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PMC532397	In the 25 years since they were first discovered, introns have puzzled molecular biologists because of their uncertain function and mysterious origin. Introns are non-coding DNA sequences that reside inside a gene, splitting it into discrete units called exons. The resulting disruption of coding sequence continuity would wreak havoc in protein assembly if eukaryotic cells did not dispose of introns in messenger RNAs---the intermediates in the decoding of gene sequences to produce protein chains---in a now well-described process known as splicing. At first glance, introns may seem like pesky parasites for which eukaryotes have cleverly evolved bypass mechanisms. But introns may also benefit their hosts. Evolutionary advantages of introns include the possibility to create new genes by cutting and pasting exons from existing genes or to diversify the protein output of a single gene by splicing the exons together in different ways. Thus, balancing intron gains and losses clearly has important evolutionary implications for a host. Yet different organisms strike that balance differently. The budding yeast Saccharomyces cerevisiae averages less than one intron per gene, whereas mammalian genes routinely have 10 or more. Whether these differences reflect different propensities for gaining or losing introns is the subject of ongoing debates. Organisms with low intron density display a bias for insertions at the beginning (5′ end) rather than the end (3′ end) of genes. A popular hypothesis is that in these organisms, genes lose their introns through a process that rewrites genomic DNA using as template the messenger RNAs purged of intron sequences. This process might preferentially remove 3′ introns because it relies on an enzyme called reverse transcriptase that can be primed to read RNAs starting at their 3′ end. The hypothesis has gained experimental support in yeast. It also presents the advantage of potentially explaining intron paucity and 5′ position bias in one stroke. In a new study, Cydney Nielsen and her colleagues present evidence that challenges this model. They address intron dynamics with a genome-wide survey of intron distribution among four Ascomycete fungi with recently completed genome sequences. The four fungi (Neurospora crassa, Magnoporthe grisea, Fusarium gramineum, and Aspergillus nidulans) form an evolutionary tree with branching points estimated at 200, 230, and 330 million years ago. While they diverged from yeast some 500 million years ago, they share with yeast a low intron density (one to two introns per gene) and a 5′ position bias. The authors\' approach is to tally intron gains and losses during the evolution of these four species and then plot their positions along the genes\' length. They identify 3,450 gene regions that are clearly conserved in all four species and harbor an intron in at least one of them. To distinguish intron gains from losses, they rely on a simple parsimony principle, which they refine with additional probability analyses. In brief, an intron present in only one species counts as a gain; an intron absent from one species but present in its closest relative and in a cousin counts as a loss. Nielsen and colleagues record between 150 and 350 intron losses in each lineage. Surprisingly, losses do not occur preferentially at the genes\' 3′ end. The authors conclude that while a 3′ reverse transcriptase-based mechanism might be a factor, it cannot be the sole reason for the introns\' 5′ bias. The other surprising result is that intron gains occur at almost the same rate as losses in all lineages. Intron gains therefore play an important role in the evolution of even intron-poor genomes. Clearly, intron distribution in fungi owes to forces more complex than simple 3′ intron elimination, forces that the authors propose may also shape evolution of other eukaryotic genomes.	PubMed Central	2024-06-05T03:55:49.750777	2004-11-30	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC532397/", "journal": "PLoS Biol. 2004 Dec 30; 2(12):e447", "authors": [] }
PMC532398	After miners unearthed a skull and bones in a Neander Valley cave in Germany in 1856---three years before the publication of On the Origin of Species---the remains were initially described as either those of a "brutish" race or of someone disfigured by disease. As Darwinian evolution caught on, so did the realization that these fossils were evidence of an earlier human species. Scientists have been debating Neanderthal\'s place in human evolution ever since. An ongoing question concerns the possibility that Neanderthals and early humans mated, since they likely crossed paths during thousands of years of European cohabitation. In a new study, Mathias Currat and Laurent Excoffier present a simulation model based on what we know about the population density and distribution of Neanderthals and Cro-Magnons. Their results complement recent genetic and morphological evidence indicating that early human and Neanderthal interbreeding was unlikely. The notion that modern Europeans directly descended from Neanderthals has mostly yielded to two competing models: One postulates that modern humans arose in Africa about 130,000 years ago and completely replaced coexisting archaic forms with no interbreeding, while the other proposes a gradual transition with interbreeding. Though mounting genetic evidence (based on mitochondrial DNA extracted from fossils) suggests Neanderthals and early humans did not breed, the evidence has been inconclusive. It\'s possible, for example, that any Neanderthal gene "leakage" could have been lost through genetic drift if the mating populations were small. And because so few fossils are available to analyze, previous studies could rule out only Neanderthal contributions over 25%. Currat and Excoffier\'s model refines various parameters---such as geographic boundaries, local population variations, range expansion, and competition for resources---based on archeological and demographic data for both populations. Evidence suggests modern humans replaced Neanderthals over 12,500 years, for example, which constrains the speed at which modern humans could expand. The authors started with a scenario based on a set of "plausible" parameter values---their basic scenario---and then varied the local interbreeding rate and, for example, the population size and location of Cro-Magnons, to produce eight alternate scenarios describing how Cro-Magnon colonization of Europe might have proceeded. They estimated the likely proportion of Neanderthal gene contributions to the modern gene pool using "coalescent simulations," which generate the genealogies and diversity of genes in local populations based on simulations of their population densities and migration histories. The simulations show that if Neanderthals bred with Cro-Magnons without constraints over thousands of years, Neanderthal contributions to the modern gene pool "would be immense." Surprisingly, the simulations also show that even a very small mixing should lead to high levels of Neanderthal DNA in modern humans.[](#pbio-0020449-g001){ref-type="fig"} ::: {#pbio-0020449-g001 .fig} ::: {.caption} ###### Reconstruction of Neanderthal woman ::: ![](pbio.0020449.g001) ::: What could account for this counterintuitive result? Given a low population density with small local breeding populations, any introduction of Neanderthal genes would decrease the frequency of Cro-Magnon genes of that population; if these Neanderthal integrations take place as the Cro-Magnon population is expanding, newly acquired Neanderthal genes would also be amplified Since no Neanderthal mitochondrial DNA has been found in modern-day Europeans, the authors modeled the maximum number of interbreeding events that would support this observation. The estimated maximum number of events, it turns out, falls between 34 and 120---extremely low values, Currat and Excoffier conclude, "given the fact that the two populations must have coexisted for more than 12,000 years." While the authors acknowledge their simulations suggest rather than reflect reality, their model does incorporate real historical data such as Cro-Magnon expansion over time and local population growth. At a value of only 0.1%, their new estimate of the rate of interbreeding is about 400 times lower than previous estimates and provides strong support that Neanderthals and Cro-Magnon didn\'t interbreed and may even have been different species.	PubMed Central	2024-06-05T03:55:49.751470	2004-11-30	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC532398/", "journal": "PLoS Biol. 2004 Dec 30; 2(12):e449", "authors": [] }
PMC532399	Long-range communication requires special technology. Although we usually think of nerve cells communicating over distances measured in millimeters, they must also stretch over centimeters and even meters to enable movement and sensation throughout our bodies. In a disorder known as spastic paraplegia, people experience stiffness and loss of function in their legs because the far ends of the longest nerves in the spinal cord degenerate. One form of this disease, autosomal dominant hereditary spastic paraplegia (AD-HSP), is most frequently caused by mutations in a gene that codes for the enzyme Spastin. Until recently, the function of Spastin has only been inferred by its similarity to another protein, Katanin, which chops up microtubules. The microtubules in neurons, as in all types of cells, provide a network to transport materials from one place in the cell to another. For neurons to communicate with each other and with muscles, neurotransmitters packaged at one end of the cell make the journey through the cell\'s long, narrow axon on the backs of microtubules. At the other end of the cell, the neurotransmitters reach small cellular projections (boutons) where they are released as a signal to the receiving cell. Since long spinal cord axons are most often affected by AD-HSP, researchers have suggested that transport through axons might be culpable. Is there a link between Spastin, microtubule severing, and AD-HSP? To address this question, Nina Tang Sherwood and colleagues studied the function of Spastin in the fruitfly Drosophila by identifying the spastin gene in this species and manipulating its expression. They found that indeed Drosophila Spastin in neurons regulates microtubule networks. Overexpressing spastin caused collapse of the embryonic central nervous system and also eliminated the microtubule network, as expected based on the related Katanin protein\'s microtubule-severing activity. But---surprisingly---knocking out spastin did not yield the opposite result. spastin-null flies had fewer microtubule bundles, particularly at the far ends of the neurons. They also exhibited smaller and more numerous boutons that were unusually clustered together. On the basis of their results, the authors speculate that Spastin cuts microtubules to a manageable size. Too much Spastin chops the microtubules into useless fragments, but too little Spastin may leave microtubule polymers too large to be efficiently moved into newly forming boutons. With an intermediate amount, microtubule pieces are the right size for transport throughout the neuron. Without Spastin, normal motor function ceases. In spastin-null flies, neurotransmitter release is impaired and flying is impossible. Flies even tend to drag their hind legs. This weakness in the legs is just one compelling parallel to human AD-HSP. The severity of symptoms in people is highly variable, similar to the variability in phenotypes exhibited by Drosophila with intermediate spastin gene mutations. The authors caution, however, that they do not prove that the fly phenotypes observed arise through the same mechanisms that cause human AD-HSP. The utility of Drosophila as a model for human AD-HSP has yet to be demonstrated, but the importance of Spastin in regulating neuronal microtubule networks in vivo is no longer in doubt.	PubMed Central	2024-06-05T03:55:49.752470	2004-11-30	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC532399/", "journal": "PLoS Biol. 2004 Dec 30; 2(12):e450", "authors": [] }
PMC532400	Four years ago in Science, Stuart Kim, a Stanford developmental biologist, made the case for laying down the broad strokes of a complex physiological process before defining its mechanisms. "A powerful, top-down, holistic approach," he wrote, "is to identify all of the components of a particular cellular process, so that one can define the global picture first and then use it as a framework to understand how the individual components of the process fit together." To get a broad view of gene expression in the aging nematode, Kim\'s lab turned to DNA microarrays and functional genomics. In a new study, Kim and colleagues apply this same approach to the decidedly more complex problem of human aging and "present a molecular portrait" of the aging kidney. Scientists have identified a wide range of molecular pathways and mechanisms associated with aging. Many have been found in evolutionarily distant organisms, suggesting they have been conserved and could shed light on human aging. Yet other studies suggest that since few animals reach old age in the wild, any aging-related physiological changes aren\'t likely to impact the fitness of a population and so aren\'t likely to be conserved. Consequently, aging pathways in worms, for example, would have little bearing on humans. To investigate the molecular pathways associated with human aging, the authors focused on human tissue---in this case, the kidney. Kidneys came from 74 patients, ranging in age from 27 to 92. Samples were extracted from donated kidneys or "meticulously harvested" from kidneys with localized disease to ensure only normal tissue was taken. Two structures that are critical to kidney function (removing toxins from blood) were removed from each sample: the renal cortex, which filters plasma, and the medulla, which alters urine composition to maintain fluid balance. Both deteriorate with age. An extensive clinical history was noted for each sample to account for any potentially confounding medical factors.[](#pbio-0020451-g001){ref-type="fig"} ::: {#pbio-0020451-g001 .fig} ::: {.caption} ###### Transcriptional profiling to study aging in the kidney ::: ![](pbio.0020451.g001) ::: Kim and colleagues then isolated RNA transcripts from the samples to determine the activity of every gene, broken down by age and kidney section, through microarray analysis. Looking for differences in gene expression across the genome, they identified genes that showed a statistically significant change in expression as a function of age. Of 33,000 known human genes on the microarray, 985 showed age-related changes, most showing increased activity. These changes are truly age-regulated, the authors conclude, since none of the medical factors impacted the observed changes in gene expression. Although cortex and medulla have different cell types and perform different functions, their genetic aging profile was very similar, suggesting a common aging mechanism operates in both structures. In fact, these mechanisms may function broadly, as most of the age-regulated kidney genes were also active in a wide range of human tissues. Other organisms appear to lack these changes, however, prompting the authors to argue that understanding aging in humans will require human subjects. Most importantly, the genetic profile of the tissue samples correlated with the physiological and morphological decline of an aging kidney. An 81-year-old patient with an unusually healthy kidney had a molecular profile typical of someone much younger, while a 78-year-old with a damaged kidney had the profile of a much older person. Using the power of functional genomics, this study has identified a set of genes that can serve as molecular markers for various stages of a deteriorating kidney and predict the relative health of a patient compared to their age group. These gene sets can also serve as probes to shed light on the molecular pathways at work in the aging kidney, and possibly on the process of aging itself.	PubMed Central	2024-06-05T03:55:49.753528	2004-11-30	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC532400/", "journal": "PLoS Biol. 2004 Dec 30; 2(12):e451", "authors": [] }
PMC533858	Introduction ============ The global burden of disease studies point to an alarming increase in the prevalence of chronic obstructive pulmonary disease (COPD) \[[@B1]\] which is predicted to be one of the major global causes of disability and death in the next decade \[[@B2]\]. COPD is characterized by a range of pathologies from chronic inflammation to tissue proteolysis and there are no drugs specifically developed for COPD so far. Cessation of cigarette smoking is accompanied by a reduction in decline in lung function \[[@B3]\] and is a most important aspect of COPD management. The mainstay medication consists of beta-adrenergic and anticholinergic bronchodilators; addition of topical corticosteroid therapy in patients with more severe COPD provides may enhance bronchodilator responses and reduce exacerbations \[[@B4]\]. In contrast to the large amount of experimental studies on allergic asthma and the detailed knowledge that exists on mediators of allergic airway inflammation \[[@B5],[@B6]\], much less has been conducted for COPD. More effort and resources have been directed into asthma research in comparison to COPD. The available insights into the pathogenesis and pathophysiology of asthma may help to improve research in COPD \[[@B7]\]. Many research centres that previously focused on asthma now also investigate mechanisms of COPD. Using molecular and genetic approaches, an increasing range of molecules has been identified that could underlie the pathogenic inflammation of chronic allergic airway inflammation \[[@B8]\]. Based on these findings and on new ways of administering drugs to the lungs \[[@B9]\], a new image of overwhelming complexity of the underlying pathophysiology of COPD has emerged (Figure [1](#F1){ref-type="fig"}). The current challenge in COPD research is to identify the role of the various mediators and molecular mechanisms that may be involved in its pathophysiology, and obtain new treatments. In addition, it is incumbent to understand the effect of smoking cessation on the pathogenetic process. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Potential pathogenetic mechanisms involved in COPDExogenous inhaled noxious stimuli such as tobacco smoke, noxious gases or indoor air pollution and genetic factors are proposed to be the major factors related to the pathogenesis of COPD. These factors may influence protease activity and may also lead to an imbalance between pro-inflammatory and anti-inflammatory mediators. ::: ![](1465-9921-5-18-1) ::: Studying the molecular pathways in human subjects is restricted to the use of morphological and molecular assessment of lung tissues obtained at surgery or performing limited in vitro studies at one single point in time \[[@B10]\]. There is a need for in vivoanimal models to examine more closely pathogenesis, functional changes and the effects of new compounds or treatments. However, animal models have limitations since there is no spontaneous model, and models do not necessarily mimic the entire COPD phenotype. The best model remains chronic exposure to cigarette smoke, since this is the environmental toxic substance(s) that cause COPD in man. However, other substances are also implicated such as environmental pollution due to car exhaust fumes. The present review draws attention to specific aspects of functional and structural features of COPD that need to be realized when interpreting molecular mechanisms identified in animal models of COPD. It identifies important issues related to the ongoing experimental COPD research which may in the future provide optimized COPD diagnosis and treatment. COPD ---- ### Clinical features Before characterizing and discussing the different animal models of COPD which have been established so far, it is crucial to reflect that within COPD, different disease stages exist and that only some of them may be mimicked in animal models. The diagnosis of COPD largely relies on a history of exposure to noxious stimuli (mainly tobacco smoke) and abnormal lung function tests. Since COPD has a variable pathology and the molecular mechanisms are only understood to a minor extent, a simple disease definition has been difficult to establish. However, the diagnosis of COPD relies on the presence of persistent airflow obstruction in a cigarette smoker \[[@B4]\]. A classification of disease severity into four stages has been proposed by the GOLD guidelines based primarily on FEV1 \[[@B4]\]. The staging on the basis of FEV1 alone as an index of severity for COPD has been criticised. A composite measure essentially based on clinical parameters (BODE) has been shown to be better at predicting mortality than FEV1 \[[@B11]\]. The natural history of COPD in terms of evolution of FEV1 remains unclear and the temptation is to regard the stages as evolving from Stage 0 to Stage 4. Just as many smokers do not develop COPD, it is possible that the disease may not progress from one stage to the next. Some patients with severe COPD are relatively young and it is not clear if early stages of their disease are similar to those found in patients with mild COPD. COPD is a heterogeneous disease and different possible outcomes may occur at each of the stages. Experimental modeling of each stage of severity may be a way of providing an answer to this issue. Animal models may also help to provide a better classification of severity by correlating biochemical, molecular and structural changes with lung function and exercise tolerance. ### Pathophysiology The presence of airflow obstruction which has a small reversible component, but which is largely irreversible is a major feature of COPD as indicated by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) guidelines \[[@B4]\]. It is proposed to be the result of a combination of small airways narrowing, airway wall inflammation \[[@B12]\] and emphysema-related loss of lung elastic recoil \[[@B13],[@B14]\]. These features differ to a large extent to findings observed in bronchial asthma (Table [1](#T1){ref-type="table"}) where airflow obstruction is usually central, while involvement of the small airways occurs in more severe disease. The degree of airflow obstruction in COPD can be variable, but loss of lung function over time is a characteristic feature. Ideally, the development of airflow obstruction which is largely irreversible but has a small reversible component should be a feature of animal models of COPD, but this has not been reproduced so far. One of the important limitations of animal models of COPD is the difficulty in: reproducing small airways pathology particularly when working in small animals, particularly the mouse and rat where there are few levels of airway branching. This is a problem inherent to small laboratory animal models but provides an advantage for developing models in larger animals such as the pig or sheep. Part of the problem of analyzing small airways is also due to the lack of sophistication of lung function measurements, particularly in mice, but there has been recent development in the methodology of lung function measurement \[[@B15]\]. A new ex-vivo method of analyzing the airway periphery is by the technique of precision cut lung slices combined to videomorphometry \[[@B16],[@B17]\]. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Currently known phenotype differences between COPD and asthma ::: Feature COPD Asthma ----------------------------------- ---------------------- -------------------- Limitation of Airflow Largely irreversible Largely reversible Parenchymal integrity destruction intact Bronchial Hyperresponsiveness Variable (small) significant Steroid response reduced or absent present ::: In addition to pulmonary alterations, other organ systems may be affected in COPD \[[@B18]\]. Systemic effects of COPD include weight loss, nutritional abnormalities and musculoskeletal dysfunction. These systemic manifestations will gain further socioeconomic importance with an increasing prevalence of COPD in the next years \[[@B19]\]. Therefore, these systemic effects should be present in animal models of COPD and further analysis of mechanisms underlying these systemic effects in experimental models may help to optimize disease management. ### Inflammatory cells An important feature of COPD is the ongoing chronic inflammatory process in the airways as indicated by the current GOLD definition of COPD \[[@B4]\]. There are differences between COPD and asthma: while mast cells and eosinophils are the prominent cell types in allergic asthma, the major inflammatory cell types in COPD are different (Table [2](#T2){ref-type="table"}) \[[@B20]-[@B22]\]. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Differences in inflammatory cells between COPD and asthma. Ranked in relative order of importance. ::: COPD Asthma ----------------------------- -------------------------- Neutrophils Eosinophils Macrophages Mast cells CD8-T-lymphocytes CD4-T-lymphocytes Eosinophils (exacerbations) Macrophages, Neutrophils ::: Neutrophils play a prominent role in the pathophysiology of COPD as they release a multitude of mediators and tissue-degrading enzymes such as elastases which can orchestrate tissue destruction and chronic inflammation \[[@B8],[@B23]\]. Neutrophils and macrophages are increased in bronchoalveolar lavage fluid from cigarette smokers \[[@B24]\]. Patients with a high degree of airflow limitation have a greater induced sputum neutrophilia than subjects without airflow limitation. Increased sputum neutrophilia is also related to an accelerated decrease in FEV~1~and sputum neutrophilia is more prevalent in subjects with chronic cough and sputum production \[[@B25]\]. The second major cell type involved in cellular mechanisms are macrophages \[[@B26]\]. They can release numerous tissue-degrading enzymes such as matrix metalloproteinases (MMPs). In an animal model of tobacco smoke-induced tissue matrix degradation, not only neutrophil enzymes but also macrophage-derived enzymes such as MMP-12 are important for the development of emphysema-like lesions \[[@B27]\]. A further key enzyme is the macrophage metalloelastase which was reported to mediate acute cigarette smoke-induced inflammation via tumor necrosis factor (TNF)-alpha-release \[[@B28]\]. Neutrophils and macrophages can communicate with other cells such as airway smooth muscle cells, endothelial cells or sensory neurons, and release inflammatory mediators that induce bronchoconstriction \[[@B29]\], airway remodelling \[[@B30]\], and mucin gene induction and mucus hypersecretion involving the induction of mucin genes \[[@B31]-[@B33]\]. Lymphocytes are also involved in cellular mechanisms underlying COPD \[[@B34],[@B35]\]. Increased numbers of CD8-positive T-lymphocytes are found in the airways of COPD patients \[[@B21],[@B22]\] and the degree of airflow obstruction is correlated with their numbers \[[@B36]\]. However, the T-cell associated inflammatory processes largely differ from those in allergic asthma, which is characterized by increased numbers of CD4-positive T-lymphocytes \[[@B7],[@B37]\] (Table [2](#T2){ref-type="table"}). Although eosinophils may only play a major role in acute exacerbations of COPD \[[@B38]\], their presence in stable disease is an indicator of steroid responsiveness \[[@B39]-[@B41]\]. Different inflammatory cell types have also been characterized in airway tissues. Epithelial neutrophilia has been seen in proximal and distal airways of patients with COPD \[[@B42],[@B43]\]. The airway wall beneath the epithelium shows a mononuclear inflammation with increased macrophages and T cells bearing activation markers \[[@B20],[@B36]\] Di Stefano 1996;. An excess od CD8+ T cells are particularly observed in central airways, peripheral airways and parenchyma \[[@B20],[@B43]\]. In the small airways from patients with stage 0 to (at risk) stage 4 (very severe) COPD, the progression of the disease is strongly associated with the accumulation of inflammatory exudates in the small airway lumen and with an increase in the volume of tissue in the airway wall \[[@B10]\]. Also, the percentage of airways containing macrophages, neutrophils, CD4 cells, CD8 cells, B cells, and lymphoid follicle aggregates and the absolute volume of CD8+ T-cells and B cells increased with the progression of COPD \[[@B10]\]. The changes are also most likely associated with an induction of mucin gene expression \[[@B44]\]. The presence of increased numbers of B cells begs the question regarding the role of these cells in the pathophysiology of COPD. In the airway smooth muscle bundles in smokers with COPD, increased localisation of T- cells and neutrophils has been reported, indicating a possible role for these cells interacting with airway smooth muscle in the pathogenesis of airflow limitation \[[@B45]\]. ### Mechanisms of COPD On the basis of the different pathophysiological mechanisms illustrated in Fig. [1](#F1){ref-type="fig"}, different animal models have been developed in past years. #### Protease-antiprotease imbalance An imbalance between protease and antiprotease enzymes has been hypothesized with respect to the pathogenesis of emphysema \[[@B46]\]. This concept derives from early clinical observations that alpha1-antitrypsin-deficient subjects develop severe emphysema and the role of protease-antiprotease imbalance was later demonstrated in animal models of COPD \[[@B47],[@B48]\]. Although alpha1-antitrypsin-deficiency is a very rare cause of emphysema \[[@B49],[@B50]\], it points to a role of proteases and proteolysis \[[@B51],[@B52]\]. Neutrophil elastase-deficient mice were significantly protected from emphysema-development induced by chronic cigarette smoke \[[@B48]\]. Depletion of the macrophage elastase gene also led to a complete protection from emphysema induced by cigarette smoke \[[@B47]\]. Each of these elastases inactivated the endogenous inhibitor of the other, with macrophage elastase degrading alpha1-antitrypsin and neutrophil elastase degrading tissue inhibitor of metalloproteinase-1 \[[@B48]\]. In tobacco smoke exposure-induced recruitment of neutrophils and monocytes was impaired in elastase gene-depleted animals and there was less macrophage elastase activity due to a decreased macrophage influx in these animals. Thus, a major role for neutrophil elastase and macrophage elastase in the mediation of alveolar destruction in response to cigarette smoke has been shown \[[@B47],[@B48]\]. This experimental evidence derived from animal models points to an important pathogenetic role for proteases that correlates well with the imbalance of proteases present in human COPD. However, many pathways of tissue destruction can be found in animal models that lead to a picture similar to human disease, and it is important to examine whether these mechanisms are operative in the human disease itself. #### Oxidative stress Oxidative stress arising from inhaled noxious stimuli such as tobacco smoke or nitrogen dioxide may be important cause of the inflammation and tissue damage in COPD. This potential mechanism is supported by clinical reports of increased levels of oxidative stress indicators in exhaled breath condensates of COPD patients \[[@B53]-[@B55]\]. Apart from elevated levels of 8-isoprostane \[[@B55]\], nitrosothiol levels were increased in COPD patients \[[@B56]-[@B58]\]. Studies in a mouse model of tobacco smoke-induced COPD also demonstrated the presence of tissue damage due to oxidative stress \[[@B59]\]. These changes could be blocked by superoxide dismutase \[[@B60]\]. Oxidative stress has also been implicated in the development of corticosteroid resistance in COPD. #### Mediators Many mediators have been identified which may contribute to COPD pathogenesis \[[@B8]\]. As in bronchial asthma, pro- and anti-inflammatory mediators of inflammation such as tachykinins \[[@B61]\], vasoactive intestinal polypeptide (VIP) \[[@B62]\], histamine \[[@B63]\], nitric oxide \[[@B64],[@B65]\], leukotrienes \[[@B66]\], opioids \[[@B67]\] or intracellular mediators such as SMADs \[[@B68],[@B69]\] have been implicated. The balance of histone acetylases and deacetylases \[[@B70]\] is a key regulator of gene transcription and expression by controlling the access of the transcriptional machinery to bind to regulatory sites on DNA. Acetylation of core histones lead to modification of chromatin structure that affect transcription, and the acetylartion status depends on a balance of histone deacetylase and histone acetyltransferase. This is also likely to play a role in the regulation of cytokine production in COPD. Cigarette smoke exposure led to altered chromatin remodelling with reduced histone deacetylase activity with a resultant increase in transcription of pro-inflammatory genes in lungs of rats exposed to smoke, linked to an increase in phosphorylated p38 MAPK in the lung concomitant with an increased histone 3 phospho-acetylation, histone 4 acetylation and elevated DNA binding of NF-kappaB, and activator protein 1 (AP-1) \[[@B70]\]. In addition, oxidative stress has also been shown to enhance acetylation of histone proteins and decrease histone deacetylase activity leading to modulation of NF-κB activation \[[@B71]\], similar to the effect of cigarette smoke. A Th2 cytokine that has been proposed to be implicated in the pathophysiology of COPD is IL-13. It is also overexpressed and related to the pathogenesis of the asthmatic Th2 inflammation and airway remodelling process \[[@B72]\]. The effects of IL-13 in asthma have been elucidated in a series of experiments that demonstrated the an airway-specific constitutive overexpression of IL-13 leads to a process of airway remodelling with subepithelial fibrosis and mucus metaplasia combined with an eosinophil-, lymphocyte-, and macrophage-rich inflammation and increased hyperresponsiveness \[[@B73]\]. Since asthma and COPD pathogenesis may be linked, similar mechanisms may contribute to the development and progression of both diseases \[[@B74]\]. In this respect, IL-13 may also play a role in COPD since the inducible overexpression of IL-13 in adult murine lungs leads to alveolar enlargement, lung enlargement and an enhanced compliance and mucus cell metaplasia \[[@B75]\] with activation of MMP-2, -9, -12, -13, and -14 and cathepsins B, S, L, H, and K in this model. Parallel to protease-based and extracellular mediator-based concepts, altered intracellular pathways may also play a role in COPD. MAPK signalling pathways i.e. p38 and c-Jun N terminal kinase (JNK) \[[@B76],[@B77]\] seem to be important signal transducers in the airways and airway-innervating neurons \[[@B78]-[@B80]\] and may therefore display an interesting target for COPD research. For some cells, the activation of p38 or JNK pathways may promote apoptosis rather than proliferation \[[@B81],[@B82]\]. #### Viral infections Previous studies showed an association between latent adenoviral infection with expression of the adenoviral E1A gene and chronic obstructive pulmonary disease (COPD) \[[@B83],[@B84]\]. It may therefore be assumed that latent adenoviral infection can be one of the factors that might amplify airway inflammation. Human data \[[@B35]\] demonstrating the presence of the viral E1A gene and its expression in the lungs from smokers \[[@B85],[@B86]\], animals \[[@B87]\] and cell cultures \[[@B88]\] support this hypothesis. A small population of lung epithelial cells may carry the adenoviral E1A gene which may then amplify cigarette smoke-induced airway inflammation to generate parenchymal lesions leading to COPD. Inflammatory changes lead to collagen deposition, elastin degradation, and induction of abnormal elastin in COPD \[[@B89],[@B90]\]. Also, latent adenovirus E1A infection of epithelial cells could contribute to airway remodelling in COPD by the viral E1A gene, inducing TGF-beta 1 and CTGF expression and shifting cells towards a more mesenchymal phenotype\[[@B84]\]. ### Genetics Since only a minority of smokers (approximately 15 to 20%) develop symptoms and COPD is known to cluster in families, a genetic predisposition has been hypothesized. Many candidate genes have been assessed, but the data are often unclear and systematic studies are currently performed to identify disease-associated genes. Next to alpha1-antitrypsin deficiency, several candidate genes have been suggested to be linked to COPD induction. Genetic polymorphisms in matrix metalloproteinase genes MMP1, MMP9 and MMP12 may be important in the development of COPD. In this respect, polymorphisms in the MMP1 and MMP12 genes, but not MMP9, have been suggested to be related to smoking-related lung injury or are in a linkage disequilibrium with other causative polymorphisms \[[@B91]-[@B93]\]. An association between an MMP9 polymorphism and the development of smoking-induced pulmonary emphysema was also reported in a population of Japanese smokers \[[@B94]\]. Also, polymorphisms in the genes encoding for IL-11 \[[@B95]\], TGF-beta1 \[[@B96]\], and the group-specific component of serum globulin \[[@B97]\] have been shown to be related to a genetic predisposition for COPD. Since it was difficult to replicate some of these findings among different populations, future studies are needed. Also, whole genome screening in patients and unaffected siblings displays a promising genetic approach to identify genes associated with COPD. Experimental models of COPD --------------------------- There are three major experimental approaches to mimic COPD encompassing inhalation of noxious stimuli, tracheal instillation of tissue-degrading enzymes to induce emphysema-like lesions and gene-modifying techniques leading to a COPD-like phenotype (Figure [2](#F2){ref-type="fig"}). These approaches may also be combined. Ideally a number of potential indicators for COPD which have been proposed by the GOLD guidelines should be present in animal models of COPD (Table [3](#T3){ref-type="table"}). Since COPD definition still rests heavily on lung function measures (airflow limitation and transfer factor), it would be ideal to have lung function measurements in experimental models \[[@B15]\]. The challenge is in the measurement of lung function in very small mammals such as mice and since the use of the enhanced pause (Penh) in conscious mice as an indicator of airflow obstruction is not ideal \[[@B98]\], invasive methods remain the gold standard and these should be correlated with inflammatory markers and cellular remodelling. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Experimental approaches to mimic COPDThere are three major experimental approaches to mimic COPD or emphysema consisting of inhalation of noxious stimuli such as tobacco smoke, tracheal instillation of tissue-degrading enzymes to induce emphysema-like lesions and gene-modifying techniques leading to COPD-like murine phenotypes. ::: ![](1465-9921-5-18-2) ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Indicators for COPD. These indicators are related to the presence of COPD and should ideally be present in animal models and available for analysis. ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Indicator Human features Experimental approach ----------------------------------------- ------------------------------------------------------------------------------------ ----------------------------------------------------------- History of exposure to risk factors Tobacco smoke.\ Exposure-based experimental protocol Occupational dusts and chemicals.\ Indoor / outdoor air pollution Airflow obstruction Decrease in FEV~1~ Lung function tests Hypersecretion Chronic sputum production Functional and morphological assessment of hypersecretion Cough Chronic intermittent or persistent cough Cough assessment Dyspnea Progressive / Persistent / worse on exercise / worse during respiratory infections Assessment of hypoxemia Emphysema Progressive impairment of lung function Morphological analysis of airspace enlargement ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ::: ### Inhalation models -- tobacco smoke A variety of animal species has been exposed to tobacco smoke. Next to guinea pigs, rabbits, and dogs, and also rats and mice have been used. Guinea pigs have been reported to be a very susceptible species. They develop COPD-like lesions and emphysema-like airspace enlargement within a few months of active tobacco smoke exposure \[[@B99]\]. By contrast, rat strains seem to be more resistant to the induction of emphysema-like lesions. Susceptibility in mice varies from strain to strain. The mode of exposure to tobacco smoke may be either active via nose-only exposure systems or passive via large whole-body chambers. The first species to be examined in detail for COPD-like lesions due to tobacco smoke exposure was the guinea pig \[[@B99]\]. Different exposure protocols were screened and exposure to the smoke of 10 cigarettes each day, 5 days per week, for a period of either 1, 3, 6, or 12 months resulted in progressive pulmonary function abnormalities and emphysema-like lesions. The cessation of smoke exposure did not reverse but stabilized emphysema-like airspace enlargement. On the cellular level, long term exposure lead to neutrophilia and accumulation of macrophages and CD4+ T-cells \[[@B83],[@B100]\]. Latent adenoviral infection amplifies the emphysematous lung destruction and increases the inflammatory response produced by cigarette-smoke exposure. Interestingly, it was shown that the increase in CD4+ T-cells is associated with cigarette smoke and the increase in CD8+ T-cells with latent adenoviral infection \[[@B83]\]. Mice represent the most favoured laboratory animal species with regard to immune mechanisms since they offer the opportunity to manipulate gene expression. However, it is more difficult to assess lung function and mice tolerate at least two cigarettes daily for a year with minimal effects on body weight and carboxyhemoglobin levels. Mice differ considerably in respiratory tract functions and anatomy if compared to humans: they are obligate nose breathers, they have lower numbers of cilia, fewer Clara cells and a restriction of submucosal glands to the trachea. Next to a lower filter function for tobacco smoke, mice also do not have a cough reflex and many mediators such as histamine or tachykinins have different pharmacological effects. The development of emphysema-like lesions is strain-dependent: enlarged alveolar spaces and increased alveolar duct area are found after 3--6 months of tobacco smoke exposure in susceptible strains such as B6C3F1 mice \[[@B101]\]. At these later time points, tissue destruction seems to be mediated via macrophages. At the cellular level, neutrophil recruitment has been reported to occur immediately after the beginning of tobacco smoke exposure and is followed by accumulation of macrophages. The early influx of neutrophils is paralleled by a connective tissue breakdown. The early stage alterations of neutrophil influx and increase in elastin and collagen degradation can be prevented by pre-treatment with a neutrophil antibody or alpha1-antitrypsin \[[@B102]\]. Rats are also often used for models of COPD. However, they appear to be relatively resistant to the induction of emphysema-like lesions. Using morphometry and histopathology to assess and compare emphysema development in mice and rats, significant differences were demonstrated \[[@B101]\]: Animals were exposed via whole-body exposure to tobacco smoke at a concentration of 250 mg total particulate matter/m3 for 6 h/day, 5 days/week, for either 7 or 13 months. Morphometry included measurements of tissue loss (volume density of alveolar septa) and parenchymal air space enlargement (alveolar septa mean linear intercept, volume density of alveolar air space). Also, centroacinar intra-alveolar inflammatory cells were assessed to investigate differences in the type of inflammatory responses associated with tobacco smoke exposure. In B6C3F1 mice, many of the morphometric parameters used to assess emphysema-like lesions differed significantly between exposed and non-exposed animals. By contrast, in exposed Fischer-344 rats, only some parameters differed significantly from non-exposed values. The alveolar septa mean linear intercept in both exposed mice and rats was increased at 7 and 13 months, indicating an enlargement of parenchymal air spaces. In contrast, the volume density of alveolar air space was significantly increased only in exposed mice. The volume density of alveolar septa was decreased in mice at both time points indicating damage to the structural integrity of parenchyma. There was no alteration in Fischer-344 rats. Morphologic evidence of tissue destruction in the mice included irregularly-sized and -shaped alveoli and multiple foci of septal discontinuities and isolated septal fragments. The morphometric differences in mice were greater at 13 months than at 7 months, suggesting a progression of the disease. Inflammatory influx within the lungs of exposed mice contained significantly more neutrophils than in rats. These results indicated that B6C3F1 mice are more susceptible than F344-rats to the induction of COPD-like lesions in response to tobacco smoke exposure \[[@B101]\]. Recent work on cigarette exposure in rats indicate that this model also achieves a degree of corticosteroid resistance that has been observed in patients with COPD \[[@B103],[@B104]\]. Thus, the inflammatory response observed after exposure of rats to cigarette smoke for 3 days is noty inhibited by pre-treatment with corticosteroids \[[@B70]\]. This may be due to the reduction in histone deacetylase activity, which could result from a defect in recruitment of this activity by corticosteroid receptors. Corticosteroids recruit hitone deacetylase 2 protein to the transcriptional complex to suppress proinflammatory gene transcription \[[@B105]\]. Modifications in histone deacetylase 2 by oxidative stress or by cigarette smoke may make corticosteroids ineffective \[[@B106]\]. Therefore, models of COPD that show corticosteroid resistance may be necessary and could be used to dissect out the mechanisms of this resistance. Generally, tobacco smoke exposure may be used to generate COPD features such as emphysema and airway remodelling and chronic inflammation. Although the alterations still differ from the human situation and many involved mediators may have different functional effects especially in the murine respiratory tract, these models represent useful approaches to investigate cellular and molecular mechanisms underlying the development and progression of COPD. As a considerable strain-to-strain and species-to-species variation can be found in the models used so far, the selection of a strain needs to be done with great caution. Animal models of COPD still need to be precisely evaluated as to whether they mimic features of human COPD, and their limitations must be appreciated. Findings obtained from these models may provide significant advances in terms of understanding novel mechanisms involved in COPD. ### Inhalation models -- sulfur dioxide Sulfur dioxide (SO~2~) is a gaseous irritant which can be used to induce COPD-like lesions in animal models. With daily exposure to high concentrations of SO~2~, chronic injury and repair of epithelial cells can be observed in species such as rat or guinea pig. The exposure to high-levels of this gas ranging from 200 to 700 ppm for 4 to 8 weeks has been demonstrated to lead to neutrophilic inflammation, morphological signs of mucus production and mucus cell metaplasia and damage of ciliated epithelial cells in rats \[[@B107],[@B108]\]. These changes are directly dependent on the exposure to the gas: signs of mucus production and neutrophilic inflammation are almost entirely reversed within a week after termination of exposure \[[@B108]\]. Acute exposure to SO~2~also leads to loss of cilia and exfoliation of ciliated cells as demonstrated in SO~2~-exposed dogs using transmission electron microscopy \[[@B109]\]. After a longer period of exposure the epithelial layer regenerates and airway wall thickening and change in cilia structure can be observed \[[@B110]\]. Long-term exposure also increases in mucosal permeability both in vivoand in vitro\[[@B111]\]. Mucus hypersecretion is an important indicator for COPD and experimental models should encompass features of hypersecretion. After chronic exposure to SO~2~in rats, visible mucus layers and mucus plugs may sometimes be observed in the large airways \[[@B107]\] and an elevation of mucus content may be found in bronchoalveolar lavage fluids \[[@B112]\]. Parallel to these findings, there is an increase of PAS- and Alcian Blue-staining epithelial cells in chronically SO~2~exposed rats \[[@B113]\] but there is substantial variation present as with human COPD \[[@B114]\]. Tracheal mucus glands are also increased in size after SO~2~-exposure \[[@B115]\] and increased levels of mucin RNA can be found in lung extracts \[[@B112]\]. The mechanisms underlying mucus hypersecretion have not been elucidated so far and also, functional studies assessing basal and metacholine-induced secretion have not been conducted so far. Airway inflammation with cellular infiltration is an important feature of COPD. After exposure to SO~2~, increases in mononuclear and polymorphonuclear inflammatory cells are present in rat airways. However, the influx is confined to large but not small airways which are important in human COPD \[[@B107]\]. Even after one day of exposure, polymorphonuclear inflammatory cells are found and their influx can be inhibited with steroid treatment \[[@B116]\]. SO~2~-based models of COPD have also been shown to be associated with an increase in pulmonary resistance and airway hyperresponsiveness \[[@B107]\] and it was hypothesized that elevated levels of mucus may account for the increased responsiveness \[[@B117]\]. Since sensory nerve fibres may function as potent regulators of chronic inflammation in COPD by changes in the activation threshold and the release of pro-inflammatory mediators such as tachykinins \[[@B61],[@B118]\] or CGRP \[[@B6],[@B119]\], this class of nerve fibres was examined in a number of studies \[[@B120],[@B121]\]. The results of these studies supported the hypothesis that rather than contributing to the pathophysiological manifestations of bronchitis, sensory nerve fibres limit the development of airway obstruction and airway hyperresponsiveness during induction of chronic bronchitis by SO~2~-exposure. In this respect, the enhanced contractile responses of airways from neonatally SO~2~-exposed capsaicin-treated rats may result from increased airway smooth muscle mass and contribute to the increased airway responsiveness observed in these animals \[[@B121]\]. To obtain coexisting expression of emphysema and inflammatory changes as seen in COPD, neutrophil elastase instillation and SO~2~-exposure were performed simultaneously \[[@B108]\]. The pre-treatment with elastase aimed to render the animals more susceptible to the inflammation induced by SO~2~. However, neither allergy-phenotype Brown Norway nor emphysematous Sprague--Dawley rats displayed an increased sensitivity to SO~2~-exposure. With regard to the observed histopathological changes, it can be concluded that SO~2~exposure leads to a more diffuse alveolar damage with a more extensive damage with destruction of lung tissue after longer exposure. Therefore, the outcome is more or less a picture of tissue destruction with close resemblance to end stages of emphysema but not a complete picture of COPD. ### Inhalation models -- nitrogen dioxide Nitrogen dioxide (NO~2~) is a another gas that may lead to COPD-like lesions depending on concentration, duration of exposure, and species genetic susceptibility \[[@B122]\]. Concentrations ranging from 50--150 ppm (94--282 mg/m3) can lead to death in laboratory animals due to extensive pulmonary injury including pulmonary oedema, haemorrhage, and pleural effusion. Short-term exposure to NO2 leads to a biphasic response with an initial injury phase followed by a repair phase. Both increased cellular proliferation and enzymatic activity occur during the repair phase. Exposure of rats to 15 ppm NO~2~for 7 days leads to an increased oxygen consumption in airway tissues. The increase in oxidative capacity reflects an increase in mitochondrial activity consistent with observations of increased DNA synthesis \[[@B123]\]. Exposure to 10 ppm NO~2~for more than 24 h causes damage to cilia and hypertrophy of the bronchiolar epithelium \[[@B124]\]. Also, exposure to 15--20 ppm NO~2~leads to a type II pneumocyte hyperplasia \[[@B125],[@B126]\]. As with the exposure to other noxious stimuli, there is also a significant inter-species variability. In comparison to mice and rats, guinea pigs exhibit changes in lung morphology at much lower NO~2~concentrations. It was shown that a 2 ppm NO~2~3-day exposure causes increased thickening of the alveolar wall, damage to cilia and pulmonary oedema \[[@B127]\]. Other changes are an influx of inflammatory cells and increases in connective tissue formation \[[@B128]\]. There is also a significant mode of inheritance of susceptibility to NO~2~-induced lung injury in inbred mice. Susceptible C57BL/6J (B6) and resistant C3H/HeJ (C3) mice, as well as F1, F2, and backcross (BX) populations derived from them, were acutely exposed to 15 parts per million NO~2~for 3 h to determine differences \[[@B122]\]. Significant differences in numbers of lavageable macrophages, epithelial cells, and dead cells were found between inbred strains: distributions of cellular responses in F1 progeny overlapped both progenitors, and mean responses were intermediate. It was shown that in C3:BX progeny, ranges of responses to NO~2~closely resembled C3 mice. Ranges of cellular responses to NO~2~in B6:BX and intercross progeny were reported to overlap both progenitor and mean responses of both populations were intermediate to progenitors. Therefore, there were likely two major unlinked genes that account for differential susceptibility to acute NO~2~exposure \[[@B122]\]. Based on the genetic background of C57BL/6 mice, a model of long-term NO~2~exposure was recently established leading to signs of pulmonary inflammation and progressive development of airflow obstruction \[[@B129]\]. ### Inhalation models -- oxidant stimuli and particulates The administration of oxidants such as ozone also causes significant lung injury with some features related to inflammatory changes occurring in human COPD \[[@B130]\] and this causes numerous effects in airway cells \[[@B131]-[@B135]\]. As a gaseous pollutant, ozone targets airway tissues and breathing slightly elevated concentrations of this gas leads to a range of respiratory symptoms including decreased lung function and increased airway hyper-reactivity. In conditions such as COPD and asthma, ozone may lead to exacerbations of symptoms. Ozone is highly reactive: the reaction with other substrates in the airway lining fluid such as proteins or lipids leads to secondary oxidation products which transmit the toxic signals to the underlying pulmonary epithelium. These signals include cytokine generation, adhesion molecule expression and tight junction modification leading to inflammatory cell influx and increase of lung permeability with oedema formation \[[@B130]\]. However, the nature and extent of these responses are often variable and not related within an individual. The large amount of data obtained from animal models of ozone exposure indicates that both ozone- and endotoxin-induced animal models are dependent on neutrophilic inflammation. It was shown that each toxin enhances reactions induced by the other toxin. The synergistic effects elicited by coexposure to ozone and endotoxin are also mediated, in part, by neutrophils. \[[@B136],[@B137]\]. Further animal models focus on the exposure to ultrafine particles, silica and coal dust \[[@B138],[@B139]\]. Ultrafine particles are a common component of air pollution, derived mainly from primary combustion sources that cause significant levels of oxidative stress in airway cells \[[@B140],[@B141]\]. The animal models are predominantly characterized by focal emphysema and it was suggested that dust-induced emphysema and smoke-induced emphysema occur through similar mechanisms \[[@B142]\]. Exposure to diesel exhaust particles (DEP) may also lead to chronic airway inflammation in laboratory animals as it was shown to have affect various respiratory conditions including exacerbations of COPD, asthma, and respiratory tract infections \[[@B143]\]. Both the organic and the particulate components of DEP cause significant oxidant injury and especially the particulate component of DEP is reported to induce alveolar epithelial damage, alter thiol levels in alveolar macrophages (AM) and lymphocytes, and induce the generation of reactive oxygen species (ROS) and pro-inflammatory cytokines \[[@B144]\]. The organic component has also been shown to generate intracellular ROS, leading to a variety of cellular responses including apoptosis. Long-term exposure to various particles including DEP, carbon black (CB), and washed DEP devoid of the organic content, have been shown to produce chronic inflammatory changes and tumorigenic responses \[[@B144]\]. The organic component of DEP also suppresses the production of pro-inflammatory cytokines by macrophages and the development of Th1 cell-mediated mechanisms thereby enhancing allergic sensitization. The underlying mechanisms have not been fully investigated so far but may involve the induction of haeme oxygenases, which are mediators of airway inflammation \[[@B145]\]. Whereas the organic component that induces IL-4 and IL-10 production may skew the immunity toward Th2 response, the particulate component may stimulate both the Th1 and Th2 responses \[[@B146]\]. In conclusion, exposure to particulate and organic components of DEP may be a helpful approach to simulate certain conditions such as exacerbations. Also, the development of lung tumours after long term exposure may be useful when studying interactions between COPD-like lesions and tumorigenesis. A further toxin is cadmium chloride, a constituent of cigarette smoke. Administration of this substance also leads to alterations in pulmonary integrity with primarily interstitial fibrosis with tethering open of airspaces \[[@B147]\]. A combination of cadmium and lathyrogen beta-aminopropionile enhances emphysematous changes \[[@B148]\]. ### Tissue-degrading approaches Emphysema-like lesions can also be achieved by intrapulmonary challenge with tissue-degrading enzymes and other compounds \[[@B149]\] (Figure [2](#F2){ref-type="fig"}). Proteinases such as human neutrophil elastase, porcine pancreatic elastase, or papain produce an efficient enzymatic induction of panacinar emphysema after a single intrapulmonary challenge \[[@B150],[@B151]\]. Since bacterial collagenases do not lead to the formation of emphysema, the effectiveness of the proteinases is related to their elastolytic activity. While these models may not be as useful as smoke exposure studies to achieve COPD-like lesions, they can lead to a dramatic picture of emphysema and may be used to study mechanisms related specifically to emphysema and to the repair of damaged lung. However, the method of inducing emphysema-like lesions by intratracheal instillation of these enzymes may not very closely relate to mechanisms found in the human situation. Among the different emphysema models, elastase-induced emphysema has also been characterized to be accompanied by pulmonary function abnormalities, hypoxemia, and secretory cell metaplasia which represent characteristic features of human COPD. Recent studies suggested that exogenous retinoic acid can induce alveolar regeneration in models of elastase-induced experimental emphysema \[[@B152]\] and that retinoic acid may have a role for alveolar development and regeneration after injury \[[@B153],[@B154]\]. However, the role of retinoic acid in relation to alveolar development has only been analysed in a rat model and models in other animals did not show similar effects \[[@B155]\]. Also, the ability of alveolar regeneration which is present in rats does not occur to a similar extent in humans; a recent clinical trial using retinoic acid in COPD did not show positive results \[[@B156]\]. The mechanisms of emphysema induction by intratracheal administration of elastase encompass an initial loss of collagen and elastin. Later, glycosaminoglycan and elastin levels normalize again but collagen levels are enhanced. The extracellular matrix remains distorted in structure and diminished with resulting abnormal airway architecture \[[@B157]\]. The enlargement of the airspaces immediately develops after the induction of elastolytic injuries and is followed by inflammatory processes which lead to a transformation of airspace enlargement to emphysema-like lesions. This progression most likely occurs due to destructive effects exerted by host inflammatory proteinases. Addition of lathyrogen beta-aminopropionile leads to an impairment of collagen and elastin crosslinking and therefore further increases the extent of emphysema-like lesions \[[@B158]\]. Effects seem to be mediated via IL-1β and TNFα receptors since mice deficient in IL-1β Type1 receptor and in TNFalpha type 1 and 2 receptors are protected from developing emphysema following intratracheal challenge with porcine pancreatic elastase. This was associated with reduced inflammation and increased apoptosis \[[@B159]\]. In general, intrapulmonary administration of tissue-degrading enzymes represents a useful tool especially when focusing on mechanisms to repair emphysematic features. However, the lack of proximity to the human situation needs to be realized since the mechanisms of emphysema induction are clearly not related to the human situation. An advantage of proteinase-based models is the simple exposure protocol with a single intratracheal administration leading to significant and rapid changes. However, extrapolating these findings to slowly developing features of smoking induced human COPD is very difficult since a large number of mediators may not be involved in the rapid proteinase approach. Therefore, these models may not encompass important features of human COPD which may be more closely mimicked by inhalation exposures and it is clear that tissue-degrading enzyme models always represent the picture of an \"induced pathogenesis\". ### Gene-targeting approaches The genetic predisposition to environmental disease is an important area of research and a number of animal strains prone to develop COPD-like lesions have been characterized \[[@B160]-[@B162]\] (Figure [2](#F2){ref-type="fig"}). Also, genetically-altered monogenic and polygenic models to mimic COPD have been developed in recent years using modern techniques of molecular biology \[[@B163],[@B164]\]. Gene-depletion and -overexpression in mice provide a powerful technique to identify the function and role of distinct genes in the regulation of pulmonary homeostasis in vivo. There are two major concepts consisting of gain-of-function and loss-of-function models. Gain-of-function is achieved by gene overexpression in transgenic mice either organ specific or non-specific while loss of function is achieved by targeted mutagenesis techniques \[[@B165],[@B166]\]. These models can be of significant help for the identification of both physiological functions of distinct genes as well as mechanisms of diseases such as COPD. A large number of genetically-altered mice strains have been associated to features of COPD and a primary focus was the assessment of matrix-related genes. As destruction of alveolar elastic fibres is implicated in the pathogenic mechanism of emphysema and elastin is a major component of the extracellular matrix, mice lacking elastin were generated. It was shown that these animals have a developmental arrest development of terminal airway branches accompanied by fewer distal air sacs that are dilated with attenuated tissue septae. These emphysema-like alterations suggest that in addition to its role in the structure and function of the mature lung, elastin is essential for pulmonary development and is important for terminal airway branching \[[@B167]\]. Also, deficiency of the microfibrillar component fibulin-5 and platelet derived growth factor A (PDGF-A) leads to airspace enlargement \[[@B168],[@B169]\]. PDGF-A(-/-) mice lack lung alveolar smooth muscle cells, exhibit reduced deposition of elastin fibres in the lung parenchyma, and develop lung emphysema due to a complete failure of alveogenesis \[[@B170]\]. The postnatal alveogenesis failure in PDGF-A(-/-) mice is most likely due to a prenatal block in the distal spreading of PDGF-R alpha+ cells along the tubular lung epithelium during the canalicular stage of lung development \[[@B170]\]. The importance of integrins in causing emphysema has been demonstrated in mouse. Epithelial restricted integrin α vβ 6-null mice develop age-related emphysema through the loss of activation of latent TGF-beta which leads to an increase in macrophage MMP-12 expression \[[@B171]\]. Fibroblast growth factors are known to be essential for lung development. Mice simultaneously lacking receptors for FGFR-3 and FGFR-4 have an impaired alveogenesis with increased collagen synthesis \[[@B172]\]. It is crucial to distinguish developmental airspace enlargement from adult emphysema which is defined as the destruction of mature alveoli. However, the identification of numerous factors influencing lung development is an important step towards identifying potential mechanisms underlying the development and progression of emphysema in human COPD. Next to developmental airspace enlargement also spontaneous emphysema may occur in genetically-modified mice strains and a gradual appearance of emphysema-like lesions has been found in mice lacking the surfactant protein D (SP-D) gene \[[@B173]\] and in mice lacking the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene \[[@B174]\]. In these strains, matrix metalloproteinases were suggested to be the primary mediators of tissue destruction. A further mechanism to induce emphysema-like lesions is to expose developmentally normal genetically-modified animals to exogenous noxious stimuli such as tobacco smoke. This also allows identifying potential molecular mechanisms involved in the pathogenesis of COPD. Using macrophage elastase (MMP-12) gene-depletion studies it was shown that in contrast to wild type mice, the lung structure of MMP-12 gene-depleted animals remains normal after long term exposure to cigarette smoke \[[@B47]\]. These animals also fail to develop macrophage accumulation in response to cigarette smoke, an effect that could be related to MMP-12 induced generation of elastin fragments that are chemotactic for monocytes \[[@B175],[@B176]\]. In summary, gene-targeting techniques display very useful tools to examine potential molecular mechanisms underlying human COPD. In combination with inhalation protocols they may identify important protective or pro-inflammatory mediators of the disease. ### Other models Various other agents have also been characterized to induce airway inflammation injury. In this respect, administration of toxins such as endotoxin leads to a recruitment of neutrophils and macrophage activation with concomitant airspace enlargement \[[@B177],[@B178]\]. Non-inflammatory emphysema-like lesions may also be accomplished by intravascular administration of a vascular endothelial cell growth factor receptor-2 (VEGFR-2) blocker \[[@B179]\]. VEGF is required for blood vessel development and endothelial cell survival and its absence leads to endothelial cell apoptosis \[[@B180]\]. An increased septal cell death in human emphysematous lungs and a reduced expression of VEGF and VEGFR-2 is found in emphysema lungs \[[@B181]\]. Also, chronic blockage of VEGFR-2 causes alveolar septal cell apoptosis and airspace enlargement \[[@B179]\]. These findings of airspace enlargement point to a role of the vascular system in the development and progression of emphysema. Conclusions =========== In contrast to the variable pathology and different stages of severity in human COPD, currently available animal models are restricted to mimicking a limited amount of characteristic features of COPD. Animal models need to be precisely evaluated based on whether they agree with features of human COPD in order to advance the understanding of mechanisms in human COPD. Based on inhalative exposure to noxious stimuli such as cigarette smoke, the administration of tissue-degrading enzymes or gene-targeting techniques, a number of experimental approaches to mimic acute and chronic features of COPD have been established in the past years. Due to the complexity of the disease, and species-specific differences they are all limited concerning their clinical significance. While the induction of the COPD lesions by tissue-degrading enzymes may appear artificial in many cases, it does not mean that these models are not valuable because they can be used to study many aspects of pulmonary pathophysiology of end-stage emphysema. Cellular mechanisms can be studied efficiently and underlying molecular mechanisms and potential therapeutic approaches can be revealed if the data is extrapolated cautiously. Combined models of inhalative exposure, proteinase-based tissue degradation to produce emphysema and gene-targeting techniques may provide models of COPD which encompass more features of the disease. However, one cannot assume that reproducing COPD with a high degree of fidelity in the animal necessarily means that the model simulates the human condition. In fact, a model that only produces a single pathologic COPD feature may be more useful as long as it produces this feature via a relevant mechanism that allows exploratory research. By contrast, a model producing all kinds of COPD features via irrelevant mechanisms may be less useful. In this respect, validation of models as being relevant is an extremely important issue in the early steps of model development. Animal models should not only assess histopathological features but also attempt to focus on functional features of human COPD such as airflow limitation, mucus hypersecretion, chronic cough and exacerbations, and also on pharmacological features such as corticosteroid resistance or diminished β-adrenergic bronchodilator responses. In conclusion, there are many benefits that can accrue from the development of animal models of COPD, most important of which is understanding of mechanisms and development of specific drugs for COPD.	PubMed Central	2024-06-05T03:55:49.754151	2004-11-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533858/", "journal": "Respir Res. 2004 Nov 2; 5(1):18", "authors": [ { "first": "David A", "last": "Groneberg" }, { "first": "K Fan", "last": "Chung" } ] }
PMC533859	Backgound ========= Eosinophils are known to be the important effector cells in asthmatic airway inflammations\[[@B1]\]. Previous studies have demonstrated that eosinophils are accumulated in the peripheral blood, the bronchoalveolar lavage fluid, and the airway of the asthmatic patients or the allergen-sensitized animals\[[@B2]\]. Eosinophil trafficking is regulated by a wide variety of chemotactic factors\[[@B3]\]. Eotaxin and RANTES ([R]{.underline}egulated on [A]{.underline}ctivation [N]{.underline}ormal [T]{.underline}-cells [E]{.underline}xpressed and [S]{.underline}ecreted) are C-C chemotaxins that can recruit eosinophils to the airway in asthma\[[@B4]\]. A variety of tissues and cell types, including lung epithelial cell, produce eotaxin and RANTES which play an important role in airway\[[@B5]\]. Pro-inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin (IL)-1 are released in the early stage of allergic inflammation. In endothelial and epithelial cells, TNF-α induces an influx of eosinophils into tissues through the increased expression of adhesion molecules\[[@B6],[@B7]\]. Although eotaxin and RANTES tend to be expressed constitutively in several cell types, their expression may also be regulated in response to TNF-α in other cell lines\[[@B8]\]. Melatonin(N-acetyl-5-methoxytryptamine) is a key regulator of circadian rhythm homeostasis in humans\[[@B9],[@B10]\]. It also appears to have an important immunomodulatory effect in allergic diseases\[[@B11],[@B12]\]. Melatonin promotes the cytokine production in the peripheral blood mononuclear cell. Pinealectomized rats sensitized to ovalbumin demonstrated that pinealectomy significantly reduces the inflammatory cell counts in the bronchoalveolar lavage fluid after ovalbumin challenge, and that melatonin administration to pinealectomized rats restores the ability of inflammatory cells to migrate to the bronchoalveolar fluid. Those results suggest that melatonin may modulate the expression of chemotaxins in airway epithelial or endothelial cells\[[@B13]\]. The circadian variations of lung function in nocturnal asthma are associated with the increased airway inflammation during night. As a key regulator in human circadian rhythm homeostasis as well as an immunomodulator in allergic diseases, melatonin may regulate the circadian airway inflammation in asthma through modulating the expression of chemotaxins in the airway epithelial cells. In order to test this hypothesis, we conducted the present study to answer two questions. First, whether melatonin is able to up-regulate RANTES and eotaxin expression in the lung epithelia cell line-A549. Second, what is the combinatory effect of melatonin and TNF-α on RANTES and eotaxin expression and whether this effect increases the eosinophils chemotactic activity (ECA) released in A549. The answers to these questions might provide new insights into the pathophysiology of asthma. Methods ======= This study was approved by the medical ethics committee of the West China Hospital of Sichuan University. Informed consents were obtained from all subjects in the study. Cell Culture ------------ A549 cells, human type II-like epithelial lung cells, were obtained from ATCC (Manassas, VA, USA). The cells were cultured in tissue flasks incubated in 100% humidity and 5% CO~2~at 37°C in DMEM medium (GIBCO BRL, Grand Island, NY) supplemented with 10% heat-inactived fetal bovine serum (GIBCO BRL) and penicillin-streptomycin (50 μg/ml, GIBCO BRL), at 1 × 10^6^cells/ml. A549 cells were then plated onto 6-well, flat-bottom tissue culture plates (Becton Dickinson and Co., NJ, USA) at a density of 1 × 10^6^cells/ well in DMEM medium. The medium was changed every 2 d until the cells became confluent and then the cells were used for the experiments. Cytokine Assays --------------- As IL-1β and TNF-α have similar effect on the expression of many chemotaxins\[[@B14],[@B15]\], we chose TNF-α as the representative pro-inflammatory cytokines in the asthmatic lung in this study. After the cells became confluent, the medium was changed to serum-free DMEM medium for 12 h. A549 cells were then exposed to increasing concentrations of melatonin (10^-10^, 10^-9^, 10^-8^, 10^-7^, 10^-6^M, the physiology concentration are 10^-9^to 10^-7^M during day and night\[[@B16]\]) (Sigma, St. Louis, MO, USA) and TNF-α (100 ng/ml) (Sigma), for 12 h. The cells were also stimulated with a combination of melatonin (10^-10^, 10^-9^, 10^-8^, 10^-7^, 10^-6^M) and TNF-α (100 ng/ml). The epithelial cell layers were then washed three times with Hanks\' balanced salt solution (GIBCO BRL) and incubated for 48 h. Cell-free culture supernatants were collected. RANTES and eotaxin were assayed using enzyme-linked immunosorbent assay (ELISA) kits according to the instructions of the manufacturers. Assay kits for RANTES and eotaxin were purchased from R&D Systems (Minneapolis, MN, USA), and the minimum detectable concentration of RANTES and eotaxin was 5 pg/ml. Experiments were performed at least three times with the similar results. RNA extraction and real-time PCR -------------------------------- RNA extraction and real-time PCR were performed as previously described\[[@B17],[@B18]\]. After the cells became confluent, the medium was changed to fetal bovine serum free DMEM medium for 12 h. A549 cells were then exposed to different concentrations of melatonin, together with or without TNF-α (100 ng/ml) (Sigma) for 12 h. Total cellular RNA was extracted using an acid guanidinium-phenol-chloroform method (Trizol; GIBCO BRL). RNA integrity was confirmed by electrophoresis on 1% agarose gels and ethidium bromide staining. Total cellular RNA, 1 μg, was reverse transcribed at 37°C for 70 min in 20 μl containing 2.5 U Superscript-II reverse transcriptase (GIBCO BRL); 10 mM dithiothreitol, 1 mM each of deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), and deoxyguanidine triphosphate (dGTP); and 5 μg/ml oligo-dT primer (Pharmacia, Piscataway, NJ). Reactions were stopped by heat inactivation for 10 min at 85°C. Primers for human eotaxin, RANTES and β-actin were synthesized, HPLC purified as GIBCO BRL Custom Primers (Hong Kong, China). Primer sequences were as follows: Eotaxin: upstream primer: 5\'- ACA TGA AGG TCT CCG CAG CAC TTC -3\', downstream primer: 5\'- TTG GCC AGG TTA AAG CAG CAG GTG -3\'. RANTES upstream primer: 5\'- GGC ACG CCT CGC TGT CAT CCT CA-3\'; downstream primer: 5\'- CTT GAT GTG GGC ACG GGG CAG TG-3\'. β-actin upstream primer: 5\'- AAG AGA GGC ATC CTC ACC CT -3\',downstream primer 5\'- TAC ATG GCT GGG GTG TTG AA -3\'. Real-time PCR was performed on the ABI Prism 7700 sequence detection system (PE Applied Biosystems) by using SYBR green (Roche Diagnostics, Somerville, NJ) as a dsDNA-specific binding dye. The PCR were cycled 40 times after initial denaturation (95°C, 2 min) with the following parameters: denaturation, 95°C, 15s; and annealing and extension, 60°C, 1 min. The threshold cycle (CT) was recorded for each sample to reflect the mRNA expression levels. The fold changes of eotaxin or RANTES gene expression were calculated as previously described\[[@B18]\]. Eosinophil Chemotaxis Assay --------------------------- Eosinophil chemotaxis assay was performed as described previously\[[@B19]\]. Briefly, eosinophils were isolated from the peripheral blood of three healthy donors by negatively selected with immunomagnetic beads. Erythrocytes in venous peripheral blood were removed by hypotonic lysis. Neutrophils and mononuclear cells were depleted with anti-CD16 and anti-CD3 immunomagnetic beads (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Eosinophils were stained with Randolph\'s stain and counted in a hemocytometer. Cytospins of each preparation were stained with Diff-Quik (International Reagent Corp., Green Cross, Osaka, Japan). The mean percentage of the eosinophil purity was 98.0 ± 0.3%. The viability measured by trypan blue exclusion was consistently greater than 95.0%. Eosinophil chemotaxis assay was measured by the Boyden\'s blind-well chamber technique using a 48-well, multiwell chamber (NeuroProbe Inc., Bethesda, MD). The bottom wells of the chamber were filled with 26.5 μl of the A549 cell supernatant stimulated by various chemicals, as described previously, in triplicate. A polycarbonate filter with a pore size of 5 μm (Nucleopore, Pleasanton, CA) was placed over the bottom wells, and isolated eosinophils were placed into each of the top wells. The chambers were then incubated at 37°C, 5% CO~2~for 90 min. After incubation, eosinophils in the top wells were removed by scraping. The filter was then stained with Diff-Quik. Eosinophil chemotactic activity (ECA) is shown as the total number of migrated eosinophils counted in 10 high-power fields under a light microscope (Olympus, Lake Success, NY) at × 400 magnification. Data analysis ------------- Data were expressed as means ± SD. Differences between groups were assessed by one-way ANOVA followed by the LDS significant difference test. A value of p \< 0.05 was considered statistically significant. Results ======= Effect of TNF-α and melatonlin on RANTES and eotaxin released from A549 cells ----------------------------------------------------------------------------- RANTES released from A549 cells increased significantly when the cells incubated with TNF-α(100 ng/ml). Melatonin alone didn\'t have this effect on A549 in dose from10^-10^to 10^-6^M. However, TNF-α induced RANTES release in A549 increased significantly by incubation with melatonin (from10^-10^to 10^-6^M). Similarly, eotaxin released from A549 cells also increased significantly when the cells incubated with TNF-α; Melatonin alone had no effect on eotaxin released from A549 at dose range from10^-10^to 10^-6^M. However, eotaxin released from A549 increased significantly when the cells incubated with melatonin and TNF-α (Figure [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### RANTES and eotaxin released from A549 cells.Melatonin(10^-6^M) alone did not change RANTES and eotaxin released from A549 cells. However, it (from10^-10^to 10^-6^M) promoted RANTES and eotaxin released from A549 cells in a dose dependent manner when co-stimulated with TNF-α (100 ng/ml). \* and \\, p \< 0.05 and 0.01, compared with control and melatonin alone (pg/ml, n = 3). \$ and \#, p \< 0.05 and 0.01, compared with TNF-α alone (pg/ml, n = 3). ::: ![](1465-9921-5-20-1) ::: Effect of TNF-α and melatonlin on the expression of RANTES and eotaxin in A549 cells ------------------------------------------------------------------------------------ To determine whether the production of RANTES and eotaxin is accompanied by the transcription of the corresponding genes, we used real-time RT-PCR to examine RANTES and eotaxin mRNA expression in A549 cells. A549 were stimulated with melatonin (10^-10^, 10^-9^, 10^-8^, 10^-7^, 10^-6^M) and TNF-α (100 ng/ml). Melatonin alone did not change the RANTES and eotaxin mRNA expression in A549. TNF-α can promote the RANTES and eotaxin expression in A549 cells. When stimulated with TNF-α, melatonin synergistically increased the RANTES and eotaxin expression in a dose dependent manner (Fig [2](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### RANTES and eotaxin mRNA expression in A549 cells.Melatonin(10^-6^M) alone did not change the RANTES and eotaxin mRNA expression in A549 cells. TNF-α (100 ng/ml) could promote the RANTES and eotaxin expression in A549 cells. Melatonin (from10^-10^to 10^-6^M) increased the RANTES expression of A549 cell in a dose dependent manner when co-stimulated with TNF-α(100 ng/ml). \\, p \< 0.01, compared with control and melatonin alone (n = 3). \#, p \< 0.01, compared with TNF-α alone (n = 3). ::: ![](1465-9921-5-20-2) ::: Effect of TNF-α and melatonlin on eosinophil chemotactic activity (ECA) released by A549 Cells ---------------------------------------------------------------------------------------------- When stimulated with TNF-α(100 ng/ml), ECA released by A549 cells increased significantly. Melatonin (from10^-10^to 10^-6^M) alone didn\'t have this effect. When stimulated with TNF-α (100 ng/ml) and melatonin, ECA released increased in A549 cells in a dose dependent manner (Fig [3](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Eosinophil chemotactic activity (ECA) released from A549 cells.Melatonin (10^-6^M) alone did not change the ECA released from A549 cells. TNF-α (100 ng/ml) could increase the ECA released from A549 cells. Melatonin (from10^-10^to 10^-6^M) increased the ECA released from A549 cell in a dose dependent manner when co-stimulated with TNF-α(100 ng/ml). \\, p \< 0.01, compared with control and melatonin alone (n = 3). \#, p \< 0.01, compared with TNF-α alone (n = 3). ::: ![](1465-9921-5-20-3) ::: Discussion ========== In this study, we examined the RANTES and eotaxin protein level and the gene expression in A549 in response to TNF-α and melatonin stimulation using ELISA and real-time RT-PCR. We also measured the ECA released by A549 in response to TNF-α and melatonin stimulation. Unexpected, we found that the eotaxin and RANTES protein level and gene expression in A549 cells were unchanged when treated with melatonin alone, and the ECA released by A549 remained unchanged too. However, when A549 cells co-stimulated with melatonin and TNF-α, eotaxin and RANTES released from the cells increased in a melatonin dose dependent manner. The gene expression of eotaxin and RANTES, and the ECA also increased at the same time. This result support our hypothesis that melatonin play an important role in airway inflammation through up-regulation of the eotaxin and RANTES expression in lung epithelial cell when the cells stimulated with pro-inflammatory cytokines. The pro-inflammatory characteristics of TNF-α have been documented extensively. Numerous studies have demonstrated that these attributes contribute to the inflammatory conditions present in airways of asthmatic subjects. TNF-α has been shown to activate the inflammatory cells, up-regulate the adhesion molecules on endothelium and circulating leukocytes, increase the production of chemotaxins\[[@B20]\], the bronchial responsiveness. TNF-α is expressed primarily by the alveolar cells and tissue macrophages, mast cells, and bronchial epithelial cells. Additionally, in most other airway cell systems studied, conditions simulating an inflammatory state result in expression of TNF-α. Thus, it is not surprising that TNF-α concentration is higher in the bronchoalveolar lavage fluid from symptomatic asthmatics compared with normal control subjects\[[@B21]\]. In this study, we found that TNF-α could promote the RANTES and eotaxin production in A549 and melatonin further exaggerated this effect of TNF-α. Lung function in a healthy individual varies in a circadian rhythm, with the peak lung function occurring near 4:00 PM (1600 hours) and the minimal lung function occurring near 4:00 AM (0400 hours). An episode of nocturnal asthma is characterized by an exaggeration in this normal variation in lung function from daytime to nighttime, with diurnal changes in the pulmonary function generally of \> 15%. A recent study showed that the circadian variability in pulmonary function in asthma was related to changes in the airway eosinophils recruitment and activation\[[@B22]\]. Although the molecular mechanism responsible for the selective infiltration of eosinophils into the inflamed tissue in asthma has not been elucidated, chemotaxin may play an important role in this process. Eotaxin is a chemotaxin that binds with high affinity and specificity to the chemotaxin receptor CCR3 and plays an important role in the pathogenesis of allergic disease. RANTES, a C-C chemotaxin, was initially shown to be chemoattractant for T cells and monocytes but has subsequently been shown to be a potent eosinophil chemoattractant\[[@B23],[@B24]\]. In other studies, an up-regulation of RANTES message was observed in the airways of asthmatic patients\[[@B25]\], and increased levels of RANTES have been detected in the nasal aspirates of children with the viral exacerbation of asthma\[[@B26]\], suggesting an important role for RANTES in this process. From the result of our study, together with the studies above, we can infer that melatonin, the most important circadian rhythm regulator, may also regulate the asthma airway inflammation by up-regulating the expression of eotaxin and RANTES in the airway epithelium in inflammatory status of asthma. RANTES and eotaxin expression are regulated by two important transcriptional factors: active protein-1 (AP-1) and nuclear factor kappa B(NFκB). Benis et al\[[@B27]\] found that melatonin could suppress the activation of NFκB and AP-1. Although NFκB and AP-1 could up-regulate the expression of many pro-inflammatory cytokines and chemotaxins, other transcriptional factors also could be involved in the regulation of RANTES and eotaxin. Further studies are needed to elucidate the mechanism of how melatonin regulates the transcription of these chemotaxins. The role of melatonin as an immunomodulator is poorly understood and, in some cases, contradictory results have been reported. For example, Shafer\'s study showed that melatonin has no effect on the activity of stimulated macrophages\[[@B28]\]. However, pinealectomy of rats significantly reduces airway inflammation after ovalbumin inhalational challenge, and melatonin administration to the pinealectomized rats seems to restore the airway inflammation, which further supports the pro-inflammatory effect of melatonin. In addition, up-regulation of the gene expression of transforming growth factor-β(TGF-β), macrophage-colony stimulating factor (M-CSF), TNF-α and stem cell factor (SCF) in peritoneal exudate cells, and up-regulation of the gene expression of IL-1β, M-CSF, TNF-α, interferon-γ (IFN-γ) and SCF in splenocytes, were observed in male C57 mice received 10 consecutive daily intraperitoneal injections of melatonin\[[@B12]\]. Further research should be directed at evaluating the mechanism of melatonin regulating the transcription of those kinds of cytokines. Conclusion ========== Melatonin alone did not change eotaxin and RANTES protein level and gene expression in A549 cells, and had no effect on ECA released by A549 cells. However, when A549 cells were stimulated with melatonin, together with TNF-α, the mRNA expression and protein release of eotaxin and RANTES increased significantly. This result suggested that combined with pro-inflammatory cytokines, melatonin may play a role in the airway inflammation through up-regulation of the eotaxin and RANTES expression in the lung epithelial cells. Authors\' contributions ======================= FML conceived of the experiment, carried out all experiments and prepared the manuscript. XJL conceived of the experiment and performed RNA extraction and real-time RT-PCR. SQL conceived of the experiment and assisted in collection and analysis of ELISA samples. CTL performed cell culture and provided expert advice and interpretation of the study\'s results. WZL participated in the study\'s design, coordination and final revisions of the manuscript. All authors read and approved the final manuscript.	PubMed Central	2024-06-05T03:55:49.758517	2004-11-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533859/", "journal": "Respir Res. 2004 Nov 10; 5(1):20", "authors": [ { "first": "FengMing", "last": "Luo" }, { "first": "XiaoJing", "last": "Liu" }, { "first": "ShuangQing", "last": "Li" }, { "first": "ChunTao", "last": "Liu" }, { "first": "ZengLi", "last": "Wang" } ] }
