Health hospitals were transferred to the Social Security Institute. Due to budgetary problems, the Colombian Ministry of Health did not employ the health educators on their return from graduate school. The health reforms would have provided a perfect opportunity to support and strengthen many of these and other autochthonous interventions. A proper course of action would have been to evaluate the projects that had been developed locally, identify their strengths and weaknesses, and establish their viability and sustainability. Through trial and error and with appropriate resources and incentives, many of them are likely to be more effective and less costly than foreign programs invented by those who hardly know the realities of developing countries and are inspired by ideological principles and questionable economic theories. There is much more that needs to be done to improve the training and management of human resources for health, and very often the solutions depend on the collaboration of a wide range of stakeholders such as those who produce health workers, those who employ them, those who pay for their services, those who negotiate working conditions and those who define the standards of professional practice. It is no easy task and can be successfully accomplished only if there is strong political will, if there is openness and trust among all stakeholders, and if sufficient resources and time are allocated to this effort. Most countries of the region have the capacity to find appropriate solutions to the problems they are facing. Dussault \[[@B55]\] argued that change is possible only on the basis of shared values, and as we have seen, the values that inspired the neoliberal reform did not coincide with those expressed in the Latin American Constitutions and in the primary health care principles that had guided the development of the health sector during the 1970s and 1980s. As Segall mentioned \[[@B4]\], it is important to recover the spirit of cooperation among health providers, and there is a need to take explicit steps to raise their motivation and patient-centered behavior. Without a motivated workforce, all other efforts to change the system may be even counterproductive. Policy makers and administrators will have to explicitly identify strategies that foster collaboration, inner motivation and work ethics, and this may require abandoning the market orientation of the neoliberal reforms and embracing the values that inspired the primary health care movement. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= Both authors have contributed equally to the design, data collection, data analysis, drafting and completion of this article. Acknowledgements ================ We are grateful to Peter Lloyd-Sherlock and René Leyva for their constructive comments on earlier versions of this manuscript.

PubMed Central

2024-06-05T03:55:52.690971

2005-1-19

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548503/", "journal": "Hum Resour Health. 2005 Jan 19; 3:1", "authors": [ { "first": "Núria", "last": "Homedes" }, { "first": "Antonio", "last": "Ugalde" } ] }

PMC548504

Background ========== In their 1986 paper Israel, Rosenberg, and Curtin \[[@B1]\] gave a sort of rallying call for researchers to consider the analytical potential for multiple cause of death data collected by the United States National Center for Health Statistics (NCHS). Beginning with the implementation of the Eighth revision of the ICD in 1968, the NCHS developed and employed several computer systems to automatically select the underlying cause for each death certificate and to produce multiple cause of death data \[[@B2]\]. The resulting multiple cause of death datasets by year are made publically available through the NCHS website. Acknowledgment of the potential for multiple cause of death data analysis is increasing in other countries as well \[[@B3],[@B4]\]. For example, the Australian Bureau of statistics point out that using multiple cause of death data allows researchers to: more comprehensively understanding and track death due to chronic disease which do not often appear as the underlying cause of death (e.g. Alzheimer\'s, diabetes, pneumonia), to provide better documentation on multi-morbid associations and the strength of associations between conditions which led to death (for example by examining the frequency of associations between diseases such as diabetes and ischaemic heart disease), and to assist in identifying problems with the process of recording and coding cause of death information \[[@B4]\]. Multiple cause of death data has been used to look at trends in certain diseases, e.g. HIV \[[@B5],[@B6]\] and lung disease \[[@B7]\], but despite its availability, surprisingly few studies have looked at it broadly. Indeed there is no annual standard summary tabulation report of the multiple cause of death data put out by NCHS. This may be due in part to the overwhelming amount of information that arises when combinations of causes of death are considered. There are an enormous number of complex combinations which could be summarized and perhaps it is not clear what tables may be of general interest. The purpose of this article is to examine some straightforward relationships of multiple causes of death as a function of factors available on the death certificate (demographics of decedent, place of death, type of certifier, disposal method, whether an autopsy was performed, and year of death). Using all death certificates issued by the state of Minnesota between 1990 and 1998 (326,332 deaths), the current study documents the relationship between these factors and the associated frequency of reporting of multiple causes of death as well as the associated frequency that a cause is considered underlying (after data processing) given that it is mentioned on the death certificate. The implication being that differences found are either due to actual differences in causes of death in these groups or due to systematic biases in the reporting of causes of death, or a combination of both. The study will not be able to discern which is the cause but hopes to contribute at the very least by providing an example of the potential relationships which can be examined with the rich multiple cause of death data. Methods ======= Data source ----------- The data used are from the Minnesota Department of Health Mortality Database and include entries from 326,332 individual death certificates, which represent all deaths occurring in Minnesota during the period of 1990--1998. Record axis codes (those codes which have been completely data processed) are used for all analyses in this paper rather than entity axis codes. A brief description of the entity and record axis coding is given here. The translation of causes of death listed on the death certificate (see Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"} for the actual certificate) to the codes used for statistical analysis goes through many steps. As seen in Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}, the medical information which focuses on the sequence of medical conditions that resulted in death is provided in a two-part format. Part I is for the conditions which directly lead to death, and Part II is for other conditions which contribute to death but are not directly related to the immediate cause of death \[[@B1]\]. The underlying cause of death is defined as the \"(a) the disease or injury which initiated the train of events leading directly to death, or (b) the circumstances of the accident or violence which produced the fatal injury\" \[[@B8]\]. The entity axis codes represent what is actually written on the death certificate by the certifier expressed in terms of ICD codes including an indicator of which line the code came from and which position on the line it came from (if more than one code was listed per line). While the conditions listed in Part I should form a causal sequence initiated by the underlying cause listed on the lowest line, errors in properly completing the form occur regularly and a reselection of the underlying cause of death is done nationally 30--40% of the time. The decision to reselect an underlying cause other than that listed on the lowest used line in Part I is governed by a set of rules developed by WHO as part of the periodic revision of the International Classification of Disease \[[@B9]\] and is incorporated, along with a complex set of decision tables, into the Automated Classification of Medical Entities (ACME) software. The record axis codes represent a further processing of the entity axis codes to be consistent with the underlying cause data and more amenable to statistical tabulation and analysis. The record axis codes distinguish the ICD code selected as the underlying cause of death and lists all additional causes of death mentioned but does not distinguish them in terms of their ordering or original location on the death certificate. For more detail on entity and record access codes, see \[[@B10]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **US standard certificate of death.**Line 27 Part I and Part II are where the causes of death are listed. ::: ![](1471-2288-5-4-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **This figure displays the backside of the certificate of death**. Details are given for filling out specific lines. ::: ![](1471-2288-5-4-2) ::: Using the record axis codes, we have for each death record: one underlying cause of death and up to 14 non-underlying causes of death (with no distinction of importance given amongst the non-underlying). When we refer to a cause of death and do not want to distinguish if it is underlying or non-underlying we will refer to it as a \"mentioned\" cause of death. In addition to listing one underlying and up to 14 non-underlying causes of death, each death certificate also contains information about the demographics of the deceased, including age, gender, race, marital status, and educational attainment. Also, other conditions related to the death are recorded -- place and time of death, who completed the death certificate, if autopsy has been performed and type of body disposal. Minnesota Laws and guidelines govern the process for who and how a death is certified under different circumstances in Minnesota. For example when an unattended death occurs (e.g. at a persons residence) a medical examiner\'s investigator must arrive at the scene. The medical examiner will contact the last attending physician asking about past medical history of the decedent and most likely cause of death. When an attending physician has seen the decedent within 90 days and the death is natural, jurisdiction is usually given to the physician to certify the death. Sudden or unexpected deaths due in part to any factor other than natural disease must be referred to the medical examiner\'s office. Autopsies are performed at the discretion of the medical examiner but can also be performed for any death at the request of the immediate family. The underlying and non-underlying causes of death derived from the death certificate, in this study, are coded according to the 9^th^revision of International Classification of Disease (ICD). The specific ICD9 codes are grouped into standard reporting of cause of death categories resulting in a total of 107 different causes of death. In this study, individuals are dichotomized as having multiple causes of death (i.e. at least one non-underlying cause) or not having multiple causes of death (i.e. only having an underlying). In addition, because heart disease is the leading cause of death and diabetes is a good example of a disease which often shows up as a non-underlying cause of death, this research investigates two sub -populations: 1) Individuals that have ischemic heart disease (ICD-9: 410 -- 414) mentioned as a cause of death (n = 79,833), and 2) Individuals that have diabetes mellitus (ICD-9: 250) mentioned as a cause of death (n = 27,181). For both sub-populations, a dichotomous variable is created to indicate whether the mentioned disease is coded as the underlying cause of death or not. Data analysis ------------- Descriptive statistics including total numbers and proportion of all deaths (n = 326,332) in each of the covariate categories are reported as well as proportions of people within each covariate category who have multiple causes of death. In order to examine the association between each covariate and the dichotomous outcome of multiple causes of death, logistic regression is used to mutually adjust each factor for the others. 95% confidence intervals of odds ratios are reported. Trends in multiple cause of death reporting across time are investigated graphically. Similarly, descriptive statistics including total numbers and proportions will be presented for the two sub-populations with ischemic heart disease (n = 79,833) or diabetes mellitus (n = 27,181) mentioned either as underlying or non-underlying cause of death. Logistic regression is used and 95% confidence intervals are reported to examine factors that are associated with each of these diseases being reported as underlying cause of death rather than non-underlying. Results ======= Overall, 68.9% of the 326,332 deaths from 1990--1998 had at least one non-underlying cause of death in addition to the underlying cause (i.e. have multiple causes). There was a noticeable decreasing trend of reporting multiple causes of death over the 9 year period from 1990 to 1998 with 74.0% in 1990 consistently dropping down to 64.8% in 1996 and remaining around 66% until 1998. Table [1](#T1){ref-type="table"} presents the marginal percentage of individuals in each demographic and death related category as well as the proportions and adjusted odds ratios of having multiple causes of death by each of the covariates. The youngest (\<25) and oldest (85+) age groups had the lowest and highest percent of multiple causes of death (61.7% and 71.8%, respectively). Interestingly, the age group from 45--64 did not have higher odds of having multiple causes than the young (\<25) group. The percentage of men with multiple causes of death reported was slightly higher (1%) than that of women. Individuals over 25 years old with less education had a higher percentage of multiple causes of death (71.3%) compared to those with higher education (66.6%). The most pronounced difference with respect to demographics was found in race categories, with Native American having the highest percentage of multiple cause of death (74.5%), compared to 68.9% of white. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Percent of all deaths (n = 326,332) by each covariate. Probability of reporting multiple causes of death given covariate, marginal percent by category, and adjusted odds ratios of reporting multiple causes of death given covariates. ::: **% of death by categories** **% with multiple COD by categories** **Odds Ratio (95% CI)**^1^ --------------------- ------------------------ ------------------------------ --------------------------------------- ---------------------------- **DEMOGRAPHIC** **Age** **0--24** 3.0 61.7 1 **25--44** 4.6 69.0 1.38(1.31--1.45) **45--64** 13.3 62.1 1.01(0.97,1.06) **65--84** 47.8 69.4 1.41(1.35,1.47) **85+** 31.3 71.8 1.58(1.51,1.65) **Sex** **Female** 50.3 68.6 1 Male 49.7 69.3 1.11(1.09,1.13) **Race** **White** 96.4 68.9 1 Black 1.7 67.5 1.15(1.078,1.22) Native American 0.9 74.5 1.54(1.41,1.69) Asian 0.5 65.0 1.04(0.94,1.16) Hispanic 0.5 66.6 1.13(1.01,1.26) **Education^2^** **High school** 32.4 68.1 1 Below High School 44.3 71.3 1.03(1.01,1.05) Above High School 23.3 66.6 0.94(0.92,0.96) **Marital Status** **Married** 41.0 67.4 1 Single 12.7 68.0 1.08(1.05,1.11) Widowed 38.5 71.1 1.09(1.07,1.11) Divorced 7.8 68.2 1.12(1.08,1.15) **DEATH RELATED** **Autopsy** **No** 77.8 69.0 1 Yes 9.4 74.4 1.57(1.52,1.62) Unspecified 12.8 64.7 0.85(0.83,0.87) **Certifier** **Physician** 82.0 69.2 1 Coroner 12.6 70.2 1.23(1.19,1.26) Osteopath 1.4 75.3 1.27(1.183,1.37) Other/Unknown 4.1 57.8 0.61(0.59,0.63) **Disposal method** **Burial** 76.3 70.1 1 Donation 0.3 69.7 1.02(0.89,1.17) Removal 1.5 47.1 0.35(0.33,0.37) Cremation 20.5 66.8 0.94(0.93,0.96) Unknown 1.4 **Death place** **Hospital Inpatient** 35.2 73.4 1 Residential 19.1 58.7 0.48(0.47,0.49) Nursing home 35.5 70.6 0.77(0.76,0.79) ER 5.5 65.5 0.64(0.61,0.66) Unknown 4.7 ^1^Odds ratios are odds of having multiple causes of death given particular category as compared to odds in reference category. Odds ratios are obtained from logistic regression and are mutually adjusted for all other variables. ^2^Education is listed only for population where age\>25. ::: For places of death, hospital in-patient (73.4%) and nursing home (70.6%) had the highest probability of reporting multiple causes of death, and residence had the lowest percentage (58.7%) (Table [1](#T1){ref-type="table"}). Between different types of body disposal methods, \"removal\", which refers to moving the body outside of the US, had the lowest percentage (47.1%) of reporting multiple cause of death. For deaths that had autopsy performed, there was an increased odds of 1.57 that multiple causes of death would be reported. In terms of different types of medical examiners, the difference was less than 1% (69.2% vs. 70.1%) marginally between physician and coroners, the two most frequently seen types of examiners, but examining this difference across time (Figure [3](#F3){ref-type="fig"}) found an interesting interaction effect in the trend. The physicians showed a decrease in multiple cause of death reporting while the coroners stayed constant or slightly increased over the decade. Table [2](#T2){ref-type="table"} provides reference for the 25 leading underlying causes of death and leading mentioned causes of death in this dataset. It also lists the leading causes of death which occur on death certificates only reporting an underlying cause of death with no non-underlying. The top four causes based on only one cause certificates are the same as the overall top four causes. But it is interesting that the fifth leading cause in this category is \"Symptoms and ill-defined conditions\" which typically are assigned as the underlying cause only if the sole cause listed. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Interaction between certifier and year. ::: ![](1471-2288-5-4-3) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Based on Minnesota death records (n = 326,332) from 1990--1998. Top 25 causes of death ranked by underlying and any mention cause of death. Top 25 causes of death for only those deaths where only one cause was listed (i.e. n = 101,423 deaths). ::: ranked by underlying cause number of deaths with underlying cause \% all deaths rankeded by any mention number of deaths with cause mentioned \% all deaths ranked by cause when only one cause mentioned number of deaths with cause as only one cause \% of deaths with only one cause mentioned ---- --------------------------------------------------------- ---------------------------------------- --------------- ----------------------------------------------------------------- --------------------------------------- --------------- ----------------------------------------------------- ----------------------------------------------- -------------------------------------------- 1 Ischemic Heart Disease 61540 0.1886 Other diseases of the Heart 88502 0.2712 Ischemic Heart Disease 13753 0.1356 2 Cerebrovascular Disease 26413 0.0809 Ischemic Heart Disease 79833 0.2446 Other diseases of the Heart 8862 0.0874 3 Other diseases of the Heart 22566 0.0692 Cerebrovascular Disease 43885 0.1345 MN of Trachea, Bronchus & Lung 8157 0.0804 4 MN of Trachea, Bronchus & Lung 18476 0.0566 Symptoms & ill-defined conditions 42196 0.1293 Cerebrovascular Disease 7602 0.0750 5 Pneumonia -- except newborn 12465 0.0382 Other mental disorders 38565 0.1182 Symptoms & ill-defined conditions 6459 0.0637 6 Other COPD 11114 0.0341 Pneumonia -- except newborn 31312 0.0960 Other mental disorders 3494 0.0345 7 Other mental disorders 10120 0.0310 Diabetes mellitus 27181 0.0833 MN of Breast 3360 0.0331 8 Diabetes mellitus 7959 0.0244 Hypertension without heart disease 26036 0.0798 MN of Intestine, not rectum 3218 0.0317 9 MN of Intestine, not rectum 7388 0.0226 Other COPD 25872 0.0793 Pneumonia -- except newborn 3131 0.0309 10 Diseases of the arteries, veins & pulmonary circulation 7181 0.0220 MN of Trachea, Bronchus & Lung 19896 0.0610 MN of Other & unspecified sites 3042 0.0300 11 MN of Breast 6646 0.0204 MN of Other & unspecified sites 19386 0.0594 MN of Prostate 2380 0.0235 12 Symptoms & ill-defined conditions 6488 0.0199 Diseases of the arteries, veins & pulmonary circulation 19180 0.0588 Other COPD 2332 0.0230 13 Transportation accidents -- Motor Vehicle 5809 0.0178 Other diseases of the digestive system 17943 0.0550 MN of Pancreas 2313 0.0228 14 Other diseases of the digestive system 5777 0.0177 Pneumoconiosis & other resp. Diseases 17728 0.0543 Diseases of arteries, veins & pulmonary circulation 1758 0.0173 15 Other disease of the Nervous System and Sense Organs 5714 0.0175 Other disease of the Nervous System & Sense Organs 16799 0.0515 Other Neoplasms of lymphatic & Hematopoietic tissue 1701 0.0168 16 MN of Prostate 5669 0.0174 Chronic and Unspec. Nephritis & renal failure & renal sclerosis 16065 0.0492 Alzeimer Disease 1536 0.0151 17 MN of Other & unspecified sites 5504 0.0169 Arteriosclerosis 13879 0.0425 Other disease of the Nervous System & Sense Organs 1477 0.0146 18 Suicide 4435 0.0136 Transportation accidents -- Motor Vehicle 10367 0.0318 Residual, Undefined 1300 0.0128 19 MN of Pancreas 4136 0.0127 Medical complications & misadventures 10299 0.0316 MN of Brain, other nervous 1292 0.0127 20 Residual, Undefined 4135 0.0127 MN of Intestine, not rectum 9116 0.0279 Leukemia, & Aleukemia 1271 0.0125 21 Pneumoconiosis & other resp. Diseases 4028 0.0123 Septicemia 8790 0.0269 Perinatal conditions 1206 0.0119 22 Accidental falls 4010 0.0123 Suicide 8740 0.0268 MN of Ovary, Fallopian tube, Broad ligament 1124 0.0111 23 Alzeimer Disease 3757 0.0115 MN of Breast 8704 0.0267 Other diseases of the digestive system 1079 0.0106 24 Other Neoplasms of lymphatic & Hematopoietic tissue 3584 0.0110 Other genito-urinary disease 8382 0.0257 Suicide 990 0.0098 25 Leukemia, & Aleukemia 3440 0.0105 MN of Prostate 8369 0.0256 MN of Kidney 955 0.0094 ::: The results presented so far explored how covariates may be correlated with multiple causes of death being reported. The following results pertain to the conditional probability that a particular cause of death (ischemic hear disease or diabetes) is selected as underlying given that it is mentioned. Results for the subpopulations with ischemic heart disease or diabetes mentioned are shown in Table [3](#T3){ref-type="table"} and Table [4](#T4){ref-type="table"}, respectively. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Population with Ischemic Heart Disease mentioned on death certificate (N = 79833). Marginal percent by category, conditional percent with ischemic heart disease as underlying given that it is mentioned by category and odds ratios of ischemic heart disease being reported as underlying cause when it is mentioned given covariates. ::: **% of deaths by category** **% with heart disease as underlying** **Odds Ratio (95% CI)**^1^ --------------------- ------------------------ ----------------------------- ---------------------------------------- ---------------------------- **DEMOGRAPHIC** **Age** **0--44** 1.7 80.0 **1** **45--64** 12.4 81.8 1.2 (1.03,1.36) **65--84** 53.0 76.1 0.84 (0.74,0.95) **85+** 32.8 76.8 0.87 (0.77,1.00) **Sex** **Female** 45.2 76.3 **1** Male 54.9 77.8 0.95(0.91,0.99) **Race** **White** 97.9 77.2 **1** Black 0.8 71.8 0.78(0.66,0.94) Native American 0.7 69.7 0.55(0.45,0.66) Asian 0.3 77.2 1.12(0.81,1.56) Hispanic 0.3 72.2 0.79(0.59,1.06) **Education^2^** **High school** 29.1 76.3 **1** Below High School 49.1 77.9 1.07(1.03,1.11) Above High School 21.8 76.5 1.02(0.97,1.07) **Marital** **Married** 44.9 77.5 **1** Single 8.2 79.7 1.25(1.16,1.33) Widowed 40.0 75.9 1.04(0.99,1.09) Divorced 6.9 77.9 1.13(1.05,1.22) **DEATH RELATED** **Autopsy** **NO** 77.8 76.9 **1** Yes 10.5 78.0 0.91(0.86,0.97) Unspecified 11.7 77.8 1.12(1.06,1.18) **Certifier** **Physician** 79.5 75.5 **1** Coroner 15.2 84.3 1.34(1.25,1.42) Osteopath 1.5 80.9 1.25(1.07,1.46) Other/Unknown 3.8 81.0 1.23(1.13,1.35) **Disposal method** **Burial** 78.9 77.3 **1** Donation 0.3 75.3 0.74(0.56,0.98) Removal 1.6 85.3 1.74(1.48,2.05) Cremation 18.0 66.8 0.95(0.91,0.99) Unknown 1.2 **Death place** **Hospital Inpatient** 36.1 72.9 **1** Residential 20.1 90.5 1.83(1.74,1.93) Nursing home 27.9 71.2 0.83(0.79,0.87) ER 11.4 83.2 1.67(1.39,1.97) Unknown 4.5 ^1^Odds ratios are odds of having ischemic heart disease as underlying rather than contributing given particular category as compared to odds in reference category. Odds ratios are obtained from logistic regression and are mutually adjusted for all other variables. ^2^Education is listed only for population where age\>25. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Population with Diabetes mentioned on death certificate (N = 27181). Marginal percent by category, conditional percent with diabetes as underlying given that it is mentioned and odds ratios of diabetes being reported as underlying cause when it is mentioned given covariates. ::: **% of deaths by category** **% with diabetes as underlying given that it was mentioned** **Odds Ratio (95% CI)^1^** --------------------- ------------------------ ----------------------------- --------------------------------------------------------------- ---------------------------- **DEMOGRAPHIC** **Age** 0--44 2.3 51.7 **1** 45--64 13.2 34.5 0.51(0.43,0.60) 65--84 59.5 27.7 0.37(0.32,0.43) 85+ 24.9 28.4 0.39(0.33,0.45) **Sex** **Female** 51.7 30.2 **1** Male 48.3 28.3 0.92(0.86,0.97) **Race** **White** 95.4 28.9 **1** Black 2.0 36.5 1.19(0.98,1.43) Native American 1.5 41.0 1.42(1.136,1.76) Asian 0.5 27.2 0.88(0.59,1.30) Hispanic 0.7 36.6 1.33(0.978,1.82) **Education^2^** **High school** 31.2 30.1 **1** Below High School 47.8 28.0 0.99(0.93,1.06) Above High School 21.0 30.8 1.03(0.96,1.11) **Marital** **Married** 43.6 27.8 **1** Single 8.5 34.3 1.25(1.13,1.38) Widowed 40.2 29.0 1.09(1.01,1.16) Divorced 7.7 33.5 1.11(0.99,1.23) **DEATH RELATED** **Autopsy** **No** 83.3 28.3 **1** Yes 5.5 22.8 0.70(0.62,0.80) Unspecified 11.3 39.4 1.49(1.37,1.62) **Certifier** **Physician** 85.0 29.7 **1** Coroner 10.0 24.2 0.69(0.62,0.77) Osteopath 1.5 30.4 1.03(0.81,1.29) Other/Unknown 3.5 34.3 1.23(1.08,1.42) **Disposal method** **Burial** 79.2 28.6 **1** Donation 0.3 25.0 0.84(0.51,1.39) Removal 1.1 37.0 1.46(1.143,1.87) Cremation 18.3 31.4 1.06(0.99,1.14) Unknown 1.1 **Death place** **Hospital Inpatient** 34.8 25.2 **1** Residential 18.8 30.9 1.29(1.19,1.41) Nursing home 37.4 32.7 1.57(1.47,1.68) ER 6.4 28.3 1.15(1.02,1.29) Unknown 2.6 ^1^Odds ratios are odds of having diabetes as underlying rather than contributing given particular category as compared to odds in reference category. Odds ratios are obtained from logistic regression and are mutually adjusted for all other variables. ^2^Education is listed only for population where age\>25. ::: Table [3](#T3){ref-type="table"} gives the odds ratio of ischemic heart disease being selected as underlying cause of death when it was mentioned as a cause, given the covariates main effect. Overall 77.1% of the time that heart disease was mentioned as a cause, it was selected as the underlying cause of death. The 45--65 year age group had the highest probability of heart disease being codes as underlying when it was mentioned (81.8%). Males had a slightly lower probability than females to have heart disease as underlying cause of death when it was mentioned on the death certificate. Furthermore, Blacks and Native Americans were less likely to have heart disease coded as underlying cause of death when it was present on the certificate as compared to Whites. Individuals that had an autopsy performed were less likely (0.91 odds ratio) to have ischemic heart disease selected as underlying when it was mentioned. If a physician is the death certifier, the probability of selecting heart disease as underlying cause of death is relatively the lowest when compared to coroner, osteopath and other and unknown certifiers. Amongst body disposal methods, the probability for heart disease to be reported as underlying cause was the lowest if bodies were donated (OR = .7 with \"burial\" as baseline category), and highest if bodies were removed (OR = 1.7). Finally, for those individuals who had heart disease mentioned on their death certificate, patients who died at a residence (not a nursing home) were most likely to have ischemic heart disease selected as the underlying cause of death (90.5% or an OR= 1.8 compare to hospital in-patient). Unlike Ischemic heart disease, only 29.3% of deaths with diabetes mentioned on the certificate had it selected as the underlying cause of death. While only 2.3% of deaths with diabetes mentioned occurred in the youngest age group (0--44 years), (Table [4](#T4){ref-type="table"}) this group has a much larger probability of having diabetes be the underlying cause compared to non-underlying (51.7% reported as underlying). Men were less likely to have diabetes selected as underlying when it is mentioned on the certificate than women. Blacks and Native Americans both have significantly higher odds (OR = 1.2 and 1.4, respectively) of diabetes being the underlying cause given that it was mentioned as compared to Whites. The role of autopsy is that it was less likely diabetes was reported as underlying (OR = 0.7) when one was performed than if one was not. Moreover, if a coroner was the death certifier, diabetes was less likely to be reported as underlying. An increase in the reporting of diabetes as underlying was found for deaths that were removed. Finally, deaths occurring outside of the hospital inpatient setting all show increased odds of diabetes being selected as the underlying cause of death when it has been mentioned. We also examined what other leading causes of death showed up as underlying when ischemic heart disease or diabetes was mentioned on the certificate. As mentioned above, 77.1% of the individual with ischemic heart disease mentioned on their death certificate had it reported as the underlying. The second most common underlying cause of death when ischemic heart disease was mentioned was, in fact, diabetes (3.4% of the time underlying), followed by cerebrovascular disease (2.7% of the time underlying), then pneumonia (1.5% of the time underlying). When we focus on the population that has diabetes mentioned on the death certificate, as mentioned above 29.3% of the time diabetes is selected as the underlying, and the second most common underlying cause selected is ischemic hear disease at 25.4%, followed by cerebrovascular disease at 7.75% then followed by Other diseases of the heart 2.9%. Discussion ========== Distinct differences in the frequency of multiple causes of death were found across time, age, race, disposal method and place of death. Definitive explanations for the differences cannot be given based on this study, but it is of interest to consider plausible explanations which may motivate further investigation. The increased reporting of non-underlying causes of death as the age of the decedent increases is likely due to actual increases in co-morbidity with age, hence would be explained by actual differences in the causes of death. The differences found in reporting of multiple causes of death for the other factors may be partly due to systematic reporting biases. According to the NCHS All Mortality Altas \[\[[@B11]\], p. 3\], the quality of cause of death determination in the US is affected by the accuracy and completeness of information -- from medical diagnosis to final coding and processing of underlying cause of death. Although since 1968 the automated selection of the underlying cause of death has helped to reduce coding and processing errors, the completeness and accuracy of the information supplied on the certificate and the decedent\'s medical diagnosis remain as potential sources of error. If the certifier enters only one underlying cause and no other causes, then that cause will have to be selected as the underlying and there will not be multiple causes of deaths for that record. It is interesting to note that \"Symptoms and ill-defined conditions\" is the 5^th^most commonly reported cause of death to be the only cause of death listed on the certificate. This reporting of it as the only cause of death pushes it up to be the overall 12^th^leading cause of death. If almost any other cause would be listed simultaneously on the death certificate, this code would not end up as underlying. The decreasing trend in reporting multiple causes over the decade may be reflective of a gradual change in the procedures of death certification. It would be of interest to consider this trend across different states and longer periods of time including shifts from one ICD coding system to the next. Previous literature offers various plausible explanations to what contributes to the inaccuracy of reporting causes of death. The cause of death reported on the death certificates depends on a person\'s disease history that leads to death \[[@B12]\]. If a person dies after a long, well-characterized illness, the cause of death on the certificate is likely to be more accurate than a sudden or unobserved death. Also, when lack of adequate information on the decedent\'s disease history, the more narrowly characterized the cause of death on the certificate, the more likely it is to be in error. If we assume that reporting multiple causes on the death certificate can be considerd a proxy for level of familiarity of the death certifier with the patient, we would expect that a death which occurs in a hospital or nursing home would be more likely to have multiple causes reported, possibly due to a better documentation of disease history. On the other hand, death at the ER and in particular at the person\'s residence, which is conceivably often sudden should show a much lower percentage of multiple cause of death reporting. Analysis results from this current study match such speculations, supporting the argument that a good understanding of disease history is crucial. Still another result that supports this conclusion is the fact that performing autopsy, which gain better understanding of the disease condition, increased the probability of reporting multiple cause of death. Gender and race can also play a role in the accuracy of reporting. Lloyd \[[@B13]\] showed that positive predictive value of the death certificate tended to be lower in women than in men. Although no large differences were seen between men and women with respect to frequency of multiple causes, there was a higher percentage of multiple causes reported for Native Americans. It is conceivable that the high percentage of multiple cause of death observed for Native Americans might be associated with the geographic factors of concentrated residence and the unique practices of local clinics. Moreover, results (not shown) indicate differences exist across counties of Minnesota in the reporting of multiple causes of death ranging from 50% to 80%. These results support suggestions for better standardized training for physicians and coroners. Similar to the case of reporting multiple cause of death, the selection of ischemic heart disease and diabetes as underlying compared to non-underlying differs across the several factors considered. The implications of these differences across demographics are that mortality rates would be differentially affected when underlying cause of death is used compared to any mention cause of death. For example, for diabetes we might conclude that diabetes is being under-reported in Whites compared to Blacks, Native Americans and Hispanics if only underlying cause of death were considered since the proportion of diabetes as underlying to mentioned is substantially lower in Whites. This is not to say there is any inaccuracy in the way it is being coded but it points out where multiple cause of death reporting will provide a different perspective than underlying. Nevertheless, studies have shown the sensitivity and positive predictive value of the death certificate are particularly poor with regard to stroke and diabetes \[[@B14]\]. Furthermore, Lloyd \[[@B13]\] concluded that physicians may use coronary heart disease as a \"default\" cause when facing some unknown cause of death cases. The fact that individuals with autopsy performed have lower probability of having heart disease selected as underlying when it is mentioned might suggest that heart disease is often over-assigned as the default disease when no further medical details are available. This is further demonstrated by the very high ratio of ischemic heart disease being coded as the underlying compared to non-underlying cause of death when the death occurred at the person\'s residence. As mentioned in the introduction, one limitation of this research is the fact that there is no outside panel of experts who decide independently what the true causes of death are for each decedent, thus whether the associations we found are due to actual differences or reporting bias cannot be discerned. Therefore, this study cannot provide sensitivity or specificity per se, but it aims to identify factors that are associated with variability in reporting multiple cause of death and that perhaps contribute to inaccuracy in reporting underlying cause. Conclusions =========== There is much to be learned from multiple cause of death data. It provides ways of looking at mortality data that go well beyond the typical examination of underlying cause of death. Future research is needed to understand further what the greatest concerns are about the accuracy of reporting causes of death. Multiple cause of death data have the potential to help point out potential concerns in the accuracy as well as provide a more complete picture of mortality for causes which are frequently not recorded as the underlying cause of death. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= MW conceived of the study and wrote most of the manuscript. JH performed the statistical analysis and wrote part of the manuscript. JO provided access to the data and collaboration regarding processes underlying data collection. DD provided details about cause of death reporting and collaboration regarding processes underlying data collection. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2288/5/4/prepub>

PubMed Central

2024-06-05T03:55:52.696435

2005-1-17

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548504/", "journal": "BMC Med Res Methodol. 2005 Jan 17; 5:4", "authors": [ { "first": "Melanie M", "last": "Wall" }, { "first": "Jinzhou", "last": "Huang" }, { "first": "John", "last": "Oswald" }, { "first": "Diane", "last": "McCullen" } ] }

PMC548505

Background ========== Obesity is the most common health problem in developed countries \[[@B1]\]. It is a chronic disease and should be treated as such. Its prevalence is increasing worldwide \[[@B2]\]. In the United States, it is estimated that 64% of the adult population is either overweight or obese with a body mass index (BMI; kg/m^2^) above 25 \[[@B3]\]. The rate of obesity is increasing \[[@B4]\] and has risen by more than 75% in the USA since 1980 \[[@B5]\]. In 2001, the prevalence of obesity (BMI ≥ 30) was 20.9% vs 19.8% in 2000, an increase of 5.6% \[[@B6]\]. In Israel, according to a survey of the Nutrition Department of the Ministry of Health, 55% of adult (ages 25--64) women and 59% of adult men have a BMI above 24.9 \[[@B7]\]. Obesity is associated with increased prevalence of many serious chronic diseases such as diabetes mellitus, hypertension, dyslipidemia, and coronary heart disease \[[@B8],[@B9]\]. It may be responsible for approximately 300,000 deaths in the USA per year \[[@B10]\]. In the Nurses Health Study, the 14-year mortality rate for women with a BMI greater than 32 was more than double that of women with a BMI of less than 19 \[[@B11]\]. Obesity now ranks second only to smoking as a cause of preventable death but, soon, obesity may surpass smoking as the leading cause of preventable death in the USA \[[@B12]\]. In the USA, 19% of deaths from coronary disease and 62% of deaths from diabetes can be attributed to obesity \[[@B13]\]. The risk of death from all causes increases in moderately and severely overweight men and women of all age groups \[[@B14]\]. Diet and exercise have limited effectiveness on long-term maintenance of weight loss \[[@B15]\]. Within five to seven years, 95% of all patients regain the lost weight or more \[[@B16]\]. Pharmacotherapy in combination with a reduced energy diet improves long-term efficacy \[[@B17]\]. Loss of 5--10% of their initial body weight substantially improves the health of obese patients and modifies their cardiovascular risk factors \[[@B8],[@B18]\]. Despite growing information on the pathophysiology of obesity and its high prevalence, obesity and obesity-related diseases are still under-diagnosed and untreated by family physicians \[[@B19]\]. Most family physicians cite lack of time, resources, reimbursement from insurance companies, or knowledge of effective interventions as significant barriers \[[@B20]\]. The intervention of primary physicians during a ten minute physician/patient encounter and telephone consultation with a community dietitian resulted in a significant decrease in the weight of patients \[[@B20]\]. Recently, several physicians\' organizations have issued recommendations for treating obesity to family physicians, including instructions in nutrition, physical activity and medications. Such recommendations were based on a number of studies that proved the effectiveness of family physician weight-reduction programs, when based on the readiness of patients to make necessary lifestyle changes and use of appropriate techniques to increase the willingness of the patient to make necessary changes \[[@B21]-[@B24]\]. The purpose of this study was to examine if more efficient and effective weight-reducing treatment can be given in the family doctor setting. The study compare a non-pharmacological intervention with drug intervention (orlistat) and compare regular management with more intensive family physician based management.. Methods ======= Study design ------------ The study was conducted in three primary care clinics in an urban area in central Israel. The family physicians who took part in this study participated in 80 hours CME course dealing with obesity treatment in Israel. The patients were divided into three groups according to their choice. Patients in groups A and B were treated with orlistat at 120 mg TID. Orlistat (Xenical ^®^) is a lipase inhibitor for obesity management that acts by inhibiting the absorption of dietary fats. The patients in group A received in addition a personal reduced-energy diet and met with a family practitioner and a clinical dietitian once every two weeks. The personal diet was created according to the daily schedule and preferred foods of the individual, emphasizing low-fat foods. The patients received instructions regarding the importance of physical activity and, at each meeting, realistic intermediate goals for achieving two or three small changes in eating habits and physical activity, based on the patient\'s desires, were determined. The obstacles to change and ways to overcome them were discussed. Support, based on improvements in the patient\'s health parameters such as an improvement in a blood test, was given. Some of the patients helped with self-criticism, by keeping food and physical activity diaries and by grading their own goal accomplishments. Patients were taught how to resist temptation and reward themselves for success. We also recruited the support and encouragement of the patient\'s family. At each meeting, the time of the next meeting was determined and it was emphasized that the most important thing for the patients to do was to attend the meetings, even if their goals were not achieved. Group B patients were treated with 120 mg orlistat t.i.d., a general formulated reduced-energy diet and follow-up by the family physician once every four weeks for weighing and prescription renewal. In groups A and B, patients were asked at each meeting if any side effects of orlistat appeared. Patients in group C were given a personal low-calorie diet, designed according to their preferences, and followed-up by a clinical dietitian once a month. The prescribed daily caloric intake was equal in the three groups and was 1200 calories per day for women and 1500 calories per day for men. Before the intervention, every patient received an explanation of the three treatments, the importance of reducing weight, and how excess weight affects their health. All were instructed about the recommended rate of weight loss and a final weight reduction goal of at least 5% of their initial weight within half a year was established. Patients who achieved the 5% reduction goal before the end of the study could choose either to stop the intervention or to complete the study period. Patients -------- Obese (BMI \> 30) patients of either sex or patients with a BMI above 27 plus two or more cardiovascular risk factors, aged 20--75 years, were eligible for the study. Patients were excluded if they were pregnant or lactating or if they had any contraindication against using orlistat (chronic malabsorption syndrome, cholestasis, pancreatic disease). Before the intervention, patients underwent an initial screening that included recording of a complete medical history, measurement of vital signs, body weight and height, and calculation of the BMI. Laboratory analysis included a lipid profile. The readiness of the patient to receive treatment was assessed. At the beginning of the intervention, all participants were in the third stage of readiness (\"the preparation stage\") according to the Transtheoretical Model of Behavior Change. An adverse event of any dose of orlistat was considered serious if it resulted in death, was life-threatening, required hospitalization, or resulted in significant disability. Efficacy measures ----------------- The main measure was weight loss. Each patient was weighed during each meeting. The primary endpoint was reduction of at least 5% of the initial weight during the study period (six months). Achievement of this goal was defined as successful treatment. Another measure was improvement in the lipid profile. The second lipid profile was done to half of the patients, only those who had dyslipidemia in the first profile had been offered a second profile. Statistical analysis -------------------- Data on patients\' background and weight-reduction results between groups were compared using the Chi square test. Continuous data of the groups, i.e., measurements at the beginning of the study and continuous background data, were compared using one-way tests (ANOVA). When significant statistical differences were found among the three groups, the Tukey Post Hoc test was used to examine the statistical difference between each group of two. ANCOVA was also used and, if there was a difference amongst the groups, the significance (for the measured parameter) was checked using the Bonferonni technique. Results ======= Two hundred and twenty-five patients participated in the study. Their demographic characteristics, history of cardiovascular disease, and initial lipid profile are shown in Table [1](#T1){ref-type="table"}. There were no significant differences between groups in average age, initial BMI or gender of the participants. The average length of follow-up and the number of meetings varied among groups. There were no cases of significant side effects that required stopping orlistat treatment of any of the participants. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Participants\' demographic data, initial lipids profile, by treatment group ::: ------------------------------------------------------------------------------------------------------------------ Group A Group B Group C P\* value -------------------------------------------------------- ----------- ----------- ----------- --------------------- Number of patients 62 112 51 Age (years +/- SD) 47.3 ± 11 46.8 ± 12 51 ± 9.6 NS Gender (% female) 71 74 61 NS Ischemic heart disease (%) 0 4 0 NS Hypertension (%) 44 51 27 P \< 0.05 Diabetes mellitus (%) 9 18 20 NS Dyslipidemia (%) 16 38 66 P \< 0.001 Initial body weight Initial body mass index (BMI; kg/m^2^+/- SD) 33 ± 3.8 34 ± 4.4 31 ± 3.6 P \< 0.01\ 34 \> 31(B \> C) Initial triglycerides (mg/dl, +/- SD) 170 ± 53 184 ± 49 255 ± 205 P \< 0.01\ 170 \<255\> 183\ (A \< B \> C) Initial low density lipoproteins (LDL; mg/dl, +/- SD) 150 ± 30 156 ± 36 152 ± 44 NS Initial high density lipoproteins (HDL; mg/dl, +/- SD) 42 ± 7.0 44 ± 6.7 47 ± 14.9 NS Average length of treatment (weeks, +/- SD) 13 ± 12.0 9 ± 4.7 23 ± 12 P \< 0.001\ 23 \> 13 \> 9\ (C \> A \> B) Number of meetings with physician/dietitian (+/- SD) 4.3 ± 2.0 3.5 ± 1.5 5.2 ± 2.9 P \< 0.001\ 5.2 \> 3.5 (C \> B) ------------------------------------------------------------------------------------------------------------------ NS = Not significant. Group A -- Orlistat, a personal reduced-energy diet and a meeting with a family practitioner and a clinical dietitian once every two weeks. Group B -- Orlistat, a general formulated reduced-energy diet and follow-up by the family physician once every four weeks. Group C -- a personal low-calorie diet and follow-up by a clinical dietitian once a month. \* When significant statistical differences were found among the three groups, we examined the statistical difference between each group of two. ::: Patient-reported adverse events in the orlistat-treated groups were all related to the gastrointestinal tract. The most commonly reported events were flatulence with discharge (9.2%), fatty or oily stool with increased defecation (9.2%), feeling of fullness in the stomach (4.6%), and constipation (1.7%). There were no statistically significant differences in side effects between groups A and B. In group A, 11 patients (17.7%) stopped the treatment for the following reasons: cost of the medication (47%), lack of time (33%), or dissatisfaction (20%). In comparison, ten patients (8.9%) in group B stopped the treatment, mainly because of the cost of the treatment (65%) or low motivation (23.5%). The reasons for stopping treatment significantly differed between groups A and B (*p*= 0.03). The percentage of patients who attained their weight reduction goals was largest in group A where patients received orlistat and intense follow-up, in addition to a personally-designed diet (Fig. [1](#F1){ref-type="fig"}). Changes in lipid profiles are shown in Table [2](#T2){ref-type="table"}. The treatment resulted in a significant reduction in triglyceride levels in all groups, a significant reduction of low density lipoproteins (LDL) in groups A and B and no significant difference between initial and final high density lipoproteins (HDL) in any group. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Weight loss by treatment group\* ::: ![](1471-2296-6-5-1) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Changes in lipid profile during treatment according to treatment group^1^(paired sample *t*test) ::: Group A Group B Group C -------------------------------------------------- ----------- ----------- ----------- Number of patients^2^ 32 55 28 Initial triglycerides (mg/dl, +/- SD) 170 ± 53 184 ± 49 255 ± 205 Final triglycerides (mg/dl, +/- SD) 139 ± 43 153 ± 35 165 ± 60 Delta (+/- SD) -31 ± 21 -31 ± 25 -90 ± 187 P value (pre-post) \<0.001 \<0.001 0.01 Initial low density lipids (LDL; mg/dl, +/- SD) 150 ± 30 156 ± 36 152 ± 44 Final LDL (mg/dl, +/- SD) 129 ± 28 143 ± 32 147 ± 34 Delta -21 ± 26 -12 ± 16 -5 ± 34 P value (pre-post) \<0.001 \<0.001 NS Initial high density lipids (HDL; mg/dl, +/- SD) 42 ± 7.0 44 ± 6.7 47 ± 14.9 Final HDL (mg/dl, +/- SD) 43 ± 6.6 45 ± 6.7 48 ± 15.3 Delta (+/- SD) 0.9 ± 2.9 0.8 ± 3.3 1.4 ± 8.8 P value (pre-post) NS NS NS NS = Not significant. ^1^See footnotes to Table 1 ^2^Patients with pre and post lipid profile analyses ::: Patients in Group A reduced 5.12 kg (range of 5--8 Kg.) of their initial body weight, patients in Group B reduced 7.8 kg (range 10--12 Kg.) from their initial body weight and patients in Group C reduced 3.12 kg (range 5--6 Kg.) of their initial body weight. Discussion ========== Obesity is a worldwide problem. Treatment of a patient for obesity involves two processes: evaluation of the severity of the obesity and general health condition of the patient, and management which includes guidance in how to gradually reduce weight and maintain the new weight together with imparting healthy lifestyle habits and keeping track of improvement. In this study, we examined weight-reduction techniques that can be carried out in the setting of a community family practice. Intense treatment, combining frequent counseling by the family physician and a dietitian with medications (group A), resulted in the best weight reduction and lipid profile improvement in the short period of this study, as was reported earlier in special weight reduction clinics \[[@B25]-[@B27]\] The effectiveness of interventions in primary care setting are controversial \[[@B28]-[@B30]\]. Beermann et al \[[@B28]\] in a community survey of 792 patients found that Orlistat was not prescribed according to the approved indication in the majority of cases. The dropout rate was high and most patients had minor gain from the treatment. Linne et al \[[@B29]\] noted that success rate of Orlistat in primary-care practice is limited by failure to follow prescribing recommendations. A simple questionnaire to 70 patients revealed that in many cases the referral physician had not observed basic rules and regulations, nor given appropriate information on Orlistat use. Hauptman et al \[[@B30]\] in a study conducted in seventeen primary care centers in the United States. The study indicates that orlistat is an effective adjunct to dietary intervention in the treatment of obesity in primary care settings. There are many advantages to a program involving cooperation between the family physician and the dietitian. The family physician is acquainted with the patient for a longer time than a dietitian. The physician is familiar with the patient\'s health condition, medications taken by the patient, the patient\'s environment and lifestyle and can recommend a treatment suitable to the patient\'s personality and lifestyle. The family physician is knowledgeable about weight-reducing drugs and possible side effects. The patient trusts and has confidence in the family doctor. Together with the dietitian, a personal diet appropriate to the drug treatment can be created. Obesity is a chronic disease. The physician and the patient must recognize that obesity treatment is a prolonged process that extends a lifetime. Since family physicians are usually familiar with their patients as well as with the patient\'s family and environment for many years, family physicians know what changes the patient can achieve. They can recruit family members to support the necessary lifestyle changes. Also, because of their training, family physicians are the most suitable professional to holistically treat obesity and its complications. There were several limitations in our study. One limitation was the non-random division of patients into groups A, B and C. It is possible that the patients who chose drug treatment differed from those who chose a diet only. Perhaps they were more ready for the weight-reduction process and to expend money for the drug (in Israel, there is a patient co-payment for medications). The rate of women amongst the patients seeking treatment was higher than the rate of men, as in other reports \[[@B26],[@B27]\]. Hence, we assume that our study represented the segment of the population more prone to try dieting and weight-reduction programs. Ways to increase the number of men participating in weight-reduction programs must be found. The study periods for groups A and B (which received orlistat) were shorter than for group C, partly because there is significant co-payment for orlistat and partly because the targeted weight had been achieved earlier. The drug was well tolerated with minimal gastrointestinal side effects. The patients were treated by family physicians that were orientated toward and trained in weight reduction. Results might have been less successful with other physicians. Hence, family physicians should be trained in the subject by increasing both their knowledge and their treatment skills. Healthcare funds must recognize the importance of weight reduction so that they will allocate the necessary additional time and resources of their physicians, clinics and multi-field staff. This study evaluated only the weight reduction period. Long-term results and whether or not the patient maintained the lifestyle change for a long period were not examined. Further studies should examine the best program for maintaining the new weight. In conclusion, this study showed that within the setting of the family practice it is possible to carry out an effective program of weight reduction and achieve significant weight loss. The addition of orlistat can further improve results. We believe that by providing family physicians with the proper tools, similar success can be achieved in many more clinics. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= AF and SP conceived and designed the study, participated in the collection, analysis and interpretation of data and drafted the manuscript. SV Participated in the statistical analysis, interpretation of data and draft of the manuscript. MS and EF participated in the design of the study, data collection and interpretetion. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2296/6/5/prepub> Acknowledgements ================ None

PubMed Central

2024-06-05T03:55:52.702473

2005-1-29

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548505/", "journal": "BMC Fam Pract. 2005 Jan 29; 6:5", "authors": [ { "first": "Amiel", "last": "Feigenbaum" }, { "first": "Shmuel", "last": "Pasternak" }, { "first": "Efrat", "last": "Zusk" }, { "first": "Miri", "last": "Sarid" }, { "first": "Shlomo", "last": "Vinker" } ] }

PMC548506

Background ========== Familial Juvenile Hyperuricemic Nephropathy (FJHN) (McKusick 162000) is an autosomal dominant disorder characterized by hyperuricemia, decreased urinary excretion of urate and the development of progressive chronic interstitial nephritis. Renal impairment usually appears between 15 and 40 years of age, leading to end-stage renal disease (ESRD) within 10 to 20 years \[[@B1]\]. A candidate gene for FJHN has been positioned in 16p11.2-12, together with evidence for genetic heterogeneity \[[@B2],[@B3]\]. The candidate gene was later found to map within the same genetic interval as MCKD2, a *locus* responsible for medullary cystic kidney disease, therefore suggesting that FJHN and MCKD2 are 2 facets of the same disease \[[@B1]\]. The marked thickening of tubular basement membranes observed in FJHN closely resembles the renal histological findings of the nephronophthisis-medullary cystic kidney disease complex (NPH-MCKD). Diseases of this group share the macroscopic feature of cyst development at the corticomedullary border of the kidney and the renal histological triad of tubular basement membrane disintegration, tubular atrophy with cyst development and interstitial cell infiltration with fibrosis \[[@B4]\]. Within this complex, clinical entities can be distinguished based on the mode of inheritance, the age of onset for ESRD and the presence of extra-renal involvement. For the recessive forms of the disease 4 different genes have been cloned. The NPHP1 gene \[[@B5],[@B6]\] and NPHP4 \[[@B7]\] are responsible for juvenile forms of NPH, while NPHP2 \[[@B8]\] and NPHP3 \[[@B9]\] account for, respectively, the infantile and adolescent forms. MCKD, the autosomal dominant disorder that presents in early adulthood, is usually accompanied by the detection of corticomedullary cysts on imaging studies. MCKD1 maps to 1q21 and remains to be cloned, while MCKD2 has been positioned in 16p12. Mutations in the uromodulin/Tamm-Horsfall protein coding UMOD gene located within the critical interval of FJHN and MCKD2 at 16p11.2-12 were recently identified in FJHN and MCKD2 families \[[@B10]\], therefore providing definite evidence that MCKD and FJHN are allelic disorders. Meanwhile, a mutation cluster in exon 4 of UMOD was reported for both diseases \[[@B11],[@B12]\]. We here describe a *novel*heterozygous *missense*mutation in affected individuals from a Portuguese FJHN family that also displays corticomedullary cysts on ultrasound examination. This mutation, c.920A→C, resides in exon 5 and is the fourth reported outside exon 4. Case presentation ================= Case report ----------- The proband is an affected female who was first evaluated at the age of 24, when she presented with a gout attack and hyperuricemia. At age 27 she was told having renal failure. However, a renal biopsy was not performed. At age 44, serum creatinine was 2.8 mg/dl and ultrasound imaging detected numerous renal cysts. Renal disease slowly progressed and the patient reached ESRD when she was 49 years old. Her father died at the age of 55 from ESRD and suffered from hyperuricemia and gout. The proband\'s only son is also affected. At the age of 18 years he had a gout attack. On the initial evaluation, serum uric acid was 15 mg/dl and serum creatinine 1.6 mg/dl. Renal cysts were also detected on ultrasound examination. Mutation analysis ----------------- Informed consent was obtained from tested individuals. Genomic DNA was isolated from peripheral blood leucocytes and the coding region of the UMOD gene was screened for mutations by direct sequencing of PCR products. We used a set of primers previously described \[[@B10]\] except in exons 4 and 5, for which different additional internal sequencing primer were designed based upon sequences from GenBank (accession numbers NT\_024776.6 and M17778). Results ------- A *novel*heterozygous *missense*mutation, c.920A→C, was detected (Figure [1](#F1){ref-type="fig"}) and found to segregate with the disease in this family. The mutation results in a Lys to Thr at position 307. This mutation was not detected in any of the 100 control chromosomes tested. In fact, no polymorphism affecting the translation of uromodulin was detected in 100 control chromosomes in a previous report \[[@B10]\] and we are, therefore, excluding the possibility of this allele being a mere polymorphism. In addition, the affected mother was found to be homozygous for the T allele in the common T to C transition synonymous polymorphism at codon C174. Discussion ---------- The UMOD gene has 12 exons and codes for the 640 amino-acid uromodulin, a glycsoyl phosphatidylinositol (GPI) anchored protein that accounts for the primary structure of the 85-kD Tamm-Horsfall glycoprotein (THP). THP is the most abundant protein in the urine and, in normal kidney, uromodulin expression is restricted to the thick ascending limb (TAL) and distal convoluted tubule. Urinary excretion occurs by proteolytic cleavage of the GPI counterpart at the luminal surface of TAL. It has been suggested that one of THP major role is of an urinary anti-adherence factor preventing type 1 fimbriated E coli from binding to the urothelial receptors \[[@B13]\]. The majority of the mutations so far published are clustered in exon 4, between codons 52 and 282, and most are *missense*mutations affecting cysteine residues (Table [1](#T1){ref-type="table"}). Exon 4 contains 3 calcium binding epidermal growth factor (cbEGF)-like domains, between residues 31 and 148. A fourth potential cbEGF-like domain extends from amino-acids 281--336, throughout exon 5. They contain 6 conserved cysteine residues responsible for the protein\'s tertiary structure, as a result of intramolecular disulfide bonding. It has been hypothesized that protein misfolding, consequence of mutations in these cbEGF-like domains, may affect uromodulin intracellular trafficking and lead to cellular protein accumulation and apoptosis \[[@B14]\]. The release of cells debris and uromodulin aggregates in the interstitium could stimulate an inflammatory response and, in addition, be responsible for tubular obstruction and medullary cyst formation. It has been proposed that hyperuricemia in these patients is secondary to a reduced TAL sodium reabsorption with volume contraction and a compensatory increase in proximal urate reabsorption. The role of hyperuricemia in the chronic interstitial nephritis remains to be clarified, since the deposition of sodium urate crystals in the medullary interstitium does not occur in these patients. The mutation c.920A→C here reported, replaces the basic amino-acid Lys for the uncharged polar Thr at position 307 (p.K307T), being the fourth mutation described in exon 5. The affected residue locates within the fourth cbEGF-like domain and is immediately preceded by a highly conserved cysteine residue. Conclusions =========== It has been referred that exon 4 sequencing should become the first diagnostic test in patients with chronic interstitial nephritis with gout or hyperuricemia, even in the absence of a family history \[[@B11]\]. In view of the increasing number of mutations in exon 5 being identified, we now recommend that exon 5 should be included in the initial sequencing effort, since otherwise nearly 12% of UMOD mutations can be missed. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JC was responsible for the study design and drafted the manuscript. JC and CC carried out the molecular analysis. AG collected the clinical data. JR participated in the study design. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2350/6/5/prepub> Acknowledgments =============== This work was supported by a grant from Roche Farmacêutica Química, Portugal. We thank all members of the affected family for their participation. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **a)**Sequence of the index patient with exon 5 c.920A→C heterozygous *missense*mutation. **b)**The same mutation sequenced with a reverse primer. ::: ![](1471-2350-6-5-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### UMOD mutations reported in the literature. ::: Mutation exon reference --------------------------------------------------- ------ ---------------- c.156T→G; p.C52W 4 \[15\] c.176A→C; p.D59A 4 \[11\] c.230G→A; p.C77Y 4 \[16\] c.278\_289del/insCCGGCTCCT; p.V93\_G97del/insAASC 4 \[12\] c.307G→T; p.G103C 4 \[10\] c.334T→C; p.C112R 4 \[11\] c.376T→C; p.C126R 4 \[16\] c.383A→G; p.N128S 4 \[16\] c.403T→A; p.C135S 4 \[15\] c.443G→A; p.C148Y 4 \[10\] c.444T→G; p.C148W 4 \[14\] c.449G→C; p.C150S 4 \[14\] c.509G→A; p.C170Y 4 \[11\] c.529\_555del; p.H177\_R185del 4 \[10\] c.553C→G; p.R185G 4 \[18\] c.553C→A; p.R185S 4 \[11\] c.563\_661del; p.E188\_L221del 4 \[11\] c.584G→T; p.C195F 4 \[15\] c.605G→C; p.W202S 4 \[15\] c.610C→G; p.R204G 4 \[11\] c.649T→C; p.C217R 4 \[10\] c.649T→G; p.C217G 4 \[11\] c.665G→C; p.R222P 4 \[11\] c.668G→A; p.C223Y 4 \[17\] c.674C→T; p.T225M 4 \[11\] c.674C→A; p.T225K 4 \[12\] c.707C→T; p.P236L 4 \[15\] c.744C→G; p.C248W 4 \[12\] c.764G→A; p.C255Y 4 \[16\] c.844T→C; p.C282R 4 \[11\] c.898T→G; p.C300G 5 \[16\] c.920A→C; p.K307T 5 present report c.943T→C; p.C315R 5 \[14\] c.950G→A; p.C317Y 5 \[14\] :::