PMC533860	Background ========== One of the most common causes of hypogonadotropic hypogonadism is Kallmann syndrome (KS). KS is a genetically heterogeneous condition that affects approximately one in 8000 males and one in 40,000--70,000 females \[[@B1]-[@B3]\]. This recent estimate is much higher than the previously estimated prevalence of Kallmann syndrome among males of 1:80,000 \[[@B4]\]. To our knowledge, no data is available on the incidence of this syndrome in Jordan or in the Arab world. In addition to the sporadic form which is the most common \[[@B2]\], KS has three modes of inheritance, X-linked, autosomal recessive and autosomal dominant \[[@B5]\]. The gene responsible for the X-linked form of the disease is KAL1 gene \[[@B6]-[@B8]\], and encodes the protein anosmin that is directly responsible for the migration of GnRH neurons and the olfactory nerves from the olfactory system to the hypothalamus \[[@B5],[@B9]-[@B11]\]. Males usually present in the second decade with delayed puberty and females present with primary amenorrhea. Prepubertal boys may present with microphallus and cryptorchidism \[[@B12],[@B13]\]. Proper management of patients with Kallmann syndrome usually allows them to attain normal reproductive health. The purpose of this paper is to demonstrate the presentation modalities, phenotypes and the modes of inheritance among 32 patients with Kallmann syndrome in Jordan, which may contribute to the recognition of the syndrome and the provision of adequate management and genetic counselling. Subjects & Methods ================== Over a period of five years, thirty-two male and female patients from twelve Jordanian and Palestinian families were referred to the National Center for Diabetes, Endocrinology and Genetics (NCDEG) in Amman, Jordan for evaluation of hypogonadism among adults, or microphallus among children. Prospective evaluation was performed including pedigree construction (fig [1](#F1){ref-type="fig"}), and complete clinical examination with special emphasis on assessment of anosmia, the presence of mirror image movements (synkinesia) and examination of external genitalia. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Pedigrees of 12 families with KS ::: ![](1742-4755-1-5-1) ::: Olfactory testing was performed through a smell identification test designed by our department using odours that can be easily identified among all social classes in the local population. These odours included cinnamon, coffee, camomile, thyme, soap, oriental perfume and tobacco. The specific odoriferous substance was approximated to the nostrils while the patient had his eyes closed. The patient is then asked to name the olfactory stimulus. According to the number of substances identified by smelling, patients were divided into anosmic and hyposmic. Those who failed to identify all odours were categorized as anosmic, while those who identified three or less odours were categorized as hyposmic. Those who identified four to all seven odours were considered normosmic. First-degree relatives of the probands were also questioned and tested for the smell sensation in a similar fashion. The same investigator carried out olfactory testing for all patients and family members. In addition to the assessment of smell sensation among relatives of probands, their answers to specific questions were recorded. These questions addressed the issues of surgery to correct cryptorchidism, administration of testosterone and whether there were any complaints of delayed puberty or infertility. The specific questions and the olfactory testing led to the identification of further cases of KS in the family. Patients were asked to perform a screwdriver motion in one hand while the examiner watches for any similar non-voluntary movement in the other hand to assess mirror image movements. Subjects were also asked to perform alternate supination and pronation of one forearm while the examiner watches for a similar movement in the other arm \[[@B14]\]. Phallus length was measured and compared to age-matched controls \[[@B15]\], and the testicle size was estimated using an orchidometer. Neurosensory hearing impairment was assessed by audiometry, renal abnormalities by renal ultrasound and congenital heart defects by echocardiography. All patients were tested for color vision and abnormal eye movements. Complete hormonal evaluation including basal gonadotrophins, sex hormones and gonadotrophin releasing hormone (GnRH) stimulation test were done for 20 patients 16 years and above. Three adult patients were not tested due to loss of contact. GnRH test involved the administration of 100 mcg of GnRH, followed by the assessment of gonadotrophin levels at 0, 15, 30, 45, 60 and 120 minutes. For individuals with no or low response to the test, priming was performed, where GnRH was given for five days, with repetition of assessment of gonadotrophin levels. Seminal fluid analysis was done for male patients aged 16 years and above. Radiological studies included brain, pituitary and olfactory tract magnetic resonance imaging (MRI). The criteria for diagnosis of KS among adults included the presence of anosmia or hyposmia with clinical signs and symptoms of hypogonadism and a testosterone level \<100 ng/dl among males 16 years and older, and estradiol level \<20 pg/dl among adult females, together with basal low gonadotrophin level. In family II, loss of contact of three brothers hindered testing their hormonal levels. They were, however, included among our patients because clinical examination revealed hypogonadism, anosmia and micropenis. Among prepubertal males, the criteria for diagnosis of KS included presence of microphallus with anosmia/hyposmia, and/or absent olfactory bulbs on MRI. Pedigree analysis was used to establish the modes of inheritance of KS in the familial cases. Inheritance in a family was classified as X-linked if only males were affected in more than one sibship connected by females, or if two or more males were affected in the sibship with a negative family history and with associated synkinesia. Inheritance was classified as autosomal recessive if all affected individuals were members of the same generation and included at least one female. Results ======= Over a period of five years (1999--2004), thirty-two patients were prospectively diagnosed with Kallmann syndrome at the National Center for Diabetes, Endocrinology and Genetics (NCDEG) in Amman, Jordan. The patients belonged to twelve Jordanian and Palestinian families and their ages at presentation ranged between 4 -- 46 years. They included 26 males and 6 females with a male/female ratio of 4.34/1. Nine male patients were aged 14 years and younger. The clinical features among male patients are presented in table [(1)](#T1){ref-type="table"}, and among female patients in table [(2)](#T2){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Clinical features of 26 males with Kallmann syndrome ::: FAMILY No Age Anosmia/hyposmia Synkinesia Hearing Impairment Renal abnormalities Azoospermia cryptorchidism Micropenis ---------- ---- ----- ------------------ ------------ -------------------- --------------------- -------------- ---------------- ------------ 1 1 46 +/H \- \+ \- \+ \+ \- 2 14 \+ \+ \+ \+ \+ \+ \- 3 27 \+ \+ \- \+ \+ \+ \- 4 20 \+ \+ \- \+ \+ \+ \- 5 20 \+ \+ \- \+ \+ \+ \- 6 19 \+ \+ \- \+ \+ \+ \- 7 16 \+ \+ \- \+ \+ \+ \- 8 14 \+ \+ \- \+ NA \+ \- 9 9 \+ \+ \- \+ NA \+ \+ 10 6 \+ \+ \- \- NA \- \+ 11 4 \+ \+ \- \- NA \+ \+ II 1 37 \+ \- \- \- \+ \+ \+ 2 24 \+ \- ND\* ND\* ND\* \+ \+ 3 22 \+ \- ND\* ND\* ND\* \- \+ 4 20 \+ \- ND\* ND\* ND\* \+ \+ III 1 14 \+ \+ \- \- NA \- \+ 2 10 \+ \+ \- \+ NA \- \+ 3 8 \+ \+ \- \+ NA \- \+ IV 1 6 \+ \+ \- \- NA \+ \+ 2 5 \+ \+ \- \- NA \+ \+ V 1 20 +/H \- \+ \- \+ \- \+ 2 19 +/H \+ \+ \+ \+ \- \+ VI 1 19 \+ \- \- \+ ND \+ \- VII 1 37 \+ \- \- \- \+ \+ \+ VIII 1 22 \+ \- \- \+ \+ \+ \+ IX 1 20 \+ \- \- \- oligospermia \+ \+ NA: not applicable. ND: not done. \: lost contact H: hyposmia ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Clinical Features of 6 females with Kallmann syndrome ::: FAMILY No Age Anosmia/hyposmia Synkinesia Hearing loss Renal abnormalities Primary Amenorrhea --------- ---- ----- ------------------ ------------ -------------- --------------------- -------------------- X* 1 23 \+ \- \- \- \+ 2 21 \+ \- \- \- \+ 3 18 \+ \- \- \- \+ IX 1 18 +/H \- \- \- \+ XI 1 30 \+ \- \- \- \+ XII 1 18 +/H \- \- \- \+ H: hyposmia ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Hormonal profile and GnRH stimulation test in adult male patients with KS. ::: FAMILY Age Inheritance Base line LH (mIU/ml) Peak LH (mIU/ml) after GnRH testing Peak LH (mIU/ml) after priming Baseline FSH (mIU/ml) Peak FSH (mIU/ml) after GnRH testing Peak FSH (mIU/ml) after priming Testosterone ng/dl ---------- --- ----- ------------- ----------------------- ------------------------------------- -------------------------------- ----------------------- -------------------------------------- --------------------------------- -------------------- 1 1 46 XR 0.5 2.26 1.09 2.22 60 2 14 XR Undetected 0.35 2.50 0.64 2.39 4.25 Undetected 3 27 XR Undetected 0.90 2.85 0.59 1.58 5.24 Undetected 4 20 XR 0.6 2.90 1.66 3.43 80 5 20 XR 0.8 3.10 2.1 5.6 50 6 19 XR Undetected 0.40 2.88 0.8 2.3 6.4 Undetected 7 16 XR 0.6 3..50 1.35 3.21 50 II 1 37 XR Undetected 0.50 3.25 0.43 2.14 6.34 Undetected 2 24 XR ND ND ND ND ND ND ND 3 22 XR ND ND ND ND ND ND ND 4 20 XR ND ND ND ND ND ND ND V 1 20 XR Undetected 0.45 2.1 0.47 1.65 3.2 Undetected 2 19 XR 0.42 4.36 0.90 5.22 40 VI 1 19 sporadic 0.5 6.2 0.9 10.1 70 VII 1 37 sporadic Undetected 1.9 0.46 3.2 Undetected VIII 1 22 sporadic 0.53 3.25 0.85 4.50 Undetected IX 1 20 AR 0.85 4.25 1.21 6.30 50 ND: not done (lost contact) XR :X-linked recessive AR :autosomal recessive ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Hormonal profile and GnRH stimulation test in adult female patients with KS. ::: FAMILY Age Inheritance Base line LH (mIU/ml) N= Peak LH (mIU/ml) after GnRH testing Peak LH (mIU/ml) after priming Base line FSH (mIU/ml) N= Peak FSH (mIU/ml) After GnRH testing Peak FSH (mIU/ml) after priming Estradiol Pg/ml -------- --- ----- ------------- -------------------------- ------------------------------------- -------------------------------- --------------------------- -------------------------------------- --------------------------------- ----------------- X 1 23 AR 0.6 7.25 2.6 19.33 5 2 21 AR 0.5 3.5 1.95 6.60 7 3 18 AR 0.6 5.2 1.2 9.5 10 IX 1 18 sporadic 0.8 5.10 2.1 5.6 15 XI 1 30 sporadic 0.7 4.2 1.5 6.2 10 XII 1 18 sporadic Undetected 2.10 2.1 5.8 Undetected AR :autosomal recessive ::: Twenty seven patients were anosmic and five patients were hyposmic (table [1](#T1){ref-type="table"}). In family V, both affected brothers were hyposmic. Cryptorchidism was found or previously operated on in 19/26 (73%) and microphallus in 17/26 (65%) male patients respectively. All patients included in this study manifested high-arched palate. Renal abnormalities including unilateral renal agenesis, malrotated kidney, and horseshoe kidney were detected in 11/19 (58%)cases with the X-linked form of KS. Two sporadic cases showed renal anomalies. A variable degree of sensorineural hearing impairment was found in 4/19 patients with X-linked KS, and in none of the other mode of inheritance or the sporadic cases. Olfactory MRI revealed olfactory tract agenesis among 19/24 cases for which the investigation was done in the series. Among females diagnosed as KS in this series primary amenorrhea was the main presenting feature. Pedigrees were constructed for all families (figure [1](#F1){ref-type="fig"}). Five cases were sporadic and 27 cases were familial belonging to seven families. Pedigree analysis assigned an X-linked mode of inheritance to 3 families with affected males linked through normal females (families I, II and IV). Family I is the largest family in our series with 11 affected males. Two further families were designated as having the X-linked form of KS because the affected males among these siblings displayed synkinesia with a negative family history (families III and V). Synkinesia has been reported to be associated only with the X-linked form of KS \[[@B16]\]. However, a recent report identifying the specific gene mutated in autosomal dominant KS pointed out that synkinesia may occur in the autosomal forms of KS \[[@B17]\]. In families III and V in this report, pedigree analysis strongly pointed to the X-linked form of KS although autosomal recessive inheritance cannot be definitely excluded. Two families were designated as having the autosomal recessive mode of inheritance. One family included affected brother and sister with normal consanguineous parents (family IX), while the other family had 3 affected sisters with normal consanguineous parents (family X). Consanguinity rate among parents of all patients was 83%, with 50% of all marriages being between first cousins. Discussion ========== This study points to the higher proportion of the X-linked form of Kallmann syndrome among all KS cases seen at an endocrine/genetic clinic in Jordan over a period of 5 years. Among 7 families with inherited KS, the X-linked form was the mode of inheritance in 5 families (71% of familial KS). None of the pedigrees was consistent with autosomal dominant inheritance in this series. In the two families with autosomal recessive inheritance, the probability of a non-penetrant autosomal dominant gene in either parent was considered remote because of absence of any relevant family history. Autosomal dominant inheritance was also considered a very remote possibility in the X-linked families because of absence of affected individuals in earlier generations and the pattern of inheritance as revealed by pedigree construction (fig [1](#F1){ref-type="fig"}) was compatible with X-linked inheritance. The X-linked form of KS has at times been reported to account for only one third of inherited cases \[[@B1]\], and at other times to be the most frequent form \[[@B2]\]. The high proportion of the X-linked form among our cases may represent a high prevalence of Kal1 gene among Jordanians and Palestinians. Consanguineous marriages in Jordan are favored culturally. Among two thousand marriages in the general population, 32% have been reported to be between first cousins \[[@B18]\]. The figure of 50% first cousin marriages among parents of our patients would thus reflect the high consanguinity rate among the population in general. Among the XR form of KS, 75% of patients were reported to show synkinesia \[[@B9]\]. Synkinesia in X-linked KS has been attributed to an abnormal projection of the corticospinal tract \[[@B19]\]. In our experience, synkinesia was present in 16/22 (73%) patients with the XR form of KS and was characteristically more pronounced in the younger age group. Intrafamilial clinical heterogeneity has been reported among family members carrying the same mutation in Kal 1 gene \[[@B9]\]. In this series, intrafamilial variability in renal anomalies was exemplified in family I, the largest family in our series, where eight out of eleven patients had renal abnormalities. Intrafamilial variability was also seen in family III, in which one patient had a malrotated kidney, his brother had a horseshoe kidney while the third brother had no renal anomalies. Sensorineural hearing loss has also been reported to be associated mainly with the X-linked form of KS \[[@B20]\]. The KAL1 gene is expressed in the inner ear from early developmental stages suggesting that the defect underlying the hearing loss in X-linked Kallmann syndrome occurs during the organogenesis period \[[@B9]\]. In our study, sensorineural hearing impairment was only diagnosed among patients with the X linked form of Kallmann syndrome. Four of nineteen tested males (21%) showed sensorineural hearing impairment with evident intrafamilial variability. In this series cryptorchidism or a history of cryptorchidism was present in 73% of patients (19/26), and was not related to a specific mode of inheritance or etiology. Microphallus was present among 17/26 (65%) patients in this study, with several other patients reporting a history of treatment with testosterone; the exact number of treated patients or the treatment profile could not be precisely determined. Seminal fluid analysis was done for all patients of 16 years and above. The test showed azoospermia for all tested males except patient 1 in family IX, where oligospermia was reported with a sperm count of 10,000,000 per ml. None of the patients with the X-linked form manifested ichthyosis, mental retardation, short stature or ocular albinism, pointing to the underlying etiology being a mutation in the Kal 1 gene rather than a contiguous-gene deletion syndrome \[[@B21]\]. Olfactory MRI revealed olfactory tract agenesis in 80% of cases for which the investigation was done in the series (19/24). Quinton et al, 1996, indicate that KS may be present with no pathology detected in the olfactory tract on MRI, and that phenotypic characterization of KS was effectively achieved by accurate estimation of olfactory sensation \[[@B22]\]. Kallmann syndrome has a favorable prognosis under proper management. Its investigation should thus be considered in any child presenting with cryptorchidism and microphallus. Since the gonad state is still dormant in childhood, gonadotrophin levels are not helpful. Olfactory MRI may be a more useful tool for the diagnosis \[[@B23]\]. Nevertheless, a normal MRI does not rule out Kallmann syndrome as normal olfactory bulbs can be present in up to 25% of cases \[[@B22]\]. The presence of anosmia/hyposmia and history of delayed puberty or infertility in the family are helpful in establishing the diagnosis. Where diagnosis remains difficult, it is indicated to follow up these children till they reach puberty. The majority of Kallmann syndrome cases in our study showed the X-linked mode of inheritance, which might indicate a high prevalence of Kal1 gene in the population. However, molecular studies for the Kal1 gene were not performed in this study. Patients in our series manifested a wide range of phenotypic heterogeneity with intrafamilial variability of clinical manifestations. We recommend an evaluation for Kallmann syndrome in our population in any child presenting with microphallus and cryptorchidism. Further studies are needed to establish the prevalence rate of Kallmann syndrome in Jordan and to define the causative mutations. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= MAJ and KA were the main researchers. HH and MAJ drafted the manuscript. All authors were part of the team that evaluated the patients	PubMed Central	2024-06-05T03:55:49.760297	2004-10-24	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533860/", "journal": "Reprod Health. 2004 Oct 24; 1:5", "authors": [ { "first": "Mousa A", "last": "AbuJbara" }, { "first": "Hanan A", "last": "Hamamy" }, { "first": "Nadim S", "last": "Jarrah" }, { "first": "Nadima S", "last": "Shegem" }, { "first": "Kamel M", "last": "Ajlouni" } ] }
PMC533861	Introduction ============ Major depressive disorder (MDD) is a recurrent, debilitating, and potentially life threatening illness. Over the last 100 years, the age of onset of major depression has decreased, and its overall incidence has increased in Western countries. The increases in depression, up to 20-fold higher post 1945, cannot be fully explained by changes in attitudes of health professionals or society, diagnostic criteria, reporting bias, institutional or other artifacts \[[@B1],[@B2]\] Despite advances in pharmacotherapy, and the increasing sophistication of cognitive/behavioral interventions, there are many patients with MDD who remain treatment resistant \[[@B3]\]. Depression is undoubtedly an extremely complex and heterogeneous condition. This is reflected by the non-universal results obtained using cognitive-behavior and antidepressant medications. As research continues to mount, it is becoming clear that neurobiology/physiology, genetics, life stressors, and environmental factors can all contribute to vulnerability to depression. While much attention has been given to genetics and life stressors, only a small group of international researchers have focused on nutritional influences on depressive symptoms. Collectively, the results of this relatively small body of research indicate that nutritional influences on MDD are currently underestimated \[[@B4]\]. Omega-3 fatty acids in particular represent an exciting area of research, with eicosapentaenoic acid (EPA) emerging as a new potential agent in the treatment of depression \[[@B5]\]. Omega-3 fatty acids =================== Omega-3 fatty acids are long-chain, polyunsaturated fatty acids (PUFA) of plant and marine origin. Because these essential fatty acids cannot be synthesized by the human body, they must be derived from dietary sources. Flaxseed, hemp, canola and walnut oils are all generally rich sources of the parent omega-3, alpha linolenic acid (ALA). Dietary ALA can be metabolized in the liver to the longer-chain omega-3 eicosapentaenoic (EPA) and docosahexaenoic acid (DHA). This conversion is limited in human beings, it is estimated that only 5--15% of ALA is ultimately converted to DHA \[[@B6]\]. Aging, illness and stress, as well as excessive amounts of omega-6 rich oils (corn, safflower, sunflower, cottonseed) can all compromise conversion \[[@B7]\]. Dietary fish and seafood provide varying amounts of pre-formed EPA and DHA as highlighted in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Various Sources of EPA and DHA ::: Fish/Seafood Total EPA/DHA (mg/100 g) ---------------- -------------------------- Mackerel 2300 Chinook salmon 1900 Herring 1700 Anchovy 1400 Sardine 1400 Coho salmon 1200 Trout 600 Spiny lobster 500 Halibut 400 Shrimp 300 Catfish 300 Sole 200 Cod 200 USDA Nutrient Database <http://www.nal.usda.gov/fnic/foodcomp/search/> ::: The dietary intake of omega-3 fatty acids has dramatically declined in Western countries over the last century, the North American diet currently has omega-6 fats outnumbering omega-3 by a ratio of up to 20:1. There are a number of reasons for this skewed ratio, most notably the mass introduction of the aforementioned omega-6 rich oils into the food supply, either directly or through animal rearing practices \[[@B8]\]. The ideal dietary ratio of omega-6 to omega-3 has been recommended by an international panel of lipid experts to be approximately 2:1 \[[@B9]\]. Given that approximately 20% of the dry weight of the brain is made up of PUFA and that one out of every three fatty acids in the central nervous system (CNS) are PUFA, the importance of these fats cannot be argued \[[@B7]\]. Considering that highly-consumed vegetable oils have significant omega-6 to omega-3 ratios (see Table [2](#T2){ref-type="table"}), it is quite plausible that, for some individuals, inadequate intake of omega-3 fatty acids may have neuropsychiatric consequences. While far from robust at this time, emerging research suggests that omega-3 fatty acids may be of therapeutic value in the treatment of depression. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Omega-6 and Omega-3 Content (%) of Dietary Oils ::: Oil Omega-6 Omega-3 ------------ --------- --------- Safflower 75 0 Sunflower 65 0 Corn 54 0 Cottonseed 50 0 Sesame 42 0 Peanut 32 0 Soybean 51 7 Canola 20 9 Walnut 52 10 Flax 14 57 USDA Nutrient Database <http://www.nal.usda.gov/fnic/foodcomp/search/> ::: Epidemiological Data ==================== A number of epidemiological studies support a connection between dietary fish/seafood consumption and a lower prevalence of depression. Significant negative correlations have been reported between worldwide fish consumption and rates of depression \[[@B10]\]. Examination of fish/seafood consumption throughout nations has also been correlated with protection against post-partum depression \[[@B11]\], bipolar disorder \[[@B12]\] and seasonal affective disorder \[[@B13]\]. Separate research involving a random sample within a nation confirms the global findings, as frequent fish consumption in the general population is associated with a decreased risk of depression and suicidal ideation \[[@B14]\]. In addition, a cross-sectional study from New Zealand found that fish consumption is significantly associated with higher self-reported mental health status \[[@B15]\]. Not all studies support a connection between omega-3 intake and mood. A recent cross-sectional study of male smokers, using data collected between 1985 and 1988, indicated that subjects reporting anxiety or depressed mood had higher intakes of both omega-3 and omega-6 fatty acids \[[@B16]\]. In a large population-based study of older males aged 50--69, there was no association between dietary intake of omega-3 fatty acids or fish consumption and depressed mood, major depressive episodes, or suicide \[[@B17]\]. The epidemiological studies which support a connection between dietary fish and depression clearly do not prove causation. There are a number of cultural, economic and social factors which may confound the results. Most significantly, those who do consume more fish may generally have healthier lifestyle habits, including exercise and stress management. Despite the limitations, the epidemiological data certainly justify a closer examination of omega-3 fatty acids in those actually with depression. Omega-3 status in MDD ===================== There are a number of methods used to determine EFA status in the human body, notably the plasma and red blood cell (RBC) phospholipids. These are a reflection of dietary fatty acid intake within the preceding few weeks. While not identical, significant correlations exist between blood and brain phospholipids. A number of studies have found decreased omega-3 content in the blood of depressed patients \[[@B18]-[@B21]\]. Furthermore, the EPA content in RBC phospholipids is negatively correlated with the severity of depression, and the omega-6 arachidonic acid to EPA ratio positively correlates with the clinical symptoms of depression \[[@B18]\]. More recently, investigators have been utilizing adipose tissue as a longer term measurement of EFA intake (1--3 years). In a study of 150 elderly males from Crete, the parent omega-3 ALA adipose tissue stores were negatively correlated with depression \[[@B22]\]. A separate study found a negative correlation between adipose tissue DHA and rates of depression. In this case, mildly depressed adults had 34.6 percent less DHA in adipose tissue than non-depressed subjects \[[@B23]\]. Relationships between omega-3 status and post-partum depression have also been investigated. In a cohort of 380 Australian women, plasma DHA was investigated at 6 months post-partum. Logistic regression analysis indicated that a 1% increase in plasma DHA was associated with a 59% reduction in the reporting of depressive symptoms \[[@B24]\]. It is well known that during pregnancy there is a significant transfer (up to 2.2 g/day) EFAs to the developing fetus \[[@B7]\]. Increased risk of post-partum depressive symptoms has recently been associated with a slower normalization of DHA levels after pregnancy \[[@B25]\]. Suicide attempts have also been associated with low levels of RBC EPA. In a study involving 100 suicide attempt cases in China compared to 100 hospital admission controls, there was an eightfold difference in suicide attempt risk between the lowest and highest RBC EPA level quartiles \[[@B26]\]. The seasonality of depression and suicide has been described by investigators, with more deaths in spring and summer vs.autumn and winter. Total serum cholesterol has been highly significantly synchronized with the annual rhythms in violent suicide deaths \[[@B27]\]. Recently, investigators found that EFA levels also vary by season, with peaks of EPA and DHA from August to September. The parent omega-3 and 6 levels did not have a seasonal variation, suggesting a seasonal interference with delta-5-desaturase conversion. The authors of this study suggest that the seasonal variation in EPA or DHA may, in part, explain seasonality of violent suicide occurrence \[[@B28]\]. The overlap between cardiovascular disease and depression has also been noted, with omega-3 status emerging as a common thread. Indeed, major depression in acute coronary syndrome patients is associated with significantly lower plasma levels of omega-3 fatty acids, particularly DHA \[[@B29]\]. In addition, elevated homocysteine levels, a known risk factor for cardiovascular disease, has been associated with the excess omega-6 fatty acids found in the Western diet \[[@B30]\]. Finally, lowered intake of the parent omega-3 ALA has been associated with depression in 771 Japanese patients with newly diagnosed lung cancer \[[@B31]\]. It is important to note that not every study supports an association between lowered omega-3 status and depression. Two studies have actually shown significant increases in plasma and RBC omega-3 status among depressed patients \[[@B32],[@B33]\]. A recent study involving depressed adolescent patients found no significant relationship between adipose tissue EFA levels and depression \[[@B34]\]. Possible mechanisms of omega-3 EFA ================================== Detailed reviews of the possible neurobehavioral mechanisms of omega-3 fatty acids have been previously published and are beyond the scope of this review \[[@B35],[@B36]\]. The influence of omega-3 fatty acids within the CNS is far from completely understood, and our current knowledge is largely based on the consequences of omega-3 deficiency within animal models. There are two major areas of omega-3 fatty acid influence worthy of further discussion. The first is the importance of omega-3 fatty acids in neuronal membranes. Omega-3 fatty acids are an essential component of CNS membrane phospholipid acyl chains and are therefore critical to the dynamic structure and function of neuronal membranes \[[@B37]\]. Proteins are embedded in the lipid bi-layer of the cell and the conformation or quaternary structure of these proteins is sensitive to the lipid components. The proteins in the bi-layer have critical cellular functions as they act as transporters and receptors. Omega-3 fatty acids can alter membrane fluidity by displacing cholesterol from the membrane \[[@B38]\]. An optimal fluidity, influenced by EFAs, is required for neurotransmitter binding and the signaling within the cell \[[@B39]\]. EFAs can act as sources for second messengers within and between neurons \[[@B35]\]. The second area where omega-3 fatty acids may exert significant influence in major depression is via cytokine modulation. A growing body of research has documented an association between depression and the production these proinflammatory immune chemicals. These cytokines, including interleukin-1 beta (IL-1β), -2 and -6, interferon-gamma, and tumor necrosis factor alpha (TNFα), can have direct and indirect effects on the CNS. Some of the documented activities of these cytokines include lowered neurotransmitter precursor availability, activation of the hypothalamic-pituitary axis, and alterations of the metabolism of neurotransmitters and neurotransmitter mRNA \[[@B40]\]. Researchers have found elevations of IL-1β, and TNFα are associated with the severity of depression \[[@B41]\]. Psychological stress can cause an elevation of these cytokines. It is worth noting that various tricyclic and selective serotonin re-uptake inhibiting antidepressants can inhibit the release of these inflammatory cytokines \[[@B40]\]. Omega-3 fatty acids, and EPA in particular, are well documented inhibitors of proinflammatory cytokines such as IL-1 β and TNFα. In addition, it has recently been suggested that the anti-inflammatory role of omega-3 fatty acids may influence brain derived neurotrophic factor (BDNF) in depression \[[@B36]\]. BDNF is a polypeptide that supports the survival and growth of neurons through development and adulthood. Serum BDNF has been found to be negatively correlated with the severity of depressive symptoms \[[@B42]\]. Antidepressant medications and voluntary exercise can enhance BDNF, while diets high in saturated fat and sucrose, and psychological stress inhibit BDNF production \[[@B36]\]. Clinical evidence ================= The epidemiological and laboratory studies, along with the research which shows depressed patients appear to have lowered omega-3 status, have naturally led to clinical investigations. A number of case reports have appeared in the literature, the first of which was over 20 years ago. In this initial series of case reports, flaxseed oil (source of the parent omega-3 ALA) at various dosages, was reported to improve the symptoms of bipolar depression and agoraphobia \[[@B43]\]. An additional case report documented an improvement in depressive symptoms during pregnancy with the use of 4 g EPA/2 g DHA per day. Interestingly, improvements in symptoms (measured via the Hamilton Rating Scale for depression -- HRDS) occurred at four weeks, and with the exception of insomnia and anxious thoughts, all symptoms resolved at six weeks \[[@B44]\]. Despite the interesting results, there are major scientific problems with case reports, most notably the placebo response. A recently published case report published took advantage of modern brain imaging to corroborate clinical improvements. In this case a patient with treatment resistant depression was placed on a daily dose of 4 g pure EPA, and after one month there were significant improvements, including a co-morbid social phobia. After nine months the patient was reportedly symptom free. It was found that over the course of the nine months of treatment, there was a 53 percent increase in cerebral phosphomonoesters and the ratio of cerebral phosphomonoesters to phosphodiesters increased 79 percent, indicating reduced neuronal phospholipid turnover. Utilizing MRI technology, the researchers found that the EPA treatment was associated with structural brain changes, including a reduction in lateral ventricular volume. This is likely to be a result of increased phospholipid biosynthesis and reduced phospholipid breakdown \[[@B45]\]. Given the recent research indicating a decrease in volume in various areas of the brain of depressed patients, this is certainly an important case study \[[@B46]\]. A series of case reports also suggest that 1 -- 4 g of pure EPA may be helpful in anorexia nervosa, a condition with the highest risk of morbidity and mortality among psychiatric disorders \[[@B47]\]. In all six of the cases, EPA was reported to improve mood to varying degrees. For some, discontinuing EPA therapy resulted in deteriorations in mood and other psychiatric symptoms. An interesting study examined fish oil vs.marine oil extracted from Antarctic krill in premenstrual syndrome. Krill is similar to fish oil, with the exception that it contains naturally-occurring phospholipids, and contains more EPA per gram than standard fish oil capsules (240 mg/g EPA in krill vs.180 mg/g in standard fish oil). In the 3-month trial, patients (n = 70) received 2 g of krill oil or 2 g fish oil daily for one month, then for eight days prior to, and two days during, menstruation for the following two months. Evaluation at 45 days and three months showed that krill oil significantly improved depressive symptoms of premenstrual syndrome. The absence of significant effects of fish oil on mood suggests that the presence of the phospholipids and/or higher amounts of EPA may be responsible for the therapeutic effect of krill oil \[[@B48]\]. There have been some controlled studies that have examined omega-3 fatty acids and a placebo intervention in depression. The first small clinical study (n = 30) showed that four months of treatment with 9.6 g of omega-3 fatty acids (6.2 g EPA/3.4 g DHA) was of therapeutic value in bipolar disorder. Specifically, this study showed a highly significant effect in treating depression (p \< 0.001 HRSD scores) \[[@B49]\]. In a separate double-blind, placebo-controlled study (n = 22), the addition of 2 g of pure EPA to standard antidepressant medication enhanced the effectiveness of that medication vs.medication and placebo. This 3-week study, involving patients with treatment-resistant depression, showed that EPA had an effect on insomnia, depressed mood, and feelings of guilt and worthlessness. There were no clinically relevant side effects noticed \[[@B50]\]. In a small pilot study (n = 30), Harvard researchers found that just 1 g of EPA could reduce aggression (modified Overt Aggression Scale) and depressive symptom scores (Montgomery-Asberg Depression Rating Scale) among borderline personality disorder patients. The results of this 2-month, placebo-controlled study are encouraging, given the difficulty in treating borderline personality disorder. It is also of note that 90 percent of participants remained in the study and no clinically relevant side effects were noticed with EPA \[[@B51]\]. In a double-blind, placebo-controlled trial over two months, high dose fish oil (9.6 g/day) was added to standard antidepressant therapy in 28 patients with MDD. In this study the patients who received the omega-3 fish oil capsules had a significantly decreased score on the HRSD compared to those taking the placebo. Once again, the fish oil, even at this high dose, was well tolerated with no adverse events reported \[[@B52]\]. Various doses of pure EPA have also been investigated in depression. In a 12-week, randomized, double-blind, placebo-controlled study, patients (n = 70) were given ethyl-EPA at doses of 1 g, 2 g or 4 g. The patients in this case had experienced persistent depression, despite ongoing standard antidepressant pharmacotherapy at adequate does. Interestingly, in this study, \"less was more.\" Those in the 1 g per day group had the best outcome. The patients who received 1 g per day of EPA were the only group to show statistically significant improvements. Among the 1 g/day group, 53 percent achieved a 50 percent reduction in HRSD scores. The 1 g EPA led to improvements in depression, anxiety, sleep, lassitude, libido, and suicidal ideation. These findings suggest that omega-3 fatty acids can augment antidepressant pharmacotherapy and/or alleviate depression by entirely different means than standard medications \[[@B53]\]. A large study examining the effects of omega-3 or placebo added to cognitive-behavior therapy would be of interest. To date, the published data on supplementation with pure EPA on MDD or depressive symptoms have been positive. With regard to DHA or a combination of EPA and DHA, there have been three negative reports. A trial on DHA alone as monotherapy in the treatment of MDD was recently reported. In this study, 2 g pure DHA or placebo was administered to 36 patients with depression for six weeks. The response differences between the groups, as measured by scores on the Montgomery-Asberg Depression Rating Scale did not reach statistical significance \[[@B54]\]. In an open label pilot study, the combination of 1.7 g of EPA and 1.2 g of DHA failed to show benefits among seven women with a past history of post-partum depression. The omega-3 monotherapy was initiated between the 34^th^-- 36^th^week of pregnancy and was assessed through 12 weeks post-partum. In these women the fish oil combination did not reduce the risk of relapse \[[@B55]\]. Finally, a pure DHA supplement, at low doses of 200 mg per day for 4 months post-partum, did not improve self-rated or diagnostic measures of depression over placebo. However, the women enrolled (n = 89) in this study were not clinically depressed as a group, which precludes interpretation that DHA is ineffective in post-partum depression \[[@B56]\]. Other dietary considerations ============================ It is important to consider the nutrients which can ultimately influence omega-3 status. Among them, four important dietary factors also relate to MDD: zinc, selenium, folic acid and dietary antioxidants. A number of studies have shown that zinc levels are lower among patients with depression and a recent study found that 25 mg zinc supplementation may improve depressive symptoms \[[@B57]\]. Interestingly, 25 mg of zinc supplemented for two months has also been shown to significantly increase omega-3 status in the plasma phospholipids at the expense of saturated fat \[[@B58]\]. Lowered levels of selenium have been associated with negative mood scores in at least 5 studies \[[@B59]\]. Selenium plays a significant role in the human antioxidant defense system. In addition, selenium deficiency can interfere with the normal conversion of ALA into EPA and DHA, and results in an increase in the omega-6:omega-3 ratio \[[@B60]\]. Regarding folic acid, a growing body of research has documented the low levels of folic acid among patients with depression \[[@B61]\]. In addition, there are small clinical trials showing a beneficial effect of folic acid in depression, and its ability to enhance the effectiveness of antidepressant medications at just 500 mcg \[[@B61],[@B62]\]. It is of relevance here because folic acid has been shown to increase omega-3 status when supplemented, and decrease omega-3 status when it is in deficiency in the animal model \[[@B63]\]. In addition, a folic acid deficient diet can enhance lipid peroxidation \[[@B64]\]. In patients with MDD there are in fact signs of oxidative stress and lipid peroxidation, and antidepressant medications may reverse the severity of oxidative stress in depressed patients \[[@B65]\]. A recent human study found that depressive symptoms are independently correlated with lipid peroxidation \[[@B66]\]. Patients with obsessive compulsive disorder (OCD) and co-morbid depression have higher levels of lipid peroxidation than those with OCD alone \[[@B67]\]. Dietary antioxidants are known to influence the antioxidant defense system, and new research suggests that dietary antioxidants can influence omega-3 status. Specifically, a diet devoid of antioxidants lowered essential fatty acid levels in the plasma of trained athletes, even though the amount and types of fats were not altered \[[@B68]\]. Omega-3 fatty acids have been shown to decrease lipid peroxidation in vivo \[[@B69]\], and antioxidant supplementation can prevent the negative influence of saturated fat on BDNF levels and cognitive function in animals \[[@B70]\]. Conclusion ========== While far from robust, there is enough epidemiological, laboratory and clinical evidence to suggest that omega-3 fatty acids may play a role in certain cases of depression. Fish oil supplements are well tolerated, and have been shown to be without significant side effects over large scale, 3-year research \[[@B71]\]. Generally, omega-3 supplements are inexpensive, which makes them an attractive option as an adjuvant to standard care. At this time, however, the routine use of omega-3 fatty acids for the treatment of MDD cannot be recommended. The research reviewed here shows that the data is far from unequivocal. Large trials are warranted to truly determine efficacy, appropriate dosing and the potentially active components -- EPA, DHA, or both. It is also clear that omega-3 intake occurs in dietary context, one that includes other important nutrients. Future research should consider the influence of zinc, selenium, folic acid and dietary antioxidant status to determine who may be a successful candidate for omega-3 supplementation. In the meantime, given the current excess intake of omega-6 rich oils, and the emerging research on omega-3 fatty acids and MDD, all mental health professionals should at least ensure adequate intake of omega-3 fatty acids among patients with MDD. The current average North American intake of EPA and DHA is approximately 130 mg per day, well short of the minimum 650 mg recommended by the international panel of lipid experts \[[@B6]\]. While it is not necessary for mental health professionals to become clinical nutritionists, consideration of a patient\'s dietary quality may be worthwhile. Hopefully future research will determine if dietary modifications or supplementation can influence the outcome of standard care.	PubMed Central	2024-06-05T03:55:49.763695	2004-11-9	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533861/", "journal": "Lipids Health Dis. 2004 Nov 9; 3:25", "authors": [ { "first": "Alan C", "last": "Logan" } ] }
PMC533862	Introduction ============ The process of fertilization is characterized by a series of complex set of events. It involves a species-specific interaction between egg and sperm activating a chain of events that leads to formation of zygote, fetus and finally a baby. However, before a spermatozoon can fertilize an oocyte, it must undergo a cascade of biochemical and physiological changes that facilitates its binding and penetration into the oocyte \[[@B1],[@B2]\]. This time-dependent acquisition of fertilizing competence has been defined as \"Capacitation\" \[[@B3],[@B4]\]. Capacitation confers upon the spermatozoon an ability to gain hyperactive motility, interact with oocyte zona pellucida (ZP), undergo acrosome reaction and initiate oocyte plasma membrane fusion \[[@B1]\]. Capacitation normally occurs in the female genital tract, however, it can also be achieved in vitro. In fact, most of the information about various aspects of sperm capacitation has emanated from in vitrostudies. Sperm cells can be capacitated in vitroby using chemically defined media containing appropriate concentrations of electrolytes, metabolic energy sources, and serum albumin (cholesterol acceptor). Although minor variations exist between these media depending on the mammalian species, most of these media contain bicarbonate, calcium and a macromolecule predominantly serum albumin. Although capacitation of a sperm cell is required before fertilization virtually in every mammalian species studied, the molecular mechanisms and signal transduction pathways involved in this process are not clearly understood. Capacitation involves an increase in membrane fluidity, cholesterol efflux, ion fluxes resulting in alteration of sperm membrane potential, increased tyrosine phosphorylation of proteins, induction of hyperactivation and the acrosome reaction. Protein phosphorylation represents a very important aspect of capacitation. Recently, many advances have been made in the study of phosphorylation of proteins during capacitation, which we will review in this article. Protein phoshphorylation ======================== Phosphorylation of proteins is a post-translational modification event that acts as one of the cell\'s regulatory mechanisms to control various processes such as cellular growth, cell cycle control, cytoskeleton assembly, ionic current modulation, and receptor regulation \[[@B5],[@B6]\]. In fact in eukaryotic cells, one of the most common mechanisms for regulating protein activity is the addition and/or removal of phosphate groups from serine, threonine, or tyrosine residues of protein moieties. Addition or removal of phosphate groups can induce allosteric modifications resulting in conformational changes in proteins leading either to their activation or inactivation. Mature spermatozoa are terminally differentiated and specialized cells. They are highly compartmentalized but are devoid of any major transcriptional and translational activity. Therefore one can justify the importance of post-translational modifications such as protein phosphorylation/dephosphorylation in regulating important phenomena such as sperm capacitation, hyperactive motility and acrosome reaction, which are required for the spermatozoon to reach, bind, penetrate and fuse with the oocyte. The phosphorylation/dephosphorylation state of phosphoproteins is controlled by the activity of protein kinases and phosphatases, and the counteracting activities of these kinases and phosphatases provide cells with a \"switch\" that can turn on or turn off the function of various proteins. Earlier studies reported the presence of various phosphoproteins, protein kinases and protein phosphatases in mammalian spermatozoa and implicated their role in sperm motility acquisition, capacitation and acrosome reaction \[[@B7],[@B8]\]. Phosphorylation can occur at serine, threonine, and tyrosine residues in proteins. Although both serine/threonine phosphorylation and tyrosine phosphorylation of proteins have been reported in spermatozoa (discussed below), the tyrosine phosphorylation is very important and may be the primary or even the exclusive indicator of a signal transduction pathway in a cell. Protein tyrosine phosphorylation in spermatozoa =============================================== 1.Tyrosine phophorylated proteins --------------------------------- Historically, in 1989, Leyton and Saling provided the first evidence for the presence of tyrosine phosphorylation in mammalian spermatozoa namely the mouse sperm \[[@B9]\]. Using anti-phosphotyrosine antibody they identified three proteins of 52, 75, and 95 kDa respectively, in mouse sperm. The 95 kDa protein showed enhancement in immunoreactivity with the antibody after sperm capacitation and interaction with oocyte ZP proteins \[[@B9]\]. In 1991, the second study by Naz and associates identified tyrosine phosphorylation in sperm of several mammalian species including human, rat, rabbit, and mouse. They reported four sets of tyrosine phosphorylated proteins in the molecular weight range of 95 kDa/94 ± 3 kDa (FA-2 antigen), 46 ± 3 kDa, 25 ± 7 kDa and 12 ± 2 kDa, respectively, in human sperm \[[@B10]\] and also identified a protein of molecular identity of 94 ± 3 kDa in mouse sperm, that was reported earlier by Leyton and Saling \[[@B9]\]. However, this protein of 94 ± 3 kDa was not identified in rat and rabbit sperm. Although it needs to be confirmed using molecular cloning and sequencing studies, it seems that 94 ± 3 kDa is not an evolutionarily conserved protein. Using ^32^P metabolic labeling and in vitrokinase assays, human sperm was found to have at least seven proteins (200, 112, 104, 48, 42, 31 and 25 kDa) that are phosphorylated and fourteen proteins (122, 105, 95, 89, 73, 62, 48, 46, 40, 33, 30, 28, 25 and 22 kDa) that are autophosphorylated \[[@B11]\]. Further studies showed that the 94 ± 3 kDa and 46 ± 3 kDa proteins are also phosphorylated at ser/thr residues besides phosphorylation at tyrosine residues \[[@B12]\]. The 46 ± 3 kDa protein was found out to be the FA-1 antigen, which has been known to play an important role in sperm-ZP binding \[[@B13]\]. FA-1 antigen also plays an important role in capacitation \[[@B14],[@B15]\]. Treatment of human spermatozoa with an anti-FA-1 monoclonal antibody (mAb) during capacitation reduces tyrosine phosphorylation of both 94 ± 3 kDa and 46 ± 3 kDa (FA-1 antigen) proteins, which indicates cross-talk between these two proteins \[[@B11],[@B15]\]. It has been observed that different compartments of human spermatozoa undergo a specific sequence of phosphorylation during capacitation and upon binding to zona pellucida \[[@B16]\]. In order to establish the link between the different phosphorylated proteins and a specific sperm function, it is necessary to differentially localize the tyrosine phosphorylated proteins in various regions of spermatozoon. The flagellum seems to be the major component of sperm cell that undergoes tyrosine phosphorylation in most species except boar \[[@B17]\]. Immunocytochemistry has been used to localize tyrosine phosphorylated proteins in flagellum of human \[[@B10],[@B18],[@B19]\], monkey \[[@B20]\], hamster \[[@B21]\], rat \[[@B22]\], and mouse \[[@B23]\] spermatozoa. In a study using immunofluorescence, Urner et al\[[@B23]\] localized the phosphotyrosine proteins in the flagellum during capacitation, zona pellucida binding and gamete fusion in mouse sperm. They observed that during capacitation there is an increase in the proportion of spermatozoa with phosphorylated proteins in the whole flagellum \[[@B23]\]. The increase in the phosphorylation in the principal-piece precedes that in the mid-piece, and phosphorylation in the principal- piece is the pre-requisite for phosphorylation in the mid-piece region. Upon binding to the zona pellucida, nearly all mouse sperm became progressively phosphorylated in both the principal-piece and the mid-piece regions \[[@B23]\]. A significant increase in phosphorylation with capacitation has also been observed in human spermatozoa but it is localized mainly in the principal piece \[[@B18],[@B19]\]. A study from our laboratory using human sperm revealed that the capacitating conditions and zona exposure increases the degree of tyrosine phosphorylation per sperm cell as well as the number of sperm cells that were phosphorylated, especially in the acrosomal regions of the sperm head \[[@B10]\]. Interestingly, with these changes, there was also a shift in the site of phosphotyrosine-specific fluorescence from the tail regions of non-capacitated sperm to the acrosomal regions of capacitated/zona-exposed sperm cells. In other cellular systems, there are reports indicating a shift in subcellular localization of various proteins after phosphorylation. In human epidermoid carcinoma A431 cells, it has been shown that the binding of epidermal growth factor (EGF) to its receptor rapidly triggers redistribution of phospholipase C-r~1~from a predominantly cytosolic localization to the membrane-bound activity, followed by phosphorylation at tyrosine residues. Since the acrosomal region of the sperm cell is involved in sperm-zona interaction, the shift in phosphotyrosine-specific fluorescence seems to have a physiological significance \[[@B10]\]. Tyrosine phosphorylation of the sperm flagellar proteins has shown to be related to the acquisition of the hyperactive motility \[[@B20],[@B24]\], which is required for the spermatozoa to penetrate the cumulus and the zona pellucida of the oocyte. In human spermatozoa, the protein A-kinase anchoring proteins (AKAPs) localized on the fibrous sheath, namely AKAP82, its precursor pro-AKAP82, and FSP95 are the most prominent tyrosine phosphorylated proteins during capacitation \[[@B18],[@B25]\]. In hamster sperm, the homolog of mouse AKAP4 has been identified as the major tyrosine phosphorylated protein in the capacitated spermatozoa \[[@B26]\]. In contrast, in the mouse sperm, AKAP4 is phosphorylated at ser/thr residues not at tyrosine residues. So there are species-specific variations in the pattern of tyrosine phosphorylation even for the same protein. Another protein that gets tyrosine phosphorylated during capacitation is CABYR (calcium-binding and tyrosine phosphorylation-regulated protein). It has been localized on the principal- piece of human spermatozoa \[[@B27]\]. It is speculated that the CABYR is involved in cross-talk between tyrosine phosphorylation and Ca^2+^in the signal transduction pathway. A 55 kDa tyrosine phosphorylated protein has been linked to motility in bovine spermatozoa \[[@B28]\]. During capacitation of mouse spermatozoa, heat shock protein (HSP)-90, a highly evolutionary conserved molecular chaperone protein, becomes tyrosine phosphorylated \[[@B29]\]. HSP-90 is also tyrosine phosphorylated in human and rat spermatozoa when incubated under conditions that induce capacitation \[[@B29]\]. In boar spermatozoa, capacitation induces tyrosine phosphorylation of plasma membrane proteins, which are believed to initiate binding to the zona pellucida and induce acrosome reaction \[[@B30]\]. It has been shown that capacitation induces tyrosine phosphorylation of three major (27 kDa, 37 kDa and 40 kDa) and three minor (34 kDa, 47 kDa and 55 kDa) plasma membrane proteins \[[@B30]\]. In a later study, two plasma membrane proteins isolated from capacitated boar sperm cells (35 kDa and 46 kDa) showed high binding affinity with zona pellucida \[[@B31]\]. These two proteins are most likely the 34 and 47 kDa proteins identified earlier. Although, phosphorylation of sperm proteins is a key feature of capacitation, it is not clear how tyrosine phosphorylation of these proteins is involved in sperm-zona recognition or interaction, and acrosomal exocytosis. However in a recent study, Asquith et al\[[@B32]\] have examined the relationship between protein phosphorylation and the ability of mouse spermatozoa to interact with zona pellucida. They have identified two chaperone proteins namely endoplasmin (erp99) and heat shock protein 60 (hsp60) expressed on the surface of mouse spermatozoa. Both erp99 and hsp60 proteins are tyrosine phosphorylated and are localized on the plasma membrane of sperm head, the region that participates in zona binding. They proposed that \"activation\" of erp99 and hsp60 proteins by tyrosine phosphorylation during capacitation may trigger conformational changes facilitating the formation of a functional zona pellucida receptor complex on the surface of spermatozoa \[[@B32]\]. On a similar line, two sperm proteins namely, ERK-1 and ERK-2 (extracellular signal-regulated kinases) have been identified to be tyrosine phosphorylated and \"activated\" during capacitation in human spermatozoa \[[@B33]\]. It is interesting to know that sperm-oviductal epithelial cell interaction in vitromodifies both the sperm tyrosine phosphorylation and capacitation. The selective sperm binding to oviductal epithelial cells \[[@B34]\] suppresses tyrosine phosphorylation of sperm proteins in boar \[[@B17]\] and canine \[[@B35]\] spermatozoa delaying capacitation. This oviductal modulation of tyrosine phosphorulation/capacitation may help to synchronize sperm function with the time of ovulation. In order to understand the molecular basis underlying capacitation, it is very important to characterize phosphoproteins involved in the signal transduction pathways. Although a large number of proteins have been reported to be tyrosine phosphorylated, very few have been characterized so far. Most of the studies at the beginning used specific inhibitors and/or phospho-specific antibodies to delineate phosphoproteins in sperm cell. Mass-spectrometric analysis provides another powerful approach to identify and characterize these phosphorylated proteins. Ficarro et al\[[@B36]\] used this approach to identify proteins phosphorylated during capacitation of human sperm. They also investigated the phosphorylation sites in these proteins. They are able to map more than 60 phosphorylated sequences in sperm cell. They also provided evidence for tyrosine phosphorylation of two proteins namely, valosin-containing protein (VCP) and AKAP3 (sperm tail protein) during capacitation \[[@B36]\]. Also, the gene knockout technology is very helpful in delineating phosphoproteins that have a role in the tyrosine phosphorylation cascade. The studies have shown that the targeted disruption of the Akap4gene causes defects in sperm flagellum and motility. In the mice lacking AKAP4 protein, that undergoes phosphorylation during capacitation, sperm numbers were not reduced but the spermatozoa failed to show progressive motility, rendering the male mice infertile \[[@B37]\]. The mice showed defect in fibrous sheath formation indicating that the AKAP4 is a scaffold protein required for the organization and integrity of the fibrous sheath. The sperm motility is lost in the absence of AKAP4 protein because signal transduction and glycolytic enzymes do not associate with the fibrous sheath \[[@B37]\]. It would be interesting to examine the gene knockouts of other proteins to delineate the missing links in signal transduction pathways involved in capacitation/acrosomal exocytosis. 2.Molecular mechanisms of signal transduction in sperm capacitation / acrosome reaction --------------------------------------------------------------------------------------- Several studies have correlated the degree of tyrosine phosphorylation with the capacitative state of spermatozoa. Visconti et al\[[@B38],[@B39]\] examined the correlation between the capacitative state and protein tyrosine phosphorylation in mouse spermatozoa. They observed a time-dependent increase in the protein tyrosine phosphorylation of a set of specific proteins in the molecular range of 40--120 kDa, which was correlated with the capacitation state of spermatozoa \[[@B38]\]. Later studies reported that the protein tyrosine phosphorylation increases in spermatozoa during capacitation in various species, including hamsters \[[@B40],[@B41]\], cats \[[@B42]\], pigs \[[@B43]\], boar \[[@B44]\], bovine \[[@B45],[@B46]\], equine \[[@B47]\], cynomolgus monkey \[[@B20]\], tammar wallaby and brushtail possum (marsupial species) \[[@B48]\] and human \[[@B49],[@B50]\]. All these studies provide evidence that the protein tyrosine phosphorylation is an important regulatory pathway in modulating the events associated with capacitation. The increase in protein tyrosine phosphorylation during capacitation has been shown to be regulated by a cAMP-dependent pathway involving protein kinase A (PKA) in sperm of various species including mouse \[[@B39]\], hamster \[[@B40]\], boar \[[@B44]\], bovine \[[@B45],[@B46]\], equine \[[@B47]\], cynomolgus monkey \[[@B20]\] and human \[[@B49],[@B50]\]. cAMP is a ubiquitous and \"central\" second messenger in all cell types. It may target cyclic nucleotide gated ion-channels, cAMP-activated guanine nucleotide exchange protein, and PKA in different signaling pathways. In sperm, cAMP has been shown to activate PKA, which regulates protein tyrosine phosphorylation. This signaling pathway is unique to sperm. It has been observed that addition of H89, a protein kinase A inhibitor, during capacitation reduces/blocks and addition of cell permeable analog of cAMP, dibutyryl cAMP, increases tyrosine phosphorylation of sperm proteins \[[@B51]\]. PKA is a tetrameric enzyme composed of two regulatory and two catalytic subunits. The activity of PKA is dependent on the activities of adenylate cyclase and phosphodiesterase. In sperm cell, PKA is compartmentalized thus ensuring specificity of function through binding of its regulatory subunit to the AKAP family of proteins. Different types of AKAPs have been characterized from sperm of different species \[[@B18],[@B52],[@B53]\]. PKA although a serine/threonine kinase, induces and causes an increase in tyrosine phosphorylation of sperm proteins, by indirect activation of tyrosine kinases \[[@B49]\]. Such kinases must be highly specific to spermatozoa, since cAMP-dependent tyrosine phosphorylation has not been reported in any other cell type examined to date. Tyrosine kinases can be divided into two classes namely the receptor tyrosine kinases (RTKs) and non-receptor protein tyrosine kinases (PTKs). RTKs are transmembrane proteins having an extracellular ligand binding domain and an intracellular tyrosine kinase domain. The localization of phospholipase C-γ(PLCγ) on the plasma membrane and its tyrosine phosphorylation-dependent activation in mouse spermatozoa \[[@B54]\] and phosphoinositide 3 kinase (PI-3 kinase) activity operating downstream of tyrosine phosphorylation in human spermatozoa \[[@B55]\], indirectly indicate the presence of RTKs. Upon extracellular ligand binding, a RTK is activated and then phosphorylate itself (autophosphorylation) or other proteins. In contrast, PTKs are located in the cytoplasm, nucleus or anchored to the inner leaflet of the plasma membrane. Presence of various tyrosine kinases (RTKs and PTKs) has been demonstrated in spermatozoa of several mammalian species. These include c-ras in human sperm cells \[[@B56]\], EGF receptor tyrosine kinase in human, mouse, rabbit and rat sperm cells \[[@B57]\] and bovine sperm \[[@B58]\], c-Abl in human sperm cells \[[@B59]\] and p190 c-met tyrosine kinase in human sperm cells \[[@B60]\]. Recently another tyrosine kinase TK-32 have been identified in pig spermatozoa and it has been demonstrated that activation of TK-32 occur concomitant with capacitation \[[@B61]\]. Insulin-like growth factor I (IGF-I) receptor has been identified in human \[[@B62]\], and bovine \[[@B63]\] sperm. IGF-1 receptor has tyrosine kinase activity and its ligand IGF-1 is present in the seminal plasma. The IGF-1 system (IGF-1/IGF-1R and IGF-1 binding protein) may be involved in the signal transduction pathway leading to sperm capacitation and acrosome exocytosis \[[@B62]\]. The src-related protein tyrosine kinase, c-yes(cellular-yamaguchi sarcoma viral oncogene) has been detected in the human sperm head \[[@B64]\]. The c-yesbelongs to PTK family. The activity of the c-yeskinase depends on cAMP, indicating again that tyrosine phosphorylation of proteins in sperm cell is a result of the cross-talk between the cAMP pathway and tyrosine kinase(s). The protein kinase C (PKC) is also present in mammalian spermatozoa and its role has been implicated in sperm motility and acrosome reaction \[[@B65]\], but its function in capacitation is poorly understood. ### a. PKA pathway The sperm proteins also undergo phosphorylation at ser/thr residues. Studies have shown capacitation-associated increase in phosphorylated ser/thr residues in human \[[@B12]\] and hamster \[[@B66]\] spermatozoa. Besides PKA (a major ser/thr kinase), the extracellular signal-regulated protein kinase (ERK1/2), which is also a ser/thr kinase, is present in sperm cell \[[@B67]\]. Earlier studies using anti-phosphoserine and anti-phosphothreonine antibodies indicated an increase in ser/thr phosphorylation of 18, 35, 43, 55, 94, 110 and 190 kDa proteins during capacitation of human sperm \[[@B12]\]. A recent study used antibody against the Arg-X-X- (Phospho-ser/thr) motif to study the PKA-dependent ser/thr phosphorylation in capacitating human sperm cell in combination with the agents that stimulates (dibutyryl cAMP, dbcAMP and 3-isobutyl-1-methylxanthine, IBMX) or inhibit (H89 and Rp-adenosine-3\', 5\'-cyclic monophosphorothionate, Rp-cAMPS) PKA \[[@B68]\]. A significant increase in phosphorylation of proteins designated as P80 and P105, respectively, was observed. This study showed for the first time that the phosphorylation of Arg-X-X- (ser/thr) motif that is characteristic of PKA substrates increases during capacitation. These ser/thr-phosphorylated proteins could provide a link between the early increase in PKA activity that is followed by protein tyrosine phosphorylation. In another report, two 36 kDa proteins (36 k-A and 36 k-B) were phosphorylated in a cAMP-dependant manner at serine residues, in hamster sperm flagella during an increase in the hyperactivated motility \[[@B69]\]. A ser/thr protein kinase, ecto-cyclic AMP-independent protein kinase (ecto-CIK) has been localized on the outer surface of mature goat spermatozoa \[[@B70]\]. Its substrate MPS (major physiological substrate) has been characterized and localized in sperm cell and is speculated to play an important role in sperm-egg interaction in this species \[[@B70]\]. ### b. MAP Kinase Pathway There is evidence that the mitogen-activated protein kinases (MAPKs), also known as extracellular signal-regulated kinases (ERKs), are present in spermatozoa and are involved in sperm motility and capacitation \[[@B56],[@B67]\] and acrosome reaction \[[@B71],[@B72]\]. The MAP kinases are ser/thr kinases that are involved in signal transduction pathways in several cell types. Their activity is regulated by a cascade of events, which starts with the activation of p21 Ras that stimulates a ser/thr kinase Raf (MAPK kinase). Raf phosphorylates and activates MEK. MEK (MAPK kinase), which is a dual specificity kinase, phosphorylates ERK1 and ERK2 (p44/p42 MAPK, respectively). The ERK pathway has been shown to be present in fowl \[[@B73]\] and human spermatozoa \[[@B71]\]. In fowl sperm, it is involved in the phosphorylation of axonemal and/or accessory cytoskeletal proteins and in the regulation of flagellar motility \[[@B73]\]. In human sperm, progesterone stimulates the ERK2 (p42ERK) and thus this kinase may have a role in capacitation and acrosomal exocytosis \[[@B71]\]. The MAPK isoform ERK2 \[[@B71]\], the adaptor protein Shc \[[@B74]\] and Ras \[[@B56]\] have been localized in human sperm head indicating that this pathway may be required for regulating protein phosphorylation in sperm. It has been speculated that MAP kinase may phosphorylate proteins that influence protein tyrosine phosphorylation indirectly. Inhibition of MAPK blocks protein tyrosine phosphorylation associated with capacitation. ### c. Factors That Affect Tyrosine Phosphorylation during Sperm Capacitation #### a. Cholesterol The lipid composition of sperm plasma membrane is different from that of somatic cell plasma membrane. The plasma membrane of sperm head has high amounts of cholesterol, which regulates the membrane fluidity and plays an important role of capacitation \[[@B75]\]. The cholesterol from sperm plasma membrane is transferred to high molecular weight proteins such as albumin and high-density lipoproteins present in the oviductal fluid as the sperm cell traverses through the female genital tract. Cholesterol efflux from sperm plasma membrane is associated with the activation of membrane signal transduction pathways related to capacitation \[[@B76]\]. β-Cyclodextrins (cyclic heptasaccharides) promote cholesterol efflux from mouse sperm plasma membrane that results in an increase in capacitation and protein tyrosine phosphorylation through the cAMP/PKA pathway \[[@B76]\]. cAMP analogues can induce protein tyrosine phosphorylation in the absence of bovine serum albumin (BSA) and can also overcome the inhibitory effect of cholesterol-3-sulphate in medium containing BSA. Rp-cAMPS, an inhibitor of PKA, decreases the degree of tyrosine phosphorylation induced by either BSA or cyclodextrins \[[@B50]\]. These findings strongly indicate an important role of cholesterol efflux in tyrosine phosphorylation, mediated through the cAMP/PKA pathway. Cholesterol efflux can also have an indirect influence on the signaling pathways. An increase in membrane fluidity after Ca^2+^efflux can increase the permeability of sperm cell plasma membrane to various ions such as Ca^2+^and HCO~3~^-^, which can then affect the downstream signaling molecules. #### b. Calcium Ca^2+^influx is one of the crucial biochemical events that occur during capacitation. There is abundant evidence to support requirement of Ca^2+^for capacitation \[[@B77],[@B78]\] and induction of acrosome reaction \[[@B79]\]. It has been demonstrated that there is an increase in concentration of Ca^2+^in sperm cell during capacitation in several mammalian species \[[@B80]-[@B85]\]. It has been observed that different species have different requirements for Ca^2+^during capacitation. There is also evidence that different aspects of human sperm function such as capacitation, acrosome reaction and ZP binding in vitrohas distinctive Ca^2+^requirements. Experiments have shown that in humans 0.22 mM of Ca^2+^ions are needed for capacitation while ≥ 0.58 mM is required for AR and ZP binding \[[@B86]\]. Although micromolar concentration of extracellular Ca^2+^is needed to achieve sperm capacitation in the mouse \[[@B77]\], millimolar concentration is required for capacitation in man \[[@B78],[@B87]\]. Contrasting reports exist on the impact of extracellular Ca^2+^on tyrosine phosphorylation in spermatozoa. The reports on mouse \[[@B38]\] and human spermatozoa \[[@B88]\] have documented that increasing amounts of extracellular Ca^2+^increase tyrosine phosphorylation. In contrast, other studies have demonstrated the opposite effect \[[@B18],[@B89]\], indicating that Ca^2+^negatively regulates tyrosine phosphorylation during sperm capacitation. A recent study was conducted by Baker et al\[[@B90]\] to investigate the impact of extracellular Ca^2+^on tyrosine phosphorylation of human and mouse spermatozoa and delineate the mechanisms by which this cation exerts its regulatory effect. They reported that Ca^2+^suppresses tyrosine phosphorylation by decreasing the availability of intracellular ATP \[[@B90]\]. Since there is an increase in the intracellular concentration of Ca^2+^during capacitation of sperm of several mammalian species \[[@B33]\], it could be speculated that such changes modulate protein tyrosine phosphorylation to regulate capacitation and acrosomal exocytosis. #### c. Bicorbonate (HCO~3~^-^) The requirement of for HCO~3~^-^in capacitation is well elucidated \[[@B91],[@B92]\]. The exact mechanism by which HCO~3~^-^regulates capacitation is not clear. However, the HCO~3~^-^influx has been associated with an increase in intracellular pH observed during capacitation, regulation of cAMP levels, reversible change in the lipid architecture of plasma membrane, and hyperpolarization of sperm plasma membrane. The presence of a Na^+^/HCO~3~^-^cotransporter has been demonstrated in the mouse sperm and it plays a significant role in capacitation \[[@B93]\]. An increase in intracellular pH during capacitation is attributed to HCO~3~^-^influx \[[@B94]\]. However, the role of pH is not clear since an increase in pH in sperm cell does not induce capacitation \[[@B95]\]. HCO~3~^-^may be involved in stimulation of adenylate cyclase activity rather than modulation of pH \[[@B39],[@B96]\]. HCO~3~^-^induces rapid changes in plasma membrane lipid architecture and in motility by a cAMP/PKA-dependent pathway in boar spermatozoa \[[@B97]\]. Harrison \[[@B97]\] and Da Ros et al\[[@B98]\] have shown the role of HCO~3~^-^in protein tyrosine phosphorylation in sperm during capacitation and fertilization. It is interesting that HCO~3~^-^levels are low in epididymis and high in seminal plasma and oviduct \[[@B99]\]. These changes in the concentration of HCO~3~^-^in the male and female reproductive tracts could play an important role in the suppression of capacitation in the epididymis and the promotion of this process in vivoin the female reproductive tract. It is observed that a protein, designated as CRISP-1, that is added to the sperm surface in epididymis is lost during capacitation \[[@B100]\]. Addition of exogenous CRISP-1 to the incubation medium inhibits tyrosine phosphorylation in a concentration-dependent manner, thus inhibiting capacitation and subsequently the acrosome reaction. A recent study investigated the effect of in vitroaddition of HCO~3~^-^on intracellular cAMP production and protein tyrosine phosphorylation in human spermatozoa \[[@B101]\]. The addition of HCO~3~^-^in the medium resulted in a significant increase in sperm motility as well as several hyperactivation parameters, mediated by an increase in cAMP production and tyrosine phosphorylation of AKAP3. The effects disappeared with the addition of 4,4\'-diisothiocyanostilbene-2, 2\'-disulfonic acid, which is a known inhibitor of bicarbonate transport. It was observed that both tyrosine phosphorylation of AKAP3 and sperm motility were blunted by the soluble adenylate cyclase (sAC) inhibitor 2OH-estradiol. These findings indicate that HCO~3~^-^stimulates human sperm motility and hyperactivation through activation of sAC and tyrosine phosphorylation of AKAP3, causing an increased recruitment of PKA to AKAP3 \[[@B101]\]. #### d. Reactive Oxygen species (ROS) Studies have suggested that ROS such as H~2~O~2~and superoxide anion are involved in the regulation of human sperm capacitation and protein tyrosine phosphorylation \[[@B19],[@B102]\]. Some reports suggest that the ROS action lie upstream from cAMP site in the signal transduction cascade \[[@B103]\], while others believe that ROS is localized downstream from cAMP \[[@B88]\]. In a recent study, Rivlin et al\[[@B104]\] suggested that H~2~O~2~activates adenylate cyclase to produce cAMP, leading to PKA-dependent protein tyrosine phosphorylation. Since adenylate cyclase present in sperm is activated by HCO~3~^-^, they proposed that H~2~O~2~and HCO~3~^-^can act in concert or partially substitute each other \[[@B104]\]. However, ROS also has detrimental effects on spermatozoa; while low concentrations of H~2~O~2~are beneficial, high concentrations can lead to sperm immobilization and death. It has been proposed that the accurate balance of amount of ROS produced and scavenged at any moment determines whether a sperm function will be promoted or jeopardized. Nicotinamide adenine dinucleotide (NADH) and NADPH are also known to promote sperm capacitation \[[@B104]\]. It has been found that exogenous NADPH enhances protein tyrosine phosphorylation in bovine sperm \[[@B104]\] and promote capacitation. NADPH acts as a coenzyme for sperm oxidase that generates superoxide anion, which is later dismutated to H~2~O~2~by superoxide dismutase. The inhibition of lactate dehydrogenase (LDH)-C4 blocks capacitation of mouse spermatozoa in vitro\[[@B105]\]. LDH-C4 is a mammalian testis-specific enzyme and is the only lactate dehydrogenase isozyme present in sperm. LDH-C4 seems to play an important metabolic role in sperm capacitation. The oxidation of NADH with the conversion of pyruvate to lactate by LDH provides ATP necessary for PKA activity \[[@B105]\]. NADH-diaphorase (enzyme that transfer electrons from NADH to an electron acceptor such as 2,6-dichlorophenol-indophenol) plays a role in spermatozoal function through ROS generation. Overproduction of ROS due to high diaphorase activity has been observed in some infertile men \[[@B106]\]. The free radical nitric oxide (NO) generated by spermatozoa has been implicated in sperm function \[[@B107]\]. Non-capacitated human spermatozoa produce low levels of NO, whereas under capacitating conditions, a time dependent increase in NO synthesis has been observed \[[@B108]\]. It has been reported that the increased levels of NO during capacitation modulate cAMP pathway that regulates the downstream protein tyrosine phosphorylation \[[@B109]\]. In vitrostudies have shown that low concentration of NO enhances the acrosome reaction of mouse \[[@B110]\] and bull \[[@B111]\] spermatozoa, as well as the zona pellucida binding ability of human sperm \[[@B112]\]. #### e. Progesterone Progesterone has been reported to affect several sperm functions especially capacitation, motility and acrosome reaction \[[@B113]\]. The effects of progesterone on spermatozoa are mediated via progesterone binding sites/progesterone receptor (PR) on the acrosomal membrane \[[@B114]\]. Different types of PRs have been shown to be present on the sperm plasma membrane. These are: plasma membrane Ca^2+^channel (PR1), a membrane-associated protein tyrosine kinase (PTK; PR2), and a plasma membrane chloride channel (PR3) \[[@B115]\]. The progesterone stimulates Ca^2+^influx in the human spermatozoa through PR1 \[[@B114]\] and voltage-dependent calcium channels (VDCC) \[[@B116]\]. In human spermatozoa, progesterone stimulates tyrosine phosphorylation of sperm proteins \[[@B117],[@B118]\] causing hyperactivation \[[@B119]\] with an increase in cAMP levels \[[@B117]\]. The tyrosine kinase-associated PR (PR2) is responsible for the effect of progesterone on the hyperactive motility and acrosome reaction. PR3 mediates the chloride influx, which takes place during the acrosome reaction \[[@B115]\]. Progesterone also increases the membrane fluidity of human sperm plasma membrane, which is an important event in sperm capacitation and tyrosine phosphorylation \[[@B115]\]. #### f. Gamma-Aminobutyric Acid (GABA) Recently GABA has emerged as a putative modulator of sperm function. GABA is the most widely distributed inhibitory neurotransmitter in vertebrate central nervous system. Three types of membrane receptors (A, B and C) mediate the inhibitory action of GABA. GABA A-receptor has been identified in human spermatozoa. In a recent study, the in vitroeffect of GABA was studied on bovine spermatozoa capacitation \[[@B120]\]. It was observed that addition of GABA to the incubation medium results in a concentration-dependent increase in the percentage of capacitated spermatozoa. A significant increase in intracellular Ca^2+^and cAMP levels was induced by GABA, and the GABA A-R antagonists, bicuculline or picrotoxin abolished the effect \[[@B120]\]. Thus, GABA seems to induce sperm capacitation through a signal transduction pathway involving Ca^2+^, cAMP and tyrosine phosphorylation. #### g. Angiotensin II (AII) AII is known to be present in seminal plasma and it modulates the adenylate cyclase (AC)/cAMP signal transduction pathway \[[@B121]\]. The existence of a class of angiotensin receptors (AT1) has been shown in bovine spermatozoa. In the capacitated sperm, AII AT1 receptors are localized in the head and tail, whereas in non-capacitated cells the receptors are localized only in the tail region \[[@B122]\]. AII significantly stimulates cAMP production in capacitated mouse spermatozoa with an associated increase in protein tyrosine phosphorylation \[[@B123]\]. #### h. Cytokines Cytokines are a family of polypeptide hormones produced primarily by the cells of the immune system in response to various stimuli including foreign antigens. Although originally identified from the immune cells, the non-immune cells also secrete them. The cytokines can have both positive and negative effects on a variety of cell types and tissues. Cytokines are present in circulation and local genital tract secretions in both men and women \[[@B124],[@B125]\]. The effects of various cytokines on sperm cell have also been studied. It has been observed that cytokines can affect sperm cell motility, capacitation, acrosome reaction, zona-pellucida binding and penetration and embryonic development in positive or negative manner \[[@B126]\]. It has been observed that cytokines namely, interferon-α (IFN-α), interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) have negative effect on sperm motility \[[@B126]\], while interleukin-6 (IL-6) enhances sperm cell capacitation and/or acrosome reaction \[[@B127]\]. However, the mechanisms by which cytokines affect sperm functions are not clear. Identification of cytokine receptors in the sperm cell is an important step in elucidating the mechanism of cytokine action. Very few cytokine receptors have been identified on sperm cells till date. A study in our laboratory, demonstrated the presence of IFN-α and IFN-γ receptors in mouse, rabbit, pig and human sperm \[[@B128]\]. The presence of interleukin-2α (IL-2α) \[[@B129]\], interleukin-2β (IL-2β) \[[@B129]\], insulin like growth factor-1 (IGF-1) \[[@B62],[@B63]\] and c-met (hepatocyte growth factor receptor) \[[@B60]\] receptors on human sperm surface has also been documented. A recent study identified the presence of CX(3)CR1 receptors on human sperm \[[@B130]\]. Fractalkine, a CX(3)C chemokine, is present in fallopian tubes. It might play an important role in maintaining the motility of spermatozoa and their ability to undergo the acrosome reaction when sperm transit through fallopian tubes \[[@B130]\]. However, the effect of various cytokines on tyrosine phosphorylation during capacitation has not been extensively studied. Since many of the cytokines affect capacitation/acrosome reaction and tyrosine phosphorylation is required for these processes, it can be envisaged that these cytokines modulate tyrosine phosphorylation in sperm cell. Conclusions =========== Before fertilization can occur, spermatozoon undergoes a series of changes to acquire the ability to bind and penetrate the oocyte. These events are regulated by the activation of intracellular signaling pathways involving various molecules such as cAMP, protein kinase A, receptor tyrosine kinases, and non-receptor tyrosine kinases. A number of molecules have been identified, that regulate these pathways such as calcium, bicarbonate, ROS, GABA, progesterone, angiotensin, and cytokines. Studies have shown that phosphorylation of sperm proteins is an important aspect of capacitation and has been shown to be associated with hyperactivated motility, zona pellucida binding and acrosome reaction. Extensive research has started to elucidate various pathways involved in protein phosphorylation during sperm capacitation. Presence of three major pathways involving cAMP/PKA, receptor tyrosine kinases, and non-receptor protein tyrosine kinases have been shown. These pathways are not mutually exclusive and may involve cross-talk among several molecules. The exact pathways have not been clearly delineated. Several questions such as how does the stimulation of cAMP/PKA pathway upregulate tyrosine phosphorylation are still to be answered. Many components of these pathways and several phosphoproteins remain to be identified. The novel technologies such as gene knockout technique will help us to elucidate the key molecules in these pathways. Based upon the present data, we have constructed a heuristic model that is shown in figure [1](#F1){ref-type="fig"}. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Heuristic model showing the tyrosine phosphorylation signaling pathways in sperm cell involved in capacitation. There seems to be three major signaling pathways operating in sperm cell, namely cAMP/PKA-dependent pathway (pathway I), receptor tyrosine kinase pathway (pathway II), and non-receptor protein tyrosine kinase pathway (pathway III). The pathway I, that is exclusive only to sperm cells, seems to be the major pathway among the three pathways. These cascades may not be mutually exclusive and include cross-talk among several molecules. Many key molecules and receptors are still to be identified to completely elucidate the molecular mechanism and signal transduction cascade involved in capacitation. G-protein coupled receptor pathway has not been included in this model. ::: ![](1477-7827-2-75-1) ::: Acknowledgements ================ This work was supported in part by NIH grant HD24425 to RKN. We thank Dr. C.Rajesh for his helpful suggestions.	PubMed Central	2024-06-05T03:55:49.766668	2004-11-9	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533862/", "journal": "Reprod Biol Endocrinol. 2004 Nov 9; 2:75", "authors": [ { "first": "Rajesh K", "last": "Naz" }, { "first": "Preeti B", "last": "Rajesh" } ] }
PMC533863	Introduction ============ Gastric cancer is the most frequently diagnosed and the second leading cause of cancer-related death in Korea. For many years, a few single agents such as 5-fluorouracil, doxorubicin, mitomycin C, and nitrosourea, have been considered to have significant antitumor activity in gastric cancer patients \[[@B1]\]. However, the response rate has been \<30% and complete remission has been rare. Several combination chemotherapy regimens such as FAM (5-fluorouracil, adriamycin, and mitomycin C) have been attempted in order to improve the treatment outcomes. In a nonrandomized Phase II study for advanced gastric cancer, the FAM regimen achieved an objective partial response rate of 42% \[[@B2]\]. Some cisplatin-based combination chemotherapy regimens have shown high response rates \[[@B3],[@B4]\]. Despite the efficacy of cisplatin against gastric cancer, there were two major problems with this agent. Firstly, there are significant side effects, such as severe nausea and vomiting, nephrotoxicity, and neurotoxicity \[[@B5]\]. Secondly, the cancer cells show a primary or acquired resistance to cisplatin \[[@B6]\]. Therefore, extensive effort to develop novel cisplatin analogs with equivalent or greater antitumor activity and a lower toxicity has been made. Among them, carboplatin has reduced renal and gastrointestinal side effects than cisplatin \[[@B7]\]. However, carboplatin has no enhanced therapeutic efficacy over cisplatin and has not circumvented the acquired resistance to cisplatin. Therefore, it is essential to develop a new platinum drug with a greater potent antitumor activity as well as being effective against resistant cells. Recently, SK Pharmaceutical (Seoul, Korea) developed many cisplatin analogs, including heptaplatin (SKI-2053R) (Fig. [1](#F1){ref-type="fig"}). Heptaplatin exhibited a greater antitumor activity against a number of human cancer cell lines including gastric cancer as well as a lower nephrotoxicity \[[@B8],[@B9]\]. In addition, heptaplatin 2053R is also effective against cisplatin-resistant L1210 leukemia cells (L1210-CPR) \[[@B10]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Structure of cisplatin analogs. ::: ![](1475-2867-4-6-1) ::: Metallothioneins (MT) are a family of stress-induced proteins with a wide variety of physiological functions, including protection against metal toxicity and oxidants. They may also assist in regulating cellular proliferation, apoptosis, and malignant progression. MT has a high affinity for heavy metals since it contains many cystein residues, which account for approximately 30% of the total amino acids in this protein molecule \[[@B11]\]. MT is a low molecular weight metal binding protein whose synthesis is induced by heavy metals, glucocorticoids and other factors \[[@B12]\]. Although the physiological role of MT is unclear, MT participates in detoxifying heavy metals or maintaining zinc and copper homeostasis \[[@B12]\]. Some reports have shown that MT has free radical scavenging ability in vitro\[[@B13],[@B14]\] and MT expression also increases the cellular resistance to radiation damage \[[@B15]\]. The cells transfected with the bovine papilloma virus expression vectors containing the DNA encoding human metallothionein-IIA were resistant to cisplatin, melphalan, and chlorambucil but not to 5-fluorouracil or vincristine \[[@B16]\]. In this study, the effect of MT on the cisplatin analog-induced cytotoxicity was investigated in the gastric cancer cell lines. Since heptaplatin is less associated with MT, it is believed to be effective against cisplatin-resistant cells related to the high level of MT. Results ======= MT cDNA, whose expression level was higher than the wild-type AML-2, were isolated from the paraquat-resistant AML-2 cells \[[@B17],[@B18]\]. The nucleotide sequence of the clone, containing an almost full-length cDNA copy of the human MT mRNA, was determined. Molecular cloning and nucleotide sequence analysis revealed that the hMT transcript is a novel hMT-I isoform, which was designated hMT-Ip, and exhibits a 98.4% homology with the hMT-Ie isoform, whereas it shows 86.9% homology with hMT-II, 83.8% homology with hMT-III and 62.9% homology with hMT-IV \[[@B18]\]. MT mRNA expression in the gastric cancer cell lines --------------------------------------------------- Using the RT-PCR assay, the MT mRNA was determined in the 8 human gastric cancer cell lines, SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-620, SNU-638, and SNU-668. The gastric cancer cell lines showed differential expression level of MT mRNA, in which the SNU-601 cells expressed the lowest MT mRNA level whereas SNU-638 expressed the highest level (Fig. [2](#F2){ref-type="fig"}). The MT mRNA level of SNU-638 was 80 times higher than that of SNU-601. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### \(A) MT mRNA expression in the gastric cancer cell lines. The expression level was determined by RT-PCR assay. β-actin was used as a control for RNA. The cDNA reverse-transcribed from the mRNA was individually amplified with each primer pair for the MTand β-actingenes. Aliquots of each PCR reaction mixture were separated on 7% polyacrylamide gel in TAE. The gel was dried and exposed on X-ray film overnight. (B) The ratio of MT/β-actin of SNU cell lines. ::: ![](1475-2867-4-6-2) ::: Comparative sensitivity of the gastric cancer cells that express MT differentially to cisplatin analogs ------------------------------------------------------------------------------------------------------- The resistance of the gastric cancer cells that express MT to cisplatin was compared. SNU-16 and SNU-601, which showed the lowest MT expression levels, were sensitive but SNU-638, which exhibited the highest MT expression levels, were resistant to cisplatin (Fig. [3](#F3){ref-type="fig"}). On the other hand, the gastric cancer cell lines expressing moderate MT expression levels (SNU-16, SNU-484, SNU-620, SNU-5 and SNU-668) showed a lower correlation between the sensitivity to cisplatin and MT expression (Fig. [3](#F3){ref-type="fig"}). In addition, 3 gastric cancer cell lines with very low (SNU-484) or moderate MT levels (SNU-1 and SNU-620) which are as resistant to cisplatin as SNU-638 (Fig. [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}), suggesting no involvement at all of MT in cisplatin resistance or the involvement of other resistance mechanisms except MT. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Cytotoxic effect of cisplatin in the gastric cancer cell lines. Cisplatin cytotoxicity was determined using an MTT assay. ::: ![](1475-2867-4-6-3) ::: Correlation of MT mRNA expression and sensitivity to the cisplatin analogs -------------------------------------------------------------------------- The cytotoxicity of the cisplatin analogs in the SNU-638 and SNU-601 cells, which expressed different MT levels, was determined by an MTT assay. The SNU-638 cells were approximately 11 times, 4 times and 2 times more resistant to cisplatin, carboplatin and heptaplatin, respectively, than the SNU-601 cells (Fig. [4](#F4){ref-type="fig"} and Table [1](#T1){ref-type="table"}), suggesting a lower involvement of MT in the resistance to heptaplatin. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Cytotoxic profiles of the cisplatin analogs in the SNU-601 and SNU-638 cell lines showing low and high MT expression levels, respectively. ::: ![](1475-2867-4-6-4) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Comparison of cytotoxicities of the cisplatin analogs in various gastric cancer cell lines ::: Ratio of IC~50~^a^ ------------ ------------------------ ------- --------- 638/601^b^ 11.23 5.10 2.00\* CIS/601^c^ 50.30 55.95 28.36\* MT/Mock^d^ 54.77^e^ 3.00 1.57\* ^a^, The 50% inhibitory concentration (IC~50~) for a particular agent was defined as the drug concentration which results in a 50% reduction in cell number to the untreated control. IC~50~values were determined directly from semilogarithmic dose-response curves. The means were obtained from the experiments carried out at least in triplicate. ^b^, The ratios of IC~50~values of SNU-638 to that of SNU-601 (Fig. 4) ^c^, The ratios of IC~50~values of SNU-601/Cis to that of SNU-601 (Fig. 5) ^d^, The ratios of IC~50~values of SNU-601/MT to that of SNU-601/Mock (Fig. 7) ^e^, The ratio calculated from extrapolated data obtained in SNU-601/MT (Fig. 7) \, P \< 0.05 versus cisplatin or carboplatin group ::: Comparison of resistance to cisplatin analogs in gastric cancer sublines resistant to cisplatin ----------------------------------------------------------------------------------------------- The cisplatin-resistant gastric cancer subline, SNU-601/Cis was selected in the presence of 2 μg/ml cisplatin. The SNU-601/Cis cells showed an approximately 49-fold, 56-fold and 29-fold increased resistance to cisplatin, carboplatin and heptaplatin, respectively (Fig. [5](#F5){ref-type="fig"} and Table [1](#T1){ref-type="table"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Cytotoxic effects of cisplatin, carboplatin and heptaplatin in the SNU-601/WT cells and its cisplatin-resistant subline SNU-601/CIS. The cytotoxicity was determined using the MTT assay. Mean ± SE of triplicate determination is given. ::: ![](1475-2867-4-6-5) ::: Sensitivity of MT-transfected SNU-601 cells to cisplatin analogs ---------------------------------------------------------------- Sensitivity to cisplatin analogs was compared between SNU-601 transfected with the vector containing the full-length MT cDNA (pIRESneo2/MT) and with the empty vector (pIRESneo2/Mock). Although the SNU-601/MT showed a 66% increase in MT expression when compared to the SNU-601/Mock cells, the SNU-601/MT cells scavenged significantly ROS generated by hydrogen peroxide or paraquat as compared with those in SNU-601/Mock using a fluorescence probe (Fig. [6](#F6){ref-type="fig"}). This result suggests that SNU-601/MT expresses not only a higher quantity of MT than does SNU-601/Mock but also the MT functions to scavenge ROS. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Comparison of ROS-scavenging activities between the SNU-601/Mock and the SNU-601/MT sublines. The reaction took place with 1 × 10^5^cells and 1 μM 2\',7\'-DCFH diacetate in 3 ml phosphate-buffered saline. The cells were exposed to H~2~O~2~and paraquat for 4 hours. The fluorescence intensity was determined using a fluorometer with excitation wavelength at 485 and emission wavelength at 530 nm. The results are expressed as means ± SE (n = 3). PQ, paraquat; \, P \< 0.05. ::: ![](1475-2867-4-6-6) ::: The sensitivities of the SNU-601/MT cells to cisplatin analogs were tested using the MTT assay. The SNU-601/MT cells showed a lower degree of resistance to cisplatin and carboplatin, but was paradoxically sensitive to heptaplatin when compared to the SNU-601/Mock cells (Fig. [7](#F7){ref-type="fig"}). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Cytotoxic effects of cisplatin, carboplatin and heptaplatin in the SNU-601/Mock and SNU-601/MT sublines. Cytotoxicity was determined using the MTT assay. Mean ± SE of triplicate determination is given. ::: ![](1475-2867-4-6-7) ::: Comparison of MT mRNA expression in SNU-601 after treatment of zinc and cisplatin analogs ----------------------------------------------------------------------------------------- To examine the effect of zinc and cisplatin analogs on MT mRNA expression, the maximum non-cytotoxic concentrations of each drug in the 3-day MTT assay were chosen and then treated for 1 day. After the treatment with 100 μM ZnCl~2~, 0.6 μg/ml cisplatin, 2 μg/ml carboplatin and 0.2 μg/ml heptaplatin for 24 hr, MT mRNA level was determined using the RT-PCR assay. The MT mRNA expression level was significantly increased by ZnCl~2~but was decreased by heptaplatin. In contrast, there were no changes in MT mRNA expression level by cisplatin and carboplatin (Fig. [8](#F8){ref-type="fig"}). ::: {#F8 .fig} Figure 8 ::: {.caption} ###### MT expression in the SNU-601 cell line after a treatment with zinc and the cisplatin analogs. The SNU-601 cells were treated with 100 μM ZnCl~2~, 0.6 μg/ml cisplatin, 2 μg/ml carboplatin, 0.2 μg/ml heptaplatin for 24 hrs. The MT mRNA expression level was determined by the RT-PCR assay. ::: ![](1475-2867-4-6-8) ::: Effect of heptaplatin on heavy metal-induced MT expression in SNU-601 cells --------------------------------------------------------------------------- Of the cisplatin analogs, heptaplatin is not only relatively less related to MT but also decreases the MT expression level. We tested whether or not heptaplatin could influence the heavy metal-induced MT expression. Each IC~50~concentration of CdCl~2~or ZnCl~2~obtained from the 3-day MTT that did not influence the growth of the SNU-601 cells for 24 hr were used in this study. The SNU-601 cells were treated with 32 μM CdCl~2~or 200 μM ZnCl~2~in the presence or absence of 200 ng/ml heptaplatin for 24 hr. After a 24-h treatment, MT expression level was determined by the RT-PCR assay and Western blot analysis. Fig. [9](#F9){ref-type="fig"} shows that heptaplatin inhibits the MT gene expression induced by cadmium but not zinc, suggesting the differential modulation of metal-induced MT expression. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Effect of heptaplatin on MT induction by heavy metals in the SNU-601 cell lines. The SNU-601 cells were treated with 32 μM CdCl~2~or 200 μM ZnCl~2~in the presence or absence of 200 ng/ml heptaplatin for 24 hr. The MT expression level was determined by (A) the RT-PCR assay and (B) the Western blotting method. ::: ![](1475-2867-4-6-9) ::: Discussion ========== The cisplatin analogs are among the most active and widely used cytotoxic anticancer drugs. However, the acquisition or presence of resistance significantly undermined the curative potential of these drugs against many malignancies. Alterations in the cellular pharmacology, including decreased drug accumulation, increased cellular thiol levels and increased repair of platinum-DNA damage, have been observed in the cisplatin-resistant cancer cell lines \[[@B19]\]. Since heptaplatin is effective against cisplatin-resistant cancer cells, its development has shed light on cisplatin-refractory cancer patients. However the mechanism by which heptaplatin is effective against cisplatin-resistant cancer cells is unclear. Since MT is involved in cisplatin resistance \[[@B26]\], the molecular mechanisms for the effect of heptaplatin against the cisplatin-resistant cancer cell lines was investigated with respect to the involvement of MT. In this study, the gastric cancer cell lines expressed different basal MT mRNA levels, whose mechanisms remain to be determined. It was suggested that DNA hypomethylation was responsible for the higher basal hMT-IIa mRNA levels in the cisplatin-resistant cells \[[@B20]\]. In this study, MT expression in the gastric cancer cell lines was not always related to the resistance to cisplatin, suggesting the involvement of other mechanisms except MT. But heptaplatin is more effective against the MT-overexpressing gastric cancer cell line selected for resistance to cisplatin than cisplatin and carboplatin were. In addition, the cytotoxicity by cisplatin and carboplatin but not heptaplatin was reduced by a pretreatment with zinc, which can induce MT remarkably (data not shown). Taken together, heptaplatin was more effective against both cisplatin-resistant gastric cancer cell subline SNU-601/CIS and MT-overexpressing SNU-638 than was cisplatin or carboplatin. These results suggest a possibility that the cytotoxicity of heptaplatin was less influenced by MT. To test the hypothesis, the function of MT involved in the cytotoxicity of the cisplatin analogs was confirmed by the transfection of MT. The cytotoxicity of heptaplatin was not influenced by the MT transfection, which indicates that the anticancer activity of heptaplatin to the cisplatin-resistant gastric cancer sublines could be less associated with MT. In addition, heptaplatin inhibited the MT expression caused by cadmiun but not zinc. This result suggests that metals differentially regulate MT, which is supported by the literature. The inhibition mechanism for H7, a protein kinase C inhibitor, may be different between the zinc- and cadmium-treated cells. H7 blocks zinc transport but not cadmium transport although rottlerin, a PKC inhibitor that can block both cadmium and zinc transport \[[@B21],[@B22]\]. Hypoxia activates MT expression through the metal response elements and that this activation involves the metal transcription factor-1 (MTF-1) \[[@B23]\]. MTF-1 was found to be present in the cytoplasm of the cells. Upon zinc stimulation, MTF-1 was translocated to the nuclei and activated MT gene expression \[[@B24]\]. However, no MTF-1 translocation was observed in cells treated with cadmium \[[@B25]\]. Whether or not MTF-1 is involved in inhibiting the cadmium-induced MT induction by heptaplatin remains to be determined. In summary, these results indicate that molecular mechanisms responsible for the effect of heptaplatin against the cisplatin-resistant gastric cancer sublines is at least in part due to the lower involvement of MT as well as its attenuation of MT induction. Materials and Methods ===================== Cell culture and the selection of the gastric cancer cell subline for cisplatin resistance ------------------------------------------------------------------------------------------ The human gastric cancer cell lines (SNU-1, SNU-5, SNU-16, SNU-484, SNU-601, SNU-620, SNU-638, and SNU-668) were obtained from the Cancer Research Center in Seoul National University (South Korea). The cells were cultured in RPMI 1640 (GibcoBRL Grand Island, NY, U.S.A.) supplemented with 10% FBS (Sigma Chemical Co. St. Louis, MO, U.S.A.). The cells were maintained as a monolayer culture and subcultured at confluence. The cisplatin-resistant gastric cancer cell subline was selected by chronic exposure to gradually increasing cisplatin concentrations ranging from 200 ng/ml (IC~50~value) to 2,000 ng/ml on an intermittent dosage schedule. Cytotoxicity assay ------------------ The in vitrocytotoxicity of the drugs was determined using an MTT (Sigma Chemical Co. St. Louis, MO, USA) assay previously described by Pieters et al. \[[@B26]\]. The 50% inhibitory concentration (IC~50~) for a particular agent was defined as the drug concentration that causes in a 50% reduction in the number of cells compared to the untreated control for 3 days. The IC~50~values were determined directly from the semilogarithmic dose-response curves. Cisplatin and carboplatin were obtained from Sigma Chemical Co. (St. Louis, MO, USA) and heptaplatin (Sunpla^®^) was generously donated by SK Pharmaceutical (Seoul, Korea). Cisplatin analogs were dissolved in phosphate buffered saline and used immediately because of their instability in aqueous solution. RNA extraction RT-PCR assay --------------------------- The total RNA was extracted from the cells using the acid guanidium thiocyanate-phenol-chloroform method \[[@B27]\]. The MT and β-actin mRNA transcripts were detected using an RT-PCR assay. The RNA from each sample was reverse transcribed using 200 units of Moloney murine leukemia virus reverse transcriptase (GibcoBRL, Grand Island, NY, U.S.A) and oligo (dT) primer for 1 h at 37°C. The resulting cDNA was diluted 1:5 with distilled water and amplified with 2.5 units of Taq polymerase (Perkin-Elmer, Foster City, CA, USA) and 10 pmole of each primer using a GeneAmp PCR2400 (Perkin-Elmer, Foster City, CA, USA). The MT expression level was detected with the sense and antisense primers corresponding to the nucleotides (5\'-ATGGACCCCAACTGCTCG) and (5\'-TCAGGCGCAGCAGCTGCA), respectively, of the published cDNA sequence \[[@B28]\] and yielded a 220-bp PCR product. The β-actin expression level, as a control of the RNA quantity, was detected with the sense and antisense primers corresponding to the nucleotides 1912--1932 (5\'-GACTATGACTTAGTTGCGTTA) and 2412--2392 (5\'-GCCTTCATACATCTCAAGTTG), respectively, of the published cDNA sequence \[[@B29]\], yielding a 501-bp PCR product. Twenty one PCR cycles for MT was carried out as follows: denaturation at 94°C for 30 s, annealing at 57°C for 60 s, and extension at 72°C for 1 min was used. Sixteen cycles of PCR for β-actin of denaturation at 95°C for 30 s, annealing at 53°C for 30 s, and extension at 72°C for 30 s. After the last cycle, all the PCR products were subjected to a final extension for 5 min at 72°C. For quantitation, 5 mCi/ml of \[α-^32^P\] dCTP was added to each reaction mixture. Subsequently, the PCR products were combined and electrophoresed on 7.5% nondenaturing polyacrylamide gels. The bands were scanned using a densitometer (Pdi, Huntington Station, NY, USA). The quantity of each mRNA transcript was normalized with that of β-actin mRNA. Autoradiographic films of the RT-PCR assay were subjected to densitometric analysis using a densitometer. Protein extraction and Western blot analysis for MT --------------------------------------------------- The total cell lysates from 2 × 10^6^cells were prepared by lysing the harvested cells in an extraction buffer (1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS in phosphate-buffered saline) supplemented with 2 mM phenylmethylsulfonyl fluoride (Sigma Chemical Co. St. Louis, MO, USA) and 10 μg/ml leupeptin (Sigma Chemical Co. St. Louis, MO, USA). The DNA was sheared by sonication. The sample buffer for MT analysis was used without 2-mercaptoethanol \[[@B30]\]. Western blotting analysis was performed using a slight modification of the method reported by Towbin et al. \[[@B31]\]. The proteins were transferred onto a nitrocellulose membrane by electroblotting at a current of 60 V overnight using a transfer buffer containing 5% mercaptoethanol. The membrane was incubated in a blocking solution (5% skim milk) for 1 hr at room temperature, washed, and then incubated with a monoclonal mouse antibody (E9 clone, 1:500, Dako Corporation, Carpinteria. CA, USA) for MT-I and MT-II. The membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibodies (diluted 1:1000) for 1 hr. The membrane was then stained using the ECL detection kit (Amersham Biosciences Corp., Piscataway, NJ, USA). Production of stable transfectant cell line ------------------------------------------- Mammalian transfectants were produced as reported previously \[[@B18]\]. The mammalian expression vector, pIRESneo2 (Clonetech, Palo Alto, CA, USA), was used to clone the MT. A 220 base pair cDNA fragment containing the full open reading frame of human MT was amplified by RT-PCR from the RNA of AML-2/PQ400 \[[@B17]\], a paraquat-resistant AML-2 cell line that highly over-expresses MT. The PCR primer pairs were as follows: the forward primer 5\'-CG[GGATCC]{.underline}ATGGACCCCAACTGCTCG-3\' to introduce a BamH I site as underlined, and reverse primer: 5\'ATAAGAAT[GCGGCCGC]{.underline}TCAGGCGCAGCAGCTGCA-3\' to introduce a NotI site as underlined. The resulting MT PCR product was cloned to a TOPO TA cloning kit (Invitrogen, Carlsbad, CA, USA). After confirmation of the sequencing, the MT was cut from the TOPO plasmid vector (pCR 2.1-TOPO), purified, subcloning to the multicloning site of the expression vector pIRESneo2 and subsequently sequenced to confirm the correct open reading frame. Transfection was performed using the LipofectAmine Plus reagents (GIBCO BRL, Grand Island, NY, USA). Approximately 1 × 10^6^of the SNU-601 cells were plated into a 60 mm tissue culture dish and cultured overnight. Prior to transfection, the growth medium was replaced with the serum free RPMI 1640 media and cultured. The LipofectAmine reagent containing 2 μg of pIRES/Mock and pIRES/MT was combined with the Plus reagent and applied to the cells. After culturing for five hours, the media was replaced with RPMI 1640 containing 10% FBS. After selection by culture in the medium containing 0.2 mg/ml of G-418 (GIBCO BRL, Grand Island, NY, USA), the emerging colonies were isolated with cloning rings. The stable transfectants, SNU-601/Mock and SNU-601/MT, were obtained. The selection medium was changed every 2--3 days. Determination of reactive oxygen species (ROS) using a fluorometric probe ------------------------------------------------------------------------- Dichlorofluorescin (DCFH) was used to measure the ROS concentration. After 2\', 7\'-dichlorofluorescin diacetate (DCFH-DA) crosses the membrane, it is de-esterified to DCFH which is oxidized to fluorescent DCF by the ROS \[[@B32]\]. Phosphate-buffered saline containing 1 × 10^5^/ml SNU-601/Mock or the SNU-601/MT cells was incubated with 1 μM DCFH-DA at 37°C for 4 hr. After incubation, the DCF fluorescence intensity was determined using a fluorometer at 485 nm for excitation and 530 nm for emission. Acknowledgement =============== The authors wish to acknowledge Ms. Y. K, Seo and Ms. H. S. Kim for their technical assistance. This work was supported by Korea Research Foundation Grant (KRF-2001-041-FOO186).	PubMed Central	2024-06-05T03:55:49.770207	2004-10-19	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533863/", "journal": "Cancer Cell Int. 2004 Oct 19; 4:6", "authors": [ { "first": "Cheol-Hee", "last": "Choi" }, { "first": "Yoon-Jung", "last": "Cha" }, { "first": "Chun-San", "last": "An" }, { "first": "Kyung-Jong", "last": "Kim" }, { "first": "Kweon-Cheon", "last": "Kim" }, { "first": "Sung-Pyo", "last": "Moon" }, { "first": "Zang Hee", "last": "Lee" }, { "first": "Young-Don", "last": "Min" } ] }
PMC533864	Background ========== The transcription factor c-Myb is regulating genes involved in proliferation and differentiation during hematopoiesis in vertebrates (reviewed in \[[@B1],[@B2]\]). c-Myb is expressed at high levels in hematopoietic progenitor cells, but becomes down-regulated when the cells reach terminal differentiation. The critical role of c-Myb in the development of hematopoietic cells is emphasized by the embryonic lethality observed in mice with a c-myb^null^mutation, caused by failure of fetal hepatic hematopoiesis \[[@B3]\]. c-Myb expression has also been detected in other rapidly proliferating tissues such as hair follicles and immature epithelial cells from colon, respiratory tract, skin and retina \[[@B4],[@B5]\]. c-Myb is essential for early T cell development \[[@B6]\] and several c-Myb target genes play an important role during T cell development, like CD4, TCRγ, TCRδ and RAG-2 \[[@B1],[@B7]\]. The best characterized c-Myb target gene is chicken mim-1, which is encoding a secretable component of granules found in normal promyelocytes \[[@B8],[@B9]\]. The Myb family of proteins is defined by the presence of a well-conserved DNA-binding domain (DBD) composed of Myb repeats \[[@B10]\]. Each repeat consists of about 50 amino acids with three regularly spaced tryptophans forming a hydrophobic core \[[@B11]-[@B13]\]. The c-Myb DBD contains three tandem imperfect Myb repeats (R~1~, R~2~and R~3~), located in the N-terminus of the protein. Determination of the structure of the c-Myb DBD has revealed that each repeat folds into three well-defined helices forming a helix-turn-helix-related structural motif, where the last helix in R~2~and R~3~makes specific DNA contacts (recognition helices) \[[@B13]-[@B15]\]. The c-Myb protein harbours two functional domains in addition to the DBD: a central activation domain and a C-terminal negative regulatory domain. The viral counterpart of the chicken c-mybgene, v-myb, found in the AMV and E26 viruses, has deletions in both ends, leading to a v-Myb protein lacking the N-terminus, most of the first Myb repeat (R~1~) and a large part of the C-terminal negative regulatory domain (reviewed in \[[@B16]\]). The c-Myb DBD binds specifically to the sequence PyAAC(T/G)G, termed the Myb recognition element (MRE) \[[@B17]-[@B19]\]. The minimal sequence-specific DBD consists of the two carboxy-terminal Myb repeats, R~2~R~3~\[[@B20],[@B21]\]. The role of the first Myb repeat, R~1~, is not fully understood. It has been shown to be dispensable for specific DNA-binding \[[@B20],[@B22]\], but bears striking similarities to the other repeats regarding sequence and structure \[[@B23]\]. Some groups have reported that R~1~stabilizes the protein-DNA complex \[[@B24],[@B25]\] and it has been proposed to allow for more flexibility in the downstream region of the Myb recognition sequence \[[@B26]\]. According to the recent three-dimensional structure of the c-Myb DBD, the R~1~repeat does not contact DNA directly \[[@B15]\]. However, a long-distance electrostatic interaction is suggested to stabilize the protein-DNA complex. Its free position in the complex makes it possible that it could be involved in other functions as well. This possibility is supported by the fact that v-myb-like truncation of the N-terminus of c-Myb (until the end of R~1~) has been shown to be sufficient for oncogenic transformation of chicken bone marrow cells \[[@B27]\]. The R~1~repeat could serve as a special regulatory module, either acting as a target for molecular interactions or being subject to post-translational modifications. In the present work we address whether the N-terminal region including the R~1~repeat confers properties beyond DNA-binding to c-Myb DBD. We sought evidence for a putative RNA-binding function, analogous with several other transcription factors harbouring dual DNA-RNA binding properties. The motivation for investigating RNA-interaction was the design of the c-Myb DBD, built of repeating modules, a design resembling the structural logic of zinc finger proteins. Some well-studied zinc fingers have been found to use subsets of the repeats as RNA- or DNA-binding units. The classical example is the Xenopuszinc finger protein TFIIIA, which acts as a DNA-binding activator of 5S ribosomal RNA genes \[[@B28],[@B29]\]. TFIIIAalso forms a stable complex with 5S rRNA in Xenopusoocytes \[[@B30],[@B31]\]. The nine zinc fingers of TFIIIA contribute differentially to DNA- and RNA-binding; the N-terminal triplet (1--3) dominating the DNA recognition event while the middle triplet (4--6) is more important for RNA-binding \[[@B32],[@B33]\]. Another example of a zinc finger with similar dual properties is the Wilms tumor suppressor gene 1 (WT1) \[[@B34]-[@B36]\]. One splice variant (the +KTS isoform) seems to be a better RNA-interacting form that co-localizes with splicing proteins in nuclear speckles, whereas another variant (the -KTS isoform) interacts stronger with DNA and co-localizes with transcription factors \[[@B34],[@B35]\]. The capability of specific interaction with both DNA and RNA is not restricted to the zinc finger family of proteins. The homeodomain protein bicoid of Drosophilaacts both as a transcription factor, activating zygotic segmentation genes during blastoderm formation, and as a regulator of mRNA translation by binding to the mRNA of another homeodomain transcription factor, caudal \[[@B37],[@B38]\]. Interestingly, the homeodomain bears structural similarities to the Myb domain, especially with respect to the presence of a helix-turn-helix-related motif \[[@B12],[@B13],[@B15],[@B21]\]. A final example is p53, which has been reported to bind to both single-stranded DNA and RNA in addition to its established role as a sequence-specific DNA-binding protein (reviewed in \[[@B39]\]). Murine p53 and human Cdk4 translation have actually been shown to be regulated by p53 mRNA binding. Based on these occurrences of dual nucleic acid interactions, we asked whether a similar design was found in c-Myb. In particular, we raised the question whether the N-terminal region including R~1~might be implicated in RNA-binding rather than DNA-binding. As a test of our proposed RNA-binding function of c-Myb, we investigated the effect of different homoribopolymers on c-Myb DNA-binding. The homoribopolymer polyguanylic acid (poly(G)) strongly inhibited the sequence-specific DNA-binding of c-Myb, while poly(A), poly(C) and poly(U) did not, indicative of an RNA-binding activity. The same phenomenon, although weaker, was observed for A- and B-Myb. An order-of-addition experiment indicated that poly(G) bound directly to c-Myb in competition with DNA. Interestingly, the DBD construct containing the N-terminus and R~1~was significantly more sensitive to poly(G) than the minimal DBD. Thus, the N-terminus including R~1~seems to be important for the RNA-sensitivity of c-Myb DBD. Results ======= Differential binding to homoribopolymers has been used as evidence for RNA-binding activity of various proteins \[[@B40]-[@B45]\]. To investigate whether c-Myb and its R~1~repeat is involved in RNA-binding we first examined the effects of homoribopolymers on c-Myb DNA-binding. The sequence-specific DNA-binding was analyzed by the electrophoretic mobility shift assay (EMSA) using two purified recombinant human c-Myb protein domains, NR~1~R~2~R~3~(amino acid 1--192) and R~2~R~3~(amino acid 89--192), in mixture. The assay was performed in the presence of increasing amounts of the homoribopolymers poly(A), poly(C), poly(G) and poly(U) (Fig. [1A](#F1){ref-type="fig"}). The DNA-binding of both proteins was strongly inhibited by poly(G) but not by the other polymers. NR~1~R~2~R~3~was most sensitive, being inhibited when as little as 1 ng poly(G) was added. We also tested two artificial RNAs, poly(I\C) and poly(I). The former is a duplex RNA, and the latter is a variant of poly(G) less prone to forming unusual structures, but retaining some of the pairing properties of guanosines. The duplex did not affect DNA-binding, while poly(I) caused some inhibition at high concentrations (Fig. [1B](#F1){ref-type="fig"}). To confirm that the observed inhibition by poly(G) was indeed due to the added RNA, we carried out the poly(G) inhibition experiment in the presence and absence of RNase T1, an endoribonuclease that specifically cuts RNA at the 3\'-end of guanosine residues. As shown in Fig. [2](#F2){ref-type="fig"}, DNA-binding was no longer inhibited by poly(G) after RNase T1 treatment, confirming the RNA-dependence of the inhibition. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Inhibition of c-Myb DNA-binding by homoribopolymers. Panel A: NR~1~R~2~R~3~(40 fmol) and R~2~R~3~(20 fmol) in mixture were incubated with 30 fmol MRE-containing DNA probe and 1 ng (lane 4,7,10 and 13), 10 ng (lane 5, 8, 11 and 14) and 100 ng (lane 6, 9, 12 and 15) of homoribopolymers poly(A) (lane 4--6), poly(C) (lane 7--9), poly(G) (lane 10--12) and poly(U) (lane 13--15). The binding reactions were incubated for 15 minutes at 25°C and subsequently analyzed by EMSA and phosphorimaging. Lane 1, 2 and 3 show binding reactions without homoribopolymer addition, with NR~1~R~2~R~3~alone in lane 1, R~2~R~3~in lane 2 and the mixture of both in lane 3. Panel B: NR~1~R~2~R~3~and R~2~R~3~were combined in a binding reaction as in panel A, but now in the presence of 1 ng, 10 ng or 100 ng of the ribopolymers poly(G) (lanes 3--5), poly(I) (lanes 6--8), poly(I-C) (lanes 9--11) and 100 ng poly(A) (lane 12), respectively. Lane 1 shows free probe, whereas lane 2 shows binding reaction without ribopolymer addition. ::: ![](1471-2091-5-15-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### RNase treatment relieves poly(G)-mediated inhibition of c-Myb DNA-binding. NR~1~R~2~R~3~and R~2~R~3~were incubated with the MRE-containing DNA probe in the presence of 20 ng poly(G) homoribopolymer (lanes 3 and 4). The sample shown in lane 4 was in addition incubated with 2000 U RNase T1. To allow for RNase T1 mediated degradation of poly(G), all samples were incubated for 30 minutes at 37°C prior to addition of Myb-protein mixture. Binding reactions were subsequently incubated for 15 minutes at 25°C and analyzed by EMSA and phosphorimaging. Lanes 1 and 2 show free probe and binding reaction without homoribopolymer addition, respectively. ::: ![](1471-2091-5-15-2) ::: To better compare the sensitivity of the two proteins, we titrated the poly(G) inhibition (Fig. [3](#F3){ref-type="fig"}). The DNA-binding of NR~1~R~2~R~3~was significantly more sensitive to poly(G) competition than R~2~R~3,~indicating that the N-terminus including the R~1~repeat was important for the inhibitory effect. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Titration of the poly(G) inhibition. NR~1~R~2~R~3~(40 fmol) and R~2~R~3~(20 fmol) were incubated separately with 20 fmol MRE-containing radiolabelled probe and increasing amounts of poly(G). The binding reactions were incubated for 15 minutes at 25°C and subsequently analyzed by EMSA and phosphorimaging. The intensities of the complex bands were quantified by phosphorimaging software and plotted as percentage of the complex band intensity when no homoribopolymers was added. ::: ![](1471-2091-5-15-3) ::: Different plausible mechanisms for the observed poly(G) inhibition of c-Myb sequence-specific DNA-binding may be operating. Poly(G) might bind to c-Myb in direct competition with DNA. Or, the binding of poly(G) to c-Myb might be allosteric, inducing structural changes in c-Myb that reduces its DNA-affinity. A third explanation might be that poly(G) in a subtle way interacts with the DNA-probe and blocks its specific interaction with c-Myb. To clarify the mechanism of inhibition, we studied the importance of the order of addition of probe and homoribopolymers to the binding reaction (Fig. [4](#F4){ref-type="fig"}). Three situations were investigated, designated R, S and D in Fig. [4](#F4){ref-type="fig"}: (R) RNA was mixed with the proteins before addition of probe, (S) RNA and probe were mixed and added simultaneously to the proteins, (D) Probe (DNA) was mixed with protein before addition of RNA. The reasoning was that if the inhibition is allosteric of nature, the magnitude of inhibition should be independent of the order of addition. If probe-interference is the mechanism, the strongest inhibition should be observed with the simultaneous addition of RNA and probe. If a competitive binding of poly(G) to c-Myb is the case, the strongest inhibition should be expected with the addition of RNA to protein first and the weakest inhibition when the probe was added first. The last scenario was in fact what we observed, indicating that c-Myb binds to poly(G) in direct competition with DNA-binding (Fig. [4](#F4){ref-type="fig"}). No inhibition at all was observed when 100 ng of poly(A) was added. It is noteworthy that the relative sensitivity of the two Myb forms also changed as a function of the order of addition. Pre-incubation with RNA enhanced the difference between NR~1~R~2~R3 and R~2~R~3~significantly, the first being fully inhibited while the latter seemed to be almost unaffected. In contrast, pre-incubation with DNA allowed the two forms to bind with similar efficiency. This supports the notion that the N-terminal region including the R~1~repeat plays an important role in conferring RNA-sensitivity to the protein. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Order-of-addition analysis. A mixture of 40 fmol NR~1~R~2~R~3~and 20 fmol R~2~R~3~was incubated with 30 fmol MRE-containing DNA probe and 1 ng poly(G) (lane 4--6), 10 ng poly(G) (lane 7--9) and 100 ng poly(A) (lane 10--12). In the cases marked R(for \"RNA first\", lane 4, 7 and 10), the proteins were incubated with homoribopolymers for 15 minutes at 25°C before addition of DNA-probe and further incubation for 15 minutes at 25°C. The cases marked S(for \`\`simultaneous addition\'\', lane 5, 8 and 11) represents reactions where homoribopolymers and DNA probe were mixed before addition of protein and 15 minutes of incubation. Finally, the cases marked D(for \`\`DNA first\'\', lane 6, 9 and 12) show reactions where the protein were incubated with DNA first and then homoribopolymers were added. The reactions in lane 1--3 contained no homoribopolymers, NR~1~R~2~R~3~protein alone in lane 1 and R~2~R~3~protein alone in lane 2. The binding reactions were subsequently analyzed by EMSA and phosphorimaging. ::: ![](1471-2091-5-15-4) ::: The homoribopolymer experiments reported above were performed with 15 minutes of incubation at 25°C. To exclude that the poly(G) effect was just a consequence of slower complex formation, we repeated the experiment with longer incubation times up to 90 minutes (results not shown). No change in the inhibition pattern was observed. This argues against the possibility that poly(G) only reduces the rate of c-Myb/DNA complex formation and indicates that the complex with RNA is highly stable. We then asked whether the poly(G) inhibition was specific for c-Myb or if other Myb proteins exhibited the same properties. Purified NR~1~R~2~R~3~forms of the vertebrate Myb relatives A- and B-Myb together with c-Myb were analyzed by EMSA in the presence of different concentrations of poly(G) and poly(A). As shown in Fig. [5](#F5){ref-type="fig"}, the DNA-binding of A- and B-Myb was clearly inhibited by poly(G), although the effect was somewhat weaker than for c-Myb. A-Myb behaved most similar to c-Myb, consistent with the close resemblance between these two transcription factors \[[@B46]\]. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Comparison of A-, B- and c-Myb\'s poly(G) inhibition. A-, B- and c-Myb NR~1~R~2~R~3~(40 fmol each) were incubated separately with 20 fmol radiolabelled MRE-containing DNA probe, 0.5 ng poly(G) (lane 2),1 ng poly(G) (lane 3), 5 ng poly(G) (lane 4) and 100 ng poly(A) (lane 5). Homoribopolymers and proteins were first incubated for 15 minutes at 25°C. Then the DNA-probe was added to the mixtures and subjected to a new incubation of the same time and temperature. The binding reactions were analyzed by EMSA and autoradiography. The extra incubation step without probe was added to the experimental setup because it enhanced the inhibitory effect (lanes marked R in Fig. 3). ::: ![](1471-2091-5-15-5) ::: Discussion ========== The DBD of c-Myb contains three tandemly arranged pseudo-repeats, R~1~, R~2~and R~3~, among which R~2~and R~3~together are responsible for sequence-specific DNA-binding. The function of the first repeat, R~1~, has remained elusive in particular because R~1~does not directly contact DNA. Its involvement in the process of oncogenic activation of Myb suggests a specific biological role of R~1~. In this report we have focused on the N-terminal region including the R~1~repeat of c-Myb, trying to find a function beyond DNA-binding. A homoribopolymer inhibition assay revealed a strong preferential inhibition of sequence-specific DNA-binding by poly(G), suggesting that c-Myb can interact with RNA in a sequence-selective fashion. The most interesting observation was the finding that the RNA-interference function of c-Myb was highly dependent on the N-terminal region including R~1~. Upon exposure to RNA before DNA, the protein domain containing this region was severely inhibited by low concentrations of poly(G) while the protein domain lacking this region was almost unaffected. What the precise biological role of this novel RNA-interaction is, remains to be elucidated. Central to our examination of the RNA-binding hypothesis is a homoribopolymer inhibition assay. Differential binding to homoribopolymers has been exploited as evidence for RNA-binding activity of several proteins, like the Ets-related transcription factor PU.1 \[[@B41]\], the neuronal KH domain containing protein Nova-1 \[[@B43]\], the chloroplast ribosomal protein CS1 \[[@B44]\], and the recently cloned RRM domain containing Ciona intestinalis protein RGC \[[@B45]\]. Whether homoribopolymer binding reflects a sequence-specific RNA binding or a more general RNA-binding, like in the case of polypyrimidine-tract or poly(A) binding proteins, requires further analysis in each case. In addition to the data presented above, the SELEX (systematic evolution of ligands by exponential enrichment) technology was applied to search for more sequence-specific RNA patterns recognized by c-Myb. The SELEX procedure did produce specific patterns, confirming the proper behaviour of the experiment, but the selected RNAs did not seem to mimic the homoribopolymer effect in terms of inhibitory efficiency and content of G-bases (results not shown). This does not argue against RNA-binding per se, but indicates that c-Myb may not interact in a strictly sequence-specific fashion with RNA. Rather, we believe that the inhibitory RNA-effect seen in the homoribopolymer experiments reflects an R~1~-dependent RNA-interaction where G-rich RNA interacts more avidly than other RNAs. Why G-rich species are so much more potent inhibitors is not obvious. G-rich RNA molecules have special folding capacities that could be recognized by c-Myb. This is illustrated by the fact that poly(G) has been reported to fold into several unique structures, including single, double and four-stranded helices \[[@B47]-[@B49]\]. To investigate the possibility that poly(dG) had a similar effect on c-Myb DNA binding as poly(G), we added DNA oligonucleotides containing deoxy-guanine stretches of varying length (1 to 5) to EMSA reactions. However, we were not able to correlate the presence of poly(dG) stretches to any inhibitory effects (results not shown). Neither did we observe any strong inhibition when poly(I\C) or poly(I) was added. It is quite probable therefore that unique structural properties of poly(G) are critical to the mechanism of inhibition. The poly(G) inhibition was surprisingly strong. Due to the undefined length of the homoribopolymers, it was not evident how to precisely determine their molar concentrations. A fictitious poly(G)-length of 23 nucleotides, which is the length of the probe, gives an RNA-concentration of about 6 nM when 1 ng is added to the binding reactions. Consequently, a six-fold estimated molar excess of poly(G) compared to the probe concentration (1 nM) was sufficient to completely abolish sequence-specific DNA-binding (Fig. [3](#F3){ref-type="fig"}). We also analyzed the effects of a series of mononucleotides (results not shown). Interestingly, specific nucleotide triphosphates did in fact inhibit DNA-binding of c-Myb with differences resembling the pattern observed with ribopolymers in the present work. GTP produced the most prominent effect among the nucleotides, while CTP or UTP had little or no inhibitory effect. However, since the inhibition was observed first in the mM concentration range, significantly higher than the amount of poly(G) producing the same level of inhibition, the GTP phenomenon seems to be only a weak reflection of the strong inhibition we see with poly(G). Still, it is intriguing that we observe the same specificity suggesting some type of specific interactions between guanosines and the DNA-binding domain of c-Myb. The reported results represent evidence for an RNA-binding function of c-Myb. The dependence on the N-terminal region including R~1~is not total, in the sense that without this region all RNA-interference disappears. Rather, the presence of this region seems to enhance the sensitivity to RNA-mediated inhibition several fold, making it possible to find conditions where a Myb DBD containing this region becomes fully inhibited while a minimal DBD remains more or less unaffected (Fig. [4](#F4){ref-type="fig"}). At higher concentrations of poly(G) RNA, however, both proteins become inhibited, suggesting that other parts of the DBD are involved too. An appealing hypothesis would be that the flexible second repeat were involved in interactions with both types of macromolecules, cooperating with R~1~ for RNA-binding and with R~3~for DNA-binding. This would explain why this repeat appears to be a more flexible protein domain than the other repeats. It is noteworthy that a DNA-bound protein seems to be resistant to RNA-mediated inhibition and that an RNA-associated protein does not bind DNA even after prolonged incubation. We have previously shown that c-Myb R~2~R~3~undergoes a conformational change upon binding to DNA \[[@B14],[@B50],[@B51]\]. It is possible that the DNA-induced conformation is resistant to RNA-interference and that RNA induces another conformation that is unable to bind DNA; in other words that the two nucleic acids lock the protein in two distinct conformations. It could be argued that we have shown mainly experiments of the RNA-interference type, not directly demonstrating RNA-binding. We have, however, several lines of evidence indicating that RNA-interference occurs through direct binding of c-Myb DBD to RNA. c-Myb NR~1~R~2~R~3~was observed to interact with RNA in a North-Western experiment where R~2~R~3~did not, and c-Myb NR~1~R~2~R~3~bound to RNA-linked beads (results not shown). The identification of an increasing number of proteins capable of both DNA- and RNA-binding challenges the established picture of DNA-bound regulators with functions confined to promoter-activation, and suggests a broader function for some transcription factors \[[@B39]\]. The precise physiological role of the novel RNA-binding property of c-Myb remains to be elucidated. An interesting possibility to investigate is whether c-Myb plays a role beyond transcriptional activation in biological processes involving RNA, like splicing, capping, polyadenylation, nuclear export or transport of RNA. A role in one or several of these processes will fit the forthcoming model of a coupling between transcription and the post-transcriptional fate of mRNA \[[@B52],[@B53]\]. Conclusions =========== We have obtained evidence that c-Myb DNA-binding is preferentially inhibited by poly(G) RNA, indicative of a sequence-selective RNA binding function. The N-terminus of c-Myb, including the R~1~repeat, was shown to contribute substantially to this RNA-sensitivity. This finding suggests a more specific function of the enigmatic first Myb repeat than having a stabilizing effect on DNA-binding only. Methods ======= Homoribopolymers ---------------- All homoribopolymers were purchased from Sigma Aldrich. Purities were higher than 98% for all polymers. The length distributions of the homoribopolymers were determined by agarose gel electrophoresis and UV-shadowing. When compared to a dsDNA ladder, poly(A), poly(C), poly(G)and poly(U) migrated with a distribution corresponding to the following ranges: 200--1600 bp, 250--1200 bp, 100--700 bp and 250--700 bp, respectively (results not shown). RNase T1 was purchased from Ambion, Inc. Expression and purification of recombinant proteins --------------------------------------------------- The following DBD subdomains were expressed in E. coli(strain BL21 (DE3) LysS) using the T7 system \[[@B54]\]: Human c-Myb residue 1--192 (NR~1~R~2~R~3~) and 89--192 (R~2~R~3~), human A-Myb 1--187 (NR~1~R~2~R~3~) and B-Myb 1--183 (NR~1~R~2~R~3~). Expression and purification were performed as previously described \[[@B46]\]. Electrophoretic mobility shift assay ------------------------------------ Sequence-specific DNA-binding was examined by electrophoretic mobility shift assay (EMSA) as previously described \[[@B55]\]. The binding reactions were performed in 20 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 10% glycerol, 0.1 mM DTT, 0.005% Triton X-100 and 50 mM NaCl in a total volume of 20 μl. RNAguard (Amersham Biosciences) RNAse inhibitor (6--20 U) was added to each reaction to avoid degradation of the homoribopolymers. The sequence of the MRE-containing DNA probe was from the mim-1promoter: 5\'-GCATTA[TAACGGTTT]{.underline}TTTAGCGC-3\'. Double-stranded DNA oligonucleotides were ^32^P- labelled by T4 polynucleotide kinase according to the specifications of the manufacturer (Ready-To-Go T4 Polynucleotide kinase, Amersham Biosciences). Radiolabelled probe was purified on G-25 MicroSpin columns (Amersham Biosciences) or by polyacrylamide gel electrophoresis with subsequent gel extraction. Phosphorimaging --------------- EMSA gels were transferred to 3 MM paper and dried for 90 minutes at 80°C in a vacuum gel drier. Radiolabelled bands were detected with Molecular Imaging Screen BI (Bio-Rad) and analyzed with a Bio-Rad GS-250 PhosphorImager. Quantitation of band intensities was performed with the Molecular Analyst 2.0.1 software (Bio-Rad). Authors\' contributions ======================= ON performed the majority of the experiments and participated in the writing of the manuscript. TØA was responsible for the experiments shown in Fig. [1B](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}. OSG designed the study and participated in the writing. All three authors read and approved the final manuscript. Acknowledgements ================ This work was supported by The Norwegian Research Council, Norwegian Cancer Society and Anders Jahres Foundation. We thank Christophe Orvain for performing North-western analysis of RNA-binding, which supported the concept of c-Myb having RNA-binding properties.	PubMed Central	2024-06-05T03:55:49.772387	2004-11-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533864/", "journal": "BMC Biochem. 2004 Nov 4; 5:15", "authors": [ { "first": "Oddmund", "last": "Nordgård" }, { "first": "Tor Ø", "last": "Andersen" }, { "first": "Odd S", "last": "Gabrielsen" } ] }
PMC533865	Background ========== d-matrix, originally designed with cardiovascular clinical and molecular genetic data in mind, is a generic database front-end that can be used to explore, visualize and analyze different typologies of datasets. Both the generation and the analysis of genome, transcriptome and proteome data are becoming increasingly widespread, and these data must be merged to generate a molecular phenotype. Moreover, the correlation between molecular and phenotypical data requires acquiring both with comparable profoundness leading to the development of large and small scale databases holding both information \[[@B1]-[@B3]\]. In the same line, we developed a CardioVascular Genetic database (CVGdb), storing the detailed clinical phenotype of patients with congenital heart diseases as well as molecular data such as gene expression analysis results \[[@B4]\] and genotypes. However, querying and analyzing the stored data to uncover the valuable information hidden in the databases are difficult tasks. With some exceptions, these are approached by a two-step procedure, in which a database specific front-end serves the query and extraction of data, which are subsequently imported in stand-alone analysis tools for visualization, mining and statistics \[[@B5]-[@B11]\]. Moreover, the visualization and mining tools frequently focus on presenting overall views of data sets for a specific task and seldom permit single-case addressability or have drill-down capability. In today\'s systems, the perceptual abilities of human users are only used to a limited extend. We believe that it is essential to make users part of the overall process through computer support of their intelligence, creativity and perceptual abilities. Hence, a major research challenge is to find human-oriented forms of representing information and enabling rapid interaction between humans and computers in the query, visualization and analysis process \[[@B12]\]. It is not the purpose of this paper to survey the various solutions available to query, visualize and mine data, but rather to illustrate how such concepts could be combined usefully within one software tool. Here, the layout should not only preserve the structure of the information, it should also convey the quality of the distribution of the values contained in the database. The features of the display should then be designed to highlight those regularities, patterns or dependencies that are not easily detectable with an ordinary front-end. One visual representation, which motivated the graphical display of the tool we describe here, is the data matrices handled in microarray studies, in which rows in the matrices typically represent genes and columns individual samples \[[@B13]\]. Rather than showing a numerical \'spreadsheet\', it is convenient to display microarray data in such matrices, which indicate varying expression levels in a grid of varying colors. With d-matrix we propose a generic front-end solution capable of extracting, exploring, visualizing and analyzing complex data. The software can be interfaced with the most common relational database management systems without any intervention on the schema or pre-processing phase. As the name suggests, the visual model proposed has the form of a matrix. Its elements are boxes whose colors show the quality of the underlying information. The granularity of the data display allows consequent drill-down, i.e. the user is able to focus the observation on a single data point. In addition, value frequency bars are available to present compact overviews. It also offers the possibility to define categories using context-sensitive rules and to assign colors to classes. The direct implementation of a broad range of descriptive and advanced statistics together with a hierarchical sorting feature permits user-defined exploration of the data. Implementation ============== Data Model ---------- The process of developing a uniform web interface for disparate data sources is a complex task because of the variability in the data models that underlie each source. To enable an effective two-dimensional display, the d-matrix model consists of a three-level tree. For the representation of a large database schema requiring a higher number of levels, several d-matrix instances can be built on the same database. Within the proposed model, the main table addressing the objects of a study is considered as the root, the first level of the tree. The second level consists of tables that are joined with the root by means of its primary key and the third level consists of tables that are further joined with the ones at level two. In particular, the dependency of the root table with the second level tables can be either one-to-many or one-to-one, while the dependency of the second level table with the third level ones can be either many-to-one or one-to-one. To apply different query and visualization rules each branch of this tree is defined as a data group characterized by the same storing strategy. In cases where Entity-Attribute-Value (EAV) tables are interfaced, the Entity must correspond to the main ID. As an example, we can refer to the CardioVascular Genetics database (CVGdb) schema set up by our group (Figure [1](#F1){ref-type="fig"}). Here, we selected the table Patients as the root of the tree, so that the CVGdb instance main ID is the Patients primary key. This selection is arbitrary and one could also choose Clones or Hybridizations, thereby focusing on different aspects of the overall dataset. In Figure [2](#F2){ref-type="fig"}, data groups and tree levels are represented. The data groups 2 to 4 address the EAV tables Invasive\_Treatments, Medications and Samples; the groups 5 to 6 contain the same table Clones joined with different tables containing gene expression analysis results \[[@B4]\]; whereas the last group is built by two tables describing sequence variations (SV). Data selection and query ------------------------ The schema is presented to the user in a structure recalling a file system selector (Figure [3A](#F3){ref-type="fig"}). Nodes represent attributes or value attributes that can be optionally divided further into more folders without any depth limitation. Collecting nodes in visually distinct entities becomes a necessity when coping with a large number of attributes. To obtain a quantitative measure of the information that is contained within groups of nodes, a summary nodecan be included in the query form. For each value of the x-axis, the values of the summary nodes are computed by counting the nonempty nodes in the respective folders. The query process consists of two steps. First, the users select all nodes they want to be included in the query (Figure [3A](#F3){ref-type="fig"}); second, these are listed in a query form where conditions and analysis features can be specified (Figure [3B](#F3){ref-type="fig"}). To visually distinguish between nodes referring to data belonging to single-table data group and two-table data groups, single-table group nodes are represented as sheet-like-icons, whereas two-tables data group nodes are represented by double-arrow-like icons: diagonally oriented for the nodes that belong to the second level tables, and vertically oriented for the nodes that belong to the third level tables (Figure [3A](#F3){ref-type="fig"}). The attribute on which the query display shall be focused can be selected by means of the three-banded icons placed on the right side of the nodes. For each of the nodes, the query form permits the definition of sorting order and direction (ascendant/descendent), values and operators for query conditions, display order and parameters for statistical evaluations. The value cell is not shown if the node itself is an attribute value. Alternatively to the matrix view of the query result, the user can optionally export the resulting dataset in form of text or XML (Figure [3B](#F3){ref-type="fig"}). Data visualization ------------------ The graphical output of d-matrix consists of two-dimensional matrices, whose colored boxes code the meaning of the underlying information, the description of the chosen nodes and a prospect of statistical evaluations (Figure [4](#F4){ref-type="fig"}). The display of the data is determined by the data dimensionality. The main ID corresponds always to the x-axis of the matrix. To permit the display of single and multiple dependencies with the main ID, the y-axis shows either node descriptions or node values. In cases of single dependency each data point is represented by one box of the matrix. If there is a multiple dependency (two-table data groups), subsequently more rows for each value of a single node are displayed. EAV data groups can lead to both single or multiple dependency; in the second case the entries are aggregated in one matrix box. In Figure [4](#F4){ref-type="fig"} the tuples of the data group \"PHENOTYPES\" addressing the table Patients are displayed in the first matrix. Each tuple corresponds to a column whereas row headers are node descriptions. The tuples of the data group \"SEQUENCE VARIATIONS\" addressing the tables SV\_Genotypes and SV\_Loci are aggregated column-wise and grouped by the main ID. Here, there is more than one tuple for each column whereas row headers are values of the node Locus ID. Hence, each column of boxes on the matrix display represents an aggregation of more than one tuple of the query result. Following data mining terminology, we can say that in d-matrix cases(and aggregations of them) are represented column-wise. When the matrix oversize the available space, the use of two distinct scrollbars lets the user move the data matrix horizontally and vertically. The general overview is given together with the advantage of single-case addressability, i.e. each case (tuple) representation is entirely visible and its components clearly distinguishable. The display is obtained as a group of images (generated using the Perl GD module and stored as temporary files), each in a separate HTML DIV container, which can be moved independently. Drill-down ---------- The matrix display represents a summarized view of the query. Each box holds three levels of detail: first, the coordinates that uniquely identify the box position and represent two units of information; second, the color that corresponds to either a single value or a category; third, the hidden content of the box obtained by drill-down, which gives all remaining information for that box. In the d-matrix display the drill-down can be obtained for each box in form of a pop-up window (Figure [4](#F4){ref-type="fig"}). The content structure of this new window varies according to the data group to which the box belongs, although it always contains the value that is substituted by its color code together with the underlying node description. Further supplementary data can be included from attributes of the same data group. It is possible to add further detail by the mean of hyperlinks to grant access to remote databases, external analysis results and multimedia documents (Figure [4](#F4){ref-type="fig"}), or even to trigger further analysis processes. Schema interface and configuration ---------------------------------- The software requires four configuration files: the data definitions file that is needed to connect d-matrix with the relational schema, a database settings file storing the information to access the database, a color file for the definition of the colors used in the matrix and a general server settings file. Every configuration file is maintained as plain text to permit easy access and modification. The structure of the data definitions file must reflect the hierarchy in which the metadata (relational schema definition) have to be organized on the screen, while its textual content depicts a level of abstraction (definitional abstraction) \[[@B14]\] between the database physical representation and the human-comprehensible view of the data. Therefore, the data definitions file reflects the subdivision of the database schema in data groups. For each group the table attributes, information about identifiers, joining conditions as well as aggregation (where needed), display settings and the content of the pop-up window have to be defined. User-defined human-intelligible terms can be assigned for any term used in the database. Besides the attributes\' names, types and descriptions, it is possible to define categories, orderings and associations with colors. It is important to notice that the rules that define categories can even involve other attributes of the same data group. This context-sensitive categorization, intended as a qualitative abstraction\[[@B14]\], allows the concurrent representation of two layers of information. For each attribute value, value range or defined category, rules can be given to assign its respective color. This leads to a common method to visualize both discrete and continuous variables. In addition, categorized numeric values can be treated as categorical in specific contexts like sorting and statistics. Furthermore, colored boxes can be composed by combining the values of two nodes, which enables, for example, the visualization of both Alleles within horizontally split boxes for sequence variations (Figure [4](#F4){ref-type="fig"}). Several data definitions files (each defining a separate d-matrix instance) can independently coexist on the same server for the same or different database systems and schemata. Visual data mining and statistical analysis ------------------------------------------- d-matrix permits consecutive data-filtering operations that -- as a whole -- can be seen as a single user-driven data mining session. A compact and information-dense graphical outcome, context-sensitive categorization, hierarchical sorting and drill-down enable this mining process. Frequency bars give an overview of the overall queried dataset whereas box plots improve the visual perception of the data distribution. A key feature within the mining process is the opportunity to obtain different views of a single data set rapidly in parallel using different browser windows. Here, the interactivity becomes a primary factor and is supported by the human-oriented representation. A wide range of descriptive statistics and statistical tests is directly accessible. This permits statistical evaluation of the correlation between attributes and determination whether it is reasonable or not to assume that a sample fits to a specific distribution. For numerical values it is possible to perform up to ten different statistical tests, while for non-numerical entities (Boolean and categorical data) the Chi-square and Fisher exact tests are available. The user interface automatically performs a selection of attributes and tests according to their respective compatibility. In addition to directly implemented tests, external data analysis environments like R \[[@B15]\] or user defined routines can be easily interfaced. The results of the tests, together with the descriptive statistics, are displayed at the side of the matrix and colors of the boxes reflect the results (e.g. significance) of the tests. CardioVascular Genetics database (CVGdb) ---------------------------------------- For interfacing d-matrix with the CVGdb, we assigned categories if appropriate and colors to more than 700 nodes. Figure [5](#F5){ref-type="fig"} shows an example of a single user-driven data mining session, which was initiated with the aim to discover cardiac phenotype features associated with shunts abroad the interventricular septum (IVS shunt). Therefore, the only query condition specified is that \"IVS shunt\" is not \"NULL\". This condition is fulfilled by 211 out of 560 IDs stored to date. In addition, a subset of nodes referring to phenotype descriptions physically surrounding the interventricular septum has been chosen to be displayed. To structure the display, hierarchical sorting has been applied to the \'IVS shunt\' and an arbitrary selection of other nodes. Viewing the entrance matrix (Figure [5A](#F5){ref-type="fig"}), one could easily recognize data clusters such as the relation of the category \'bidirectional\' of the \'IVS shunt\' (blue boxes) to categories of interatrial septum shunts (IAS shunt) and right ventricular systolic pressure \'RV sys pressure\'. Almost all patients with a bidirectional \'IAS shunt\' are also characterized by a bidirectional \'IVS shunt\'. Furthermore, the majority of bidirectional \'IVS shunt\' is associated with severe \'RV sys pressure\', whereas the non-sorted nodes pulmonary valve morphology (PV morphology), pulmonary valve systolic pressure gradient (PV Psys gradient) and right ventricular anatomy (RV anatomy) are distributed in a questionable co-occurrence to each other in this first matrix. For further evaluation, we focus on the \'RV anatomy\' or the \'PV morphology\' chosen as the first sorted nodes in the second and third matrix (Figure [5B,5C](#F5){ref-type="fig"}), respectively. By using the tree save/reload option to retrieve these new matrices, only the sorting criteria needed to be modified to obtain different views on the same data set in parallel using three browser windows. Hence, the frequency bars remain the same in all visualization sessions. Now it becomes clear that more than half of the patients with infundibular stenosis (RV anatomy) show a stenotic \'PV morphology\', which by itself is highly associated with an extreme \'PV Psys gradient\'. Applying the correlation analysis implemented in d-matrix, the significance of the correlation of the \'PV Psys gradient\' with the \'RV sys pressure\' could be verified (Figure [5D](#F5){ref-type="fig"}). The described data are available for the exploration using d-matrix at the web supplement. Finally, the session explained is just one out of several examples in which d-matrix proved to be highly effective for the visualization of regularities and dependencies within the CVGdb data. Moreover, based on the general visualization concept, d-matrix provides an integration between clinical and genetic information that is crucial for the correlation of phenotypical and molecular data (Figure [4](#F4){ref-type="fig"}). Other applications ------------------ With respect to an ongoing project on gene regulation, we found it very convenient to visualize potential transcription factor binding sites (TFBS) in promotor sequences by interfacing d-matrix \[[@B16]\]. Here, the nucleotides are used as the main ID (x-axis) and the TFBS are consequently displayed at the y-axis. This allows a much higher level of interactivity than a usual figure output. One could easily have different views of the data set by sorting or parallel display of different information, like color coded core or matrix match similarities. To demonstrate the versatility of the software, we further interfaced d-matrix with a database that represents the periodic table of the elements \[[@B16]\]. Although we did not expect unusual or unexpected regularities in such a simple case, it was easy to obtain a matrix that shows the well-known dependency between Atomic Number, Atomic Mass and Energy Levels and the obvious lack of available information about elements with seven energy levels, which are the most unstable and rare. The interfacing with both dataset required only one working day for each. Results and discussion ====================== We have presented d-matrix, a non-task-specific database front-end with a new visualization strategy with embedded analysis features. The graphical outcome of d-matrix consists of colored boxes arranged in matrices; it permits single-case addressability with further drill-down capability. Together with the hierarchical sorting and statistical feedbacks, d-matrix enables consecutive data-filtering operations that -- as a whole -- can be considered as a single data mining session. Also, the result of such a session can be exported for further study. For a qualitative evaluation of d-matrix, one should not only focus the attention on the final display, which only represents the end product of a sequence of user-driven data exploration sessions. The high level of interactivity that our approach offers is indeed a primary factor; with d-matrix, the communication between human and computer is a rapid interaction. The future development of d-matrix will focus on the implementation of clustering algorithms to be executed before display. Furthermore, we envisage the design of instruments to inquire metadata to maximize the quantity of information that will be eventually displayed and analyzed \[[@B17]\]. In addition, a user-friendly way to interact with configuration files will be granted by specific CGI scripts leading to a further reduction of the time to interface d-matrix with relational schemata. An inquiry of the solutions reported to date for data exploration, visualization and analysis resulted in an approximate distinction between reports about efforts for database development with their task specific front-end solutions and stand-alone data analysis, visualization and mining tools. In our view, d-matrix stands in between those two groups and aims to combine features of both efforts, which we believe can be very effective and useful in general and especially for the association of distinct data types such as phenotypical and molecular data. As a front-end, it does not require complex installation processes or maintenance, and it is suitable for multi-user remote access. As a visual data mining tool, it gives an effective display that allows the detection of exceptions, trends, regularities, clusters and dependencies, as well as incomplete or erroneous data. Availability and requirements ============================= Project name:d-matrix Project home page:<http://www.molgen.mpg.de/~heart/index_dmatrix.html> Operating system(s):Platform independent Programming language:Perl Other requirements:d-matrix was successfully interfaced to Oracle 8i, MySQL, Microsoft Access and text-based databases and is compatible with recent JavaScript-enabled browsers. License:d-matrix is available on request from the author. To academic institutions d-matrix is available for a fee of 250 Euro that is intended to cover our costs of distribution and maintenance. Authors\' contributions ======================= DS developed the first generation of d-matrix and carried out the main programming work. RG is the current maintainer and carried out the main implementation. SM and HPS participated in the design, testing and quality control. HH participated in the conceptual design. SS conceived the development of d-matrix, managed and participated in its design and implementation. Acknowledgements ================ We thank Ulrike Klehmet for testing d-matrix at CVGdb as a non-biomedical scientist and Anja von Heydebreck for insightful discussions. We gratefully acknowledge the support of this work from the Max-Planck-Society and the BMBF grant 01GR0155. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Relational database schema of the CardioVascular Genetics database (CVGdb)Tables are represented as boxes and foreign keys constraints as arrows. Grey boxes mark the schema subset interfaced in d-matrix. SV -- Sequence Variations; RV\_versus\_LV, A\_versus\_V, RVH\_in\_RV, VSD\_in\_RA and TOF\_in\_RV are tables containing gene expression results \[4\]. ::: ![](1471-2105-5-168-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Data ModelShown is an excerpt of the three-level tree for interfacing d-matrix with CVGdb. The table \"Patients\" defines the root of the tree. Each branch refers to a defined data group consisting of one or two tables, respectively. ::: ![](1471-2105-5-168-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Data selection and queryWithin the data selection schema (A) users can choose all nodes they want to be included in the query. If a data group consists of two tables, the nodes are represented by vertical arrows for the first table and diagonal arrows for the second. The attribute on which the query display is focused can be selected by the three-banded icons, which switch from black-white to color and vice versa upon selection. Furthermore, trees can be saved and reloaded for subsequent analysis. Upon selection all nodes are listed in a secondary form (B), where query conditions, display and sorting order as well as the implementation of descriptive and advanced statistic can be specified. In addition to the graphical output, the query can be exported as a text of XML file. ::: ![](1471-2105-5-168-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Graphical output of d-matrixThe graphical output consists of the matrices itself, the description of the nodes displayed, a prospect of statistical evaluations and hyperlinks to external resources. Each matrix corresponds to a single data group (Phenotypes; Sequence variations). The x-axis of the matrix is defined by the main ID (Record) and the y-axis by the nodes displayed. The terms like \"Gender\", \"Age (Years)\" and \"IVS Shunt\" are descriptive names for the respective column names GENDER, AGE\_YEARS and IVS\_SHUNT of table PATIENTS; terms like \"Ichd0001\" and \"Ichd0002\" refer to locus names, values of the column LOCUS\_ID of table SEQ\_VAR\_LOCI. The matrix is built by colored boxes coding for the meaning of the information itself, which is further described in the pop-up window (as shown for Record 366 and Ichd0009). Frequency bars and boxes for descriptive statistics are displayed. Numbers are reflecting the sorting order, whereas blue boxes at the left border hold the hyperlinks. ::: ![](1471-2105-5-168-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Example of a data exploration session for CVGdbShown are the first 61 of 211 records that meet the query condition \"IVS shunt\" is not \"Null\" focusing on different views of the data given by different sorting options (A, B, C). To provide information about the color code as well as the overall query output, pop-up windows for frequency bars of sorted nodes are shown (D). Further, the pop-up window for the correlation analysis between \'RV sys pressure\' and PV Psys gradient\' is displayed (D). See text for detailed description of the observed cluster. ::: ![](1471-2105-5-168-5) :::	PubMed Central	2024-06-05T03:55:49.775265	2004-10-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533865/", "journal": "BMC Bioinformatics. 2004 Oct 28; 5:168", "authors": [ { "first": "Dominik", "last": "Seelow" }, { "first": "Raffaello", "last": "Galli" }, { "first": "Siegrun", "last": "Mebus" }, { "first": "Hans-Peter", "last": "Sperling" }, { "first": "Hans", "last": "Lehrach" }, { "first": "Silke", "last": "Sperling" } ] }
PMC533866	Background ========== The skin contains a system for producing serotonin as well as serotonin receptors. Serotonin can also cause pruritus when injected into the skin. SSRI-drugs increase serotonin concentrations and are known to have pruritus and other dermal side effects e.g.exanthema, purpura, urticaria and pruritus \[[@B1]\]. In contrast, SSRI-medication has also been used to treat pruritus associated with cholestasis \[[@B2]\] and polycythemia vera \[[@B3]\]. In this report we describe a patient who developed pruritus and skin rash from chocolate, but only when he was under SSRI-treatment. The case is presented and we provide a putative biological rationale for the described phenomenon. Case presentation ================= A 46-year-old man consulted his doctor in September 2003 due to depression. He had then experienced symptoms for a few years that had aggravated during the last six to eight months. Using the Montgomery-Åsberg Depression Rate Scale (MADRS) the patient scored 24 points and was diagnosed as having a clinical depression. He did not take any medication and had no regular medical contact. The patient did not have any history of allergy or dermatological diseases. However, he sometimes suffered from vasomotor rhinitis after drinking red wine. The doctor prescribed fluoxetine 20 mg daily as antidepressive treatment. At the revisit three weeks later the patient was very pleased with the fluoxetine treatment and reported that he \"had not felt better in 20 years\" although he initially had experienced slight nausea and insomnia. A week later, he visited his doctor due to an itching rash that had started the day before. The doctor noted partly confluent urticae on the abdomen, a modest periorbital oedema and red, warm palms and wrists. An ADR induced by fluoxetine was suspected and fluoxetine treatment was discontinued. The symptoms were treated with 2 mg clemastine and 6 mg betametasone orally and disappeared within 48 hours. However, the symptoms of depression returned. Sertraline medication was initiated 10 days after the cessation of fluoxetine treatment since SSRI medication had shown good effect. During the weeks of sertraline treatment no urticarial symptoms appeared. The patient improved in his depression although full recovery was not achieved this time. After approximately two weeks of sertraline treatment he noted an intense itching sensation in his scalp after eating a piece of chocolate cake. The itch spread to the arms, abdomen and legs within a few hours. This time the patient did not seek his doctor but treated himself with clemastine and the itch disappeared during the night. He now remembered that he had had a chocolate mousse dessert before the first episode. Since he had never had any reaction from eating chocolate before, he found this observation so striking that he reported it to his doctor. The patient, himself a scientist, later tried small doses of chocolate and skin rash and itch appeared at an intensity that to him seemed dependent on the \"dose\" of chocolate ingested. It has been known for 30 years that serotonin can stimulate cutaneous C-fibres \[[@B4]\], the type of fibres that is also known to transmit itch \[[@B5]\]. Moreover, serotonin injections into the skin can induce itch \[[@B6]\] and pruritus is a component in 24% of reported skin reactions to fluoxetine in Sweden, the corresponding figure for sertraline is 15 % \[[@B1]\]. However, attempts to treat pruritus using 5-HT3-receptor-antagonists have not given clear-cut results \[[@B6]-[@B8]\]. The enzymes necessary for conversion of tryptophan to serotonin are expressed in human skin \[[@B9]\]. In addition, 5-HT2AR are present in one third of unmyelinated axons at the dermal and epidermo-dermal junctional area \[[@B10]\]. An altered localisation pattern of serotonin receptors 5-HT1AR, 5-HT2AR and 5-HT3R has been reported in contact eczematous skin together with increased serotonin concentrations \[[@B11],[@B12]\] indicating the presence of a serotonin system in the skin that can be altered in pathologic conditions. Moreover, a cross-sensitivity has been reported when skin rash developed after both paroxetine and sertraline medication \[[@B13]\]. Since these substances are structurally different, one interpretation is that the skin can react to an SSRI-induced increase in serotonin concentrations. In the present case the patient experienced skin symptoms from two different SSRIs. However, these symptoms occurred only when he had eaten chocolate. Chocolate contains serotonin, at concentrations which depend on the type of chocolate \[[@B14]\]. A concentration of 1.4 -- 5 μg / g has been reported in dark chocolate \[[@B14]\]. The present report suggests an interaction between SSRI-medication and chocolate leading to pruritus and rash. A plausible explanation is that SSRI together with serotonin-containing chocolate has increased serotonin concentration to a level where 5-HT receptors system at the dermal and epidermo-dermal junctional area are affected. Moreover, the patient in this case had previously noted nasal congestion and cough when he was drinking red wine. Red wine can induce release of serotonin from platelets \[[@B15]\] and from the gut \[[@B16]\]. Serotonin can induce nasal itch, sneeze and hypersecretion \[[@B17],[@B18]\]. Conclusions =========== Apart from the SSRI -- chocolate interaction this patient had another possible sign of sensitivity to serotonin. The present case thus suggests that there may be individuals that are very sensitive to increases in serotonin concentrations. Skin side reactions to SSRI-drugs in these patients may be due to high activity in the serotonergic system system at the dermal and epidermo-dermal junctional area rather than a hypersensitivity to the drug molecule itself. However, the reaction of skin to serotonin from food is poorly studied and further studies are necessary to determine how much alimentary serotonin can increase serum serotonin concentrations and to what extent SSRI-medication affects this process. More knowledge in this field could be of help for physicians who encounter patients with dermal reactions to SSRI-drugs and there might be food and beverages containing serotonin that these patients should avoid. Moreover, possible individual differences in the serotonergic system at the dermal-epidermal junction remain to be studied. What happened to the patient and his depression? Due to poor anti-depressive effect of sertraline, the treatment was altered back to fluoxetine. He is now free from his depression and experiences no rash or oedema-like adverse reactions as long as he is avoiding chocolate. List of abbreviations ===================== 5-HT: 5-hydroxytryptamine, ADR: Adverse Drug Reaction, SSRI: serotonin selective reuptake inhibitors Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= SS first described the case, JC and HM performed literature searches and JC first drafted the manuscript. HM and SK took part in the scientific discussion and in finalising the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-244X/4/36/prepub> Acknowledgements ================ Written consent was obtained from the patient for publication of study.	PubMed Central	2024-06-05T03:55:49.778014	2004-11-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533866/", "journal": "BMC Psychiatry. 2004 Nov 2; 4:36", "authors": [ { "first": "Jonas", "last": "Cederberg" }, { "first": "Stefan", "last": "Knight" }, { "first": "Svante", "last": "Svenson" }, { "first": "Håkan", "last": "Melhus" } ] }
PMC533867	Background ========== The advent of large genome projects has led to an explosion of sequence data in public databases. Analysis of protein families, understanding their evolutionary trends and detection of remote homologues are now the primary objectives. Genome annotation and analysis tools like fold prediction, homology modelling, protein-ligand docking and clustering algorithms rely heavily on accurate multiple alignments to provide a genome-wide perspective. The most popular approach for multiple sequence alignment has been the progressive alignment method \[[@B1]\]. A multiple alignment is built up gradually by aligning the closest sequences first and successively adding in more distant relatives. A number of alignment programs imply this algorithm, for example MULTALIGN \[[@B2]\], MULTAL \[[@B3]\] and CLUSTALX \[[@B4]\]. They employ a global alignment algorithm to construct an alignment over the entire length of the sequences and differ mainly in the procedure employed to determine the order of alignment of the sequences. The most common usage is the sequential branching method to identify two closest sequences first and subsequently align the next closest sequence to those already aligned. MULTALIGN \[[@B2]\] constructs a guided tree using UPGMA \[[@B5]\] method. A consensus method is then used to align larger and larger groups of sequences according to the branching order of the tree. CLUSTALX uses the alternative neighbour-joining algorithm \[[@B6]\] to construct the tree. In contrast to the above global method, PIMA \[[@B7]\] uses a local dynamic programming algorithm to align only the most conserved motifs. In addition, numerous new alignment algorithms have recently been developed which offer fresh approaches to the multiple alignment problem. A common point of interest has been the application of iterative strategies to refine and improve the initial alignment. A local alignment approach is implemented in the DIALIGN program \[[@B8],[@B9]\] to construct multiple alignments based on segment-to-segment comparison rather than residue-to-residue comparison using an iterative strategy to improve alignment accuracy. Alignment programs like MATCH-BOX \[[@B10]\] utilize statistical similarity measures to delineate sequentially conserved regions and the final alignments are derived by those of the conserved \"box\" regions. The regions outside the \"boxes\" are not aligned. METAMEME \[[@B11]\] is a motif based search engine that aligns motif regions found in the target sequences. DbClustal \[[@B12]\] combines the advantages of both local and global alignment algorithm in a traditional tree-based progressive alignment. Starting from ClustalW \[[@B22]\], which is a global alignment program, local alignment data or anchor points are incorporated in the dataset. The global alignment is then weighted towards, but not constrained, to the locally conserved segments and the alignment is not subject to iterative refinements. MACAW \[[@B13]\] provides a user interactive interface to select conserved segments from the alignment but these segments are not utilized further to refine the resulting alignment. In this paper, we present an alignment algorithm that combines the properties of both progressive alignment methods and iterative refinement algorithms. This algorithm offers the user the selective advantage of guiding the course of alignment by simultaneous inputs of multiple conserved motif regions that in turn guarantees retention of structure/function in the final alignment. FMALIGN is an alignment server that provides the user to obtain a control over the alignment by providing important conserved regions as input to the alignment program to achieve a more structurally relevant and functionally useful alignment of protein sequences. It employs the sequential branching method to identify the closest pair of sequences and subsequently includes the next closest sequences to generate a guided tree using UPGMA \[[@B5]\], which in turn dictates the sequential order of the alignment. FMALIGN also considers the local similarity of the sequences in the conserved motif regions; as the name implies, it allows local conserved regions of the sequences to be fixed and aligns the rest based on normal progressive alignment. The chances of global misalignment are thereby reduced and the possibility of obtaining overall better alignment is increased. The FMALIGN server also offers an iterative refinement option where a routine (FINDMOTIF) identifies more conserved regions in the derived alignment and allows the user to provide fresh \'equivalences\' to obtain an overall better alignment. Benchmarking studies on difficult alignments, examined in BAliBASE \[[@B14]\], show promising prospect for the FMALIGN server to be an useful alignment algorithm. Implementation: methodology and description of FMALIGN server ------------------------------------------------------------- The algorithm of FMALIGN (Fixed Motif ALIGNment) program combines three main criteria: (a) Progressive global alignment method, (b) substructure (local) conserved region fixation and (c) iterative refinement of alignment with identification of more conserved regions. The procedure involves three steps: (i) identification and fixing of the specified sequential conserved regions. This alignment method requires specific regions of the sequences to be aligned as anchor and these anchors are generally meant for sequentially conserved parts which do not undergo many changes. (ii) derivation of the progressive multiple sequence alignment guided by the tree. During this step, excluding the fixed anchored regions, the rest of the sequences are divided into several sub-segments that are aligned employing a dynamic alignment algorithm in a sequential order from N to C-terminus. The phylogenetic guide tree has been derived using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) \[[@B5]\] which is the simplest method of tree construction. The UPGMA method was adopted since the whole length of the protein is divided into several segments considering them as ultrametric After all the sub-segment alignments are performed, the aligned parts as well as the selected, fixed motif regions are combined to produce the full-length alignment for a group of proteins under the hierarchical cluster. This process continues until all the protein sequences are aligned multiply. (iii) The iterative refinement of alignment subsequent to the identification of more motifs. In this step, more conserved regions are identified by observing the amino acid exchanges in the resultant alignment derived from the previous iteration. These conserved regions are then used as anchors together with previously identified motifs and the whole process is repeated until an optimal alignment having maximum number of conserved regions and alignment score is obtained. The algorithm thus combines the progressive dynamic algorithm for global multiple alignment and selected conserved regions or local alignments. Once the primary alignment is derived, the second step can be repeated by including more conserved regions from this alignment as motifs to derive a better alignment. Figure [1](#F1){ref-type="fig"} shows a cartoon representation of the methods in a flowchart diagram. FMALIGN server can accept amino acid patterns for multiple motifs provided by the user. It also provides option to the user to search motifs within their sequences using FINDMOTIF routine or to obtain them for the alignment by providing a link to SMoS \[[@B15]\], a structural motif database for aligned protein superfamilies. The FINDMOTIF routine in the server provides sequential conserved regions for a set of proteins on the basis of sequence similarity and a 20 × 20 substitution matrix by consulting large number of structure-based sequence alignments of homologous families \[[@B16]\]. Amino acid exchange scores at every alignment position are assigned the same as the element in this matrix for all possible pairs of proteins and averaged over the number of pairs. Contiguous alignment positions with an average amino acid exchange score over 50 (for homologues) or 40 (for superfamilies) are recognised as motifs. FMALIGN also offers an option for the user to refine the derived alignment by generating more sequential conserved regions through FINDMOTIF option. The inter-motif regions are aligned by normal progressive alignment using standard substitution matrices like BLOSUM62. The gap penalties used in this version of FMALIGN are all maintained according to standard multiple alignment parameters. ### Alignment scores To assess the performance of FMALIGN in comparison to other programs, Sum-of-Pair-score (SPS) and Column-Score (CS) alignment scoring scheme \[[@B14]\] are applied on FMALIGN derived alignments to assess the quality of alignments compared to BAliBASE reference alignments. SPS and CS are calculated such that the score increases with the number of sequences aligned accurately and is used to determine the extent to which the programs succeed in aligning some, if not all, of the sequences in an alignment correctly \[[@B14]\]. The scores used to measure the performance of the various alignment programs may not be appropriate for all the datasets. Therefore, for each reference test the most suitable scoring function have been selected according to the nature of the benchmarking. Results and discussion ====================== Benchmarking at family level ---------------------------- FMALIGN derived alignments retain high degree of conservation in secondary structures. Six members from the globin family were selected comprising a wide range of sequence identity between them (7% to 61%). Conserved regions for six aligned globin sequences were identified by the FINDMOTIF routine starting from CLUSTALX \[[@B4]\] alignment with default parameters and aligned by the FMALIGN server. The resultant alignment shows high degree of secondary structural conservation despite the difficulty of aligning a set of sequences having very wide range of sequence identity (Figure [2](#F2){ref-type="fig"}). In order to evaluate the FMALIGN server, the results have been compared with 10 other alignment programs and objective criteria were employed to assess the quality of an alignment. We selected the BAliBASE benchmark alignment database \[[@B14]\] to compare the performance of the FMALIGN alignment server. The BAliBASE benchmark alignment database contains 142 reference alignments, divided into five reference sets each containing at least 12 representative alignments. Performance of FMALIGN is checked on all five reference sets provided in BAliBASE datasets. Reference 1 alignments consist of a small number of equidistant sequences of similar length, i.e. the percent residue identity (% ID) between any two sequences is within a specified range and no large extensions or insertions have been introduced. Reference 2 contains alignments of a family of closely related sequences with \>25% ID, plus up to three \'orphan\' sequences (distant members of the family with \<20% ID, sharing a common fold). It is designed to evaluate program accuracy according to two criteria: (i) the stability of the family alignment when orphans are introduced into the sequence set and (ii)the quality of the alignment of the orphan sequences. Reference 3 checks the ability of the programs to correctly align equidistant divergent families into a single alignment. The reference alignments consist of up to four families, with \<25% ID between any two sequences from different families. Reference 4 and 5 contain alignments of upto 20 sequences including N/C-terminal extensions (upto 400 residues) and insertions (upto 100 residues), respectively. ### Reference 1: a small number of approximately equidistant sequences This dataset is designed to study the effect of sequence length and percentage identity on the performance of the alignment program and provides a basis for the remaining tests. The overall performance of FMALIGN server for this dataset is comparable to the two best performing alignment programs, like PRRP \[[@B17]\] and CLUSTALX \[[@B4]\], in all three categories (VI, V2, and V3) based on sequence identity and alignment lengths (short, medium and long) as shown in Figure [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}. ### Reference 2: a related family with divergent, orphan sequences It is possible to assess the performance of the methods to align divergent \'orphan\' sequences (10--20% ID with the family and between orphans) with a family of highly related (\>25% ID) sequences using this data set. It is also interesting to observe the disruption of the family alignment due to the introduction of orphans. Figure [6](#F6){ref-type="fig"} shows SPS for the alignment of a single orphan against a closely related family. The global alignment programs again perform better than the local ones in this test. However, CLUSTALX and SAGA \[[@B18]\] now rank above PRRP. The performance of FMALIGN server is significantly better than other programs for all the three length categories. ### Reference 3: families of related sequences This allows the assessment of the programs to correctly align approximately equidistant divergent families (\<20% ID) composed of highly related sequences (\>25% ID) into a single multiple alignment. Figure [7](#F7){ref-type="fig"} shows the scores for the programs in the order. The iterative strategies of PRRP \[[@B17]\] and SAGA \[[@B18]\] perform better in this test than the traditional progressive alignment methods. However, FMALIGN performs better than the other progressive methods, with the global methods generally ranking higher than the local methods. ### Reference 4: N/C-terminal extensions This dataset includes large N/C-terminal extensions to investigate whether the programs are capable of aligning the core blocks flanking the extensions. No large internal insertions are introduced at this stage. Mostly local alignment strategies out-perform the global methods. PILEUP (Wisconsin Package v.8; Genetics Computer Group, Madison, WI) is the only program based on a global alignment method which does reasonably well compared to other global methods. Performance of FMALIGN is comparable to the best three methods (DIALIGN \[[@B8]\], SB\_PIMA \[[@B19]\] and PILEUP) as shown in Figure [8](#F8){ref-type="fig"}. ### Reference 5: internal insertions In contrast to reference 4, in this dataset the insertions are internal to the homologous domains and not at the N/C-terminus as overhangs. FMALIGN also performs well in this category and results are comparable to the other better performing global alignment methods like PRRP and CLUSTALX as shown in Figure [8](#F8){ref-type="fig"}. Benchmarking at superfamily level --------------------------------- ### Utilization of structural motifs to improve alignment accuracy The performance of FMALIGN has been tested on a dataset of representative superfamilies of proteins belonging to different structural classes in PASS2 \[[@B20]\] and SMoS \[[@B15]\] databases. The structural motifs from the SMoS database have been utilized as conserved regions for the member of the superfamily (average sequence identity less than 30%). All the proteins of a superfamily have been aligned. Each alignment is assigned a quality score by averaging the amino acid exchange score of each column over the length of the alignment (see method for details). The alignments derived from FMALIGN have been compared against the CLUSTALX-derived sequence alignment as well as the sequence-structure alignment derived from COMPARER \[[@B21]\]. Figure [9](#F9){ref-type="fig"} shows an equivalent or better accuracy of the FMALIGN server compared to CLUSTALX and COMPARER-derived alignment. This indicates that FMALIGN is particularly efficient for specific sets of sequences for which the degree of conservation is known. The initial results also indicate that FMALIGN can perform very well and provide an alignment which is very similar or better than a structurally derived alignment. ### Utilization of sequence motifs to improve alignment accuracy Representative superfamily alignments belonging to different structural classes from the PASS2 \[[@B20]\] superfamily alignment database are taken for a benchmarking test (Table [1](#T1){ref-type="table"}). These superfamily alignments are of different lengths (short, medium, and long) and possess an average sequence identity that ranges from 10% to 26%. Sequential conserved regions or motifs are identified for each superfamily alignment and utilized in the FMALIGN server to realign the superfamily members. Similarly, the superfamily members are also aligned by the two best performing multiple alignment methods, CLUSTALW \[[@B22]\] and T-Coffee \[[@B23]\]. These alignments are then compared against the structure-based COMPARER alignments provided by PASS2 database using the same alignment scoring scheme (Sum-of-Pair score) of the BAliBASE benchmarking database. FMALIGN-derived alignments performed better for most of the cases compared to the other two methods as shown in Figure [10](#F10){ref-type="fig"}. Conclusions =========== FMALIGN server provides a web interface for pairwise and multiple sequence alignments of proteins. FMALIGN provides an alignment by combining progressive dynamic algorithm, local substructure alignment and iterative refinement presenting an improved, user interactive alignment procedure. FMALIGN server allows the user to fix conserved regions in equivalent positions amongst the sequences to be aligned leading to alignments that are reliable and biologically more meaningful. Additional options for the users to choose substitution matrices and gap penalty values may be incorporated in future. All the alignments provided in BAliBASE are realigned by FMALIGN and the resulting scores (both SPS and CS) are calculated using a standard program kindly provided by the authors of BAliBASE. The inter-motif regions are aligned in FMALIGN by normal progressive alignment using standard substitution matrices like BLOSUM62. The gap penalties used in this version of FMALIGN are all maintained according to standard multiple alignment parameters. Benchmarking at the superfamily level has also been done utilizing both structural and sequential conserved regions. The dataset is wide enough to include proteins from different structural classes and of different length. The average sequence identity for this dataset was less than 30% since the proteins are related at the superfamily level. The performance of FMALIGN has been tested against one of the best performing multiple sequence alignment (T-Coffee) as well as structure based alignment tool (COMPARER). Studies on large and different datasets revealed an overall better performance of FMALIGN server for all categories in BAliBASE benchmarking database. It works especially well at lower sequence identity range, such as superfamily level, where no two proteins are more than 25% identical to each other in comparison to other popular methods like CLUSTALX and T-Coffee. It is well-known that automatic multiple sequence alignments at poor sequence identity are often subject to careful manual validations and improvements to avoid offsets of critical functionally important residues. FMALIGN can sensitively address this issue to avoid manual intervention subsequent to final alignment. FMALIGN-derived alignments also show a high conservation of secondary structural elements and provide better alignments for comparative modelling. The implementation of this alignment algorithm can be used to include new members into an existing protein superfamily with the help of motif regions that provide a reliable approach to connect protein sequences with their structural homologues within a particular superfamily. FMALIGN is available via the following URL <http://caps.ncbs.res.in/FMALIGN/Home.html> Authors\' contributions ======================= S.C. had carried out the benchmarking, was involved in the development of the server and has written the first draft of the manuscript. N.B. had written the initial part of the code and P.A. has written the latter part of the code and developed a web server. R.S. had initiated the idea, was involved in discussions and in the critical reading of the manuscript. List of abbreviations ===================== FMALIGN [F]{.underline}ixed [M]{.underline}otif [ALIGN]{.underline}ment SPS Sum-of-Pairs Score Acknowledgements ================ R.S. is a Senior Research Fellow funded by the Wellcome Trust Grant. We would also like to acknowledge National Centre for Biological Sciences (TIFR) for financial support. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Cartoon representation of the flowchart of FMALIGN methodology. Coloured parts are meant to be important segments of protein sequences. ::: ![](1471-2105-5-167-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Six members from globin family are selected and aligned by FMALIGN utilizing sequentially conserved regions. The structure-annotated JOY \[24\] representation of the alignment shows high conservation of structural features retained in this alignment. Capital and small letters represent solvent-buried and solvent-exposed residues, respectively. Helices and β-strands are represented in red and blue colours, respectively. Concensus secondary structures are marked. Hydrogen bonding patterns are also annotated: hydrogen bond to main chain and side chain are indicated in bold and underlined, respectively. Residues with positive-φ conformation are italised. ::: ![](1471-2105-5-167-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### SPS for reference 1 for all three categories. a) SPS scores are shown for VI (\<25% sequence identity) (b) for V2 (20%--40% sequence identity), and (c) V3 (\>35% sequence identity). Alignments of different length categories are shown by different line plot. ::: ![](1471-2105-5-167-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### SPS scores for reference 1 for V2 (20% -40% sequence identity). Alignments of different length categories are shown by different line plot. ::: ![](1471-2105-5-167-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### SPS scores for reference 1 for V3 (\>35% sequence identity). Alignments of different length categories are shown by different line plot. ::: ![](1471-2105-5-167-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Alignment scores (SPS) are compared for reference 2. Alignments of different length categories are shown by different line in the plot. ::: ![](1471-2105-5-167-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Alignment scores (CS) are compared for reference 3. Alignments of different length categories are shown by different line in the plot. ::: ![](1471-2105-5-167-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Alignment scores (CS) are compared for reference 4 and 5. ::: ![](1471-2105-5-167-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Comparison of alignment accuracy on the basis of sequence similarity. Conserved motifs are identified for superfamilies like, Cytochromes (code: 02.01.050), ACP-like (02.01.060), C2 domain (Calcium/lipid-binding domain, CaLB) (code: 02.02.027), ISP domain (code: 02.02.058), Phosphatidylinositol-specific phospholipase C (PI-PLC) (code: 02.03.018), Esterase/acetylhydrolase (02.03.054), Chromo domain-like (code: 02.04.010) and BPTI-like (02.07.023). These motifs are utilized to realign the sequences by FMALIGN. Alignments are scored using sequence similarity scores based on a 20x20 substitution matrix. FMALIGN derived alignments are compared against CLUSTALX \[4\] and sequence-structure alignment, COMPARER \[21\]. ::: ![](1471-2105-5-167-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### Comparison of alignment accuracy. FMALIGN derived alignments of the PASS2 \[20\] superfamily members are compared against the same derived by CLUSTALW \[22\] and T-Coffee \[23\]. ::: ![](1471-2105-5-167-10) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### List of superfamilies used for FMALIGN benchmarking. ::: Superfamily code Superfamily name Structural Class Average length of proteins Average sequence identity(%) ---------------------- ---------------------------------------------------------- ---------------------- -------------------------------- ---------------------------------- 02.01.001 Globin-like All α 142 15.2 02.01.023 Putative DNA-binding domain All α 76 10.0 02.01.050 Cytochromes All α 122 22.0 02.01.060 ACP-like All α 80 23.5 02.01.101 SAM/Pointed domain All α 84 13.3 02.02.027 C2 domain(Calcium/lipid-binding domain, CaLB) All β 75 11.4 02.02.042 Galactose-binding domain-like All β 185 12.6 02.02.058 ISP domain All β 136 23.4 02.02.094 Acid proteases All β 217 20.4 02.02.152 Hedgehog/intein (Hint) domain All β 177 16.9 02.03.018 Phosphatidylinositol-specific phospholipase C (PI-PLC) α and β 286 13.6 02.03.059 Ferredoxin reductase-like, C-terminal NADP-linked domain α and β 132 21.6 02.03.073 Thiamin diphosphate-binding fold (THDP-binding) α and β 236 12.1 02.03.148 \"Helical backbone\" metal receptor α and β 320 14.4 02.04.010 Chromo domain-like α and β 68 26.3 02.04.088 Regulatory domain in the amino acid metabolism α and β 90 12.2 02.04.218 Ribosome inactivating proteins (RIP) α and β 253 22.1 02.07.017 Leech antihemostatic proteins α and β 47 19.1 :::	PubMed Central	2024-06-05T03:55:49.778975	2004-10-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533867/", "journal": "BMC Bioinformatics. 2004 Oct 28; 5:167", "authors": [ { "first": "Saikat", "last": "Chakrabarti" }, { "first": "Nitin", "last": "Bhardwaj" }, { "first": "Prem A", "last": "Anand" }, { "first": "Ramanathan", "last": "Sowdhamini" } ] }