PubMed Central

2024-06-05T03:55:52.705154

2005-1-27

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548506/", "journal": "BMC Med Genet. 2005 Jan 27; 6:5", "authors": [ { "first": "Joaquim", "last": "Calado" }, { "first": "Augusta", "last": "Gaspar" }, { "first": "Carla", "last": "Clemente" }, { "first": "José", "last": "Rueff" } ] }

PMC548507

Background ========== In the last few years, exciting advances in the biology and molecular genetics of the development of *Plasmodium*parasites in their mosquito vectors \[[@B1],[@B2]\] have led to the creation of transgenic mosquitoes that are partially resistant to malaria infection \[[@B3]\], bringing the efforts to control malaria with the techniques of transgenesis a major step forward \[[@B4],[@B5]\]. A crucial aspect of these advances is, of course, the fact that the mosquito\'s genetic make-up determines, at least partly, its resistance to malaria infection \[[@B6],[@B7]\], giving hope for the possibility that key genes controlling resistance may be identified. This hope has been reinforced by the recent identification, in a rodent model of malaria, of several mosquito immune genes that affect parasite development \[[@B8],[@B9]\]. Unfortunately, several aspects of the current knowledge make it difficult to estimate the relevance of such laboratory-based studies to control malaria in natural conditions \[[@B10]\]. One of the crucial aspects is that most studies on the genetics of resistance have considered the response of a mosquito line or colony to a single malaria genotype, while any malaria control programme based on the release of resistant mosquitoes in highly endemic areas can be effective only if mosquitoes are resistant to all the genotypes of the parasite \[[@B11]\]. Because of the limited genetic variation in laboratory colonies compared to natural populations of mosquitoes \[[@B12]\] and the large diversity of natural populations of malaria parasites \[[@B13]\], it is currently far from clear whether this will be possible. One potential problem of the genetic diversity in natural populations could be that, as in many other host-parasite systems (e.g. plant-fungus \[[@B14]\], snail-schistosome \[[@B15]\], bumble-bee-trypanosome \[[@B16]\], *Daphnia*-bacterium \[[@B17]\]), the outcome of the interaction is determined by an interaction between host and parasite genotypes. In systems governed by such genotype by genotype interactions, individual hosts are resistant to only a portion of the parasite genotypes and, reciprocally, individual parasites can infect only particular host genotypes \[[@B18]\]. In other words, no parasite is best at infecting all hosts, and no host is best at resisting all parasites, so that the success of infection depends on the specific combination of the two opponents. Despite its potentially important role for malaria epidemiology and control, such a genetic specificity of host-parasite compatibility between malaria parasites and their insect vectors have never been investigated. This study examines the potential for genotype by genotype interactions in the combination that is most important for the epidemiology of malaria -- *Plasmodium falciparum*and *Anopheles gambiae*. Malaria genotype by mosquito genotype interactions were tested with a standard procedure of quantitative genetics from measurements of the resistance of different genetic backgrounds of the mosquito *A. gambiae*, a major malaria vector in sub-Saharan Africa, to different isolates of the human malaria parasite *P. falciparum*. The parasite isolates were obtained from naturally infected children in western Kenya that harboured gametocytes, the infective stage of the parasite. The genetic backgrounds of mosquitoes were \'mosquito families\' generated as the F1 offspring of single egg-laying females. Each mosquito family was challenged with each parasite isolate, and all mosquitoes were simultaneously fed on the blood of the gametocyte carriers via membrane-feeding. This basic design was repeated three times throughout three successive experimental blocks that involved different families and isolates, giving a total of 18 mosquito families, 11 parasite isolates, and 62 specific interactions. As in previous studies \[[@B7]\], the resistance of mosquitoes was quantified with the proportion of blood-fed females that developed oocysts and with the number of oocysts. The genotype by genotype interaction on mosquito resistance was estimated according to standard quantitative genetic methods as the interaction effect in a statistical analysis between the parasite isolate and the mosquito family \[[@B17],[@B19]\]. These methods are based on the idea that sibs are genetically more similar that non-sibs. Therefore, partitioning the variance of any trait (e.g. number of oocysts) among families (individuals sharing a mother) and within groups of sibs give an indication of the extent to which the trait has a genetic basis \[[@B20]\]. Methods ======= Mosquitoes ---------- The mosquitoes used in this study came from a colony that had been established in 2001 from *A. gambiae s.s*. caught in the area surrounding Mbita, a small village on the shore of Lake Victoria in Suba District (western Kenya). These mosquitoes had been initially adapted to feed on a Parafilm^®^membrane, and then maintained in standard insectary conditions using a rabbit as a blood source for routine maintenance. Females of the colony were blood-fed on a rabbit and allowed to lay eggs in individual vials. Immediately after hatching, each larva was individually placed in one well of a 12-well plate with three mL of filtered lake water. They were fed daily on a standard diet of Tetramin^®^fish food (0.06 mg per larva on day 0; 0.12 mg on day 1; 0.24 mg on day 2; 0.36 mg on day 3; 0.48 mg on day 4; 0.6 mg on the following days). Adults were kept in an insectary and supplied with a 6% glucose solution and cotton soaked with distilled water. The temperature and humidity in the insectary followed the daily environmental fluctuations. So that mosquito age at pupation did not affect the success of infection, only females that pupated seven days after hatching were used. The wing length of the mosquitoes, measured from the tip (excluding the fringe) to the distal end of the allula with a precision of 0.02 mm, was used as an indication of body size \[[@B21]\]. Where both wings could be measured, the mean of the two lengths was used. Gametocyte carriers ------------------- *P. falciparum*carriers were recruited from the two- to 10-year old children in the rural area around Mbita, from December 2003 to January 2004. Finger-prick blood samples were collected and thick blood smears were air-dried, stained with 8% Giemsa during 15 minutes, and examined microscopically for the presence of *P. falciparum*. Children with asexual parasitemia (\>1,000 parasites/μL) were immediately treated with sulfadoxine-pyrimethamine according to national guidelines. Asymptomatic gametocyte-positive children were recruited for the study after their parents or guardians had signed an informed consent form. The Kenyan and the United States National Institute of Health ethical review committees approved this recruitment procedure. Experimental infections ----------------------- For logistic reasons, the experiment was repeated three times, and within each experimental block infections were done simultaneously. For each of the three blocks, *P. falciparum*isolates were collected from gametocyte carriers that had been identified one or two days before, and used to feed the mosquitoes on the same single day (block 1: December 14, 2003; block 2: January 23, 2004; block 3: January 28, 2004). The gametocyte densities were assessed just before blood withdrawal on a blood smear (as described above) by counting against 500 leukocytes, and converted to numbers of parasites per μL by assuming a standard leukocyte count of 8,000/μL. Although gametocyte densities in the venous blood and the peripheral finger-prick blood might differ, potential differences were assumed to be proportional among the isolates. A sample of five mL of venous blood was collected from each gametocyte carrier in a heparinized tube, 400 μL of which were stored at -20°C for further parasite genotyping. So that the importance of human factors such as transmission-blocking immunity \[[@B22]\] was reduced, the blood was centrifuged at 37°C for three minutes at 2,000 *g*and the autologous serum was replaced with the same volume of a pool of AB serum from two French blood donors without any malaria exposure (the same pool of AB serum was used for all experimental blocks). The mixture was used to feed mosquitoes, which had been starved for 12--16 h before blood feeding, with a standard membrane-feeding system \[[@B23]\]. For each mosquito family, i.e. each group of mosquitoes that was derived from the eggs of a single female, equal groups of three-day old females were randomly chosen and fed separately with each isolate. Depending on the size of the family, each feeding cage contained between four and 15 females. Mosquitoes were allowed to take a blood meal for 40 minutes, after which unfed and partially fed mosquitoes were discarded. Seven or eight days after the infective blood meal, mosquitoes were dissected and their midguts were stained with 2% mercurochrome in distilled water in order to detect the presence and number of oocysts by light microscopy. Microsatellite genotyping ------------------------- *P. falciparum*DNA was extracted from the blood samples using the QIAamp DNA blood kit following the manufacturer\'s instructions (Qiagen, CA). The isolates were typed using the semi-nested PCR method slightly modified from a previous study \[[@B24]\] (details are available upon request to PD) and the markers used \[[@B25]\] and their GenBank accession number in parenthesis are as follows: PJ2 (G37826), UIDG (G37823), Polyα (L18785), TA60 (AF010556), ARA2 (G37848), Pfg377 (L04161), PfPK2 (X63648), TA87 (AF010571), TA109 (AF010508). The microsatellite PCR products were size-genotyped using a standard size Genescan 500 LIZ on an ABI Prism 310 Genetic Analyser (PE Applied Biosystems, CA). Data analysis ------------- Only those mosquito families in which at least four individuals had been fully fed and had survived infection with each isolate were included in the analyses. The likelihood that a mosquito had been infected was analysed with a nominal logistic analysis. The intensity of infection was analysed with an analysis of variance (ANOVA). In this analysis, the square root of the number of oocysts was used, so that the assumptions of the statistical tests (in particular, normality of the residuals) were satisified. As the study was run in three successive experimental blocks, both analyses included the effect of block, and the effects of family, isolate and their interaction. The effect of wing length was also included as a potential confounder \[[@B26]\]. As different families and isolates were used in each experimental block, the factors family, isolate and their interaction were nested within block. Block, family and isolate were considered as random factors. Results ======= The three successive experimental blocks involved three, five and three parasite isolates, and nine, four and five mosquito families, respectively. A third (151) of the 455 mosquitoes of the study were infected by *P. falciparum*oocysts, and the number of oocysts in infected mosquitoes ranged from one to 97 (mean 11.0, median 3). The prevalence and the number of oocysts differed among blocks (block effect, Table [1](#T1){ref-type="table"}), and were lower in larger mosquitoes (wing length effect, Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Statistical analysis of the effects of mosquito family and parasite isolate on the success of infection. The proportion of infected mosquitoes (a, nominal logistic analysis) and the square root of the number of oocysts (b, ANOVA) were analysed as a function of the mosquito family, the parasite isolate, and their interaction. In both analyses, the mosquito\'s wing length was included as a confounder. As the study was run in three experimental blocks using different families and isolates, the factors family, isolate and their interaction were nested within block. Block, family and isolate were considered as random factors. ::: \(a) Proportion infected \(b) Intensity of infection -------------------------------- ------ -------------------------- ----------------------------- ---------------- ------- --------- Source d.f. χ^2^ P Sum of Squares F P Experimental Block 2 85.5 \<0.001 265.2 2.40 0.155 Wing Length 1 3.1 0.029 6.4 7.14 0.008 Family (within Block) 14 3.8 0.927 29.5 0.66 0.800 Isolate (within Block) 8 15.5 0.482 462.5 19.40 \<0.001 Family\*Isolate (within Block) 34 127.2 \<0.001 110.2 3.59 \<0.001 Error (for analysis b) 395 356.8 ::: Family by isolate interaction ----------------------------- While the crude variation among families (averaged across all parasite isolates) was substantial (mean infection rate ranging from four to 83%; mean number of oocysts ranging from 0.1 to 16.4), most of this variation was due to differences among blocks (Fig. [1](#F1){ref-type="fig"}), so that there was no evidence that families differed in the overall proportion of infected individuals or in the intensity of infection (family effect, Table [1](#T1){ref-type="table"}). Similarly, most of the crude differences among isolates (averaged across mosquito families) were due to differences among blocks. Thus, isolates did not vary in the proportion of mosquitoes they infected (ranging from four to 94%), although they did vary in the number of oocysts they produced (median ranging from zero to 35) (isolate effect, Table [1](#T1){ref-type="table"}). More importantly in the context of our study, while neither the families of mosquitoes nor the parasites differed in their average responses to all of their partners, the interaction between mosquito family and parasite isolate (an estimation of the genotype by genotype interaction) had a highly significant effect on the likelihood and the intensity of infection (family by isolate effect, Table [1](#T1){ref-type="table"}). Thus, no parasite isolate was most infectious to every host genotype. Rather, isolates that were most infectious on one host tended to be less infectious than the other isolates on other hosts (Fig. [1](#F1){ref-type="fig"}). Similarly no host genotype was most resistant to every parasite isolate (Fig. [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Graphic representation of the mosquito family by parasite isolate interactions underlying (a) the probability and (b) the intensity of infection**. Each point represents the proportion of infected mosquitoes (in a) or the mean of the square root of the number of oocysts (in b) for a given combination of family and isolate. The families are indicated on the x-axes, and are separated into the three experimental blocks of the study with vertical lines. Different colours represent different isolates (squares: isolates containing two clones; circles: isolates containing three clones), and the lines connect points representing the same isolate. Crossing lines give an indication of family by isolate interactions. ::: ![](1475-2875-4-3-1) ::: Genetic characterization of *P. falciparum*isolates --------------------------------------------------- While the quantitative genetic analysis of the data gives an adequate representation of the genetic basis of the mosquito\'s resistance \[[@B20]\], the use of natural isolates may complicate the interpretation, as (i) they do not necessarily consist of different malaria clones and (ii) isolates often contain several clones in areas where transmission is high \[[@B27],[@B28]\]. However, genotyping the blood samples at nine microsatellite markers showed that the isolates differed. The overall genetic diversity was high, ranging from four to 15 allelic variants per locus. Each isolate had an allelic pattern that differed from all other isolates at, at least, one locus (data not shown), showing that the isolates were genetically distinct. Using the maximum number of alleles at a single locus as a conservative estimate of the number of clones, each isolate was found to contain either two or three distinct clones of *P. falciparum*(Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Description of *P. falciparum*isolates. The number of gametocytes per 500 leukocytes, converted to numbers of parasites per μL (assuming a standard leukocyte count of 8,000/μL) and the maximum number of alleles at a single locus found for 9 microsatellite markers (a conservative estimate of the number of clones) are given for each isolate. ::: Experimental block Isolate Gametocyte density (parasites/μL) Number of clones -------------------- --------- ----------------------------------- ------------------ 1 A 176 3 1 B 32 3 1 C 32 2 2 D 32 3 2 E 16 2 2 F 16 3 2 G 32 3 2 H 32 2 3 I 48 2 3 J 16 2 3 K 16 3 ::: Potential confounding effects ----------------------------- Separate analyses of the data for the two numbers of clones (two or three) ensured that the number of clones contained in each isolate did not confound the interpretation. The effect of the mosquito family by parasite isolate interaction on the likelihood of infection was significant in both cases (two clones: *P*= 0.050; three clones: *P*= 0.003) and the effect of the interaction on the number of oocysts was significant in one of the cases and showed a tendency in the other case (two clones: *P*= 0.219; three clones: *P*= 0.008). In addition, differences in gametocyte density between isolates (Table [2](#T2){ref-type="table"}) may be expected to bias infection success \[[@B23]\]. There were sufficient data to analyse the effects of the mosquito family by parasite isolate interaction separately for the isolates with 16 or 32 gametocytes/μL (i.e. one or two gametocytes per 500 leukocytes). At both gametocyte densities, the interaction significantly influenced the probability of infection (16 gametocytes/μL: *P*\< 0.001; 32 gametocytes/μL: *P*\< 0.001) and the number of oocysts (16 gametocytes/μL: *P*\< 0.001; 32 gametocytes/μL: *P*\< 0.001). In conclusion, the two potential confounders -- number of clones per isolate and gametocyte density -- had no qualitative influence on the results of the analysis. Discussion ========== While the specificity of mosquito-malaria interactions at the species level is well documented \[[@B29]\], the present results are the first experimental evidence of the genetic specificity of mosquito infection by malaria parasites at the intraspecific level. This finding corroborates an earlier study, where a mosquito line selected for resistance to malaria infection varied considerably in its response against different *Plasmodium*species and strains \[[@B6]\]. The present study goes one step further by suggesting that mosquito resistance to malaria is at least partly determined by the specific interaction between its own and the parasite\'s genotype. This idea is supported by two other studies showing that, in a strain of mosquitoes selected to resist infection by a wide variety of malaria species, different genetic loci are involved in the responses against different *Plasmodium*parasites \[[@B30],[@B31]\]. The present study suggests, moreover, that the genes conferring resistance to a particular parasite depend on the genetic background of the mosquito. The specificity of host-parasite interactions is often postulated to occur at the level of parasite recognition. While the molecular mechanisms of *Plasmodium*recognition by mosquitoes are still largely unknown, a group of thioester-containing proteins (TEPs) represents a promising family of candidate recognition molecules. One of them, the complement-like protein TEP1, has recently been shown to bind to and mediate the killing of the rodent malaria parasite *P. berghei*by the mosquito *A. gambiae*\[[@B9]\]. Moreover, two allelic variants of the *TEP1*gene are associated to susceptible and refractory strains of *A. gambiae*\[[@B9]\]. It is therefore tempting to speculate that this protein may be involved in the specific recognition of particular malaria genotypes by the insect\'s immune system. The mosquito genotype by parasite genotype interactions shown in this paper may help to understand some puzzling aspects of the epidemiology of malaria. Thus, even in areas with intense transmission, the probability that a mosquito becomes infected is generally low \[[@B26],[@B32]\]. Furthermore, the probability of infection is generally low even when, as in our study, mosquitoes fed on a blood-meal known to contain infectious gametocytes \[[@B33],[@B34]\]. This could have several explanations: mosquitoes fail to pick up infective gametocytes, transmission-blocking immunity in the human hosts prevents the parasite\'s development within the mosquito \[[@B22],[@B35]\], or parasites are cleared by mosquitoes that mount a sufficiently effective immune response \[[@B2]\]. The present results indicate an additional reason: that many parasites are incompatible with many of the mosquitoes in a natural population. The epidemiological consequences of mosquito genotype by malaria genotype interactions are perhaps most obvious in the context of malaria control with mosquitoes transformed to be resistant against malaria. The strong genetic specificity of compatibility between parasite isolates and individual insect vectors suggests that most studies on the mechanisms underlying the resistance of mosquitoes against *Plasmodium*might be misleading for the development of malaria control strategies. Indeed, most of these laboratory-based studies focus on the response of one mosquito line or colony against a single parasite strain and thus do not represent the genetic diversity of mosquitoes and parasites in natural populations \[[@B12],[@B13]\]. However, any malaria control programme based on the release of mosquitoes harbouring \'resistance genes\' is unlikely to be effective if resistance is expressed against only a subset of the parasite genotypes of the local population. Indeed, as parasites facing resistant mosquitoes will be under strong selective pressure to avoid mosquito defence mechanisms, genotypes that are eliminated by the resistance genes might be replaced rapidly by genotypes that cannot be controlled. Thus, before a possible release of transgenic mosquitoes, it will be crucial to ensure that the transformed mosquitoes are resistant to all of the parasite genotypes in the local population. This reinforces the idea that any release of genetically modified mosquitoes for reducing transmission of mosquito-borne diseases must be preceded by studies that have moved from the laboratory to the field \[[@B10]\]. Conclusions =========== This study demonstrated that the resistance of an anopheline mosquito to *P. falciparum*development, a major component of its vector competence, varies considerably between different combinations of parasite isolates and individual, genetically variable, vectors. Optimal transmission may thus require some specific compatibility between the insect\'s and the parasite\'s genotypes. This result has important consequences for the epidemiology of malaria. Overall, it suggests that conclusions from a particular subset of mosquito and malaria genotypes will not necessarily hold for other combinations of genotypes. Therefore, field studies taking into account the full diversity of mosquito and parasite populations are necessary to reach valid conclusions concerning the technologies developed in laboratories for the control of malaria. Authors\' contributions ======================= LL participated in the design and the coordination of the study, carried out the fieldwork, participated in the molecular analysis, performed the statistical analysis, and wrote the manuscript. JH participated in the fieldwork. PD carried out the molecular analyses and helped to draft the manuscript. LCG supervised and coordinated the fieldwork. JCK conceived and designed the study, performed the statistical analysis, and wrote the manuscript. Acknowledgments =============== The authors thank J. Kongere, D. Oulo, L. Omukuba, J. Wauna, S. Orao, S. Ombonya and S. Otieno for their assistance during the fieldwork in Mbita, F. Renaud for helpful discussions, O. Kaltz for statistical advice, and two anonymous reviewers for their comments on a earlier version of the manuscript.