PMC533868	Background ========== Expressed sequence tag (EST) collections represent partial descriptions of the transcribed portions of genomes. They are generated from single-pass cDNA library sequencing that is often carried out by small or mid-size sequencing centers and research groups. The research may be aimed at transcriptome expression analysis using microarray, RT-PCR, or hybridization techniques. An increasing number of research centers are involved in sequencing multiple cDNA libraries. This is manifest by the exponential growth in the number of ESTs deposited to dbEST at NCBI (National Center for Biotechnology Information), and as of the May 21, 2004 update, that number is at over 21.2 million ESTs from 686 species. Small sequencing centers are faced with searching for cost-effective and convenient tools for EST management and querying, visualization, public and private user access, and functional classification. EST processing pipelines that transform raw chromatograms into high-quality filtered sequences, and software to cluster and assemble them into contigs are convenient to implement, and various tools are now available to researchers (StackPACK \[[@B1]\], ESTWeb \[[@B2]\], ESTAP \[[@B3]\], PipeOnline2.0 \[[@B4]\]). The TIGR gene indices \[[@B5]\], for example, are generated by performing an all-against-all pairwise similarity search of the ESTs, and then clustered via single-node transitive closure. The clusters are fed into CAP3 \[[@B6]\] for assembly. We have used the following assembly protocol for in-house sequencing projects such as cattle \[[@B7]\] and honeybee \[[@B8]\]. High quality sequences are pooled and run through BlastClust \[[@B9]\] to form clusters of similar sequences. Then the BlastClust output is run through CAP3. Commercial software (such as Paracel \[[@B10]\]) that is targeted for bioinformatics centers equipped with high-performance computing machines is also available. We describe ESTIMA (EST Information Management and Annotation) software that provides a database schema for management of raw and annotated ESTs, and is coupled with a suite of custom web-based tools that facilitate searching various aspects of ESTs and contigs, visualization, pairwise searching by BLAST \[[@B9]\], and functional classification based on the controlled vocabulary defined by the Gene Ontology (GO) Consortium \[[@B11]\]. ESTIMA accepts assembled sequences from any EST processing and clustering software, and has been equipped with password protection so that project researchers can use the ESTIMA tool confidentially with academic or industry collaborators. To form an association between a GO term and an EST, an organism that has already been annotated with the GO terms is selected. The ESTs are searched against this reference organism\'s annotated sequences, and the resulting alignments are used to ascribe putative function. The ESTIMA database schema provides users the flexibility to use any reference database that has GO term association, and any number of external databases, such as NCBI\'s non-redundant (nr) database or EMBL\'s Swissprot collections, to annotate ESTs and their contigs. This flexibility in the schema is critical given the increasing number of sequenced genomes, and specialized reference databases that researchers can download and integrate with existing annotations. Other database schemas that house EST project and analysis data have been reported \[[@B3],[@B12]\]. But the inherent flexibility of multiple EST project management that ESTIMA affords, by allowing users to create multiple instances of the PROJECT schema within the GENOME schema (see section on databases), and allowing any number of reference genomes to be added, is unique to ESTIMA. The inputs to ESTIMA are the chromatograms, raw and processed ESTs, clusters and assembled EST contigs from any source, reference database annotations of ESTs, and contigs. All of these get loaded into the tables generated from the ESTIMA database schema and loading scripts. The raw and processed data, with their annotations, are made available to users through the ESTIMA query interfaces, for exporting, visualization, and further research. Implementation ============== ESTIMA is composed of three major components: an ODBC-compliant database, loading scripts and a web application. Figure [1](#F1){ref-type="fig"} shows the relationship of these components. Databases --------- The heart of the system is a pair of database schemas. Figure [2](#F2){ref-type="fig"} shows an ER diagram describing both schemas. The first schema, GENOME is common to all projects in the installation. It houses tables containing the GO structure, gene association information, annotation, and project security. GO terms are stored in two tables. Each GO term has a record in the Term table. The Term2Term table contains one record for each edge in the directional graph linking the terms. The graph can then be searched by starting with a term identifier (ID), finding all child IDs, then finding all records where each of the child terms appear as a parent. In this way, the entire graph can be searched with one call per tier below the original term. Term\_Seq\_Count contains the pre-calculated counts of ESTs associated with each term, and all the terms\' child terms. Pre-calculation saves time at execution, and is handled by a Perl script that is rerun each time the GO tables are updated. The Gene\_Association and Annotation table contains information that links the ESTs to the reference genomes. The Gene\_Association table has one record for each reference gene that is associated with an EST or contig. The reference gene is then related to a GO term in the Term table. GENOME also contains the Blast\_All table that holds information regarding the association between the ESTs and any project-specific DNA sequences. The ESTs are searched against sequences of interest, such as NCBI\'s nr, and the results are parsed and loaded into the Blast\_All table. In addition to the common data, each project has its own schema that contains information such as DNA sequences, contig assemblies and paths to the chromatogram files. The PROJECT schema is centered on the Project table that contains a simple link between the other PROJECT tables and the GENOME schema. This organization has proven useful when the contents of the PROJECT schema are to be transferred directly from a sequencing facility to a Laboratory Information Management System (LIMS). Each project may have several different contig assemblies deriving from different assembly techniques. The assemblies are all housed in the Assembly\_Project, Contig, Consensus\_Link and Singlet tables. Each assembly has a single ID that is used by the web application to determine which is \"live\". After a new assembly is completed and loaded, it can be taken live by simply changing a single number in the web applications configuration file. Finally, the sequence data itself is stored in the DNA\_Sequence table, while related GenBank accession numbers are stored in their own table. Although accession numbers are not required by the web application, if they are available, then the web application will accept them where a sequence ID would be used. Loading ------- The second major component of the ESTIMA system is a series of Perl and JAVA applications used to parse and load data into the database. Because each project will have its own needs, the loading system was not automated. Instead, several dozen separate scripts and applications have been developed to allow researchers to manipulate and analyze large data sets using standard analysis applications. Web application --------------- The third component of ESTIMA is the web-application that queries and reports the EST information to the end-users. The ESTIMA web application is organized around seven points of entry into the system -- the start screen and six query applications. Each query application interfaces with the databases, and allow users to query raw and annotated ESTs and contigs. ESTIMA supports password-protection, multiple-projects, and multiple libraries within a project. This is achieved with an XML configuration file with project-specific information that is called by the various applications when project-related information is needed. Figure [3](#F3){ref-type="fig"} shows the relationship between the components of the web application. Their implementation is discussed in detail below. ### The start screen The start screen provides a convenient point of reference to the system. The page contains information about each library (genomic, cDNA, etc) in the project. All of the elements of the start page are contained within the configuration file. ### GO Browser The GO Browser (Figure [4](#F4){ref-type="fig"}) allows a researcher to start with a GO term, and find all ESTs associated with the term, and all of the descendent terms. The browser has a term search option that locates terms based on user-defined strings; for example, \'DNA%\' will locate all GO terms starting with DNA. Once a term has been identified, the browser will provide a map of the GO tree both above and below any term, as well as a count of the ESTs associated with each of the terms displayed. For each term the browser also provides the option of either downloading the sequences of all ESTs and contigs associated with the term in a single FASTA file, or download a spreadsheet of the EST identifiers, associated GO terms and information about the linkage between the two. Alternately, the spreadsheet can be viewed as a web page. In this form the sequence identifiers will link to the appropriate ESTIMA page, ESTs to the Sequence ID page and contigs to the contig viewer. Further, the reference sequence used to form the association will link to the appropriate external web site. The GO Browser output can be filtered so that only ESTs from a single library are displayed. ### Sequence ID ESTs can be accessed directly from the sequence ID interface. More commonly, results from queries on annotations or contigs are dynamically linked to the EST sequence information. From these screens users can access chromatograms, and both raw and filtered sequences. ### Gene association Information about the association between the ESTs and the reference genome sequences can be accessed through these pages. ### BlastUI ESTIMA provides a Blast User Interface that allows FASTA-formatted sequences to be searched against the EST libraries. The libraries that are available for BLAST \[[@B9]\] can be defined for each project. We usually allow at a minimum, the raw sequences, the clean sequences, and the unique assembled sequences (contigs plus singletons). This allows researchers to rapidly identify those ESTs associated with any particular sequence of interest. The data sets available for BLAST searches can be easily extended for specific projects. For example, we make the Baylor University Honeybee Genome assemblies \[[@B13]\] available to the honeybee site \[[@B8]\]. ### Annotations The Annotation page is an optional page, the presence or absence of which is controlled from a configuration file. This page allows additional BLAST-derived annotations, beyond GO annotations, to be displayed and queried in a number of different ways. The web application creates an interface that allows users to query the Blast\_All table directly. For example, songbird ESTs \[[@B14]\] were searched against the nr from NCBI, Swiss-Prot, and chicken database from TIGR. A researcher can use this to find all the ESTs and contigs in the songbird collection that are associated with any term in the sequence description. The term RNA binding, for instance, returns 38 hits, of which 19 are to nr proteins, 11 to Swiss-Prot, and 8 to TIGR chicken. ### Contig Viewer The Contig Viewer provides an image of the assembled contigs showing the relationship of the contig with the ESTs it contains as members. It provides the contig\'s consensus sequence, and links to each of the member ESTs. The web application maintains a common look and feel to all the pages within a project. This is implemented by having a single block of HTML stored within a configuration file. As each page is rendered, the same HTML is called to generate the header block of the page. Each project has its own HTML, so the look of each project\'s web site can be customized. The inter-connectivity of the different ESTIMA modules allows researchers to engage in a \"free form\" exploration of the data. Users can query the GO Browser, for example, to find a contig associated with a term of interest, drill down to see the structure of the contig, and then, if desired, drill down to get specific information on each EST in the contig. Just as easily, the users could take a sequence of interest from their research, and similarity-search it against the assembled ESTs. Then they could drill down on an EST that aligned with their sequence, and see the additional information about the sequence, including any annotations. Chromatograms ------------- ESTIMA allows users to view the actual chromatograms of the ESTs. Chromatogram files are stored in a file system visible to the web server, while file paths are stored in the database. When a user requests a chromatogram, Phred \[[@B15],[@B16]\] is called to convert the chromatogram to SCF format and this is sent to Traceviewer \[[@B17]\] that displays the trace in the user\'s web browser. Results ======= ESTIMA is independent of an EST processing pipeline --------------------------------------------------- ESTIMA is unlinked from the backend EST processing pipeline, clustering, and assembly of ESTs. It serves as a stand-alone web application that allows users to store, access, research, and visualize the raw and annotated ESTs and contigs, including GO annotation. The output from a sequencing center\'s EST processing pipeline (base-called high-quality ESTs, assembled contigs, BLAST results against a reference genome) gets loaded into databases, and serves as input to ESTIMA. (The W.M. Keck Center will gladly share the source code to its pipeline with any interested academic institution). Additionally, researchers may choose to use any reference genome to annotate their sequences. ESTIMA comes packaged with a flexible database schema that supports the linking of sequences to GO terms, and other user-supplied sequences. The flexibility of the ESTIMA database schema becomes more relevant with the increasing number of sequenced genomes. ESTIMA provides a flexible, password-protected, multi-project environment to researchers. It facilitates analysis of an unlimited number of ESTs and contigs linked to GO and non-GO annotations, and the download of annotated sequences related to any GO term. ESTIMA comes with an implementation of a GO browser that allows users to view the entire child term tree for any term, conveniently from a single query interface. There can be multiple installations of ESTIMA --------------------------------------------- ESTIMA is designed to be a stand-alone application. Each installation of the web application has all system dependent information in its own configuration files, including the information needed to connect to the databases. We maintain two instances of the interface, a development and a production copy. As new projects are introduced, they can be tested, and any interface modifications that are needed can be perfected on the development machine, concurrent with the creation of the production version of the databases. The production database can be examined using the development web application and any errors corrected before taking the system \"live\" to the production version. ESTIMA maintains multiple projects and supports multiple libraries in a project ------------------------------------------------------------------------------- ESTIMA is designed to accept new projects. As new EST sequencing projects are finished, they can be easily added to an existing ESTIMA installation. A new schema is created for the new project and the loading scripts are used to populate the database. All that is required to activate the web application is the addition of a block of XML code to the configuration file, and a connection string and some HTML to the system configuration file. XML tags within a configuration file control project- specific issues such as whether the data is password protected, or which BLAST databases can be accessed from the Blast User Interface. There are three public ESTIMA projects currently administered through the W.M. Keck Center for Comparative and Functional Genomics at the University of Illinois. The bee-ESTdb \[[@B18]\] is a resource created from a normalized unidirectional cDNA library from which 21,408 cDNA clones were partially sequenced. These sequences were assembled into 8,966 putatively unique sequences. The contigs were then tested for similarity to sequences in the Drosophilagenome, and based on these similarities the sequences were tentatively assigned one or more molecular functions and biological processes. Likewise, BOVEST, the cattle EST database \[[@B19]\], contains 17,452 cDNAs from a bovine placenta library and 6,144 from a spleen library, all of which were annotated against the human UniGene \[[@B20]\]. The songbird project contains 14,461 sequences from a songbird brain library \[[@B14]\] that were assembled into 8,526 unique sequences. ESTIMA allows multiple libraries within each project. Information about each library is stored in the configuration file, and the interface elements dynamically generate the start page and the library filters within the GO Browser. Practical examples that demonstrate research utility of ESTIMA -------------------------------------------------------------- The presence of multiple projects in ESTIMA allows for efficient cross-tissue or cross-species homology searches. For example, a mouse brain EST may be used to interrogate the honeybee brain or songbird brain library to test the hypothesis that the gene is expressed as a brain-specific transcript. Thus, a mouse brain transcript, NCBI accession number BM875176, similar to human tubulin alpha-1 chain protein may be used to do a TBLASTX against the honeybee brain assembled ESTs from the BlastUI interface in ESTIMA. The BLAST results retrieve a significant hit, Contig2466, to the honeybee brain database (Figure [5](#F5){ref-type="fig"}). The contig identifier is hyperlinked to the Sequence ID interface in ESTIMA from where the consensus sequence may be downloaded. The chromatograms for the ESTs that make up the contig, may also be checked for quality from the same interface in ESTIMA as shown in Figure [5](#F5){ref-type="fig"}. Both mouse and honeybee brain sequences, may then be used to do a deeper phylogenetic search with a BLASTX against non-redundant protein database to test the tissue-specificity hypothesis. ESTIMA projects, as compared to other public web-applications such as TIGR gene indices \[[@B5]\], allow access to singlets from the EST assemblies, and chromatogram retrieval. These singlets would include rare, novel transcripts and divergent homologs that are increasingly the sole motivation for a research project. Since ESTIMA includes only high quality sequences in the databases, users may search for and download these novel transcripts, and also efficiently implement a homology search strategy using the web-application. Another strength of ESTIMA is in facilitating chromatogram and contig viewing from a common interface (Sequence ID). Any contig may be displayed and chromatograms of the member ESTs checked for errors in base-calling that may result in a premature stop-codon, or frameshift indels. Thus, ESTIMA is a valuable genome research tool. Conclusions =========== ESTIMA is a full-featured web-application and database, designed to simplify exploring and sharing EST libraries and databases. It can be easily adapted to a wide variety of system configurations, and back-end database engines. Our installation of ESTIMA easily supports three public projects, with five different EST libraries, and additionally a growing number of private projects. Availability and requirements ============================= ESTIMA is available free to academic users at <http://titan.biotec.uiuc.edu/ESTIMA/>. Under \'Downloadable Software\' section of the web page, detailed installation instructions and a user manual have been included as well. ESTIMA is still in active development. New features are constantly being developed to meet the changing needs of the research projects that use it. Further, new projects are being added to our ESTIMA installation. The system has been written to facilitate its own change, and as such, researchers should find it approachable with a good working knowledge of Perl, SQL, and HTML. System requirements ------------------- ESTIMA requires Perl and a database. All communication with the database is handled through Perl DBI, which is extensible to any ODBC compliant database. The use of Perl DBI and ODBC allow the databases to reside on separate servers from the web interface. Although many databases may be used, in practice, there are several complex joins in the code that could result in slow performance on large EST sets unless a well-optimized database was selected. ESTIMA requires certain additional Perl modules, specifically BIO, CGI, DBI, and GD. Bioperl, the BIO module \[[@B21]\] is used extensively. All BLAST and FASTA file parsing, as well as all references to sequence objects in the user interface are handled with BIO methods. GD \[[@B22]\] is used to generate the Portable Network Graphics (PNG) files displayed in the contig viewer. We have been using ESTIMA throughout its development. Our schemas are housed in an Oracle 8I database on a Silicon Graphics Origin 2000 16 × 250 MHz machine running IRIX 6.5.20. The web application, including the user requested BLAST jobs are run on a Sun Microsystems SunFire 280 R with dual 700 SPARK V9 CPUs. Authors\' contributions ======================= GG and LL developed the database schemas. CGK, LL and LR developed the initial web application prototype. HAL managed project development and contributed to design concepts. RL modified the web application and optimized the code. All authors read and approved the manuscript. Acknowledgements ================ We would like to thank Keith Frazier, systems administrator, who has ably administered all of the servers. We would also like to acknowledge Stephen S. Davis, Shreedhar Natarajan, Joe Sola and Andrew Birck for the contributions to the ESTIMA code base. We would like to thank the generous support of the Biotechnology Center and W.M.Keck Foundation at the University of Illinois for supporting this work. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### ESTIMA is organized around three major components. A single installation of the ESTIMA web application can provide a front-end for any number of projects; in this case, three different projects are shown. The web application connects to a different project database for each project. All projects share the GENOME database, and a common repository for the blastable databases, although project users can only \"see\" those databases associated with their project. ::: ![](1471-2105-5-176-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### This ER diagram shows both the GENOME schema and a single PROJECT schema. In practice, each project schema is given a unique name associated with the organism under study, thus the songbird project information is stored in the \"songbird\" schema. ::: ![](1471-2105-5-176-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### The seven elements of the web application (the start screen and six query applications shown as rectangles) interact with each other in a complex manner. A single headed arrow means that the element at the tail of the arrow creates hyperlinks in its output that automatically calls the element at the arrowhead. For example, whenever the contig viewer refers to an EST sequence, it links the ID to information about the EST from the Sequence ID element. The GO Browser and the Sequence ID elements allow users to download the appropriate FASTA files. Additionally, the GO Browser and Gene Association elements provide links to external information about reference sequences. ::: ![](1471-2105-5-176-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### A screenshot of the custom GO Browser. The left panel is the query page, and the right panel displays the parent-term tree at the top (not visible), and a child-term tree that indicates the number of ESTs associated with each term. Detailed EST annotation reports may be displayed or downloaded, as also the sequences of these annotated ESTs. ::: ![](1471-2105-5-176-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### An example of the use of ESTIMA in research. The top panel shows the results of a TBLASTX of a mouse brain mRNA similar to human tubulin alpha-1 protein against honeybee brain assembled ESTs. The resulting hit Id, Contig2466, is linked to the Sequence ID interface in ESTIMA from where the consensus sequence of the honeybee contig may be retrieved. The chromatogram for a member EST in the contig is displayed. ::: ![](1471-2105-5-176-5) :::	PubMed Central	2024-06-05T03:55:49.781464	2004-11-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533868/", "journal": "BMC Bioinformatics. 2004 Nov 4; 5:176", "authors": [ { "first": "Charu G", "last": "Kumar" }, { "first": "Richard", "last": "LeDuc" }, { "first": "George", "last": "Gong" }, { "first": "Levan", "last": "Roinishivili" }, { "first": "Harris A", "last": "Lewin" }, { "first": "Lei", "last": "Liu" } ] }
PMC533869	Background ========== The Coxsackie and adenovirus receptor (CAR) \[[@B1],[@B2]\], a transmembrane protein of the immunoglobulin super-family, serves as a receptor for adenovirus (Ad) subgroups A, C, D, E and F \[[@B3]\] as well as Coxsackie B viruses (CVB) \[[@B4]\]. CAR is a highly conserved protein with two predominant isoforms, produced through differential splicing, and having cytoplasmic domains of either 107 residues (ending in SIV) or 94 residues (ending in TVV) \[[@B2],[@B5]\]. The extracellular domain mediates homophilic cell adhesion \[[@B6]-[@B8]\] and ectopically-expressed CAR localizes to homotypic intercellular contacts \[[@B8]\]. The expression of CAR is regulated developmentally \[[@B6],[@B9]-[@B12]\] as well as in a tissue-specific manner \[[@B2],[@B5]\]. To date, most studies on CAR expression in the adult have resorted to analysis of transcript levels. These have revealed that the pattern of tissue-specific expression differs between humans and mice. In humans, a predominant transcript of \~6 -- 6.5 kb is observed in heart, testis, prostate and pancreas while much less expression is detected in liver, brain, colon and small intestine. In the mouse on the other hand, the most abundant expression is in liver, kidney, lung and heart. Interest in CAR stems from its function as the primary high affinity receptor for Ad serotype 5, the most commonly used adenoviral vector in gene therapy protocols. CAR expression is the main determinant in gene transfer to normal tissue as ectopic expression of CAR in transgenic mice leads to several magnitudes of increase in adenovirus transducibility of tissues that are otherwise refractory to Ad-mediated gene expression \[[@B13]-[@B17]\]. As well, although decay accelerating factor (DAF, CD55) was the first described CVB receptor \[[@B18],[@B19]\], CAR is necessary and sufficient for CVB infection in vitro\[[@B20]\]. Thus, the expression levels of CAR may also govern the susceptibility to CVB diseases and the pathological consequences of CVB viral infection. In this context, acute viral myocarditis and myositis are inflammatory diseases affecting cardiac and skeletal muscle that can result from infection by the Coxsackie B virus. In both humans and rodents, heart is among the tissues showing the greatest abundance of CAR transcript while its transcripts are barely detectable in skeletal muscle even with the more sensitive reverse-transcriptase (RT)-PCR-based assay \[[@B21]\]. In contrast to heart, DAF expression is absent in mature skeletal muscle \[[@B22]\]. Despite the absence of DAF and low CAR transcript levels, skeletal muscle is nevertheless susceptible to Coxsackie virus-induced myositis. Indeed, human patients suffering from inflammatory muscle diseases have tested positive for CBV RNA \[[@B23]\]. This suggests that the low CAR transcript level in skeletal muscle may produce functional receptor. Therefore, to examine CAR localization in skeletal and cardiac muscle, we used antibodies directed against the extracellular domain of CAR \[[@B21]\] as well as antibodies that can differentiate between the two major CAR isoforms \[[@B24]\] with alternate 3\' splicing \[ending in the amino acids SIV or TVV\] \[[@B2],[@B5]\] \[Fig. [1](#F1){ref-type="fig"}\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Depiction of the CAR sequences recognized by the anti-C-terminal antibodies.The chicken antibody ChCT was raised against a fusion protein containing C-terminal sequence common to both CAR isoforms (from aa 259 -- 339). The rabbit antibodies (RP291 and RP194) were raised against peptides that were specific to each isoform. The diagram does not show the immunogen of the rabbit antibody 2240 which is the extracellular N-terminal domain of murine CAR. Note that each antibody has a distinct recognition sequence. TM -- transmembrane sequence. ::: ![](1471-2121-5-42-1) ::: Results and Discussion ====================== To immunolocalize CAR, frozen sections of normal human muscle biopsies were probed with polyclonal antibodies raised against the extracellular domain of CAR \[[@B21]\] \[ab 2240\], or against the cytoplasmic tail, antibodies which specifically recognize the two predominant isoforms of human CAR (referred to as SIV \[ab RP291\] and TVV \[ab RP194\] respectively) \[[@B24]\]. We localized CAR exclusively to the neuromuscular junction in human skeletal muscle (Fig. [2](#F2){ref-type="fig"}) where CAR immunoreactivity coincided with acetylcholine receptors as revealed by α-bungarotoxin binding. Similarly, in mature murine skeletal muscle, CAR expression was restricted to the neuromuscular junction (Fig. [3](#F3){ref-type="fig"}). Interestingly, we find CAR expression at the neuromuscular junction to be isoform specific, with the CAR SIV isoform accounting for all CAR immunoreactivity (Fig. [2G](#F2){ref-type="fig"}). In contrast, the TVV isoform is present in the vasculature (Fig. [2I](#F2){ref-type="fig"}). CAR is therefore identified as a novel component of the adult neuromuscular junction, joining other homotypic cell adhesion molecules such as N-cadherin and neural cell adhesion molecule (N-CAM) which have been localized previously to this specialized site. As with NCAM \[[@B25]\], although CAR is uniformly expressed in immature mouse muscle (data not shown), its expression becomes downregulated within a few weeks of birth \[[@B21]\] and its localization is confined to the neuromuscular junction. As well, in skeletal muscle undergoing regeneration, as is the case in the dystrophic mdxmouse, CAR is re-expressed \[[@B21]\] as is NCAM \[[@B26]\]. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Immunolocalization of CAR to the neuromuscular junction in human skeletal muscle. Frozen sections of human muscle were incubated with Alexa Fluor-conjugated α-bungarotoxin and antibodies to CAR as described in Materials and Methods. Panels Aand Dshow α-bungarotoxin staining of acetylcholine receptors at neuromuscular junctions (green). Panel Bshows immunofluorescent staining of the section in panel Awith a polyclonal antibody (ab 2240) to the extracellular domain of CAR (red). Panel Eshows immunofluorescent staining of the section in panel Dwith a mixture of the isoform-specific C-terminal antibodies RP194 and RP 291 (red). Panel Cis a merge of panels Aand B. Panel Fis a merge of panels Dand E. These merges \[C, F\] show that CAR colocalizes with α-bungarotoxin at neuromuscular junctions (in yellow). Of the two C-terminal antibodies, only RP291 (G) demonstrates the typical neuromuscular junction staining while signal is absent when sections are incubated under similar conditions with RP194 (H) although RP194 does label blood vessels (I). Sections incubated with secondary antibody alone did not reveal any signals (J). Bar = 25 μm (A, D); 50 μm (G, I). ::: ![](1471-2121-5-42-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Immunolocalization of CAR to the neuromuscular junction in mouse skeletal muscle. Frozen sections of mouse muscle were incubated with Alexa Fluor-conjugated α-bungarotoxin and antibodies to CAR as described in Materials and Methods. Panels Aand Dshow α-bungarotoxin staining of acetylcholine receptors at neuromuscular junctions (green). Panel Bshows immunofluorescent staining of the section in panel Awith a polyclonal antibody (ab 2240) to the extracellular domain of CAR (red). Panel Eshows immunofluorescent staining of the section in panel Dwith a chicken polyclonal antibody (ChCT) directed against a C-terminal portion of CAR conserved in both CAR isoforms (red). Panel Cis a merge of panels Aand B. Panel Fis a merge of panels Dand E. These merges \[C, F\] show that CAR colocalizes with α-bungarotoxin at murine neuromuscular junctions (in yellow). Sections incubated with the secondary antibodies alone did not give any signal -- anti-rabbit IgG \[G\], anti-chicken IgY \[H\]. Bar = 35 μm ::: ![](1471-2121-5-42-3) ::: CAR has been suggested to serve as a structural component at the intercalated discs in murine cardiac muscle \[[@B8]\] and recently confirmed to reside primarily at the intercalated discs in both rat heart and neonatal cultured cardiomyocytes of the rodent \[[@B12]\]. Using antibodies directed to the specific SIV and TVV isoforms of the receptor as described above, we localized CAR to the intercalated discs (Fig. [4](#F4){ref-type="fig"}), demonstrating for the first time expression of both receptor isoforms in human and murine heart. To confirm the presence of both isoforms, murine cardiac muscle homogenates were analyzed by SDS-PAGE followed by electrotransfer to nitrocellulose membranes. This immunoblot analysis revealed the presence of both the SIV and TVV isoforms (Fig. [5A](#F5){ref-type="fig"}). Furthermore, when murine cardiac muscle homogenates were immunoprecipitated with either RP194 or RP 291 rabbit polyclonal antibodies, Western blot analysis of the resultant fractions with a chicken antibody against CAR C-terminal domain showed a single 46 kDa band (Fig. [5B](#F5){ref-type="fig"}), confirming the specificity of the antibodies for CAR. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Immunolocalization of CAR to intercalated discs in human and murine cardiac muscle.Human \[A, B\] and murine \[C, D\] cardiac muscle sections were reacted with the polyclonal anti-C-terminal antibodies RP194 \[A, C\] and RP291 \[B, D\]. Note the intense staining of both isoforms at the human intercalated discs (arrow) \[A, B\] and murine intercalated discs \[C, D\]. Neither the human cardiac tissue \[E\] nor the murine cardiac muscle \[F\] gave any signal when incubated with the anti-rabbit IgG. Bar = 10 μm (A, E); 30 μm (C, F). ::: ![](1471-2121-5-42-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Murine cardiac muscle homogenates express both isoforms of CAR.A.Immunoblot analysis of cardiac muscle homogenates (10 μg) reveals that a single polypeptide of 46 kDa is detected by the anti-C-terminal antibodies recognizing the two different CAR isoforms (RP194 and RP291). B.Murine cardiac homogenates were immunoprecipitated with either RP194 (lanes 1,2) or RP291 (lanes 3,4) followed by immunoblotting with the chicken ChCT antibody that recognizes the C-terminal portion of CAR that is common to both isoforms. The ChCT antibody detected as a single band the increasing amounts of CAR that was immunoprecipitated with increasing amounts of the rabbit polyclonal antibodies (2 μl, lanes 1,3; 4 μl, lanes 2,4). ::: ![](1471-2121-5-42-5) ::: Both of the cytoplasmic variants contain a PDZ recognition motif at their distal end, implicating CAR as a putative member of multiprotein complexes. This is indeed the case in polarized epithelial cells in which CAR is expressed at the tight junction where it associates with the tight junction scaffolding protein ZO-1 \[[@B8]\] and contributes to maintenance of transepithelial resistance. The localization of CAR to cardiac intercalated discs is in agreement with CAR having a structural or regulatory role as a transmembrane member of junctional complexes. The intercalated discs are composed of at least three structurally distinct cellular junctions -- desmosomes, the adherens junctions, and gap junctions \[[@B27]\]. The SIV and TVV isoforms may localize to separate components of the intercalated disc, or given that CAR has been implicated as a transmembrane component in tight junctions \[[@B8]\], both isoforms may be localized to the adherens junction, a structure analogous to tight junctions. It is interesting that a correlation can be drawn between disease incidence and expression levels of the CAR receptor. Cases of viral myocarditis in the human population outnumber those of myositis. This may be attributable to the difference of endogenous CAR expression between the two tissues. Considering that induction of CAR accompanies myocarditis \[[@B28]\] and its dramatic upregulation has recently been demonstrated in patients suffering from multiple diseases of the heart \[[@B29]\] including dilated cardiomyopathy, a pathological phenotype linked to persistent acute myocarditis, upregulation of the receptor may render the heart even more susceptible to further viral infection. Conversely, the low level of endogenous CAR expression in skeletal muscle may safeguard against the wide-spread viral infection seen in myocarditis and may be responsible for the less severe clinical features of myositis. Conclusions =========== CAR is a novel member of the neuromuscular junction. In cardiac muscle, both CAR isoforms are found at the intercalated discs. The localization of CAR to these important junctional complexes suggests that CAR may play both a structural and a regulatory role in skeletal and cardiac muscle, and that these complexes may serve as a point of entry for Coxsackie B virus. Methods ======= Antibodies ---------- Figure [1](#F1){ref-type="fig"} depicts the sequences used as immunogen to generate the various antibodies. There was no overlap (i.e. common epitopes) between any of the antibodies. The rabbit polyclonal antibodies used in this study were described previously \[[@B21],[@B24]\]. Briefly, the N-terminal polyclonal antibody (ab 2240) was prepared against a His-tagged fusion protein which encoded amino acid residues 22--208 of the extracellular domain of mouse CAR \[[@B21]\]. This antiserum cross-reacts with human CAR on Western blots and in indirect immunofluorescence. The two C-terminal polyclonal antibodies \[[@B24]\] were generated using peptides encompassing the last 13 amino acids of the two predominant human CAR isoforms \[[@B2],[@B5]\]. Antiserum RP194 was raised against the sequence FKYAYKTDGITVV while RP291 was raised against the sequence VMIPAQSKDGSIV. Both these antisera cross-react with the mouse CAR homologs (the peptides are conserved 100% between the two species \[[@B30]\]). All antisera were affinity purified prior to use. To raise the chicken anti-CAR antibody (ChCT), purified His-tagged fusion protein encoding the C-terminal portion of CAR that is common to both isoforms (amino acids 259--339) was emulsified in an equal volume of TiterMax Gold adjuvant (CytRx Corp., Norcross, GA) and injected intramuscularly into chickens. One month post-injection, IgY antibodies to CAR were obtained from the eggs of injected chickens and subjected to affinity purification. Immunocytochemistry ------------------- Immunolabelling was performed using standard techniques. Briefly, frozen sections (5 μm) of normal human skeletal and cardiac muscle biopsies, murine skeletal and cardiac muscle were fixed in 2% paraformaldehyde (pH 6.8) for 1--2 minutes, followed by overnight incubation at 4°C with the primary antibodies (a 1:30 dilution was used for ab 2240, and 1:200 dilution for the abs RP291 and RP194, in blocking solution made of 3% bovine serum albumin and 0.05% Tween-20 in phosphate-buffered saline). Incubation with a mouse anti-rabbit biotin-conjugated secondary ab (1:120; Jackson Immunoresearch Laboratories, West Grove, PA) was followed by Cy-3-conjugated streptavidin (1:1000; Jackson Immunoresearch Laboratories). Controls consisted of sections treated in the absence of primary antibody. Neuromuscular junctions were revealed with Alexa-488-conjugated-α-bungarotoxin \[α BTX\] (1:40) (Molecular Probes, Eugene, OR). Slides were viewed on a Leica microscope-based imaging system using OpenLab imaging software (Quorum Technologies, St Catharines, ON). Western blot analysis and immunoprecipitation --------------------------------------------- Cardiac muscle tissue was homogenized in extraction buffer \[1% Triton X-100; 0.1 mM EDTA; 0.1 mM EGTA; 50 mM Tris-HCl; pH 8.0; with protease inhibitors (Roche)\] at 4°C. After a 30 second sonication, samples were centrifuged at 3000 × g for 30 seconds at 4°C. Protein samples (10 μg) were anayzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 10% (w/v) polyacrylamide gels, followed by electrotransfer to nitrocellulose. The blots were blocked in 10% BLOTTO (skim milk powder) in Tris buffered saline -- Tween 20 (TBS-T) for 45 minutes at room temperature. Anti-CAR antibody was added in 10% BLOTTO at a dilution of 1:2500. Following incubation with peroxidase-labeled goat-anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories), the signal was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Baie d\'Urfe, QC). Immunoprecipitation was carried out on cardiac muscle homogenates that had been pre-cleared with Protein-A Agarose slurry (Sigma), followed by overnight incubation at 4°C with RP291 or RP194. The samples were further incubated with Protein-A Agarose for 2 hours, washed twice with extraction buffer and then eluted with 2 X Laemmli SDS sample buffer with 5% mercaptoethanol. Following SDS-PAGE and electrotransfer, nitrocellulose membranes were probed with a primary polyclonal chicken anti-CAR (ChCT) in blocking solution (diluted 1:500) overnight at 4°C. Signal was revealed following incubation with a peroxidase-conjugated donkey anti-chicken IgY (Jackson Immunoresearch Laboratories) at a dilution of 1:2500 for 40 minutes, and enhanced chemiluminescence. Authors\' contributions ======================= CAS performed the immunoprecipitation, the immunoblotting, participated in the immunofluorescent staining experiments and drafted the manuscript. PCH and JN conceived the study, participated in its design and interpretation as well as drafting the manuscript. CA participated in the immunofluorescent staining experiments. KS prepared and characterized the isoform-specific antibodies. MS and GK participated in the design and interpretation of the studies. All authors read and approved the final manuscript. Acknowledgements ================ This work was supported by a grant from the Canadian Institutes of Health Research and the Muscular Dystrophy Association (USA). J. N. is a National Research Scholar of the FRSQ and a Killam Scholar.	PubMed Central	2024-06-05T03:55:49.784306	2004-11-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533869/", "journal": "BMC Cell Biol. 2004 Nov 8; 5:42", "authors": [ { "first": "Christian A", "last": "Shaw" }, { "first": "Paul C", "last": "Holland" }, { "first": "Michael", "last": "Sinnreich" }, { "first": "Carol", "last": "Allen" }, { "first": "Kerstin", "last": "Sollerbrant" }, { "first": "George", "last": "Karpati" }, { "first": "Josephine", "last": "Nalbantoglu" } ] }
PMC533870	Background ========== Since the initial description of the Drosophilasegmentation gene runtover a decade ago \[[@B1]\], a small family of runt domain (RD) genes has been described and extensively analyzed in several species. The 130 amino acid long runt domain is very highly conserved and is readily identifiable computationally. RD transcription factors are developmental regulators involved both in cell proliferation and differentiation, and their disruption usually leads to disease \[[@B2]\]. In humans, three different RD genes were identified \[[@B3]\] and named according to various schemes, currently standardized by the human gene symbols RUNX1, RUNX2and RUNX3. RUNX genes have two promoters (P1 and P2, also called distal and proximal, respectively) \[[@B4]-[@B7]\] separated by a long intron; the proximal promoter (P2) is always located within a large CpG island \[[@B8]\]. Extensive alternative splicing giving rise to many isoforms has been described for all RUNX genes \[[@B9]-[@B11]\]. Orthologs of all three human RUNXgenes were identified in mouse. A single RD gene was described in Xenopus, presumed to be orthologous to RUNX1\[[@B12]\]. An experimental search for RD genes in fugu showed the existence of a fugu ortholog of human RUNX2, and suggested the existence of a single additional RD gene in fugu \[[@B13]\], while a computational search of the fugu genomic sequence revealed three RUNXgenes \[[@B14]\]. Four RD genes were identified in Drosophila\[[@B14],[@B15]\], while a single RD gene exists in C. elegans\[[@B16]\], sea urchin and amphioxus \[[@B17]\]. Based on these data, current thought on the evolution of the RD gene family posits that a single RD gene was present in the common ancestor of chordates \[[@B17]\], and this ancestral gene triplicated during early vertebrate evolution, giving rise to the modern RUNXgene complement. The proposed mechanism of expansion involved large-scale genomic duplications, identifiable today as large paralogous segments \[[@B18]\]. The proper identification of true orthology relationships is often helpful for inferring gene function and translating knowledge between model organisms and more complex species. Under the current model, simple orthology relationships should be expected among vertebrate RUNXgenes, but their functional relationship to the ancestral RD gene is unknown. The single known RD gene in C. eleganshas been shown to be required for the formation of a functional gut; this role has been claimed to be conserved with mouse Runx3\[[@B19]\]. The current availability of genome drafts for several vertebrate species, including Homo sapiens, Mus musculus, Rattus norvegicus, Canis familiaris, Gallus gallus, Takifugu rubripes, Tetraodon nigroviridis, Danio rerioand Xenopus tropicalis, allows us to explore a comprehensive set of vertebrate RD genes and characterize their genomic environments, shedding light on the structure and evolution of this important gene family. Results ======= The fugu genome has at least four runt domain genes --------------------------------------------------- Our search for RD genes in the fugu draft yielded four distinct genomic scaffolds (Fig. [1](#F1){ref-type="fig"} and Table [1](#T1){ref-type="table"}), each containing a single, complete RD gene. Each scaffold had one or more sequence gaps, some within the RD genes, others between them and their neighbors. We employed a directed sequencing approach to obtain the additional sequence needed to close the gaps in these four scaffolds and to improve sequence quality. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The four fugu scaffolds analyzed and visualized using the GESTALT Workbench. The RD genes are aligned by the position of the proximal promoter. For each scaffold, graphs are shown of the CpG contrast values (observed/expected, typically \~0.5 for the fugu genome), G+C percentage (green -- below 43%, blue -- between 43% and 50%, red -- above 50%), interspersed repeats (SINEs in red, LINEs in green, DNA and LTR elements in brown, other repeats in purple), and gene annotations. Genes and repeats displayed above each black midline are in the forward strand, while those displayed under the midline are in the reverse strand of the sequence. All scaffolds are shown at a resolution of 200 bp/pixel, except for scaffold 835, which includes the tiny FrRUNTgene. For clarity, only the last 17 kb of this scaffold are shown at 20 bp/pixel. The much longer scaffold scaffold 183 is also shown truncated at 182 kb; the LCK locus has been studied in detail elsewhere \[42\]. ::: ![](1471-2148-4-43-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The four fugu RD genes and their human orthologs. ::: Fugu gene Human ortholog ----------- ---------------- ------ ------- ---------- ----------- ------- Name Location Size \%G+C location Gene size \%G+C FrRUNT scaffold\_835 3.0 41.2% N.O. N.O. N.O. FrRUNX1 scaffold\_682 25.2 42.5% chr21 262 43.6% FrRUNX2 scaffold\_260 32.6 45.7% chr6 219 39.9% FrRUNX3 scaffold\_183 36.8 47.4% chr1 66 54.8% Sizes are expressed in kb. N.O.: No ortholog. ::: We studied the four scaffold sequences using the GESTALT Workbench \[[@B20]\] and constructed hypothetical gene structures for the fugu RD genes by maximizing similarity to known vertebrate RD proteins. Three of the four RD genes found in the fugu genome have clear one-to-one similarity relationships with the three mammalian RUNXgenes (see phylogenetic analysis below). They have been assumed to be their orthologs \[[@B14],[@B17]\]; we call them FrRUNX1, FrRUNX2and FrRUNX3(Fig. [1](#F1){ref-type="fig"}). Their genomic structures are similar to those of their human counterparts, but their sizes have evolved differently. RUNX3is the smallest of the three human RUNX genes, while in fugu FrRUNX3is the largest (Table [1](#T1){ref-type="table"}), and FrRUNX2is significantly larger than FrRUNX1. FrRUNX1has acquired an additional intron \[[@B17]\] that is not present in human RUNX1or in any other RD gene. This intron is just 65 bp long, has canonical splice signals, and is in phase 0 with respect to the protein reading frame, at the beginning of the runt domain. An additional intron has been described at the 5\' end of the coding region, yielding a short form that would be locally non-homologous to the other RD genes \[[@B14]\]. A detailed comparison of human RUNX2and FrRUNX2has been published \[[@B13]\]. In both human and in fugu, RUNX3has the highest G+C content of the RD genes, while the G+C content of RUNX2differs significantly between the two species (Table [1](#T1){ref-type="table"}). The fugu RUNT gene ------------------ In addition to the three RUNXgenes, the fugu genome has a fourth and more divergent runt-domain gene, that we named FrRUNT. FrRUNTis an extremely compact gene, spanning just 3 kb of genomic sequence (Fig. [2](#F2){ref-type="fig"}). Based on sequence analysis only, FrRUNTappears to have two promoters, with an intron separating the hypothetical distal promoter (P1) and first exon from the main body of the gene. This intron is usually very long in RUNXgenes. It is indeed the longest intron observed in FrRUNT, but it is nevertheless very short, spanning just 1372 bp. There is a local concentration of CpG dinucleotides 200--300 bp upstream of exon 2 (Figs. [1](#F1){ref-type="fig"}, [2](#F2){ref-type="fig"}), suggesting that an incipient CpG island might function as a proximal promoter (P2). The G+C content is not elevated in this area, in similarity to the CpG islands of the fugu RUNXgenes (Fig. [1](#F1){ref-type="fig"}). The main body of the gene is split into five exons, separated by much shorter introns (69--190 bp long), all of which have canonical splice signals. The longest predicted FrRUNT product is 294 amino acids long, in contrast with the 496 aa, 463 aa and 421 aa observed for FrRUNX1, FrRUNX2 and FrRUNX3, respectively. The small number of exons in FrRUNTleaves little room for alternative splicing by exon skipping, without compromising functionally important domains of the protein. The overall compactness of the gene makes the incorporation of yet undetected exons improbable. Several cryptic splice sites within the exons could enable splicing variants altering exon length. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### FrRUNTsequence detail, showing its compact organization and its interlocking with the SUPT3Hgene. The two genes are disposed in opposite orientations. The inverted lettering for SUPT3H denotes translation from the reverse strand. The two putative promoters and the extent of the runt domain (RD) are indicated; x1--x6 denote the six exons. The black triangle points at the asparagine residue absent from all previously known runt domain proteins. ::: ![](1471-2148-4-43-2) ::: FrRUNT is exceptional in that the length of the runt domain (131 residues) varies from the universally conserved 130 amino acids, due to the introduction of an asparagine residue after position 47 in the RD (Fig. [2](#F2){ref-type="fig"}). This appears to be the first report of such a mutation within this highly conserved domain. We also noted that the RD sequence of the tunicate Oikopleura dioica(AAS21356 in GenBank) has an insertion at the same position (of two amino acids, a proline and an isoleucine). Comparison to the published structure of this domain \[[@B21]\] shows that this variable region is located in loop L4, opposite the DNA-binding region, i.e. in the location least likely to disrupt the structure of the protein. A surprising observation is that FrRUNTis \"interlocked\" with FrSUPT3H(Fig. [2](#F2){ref-type="fig"}), the gene orthologous to the human transcription factor SUPT3H\[[@B22]\]: The first exon of FrRUNTis located within the first intron of FrSUPT3H, and vice-versa. In the human genome, though, SUPT3His interlocked with RUNX2, as shown in \[[@B13]\]. We discuss the puzzles posed by these differences in genomic organization below. Four RD genes in Tetraodon and in zebrafish ------------------------------------------- A genome draft of another pufferfish species, Tetraodon nigroviridis, has been released \[[@B23]\]. A computational search into this draft reveals four RD genes with clear orthologous relationships with the four fugu RD genes. We call them TnRUNX1, TnRUNX2and TnRUNX3, and TnRUNT(Table [2](#T2){ref-type="table"}). The RD portion of TnRUNT has been deposited in the EMBL database (accession CAG00330); in this work we report the complete gene structure of TnRUNT, which is similar to that of FrRUNT. In further similarity with the fugu genomic organization, TnRUNTis \"interlocked\" with the Tetraodon ortholog of SUPT3H(not shown). The three RUNXgene pairs are conserved between fugu and Tetraodon (Table [2](#T2){ref-type="table"}) and display a larger percentage of protein identity than nucleotide identity, indicating a prevalence of conservative substitutions. In contrast, TnRUNT is only 83.3% identical to FrRUNT at the protein level, less than their nucleotide identity. Indeed, a striking series of non-synonymous mutations has created a highly divergent segment (only eight identical amino acids out of twenty-two) including the N-terminus of the runt domain (Fig. [3](#F3){ref-type="fig"}). The strong and unexpected divergence is not the result of local low sequence quality, as tested by examining the relevant Tetraodon entries from the NCBI Trace Archive, and by resequencing the fugu gene. The differences between TnRUNT and FrRUNT do not modify the Ig-like β-sandwich core of the runt domain \[[@B21]\], which is highly conserved as expected. Therefore, the structural integrity of the runt domain appears not to be compromised. We can only speculate that the extensive variation in its N-terminus may reflect species-specific constraints. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The four Tetraodon nigroviridisRD genes, and their identity levels to their respective fugu orthologs. ::: Tetraodon gene Identity ---------------- ----------- ----------- ------- ------------ --------- Name Location Size \%G+C Nucleotide Protein TnRUNT SCAF14597 2.5 42.3% 87.5% 83.3% TnRUNX1 SCAF15084 (\) 6.0 44.1% 93.3% 97.0% TnRUNX2* SCAF14590 (\) 31.8 44.4% 96.4% 99.4% TnRUNX3* SCAF15009 (\) 36.6 45.3% 94.6% 98.1% Sizes are expressed in kb. Asterisks denote minimal sizes due to the presence of gaps within the genes; the gene structure for TnRUNX1is incomplete at its 3\' end. ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Localized high divergence between TnRUNT and FrRUNT. This alignment between the first 171 bp of the predicted TnRUNTcoding sequence, with the first 180 bp of FrRUNT, shows a cluster of mutations (curly brackets) around the N-terminus of the runt domain (gray background). Non-conservative mutations (negative BLOSUM scores) are surrounded with thick black frames; thin frames enclose conservative mutations, and synonymous mutations are not indicated. The first sixteen codons derive from exon 1, the rest of the alignment from exon 2. The secondary structures indicated (alpha helix, beta strands and loops) are after \[21\]. ::: ![](1471-2148-4-43-3) ::: We also searched for RD genes in the genome draft of the zebrafish, Danio rerio\[[@B24]\]. Orthologs of RUNX1-3 are present, but no ortholog of FrRUNTcould be found. This could be due to the incompleteness of the draft sequence. On the other hand, there are two copies of RUNX2, RUNX2Aand RUNX2B, which have been shown to have somewhat different patterns of expression \[[@B25]\]. We next analyze the phylogenetic relationships between all the observed RD genes. Phylogenetic distribution of runt domain genes ---------------------------------------------- We performed exhaustive computational searches for RD genes, and in particular for potential orthologs of FrRUNT, using the available drafts of the human, chimp, mouse, rat, dog, chicken and frog genomes. In all cases, we identified three clear matches corresponding to orthologs of the three RUNXgenes. None of these genomes included a potential ortholog of the fugu/Tetraodon RUNTgene. In principle, such orthologs might be found in the future within current sequencing gaps or heterochromatic regions, but considering the virtually finished human genome, and the combined coverage of all the genome drafts, we can infer that the RUNTgene is absent in mammals, and probably in all tetrapods. Using representative protein sequences of the RUNX and RUNT genes (see Methods), we reconstructed a molecular tree (Fig. [4](#F4){ref-type="fig"}, top left) showing the relationship between the three RUNX proteins, FrRUNT and TnRUNT, and the runt proteins of Ciona intestinalisand Branchiostoma floridae(amphioxus). In this analysis, the FrRUNT protein is nearly equidistant from the three RUNX proteins and amphioxus RUNT (\~72% identical, see Table [3](#T3){ref-type="table"}). In comparison, the identity level between the RUNX proteins is 90%--98% in the same region (Table [3](#T3){ref-type="table"}), and they are 90%--95% identical to amphioxus RUNT. Therefore, while the amphioxus RUNT protein is very closely related to the vertebrate RUNX, the pufferfish RUNT proteins are significantly more divergent. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Phylogenetic reconstruction of runt domain evolution. Top left: unrooted tree of RD proteins based on the runt domain; X1, X2 and X3 denote the RUNX clades, BfRUNT, FrRUNT, TnRUNT, CiRUNT, OdRUNT, SpRUNT and CeRNT1 represent RUNT proteins from amphioxus, fugu, Tetraodon, Ciona, Oikopleura dioica, sea urchin and C. elegans, respectively, while taxa named Dm- represent D. melanogasterRD proteins. Top right: unrooted tree of first exons of human and fugu runt domain genes. Bottom: expanded species trees for the three RUNX orthologous groups. For all trees, numbers indicate percent bootstrap support; the horizontal bars indicate 10% divergence along each branch. White and gray backgrounds indicate comparisons at the nucleotide and amino acid level, respectively. The dashed branch in the RUNX2 panel represents the position of zebrafish (A) prior to AsaturA analysis. The arrows indicate the change effected by this correction. ::: ![](1471-2148-4-43-4) ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Identity matrix. ::: FrRUNX2 FrRUNX3 HsRUNX1 HsRUNX2 HsRUNX3 FrRUNT --------- --------- --------- --------- --------- --------- -------- FrRUNX1 89.7% 91.4% 95.7% 89.7% 89.7% 72.7% FrRUNX2 95.7% 92.3% 96.6% 96.6% 71.2% FrRUNX3 94.8% 97.4% 97.4% 72.7% HsRUNX1 94.0% 94.0% 72.7% HsRUNX2 98.3% 72.7% HsRUNX3 72.7% Percentages of identity between the human and fugu runt domains. ::: A difficulty has been documented in phylogenetic reconstruction of gene families with anciently duplicated genes \[[@B26]\], in which saturation of frequently-mutating amino acids leads to erroneous \"outgroup topologies\". In these incorrect topologies, the duplication event appears to be more ancient than supported by the data, which in turn suggest the existence of lineage-specific gene loss events. We tested our phylogenetic reconstruction using the program ASaturA \[[@B26]\], which identifies and suppresses saturated amino acids, thereby correcting the affected tree topology. This analysis did not modify the location of FrRUNT and TnRUNT in our reconstruction, suggesting that mutational saturation is not causing the observed divergence age of the pufferfish RUNTgenes. In this protein-level comparison of the conserved runt domains, the pufferfish RUNT proteins appear to be surprisingly ancient, predating the divergence between craniates (including vertebrates) and cephalochordates (including amphioxus). On the other hand, when comparing the nucleotide sequences of the first exon from each human and fugu runt domain gene, we observed that FrRUNTis more closely related to RUNX2(Fig. [4](#F4){ref-type="fig"}, top right), suggesting the possibility of a recombination event between these genes (see discussion below). We also studied the relationships between the RUNX1, RUNX2 and RUNX3 orthologs in several vertebrates and found the species trees to be largely as expected (Fig. [4](#F4){ref-type="fig"}, bottom row). One of the two zebrafish RUNX2 protein sequences (RUNX2A) appeared to be slightly more closely related to tetrapod RUNX2 genes than to the other RUNX2 genes in fish species, including zebrafish RUNX2B (dashed branch in Fig. [4](#F4){ref-type="fig"}, RUNX2 panel). We tested this result using ASaturA \[[@B26]\], and found it to be an artifact of mutational saturation: in the corrected tree (Fig. [4](#F4){ref-type="fig"}), RUNX2A is more closely related to the other fish RUNX2 genes. Comparative genomics of runt domain genes ----------------------------------------- Four RD genes have been identified in Drosophila\[[@B14]\], more similar to each other than to the vertebrate RUNXgenes: they represent an independent family expansion in insects. The four DrosophilaRD genes are all linked on chromosome X. Moreover, three of these genes are clustered within a 150 kb region. There is no linkage of RD genes, however, in the human genome: each gene is on a different chromosome. Their genomic environments usually show some conservation: the three human RUNXgenes are followed by CLICgenes in the complementary strand (Fig. [5](#F5){ref-type="fig"}), and linked to members of the DSCR1family. The RD genes appear not to be clustered in the fugu genome, though this conclusion is limited by the fragmentary nature of the current genome draft. All four fugu RD genes are flanked by at least one non-RD gene on each side. Fugu RUNXgenes are followed by CLICgenes except for FrRUNX1, but a CLICgene is located \~55 kb upstream of FrRUNX1and in the same orientation. This organization could have arisen by an inversion event in the fugu lineage. To ensure a misassembly did not cause this apparent inversion, we performed a 3x shotgun sampling of the BAC clone OML73850, which spans the range 1--97413 of scaffold 682. This quality control step failed to uncover any misassemblies, and confirmed the genomic organization observed in this fugu scaffold. FrRUNTdoes not appear to be linked to any CLICgene, and no DSCR1 family members can be discerned near any of the fugu RD genes. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Comparative genomic organization. The genomic neighborhoods of the human and fugu RD genes are compared and contrasted, highlighting synteny conservation (same color between the two species) and gene loss (green features). Feature widths represent rough gene size but are not exactly proportional to gene lengths; likewise intergenic distances are not meant to be precise. Arcs indicate larger genomic distances with one or more intervening genes (not displayed). The numbers associated with the arcs represent the distance in Mb. ::: ![](1471-2148-4-43-5) ::: When studying the wider genomic environments of fugu and human RD genes, we observed significant synteny conservation, in agreement with the observations reported for chromosome X genes \[[@B27]\]. Indeed, when comparing each of the genes neighboring fugu RUNXgenes to the human genome, we find that their orthologs tend to be located in the corresponding human chromosome, e.g. most of the genes linked to FrRUNX3have orthologs on human chromosome 1, where human RUNX3resides (Fig. [5](#F5){ref-type="fig"}). Some inversion events can be inferred, e.g. one involving the genes PHIPand IRAK1BP1and another involving MUT. Gene order and orientation has changed, and intergenic distances have changed drastically, but the overall gene synteny is largely preserved, lending support to the assignments of orthology between the human and fugu RUNXgene pairs. An exception to the conservation of synteny involves the genes CDC5Land SUPT3H, which in the human genome are found immediately upstream of RUNX2, but in fugu are instead located upstream of FrRUNT, including the first-exon interlocking with SUPT3Hmentioned earlier. Evidence points at a larger duplication in fishes, encompassing at least CDC5L, SUPT3H, RUNX2, CLIC5, ENPP4and ENPP5, followed by differential gene loss. One duplicate copy would have retained FrRUNX2, CLIC5and ENPP5(see Fig. [5](#F5){ref-type="fig"}), while the other copy (currently represented by scaffolds 835 and 376) would have retained CDC5L, SUPT3H, a second RD gene, a second copy of CLIC5(CLIC5L) and ENPP4. The presence of remnants of SUPT3Hupstream of FrRUNX2\[[@B13]\], and of two copies of RUNX2in zebrafish, lends further support to this hypothesis. Thus, comparative genomic analysis of human, fugu and zebrafish suggests that FrRUNTmay be a derivative form of a duplicated RUNX2gene. However, this contradicts the conclusions from phylogenetic analysis or the protein sequences; we discuss this contradiction below. A new family of follistatin-like genes -------------------------------------- We find several fugu genes for which no human ortholog can be discerned (green features in Fig. [5](#F5){ref-type="fig"}), among them FrRUNT. Immediately downstream to FrRUNTwe identified a novel gene (FrFSTL6) from the follistatin family, most closely related to FSTL1. A detailed computational search for additional sequences of this family identified two novel, large human follistatin-like genes, which we called FSTL4and FSTL5. We then found clear orthologs for both in the fugu genome (FrFSTL4and FrFSTL5, respectively). The genes in this family share a Kazal-type cysteine-rich domain (Fig. [6a](#F6){ref-type="fig"}) and a calcium-binding EF-hand domain (not shown). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Comparison of selected follistatin-like proteins. FST: follistatin. (a) Multiple alignment of the cysteine-rich domain. Darker shading indicates perfect conservation, lighter shading indicates positions that can be explained assuming one or two mutations. (b) Neighbor-joining tree rooted using human osteonectin/SPARC (NP\_003109) as outgroup. Numbers on branches indicate the percent support in 1000 bootstrap replicates. The horizontal bar indicates 10% divergence along each branch. The column on the right indicates the scaffold in which each gene is located (fugu) or its genomic location in Mb coordinates and cytogenetic band (human). ::: ![](1471-2148-4-43-6) ::: Having characterized the complete gene family in both human and fugu, we performed a phylogenetic reconstruction based on the conserved Kazal-type domain (Fig. [6b](#F6){ref-type="fig"}). FrFSTL6appears to have no ortholog in the human genome, nor could we identify a potential ortholog in the mouse and frog genomes. This suggests that FrFSTL6was also lost in the tetrapod lineage. It is reasonable to hypothesize that the neighboring FrRUNTand FrFSTL6genes were lost in a single deletion event. Discussion ========== We have taken advantage of the availability of genomic drafts for several vertebrate species, including the finished human genome, to identify the orthologs of all currently known runt-domain (RD) genes, as well as a novel member of this small gene family. Both pufferfish species (Takifugu rubripesand Tetraodon nigroviridis) have four RD genes; since these genomes are only available as draft assemblies, additional RD genes might be found when the finished genomic sequences are made available. The function of the novel FrRUNT/TnRUNTgene is currently unknown. Based on the phylogenetic analysis, this novel gene appears to represent an ancestral form of the RD family in vertebrates, subsequently lost in the tetrapod lineage. It is therefore surprising that its gene structure, and not that of RUNX2as in humans, is interlocked with the SUPT3Hgene. Based on the comparative genomics analysis alone, one could hypothesize that FrRUNTis simply a derivative form of RUNX2, i.e. the ortholog of zebrafish RUNX2A. In this case, though, one would expect FrRUNT to be more similar to FrRUNX2 than it is to either FrRUNX1 or FrRUNX3, but in terms of amino acid sequence similarity, it appears to be equidistant from the three RUNXgenes. This discrepancy might be explained by invoking accelerated evolution of the pufferfish RUNT genes, perhaps as a lineage-specific adaptation. Typically, nucleotide sequences diverge much faster than amino acid sequences, and the first exons of RD genes are significantly less conserved than the runt domain itself, on which we based our phylogenetic analysis. Therefore, we find it hard to sustain that, while the first exon of FrRUNTmaintains its nucleotide similarity to the first exon of RUNX2, the amino acid sequence of the (normally highly conserved) runt domain itself has diverged at such an accelerated pace. Furthermore, we found this not to be an artifact of mutational saturation \[[@B26]\]. A similar situation is observed for the neighboring FSTL6gene: parsimony considerations could lead one to assume that FSTL6is a fish-specific duplicate of FSTL1, though contradicting the phylogenetic reconstruction of the evolution of this gene family (Fig. [6](#F6){ref-type="fig"}). This situation could again be explained by assuming accelerated evolution of FSTL6, but we consider this to be a remarkable coincidence. The conundrum is whether FrRUNTis an ancestral form, or is derived from RUNX2. Both hypotheses contradict part of the available data. We propose here a third hypothesis, in the form of an evolutionary history (see Fig. [7](#F7){ref-type="fig"}): An ancestral RD gene duplicated in chordates, after divergence from sea urchin, which has a single RD gene \[[@B17]\]. One of the two resulting RD genes became the RUNXfamily founder, which expanded by triplication, and one of the three RUNXgenes (namely RUNX2) became interlocked with SUPT3H. After the teleost/tetrapod divergence, a regional duplication in teleosts created a second copy of RUNX2and its neighboring genes. In the tetrapod lineage, the ancestral RUNTgene was lost, in conjunction with FSTL6. In the pufferfish lineage, the RUNTgene replaced most of RUNX2A, perhaps by recombination (Fig. [8](#F8){ref-type="fig"}). This is supported by the clear similarity between the first exons of FrRUNTand RUNX2. The copy of SUPT3Hinterlocked with RUNX2B, apparently superfluous, is being lost by gradual degradation, and only small fragments of it remain \[[@B13]\]. This scenario is compatible with all the data observed. While it posits a small number of additional evolutionary events, it does not involve highly improbable events like the accelerated evolution of a normally highly conserved protein structural domain. Interestingly, the first duplication event could correspond to the first round of vertebrate genome tetraploidization \[[@B28]\]. A second round of tetraploidization in the ancestral vertebrate could have produced a set of four paralogous runt domain genes, and a hypothetical gene conversion event may have led to the current complement of three RUNX genes (Fig. [7](#F7){ref-type="fig"} inset). Conversion between paralogous copies of genes derived from tetraploidization events has been demonstrated \[[@B29]\]. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Hypothesis for the evolution of vertebrate RD genes. Features with thick edges represent RD genes interlocked with SUPT3Hfamily members. The closed padlock icon represents the interlocking event between the RUNX2and SUPT3H, and the open padlock represents the degradation of a copy of SUPT3H, releasing RUNX2from interlocking. Not enough information is yet available to establish which of the zebrafish RUNX2genes is interlocked with a SUPT3Hgene. \"dup\": duplication. Top right inset: Hypothesis of how the ancestral RD duplication could map to the first vertebrate tetraploidization event. RT and RX represent the ancestral forms of RUNT and RUNX genes. ::: ![](1471-2148-4-43-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### The hypothesized recombination event between the ancestral RUNX2Aand RUNTgenes. After the recombination, the RUNTgene is linked to SUPT3Hand has a RUNX2-like first exon. The derivative RUNX2Agene has not yet been observed in pufferfishes, and may have been lost in evolution. ::: ![](1471-2148-4-43-8) ::: Under the proposed scenario, none of the three extant RUNXgenes in mammals represents the ancestral vertebrate RD form. Rather, these derivative genes coexisted with an additional RUNTgene and still do so in teleost genomes. The single known RD gene in amphioxus is more similar to the vertebrate RUNXgenes than it is to FrRUNT. It is possible, therefore, that cephalochordates (including amphioxus) have a second RD gene, short and divergent enough to escape experimental detection by DNA hybridization \[[@B17]\]. Why was one of the ancestral RD genes lost in the tetrapod lineage? The three RUNXgenes bind to the same DNA motif and modify the expression of target genes through recruitment of transcriptional modulators, which are also shared \[[@B30]\]; functional differences between the three RUNXgenes are attained by way of tightly regulated spatiotemporal expression patterns. We hypothesize that the ancestral RUNTgene became inessential to amniotes by functional reprogramming of the remaining three RUNXgenes. Its loss would therefore represent an example of evolution by reduction in complexity. In pufferfishes, the hypothesized recombination event would have placed the RUNTgene under the regulatory control of the former RUNX2Apromoter. The viability of such a sudden regulatory change would in turn suggest a significant level of functional redundancy among the RD genes. Conclusions =========== We identified a fourth runt domain gene in the fugu genome, which appears to represent either a pufferfish-specific, fast-evolving derivative of RUNX2, or a direct descendant of the ancestral chordate RUNTgene. We find the latter hypothesis more reasonable. This novel gene evolved in parallel with the vertebrate RUNXgenes, and while it has been preserved in pufferfishes, it appears to have been lost entirely in tetrapods. This suggests that the ancestral vertebrate was more complex than previously suspected. By studying a very limited set of fugu genomic regions, namely the scaffolds related to RD genes, we have identified seven apparently functional fugu genes that are absent from the human genome (Fig. [5](#F5){ref-type="fig"}), and were probably lost early in tetrapod history. In the process of identifying relevant homologs for one of these genes (FrFSTL6), we have identified a new family of follistatin-like genes in the human genome. Phylogenetic analysis of the RD protein sequences led to results that contradict those derived from comparative genomics, but we showed that the two could be reconciled into a coherent evolutionary model. These results underscore the importance of obtaining complete genomic sequences of strongly divergent vertebrates, and the value to be derived by performing detailed and integrated analyses of their gene complements. Methods ======= Search for RD genes ------------------- We used the human RUNX1, RUNX2 and RUNX3 proteins (SwissProt entries Q01196, Q13950 and Q13761, respectively) as queries in a TFASTY \[[@B31]\] search into the Takifugu rubripes\"assembly3\" genome draft \[[@B32]\] released after publication of the fugu genome \[[@B33]\]. These data have been provided freely by the Fugu Genome Consortium for use in this publication only. This search resulted in the unambiguous identification of four complete RD genes in scaffolds 183, 260, 682 and 835. Scaffold 25789 is nearly identical with range 115299--115845 of scaffold 183, partially overlapping the last exon of FrRUNX3. No further evidence was found for an additional RUNX3gene: we conclude that scaffold 25789 is an assembly artifact. We similarly searched the genome drafts for Tetraodon nigroviridisproduced by the Whitehead Institute and the Genoscope \[[@B23]\], and Danio rerio(Zv1/06 assembly, which was produced by the Zebrafish Sequencing Group at the Sanger Institute \[[@B24]\]. We analyzed, visualized and annotated all resulting genomic sequences using the GESTALT Workbench \[[@B20],[@B34]\], and produced a detailed gene model for each RD gene. Lacking cDNA or EST data, we reconstructed the putative gene structures by maximizing similarity to known RD proteins. Genomic sequence data have been submitted to GenBank with accessions AY739093-AY739096; the predicted sequences for fugu RD proteins have accessions AAU14190-AAU14193. In a second round of analysis, we used the newly identified RD genes as queries for renewed TFASTY searches of the genome drafts of human (July 03), mouse (February 03), Xenopus \[[@B35]\] (December 03 assembly) and Ciona intestinalis\[[@B36]\]. We also used BLAT to search into the updated \"freezes\" of human (May 04), chimp (November 03), mouse (May 04), rat (June 03), dog (July 04), and chicken (Feb 04). Sequence finishing ------------------ Large insert clones spanning the gaps in scaffolds from the version 3 assembly were identified by BLAST searches against the BAC/cosmid database in the v.3.0 JGI website \[[@B37]\]. Cosmids (cloned in Lawrist4) were grown in LB media with kanamycin at 37 ° C for 14 hrs, and DNA was prepared on the Autogen 740 DNA Isolation system in accordance with the manufacturer\'s instructions. BACs (cloned in pBeloBAC 11) were similarly prepared by growing in media with chloramphenicol. Primers were designed in both directions, across all gaps. Oligonucleotide-directed sequencing from clones and Polymerase Chain Reaction (PCR) methods were used to fill the gaps. PCR amplification was performed on spanning BAC/cosmid or genomic DNA of Takifugu rubripes, generously provided by Dr. Greg Elgar. PCR products were purified with sephacryl (Amersham Pharmacia) and sequenced directly using Applied Biosystems Big dye terminator kit reagents. Whenever necessary, additional pairs of primers were designed for oligonucleotide-directed sequencing to close gaps. Shotgun sequencing data was obtained from the BAC clone OML73850 (b193C08) for part of scaffold 682. OML73850 was fragmented by sonication, end-repaired and electrophoresed to select insert size of 2--5 kb. Insert was ligated into pUC18 vector, transformed and plasmid DNA was made using Eppendorf -- 5 Prime PERFECTprep robot and sequenced from both ends. Assembly was carried out using Phrap \[[@B38]\]. Analysis of the resulting sequence shows that OML73850 spans the first 97413 bp of scaffold 682, and links it to scaffold 4260. The additional sequence data generated in-house were combined with the consensus sequences of the scaffolds produced by the JGI WGS assembly v.3 for the purpose of producing a contiguous sequence for each scaffold. Phylogenetic reconstruction --------------------------- The sequences were aligned using ClustalW \[[@B39]\]. Phylogenetic trees were built using the neighbor-joining algorithm \[[@B40]\] and tested with 1000 rounds of bootstrapping. Graphics were produced with TreeView \[[@B41]\]. Since full-length protein sequences cannot be reliably aligned for extremely divergent RD genes, we used only the runt domain to reconstruct the relationship between the pufferfish RUNT, human RUNX1, RUNX2 and RUNX3 (NP\_00175, NP\_033950 and NP\_004341, respectively), ciona (C. intestinalis) RUNT, Oikopleura dioicaRUNT (AAS21356), amphioxus (B. floridae) RUNT (AY146617), sea urchin S. purpuratusRUNT (NP\_999779) and the four Drosophila melanogasterRD sequences (NP\_523424, NP\_511099, NP\_572693 and NP\_608398). The tree was rooted using the C. elegansRUN protein (AB027412) as outgroup. We further excluded the first thirteen amino acids of the runt domain, to avoid the topological distortion expected in this region from the highly divergent pufferfish RUNT sequences. For the separate phylogenetic trees of the three RUNX genes, we used the complete protein sequences, with gap opening and extension penalties of 5 and 0.1, respectively. ASaturA analyses were performed using PAM250, Kimura\'s correction and a cutoff value of 9, with 1000 rounds of bootstrap. The first exons of human and fugu runt domain genes were compared at the nucleotide level. For each exon, we selected the range from 30 nucleotides upstream of the ATG codon, to 15 downstream of the splicing donor site, i.e. 103 nucleotides for each RUNXgene and 94 nucleotides for FrRUNT*. Authors\' contributions ======================= GG conceived of the study, performed the bioinformatics analyses and prepared the manuscript. AK generated the sequence data required for finishing. LH, the laboratory director, provided guidance and contributed to the preparation of the manuscript. LR supervised the Institute for Systems Biology\'s contributions to the Pufferfish Finishing Consortium and contributed to the preparation of the manuscript. All authors read and approved the final manuscript. Acknowledgments =============== We wish to thank Yoram Groner, Jared Roach, Irit Rubin, Arian Smit and Raafat El-Gewely for helpful discussions and support, Sydney Brenner for organizing the Pufferfish Finishing Consortium, the Joint Genome Institute for their public release of fugu assembly v.3 and for providing BAC and cosmid libraries, and the Institute for Molecular and Cell Biology at Singapore for funding fugu sequencing in our laboratory.	PubMed Central	2024-06-05T03:55:49.786117	2004-11-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533870/", "journal": "BMC Evol Biol. 2004 Nov 4; 4:43", "authors": [ { "first": "Gustavo", "last": "Glusman" }, { "first": "Amardeep", "last": "Kaur" }, { "first": "Leroy", "last": "Hood" }, { "first": "Lee", "last": "Rowen" } ] }