PubMed Central

2024-06-05T03:55:52.706672

2005-1-11

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548507/", "journal": "Malar J. 2005 Jan 11; 4:3", "authors": [ { "first": "Louis", "last": "Lambrechts" }, { "first": "Jean", "last": "Halbert" }, { "first": "Patrick", "last": "Durand" }, { "first": "Louis C", "last": "Gouagna" }, { "first": "Jacob C", "last": "Koella" } ] }

PMC548508

Background ========== This is the second presentation from a project for the International Programme on Chemical Safety (IPCS) evaluating the possible use of transgenic animal mutagenicity assays in toxicity testing and mechanistic research. Part I, preceeding this article, discussed comparison of effects of chemicals using certain transgenic assays with results using the bone marrow micronucleus test. The assessment of the potential genotoxicity of chemicals *in vivo*is important for both the verification and confirmation of intrinsic mutagenicity and for establishing the mode of action of chemical carcinogens. Although the present trend is to reduce animal testing, *in vitro*data must be confirmed by testing in *in vivo*conditions which take into account whole animal processes like absorption, tissue distribution, metabolism and excretion of the chemical and its metabolites, and overall toxicity \[[@B1]\]. In the mid 1980s, the mouse spot test \[[@B2]\] was suggested as a complementary *in vivo*test to the bacterial mutagenicity assay for detection of mutagenic substances and as a confirmatory test for the identification of carcinogens \[[@B3]\]. The mouse spot test has been used to assess a number of chemicals (see e.g. [Additional file 1](#S1){ref-type="supplementary-material"}, see separate file). It is at present the only *in vivo*mammalian test system capable of detecting somatic gene mutations according to OECD guidelines (OECD guideline 484 \[[@B4]\]). However to achieve an acceptable sensitivity, a large number of animals are necessary and it is therefore an expensive type of test and seldom used. More recently assays using transgenic animals have been developed for testing *in vivo*gene mutagenicity. The two transgenic mouse models with the best data base available are the *lacI*model (commercially available as the Big Blue^®^mouse), and the *lacZ*model (commercially available as the Muta™ Mouse). The present study compares the results of *in vivo*testing of a number of chemicals using the mouse spot test and compares it with results from these two transgenic mouse models. Descriptions of test systems ---------------------------- ### Mouse spot test In the spot test, mouse embryos which are heterozygous for different recessive coat colour genes, are treated *in utero*at gestation day 9--11 with the test substance. The exposed embryo at gestation day 10 contains about 150--200 melanoblasts and each melanoblast has 4 coat colour genes under study \[[@B2],[@B5]\]. The *in utero*exposure may result in an alteration or loss of a specific wild-type allele in a pigment precursor cell resulting in a colour spot in the coat of the adult animal. The frequency of spots is compared with the frequency in sham-exposed controls \[[@B2],[@B4]\]. In the mouse spot test there are 4 possible mechanisms by which the recessive coat-colour alleles can be expressed: 1) gene mutation in the wild-type allele, 2) deficiency (large or small) of a chromosomal segment involving the wild-type allele, 3) nondisjunctional (or other) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) \[[@B5]\]. Gene mutagenic but also clastogenic effects are detected by this test system. ### Transgenic mouse models The transgenic mutation test systems the *lacI*model (Big Blue^®^mouse), and the *lacZ*model (Muta™ Mouse) are described in detail in the preceding article: Mutagenicity testing with transgenic mice. Part I: Comparison with the mouse bone marrow micronucleus test Methods ======= Data presented in this documentation are the results of an extensive literature research. Concerning data on transgenic mouse assays only primary literature was used. Data on the mouse spot test were extracted from reliable reviews on this item or from primary literature. For all other data informations from secondary literature or data banks were used. Results and Discussion ====================== Comparison of the mouse spot test with transgenic mouse model systems --------------------------------------------------------------------- In the literature search chemicals have been identified that had been tested using the spot test **and**the Muta™ mouse assay (n = 20) or the Big Blue^®^mouse assay (n = 9) or both transgenic mutation assays (n = 8). The results (including references) are given in [Additional file 1](#S1){ref-type="supplementary-material"}. The results on 15 out of 20 substances (2-acetylaminofluorene, acrylamide, benzo\[*a*\]pyrene, 1,3-butadiene, cyclophosphamide, ethylmethanesulfonate, *N*-ethyl-*N*-nitrosourea, *N*-methyl-*N*\'-nitro-*N*-nitrosoguanidine, *N*-methyl-*N*-nitrosourea, 4-nitroquino-line-1-oxide, *N*-nitrosodiethylamine, *N*-nitrosodimethylamine, procarbazine, 4-acetylaminofluorene and *N*-propyl-*N*-nitrosourea) showed agreement between the Muta™ mouse and the mouse spot test. No agreement was seen with 5 out of 20 substances (4-acetylaminofluorene, 2-amino-3-methylimidazo(4,5-f)quinoline (IQ), hydrazine, mitomycin C, trichloroethylene). The positive results obtained with the Big Blue^®^mouse assay agreed with results in the mouse spot test for 7 out of 9 substances (2-acetylaminofluorene, benzo\[*a*\]pyrene, 1,3-butadiene, cyclophosphamide, *N*-ethyl-*N*-nitrosourea, *N*-methyl-*N*-nitrosourea, *N*-nitrosodimethylamine); one (di-(2-ethylhexyl)phthalate) was negative in both test systems and only one (methyl methanesulfonate) showed no agreement between the two test systems. With two exceptions, 4-acetylaminofluorene and N-propyl-N-nitrosourea (discussed later), all of the tested substances showed also clearly positive results in *in vitro*gene mutation assays (exception of 1,3-butadiene, negative results) and in the majority of *in vivo*studies on this endpoint. Further they induced carcinogenic effects in long-term studies on mice. Although no data on carcinogenicity on mice is available on *N*-propyl-*N*-nitrosourea, this substance might also be included in the category mentioned above, since carcinogenic effects were reported in rats \[[@B113]\] and *in vitro*gene mutation assays revealed clearly positive results. The following substances did [not]{.underline} show agreement between results in the mouse spot test and transgenic mouse assays or negative results were reported in both test systems (see [Additional file 1](#S1){ref-type="supplementary-material"}). These are therefore discussed in more detail here; for references see Additional file [1](#S1){ref-type="supplementary-material"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of the Muta™ mouse assay and the Big Blue^®^mouse assay for predicting mouse carcinogenicity in comparison with the mouse spot test ::: Term\# Calculation\* for the mouse spot test Calculation\* for Muta™ and/or Big Blue^®^mouse combined \*\* ------------------------- --------------------------------------- --------------------------------------------------------------- Sensitivity 84% (16/18) 79% (15/18) Specificity 0 (0/0) 0 (0/0) Positive predictability 100% (16/16) 100% (15/15) Negative predictability 0 (0/2) 0 (0/3) Overall accuracy 84% (16/18) 79% (15/18) \# Sensitivity = % of carcinogens with a positive result in the specified test system (STS) Specificity = % of noncarcinogens with a negative result in the STS Positive predictivity = % of positive results in the STS that are carcinogens Negative predictivity = % of negative results in the STS that are noncarcinogens Overall accuracy = % of chemicals tested where STS results agree with the carcinogenicity results \*: carcinogens with genotoxic and nongenotoxic mechanisms were considered but not substances without data on carcinogenicity; only data on mice were used \*\*: judged as positive in transgenic assays if positive in one of the two test systems For methylmethanesulfonate, the weak positive results were judged as positive. Trichloroethylene was not included in the calculation (inconclusive results in the mouse spot test). ::: ### 4-Acetylaminofluorene This substance showed mutagenic activity in the Muta™ mouse assay \[[@B19]\] but negative results in the mouse spot test \[[@B12],[@B13]\]. No data on carcinogenicity are available on 4-acetylaminofluorene. However, data on two *in vitro*test systems indicated gene mutagenic activity supporting results in the transgenic assay \[[@B15]-[@B18]\]. ### 2-Amino-3-methylimidazo(4,5-f)quinol (IQ) IQ is mutagenic in the Muta™ mouse assay \[[@B28]\] but negative results were obtained in the mouse spot test \[[@B29]\]. This negative result in the mouse spot test is in contrast to all other *in vivo*gene mutation assays on rodents and insects which revealed positive results \[[@B27]\]. Furthermore, gene mutagenic activity was detected in *in vitro*test systems and carcinogenic effects were observed in long-term studies on mice \[[@B27]\]. The results in the Muta™ mouse assay are in accordance with these data. ### Di-(2-ethylhexyl)phthalate Negative results in the mouse spot test \[[@B51]\] are in agreement with the negative Big Blue^®^assay \[[@B11]\]. Furthermore no gene mutagenic or questionable activity was reported in *in vitro*tests and in tests on Drosophila. Carcinogenic effects were obtained in studies on mice but nongenotoxic mechanisms are presumed. ### Hydrazine This substance induced mutagenic effects in the mouse spot test \[[@B72]\] but negative results were observed in the Muta™ mouse assay \[[@B71]\]. Other *in vivo*as well as *in vitro*test systems revealed gene mutagenic effects \[[@B70]\]. Increased tumor incidences were observed in carcinogenicity studies on mice. Overall, the mouse spot test but not the Muta™ mouse assay reflects data on genotoxicity and carcinogenicity. However, a single exposure was used in the Muta™ mouse assay \[[@B71]\]. Studies on other *in vivo*genotoxicity endpoints have shown generally negative results after single exposure but genotoxic activity after repeated application, for example the mouse bone marrow micronucleus assay was positive \[[@B20]\]. It is possible that positive results may be found using another experimental design in the Muta™ mouse assay e.g. repeated exposure. ### Methyl methanesulfonate Only weak mutagenic effects were observed in the Muta™ mouse \[[@B19],[@B57],[@B75]-[@B77]\] and negative results in the Big Blue^®^mouse \[[@B63]-[@B65],[@B78]\]. In the mouse spot test this carcinogenic substance is mutagenic \[[@B3]\] as well as in other gene mutation assays *in vitro*and *in vivo*\[[@B73],[@B74]\]. However, there is evidence that the chromosome mutagenic activity is detectable at much lower doses than the gene mutagenic activity. Tinwell et al. \[[@B19]\] have shown in Muta™ mice a weak gene mutagenic effect in the liver but no effect in the bone marrow. The same dose induced in these animals a significant increase in bone marrow micronuclei indicating clear clastogenic activity. However, the transgenic mutation assay is less suitable for detection of these effects \[[@B1]\]. ### Mitomycin C No mutagenic activity was observed in the Muta™ mouse assay after single application and ambiguous results after repeated exposure \[[@B93]\] but positive results were obtained with the mouse spot test \[[@B2],[@B3]\] and other gene mutation assays *in vitro*and *in vivo*with this carcinogenic substance \[[@B90]-[@B92]\]. The reason for this discrepancy is similar to that presumed for methyl methanesulfonate above. Clastogenicity in bone marrow but no gene mutagenic activity in liver and bone marrow has been shown in the same animals in the Muta™ mouse assay combined with a micronucleus assay \[[@B93]\]. However, using another experimental design for detection of gene mutations in the Muta™ mouse assay (dose level up to the MTD, repeated exposure) positive results might be obtained. ### Trichloroethylene Also with this carcinogenic substance, no mutagenicity was detected in the Muta™ mouse assay \[[@B117]\], the mouse spot test was positive \[[@B3]\], but this result is possibly related to contaminations with epoxides \[[@B116]\]. Further *in vitro*and *in vivo*assays on gene mutation resulted in weak positive, questionable, or negative effects \[[@B116]\]. Results in chromosome mutation assays are equivocal. However, a further (simple) reason for this discrepancy between the Muta™ mouse assay and the mouse spot test might be that the MTD was not reached in the Muta™ mouse assay presented by Douglas et al. \[[@B117]\]. In general, from the studies on genotoxic carcinogens given above, the results do not seem to give a preference for either the spot test or transgenic mouse model system. However, considering the mechanisms of action of specific substances there is some evidence, that the mouse spot test detects gene mutations as well as chromosome mutations whereas the transgenic mouse assays are restricted to gene mutations. Evidence for this hypothesis has been shown with the examples methyl methanesulfonate, mitomycin C, and trichloroethylene. In the mouse spot test, there are four possible mechanisms by which the recessive coat-colour alleles can be expressed (see introduction) including gene and chromosome mutations. Although the chromosome mutations have to survive several mitoses to cause the expression of the recessive allele \[[@B118]\], there is evidence that also predominantly clastogenic substances might result in a positive mouse spot test. In contrast, the transgenic mutation assays detected point mutations and maximal small deletions and insertions \[[@B1]\]. Predictivity of the transgenic animal assays and the mouse spot test for carcinogenicity ---------------------------------------------------------------------------------------- The sensitivity, specificity and predictivity of carcinogenicity for the transgenic mouse model (Muta™ mouse assay and the Big Blue^®^mouse assay combined) and the mouse spot test are documented in Table [1](#T1){ref-type="table"}. Data on 18 substances (see [Additional file 1](#S1){ref-type="supplementary-material"}) are available on carcinogenicity in mice [and]{.underline} mutagenic effects in transgenic mice as well as mutagenic effects in the mouse spot test (trichloroethylene not included because of inconclusive results in the mouse spot test). Although the data pool is not sufficient for a comprehensive comparison, there is some indication, that no significant differences were detectable between the two test systems. Advantages and disadvantages of both test systems ------------------------------------------------- ### Sensitivity of the test system In comparison to models using endogenous genes like the target genes in the mouse spot test, the spontaneous mutant frequency in transgenic animals is relatively high. This might be due to the fact that bacterial DNA is the target gene (high methylation rate) and/or the transgene is silent and no transcription related repair occurs as in endogenous genes which are more efficiently repaired \[[@B1]\]. However, comparing the number of cells and genes at risk at the time of exposure, the mouse spot test is numerical inferior to the transgenic mouse mutation assays. In the mouse spot test, the exposed embryo at gestation day 10 contains about 150--200 melanoblasts and each melanoblast has 4 coat colour genes under study \[[@B2],[@B5]\]. In the transgenic Big Blue^®^mouse, for example, 30--40 copies of the target gene (the constructed λLIZα shuttle vector) are integrated on chromosome 4 of **each**cell of the animal \[[@B1]\]. ### Other factors To achieve an acceptable sensitivity, a large number of animals are necessary in the mouse spot test. Many pregnant dams have to be in one treatment group to get a sufficient number of surviving F1-animals, since the test substance may induce maternal and developmental toxicity. Fahrig \[[@B2]\] suggested that 30--40 pregnant mice are needed per treatment group for evaluation of spots in the progeny. At least 150 F1-mice are recommended for the concurrent vehicle control \[[@B5]\] and at least two dose groups are used (OECD guideline 484 \[[@B4]\]). Therefore, the mouse spot test is an expensive type of *in vivo*test. In contrast, in transgenic mutation assays ca. 20 animals (3 dose groups and 1 concurrent vehicle control group in laboratories which already established this test system) are recommended per species and gender \[[@B119]-[@B121]\]. In the mouse spot test the discrimination between spots of mutagenic and non-mutagenic origin may be problematic \[[@B2]\]. A comparison of both test systems is presented in Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Advantages and Disadvantages of mouse spot test compared to the transgenic Big Blue^®^and Muta™ mouse assays ::: **Mouse spot test ^\[2-5\]^** **Transgenic mouse mutation assay^\[1,\ 122\]^** ---------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ [Age restriction]{.underline} Exposure restricted to embryos at gestation day 9--11 Usually less than 3 months [Toxicokinetics and metabolism]{.underline} Restrictions in toxicokinetics: test substance reaches the fetal melanoblasts after administration to the dams and absorption of the test substance itself or the toxic metabolites via the placenta No further barrier like the placenta after absorption and distribution [Target tissue]{.underline} Restricted to melanoblasts No tissue restriction; analysis of mutagenic potency in different organs [Type of mutation]{.underline} Detects 1) gene mutation, 2) large or small deletions, 3) loss of the chromosome carrying the wild-type allele and 4) somatic recombination (marker gene then homozygous) Detects 1) gene mutation, 2) small deletions or insertions [Dependency of effects on application route]{.underline} Only systemic effects can be detected; no application route specific effects For different routes systemic as well as local mutagenic effects can be detected [Target gene/cell]{.underline} 4 genes per cell in ca. 200 melanocytes Ca. 40 (Big Blue) or ca. 80 (Muta™ mouse) copies of the transgene per nucleus of each cell of the organismk [Number of animals]{.underline} Animal consuming test system Not more than 5 animals per gender per dose necessary [Specificity of test system]{.underline} Discrimination between spots of mutagenic and non-mutagenic origin may be problematically Identifying and isolating mutated genes with a high specificity [Characterisation of mutations by molecular methods]{.underline} Less suitable for identification of mutations in DNA analysis due to size of the genes detection of the \"molecular signature\" of a particular mutagen by DNA sequence analysis with standardized methods [Possibility of parallel investigation of several genetic endpoints]{.underline} No combination with other genotoxic endpoints possible The transgenic mouse assay can be combined with other *in vivo*genotoxic endpoints in the same animal: e.g. micronuclei, chromosomal aberration, unsheduled DNA synthesis, sister chromatid exchange [Endogenous versus foreign target gene]{.underline} The mouse spot test shows an in situ end point (expression of the target genes) Target genes are integrated parts of foreign DNA and consequently no \"normal\" mutational target [Costs]{.underline} Expensive type of *in vivo*test Less expensive ::: Conclusions =========== Although the mouse spot test is a standard genotoxicity test system according to the OECD guidelines, this system has seldom been used for detection of somatic mutations *in vivo*in the last decades. This is partly due to considerations of cost effectiveness and number of animals needed for testing but also for toxicological considerations. The usefulness of the mouse spot test in toxicology is limited by restrictions in toxicokinetics, sensitivity, target cell/organ, and molecular genetics. From the limited data available, it seems that the transgenic mouse assay has several advantages over the mouse spot test and may be a suitable test system replacing the mouse spot test for detection of gene but not chromosome mutations *in vivo*. Author\'s contributions ======================= UW was the main author. The other authors were involved in the discussions, writing small parts of text and in final preparation of the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Results in the transgenic mouse assay versus mouse spot test ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This paper is based on work performed by the authors in preparation of an Environmental Health Criteria document on \'Transgenic Animals in Mutagenicity Testing\' for the International Programme on Chemical Safety (IPCS). However, opinions expressed in this paper are the sole responsibility of the authors. We acknowledge the financial support of the German Federal Ministry for the Environment, Nature Conservation and Nuclear Safety.

PubMed Central

2024-06-05T03:55:52.709135

2005-1-27

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548508/", "journal": "J Carcinog. 2005 Jan 27; 4:4", "authors": [ { "first": "Ulrich", "last": "Wahnschaffe" }, { "first": "Annette", "last": "Bitsch" }, { "first": "Janet", "last": "Kielhorn" }, { "first": "Inge", "last": "Mangelsdorf" } ] }

PMC548509

Background ========== Injuries have been recognised as a major public health problem in both developed and less developed countries \[[@B1]\]. It is generally acknowledged that this problem is growing rapidly in sub-Saharan Africa \[[@B2]\]. Studies in Tanzania show that injuries are an important cause of death among adults \[[@B3]\], and accounted for 12% of all admissions at the national hospital in the country\'s largest city \[[@B4]\]. Studies on the magnitude of injuries and the groups at risk, have been conducted world-wide, and especially in developed countries. Hospital based studies, which are commonly reported from developing countries, presumably provide a representative picture of the prevalence and incidence of serious injury, but only a partial picture of the circumstances in which injuries occur. Given the limited access to hospital care in poor countries, however, data based on health facility data are not likely to be representative. In contrast, population-based studies are costly and rarely carried out particularly on topics such as injury, which are not high on the public health agenda in developing countries at present. Several studies have been conducted in high-income countries to examine factors associated with injury morbidity \[[@B5]-[@B7]\]. In developing countries, a number of population-based studies on nonfatal injuries have been done \[[@B8]-[@B14]\]. In order to understand the circumstances and risk factors associated with nonfatal injuries, we conducted a community-based study in an urban and a rural location of Tanzania. We describe injury patterns in both settings. We also investigate demographic and socioeconomic factors associated with nonfatal injuries. Methods ======= Study area ---------- The survey was conducted in Dar es Salaam city (an urban area) and Hai District (a rural area). These areas are part of a health and demographic surveillance system carried out by the Adult Morbidity and Mortality Project (AMMP) in six districts in Tanzania from 1992 to 2004. One aim of the project was to measure rates and causes of morbidity and mortality. At the time this investigation was carried out, the areas were being prospectively monitored through repeated censuses to ascertain the resident population at risk. Deaths were recorded through an active reporting system and probable cause of death determined by a validated verbal autopsy \[[@B15],[@B16]\]. Dar es Salaam city lies on the east coast of Tanzania. The AMMP demographic surveillance population was situated in two of the city\'s three municipalities. These areas contained 8 \'branches\' (an urban administrative unit) and covered 63,330 persons in 15,269 households living in urban and peri-urban neighbourhoods. Hai District lies on the South-Western slopes of Mount Kilimanjaro in Northern Tanzania. The AMMP demographic surveillance area in Hai covered 51 out of 61 villages in the district and around 62% of the total district population (159,906 persons in 40,238 households). Agriculture, livestock keeping and commercial mining are the main economic activities there. Details of the study population have been described elsewhere \[[@B15],[@B16]\]. Sampling procedure ------------------ In the urban surveillance area, an initial cluster sample of 500 households was randomly selected. Because initial data collection yielded fewer injuries than expected in the first two enumeration areas, the sample size was increased to 2,000 households with the difference made up of households selected at random from the remaining six surveillance branches. Thus, the final sample under-represented the first two branches. Information was sought on all individuals residing in the selected households. A two-stage cluster sampling method was adopted in selecting the rural sample. In the first stage, using existing AMMP data on mortality and poverty, six out of 51 villages were selected to represent different levels of socio-economic status and injury mortality. A random sample of 2,000 households was obtained from the selected villages in the second stage. All individuals in the selected households were included in the survey. Ethical clearance and informed consent -------------------------------------- Ethical clearance for this study was given by the Tanzania Commission for Science and Technology and the Regional Committee for Medical Research Ethics in Norway. Informed verbal consent was sought from each family. For children below age 15, parents or guardians were interviewed; for adolescents aged 15 to 18, consent was obtained from both the parent and the child. Data collection --------------- The survey tool was translated into Swahili, back translated into English, and pre-tested before use in the field. Two questionnaires were used in the study. Questionnaire 1 was a screening form used to identify whether a household member had an injury in the past one year that resulted in losing one or more days of \'normal\' activity (e.g. not being able to work or go to school). The head of household or any other responsible person was interviewed to obtain information about the household members. Variables included were age, sex, relationship to head of household, level of education, religion, marital status and occupation. Questionnaire 2 was used to record the circumstances in which the injury occurred. Some of the variables included were: month and year when the injury occurred; cause of the injury; place of occurrence; activity at time of injury; length of disability; and health facility use. Efforts were made to interview the injured person if an adult, otherwise we interviewed an informed member of the injured person\'s household. The number of days with restricted activity (disability days) was considered as a measure of severity of injury. Poverty was assessed at the household level using data from the 2000--2001 National Household Budget Survey and variables from AMMP data. The measure of poverty used was a predicted value of monthly consumption expenditure per adult equivalent for each household included in the study \[[@B17],[@B18]\]. Statistical analysis -------------------- Data analysis was done using STATA (version 7). Bivariate analyses were performed by cross tabulations and the chi-squared test was used to test for homogeneity. In the case of multiple injuries, the most recent injury episode was considered in all the analysis. Multiple logistic regression was used to examine the influence of socio-demographic and socio-economic factors on the risk of being injured, controlling for potential confounding variables. Odds ratios are reported with 95% confidence intervals. Tests for trend in associations over groups defined by other factors were performed where appropriate. Preliminary analysis indicated that a long recall period underestimated annual injury rates, with the effect being greater for injuries resulting in \<30 disability days while the rates for injuries resulting in 30 or more disability days were quite stable\[[@B19]\]. We therefore categorized severity of injury as \'minor\' if resulting in less than 30 days of lost activity and \'major\' if resulting in 30 or more days of lost activity. This kind of categorization has also been used in the Ghana study \[[@B13]\]. The category for major injuries is less likely to include actual minor injuries, and therefore constitutes a well-defined small group. However, the category for minor injuries might include some injuries that were actually severe. About 37 (7.3%) individuals who sustained an injury reported between 15 and 21 disability days, with only 3 reporting 22 to 29 days of restricted activity. Poverty quintiles were classified as most poor, very poor, poor, less poor, or least poor in terms of socioeconomic status. Adjustment for clustering was performed with standard STATA commands for analysis of survey data. Results ======= Data were gathered on a total of 15,223 individuals residing in 3,653 households. The urban sample included 8,188 individuals while the rural sample included 7,035 persons. The response rates were 89% and 92% for the urban and rural areas respectively. The rural sample had a larger proportion of individuals aged 44 years and above (24%) compared to the urban area (11%). This was comparable to national averages as reported by the 2002 national census in Hai (17%) and Dar es Salaam (10%)\[[@B20]\]. Educational status was higher in the urban area. Of the total sample, 509 persons reported to have sustained an injury in the past one year preceding the survey, representing an injury incidence of 32.7 per 1,000 persons per year (95% CI= 29.9 -- 35.7). The incidence for all, minor and major injuries was 24.5, 16.4 and 8.1 per 1,000 persons per year for Dar es Salaam, and 42.5, 32.8 and 9.7 for Hai district. The mean age of the injured was 27.6 years (standard deviation 20) and 62% were males. Almost all injuries were unintentional (96%). On average, 14 days of normal activity were lost per person because of an injury. In Dar es Salaam, the most common cause of injury reported in both males and females was transport injuries, followed by falls and cuts (Table [1](#T1){ref-type="table"}). In Hai, cuts ranked first, followed by falls and transport injuries. The proportion of individuals who sustained transport injuries in the urban area was four times higher than in the rural area (33.0% vs 7.6% respectively; p \< 0.001). Cuts and stabs accounted for 49% of the injuries in Hai compared to only 18% in Dar es Salaam (p \< 0.001). There was no statistical difference in the distribution of injury categories between males and females in the urban area (p = 0.37). In the rural area, males and females differed with respect to the most common causes of injuries (p \< 0.001), with transport injuries experienced almost exclusively by males, and cuts being more frequent in females. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Cause of nonfatal injury by sex in the urban and rural areas ::: **Cause of injury** **Total** **Males** **Females** ----------------------------- ----------- ----------- ------------- ----------- ----------- ----------- ***Urban (Dar es Salaam)*** ***Total*** ***206*** ***100*** ***136*** ***100*** ***70*** ***100*** Transport injuries 68 33.0 43 31.6 25 35.7 Falls 56 27.2 35 25.7 21 30.0 Cuts/stabs 38 18.5 26 19.1 12 17.1 Burn 12 5.8 6 4.4 6 8.5 Struck by object 12 5.8 10 7.4 2 2.9 Animal bites 4 1.9 3 2.2 1 1.4 Assault 7 3.4 4 2.9 3 4.3 Other 9 4.4 9 6.7 0 0 ***Rural (Hai)*** ***Total*** ***303*** ***100*** ***177*** ***100*** ***126*** ***100*** Transport injuries 23 7.6 22 12.4 1 0.8 Falls 83 27.4 49 27.7 34 26.9 Cuts/stabs 149 49.2 77 43.5 72 57.1 Burns 18 5.9 8 4.5 10 7.9 Struck by object 11 3.6 11 6.2 0 0.0 Animal bites 6 1.9 4 2.3 2 1.6 Assault 3 0.9 3 1.7 0 0 Other 10 3.3 9 5.1 1 0.8 ::: As expected, the cause of injury varied by age (Figure [1](#F1){ref-type="fig"}). In the urban area, transport injuries were most common among adults aged 15 years and above while burns were common among children under 5 years. Cuts and stabs ranked second as a cause of injury among the 5 to 14 year olds. In Hai, cuts were the commonest cause of injury in all age groups except among 0--4 year olds where burns and falls were most frequent. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### 1a: Cause of injury by age in Dar es Salaam (urban area) 1b: Cause of injury by age in Hai (rural area) ::: ![](1471-2458-5-11-1) ::: Major injuries accounted for 33% and 23% of all injuries in Dar es Salaam and Hai respectively (Table [2](#T2){ref-type="table"}). The percentage of transport injuries resulting in a major injury was 41% and 30% in the urban and rural areas respectively. Of cuts or stabs in the rural area, 13% were major whilst in the urban area, 21% of the cuts were major. More than one third of the falls were categorised as major injuries in both areas. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Major injuries as a percentage of all injuries (≥ 30 disability days) by cause and sex in the urban and rural areas ::: ----------------------------- ------------------------- --------------------------- ----------- ------------- **Cause of injury** **No. of all injuries** **Both sexes** **Males** **Females** Percent of major injuries ***Dar es Salaam (Urban)*** **Total** **206** **33.0** **28.7** **41.4** Transport injuries 68 41.2 34.9 52.0 Falls 56 35.7 28.6 47.6 Cuts/stabs 38 21.1 23.1 16.7 Burn 12 33.3 16.7 50.0 Struck by object 12 33.3 30.0 50.0 Other 20 20.0 30.8 0 ***Hai (Rural)*** **Total** **303** **22.8** **23.7** **21.4** Transport injuries 23 30.4 27.3 100.0 Falls 83 38.6 36.7 41.2 Cuts/stabs 149 12.8 13.0 12.5 Burns 18 16.7 25.0 10.0 Struck by object 11 45.5 45.5 0 Other 19 15.8 10.0 28.6 ----------------------------- ------------------------- --------------------------- ----------- ------------- ::: After controlling for age, sex and education, persons residing in Hai were 1.7 times as likely to have had an injury in the past one year as compared to those residing in Dar es Salaam (Table [3](#T3){ref-type="table"}). Males had a higher risk of being injured than females. Those with primary education only had an increased risk of having an injury compared to their counterparts who had no formal education. Children aged 5 to 14 had slightly higher odds of sustaining a minor injury compared to adults, while for major injuries adults aged 45 years and above were at an increased risk (p \< 0.01 comparing trends with age for minor and major injuries). After adjusting for age, sex, education and area, there was no significant association between poverty and risk of nonfatal injury. Male sex turned out to be the only significant risk factor for major injuries. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Adjusted odds ratios (OR) for all, minor (\<30 disability days) and major (≥ 30 disability days) injuries by demographic and socio-economic factors ::: **Factors** **Total** **All injuries** **Major injuries** **Minor injuries** ----------------------------------------- ------------ ------------------ --------------------- -------------------- --------------------- --------- --------------------- **Sample** **No.** **OR^a^(95% CI)** **No.** **OR^a^(95% CI)** **No.** **OR^a^(95% CI)** **Area** Dar es Salaam (Urban) 8188 206 1.0 68 1.0 138 1.0 Hai (Rural) 7035 303 1.66 (1.37 -- 2.02) 69 1.09 (0.75 -- 1.58) 234 1.94 (1.54 -- 2.44) *p \< 0.001* *p = 0.65* *p \< 0.001* **Age\*** 0--4 1720 49 1.28 (0.83 -- 1.99) 13 0.85 (0.38 -- 1.91) 36 1.53 (0.91 -- 2.57) 5--14 3711 140 1.23 (0.98 -- 1.56) 29 0.88 (0.56 -- 1.40) 111 1.37 (1.04 -- 1.79) 15--44 7140 212 1.0 59 1.0 153 1.0 45+ 2651 108 1.25 (0.97 -- 1.59) 36 1.57 (0.99 -- 2.47) 72 1.12 (0.84 -- 1.50) *p = 0.20* *p = 0.10* *p = 0.11* **Sex** Females 7844 196 1.0 56 1.0 140 1.0 Males 7379 313 1.75 (1.46 -- 2.12) 81 1.57 (1.11 -- 2.21) 232 1.81 (1.46 -- 2.25) *p \< 0.001* *p \< 0.01* *p \< 0.001* **Education\*\*** None 3546 97 1.0 30 1.0 67 1.0 Primary 9674 362 1.50 (1.09 -- 2.06) 92 1.02 (0.59 -- 1.74) 270 1.77 (1.21 -- 2.59) Secondary+ 2001 50 1.18 (0.77 -- 1.82) 15 0.79 (0.37 -- 1.71) 35 1.41 (0.84 -- 2.36) *p = 0.01* *p = 0.67* *p \< 0.01* **Poverty quintiles (n = 12320)\*\*\*** 1 (Most poor) 2471 95 1.13 (0.82 -- 1.56) 22 1.05 (0.56 -- 1.96) 73 1.15 (0.80 -- 1.66) 2 2462 78 0.92 (0.66 -- 1.29) 21 1.01 (0.52 -- 1.97) 57 0.89 (0.61 -- 1.32) 3 2466 81 0.96 (0.69 -- 1.32) 23 1.11 (0.61 -- 2.01) 58 0.91 (0.62 -- 1.32) 4 2462 72 0.86 (0.61 -- 1.22) 25 1.24 (0.67 -- 2.29) 47 0.74 (0.49 -- 1.12) 5 (Least poor) 2459 83 1.0 20 1.0 63 1.0 *p = 0.49* *p = 0.96* *p = 0.19* \* 1 missing subject, \*\* 2 missing subjects ^a^Adjusted odds ratios from logistic regression models that included all variables in table except poverty \*\*\* Adjusted for age, sex, area and education p-value for test of heterogeneity ::: We investigated associations between different factors and some of the most frequent causes of injury (Table [4](#T4){ref-type="table"}). Rural residents were significantly less likely to have transport injuries compared to urban dwellers (OR = 0.39; 95% CI 0.23 -- 0.66). Children aged between 5 and 14 years were less likely to sustain transport injuries compared to adults aged 15--44 years. Males had a significantly increased risk of having transport injuries compared to females. However, household consumption expenditure was not associated with risk of transport injuries. In the rural area, the commonest type of transport involved was bicycle (52%) while in the urban area cars and trucks (46%) and commercial buses (22%) were frequently involved (Table [5](#T5){ref-type="table"}). In Dar es Salaam, 41% of those involved in transport injuries were pedestrians who were struck by motor vehicles or bicycles, whereas in Hai, the largest proportion of those with a transport injury were vehicle occupants (52%) followed by cyclists (35%). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Adjusted odds ratios (OR) for transport injuries, falls and cuts or stabs by demographic and socio-economic factors ::: **Factors** **Total Sample** **Transport injuries** **Falls** **Cuts/stabs** ----------------------------------------- ------------------ ------------------------ --------------------- ---------------- --------------------- --------- --------------------- **No.** **OR (95% CI)^a^** **No.** **OR (95% CI)^a^** **No.** **OR (95% CI)^a^** **Area** Dar es Salaam (Urban) 8188 68 1.0 56 1.0 38 1.0 Hai (Rural) 7035 23 0.39 (0.23 -- 0.66) 83 1.56 (1.09 -- 2.25) 149 4.27 (2.96 -- 6.15) *p \< 0.001* *p = 0.01* *p \< 0.001* **Age\*** 0--4 1720 5 0.52 (0.14 -- 1.97) 15 2.21 (0.98 -- 4.98) 5 0.35 (0.12 -- 0.99) 5--14 3711 8 0.28 (0.13 -- 0.63) 51 2.44 (1.58 -- 3.79) 54 1.12 (0.77 -- 1.62) 15--44 7140 60 1.0 41 1.0 80 1.0 45+ 2651 18 1.01 (0.56 -- 1.83) 32 1.98 (1.22 -- 3.21) 48 1.19 (0.82 -- 1.74) *p \< 0.01* *p \< 0.001* *p = 0.07* **Sex** Females 7844 26 1.0 55 1.0 84 1.0 Males 7379 65 2.66 (1.64 -- 4.29) 84 1.62 (1.14 -- 2.30) 103 1.38 (1.03 -- 1.85) *p \< 0.001* *p \< 0.01* *p = 0.03* **Education\*\*** None 3546 10 1.0 30 1.0 23 1.0 Primary 9674 67 1.76 (0.63 -- 4.93) 96 1.56 (0.90 -- 2.69) 151 1.61 (0.98 -- 2.66) Secondary+ 2001 14 1.10 (0.34 -- 3.56) 13 1.50 (0.67 -- 3.38) 13 1.03 (0.48 -- 2.21) *p = 0.15* *p = 0.27* *p = 0.05* **Poverty quintiles (n = 12320)\*\*\*** 1 (Most poor) 2471 15 1.18 (0.56 -- 2.49) 19 0.67 (0.36 -- 1.22) 41 1.36 (0.81 -- 2.28) 2 2462 12 0.90 (0.41 -- 2.01) 24 0.85 (0.48 -- 1.51) 35 1.14 (0.68 -- 1.91) 3 2466 12 0.93 (0.43 -- 2.01) 24 0.84 (0.48 -- 1.49) 27 0.87 (0.51 -- 1.48) 4 2462 13 0.99 (0.47 -- 2.11) 21 0.76 (0.41 -- 1.39) 26 0.86 (0.50 -- 1.50) 5 (Least poor) 2459 14 1.0 27 1.0 30 1.0 *p = 0.96* *p = 0.74* *p = 0.31* \* 1 missing subject, \*\* 2 missing subjects ^a^Adjusted odds ratios from logistic regression models that included all variables in table except poverty \*\*\* Adjusted for age, sex, area and education p-value for test of heterogeneity ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Vehicles involved in crashes causing traffic injuries in the urban and rural area ::: **Type of vehicle** **Dar es Salaam (n = 68)** **Hai (n = 23)** --------------------- ---------------------------- ------------------ Car/truck 46 26 Bus 22 9 Bicycle 16 52 Motorcycle 13 4 Train 3 \- Cart \- 9 ::: Our results show that rural residents were 1.6 times as likely as urban dwellers to experience a fall resulting in an injury. Falls were more likely in children aged below 15 years and adults 45 years and above (Table [4](#T4){ref-type="table"}). They were reported to occur mainly in and around homes in the urban area (Table [6](#T6){ref-type="table"}). In the rural area, outside home, on the roads and farms were reported to be the most frequent places of occurrence for falls. ::: {#T6 .table-wrap} Table 6 ::: {.caption} ###### Place of injury by area and cause ::: **Place of injury** **All injuries** **Falls** **Cuts** ---------------------------------- ------------------ ----------- ---------- ------ ------ ------ Home Inside 16.9 12.5 23.2 6.0 13.2 8.1 Outside 24.3 24.8 41.1 31.3 39.5 24.8 Workplace/factory 8.3 2.3 3.6 3.6 23.7 1.3 Farm 0 33.3 0 22.9 0 53.0 On the road 38.8 19.5 5.4 25.3 15.8 6.7 Recreation area including sports 9.7 1.9 21.4 2.4 7.9 1.3 School 1.9 4.6 5.4 8.4 0 4.0 ::: Table [4](#T4){ref-type="table"} shows that rural inhabitants had a four fold risk of experiencing injuries due to cuts or stabs compared to urban residents (OR = 4.27; 95% CI = 2.96 -- 6.15). It was noted that 68% (102/149) of injuries due to cuts in the rural area were related to agricultural activities, of which 81% occurred in adults aged 15 years and above. The farm and outside homes were where the injuries occurred most (Table [6](#T6){ref-type="table"}). In the rural area, about 50% of children aged 5--14 years were injured when working on farms or around their homes and one third of the injuries were related to play. In the urban area, most (70%) of the children aged 5--14 years were injured while playing. Burns accounted for about 6% (30/509) of all injuries in both areas. Children aged less than five years were 8 times as likely to sustain injuries due to burns as adults aged 15 to 44 years (OR = 8.58; 95% CI = 1.73 -- 42.5). Although the magnitude of association appears to be large, the estimate was based on small numbers (16 and 5 injuries respectively; table not shown). Discussion ========== In this study, we found major differences between urban and rural residents with respect to cause and severity of injury and the circumstances in which they occurred. This has great implications in setting priorities when planning for intervention strategies. Transport injuries formed the most common injury category in the urban area. The low risk of transport injury in the rural areas is probably a reflection of the relatively lower level of motorization in this mainly agricultural area. However, they will often have more serious consequences than other types of injury. A number of studies have reported similar findings. In a study from Pakistan, farmers were found to be at a lower risk of traffic injury than labourers and vendors \[[@B11]\]. A study from Bangladesh found a low incidence of traffic injury in a rural population \[[@B14]\]. Our data revealed that in the rural area, bicycle injuries predominated while in the urban area motorized vehicles accounted for a large proportion of transport injuries. Bicycles play a very important role in rural areas of Tanzania as a means of transport. In the urban area, most of the transport injury victims were passengers on public transport and pedestrians. Previous studies from developing countries have also reported the dominance of pedestrians, passengers of commercial vehicles and cyclists as vulnerable road users to transport injuries \[[@B10],[@B11],[@B21]\]. A hospital-based study conducted in an urban area in Tanzania reported that 42% of those subjected to transport injuries were pedestrians \[[@B4]\]. Strategies for prevention of transport-related injuries should take into account the local patterns. Cuts and stabs by instruments such as axes and machetes constituted the most frequent injury category in the rural area, which is due to the fact that rural residents engage in agricultural activities using unprotected equipment. Cuts and stabs also contributed significantly among children aged 5 to 14 years with farm work being the common activity in the rural setting while play was the main contributing factor in the urban area. As emphasized in other studies, there is a need for safe space for play among children. In addition, the issue of a working child in developing countries like Tanzania needs to be addressed. Falls were also a significant contributor among young children and older adults. A better understanding of the circumstances in which falls occur would assist in planning for fall-prevention programmes in Tanzania. Injuries due to accidental poisoning were infrequently reported in both areas and hence grouped under \'others\'. It is possible that people were not comfortable reporting such events. In addition, near drowning did not feature among the causes of nonfatal injuries although the urban area has a port. Except for transport injuries which were the commonest cause of fatal and nonfatal injuries in the same surveillance areas, the distribution of nonfatal injuries differed essentially from those of fatal injuries reported for 1992--1998 in the same surveillance areas \[[@B22]\]. In addition to transport injuries, the commonest causes of injury death were suicide, assault, accidental poisoning and drowning while the main causes of nonfatal injuries were falls, cuts and burns in these settings. A significant risk factor for injury turned out to be the place of residence. The likelihood of self reported injury was 66% higher for rural residents compared to urban residents. Similar findings have been reported from other studies \[[@B13],[@B23]\]. However, a study from Uganda found a high incidence of injury in the urban setting \[[@B12]\]. For severe injuries, we observed similar rates in the urban and rural areas. The main explanation is that transport injuries are more common and more severe in the urban areas whereas cuts and stabs are less common but more severe than in the rural area. Age is an important risk factor for many injuries but its influence varies between specific injury groups. Our findings show that adults aged 15--44 years are at a high risk of transport injuries. This has great economic impact since these are people in their most productive years and the injuries impose a considerable burden on their families and the society as a whole. Children below 15 years were at greater risk of injuries due to falls. This may be due to high risk environments such as lack of proper play facilities. This is in keeping with studies from other developing countries whereby falls among children have been reported to be a common cause of injury seen in hospitals \[[@B24]\]. As expected, males were found to have an increased overall risk of injury which was more pronounced for transport injuries. A possible explanation may be that men spend more time on the roads than females and therefore they are more prone to high risk behaviours or unsafe road practices \[[@B25]\]. Socio-economic status has been documented to be an important determinant of injury, although the effect depends on the socio-economic indicator considered, the cause and severity of injury \[[@B7]\]. We found no significant relationship between income poverty and nonfatal injuries. This is consistent with findings from other studies \[[@B26],[@B27]\]. Persons with primary education were at a greater risk of injuries than those with no formal education. This finding conforms to results from a previous study done in India \[[@B28]\]. The findings of this study are subject to a number of limitations. The information is based on self-reported data elicited through interviews, which is subject to recall bias \[[@B19],[@B29]\]. A 12 month recall period was used in this study in order to include as many injuries as possible. Although long recall periods underestimate injury rates, they can be useful in investigating associations between injuries and different risk factors. Initial multiple logistic regression analysis was done using injuries reported in the three months prior to the interview only. We found that the pattern of the associations was similar to that based on all injuries except for area of residence where the size of the effect was stronger when a short recall period was used (results not shown). Other studies have demonstrated that relative risks are not affected when a long recall period is employed \[[@B30]\]. In a previous report, we found that memory decay was greater in the rural area than in the urban area \[[@B19]\]. Therefore, the actual difference in rates between the urban and rural area may have been underestimated. Intentional injuries such as assaults and domestic violence are probably underreported since they would not be adequately captured in such a survey. This may lead to injury rates being underestimated. In this study, a clinical injury severity assessment was not possible. Disability days were used instead as a measure of severity of injury. One should be careful in generalizing the findings to other urban and rural settings of the country. Dar es Salaam is in many ways different from other urban areas of Tanzania and Hai is a relatively wealthy rural area. Furthermore, it might be difficult to generalize the findings to the surveillance areas due to the selection process that was employed. However, from our knowledge of the areas, we see no obvious reasons indicating that our samples should be very different from the urban and rural surveillance areas. Despite its limitations, this study has generated information that could be useful for targeted prevention at the local level. Conclusions =========== This study, the first of its kind in Tanzania, describes the patterns of nonfatal injuries and associated socio-demographic and socio-economic factors. It has attempted to identify specific groups of individuals as having a greater risk of experiencing certain types of injuries. This information is important for raising the level of awareness among policy makers and the public in general since the problem of injury receives little attention in most of the developing world including Tanzania. It is also useful in setting priorities for cause-specific prevention strategies. More detailed qualitative studies are required, however, on sensitive events such as assault. A nationally representative sample is also essential to measure the health burden due to nonfatal injuries. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= CM designed and conducted the study, performed statistical analysis, wrote the initial draft and revisions of the manuscript after consultation with other authors. IH, AN and GK participated in the design of study and revision of the manuscript. PS and YH participated in study design and co-ordination, and in revision of manuscript. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/5/11/prepub> Acknowledgements ================ The Norwegian State Educational Loan fund and the Norwegian Research Council funded this work. The Centre for International Health in University of Bergen carried out this research in collaboration with the Adult Morbidity and Mortality Project (AMMP) of the Tanzanian Ministry of Health. We thank David Whiting and Gregory Kabadi for the coordination and conduct of the survey. We also thank Dr. Deo Mtasiwa and Dr. Gabriel Masuki for providing assistance during data collection. The efforts of our research assistants are greatly acknowledged.

PubMed Central

2024-06-05T03:55:52.711079

2005-1-28

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548509/", "journal": "BMC Public Health. 2005 Jan 28; 5:11", "authors": [ { "first": "Candida", "last": "Moshiro" }, { "first": "Ivar", "last": "Heuch" }, { "first": "Anne Nordrehaug", "last": "Åstrøm" }, { "first": "Philip", "last": "Setel" }, { "first": "Yusuf", "last": "Hemed" }, { "first": "Gunnar", "last": "Kvåle" } ] }