PMC533871	Background ========== The evolutionary history of prokaryotes includes both horizontal and vertical inheritance of genes \[[@B1]-[@B3]\]. Horizontal gene transfer (HGT) events are of great interest in themselves, for their roles in creating functionally new combinations of genes \[[@B4]\], but they pose problems for investigating the phylogenetic history and divergence times of organisms. The existence of a core of genes that has not been transferred is still under debate as HGTs have been detected in genes previously considered to be immune to these events \[[@B2],[@B5]-[@B11]\]. Although a complete absence of HGT appears to be unlikely, genes belonging to different functional categories seem to be horizontally transferred with different frequencies \[[@B11]-[@B13]\]. Genes forming complex interactions with other cellular components (e.g. translational proteins) have a lower frequency of HGT and are generally more conserved among organisms. Recent studies based on analyses of these genes have obtained similar phylogenies suggesting an underlying phylogenetic signal \[[@B3],[@B14]-[@B17]\]. If we accept the use of core genes for phylogeny reconstruction then they should also be of use for time estimation with molecular clocks. Moreover the increasing number of prokaryotic genomes available has facilitated the detection of HGT through more accurate detection of orthology, paralogy, and monophyletic groups, and the concatenation of gene and protein sequences has helped increase the confidence of nodes and decrease the variance of time estimates \[[@B14],[@B16],[@B18],[@B19]\]. Temporal information concerning prokaryote evolution has come from diverse sources. For eukaryotes, the fossil record provides an abundant source of such data, but this has not been true for prokaryotes, which are difficult to identify as fossils \[[@B20],[@B21]\]. Limited information on specific groups or metabolites has been obtained from analyses of isotopic concentrations \[[@B22]\] and detection of biomarkers \[[@B23],[@B24]\]. By making some simple assumptions -- e.g., that aerobic organisms evolved after oxygen became available \[[@B25]\]- it is possible to constrain some nodes in the prokaryote timescale, but only in a coarse sense. However, most information on the timescale of prokaryote evolution has come from analysis of DNA and amino acid sequence data with molecular clocks \[[@B26]-[@B30]\]. The detection of evolutionary patterns in metabolic innovations, as a consequence of a phylogeny not dominated by HGT events, allows more detailed constraints on a prokaryote timescale. In contrast to conventional interpretations of cyanobacteria as being among the most ancient of life forms on Earth \[[@B31]\], these studies have consistently found a late origin of cyanobacteria \[[@B28],[@B30]\], nearly contemporaneous with the major Proterozoic rise in oxygen at 2.3 billion years ago (Ga), termed the Great Oxidation Event (GOE) \[[@B32]\]. In this study we have assembled a data set of amino acid sequences from 32 proteins common to 72 species of prokaryotes and eukaryotes and estimated phylogenetic relationships and divergence times with a local clock method. These results in turn have been used to investigate the origin of metabolic pathways of importance in evolution of the biosphere. Results ======= Data set -------- The majority (81%) of the 32 proteins that were used are classified in the \"information storage and processes\" functional category of the COG. The other categories represented are \"cellular processes\" (10%), \"metabolism\" (3%), and \"information storage and processing\" + \"metabolism\" (proteins with combined functions; 6%). Other studies that have analyzed prokaryote genome sequence data for phylogeny have found a similar high proportion of proteins in the \"information storage and processes\" functional category, presumably because HGT is more difficult with such genes that are vital for the survival of the cell \[[@B3],[@B18],[@B33],[@B34]\]. The concatenated and aligned data set of 32 proteins contained 27,205 amino acid sites (including insertions and deletions). With alignment gaps removed, the two data sets analyzed were 7,338 amino acid sites (Archaebacteria) and 7,597 amino acid sites (Eubacteria). The data sets were complete in the sense that sequences of all taxa were present for all proteins. Phylogeny --------- The phylogeny of eubacteria (Fig. [1](#F1){ref-type="fig"}) shows significant bootstrap support for most of the major groups and subgroups. All proteobacteria form a monophyletic group (support values 95/47/99 for ME, ML and Bayesian respectively) with the following relationships of the subgroups: (epsilon (alpha (beta, gamma))). There has been debate about the effect of base composition and substitution rate on the phylogenetic position of the endosymbiont Buchneraamong γ-proteobacteria \[[@B35],[@B36]\]. Its position here (Fig. [1](#F1){ref-type="fig"}) differs slightly from both studies; accordingly, any conclusions concerning its divergence time should be treated with caution. Spirochaetes cluster with Chlamydiae, Actinobacteria with Cyanobacteria and Deinococcus(support values for Cyanobacteria + Deinococcusare 92/80/99) and the hyperthermophiles (Thermotoga, Aquifex) branch basally in the tree. These groups and relationships are similar to those found previously with analyses of prokaryote genome sequences \[[@B3],[@B18],[@B33],[@B34]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Phylogenetic tree (ME; α = 0.94) of eubacteria rooted with archaebacteria, using sequences of 32 proteins (7,597 amino acids). Bootstrap values are shown on nodes; asterisks indicate support values \> 95%. For major groups, support values from three phylogenetic methods (ME/ML/Bayesian) are indicated in italics (dash indicates a group was not present). ::: ![](1471-2148-4-44-1) ::: The phylogeny of archaebacteria (Fig. [2](#F2){ref-type="fig"}) agrees with some but not all aspects of previous phylogenetic analyses of prokaryote genomes using sequence data \[[@B3],[@B14],[@B18],[@B30],[@B37],[@B38]\] and the presence and absence of genes \[[@B37],[@B39]-[@B41]\]. For example, each of the two major clades of Archaebacteria (excluding Korarchaeota, which was not represented) is monophyletic. This is consistent with some analyses \[[@B14],[@B18]\] but not others \[[@B3]\]. Also, the position of Crenarchaeota as closest relatives of eukaryotes (Fig. [2](#F2){ref-type="fig"}), instead of Euryarchaeota, has been debated \[[@B14],[@B18],[@B30],[@B42],[@B43]\]. The faster rate of evolution in eukaryotes (Fig. [2](#F2){ref-type="fig"}), as noted elsewhere \[[@B30],[@B44]\], requires some caution in drawing conclusions regarding their phylogenetic position. Methanogens were found to be monophyletic in some previous analyses \[[@B3],[@B41]\] but were paraphyletic in other analyses \[[@B38],[@B45],[@B46]\] and in our analysis (Fig. [2](#F2){ref-type="fig"}). The phylogenetic position of one species of methanogen in particular, Methanopyrus kandleri, has differed among previous studies \[[@B47]-[@B49]\]. However, it is difficult to make direct comparisons among various studies because they have included different sets of taxa. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Phylogenetic tree (ME; α = 1.20) of archaebacteria rooted with eubacteria, using sequences of 32 proteins (7,338 amino acids). Bootstrap values are shown on nodes; asterisks indicate support values \> 95%. For major groups, support values from three phylogenetic methods (ME/ML/Bayesian) are indicated in italics. ::: ![](1471-2148-4-44-2) ::: Time estimation --------------- Times of divergence were estimated for all nodes in the phylogenies of eubacteria (Fig. [1](#F1){ref-type="fig"}) and archaebacteria (Fig. [2](#F2){ref-type="fig"}) using the alternative constraints (calibrations) described in the Methods. The eubacteria time estimates show an average 7% increase from the molecular to the geologic (2.3 Ga minimum) calibration point. Two other additional geologic calibration points were used in the analyses (see Methods), 2.3 Ga fixed and 2.7 Ga minimum, which showed respectively 10% younger and 11% older time estimates compared with the 2.3 Ga minimum calibration point. The times estimated with the fossil calibration point in the archaebacteria data set were on average only 10% younger than the ones estimated with the molecular calibration. Moreover there was even a smaller effect on the time estimates of the deepest nodes, which were the ones of interest in this study (node M 3.2%, node N 2.1%, node O 1.8% and node P 1.3%). This variation is due not only to the different calibration times but also to the type of constraints used (i.e. minimum boundaries only vs. minimum and maximum bounds). A single timetree (Fig. [3](#F3){ref-type="fig"}) was constructed from the phylogenetic and divergence time data. The time estimates summarized in that tree derive only from the best-justified calibrations. For eubacteria, the 2.3 Ga minimum calibration (constraint), from the geologic record, was chosen because it encompasses all of the hypothesized time estimates for the origin of cyanobacteria. For archaebacteria, the 1.2 Ga calibration (minimum 1.174 Ga, maximum 1.222 Ga), from the red algae fossil record, was selected because it provides a conservative constraint on the divergence of plants and animals. Time estimates and 95% credibility intervals for all nodes under all calibrations are presented elsewhere \[see [Additional file 1](#S1){ref-type="supplementary-material"}, [Additional file 2](#S2){ref-type="supplementary-material"}, and [Additional file 3](#S3){ref-type="supplementary-material"}\], and those data are summarized for selected nodes and calibrations for eubacteria and archaebacteria (Table [1](#T1){ref-type="table"}). Although some undetected HGT could be a source of bias in the time estimates, the direction of the bias (raising or lowering the estimate) would depend on the specific node and groups involved, and it is unlikely to have had a major affect on the results, even if present. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### A timescale of prokaryote evolution. Letters indicate nodes discussed in the text. The last common ancestor was arbitrarily placed at 4.25 Ga in the tree, although this placement was not part of the analyses. The grey rectangle shows the time prior to the initial rise in oxygen (presumably anaerobic conditions). Mtb: Methanothermobacter, Tab: Thermoanaerobacter, Tsc: Thermosynechococcus. ::: ![](1471-2148-4-44-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Time estimates for selected nodes in the tree of eubacteria (A-K) and archaebacteria (L-P). Letters refer to Fig. 3. ::: Time (Ma)^a^ CI^b^ ------------ ------------------ ------------ Node A 102 57--176 Node B 2508 2154--2928 Node C 2800 2452--3223 Node D 1039 702--1408 Node E 2558 2310--2969 Node F 2784 2490--3203 Node G 2923 2587--3352 Node H 3054 2697--3490 Node I 3186 2801--3634 Node J 3644 3172--4130 Node K 3977 3434--4464 Node L 233 118--386 Node M 3085 2469--3514 Node N 3566 2876--3948 Node O 3781 3047--4163 Node P 4112 3314--4486 ^a^Averages of the divergence times estimated using the 2.3 Ga minimum constraint and the five ingroup root constraints (nodes A-K) and using the 1.198 ± 0.022 Ga constraint and the five ingroup root constraints (nodes L-P). ^b^Credibility interval (minimum and maximum averages of the analyses under the five ingroup root constraints) ::: Divergence times within eubacteria (Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}, nodes A-K) show a pattern seen previously \[[@B30]\] whereby most major groups diverge from one another (nodes B-I excluding node D) in a relatively limited time interval, approximately between 2.5--3.2 Ga. The position of the hyperthermophiles has been debated, with some studies showing them in a basal position whereas others place them more derived. The high G-C composition of these taxa is believed to be responsible for this difficulty in phylogenetic placement. Here, they branch basally (node J, 3.17--4.13 Ga and node K, 3.43--4.46 Ga), but this should be interpreted with caution for this reason. The divergence of Escherichia colifrom Salmonella typhimurium(Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}, node A; 0.06--0.18 Ga) is consistent with the time estimated previously from consideration of mammalian host evolution (0.12--0.16 Ga) \[[@B26]\]. On the other hand an inconsistency with the fossil record is represented by the divergence of unicellular (Thermosynechococcus elongatus) and heterocyst-forming (Nostoc sp.) cyanobacteria. Our time estimate for this divergence is 0.70--1.41 Ga (Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}, node D) while microfossils of both groups have been identified in Mesoproterozoic (1.5--1.3 Ga) and Paleoproterozoic (2.12--2.02 Ga) rocks \[[@B50]-[@B52]\]. However the identification of these latter fossils has been debated \[[@B51]\]. Branch lengths of cyanobacteria in our protein tree and in 16S ribosomal RNA trees \[[@B34]\] do not suggest obvious substitutional biases or rate changes as they are neither unusually long nor unusually short. The reason for the discrepancy between the molecular and fossil times remains unclear but a possible misinterpretation of the fossil record cannot be dismissed. Divergence times of most internal nodes among archaebacteria (Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}, nodes L-P) are closely spaced in time and relatively ancient, approximately between 3.1--4.1 Ga, regardless of the initial setting (prior) for the ingroup root. Node P is the earliest divergence, separating Euryarchaeota from Crenarchaeota+eukaryotes. Node O represents the common ancestor of the methanogens in our analysis (Methanopyrus kandleri, Methanothermobacter thermoautotrophicus, Methanococcus jannaschii, Archaeoglobus fulgidus, Methanosarcina mazeiand M. acetivorans). Therefore, methanogenesis presumably arose between nodes P and O, or between 4.11 Ga (3.31--4.49 Ga) and 3.78 Ga (3.05--4.16 Ga) (Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}). If the position of Methanopyrus kandleriis not considered, in lieu of the current debate concerning its relationships (noted above), node N (Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}), the minimum time for the origin of methanogenesis drops only slightly, from 3.78 Ga (3.05--4.16 Ga) to 3.57 Ga (2.88--3.95 Ga). Discussion ========== Origin of life on Earth ----------------------- Neither the time for the origin of life, nor the divergence of archaebacteria and eubacteria, was estimated directly in this study. Nonetheless, one divergence within archaebacteria was estimated to be as old as 4.11 Ga (Node P), suggesting even earlier dates for the last common ancestor of living organisms and the origin of life. This is in agreement with previous molecular clock analyses using mostly different data sets and methodology \[[@B28],[@B30]\]. A Hadean (4.5--4.0 Ga) origin for life on Earth is also consistent with the early establishment of a hydrosphere \[[@B31],[@B53]\]. Nevertheless, the earliest geologic and fossil evidence for life has been debated \[[@B21],[@B54]-[@B59]\] leaving no direct support for such old time estimates. Methanogenesis -------------- The lower luminosity of the sun during the Hadean and Archean predicts that surface water would have been frozen during that time. Instead there is evidence of liquid water and moderate to high surface temperatures \[[@B60],[@B61]\]. The long term carbon cycle (carbonate-silicate cycle), which acts as a temperature buffer, combined with greenhouse gases, probably explain this \"Faint Young Sun Paradox\" \[[@B61]\]. Arguments have been made in support of either methane \[[@B62]-[@B64]\] or carbon dioxide \[[@B65]\] as the major greenhouse gas involved. If methane was important, it would have necessarily come from organisms (methanogens), given the volume required. Archaebacteria are the only prokaryotes known to produce methane. Our time estimate of between 4.11 Ga (3.31--4.49 Ga) and 3.78 Ga (3.05--4.16 Ga) for the origin of methanogenesis suggests that methanogens were present on Earth during the Archean, consistent with the methane greenhouse theory \[[@B64]\]. Nonetheless, this does not rule out the alternative (carbon dioxide) explanation \[[@B65]\]. Anaerobic methanotrophy ----------------------- Anaerobic methanotrophy, or anaerobic oxidation of methane (AOM), is a metabolism associated with anoxic marine sediments rich in methane. This metabolism is characterized by the coupling of two reactions, oxidation of methane and sulfate reduction. The methane oxidizers are represented by archaebacteria phylogenetically related to the Methanosarcinales, while the sulfate reducers, when present, are eubacterial members of the δ-proteobacteria division \[[@B66]\]. These two groups of prokaryotes have been found associated in syntrophies, thus suggesting the coupling of these two pathways \[[@B66]-[@B69]\]. Archaebacteria have been found also isolated in monospecific clusters, oxidizing methane through an unknown reaction. It has been suggested that they may use elements of both the methanogenesis and sulfate-reducing pathways \[[@B70]\]. An example of coexistence of genes from both of these pathways is Archaeoglobus fulgidus. The particular condition of this archaebacterium has been explained with an ancient horizontal gene transfer from an eubacterial lineage, most likely a δ-proteobacterium \[[@B71],[@B72]\]. The phylogenetic position of the anaerobic methanotrophs with the Methanosarcinales places the maximum date for the origin of this metabolism at 3.09 (2.47--3.51) Ga (Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}, node M). The minimum time estimate of 0.23 Ga (0.12--0.39 Ga) (Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}, node L), probably a substantial underestimate of the true time, results from the limited phylogenetic sampling available for this group. Aerobic methanotrophy --------------------- Aerobic methanotrophs are represented in the α and γ divisions of the proteobacteria. This suggests an origin for this metabolism between node C (2.80 Ga; 2.45--3.22 Ga) and node B (2.51 Ga; 2.15--2.93 Ga) (Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}). Shared genes from this pathway and from methanogenesis also have been found in the Planctomycetales \[[@B73]\]. This has suggested a revision of the direction of the HGT, usually considered from archaebacteria to eubacteria \[[@B1]\], that presumably has spread these genes in the two domains. However the absence of Planctomycetales from our dataset and its controversial phylogenetic position \[[@B74]\] does not allow us to discriminate among these possibilities. Both anaerobic and aerobic methanotrophy have been used to explain the highly depleted carbon isotopic values found in 2.8--2.6 Ga geologic formations \[[@B22],[@B75]\]. Our time estimates for these two metabolisms are both compatible with the isotopic record. Molecular clock methods have estimated the origin of cyanobacteria at 2.56 Ga (2.04--3.08 Ga) \[[@B30]\]. Because oxygenic photosynthesis would have been necessary for aerobic methanotrophy \[[@B75]\], an anaerobic metabolism seems more likely to explain the isotopic record. Phototrophy ----------- The ability to utilize light as an energy source (phototrophy, photosynthesis) is restricted to eubacteria among prokaryotes. Phototrophic eubacteria are found in five major phyla (groups), including proteobacteria, green sulfur bacteria, green filamentous bacteria, gram positive heliobacteria, and cyanobacteria \[[@B4],[@B76]\]. Only cyanobacteria produce oxygen. There are three explanations for this broad taxonomic distribution of phototrophic metabolism; it evolved in one lineage of eubacteria and spread at a later time to other lineages by horizontal transfer, the common ancestor of these groups possessed this metabolism and genetic machinery, or there was a combination of horizontal transfer and vertical inheritance \[[@B4]\]. Because two of the three explanations require a phototrophic common ancestor, and because some features of the Archean geologic record require this metabolism if biologically produced \[[@B77]\], we have assumed here that the common ancestor (Node I) was phototrophic. Therefore, we estimate that phototrophy evolved prior to 3.19 (2.80--3.63) Ga (Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}, node I). Because the hyperthermophiles Aquifexand Thermotogaare not phototrophic and branch more basally, 3.64 (3.17--4.13) Ga (Node J) can be considered a maximum date for phototrophy. However, if those hyperthermophiles instead occupy a more derived position on the tree, as some analyses have indicated \[[@B33]\], then the maximum date is no longer constrained in this analysis. The colonization of land ------------------------ The evolution of phototrophy was most likely linked to the evolution of other features essential to survival in stressful environments. Considerable biological damage can occur from exposure to ultraviolet radiation, especially prior to the GOE and later formation of the protective ozone layer \[[@B78]\]. The synthesis of pigments such as carotenoids, which function as photoprotective compounds against the reactive oxygen species created by UV radiation \[[@B79]\], is an ability present in all the photosynthetic eubacteria and in groups that are partly or mostly associated with terrestrial habitats such as the actinobacteria, cyanobacteria, and Deinococcus-Thermus. Pigmentation was probably a fundamental step in the colonization of surface environments \[[@B80]\]. Besides the sharing of photoprotective compounds, these three groups (cyanobacteria, actinobacteria, and Deinococcus) also share a high resistance to dehydration \[[@B81]-[@B84]\], which further suggests that their common ancestor was adapted to land environments. Therefore we propose the name Terrabacteria (L. terra, land or earth) for the group that includes the bacterial phyla Actinobacteria, Cyanobacteria, and Deinococcus-Thermus. An early colonization of land is inferred to have occurred after the divergence of this terrestrial lineage with Firmicutes (Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}, node H), 3.05 (2.70--3.49) Ga, and prior to the divergence of Actinobacteria with Cyanobacteria + Deinococcus(Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}, node F), 2.78 (2.49--3.20) Ga. These molecular time estimates are compatible with time estimates (2.6--2.7 Ga) based on geological evidence for the earliest colonization of land by organisms (prokaryotes) \[[@B85]\]. Many groups of prokaryotes currently inhabit terrestrial environments, indicating that land has been colonized multiple times in different lineages. Oxygenic photosynthesis ----------------------- From the above analyses and discussion, some of the early steps leading to oxygenic photosynthesis apparently were acquisition of protective pigments, phototrophy, and the colonization of land. Currently, hundreds of terrestrial species of cyanobacteria are known, broadly distributed among the orders, with species occurring in some of the driest environments on Earth. It is possible that a terrestrial ancestry of cyanobacteria, where stresses resulting from desiccation and solar radiation were severe, may have played a part in the evolution of oxygenic photosynthesis. Nonetheless, there is ample evidence that horizontal gene transfer also has played an important role in the assembly of photosynthetic machinery \[[@B4]\]. Although we have used the origin of cyanobacteria as a calibration (2.3 Ga, geologic time based on GOE), such minimum constraints permit the estimated time to be much older in a Bayesian analysis. However, in this case, the time estimated for node E (2.56 Ga; 2.31--2.97 Ga; Fig. [3](#F3){ref-type="fig"}, Table [1](#T1){ref-type="table"}) was not much older than the constraint itself. It also agrees with an earlier molecular time estimate (2.56 Ga; 2.04--3.08 Ga) based on a largely different data set and methods \[[@B30]\]. When we used the older minimum constraint of 2.7 Ga, corresponding to 2α-methyl-hopane evidence considered to represent a biomarker of cyanobacteria \[[@B86]\], the estimated time was likewise only slightly older \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. The oldest time estimates for oxygenic photosynthesis that we obtained are still considerable younger than has been assumed -- generally -- in the geologic literature \[[@B31],[@B32],[@B87]\]. This suggests that carbon isotope excursions, microfossils, microbial mats, stromatolites, and other pre-3 Ga evidence ascribed to cyanobacteria should be re-evaluated. Conclusions =========== The analyses presented here are based on the assumption, still under debate, that historical information (phylogenies and divergence times) can be retrieved from genes in the prokaryote genome that have not been affected by horizontal gene transfer. Our prokaryotic timeline shows deep divergences within both the eubacterial and archaebacterial domains indicating a long evolutionary history. The early evolution of life (\>4.1 Ga) and early origin of several important metabolic pathways (phototrophy, methanogenesis; but not oxygenic photosynthesis) suggests that organisms have influenced the Earth\'s environment since early in the history of the planet (Fig. [4](#F4){ref-type="fig"}). An inferred early presence of methanogens (3.8--4.1 Ga) is consistent with models suggesting that methane was important in keeping the Earth\'s surface warm in the Archean but does not rule out the possibility that carbon dioxide may have been equally (or more) important. In contrast to many classical interpretations of the early evolution of life, we find no compelling evidence for a pre-3 Ga evolution of cyanobacteria and oxygenic photosynthesis. This unique metabolism apparently evolved relatively late in the radiation of eubacterial clades, shortly before the Great Oxidation event (\~2.3 Ga). The evolution of oxygenic photosynthesis may have involved a combination of adaptations to stressful terrestrial environments as well as acquisition of genes through horizontal transfer. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### A time line of metabolic innovations and events on Earth. The minimum time for oxygenic photosynthesis is constrained by the Great Oxidation Event (2.3 Ga) whereas the maximum time for the origin of life is constrained by the origin of Earth (4.5 Ga). Horizontal lines indicate credibility intervals, white boxes indicate minimum and maximum time constraints on the origin of a metabolism or event, and colored boxes indicate the presence of the metabolism or event. ::: ![](1471-2148-4-44-4) ::: Methods ======= Data assembly ------------- We assembled a dataset that maximized the number of taxa and proteins from available organisms with complete genome sequences of prokaryotes and selected eukaryotes. In doing so, we omitted a few taxa (e.g., Agrobacterium tumefaciens Cereon str C58and Halobacterium sp. NRC-1) whose addition to the data set would have resulted in a substantial reduction in the total number of proteins. Data assembly began with the Clusters of Orthologous Groups of Proteins (COG) \[[@B88]\], which consisted of 84 proteins common to 43 species. With that initial dataset we added other species from among completed microbial genomes (NCBI; National Center for Biotechnology Information), assisted by BLAST and PSI-BLAST \[[@B89]\]. In total 72 species were included in the study (54 eubacteria, 15 archaebacteria and three eukaryotes). The species of Archaebacteria and their accession numbers are: Aeropyrum pernix K1(NC\_000854), Archaeoglobus fulgidus(NC\_000917), Methanothermobacter thermoautotrophicus str. Delta H(NC\_000916), Methanococcus jannaschii(NC\_000909), Methanopyrus kandleri AV19(NC\_003551), Methanosarcina acetivorans str. C2A(NC\_003552), Methanosarcina mazei Goe1(NC\_003901), Pyrobaculum aerophilum(NC\_003364), Pyrococcus abyssi (NC\_000868), Pyrococcus furiosus DSM 3638(NC\_003413), Pyrococcus horikoshii(NC\_000961), Sulfolobus solfataricus(NC\_002754), Sulfolobus tokodaii(NC\_003106), Thermoplasma acidophilum(NC\_002578), Thermoplasma volcanium(NC\_002689). The species of Eubacteria are: Aquifex aeolicus(NC\_000918), Bacilllus halodurans(NC\_002570), Bacillus subtilis(NC\_000964), Borrelia burgodorferi(NC\_001318), Brucella melitensis(NC\_003317, NC\_003318), Buchnera aphidicola str. APS (Acyrthosiphon pisum)(NC\_002528), Campylobacter jejuni(NC\_002163), Caulobacter crescentus CB15(NC\_002696), Chlamydia muridarum(NC\_002620), Chlamydia trachomatis(NC\_000117), Chlamydophila pneumoniae CWL029 (NC\_000922), Chlorobium tepidum str. TLS(NC\_002932), Clostridium acetobutylicum(NC\_003030), Clostridium perfringens(NC\_003366), Corynebacterium glutamicum ATCC 13032(NC\_003450), Deinococcus radiodurans(NC\_001263, NC\_001264), Escherichia coli O157:H7 EDL933(NC\_002655), Fusobacterium nucleatum subsp. nucleatum ATCC 25586(NC\_003454), Haemophilus influenzae Rd(NC\_000907), Helicobacter pylori 26695(NC\_000915), Lactococcus lactis subsp. lactis(NC\_002662), Listeria innocua(NC\_003212), Listeria monocytogenes EGD-e(NC\_003210), Mesorhizobium loti(NC\_002678), Mycobacterium leprae(NC\_002677), Mycobacterium tuberculosis H37Rv(NC\_000962), Mycoplasma genitalium G-37(NC\_000908), Mycoplasma pneumoniae(NC\_000912), Mycoplasma pulmonis(NC\_002771), Neisseria meningitidis MC58(NC\_003112), Nostoc sp. PCC7120(NC\_003272), Pasteurella multocida(NC\_002663), Pseudomonas aeruginosa PA01(NC\_002516), Ralstonia solanacearum(NC\_003295), Rickettsia conorii(NC\_003103), Rickettsia prowazekii(NC\_000963), Salmonella enterica subsp. enterica serovar Typhi(NC\_003198), Salmonella typhimurium LT2(NC\_003197), Sinorhizobium meliloti(NC\_003047), Staphylococcus aureus Mu50(NC\_002758), Streptococcus pneumoniae TIGR4(NC\_003028), Streptococcus pyogenes M1 GAS(NC\_002737), Streptomyces coelicolor A3(2)(NC\_003888), Synechocystis PCC6803(NC\_000911), Thermoanaerobacter tengcongensis(NC\_003869), Thermosynechococcus elongatus BP-1(NC\_004113), Thermotoga maritima(NC\_000853), Treponema pallidum subsp. pallidum str. Nichols(NC\_000919), Ureaplasma parvum serovar 3 str. ATCC 700970(NC\_002162), Vibrio cholerae O1 biovar eltor str. N16961(NC\_002505, NC\_002506), Xanthomonas campestris pv. campestris str. ATCC 33913(NC\_003902), Xanthomonas axonopodis pv. citri str. 306(NC\_003919), Xylella fastidiosa 9a5c(NC\_002488), Yersinia pestis(NC\_003143). The eukaryotes were Arabidopsis thaliana, Drosophila melanogaster, Homo sapiens. Accession numbers for eukaryote proteins are presented elsewhere \[[@B90]\]. This dataset consisted of 60 proteins that were individually analysed as a step in orthology determination. The proteins were aligned with CLUSTALW \[[@B91]\]. Then phylogenetic trees of each protein were built and visually inspected. Initial trees were constructed using Minimum Evolution (ME), with MEGA version 2.1 \[[@B92]\]. The major criterion that we used in determining which genes to include or exclude was the monophyly of domains. We rejected genes with domains (archaebacteria and eubacteria) that were non-monophyletic, as these would be the best examples of HGT; this amounted to 61% of the genes rejected. Some other genes were omitted if there were detectable cases of HGT within a domain, such as the deep nesting of a species from one Phylum within a clade of another Phylum. Otherwise we did not eliminate genes that had a different branching order of phyla within a domain or different relationships of groups of lower taxonomic categories. Admittedly, ancient cases of HGT might be an explanation for some of those topological differences, but they are not detectable. However, we further tested the effectiveness of our criteria by examining the stability of individual protein trees, using different gamma values (α = 1, 0.5 and 0.3). We kept only the genes that were stable to such perturbations (in terms of remaining in that category of non-HGT genes). The position of eukaryotes, which varies depending on the gene, was not considered in assessing monophyly of eubacteria and archaebacteria. The 32 remaining proteins were concatenated for analysis. The α parameters used during the tree building process were estimated with the program PamL (JTT+gamma model) \[[@B93]\]. From the concatenation, trees were constructed with ME, Maximum Likelihood (ML) \[[@B94]\] and Bayesian \[[@B95]\] methods. The phylogenies obtained with ME, ML and Bayesian were similar, differing only at non-significant nodes assessed by the bootstrap method \[[@B96]\], with one only significant exception on the position of M. kandleriin the Bayesian phylogeny. The sequence alignments and other supplementary data are presented elsewhere \[[@B90]\]. ### Time estimation Time estimation was conducted separately within each domain (Archaebacteria and Eubacteria) using reciprocal rooting and several calibration points. All time estimates were calculated with a Bayesian local clock approach \[[@B97]\] utilizing concatenated data sets of multiple proteins and a JTT+gamma model of substitution \[[@B19],[@B98],[@B99]\]. The following settings were used: numsamp (10,000), burnin (100,000), and sampfreq (100). This method permitted rates to vary on different branches, which was necessary given the known rate variation among prokaryote and eukaryote nuclear protein sequences \[[@B30],[@B44]\]. Calibration of rate in this method was implemented by assigning constraints to nodes in the phylogeny. Five different initial settings (prior distributions) were used in each domain \[see [Additional file 4](#S4){ref-type="supplementary-material"}\]. These were chosen at intervals of 0.5 Ga starting from 4.5 Ga, which is approximately the age of the Earth and Solar System, to 2.5 Ga, which is slightly before the major rise in oxygen (Great Oxidation Event; GOE) as recorded in the geologic record \[[@B32]\] and related to the presence of oxygenic cyanobacteria. Those constraints pertained to the ingroup root, or deepest divergence in the tree excluding the outgroup. Because of the relatively small number of duplicate genes available for rooting the tree of life, we were unable to estimate the time of the last common ancestor (the divergence of eubacteria and archaebacteria). For the archaebacterial data set, we included eukaryotes for calibration purposes because reliable calibration points were unavailable among those prokaryotes. In doing so, only proteins in which eukaryotes clustered with archaebacteria were included \[[@B30]\]. An outgroup was used that consisted of representatives of the major groups of eubacteria \[[@B90]\]. We used the fossil and molecular times (separately) of the plant-animal divergence as calibration points, for comparison. The fossil calibration was the first appearance of a representative of the plant lineage (red algae) at 1.198 ± 0.022 Ga \[[@B100]\]. The molecular time estimate for this divergence was 1.609 ± 0.060 Ga from a study of 143 rate-constant proteins \[[@B98]\]. We used the minimum and maximum bounds for these calibration times as constraints in the Bayesian analysis. Although the results of these two different calibrations are provided for comparison, our preferred calibration is the 1.2 Ga fossil calibration because it has the best justification (supporting evidence). Therefore, our summary time estimates for archaebacteria, presented in the timetree (Fig. [3](#F3){ref-type="fig"}), use only this fossil calibration. For the eubacterial data set, we used four internal time constraints in separate analyses, all involving the origin of cyanobacteria. The first and most conservative constraint was a fixed origin (minimum and maximum bounds) at 2.3 Ga, which corresponds to the GOE. For the second constraint we used 2.3 Ga as a minimum bound, with no maximum bound. For the third constraint we used a previous molecular time estimate (2.56 Ga) for the divergence of cyanobacteria from closest living relatives among eubacteria, and fixed the minimum (2.04 Ga) and maximum (3.08 Ga) values to the 95% confidence limits of that time estimate \[[@B30]\]. The fourth constraint for the origin of cyanobacteria was set at 2.7 Ga (minimum constraint) based on biomarker evidence for the presence of 2α-methylhopanes \[[@B86]\]. We did not consider the fossil record of cyanobacteria because the earliest indisputable fossils \[[@B52]\] are younger (2000 Ma) than the indirect evidence (GOE) for the presence of these oxygen-producing organisms. Older fossils of cyanobacteria are known but are disputed \[[@B52],[@B101]\]. The use of these four alternative constraints for the origin of cyanobacteria considers most of the widely discussed hypotheses but does not rule out an origin prior to 2.7 Ga. Although the results of the four different calibrations are provided for comparison, our preferred calibration is the 2.3 (minimum) geologic calibration because it has the best justification (supporting evidence). Therefore, our summary time estimates for eubacteria, presented in the timetree (Fig. [3](#F3){ref-type="fig"}), use only this geologic calibration. For each of these calibration points, all five initial settings were applied, resulting in 15 and 20 analyses for the Archaebacteria and Eubacteria (respectively). The effects of the different initial settings on the analyses were found to be minimal. A 44% difference in the priors, in fact, generated a maximum 2.7% (average of all significant nodes) difference in the time estimates (fossil calibration point) in the archaebacteria and a maximum 3.5% (average of all significant nodes) difference in the eubacteria (molecular calibration point) \[see [Additional file 5](#S5){ref-type="supplementary-material"}\]. Authors\' contributions ======================= AF assembled and aligned the dataset and conducted initial analyses. FUB conducted phylogenetic and molecular clock analyses and co-drafted the manuscript. SBH directed the research and co-drafting the manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Complete time estimation analyses. Estimated times for each node and calibration for Eubacteria and Archaebacteria. The node numbers refer to additional files 1 (eubacteria) and 2 (archaebacteria). ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Eubacteria tree. Phylogenetic tree of eubacteria (ME; α = 0.94). Node numbers assigned during the time estimation analyses are represented in italics. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 Archaebacteria tree. Phylogenetic tree of archaebacteria (ME; α = 1.20). Node numbers assigned during the time estimation analyses are represented in italics. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 4 Prior distribution values. Mean of the prior distribution for the rate of molecular evolution of the ingroup root node (rtrate) in Eubacteria and Archaebacteria. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 5 Percentage difference. Divergence time estimates and percentage difference due to different ingroup root constraints used under each calibration point. Node numbers refer to additional file 2 (eubacteria) and additional file 3 (archaebacteria). ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We thank Prachi Shah for programming assistance, Hidemi Watanabe for providing alignment tools, and Jaime E. Blair, Robert E. Blankenship, James G. Ferry, Davide Pisani and Fabienne Thomarat for discussion. This work was supported by grants to SBH from the NASA Astrobiology Institute and the National Science Foundation. AF was supported by a Director\'s Travel Scholar grant from NASA Astrobiology Institute.	PubMed Central	2024-06-05T03:55:49.789568	2004-11-9	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533871/", "journal": "BMC Evol Biol. 2004 Nov 9; 4:44", "authors": [ { "first": "Fabia U", "last": "Battistuzzi" }, { "first": "Andreia", "last": "Feijao" }, { "first": "S Blair", "last": "Hedges" } ] }
PMC533872	Background ========== The honey bee, Apis mellifera, occupies a prominent place in biological research due to its social behavior, learning capabilities, haplodiploid mechanism of sex determination, and plasticity in phenotype (caste) and longevity. Thus, it is a model organism for classical and sociogenetic studies. In addition, bees drive a large-scale apicultural industry, and also generate important income in small-scale subsistence beekeeping. And finally, bees are of great economic and ecological relevance for their role as generalist pollinators. The decision to include the honey bee amongst the current organisms for complete genome sequencing, <http://www.hgsc.bcm.tmc.edu/projects/honeybee/> was, therefore, well founded, yet information on its transcriptome is still meager. When starting this study, little over 250 genes were annotated as partial or full length coding sequences, and only about 15,500 expressed sequence tags (mainly 5\'-ESTs generated from a normalized bee brain cDNA library \[[@B1]\]) were available in public databases. Thus, even after sequencing of the honey bee genome will be completed a considerable transcriptome sequencing effort will still be required for unequivocal genome annotation, gene identification, and subsequent functional studies. We used the ORESTES (Open Reading frame Expressed Sequence Tags) strategy to generate ESTs from different life cycle stages of the honey bee, such as appropriate for a genome annotation initiative. This strategy preferentially generates ESTs of the central, and thus most informative portion of the transcript \[[@B2]\], and frequently also identifies less abundant mRNAs \[[@B3]\]. The efficacy of the Open Reading frame ESTs strategy, in the context of an organism for which there is limited genomic information, has recently been demonstrated for Schistosoma mansoni\[[@B4]\]. This cost-efficient approach increased the already existent ApisEST database by 30% new reads. Of the 5,021 ORESTES, only 35.2% matched with previously deposited ApisESTs. When assembled with the existent ApisESTs in the NCBI database, the ORESTES sequences extended 66% of the mixed contigs. Together these data indicate that the ORESTES methodology could effectively complement the current efforts towards the definition of the Apistranscriptome. Results and discussion ====================== Honey bee Open Reading frame ESTs --------------------------------- We generated a total of 87 mini-libraries from the four major life cycle stages of honey bee workers (embryo, larva, pupa, adult) by the use of arbitrary primers and a low-stringency RT-PCR protocol \[[@B2]\]. From these libraries we obtained 5,021 sequences of appropriate standard quality (sequence \> 100 bases; Phred 15) and with an average size of 373.9 bp. These sequences were deposited in the GenBank EST database (accession numbers CK628548 to CK633568). In the annotation pipeline, these were first submitted to BLASTN searches against Apis melliferasequences deposited in the NCBI EST database (dbEST). At this step, 35.2% (1,769) of the validated sequences matched ApisESTs (Table [1](#T1){ref-type="table"}). In a subsequent step, a BLASTX comparison of the remaining sequences against the nr-NCBI database permitted the annotation of an additional 22.4% (1,123) of the honey bee ORESTES, while the remaining 42.4% (2,129) did not match any known sequence. This rather large set of ESTs that did not result in significant alignment with any sequence deposited in non-redundant databases contains candidates for novel honey bee genes. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Apis melliferaOpen Reading frame ESTs. ::: Sequencing results Number of reads ----------------------------------------- --------------------- Total analyzed reads 5021 \- Embryos 1358 \- Larval stages 720 \- Pupae 1219 \- Adults 1479 \- Stage mix 245 Local alignment matches \- ApisESTs from dbEST 1769^a^ \- Apis melliferasequences in GenBank 16\^b^ \- Genes of other organisms (orthologs) 1123^b^ \- No matches in GenBank 2129 Clusterization results* \- Number of contigs 488 \- Number of singlets 893 \- Total number of clusters 1381 ^a^BLASTN against dbEST using an E-score of 10^-30^as cutoff value; ^b^BLASTX against the nr database used \<10^-15^as the cutoff. \This set is also represented by ESTs in the dbEST database. It is included here as additional information only and is not to be summed up with the other matches. ::: The 5,021 ApisORESTES were assembled by CAP3 into 488 contigs of a mean size of 519 bp, leaving 893 singlets. In a second round of BLASTX comparisons against the nr-NCBI database, 28.5% of the contigs and 9.2% of the singlets were classified as putative orthologs. When the respective best matches were classified according to species or higher order taxa (Figure [1](#F1){ref-type="fig"}), 89.6% were from the arthropod clade (including fully or partially sequenced Apis melliferagenes). The largest fraction of these putative orthologs showed best matches with predicted Anophelesgenes (43.9%), followed by ORESTES that were classified as putative orthologs of Drosophila(29.5%). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Distribution of Best-BLASTX-matches for assembled Apis melliferaOpen Reading frame ESTs. After assembly into contigs and singlets the sequences were submitted to a search against a non-redundant protein database (NCBI). Independent of its E-score, the best match in each BLASTX result was listed according to organism category. ::: ![](1471-2164-5-84-1) ::: ### Gene Ontology classification We assigned level 3 Gene Ontology (GO) classifications to 326 of the total of 488 assembled contigs; 162 contigs did not match any sequence in the nr-protein database. In the manual annotation preceding the GO analysis we preferentially assigned the contigs with respect to their Drosophilaorthologs. The cellular component, biological process, and molecular function classifications of the honey bee sequences are shown in Table [2](#T2){ref-type="table"}. In the biological process categories there is a clear prevalence for ESTs representing cell communication, cell growth and maintenance, metabolism and morphogenesis. For molecular function, the dominant assignments were to enzymatic activity and to nucleic acid binding and related functions (translation factor, transcription factor). When compared to the corresponding GO results obtained for the bee brain ESTs \[[@B1]\], we noted a similar distribution in category dominance structure, except for the molecular functions \'transporter and ligand binding/carrier\' which have a higher representation in the bee brain ESTs than in our ORESTES contigs. This discrepancy most probably reflects functional differences in the tissues used in these two studies. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Gene Ontology classification of Apis melliferaORESTES contigs according to the Drosophilagenes that they represent. ::: Gene Ontology* Number of genes ------------------------------------------------- --------------------- Cellular Component extracellular matrix 4 extracellular space 5 intracellular 99 membrane 29 others 8 Biological Process reproduction 18 cell motility 7 response to stress 6 cell communication 25 pattern specification 10 cell growth and/or maintenance 55 metabolism 79 response to external stimulus 10 morphogenesis 24 embryonic development 9 cell differentiation 9 others 41 Molecular Function nucleotide binding 14 nucleic acid binding 40 RNA polymerase II transcription factor activity 7 antimicrobial peptide activity 3 helicase activity 4 receptor signaling protein activity 5 structural constituent of cytoskeleton 5 microfilament motor activity 5 transcription factor activity 6 kinase activity 14 oxidoreductase activity 22 transferase activity 23 hydrolase activity 38 protein binding 29 metal ion binding 9 ion transporter activity 8 others 70 GO levels were set at 3. In either of the GO categories, individual contigs may be listed in more than one category. This GO classification only includes Apis mellifera orthologs to Drosophila genes that are represented by a Flybase code. ::: ### Clustering of honey bee ESTs We clustered the contigs generated in this study (AmORESTES contigs) with the Apis melliferaESTs already present in the NCBI dbEST database (further referred to as AmNCBI contigs). Clustering performed by CAP3 resulted in a total of 3,408 contigs and led to a general increase in read depth (Figure [2A](#F2){ref-type="fig"}). This increase in read depth is reflected in the CAP3 assembled mixed sequences of the two databases. Mean length is 696 bp for the AmNCBI contigs and 496 bp for the AmORESTES contigs (Figure [2B](#F2){ref-type="fig"}). For the mixed contigs we noted a mean increase of about 150 bp in contig length, thus documenting that the ORESTES sequences add considerable information to the characterization of the honey bee transcriptome and for subsequent studies of specific genes. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### CAP3 assembly of Apis melliferaOpen Reading frame ESTs (AmORESTES) with Apis melliferaESTs previously deposited in dbEST (AmNCBI). A) Read depth distribution of pure AmNCBI or AmORESTES and of mixed contigs; B) EST size distribution of these contigs, C) Details of individual mixed contigs showing the extension and gap-closing characteristics. In all graphs, AmORESTES sequences are in blue, AmNCBI contigs are in red, and mixed contigs are in green. ::: ![](1471-2164-5-84-2) ::: Within the total contig population, 9.5% of the assembled sequences (323) are represented by mixed contigs of both AmORESTES and AmNCBI sequences, and within this group 66.3% (214) of the original AmNCBI contigs were considerably extended, or were joined across gaps by the AmORESTES contigs, as illustrated in Figure [2C](#F2){ref-type="fig"}. The fact that the number of mixed contigs is relatively low compared to total contig number may be attributed to two aspects. First, most of the AmNCBI contigs were obtained from a single tissue (brain) library, whereas the AmORESTES sequences represent whole body transcripts of all life cycle stages of the honey bee. Second, the AmNCBI sequences are mainly 5\'-ESTs, whereas the AmORESTES sequences are expected to cover more central cDNA regions. ### Genome comparison Even though the total number of ESTs available for Apis melliferais still low when compared to established genomic model organisms, we performed an across genome analysis with the set of 3,408 honey bee contigs. This involved sequential BLASTX searches, using the honey bee sequences as query entries against protein databases of Drosophila melanogaster, Anopheles gambiae, Caenorhabditis elegans, human, protozoan and fungal origin. With this selection of organisms we intended to extract information on the percentage of genes that Apisshares (i) with all organisms, (ii) with animals, (iii) with different sets of metazoans, (iv) and exclusively with insects. The cutoff E-value in these comparisons was set at 10^-6^, as used in comparisons of similar nature \[[@B4]\], and the representation of the respective putative orthologs was listed across taxonomic levels. We found that 1,629 Apiscontigs presented significant match with sequences belonging to at least one of the taxa genomes. From these, 460 contigs (28.2%) correspond to genes with a representation in all the above taxa (Figure [3](#F3){ref-type="fig"}). In addition, further 211 contigs (12.9%) could also be classified as common to all organisms since they were represented in all but in one of the members of this set of taxa (at this level, Anophelesand Drosophilawere considered as a single group representing Diptera). This increases the set of EST contigs that the honey bee may share with all organisms to 41.2%, or, when considering the entire set of 3,408 contigs, to 19.7%. The second largest set of ESTs (312 + 37 contigs) is the one that is represented as genes common to the bilaterian clade (or metazoans in general), and only the third largest set (198 + 68 ESTs) contains genes that are represented solely in hymenopterans and dipterans, and thus in the insect clade. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Similarity and representation pattern of assembled Apis melliferaESTs (ORESTES + NCBI dbESTs) with predicted proteins of other organisms. In this comparison we included eukaryotes with completely sequenced genomes (Drosophila melanogaster, Anopheles gambiae, Caenorhabditis elegansand human), plus higher taxon groups, such as protozoans (primarily represented by Plasmodium falciparumand P. yoelii) and fungi (primarily represented by Saccharomyces cerevisiae, Schizosaccharomyces pombeand Neurospora crassa). These BLASTX comparisons were performed with an E-value cut-off level set at 10^-6^. Subsequently, the representation pattern of each of the ApisESTs in each of the eukaryotic genomes was listed. Out of the total 3,408 ApisEST contigs, 1,629 could be classified as putative orthologs, and these were grouped according to the representation of these genes at the different taxonomic levels. ::: ![](1471-2164-5-84-3) ::: Since deep-level phylogeny relationships within the bilateria are still a matter of debate, we separated our dataset according to the two prevalent hypotheses. The traditional view clusters arthropods within the coelomate clade. In our set of genomes, this tree architecture would be represented by genes shared between insects and the human genome. The alternative, more recently proposed hypothesis joins arthropods with nematodes to form an ecdysozoan clade \[[@B5]\]. The result of our comparison, which places emphasis on shared genes and not on the frequency of gene losses, is more consistent with the traditional view, since the coelomate clade is represented in this analysis with almost five times more shared genes than the ecdysozoan clade. To infer on functional aspects within this pattern of genes that different clades appear to have in common we performed a Gene Ontology classification on biological process. In the set of ApisESTs that stands for genes putatively shared with all organisms, the majority was classified as having a role in metabolism, and thus can be considered to represent basic functions. In contrast, the majority of ApisESTs that are shared within the insect clade was represented in the biological process categories of cell growth and/or maintenance and cell communication. The corresponding insect-specific genes are therefore supposedly involved in more specialized functions. A similar conclusion can be reached from the micro- and macroarray analyses of transcripts detected in adult honey bee workers performing different tasks during their adult life cycle \[[@B6],[@B7]\]. A total of 70 putative ortholog ESTs did not comply with any of the plausible phylogenies, yet nevertheless, this set may contain ESTs of interesting information content, especially when considering that the main set of genes within this group consists of Apis melliferacontigs that overlap with a mammalian genome. A manual analysis of these 23 contigs by BLASTX against the nr-NCBI database revealed that they are (at least by three orders of magnitude in E-values) more similar to mammals (especially to humans) than to other vertebrates and even other insects. This suggests that these genes may have diverged less in Apisand mammals and, therefore, may be subject to related selection pressure. Alternatively, at least some of them may have been modified in Diptera, and thus would show up as insect genes only once further non-dipteran insect genomes or transcriptomes have been sequenced and annotated. As shown in Table [3](#T3){ref-type="table"}, this set of bee/human contigs contains a considerable number of predicted proteins related to cell signaling/signal transduction and transcription factors. Such a bias to information processing in our dataset of genes shared between honey bees and a mammalian genome may reveal system properties related to complex functions. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Annotation and Gene Ontology characteristics of 11 honey bee EST contigs sharing significant similarity with mammalian but not with other vertebrate or invertebrate sequences. In all cases, the best match was with human proteins. For these 11 out of 23 contigs we could retrieve functional information. ::: **ApisEST contig E-score value^a^ GO, Biological process^b^ Human LOCUS ID GenBank annotation Additional information** ---------------------- ------------------------ --------------------------------- --------------------------------------- -------------------------------------------------------- --------------------------------------------------- 437 2e-67 NA without result NM\_019116: ubiquitin binding protein ubiquitin-specific protease domain 663 3e-10 NA without result NM\_182830: MAM domain contactin 5; neural adhesion molecule 1081 5e-07 9.7 without result NM\_013041: RAB3A interacting protein (rabin3)-like1 guanin nucleotide exchange factor domain 1425 8e-27 NA without result NM\_182565: hypothetical protein MGC29814 TBP-associated factor 4; TATA box binding protein 1674 4e-82 NA without result NM\_172374: interleukin 4 induced 1 none 1953 7e-11 NA without result NM\_024707: gem (nuclear organelle) associated protein spliceosomal snRNP biogenesis 2807 8e-07 5e-04 regulation of physiological process NM\_138457: forkhead box P4 transcription factor activity 2896 4e-05 0.002 reproduction, metabolism NM\_004654: ubiquitin-specific protease 9 ubiquitin thiolesterase activity 3167 2e-27 7e-05 cell communication NM\_033046: rhotekin signal transduction 3347 5e-26 7.5 without result NM\_014006: PI-3-kinase-related kinase SMG-1 involved in nonsense-mediated mRNA decay 3374 1e-10 0.18 cell communication, NM\_014035: sorting nexing 24 intracellular signaling cascade ^a^E-value of the contig alignment with human or known insect sequences, NA: did not show any alignment; ^b^Gene Ontology results on Biological Process, FatiGO level 3, ^c^additional information obtained from Entrez Gene -- NCBI and GOA link (GOAnnotations@EBI -- European Bioinformatics Institute). ::: Finally, we found that 1,779 (52,2%) of the assembled EST contigs did not match with any sequence of the analyzed organisms. Such a large proportion of Apis-specific contigs is likely to be an overestimate. As noted in a previous study \[[@B1]\], this might be partly due to technical problems, such as, sequencing of cDNA inserts consisting mainly of 3\'-untranslated regions, the presence of unspliced intron sequences, cDNAs with a negative reading frame, or chimaeric cDNAs. However, the major portion of the Apis-specific contigs may have become classified as species-specific due to their relatively short ORFs. We performed an ESTScan analysis <http://www.ch.embnet.org/software/ESTScan.html> on the Apis-specific contigs which detected ORFs in 56% of the assembled ESTs. These ORFs are, however, relatively short, with a mean ORF length around 280 bp. Short ORF length represents a notorious problem to alignment algorithms resulting in low match scores, and consequently, a more frequent classification of short ORF ESTs as species-specific transcripts. For the honey bee, this has been shown for the brain cDNA library where 84% of the ESTs with ORFs shorter than 450 bp were classified as species-specific, against 24% in the EST set that had ORFs larger than 450 bp \[[@B1]\]. In order to gain a general perspective on the representation of species-specific ESTs we also directed our attention to estimates obtained in whole-genome cross-species analyses. For instance, a figure of 18.6% of species-specific genes was ascertained for Drosophila melanogasterin a genome comparison which included Anopheles gambiaeas the other insect representative \[[@B8]\]. Based on this information, and taking advantage of a set of Drosophila melanogasterORESTES, generated in a parallel project, we calculated the frequency of Drosophila-specific ORESTES sequences to obtain a more realistic estimate on Apis-specific genes in relatively small sets of ESTs. In this analysis, a set of 5,000 CAP3 assembled DrosophilaORESTES (409 contigs) was submitted to sequential BLASTX searches against protein databases of Drosophila melanogaster, Anopheles gambiae, Caenorhabditis elegans, human, protozoan and fungal, as described for the Apiscontigs. For comparison, this same analysis was also performed with ApisESTs, using separately 5,000 AmORESTES (486 contigs) and 5,000 AmNCB (632 contigs). The Drosophilaand ApisEST contigs consistently showed relatively low proportions of insect-specific genes (6--13%). Still lower (ca. 1% each) was the proportion of ESTs that had significantly higher similarity scores with eukaryotes other than Insecta. In all EST sets we found a large fraction of sequences that were classified as either Drosophila-specific (40%) or Apis-specific (51% for AmNCBI dbESTs and 47% for AmORESTES). This high proportion of species-specific genes, therefore appears to be generated independent of the method used in EST sequencing, as it is represented in similar proportions in both the ORESTES set and the conventional 5\'-EST set (Figure [4](#F4){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Percentage of honey bee and DrosophilaESTs representing putative species-specific genes (blue bars) in relation to ESTs that represent genes solely shared within the insect clade (pink bars), or that have higher similarity with eukaryotes other than the insect clade (yellow bars). In separate comparisons, the Apis melliferacontigs (ORESTES + NCBI dbESTs, n = 5,000), AmORESTES (n = 5,000), AmNCBI dbESTs (n = 5,000), and Drosophila melanogasterORESTES contigs (n = 5,000) were analyzed against protein databases of an insect (Anopheles gambiae) and several non-insect species (C. elegans, protozoans, fungi and H. sapiens) with completely sequenced genomes. The cut-off E-value in these comparisons was set at 10^-6^. ::: ![](1471-2164-5-84-4) ::: The figure of 40% Drosophila-specific genes obtained for our DrosophilaORESTES set can be directly set in contrast with the estimate of 18,6% species-specific genes reported in the Drosophilagenome based study \[[@B8]\], and this would predict an overestimate factor of 2.15 for species-specific genes in the EST sets. When this factor is applied to the honey bee ORESTES, the 47% estimate for species-specific AmORESTES can thus be corrected to a more realistic figure of 22%. This estimate is in agreement with the results of Whitfield et al. \[[@B1]\] who observed that 24% of the honey bee genes represented by ESTs with ORFs larger than 450 bp did not have matches to any known protein sequences. This Apis-specific gene estimate is also in range when considering that the two dipteran species are thought to have separated from a common ancestor approximately 250 million years ago, whereas the postulated sister-group relationship of Hymenoptera and Mecopteroidea \[[@B9]\] suggests a pre-permian divergence, with a predicted separate lineage evolution of over 280 million years for honey bees and dipterans \[[@B10]\]. Conclusions =========== The generation of a relative small set of Open Reading frame ESTs (ORESTES) that match and complement the already existent ApisEST database shows that this approach is sufficiently robust and favorably complements other strategies, such as ESTs prepared from normalized cDNA libraries. Its inherent properties of detecting transcripts of low abundance and aligning with central regions of transcripts \[[@B2],[@B3]\] also make it a suitable tool in searches for novel honey bee genes and their annotation in parallel with ongoing genome sequencing projects. Furthermore, the genome comparisons performed in this and other studies \[[@B1],[@B11]\] highlight that the elevated number of putative Apis-specific genes will still require extensive transcriptome sequencing for high quality genome annotation, and will play an important role in the question of insect genome organization and model systems in comparative studies \[[@B12]\]. Methods ======= Biological samples and RNA extraction ------------------------------------- Samples of the four major stages of the honey bee life cycle were collected from Apis melliferacolonies (Africanized hybrids) kept in the experimental apiary of the Dept. Genetics, Univ. São Paulo, Campus Ribeirão Preto, Brazil. Each embryo sample contained approximately 300 eggs retrieved from a frame on which the queen had been caged for up to 72 hours. This assured that we covered the entire embryonic period. The larval sample was a representation of all five instars and included also spinning-stage larvae. Prepupae and pupae, including white-eyed, pink-eyed, brown-eyed and pigmenting pupae, were pooled into the pupal samples. For the adult sample we collected newly emerged bees, a random sample of hive bees (picked from a brood frame), and returning foragers. All these samples were snap frozen in liquid nitrogen. Total RNA was isolated using TRIzol reagent (Invitrogen). The lipid-rich larval and pupal samples required two additional extraction steps with phenol/chloroform and chloroform to obtain RNA of adequate purity. In the case of Drosophila melanogaster, dechorionated embryos, larvae plus prepupae and pupae, as well as adult flies were collected from an isogenic y, w^1118^stock of Drosophila melanogaster. These were immediately frozen in liquid nitrogen and stored at -80°C until use. Total RNA was extracted with TRIzol, as described for Apis mellifera. Generation of Open Reading frame ESTs (ORESTES) ----------------------------------------------- From high quality DNA-free total RNA samples we isolated poly(A)^+^RNA using an Oligotex II (Qiagen) kit. To assess poly(A)^+^RNA quality of the samples we performed Northern blot hybridizations with an actin (Apis mellifera) or tubulin (Drosophila melanogaster) probe. The probes were labeled by a random priming reaction in the presence of \[α-^32^P\]dCTP. The actin fragment was amplified using the primers described in Table [4](#T4){ref-type="table"}. The Drosophilatubulin probe was already available from previous studies. High quality total RNA preparations were subjected to a DNase I treatment, and the absence of DNA contaminants was assessed by Southern blot hybridization of PCR products amplified with Apisor Drosophila16S mitochondrial DNA primers, respectively. High quality poly(A)^+^RNAs were aliquoted and stored at -80°C. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Specific primers used to assess quality and absence of DNA contaminants of the RNA samples, and randomly selected primers used to generate cDNA profiles. ::: Primer code Sequence ---------------------------------- --------------------------------------- actin F (Apis) 5\' AGCTATGAACTTCCAGATGGT 3\' actin R (Apis) 5\' CCACATCTGTTGGAAGGT 3\' 16S mitochondrial F (Apis) 5\' TTATTCACCTGTTTATCAAAACAT 3\' 16S mitochondrial R (Apis) 5\' \'TATAGATAGAAACCAAYCTG 3\' 16S mitochondrial F (Drosophila) 5\' CCGGTCTGAACTCAGATCACGT 3\' 16S mitochondrial R (Drosophila) 5\' CGCCTGTTTAACAAAAACAT 3\' p3\_2 5\' TTGGGGATCGTATGTAGTATG 3\' pA82\_1 5\' CACTTCAGGATCCCTTGTAAGC 3\' pA82\_2 5\' CCAACATTGAATTCTCTTTGAC 3\' pA82\_4 5\' CAATAACAATGAATTCCAGAATCTCG 3\' pPT7C4\_B 5\' GCTTACAAGGGATCCTGAAGTGTTTCC 3\' pPT7C4\_XS 5\' GCAGGTAAACTCTACTCGAGTTACG 3\' M-RON-AS 5\' CCAGGATGTTTGGGTGATGTA 3\' CREB-S 5\' TCATGCAACATCATCTGCTCC 3\' H-SPARC-S 5\' CTAACCCAAGACATGACATTC 3\' M-CD151-S 5\' AAAGCTCGGAGGCAGCGAACT 3\' H-CD151-AS 5\' CATGTGGCTGCAAGGCAAAGC 3\' M-SPARC-AS 5\' GCCCAATTGCAGTTGAGTGAT 3\' M-ETS1-AS 5\' GTCTTGATGATGGTGAGAGTC 3\' FUT-3-S 5\' TCATGTCCAACCCTAAGTCAC 3\' FUT-3-AS 5\' TCCAGCAGGCCTTGCAGAAAT 3\' M-CMET-S 5\' TATCTCAAACGATCGAGAGAC 3\' M-CMET-AS 5\' GCACATCTATTACCAGCTTTG 3\' H-CMET-S 5\' TTTCAAATGGCCACGGGAC 3\' H-CMET-AS 5\' GCACATTTATGACCATTCTCG 3\' H-Rhoc-AS 5\' AGAAACAACTCCAGGGGCCTG 3\' M-Rhoc-AS 5\' CTACCCAAAGCAGAAACCCCA 3\' H-Sparc-AS 5\' CCAAAACCATCCTTGACAACA 3\' H-RON-AS 5\' TGATGAGGTCCTTCACGGTG 3\' B237-2 5\' CGGAATTCACCAGATTTGAACAGAAGAG 3\' B237-3 5\' AACTGCAGTTAACCAGATTTGAACAGAAA 3\' GST\_(PGEX)\_NHE\_I-S 5\' CCGCTAGCATGTCCCCTATACTAGGTTA 3\' HOXA\_I-F 5\' CGCTCCCGCTGTTTACTCT 3\' P21-RasaI-F 5\' GACCGCTCCTCCAACTAACC 3\' P21-RasaI-R 5\' CCGGCCCACCTCTTCTACTA 3\' SRY8299.2 5\' TCTCTTTATGGCAAGACTTACG 3\' SRY1532.1 5\' TCCTTAGCAACCATTAATCTGG 3\' 92R7.2 5\' GCCTATCTACTTCAGTGATTTCT 3\' TAFIEX.1R 5\' ATCCAAGGTTCTCCCAATA 3\' ::: ORESTES profiles were generated according to Dias-Neto et al. \[[@B2]\]. Briefly, aliquots of 15 ng of purified mRNA were subjected to reverse transcription reactions utilizing SuperScript II Reverse Transcriptase (Invitrogen) and a set of randomly selected primers (Table [4](#T4){ref-type="table"}). First-strand cDNA synthesis occurred at 37°C for 60 min in a total volume of 20 μl. The products of this reaction were diluted 1:5 in water and stored at -20°C. The cDNAs contained in 1 μl of each diluted RT-product were then amplified by PCR using the same or a single alternative random primer in a PCR mix (Ready-to-Go PCR bead, Amersham Biosciences). The amplification protocol consisted of an initial step at 75°C for 5 min, followed by a 45 cycles touchdown series (95°C for 30 s, a gradually decreasing annealing temperature from 66 to 44°C lasting 10 s per step and a decrease of 2°C per step, 72°C for 1 min), and a final extension reaction at 72°C for 7 min. Aliquots of the PCR products (3--5 μl) were run on 1% agarose gels and stained with ethidium bromide. From profiles that presented near-even smears we excised two sets of amplification products, one covering a size range from 300 to 700 bp and a second one from 700 to 1500 bp. For cloning, these were extracted from the agarose gels (QIAquick Gel Extraction kit, Qiagen) and ligated into pUC18 (SureClone Ligation kit, Amersham Biosciences) for transformation of competent E. coliDH5α-cells by heat shock. Bacteria were grown in 2 × YT medium before aliquots were plated on 2 × YT agar containing ampicillin. Blue-white selected positive colonies were picked, grown overnight in 2 × YT medium in 96-well plates, and used as templates for PCR using vector primers (M13 forward and reverse). An aliquot of each amplification product was analyzed on a 1% agarose gel before another 1 μl aliquot was submitted to DNA sequencing using standard protocols of the DYEnamic™ ET Terminator kit (Amersham Biosciences). The reaction products were analyzed in a MegaBACE™ 1000 automated sequencer. Only profiles with more than 80% positive PCR reactions were sequenced. Sequence analysis ----------------- After passing through the Base Caller Cimaron 1.53 Slim Phredfy(insert size \> 100, \"N\" nucleotides less than 20%, and \"N\" repetitions of less than 6 nucleotides) and ScoreCard procedure (MegaBACE™) to check sequences quality, reads that were larger than 100 nt were submitted to an automated protocol for data analysis (Gene Annotation Pipeline) of the Apis melliferaor Drosophila melanogasterORESTES. The protocol consisted of the following steps: conversion of electropherograms (Phred, to formats .fasta, .phd and .qual), primer and vector detection and trimming (Cross\_match) and masking of repeats (RepeatMasker). Validated fasta format sequences were then submitted to a general BLASTN search against GenBank entries for mitochondrial and rRNA, as well as bacterial and fungal RNA to detect and eliminate contaminant sequences. For the Apis melliferaORESTES, subsequent BLASTN searches were performed against the approximately 15,500 Apis melliferaEST sequences deposited in GenBank dbEST. In this case, significant E values were set at 10^-30^. Searches against the non-redundant protein database entries used the BLASTX option with E-values set at 10^-10^as significance cut-off level. CAP3 was used to clusterize the ORESTES sequences of both species. For Apis mellifera, the annotation of the 488 contigs was manually checked, giving preference to Drosophilasequences in the Unigene assignment. Subsequently, the contigs were batch submitted to a Gene Ontology procedure utilizing the FatiGO tools \[[@B13]\]<http://fatigo.bioinfo.cnio.es/>. Clusterization of the ApisORESTES contigs and singletons with the Apis melliferaESTs deposited in GenBank dbEST was also performed using a CAP3 routine (standard parameters). Authors\' contributions ======================= [ApisORESTES:]{.underline} FMFN and JFS participated in all steps of library preparations data and analyses; MAVC and DGP performed the bioinformatics analyses; RMM, PMVP and MFRS participated in the library preparations and GO analysis; MCRC sequenced libraries; AMN performed validation PCRs on selected ORESTES; AEE participated in the design of the study and preparation of biological material; MMGB, EME, FSE and ZLPS participated in the design of the study, library preparations and conceptual data analysis; MLPL, VV and KH participated in the design of the study, library preparations and prepared the manuscript; WASjr coordinated the design of the study and the bioinformatics analysis. [DrosophilaORESTES:]{.underline} VV, JFS, DDA, RMM and EDN participated in all steps of library preparations and analyses; NM and RGRP participated in the design of the study and preparation of biological material; LFLR, WKM and AFC participated in RNA sample preparation; SJS, MAVC and WASjr participated in the design of the study and performed the bioinformatics analyses; AJGS, MAZ, EME and MLPL conceived and coordinated the study. All authors read and approved the final manuscript. Acknowledgements ================ The production of ApisORESTES was supported by grants from FAPESP (99/00719-6 and 98/142476) and that of DrosophilaORESTES received supported from a grant from Ludwig/FAPESP (99/03677-2). MCRC and NM were research fellows of FAPESP; KH received a CAPES/DAAD visiting professor fellowship, VV, DDA and JFS were supported by fellowships from FAPESP; FMFN, RMM, and MFRS received fellowships from CAPES and PMVP from CNPq. EDN is supported by ABADHS and CNPq grants. We thank Amélia G. Araujo, Fernanda Barbuzano, Cristiane A. Ferreira, Roberto Focosi Jr., Adriana A. Marques, Camila C.B.O. Menezes, Gislaine S.P. Pereira, Marlus C.O. Rocha, Anemari R.D. Santos, Cirlei A.V. Saraiva, Israel T. Silva and Benedita O. de Souza for their dedicated technical assistance.	PubMed Central	2024-06-05T03:55:49.793398	2004-11-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533872/", "journal": "BMC Genomics. 2004 Nov 3; 5:84", "authors": [ { "first": "Francis MF", "last": "Nunes" }, { "first": "Valeria", "last": "Valente" }, { "first": "Josane F", "last": "Sousa" }, { "first": "Marco AV", "last": "Cunha" }, { "first": "Daniel G", "last": "Pinheiro" }, { "first": "Rafaela M", "last": "Maia" }, { "first": "Daniela D", "last": "Araujo" }, { "first": "Maria CR", "last": "Costa" }, { "first": "Waleska K", "last": "Martins" }, { "first": "Alex F", "last": "Carvalho" }, { "first": "Nadia", "last": "Monesi" }, { "first": "Adriana M", "last": "Nascimento" }, { "first": "Pablo MV", "last": "Peixoto" }, { "first": "Maria FR", "last": "Silva" }, { "first": "Ricardo GP", "last": "Ramos" }, { "first": "Luis FL", "last": "Reis" }, { "first": "Emmanuel", "last": "Dias-Neto" }, { "first": "Sandro J", "last": "Souza" }, { "first": "Andrew JG", "last": "Simpson" }, { "first": "Marco A", "last": "Zago" }, { "first": "Ademilson EE", "last": "Soares" }, { "first": "Marcia MG", "last": "Bitondi" }, { "first": "Enilza M", "last": "Espreafico" }, { "first": "Foued S", "last": "Espindola" }, { "first": "Maria L", "last": "Paco-Larson" }, { "first": "Zila LP", "last": "Simoes" }, { "first": "Klaus", "last": "Hartfelder" }, { "first": "Wilson A", "last": "Silva" } ] }
PMC533873	Background ========== The nematode Caenorhabditis eleganshas been used as a simple model for understanding animal biology for nearly four decades. After the sequencing of entire genomes from several metazoans, we are now in an excellent position to take a gene-centric approach to the worm as a model organism. A majority of human genes have homologs in C. elegans. In a comparative proteomics study, 83% of the worm proteome was found to have human homologous genes \[[@B1]\]. Only 11% or less contains nematode specific genes. This makes the worm a suitable model organism for delineating human gene function \[[@B2]-[@B4]\]. In a previous study, all transmembrane protein families in the C. elegansgenome were classified and the human orthologs identified \[[@B5]\]. Predicted proteins with two or more membrane domains were clustered and for each cluster a multiple alignment was created. From the alignments, HMMs (Hidden Markov Models) were built and subsequently used to search for mammalian homologs. The consensus of nine different phylogenetic methods and BLAST were used to assign orthology. This resulted in a total of 174 worm-human orthology assignments with a high confidence. Orthologs are sequences that arose from a common ancestor gene and were separated by a speciation event \[[@B6]\]. Identification of orthologs is important, since they might share functionality. In closely related species, such as human and mouse, orthologs are normally trivial to find. However, when comparing distantly related species, e.g. human and worm, this is no longer the case because the similarity levels overall are low. Instead, one needs to rely on sophisticated phylogenetic reconstruction techniques to infer whether two genes stem from a node that corresponds to a speciation split or to a duplication event within a lineage. Close orthologs are likely to have the same biological role in the two organisms. Distant orthologs on the other hand, are less likely to have the same phenotypical role, but may have the same role in the corresponding pathway. Consequently, by studying true C. elegansorthologs to human genes experimentally in the worm, one can potentially learn more about the gene function also in humans. Depending on whether duplication(s) have occurred in one or both lineages since the speciation event, orthologs can form one-to-one, one-to-many or many-to-many relationships. Paralogs arise from a duplication event. A common scenario when genes are duplicated is that one of the gene copies is under negative selective pressure and therefore retains the function of the ancestor. The other copy might then be more free to evolve a new function different from the ancestral function. This is the reason why paralogs in different species are less likely to share functionality compared to orthologs. Paralogs can be divided into two subtypes -- outparalogs and inparalogs \[[@B7]\]. Outparalogs are paralogs that evolved by gene duplications that happened before the speciation event and therefore they do not form orthologous relationships. Inparalogs, on the other hand, form co-orthologous relationships, since they are paralogs that evolved by gene duplications that happened after the speciation event. Here we present an initial functional characterization in C. elegansof seventeen genes. The criteria for selecting these genes were that they are high confidence candidate orthologs to human genes \[[@B5]\] and that their function is unknown. They are all predicted to encode transmembrane proteins, which imply that they could constitute as yet unknown receptors, channels or transporters playing important roles in various biological processes in multicellular organisms. We are particularly interested in studying those genes that might have a neuronal function. The phylogeny, membrane topology and domain organization were analyzed. Gene function was explored experimentally in the worm by means of RNA interference induced knock-down phenotypes and gene expression patterns. Results ======= Membrane topology predictions ----------------------------- The consensus of nine different methods was used to predict membrane topologies for the putative C. elegansproteins, and two different methods were used to predict signal peptides (see Methods for details). Each predicting method has some margin of error; therefore the consensus from several different predictors is more likely to give a better estimate of the true topology. Results were viewed using the SFINX tool \[[@B8],[@B9]\], an example of output can be seen in Fig. [1](#F1){ref-type="fig"}. The number of transmembrane (TM) regions ranges between six and ten (except for one of the splice variants of R155.1), with a majority of proteins having six or seven TM regions (see Table [1](#T1){ref-type="table"}). Such proteins are likely to be receptors, channels, or transporters. One case, however, (C36B1.12) is likely to be an intramembrane protease. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Output from SFINX for (A)C. elegansprotein C36B1.12 and (B)one of its assigned human orthologs Q8TCT8. The overall membrane topology of the two proteins is very similar. The consensus from the different topology predictors is nine transmembrane (TM) regions, a N-terminal signal peptide, and a \>150 amino acids non-cytoplasmic N-terminal region. Conserved aspartic acid residues are marked with an asterisk (residues 433 and 516 for C36B1.12; residues 351 and 412 for Q8TCT8). Phobius is the only program used that predicts both TM regions and N-terminal signal peptides. The other programs are designed to only detect TM regions, and therefore, they commonly mistake the signal peptide for a TM region. Numbers on the horizontal axis indicate amino acid positions. Color coding: black = TM region, white = non-cytoplasmic, gray = cytoplasmic, striped = N-terminal signal peptide predicted by Phobius. ::: ![](1471-2164-5-85-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Description of C. elegans-- human orthologs. The Swiss-Prot accession numbers are given for the worm sequences and their human orthologs. TM: the number of transmembrane regions predicted using the consensus of nine different methods in the SFINX tool. Bootstrap support (%) is given for the inferred speciation node in the phylogenetic tree constructed using PHYLOWIN with PAM distances. Identity (%): sequence percentage identity from the Blastp output between the C. elegansgene and the nearest human ortholog. Four of the worm genes are predicted to have two splice variants (a and b). However, there are only small differences in the protein sequences between the two variants, except for R155.1. Splice variant R155.1b is predicted to have a truncation of more than one hundred amino acids in the N-terminus compared to R155.1a. The PF03062 domain (MBOAT) is still present in both splice variants. The protein sequence for R155.1a was used in the phylogenetic analysis. ::: **C. elegansorthologs TM Pfam-A domains Human orthologs Bootstrap support (%) Identity (%) Nearest human ortholog with putative function -------------------------------------------- -------- --------------------------- -------------------------------- --------------------------- ------------------ ----------------------------------------------------------------------------- C30H6.2(Q9XVR4) 6 PF02535 (ZIP) Q9H6T8, Q9NXC4, Q96NN4 57 50 Q9H6T8: SLC39A4, involved in intestinal absorption of zinc. T11F9.2a(Q8I4G0), T11F9.2b(Q22395) 7 PF02535 (ZIP) Q15043, Q96SM9, Q9C0K1, Q96BB3 91 30 Q9C0K1: BIGM103, involved in intracellular zinc retention and accumulation. H13N06.5(Q9XTQ7) 9 PF02535 (ZIP) Q92504 98 52 Transport of zinc out of ER^2^and other intracellular stores. T28F3.3*^1^(Q9XUC4) 7\ PF02535 (ZIP) Q92504 79 38 Transport of zinc out of ER^2^and other intracellular stores. T01D3.5(Q9XVJ5) 8 PF02535 (ZIP) Q9NUM3 99 38 Function unknown. F40F9.1a(Q8MQ56), F40F9.1b(Q8MQ55) 7 PF01027 (UPF0005) Q9BWQ8, Q969X1 70 39 Q9BWQ8: Lifeguard protein, protects cells from Fas-mediated cell death. F40F9.2(Q20241) 7 PF01027 (UPF0005) Q9BWQ8, Q969X1 70 40 Q9BWQ8: Lifeguard protein, protects cells from Fas-mediated cell death. F08F1.7(O17388) 9\* PF02990 (EMP70) Q99805 100 62 Endosomal integral membrane protein. ZK858.6a(Q94422) 9 PF02990 (EMP70) Q92544 100 53 Function unknown. ZK858.6b(Q7YTF9) 9\* F14F3.3(Q19468) 9 PF03062 (MBOAT) Q96N66, Q99908 100 27 Q99908: BB1 protein, malignant cell expression-enhanced gene. R155.1a(O01925) 8 PF03062 (MBOAT) Q92980 100 32 Function unknown. R155.1b(Q86DC4) 4 C36B1.12(Q93346) 9\* PF04258 (Peptidase\_A22B) Q8TCT7, Q8TCT8, Q8IUH8 100 30 Q8TCT7: Presenilin-like protein, may act as intra-membrane protease. *sft-4(Q18864) 7 PF02077 (SURF4) O15260 100 55 Surfeit locus protein 4, probable ER^2^integral membrane protein. D2013.10(O62126) 6 None Q15055 98 53 Function unknown. T04A8.12(Q22141) 6 None Q9UHJ9 50 35 FRAG1 (FGFR (fibroblast growth factor receptor) activating gene 1). Y6B3B.10(Q9XWE9) 6 PF03798 (LAG1) P27544 99 37 Function unknown. ZK721.1(Q9GYF0) 10\ None Q9NXL6, Q9Y357 97 34 Function unknown. ^1^T28F3.3 was initially included because of strong similarity to Q92504; however, the phylogenetic analysis showed that it is probably an outparalog to the human gene. ^2^ER = endoplasmic reticulum. \* predicted to have a N-terminal signal peptide. ::: Phylogenetic analysis --------------------- The results from the phylogenetic analysis are presented in Table [1](#T1){ref-type="table"}. The previous orthology assignments are still valid \[[@B5]\], although for some C. elegansgenes, additional human orthologs have emerged from the sequencing efforts. At present, 29% (5 of 17) of the worm genes have one-to-many ortholog relationship with human genes, which means that there has probably been an expansion in the human lineage. This is the case for C36B1.12 (Q93346) and ZK721.1 (Q9GYF0) (see Fig. [2A](#F2){ref-type="fig"} and [2D](#F2){ref-type="fig"}, respectively). 53% (9 of 17) showed a one-to-one relationship with its human ortholog, for example sft-4(Q18864) (see Fig. [2C](#F2){ref-type="fig"}). Two of the worm genes, F40F9.1 (Q8MQ55, Q8MQ56) and F40F9.2 (Q20241), seem to have a many-to-many relationship (see Fig. [2B](#F2){ref-type="fig"}). The phylogenetic tree in Fig. [2B](#F2){ref-type="fig"} show somewhat inconclusive support for where the speciation event could have taken place. It is possible that the human gene Q8IVW7 is also an ortholog to the two worm genes. To investigate the ortholog relationship further, Orthostrapper was used \[[@B10]\]. Orthostrapper analyzes a set of bootstrap trees instead of the optimal tree for orthologs. The algorithm detects orthologous relations between two (groups of) species. The frequency of orthology assignments in the bootstrap trees can be interpreted as a confidence value for the possible orthology of two proteins. Orthology assignments in the optimal phylogenetic tree that might be incorrect can be identified by their low ortholog bootstrap value. This makes it possible to resolve complicated many-to-many orthologous relationships. When analyzing the multiple alignment for the phylogenetic tree in Fig. [2B](#F2){ref-type="fig"} using Orthostrapper, the results showed a stronger support for the human genes Q9BWQ8 and Q969X1 to be the orthologs compared to Q8IVW7 (65% vs. 23%). It seems that the orthologous relationship in this particular case is complicated to elucidate. Still, Q9BWQ8 and Q969X1 are the best candidate human orthologs for the C. elegansgenes F40F9.1 and F40F9.2. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Phylogenetic trees for genes (A)C36B1.12 (Q93346), (B)F40F9.1a (Q8MQ56), F40F9.1b (Q8MQ55) and F40F9.2 (Q20241), (C)sft-4(Q18864), and (D)ZK721.1 (Q9GYF0). The trees were constructed using PHYLOWIN with neighbor-joining method and PAM distances. 500 bootstrap replicates were run. All gene identifiers are Swiss-Prot accession numbers, except in (D) where XP\_148505 is the NCBI accession number. The C. elegansgenes studied here are marked with asterisks. F40F9.1 is predicted to have two splice variants; however, the putative proteins have the same length and only differ in the two most C-terminal amino acids. Species abbreviations: Arabidopsis thaliana(AT), Caenorhabditis elegans(CE), Drosophila melanogaster(DM), Fugu rubripes(FR), Homo sapiens(HS), Mus musculus(MM), Oryza sativa(OS), Rattus norvegicus(RN), Saccharomyces cerevisiae(SC), Schizosaccharomyces pombe(SP), Xenopus laevis(XL). ::: ![](1471-2164-5-85-2) ::: H13N06.5 and T28F3.3 both show sequence similarity to the human gene Q92504. However, the phylogenetic tree and results from Orthostrapper (data not shown), suggest that H13N06.5 is the putative ortholog to Q92504, whereas T28F3.3 may be an outparalog. The bootstrap support for the speciation node between T04A8.12 and its human ortholog Q9UHJ9 barely made the cutoff of 50% when PAM distances was used. With observed divergence and Poisson correction as distance methods, the bootstrap support improved to 99% and 67%, respectively. When analyzing the phylogenetic tree using Orthostrappper, there was a very strong support for this orthology assignment (94%). Therefore, we conclude that T04A8.12 is probably the ortholog to Q9UHJ9. C30H6.2 has three potential human orthologs, Q9H6T8, Q9NXC4 and Q96NN4. The bootstrap support for the speciation node with PAM distances was 57%. This improved to 87% and 88% with observed divergence and Poisson correction, respectively. Orthostrapper results showed a strong support for Q9H6T8 and Q9NXC4 as orthologs to C30H6.2 (84%), whereas the support for Q96NN4 was weaker (57%). Considering these results, we believe that all three human genes are orthologs to the worm gene; however, the ortholog relationship seems to be weaker between Q96NN4 and C30H6.2. Putative domain assignments --------------------------- The domain organization of the predicted proteins was analyzed using the Pfam database \[[@B11],[@B12]\] (see Table [1](#T1){ref-type="table"}). Conclusions about possible functions cannot be drawn from the mere presence of a putative domain, although it can give some indication. Five of the proteins (C30H6.2, T11F9.2, H13N06.5, T28F3.3 and T01D3.5) may have a PF02535 domain, which is annotated as being a ZIP domain. The ZIP family is believed to include zinc and other metal transporters. The ZIP proteins have been classified into four groups based on sequence conservation \[[@B13]\]; the ZIP subfamily I and II, the gufA subfamily and the LIV-1 subfamily (also called the LZT subfamily). The ZIP I subfamily contains mostly plant and yeast sequences; however, it also includes T01D3.5 and its putative orthologs in Drosophila melanogaster, mouse and human (Q9V4C6, Q8BFU1 and Q9NUM3, respectively). The other four worm genes appear to belong to the LIV-1 subfamily. This subfamily has a unique metalloprotease motif that raises the possibility that they might have protease activity \[[@B14]\]. Within the LIV-1 subfamily there is a subgroup called the KE4 group, to which H13N06.5 and its human ortholog hKE4 (Q92504) belong. C36B1.12 was predicted to have a PF04258 domain, a probable signal peptide peptidase (SPP) domain. SPP catalyzes intramembrane proteolysis of some signal peptides after they have been cleaved from a preprotein. This processing by SPP is related to protein cleavage by presenilins. Homologs to SPP are divided into five subfamilies based on phylogenetic analysis (subfamily SPP and subfamilies SPPL1-4, for SPP like) \[[@B15]\]. C36B1.12 and its putative human orthologs Q8TCT7, Q8TCT8 and Q8IUH8 belong to the SPPL2 subfamily. The members of subfamilies SPPL1-4 only show homology to SPP in the C-terminal half of the protein and in the N-terminus there is substantial variation. This suggests that the C-terminal part may constitute the proteolytic subdomain, whereas the N-terminus defines the specific function of the respective proteins. F40F9.1 and F40F9.2 seem to have a PF01027 domain (UPF0005), which is an uncharacterized protein family. Both F08F1.7 and ZK858.6 may belong to the PF02990 domain family (EMP70). Proteins in this family might be located to endosomal membranes \[[@B16]\]. F14F3.3 and R155.1 were predicted to have a PF03062 domain, which is annotated as a MBOAT (Membrane bound O-acyl transferases) domain. Biochemically characterized proteins of this group encode enzymes that transfer organic acids onto hydroxyl groups of membrane-embedded targets \[[@B17]\]. SFT-4 most likely has a PF02077 (SURF4) domain. Members of this family are believed to encode integral membrane proteins located to the endoplasmic reticulum \[[@B18]\]. A PF03798 domain (LAG1) was found in Y6B3B.10. This domain is associated with longevity in yeast (Jiang et al. 1998). Three of the seventeen putative proteins (D2013.10, T04A8.12 and ZK721.1) do not match to any Pfam-A domain. RNA interference studies ------------------------ Out of the seventeen genes studied, sft-4and R155.1 exhibited phenotypes when both the N2 (wildtype) and the RNAi sensitive rrf-3(pk1426)II \[[@B19],[@B20]\] strains were subjected to RNAi by feeding (see Table [2](#T2){ref-type="table"}). The phenotypes were enhanced with strain rrf-3, although the Dpy (dumpy) phenotype seen for R155.1 was still low penetrant and relatively weak. The Lva (larval arrest) observed for sft-4occurred at larval stages L2--L3 and there was an almost complete penetrance with the sensitive strain. The RNAi phenotypes for both genes were detected at all temperatures, although, for sft-4they were more severe at higher temperatures. The positive results were verified with RNAi by injection in strain N2. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### RNAi phenotype and major tissues of gene expression for C. elegansorthologs. Abbreviations: RNAi phenotype:clear (Clr), dumpy (Dpy), larval arrest (Lva), ruptured (Rup), sterile (Ste), wildtype (WT). Gene expression:body wall muscle (bwm), commissures (c), excretory system (exc), gonad (g), hypodermis (h), hypodermal seam cells (hs), intestine (i), neuronal (n), pharyngeal muscle (phm), rectal epithelial cells (re), spermatheca (s), vulva (v), ventral nerve cord (vnc). A limitation when extrachromosomal array transgenes are used is that expression in the germ line is not possible to evaluate. No transgenic lines could be generated for T11F9.2, H13N06.5 and T04A8.12. Possible reasons for this could be that the injected DNA concentration was too low or that the sequence was toxic. In either case, the extrachromosomal array formed may not have been sufficiently large to be inheritable \[46\]. The transgenic lines for ZK858.6 and F14F3.3 showed no expression of gfp. This might be caused by conditional gene expression, germline silencing or absence of the promoter::gfpfusion from the inheritable extrachromosomal array \[46\]. ::: **C. elegansorthologs RNAi phenotype Major tissues of gene expression** --------------------------- -------------------- -------------------------------------- C30H6.2 WT h, phm T11F9.2a, T11F9.2b WT No transgenic line H13N06.5 WT No transgenic line T28F3.3 WT h, i, n, v, vnc T01D3.5 WT hs F40F9.1a, F40F9.1b WT bwm, c, h, n, phm, vnc F40F9.2 WT exc, n, phm F08F1.7 WT h, n, phm, re, s, v ZK858.6a, ZK858.6b WT No expression F14F3.3 WT No expression R155.1a, R155.1b Dpy bwm, h, i, phm C36B1.12 WT i, n sft-4 Clr, Lva, Rup, Ste bwm, h, i, n, phm, v D2013.10 WT bwm, h, i, n, s, v T04A8.12 WT No transgenic line Y6B3B.10 WT phm ZK721.1 WT bwm, g, h, i, n, phm, s, v ::: Analysis of gene expression --------------------------- Transcriptional fusions with gfpwere established for fourteen genes and the resulting gene expression was analyzed. The results are presented in Table [2](#T2){ref-type="table"}. Because the arrays are extrachromosomal and not integrated; mosaic patterns of expression were observed. Also, germ line expression could not be analyzed, due to germ line silencing. For 18% (3 of 17) of the genes no transgenic lines could be established despite several attempts, and out of the lines established, 14% (2 of 14) showed no expression. This could be due to several reasons (see Discussion). In half of the transgenic lines established, expression was found in more than three different tissues. The most prevalent major tissues of expression were hypodermis (9 of 14 transgenic lines), nervous system and pharyngeal muscle (8 of 14) and intestine (6 of 14). Examples of gene expression patterns observed are presented in Fig. [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}, [6](#F6){ref-type="fig"}. C36B1.12 shows expression restricted to head neurons and intestine (see Fig. [3](#F3){ref-type="fig"}). The intestinal expression was stronger during larval stages compared to the adult stage, and it was predominantly located to posterior intestinal nuclei. F40F9.1 and F40F9.2 demonstrate some overlapping expression in nervous system and pharyngeal muscle (see Fig. [4A,4G](#F4){ref-type="fig"}); however, F40F9.1 appear to be more widely expressed in the nervous system with expression in more neuronal cell bodies and in commissures and ventral nerve cord (see Fig. [4C](#F4){ref-type="fig"}). Expression of F40F9.1 is also located to body wall muscle and hypodermal cells in the tail (see Fig. [4E](#F4){ref-type="fig"}). F40F9.2 is also expressed in the excretory system, although it is weaker compared to the other tissues (see Fig. [4G](#F4){ref-type="fig"}). Widespread expression patterns were observed for sft-4and ZK721.1 both during larval and adult stages (see Fig. [5](#F5){ref-type="fig"} and [6](#F6){ref-type="fig"}, respectively). For sft-4it was highly mosaic with pharyngeal muscle as the most consistent tissue of expression. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Major tissues of expression for C36B1.12. (A)Fluorescence micrograph of an L4 larvae hermaphrodite carrying a transcriptional fusion between gfpand a putative promoter of C36B1.12 expressed in neurons in the head. (C)Fluorescence micrograph of a young adult hermaphrodite carrying the same construct expressed in intestine. The observed intestinal expression is mostly located to posterior intestinal nuclei and is more prominent in younger worms. (B and D)Corresponding DIC images. Scale bars, 20 μm. ::: ![](1471-2164-5-85-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Major tissues of expression for F40F9.1 and F40F9.2. Fluorescence micrographs of an adult hermaphrodite carrying a transcriptional fusion between gfpand a putative promoter of F40F9.1 expressed in (A)neurons and pharyngeal muscle, (C)commissures (c) and the ventral nerve cord (vnc), and (E)body wall muscle (bwm) and hypodermis (h). (G)Fluorescence micrograph of an L4 hermaphrodite carrying a transcriptional fusion between gfpand a putative promoter of F40F9.2 expressed in the excretory system (exc), neurons, and pharyngeal muscle. (B, D, F, and H)Corresponding DIC images. Scale bars, 20 μm. ::: ![](1471-2164-5-85-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Major tissues of expression for sft-4. Fluorescence micrographs of an adult hermaphrodite carrying a transcriptional fusion between gfpand a putative promoter of sft-4expressed in (A)pharyngeal muscle, (C)vulva and (E)intestinal nuclei (i). The intestine shows some unspecific autofluorescence, but there is also specific expression in the intestinal nuclei. Expression in body wall muscle, hypodermis and neurons is not shown. (B, D, and F)Corresponding DIC images. Scale bars, 20 μm. ::: ![](1471-2164-5-85-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Major tissues of expression for ZK721.1. Fluorescence micrographs of an adult hermaphrodite carrying a transcriptional fusion between gfpand a putative promoter of ZK721.1 expressed in (A)body wall muscle, (C)hypodermis and (E)gonad. Gene expression in hypodermis is weaker compared to expression in other tissues. Expression in intestine, neurons, pharyngeal muscle, spermatheca, and vulva is not shown. (B, D and F)Corresponding DIC images. Scale bars, 20 μm. ::: ![](1471-2164-5-85-6) ::: Putative function assignments ----------------------------- ### C36B1.12 The three putative human orthologs (Q8TCT7, Q8TCT8 and Q8IUH8) to the C. elegansgene C36B1.12 are thought to be presenilin-like (PSL) proteins (also called PSH proteins for presenilin homologs). Presenilins are an important group of proteases acting in the nervous system. Abnormal proteolytic cleavage may result in accumulation of pathogenic insoluble proteins, implied in e.g. Alzheimer\'s disease. We have shown that C36B1.12 is probably expressed in head neurons and intestine; however, the intestinal expression might be ectopic (see Discussion for details), which would imply that the gene is exclusively expressed in neurons. This suggests that the three human orthologs may also encode neuronal functions. The membrane topology of human proteins belonging to the presenilin-like family has been analyzed previously. Q8IUH8 was predicted to have seven transmembrane (TM) segments and a cytoplasmic C-terminus \[[@B21]\], and the same was predicted for HM13\_HUMAN (Swissprot: Q8TCT9) \[[@B15]\]. However, it should be noted that although the number of TM segments of these predictions is the same, the topologies are in fact different. The fourth segment in the Q8IUH8 prediction is missing from the HM13\_HUMAN prediction, and the C-terminal segment in the HM13\_HUMAN prediction is missing from the Q8IUH8 prediction. This means that the four C-terminal TM segments are not in register between the predictions, and consequently the loops are on opposite sides. This includes the loop between the putatively catalytic aspartic acid residues also present in presenilins that was predicted cytoplasmic by Ponting et al., and non-cytoplasmic by Weihofen et al. Because of the TM segment disagreement, these aspartic acid residues were predicted to be located in TM5 and TM6 in the Ponting et al. prediction, but in TM4 and TM5 in the Weihofen et al. prediction. Merging these two proposed topologies by accepting all TM segments predicted by one or the other study would yield a topology with nine TM segments. Our own analysis of the proteins in question using the SFINX tool \[[@B8],[@B9]\] provides strong support for this topology, with the C-terminus in the cytoplasm (data not shown). We therefore propose that both previous TM topologies had incorrectly left out one TM segment, which would correspond to segments 4 and 9 in the 9-TM segment model. We further analyzed the other members of this family with SFINX \[[@B8],[@B9]\], and consistently found a 9-TM topology model with C-terminus in the cytoplasm. The conserved aspartic acid residues would be located in TM6 and TM7. As an example, the SFINX output for C36B1.12 and one of its human orthologs Q8TCT8 is shown in Fig. [1](#F1){ref-type="fig"}. The overall topology is very similar between the putative worm protein and all of its orthologs in both human and mouse, as well as the other presenilin-like proteins. One difference, however, is that C36B1.12 and two of its human (Q8TCT8 and Q8IUH8) and mouse orthologs (Q9JJF9 and Q8BHP0) are predicted to have a N-terminal signal peptide, a feature that seems to be missing from the other presenilin-like proteins. ### F40F9.1 and F40F9.2 F40F9.1 and F40F9.2 are 48% identical to each other on the protein sequence level and they also appear to have similar membrane topologies. They are close in the genome (\<100 bp apart), but on opposite strands. It has been shown that genes closer than 500 bp on opposite strands are likely to have a shared control region \[[@B22]\], which means that these two genes might be coexpressed. The expression patterns observed are indeed overlapping, although not to a full extent (see Table [2](#T2){ref-type="table"}). One of the human orthologs (Q9BWQ8) identified has been shown to protect cells from Fas-mediated cell death \[[@B23]\], suggesting that F40F9.1 and F40F9.2 might be involved in apoptosis. ### sft-4 The sft-4gene (C54H2.5) is highly conserved throughout evolution with orthologs in both vertebrates and non-vertebrates. All orthologous relationships are one-to-one with a high bootstrap support. The tree in Fig. [2C](#F2){ref-type="fig"} indicates that there exists a worm homolog (O45731) to sft-4. O45731 (T02E1.7) was found to be 33% identical to sft-4on the protein sequence level and it was also predicted to have a PF02077 (SURF4) domain. However, our RNAi screen indicates that there is no or little functional redundancy between the two genes, since sft-4has a very strong RNAi phenotype, showing an almost complete larval arrest at stages L2--L3. The RNAi phenotype for T02E1.7 is wildtype according to previous studies \[[@B24]\]. Our gene expression analysis revealed a wide spread expression of the reporter construct for sft-4(see Table [2](#T2){ref-type="table"}). Taken together, these data suggests that sft-4may play an essential role during development acting in many tissues. ### ZK721.1 ZK721.1 is most probably orthologous to the human genes Q9NXL6 and Q9Y357 (CGI-40 protein). The CGI-40 protein was found in a screen where novel human genes evolutionary conserved in C. eleganswere identified \[[@B1]\]. The function of both CGI-40 and Q9NXL6 is unknown. ZK721.1 has several worm homologs, one of which is sid-1(Q9GZC8). SID-1 has been identified as a protein that is required for systemic RNAi \[[@B25],[@B26]\]. It was predicted to have eleven transmembrane (TM) regions and some of them have been experimentally verified \[[@B27]\]. The high number of TM regions suggests that SID-1 forms a channel. Double stranded RNA is thought to diffuse through this channel, leading to spreading of the RNA and hence, a systemic RNAi effect. This idea is also supported by the fact that no homolog of sid-1has been found in Drosophila, which can explain the observed absence of systemic RNAi in this organism \[[@B28],[@B29]\]. Our analysis of ZK721.1 predicts that it has ten TM regions, which makes it a likely candidate for forming a channel. The phylogenetic tree indicates that ZK721.1 is the best candidate ortholog to human genes Q9NXL6 and Q9Y357, whereas sid-1is a probable outparalog to the human genes (see Fig. [2D](#F2){ref-type="fig"}). The tree also supports the previous finding that there is no homolog to ZK721.1 or sid-1in the Drosophilagenome. We have observed a wildtype RNAi phenotype for ZK721.1, which is consistent with results from other studies \[[@B30],[@B31]\]. Further analysis might reveal if ZK721.1 also is involved in systemic RNAi or if it has some other function. Four additional genes required for systemic RNAi have been reported (rsd-2, -3, -4and -6) \[[@B26]\], but none of them map to locus ZK721.1. ### C30H6.2, H13N06.5, T01D3.5, T11F9.2 and T28F3.3 SLC39A4 (Q9H6T8) was identified as one of three possible human orthologs to C30H6.2. The human gene has been implicated in the rare inherited condition acrodermatitis enteropathica, which results from a defect in the absorption of zinc \[[@B32]\]. It is believed that SLC39A4 might encode a zinc transporter responsible for intestinal absorption of zinc. Therefore, C30H6.2 may also be a zinc/metal transporter. The predicted human ortholog (Q92504) to H13N06.5 in C. eleganshas been shown to be a zinc transporter localized to intracellular membranes \[[@B33]\]. Q92504 probably transports zinc out of the endoplasmic reticulum and other intracellular stores. The Drosophilaortholog to H13N06.5 is Catsup(Catecholamines up, Q9V3A4), which encodes a negative regulator of tyrosine hydroxylase (TH) activity \[[@B34]\]. TH is a rate-limiting enzyme for production of dopamine in the brain. The Arabidopsis thalianagene IAR1(Q9M647) is also an ortholog to H13N06.5. It is proposed to be involved in auxin metabolism or response \[[@B35]\]. Interestingly, the mouse ortholog (Q31125) to H13N06.5 and IAR1was shown to functionally substitute for the Arabidopsisgene. These data indicate that there is functional conservation among these orthologs and it is likely that H13N06.5, and possibly also T28F3.3, could play similar roles in the corresponding pathways in the worm. BIGM103 (Q9C0K1) was identified as one of four candidate human orthologs to T11F9.2. The human gene was found to be induced during the infection and inflammatory response. It was also shown to play a role in intracellular zinc ion accumulation and retention \[[@B36]\]. Consequently, it is possible that T11F9.2 might be an integral membrane zinc/metal transporter. The human ortholog to T01D3.5 has no known putative function. We observed a wildtype RNAi phenotype for T01D3.5 as well as for the other four PF02535 (ZIP) domain containing putative proteins (C30H6.2, H13N06.5, T11F9.2 and T28F3.3). This indicates that there might be some functional redundancy between these genes. Considering the information available, it is conceivable that T01D3.5 may also be a zinc/metal transporter. ### F08F1.7, T04A8.12 and ZK858.6 F08F1.7 and ZK858.6 show 47% identity on the protein sequence level and they have similar membrane topologies with a large N-terminal non-cytoplasmic region and nine transmembrane regions in the C-terminal part. F08F1.7 and one of the splice variants of ZK858.6 were predicted to have a N-terminal signal peptide. The phylogenetic analysis indicated that the human gene p76 (Q99805) is the ortholog to F08F1.7 and it appears to localize to endosomes \[[@B37]\]. The function of the probable human ortholog (Q92544) to ZK858.6 is unknown. Both F08F1.7 and ZK858.6 are predicted to be in operons, as is T04A8.12 \[[@B38]\]. F08F1.7 is probably in an operon with tth-1(F08F1.8, O17389). TTH-1 is likely to belong to the PF01290 domain family (thymosin beta-4), which includes actin-binding proteins, implicating a possible role in cytoskeleton organization. ZK858.6 is predicted to be in an operon with ZK858.5 (Q94421) and ZK858.7 (Q94416). A PF05154 (TM2) domain with unknown function is likely to be present in ZK858.5. ZK858.7 might have a PF04189 domain (eIF3gamma; eukaryotic initiation factor 3, gamma subunit), suggesting that it could be involved in translation. T04A8.12 may be in an operon with T04A8.11 (Q22140) and T04A8.13 (Q22142). T04A8.11 might be a ribosomal protein, since it appears to have a PF00252 (ribosomal L16) domain. T04A8.13 was predicted to have a PF00646 (F-box) domain, which is known for forming structural complexes with other proteins. There are no matching Pfam-A domains for T04A8.12. The best candidate human ortholog to T04A8.12 is FRAG1 (fibroblast growth factor receptor activating gene 1, Q9UHJ9). FRAG1 seems to be ubiquitously expressed in adult human tissues and it has also been detected in several human tumor cell lines \[[@B39]\]. Our results from the gene expression analysis demonstrated that F08F1.7 is probably expressed in several tissues in C. elegans, suggesting an important biological role. Previous studies have shown that the human ortholog p76 (Q99805) is ubiquitously expressed \[[@B37]\]. There was no expression observed for ZK858.6 and for gene T04A8.12, we failed to generate a transgenic line. All three genes exhibited a wildtype RNAi phenotype. For F08F1.7 and ZK858.6, the wildtype phenotype could possibly be explained by functional redundancy between the two genes and a third C. elegansEMP70 domain containing protein Y41D4A.4 (Q95Y24). We found that Y41D4A.4 is homologous to F08F1.7 and ZK858.6, and most likely an ortholog to the human gene Q9HD45. Taken together, these findings point to that F08F1.7, ZK858.6 and T04A8.12 might play fundamental biological roles, and that they may be involved in processes such as cellular organization (F08F1.7) and translation (ZK858.6 and T04A8.12). ### F14F3.3 and R155.1 The human genes Q99908 (BB1) and Q96N66 are probable orthologs to F14F3.3. BB1 has been shown to be overexpressed in breast and bladder carcinoma \[[@B40]\], suggesting that it might have a role in tumor progression. The function of Q96N66 is unknown. The likely Drosophilaortholog to R155.1 is Nessy(Q9XYV9), a putative Hox gene target \[[@B41]\], indicating a possible developmental role. The Dpy (dumpy) RNAi phenotype detected for R155.1 could be due to some developmental/body size regulatory error in possibly the hypodermis and/or body wall muscle; tissues in which the gene is expressed according to our analysis. The best human ortholog candidate (Q92980) to R155.1 has not yet been functionally characterized. ### Y6B3B.10 Y6B3B.10 is most probably orthologous to the human gene P27544 and they both seem to belong to the PF03798 (LAG1) domain family. LAG1 is a longevity gene that was cloned from yeast \[[@B42]\]. Members of the LAG1 family are thought to be involved in determining lifespan. However, the phylogenetic tree revealed that Y6B3B.10 and its human and mouse ortholog (P27545) form a tight cluster in the tree, separated from the other LAG1 domain containing proteins, indicating that they may have evolved a slightly different function. Y6B3B.10 showed a wildtype RNAi phenotype and it appears to have an expression restricted to the pharyngeal muscle. ### D2013.10 D2013.10 is orthologous to Q15055 (human), Q8K1A5 (mouse) and Q9VX39 (Drosophila). Neither of these genes has any putative function assigned to them and they have no matching Pfam-A domains. D2013.10 is expressed in several tissues in C. elegansand it exhibits a wildtype RNAi phenotype. Discussion ========== This study illustrates how bioinformatic and experimental analysis can be combined to elucidate putative gene function. We have predicted worm-human orthologs and performed an initial functional characterization of the worm genes. Since orthologs are likely to have the same biological function, a better understanding of the function of the human genes can be accomplished through analysis in C. elegans. The genes explored in this study were selected from a previous study \[[@B5]\] and they are all predicted to encode transmembrane proteins. Such proteins are attractive to study since many interesting receptors, channels, transporters and signaling proteins are found among them, making them likely to be involved in important regulatory processes in multicellular organisms. The number of transmembrane (TM) regions predicted for each protein, is similar to the number predicted for each cluster of putative TM proteins from the former study (± 1 TM region) \[[@B5]\]. For the putative proteins H13N06.5, F14F3.3 and ZK721.1, however, the difference is larger (+2-3 TM regions). This divergence could be due to Remm and Sonnhammer having performed predictions on a cluster and not on individual genes. In addition, they used only the program TMHMM \[[@B43]\] for analyzing membrane topology. TMHMM, when using default settings, may miss weak TM regions, leading to a possible underestimation of the number of TM segments. A better estimate of the true topology can be achieved through the use of several different prediction programs. In this study, we used the consensus of nine different methods provided by the SFINX tool \[[@B8],[@B9]\] to assign membrane topology. We observed an RNAi phenotype for 11.8% (2 of 17) of the genes when using both strains N2 (wildtype) and rrf-3(RNAi sensitive), respectively. This is in agreement with previous experiments, where 10.3% (N2) and 12.8% (rrf-3) phenotypes have been detected \[[@B30],[@B44]\]. The RNAi phenotypes for sft-4are consistent with previous results \[[@B30],[@B31]\]. However, the two groups have reported non-overlapping phenotypes, but in this screen we have observed all of them. The Lva (larval arrest) phenotype has also been reported from the genome wide screen using strain rrf-3\[[@B44]\]. The Dpy (dumpy) phenotype for R155.1 has not been reported before. The gene was downregulated using RNAi by injection in a screen of chromosome III \[[@B45]\] and the phenotype was found to be wildtype. However, the focus of that analysis was to identify genes involved in cell division and therefore only a few post-embryonic phenotypes were scored. The Dpy phenotype observed is also low penetrant and relatively weak and could therefore be missed. Furthermore, differences in results from RNAi screens have been shown to exist. A 10--30% difference between experiments done in both different and in the same laboratories has been reported \[[@B44]\]. When analyzing expression patterns using transcriptional reporter fusions, one issue of concern is whether the pattern observed is the expression pattern of the native gene or not. Ectopic or lack of expression can occur if the putative promoter used does not include all the regulatory elements. Expression in several different cell types in the pharynx and in the posterior intestinal cells of young animals has been attributed to the use of incomplete promoters \[[@B46]\]. Another limitation when using extrachromosomal arrays is that analysis of expression in the germ line is not possible, due to germ line silencing. The putative promoter used in the transcriptional fusion for C36B1.12 is only 1 kb, due to the presence of an upstream gene. Therefore, it is possible that the intestinal expression seen predominantly in young worms and mostly located to posterior intestinal nuclei, is an artifact of the use of an incomplete promoter region \[[@B46]\]. If this is the case, C36B1.12 might be expressed exclusively in neurons in the head (see Fig. [3](#F3){ref-type="fig"}). This finding provides support to the idea that C36B1.12 and its three human orthologs encode neuronal functions. A possible consequence of this could be that they act in a fashion analogous to presenilin, or even that they could be involved in β-amyloid precursor protein processing. It would be interesting to study their role in nervous system development and function, and whether they are linked to neurological disorders The transcriptional fusion for T28F3.3 also showed a similar intestinal expression, apart from the specific expression in neurons in the head, ventral nerve cord, vulva and a weak expression in hypodermis. The putative promoter region used is only 0.8 kb, due to the presence of an upstream gene. Thus, the intestinal expression seen for T28F3.3 may once again be related to the use of an incomplete promoter region \[[@B46]\]. Two of the transcriptional fusions (for the genes F14F3.3 and ZK858.6) showed no expression of the reporter gene. This is unlikely due to the use of an incomplete promoter region, since the upstream region included was 2.9 kb and 3.0 kb, respectively. Instead, it might be caused by conditional gene expression, germline silencing or absence of the promoter::gfpfusion from the inheritable extrachromosomal array \[[@B46]\]. For three of the genes in this study we failed to establish transgenic lines. Possible reasons for this could be that the injected DNA concentration was too low or that the sequence was toxic. In either case, the extrachromosomal array formed may not have been sufficiently large to be inheritable \[[@B46]\]. Out of the seventeen genes in this study, three are predicted to be in operons (18%). This is equivalent to the number of genes in the C. elegansgenome that are believed to be in operons (15%). Whether C. elegansoperons contain genes of related function or not is still unknown. There are, however, some indications that genes encoding proteins of fundamental biological importance might be clustered into operons. For example, genes for mitochondrial proteins have a strong tendency to be together in operons and also genes encoding splicing proteins \[[@B38]\]. Conclusions =========== This study has shed some light upon the putative function of a few predicted worm-human orthologs. Our aim was to identify genes that could play a role in the nervous system and indeed we have been able to find eight genes that appear to be expressed in neurons. C. elegansis an excellent model organism for pursuing the functional characterization of these genes, considering its well mapped and relatively sophisticated nervous system. Investigating the function of orthologous proteins using a simple multicellular organism is a suitable approach for the possibility of learning more about the function of a gene not only in one species but also hopefully in several. This approach becomes even more valid as several genomes are being sequenced at the moment with additional ones already in the pipeline. With the enormous amount of data that these sequencing efforts are generating, it is very useful to be able to start delineating the gene function based on functional characterization of the ortholog in another species, before initiating studies in more complex organisms. Methods ======= Membrane topology predictions ----------------------------- The membrane topology was predicted with nine different methods, and the SFINX tool \[[@B8],[@B9]\], was used to display the results. Eight membrane topology predictors were used: Phobius \[[@B47]\], TMHMM2.0 \[[@B48]\], TMHMM1.0 \[[@B43]\], PHDhtm \[[@B49]\], HMMTOP2.1 \[[@B50]\], HMMTOP1.0 \[[@B51]\], MEMSAT \[[@B52]\] and TOPPRED \[[@B53]\]. In addition, a Kyte-Doolittle hydrophobicity curve \[[@B54]\] was constructed for each putative protein sequence. Transmembrane regions were considered positive if they were predicted by a majority of the methods, or by four methods and having a supporting Kyte-Doolittle hydrophobicity curve. Phobius also predicts N-terminal signal peptides. The signal peptides predicted by Phobius were verified with SignalP1.1 \[[@B55]\]. Each program was used with default settings. Databases --------- The Pfamseq database version 10.0 \[[@B56]\] was used for searching for homologous sequences. It is based on the Swiss-Prot 41.10 and SP-TrEMBL 23.15 databases. The Pfam database \[[@B11]\] version 11.0 \[[@B12]\] was used for domain assignments. Phylogenetic analysis --------------------- The Pfamseq database \[[@B56]\] was searched for homologs using the Blastp 2.2.5 program \[[@B57]\] with default settings and with the putative worm proteins as query. Multiple alignments of full-length sequences were created using POA \[[@B58]\] with default settings. Gappy sequences and columns (\>50% gaps) and redundant sequences (\>99% identical) were removed. The program PHYLOWIN \[[@B59]\] with tree building method neighbor-joining \[[@B60]\] and PAM distance was used for constructing phylogenetic trees. Trees were also built with observed divergence and Poisson correction as distance methods, however, the results from that analysis are only discussed for genes where there were major differences in bootstrap support. If available, a yeast sequence was used as an outgroup. A total of 500 bootstrap tests were run on trees to assess the significance of the branching order. Only bootstrap values ≥ 50% were considered positive. Domain assignments ------------------ Pfam-A domains were assigned using the Pfam database \[[@B11],[@B12]\]. Pfam-B domains were not considered, since they are automatically generated and non-curated and therefore of lower quality. Nematode strains and culture conditions --------------------------------------- Maintenance and handling of C. elegansstrains were as previously described \[[@B61]\]. Strains used were CGC N2 (wildtype) and CGC NL2099 (rrf-3(pk1426)II) \[[@B19]\] (Caenorhabditis Genetics Center \[[@B62]\]). The rrf-3mutant strain has an increased sensitivity to RNAi, also for neuronal genes \[[@B20]\], which otherwise are more refractory towards RNAi compared to other tissue types. Strain CB00907 (dpy-5(e907)I) was used for generating the transgenic lines \[[@B63]\]. RNAi screening -------------- ### Generation and cloning of PCR products Total RNA extracted and purified from C. elegansusing TRIzol (Invitrogen Life Technologies, Carlsbad, CA) was reverse transcribed using Reverse Transcription System (Promega, Madison, WI) and then PCR products were generated using Advantage™ 2 PCR Enzyme System (Clontech, Palo Alto, CA) with gene specific primers as well as primers for spliced leader 1 (SL1) and SL2 (Invitrogen Life Technologies): 95°C 60 s, 35 cycles of (95°C 30 s, 55°C 30 s, 68°C 4 min) followed by an additional extension at 68°C 4 min. See [additional data file 1](#S1){ref-type="supplementary-material"} for the primer sequences used for the RNAi studies. Products were ligated into linearized (XmaI) (New England Biolabs, Frankfurt am Main, Germany) and dephosphorylated L4440 vector (Fire Laboratory \[[@B64]\]) using Rapid DNA Ligation Kit (Roche, Mannheim, Germany) and transformed into JM109 E. colibacterial strain (Promega). Colonies were screened using XmaI, correct colonies were grown in overnight cultures and DNA was extracted using QIAfilter Plasmid Kit (QIAGEN, Hilden, Germany). The vector with the insert was sequenced using ABI PRISM^®^Big Dye™ Terminator Cycle Sequencing Ready Reaction Kits (Applied Biosystems, Foster City, CA). ### RNAi by feeding Strains N2 and rrf-3were used for RNAi screening by feeding \[[@B30],[@B65]\]. CGC \[[@B62]\] bacterial strain E. coliHT115(DE3) was transformed with the L4440 vector (Fire Laboratory \[[@B64]\]) containing the cloned gene fragment using standard methods. The vector contains an ampicillin (Amp) resistance and strain HT115 is tetracycline (Tet) resistant, so bacteria were selected on Amp (75 μg/ml) and Tet (12.5 μg/ml) plates. Single colonies were picked and grown in cultures of LB with Amp (60 μg/ml) and Tet (12.5 μg/ml) for 14--17 h. The bacterial solution was seeded onto NGM plates containing 1 mM IPTG and 25 μg/ml carbenicillin. Seeded plates were allowed to dry at room temperature. Eggs were prepared with standard bleaching method and transferred to the plates. N2 strain was incubated at 15, 20 and 25°C. rrf-3was incubated only at 15°C and 20°C, since the strain has a temperature-sensitive decrease in broodsize \[[@B20]\]. The hatched worms and their progeny were scored for a number of different phenotypes \[[@B30],[@B44]\]. The phenotypes assayed were: Adl (adult lethal), Bli (blistering of cuticle), Bmd (body morphology defect), Brd (low broodsize), Clr (clear), Dpy (dumpy), Egl (egg laying defect), Emb (embryonic lethal), Gro (slow post-embryonic growth), Him (high incidence of males), Lon (long body), Lva (larval arrest), Lvl (larval lethal), Mlt (molt defect), Muv (multivulva), Prz (paralyzed), Pvl (protruding vulva), Rol (roller), Rup (ruptured), Sck (sick), Sma (small), Ste (sterile), Stp (sterile progeny), Unc (uncoordinated). Emb was defined as greater than 10% dead embryos for N2 and greater than 30% dead embryos for rrf-3. Ste and Stp required a brood size of fewer than ten for N2 and fewer than five for rrf-3. Each postembryonic phenotype was required to be present among at least 10% of the analyzed worms. The experiment was ongoing for about 4 generations and the phenotypes were scored on a daily basis. A constant supply of transformed HT115 bacteria was ensured. The experiments were performed in duplicates at each temperature for each gene and worm strain. For the postembryonic phenotypes typically at least 20 worms per plate were scored. As a positive control the gene unc-22(\"twitchin\") was used (Fire Laboratory vector pPD34.09 \[[@B64]\]). Empty L4440 vector was used as negative control. ### RNAi by injections The L4440 vector (Fire Laboratory \[[@B64]\]) containing the cloned gene fragment was linearized in two separate reactions using restriction enzymes NcoI and XhoI (New England Biolabs, Frankfurt am Main, Germany), respectively. The reactions were purified and single stranded RNA was synthesized using T7 RNA polymerase (Promega, Madison, WI). The two reactions were mixed and annealing was performed to produce double stranded (ds) RNA, which was subsequently purified. The dsRNA was injected undiluted into twelve young adult N2 hermaphrodites for each gene. The injected worms were put on individual plates and split between three different incubation temperatures (15, 20 and 25°C). Phenotypes were scored for both the injected worms and two subsequent generations on a daily basis. Phenotypes scored and criteria for scoring were the same as for the RNAi by feeding of strain N2. ### Generation of transgenic lines The transgenic lines were constructed at the Baille Laboratory, Simon Fraser University, Canada \[[@B66]\]. Transcriptional expression constructs for gonadal injection were generated using fusion PCR, also known as \"PCR-stitching\" \[[@B67]\]. Typically, about 3 kb of genomic DNA sequence immediately upstream of the predicted ATG initiator site, was used as the putative promoter (see supplementary material for primer sequences). When an upstream gene was within the 3 kb, the size of the putative promoter was adjusted downwards. For genes in operons, the sequence upstream of the first gene in the operon was used. The putative promoter was fused with another DNA fragment containing gfp(green fluorescent protein) and unc-543\'UTR amplified from vector pPD95.67 (Fire Laboratory \[[@B64]\]). See [additional data file 1](#S1){ref-type="supplementary-material"} for the primer sequences used for generating the fusion PCR products. The resulting fusion PCR product was injected without purification into the gonad of young adult hermaphrodites of strain CB00907 at a concentration of 10 ng/μl together with 100 ng/μl dpy-5(+) plasmid (pCeh361) in 1xTE buffer to generate an extrachromosomal array. Analysis of the expression patterns of the different transgenic lines was performed at the Vaz Gomes Laboratory, Karolinska Institutet, Sweden. Author\'s contributions ======================= AH carried out the membrane topology predictions, phylogenetic analysis, domain assignments, RNAi studies and analysis of gene expression in the transgenic worm strains. ES participated in the membrane topology predictions, phylogenetic analysis and domain assignments. DB contributed in making the transgenic worm strains. AVG participated in the RNAi studies and analysis of gene expression. All authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Primer sequences used in RNAi and gene expression studies. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We thank Robert Johnsen, Domena Tu, Allan Mah and Lily Fang from the David Baillie Laboratory for making the gene fusion constructs and injecting them to make transgenic worm strains for the genes in this study. Other strains were kindly provided by the Caenorhabditis Genetics Center, which is funded by the NIH National Center for Research Resources. We thank the Fire Laboratory for plasmids, Elizabeth Valenzuela for technical assistance and Ivica Tamas for critical reading of the manuscript. This work was supported by a grant from the Swedish Knowledge Foundation viathe Research School of Medical Bioinformatics and Pfizer Corporation to A.H., by grants from Pfizer Corporation to E.S and A.V.G., and by grants from Genome Canada and Genome BC to D.B.	PubMed Central	2024-06-05T03:55:49.796734	2004-11-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533873/", "journal": "BMC Genomics. 2004 Nov 8; 5:85", "authors": [ { "first": "Anna", "last": "Henricson" }, { "first": "Erik LL", "last": "Sonnhammer" }, { "first": "David L", "last": "Baillie" }, { "first": "Ana Vaz", "last": "Gomes" } ] }
PMC533874	Background ========== Microarray technology has become an important tool for studying gene expression levels on the whole genome scale \[[@B1]\]. One important objective of many microarray studies is to identify differentially expressed genes between different conditions. Despite the effectiveness of the technology, microarray experiments are usually done with very few replicates due to budgetary constrains, which often results in high false positive (Type I error) and false negative rates (Type II error). For many microarray experiments, once a list of genes has been identified, intensive follow-up investigations of these genes using traditional molecular tools are often pursued. Hence, valuable resources can be wasted in pursuing genes from experiments with a high false positive rate. Increasing the sample size increases the statistical power to detect expression differences in microarray analysis while also decreasing the error rate. However, it is important to balance sample size with other experimental goals so as not to waste resources. An important issue that concerns many biologists is, therefore \"how many replicates are needed to obtain a given type of result?\" In general, the required sample size depends on the magnitude of the variability of the population, the magnitude of the expression change that is biologically meaningful (or desirable to detect), the power to detect the expression change, and the P-value/significance level/false positive rate. However, power and sample size have been viewed as complicated and difficult issues for microarray studies due to the large number of genes being investigated and little knowledge of the degree of natural expression variation within a population. To date, very few studies have assessed power and sample size requirements in microarray experiments. Pan et al. \[[@B2]\] proposed a normal mixture model to calculate the number of replicates required. In this study, the parameters were estimated using a subset of a real data set generated by cDNA arrays. This paper assumed that the replicates were independent of each other, whether they were drawn from the same individual or multiple individuals. Lee and Whitmore \[[@B3]\] discussed conceptual issues and presented computational methods (Analysis of Variance) for statistical power and sample size for different types of experimental designs, taking multiple testing into account. However, the data sets used to demonstrate these models contained a single pooled sample for each treatment/time point but not true biological replicates. Zien et al. \[[@B4]\] proposed a complex model that applies only to Affymetrix data to estimate biological variation and measurement error (normal additive and multiplication measurement error) for two sample comparisons. This study was based on 5 real Affymetrix data sets where the minimum required sample size was estimated based on a simulation study. Zien et al. \[[@B4]\] assumed a normal distributed additive measurement error and lognormal distributed measurement error. However, in real data, the functional form of the distribution of gene expression levels is generally unknown. The most common practice in microarray experiments is to assume normality of log transformed intensities or ratios. Pavlidis et al. \[[@B5]\] used a random sampling approach to evaluate the stability of the genes found to be differentially expressed between two groups from 16 published data sets; they found that the stability of some of the smaller data sets with fewer than 10 replicates was inconclusive. This approach is sound for the purpose of planning a study when pilot data is available with a large number of replicates. However, a pilot study is usually done with a small number of replicates where this is not feasible. Pair-wise comparisons between conditions/groups/treatments are frequently used in microarray studies. Parametric and nonparametric statistical methods have been proposed to identify differentially expressed genes, among which t-tests are most commonly used. This paper is intended to provide some guidelines for sample size planning for pair-wise comparisons. Normalization is an essential and important pre-processing step in microarray data analysis. To our knowledge, no previous studies using multiple data sets have pre-processed the data sets in a comparable way. In addition, previous studies did not look at the effect of inbred vs outbred populations on the variation of gene expression. In order to make the results more comparable, we make use of 7 cDNA microarray data sets and apply the same normalization method (spatial lowess). We estimate the variance by one sample t-test, paired t-tests or two sample t-tests on a gene-by-gene basis using several large expression data sets from both human, rats and mice. We then calculate the sample size required to detect a 1.5-, 2-, and 4-fold change in expression levels for the 90^th^, 75^th^, 50^th^and 25^th^percentile of genes ranked by variability at fixed settings for false positive and false negative rates. The sample size calculation provides the approximate but not exact number of replicates required for a given set of criteria. Results ======= Data sets --------- We estimate the standard deviation and required sample size from 1 unpublished and 6 published cDNA data sets (Table [1](#T1){ref-type="table"}). Data set A-C and E are from human samples. Data sets D and F-G are from mouse or rat samples. Data set F is from a study that is not yet published. The raw and/or pre-processed data for the unpublished dataset with gene order randomized and without the original gene/probe identifications can be downloaded from <http://expression.washington.edu/publications/caimiao/power&samplesize/samplesize.html>. Removing the gene IDs does not affect the analysis in this paper in any way. The same website also provides the links for the published data sets. In addition, all the control genes were removed from the data analysis for the cDNA data sets. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### cDNA microarray data sets used in the study ::: Data set Reference \# Rep \# Genes Tissue type Description Hybridization ---------- ----------------------- ------------------------ ---------- ------------------------ --------------------------------------------- -------------------------------------------- A Smith et al. 2003 20 15,592 Human liver Paired HCC tumor vs adjacent non-tumor Direct hyb between tumor and non tumor B Lapointe et al. 2004 41 38627 Human prostate Paired prostate tumor vs adjacent non-tumor Indirect hyb using common reference C Chen et al 2002 48 22618 Human liver Paired HCC+HBV vs HBV Indirect hyb using common reference D Pritchard et al. 2001 6 5281 Mouse liver and kidney Paired liver vs kidney Indirect hyb using common reference E Zhao et al. 2004 36 ductal + 21 lobular 44549 Human breast lobular and ductal tumor tissue Indirect hyb using common reference F NA 6 13, 056 Mouse liver One third vs two thirds hepatectomy Indirect hyb using individual baseline G Callow et al. 2000 8 5548 Mouse liver ApoAI knock-out vs normal Indirect hyb using common reference (pool) ::: Data set A comprises data generated from 40 liver RNA samples isolated from paired liver hepatocellular carcinoma (HCC) tumor and adjacent cirrhotic non-tumor tissue from 20 HCV infected Caucasian patients \[[@B6]\]. The objective of this study was to identify potential hepatocellular carcinoma markers. The microarray analysis was performed using in-house spotted human cDNA arrays containing 15,592 genes split between and spotted in duplicate on arrays HHD1 and HHD2. The duplicate sets of cDNAs within each slide were spotted side by side (panels A, B). RNA from tumor and adjacent non-tumor tissue from individual patients was co-hybridized to 2 slides (one was a dye flip of the other). Data set B was generated from 41 matched pairs of prostate tumor and non-tumor tissue hybridized to arrays spotted with 38627 cDNAs. All samples were labeled with Cy5 and co-hybridized with a common reference labeled in Cy3 \[[@B7]\]. The purpose of this study was to identify the difference in expression levels between normal and prostate tumor tissues. Data set C was generated from RNA isolated from paired HCC tumor and adjacent non tumor liver from 41 HBV infected patients \[[@B8],[@B9]\] hybridized to cDNA arrays. Each RNA sample was labeled with either Cy5 and co-hybridized with Cy3-labeled reference RNA. The purpose of this study was to identify gene expression differences between HCC tumor and non-tumor liver tissue. Data set D was generated from RNA isolated from paired liver and kidney tissue from 6 male C57BL6 mice \[[@B9]\] hybridized to cDNA arrays. Each RNA sample was labeled with Cy5 and co-hybridized with Cy3-labeled amplified RNA from Universal Human Reference to total RNA (Stratagene). The purpose of this study was to identify gene expression differences between tissue types. Data set E was generated from RNA isolated from 36 breast ductal tumor and 16 lobular tumor tissues \[[@B10]\] hybridized to cDNA arrays. Each RNA sample was split in two and labeled with either Cy5 or Cy3, and co-hybridized with a common reference RNA as color flips with two replicates (4 arrays/tissue/mouse). The purpose of this study was to identify gene expression differences between ductal carcinoma and lobular carcinoma. Data set F consists of data generated from 24 liver tissue samples from 12 inbred mice (unpublished data). One third or two thirds of the liver was removed from each mouse and used as the baseline samples. At 12 hours post operation, the mice were sacrificed and the remaining liver tissue was used as the experimental sample. The aim of this study was to screen for genes potentially related to liver regeneration after hepatectomy. RNA samples from the 12 hour post-operation livers were co-hybridized with their own baseline liver samples. A total of four DNA arrays were used for each sample comparison. Two sets of arrays (MOD1 and MOD2), each containing 6528 different cDNAs spotted in duplicate (A and B) on each array were used. In addition, each comparison was done with a dye flip pair of slides. This data set made use of arrays generated at the University of Washington Center for Expression Arrays. The goal of data set G was to identify genes with altered expression in the liver tissues of two mouse models with very low HDL cholesterol levels (treatment groups) as compared to inbred control mice. The mouse model considered in this study is the Apolipoprotein AI (ApoAI) knock-out, where ApoAI is a gene known to play a pivotal roles in HDL metabolism \[[@B11],[@B12]\]. Each cDNA array contained 5548 non-control genes or ESTs. A pool of normal RNA samples labeled with Cy3 served as the reference for all the arrays. In summary, three of the data sets (D, F-G) are from inbred mouse and rat strains respectively, and the other four data sets (A-C, and E) are from large scale studies of gene expression in humans. If all other factors (assay protocol microarray platform, data pre-processing) were equal, one might anticipate that fewer individuals would be needed for the same statistical power using inbred animals as opposed to unrelated human subjects. Background adjustment and normalization --------------------------------------- Background adjustment and normalization is necessary to remove systematic biases of non-biological origin in microarray studies. A number of methods of background correction and normalization have been proposed \[[@B13],[@B14]\]. We used the locally written program \"spot-on Image\" to analyze the cDNA array data for data sets A and F. Spot-on uses a local background for each spot. The background subtracted intensity of all cDNA data sets were normalized by the spatial lowess method using the R add on package MAANOVA written by the Jackson Lab, which is available at <http://www.jax.org/staff/churchill/labsite/software/index.html>. Estimates of standard deviation and sample size calculation ----------------------------------------------------------- The distribution of the standard deviations estimated from these 7 data sets are presented in Figure [1](#F1){ref-type="fig"}. All data are log~2~transformed prior to data analysis. Figure [1A](#F1){ref-type="fig"} shows the standard deviation of the log ratio of the 4 paired cDNA data sets (A-D). The standard deviations of data sets E-G in Figure [1B](#F1){ref-type="fig"} are the common standard deviation of the log~2~ratio (sample/reference) of two independent groups. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Histogram of standard deviation The X axis is the standard deviation, and the Y axis is the percentage of genes that has standard deviation below the value of X. All data sets were normalized by spatial lowess; (A) Data set A-standard deviation of log ratio of two groups (direct hybridization); data set B-D standard deviation of the difference of log (sample/reference) of the two groups (indirect hybridization); (B) Data sets E-G common standard deviation of (sample/reference) of the two independent groups (indirection hybridization). ::: ![](1471-2164-5-87-1) ::: The required sample size of an experiment depends on the variance component (σ), the desired detectable fold change (δ), the power to detect this change (1-β, the likelihood of detecting the change or the true positive rate), and a chosen type I error rate (α). For microarrays, a combination of fold-change and test p-value is commonly used for selecting differentially expressed genes between two groups or conditions. In this study, sample sizes were calculated in R using the function of power.t.test. The required input parameters are the log scale fold change of interest δ(δ= 1 in log transformed data translates into a 2 fold change in expression level, δ= 2 in log transformed data translates into a 4 fold change in expression level, etc), significance level, power, and the standard deviation (common standard deviation for two sample t-test, the standard deviation of the difference within subject for paired t-test, or the standard deviation of one sample t-test), the type of t-tests (one sample, two sample, or paired t-test), and the type of test (two sided or one sided). For example, in the case of data set A (one sample t-test), if we wish to find out the approximate sample size to detect a 2 fold change (δ= 1) in expression level between tumor and non-tumor tissue in the 75% least variable genes (σ\<= 0.5884) with a two sided 0.001 significance level test with 90% power, we could use the following R function power.t.test(n = NULL, delta = 1, sd = 0.5584, sig.level = 0.001, power = 0.9, type = \"one.sample\", alternative = \"two.sided\") Where sd = 0.5584 is the 75^th^percentile of the standard deviation of log ratio. In the case of data set G (two sample t-test), if we wish to find the approximate sample size to detect a 2 fold change (δ= 1) in expression level between knock-out and control mice in the 75% least variable genes (σ\<= 0.3102) with a two sided 0.001 significance level test with 90% power, we could use the following R function power.t.test(n = NULL, delta = 1, sd = 0.3102, sig.level = 0.001, power = 0.9, type = \"two.sample\", alternative = \"two.sided\") Where sd = 0.3102 is the 75^th^percentile of the common standard deviation of log (sample/reference). In R, for a one sample t-test or a paired t-test to have power 1-βto reject for a two sided testing and strict interpretation of tail probability with significance level αfor detecting a difference of δ, the minimum number of samples or pairs is obtained by solving the following equation iteratively Power = Pr(t~v,\ ncp~\< t~v,\ α/2~) + Pr(t~v,\ ncp~\> t~v,\ 1-α/2~) Where ncp is the noncentrality parameter of the non-central t-distribution, and is estimated by ![](1471-2164-5-87-i1.gif) t~v,\ α/2~is the α/2 quantile of a central t-distribution with v degrees of freedom and v = n-1. t~v,\ ncp~follows a non-central t-distribution with v degrees of freedom and a non-centrality parameter of ncp. For a two sample t-test with equal sample sizes, if we wish to have a large enough sample to detect a difference δ(with a two-sided test and strict interpretation of tail probability with αsignificance level test with 1-βpower), then the sample size (n) for each group is obtained by solving the following equation iteratively Power = Pr(t~v,\ ncp~\< t~v,\ α/2~) + Pr(t~v,\ ncp~\> t~v,\ 1-α/2~) Where ncp is the noncentrality parameter of non-central t-distribution, and is estimated by ![](1471-2164-5-87-i2.gif) t~v,\ α/2~is the α/2 quantile of a central t-distribution with v degrees of freedom and v = 2n - 2. t~v,\ ncp~follows a non-central t-distribution with v degrees of freedom and a non-centrality parameter of ncp. Microarray experiments usually involve a large number of genes, with variance components varying greatly across the genes. In general, the variance is higher for low expressors which make up of a large percentage of the genes (Figure [2](#F2){ref-type="fig"}). Approximately 50% of genes are called absent on the Affymetrix full genome GeneChips. It is reasonable to choose a value of variance, e.g. the median or the upper 75^th^percentile of variance across all genes, and to use this as the value in the power calculations. For example, if we use the variance for the 50^th^percentile, then the sample size calculations will assure us of having the desired power to detect a chosen n-fold change for all but the 50% most variable genes. In Figure [1](#F1){ref-type="fig"}, we show horizontal lines at the 25^th^, 50^th^, 75^th^and 90^th^percentiles. The intersection of these lines with the \"cumulative percentage of genes\" provides the value of αfor each data set. [Additional file 1](#S1){ref-type="supplementary-material"} shows the estimated sample size required to detect a 1.5-, 2-, and 4-fold change in expression level for the 90^th^, 75^th^, 50^th^, and 25^th^percentile genes for a given setting of false positive rate and power. As is expected, the required sample size increases with increasing variance, increasing power, and decreasing fold-change and false positive rate. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Standard deviation versus log intensity Standard deviations are based on one sample t-test (data set A), paired t-test (data sets B-D), or two independent t-test (data sets E-G). ::: ![](1471-2164-5-87-2) ::: A significance level (the probability of making a type I error, that is getting a false positive) of 0.05 is often employed in hypothesis testing. Thousands of genes are usually studied in microarray experiments. When more than 10,000 genes are tested independently, we would expect more than 500 genes to appear as false positives when the 0.05 significance level is applied. Hence, a smaller cut-off p-value should be used in order to reduce the number of false positives. Many multiple testing correction methods have been proposed. The simplest one is the Bonferroni correction (family wise error control) where the nominal significance level is divided by the number of tests. The Bonferroni correction is very stringent. False discovery rate (FDR) \[[@B15]\], the proportion of false positives among the genes that are identified as differentially expressed, is a post-data measure for controlling false positive. For the purpose of sample size planning, we suggest using the family wise type of error control. A higher false positive rate (lower false negative rate) can be employed for studies aiming to eliminating non-significant differentially expressed genes, and a smaller false positive rate can be used for those studies involving costly follow-up research. For reference, Table [2](#T2){ref-type="table"} lists the number of genes/ESTs/probes found to be differentially expressed between two conditions by different significance levels and fold changes. The number of significant genes in data sets F and G are small, possibly due to the relatively fewer number of genes included in the study and the homogeneity between conditions (data set F compare in-bred mice with two different volumes of hepatectomy; data set G compares ApoAI knock-out versus normal mice). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Significant genes/ESTs/probes called by methods used in the studies using different criteria (combination of significance level and fold changes) ::: Data set Reference P \<= 0.001 and the estimated fold change \>=2 P \<= 0.001 only P \<= 0.01 only ---------- ----------------------- ------------------------------------------------ ------------------ ----------------- A Smith et al. 2003 183 1783 3590 B Lapointe et al. 2004 609 6549 10153 C Chen et al 2002 1253 4187 6197 D Pritchard et al. 2001 479 1557 1845 E Zhao et al. 2004 270 1050 3821 F NA 16 145 723 G Callow et al. 2000 6 11 77 ::: Discussion ========== Factors that affect sample size calculation include the magnitude of the variability of the population, the magnitude of the desired detectable expression change, the chosen power to detect the expression change, and the cut-off P-value/significance level/false positive rate. For a given study, the variability of the population being studied is fixed, and once researchers have identified the desired detectable expression change, the required sample size depends on the chosen false positive and false negative rates. The variability of human subject data is typically larger than that seen with laboratory animals and cell lines due to genetic influences on gene expression. Hence, more replicates are needed for studies that involve human subjects (or any other outbred population) than for studies with samples from an inbred population. This is readily apparent in the cDNA data in [Additional file 1](#S1){ref-type="supplementary-material"} (data sets A-C, and E are human samples while D and F-G are from mice). With the cDNA array data, one needs roughly 5 times as many human samples relative to mouse to detect the same magnitude of change with the same statistical power at the same significance level. This increase in the required number of samples for an outbred population has not been discussed before and has practical implications for those wishing to translate gene expression work from animal models to studies in human populations. Multiple levels of replicates are common in two color microarray experiments. Multiple arrays probed with RNA samples isolated from multiple individuals of a population/treatment/group are referred to as biological replicates. Multiple arrays hybridized using the same RNA or multiple replicates of the same gene within an array are referred to as technical replicates. Although technical replicates can improve the precision and the reliability of the measurement and provide information for quality control, biological replicates are most effective in reducing the variance of the estimate of mean difference. Biological replicates therefore increase the power to detect biologically significant gene expression differences. More importantly, when trying to identify differences between a treatment and a control group, accurate estimates of the biological variability within the groups is essential to determine if the between group differences are meaningful (by a t-test, Analysis of Variance (ANOVA) or other method). Careful experimental design is necessary to maximize the statistical power of the test \[[@B16]-[@B18]\] while balancing resource allocation. For example, dye swapping (in which pairs of RNA samples are hybridized twice with reverse dye labeling) is common in two color array experiments and is a great help in removing dye bias. However, if the experiments involve a common reference sample, which is not of biological interest and the goal is to identify gene expression differences between two groups (both of which are co-hybridized again the common reference), using twice as many independent biological replicates is preferable to dye swapped technical replicates. Caveats ------- This paper is intended to give some guidance to those planning microarray experiments. The sample size calculations we performed provide an approximate number of replicates for a given set of criteria. Our studies were limited to a small number of published microarray studies for which the following criteria were true: 1\) A reasonably large number of biological replicates were analyzed. 2\) Raw data was readily available so that we could reprocess all data with the same algorithms. 3\) Other potentially large sources of variability such as flow sorting, laser micro-dissection and/or multiple rounds of amplification were not present. We have only analyzed date from a limited number of tissue types -- liver, prostate, breast and blood in human, liver and kidney in mouse, and mammary gland in rat. It is entirely possible that different tissue types will have larger or smaller degrees of biological variation and hence will require more or fewer samples to reach a given conclusion. In addition, lab or experiment specific methods of obtaining and processing samples may induce greater degrees of expression variation than seen in our sample data. As more large data sets become available, it will be useful to extend these studies to better define the magnitude of gene expression variation in purebred animals and in outbred humans across a variety of tissues. However, the data shown in [Additional file 1](#S1){ref-type="supplementary-material"} however should be sobering to those planning or reviewing an experimental protocol for microarray analysis. In these limited data sets with human samples hybridized to cDNA arrays with a common reference (B-C, E), we show that 32 samples for each group are required to detect a 2-fold change in the 75% least variable genes with 90% power and a p-value of 0.001. With a p-value of 0.01 and 90% power, at least 20 samples are required to detect a 2-fold change in the 75% least variable genes. This is a much larger number of samples than is frequently used in human microarray case-control studies designed to identify gene expression differences between two groups. Methods ======= Data set selection, pre-processing and normalization ---------------------------------------------------- Background adjustment and normalization is needed in microarray data analysis in order to remove non-biological variation. Intensity based normalization methods such as locally weighted least square polynomial regression (lowess) is commonly used in cDNA microarray experiments. The background subtracted intensities were normalized by the spatial lowess method using the R add on package MAANOVA written by the Jackson Lab. For the two cDNA experiments with replicate panels within each array, we normalized the two panels separately. All control genes were excluded from data analysis for data sets A-G. Estimate of variance components ------------------------------- Pair-wise comparisons among conditions/groups/treatments of gene expression levels are common goals of microarray studies. Simultaneous comparison of more than two treatments/conditions using one way ANOVA can be advantageous. However, a significant F for a comparison of several treatments does not provide information about which particular groups differ from each other. In addition, one way ANOVA is not sensitive to treatment effects when only one or two samples out of many are quite different. T-tests are commonly used to compare individual treatments in pairs. In order to calculate power and plan sample size, one must first estimate the variance. We applied paired or two sample t-tests in this study based on the correlation between the two groups. For data set A, as the pairs of tumor and adjacent non-tumor tissue are highly correlated, we used two tailed one sample t-tests with the normalized log~2~ratio of tumor/non-tumor as the response variable. Data sets B-D were generated from paired samples using a reference design on cDNA arrays; Paired t-tests are appropriate for these three data sets. We performed two tailed, two sample (or independent) t-tests on data sets E-G with normalized log ratio as the response variable. The two sample t-tests are based on unequal variances for the two groups of samples. The variances of the data sets with paired samples are the variance of the difference. The common variance of the datasets with independent samples was estimated by the following formula: ![](1471-2164-5-87-i3.gif) Where n~1~, n~2~are the number of observations for group 1, and group 2, respectively; and S~1~and S~2~are the standard deviation for group 1, and group 2, respectively. To simplify power and sample size calculation, and to focus our calculation on biologicalvariance, the log ratios of the 4 technical replicates of data sets A, E, and G were averaged for each RNA pair (data set A) or sample (data sets E and G). The standard deviations across biological replicates were estimated on a gene-by-gene basis. Sample sizes were calculated using R for detecting a 1.5-, 2- or 4-fold change for the 90%, 75%, 50%, and 25% least variable genes with a range of power (0.70, 0.80, 0.90) and confidence level (0.01, 0.001, 0.0001), assuming equal sample size for the two groups. The numbers for sample size are rounded to integers. Authors\' contributions ======================= JL provided the mouse liver microarray data set F prior to publication. CW performed all the analysis in this paper. RB supervised JL and CW and contributed to the design, coordination and writing. All authors read and approved of the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Sample size required to detect a 1.5-, 2-, and 4-fold changes of expression level for the 90%, 75%, 50%, and 25% least variable genes for a given settings of false positive rates (α) and power (1-β). This additional file shows the estimated sample size to a 1.5-, 2, and 4-fold changes of expression level for the 90%, 75%, 50%, and 25% least variable genes for a given settings of false positive rates (α) and power (1-β) for all of the data sets referred in Table 1. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ Roger Bumgarner receives funding from the following grants: NHBLI-5R01HL072370and 1P50HL07399, NIDDK-5U24DK058813, NIEHS-1U19ES011387and NIAID-1R21AI052028 and 5P01AI052106. In addition he was partially funded by NIDA-5P30DA015625during this work. Caimiao Wei is funded from grant NHBLI-5R01HL072370. Jiangning Li was funded from NIDA grant 5P30DA015625.	PubMed Central	2024-06-05T03:55:49.802157	2004-11-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533874/", "journal": "BMC Genomics. 2004 Nov 8; 5:87", "authors": [ { "first": "Caimiao", "last": "Wei" }, { "first": "Jiangning", "last": "Li" }, { "first": "Roger E", "last": "Bumgarner" } ] }
PMC533875	Background ========== The survey is a key method in health services research \[[@B1]\]. The majority of survey questionnaires consist of closed questions where respondents are asked to choose from a fixed number of options. These are considered to be efficient because data are easy to collect, code and analyse \[[@B2]\]. Efficiency is important in survey methodology where researchers attempt to obtain the attitudes or experiences of a representative sample for generalisation to a wider population, and may need to gather information from large numbers to ensure precision of estimates. In addition to closed questions, it is not uncommon to include an \'open\' question where respondents are invited to provide information in free text format, for example \'Is there anything else you would like to say\' at the end of a questionnaire. When the questionnaires are returned and being prepared for analysis, the researcher may face the dilemma of whether or not to analyse and report the written responses to this open question. In this paper we draw on expert opinion in key texts, and examples of the use of open questions in predominantly closed question questionnaires, to consider whether there is value in including such questions, and if so, how best to optimise the quality of the data and analysis. Discussion ========== Different types of open questions in surveys -------------------------------------------- There are four types of questions which might require an open rather than closed response (see Figure [1](#F1){ref-type="fig"}). The general open question, typically \'any other comments?\', used at the end of a structured questionnaire is the type we focus on in this paper. We believe that the use of this type of open question is common, and we consider it to be the type that is most likely to pose a dilemma for researchers around whether and how to analyse any responses. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Different types of open questions in surveys ::: ![](1471-2288-4-25-1) ::: The potential benefits of general open questions ------------------------------------------------ General open questions offer a number of benefits when piloting a questionnaire. Responses to them can reassure the researcher that all relevant issues have been covered \[[@B3]-[@B5]\]. Responses may also be used to corroborate answers to closed questions, offering reassurance to the researcher that the questionnaire is valid, or highlighting problems with particular questions. The benefits of using general open questions in the main study are less clear. They have been recommended to help make a dull statistical report more interesting \[[@B6]\], by providing the reader with quotes to illustrate important points, and in self administered questionnaires because there is some evidence that they increase response rates \[[@B5]\]. Increasing the response rate is a considerable benefit in survey methodology, but it is not necessarily the issue which drives researchers to use general open questions. Researchers may use general open questions without giving much thought to why they are doing so, simply including the question because it is usual practice. Or they may be driven by a desire to offer respondents an opportunity to voice their opinion. Closed questions represent the researchers\' agenda, even if they have been developed through listening to people\'s views in focus groups and depth interviews. The use of \'any other comments\' may redress the power balance between researchers and research participants. Respondents may take this opportunity to ask for clarification or information about a health issue or health service, or voice concern about the research. If researchers include a general open question for this reason then they will need to consider how best to respond to individuals about such queries and concerns. Another possible driver for including a general open question is a concern about missing an important issue, even if the questionnaire has been developed using a considerable amount of qualitative research and piloting. There may be issues which respondents want to give more details about than the structured questions allow. There may be issues which qualitative methods and piloting fail to uncover because they affect a small number of people only, or they are specific to sub groups which have not been included in the development work, or they have occurred since the design of the questionnaire. Thus general open questions may act as a \'safety net\' and help the researcher to identify issues not covered by the closed questions, either by elaborating and explaining some of the findings from closed questions, or identifying new issues. For example, the purpose of a survey of NHS Direct nurses was to describe the nurses\' qualifications, experience and reasons for joining the service \[[@B7]\]. A small number of closed questions asked about nurses\' views of working for this new service, and respondents used the general open question to expand in considerable detail on this issue \[[@B8]\]. In other studies, responses to general open questions have elaborated on answers to a closed question, identifying the aspects of the service which contributed to NHS Direct users feeling reassured by the advice offered \[[@B9]\], explaining why junior doctors felt that training had not prepared them for their job \[[@B10]\], and illuminating why people were more satisfied with an emergency ambulance call taker making use of a priority dispatch system \[[@B11]\]. An example of a new issue emerging after the design of the questionnaire was the emergence of media criticism as a concern of doctors in one of a series of annual surveys \[[@B12]\]. Why are general open questions a problem? ----------------------------------------- Having asked a general open question, researchers may face the dilemma of whether to analyse responses or not. Practical constraints may contribute to a decision not to do so because data input and analysis require considerable resources \[[@B5],[@B6]\] and these may not have not been allocated during the study design. However, ignoring this data can feel unethical and it has been recommended that researchers should not ask open questions unless they are prepared to analyse the responses \[[@B13]\]. Another barrier may be the lack of clarity around the status of the responses. They tend to fall between two stools, being neither strictly qualitative nor quantitative data, and this can make them uncomfortable to work with. This lack of clarity of status may result in them not being analysed, or being analysed and published in the body or appendix of a report but not within any peer reviewed articles emerging from the study. Are responses to general open questions qualitative or quantitative data? ------------------------------------------------------------------------- Some researchers consider responses to general open questions to be qualitative data \[[@B14],[@B15]\], some do not \[[@B16]\], and others describe them as \'quasi-qualitative data\' \[[@B17]\]. General open questions have some of the features of qualitative approaches: they appear to allow respondents to write whatever they want in their own words, with little structure imposed by the researcher; the output is words rather than numbers or ticks; the analysis may use techniques associated with qualitative research; and publication can involve the display of verbatim quotes so that it looks like qualitative data. However, data from general open questions can lack some of the key strengths of qualitative research. One could argue that the closed questions indicate the legitimate agenda for the responses to the general open question, and thus may impose constraints on responses. More importantly, there is a lack of attention to context, and a lack of conceptual richness, because the data on each case often consist of a few sentences or less. Typically, recipients are asked non-directive questions such as \'Is there anything else you would like to say\' or \'Any other comments?\', with a small amount of space for responses. The key to determining the status of data derived from general open questions may therefore be their depth; both the amount recipients are prompted to write (either through the instructions given or the amount of space allocated), and the amount they actually write. Thus researchers may be able to determine the status of a general open question at the design stage of a study by having a strategy to generate depth and treat the data qualitatively, or by having a strategy to generate shorter responses as a \'safety net\' for complementary or new issues. Having such a strategy may help researchers to devise a strategy for analysis and publication. Generating qualitative data by design ------------------------------------- Researchers can determine the status of a general open question at the design stage of a study by having a strategy to generate depth and treat the data qualitatively. For example, at the end of a structured questionnaire about use of Chinese medicine, one researcher invited respondents to tell the \'story\' of their use of Chinese medicine, leaving a full one and a half pages of white space, and offering a example of the detail required. The following instructions were given: \'Now tell us your own story, using the space on the next page. We\'ve provided one true patient story to give you an idea of the kinds of details we need. The important subjects are repeated in the list above the space we\'ve provided for you to write in. Also use the back of the page if you wish. Please remember to write clearly.\' \[[@B18]\]. This approach produced 460 accounts from 575 respondents (80%). These \'handwritten stories\' were treated as qualitative data, and analysis focused on the language used by respondents, as well as emerging themes, to show the holistic nature of the health care delivery as experienced by the respondents. In the above example, the 80% response suggests that the stories obtained could be viewed as representative of the population surveyed. However, in qualitative research the validity of the study does not rest on the researcher\'s ability to demonstrate representativeness with respect to the total population. Rather, it rests on transferability whereby the researcher offers detailed description of the setting in which the research was undertaken \[[@B19]\]. Thus what is required is that the characteristics of the sample are clearly presented, such that the reader is informed about the likely transferability of the beliefs and experiences expressed. With data obtained from a structured survey, it is always possible to use the quantitative responses to characterise the nature of the group providing comments, and to make their relationship to the wider population apparent. This means that comments from a subset of responders are still valuable data even when they do not represent the entire sample. One important corollary of this is that open questions can be designed expressly to elicit comments from a subset of the population surveyed, using the principles of purposive sampling \[[@B20]\]. An example of this would be to encourage all respondents reporting a particular type of experience in a closed question to tell their story. An alternative approach would be to sample post hoc from the full range of responses received, for example sampling information rich cases or extreme cases. If the open question is used to generate qualitative data, then researchers will need to use qualitative analysis techniques and possibly qualitative software as used in the Chinese medicine example discussed previously \[[@B18]\], and will need to consider issues important to good quality qualitative research, such as clear exposition of data collection and analysis, the search for disconfirming evidence and reflexivity \[[@B17],[@B21],[@B22]\]. Qualitative researchers expect analysis to be challenging and time consuming and will ensure that they have the resources required to undertake it if the intention to collect such data is explicit in the research proposal. When reporting research findings from studies using face-to-face interviews, it is good practice to indicate the length of the interviews. Similarly, when reporting the data from these open questions, it might be helpful to indicate the potential depth of data to the reader by detailing the average number of lines of text available from respondents \[[@B18]\]. Generating quantifiable data by design -------------------------------------- General open questions may produce little more than the closed questions on the questionnaire \[[@B23]\] and rather than considering it unethical to analyse these responses, a more appropriate strategy might be preliminary analysis involving reading the responses so that the researcher can consider the contribution they make to the study overall. If the comments merely corroborate or slightly elaborate upon the answers to closed questions, then formal analysis may not be worthwhile \[[@B23]\]. It may be good practice to report within publications that the responses to the general open question did not provide additional information to the closed questions. It is where they offer insights or issues not available in the closed questions that formal analysis could be considered good practice, even if the role of this analysis is to identify hypotheses or questions for further study. Formal analysis may be prompted by either the strength of numbers making particular comments, or the strength of feeling within a small number of the comments. For example, in a survey of NHS Direct nurses, the large number of detailed comments and the emotional content of some of them, prompted a formal analysis \[[@B8]\], and in a survey of junior doctors, the strength of feeling expressed by a small number of doctors around one issue prompted formal analysis \[[@B12]\]. From a quantitative perspective, the strength of a survey approach is representativeness, and thus non-response bias should be a concern. Respondents are less likely to complete a general open question than a closed one on a postal questionnaire \[[@B4]\]: 81% of the 71% of respondents to a survey of NHS Direct users, that is 58% of the sample \[[@B9]\]; 67% of the 74% of respondents to a survey of NHS Direct nurses, that is 50% of the sample \[[@B8]\]; and 40% of the 74% of respondents to a survey of junior doctors, that is 30% overall \[[@B12]\]. Those who choose to answer the general open question could be different from respondents overall, either being more articulate or having a greater interest in the survey topic. It is important to consider and report on who has made written comments so that bias can be considered. In a patient satisfaction survey, females were more likely to make comments than males but interestingly there were no significant differences by age group or educational status \[[@B23]\]. In a survey of NHS Direct nurses, the proportion of nurses making written comments varied by their job satisfaction levels, with nurses who felt that their job satisfaction had \'not really changed\' under-represented in the written comments and those who felt it had \'worsened a lot\' over-represented in the written comments \[[@B8]\]. The comments were reported in this context. Formal analysis must be rigorous so that the findings are useful and convincing. Content analysis may be undertaken \[[@B2],[@B3]\], where the researcher takes the following steps: 1\. Reads a sub-set of the comments. 2\. Devises a coding frame to describe the thematic content of the comments. 3\. Assigns the codes to all the comments. The coding frame can be applied using software designed for this purpose \[[@B24]\] or manually. Two coders may be needed to test the reliability of assigning codes \[[@B2]\]. 4\. The codes can be entered into a statistical package alongside the data from the closed questions and treated as variables in a quantitative analysis. The coding process is time consuming and requires expertise \[[@B2],[@B4],[@B6]\]. The skills of a qualitative researcher are not needed, but the coding is similar to the early stages of qualitative analysis \[[@B25],[@B26]\] and researchers may wish to seek the advice of a qualitative researcher. Any decisions made will affect the results and thus the coding process is suited to a skilled researcher. When reporting the responses to general open questions it is important that the numbers of respondents making each comment are displayed, with recognition that although a specific number of people mentioned an issue it might be relevant to many more who did not choose to mention it. Although numbers rather than percentages tend to be used when reporting responses to open questions \[[@B8],[@B12],[@B15],[@B26]\], percentages will sometimes be the most appropriate way of presenting the results, for example, in a before and after design with different numbers of comments in each time period \[[@B11]\]. Verbatim comments can be displayed to illustrate the themes \[[@B8],[@B26]\], because it is the comments themselves which have convinced the researcher of the importance of the dissemination of the information \[[@B8]\]. When doing this, attention to confidentiality is important, taking care not to report comments which might identify an individual. Publication of responses to open questions ------------------------------------------ Publication of responses to open questions can occur within a paper reporting the main findings of the questionnaire or as a separate publication. Where comments elaborate and explain findings from closed questions, it may be most appropriate to publish them in the same paper \[[@B9]\]. Where a new issue emerges \[[@B8],[@B12]\], a separate publication may be appropriate. For publication, both the data and analysis need to be robust enough to stand up to scrutiny and peer review. Advantages and disadvantages of having an explicit strategy ----------------------------------------------------------- An explicit strategy requires that researchers consider the role of a general open question in the context of their survey, and its status in terms of generating either qualitative or quantifiable data. If the role of the question is to give a voice to participants then the researcher can ensure that comments will be read to identify any concerns and queries expressed by individuals, and that appropriate action is taken with individual comments. If the role is to generate qualitative data then attention can be paid to generating depth of data and the quality issues associated with qualitative research. If the role is to act as a safety net and generate quantifiable data then resources will need to be allocated for reading the comments, and if there appears to be added value, for formally analysing the data with attention to non-response bias and reliability of coding. Having a strategy may reduce any dilemma faced by researchers about whether and how to analyse these questions, may help the researcher to allocate the appropriate time and expertise to this data, and may produce an analysis robust enough for publication in peer reviewed journals. A potential disadvantage of having such a strategy may be that some flexibility is lost and that some important issues are missed. Finally, researchers cannot assume that they know how best to facilitate a respondent to complete a general open question and may need to consider using cognitive aspects of survey methodology to construct the question \[[@B27]\]. Summary ======= • General open questions at the end of structured questionnaires can present a problem to researchers who may face the dilemma of whether or not to analyse them. • They are necessary when piloting questionnaires because they identify further issues for inclusion in the survey, and may be a bonus in the main study because they may increase response rates and may identify issues which complement responses to closed questions. • The value of such questions, and the quality of the data and analysis, may be optimised if researchers make more strategic use of them by being clear about their role, and understanding the type of data they wish to generate when they design their study. • An explicit strategy for generating qualitative data will encourage attention to depth of data and issues important to the analysis of qualitative data such as reflexivity. • An explicit strategy for generating quantifiable \'safety net\' data, that is important issues missed by the closed questions, will encourage attention to non-response bias and reliability of coding. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= AOC conceived the paper and wrote the first draft. KJT reviewed it critically, and developed the sections on qualitative research. Both authors produced the final draft and read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2288/4/25/prepub> Acknowledgments =============== Reviewers offered helpful advice which resulted in clarification of some points and the addition of further arguments. AOC is a Medical Research Council Fellow. The work was undertaken within the Medical Care Research Unit which is supported by the Department of Health. The views expressed here are those of the authors and not necessarily those of the Department.	PubMed Central	2024-06-05T03:55:49.804631	2004-11-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533875/", "journal": "BMC Med Res Methodol. 2004 Nov 8; 4:25", "authors": [ { "first": "Alicia", "last": "O'Cathain" }, { "first": "Kate J", "last": "Thomas" } ] }
PMC533876	Background ========== An important aspect of any trial that should be clearly stated in the final report is the method used to assign treatments (or other interventions) to participants \[[@B1]\]. In the final report of the trial, authors should specify the method of sequence generation, i.e. whether they have used mechanical means, a computer generated random list or random number table. After preparing a random sequence, subjects will be allocated to the trial groups using an implementation method such as numbered containers, central telephone line, or allocation by a person who is not involved in the main research and patient care (the encoder). During the process of allocation each subject will be given a unique identification code (Unique Identifier, UI). This UI will be used as a label to uniquely identify the patient\'s group after completion of the study. During the study period this UI will be given to the main researchers together with the necessary treatment (e.g. drug or placebo). Usually these treatments are prepared by the encoder with the same physical characteristics (shape, color, size, etc), differing only by the UI labels to blind the main researchers about the actual patient group. Following enrollment of all subjects into the study, these UIs are decoded to determine the patient group. Depending on the preference of the researchers or facilities of the research environment, subjects are randomly allocated to intervention groups using either a random list prepared before the study (In Advance method) or a randomized allocation at the moment of intended intervention (Just In Time method; JIT). Both the JIT and In Advance methods produce acceptable allocations, and the actual choice depends on the availability of certain facilities for each method. Usually the randomization components of these two methods are produced by running a randomization software on either an Internet service provider or a local computer. The local encoder will obtain the next allocation (JIT method) or the entire random list (In Advance method) from the service provider or from the software on local computer and prepare the necessary blinded equipments. Without the use of computer software or Internet services the maintenance of the whole process of randomization and allocation is difficult. In addition, in the case of any necessary restrictions on the process of randomization (i.e. block randomization \[[@B2]\]) the complexity of the process will be increased even further and be prone to errors. Randomization software may run on a local computer or may be hosted by an Internet server. A complete list of these software and services can be found on Martin Bland\'s web site \[[@B3]\]. Most of randomization software are hosted by websites for both JIT and In Advance methods, which require access to the Internet \[[@B4]-[@B8]\]. However, most of these Internet services have restricted capabilities with respect to the block design specification, control over the output format and flexibility of UIs. Among these available services, the tool in Randomization.com \[[@B4]\] seems to be more advanced than the others. It allows users to specify the number of subjects per block, the number of blocks and up to 20 treatment labels. Therefore, this service produces simple and block randomization using fixed and equal block sizes. Unfortunately, this service does not allow further restriction on block design (e.g., multiple block lengths or random variation in block number or size). The generated random list is in the form of UI and group name pairs, formatted in a single column, which in cases of large sample sizes may require further work to fit it in multiple columns for fine printing. Moreover, the block borders are not visible to allow for easy visual inspection of block sizes and equality of cases. Although a minor problem, Randomization.com only produces sequential numeric UIs with variable lengths (e.g. 1, 10, 100). The variability in lengths of UIs may disturb the visual impact of the generated list compared to the fixed length UIs (e.g. 001, 010, 100). Some researchers prefer to use random UIs in mixed alphanumeric format to decrease the likelihood of memorization and to improve the blindness of the study and concealment of allocations. Available randomization software have more restrictions in their capabilities than the Internet randomization services. They have limitations in their output format \[[@B9]\] and users can not specify the number and naming of the treatment groups \[[@B10],[@B11]\]. In addition, these software are not designed with the capability to produce flexible UIs for participants. Therefore, the main objective of Random Allocation Software was to construct a randomization software for parallel group trials with the following characteristics: 1\. Independent running on a local computer without any need to access the Internet 2\. Different types of program output: to file (html or text files), window and system clipboard 3\. Provisions for different block design 4\. Capability to deal with a larger number of groups 5\. Specifying a name for each group 6\. Control over the format, length and ordering of the generated UIs 7\. Control over the format of generated sequence 8\. Saving or loading the randomization settings 9\. Viewing previously generated randomized sequences Implementation ============== Random Allocation Software is a program created in Microsoft Visual Basic 6, and it installs in the same way as ordinary Windows software (i.e. running setup.exe and following on screen instructions). Once installed and run, there are some controls in the main window for specifying the number of groups (2 to 16), sample size and the name of each group. It also contains menu items to determine the program output and randomization settings. The default program output is saved into either html or text files, and it may also have output to a window or to the system clipboard. A variety of randomization options can be set in the options window. The length of generated UIs (named as Code in the program) can be between 3 to 10 characters and there are options for different alphanumeric structures. In addition, these UIs can appear in sequential or random order in the generated random list. The program can generate simple or block randomization in different types, including equal size blocks, multiple block lengths with random variation among the specified block sizes and complete randomized blocks (random number and size of blocks). The generated sequence will appear in a multiple columns format and the number of columns (1 to 10) can be changed in the options window. Output to html file will be formatted in the form of one block per table. Borders of the tables may be shown or hidden. By clicking the \'Generate\' button in the main window the random sequence will be generated and opened by the default viewer for the output file (e.g. Internet Explorer for html files). Previously created output files can also be viewed from inside the program. Additional options include saving the current randomization settings, loading a previous setting and enabling the program to save the last setting upon program exit. During execution, the program produces a random sequence of allocation using the Rnd function that generates a floating point random number. The Rnd function uses the linear-congruent method for pseudo-random number generation as depicted by the following formula: x1 = (x0 \* a + c) MOD (2\^24) \[12\] where: x1 = new value x0 = previous value (an initial value of 327680 is used by Visual Basic unless the Randomize X function is used to specify a different seed as X) a = 1140671485 c = 12820163 The seed of the Rnd will be the Timer function, which will return the number of seconds elapsed since midnight. Although this version of the program does not produce repeatable lists, it is possible to revise the program in subsequent versions to save the value of the seed to reproduce the same random list. The output consisted of shuffled allocations each of which is a UI, group name pair. The program checks for the uniqueness of the UIs and generates an error message if the specified UI length is insufficient to hold the entire sample size. Runs test was used to check randomness of the output list with sample sizes from 10 to 190 (10, 30, 50, \..., 190) and from 200 to 3000 (200, 600, 1000, \..., 3000). Each runs test was carried out for the group number of 2, 3, 5 and 6. SPSS 10 software was used to perform the runs test. Results ======= The program starts running with the default settings. Users may run the program with the default settings or set the number of groups, the name of each group and the sample size. Clicking the \'Generate\' button (figure [1](#F1){ref-type="fig"}) produces the random sequence. Before generating the random sequence, the option window will be displayed and different randomization settings can be entered (figure [2](#F2){ref-type="fig"} and [3](#F3){ref-type="fig"}). Consider, for example, that we want to produce a simple randomized list for a sample size of 30 subjects into three groups of Case, Control and Placebo with numeric sequential UIs. After setting different options and clicking the \'Generate\' button, the generated list will appear in columns (Table [1](#T1){ref-type="table"}). Each entry in the list consists of a UI, and a group name pair. Alternatively numeric UIs may appear in random order (Table [2](#T2){ref-type="table"}). Table [3](#T3){ref-type="table"} shows the output of the program for a block randomization with blocks of equal sizes. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Main window.The main window showing different options for number of groups, sample size, and group names. ::: ![](1471-2288-4-26-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Options window: Blocks.Options window, settings for block design. ::: ![](1471-2288-4-26-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Options window: Code.Options window, setting the format of unique identifier (UI) specified in the program as Code. ::: ![](1471-2288-4-26-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### A simple randomized list produced by the software for a sample size of 30 subjects into three groups of Case, Control and Placebo with numeric sequential unique identifiers ::: -------------- -------------- -------------- -------------- 001: Case 009: Placebo 017: Control 025: Placebo 002: Control 010: Control 018: Control 026: Case 003: Case 011: Placebo 019: Case 027: Case 004: Case 012: Control 020: Control 028: Placebo 005: Control 013: Case 021: Placebo 029: Placebo 006: Placebo 014: Case 022: Case 030: Control 007: Placebo 015: Placebo 023: Case 008: Control 016: Control 024: Placebo -------------- -------------- -------------- -------------- ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### The same setting as in table 1, but with the numeric UIs in random order ::: -------------- -------------- -------------- -------------- 288: Case 200: Control 462: Placebo 775: Case 644: Control 437: Case 448: Case 622: Control 278: Placebo 364: Control 523: Control 327: Control 427: Case 525: Control 837: Case 514: Placebo 146: Placebo 796: Case 804: Placebo 610: Case 383: Placebo 208: Control 581: Control 167: Placebo 493: Placebo 862: Placebo 181: Control 484: Case 079: Case 254: Placebo -------------- -------------- -------------- -------------- ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### A block randomization list with four blocks of equal sizes ::: -------------- -------------- -------------- -------------- -------------- 504: Placebo 671: Placebo 767: Case 442: Control 094: Placebo 256: Case 636: Case 200: Control 677: Control 669: Placebo 355: Control 334: Case 765: Control 073: Control 377: Case 537: Placebo 527: Placebo 485: Case 183: Placebo 658: Control 612: Case 875: Control 888: Case 733: Case 127: Placebo 864: Placebo 476: Control 552: Control 138: Case 548: Case 938: Placebo 592: Placebo 810: Control 213: Control 584: Placebo 438: Case -------------- -------------- -------------- -------------- -------------- ::: Figure [4](#F4){ref-type="fig"} is the printed output of the program for a block randomization with random block sizes. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Sample output.Sample output for a block randomization with random block sizes. ::: ![](1471-2288-4-26-4) ::: In block randomization the final sample size is usually larger than the specified one. A total of 18 runs test were performed to check the randomness of the program output, which resulted in P values of 0.22 to 0.81. Discussion ========== The main use of Random Allocation Software is to produce simple or block randomized sequences for parallel group trials. Its use is restricted to parallel group randomized trials. Compared with similar software, it enables the user to control the length, order and format of the UIs; and the type and format of the output. It allows specifying up to 16 groups for parallel trials. Conclusions =========== Random Allocation Software has been designed to produce random sequences consisting of UI, group name pairs with additional control over the output format and type. Available randomization software generally has limitations in the number of groups, naming each group, generating UIs and control over the output. Many of these problems have been addressed in the present software. As has been stated in previous sections, the main use of this software is for randomization in parallel group trials. The software can be revised to support crossover and other types of randomized trials. The experienced user may test the randomness of the program output by selecting numeric labels for group names and then exporting the generated list into a statistical software such as SPSS to execute a runs test on the exported data. Availability and requirements ============================= Project name: Random Allocation Software Public use access: <http://www.msaghaei.com/Softwares/dnld/RA.zip> (Latest version) <http://ftp.mui.ac.ir/RA.zip> Operating systems: Windows 98, Me, 2000, XP. It should be noted that on some Windows operating systems (especially Windows 2000) during installation of the program an error message like \"Setup Cannot Continue\... System Files Are Out of Date\" may be displayed. If this happens, click OK and restart the system. Then run the setup.exe again. This is due to a known bug in the installation programs of Microsoft Visual Basic \[[@B13]\]. This problem has been removed from the newer versions of the program. Users are recommended to download the latest version from the first address. Programming Language: Visual Basic 6 Other requirements: Internet Browser (Internet Explorer 5 or higher is recommended) License: Free for academic use. Abbreviations ============= JIT = Just in Time UI = Unique Identifier Competing interests =================== The author declares that he has no competing interests. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2288/4/26/prepub> Acknowledgements ================ The author thanks Azra Kianinejad, Department of Human Development, Kobe University, Kobe, Japan for her kind review of the manuscript.	PubMed Central	2024-06-05T03:55:49.806813	2004-11-9	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533876/", "journal": "BMC Med Res Methodol. 2004 Nov 9; 4:26", "authors": [ { "first": "Mahmood", "last": "Saghaei" } ] }