PMC548510

Background ========== As a barrier and immune organ, the gastrointestinal tract, lung and skin play a key role in protecting the body from a hostile environment \[[@B1]\]. The low incidence of infection at normal epithelial surfaces reflects the presence of innate, broad-spectrum antimicrobial defense mechanisms \[[@B2]\]. Host defense peptides (HDPs) of the innate immune response play an important role in the protective barrier function of the epithelia \[[@B3]\]. Host defense peptides have been isolated from diverse organisms, including plants, insects, bacteria and vertebrates \[[@B4]\]. Several classes of mammalian peptide antibiotics have been ascribed pivotal roles in innate immunity \[[@B5]\]. Among these are various cysteine-rich peptides such as defensins \[[@B6],[@B7]\] and the more structurally diverse cathelicidins \[[@B8]\]. Produced as precursors, they require proteolytic processing to liberate the mature functional antimicrobial peptide. Cathelicidins contain a conserved N-terminal cathelin domain, and a structurally diverse C-terminal domain that possesses the peptide\'s antimicrobial activity. Rabbit CAP18 was the first cathelicidin precursor described, and its mature peptide has broad-spectrum antimicrobial activity \[[@B9]\]. Cathelicidins have since been identified in many other species including hCAP18/LL37 in humans \[[@B10]\], protegrins in swine \[[@B11]-[@B13]\], CRAMP in mice \[[@B14],[@B15]\] and SMAP29 in sheep \[[@B16]\]. Many of these peptides demonstrate extremely broad-spectrum antimicrobial activity, including Gram positive and Gram negative bacteria and fungi \[[@B4],[@B15]\]. In addition, they achieve bacterial killing much more rapidly than any commercially available antibiotic \[[@B17]\]. Recently, a new family of synthetic, α-helical HDPs called \"ovispirins\" was described \[[@B18]-[@B20]\]. Although some of these modified peptides had similar antimicrobial activity of naturally occurring peptides, they manifested appreciable cytotoxicity. We have demonstrated recently that variants of Ovispirin, the so called Novispirin peptides, displayed more favorable toxic/ therapeutic ratios *in vitro*and broad spectrum activity in infected rat burn model \[[@B21],[@B22]\]. Some of these peptides are induced at epithelial surfaces in response to invading organisms \[[@B23]-[@B25]\]. Many HDPs kill microorganisms by causing membrane permeabilization, although not necessarily as their sole mode of action \[[@B26]\]. Some HDPs also direct chemotaxis, promote wound healing, angiogenesis and contribute to adaptive immunity by mobilizing memory T cells and immature dendritic cells \[[@B25],[@B27]\]. Recent studies have also demonstrated antitumor activity after treatment with HDPs \[[@B28]\]. In addition, several antiviral activities were reported. Recently it has been demonstrated that rabbit neutrophil peptide alpha-defensin NP1 protects cells from infection with HSV-1 and 2 \[[@B29]\]. Other studies revealed that human neutrophil peptide HNP1 to 3 and Theta-defensins also inhibit HSV infection although by different mechanisms \[[@B30]-[@B32]\]. The ancestral human theta-defensins retrocyclin blocked HSV attachment \[[@B33]\]. The inhibition of adenovirus replication by the antimicrobial peptide awaits identification of a mechanism of action \[[@B30]\]. Anti-HIV activity of defensins were first reported 1993 by Nakashima and coworkers \[[@B34]\]. The alpha-defensins exhibited anti-HIV activity on at least two levels: directly inactivating virus particles; and affecting the ability of target CD4 cells to replicate the virus \[[@B35]-[@B37]\]. Binding to gp120 of HIV-1 and inhibition of HIV entry has also been identified as the mechanism of inhibition of HIV infection by theta defensins \[[@B38]\]. Due to their inhibitory effect on HIV-1 replication and due to an association of a single-nucleotide polymorphism in a beta defensin gene human beta-defensins might also play an important role in host defense against HIV-1 \[[@B39]\]. The antibacterial activity of HDPs is largely mediated by pore formation leading to permeablization of the bacterial membrane. Although some selectivity for bacterial membranes has been described, the lipid membrane of enveloped viruses might also be a target of antimicrobial peptides \[[@B32],[@B40]\]. This might allow development of antiviral effector molecules for topical application against a broad spectrum of enveloped viruses. Targeting host cell-derived membrane components might be a particularly interesting approach to inhibit viruses that rapidly develop resistance to compounds directed against viral proteins such as HIV. Therefore, we screened a panel of different natural occurring and designer HDPs for Env-independent inhibition of HIV infection at an early step in the viral replication cycle. Results ======= Given the potential cytotoxicity of HDPs, it was important to discriminate between modulation of cell metabolism and direct antiviral effects. We were concerned that HDPs could modulate host cell metabolism without affecting cell viability as assayed for example by standard MTT assays. We therefore decided to use immunodeficiency virus-based vectors transferring the luciferase gene to determine both, the HDP antiviral activity and modulation of cell metabolism. The luciferase activity of target cells, stably transduced with the lentiviral vector prior to HDP treatment should reveal any effect of HDPs on cellular transcriptional and translational efficacy. Treating cells with HDPs during infection with the lentiviral vector and comparison with results obtained with the stably transduced cells should then allow identifying HDPs that inhibit early steps in the viral replication cycle. To validate the luciferase-based assay for modulation of cell metabolism, 293 cells were stably transduced with a lentiviral vector transferring the luciferase gene. Incubation of transduced cells with increasing concentrations of Protegrin-1 and LL37 led to a dose-dependent inhibition of luciferase activity (Fig. [1](#F1){ref-type="fig"}). Comparison to the MTT test revealed a similar dose response curve, although the MTT test might be less sensitive at lower concentrations of the HDPs. Testing cell proliferation by a BrdU incorporation assay revealed a threshold level above which proliferation is strongly reduced. To allow side by side evaluation of cytotoxic and antiviral effects of HDPs with the same read-out the luciferase-based assay was used in most subsequent experiments. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### **Comparison of different cytotoxicity assays.**293A target cells stably transduced with the luciferase gene were incubated for 48 hours in the indicated concentrations of LL37 or Protegrin-1. Viability, cell proliferation and cell metabolism of parallel cultures were assessed by a standard MTT assay, Brd-U incorporation and the luciferase assay, respectively. Values are expressed as percentage of the values obtained from cultures without HDPs. The mean and the standard deviation of triplicates are given. ::: ![](1742-4690-2-2-1) ::: A panel of HDPs containing members of the major classes of antimicrobial peptides were analyzed for inhibitory effects against lentiviral vectors. Given the variability of the viral envelope protein, we focused on identifying Env-independent inhibitory activities by using VSV-G pseudotyped lentiviral vectors. All antimicrobial peptides used in this study, human cathelicidin LL37, recombinant human β-Defensin-2, porcine Protegrin-1 (PG-1), fungal Plectasin and Novispirin G10, inhibited gram positive and gram negative bacteria revealing antimicrobial activity in the expected range (Table [1](#T1){ref-type="table"}) and confirmed bioactivity of the peptides used. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Summary of the radial diffusion assay results comparing host defense peptides with a clinically used antibiotic (Ampicillin) ::: M E C (μg/ ml) ------------------ ---------------- -------------- ------------ ------------- ------------ ------------- *S. aureus* 0,91 ± 0,04 4,74 ± 0,1 9,13 ± 0,5 2,42 ± 0,4 4,2 ± 0,4 10,50 ± 0,2 *S. epidermidis* 4,48 ± 0,2 13,09 ± 0,6 8,96 ± 0,2 8,20 ± 0,5 2,8 ± 0,02 ND *E. faecalis* 4,46 ± 0,3 13,61 ± 0,7 \- 10,60 ± 0,7 4,3 ± 0,3 28,85 ± 0,6 *P. aeruginosa* 3,3 ± 1,1 12, 28 ± 0,5 6,87 ± 0,8 \> 128 1,56 ± 0,4 ND *E. coli* 2 ± 0,1 11,66 ± 1,5 3,66 ± 0,3 79,10 ± 0,3 1,63 ± 0,3 18,9 ± 0,9 *A. baumanii* 5,01 ± 0,2 13,13 ± 0,2 3,19 ± 0,5 \> 128 4,5 ± 0,02 ND All clinical isolates from human wounds showed significant sensitivity to HDPs. ND: not determined. MEC: minimal effectory concentration. MEC \> 128 means that the tested bacteria is not susceptible to the drug tested. Depicted data consists of 3 individual experiments; each condition was performed in quadruplicates ::: The inhibitory effect against the lentiviral vector was determined by preincubation of the vector with human cathelicidin LL37, recombinant human β-Defensin-2, porcine PG-1, fungal Plectasin and Novispirin G10 for 30 minutes in increasing concentrations of peptide prior to the addition of vectors with antimicrobial peptide to the target cells. Stably transduced target cells were incubated in parallel with the HDPs to detect effects on cell metabolism. Luciferase activities were determined 2 days after infection. All HDPs led to a reduction in luciferase activity of cells transduced with the lentiviral vector (Fig. [2A--E](#F2){ref-type="fig"}), but most of them also reduced the luciferase activity of stably transduced target cells. Specific inhibition of early steps of infection was only seen for the cathelicidin LL37 and PG-1. As an additional control for the specificity of inhibition, a non-enveloped adenoviral vector transferring the luciferase gene was also incubated with the same panel of HDPs. None of the HDPs led to a dose-dependent inhibition of early steps of the adenoviral replication cycle (Fig. [2F--J](#F2){ref-type="fig"}). Two-fold serial dilutions were used to determine 50% inhibitory concentrations (IC~50~) of LL37 and PG-1, resulting in IC~50~s of approximately 30 μg/ml and 16.8 μg/ml, respectively (Fig. [3](#F3){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### **Inhibitory activity of HDPs against lentiviral and adenoviral vectors.**Percent luciferase activity of 293A target cells transduced in the presence of the indicated amounts of HDP with the VLΔBH lentiviral vector (A to E) or an adenoviral vector (F to J) both transferring the luciferase gene is shown. Modulation of cell metabolism was investigated in parallel by incubating 293A target cells stably transduced with a luciferase gene with the indicated amounts of HDP. The luciferase activity is expressed as percentage of the luciferase activity of cells cultured in the absence of HDP. The mean and the standard deviation of triplicates are given. ::: ![](1742-4690-2-2-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### **50% inhibitory concentrations of LL37 and Protegrin-1.**Two-fold serial dilutions were used to determine the IC~50~s of LL37 (A) and Protegrin-1 for the VLΔBH vector (B). Modulation of cell metabolism was investigated in parallel by incubating 293A target cells stably transduced with a luciferase gene with the indicated amounts of HDP. The luciferase activity is expressed as percentage of the luciferase activity of cells cultured in the absence of HDP. The mean and the standard deviation of triplicates are given. ::: ![](1742-4690-2-2-3) ::: In initial attempts to characterize the mechanism of inhibition of lentiviral vector infectivity, LL37 and PG-1 were added at different time points during infection: both HDPs were either preincubated with the vector preparation for 30 minutes prior to addition to the cells or the HDPs and the vector were added simultaneously to the cells (Fig. [4A+B](#F4){ref-type="fig"}). In addition, cells were first infected with the lentiviral vector for two hours prior to addition of the HDPs. LL37 and PG-1 exerted the strongest inhibition after preincubation of HDPs and the lentiviral vectors, indicating a direct effect on infectivity of the vector particles. However, adding LL37 and PG-1 two hours after incubation of cells with the lentiviral vector also led to a stronger reduction of luciferase activity than observed after incubation of cells stably transduced with the luciferase gene suggesting a second target in the infection cycle that is affected by LL37 and PG-1. To further discriminate between direct inhibitory effects on vector particles and effects mediated by potential HDP cell interactions lentiviral vector particles were first incubated with LL37 and PG-1 for 30 minutes and then added either undiluted or at a 1:10 dilution to the target cells. Due to a 10-fold lower HDP concentration in the latter cultures, vector infectivity should be reduced to a lesser extent, if inhibitory effects are mediated by cellular targets. Comparable dose-dependent inhibition curves (Fig [4C,D](#F4){ref-type="fig"}) of diluted and undiluted vectors demonstrate that the inhibitory effects of LL37 and PG-1 depend on the HDP concentration during preincubation of the vector particles and not on the HDP concentration during subsequent cell culture. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### **Time and concentration dependent inhibition of lentiviral vectors by LL37 and Protegrin-1.**The lentiviral vector VLΔBH transferring the luciferase gene was either preincubated with 200 μg/ml of LL37 (A) or 50 μg/ml of Protegrin-1 (B) for 30 minutes (-30) or added simultaneously (0) with LL37 and Protegrin-1 to 293A target cells. Target cells were also preincubated for 120 minutes with the lentiviral vector prior to addition of LL37 and Protegrin-1. Two days after infection luciferase activities were determined as percentage of luciferase activities of cells cultured in the absence of HDPs. Cells stably transduced with the luciferase gene were also cultured in the presence and absence of LL37 and PG-1 to determine the effect of HDPs on the cell metabolism. The mean and the standard deviation of triplicates is given. A lentiviral vector transferring the GFP gene (HIV-CSCG) was incubated for 30 minutes at the indicated concentrations of LL37 (C) or Protegrin-1 (D). The vector was then added directly to 293A target cells (undiluted) or after a 1:10 dilution in medium lacking the HDPs. The number of GFP positive cells at each HDP concentration is given as percentage of GFP-positive cells of cultures transduced with diluted and undiluted vectors in the absence of HDPs. The mean and standard deviation of triplicates are shown. ::: ![](1742-4690-2-2-4) ::: The lentiviral vector used in this study had been pseudotyped with the G protein of vesicular stomatitis virus (VSV-G). To evaluate whether LL37 and PG-1 directly target VSV-G or a lentiviral protein, the inhibitory effect of these HDP against the lentiviral vector was compared side by side to their effect on a retroviral vector containing the amphotropic MLV Env for entry into target cells. The dose dependent reduction in the luciferase activity in the target cells was very similar in cells transduced with the lentiviral or the MLV vector (Fig. [5](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### **Comparative analysis of inhibition of lentiviral and retroviral vector infectivity.**Percent luciferase activity of 293A target cells transduced in the presence of the indicated amounts of HDP with a lentiviral vector (VLΔBH) or a retroviral vector (pRV-172) both transferring the luciferase gene is shown. Modulation of cell metabolism was investigated in parallel by incubating 293A target cells stably transduced with a luciferase gene with the indicated amounts of HDP. The luciferase activity is expressed as percentage of the luciferase activity of cells cultured in the absence of HDP. The mean and the standard deviation of triplicates of two independent experiments are shown. ::: ![](1742-4690-2-2-5) ::: The inhibition of HIV vectors containing the HIV-1 envelope by LL37 and PG-1 were studied on P4CCR5 cells expressing CD4 and coreceptors. IC~50~s of 25 μg/ml and 14 μg/ml were observed for LL37 and PG-1, respectively (Fig [6A,B](#F6){ref-type="fig"}), while only minimal inhibitory effects on cell proliferation were detected at these concentrations. Inhibition of early steps of wild type HIV-1 infection by LL37 and PG-1 was also evaluated on P4CCR5 indicator cells, which produce beta-Galactosidase upon expression of the viral tat gene after infection. The BrdU incorporation assay was used to evaluate modulation of cell function. A dose dependent reduction of the titer of HIV-1 on P4CCR5 cells was observed for both HDPs. However, the IC~50~of LL37 was approximately 3-fold higher than the IC~50~previously determined for the lentiviral vectors resulting in a narrow gap between antiviral and antiproliferative effects of LL37. In contrast, the IC~50~of PG-1 was below 10 μg/ml, while inhibitory effects on cell proliferation were not observed up to concentrations of 50 μg/ml. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### **Inhibitory effects of Protegrin-1 and LL37 on HIV-1 Env mediated vector entry (A, B) and HIV-1 infection (C,D).**A lentiviral vector transferring the GFP gene (VGΔBH-SIN) was incubated at increasing concentrations of LL37 (A) or Protegrin-1 (B) prior to transduction of P4CCR5 cells. The vector titer is given as percentage of the titer of the vector incubated in the absence of HDPs. Wild type HIV-1 was incubated with increasing concentrations of LL37 (C) and Protegrin-1 (D). The virus titer was subsequently determined on P4CCR5 indicator cells and is expressed as percentage of the titer of the untreated HIV-1 virus stock. The toxicity of LL37 and Protegrin-1 was determined in parallel using the BrdU incorporation assay. ::: ![](1742-4690-2-2-6) ::: Discussion ========== From the panel of five HDP studied, the cathelicidin LL37 and PG-1 were found to specifically inhibit lentiviral and retroviral vector, but not adenoviral vector infectivity. The strongest inhibition was seen if the lentiviral vectors were preincubated with LL37 and PG-1. This suggests that these HDPs directly interacted with the vector particles, which is consistent with our observation that inhibition was dependent on the HDP concentration during preincubation of the vectors with HDPs, but not on the HDP concentration during infection of the cells. Since lentiviral vectors and retroviral vectors were inhibited to a similar degree although they do not share any viral protein, the target for the HDP on the particles is probably cell-derived. This could either be the lipid membrane derived from the cell, which surrounds the vector particles or cellular membrane proteins that are frequently incorporated in lentiviral and retroviral particles during budding \[[@B41]\]. A permeabilizing effect of LL37 and PG-1 on the viral particles would be consistent with our data, but other mechanisms of inhibition cannot be excluded. While the inhibitory effect of PG-1 was also detected with wild type HIV-1 on P4CCR5 cells, LL37 inhibited HIV-1 to lesser degree then the lentiviral vectors. Due an IC~50~of 88 μg/ml against wild type HIV-1, it is questionable whether LL37 concentrations are sufficiently high at mucosal membranes to play a role in host defense against HIV-1. Conclusions =========== Modulation of cell metabolism was generally seen at concentrations of HDPs exceeding 50 μg/ml, while the MEC of the antibacterial activity ranged from 1 to 10 μg/ml. This might leave a sufficient window for therapeutic intervention of bacterial infection. However, for the treatment of HIV-1, the therapeutic window of LL37 and PG-1 is rather narrow. It should also be noted that the HDP-induced modulation of cell metabolism and cytotoxicity can be cell type dependent. Therefore, increasing the selectivity of HDPs for early steps in the viral replication cycle seems to be necessary for further development of the human cathelicidin LL37 and the porcine Protegrin-1 as antiviral agents for systemic or topical applications. Methods ======= Preparation of vectors transferring the luciferase or GFP genes --------------------------------------------------------------- To generate lentiviral vector particles transferring the luciferase gene, a codon-optimized (Geneart GmbH, Regensburg, Germany) HIV-1 *gag-pol*expression plasmid (Hgp^syn^) \[[@B42]\] and a VSV-G expression plasmid (pHIT-G) \[[@B43]\] were used to package the SIV-based vector VLΔBH. This vector contains the luciferase gene replacing the GFP gene of VGΔBH \[[@B44]\].5 μg of Hgp^syn^, 2 μg of pHIT-G and 5 μg of VLΔBH were transiently cotransfected by the CaPO~4~coprecipitation method into 293T cells as previously described \[[@B45]\]. An HIV vector construct containing the GFP reporter gene (HIV-CSCG) \[[@B46]\] was also used to prepare lentiviral vector particles by cotransfection with Hgp^syn^, pcTat \[[@B47]\], pcRev \[[@B47]\] and pHIT-G or pSVIIIenv3-2, an HIV-1 envelope expression plasmid \[[@B48]\]. The MLV vectors were prepared by cotransfection of pHIT-456, pHIT-60 and pRV-172 \[[@B49]\]. Two days after transfection, the supernatants were cleared from cellular debris by low speed centrifugation (10 minutes, 1000 × g) and filtration through 0.2 μm filters from Roth (Karlsruhe, Germany). Aliquots were stored at -80°C. Construction and Production of Ad.OW126 Vector ---------------------------------------------- Beginning with a first generation E1- and E3-deleted adenoviral vector, we generated a replication-competent adenoviral vector Ad.OW126, which harbors in the E1 region the firefly luciferase cDNA (subcloned from pGEM-Luc (Promega, Madison, WI)), a IRES element \[[@B50],[@B51]\], and an Ad5 E1A ΔE1B-55K gene. The entire expression cassette is driven by the human CMV-IE promoter in parallel to the transcriptional orientation of the adenovirus E1 gene products and terminated by the bovine growth hormone polyadenylation site. The expression cassette was flanked upstream by the Ad5 packaging sequence and downstream by the Ad5 pIX. The Ad.OW126 vector was generated by *in vitro*ligation \[[@B52]\] to H5*dl*327 (kindly provided by T. Shenk, Princeton University, Princeton, NJ), utilizing the unique *Bst*1107 I restriction site. The vector was propagated in 293 cells and purified by two rounds of CsCl density centrifugation \[[@B53]\], dialyzed (Slide-A-Lyzer, Pierce, Rockford, IL) against 1500 ml of PBS with 1 mM MgCl~2~and 10% glycerol four-times (1 hour each) at 4°C, and stored at -80°C until use. The concentration of the vector was determined by measuring absorbency at 260 nm \[[@B54]\], and the infectious titer was determined by plaque assay on 293 cells \[[@B55]\]. The ratio of infectious to non-infectious virus particles was approximately 1:80. Generation of 293A cells stably transduced with a luciferase gene ----------------------------------------------------------------- To stably transduce the luciferase gene into 293A cells, a self-inactivating version of VLΔBH, VLΔBH-SIN similar to VGΔBH-SIN \[[@B44]\] was packaged by cotransfection with Hgp^syn^and pHIT-G. 293A cells were plated in 24 well plates at a density of 50.000 cells / well and transduced with 200 μl of VLΔBH-SIN vector for two hours. Two days after plating cells were transferred to one well of a six well plate and transduced again with 1 ml of VLΔBH-SIN vector. Cells were subsequently expanded resulting in 293-Luc cells. Luciferase assay ---------------- The supernatant of infected 293A or 293-Luc cells, cultured in 96 well plates was removed and cells were lysed in 50 μl of cell lysis buffer (Promega, Pittsburgh, PA). 20 μl of the cell lysates were used in the firefly luciferase assay system of Promega as described by the manufacturer. Each single value of the triplicates was expressed as percent of the mean of triplicates of control cultures infected with the same vector in the absence of HDPs and the mean and the standard deviation of the percent values was calculated for each triplicate. Host defense peptides --------------------- The antimicrobial peptides (human LL37, porcine PG1-1, mutants from the ovine SAP29: Novispirin G10 and fungal Plectasin) used in this study were prepared by solid phase synthesis and purified by RP-HPLC. Recombinant human β-Defensin-2 was produced by a molecular farming approach in transgenic potato tubers and purified by perfusion chromatography (data not shown). The peptides (≥ 98% pure) were dissolved in 0.01% acetic acid and used for all *in vitro*and *in vivo*studies. Potential endotoxin contamination was monitored with the chromogenic *Limulus*amoebocyte lysate assay (BioWhittaker, Walkersville, MD) using *Escherichia coli*endotoxin (supplied with the kit) as the standard. Endotoxin levels for the peptides were not detectable. Bacteria -------- The following strains were used in this study: Gram-negative strains: *Acinetobacter baumannii*(ATCC 19606), *Escherichia coli*(ATCC 25922) and *Pseudomonas aeruginosa*(ATCC 27853) Gram-positive strains: *Staphylococcus aureus*(ATCC 25923), *Staphylococcus epidermidis*(ATCC 12228) and *Enterococcus faecalis*(ATCC 29212). All bacterial strains were analyzed with API test strips (BioMerieux, Hazelwood, MO) to confirm identity and aliquots were stored frozen in 50 % skim milk at -80°C. Bacteria were grown overnight in trypticase soy broth (Becton Dickinson, Franklin Lakes, NJ) at 275 rpm and 37°C. An aliquot of the resulting stationary phase cultures was then transferred to 20 ml of trypticase soy broth and incubated at 37°C for 2.5 hours to reach log phase. This subculture was transferred to a 50 ml conical polystyrene tube and centrifuged for 10 min at 4°C at 880 g. The bacterial pellet was washed once with chilled phosphate buffered saline, pH 7.4, and resuspended in 5 ml of the same cold buffer. One milliliter was removed to measure its optical density at 620 nm. The bacterial concentration was calculated from the following formula: CFU/ml = OD~620~× 2.5 × 10^8^. Growth Inhibition Assay ----------------------- To monitor bacterial growth inhibition *in vitro*a radial diffusion assay was performed as previously described \[[@B56]\]. Briefly, the underlay agar consisted of 1% agarose (A-6013, Sigma Chemical, St. Louis, MO) and 0.3 mg/ml trypticase soy broth (TSB) powder in 10 mM sodium phosphate with 100 mM NaCl (normal salt medium), pH 7.4. Bacteria (approximately 5 × 10^6^CFU) were mixed with 10 ml of underlay gel (43°C) and immediately poured into square 9 × 9 cm petri dishes. A series of 3 mm wells was punched after the agarose solidified. After appropriate serial dilutions were done, 5 μl of HDP, vancomycin (Abbott Labs, Chicago, IL), gentamicin, ciprofloxacin, or fluconazole (Sigma-Aldrich, St. Louis, MO) were added to the designated wells. Plates were incubated at 37°C for 3 hours. The bacteria-containing layer was covered with a 10 ml overlay of the nutrient rich agar. The overlay agar consisted of 6% (w/v) TSB and 1% agarose in PBS for all assays. After 18 h of incubation at 37°C, the plates were stained with 0.001% Coomassie blue for 10 h. The clear zones (bacterial growth inhibition) around the punched wells indicated antibacterial activity. The diameters of the clear zones were converted into units by subtracting the well diameter and multiplying the difference by 10. Results were plotted using a semi log scale and correlation coefficients and X-intercepts obtained from linear regression analysis. The minimal effective concentration (MEC) corresponded to the X-intercept value. All assays were performed in triplicates and repeated at least once. Cytotoxicity and proliferation Assay ------------------------------------ 293-Luc cells were plated in 96 well plates at a density of 2 × 10^3^cells / well. After 48 hours, 50 μl of MTT-solution (3 mg/ml) was added and incubated at 37° C under 5% CO~2~for 1 hour. After this time medium was removed and 100 μl 0.04 N HCL + 10% SDS was used to dissolve the resulting blue formazan crystals in living cells. The optical density was determined at 550 nm. Each single value of the triplicates was expressed as percent of the mean of triplicates of control cultures infected with the same vector in the absence of HDPs and the mean and the standard deviation of the percent values was calculated for each triplicate. In addition the BrdU Cell proliferation ELISA with chemiluminescence detection (Roche Diagnostics GmbH, Mannheim, Germany) was performed. After 293-Luc cells or P4CCR5 cells were plated in 96 well plates at a density of 2 × 10^3^cells/well BrdU (5-bromo-2\'-deoxyuridine) was added to the cells with a resulting concentration of 10 μM for the last 22 h of the incubation period. After removing the culture medium, the cells were fixed and DNA was denatured in one step with Fixdenat. Thereafter the cells were incubated with Anti-BrdU-POD for 1 h at room temperature. The chemiluminescence detection was measured after automatic injection of substrate solution with a microplate-luminometer (Orion, Berthold detection systems, Pforzheim, Germany). Inhibition of vector infectivity -------------------------------- To determine the effect of HDPs on vector infectivity and cell metabolism, 293A target cells were plated in triplicates into 96-well plates at 2 × 10^3^cells / well. After overnight incubation, the supernatant of the wells were removed and replaced by 25 μl vector preparation and 25 μl HDP at twice the final concentration indicated. 50 μl fresh medium with HDP at the final concentration indicated was added after two hours. One (adenoviral vector) or two (lentiviral and retroviral vector) days after infection, the supernatant was removed and 50 μl of cell lysis buffer (Promega) was added. Lysates were stored at -80°C until determination of the luciferase activity of the extracts. The affect of HDPs on cell metabolism was determined in parallel by plating 293-Luc cells exactly the same way as 293A cells and the luciferase activity was determined after one (as a control to inhibition of adenoviral vector infectivity) or two (as a control of lentiviral infectivity) days of incubation with HDPs. For GFP-expressing vectors, vector titers were calculated from the number of GFP positive cells per well as previously described \[[@B45]\] and the BrdU incorporation assay was used to monitor cytotoxic effects. Inhibition of HIV-1 ------------------- Stocks of HIV-1 were generated by transient transfection of 293T cells with the molecular clone pNL4-3. 25 μl of the virus stock were incubated for 30 minutes at room temperature with 25 μl of LL37 or PG-1 adjusted to twice the final concentration indicated. The mixture was added to P4CCR5 cells plated the day before at a density of 2 × 10^3^cells / well of 96 well plate. 50 μl fresh medium with HDP at the final concentration indicated was added after two hours. Two days after infection, the supernatant was removed and cells were stained by X-Gal. The number of infected cells per well were counted in the microscope. Competing interest ================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= LS, BT and JM performed most of the experiments. LS, OW, ML, EL, HH, HS and KU participated in the experimental design, data interpretation and writing of the manuscript. Acknowledgement =============== Recombinant human β-Defensin-2 was kindly provided by Michael Kleine at Planton GmbH (Kiel, Germany). Novispirin G-10 and Plectasin (WO 03/044049) were kindly provided by Hans-Henrick Christensen and the AMP team at Novozymes A/S (Copenhagen, Denmark). We would like to thank Ralf Wagner for providing Hgp^syn^, Ulrike Blömer for HIV-CS-CG, Joachim Hauber for pcTat and pcRev, Michael Malim for pHIT-G, and Paula Cannon for pHIT456, pHIT60 and pRV172. This work was supported by a grant from the FORUM program of the Ruhr-University Bochum.

PubMed Central

2024-06-05T03:55:52.717455

2005-1-18

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548510/", "journal": "Retrovirology. 2005 Jan 18; 2:2", "authors": [ { "first": "Lars", "last": "Steinstraesser" }, { "first": "Bettina", "last": "Tippler" }, { "first": "Janine", "last": "Mertens" }, { "first": "Evert", "last": "Lamme" }, { "first": "Heinz-Herbert", "last": "Homann" }, { "first": "Marcus", "last": "Lehnhardt" }, { "first": "Oliver", "last": "Wildner" }, { "first": "Hans-Ulrich", "last": "Steinau" }, { "first": "Klaus", "last": "Überla" } ] }