PMC533877	Background ========== Early in the epidemic, Human Immunodeficiency Virus (HIV) infection primarily affected young adults; later on, it occurred with increasing frequency in middle-aged and in older individuals, too. In fact, a rate of 12.3% of AIDS patients aged 50 years or older, with subjects ≥ 60 years equalling 3.2%, has been recently reported by the Centres for Diseases Control and Prevention (CDC) \[[@B1]\]. Most of the known epidemiological features about older HIV-infected subjects, have been defined before the introduction of Highly Active Antiretroviral Therapy (HAART), as the standard therapy for HIV-positive subjects, in 1996. A low level of T CD4+ cells at HIV diagnosis \[[@B2],[@B3]\], a rapid decline of T CD4+ cell count \[[@B4],[@B5]\] and a high level of HIV viral load after seroconversion \[[@B6],[@B7]\] have been reported in older HIV-infected subjects. In addition, these individuals presented a clinical condition of AIDS at the time of diagnosis of HIV infection more frequently than younger \[[@B2]\] and they were more likely to die within one month after HIV diagnosis \[[@B8]\]. Several studies have indicated that older patients had a shorter survival than younger \[[@B2],[@B9],[@B10]\], independently of HIV-risk behaviour \[[@B5],[@B11],[@B12]\] and AIDS-related illnesses \[[@B13]\]. Either the more rapid course \[[@B4],[@B9]\] or the decreased survival \[[@B10],[@B14]\] observed in older subjects were considered to be determined by the more frequent presence of non HIV-related comorbidities. As HAART became available, a less beneficial effect on immunological outcome in older in respect of younger subjects has been reported \[[@B15]-[@B17]\]. In fact, both the intensity \[[@B15],[@B16]\] and the rapidity \[[@B17]\] of the immunological response appeared to be reduced in older subjects. On the contrary, only few reports have indicated a similar immunological outcome both in older and younger HIV-positive subjects \[[@B18],[@B19]\]. Interestingly, older age did not seem to significantly affect the long-term virological outcome of HAART treated subjects \[[@B15],[@B18],[@B20]\]. Two were the aims of this study: 1\. To characterise the epidemiological and clinical features of older subjects at the time of HIV diagnosis in the HAART era. 2\. To better define the impact of HAART on virological and immunological response in a cohort of older HIV-positive patients when confounding variables such as adherence to therapy, side effects and non HIV-related comorbidities were evaluated. Methods ======= Study setting ------------- The Catholic University teaching hospital is a 1,900-bed tertiary care centre located in Rome, Italy, with approximately 67,000 patient admissions each year. There is a 60-bed unit for the admission of HIV-infected subjects and a day-hospital for out-patient care. Study design ------------ Prospective case-control study. Study population ---------------- All patients presenting the first HIV positive test from January 1997 to June 2003, admitted to our in- and out-patient care, were considered. For the first aim of the study, subjects aged ≥ 50 years (older) and between 20 and 35 years (younger) were identified. For the second aim of the study, older and younger patients who were given HAART regularly and with a follow-up of at least six months, were included as cases and controls, respectively (ratio 1:2). The control group was matched by sex, year of HIV diagnosis, presence of AIDS defining conditions and type of HAART regimen (i.e. NNRTI containing vs. PI containing regimens). A patient (either case or control) was considered as regularly HAART-treated if he/she was under antiretroviral therapy with a combination of two nucleoside reverse transcriptase inhibitors (NRTI) plus either a non nucleoside reverse transcriptase inhibitor (NNRTI) or a protease inhibitor (PI), as single drug or with the addiction of a low dose of ritonavir (i.e. 100 mg bid), for at least three months. Parameters evaluated -------------------- Patients\' records were reviewed by one of us, using a standardised data collection form with predefined criteria. For each subject enrolled in the study, the following data were obtained at inclusion after patient\'s informed consent: age, gender, HIV-risk behaviour, date of the first available HIV test, reason for HIV testing, CDC stage of HIV infection \[[@B21]\], HIV-related conditions, date of AIDS diagnosis, number of hospitalisations after HIV diagnosis, observed AIDS free interval (i.e. time from HIV infection to AIDS diagnosis), type and use of prophylaxis for Pneumocystis cariniipneumonia (PCP) and toxoplasmosis, antiretroviral therapy (type, duration, adherence and side effects) and other medications (antituberculous drugs, antivirals, corticosteroids, antacids), time of follow-up, time from AIDS diagnosis to death, cause of death. Data collected from the laboratory records included: numbers of circulating CD4+ T cells (/mm^3^), CD8+ T cells (/mm^3^) and plasma HIV RNA viral load (copies/ml) with a limit of detection below 50 copies/ml. The time of the beginning of HAART was considered as time zero in the analysis. All subjects underwent periodical clinical evaluations including determination of CD4+ T cells and HIV viral load every six months. Adherence was defined through a self-reported evaluation by the patient and registered as percentage by the physician. An evaluation higher than 80% was classified as \"adherent\", whereas lower than 80% was considered as \"non-adherent\". The following adverse effects were considered: anaemia (haemoglobin \<9.0 gr/dl), digestive intolerance, dyslipidemia (serum cholesterol \>240 mg/dl and/or triglycerides \>200 mg/dl), hepatitis, hyperglycaemia (glucose \>150 mg/dl), lipodystrophy, pancreatitis, peripheral neuropathy, rash, renal colic and thrombocytopenia (platelets \<50,000 cells/mm^3^). Three outcomes were considered for the study: 1. Immunological success (IS): defined as T lymphocytes CD4+ cell count \>200/mm^3^at the end of the follow-up; 2. Virological success (VS) defined as a HIV viral load undetectable (\<50 copies/ml) at the end of the follow-up; 3. Viro-immunological success (VIS) defined as either undetectable HIV viral load and T lymphocytes CD4+ cell count \>200/mm^3^at the end of the follow-up. Evaluation of comorbidity ------------------------- In order to evaluate the significance of non HIV-related comorbid conditions, we used a modified version of the Charlson comorbidity index which excludes AIDS from the diagnosis \[[@B22]\]. Cardiovascular, pulmonary, neurological, endocrine, renal, hepatic, gastrointestinal, rheumatologic diseases, coagulation disorders and cancer were considered as comorbid non HIV-related conditions. Statistical analysis -------------------- Quantitative variables were tested for normal distribution and compared by means of Student\'s two tailed t-test. Differences in group proportions were assessed by use of the χ^2^test or, for small numbers, Fisher\'s exact test. Stepwise logistic regression models were used for each factor to adjust for the effects of confounding variables. Two tailed tests of significance at the p \< 0.05 level were used to determine statistical significance. Statistical analysis was performed using the software program Intercooled Stata 6.0 (Stata Corporation, Texas). Results ======= Epidemiological and clinical features of older patients at the time of HIV diagnosis ------------------------------------------------------------------------------------ The rate of HIV diagnosis in patients aged ≥ 50 years admitted to our in- and out-patient care remained constant (about 10%) over the study-period. From a total of 1265 subjects with new HIV-positive test, 129 (10,1%) subjects aged ≥ 50 years were identified. Nine of them were not further considered because of missing data. One hundred twenty older subjects were evaluated. Data were available for 476 younger subjects. Among older subjects there were more males, less intravenous drug abusers, more advanced HIV disease, more frequent C stage of HIV infection and higher mortality, (Table [1](#T1){ref-type="table"}). The reasons for the first HIV test were comparable both for older and younger subjects. In particular, 30 (25%) older subjects were asymptomatic. For those who were symptomatic, the more frequently observed symptoms were: fever (25%), haematological dysfunctions (17%), weight loss (14%), neurological (8%), gastrointestinal (6%) or hepatic (7%) disorders. Regarding AIDS-defining conditions, a significantly higher frequency of PCP, HIV-encephalopathy, Kaposi\'s sarcoma and cryptococcosis was observed in older HIV-infected patients than in younger. Matched case-control study -------------------------- Thirty nine out of 120 HIV-positive older subjects were not further considered: 29 because the duration of follow-up was below six months and ten because the case-control matching was not possible. A final number of 81 older patients under regular HAART and with a follow-up of at least six months (cases) were thus compared with 162 younger subjects (controls). ### Baseline characteristics and evolution Cases had a median age of 56.9 ± 5.2 years and controls of 31.3 ± 3.2 years. As a result of the matching, 73% of subjects in both groups were males and 49% were in CDC stage C of HIV infection. HIV infection risk factors were similar in the two groups, but eighteen (22%) cases referred an unknown risk factor compared to 26 (16%) controls (p = 0.1). The mean of CD4+ T cells was significantly lower in cases compared to controls (107 ± 109 cell/mm^3^vs. 178 ± 189 cell/mm^3^; p \< 0.01), such as the mean CD4 percentage (9% vs. 14%; p \< 0.01). The mean of CD8+ T cells at baseline was similar in cases and controls (757 ± 380 cell/mm^3^vs. 818 ± 362) (p = ns). Mean of HIV viral load log was similar in the two groups (4.65 ± 0.57 vs. 4.74 ± 0.76 copies/ml; p = ns). No statistical significant difference was observed for haematological parameters, serological markers of previous viral hepatitis (type B or C) and syphilis. Laboratory tests only indicated a significant increase of glycaemia in older patients (103.3 ± 18.7 vs. 89.0 ± 10.6 mg/dl; p \< 0.01). Follow-up was similar in the two groups (40 ± 13 vs. 42 ± 11 months; p = ns), ranging from 6 to 78 months. No differences were observed between cases and controls in the number of hospitalisations (1.2 ± 2.1 vs. 1.3 ± 2.8; p = ns). Seven (9%) and six (4%) patients died in the case and control groups, respectively (p = ns). All causes of death in both groups were HIV-related conditions with the exception of one case who died for an acute mesenteric vascular episode. ### Comorbid conditions Cases had more comorbid conditions than controls (45.6% vs. 15.9%; p \< 0.01). One third of the cases suffered from cardiovascular diseases, a rare condition among controls (32.2% vs. 2.1%; p \< 0.01). Gastrointestinal disorders (9.4% vs. 1.2%; p = 0.04) and diabetes (11.0% vs. 2.1%; p = 0.02) were also significantly more frequent among cases than controls. No statistically significant differences were found for cancer, pulmonary, renal or endocrine diseases, chronic hepatitis and coagulation disorders. The total comorbid points were 37 for cases and 20 for controls. Cases had also a higher mean Charlson index in respect to controls (0.57 ± 0.92 vs. 0.12 ± 0.44; p \< 0.01). ### Antiretroviral therapy No statistically significant difference between cases and controls was observed in type, number and duration of HAART regimens. The first line antiretroviral therapy included PI in more than 80% of cases and controls (Table [2](#T2){ref-type="table"}). The patients who required a change in antiretroviral therapy (second line therapy) received a therapeutical cocktail including PI in 40% vs. 48% (p = ns) and NNRTI in 57% vs. 50% (p = ns) of cases and controls, respectively. Similar percentages were observed in the patients who required another change of antiretroviral therapy (third line therapy) during the follow-up. In the first line drug regimen, side effects were more frequently responsible of the change of the drug regimen in cases compared with controls, although not reaching a statistically significant level (p = 0.1). A high level of adherence to HAART was observed in both groups (82% vs. 75%; p = ns). The more frequent adverse reactions were dyslipidemia, digestive intolerance and lipodystrophy in both groups (p = ns). A significantly higher frequency of hyperglycaemia and anaemia was observed in cases than in controls (Table [2](#T2){ref-type="table"}). ### Immunological and virological response #### HAART related immunoreconstitution Comparing the mean values obtained at baseline in both cases and controls with those obtained at the end of the follow-up, a statistically significant increase in T CD4+ cell number was observed. In particular, T CD4+ cell count increased in 48 months from 107 ± 109 to 476 ± 258 for cases (p = 0.04), and from 178 ± 189 to 584 ± 252 for controls (p \< 0.01). There was always a progressive linear enhancement in both groups at any individual time of the follow-up. The lower values observed in cases in respect to controls at baseline, persisted until the end of the follow-up (Figure [1](#F1){ref-type="fig"}). The increasing value of T CD4+ cell count every six months in respect to baseline was comparable in the two groups (Figure [2](#F2){ref-type="fig"}). According to the above mentioned outcome definition, IS was observed in 59 (73%) of the cases and 128 (79%) of the controls (p = ns). The following variables were significantly associated with IS at univariate analysis: CDC stage A (p = 0.01), low Charlson index (p = 0.01), CD4+ T cell count at the beginning and at 6,12,18,24,30,36 months of HAART (p \< 0.01), months to achieve CD4+ T cell \>200/mm^3^(p \< 0.01), enhancement of CD4+ T cell count in the first six months (p \< 0.01), HIV viremia at 30 months (p \< 0.01). #### HAART related viral suppression A statistically significant reduction of HIV viral load was observed in both cases and controls when comparing baseline with the end of the follow-up values. In particular, means of HIV-RNA viral load log decreased in 48 months from 4.74 ± 0.57 to 2.01 ± 0.4 copies/ml in cases (p \< 0.01) and from 4.69 ± 0.76 to 1.6 ± 0.2 copies/ml in controls (p \< 0.01). HIV viral load reduced under antiretroviral therapy in both groups in a comparable manner. In fact, there was no statistically significant difference between cases and controls at any individual time of the follow-up (Figure [3](#F3){ref-type="fig"}). According to the above mentioned outcome definition, VS was observed in 66 (82%) of cases and 121 (75%) of controls (p = ns). The following variables were significantly associated with VS at univariate analysis: CDC stage A (p = 0.02), high adherence to HAART (p = 0.01), dyslipidemia as adverse effect of HAART (p = 0.01), HIV viral load at 6 (p \< 0.01) and 24 months (p = 0.02). #### HAART related virological and immunological outcome According to our definition of the outcome, VIS was observed in 53 (66%) of cases and 100 (62%) of controls (p = ns). The following variables were significantly associated with VIS at univariate analysis: CDC stage A (p \< 0.01), high adherence to HAART (p = 0.02), dyslipidemia as adverse effect of HAART (p \< 0.01), CD4+ T cell number at baseline and at 6,12,18,24,30,36 months of HAART (p \< 0.01), enhancement of CD4+ T cellcount in the first six months (p \< 0.01), HIV viral load at 6 (p = 0.04) and 30 months (p = 0.01). No statistical difference in immuno-virological response was found between cases and controls when, as cases, only a subgroup of patients aged \> 60 years was considered (data not shown). ### Multivariate analysis In a multivariate logistic regression model, after adjustment for sex and Charlson index, there was no statistically significant difference between cases and controls for IS, VS and VIS (p = ns). Similar results were obtained when CDC stage A and CD4+ T cell count at HIV diagnosis were added to the model. Another model including sex, Charlson index, CDC stage A, CD4+ T cell count at the beginning of HAART, adherence and dyslipidemia as adverse effect of HAART did not show any statistically significant difference between cases and controls for IS, VS and VIS. Further adjustment for sex, Charlson index, CDC stage A, adherence, dyslipidemia as adverse effect of HAART, CD4+ T cell count at beginning of HAART and after six months of therapy, enhancement of CD4+ T cell count in the first six months, months to achieve CD4 + T cell count \>200/mm^3^, HIV viral load at six months did not modify the results. Discussion ========== This study, conducted in an Italian population, shows that in the HAART era, the percentage of older patients out of the new HIV infections is approximately 10%, slightly higher than previously reported \[[@B23]\]. The impact of long term HAART-related immunoreconstitution in this sub-population of HIV patients could reduce the incidence of older patients with AIDS within the next years to less than the 12% recently reported \[[@B1]\]. The first part of our study confirms previous pre-HAART era observations, about more severe HIV infection in older subjects \[[@B2]-[@B10]\]. In fact, higher frequency of patients with AIDS at HIV diagnosis, lower T CD4+ cell count and higher mortality in older subjects in respect of younger were observed. A late diagnosis or a more aggressive impact of HIV infection in older population could explain these results. The first possibility could reflect low level information and/or physicians\' insufficient suspicion \[[@B2],[@B24]\], despite the world-wide durable informative campaigns and preventive efforts on this issue. Alternatively, a shorter or less symptomatic pre-AIDS phase, as already reported in older HIV-positive subjects, could contribute to the late diagnosis \[[@B2]\]. On the other hand, the more aggressive impact of HIV infection in older population has been related to the hypothesis that HIV is an additional aggravating factor to an already debilitated host potentially presenting numerous comorbidities, less physiological reserves \[[@B10],[@B14]\] and a less responsive immune system \[[@B2],[@B25]\]. In fact, in HIV-negative older subjects, important changes in cellular and humoral components of the immune system including defects in T CD4+ lymphocyte function, have been described \[[@B26]\]. Regarding HIV-risk behaviour, our data indicate the heterosexual route as the most frequent in older patients, followed by no risk reported, homosexual contacts, intravenous drug abuse. In particular, our data confirm recent reports \[[@B8],[@B27]\] indicating an increased incidence of HIV subjects with no risk reported. This aspect could contribute to the above mentioned low level of suspicion of HIV diagnosis in older subjects. It is of interest that a large epidemiological survey in the US general population has reported a prevalence of at least one risk factor for HIV infection in about 10% of subjects aged 50 years or older \[[@B28]\]. To date, no studies have indicated that older AIDS patients should be treated differently for HIV disease. Clinical drug trials often exclude older patients because of multiple medical problems, comorbidities or co-existing non HIV-related medication regimens \[[@B29]\]. The first available literature data obtained in a large population of HIV-positive patients treated with zidovudine in the early nineties showed that age was an independent predictor of rapid progression to AIDS and shorter survival \[[@B30]\]. On the contrary, antiretroviral therapy was the only independent predictor of survival after the diagnosis of AIDS in older HIV-positive subjects in the pre HAART era \[[@B31]\]. Very little literature and study has been devoted specifically to the role of protease inhibitors and aggressive antiretroviral triple therapy in elderly patients. Few data have been published concerning the specific response of older patients to new HIV antiretroviral treatments, and currently there are no guidelines for specific antiretroviral treatment modalities for patients\>50 years of age. However, a poor immunological response under HAART was observed in small groups of patients \[[@B15],[@B16]\] possibly due to the well known depressed thymic function of the elderlies \[[@B15]-[@B17]\]. Only few reports showed a similar virological and immunological outcome in older HIV-positive compared with younger subjects \[[@B18],[@B19]\]. This six year prospective matched case control study not only confirms this observation but indicates that older patients under HAART experienced a successful immunological response comparable to younger subjects also in a long term follow-up, in line with preliminary reports \[[@B32]-[@B34]\]. In fact, our older patients, starting HAART with a lower level of T CD4+ cells than younger, obtained a significant increment in their T CD4+ cell count, with the same rapidity, intensity and persistence in time as observed in younger patients. Meticulous adherence to HAART is necessary to achieve an optimal clinical and virological response. A variety of factors may predict medication adherence among HIV-infected adults. Although older age is usually associated with higher rates of antiretroviral adherence, older participants who were cognitively impaired could have difficulty in adequately adhering to their medication regimen \[[@B35]-[@B37]\]. Our findings indicate that this goal could be achieved independently from age. In fact, although thymic functions decline with age, substantial output of T CD4+ cells can be maintained into late adulthood, thus contributing to HAART-related immune recovery in older HIV-positive patients \[[@B38]-[@B40]\]. Early work also demonstrated a positive association between age and the plasma HIV-1-RNA copy number at the time of identification of HIV-1 serostatus among recently diagnosed older HIV-1 positive individuals \[[@B41]\]. However, this could have been caused by a lead-time diagnostic bias, with a greater delay among older individuals. No differences were observed in virological response as indicated by viral load reduction in older patients with respect to younger. This result is in line with previous reports \[[@B15],[@B18]\] including a large study which indicated that older age decreases the risk to have viral load greater than 1000 copies/ml in HIV-positive subjects under HAART \[[@B2]\]. In a recent report, older age was associated with lower levels of HIV-1 replication, independent of antiretroviral therapy usage, regimen adherence, and diseases stage. It is suggested that the effect may be caused by changes in viral evolution or immunological monitoring specific to older individuals with HIV-1 infection \[[@B42]\]. In our study both the frequency and the distribution of adverse effects of HAART were similar between the two groups, with the exception of anaemia and hyperglycaemia which were more frequent in older patients. Hyperglycaemia can be explained because diabetes is a frequent comorbid condition among older. Older subjects were more susceptible than younger to adverse effects in the first line therapy; consequently they had to change therapy more frequently. Univariate analysis showed an association of low Charlson index with immunological success. However, considering that the Charlson index was higher in older patients, none of the models of multivariate analysis indicated an association between comorbidity and virological and/or immunological response. Nonetheless previous studies have suggested a more rapid course and a decreased survival in older HIV-positive patients because of the presence of comorbidities \[[@B10]\], our data did not confirm this association (data not shown). Interestingly, cardiovascular disease and diabetes were the more frequent comorbidities in our older population. Dyslipidemia and lipodystrophy in association with gastrointestinal disorders were the more frequent adverse reactions. In view of these results, of the recent reports of endothelial vascular damage, premature atherosclerosis and coronary events in younger HAART-treated patients \[[@B43]-[@B45]\], an important increase of cardiovascular event incidence in older HIV-positive patients under HAART could not be excluded to occur in the forthcoming years. Conclusions =========== In conclusion, we strongly underline the need for an early diagnosis of HIV infection in older subjects through implementation of educational campaigns specifically targeted to this population. In relation to older people, physicians should consider more often the possibility of HIV diagnosis, through more screening and counselling. An early diagnosis is mandatory because, although older subjects present with more severe HIV infection, the use of HAART allow them to achieve the same viro-immunological response as younger individuals. At present, there is an increasing number of older subjects living with HIV either because of new HIV diagnosis in older population or because who have previously acquired HIV infection now become older due to HAART improved survival. It is reasonable to predict that the average age of chronically treated HIV-positive patients will progressively increase in the forthcoming years. Based on this reality, ageing with HIV is a newly manifested chronic disease with a complex long-term management in consideration also of the impact of HIV and HAART on the natural history of other chronic diseases typically associated with older age. List of abbreviations ===================== CDC Centers for Diseases Control HAART Highly Active Antiretroviral Therapy NRTI Nucleoside Reverse Trascriptase Inhibitor NNRTI Non Nucleoside Reverse Trascriptase Inhibitor PI Protease Inhibitor VIS Viro Immunological Success VS Virological Success IS Immunological Success Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= MTconceived the study, performed the statistical analysis and wrote the manuscript RRparticipated in the design of the study, in the collection of the data, in the statistical analysis and in the writing of the manuscript KdGDparticipated in the design of the study, in the collection of the data and in the writing of the manuscript SBparticipated in the design of the study and in the collection of the data EMparticipated in the collection of the data ETparticipated in the collection of the data ETparticipated in the design of the study RCdid the supervision and critically read the last version of the paper 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/1471-2334/4/46/prepub> Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### T CD4+ cell count/mm^3^(mean ± SD) at the beginning of HAART and each six months of follow-up in cases and controls. ::: ![](1471-2334-4-46-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### T CD4+ cell count/mmc six month increment (mean ± SD) from the beginning of HAART to the end of the follow-up in cases and controls ::: ![](1471-2334-4-46-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Comparison of HIV-RNA log 10 (mean ± SD) between cases and controls from baseline to the end of follow-up. ::: ![](1471-2334-4-46-3) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographic and clinical characteristics of older (≥ 50 years) and younger (20--35 years) HIV-positive subjects in the period 1997--2003. ::: Older n = 120 (%) Younger n = 476 (%) p ----------------------------------------------------- ------------------- --------------------- -------- Male sex 91(76) 285 (60) \<0.01 Age (mean ± SD), years 58 ± 5.1 31 ± 3.2 \<0.01 Risk factor of HIV infection Heterosexual 62(52) 199(42) 0.1 No risk reported 32(27) 119 (25) 0.6 Homosexual 19(16) 57 (12) 0.2 Injection drug users 6(5) 104(22) \<0.01 Months between first HIV+ test and AIDS (mean ± SD) 1.8 ± 7.8 3.3 ± 6.7 0.4 Stage C of HIV infection\* 62 (52) 114 (24) \<0.01 PCP 9 (8) 14 (3) 0.02 HIV encephalopathy 9 (8) 9 (2) \<0.01 Kaposi 8 (7) 5 (1) \<0.01 Candidiasis 8 (7) 43 (9) 0.5 Cryptococcosis 5 (4) 1 (0.3) \<0.01 Mycobacteriosis 4 (3) 5 (1) 0.2 Cryptosporidiosis 4 (3) 5 (1) 0.06 Tuberculosis 4 (3) 14 (3) 0.7 Isosporiasis 1 (1) 0 0.2 Bacterial pneumonia 1 (1) 0 0.2 Wasting syndrome 1 (1) 9 (2) 0.5 Toxoplasmosis 0 9 (2) 0.1 Cytomegalovirus retinitis 0 1 (0.3) 0.6 Lymphoma 7 (6) 9 (2) 0.05 CD4+ T cell/mm^3^(mean ± SD)\\ 59.1 ± 65.3 105.3 ± 127.4 0.04 Deaths 16 (13) 14 (3) \<0.01 \see Ref. n° 21; \\*at AIDS diagnosis; ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Antiretroviral therapy in 81 cases and 162 controls ::: Cases n (%) Controls n (%) p ------------------------------------------ --------------- ---------------- ------ First line therapy 81 (100) 162 (100) Combinations -NRTI+PI 67 (83) 138 (85) 0.7 -NRTI+NNRTI 14 (17) 26 (16) 0.7 Duration; months (mean ± SD) 13.46 ± 10.23 16.67 ± 8.45 0.06 N° of patients who suspended 53 (66) 91 (56) 0.2 Cause of suspension -side effects 30 (56) 37 (41) 0.1 -viro-immunological failure 8 (15) 15 (17) 0.6 Second line therapy 45 (56) 91 (56) 1 Third line therapy 11 (14) 31 (19) 0.4 Total number of drug changes (mean ± SD) 2.1 ± 0.9 2.0 ± 0.6 0.8, Adherent to the last drug regimen 68 (84) 118 (73) 0.08 Side effects to HAART dyslipidemia 32 (40) 55 (34) 0.5 digestive intolerance 26 (32) 58 (36) 0.6 lipodystrophy 12 (15) 23 (14) 0.7 hyperglycaemia 8 (10) 3 (2) 0.01 anaemia 8 (10) 3 (2) 0.01 :::	PubMed Central	2024-06-05T03:55:49.808366	2004-11-6	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533877/", "journal": "BMC Infect Dis. 2004 Nov 6; 4:46", "authors": [ { "first": "Mario", "last": "Tumbarello" }, { "first": "Ricardo", "last": "Rabagliati" }, { "first": "Katleen", "last": "de Gaetano Donati" }, { "first": "Silvia", "last": "Bertagnolio" }, { "first": "Eva", "last": "Montuori" }, { "first": "Enrica", "last": "Tamburrini" }, { "first": "Evelina", "last": "Tacconelli" }, { "first": "Roberto", "last": "Cauda" } ] }
PMC533878	Background ========== Screening for colorectal cancer lessens the risk of dying from that disease \[[@B1]\]. Knowledge of this fact has not solved all the problems related to screening. The optimal modality of screening is still the subject of debate \[[@B1]-[@B3]\]. More problematic is the very low participation of the general public in recommended screening \[[@B4]\]. In contrast to breast cancer screening, in which the Healthy People 2000 Goal of the U.S. National Institutes of Health was surpassed, at 64% participation by women over 40 years of age, only 20% of Americans over age 50 had fecal occult blood testing within the past year (This is the best estimate of actual screening, rather than diagnostic endeavors for symptoms for which endoscopy or radiologic imaging might be done.), and 34% had a sigmoidoscopy within the past 5 years \[[@B5],[@B6]\] Even if screening is appropriately performed, it is far from certain that a positive screen will be followed by appropriate diagnostic testing, as has been shown in follow-up surveys of fecal occult blood testing \[[@B7]\]. Most publication concerning colorectal cancer screening relates to the choice of screening modality; discussing accuracy, efficacy and cost, since the most inexpensive technique, faecal occult blood testing, is inaccurate in the detection of colorectal neoplasia, though effective in significantly diminishing disease specific mortality \[[@B8]\], and the most accurate technique, colonoscopy, is expensive and not without danger \[[@B3]\]. The choice is not an easy one for clinicians, much less patients or the asymptomatic public. Therein may lie one of the problems with public participation in screening. Unlike cancers of the breast, cervix, prostate or lung, where a single screening modality dominates current recommendations for each, there are four different and relatively independent screening tests for colorectal cancer that are currently recommended by the American Cancer Society, National Cancer Institute and United States Preventative Services Task Force: faecal occult blood test (FOBT), fiberoptic sigmoidoscopy (FS), barium enema (BE), and colonoscopy (C) \[[@B1]\]. The absence of a single recommendation may lead from indecision to inaction on the part of clinicians or patients. However the greatest problem related to screening remains the low level of participation by those for whom it is intended: asymptomatic individuals over the age of 50 years with no specific risk factors for colorectal cancer, i.e., no past history of colorectal polyps, cancer, rectal bleeding, colitis, change in bowel habits, iron deficiency anemia, weight loss or a close family member with colorectal cancer. We agree with Dr. Woolf \[[@B2]\], that strategies to improve public compliance with recommended colorectal cancer screening might be more effective if they include an awareness of what the public thinks about the tests being recommended. Previous studies have not surveyed asymptomatic participants\' preference over the whole range of screening choices, focusing instead on symptomatic patients undergoing diagnostic evaluation such as colonoscopy and barium enema \[[@B9]-[@B11]\] or patients ailing from extracolonic diseases whose motivation for screening might be very different than the healthy population for whom screening is intended \[[@B12]-[@B18]\]. Among these latter studies there has been a general preference noted for FOBT (table [1](#T1){ref-type="table"}). We have in this report chosen to focus our survey differently and uniquely; first to inform healthy, ambulatory and younger people, and not ailing patients, concerning only the preparation and conduct of each screening test. Secondly, in order to determine how their perceptions of the conduct of each test might affect their participation, participants were then asked to rank not just their overall preference based upon the preparation and conduct of the tests alone, but four other domains of preference for each screening modality: perceived physical discomfort, inconvenience, embarrassment and danger. Test accuracy was not included in the preamble on test performance, first, because we wanted to isolate perceptions of the physical conduct of the screening test, and second, because test accuracy has been part of many of the previous surveys, often presented with considerable bias. Randomized trials of decision aids have also shown that description of a test\'s ability to detect colorectal cancer has not been successful in increasing participation in screening \[[@B15]-[@B17]\]. Lastly, despite the current enthusiasm for screening colonoscopy by organizations that do colonoscopy as the complete screening test \[[@B19]\], as mentioned above, the choice of screening modality is still regarded as controversial. Methods ======= Participants were a convenience sample of parents or grandparents of children visiting a general pediatrics office (usually for well child visits or minor ailments), personnel working in that office, or parishioners attending a church social gathering, all aged 18 and over. An introductory letter described the purpose of the survey. This was followed by a brief description of the preparation and performance of each commonly used screening test for colorectal neoplasia: faecal occult blood testing (FOBT), fiberoptic sigmoidoscopy (FS), air contrast barium enema (BE) and total colonoscopy (C). The relative accuracy of each exam was not discussed. Six questions followed. The first asked the participant to rank each test in order of overall preference. The second asked the participant to rank each test according to how much that test might cause physical discomfort, the third, inconvenience; the fourth embarrassment, the fifth, the relative danger of the exam. The sixth question asked participants which of the four tests they had previously experienced, along with their gender and age. No further symptom or medical history was obtained and surveys were only numbered consecutively with no personal identifiers. (see appendix for letter and survey) Based upon a related survey concerning subject preference for tests of colonic inflammation \[[@B20]\], a sample size of 50 individuals was estimated. Eighty four questionnaires were distributed in order to assure receipt of an adequate number of usable responses from individuals who had experienced none of the screening tests. The questionnaire is shown in the Appendix. Analyses -------- Data were analyzed using SPSS 11.0. Analyses focused on comparisons between ranks assigned each test on preference and the other assessed attributes, and included Friedman\'s test for ranks (to test the hypothesis that ranks differed for different tests) and the Wilcoxon signed-ranks test (to test the hypothesis that pairs of tests were differently ranked.) We also considered whether those rank orders might differ between participants who have and have not received any of these tests, and how gender and age affected preferences. Results ======= 80 of 84 surveys were available for analysis; twenty nine from men and 51 from women. The mean age of the participants was 38.3 years (range 18 -- 54 years; St. Dev. 8.19 years; median 40 years). Eight subjects had previously had a colonoscopy, five a barium enema, seven a sigmoidoscopy and 17 had stool collected for various reasons. Fifty seven subjects had experienced none of the screening tests. The mean rankings for preference among the entire sample are presented in Table [2](#T2){ref-type="table"} and among only those individuals who had experienced none of the tests are presented in the Table [3](#T3){ref-type="table"}, score \"1\" being the most preferred and \"4\" the least. In each case, mean rankings were found to vary by test (Friedman\'s test, 3 df), and FOBT was significantly preferred over the second-ranked test (FS) by Wilcoxon signed-ranks test. Median scores were determined for each domain for both the whole survey group and the naive subgroup. For each domain and in each group the results were the same, with ranks of 4,3,2 &1 for C, BE, FS and FOBT respectively, 1 being most preferred, except for embarrassment in both groups in which C and BE each had a median rank 3. The results hold up for each gender subgroup in all cases except that men didn\'t consider FOBT significantly less inconvenient than FS. Age was not significantly correlated with ranking of FOBT (that is, the ranking given didn\'t change with age) by Spearman\'s rho. Rho values ranged from -0.10 to +0.16, none significant. Splitting the groups into ages 18--39 (n = 39) and 40+ (n = 40), the results are the same for both groups except that for those over 40, preference for FOBT vs. FS and inconvenience of FOBT vs. FS did not reach significance by two-tailed test (p = .079 and p = .057 respectively) Discussion ========== A recent review of colorectal cancer screening stated that, \"At present there is no preferred CRC screening strategy\"\[[@B1]\]. This presents the perspective of a group of impartial physicians. However from the perspective of those who should take part in CRC screening in the future, a clear preference for FOBT over each other screening modality is expressed in this survey. Each domain of preference similarly ranks FOBT as significantly most preferred. Among previous surveys there are four randomized controlled trials of the use of decision aids that were designed with the intent of altering participation in screening. Three of these presented choices of screening modality or scenario to both intervention and control groups \[[@B15]-[@B17]\]. These studies therefore provided information of participant preference for specific screening modality, though again the participants, primary care patients, were quite different from the group reported herein. Only one of those reports offered all four of the screening modalities that we did in our study \[[@B16]\], the other two offering only a choice between FOBT and FS \[[@B15],[@B16]\]. Nevertheless a uniform preference for FOBT was reported in these studies as well (Table [1](#T1){ref-type="table"}). None of the test interventions were particularly effective in increasing participation in screening, an endpoint not assessed in our study. The fourth randomized trial randomized non-patients, relatives of gastroenterology patients, to be offered either FS or C and measured differential participation, which was equal in the two groups \[[@B20]\]. In the survey most similar to the present study, Pignone surveyed 146 patients in a general medicine clinic \[[@B18]\] and questioned participants after four sequential levels of information were given. Only two screening options were presented, FOBT & FS. Information included in sequence 1) the risk of colorectal cancer, 2) description of the conduct of the test, 3)accuracy of the tests, 4) cost. Previous screening participation was queried but not an exclusion. Less than 5% of those approached refused participation and no data were presented on the screening naïve participants in his sample. FOBT was preferred at each level of investigation, though both tests together were preferred after level 2 (Table [1](#T1){ref-type="table"}). Participants were also asked for reasons for their preferences. The reasons most often given related to cost, ease of performance and being done alone. Among some physicians there is a growing popularity for the use of definitive diagnostic testing as a screening tool, that is, colonoscopy \[[@B19]\]. Though expensive and not without danger, reimbursement for the test is declining and the procedure is getting safer. It has obvious theoretical advantages of offering precise diagnostic capabilities, through biopsy, for those with positive screens. Most important, colonoscopy has the best potential for cancer prevention by adenoma removal -- which is not possible with any other test \[[@B22],[@B23]\]. This, properly applied, might even result in cost savings in the global cost of caring for colorectal cancer. But the public has to want to participate in this program and there is little evidence in this current survey and previous studies, especially those done in primary care settings \[[@B13]-[@B18]\], that this is likely. The concerns expressed herein about safety, embarrassment, inconvenience and discomfort all must be addressed in future efforts to increase screening participation. A potentially significant development related to these issues is that the principal disadvantage of FOBT, its inaccuracy in detecting colorectal neoplasia, might be overcome. Recently developed stool tests show an ability to diagnose cancer with much greater reliability \[[@B24]\]. Perhaps these gene based stool tests may establish the potential for adenoma discovery by non-invasive testing as well. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= RLN conceived of the study, designed the questionnaire and supervised its administration. AS organized the domains of preference and performed the statistical analyses. Both authors participated in the writing of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/4/76/prepub> Acknowledgments =============== The authors are grateful to Jacqueline Campbell, R.N. and Susan Nelson, M.B.B.S. for their recruitment of participants and survey administration. Figures and Tables ================== ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Surveys of screening preference for colorectal cancer ::: First Author Subjects Age Range N = Comparison Preference Comment ------------------ ----------------------------------- --------- ----------------- ------------------------------------------ ---------------------------------------------------------------------------------------------------------------- Steine^9^ GI Patients 45--79 y 190 BE v C BE Post hoc Durdey^10^ GI Patients 19--88 y 66 BE v C C Post hoc VanNess^11^ GI Patients 20--84 y 189 BE v C C Post hoc Elwood^20^ Relatives of GI Patients 45--70 y 232 FS v C FS = C RCT; Subjects offered FS or C and differential compliance measured Dominitz^12^ VAOP 50--75 y 62 FS v C FS Time trade off measure Least for FS. Frew^13^ PCOP \>25 y 2294 FOBT v FS FOBT Willingness to pay primary endpoint. Preference also collected. Leard^14^ PCOP 50--75 y 100 FOBT, FS, BE, C C preferred FOBT more likely to be done. Post hoc 93% previously screened Dolan^17^ PCOP \>50 y 96 FOBT, FS, BE, C FOBT DARCT Pignone^15^ PCOP 50--75 y 227 FOBT v FS FOBT DARCT Pignone^18^ PCOP 50--75 y 146 FOBT v FS FOBT 4 levels of survey after varying quantities of information on colon cancer risk, conduct, test accuracy, cost. Wolf^16^ PCOP \>65 y 57 FOBT v FS FOBT DARCT Nelson Non-patients 18--54 y 80 FOBT, FS, BE, C FOBT GI Patients; Gastroenterology patients BE; Barium enema. C; Colonoscopy FS; Fiberoptic sigmoidoscopy FOBT; Fecal Occult Blot Testing Post hoc; Preference measured after undergoing one or more of the above screening tests. RCT; Randomised Controlled Trial VAOP; Veteran\'s administration hospital outpatients PCOP; Primary care outpatients DARCT; Randomised trial to investigate the effectiveness of decision aids in increasing screening participation ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Mean test ranks for each domain of preference of colorectal cancer screening test ::: Dimensions --------------------------------------- ------------ --------------------- --------------- --------------- --------- Test Modality Preference Physical Discomfort Inconvenience Embarrassment Danger Colonoscopy 3.14 3.37 3.47 3.14 3.56 Barium Enema 2.87 3.09 2.97 3.12 3.56 Fiberoptic Sigmoidoscopy 2.38 2.46 2.04 2.40 2.32 Fecal Occult Blood Test 1.61 1.09 1.52 1.34 1.09 Friedman\'s test χ^2^(3 df) 62.7\* 146.6\* 110.9\* 102.4\* 162.8\* Wilcoxon signed-ranks Z (FOBT vs. FS) 4.1\* 7.2\* 3.2\* 5.3\* 7.5\* N = 77 79 79 78 79 Notes: Mean ranks for each test on each of the dimensions. Lower mean ranks refer to greater preference, and less discomfort, inconvenience, embarrassment, or danger. A \* indicates test statistics that are significant at p \< 0.05. N = ; Less than 80 responses due to blank forms. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Mean test ranks for each domain of preference of colorectal cancer screening test:: individuals who have experienced none of the tests ::: Dimensions --------------------------------------- ------------ --------------------- --------------- --------------- --------- Test Modality Preference Physical Discomfort Inconvenience Embarrassment Danger Colonoscopy 3.13 3.42 3.56 3.07 3.57 Barium enema 2.83 3.11 2.98 3.15 3.05 Fiberoptic sigmoidoscopy 2.35 2.45 1.91 2.38 2.32 Faecal occult blood test 1.69 1.02 1.55 1.40 1.05 Friedman\'s test χ^2^(3 df) 38.7\* 112.6\* 86.6\* 65.9\* 118.4\* Wilcoxon signed-ranks Z (FOBT vs. FS) 3.2\* 6.5\* 2.0\* 4.3\* 6.6\* N = 54 55 55 54 55 Notes: Mean ranks for each test on each of the dimensions. Lower mean ranks refer to greater preference, and less discomfort, inconvenience, embarrassment, or danger. A \* indicates test statistics that are significant at p \< 0.05. N = ; Less than 57 due to blank responses :::	PubMed Central	2024-06-05T03:55:49.811417	2004-11-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533878/", "journal": "BMC Cancer. 2004 Nov 8; 4:76", "authors": [ { "first": "Richard L", "last": "Nelson" }, { "first": "Alan", "last": "Schwartz" } ] }
PMC533879	Background ========== The use of mobile phones is increasing worldwide, although electromagnetic fields emitted by mobile phones and base stations are a source of great concern. However, so far it is unclear, if non-thermal exposure has a direct influence on public health. French et al. \[[@B1]\] developed a theoretical model, by which radiofrequency radiation from mobile phones could induce cancer, via the chronic activation of the heat shock response. Non-thermal exposure to electromagnetic fields can result in an increased expression of heat shock proteins (hsp) \[[@B2],[@B3]\]. This is a normal defense response to cellular stress. However, chronic expression of hsp is known to induce or promote oncogenesis, metastasis and/or resistance to anticancer drugs \[[@B1]\]. Additionally, 72 hours exposure of human lymphocytes to continuous 830 MHz electromagnetic fields caused a linear increase in chromosome 17 aneuploidy with rising specific absorption rates (SAR: 1.6--8.8 W/kg). This is a signal for genetic instability and may thereby lead to cancer development \[[@B4]\]. In principal agreement, few epidemiological studies suggest a relationship between the use of mobile phones and uveal melanoma \[[@B5]\] or malignant brain tumors \[[@B6]-[@B9]\] However, the overall literature does not provide persuasive epidemiological evidence that mobile-phone-related emissions are carcinogenic, although mobile phones have not been in use long enough to exclude long-term impact on health \[[@B10]\]. It was suggested that non-thermal exposition to high-frequency electromagnetic fields may rather have a tumor promoter than an initiator effect \[[@B9]\], since DNA, generally, does not appear to be significantly altered or damaged by electromagnetic fields \[[@B11]\]. In this respect it was discussed, if a possibly reduced excretion of the oncostatic hormone melatonin by electromagnetic fields may facilitate the development of estrogen dependent tumors \[[@B12]\]. However, the results of different rodent studies concerning tumor promotion are not consistent. On the one hand, no difference in radiation or chemically induced tumor growth could be found after long-term exposure to electromagnetic fields (860--900 MHz) in rats or mice \[[@B13]-[@B15]\] Additionally, exposure of human leukaemia cells to electromagnetic fields failed to induce any changes in apoptosis, micronucleation or differential gene expression \[[@B16]\]. On the other hand, long-term exposure to pulse-modulated electromagnetic fields similar to those used in digital mobile telecommunication significantly increased in one study the incidence of lymphoma in Eμ-Pim1 transgenic mice \[[@B17]\], which are genetically predisposed to develop lymphoma spontaneously, but not in another \[[@B18]\]. The differences between the studies may indicate that various species or strains as well as cancer types differ in their sensitivity to electromagnetic field exposure. The sensitivity to electromagnetic fields may result from an acquired lower resistance against adverse effects or a genetic predisposition \[[@B19]\]. Different proportions of a sensitive subpopulation within an epidemiological or experimental study would influence the interpretation of a possible role in carcinogenesis. However, national or international thresholds for electromagnetic field intensities must ensure adequate health protection also for susceptible people. AKR mice are widely used in cancer research for their high leukaemia incidence (60--100%) \[[@B20]\]. Mice of this strain are viremic from birth and express in all tissues the retrovirus AKV, which is associated with spontaneous leukaemia development in mice \[[@B21]-[@B23]\]. Generally, leukaemia induced by a given virus is restricted to a single histopathological type; most common is a lymphocytic leukaemia originating in the thymus. However, the type of leukaemia induced can sometimes be altered by age or experimental manipulation \[[@B24],[@B25]\]. Using AKR mice, we studied the incidence of tumors and survival rates under chronic influence of high-frequency electromagnetic fields. Despite some physiological differences between mice and humans, a good correlation between known or assumed human carcinogens and test results in rodent studies has been described, often with the same organs affected in humans and in rodents \[[@B26]\]. Therefore, the results of this study shall help to evaluate a possible health risk of mobile phones. Methods ======= Animals and animal husbandry ---------------------------- 320 female AKR/J mice were airfreighted from the Jackson Laboratory (Bar Harbor, ME, USA), at an age of 4--5 weeks. After arrival, animals were randomized and housed in groups of 6 or 7 on softwood bedding (Altromin, Lage, Germany) in polycarbonate cages (40 × 25 × 15 cm, W × L × H, Ebeco, Castrop-Rauxel, Germany), enriched with paper. Mice were allowed free access to mice standard food pellets (type 1324, Altromin) and tap-water. Twice a week cages were cleaned and water changed. Temperature was controlled at 21°C ± 2°C. The light was on a 12 hours light-12 hours dark cycle, with light on at 8 am. No sterilization measurements were taken, since AKR/J mice are not especially sensitive for pathogens. However, to prevent a possible transfection from humans to mice or mice to mice, respectively, masks and gloves were worn, which were sterilized between handling of different cages. Animals were inspected daily for signs of moribundity and were weighed (accuracy ± 0.1 g) and palpated weekly. Starting with an age of 6 months, blood samples were taken monthly from the tail. A tattoo in the ear allowed individual identification. The Bremen state commission for animal welfare according to §8a of the German animal welfare legislation approved the experiments (522-27-11/2-0). Exposure setup -------------- In order to accomplish whole body exposure of a large number of non-restrained animals, one approach is to design exposure setups based on radial waveguides. Inside the waveguide the animals are kept in cages which are arranged at a constant distance from a radiating antenna in the centre, thus uniform exposure of the cages can be achieved. Another advantage is that the radial waveguide is an electromagnetic shielded system, on that score no costly shielding of any laboratory is necessary. The height of the waveguide is preferably chosen smaller than half a wavelength \[[@B28]\]. Thereby, single-mode operation of the TEM-mode is possible and by this homogeneous field distribution inside every cage can be guaranteed. However, in the actual study the plate distance of the radial waveguide must be chosen larger than half the wavelength of the exposure frequency due to the prescribed height of the cages of about 16 cm. Consequently, higher order modes are able to propagate in addition to the TEM-wave, which have inhomogeneous field distributions in the waveguide\'s cross-section. Furthermore, the simultaneous propagation of several modes leads to interference effects and thus to an unstable exposure field. Therefore, special modifications of the fundamental geometry of radial waveguides had to be performed to avoid the propagation of the unwanted higher order modes. In order to produce a well defined field distribution the cage region was excited only by the fundamental TEM-mode in a first step, i.e. for small radii the plate distance was kept below 14 cm and for larger radii the height was increased to the required value of 17 cm (Figure [1a](#F1){ref-type="fig"}). Since it was still possible that the additional modes were excited by the field scattered of the mice, metal rib structures were attached to the upper and lower plate between the cages in order to shift the cut off frequencies of these unwanted modes to higher values, so that they could not propagate at the exposure frequency (Figure [1b](#F1){ref-type="fig"}). The optimum size of the ribs was determined by numerically solving the eigenvalue problem of the modified waveguide. It yielded that a maximum attenuation of the higher order modes is reached for specific heights and widths of the ribs. Therewith, it was shown that the unwanted modes can be forced to become evanescent and thus a stable field distribution is achievable. Moreover, the rib structure does not alter the propagation constant of the TEM-mode, and the perturbation of the field distribution of the fundamental mode is negligible within the cage volume. Both implemented waveguides (one for exposure, one for sham-exposure) of ca. 4 m diameter and 17 cm vertical plate distance were placed within the same room and carried up to 24 cages measuring 425 mm × 265 mm × 160 mm (L × W × H) each of them housing 6--7 mice. The cage area was covered with trapezoidal lids (3 cages per opening) with wire mesh (Figure [2](#F2){ref-type="fig"}). This design ensured easy maintenance and also that both light and gas could penetrate the lids while electromagnetic radiation was effectively shielded. At the outer boundaries of the units, absorbers were installed which caused minimal reflection and \"hot spots\". A signal generator (SMT 06, Rohde & Schwarz, Munich, Germany) and an amplifier (HLV-500, BEKO, Munich, Germany) were connected to the cone antenna of one unit via a \"black box\" so that it could not be seen which group of animals was exposed (blind design). The signal of the generator was modulated (BS 825F, BUGH Wuppertal, Germany) in a way which simulated a situation near a mobile communication base station (downlink) combined with a contribution from an mobile phone DTX operation mode (uplink), thus including 2 Hz, 8 Hz, 217 Hz, and 1733 Hz frequency components (Figure [3](#F3){ref-type="fig"}). For additional details see also \[[@B27]\]. Animals were exposed 24 hours per day with the exception of approximately 1 hour weekly when animals were weighed and palpated, and during which the cages were cleaned. The experiment was performed at a mean value of 0.4 W/kg of the whole body specific absorption rates. This value, which was stipulated by the financial backer, is five times higher than the limit of whole-body exposure for the general population and is based on the limit value for occupational exposure \[[@B29]\]. Since the mice can move freely, the whole body SAR varies with their postures and positions inside their cage. Therefore, the specific absorption rates in the mice were analyzed by numerical computations of the electromagnetic field distribution inside the radial waveguide for five different configurations of the animals, which were assumed to be uniformly distributed in time. Configurations account for groups of mice in the front and rear section of the cage as well as mice with head, left/right side toward the incident wave and upright posture. Since only the variation of the whole body SAR is subject of interest, it is sufficient to use simple homogeneous models (ellipsoids, length 6 cm, diameter 3 cm, appr. 32 g) filled with muscle tissue for the mice (Figure [4a](#F4){ref-type="fig"}). After evaluation of all absorption rates per rodent it turned out that the standard deviation of the whole body SAR was ± 40%. The assessment of maximum localized SAR was performed by use of an anatomical mice model which was placed into the group of ellipsoids. (Figure [4b](#F4){ref-type="fig"}). The required time-averaged input power of the exposure unit was 35 W. The presence of the field was monitored continuously, again via a \"black box\". In Streckert et. al \[[@B30]\] features of the exposure facility, which was previously used for experiments with rats, are described in detail. Noise levels provoked by the integrated ventilation system were measured in close proximity to both units and were found to be identical (sound meter model 2218 with 1/3 octave filter set model 1616, Brüel & Kjaer, Naerum, Denmark). The total level was 69 dB (lin), and less than 25 dB at frequencies between 8 and 40 kHz. Thus possible disturbing effects of ultrasound were excluded. Pathology --------- Animals were sacrificed by CO~2~gas when signs of a developing disease became evident or at an age of about 46 weeks, after a last blood sample was taken. A gross necropsy was performed focusing on main tissues of disease involvement (spleen, thymus and lymph nodes) and tumor infiltration (liver, kidney, lung, brain). Tissues were immersion-fixed in Bouin\'s solution for up to 24 h and subsequently in ethanol (70%), embedded in paraffin and sectioned at 5 μm. Blood smears were stained with Pappenheim\'s stain and tissue slices using hematoxylin and eosin. When a mouse was found dead in its cage (5 exposed, 7 sham-exposed mice) a necropsy was performed, but no tissues were fixed. Statistics ---------- Group mean body weights were tested for a possible exposure influence in dependence of time, using multiple regression analysis (InStat 3.05, GraphPad). An unpaired t-test was applied to compare the loss of weight associated with lymphoma development. Survival curves and lymphoma incidence were plotted according to the method of Kaplan and Meier. Differences between curves were compared using the logrank test, with animals censored, which were still alive at the end of the study (Prism 4.01, GraphPad). Two-way ANOVA was used to test for possible changes in differential leucocytes counts with time and exposure. Statistical significance of differences was tested two-sided at the p ≤ 0.05 level. If not indicated otherwise, data are given as means ± standard error of the mean. The exposure code was broken only after completion of the analyses. Results ======= Body weight and water consumption --------------------------------- Chronic exposure to 900 MHz electromagnetic fields had no influence on the absolute body weight of female AKR/J mice; however, its influence on the relative weight change was significant (p \< 0.001) (Figure [5](#F5){ref-type="fig"}). The rapid development of lymphoma in this strain of mice was associated with a loss of individual body weight of about 9.2% in exposed and 8.5% in sham-exposed mice, but the group difference was not statistically significant. Water consumption was approximately 4 g per day and mouse, and not different between exposed or sham-exposed animals (data not shown). This value is in accordance to the water intake measurements published by the Jackson Laboratory \[[@B31]\], and obviously not influenced by the experimental set-up. Survival and incidence of lymphoma ---------------------------------- Similar survival rates were seen in both groups of AKR/J mice (Figure [6](#F6){ref-type="fig"}). The first exposed mouse died of lymphoma after 60 days and the first sham-exposed animal after 88 days. Median survival time was 183 (sham-exposed) and 190 days (exposed mice), and not significantly different according to the logrank test, with animals censored which were still alive at the end of the study. Patterns of tumor-related mortality in the sham-control group were consistent with those observed in a previous study conducted in this laboratory with AKR/J mice \[[@B32]\]. As seen in our previous study, essentially all mortality observed in AKR/J mice was related to the development of lymphoblastic lymphomas \[[@B33],[@B34]\] (Figures [7](#F7){ref-type="fig"}, [8](#F8){ref-type="fig"}). Exceptions were 3 animals with rectal eczema (2 exposed, 1 sham-exposed), one animal with unclear findings and two sham-exposed animals with protruded vagine. These findings were considered random findings and not related to the exposure. Clinical picture ---------------- In female AKR/J mice lymphoma developed rapidly, usually associated with lymphadenopathy. In 28.2% (exposed) and 30.3% (sham-exposed) of all animals, lymphomas were restricted to the thymus, followed by respiratory distress and protrusion of the eyes. 13 animals (8 exposed, 5 sham-exposed) died in their cages without any earlier sign of distress, although autopsy revealed enlarged mediastinal mass compressing the thoracic space. The lung was affected in 10% of the exposed and sham-exposed animals; 8 exposed and 6 sham-exposed animals developed macroscopically visible metastatic tumors in the liver or spleen (Figure [9](#F9){ref-type="fig"}). Other clinical observations like splenomegaly and ruffled fur were considered to be associated with neoplastic development and did not correlate with the electromagnetic field exposure. Tumors of other sides such as mammary gland or intestine could not been observed. When the animals reached an age of about half a year, differential counts of leucocytes were performed monthly using blood smears from tail blood. As seen in Figure [10](#F10){ref-type="fig"}, exposure to GSM-modulated electromagnetic fields had neither influence on the ratios of lymphocytes to neutrophilic granulocytes nor on counts of monocytes, eosinophilic or basophilic granulocytes. However, the development of lymphoma was associated with changes in the red and white blood picture. Symptoms were changes in the counts of lymphocytes and neutrophilic granulocytes, toxic granulation in neutrophilic granulocytes, the occurrence of juvenile cells or blasts, blastoid or atypical lymphocytes, Gumprecht\'s shadows, leucopenia, anisocytosis, poikilocytosis or enhanced polychromasia. These changes were not related to the exposure. Discussion ========== The present study was designed to test the hypothesis that 900 MHz electromagnetic fields, modulated according to the global system for mobile communication (GSM), increases the risk of lymphoma incidence in female AKR/J mice. According to the results this hypothesis has to be rejected, since compared with sham-exposed females of this strain, a mean exposition of 0.4 W/kg SAR neither influenced the risk to develop lymphoma, nor the malignancy of the disease, only an influence on the growth pattern of the animals was observed. However, the present experiment does not allow any conclusions about tumor onset or the kinetics of tumor development, since for such type of study animals would have to be sacrificed and examined at fixed intervals irrespective of clinical symptoms. Exposure to electromagnetic fields increased the growth rate in the nematode Caenorhabditis elegans\[[@B35]\], but decreased the birth weight of albino rat offsprings \[[@B36]\]. In contrast, the growth pattern of Eμ-Pim1 mice was not changed due to exposure to electromagnetic fields \[[@B17],[@B18]\]. During the progression of the present experiment, female AKR/J mice showed a tendency to obesity. Accumulation of weight was significantly related to the exposition (see Figure [5](#F5){ref-type="fig"}), leading to a higher body weight gain in exposed compared with sham-exposed mice. Food was available ad libitum, and various studies described effects of electromagnetic fields on the nervous system in humans or rodents \[[@B37]-[@B39]\]. It may, therefore, be possible that the exposure to the electromagnetic field influenced the appetite, leading to higher food intake. However, the exposure-independent water consumption does not indicate that major changes in the intake of food occurred. Mitochondrial heat production is one of the main energy consuming processes in endotherms like mice \[[@B40],[@B41]\]. The Interaction of electromagnetic fields with water molecules in cells results also in heat production. Although the continuous exposure in our study should not have increased the mice\'s body temperatures, it may have contributed in keeping the animal\'s body temperature above ambient, therewith economizing mitochondrial heat production. If mitochondrial functions were not changed due to the electromagnetic field exposure, unchanged food intake may so have let to increased fat accumulation. In this context it is important to compare the SAR value of 0.4 W/kg with the total energy consumption of mice (approximately 5 W/kg), thus on the order of 10%. Since the electromagnetic energy is absorbed passively (i.e., not originating from metabolizing food), the increased body weight might be a consequence of a shift in energy utilization. This hypothesis, however, must be examined by specific studies of the mice\' metabolism. Anyhow, such an effect would be visible only in long-term exposure studies and probably insignificant with only 1 hour exposure per day \[[@B17],[@B18]\]. A demonstration that long-term exposure to electromagnetic fields derived from mobile phones or base stations increases the incidence of tumors in animals would provide direct evidence that such radiation is carcinogenic. The most positive evidence of an effect of exposure to high frequency electromagnetic fields similar to that used by mobile phones was reported by Repacholi et al. \[[@B17]\], using Eμ-Pim1 transgenic mice, which are known to develop spontaneous lymphoma with a high incidence rate. Lymphoma risk was found to be significantly higher in the exposed Eμ-Pim1 mice than in the controls, mostly pronounced for non-lymphoblastic lymphoma. Humans are presently not known to carry an activated Pim1 gene, but other inherited gene defects are known that predispose carriers to develop cancer \[[@B19]\]. However, an investigation within the electromagnetic energy program of the National Health and Medical Research Council of Australia, using the same Eμ-Pim1 mice from the same supplier (Taconic Farms, New York) as Repacholi et al. \[[@B17]\], did not find a significant effect of 898.4 MHz GSM radiofrequency radiation at SAR values of up to 4.0 W/kg \[[@B18]\]. Because of the importance of these studies, a replication started according to plan in spring 2002 in Italy. First results are expected 2005. Nevertheless, the inconsistent results already indicated the need of further assessment of the relevance of such findings for human health \[[@B10]\]. A Japanese study showed that neither 1.5 GHz nor 929.3 MHz electromagnetic field exposure promotes liver carcinogenesis in a medium-term bioassay system, using partially hepatectomised F344 rats \[[@B42]\]. A monopole antenna close to the constrained animals emitted a \"near-field\" radiation that resulted in SAR values of 0.45--0.68 W/kg as a whole-body average and of 0.9--1.9 W/kg in the liver. It was suggested that the applied \"near-field\", which is more in line with the actual exposure conditions of cellular telephone users, explains the differences to the study of Repacholi et al. \[[@B17]\], who employed a \"far-field\" and mean whole-body SAR values of 0.13--1.4 W/kg. However, the confinement may also affect the results \[[@B43],[@B44]\], as well as the different animal models: long-term exposure of genetically predisposed animals on the one hand and a medium-term bioassay of chemically induced carcinogenesis on the other hand. Therefore, it is difficult to decide which results are more relevant for human mobile phone users. In the present study non-restrained mice that are genetically predisposed to develop lymphoma with a high incidence were exposed to a \"far-field\" similar to both Australian studies \[[@B17],[@B18]\], with the difference that our mice were exposed for 24 h, whereas their mice were exposed for 1 hour per day only. In contrast to Repacholi et al. \[[@B17]\], we could not observe an increased lymphoma risk. However, our study\'s results are consistent with the investigation within the electromagnetic energy program of the National Health and Medical Research Council of Australia \[[@B18]\]. The authors also did not find a significant effect of 898.4 MHz GSM radiofrequency radiation at SAR values of up to 4.0 W/kg when compared to sham-irradiated animals. The overall conclusion of the present study as well as of data from the literature is that they lack evidence that electromagnetic field exposure increases the incidence of leukaemia in rodents. Conclusions =========== The present study does not support the hypothesis that chronic exposure to high-frequency electromagnetic fields, similar to those emitted by mobile phones and base stations promote neoplastic development in the hematopoietic system in genetically predisposed mice. However, we cannot rule out that exposure to electromagnetic fields may be risk factors for other neoplastic development. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= AS carried out the study, performed the statistical analyses and drafted most of the manuscript. AL conceived the study, participated in its design and coordination, and drafted some parts of the manuscript. JS, AB and VH developed the technical set up and delivered the dosimetry for the experiment. 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/1471-2407/4/77/prepub> Acknowledgements ================ A special thanks goes to K. Grote and T. Ströhlein for their assistance with the animal experimentation and to T. Simon for her help in the histological and hematological analyses (all International University Bremen). This study was part of a project supported by the Bundesamt für Strahlenschutz, St. Sch 4315: \"Wirkung chronischer Exposition mit einem athermisch wirkenden GSM-Mobilfunksignal auf die Entwicklung spontaner lymphatischer Leukämie bei frei beweglichen weiblichen Mäusen des AKR/J-Stammes\". Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Measures to guarantee stable and reproducible exposure fields.a) Single mode excitation of the cage region b) Metal ribs used to shift the cut off frequency of unwanted higher order modes (e.g. TEz01, TMz01) out of the operation frequency range ::: ![](1471-2407-4-77-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### One of the two identical exposure units viewed from top.From an antenna in the middle (under the central plate; not visible in this picture) electromagnetic fields were fed into the radial waveguide and absorbed by either the animals in the cages or by absorbers at the boundaries of the waveguides. Each of the upper segments had a trapezoidal opening to allow for removing and inserting the cages. The openings were closed with wire mesh lids in order to guarantee penetration of light and gases and to prevented high-frequency electromagnetic radiation to leave or enter the exposure unit. Water bottles were placed outside the waveguides to prevent unwanted absorption. ::: ![](1471-2407-4-77-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Synthetic GSM modulation pulse pattern and frequency spectrum.The exposure frequency was 900 MHz with a modulation simulating a typical GSM ([G]{.underline}lobal [S]{.underline}ystem for [M]{.underline}obile Communication) scenario, namely a situation combining the emissions from a base station (downlink) with those from an uplink operating mobile phone, thus including 2.1 Hz, 8.3 Hz, 217 Hz, and 1733 Hz frequency components. ::: ![](1471-2407-4-77-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Dosimetry.a) Examples for groups of 7 mice inside a cage (P~in~= 35 W): Electric field distribution for a 15° sector of the radial waveguide. Higher field values occur due to the field concentration at the metal ribs between the cages (cf. fig. 1b). b) Example for insertion of an anatomical mice model into the group of ellipsoids: Localized SAR distribution (SAR~wb~= 366 mW/kg, resolution: 1.2 mm (voxel size)). The overall maximum of SAR~voxel~= 5.9 W/kg is not located in the cuts shown here. ::: ![](1471-2407-4-77-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Growths curves for female AKR/J mice exposed or sham-exposed to 900 MHz electromagnetic fields.The relative weight increase of female AKR/J mice was more pronounced in exposed than in sham-exposed animals. Therefore, the mice\'s tendency to obesity seems not only to be strain-specific but was also significantly (p \< 0.001) related to the exposure. Data are given as % of the animals weight (with 100%) at the beginning of the study ± standard error of the mean, n = 160 at the beginning of the experiment. ::: ![](1471-2407-4-77-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Survival rates in AKR/J mice exposed to 900 MHz electromagnetic fields.No significant differences in the survival proportion or mean survival time were seen between exposed and sham-exposed animals when average whole body specific absorption rates were 0.4 W/kg. Data are given as % of 160 animals ± standard error of the mean. ::: ![](1471-2407-4-77-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Lymphoma incidence in AKR/J mice.Essentially all mortality observed in AKR/J mice was related to the development of hematopoietic diseases independent of electromagnetic field or sham-exposure. Median time for lymphoma development was 183 days (exposed mice) or 193 days (sham-exposed mice), and not significantly different according to the logrank test, with animals censored which were still alive at the end of the study or died of other reasons than lymphoma. Data are given as % of 160 animals ± standard error of the mean. ::: ![](1471-2407-4-77-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Lymphoblastic lymphoma in AKR/J mice.The architecture of spleen (seen here) or other involved organs was not maintained due to the development of lymphoblastic lymphoma. Often starry-sky appearance was present and mitotic figures were numerous. ::: ![](1471-2407-4-77-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Malignant lymphoma metastatic to liver in AKR/J mice.Extensive proliferation of lymphoma cells around portal tracts was seen in liver. Infiltrations resulted in few cases in macroscopically visible tumors in liver or spleen of up to 1 cm in diameter. ::: ![](1471-2407-4-77-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### Differential leucocyte count.Differential leucocyte counts of peripheral blood of healthy female AKR/J mice did not differ significantly between animals sham-exposed or exposed to 900 MHz electromagnetic fields, although time influenced the ratio of lymphocytes to neutrophilic granulocytes (A) as well as the percentage of monocytes (B) and eosinophilic granulocytes (C). Percentage of basophilic granulocytes was in all cases below 1. 100 cells were counted per slice. Mean ± standard error of the mean, n = 138 (exposed) or 137 (sham-exposed) after 21 weeks of exposure. ::: ![](1471-2407-4-77-10) :::	PubMed Central	2024-06-05T03:55:49.814411	2004-11-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533879/", "journal": "BMC Cancer. 2004 Nov 11; 4:77", "authors": [ { "first": "Angela M", "last": "Sommer" }, { "first": "Joachim", "last": "Streckert" }, { "first": "Andreas K", "last": "Bitz" }, { "first": "Volkert W", "last": "Hansen" }, { "first": "Alexander", "last": "Lerchl" } ] }
PMC533880	Background ========== Dementia, an acquired persistent impairment of cognitive functioning is an increasingly common problem in Korea, and is associated with increased morbidity and mortality, functional loss, caregiver burden, and institutionalization \[[@B1]\]. Nevertheless, most patients with dementia are not detected by family members or clinicians at an early stage, even though such patients have memory problems \[[@B2],[@B3]\]. Screening for dementia has been recommended to increase detection of dementia. The Mini-Mental State Examination (MMSE) is a brief screening test that quantitatively assesses the cognitive status of elderly people \[[@B4],[@B5]\]. Although it has shown good validity, the MMSE has been found to be influenced largely by racial differences and educational status \[[@B6]-[@B10]\]. Korean elderly show very low levels of educational background, and it is necessary to develop appropriate cognitive assessment method that is not influenced by education. The Time and Change (T&C) test was developed by Inouye et al. \[[@B11]\] to assess (i) how well a patient understands time and (ii) the ability of the patient to calculate using money. Several studies have found that the T&C test allows direct assessment of two important activities of daily living and supplements the testing of calculation, conceptualization, and visuospatial cognitive domains irrespective of race and education \[[@B11]-[@B13]\]. We have therefore introduced a T&C test, and assessed the applicability of the T&C test as a convenient and accurate means to detect early stage dementia in primary care and community settings. Methods ======= Subjects -------- One study group initially comprised 60 participants who either visited or were admitted to a hospital that was located in an urban area, namely Gwangju city (referred to forthwith as area A) between November and December 2001. Of these 60 participants, 37 were diagnosed with Alzheimer\'s disease or vascular dementia, 11 had mental illness such as schizophrenia or depression, 7 had organic brain disease such as cerebral apoplexy or Parkinson\'s disease, and 5 were alcoholics. Another study group initially comprised 412 participants who were recruited from all residents of Jangseong-county, Jeonnam province, South Korea, aged 65 and over in 2002(referred to forthwith as area B). The area consists of 11 towns and had an estimated population 54,528 of whom about 16.5% were aged 65 and over. The subjects were selected from each stratum (town) using cluster sampling. All subjects in whom vision or hearing was impaired were excluded from the study. We selected 59 (of 60) and 405 (of 412) participants from area A and B, respectively. All participants gave informed consent, and when participants with dementia cannot provide informed consent, caregivers were asked to provide it. The Time and Change test ------------------------ The \'time\' component of the T&C test evaluated the ability of a subject to comprehend that the hands of a clock indicated 11:10. The diameter of the clock was 15 cm, and the distance between the clock and the participant was 25--35 cm (in consideration of the visual deficits of the older subjects). When the subject responded incorrectly on the first attempt, the interviewer posed the same question, i.e., a second attempt was permitted. The time (in seconds) that it took for the participant to respond correctly was recorded. A time limit of 60 s for a response was imposed. In the \'change\' component of the T&C test, the participants were required to make 1,000 Won from a group of coins of several smaller denominations, namely one 500, seven 100, and seven 50 Won coins, that were placed on a table in front of the participant. The participants were given 120 s to complete the task, and an additional 120 s was granted if the subject failed to complete the task at the first attempt. The time (in seconds) that it took for the participant to complete the task was recorded by the interviewer. The participants who completed both of the aforementioned tasks of the T&C test successfully were determined to be negative for suspected dementia, whereas participants who failed to complete either or both of the tasks were determined to be positive for suspected dementia. The T&C test was performed prior to the other cognitive tests by an interviewer who was unaware of the results of the other tests. Measurement ----------- In the participants from area A, the T&C test was conducted by a nurse, while a physician who were blinded to the result of the T&C test performed a clinical examination and neuropsychiatric inventory. The diagnostic criteria for dementia were based on those of the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) \[[@B14]\]. To detect the causative diseases of dementia, the following were carried out: general blood and chemical tests (including VDRL, Vitamin B12, T3, T4, TSH); a chest radiograph; an electrocardiogram; a brain computerized tomography scan; and psychometric tests including the K-MMSE \[[@B15]\]. Alzheimer\'s dementia was diagnosed based on the guidelines of the National Institute of Neurological and Communicative Disorders and Stroke and Alzheimer\'s disease and Related Disorders Association (NINCDS-ADRDA) \[[@B16]\]. Vascular dementia was diagnosed based on the guidelines of the National Institute of Neurological Disorders and Stroke and the Association Internationale pour la Recherche et l\'Enseignement en Neurosciences (NINDS-AIREN) \[[@B17]\]. The CDR developed by Hughes et al. \[[@B18]\] was used to determine the progression of dementia. The participants from area B were interviewed by interviewers who had undergone sufficient training to be able to conduct the K-MMSE and T&C test. All participants were interviewed for data on social and demographic factors such as address, age, sex, educational status, and the number of family members living together. Assessment of the validity of the T&C test ------------------------------------------ To assess the validity of the T&C test as a method to screen for dementia in the participants from area A, we compared the results of the test to a reference standard (diagnosed by a physician), and evaluated sensitivity, specificity, and positive and negative predictive values. To assess the test-retest reliability, the one type of interviewer (specifically, physicians) conducted the same test twice at an interval of 24 h (n = 22 participants). To assess the interobserver reliability, two different types of interviewer (specifically, physicians and psychometrists) conducted the same test, at an interval of 24 h (n = 22 participants). To assess the applicability of the T&C test as a method to screen for dementia in the participants from area B, the association between the T&C and K-MMSE test scores was evaluated. Statistical analysis -------------------- The sensitivity, specificity, and positive and negative predictive values and 95% Confidence interval of the T&C test were analyzed. After classifying the participants from area B into a group in which dementia was suspected, and another group in which dementia was not suspected based on the results from the T&C test, a Student\'s t-test was used to compare the total K-MMSE score with the scores for each of the components of the K-MMSE test, and Spearman\'s rank correlation was used to analyze the correlation between the T&C and K-MMSE test scores. SPSS for Windows (version 10.0; SPSS Inc., Chicago, IL, USA) and Stata Software 6.0 (Stata Corporation, College Station, Texas) were used to analyze data and statistics. Results ======= Characteristics of subjects --------------------------- For the participants from area A, the average age was 73.2 ± 7.9 years, 55.9% of the group was female, the average number of years of education was 4.2 ± 5.4, and 62.7% lived alone. For the participants from area B, the average age was 73.1 ± 6.1 years, 58.5% of the group was female, the average number of years of education was 3.1 ± 4.0, and 29.0% lived alone (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of the study groups. ::: Characteristic Hospital\* (n = 59) Community^†^(n = 405) ----------------------------- --------------------- ----------------------- Age: years, mean ± SD 73.2 ± 7.9 73.2 ± (range) (58--90) (65--97) Gender: female, n (%) 33 (55.9) 238 (58.5) Education: years, mean ± SD 4.2 ± 5.4 3.1 ± 4.0 (range) (0--16) (0--16) Living alone: n (%) 32 (62.7) 118 (29.0) SD = standard deviation. \Referred to in main text as area A (see Methods). ^†^Referred to in main text as area B (see Methods). ::: Validity of the T&C Test ------------------------ Table [2](#T2){ref-type="table"} shows the results of an analysis of the validity of the T&C test. In a comparison of the T&C test and a diagnosis of dementia by physicians (which served as a reference), 27 of 37 patients that had been diagnosed as demented were classified as positive for dementia according to the T&C test, and the sensitivity was 73.0%. Of the subjects who were diagnosed as not demented, 20 of 22 were classified as negative according to the T&C test, and the specificity was 90.9%. The positive predictive value that indicated the probability of dementia, as determined by a positive classification according to the T&C test, was 93.1%, while the negative predictive value that indicated the probability of not being demented, as determined by a negative classification according to the T&C test, was 66.7%. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Concurrent validity of the Time and Change (T&C) test in a sample of elderly hospital patients. ::: Reference standard\ Total -------------------------------------------------------------- ------- ---------------------- ------- ---- T&C \+ 27 2 29 assessment \- 10 20 30 Total 37 22 59 Sensitivity = 27/37 (73.0%) (CI = 61.6--84.3%) Specificity = 20/22 (90.9%) (CI = 83.6--98.2%) Positive predictive value = 27/29 (93.1%) (CI = 86.6--99.6%) Negative predictive value = 20/30 (66.7%) (CI = 54.6--78.7%) Accuracy = 47/59 (79.7%) CI = 95% confidence interval for sensitivity and specificity values. \Diagnosis of dementia made by a clinician. +, positive for dementia according to T&C test. -, negative for dementia according to T&C test. ::: Reliability of the T&C test --------------------------- The rate of agreement of the test-rest & interobserver variability was analyzed to assess the reliability of the T&C test. The rate of agreement of both the test-retest and interobserver was 95% (κ = 0.91) (time interval, 24 hours). Comparison of the T&C and K-MMSE test ------------------------------------- There was a significant difference between the total K-MMSE score and the scores for each of the components of the K-MMSE test between participants that were classified as positive and those that were classified as negative for dementia according to the T&C test (Table [3](#T3){ref-type="table"}). The association between the T&C test and K-MMSE test revealed modest, while significant (r = 0.422, p \< 0.001) (Table [4](#T4){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Korean Mini-Mental State Examination (K-MMSE) scores according to T&C test performance in a sample of elderly community population. ::: K-MMSE components --------------- -------------- ------------ ------------------- ----------- ----------- ----------- ----------- T&C test\ +^†^(n = 65) 14.8 ± 5.8 6.1 ± 2.8 2.1 ± 1.1 0.5 ± 1.4 0.8 ± 1.1 5.4 ± 1.7 \- (n = 340) 22.4 ± 5.0 8.9 ± 1.8 2.8 ± 0.6 2.2 ± 1.9 1.6 ± 1.1 7.0 ± 1.6 Time test\* +^‡^(n = 53) 14.3 ± 5.3 6.0 ± 2.6 2.0 ± 1.1 0.4 ± 1.2 0.7 ± 1.1 5.2 ± 1.6 \- (n = 352) 22.3 ± 5.1 8.8 ± 1.9 2.8 ± 0.6 2.2 ± 1.9 1.6 ± 1.2 7.0 ± 1.6 Change test\* +^§^(n = 31) 14.6 ± 6.9 6.0 ± 3.0 2.0 ± 1.2 0.8 ± 1.7 0.5 ± 1.0 5.3 ± 2.0 \- (n = 374) 21.8 ± 5.4 8.6 ± 2.0 2.7 ± 0.6 2.0 ± 1.9 1.5 ± 1.2 6.8 ± 1.7 () : number mean ± SD. \p \< 0.01 for K-MMSE score (or a component thereof) versus T&C test score (Student\'s t-test). +, positive for dementia according to T&C test. -, negative for dementia according to T&C test. ^†^Incorrect response for either or both the time and change task of the T&C test (see Methods for details). ^‡^Incorrect response for the time task. ^§^Incorrect response for the change task. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Convergent validity of the T&C test in a sample of elderly community population. ::: Correlation coefficient (r)\ ------------------------------ ------------------------------- K-MMSE (overall score) 0.422 K-MMSE component scores: Orientation 0.431 Registration 0.331 Concentration & Calculation 0.358 Recall 0.258 Language & Diagram 0.314 \*p \< 0.001 for all values (Spearman\'s rank correlation analysis). ::: Association between the T&C test and educational status ------------------------------------------------------- The results of a logistic regression in which the T&C test as a dichotomous and dependent variable was performed with education status adjusting for age and sex revealed no association between them. The odds ratio for T&C test associated with educational status is 0.877 (95% CI = 0.766--1.004). Response times in the T&C test ------------------------------ For the time task in the T&C test, 75.8% of the participants produced a correct response on the first attempt after 6.3 ± 6.7 s, and 45 participants (11.1%) produced a correct response on the second attempt. For the change task in the T&C test, 81.2% of the participants produced a correct response on the first attempt after 12.7 ± 14.2 s, and 43 participants (10.6%) produced a correct response on the second attempt. 34 participants(8.4%) were tested twice for both the time and change task. None of the subjects refused to respond during the tests. Discussion ========== Interracial variability in both the etiology of dementia and the accuracy of cognitive testing suggests that there is an urgent need to develop racially appropriate methods of cognitive assessment. The rate of vascular dementia due to cerebrovascular disease is much higher in Koreans than in other races; this is due to insufficient prevention, diagnosis, and treatment of hypertension, diabetes, and hyperlipidemia in Korea. Vascular dementia, unlike Alzheimer\'s disease, can be prevented by appropriate treatment and prevention for the risk factors of cerebrovascular diseases, can be treated to improve symptoms and inhibit progression of the disease. In the present study, the original T&C test of Inouye et al. \[[@B11]\] was modified to apply specifically to Korean elderly sample, and was used to screen for dementia. We found that the sensitivity of the T&C test was 73.0%, specificity was 90.9%, and the positive and negative predictive values were 93.1% and 66.7%, respectively. The disparity between the sensitivity and the specificity might mean the insensitivity to mild stage or pre-dementia. We advocate using the T&C test sequentially with other dementia measures (with increased sensitivity). When compared to the results of Kawamato \[[@B19]\], in which sensitivity and specificity was 49.1 and 95.2%, respectively; the sensitivity of the T&C test in the present study was remarkably high, while specificity was relatively low. In a study by Inouye et al. \[[@B8]\] in which the MMSE and modified Blessed Dementia Rating Scale (mBDRS) served as standards, sensitivity was 86%, specificity was 71%, and the negative predictive value was 97% when the subjects were hospital patients \[[@B8]\], while sensitivity was 63%, specificity was 96%, and the negative predictive value was 93%, for outpatients \[[@B12]\]. This discrepancy might be due to the characteristics of different samples or the diagnostic standards for dementia. The rate of dementia among the participants of the present study (62.7%) was higher than in the studies by Inouye and colleagues (14 and 16% for admitted and outpatients, respectively). Therefore, there are limitations when it comes to comparing the predictive values of screening tests, which would appear to be influenced by the prevalence. In elderly patients, the assessment of cognitive function is affected by psychological factors and by the circumstances under which the tests are conducted. In our measurement of the reliability of the T&C test, the test-retest and interobserver agreement rates were both remarkably high (95% for each, κ = 0.91). In a study by Inouye et al. \[[@B8]\] the test-retest agreement rate was 88%, and the interobserver agreement rate was 78% when the subjects were hospital patients \[[@B8]\]. However in the present study test-rest was conducted in the short interval, therefore it might to be influenced by carry-over effects and learning effects. In the present study, no association was observed between T&C test scores and educational status. This result may be explained by the fact that interpreting the hands of a clock and calculation using change are behaviors that are common to all people during daily life, irrespective of race and education. In addition, unlike the language-focused MMSE, the questions in the T&C test cannot be misinterpreted or misunderstood, and are effective for assessing calculation ability and attention. Clock test (similar to T&C test) is less likely to be confounded by educational attainment \[[@B20]\], and measures various facets of cognitive functioning at varying levels of difficulty \[[@B21]\]. This study has several important limitations. First, while the hospital patients underwent an evaluation of their medical history as well as neurological and physical examinations before a diagnosis of dementia was made, the elderly community sample were evaluated using only the K-MMSE as a reference. Second, the hospital patients would appear to be a rather unusual sample in that there was a high prevalence of psychiatric morbidity in the non-demented subjects. Third, dementia in the present study was not classified as vascular or Alzheimer\'s-type, nor did we consider the severity of symptoms. Finally, the T&C test has limitations in assessment of a wide range of deficits associated with dementia. However, because T&C test is simple, rapid, and easy to use, the T&C test may pose particular advantages in primary care and community settings where frequent assessment of cognitive functioning is required. The T&C test adds supplemental testing of important domains (e.g., calculation, conceptualization, visuospatial) to traditional measures such as MMSE. In addition, because the T&C test is less influenced by educational status, it may be particularly useful in populations with diverse educational and cultural backgrounds. Conclusions =========== We conclude that the T&C test is useful as a supplemental testing of important domains (e.g., calculation, conceptualization, visuospatial) to traditional measures such as the MMSE, because sensitivity of T&C test is not great and the association between the T&C test and MMSE is modest. However, because T&C test is simple, rapid, and easy to use, it can be applied conveniently to elderly subjects by non-specialist personnel who receive training. In addition, the T&C test is less influenced by educational status Funding ======= This work was supported by grants from Chonnam National University Hospital (CUHRI-U-200241). Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JAR conceived of the study, collected data and drafted the manuscript. EKC participated in its design, performed data collection, and reviewed the manuscript. MHS participated in data analysis and reviewed 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/1471-2458/4/52/prepub>	PubMed Central	2024-06-05T03:55:49.816762	2004-11-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533880/", "journal": "BMC Public Health. 2004 Nov 4; 4:52", "authors": [ { "first": "Jung-Ae", "last": "Rhee" }, { "first": "Eun-Kyung", "last": "Chung" }, { "first": "Min-Ho", "last": "Shin" } ] }