PMC548511

Introduction ============ Migraines and tension-type headaches impose an important burden upon society and the working public. According to the National Headache Foundation, some 45 million Americans suffer from chronic, recurring headaches and 28 million of these suffer from migraine headaches annually \[[@B1]\]. Furthermore, the work force loses approximately 50 billion dollars per year due to absenteeism and medical expenses caused by headaches, with more than 157 million workdays lost each year to migraine sufferers alone \[[@B1]\]. Even though advances have been made with regard to the treatment of acute migraine headaches (i.e., the triptan formulations), many patients often discontinue their migraine interventions due to treatment dissatisfaction \[[@B2]\]. Among individuals seeking treatment for tension-type headaches, the frequency of such headaches is often daily or almost every day \[[@B3]\]. Unfortunately, chronic tension-type headaches are associated with analgesic abuse \[[@B4]\], and are difficult to manage in a primary care setting due to frequent comorbid psychiatric or analgesic use problems \[[@B5]\]. Thus, it is imperative that other methods of treatment be researched and developed in order to increase satisfaction, therapeutic response, and compliance amongst these patients. One novel, but not really new treatment option, is the administration of niacin (nicotinic acid) through intravenous and/or oral routes. Niacin is a well-known over-the-counter (OTC) supplement primarily used for its ability to favorably influence cholesterol levels. Recently, there have been anecdotal reports demonstrating the effectiveness of niacin for aborting acute migraine attacks \[[@B6]\], and for preventing migraine headaches \[[@B7]\]. To assess the therapeutic spectrum of niacin\'s clinical effectiveness, we conducted a systematic review of the literature to examine the evidence of intravenous and/or oral niacin as a treatment for migraine headaches, tension-type headaches, and for headaches of other etiologic types. Methods ======= Literature Search ----------------- We conducted a systematic search of English and non-English language articles in the following databases: MEDLINE (1966--February 2004), AMED (1995--February 2004) and Alt HealthWatch (1990--February 2004). Articles were searched with the key search terms \"Migraine\" combined with the Boolean Operator \"AND \"Niacin\" OR \"Nicotinic Acid.\" Additional searches were conducted with the search terms \"Headache\" and \"Tension.\" To supplement the search, we searched through the references of the articles we found from the databases. Selection of Articles --------------------- To be included in our final review, articles had to report on the use and administration of niacin for migraine or any other types of headache. We included articles assessing original reports in both peer-reviewed and non-peer-reviewed journals. Quality assessment ------------------ We determined the evidence grade of each report found, based on the hierarchy of evidence developed by the Oxford Centre for Evidence Based Medicine \[[@B8]\]. Table [1](#T1){ref-type="table"} displays the hierarchy of evidence. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Grades of Evidence ::: ------- ---------------------------------------------------------------------------------------------------------------------------------- **A** Systematic reviews of randomized controlled trials and/or randomized controlled trials. **B** Systematic reviews of observational studies and/or high-quality observational studies including cohort and case-control studies. **C** Case-series, case-reports and/or poor quality cohort and case-control studies. **D** Expert opinion without explicit critical appraisal, or based on physiology, bench research or \"first principles.\" ------- ---------------------------------------------------------------------------------------------------------------------------------- ::: Search Results -------------- A total of 14 articles were screened \[[@B6],[@B7],[@B9]-[@B20]\]. Five articles were excluded in total; three because niacin was not the sole therapeutic agent used for the treatment of headache \[[@B16]\], histaminic cephalgia \[[@B17]\], and migraine \[[@B20]\]; and two because the reports were opinion pieces without any objective or subjective data to support the assertions made \[[@B13],[@B19]\]. In total, nine articles were found to meet the inclusion criteria and were included in this systematic review \[[@B6],[@B7],[@B9]-[@B12],[@B14],[@B15],[@B18]\]. Table [2](#T2){ref-type="table"} displays the characteristics of the studies included in this review. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Summary of Articles Demonstrating Niacin\'s Effectiveness for the Treatment of Migraine Headaches, Tension-type Headaches, and Headaches of other Etiologies ::: **Reference** **Condition** **n** **Protocol** **Outcome** **Evidence Grade** --------------- ----------------------------------------------- ------- ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------- 9 Migraine headaches 21 One initial intramuscular injection (IM) followed by a series of 6 or 8 intravenous (IV) treatments (maximum 50 mg), then regular IM injections (25--50 mg) combined with 50--150 mg of oral administration. 17 of the 21 subjects had a positive response. C: case-series 10 Headaches of different etiologic types 100 100 mg of IV sodium nicotinate or niacin. 75 of the 100 subjects had complete relief. C: case-series 11 Migraine headaches 15 100 mg of IV niacin, and an additional 50--200 mg if necessary to ensure a flushing response of more than 15-minutes. 13 of the 15 subjects had a positive response. The headaches were relieved in 27 of the 31 times when niacin was administered by IV administration. C: case-series 12 Tension headaches 35 22 subjects received 100--200 mg of IV niacin for a total of 53 times. 13 of the 22 subjects had a positive response. The headaches were relieved in 41 of the 53 times when niacin was administered by IV administration. C: case-series 14 Emotional or tension headaches 5 100 mg of IV niacin regularly for 12 weeks combined with a graded schedule of oral dosing, beginning at 300 mg daily, increasing to 900 mg daily, and tapering down to 300 mg daily. All 5 cases of emotional or tension headaches were very responsive to both IV and oral niacin. C: case-series 15 Tension headaches accompanied with depression 50 100 mg of IV niacin regularly for 23 weeks and then continued once every 2 months and as needed. This was combined with a graded schedule of oral dosing, beginning at 300 mg daily, increasing to 900 mg daily, and tapering down to 300 mg daily. In 44 of the 50 subjects the results with niacin therapy were very satisfactory or favorable. C: case-series 18 Migraine headaches 1 300--500 mg of niacin were chewed and allowed to slightly dissolve in the mouth. Resolution of migraine headaches. C: case-report 6 Migraine Headaches 2 500 mg of oral niacin taken at the onset of acute symptoms. In 2 of the 2 subjects, niacin aborted the acute migraine symptoms. In the first subject, niacin resolved the acute attacks in 4 of 4 occasions. In the other subject, niacin resolved the attack on one occasion. C: case-report 7 Migraine headaches 1 375 mg of oral sustained-release niacin twice daily for 1 month, and 375 mg once daily for 2 months. Migraine-free for the first month, and a marked reduction in migraine headaches over the next 2 months. C: Case-report ::: Limitations =========== Article \#1 \[[@B9]\] --------------------- This case series of 21 patients was limited as it was uncontrolled and involved a small number of patients. The methods that were used to evaluate efficacy of the treatment were primarily based upon a subjective questionnaire or medical record. The results would have been more meaningful if all the patients used the questionnaire to evaluate their treatment responses. Finally, although the symptomatic changes were witnessed immediately following niacin injection, the lifestyle recommendations may have had therapeutic benefits as well. Article \#2 \[[@B10]\] ---------------------- This study involved 100 patients having headaches of multiple etiologies. This study was not properly controlled, but did at least provide some comparison against a group of patients that were not administered the flush forms of intravenous niacin. The fact that the control group did not substantially benefit from the intravenous niacinamide lends more therapeutic efficacy to the ability of intravenous sodium nicotinate or niacin to have a marked therapeutic effect. The sample size was sufficient in number (n = 100), but the determination of therapeutic effectiveness was purely subjective and did not include any questionnaire or standardized method of evaluation. Side effects were minimal; 2 patients experienced mild abdominal cramps, 1 patient vomited but was suspected as having a pathologic condition of the stomach, and 1 other patient with migraine vomited forty-five minutes after the injection. A few patients found the treatment to be worse than their headaches. The authors concluded that the relief of headaches seemed to be correlated with the degree of flushing from the sodium nicotinate or niacin, and that this therapy was most useful among the migraine, spinal tap, and idiopathic groups. Article \#3 \[[@B11]\] ---------------------- A number of shortcomings were evident in this article. The first of which was that the study was not properly controlled and did not contain a placebo group or a group of patients acting as self-controls. However, some of the patients were given intravenous treatments on more than one occasion. This procedure helped in determining treatment reliability and reproducibility since niacin appeared to achieve therapeutic benefits on several occasions. There were no standardized methods of evaluating efficacy since the treatment responses were based upon the medical record and subjective reports. The results would have been more meaningful if all the patients used a standardized questionnaire prior to and after each treatment. In addition, there was only one male subject and 14 females in the study. Article \#4 \[[@B12]\] ---------------------- In this study of 35 patients were given treatments of intravenous dihydroergotamine methanesulfonate, intravenous niacin, or oral combination tablets of ergotamine tartrate and caffeine. Although no control group was used in this study, the patients were given multiple treatments on several occasions. This offered an interesting comparison to be made between niacin and other treatments. Since no control or placebo group was included, it cannot be determined if the therapeutic results were due, in part, to chance or placebo effects. The results might have been more meaningful if all the patients were given a standardized questionnaire prior to and after each treatment, and if an objective measure was incorporated to further substantiate patient responses. Article \#5 \[[@B14]\] ---------------------- These cases reported were well described and clearly demonstrated therapeutic responses during the intravenous and oral niacin therapy. The results would have been more reliable if a comparison had been made to a control group or to a similar patient cohort that were not given the same treatments. Even if the patients served as self-controls, and were told to stop the niacin treatment for a specified period of time, more information could have been gained from their therapeutic responses to niacin. Overall, this report of five cases provides an interesting approach to patients having chronic tension headaches and depression. Its value is limited by the difficulty in extrapolating these findings to a greater number of patients. Article \#6 \[[@B15]\] ---------------------- In this study involving 50 patients there was no data that listed pertinent identifying information, treatment response, past treatments, and duration of treatment for each patient. This would have strengthened the report by being more descriptive, and thus more amenable to critical analysis. If a comparison were made to a control group or a similar patient cohort not given the same treatments, more validity could be have been ascribed to this method of treatment. The patients could have also served as their own controls, thus providing more information about the therapeutic responses to niacin. It cannot be determined if the therapeutic results were due, in part, to chance or placebo. No method of evaluating efficacy was mentioned, except that the responses to niacin were \"very satisfactory\" in 44 of 50 cases. The results would have been more meaningful if all the patients were given a standardized questionnaire prior to and after each treatment, and if an objective measure was incorporated to substantiate their responses to niacin. Article \#7 \[[@B18]\] ---------------------- In this report, Hall describes the use of niacin for his migraine headaches remarking that the migraines resolved when intense flushing occurred. According to Hall, niacin\'s benefits and side effects might be due to its ability to release serotonin and histamine from the stomach. There is no reason to doubt Hall when describing his therapeutic response to niacin. However, his report was brief, had no control and was entirely subjective as he was the participant as well as examiner. Article \#8 \[[@B6]\] --------------------- In this report of 2 cases, it was found that in both cases migraines were relieved with oral niacin. The report would have been more rigorous if the patients had acted as their own self-controls. The value of the report is further diminished by the difficulty in extrapolating these findings to a greater number of patients. Article \#9 \[[@B7]\] --------------------- This case report was of a 62-year-old woman with a 40-year history of migraine headaches. The patient never acted as her own self-control, which would have made the findings of the case report more meaningful. The fact that the patient\'s migraine headaches increased in severity after a reduction in dosage does lend more support to niacin as being a migraine preventive agent. The hypothesized increase in serotonin from niacin administration cannot be proven given the limited amount of data contained in the case report. Like all case reports, its value is diminished by the difficulty in extrapolating these findings to a greater number of patients. Discussion ========== The results of this systematic review indicate that niacin may have a therapeutic effect on migraine headaches, tension-type headaches, and headaches of other etiologies. The quality of the evidence at this point, however, is only hypothesis generating, and randomized trials are required to determine the clinical implications of this novel treatment. There are several important limitations to consider in the interpretation of this review. We did not find any randomized or controlled trials of niacin on these headaches. We cannot determine to what extent publication bias has on the results of this review. We are unable to draw clinical inferences on the results of the included studies as they were of low quality and have a low level of external generalizability. Despite these limitations, we attempted to conduct an exhaustive search and included all reports of relevance. Reasons for niacin\'s effectiveness can only be considered hypothetical, and require clarification from future randomized controlled trials. In acute migraine headaches some of the symptoms arise from activation of the trigeminovascular complex. Activation of this complex leads to intracranial vasoconstriction causing the migraine aura, followed by headache due to vasodilation of the extracranial vessels and activation of the perivascular nociceptive nerves \[[@B21]\]. When taken intravenously or orally, niacin causes cutaneous flushing that might abort the acute symptoms of migraine by vasodilating the intracranial vessels, thus preventing the subsequent vasoconstriction of the extracranial vessels. There is evidence that niacin is an effective peripheral vasodilator, but its ability to influence central mechanisms (i.e., cerebral blood flow and cranial hemodynamics) involved in migraine headaches have not been well studied. Niacin causes peripheral vasodilation and cutaneous flushing by inducing the production of prostaglandin D~2~(PGD~2~) in the skin, leading to a marked increase of its metabolite, 9α, 11β-PGF~2~, in the plasma \[[@B22]\]. When niacin is administered orally in amounts of 500 mg or topically via a 6-inch patch of 10^-1^M aqueous methylnicotinate on the forearm, PGD~2~is markedly released in the skin and its metabolite appears in high amounts in the plasma \[[@B22],[@B23]\]. It is not known if PGD~2~causes vasodilation of the intracranial arteries, but niacin\'s ability to abort acute migraine headaches suggests that this might be what is occurring. Old reports cited by Bicknell and Prescott \[[@B24]\], demonstrate that niacin does indeed cause vasodilation of the cerebral and spinal vessels, and that intravenous administration increases the rate of intracranial blood flow in human beings for 20--60 minutes without any significant change in blood pressure. Unfortunately, there have not been more recent reports examining the effects that niacin has upon cerebral blood flow in human subjects. In terms of tension-type headaches, it appears that intravenous niacin is of benefit acutely due to its presumed central vasodilatory properties. Like migraines, part of the underlying pathophysiology of chronic tension-type headaches involves central mechanisms, such as the trigeminal system \[[@B26]\]. Chronic tension-type headaches are also associated with cerebrospinal pressure or intracranial venous pressure (or both) \[[@B26]\]. In fact, tension-type headaches are more similar to migraine headaches than they are dissimilar, in that they seem to progress into migraine headaches due to an escalating pathophysiological process \[[@B27]\]. Thus, niacin might mitigate the acute phase of tension-type headaches through the same hypothesized mechanism of action described earlier. Some of the reports did demonstrate prophylactic benefits when niacin was administered orally every day. It is now recognized that a deficit of mitochondrial energy metabolism (i.e., impaired mitochondrial phosphorylation potential) plays a role in the pathogenesis of chronic migraine headaches \[[@B28]\]. Niacin maintains adequate mitochondrial energy metabolism by increasing substrate availability to complex I \[[@B29]\], and this is how it might function as an effective prophylactic agent for migraine prevention. Two other nutritional agents (riboflavin and coenzyme Q10) augment complex I of the mitochondrial respiratory chain, and have been subjected to clinical trials demonstrating their effectiveness for the prevention of migraine headaches \[[@B30]-[@B32]\]. A deficit of mitochondrial energy metabolism may play a role in the pathogenesis of migraine. Since niacin improves mitochondrial energy metabolism by increasing substrate availability to complex I, it might also be an effective agent for migraine prevention. Niacin might also prevent tension-type headaches by improving mitochondrial energy metabolism within the skeletal muscles, and by increasing blood flow and oxygenation to the skeletal muscles. The overall net-effect could be a reduction in lactic acid concentrations, leading to reduced episodes of muscular tension and soreness. Niacin may reduce lactic acid concentrations since supplemental niacinamide (the amide of niacin) has been shown to reduce blood lactate and pyruvate concentrations by more than 50% in a patient with MELAS (mitochondrial encephalopathy, myopathy, lactic acidosis, and stroke-like episodes) syndrome by the third day of treatment \[[@B33]\]. This possible mechanism might only relate to migraine sufferers, however, since plasma levels of lactic and pyruvic acids were found to be significantly higher in migraine patients compared to patients with tension-type headaches and normal controls \[[@B34]\]. The side effects of intravenous niacin were found to be minimal from the summarized articles. The most common side effects were abdominal cramping, vomiting, and uncomfortable sensations of the skin and burning. A few of the patients described found the treatments to be worse than their headaches. In the articles where blood pressures were evaluated, little-to-no change occurred among the individuals treated with intravenous niacin. In a more recent study, parenteral niacin given to hypertensive and normotensive patients demonstrated a significant decrease in systolic, diastolic, and pulse pressures among the hypertensive subjects \[[@B35]\]. With respect to the oral administration of niacin, very few patients reported side effects. Even though niacin was well tolerated orally, we previously reported in a randomized placebo-controlled trial examining the safety of immediate-release or crystalline niacin, that it can be associated with intolerable side effects \[[@B36]\]. The most common side effects found using 500 mg of immediate-release niacin were unpleasant warmth or flushing, pruritis, chills, tingling, nausea, and vomiting. Approximately 75% of the subjects who were given niacin found it tolerable or difficult to tolerate, but did not indicate that they would never take niacin again. Some 18.2% of the niacin subjects indicated that they found niacin to be intolerable and would never take it again \[[@B36]\]. By contrast, very few of the patients from the summarized articles discontinued treatment due to the side effects of oral niacin. The side effects of greater concern from oral niacin have to do with sustained- or slow-release preparations. These preparations are better in terms of compliance since patients experience less cutaneous flushing with them. However, the use of these preparations have been associated with reversible hepatic toxicity in doses equal to or greater than 1500 mg per day with an acute onset of clinical symptoms of hepatitis in a relatively short period of time (2 days to 7 weeks) \[[@B37]\]. Other reports have demonstrated clinical symptoms of hepatitis when much larger doses of sustained-release niacin (greater than or equal to 3 grams per day) were used for months to years \[[@B38]-[@B42]\]. Sustained-release preparations have a higher incidence of hepatic toxicity in doses comparable to the immediate-release preparations \[[@B43]\], but these important differences are not typically mentioned in reviews of niacin\'s lipid lowering properties. Conclusion ========== Even though niacin\'s mechanisms of action have not been substantiated from controlled clinical trials. It is possible that this agent has a beneficial effect upon migraine and tension-type headaches and the prophylaxis of these headaches. It is imperative that properly designed controlled trials are developed in order to determine niacin\'s true therapeutic effects and adverse effect profile. In terms of its ability to abort acute migraine headaches, a placebo-controlled trial of parenteral niacin and sumatriptan seems to be worthy of consideration. In terms of prophylaxis, a placebo-controlled trial of oral niacin and riboflavin or coenzyme Q10 also seems worthy of investigation. Competing interests =================== Dr. Jonathan Prousky is associated with Swiss Herbal Remedies, Ltd., a company that sells nutritional supplements including niacin. They had knowledge of this manuscript and have not seen this manuscript. Authors\' contributions ======================= JP wrote the manuscript. DS contributed to the text, revised the results section, and assisted with the tables.

PubMed Central

2024-06-05T03:55:52.720346

2005-1-26

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548511/", "journal": "Nutr J. 2005 Jan 26; 4:3", "authors": [ { "first": "Jonathan", "last": "Prousky" }, { "first": "Dugald", "last": "Seely" } ] }

PMC548512

Background ========== Recent reports using murine models of allergic asthma have shown that the Th2 type cytokine IL-13 may play a critical role in the pathogenesis of asthma, either by regulating airway inflammation, mucus hyper-secretion or airway hyper-responsiveness \[[@B1]-[@B5]\]. Evidence suggests that the potential role of IL-13 in asthma may come from its aptitude to directly interact with airway resident cells, such as epithelial cells or airway smooth muscle (ASM) cells, as shown by the ability of IL-13 to stimulate a set of different pro-asthmatic genes including inflammatory cytokines such as thymus and activation-regulated chemokine (TARC), eotaxin, monocyte chemotactic protein-1 (MCP-1) as well as growth factors such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) \[[@B6]-[@B10]\]. The ability of IL-13 to modulate ASM responsiveness to G-protein coupled receptor (GPCR) agonists, either by increasing contractile agonist-evoked calcium responses \[[@B11]\], and/or by impairing ASM responsiveness to β2-adrenoceptor stimulation \[[@B6]\], may also explain, at least in part, the putative role of IL-13 in allergen-associated BHR reported in animal models \[[@B1]-[@B4]\]. Previous reports have shown that other cytokines such as tumor necrosis factor alpha (TNFα) or interleukin (IL)-1β, may also participate in airway hyper-responsiveness by modulating ASM responsiveness to contractile GPCR agonists \[[@B12]-[@B14]\]. These data strongly support the current concept that cytokine modulation of ASM, an effector cell thought to solely regulate bronchomotor tone \[[@B12]\], may play an important role in the development of airway inflammation and bronchial hyper-responsiveness, the two main features of asthma. The molecular mechanisms by which IL-13 induces \"pro-asthmatic responses\" in ASM have not been clearly established. Identifying the expression profile of \"pro-asthmatic\" genes activated by IL-13 in ASM cells may therefore provide new insight into the design of novel therapeutic approaches for asthma. Using complementary molecular approaches, we investigated the effect of IL-13 on the transcription of \"pro-asthmatic\" genes in human airway smooth muscle cells (HASMC). The effect of IL-13 was compared to that of IL-13R130Q, a naturally occurring isoform resulting in a change from glutamine to arginine residues in the coding region that is associated with high serum IgE levels \[[@B15]\]. Interestingly, no report has yet investigated whether both IL-13 and IL-13R130Q share the same or have different biological activities. We found that both IL-13 and IL-13R130Q stimulate the same set of important genes that encode for proteins which may be clinically relevant for regulating airway hyper-responsiveness, airway inflammation and airway remodeling, key characteristics of asthma. Methods ======= Cell Culture ------------ Human tracheas were obtained from lung transplant donors, in accordance with procedures approved by the University of Pennsylvania Committee on Studies Involving Human Beings. A segment of trachea just proximal to the carina was removed under sterile conditions and the tracheal muscle was isolated. The muscle was then centrifuged and resuspended in 10 ml of buffer containing 0.2 mM CaCl~2~, 640 U/ml collagenase, 1 mg/ml soybean trypsin inhibitor and 10 U/ml elastase. Enzymatic dissociation of the tissue was performed for 90 min in a shaking water bath at 37°C. The cell suspension was filtered through 105 μm Nytex mesh, and the filtrate was washed with equal volumes of cold Ham\'s F12 medium (Gibco BRL Life Technologies, Grand Island, NY) supplemented with 10% FBS (HyClone, Logan, UT) 100 U/ml penicillin (Gibco), 0.1 mg/ml streptomycin (Gibco), and 2.5 μg/ml fungizone (Gibco). Aliquots of the cell suspension were plated at a density of 1.0 × 10^4^cells/cm^2^. The cells were cultured in Ham\'s F12 media supplemented with 10% FBS, 100 U/ml penicillin, 0.1 mg/ml streptomycin and this was replaced every 72 h. Human ASM cells in subculture during the second through to fifth cell passages were used because, during these cell passages, the cells retain native contractile protein expression, as demonstrated by immunocytochemical staining for smooth muscle actin and myosin \[[@B16]\]. Unless otherwise specified, all chemicals used in this study were purchased from Sigma/Aldrich (St. Louis, MO). RNA isolation ------------- Total cellular RNA was isolated from IL-13 (50 ng/ml), IL-13R130Q (50 ng/ml) or control treated HASMC using the RNeasy mini kit (Qiagen, Inc. Valencia, CA) as per manufacturer\'s instructions. The IL-13 was purchased from R&D Systems (Minneapolis, MN) and the IL-13R130Q was generated in house at Centocor Inc. The quality and quantity of RNA was assessed using the Agilent 2100 Bioanalyzer (South Plainfield, New Jersey). Samples that demonstrated high quality (ratio of 28S rRNA and 18S rRNA is greater than 1.7) were submitted for microarray analysis. Microarray Processing --------------------- A complimentary DNA (cDNA) microarray, or DNA chip (Target B), containing a total of 8159 human gene cDNA clones from Research Genetics (IMAGE consortium, Huntsville, AL), Incyte Genomics (Santa Clara, CA) and internal sources was used in this study. All clones have been verified by DNA sequencing and are printed as 2 independent spots on a given chip. Duplicate chips were used for each RNA sample. Non-linear normalization between duplicate chips allowed each clone to be averaged to a single intensity value for each RNA sample. RNA amplification, probe synthesis and labeling, cDNA chip hybridization and washing were performed as described previously \[[@B17]\]. Agilent Image Scanner was used to scan the cDNA chips (Agilent Technologies, Palo Alto, CA). Fluorescence intensity for each feature of the array was obtained by using ImaGene software (BioDiscovery, Los Angeles, CA). Microarray data analysis ------------------------ In this study, fifty one-color cDNA microarrays were used to profile gene expression in human airway smooth muscle cells from 3 donors stimulated with IL-13, or its variant IL-13R130Q at 2 time points (6 hr and 18 hr). Untreated samples from the same group of donors were used as control. The samples being analyzed are listed in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Summary of number of samples from each donor and treatments ::: Time Donor Untreated IL-13 IL-13R130Q ------- ----------- ----------- ------- ------------ 6 hr Donor 1\* \- \- \- Donor 2 1 2 2 Donor 3 1 2 2 18 hr Donor 1 1 2 2 Donor 2 1 2 2 Donor 3 1 2 2 \*The samples from Donor 1 at the 6 hr time point were not included due to poor quality of RNA. ::: Purified cDNA probes were hybridized to two microarrays, each containing two spots for each cDNA. Raw intensity data from the cDNA arrays were first normalized within each sample. Linear normalization and then nonlinear normalization was performed within each sample. Outlier intensity data points (greater than 1.4 fold away from the median of replicate measurements) were identified and removed from the data sets. The average intensity was generated by calculating the arithmetic mean of nonoutlier intensity values. The averaged intensity for each clone was further normalized across all samples. Chip-to-chip normalization was performed by dividing the averaged intensity of each clone by the 50.0^th^percentile of all measurements in that sample. The intensity of each clone was then normalized to the median intensity of that clone in the untreated control group. The normalized intensity was then log transformed. Using Partek Pro™ 5.1, sources of variance, such as batch effects, were identified by Principle Component Analysis (PCA) and appropriate factors were taken into account in the Analysis of Variance (ANOVA). ANOVA was performed to identify the genes that were differentially expressed by cytokine stimulation. Treatment (IL-13 and IL-13R130Q), time (6 hour and 18 hour), and donor (1, 2, and 3) were the three main effects considered in ANOVA. P-value cutoff was 0.05. Benjamini and Hochberg false discovery rate (FDR) was performed for multiple testing correction. After comparing the gene lists from IL-13 and IL-13R130Q treatments, it was clear that these two treatments resulted in the regulation of the same set of genes. Subsequently, samples from these two treatments were combined and regarded as replicates in ANOVA. Furthermore, outliner samples in the data set were detected by PCA and removed to improve the detection power. As an alternative approach, fold change comparisons (cutoff = 1.5 fold) between a treated condition and the control were carried out within each donor by using GeneSpring™ 6.2 \[[@B18]\]. A gene was considered as reliably detected in a given condition if more than half of the replicates representing the same condition had a raw expression intensity of more than 50, CV smaller than 25%, and raw intensity being generated from 2 or more of the duplicate spots representing the clone. A pair-wise comparison between a treatment and its untreated control was performed only on the genes that were reliably detected in at least one condition of the pair. The genes that showed at least 1.5 fold differential expression in two or more donors were identified for each cytokine treatment at a time. Reverse Transcription and Real Time PCR --------------------------------------- 1 μg of total RNA from each of the IL-13 (50 ng/ml) or IL-13R130Q (50 ng/ml) or control treated HASMC were used for the reverse transcription (RT) reaction. The RT reaction was performed as per protocol using TaqMan^®^RT reagents (Applied Biosystems) at 37°C for 120 min followed by 25°C for 10 min. Forty ng of cDNA per reaction were used in the Real Time PCR using the ABI Prism^®^7900 sequence detection system (Foster City, California). In the presence of AmpliTaq Gold DNA plolymerase (ABI biosystem, Foster City, California), the reaction was incubated for 2 min at 50°C followed by 10 min at 95°C. Then the reaction was run for 40 cycles at 15 sec, 95°C and 1 min, 60°C per cycle. Assays-on-Demand™ primers and probes (Applied Biosystems) were used in the PCR. The Real Time PCR data was analyzed using the standard curve method. Flow Cytometry -------------- Flow cytometry was performed as described previously with slight modifications \[[@B19]\]. Briefly, adherent cells treated with IL-13 for 24 hr were washed with PBS, detached by trypsinization (2 min, 37°C) and then washed with Ham\'s-F12 (10% FCS) media, centrifuged, and transferred to microfuge tubes (1.5 ml). Cells were incubated with anti-IL-13Rα2 (5 μg/ml, Santa Cruz Biotech) antibody followed by 1 hr incubation with a fluorescein isothiocyanate-conjugated secondary antibody (Jackson ImmunoResearch Laboratories; West Grove, PA). In parallel experiements, cells were incubated with the FITC-conjugated mouse anti-VCAM-1 antibody (2 μg/ml, Santa Cruz Biotech) for 1 h at 4°C. The cells were then centrifuged and resuspended in cold PBS in microfuge tubes. Samples were then analyzed using an EPICS XL flow cytometer (Coulter, Hialeah, FL). VCAM-1 and IL-13Rα2 levels were expressed as the increase in mean fluorescence intensity over un-stimulated cells. Results ======= IL-13 regulates gene expression of HASMCs ----------------------------------------- IL-13 may exert its deleterious effects in asthma by directly altering gene expression in airway resident cells such as epithelial cells or ASM cells \[[@B5]-[@B7],[@B20]\]. In order to determine which genes are regulated by IL-13 in airway smooth muscle cells, we employed the cDNA microarray technology. We also wanted to ascertain if the effect of IL-13R130Q, a naturally occurring isoform of IL-13 and associated with high serum IgE levels \[[@B15]\], was any different than IL-13 in terms of modulating gene expression. The concentrations of IL-13 (10--100 ng/ml) used in our study were shown previously to stimulate gene expression in human ASM cells \[[@B7],[@B8],[@B10]\], although the *in vivo*relevance of these particular concentrations remains unknown. Three donors were used and two types of analyses were carried out (Fold change analysis; Statistical Analysis). Both IL-13 and IL-13R130Q generated a similar expression profile i.e., genes regulated by IL-13 were the same as those regulated by IL-13R130Q at the 1.5 fold cutoff. Table [2](#T2){ref-type="table"} lists genes of interest that were identified from analyzing the data and divides them into one of three categories. Genes involved in all three characteristics of asthma (airway inflammation, remodeling and bronchial hyper-responsiveness) were identified. Of particular interest are vascular cellular adhesion molecule (VCAM)-1, Tenascin C, IL-13Rα2 and Histamine Receptor H1. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Summary of genes up regulated by IL-13 and IL-13R130Q. ::: Category Gene(s) Fold change ------------------------------------ -------------------------------- ---------------- **Airway Inflammation** Adhesion Molecules **VCAM-1** ↑ **2 fold** ALCAM Selectin P ligand Laminin B1 Chemokines Chemokine Ligand 2 Chemokine Ligand 11 Chemokine Ligand 26 Chemokine Ligand 27 Cytokine receptors **IL-13 Rα2** ↑ **1.6 fold** Interleukin 1 receptor **Airway Remodeling** Extracellular matrix **Tenascin C** ↑ **2 fold** Tenascin R Collagen Type I Collagen Type VI Collagen Type III Fibulin 1 CD44 Cell proliferation Pim-1 eEF1A Cytokines PDGFC Retinoic acid Receptor Interferon beta 1 **Bronchial Hyper-responsiveness** Cytoskeletal constituants Vimentin Tropomyosin 1 Tropomyosin 2 Actin Calcium regulators Phospholipase D Calreticulin hGIRK1 TRPC4 TRPC6 Sphingosine kinase 1 Rho GDP dissociation inhibitor FKBP1A Receptor **Histamine H1 receptor** ↑ **1.3 fold** The fold changes correspond to the genes in bold. ::: Real Time PCR validation ------------------------ TaqMan™ Real Time PCR was used to validate VCAM1, IL-13Rα2, Tenascin C and Histamine Receptor H1. As shown in Figure [1A](#F1){ref-type="fig"}, VCAM1 was upregulated between 2 and 2.5 fold upon IL-13 or IL-13R130Q treatment at the 6 and 18 hour time points in both donors. This is comparable to the microarray data (Table [2](#T2){ref-type="table"}). In Figure [1B](#F1){ref-type="fig"}, IL-13Rα2 mRNA is upregulated with IL-13 or IL-13R130Q. However, the upregulation is more pronounced at the 18 hour time point compared to 6 hour. In Figure [2A](#F2){ref-type="fig"} Tenascin C is upregulated with IL-13 and IL-13R130Q and in Figure [2B](#F2){ref-type="fig"}, Histamine Receptor H1 shows an upregulation of about 1.5 fold in both donors at both time points and with both treatments. Again, this is comparable to the microarray data (Table [2](#T2){ref-type="table"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Real Time PCR (Taqman^®^) analysis showing the level of A) VCAM1 B) IL-13Rα2 upon treatment of ASM from two donors with IL-13 or IL-13R13Q for 6 or 18 hrs. The quantity of each gene is normalized to 18S and relative to the untreated sample. Values shown are mean ± standard deviation from an n = 6. ::: ![](1465-9921-6-9-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Real Time PCR (Taqman^®^) analysis showing the level of A) Tenascin C and B) Histamine receptor H1 upon treatment of ASM from two donors with IL-13 or IL-13R13Q for 6 or 18 hrs. The quantity of each gene is normalized to 18S and relative to the untreated sample. Values shown are mean ± standard deviation from an n = 6. ::: ![](1465-9921-6-9-2) ::: Validation of VCAM-1 and IL-13Rα2 at the protein level ------------------------------------------------------ In order to validate the modulatory effect of IL-13 on VCAM-1 and IL-13Rα2 genes at their protein level, flow cytometry was performed to confirm the up regulation of VCAM-1 and IL-13Rα2 in HASMC by IL-13. As shown in Figure [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}, IL-13 (10--100 ng/ml, 24 hr) differentially stimulates the expression of VCAM-1, with levels increasing in a dose-dependent manner, while IL-13Rα2 levels were identical at 10, 30 and 50 ng/ml. At 100 ng/ml IL-13, VCAM-1 and IL-13Rα2 levels were significantly increased by 20% and 35% over basal, respectively (n = 3, p \< 0.05). Increases in VCAM-1 and IL-13Rα2 proteins by IL-13 nicely correlate with changes in mRNA levels (Figure [1A](#F1){ref-type="fig"} and Figure [1B](#F1){ref-type="fig"}), suggesting that the IL-13 regulates the expression of inflammatory proteins via transcriptional mechanisms. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effect of IL-13 on VCAM-1 expression. ASM cells were incubated with the indicated concentrations of IL-13 for 24 hr. VCAM-1 expression was assessed by flow cytometry as described in Methods. Values shown are mean ± SEM and are significantly different from basal, n = 3 different experiments. \*P \< 0.05 significant from untreated cell. ::: ![](1465-9921-6-9-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effect of IL-13 on IL-13Rα2 expression. ASM cells were incubated with the indicated concentrations of IL-13 for 24 hr. IL-13Rα2 expression was assessed by flow cytometry as described in Methods. Values shown are mean ± SEM and are significantly different from basal, n = 3 different experiments. \*P \< 0.05 significant from untreated cell. ::: ![](1465-9921-6-9-4) ::: Discussion ========== Recent evidence using various experimental approaches such as gene-deficient mice, soluble inhibitors or transgene overexpressing IL-13 in the airways have highlighted the critical role of IL-13 in the pathogenesis of allergic asthma, possibly due to its ability to regulate goblet cell metaplasia, mucus hypersecretion and airway hyper-responsiveness \[[@B1],[@B3],[@B21]\]. Few reports that used microarray analyses applied to cultured human ASM cells demonstrated that IL-13 differentially regulates a number of important genes that are relevant for the pathogenesis of asthma \[[@B7],[@B10]\]. Although the functional relevance of such gene microarray analyses remains yet uncertain, these studies strongly suggest that IL-13 may be involved in the pathogenesis of asthma by directly modulating physiological responses of the ASM. Compared to the previous gene microarray reports \[[@B7],[@B10]\], we did confirm the physiological relevance of the microarray data using two different experimental approaches. At least for four different genes, VCAM-1 (an adhesion protein), IL-13Rα2, Histamine H1 receptor (a G protein-coupled receptor) and Tenascin C (an extracellular matrix glycoprotein), there was a close correlation between the data obtained from gene microarray with those obtained by real time PCR analyses. In addition, we showed that IL-13 stimulates the expression of VCAM-1 and IL-13Rα2 at the protein level, showing the physiological relevance of the gene array data. It is interesting to note that no difference in gene expression profile were noticeable between cells exposed to IL-13 or IL-13R130Q, an IL-13 polymorphism recently found to be associated with elevated serum and allergen-specific IgE \[[@B15],[@B22]\]. Our report is the first to suggest that this particular IL-13 polymorphism is equally effective as IL-13 in the transcriptional regulation of the genes examined in the present study. Our present study further supports the concept that IL-13 regulates the expression of different \"pro-asthmatic\" genes that are potentially important in the regulation of all three key features of asthma, i.e., airway inflammation, airway remodeling and bronchial hyper-responsiveness (for details see Table [2](#T2){ref-type="table"}). Previous reports using cultured ASM cells also demonstrated that IL-13 can stimulate the expression of other pro-inflammatory proteins, such as eotaxin \[[@B8],[@B23],[@B24]\], TARC \[[@B9]\] or VEGF \[[@B25]\] and tenascin \[present report and \[[@B10]\]\]. Upregulation of tenascin C and R, glycoproteins that contribute to extracellular matrix structure \[[@B26]\], may play an important role in airway remodeling, a characteristic of chronic asthma. The stimulatory role of IL-13 on VCAM-1 may be important in asthma since VCAM-1 has been regarded as a key player in the development of airway inflammation \[[@B27]\]. The ability of IL-13 to increase the expression of different G-protein coupled receptors (GPCR), such as Histamine H1 \[present report and \[[@B10]\]\] or CysLT1 receptor \[[@B28]\] represents one potential mechanism by which IL-13 promotes airway hyper-responsiveness to GPCR agonists previously described both *in vivo*\[[@B1],[@B3],[@B21]\] or *in vitro*in isolated airways preparations \[[@B11],[@B29],[@B30]\]. Additional studies are needed to determine whether IL-13 also modulates ASM responsiveness to Histamine. The receptor complex by which IL-13 regulates cellular function comprises the IL-13Rα1, which binds IL-13 and forms a complex with the IL-4Rα to initiate signal transduction via the JAK/STAT6 pathway \[[@B31]\]. IL-13Rα2, the other cell surface protein binds IL-13 with high affinity but the complex is not functionally active. One previous report using transgenic mice showed that overexpressing IL-13 in the airways induced a marked increase in both IL-13Rα1 and IL-13Rα2 mRNA levels, mostly in epithelial cells and macrophages \[[@B32]\]. Our study is the first to show that the expression of IL-13Rα2 is also transcriptionally increased by IL-13 in ASM cells. Although the functional significance of such regulation remains unknown, it is possible that the newly induced IL-13Rα2 could function as a decoy receptor to limit IL-13 signaling in ASM cells. Additional studies are needed to further support this hypothesis. Conclusions =========== These data further support the hypothesis that gene modulation by IL-13 in ASM may be essential for the events leading to the development of allergic asthma. Additional studies are clearly needed to define the transcriptional regulation of the different \"pro-asthmatic\" genes by IL-13, which may lead to novel therapeutic approaches for the treatment of allergic asthma. Authors\' contributions ======================= FS, LL, YA and RAP participated in the conception and design of the study. FS coordinated the study and along with YA drafted the manuscript. OT performed the RNA isolation and flow cytometry experiments. CH and KL performed the microarray data analysis. MB performed the Real Time PCR experiments. DG and BA proof read the manuscript. All authors read and approved the final manuscript. Acknowledgements ================ This study was supported through grants from the National Institutes of Health (RO1-HL55301 to RAP and 2RO1-HL064063 to YA), and American Lung Association grant RG-062-N (YA). Yassine Amrani is a Parker B. Francis Fellow in Pulmonary Research.

PubMed Central

2024-06-05T03:55:52.722811

2005-1-20

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548512/", "journal": "Respir Res. 2005 Jan 20; 6(1):9", "authors": [ { "first": "Farhat", "last": "Syed" }, { "first": "Reynold A", "last": "Panettieri" }, { "first": "Omar", "last": "Tliba" }, { "first": "Chris", "last": "Huang" }, { "first": "Katherine", "last": "Li" }, { "first": "Michelle", "last": "Bracht" }, { "first": "Bernard", "last": "Amegadzie" }, { "first": "Don", "last": "Griswold" }, { "first": "Li", "last": "Li" }, { "first": "Yassine", "last": "Amrani" } ] }

PMC548513

Background ========== Obesity is becoming increasingly common and is recognized as a major public health problem worldwide \[[@B1]-[@B3]\]. In the UK, the overweight and obese population increased by almost 15% between 1980 and 1992. Similar increases have been noted in many countries, including the USA \[[@B4]\], Sweden \[[@B5]\] and the Netherlands \[[@B6]\]. In Israel, 55% of females and 58% of males aged 25--64 were reported to be overweight or obese (Body Mass Index \>25 kg/m^2^) in a Mabat survey \[[@B7]\] in 2000. Guidelines published in 1996 for the management of obesity recommended setting a modest weight loss and weight maintenance, rather than a targeted ideal weight, as goals \[[@B8]\]. Lately, pharmacological therapies have been proposed as adjuncts to diet and lifestyle changes to improve long-term weight loss \[[@B9]\]. The World Health Organization declared obesity an epidemic in developed countries. Awareness of physicians of their important role in defusing the obesity epidemic has increased. Yet, the number of family practitioners (FP) treating obesity problems continues to be low \[[@B10]\]. Many chronic health problems are exacerbated by unhealthy behaviors and harmful environmental conditions. From the psychological perspective, healthful lifestyles and environmental conditions may yield large health benefits. The widespread adoption of a healthier lifestyle rather than medical technologies has resulted in a substantial decline in premature mortality and morbidity \[[@B11]\]. The media plays a major role in informing the public about health risks. Efforts to convince people to adopt health practices that prevent disease have relied heavily on persuasive communications in health education campaigns \[[@B11]\]. Health benefits are accelerated by community-wide efforts to reduce habits that impair health \[[@B12]\]. Self-efficacy refers to the belief in one\'s ability to organize and execute the courses of action required to produce a given attainment \[[@B11]\]. Such beliefs influence the courses of action people choose to pursue, how much effort they put into a given endeavor and how long they will persevere in the face of obstacles and failure. If people believe they have no power to produce results, they will not attempt to effect changes. Self-efficacy is not a fixed ability that one has or lacks in one\'s behavioral repertoire. Rather, it is a thinking process, a generative capability in which cognitive, social, emotional and behavioral sub-skills are organized and effectively orchestrated to serve innumerable purposes. Self-efficacy is concerned not with the number of skills one has, but with what one believes one can do with the skills under a variety of circumstances. Efficacy beliefs operate as a key factor in a generative system of human competence \[[@B11],[@B13]-[@B15]\]. Self-efficacy is an important contributor to performance accomplishments, whatever the underlying skills might be \[[@B11]\]. The greater a person\'s efficacy beliefs, the greater the academic challenges one sets for oneself and the greater their intrinsic interest \[[@B16]\]. Personal efficacy beliefs influence the level of interest in occupational pursuits even when the influence of ability is removed \[[@B17],[@B18]\]. A sense of personal efficacy is constructed through a complex process of self-persuasion. Efficacy beliefs are the product of cognitive processing of diverse sources of information conveyed inactively, vicariously, socially and physiologically. Once formed, efficacy beliefs contribute to the quality of human functioning \[[@B11]\]. Self-efficacy is measured by the strength of a subject\'s beliefs in the ability to execute requisite activities. Social cognitive theory distinguishes among three basic processes of personal change: the adoption, general usage and maintenance over time of new behavioral patterns \[[@B19]\]. Efficacy beliefs affect each of these phases \[[@B11]\]. Self-efficacy conceptualizes a person\'s perceived ability to perform on a task as a mediator of performance on future tasks \[[@B11],[@B13]\]. A change in the level of self-efficacy can predict a lasting change in patients\' behavior if there are adequate incentives and skills \[[@B13]-[@B15]\]. People\'s beliefs that they can motivate themselves and regulate their own behavior play a crucial role in whether they even consider acting. Thus, practitioners who judge themselves incapable of treating obesity do not even try. Obesity has not been considered a disease till recent years. Physicians did not treat obesity unless they were asked to do so by patients. To our knowledge and after a literature search, no intervention studies have yet been conducted to change the self-efficacy of FPs in Israel towards treating obese people. Among other courses for which physicians usually receive credits towards their annual professional training, a course was offered by the Israeli Academic Medical Council. The course was initiated by the Israeli Association of FPs and recommended by the Medical Professional Journal of FPs in Israel. Registration in the course was open to all FPs. The present research was a preliminary study. This being the case, the first group of FPs who attended the new course participated in it. Though that group contained a small number of subjects, the importance of investigating a new program contribution to FPs self-efficacy for future research was evident. The objectives of the course were to enrich the knowledge of FPs with up-to-date information on obesity and to raise their motivation to treat it. The study objective was to determine if an interactive course would raise the self-efficacy of FPs to treat obesity. It was hypothesized that the self-efficacy of FPs would be enhanced as a result of participating in an interactive obesity-treatment course. Methods ======= Subjects -------- Twenty-nine FPs (62% female) chose to participate in the course along with other Continuing Medical Education (CME) courses. All participants work as FPs in public health care clinics throughout the country. Design ------ This study was based on a one group, pre-course -- post-course test design, without a control group. It was accompanied by qualitative interviews to validate the results of the data analysis. Research tools -------------- The strength of the efficacy beliefs of the FPs to treat obesity was estimated by using a five point scale Likert type questionnaire containing nine items (Table [1](#T1){ref-type="table"}). The subjects were asked how confident they were in the ability to treat obesity across problem situations. The levels of confidence were as follows: 1. not at all confident, 2. somewhat confident, 3. moderately confident, 4. very confident, 5. completely confident. The questionnaire was filled out before and after the course. The items were generated from the criteria on the domain map that was constructed on the basis of theoretic analyses of knowledge accumulated in the domains of self-efficacy and health behavior change \[[@B11],[@B13]-[@B15]\] and consultation with experts. The researchers used the analytic induction method for the theoretic analysis of knowledge. The map\'s main criteria were: obesity -- a serious disease; up-to-date clinical and behavioral knowledge; processes of change, e.g: decision making, planning, monitoring and behavior controlling; resilience; lack of time and social involvement. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Original domain map criteria and the sequence of criteria in the questionnaire regarding the self-efficacy beliefs of family practitioners to manage obesity ::: Original sequence of criteria Sequence in the questionnaire ----------------------------------------------------------------------------------------------- ------------------------------- Efficacy to treat a problem of high priority 1 Efficacy to give up-to-date and correct information 7 Efficacy to persuade, support and help patients make decisions 3 Efficacy to make patient plan behaviors and situations 6 Efficacy to make patient monitor his behavior 4 Efficacy to make patient control behaviors and situations 9 Efficacy to treat obesity regardless of previous failure or unsuccessful experiences 2 Efficacy to treat obesity regardless of lack of time 5 Efficacy to bring about involvement of other people in the patient\'s behavior change process 8 ::: The Alpha Cronbach for the reliability of the tests was α = 0.88 for the pre-course test and α = 0.90 for the post-course test (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Pre-course (α = 0.88) and post-course (α = 0.90) scale mean, SD, item total correlation, and α if item deleted (n = 29) ::: Item no. Mean SD Item total correlation α, if item deleted ------------- ------ ------ ------------------------ -------------------- Pre-course 1 0.77 0.13 44.03 0.88 2 0.61 0.15 43.62 0.87 3 0.53 0.16 43.40 0.86 4 0.59 0.16 42.83 0.86 5 0.74 0.15 39.85 0.87 6 0.84 0.15 41.39 0.87 7 0.44 0.13 46.61 0.88 8 0.77 0.16 39.89 0.86 9 0.72 0.14 39.36 0.85 Post-course 1 0.67 0.16 20.10 0.88 2 0.75 0.16 20.67 0.88 3 0.67 0.16 20.11 0.88 4 0.46 0.14 21.17 0.90 5 0.59 0.15 19.82 0.90 6 0.72 0.15 20.29 0.88 7 0.68 0.15 21.50 0.89 8 0.71 0.16 19.21 0.88 9 0.81 0.16 19.99 0.88 ::: The following aspects of structure validity were examined: (a) for the content aspect, the items matched the domain concept map. Final version was rewritten on the basis of experts\' and researchers\' comments. The items were finally presented in an nonsequential order to make subjects think while reading the questions and not relate to earlier answers; and (b) for the substantive aspect, FPs were interviewed regarding their self-efficacy to assure that the questions were clear and did not require rewording. The interview was a 30 min. open interview. A physician was given open questions such as: \"Describe your feelings and thoughts of efficacy to treat obesity\" or \"How the self-control lectured help your efficacy perceptions\". The subject was free to speak openly on the issue. The interview was actually a verbal reflection of thoughts and feelings of the subjects. It was recorded and later on transcribed by the researchers. The interview was analyzed by the constant comparative qualitative method of analysis \[[@B20],[@B21]\]. Every sentence said by the subject in the interview was considered a unit to be taken into account for the content analysis. The present study design used paired *t*-test in order to compare pre and post strength of efficacy beliefs for treating obesity. Procedure --------- Participants answered the questionnaire before the start of the course and at the end of the last session, and a few days later participated in an open interview. The course was interactive and contained 12 clinical and psychological lectures given by experts in all subjects relating to obesity (Table [3](#T3){ref-type="table"}). Every lecture was followed by a workshop. The program consisted of six monthly sessions. Each session (from 17:00 to 21:00) started with two medical lectures followed by a 90 min workshop, and ended with a panel discussion. In the first workshop FPs said they did not address the problem of obesity unless they were asked to do so by patients. Even if they wanted to relate to it they would feel uncomfortable with it, as if that was none of their business. They reported between 1--3 obesity treatments a day. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Table of contents of the interactive course: \"Monitoring and treating obesity by the family practitioner\" ::: ---- ------------------------------------------------------------------------------------------------------------------------------------------------ 1 Obesity -- the epidemiological perspective 2 The pathogenesis and metabolic factors of obesity 3 The clinical approach of the family physician to the obese patient 4 Nutrition, diet and treatment of overweight 5 Self-control and behavior modification -- diagnosing and managing the stages of change: the trans-theoretical model, the self-regulation model 6 Physical activity and exercise, lifestyle and energy expenditure 7 Drug treatment of obesity 8 Surgical treatment of obesity 9 The metabolic syndrome 10 The mechanism of hypertension in obesity 11 The diabetic obese patient 12 Infertility of obese women ---- ------------------------------------------------------------------------------------------------------------------------------------------------ ::: FPs filled in clinical report forms and presented the cases they treated and tools they used. The cases were discussed during the course and feedback was given by colleagues and experts. The course supplied the FPs with knowledge, skills and psychological tools such as: food and physical activity diaries, decision making tools, self-evaluation tools, self-report tools, self-monitoring tools, persuading tools, stimulus control tools and counter-conditioning tools, to treat obesity. Medical knowledge and skills were not examined. The course attempted to raise the self-efficacy of FPs by creating a supportive atmosphere, providing feedback, recalling successful experiences, learning from models, bringing patients into the workshops, discussing sociological and psychological problems and allowing reflection of thoughts and emotions. Studies have shown that reflection enhances metacognitive processes such as: self-monitoring, self-evaluation, self-reaction and attribution \[[@B11],[@B22]-[@B25]\]. Since self-appraisal of efficacy is a form of metacognition and efficacy beliefs are structured by experience and reflective thoughts, we viewed reflection on obesity issues and on FPs self-efficacy as a forethought process so that the mental process the FPs went through had an effect on the processing of their efficacy appraisals. We expected their appraisals to go through a change \[[@B13]-[@B15]\]. Results ======= The differences between FP pre and post course efficacy appraisals was tested by paired *t*-test (Table [4](#T4){ref-type="table"}) and significant differences were found (Effect size .317). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Difference between pre and post course efficacy beliefs to treat obesity ::: Item no. Mean pre-course Mean post-course *t* df N ---------- ----------------- ------------------ ---------- ---- ---- 1 3.93 4.48 2 3.86 4.48 3 4.21 4.41 4 3.48 4.17 5 3.00 3.79 6 3.34 4.07 7 4.17 4.55 8 3.31 4.31 9 3.41 4.10 -3.606\* 28 29 \*(significant 1-tailed) *p*\< 0.0005 ::: All subjects reported enhanced efficacy beliefs to treat obesity. Negative feelings towards the course were not said or written. Yet, efficacy belief enhancement of the group differed among the items (see Table [4](#T4){ref-type="table"}). Three FPs left the course right after the first meeting claiming a lack of time in learning and dealing with new perceptions and no time to treat the family holistically. They also said that the time slot of the course was inconvenient. Some FPs presented cases which had failed to bring about a change in the patient\'s behavior. Those cases were discussed and the FPs were given strategies and tips for further treatment. In addition, the interviews resulted in reflection by the FPs on the process of their self-efficacy. Coded sentence units of the interviews were constantly compared while evidence accumulated. Nine main criteria were generated through that systematic analysis. These criteria matched the questionnaire items. The four criteria that received the most evidence formed four core constructs. The first was the contribution of the course to the FPs. FPs appreciated the importance of up-to-date information, the various treatment perspectives, and the skills they acquired. Knowledge was a precondition for change. FPs talked about the practical tools and tips they acquired. This is illustrated in the example: *\"It is **his**will and actions and **our**help\"*. The second criterion was the efficacy to persuade patients to treat obesity. FPs described how they could persuade patients to treat obesity in a variety of ways. The third criterion was the FP performance reports. FPs presented their clinical report forms and described how they successfully used the new knowledge, skills, tools and tips they acquired in the course to manage obesity in their clinics. The fourth criterion was efficient time management. Time management scored the lowest on the pre-course questionnaire. Whenever the issue was raised during the course, FPs claimed that time was the main obstacle in treating obesity. By the end of the course, a significant change in their perception of time was noted. Prior to the beginning of the course, only 1--3 clinical report forms on obesity treatment were reported by the FPs; at the end of the course, that number increased to 8--37. After having taken the course, obesity problems were not ignored anymore. Table [5](#T5){ref-type="table"} shows the criteria listed in the questionnaire, and the criteria generated from coded sentence units mentioned during interviews. The criteria derived from the interviews are illustrated by characteristic examples. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Questionnaire criteria, criteria coming out of interviews, and examples ::: -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Questionnaire criteria Criteria coming out of interviews Examples from interviews --------------------------------------------------------------------------------------------------- ----------------------------------------------------------------------------------------------------------------- ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- 1\. Efficacy to treat a problem of high priority a\. Course contribution: motivation to learn, and to work harder \- *\"I used to treat obesity as something that needed cosmetics now I know it\'s a disease that needs a cure\...and it is my job\"*\ - *\"I\'ve a lot more to learn\...I wish I had more courses like this one\...It\'s very important\...I\'m ready to work hard\...to do a lot more\...\"* 2\. Efficacy to give up to date precise information b\. Course contribution: enriched important knowledge, treatment perspectives, skills, practical tools and tips \- *\"Now I have the tools, a lot of information\...now I have the right perspective\...I shouldn\'t be angry with him but supportive\...with the tools it\'s much easier\...It\'s **his**will and actions and **our**help. I feel I know how to help him\...the course was helpful\"* 3\. Efficacy to persuade, support and help patients make decisions c\. Efficacy to persuade patients to treat obesity \- *\"Today I\'m more patient\...I know when it\'s the right moment to bring up the issue\...and I know how to do it\"*\ - *\"I can use emotions to persuade him\"*\ - *\"Before taking the course, I wasn\'t self-confident enough to do it, now I feel free to talk about this\...I can show him statistics on obesity\...\"* 4\. Efficacy to make patients plan behavior and situations d\. Efficacy to make patients plan, monitor and control health behavior \- *\"Since he is aware of his expectations I can make him plan or act\...\"*\ - *\"under my guidance he can see what\'s right and what\'s wrong\... and he can change things\...\"*\ - *\"when he understands the process he can initiate behavior\"* 5\. Efficacy to make patients monitor behavior 6\. Efficacy to make patients control behavior and situations 7\. Efficacy to treat obesity regardless of previous failures or unsuccessful experiences e\. FP performance reports \- *\"What you taught in the course works!\"*\ - *\"Now that I know that taking small steps is better than expecting a dramatic weight loss -- it is easier\...and the patient is happier.*\ *I now treat 10 obese people\...\"*\ - *\"I now treat 8 people, before taking the course, I did not treat any\"*\ - *\"I have 9, I was given feedback by a patient\... she said: I lost two pounds, thank **you**!\...you are great\...\"* 8\. Efficacy to treat obesity regardless of lack of time. f\. Efficient time management \- *\"I\'ll tell you my secret, everybody starts work at 8.00, I com at 7.00.*\ *I\'m ready to do it, I want to succeed, after all, it is my job\"*\ - *\"I make a double appointment for an obese patient\...\"*\ - *\"If I treat obesity now, I save time, I won\'t have to treat other diseases in the future\"*\ - *\"I keep thinking about how to be more efficient, I have to do something about the time!\"*\ - *\"I know that when I want something I find the time\"* 9\. Efficacy to bring about other people\'s involvement in the patient\'s behavior change process g\. Better done with someone\'s help \- *\"I tell her, doing it alone is too difficult. You should bring your husband or a friend to the next visit\"* h\. FP weight loss \- *\"I lost weight as a result of the course\"* i\. Enhancing efficacy beliefs by reflection, feedback and supportive climate \- *\"Did you hear the FPs\' reaction to my presentation?\...Wow!\... that was great\...I understood I had great success\"*\ - *\"I was given feedback by patients, now I know I can!\"*\ - *\"The best thing that happened to me in this course is that it made me think\"*\ - *\"I could open myself to talk about things that I hadn\'t done before\...I think it\'s because of the warm climate\"* -------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: Discussion ========== The purpose of the study was to discover whether participation in an interactive course would result in a change in the efficacy beliefs of FPs to treat obesity. Results show a significant difference between pre and post course beliefs. The increase was satisfactory (\>4 in a scale of 1--5) and the results of the qualitative analysis indicate that the criteria derived from the interviews matched those of the questionnaire. When speaking openly, FPs addressed the same issues that made up the domain theoretical concept map. Several researchers have suggested that quantitative efforts in the study of self-efficacy should be complemented by qualitative studies aimed at gaining a deeper understanding of attitudes and emotions \[[@B22]-[@B24]\]. The criteria obtained from the interviews not only matched the criteria in the questionnaire but also enriched our knowledge with a deeper understanding of the attitudes and thoughts of the FPs about the course and the way they looked at obesity management after the course. All sentence units of the interviews were taken into account for content analysis. The study showed that acquisition of knowledge and skills enable a person to meet personal standards of merit that tend to heighten beliefs of personal efficacy \[[@B11]\]. In judging their efficacy, individuals necessarily unite personal agency with means. They act on beliefs of how well they can use prescribed means \[[@B10]\]. The FPs felt the course equipped them with appropriate knowledge and means for treating obese patients. This was expressed in many sentence units. Another important psychological role in all phases of behavior change was the efficacy to persuade patients to treat obesity. The FPs\' presentations of clinical report forms and descriptions of success to manage obesity in their clinics support research findings that efficacy beliefs predict both intentions and behavior \[[@B10]\]. The FPs challenged time management as efficiently as they could, which is another important change in the perception of their role as FPs. The impact of the questions analyzed in this study extends beyond the issues asked by the questionnaire. New insights were gained through qualitative analysis: FPs analyzed the process they experienced during the course. They described how their efficacy beliefs were enhanced through reflection, feedback and the supportive climate of the course. Studies have shown that reflection enhances self-efficacy processes since self-appraisal of efficacy is structured by experience and reflective thought \[[@B25]\]. FPs reported that reflection on thoughts and emotions helped them construct their beliefs. Feedback regarding the quality of one\'s work progressively raises perceived efficacy, which, in turn, predicts subsequent performance. This is illustrated in studies of self-regulated productivity \[[@B26]\]. The feedback the FPs received from their colleagues and the experts on the quality of their reported experiences enhanced their efficacy beliefs. The FPs feedback on the course showed that the workshops contributed most to their self-efficacy. FPs explained that they had got the chance to \"bring the clinic into the workshop\", to discuss their performance, to get feedback on it, and to be stimulated to reflect on the treatment and on themselves as physicians. Incentives for mastering activities contribute to the growth of interest and perceived efficacy\[[@B10]\]. The credit points FPs received for their professional training served as an incentive that fostered performance accomplishments. The FPs reported on an increased number of obesity treatments which was an important gain of this educational program. There were no other absences except for the 3 who had left after the first session. At the end of the course FPs requested that the course be continued. In summary, an effective program of widespread change in health practices includes four major components. The first is informational and intended to increase the physician\'s awareness and knowledge of the subject. The second involves development of skills needed to translate informed concerns into effective action. The third is aimed at building a robust sense of self-efficacy to support the exercise of control in the face of difficulties that inevitably arise. This is achieved by providing repeated opportunities for guided practice and corrective feedback in applying the skills in simulated situations that people are likely to encounter. The final component involves creating social support for desired changes. The present program contained all these components. The limitations of the study were the small number of participants and the reliance on one motivated group of FPs. These limitations result from the fact that this was the first course organized by the Israeli Association of Family Physicians on the subject of obesity. It was important to study the effect of the program on FPs self-efficacy for future continuing education and research. We recommend studying the effect of interactive courses on the lifestyle and weight loss of FPs. Future research should consider randomized samples from larger courses and analyses of the correlation between course success and actual FPs performance, FPs\' self-efficacy and treatment outcomes e.g: BMI change, Lipids and BP. We also recommend studying the differences in obesity treatment outcomes and in general health feelings between patients whose FPs attended obesity courses and patients whose FPs did not. Practical recommendations for Continuing Medical Education planners would be to focus on workshops rather than on lectures, to enhance those processes the FPs felt less efficacious to go through and to have guidance of Endocrinology experts\' as well as Psychology and Education specialists to improve communication with patients and their families in an effort to enhance motivation to loose weight. It is also recommended to bring patients to the workshops to reflect on the treatment they have received. Competing interests =================== The author(s} declare that they have no competing interests. Authors\' contributions ======================= SK and AF conceived and designed the study, participated in the collection, analysis and interpretation of data and drafted the manuscript. SV Participated in the statistical analysis, interpretation of data and draft of the manuscript. SP participated in the design of the study, data collection and interpretation. All authors read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6920/5/4/prepub> Acknowledgment ============== Prof. Elliot Berry, Director of the Department of Human Nutrition and Metabolism of the Hebrew University-Hadassah Medical School of Jerusalem, is acknowledged for his helpful comments.

PubMed Central

2024-06-05T03:55:52.725076

2005-1-29

{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC548513/", "journal": "BMC Med Educ. 2005 Jan 29; 5:4", "authors": [ { "first": "Sara", "last": "Katz" }, { "first": "Amiel", "last": "Feigenbaum" }, { "first": "Shmuel", "last": "Pasternak" }, { "first": "Shlomo", "last": "Vinker" } ] }