PMC533881	Background ========== The neurological consequences of diabetes mellitus in the Central Nervous System (CNS) are now receiving greater attention. Cognitive deficits, along with morphological and neurochemical alterations illustrate that the neurological complications of diabetes are not limited to peripheral neuropathies \[[@B1]\]. The central complications of hyperglycemia also include the potentiation of neuronal damage observed following hypoxic/ischemic events, as well as stroke \[[@B2]\]. Glucose utilization is decreased in the brain during diabetes \[[@B2]\], providing a potential mechanism for increased vulnerability to acute pathological events. Oxidative stress, leading to an increased production of reactive oxygen species (ROS), as well as lipidperoxidation, is increased in diabetes \[[@B3]\] and also by stress in euglycemic animals \[[@B4]\]. Similarly, oxidative damage in rat brain is increased by experimentally induced hyperglycemia \[[@B5]\]. Under experimental conditions, hyperglycemia dramatically increases neuronal alterations and glial cell damage caused by temporary ischaemia \[[@B6]\]. Several lines of evidence indicate that the modified oxidative state induced by chronic hyperglycemia \[[@B7]\] may contribute to nervous tissue damage: free radical species impair the central nervous system, attacking neurons and schwann cells \[[@B8]\] and the peripheral nerves \[[@B9]\]. Due to their high polyunsaturated lipid content, schwann cells and axons are particularly sensitive to oxygen free radical damage: lipidperoxidation may increase cell membrane rigidity and impair cell function. Increases in superoxide production are observed in the serum of Type 1 diabetic patients and was reduced with improved glycemic control \[[@B10]\]. Lipidperoxidation products are also increased in the brains of Type 1 diabetic rats \[[@B11]\] and Type 2 diabetic mice \[[@B8]\]. Diabetes and stress mediated increases in oxidative stress, as well as decreases in antioxidant activity, may make the brain more vulnerable to subsequent pathological events. Nowadays, the use of complementary/alternative medicine and especially the consumption of botanicals have been increasing rapidly worldwide, mostly because of the supposedly less frequent side effects when compared to modern western medicine \[[@B12]\]. Scoparia dulcisL (Scrophulariacae), a folk-medicinal plant known as sweet broomweed, has been used as a remedy for diabetes mellitus in India \[[@B13]\] and for hypertension in Taiwan \[[@B14],[@B15]\]. A number of active principles from Scoparia dulcisinclude scoparic acid A, scoparic acid B and scoparic acid D \[[@B16]\], scopadulcic acid A and B, scopadulciol \[[@B17]\] and Scopadulin \[[@B18]\] that have been identified as contributor to the observed medicinal effect of the plant. Among them, scopadulcic acid B (SDB) and scopadulciol (SDC) were found to be unique biomolecules with inhibitory effects on replication of herpes simplex virus type 1 (HSV-1) \[[@B16]\], gastric proton pump and bone resorption stimulated by parathyroid hormone (PTH) \[[@B18]\]. In addition, SDB showed antitumour promoting activities \[[@B17]\]. Because of their unique carbon skeleton and many sided biological activities, they were paid much attention as chemical synthetic targets by organic synthetic chemists. In a previous study, Nath (1943) studied the antidiabetic effect of Scoparia dulcisand obtained a glycoside, amellin from fresh plant and reported that it brought relief in other complications accompanied with diabetes (ie., pyorrhoea, retinopathy, joint pain, susceptibility to cold etc.) within a very short period \[[@B19]\]. Administration of Scoparia dulcisto STZ diabetic rats led to reduction in blood glucose \[[@B20]\]. In Recent studies on this plant, we have demonstrated a defective metabolism of lipid peroxides in tissues (liver, kidney and brain) of STZ diabetic rats \[[@B21]\] for 3 weeks treatment. Since increases in oxidative stress are associated with both long standing diabetes and stress, the present investigation was to assess the antioxidant efficacy of Scoparia dulcisin STZ diabetic rats after 6 weeks treatment and the effect produced by Scoparia dulciswas compared with Glibenclamide. Methods ======= Animals ------- Adult male albino Wistar rats (8 weeks), weighing 180--200 g bred in the Central Animal House, Rajah Muthiah Medical College, Annamalai University, were used. All animal experiments were approved by the ethical committee (Vide. No: 73, 2002), Annamalai University and were in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India. The animals were fed ad libitumwith normal laboratory pellet diet (Hindustan Lever Ltd., India) and water. Animals were maintained under a constant 12 h light and dark cycle and at an environmental temperature of 21--23°C. Drugs and chemicals ------------------- All the drugs and biochemicals used in this experiment were purchased from Sigma Chemical Company Inc., St Louis, Mo, USA. The chemicals were of analytical grade. Plant material -------------- Whole plants of Scoparia dulcisL. (40 -- 60 cm in height) were collected from Neyveli, Cuddalore District, Tamil Nadu, India in September 2001. The plant was identified and authenticated at the Herbarium of Botany Directorate in Annamalai University. A voucher specimen (No.3412) was deposited in the Botany Department of Annamalai University. Preparation of Scoparia dulcisplant extract (SPEt) ---------------------------------------------------- Five-hundred grams of fresh whole Scoparia dulcisplants were extracted with 1.5 l of water by the method of continuous hot extraction at 60°C for 6 h according to Jain (1968) and the filtrate was concentrated at 40°C to constant weight in a rotavapor apparatus (Buchi Labortechnik AG, Switzerland). The residue collected (yield 31 g) were thick paste, green in color and gumaceaous in nature and stored at -20°C, when needed the extract was dissolved in sterile water and used in the investigation \[[@B22]\]. Induction of experimental diabetes ---------------------------------- STZ, freshly prepared in 10 mmol/l citrate buffer, pH 4.5, was injected to experimental animals (30 rats) intraperitoneally at a dose of 45 mg/kg body weight \[[@B23]\]. 48 h after STZ administration, rats with moderate diabetes having glycosuria and hyperglycemia (i.e with blood glucose of 200 -- 300 mg/dl) were taken for the experiment. Experimental design ------------------- In the experiment, a total of 30 rats (18 diabetic surviving rats, 12 normal rats) were used. The rats were divided into 5 groups of 6 rats each. Group 1: Normal rats. Group 2: Normal rats given Scoparia dulcisplant extract (SPEt) (200 mg/kg body weight) in aqueous solution daily using an intragastric tube for 6 weeks. Group 3: Diabetic control rats. Group 4: Diabetic rats given SPEt (200 mg/kg body weight) \[[@B21]\] in aqueous solution daily using an intragastric tube for 6 weeks. Group 5: Diabetic rats given Glibenclamide (600 μg/kg body weight) in aqueous solution daily using an intragastric tube for 6 weeks \[[@B24]\]. All doses were started after 48 h STZ injection. No detectable irritation or restlessness was observed after each drug or vehicle administration. No noticeable adverse effect (i.e., respiratory distress, abnormal locomotion and catalepsy) was observed in any animals after the drug administration. Blood samples were drawn at weekly intervals till the end of study (ie. 6 weeks). At the end of 6^th^week, all the rats were killed by decapitation (Pentobarbitone sodium) anaesthesia (60 mg/kg). Blood was collected in two different tubes (i.e.,) one with anticoagulant -- potassium oxalate and sodium fluoride for plasma and another without anticoagulant for serum separation. Plasma and serum were separated by centrifugation. Whole Brain was immediately dissected out, washed in ice cold saline to remove the blood. The brains were weighed and 10% tissue homogenate was prepared with 0.025 M Tris -- HCl buffer, pH 7.5. After centrifugation at 200 rpm for 10 min, the clear supernatant was used to measure thiobarbituric acid reactive substances (TBARS), hydroperoxides and GPx activity. For the assay of SOD, CAT, GST and GSH, the brains were weighed and 10% homogenate was prepared with 0.2 M, phosphate buffer pH 8.0. After centrifugation, the clear supernatant was used for the assay of enzyme activities. Biochemical analysis -------------------- ### Estimation of blood glucose and plasma insulin Blood glucose was determined by the O-toluidine method \[[@B25]\]. Plasma insulin was assayed by ELISA, using Boeheringer-Mannheim Kit with a Boeheringer analyser ES300. ### Estimation of lipid peroxidation Lipid peroxidation in brain was estimated colorimetrically by thiobarbituric acid reactive substances TBARS and hydroperoxides by the method of Niehius and Samuelsson \[[@B26]\] and Jiang et al. \[[@B27]\], respectively. In brief, 0.1 ml of tissue homogenate (Tris-Hcl buffer, pH 7.5) was treated with 2 ml of (1:1:1 ratio) TBA-TCA-HCl reagent (thiobarbituric acid 0.37%, 0.25 N HCl and 15% TCA) and placed in water bath for 15 min, cooled. The absorbance of clear supernatant was measured against reference blank at 535 nm. 0.1 ml of tissue homogenate was treated with 0.9 ml of Fox reagent (88 mg butylated hydroxytoluene (BHT), 7.6 mg xylenol orange and 9.8 mg ammonium ion sulphate were added to 90 ml of methanol and 10 ml 250 mM sulphuric acid) and incubated at 37°C for 30 min. The color developed was read at 560 nm colorimetrically. Hydroperoxides was expressed as mM/100g tissue. ### Assay of catalase (CAT) and superoxide dismutase (SOD) CAT was assayed colorimetrically at 620 nm and expressed as μmoles of H~2~O~2~consumed/min/mg protein as described by Sinha \[[@B28]\]. The reaction mixture (1.5 ml, vol) contained 1.0 ml of 0.01 M pH 7.0 phosphate buffer, 0.1 ml of tissue homogenate (supernatant) and 0.4 ml of 2 M H~2~O~2~. The reaction was stopped by the addition of 2.0 ml of dichromate-acetic acid reagent (5% potassium dichromate and glacial acetic acid were mixed in 1:3 ratio). SOD was assayed utilizing the technique of Kakkar et al. \[[@B29]\] based on inhibition of the formation of nicotinamide adenine dinucleotide, phenazine methosulfate and amino blue tetrazolium formazan. A single unit of enzyme was expressed as 50% inhibition of NBT (Nitroblue tetrazolium) reduction/min/mg protein. ### Determination of glutathione peroxidase (GPx) and reduced glutathione (GSH) GPx activity was measured by the method described by Rotruck et al. \[[@B30]\]. Briefly, reaction mixture contained 0.2 ml of 0.4 M Tris-HCl buffer pH 7.0, 0.1 ml of 10 mM sodium azide, 0.2 ml of tissue homogenate (homogenised in 0.4 M, Tris-HCl buffer, pH 7.0), 0.2 ml glutathione, 0.1 ml of 0.2 mM hydrogen peroxide. The contents were incubated at 37°C for 10 min. The reaction was arrested by 0.4 ml of 10% TCA, and centrifuged. Supernatant was assayed for glutathione content by using Ellmans reagent (19.8 mg of 5,5\'-dithiobisnitro benzoic acid (DTNB) in 100 ml of 0.1% sodium nitrate). GSH was determined by the method of Ellman \[[@B31]\]. 1.0 ml of supernatant was treated with 0.5 ml of Ellmans reagent and 3.0 ml of phosphate buffer (0.2 M, pH 8.0). The absorbance was read at 412 nm. Glutathione peroxidase activity was expressed as μg of GSH consumed/min/mg protein and reduced glutathione as mg/100g of tissue. ### Determination of glutathione-S-transferase (GST) The GST activity was determined spectrophotometrically by the method of Habig et al. \[[@B32]\]. The reaction mixture (3 ml) contained 1.0 ml of 0.3 mM phosphate buffer (pH 6.5), 0.1 ml of 30 mM 1-chloro-2, 4-dinitrobenzene (CDNB) and 1.7 ml of double distilled water. After pre-incubating the reaction mixture at 37°C for 5 min, the reaction was started by the addition of 0.1 ml of tissue homogenate and 0.1 ml of glutathione as substrate. The absorbance was followed for 5 min at 340 nm. Reaction mixture without the enzyme was used as blank. The activity of GST is expressed as μmoles of GSH-CDNB conjugate formed/min/mg protein using an extinction coefficient of 9.6 mM^-1^cm^-1^. ### Estimation of protein Protein was determined by the method of Lowry et al. \[[@B33]\] using Bovine Serum Albumin (BSA) as standard, at 660 nm. Statistical analysis -------------------- All data were expressed as mean ± SD of number of experiments (n = 6). The statistical significance was evaluated by one-way analysis of variance (ANOVA) using SPSS version 7.5 (SPSS, Cary, NC, USA) and the individual comparison were obtained by Duncan\'s Multiple Range Test (DMRT). A value of p \< 0.05 was considered to indicate a significant difference between groups \[[@B34]\]. Results ======= Table [1](#T1){ref-type="table"} shows the level of blood glucose and plasma insulin in normal and experimental groups. The level of blood glucose was significantly increased whereas the level of plasma insulin was significantly decreased in diabetic control rats. Oral administration of SPEt and glibenclamide to diabetic rats significantly reversed all these changes to near normal levels. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of Scoparia dulcison blood glucose and plasma insulin in normal and experimental rats ::: Groups Fasting blood glucose (mg/dl) Plasma insulin (μu/ml) -------------------------------------- ----------------------------------- ---------------------------- Normal 84 ± 4^a^ 12 ± 1^a^ Normal + SPEt (200 mg/kg) 77 ± 3^a^ 15 ± 1^b^ Diabetic control 270 ± 15^b^ 4 ± 0.3^c^ Diabetic + SPEt (200 mg/kg) 98 ± 3^c^ 11 ± 4^d^ Diabetic + Glibenclamide (600 μg/kg) 114 ± 9^d^ 9 ± 0.5^e^ Values are given as mean ± SD from 6 rats in each group. Values not sharing a common superscript letter differ significantly at p \< 0.05 (DMRT); Duncan Procedure; Ranges for the level: 2.95, 3.09, 3.20, 3.22. ::: Table [2](#T2){ref-type="table"} illustrates markers of lipidperoxidatioon namely, TBARS and hydroperoxides from brain of normal and experimental rats. The levels of TBARS and hydroperoxides were significantly increased in diabetic control rats. Administration of SPEt to diabetic rats significantly decreased the levels of lipidperoxidative markers. Treatment of normal rats with SPEt did not show significant changes in lipidperoxidation. The effect produced by SPEt was significant than glibenclamide. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Change in the levels of brain TBARS and hydroperoxides in normal and experimental rats ::: Groups TBARS (mM/100g tissue) Hydroperoxides (mM/100g tissue) -------------------------------------- ---------------------------- ------------------------------------- Normal 1.10 ± 0.08^a^ 113.20 ± 4.10^a^ Normal + SPEt (200 mg/kg) 0.90 ± 0.05^a^ 108.70 ± 2.20^b^ Diabetic control 1.85 ± 0.07^b^ 130.90 ± 1.50^c^ Diabetic + SPEt (200 mg/kg) 1.18 ± 0.06^c^ 117.22 ± 3.26^d^ Diabetic + Glibenclamide (600 μg/kg) 1.32 ± 0.06^d^ 120.40 ± 4.05^e^ Values are given as mean ± SD for 6 rats in each group. Values not sharing a common superscript letter differ significantly at p \< 0.05 (DMRT). Duncan procedure, Range for the level 2.95, 3.09, 3.20, 3.22. ::: For studying the effect of SPEt on antioxidant status, the activities of enzymic antioxidants SOD, CAT, GPx, GST and non-enzymic antioxidant GSH were measured (Table [3](#T3){ref-type="table"} and [4](#T4){ref-type="table"}). The activities of enzymic and the levels of non-enzymic antioxidant were significantly decreased in diabetic control rats. They presented significant increases in diabetic rats treated SPEt. Administration of SPEt to normal rats increased the antioxidants levels with no significant differences. The effect produced by SPEt was comparable with that of glibenclamide. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Changes in activities of catalase and superoxide dismutase in brain of normal and experimental rats ::: Groups Catalase (Units^A^/mg protein) Superoxide dismutase (Units^B^/mg protein) -------------------------------------- ------------------------------------ ------------------------------------------------ Normal 3.12 ± 0.29^a^ 7.75 ± 0.38^a^ Normal + SPEt (200 mg/kg) 4.00 ± 0.20^b^ 7.05 ± 0.28^a^ Diabetic control 0.86 ± 0.05^c^ 5.17 ± 0.30^b^ Diabetic + SPEt (200 mg/kg) 2.75 ± 0.20^d^ 7.32 ± 0.46^c^ Diabetic + Glibenclamide (600 μg/kg) 1.98 ± 0.11^e^ 6.32 ± 0.30^d^ Values are given as mean ± SD for 6 rats in each group. Values not sharing a common superscript letter differ significantly at p \< 0.05 (DMRT). Duncan procedure, Range for the level 2.95, 3.09, 3.20, 3.22. A -- μmole of H~2~O~2~consumed/minute. B -- One unit of activity was taken as the enzyme reaction, which gave 50% inhibition of NBT reduction in one minute. ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Changes in activities of glutathione peroxidase, glutathione-S-transferase and the levels of reduced glutathione in brain of normal and experimental rats ::: Groups Glutathione peroxidase (Units^A^/mg protein) Glutathione-S-transferase (Units^B^/mg protein) Reduced glutathione (mg/100g tissue) -------------------------------------- -------------------------------------------------- ----------------------------------------------------- ------------------------------------------ Normal 3.4 1± 0.20^a^ 5.62 ± 0.28^a^ 35.19 ± 2.21^a^ Normal + SPEt (200 mg/kg) 3.80 ± 0.18^a^ 5.90 ± 0.28^a^ 37.12 ± 2.14^a^ Diabetic control 1.01 ± 0.05^b^ 0.81 ± 0.02^b^ 15.20 ± 1.44^b^ Diabetic + SPEt (200 mg/kg) 2.62 ± 0.15^c^ 2.10 ± 0.13^c^ 26.02 ± 2.01^c^ Diabetic + Glibenclamide (600 μg/kg) 1.97 ± 0.12^d^ 2.04 ± 0.13^c^ 25.50 ± 2.10^c^ Values are given as mean ± SD for 6 rats in each group. Values not sharing a common superscript letter differ significantly at p \< 0.05 (DMRT). Duncan procedure, Range for the level 2.95, 3.09, 3.20, 3.22. A -- μg of GSH consumed/min. B -- μmoles of CDNB -- GSH conjugate formed/min. ::: Discussion ========== This work is one of a series of studies showing that chronic hyperglycemia causes an imbalance in the oxidative status of the nervous tissue and that the resulting free radicals damage the brain through a peroxidative mechanism. The STZ diabetic rat serves as an excellent model to study the molecular, cellular and morphological changes in brain induced by stress during diabetes \[[@B7]\]. Under normal conditions, the generation of free radicals or of active species in the brain, as in other tissues, is maintained at extremely low levels \[[@B4]\]. Diabetes also contributes to cerebrovascular complications, reductions in cerebral blood flow, disruption of the blood brain barrier and cerebral edema \[[@B5]\]. All of these neurochemical and neurophysiological changes ultimately contribute to the long-term complications associated with diabetes, including morphological abnormalities, cognitive impairments and increased vulnerability to pathophysiological event \[[@B6]\]. In the present study, treatment with aqueous extract of Scoparia dulcisshowed significant antihyperglycemic activity. The antihyperglycemic activity of this plant may be, at least in part, through release of insulin from the pancreas in view of the measured increase in the plasma insulin concentrations. Earlier studies in this lab have demonstrated a defective metabolism of lipid peroxides in other tissues of diabetic animal \[[@B35],[@B36]\]. TBARS and hydroperoxides (lipid peroxidative markers) showed high lipidperoxidation. This may be because; the brain contains relatively high concentration of easily peroxidizable fatty acids \[[@B37]\]. In addition, it is known that certain regions of the brain are highly enriched in iron, a metal that, in its free form, is catalytically involved in production of damaging oxygen free radical species \[[@B38]\]. It has been suggested that free radical species responsible for STZ toxicity is the hydroxyl radical, formed via the metal catalyzed Haber-weiss reaction or Fenton reaction. In this process, the ferric iron is reduced by superoxide, with subsequent oxidation of ferrous iron by H~2~O~2~forming hydroxyl radical: Fe^3+^+ O~2~^•-^→ Fe^2+^+ O~2~ Fe^2+^+ H~2~O~2~→ Fe^3+^+ OH\* + OH^-^ The destruction of superoxide radical or H~2~O~2~by SOD or CAT would ameliorate STZ toxicity, as would substances able to scavenge the hydroxyl radical \[[@B39],[@B40]\]. Vulnerability of brain to oxidative stress induced by oxygen free radicals seems to be due to the fact that, on one hand, the brain utilizes about one fifth of the total oxygen demand of the body and on the other, that it is not particularly enriched, when compared with other organs, in any of the antioxidant enzymes. Relatively low levels of these enzymes may be responsible in part for the vulnerability of this tissue \[[@B41]\]. The altered balance of the antioxidant enzymes caused by decrease in CAT, SOD, GPx, GST and GSH activities may be responsible for the inadequacy of the antioxidant defenses in combating ROS mediated damage. The decreased activities of CAT and SOD may be a response to increased production of H~2~O~2~and O~2~by the autoxidation of glucose and non-enzymatic glycation \[[@B5]\]. These enzymes have been suggested as playing an important role in maintaining physiological levels of oxygen and hydrogen peroxide by hastening the dismutation of oxygen radicals and eliminating organic peroxides and hydroperoxides generated from inadvertent exposure to STZ \[[@B42]\]. Treatment with SPEt increased the activity of enzymes and may help to control free radicals, as Scoparia dulcishas been reported to be rich in alkaloids and terpenoids \[[@B16]-[@B18],[@B43],[@B44]\], well-known antioxidants, which scavenge the free radicals generated during diabetes. The increase in SOD activity may protect CAT and GPx against inactivation by O~2~^•-^anions as these anions have been shown to inactivate CAT and GPx \[[@B45]\]. Under in vivo conditions, GSH acts as an antioxidant and its decrease was reported in diabetes mellitus \[[@B46]\]. We have observed significant decrease in GSH levels in brain during diabetes. The decrease in GSH levels represents increased utilization due to oxidative stress \[[@B47]\]. The depletion of GSH content may also lower the GST activity \[[@B48]\]. Depression in GPx activity was also observed brain of diabetic rats. GPx has been shown to be an important adaptive response to condition of increased peroxidative stress \[[@B46]\]. The increased GSH content in the brain of the rats treated with SPEt and glibenclamide may be a factor responsible for inhibition of lipidperoxidation. The elevated level of GSH protects cellular proteins against oxidation through glutathione redox cycle and also directly detoxifies reactive oxygen species generated from exposure to STZ \[[@B48]\]. The significant increase in GSH content and GSH dependent enzymes GPx and GST in diabetic rats treated with SPEt indicates an adaptive mechanism in response to oxidative stress. Significantly lower levels of lipid peroxides in brain of SPEt treated diabetic rats and increased activities of enzymic and non-enzymic antioxidants in brain suggest that the extract reduce oxidative stress by quenching free radicals. Terpenoids and alkaloids were reported to have free radical scavenging activity and antioxidant capacity in diabetes \[[@B49],[@B50]\]. SPEt was reported to be rich in an alkaloid-6-methoxybenzoxazolinone \[[@B51]\] and terpenoids such as scoparic acids A, B, C and scopadulcic acids A and B \[[@B16]-[@B18]\], which may be responsible for scavenging free radicals liberated by STZ and thus enhance both enzymic and non-enzymic antioxidants in diabetic rats treated with SPEt. Any compound, natural or synthetic with antioxidant properties that might contribute towards the partial or total alleviation of this damage may have a significant role in the treatment of diabetes mellitus. The antioxidant responsiveness mediated by Scoparia dulcismay be anticipated to have biological significance in eliminating reactive free radicals that may otherwise affect the normal cell functioning. The disfunctioning of these antioxidant enzymes has been implicated in several disorders including rheumatoid arthritis, reperfusion injury, cardiovascular diseases, immune injury as well as diabetes mellitus \[[@B52]\]. It may be concluded that in diabetes, brain tissue was more vulnerable to oxidative stress and showed increased lipidperoxidation. The above observation shows that the aqueous extract of Scoparia dulcisplant possesses antioxidant activity, which could exert a beneficial action against pathological alterations caused by the presence of free radicals in STZ diabetes. Conclusions =========== The brain exhibits numerous morphological and functional alterations during diabetes. Oxidative stress, a factor implicated in the pathogenesis of diabetic complications may contribute towards some of these alterations. Treatment of diabetic rats with Scoparia dulcisplant extract significantly decreased the lipidperoxidation and significantly increased the antioxidant status. Since the study of induction of the antioxidant enzymes is considered to be a reliable marker for evaluating the antioxidant efficacy of the medicinal plant, these findings are suggestions of possible antioxidant role played by Scoparia dulcisplant extract in addition to its antidiabetic effect. Competing interests =================== The author(s) declare that they have no competing interests Authors\' contributions ======================= LP -- supervised the design and co-ordination of the study ML -- Practically conducted the design of the study and drafted the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6882/4/16/prepub>	PubMed Central	2024-06-05T03:55:49.818933	2004-11-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533881/", "journal": "BMC Complement Altern Med. 2004 Nov 2; 4:16", "authors": [ { "first": "Leelavinothan", "last": "Pari" }, { "first": "Muniappan", "last": "Latha" } ] }
PMC533882	Background ========== The assessment of problem-solving skills, and specifically diagnostic skills, was once reserved for examination formats such as free-response questions, patient management problems (PMPs) or oral examinations. These evaluation methods, however, are all resource-intensive, thus making it difficult to provide the representative sampling of problems necessary to circumvent the problem of case specificity, which predicts that success in solving one clinical presentation does not predict success in another \[[@B1]\]. As a consequence of case specificity, reliability and content validity of an examination are dependent on a broad sampling of problems. Such extensive sampling is more easily done with pencil-and-paper type of tests. This study will examine two pencil-and-paper formats specifically in regards to their relative problem-solving testing abilities. Previous literature has demonstrated that altering item stems tends to determine clinical challenge, while psychometric properties such as discrimination and difficulty tend to be affected by the number of answer options \[[@B2]\], hereby referred to as \'number of alternatives\'. The central focus of this paper surrounds whether altering the number of alternatives within a pencil-and-paper format alters diagnostic higher order thinking and/or format psychometric properties. Two formats were studied, both with a stem consisting of a long vignette with distracters, but with different number of alternatives. The format presenting five options to the examinee will henceforth be referred to as the \"multiple-choice question\" or MCQ format, while the second format, presenting greater than ten options to the examinee, will be referred to as \"extended-matching\" or EMQ format. The first examination format studied is the five-option MCQ (see Appendix A for example). Although MCQs have always been considered an efficient and reliable testing tool, they have not always been perceived as ideal for the evaluation of higher-order thinking skills such as problem solving. Prevailing perceptions that MCQs assess lower levels of knowledge such as recall of isolated facts, and/or encourage trivialization, do exist in the medical education community \[[@B3]\]. To the extent that some clinicians question whether MCQs can test problem-solving skills, suggests that this format may have low validity \[[@B4]\]. However, as discussed by Case and Swanson \[[@B5]\] well constructed MCQs could challenge students to problem solve. Maguire et al also recognized that MCQs could yield valid information of clinical reasoning skills, providing that stems and alternatives are well constructed \[[@B6]\]. Evidence does exist that MCQs have predictive value for more recognized problem-solving tasks \[[@B7]\] and can elicit higher order problem solving such as forward reasoning \[[@B8]\]. The second examination format the EMQ format, initially designed in response to some of the criticisms of the MCQs. EMQs (see Appendix A for example) were introduced in the 1990s in both the NBME and USMLE, amongst others. Case and Swanson \[[@B5]\] have been instrumental in the development of these questions, which are defined as any matching format with more than the five alternatives traditionally used by MCQs. From its conception, the question preparation of the EMQs has been very careful in designing stems that test higher cognitive levels such as problem solving. The first study that examined the psychometric features of Extended-matching \[[@B5]\] questions showed that Extended-matching items were more difficult, more discriminating, had higher reliability and needed significantly less testing time to achieve reproducible scores than traditional MCQs. Other studies have shown that EMQs, by increasing the number of alternatives used, increased mean item difficulty as well as, perhaps by reducing guessing, provided improvement in item discrimination over the five-option MCQ \[[@B9]\]. By increasing item discrimination, EMQs offer comparable levels of reproducibility with 30% fewer items than the MCQ with five options \[[@B9]\]. Reliability coefficients were also markedly higher with Extended-matching \[[@B5]\]. Positive psychometric outcomes have been found in other studies using the format \[[@B10]-[@B14]\]. These studies have focused on psychometrics, whereas potential benefits, and possible reasons for such benefits, of the EMQ format over standard MCQs in eliciting higher order problem solving remain unclear. No study has formally used think-aloud protocols to assess whether a well-written MCQ differs from EMQs in challenging examinees to problem solve. There is little doubt that poorly written MCQs can encourage students to learn isolated facts by rote. In fact, all available evaluation methods potentially yield information on clinical reasoning if the content is appropriate, suggesting that content is more important than question type \[[@B15]\]. The two examination formats will be tested for their ability to elicit the three different diagnostic reasoning strategies generally available to learners: hypothetico-deductive reasoning, pattern recognition, and scheme-inductive reasoning. Deductive reasoning (hypothetico-deductive) \[[@B16]\] is a \"to-and-fro\" strategy of problem solving, also termed \"backward reasoning\". The method is generally used by novices or experienced diagnosticians to include or exclude a single diagnosis, when faced with a particularly complex problem, or as a fallback strategy when faced with clinical problems that are outside their domains of expertise. Pattern recognition has been identified by other research as a very successful approach used by experts to solve clinical problems \[[@B17]-[@B19]\]. Before becoming more expert in problem solving, learners progress through several transitional stages characterized by different knowledge structures: elaborated causal networks, abridged networks, illness scripts, and instance scripts \[[@B18]\]. Extensive experience eventually leads to acquisition of a repertoire of problems common to the domain of expertise termed \"illness scripts\". This repertoire permits problem resolution by recognition of new problems as ones that are similar or identical to old ones already solved, and the solutions are recalled. The third strategy is scheme-inductive reasoning. \"Schemes\" are defined as a mental categorization of knowledge that includes a particular organized way of understanding and responding to a complex situation. They are drawn on paper like \"inductive trees\" or \"road maps\" to recreate the major divisions (or chunks) used by expert clinicians for both storage of knowledge in memory and its retrieval for solving problems \[[@B19],[@B20]\] (see Figure [1](#F1){ref-type="fig"} for an example of the scheme for \"dysphagia\"). Decisions are explicitly at the forks in the road or branching of the tree. The organizational structure, or \"scheme\", proceeds from alternative causes in a forward direction, through crucial \"tests\", to exclusion of some alternative causes and adoption of what is left. These tests may be based on an evaluation of symptoms, signs, or results of investigations, singly or in any combination. Scheme-inductive reasoning is a strategy used by experts when pattern recognition is not possible \[[@B21]\]. This type of problem solving represents the \"climbing of a conditional inductive tree\" \[[@B22]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Example of the scheme for \"dysphagia\". ::: ![](1472-6920-4-23-1) ::: By directly comparing the problem-solving strategies elicited by the two pencil-and-paper formats, using the think-aloud method previously described, two major questions will be addressed. The first question is whether pencil-and-paper formats such as EMQ and MCQ can in fact assess problem-solving skills. The examination formats\' capacity to evoke more \'expert\' methods of problem solving, such as scheme-inductive reasoning or pattern recognition, will be taken as evidence of their ability to assess problem-solving skills. The second question relates to the impact of the alternatives number on psychometric properties and diagnostic higher order thinking, considering that a shift to hypothetico-deductive reasoning could conceivably occur with the shorter alternatives lists of the MCQ format. A corollary to these questions is whether in testing problem solving, it is the construction of question stems that is important, as opposed to the number of alternatives or examination format. Methods ======= Examination construction ------------------------ An examination for four clinical presentations, each representing a different domain in gastrointestinal medicine, was constructed: dysphagia, chronic diarrhea, nausea and vomiting, and elevated liver enzymes. The examination consisted of eight pencil-and-paper questions, with two questions, one of the MCQ type and another of the EMQ type, created for each of the four clinical presentations. While completing the questions, the examinees were permitted to write notes. The two question stems written for each of the clinical presentations were long vignettes created with a problem-solving task in mind. Furthermore, the stems within each clinical presentation (see Appendix A for the two stems for clinical presentation \'diarrhea\') were designed to be as similar as possible in length, difficulty, and the presence of distracters. The stems differed only in the presence of a few key different pieces of information that led to a different diagnosis. The stems were then randomly assigned to one of the examination formats described above, MCQ or EMQ. The alternatives list included the correct diagnosis, and two plausible \'competing alternatives\' to the correct answer. Subjects -------- The examination was administered to twenty experts in Gastroenterology in two centers, Calgary (15) and Ottawa (5), as well as twenty non-experts, final-year medical students at the University of Calgary. Candidates were considered experts if they were specialists who spent more than 80% of their clinical time in the practice of Gastroenterology. Data collection --------------- The subjects were first asked to answer the eight questions. The examinees were not given a time constraint to complete the examination, though most completed it in 45 minutes. After the completion of the eight questions, the subjects, with the examination paper in hand and any written notes made during the examination, were asked to explain how they arrived at each diagnosis. A panel of two judges (experts in the Gastroenterology presentations being tested and in the recognition of the diagnostic reasoning process) interviewed the examinees. With as little prompting as possible, the examinees were asked to think-aloud \[[@B23]\] and describe how each diagnosis was derived. Based on the examinees\' verbal discourse for that question, the two judges assigned a discrete \'Process Score\' of 1, 2, or 3, depending on the predominant diagnostic process used. Once the score was assigned, the examinee was encouraged to proceed to the next question, until a diagnostic process score had been assigned for all eight questions. A \'Process Score\' of 3 was assigned if pattern recognition was used. Determination that \"pattern recognition\" was used occurred when the subject directly reached a single diagnosis with only perfunctory attention to the alternatives. A \'Process Score\' of 2 was assigned if a well-structured and accurate scheme was predominantly used to guide the inductive inquiry. Determination that a scheme-directed diagnostic reasoning strategy was used occurred by analysis of the verbal discourse using modified propositional analysis \[[@B24]\]. A proposition is defined as \"the smallest unit of meaning that underlies the surface structure of a text\" \[[@B25]\]. This analysis consisted of searching the examinees\' discourse for key predetermined propositions that linked categories and thus provided evidence for chunking (i.e. scheme use). These key chunking propositions were determined by the authors based on information from texts, databases, consultation with experts not participating in the study, and personal experience. A recall method was utilized and felt appropriate given that of major interest to the present study was global description of representations in memory, as opposed to exact numbers of recall or specific inferences made from recalled texts \[[@B26]\]. The key propositions are shown in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Propositions demonstrating evidence of chunking. ::: -------------------------------------------------------------------------------- Clinical presentation Key chunking propositions ------------------------ ------------------------------------------------------- Dysphagia \- Oropharyngeal vs. esophageal\ - Mechanical vs. motility Elevated liver enzymes \- Hepatocellular vs. cholestatic\ - Intra vs. extrahepatic cholestasis Nausea and vomiting \- GI vs. non-GI causes\ - GI vs. metabolic vs. CNS vs. drugs Diarrhea \- Small bowel vs. large bowel\ - Steatorrhea (malabsorption) vs. non-steatorrhea\ - Osmotic vs. secretory vs. inflammatory vs. motility -------------------------------------------------------------------------------- ::: A \'Process Score\' of 1 was assigned if the examinee relied on hypothetico-deductive reasoning exclusively or predominantly. It was determined that hypothetico-deductive reasoning was the diagnostic strategy utilized when the subjects analyzed one by one each alternative diagnosis presented with the clinical vignettes prior to selecting the most likely diagnosis. The interviews were audio taped or videotaped for later review. Such reviews were required infrequently, but were found necessary when the two judges identified different reasoning strategies. The most frequent cause for differences in identification of diagnostic reasoning strategy was examinees\' use of more than one strategy. For example, the candidate might initiate the diagnostic reasoning process using scheme-inductive inquiry, but resort to deductive reasoning immediately after. Disagreement between the two judges was resolved by discussion until concurrence about the diagnostic reasoning strategy was reached. The final assigned mark reflected the predominant diagnostic reasoning strategy utilized. A dichotomous score (0 for incorrect answer, 1 for correct answer) was assigned in order to compute the format psychometric properties. Data analysis ------------- Reliabilityof the process scores and formats was estimated using Cronbach\'s alpha coefficient. Item statistics were generated for each item including a discrimination index. Inter-rater reliability of diagnostic reasoning scores was estimated by a Pearson correlation coefficient. ### Effects of expertise, examination format, and clinical presentations on diagnostic reasoning or \'process score\' A logistic regression analysis was used to determine which of the three independent variables being studied (examination format, expertise, and clinical presentation) had an impact on diagnostic reasoning or \'process score\' (the dependent variable). Specifically, the analysis will model the odds of using an \'expert\' method of problem-solving, that is scheme-inductive or pattern recognition (in other words, odds of not using hypothetico-deductive reasoning) in relation to the three independent variables of format, expertise and clinical presentation. An expertise effect, which would be expected, will lend evidence of construct validity to the \'process score\'. Analysis was carried out using the Stata software system \[[@B27]\]. Results ======= A. Reliability of \'Process Score\' ----------------------------------- The two judges found it easy to agree on the broad type of strategy used by the subjects (hypothetico-deductive, scheme-directed, and pattern recognition). However, there was less agreement when the same subject used more than one diagnostic strategy. The initial diagnostic reasoning scores resulted in an agreement between the two judges of 0.84. B. Reliability and discrimination of examination formats -------------------------------------------------------- Both formats demonstrated quite acceptable reliability and discrimination, as per Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Cronbach alpha reliabilities and discrimination indices based on question format over all subjects. ::: Question format Alpha coefficient Average disc. index ------------------- ------------------- --------------------- Multiple-choice 0.76 0.63 Extended-matching 0.66 0.58 ::: C. Relationship of examination format to cognitive process ---------------------------------------------------------- The results of the logistic regression analysis are as follows, in Table [3](#T3){ref-type="table"}. There was no difference in the odds of using \'expert\' methods of problem solving (scheme-inductive or pattern recognition) across the two examination formats (MCQ or EMQ). As would be expected, experts had approximately threefold higher odds of using either of these two problem-solving methods as opposed to novices (p 0.00). There was a negative odd of using scheme-inductive and pattern recognition (-1.55) within the diarrhea and nausea/vomiting clinical presentations (i.e. more likely to use hypothetico-deductive) as opposed to the elevated liver enzymes presentation. Explanation for this lies in the fact that the diarrhea questions were the most complex for both novices and experts (in which case experts and non-experts resorted to hypothetico-deductive reasoning, as has been described in the literature \[[@B28]\]), while the nausea and vomiting questions were complex for the experts especially, given that the experts were gastroenterologists, but the diagnoses for this clinical presentation were \'metabolic\' causes of nausea and vomiting. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Logistic regression of the odds of using an \'expert\' process (either pattern recognition or scheme-inductive) ::: *Independent variable* *Baseline Level* *OR (95% CI)* *p value* ---------------------------- --------------------- ---------------------- ---------------------- --------------- Examination format Extended-matching Multiple-choice -0.59 (-1.73, 0.56) 0.31 Expertise Expert group Non-expert group 2.69 (1.64, 3.75) 0.00 Clinical presentation Nausea and vomiting Liver enzymes -1.55 (-2.67, -0.43) 0.01 Diarrhea Liver enzymes -1.55 (-2.67, -0.43) 0.01 Dysphagia Liver enzymes 1.11 (-1.21, 1.21) 1.00 ::: D. Ability of the two formats to evoke higher-order thinking ------------------------------------------------------------ Table [4](#T4){ref-type="table"} and Table [5](#T5){ref-type="table"} are frequency tables for the Expert and Non-expert Process Scores, across the two examination formats and four Clinical Presentations. They demonstrate that experts utilized either scheme-inductive or pattern recognition more than 90% of the time for both pencil-and-paper examination formats, while non-experts utilized these two reasoning strategies less often than experts, but still greater than 50% of the time for both formats. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Frequency table for the expert (n = 20) process scores, across two formats and four clinical presentations ::: Question format Process score Liver enzymes Nausea and vomiting Diarrhea Dysphagia Total ------------------- -------------------------- --------------- --------------------- ---------- ----------- ------- Multiple-choice 1: Hypothetico-deductive 0 3 2 0 5 2: Scheme 9 4 10 13 36 3: Pattern recognition 11 13 8 7 39 Total 20 20 20 20 80 Extended-matching 1: Hypothetico-deductive 1 2 1 1 5 2: Scheme-inductive 8 3 5 14 30 3: Pattern recognition 11 15 14 5 45 Total 20 20 20 20 80 ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Frequency table for the non-expert (n = 20) process scores, across two formats and four clinical presentations ::: Question Format Process score Liver enzymes Nausea and vomiting Diarrhea Dysphagia Total ------------------- -------------------------- --------------- --------------------- ---------- ----------- ------- Multiple-choice 1: Hypothetico-deductive 6 12 13 6 37 2: Scheme-inductive 10 2 6 10 28 3: Pattern recognition 4 6 1 4 15 Total 20 20 20 20 80 Extended-matching 1: Hypothetico-deductive 8 4 14 6 32 2: Scheme inductive 8 1 4 13 26 3: Pattern recognition 4 15 2 1 22 Total 20 20 20 20 80 ::: Discussion ========== The present study had two major goals. The first was to determine whether the two pencil-and-paper formats studies, the MCQ and EMQ, could in fact assess problem-solving skills. In Table [4](#T4){ref-type="table"} and Table [5](#T5){ref-type="table"}, the two pencil-and-paper formats demonstrated high preponderance of scheme-inductive and pattern recognition utilization, in both experts and non-experts, thus suggesting that these question types can potentially elicit higher order clinical reasoning strategies. Another aim was to assess, by using think-aloud protocols, the impact of the alternatives number on psychometric properties and reasoning strategies employed. The logistic regression analysis shown in Table [3](#T3){ref-type="table"} demonstrates that the number of alternatives, in the form of the two examination formats used (MCQ and EMQ), did not exert an independent effect on reasoning strategy utilized. Table [2](#T2){ref-type="table"} demonstrates that both formats had good and comparable psychometric properties. The first research question of this paper was to investigate whether the examination formats used in this study, the standard five-option Multiple-choice and Extended-matching questions, were capable of testing problem-solving abilities. The observation from the data is that the two formats can potentially evoke more \'expert\' methods of diagnostic reasoning processes such as scheme utilization or pattern recognition. Table [4](#T4){ref-type="table"} and Table [5](#T5){ref-type="table"}, demonstrate preponderance in both experts (greater than 90%) and non-experts (greater than 50%) of scheme-inductive and pattern recognition utilization in answering the questions. It can be concluded that by evoking these \'expert\' methods of clinical reasoning, the two pencil-and-paper formats used in this study have the capability to assess diagnostic higher order thinking, assuming the question stems are constructed with a problem-solving task in mind, as was done in this study. In regards to the second main research question, the two question formats, with different number of alternatives, did not exert an independent effect on diagnostic reasoning strategy, as shown in the logistic regression analysis (Table [3](#T3){ref-type="table"}). Shortening the number of alternatives in the MCQ format to five did not lead to an examinee \'shift\' of relying on hypothetico-deductive reasoning. Explanation of this result may be found in the view raised by several authors \[[@B6],[@B15]\] that it is not the examination format, or the number of alternatives, that dictates the cognitive level of the testing, but rather the specific construction of the question stems. We have demonstrated that a well-constructed Multiple-choice question, designed specifically to target problem solving, can achieve the purpose of testing higher order cognitive reasoning. Critics of the Multiple-choice format, who believe that it only tests recall of isolated facts, need to consider altering the construction of the stems rather than the format. That no difference exists between MCQs\' and EMQs\' relative ability to test for problem-solving may lie in the notion that a person\'s problem-solving strategy is a trait attribute and not dependent on the item format or number of alternatives. In other words, a given diagnostician will use a given strategy, such as scheme-inductive, on all questions which ask for a problem-solving task (i.e. diagnosis), regardless of the format. A well-created problem-solving question stem will challenge the examinee to use, in many cases, \'expert\' (scheme-inductive, pattern recognition) diagnostic reasoning strategies to arrive at an answer, prior to looking at the alternatives. This minimizes the impact of alternatives number on the diagnostic reasoning strategy utilized, and specifically minimizes any shift to hypothetico-deductive reasoning that could have been feared occurring with the smaller number of alternatives presented in the MCQ format. The key is to create the stem with a problem-solving task in mind, and not looking for rote memorization of facts. While the findings presented do support ongoing use of the MCQ format, there is no denying that the EMQ format has demonstrated superior psychometric properties over the MCQ format in a number of studies mentioned earlier in this paper. Furthermore, in our own study, several non-expert and some expert examinees did comment that Extended-matching questions made it more difficult to go through the list of alternatives prior to answering the question. For examinees relying on hypothetico-deductive reasoning, the Extended-matching format, because of the inherent difficulty of reading through an extended alternatives list, may, at least subjectively, provide a better challenge than the Multiple-choice format. A significant limitation to the study is the manner in which the cognitive problem-solving process selected by the subjects was ascertained. Thinking aloud was used. After the completion of the examination, subjects were asked to verbally report their thinking method to two judges. The two judges independently noted the cognitive problem solving process the subjects had used in arriving at a diagnosis. Although agreement between the two judges on the process selected was identical in more than 85% of the think-aloud interviews, in the remaining 15% there was disagreement. The cognitive process was then decided by reviewing audiotapes and videotapes, so that 100% agreement could result. In other words, consensus and not initial judgements were used. Conclusions =========== This is the first study that has used this type of think-aloud analysis to directly assess the ability of pencil-and-paper examination formats to test higher order problem solving. The results failed to show a significant difference between the two formats used, but did show that both formats can potentially evoke higher order diagnostic thinking. The results have several potential implications for medical education. Firstly, the results are important to examination construction, by demonstrating direct evidence that problem solving can be tested by pencil-and-paper formats, and specifically change some of the presented misperceptions about the standard five-option MCQ format. Secondly, demonstrating that the two formats can evoke scheme utilization is important. There is evidence \[[@B29]\] that the odds of diagnostic success are greater in examinees using scheme-inductive (and pattern recognition) as opposed to hypothetico-deductive reasoning. However, over and above their potential advantage in problem solving, schemes can be a very powerful tool for knowledge organization in an undergraduate curriculum. In this light, showing that MCQs and EMQs can test for scheme utilization is an important step for medical schools planning to include schemes as a teaching tool in their curricula. Lastly, this study demonstrates that testing higher order problem solving requires careful attention to question stem rather than question format or number of alternatives. A well-constructed stem will challenge examinees to choose the correct response, potentially using more expert reasoning strategies, prior to examining the alternatives. This has great potential impact on examination writers, who need not feel obliged to provide more than five alternatives, once they have carefully constructed a stem with a problem-solving task in mind. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= SC conceived of the study, participated in its design and coordination, and drafted the final manuscript. HM participated in the study conception, design, and revised the initial manuscript draft. PH participated in the study design and performed the statistical analysis. GF participated in the statistical analyses. All authors read and approved the final manuscript. Appendix A: The two examination formats ======================================= Format: Multiple-choice question -------------------------------- A 35 year-old woman presents with a one year history of diarrhea. She describes her stools are 10 -- 12 profuse, watery, non-bloody bowel movements per day. She is eating well but has lost 7 kg over the last year. She has no abdominal pain. She is unsure if her stools are oily, but they are difficult to flush. She is otherwise perfectly well, with no previous surgeries. She smokes 1/2 pack a day but does not drink alcohol. She has never traveled, camped or drank well water. Her family history reveals an aunt with ulcerative colitis. Examination is unremarkable except for pallor. Stool C & S, O & P and C. difficile are all negative. Laboratory work shows a microcytic anemia (Hb 95, MCV 63), with low ferritin (4), but normal B12 and folate levels. 1\) What is the most likely diagnosis for this patient? A\) Celiac disease B\) Crohn\'s colitis C\) Villous adenoma of rectum D\) Pancreatic insufficiency E\) Bacterial overgrowth ANS:\_\_\_\_\_\_\_\_\_\_ Format: Extended-matching question ---------------------------------- A 33 year-old woman presents with a one year history of diarrhea. She describes her stools as 10 -- 12 profuse, watery, non-bloody bowel movements per day. She is eating well, but has lost 9 kg over the last year. She has no abdominal pain. She sometimes sees oil droplets in her stool, and they are very difficult to flush. She had surgery for stomach ulcers at age 20, and had repeat surgery five years later for \"bile gastritis\". She is otherwise healthy. She smokes 1/2 pack per day but does not drink alcohol. She has not drank well water, and has not traveled or gone camping recently. Her family history is significant for two cousins with Crohn\'s disease. Examination is unremarkable. Stool C & S, O & P and C. difficile are all negative. Her CBC shows a macrocytic anemia (Hb 108, MCV 110) with a normal ferritin, but low B12 and elevated folate levels. Select the most likely diagnosis from the list below: \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ A\) Bacterial overgrowth B\) Celiac disease C\) Collagenous colitis D\) Crohn\'s colitis E\) Crohn\'s ileitis F\) Colonic carcinoma G\) Factitious diarrhea H\) Giardiasis I\) Ischemic colitis J\) Irritable bowel syndrome K\) Lactose intolerance L\) Pancreatic insufficiency M\) Shigella dysentery N\) Villous adenoma of rectum O\) Viral gastroenteritis Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6920/4/23/prepub> Acknowledgements ================ The authors would like to acknowledge and thank the Medical Council of Canada for its financial support of this work.	PubMed Central	2024-06-05T03:55:49.821307	2004-11-5	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533882/", "journal": "BMC Med Educ. 2004 Nov 5; 4:23", "authors": [ { "first": "Sylvain P", "last": "Coderre" }, { "first": "Peter", "last": "Harasym" }, { "first": "Henry", "last": "Mandin" }, { "first": "Gordon", "last": "Fick" } ] }
PMC533883	Background ========== The term pervasive developmental disorder (PDD) refers to a range of disorders with onset in childhood characterised by abnormalities in the domains of language development, communication abilities and social interactions and by a rigid, repetitive pattern of behaviours and interests \[[@B1]\]. Within PDDs, autism refers to children meeting full diagnostic criteria for the above three domains of developmental impairments and onset before the age of three years. The other PDDs are Asperger\'s syndrome (a less severe form of PDD with the features described above but without language delay and with intelligence within the normal range), pervasive developmental disorder not otherwise specified (not fulfilling complete criteria for autism), and childhood disintegrative disorder (a severe form of autism developing in previously normal children who undergo massive regression between the ages of 2 and 10 years) \[[@B2]\]. Rett\'s disorder (occurring only in girls, characterised by acquired microcephaly, regression and neurological signs) is also classified as a PDD in ICD-10 but has a known genetic aetiology \[[@B3]\] and is not included in the incidence estimates presented in this paper. There have been a number of reports that the incidence of PDD is increasing \[[@B3]\] and the prevalence for all PDDs appears to be around the 60/10,000 in recent studies in the United Kingdom \[[@B4]\] and the United States \[[@B5]\]. There is some evidence that increased prevalence rates are due to a broadening of the concept of PDD and changes in diagnostic practice \[[@B6],[@B7]\]. This has not gone unchallenged, however, and it is likely that increased awareness and changes in educational and social policies account for some of the recent upward trend in rates\[[@B6],[@B8]\]. However, a real increase in the incidence cannot be ruled out \[[@B3]\]. As part of a case-control study to identify risk factors for autism and other PDDs \[[@B9]\], we have investigated time trends in the incidence of the diagnosis of PDDs using data obtained from the United Kingdom General Practice Research Database (GPRD). Methods ======= The General Practice Research Database -------------------------------------- The GPRD was set up in 1987, then known as the VAMP (Value Added Medical Products) Research Bank \[[@B10]\]. It consists of the electronic clinical records of patients registered with contributing general practices and aims to include complete prescribing and diagnostic information. The practices included in the GPRD are broadly representative of all practices in England and Wales in terms of geographical distribution, practice sizes and the age and gender distributions of registered patients \[[@B11]\]. Contributing practices must meet a range of data quality criteria before they are included in the GPRD. The quality of the information in the database, including the completeness of recording of diagnoses made in medical facilities outside the practice, has been validated in a number of independent studies and has been found to be high \[[@B10]\]. There is excellent agreement between prescribing data from the GPRD and national data from the Prescription Pricing Authority \[[@B12]\]. General practices contributing to the GPRD originally used a software system called VAMP but during the late 1990s most practices switched to a new software system called VISION. At the start of the study period, general practitioners contributing to the GPRD used a modified version of the Oxford Medical Information Systems coding system (OXMIS) to record diagnoses \[[@B13]\], but in later years most practices used the READ coding system \[[@B14]\]. Each individual is assigned a unique identification number and all data that could identify individuals are removed from records before they are incorporated into the GPRD. ### Study population and denominators The information analysed in the present study comprised the electronic medical records of children born in 1973 or later and registered with general practices contributing data to the GPRD during the period 1^st^January 1988 to the 31^st^December 2001. Such children will have been eligible at some time in their life for MMR vaccination, an exposure of particular interest in the case-control study \[[@B9]\]. For a specific general practice, the start of the observation period for this study was taken as the later of either the 1^st^January 1988 or the date at which the practice started contributing data to the GPRD. The end of the observation period was the date at which the practice stopped contributing data to the GPRD, or the 31^st^December 2001 if this was earlier. Individual patients were included in the study during the times within the observation period that they were registered with a practice contributing to the GPRD. The number of practices included in the GPRD varied during the study period, rising from 34 in 1988 to 557 in 1996 then falling to 380 by 2001. Age- and gender-specific rates were calculated for people born in 1973 or later and registered with contributing general practices during each calendar year. Those registered for part of a particular year contributed time at risk for the period they were registered. People were classified by geographical location of their general practice by country for Wales, Northern Ireland and Scotland, and by the eight former administrative National Health Service regions for England. Identification of cases ----------------------- When a patient first registers with a practice contributing to the GPRD, past medical events and diagnoses judged to be clinically important by the general practitioner are recorded in the electronic record, with the date of each diagnosis where available. While a patient is registered with a contributing practice, all new diagnoses and all new drug therapies or changes in existing therapies are recorded contemporaneously. Cases were defined as people born in 1973 or later who had a first diagnosis of PDD entered into their general practice record while registered with a practice contributing to the GPRD during the study period. Cases were identified by searching the complete medical record of all people registered with participating practices during the study period for either OXMIS or READ clinical codes indicating a diagnosis of PDD: Appendix \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]. People with a diagnosis of a PDD made prior to their observation period, and people whose PDD diagnosis was the first entry recorded when they joined a practice, were classified as prevalent cases and were excluded from the incidence estimates. On the basis of the recorded diagnoses patients were classified as \'autism\' or \'other PDD\'. Those with autistic disorders and similar presentations were grouped in the autism category and other descriptions (such as Asperger\'s syndrome) were grouped in the other PDD category. ### Analysis Crude rates and age standardised incidence ratios for first diagnosis of all PDDs, autism, and other PDDs were calculated by year of diagnosis and gender and for different geographical regions. Because of low numbers of cases in some years, indirect standardisation was used with rates during the whole study period as the standard rates. The age-standardised incidence ratios presented take into account variations in the age structure of the population in different time periods and different geographical regions. Rates of first diagnosis were calculated by year of diagnosis and year of birth, both in two year intervals. Ethical approval ---------------- Ethical approval for the study was obtained from the Scientific and Ethical Advisory Group of the GPRD and from the ethics committee of the London School of Hygiene and Tropical Medicine. Results ======= We identified 1410 persons with a first recorded diagnosis of PDD during the study period: 1097 were categorised as autism and 313 as other PDD, for 294 (94%) of whom the diagnosis was Asperger\'s syndrome (appendix). Table [1](#T1){ref-type="table"} shows the crude rates of first diagnosis of PDD and age standardised incidence ratios by year of diagnosis and gender. The person-years at risk varied from year to year because of changes in the number of contributing practices, which increased until the mid 1990s and then fell. During the study period incidence rates increased progressively for both males and females. The patterns of crude rates and of standardised incidence ratios were very similar. The overall rate increased seven-fold from 0.40/10,000 person-years (95% CI 0.30 to 0.54) in 1991 to 2.98/10,000 person-years (95% CI 2.56 to 3.47) in 2001. A similar change occurred in the age standardised incidence ratios, from 35 (95% CI: 26--47) in 1991 to 365 (95% CI: 314--425) in 2001. The overall male to female ratio was 4.8 and during 1991 to 2001 ranged from 2.7 to 8.4 (chi-squared test for heterogeneity, p = 0.21), with no clear temporal trend. Table [2](#T2){ref-type="table"} shows the number of cases, person-years of follow-up and rate of first recorded diagnosis by date of diagnosis and by date of birth, both in two year intervals. The diagonally linked numbers identify rates among children of the same age in different years. The temporal increase observed in rates of diagnosis is not limited to children born during specific years or to children diagnosed in a specific time period. The rates of diagnosis for children at the same age, born in successive birth cohorts, increased in each successive cohort, including for relatively old ages at diagnosis. For example, for diagnoses at the ages of two to four years, the rates of new diagnosis per 10 000 person-years from 1992--93 to 2000--01 were 1.41, 2.70, 3.07, 5.56 and 7.74. Corresponding rates at ages eight to ten years were 0.36, 0.40, 0.67, 2.46 and 4.46. Table [3](#T3){ref-type="table"} shows the rates of first recorded diagnosis and age standardised incidence ratios of autism and other PDDs by year of diagnosis. There was a striking rise in the rate of diagnoses of other PDDs: from zero for the period 1988 to 1992 rising to 1.06/10,000 person-years by 2001. Over the study period the rate of diagnosis of autism also increased substantially. The patterns were broadly similar for males and females (data not shown). Few diagnoses of other PDDs were made before 1997, but by 2001 over one third of all diagnoses were PDDs other than autism. Table [4](#T4){ref-type="table"} shows the rates of first recorded autism and other PDDs by geographical area. There was marked geographical variation in rates of first diagnosis, with the lowest rate in Wales being less than half the rate in the South East of England (chi-squared test for heterogeneity in SIRs by region, p \< 0.001). In general, the regional variations were less marked for autism than for other PDDs and the ratio of the diagnoses of autism compared with other PDDs varied greatly between areas (chi-squared test for heterogeneity, p = 0.0006). Discussion ========== There was around a 10-fold increase in the rate of first recorded diagnoses of PDDs in United Kingdom general practice medical records from 1988--92 to 2000--01 (table [1](#T1){ref-type="table"}). The increase was more marked for PDDs other than autism but the increase in autism was also striking (table [3](#T3){ref-type="table"}). If these changes indicate a true increase in the incidence of the conditions it is of great public health importance. However, it is probable that the increase is due, at least in part, to changes in the ascertainment and diagnosis of the conditions. Factors that could have affected the results -------------------------------------------- Some of the general practices contributing data to the GPRD will provide anonymised copies of hospital letters and specialist reports on individual patients. Of patients with a recorded diagnosis of PDD in the GPRD, including prevalent cases when first registered, 446 were registered with 203 general practices willing to provide this service. For 80 of these, medical records were not available as the patient was no longer registered with the general practitioner. We obtained complete case records including copies of hospital clinic letters and specialist reports for 318 (87%) of the remaining 366 people. These were reviewed by a psychologist (LH) and a random sample of 50 records were also reviewed by a child psychiatrist (EF), both of whom have long experience in the field of autism. They judged that a PDD was likely to be present in 294 children (92.5%)\[[@B15]\]. For the 211 patients (of 318) who were first diagnosed with a PDD after they entered the GPRD (and thus are included in this paper), a diagnosis of PDD was confirmed for 193 (91.5%). Thus the positive predictive value of a recorded diagnosis of PDD in the electronic record was high, but we were not able to assess the sensitivity of the ascertainment of cases, that is how often the diagnosis of a PDD may have been missed or not recorded. Of the 211 cases reviewed and included in this paper, 5 (2.4%) were classified as \'other-PDD\' on the basis of their electronic record only, whereas in the validation, 32 (15.1%) met diagnostic criteria for a PDD other than autism. Thus it is likely that a proportion of people in the \'autism\' diagnostic category in this paper have a form of PDD other than autism. The inaccuracy of diagnostic descriptions of different PDDs within the GPRD is likely to reflect changes in the definition of PDD over the past two decades, in particular a broadening of PDDs other than autism \[[@B1],[@B16],[@B17]\]. Many children with Asperger\'s syndrome or pervasive developmental disorder not otherwise specified would only have been assigned a diagnosis of PDD from the latter half of the 1990s. In the earlier period such children may either not have received a diagnosis of PDD at all or have been diagnosed as autism. Inflation in the number of cases in later years could have occurred as other PDD diagnoses came into widespread use and some previously undiagnosed children were diagnosed. For example the OXMIS coding dictionary, used by most general practitioners contributing to the GPRD until the mid-1990s, has only two possible codes for autism and no clinical code for Asperger\'s syndrome, and this diagnosis could only be assigned when practices started to use the READ coding system. These changes explain the low level of diagnoses of other PDDs until the mid 1990s (table [3](#T3){ref-type="table"}). During the study period there is likely to have been an increase in the diagnosis of high-functioning autism, as professionals have become more aware that autism can occur in people of normal intelligence \[[@B3]\]. Greater ascertainment of high-functioning autism may partly explain the increased incidence of autism as well as in the other-PDD diagnoses. That there was also a marked increase in the rate of diagnosis of severe disorders, however, suggests that better detection of less severe cases alone can not explain all of the increase. Two previous studies have demonstrated falls in the rates of diagnosis of mental retardation \[[@B6]\] and of non-specific developmental disorders \[[@B18]\] during the 1990s as the rate of diagnosis of autism increased. These patterns could be partly due to improvements in the detection and diagnosis of autism. In the late 1980s and early 1990s autism was only likely to be diagnosed by specialist child psychiatrists who increased in number by 40% between 1988 and 2001 (personal communication: Royal College of Psychiatrists). Through the 1990s developmental and community paediatricians began to diagnose PDDs, and this, combined with increased awareness of autism and PDDs among the general public, may have contributed to increased ascertainment of the disorders \[[@B19]\]. The marked geographical variation in rates of diagnosis and in the ratio of diagnoses of autism to other PDDs (table [4](#T4){ref-type="table"}) may reflect differences in service provision and parental awareness in different regions. It is likely that children with PDDs other than autism will generally be first diagnosed at later ages than children with autism. Greater ascertainment of other PDDs during the latter part of the study period (table [3](#T3){ref-type="table"}) could have led to the observed increase in incidence being largely restricted to older age groups. However, the increased rates were observed for all age groups (table [2](#T2){ref-type="table"}). Because of low numbers of cases in some years, indirect standardisation was used to calculate SIRs. When comparing SIRs, marked differences in the age distribution of the populations being compared can lead to a biased comparison \[[@B20]\]. However, the differences in age distributions were not great in our study and the patterns seen in the crude rates and the age standardised rates did not differ materially, suggesting the comparison of SIRs was valid. The accuracy of the denominator data may have changed during the study period. When patients move geographical area they may delay registering with a new general practice until they have a specific reason to visit them. Thus our estimates of person-years at risk may be too low for those moving into a practice and too high for those moving out. Person-years at risk may also be inflated as patients who emigrate may omit to inform their general practitioner and, furthermore, administrative delays and errors may result in individuals being registered with more than one general practitioner. For example, in 1997 in England 51 million people were registered with a general practitioner \[[@B21]\] whereas the total population size was only around 49 million people\[[@B22]\]. It has also been suggested that in recent years the period of time for which patients who have moved out of an area remain registered with a general practice in that area has shortened as health authorities and general practices have streamlined procedures \[[@B23]\]. This may mean that the inflation of denominators could have been greater in the early years compared to later years. However, these factors could explain only a very small part of the increased rates observed. An additional issue is that the person-years used as denominators in our analysis did not exclude the period following diagnosis. However, given the relative rarity of the disease, this will have produced only a very small inflation in the denominators. Comparison with other studies ----------------------------- A previous study based on the GPRD assessed time trends in the diagnosis of autism from 1988 to 1999 \[[@B24],[@B25]\]. The study did not examine other PDDs, was restricted to children aged less than 13 years and included a total of 305 cases. The previous paper was based on a sub-set of the GPRD data held by the Boston Collaborative Drug Surveillance Program. This is reflected in the person-years of observation contributing to the two studies -- just under 3.1 million person years in the previous study compared with over 14 million person years in our paper. The two studies were undertaken in substantially different populations. However, the extent of the rise in incidence in the two studies was similar: from around 0.3/10,000 person-years in 1989/1990 to around 2.0/10,000 person-years by 1999 \[[@B24]\]. In addition, the increased risks of autism observed in successive birth cohorts were similar to those observed in our study \[[@B25]\]. A study in the West Midlands area of the United Kingdom based on diagnoses made at child development centres found cumulative incidence rates of autism for children between the ages of one to four years of 2.22/10,000 person-years in 1991--3 (based on 20 cases), rising to 4.75/10,000 person-years in 1994--6 (based on 42 cases) \[[@B26]\]. The corresponding cumulative rates from our study were 1.00/10,000 person-years in 1991--3 rising to 1.97/10,000 person-years for 1994--6. Restricting our data to the West Midlands region we observed rates of recorded diagnosis of autism of 1.57/10,000 person-years in 1991--3 and 1.56/10,000 person-years in 1994--6, based on 14 and 16 cases respectively. It is unclear why the rates observed in the West Midlands study were different from the rates we observed. However, these area and time period specific rates are based on small numbers of cases. The validation of cases in the GPRD can not necessarily be assumed to apply to all geographical areas. A recent study in North London estimated the prevalence of autism by year of birth and concluded there was a rise from 1979 to 1992 after which there was a plateau between 1992 and 1996 \[[@B27]\]. These authors excluded children with Asperger\'s. The review of clinical records of children in our study indicated that some children who would have been diagnosed as having autism in the early 1990s actually had Asperger\'s and would probably not have been classified as \'autism\' in later years. This shift in diagnostic labelling from autism to Asperger\'s may explain, at least in part, the plateau in incidence described by Lingam et al. and would be compatible with the reduction in average age at diagnosis observed in that study. Conclusions =========== This is one of the largest studies undertaken of trends in the incidence of autism and other PDDs. We describe striking increases in the rates of diagnosis of these conditions. However, much of the increase may be due to better ascertainment related to changes in diagnostic practice and improved recognition of the conditions. While review of clinical records confirmed that over 90% of diagnoses of PDDs recorded by general practitioners were likely to be correct, the nature of the study precluded us from assessing how often children with PDDs were not diagnosed. Thus the extent to which the increases in incidence we document are true increases in disease is uncertain. Abbreviations ============= PDDpervasive developmental disorder GPRDGeneral Practice Research Database Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= AH, LS, EF, LR and PS designed the study. LH and EF undertook the validation of case reports. CC analysed the data. LS drafted the paper. All authors commented on drafts and approved the final version of the paper. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1741-7015/2/39/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Appendix: Codes used to identify cases, numbers identified, and diagnostic classification used in this paper ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ The study was funded by the Medical Research Council. LS is supported by a Medical Research Council Clinician Scientist Fellowship. Figures and Tables ================== ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### First diagnoses of PDD by year of diagnosis and gender ::: Year Number of cases Person years at risk Crude incidence rate (per 10000 person-years) Male:Female incidence rate ratio Age standardised incidence ratio (S.I.R) ---------- --------------------- -------------------------- --------------------------------------------------- -------------------------------------- ---------------------------------------------- ------------- ---------- ---------- ---------- --------- --------- --------- --------- All M F All M F All M F All M F 1988 1 1 0 94474 49353 45121 0.11 0.20 0 ∞ 8 10 0 1989 6 6 0 260978 135542 125436 0.23 0.44 0 ∞ 19 22 0 1990 21 20 1 643830 332819 311012 0.33 0.60 0.03 18.7 28 32 8 1991 45 36 9 1118577 576722 541855 0.40 0.62 0.17 3.8 35 34 42 1992 45 37 8 1311605 675188 636417 0.34 0.55 0.13 4.3 31 31 33 1993 69 51 18 1376111 707674 668437 0.50 0.72 0.27 2.7 47 42 72 1994 77 61 16 1432528 735395 697134 0.54 0.83 0.23 3.6 52 49 64 1995 108 97 11 1476627 756200 720427 0.73 1.28 0.15 8.4 74 79 45 1996 102 85 17 1381242 705526 675716 0.74 1.21 0.25 4.8 78 78 77 1997 133 115 18 1283151 653791 629359 1.04 1.76 0.29 6.2 114 119 89 1998 192 152 40 1228058 624723 603335 1.56 2.43 0.66 3.7 180 173 218 1999 231 193 38 1121383 568937 552446 2.06 3.39 0.69 4.9 242 244 231 2000 211 179 32 935906 474344 461562 2.26 3.77 0.69 5.4 277 284 244 2001 169 139 30 567058 287574 279483 2.98 4.83 1.07 4.5 365 361 388 All 1410 1172 238 14231526 7283787 6947739 0.99 1.61 0.34 4.8 100 100 100 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Number of cases, person-years follow-up and rates of first recorded diagnosis of PDD (per 10,000 person-years) by year of diagnosis and year of birth (in two year categories). The diagonally linked numbers identify rates among children of the same age in different years ::: --------------- ----------------------------- -------------- -------------- -------------- -------------- ---------- ---- -------- ---------- ---- -------- ---------- ---- -------- ---------- ------------ Year of first diagnosis 1992--93 1994--95 1996--97 1998--99 2000--01 Birthyear n PY rate n PY rate n PY rate n PY rate n PY rate 73--74 5 257638 0.19 5 263712 0.19 1 233838 0.04 4 194554 0.21 4 118997 0.34 age 26--28 75--76 0 233164 0.00 4 232393 0.17 5 208111 0.24 4 175331 0.23 2 107426 0.19 age 24--26 77--78 5 233093 0.21 2 227191 0.09 7 191785 0.36 8 162057 0.49 0 100419 0.00 age 22--24 79--80 1 265530 0.04 2 257752 0.08 9 211888 0.42 13 171491 0.76 3 104136 0.29 age 20--22 81--82 6 268477 0.22 3 262006 0.11 6 217307 0.28 20 174989 1.14 10 104093 0.96 age 18--20 83--84 10 275918 0.36 9 270128 0.33 4 226897 0.18 12 184013 0.65 5 110565 0.45 age 16--18 85--86 14 283299 0.49 11 277108 0.40 21 231591 0.91 28 187229 1.50 12 112080 1.07 age 14--16 87--88 26 294006 0.88 18 285548 0.63 16 237945 0.67 32 191788 1.67 31 114057 2.72 age 12--14 89--90 42 298297 1.41 45 289890 1.55 30 241271 1.24 48 194806 2.46 41 116246 3.53 age 10--12 91--92 5 248952 0.2 77 285511 2.70 53 238318 2.22 56 192373 2.91 51 114277 4.46 age 8--10 93--94 0 29342 9 231555 0.39 70 227807 3.07 89 184757 4.82 67 110641 6.06 age 6--8 95--96 0 26360 13 177284 0.73 98 176267 5.56 69 106292 6.49 age 4--6 97--98 0 20351 11 144307 0.76 81 104606 7.74 age 2--4 99--00 0 15479 4 79129 0.51 age 0--2 --------------- ----------------------------- -------------- -------------- -------------- -------------- ---------- ---- -------- ---------- ---- -------- ---------- ---- -------- ---------- ------------ ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Crude rates of first diagnosis (per 10 000 person-years) and age standardised incidence ratios for autism and other PDDs by year of diagnosis ::: year Total cases Cases with \'other PDD\' diagnosis % cases with \'other PDD\' diagnosis incidence other PDDs incidence autism SIR other PDDs SIR autism ---------- ----------------- ---------------------------------------- ------------------------------------------ -------------------------- ---------------------- -------------------- ---------------- 1988 1 0 0 0 0.11 0 10 1989 6 0 0 0 0.23 0 23 1990 21 0 0 0 0.33 0 35 1991 45 0 0 0 0.40 0 44 1992 45 0 0 0 0.34 0 39 1993 69 2 3 0.01 0.49 6 58 1994 77 2 3 0.01 0.52 6 64 1995 108 3 3 0.02 0.71 9 91 1996 102 8 8 0.06 0.68 27 92 1997 133 18 14 0.14 0.90 67 128 1998 192 51 27 0.42 1.15 204 173 1999 231 87 38 0.78 1.28 369 200 2000 211 82 39 0.88 1.38 435 225 2001 169 60 36 1.06 1.92 495 319 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Crude incidence rates (per 10 000 person-years) and age standardised incidence ratios (SIR) for the period 1988 to 2001 by geographical area of the United Kingdom (listed in order of SIR) ::: Autism Other PDDs ALL ---------------------- --------------------- -------------------- --------- --------------------- -------------------- --------- --------------------- -------------------- ----------------- Region Number of cases Incidence rate SIR Number of cases Incidence rate SIR Number of cases Incidence rate SIR (95%CI) WALES 40 0.64 83 1 0.02 7 41 0.65 66 (49,90) NORTH WEST 121 0.60 78 32 0.16 72 153 0.76 77 (66,90) NORTHERN & YORKSHIRE 86 0.74 98 11 0.10 43 97 0.84 85 (70,104) N. IRELAND 29 0.71 97 7 0.17 79 36 0.88 93 (67,129) EASTERN 132 0.76 97 35 0.20 90 167 0.96 95 (82,111) SCOTLAND 32 0.62 84 15 0.29 135 47 0.91 96 (72,128) LONDON 112 0.79 103 24 0.17 78 136 0.96 97 (82,115) TRENT 94 0.79 100 24 0.20 90 118 0.99 98 (82,117) SOUTH WEST 99 0.72 92 44 0.32 144 143 1.03 103 (87,121) WEST MIDLANDS 146 0.85 113 36 0.21 97 182 1.06 109 (94,126) SOUTH EAST 206 1.00 128 84 0.41 184 290 1.40 141 (126,158) :::	PubMed Central	2024-06-05T03:55:49.824204	2004-11-9	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533883/", "journal": "BMC Med. 2004 Nov 9; 2:39", "authors": [ { "first": "Liam", "last": "Smeeth" }, { "first": "Claire", "last": "Cook" }, { "first": "Professor Eric", "last": "Fombonne" }, { "first": "Lisa", "last": "Heavey" }, { "first": "Laura C", "last": "Rodrigues" }, { "first": "Peter G", "last": "Smith" }, { "first": "Andrew J", "last": "Hall" } ] }
PMC533884	Background ========== Treatment of low back pain in the military setting: a \'minimal intervention approach\' --------------------------------------------------------------------------------------- For the last ten years, clinical researchers from the Royal Netherlands Army (RNLA) have studied the potential of physical training modalities in preventing and alleviating nonspecific low back pain (LBP) in their working population. Many military and civilian job functions in the RNLA involve heavy manual material handling and, therefore, spine-loading activities. In general, the incidence of back problems is higher in physically demanding tasks than in sedentary activities \[[@B1]\]. Concordantly, the incidence of LBP in the RNLA is high. Acute LBP is the primary reason for soldiers to visit the general practitioner at a military health center. Chronic nonspecific LBP, defined as having complaints for at least 12 weeks, is one of the three most diagnosed disorders during consulting hours of Dutch military company doctors, and takes on average 15% of their weekly consulting hours time. Currently, there is strong evidence that exercise therapy is more effective than usual care \[[@B2]\]. Exercise therapy is a major part of the standard treatment by physiotherapists in the RNLA, involving an active role of the patient. In the experience of our health professionals (practitioners and physiotherapists), this active approach fits in well with the attitudes and beliefs of the target population: soldiers are taught to be aware of their physical abilities and limitations from the moment they enlist for the army. After all, recruits who do not successfully complete their basic training cannot progress on to a career as a soldier. The specific character of military tasks nowadays, e.g. (preparation for) crisis management operations abroad, interferes with the schedules of rehabilitation programs for back-injured soldiers which, in general, take several days per week over a considerate number of consecutive weeks. Therefore, the search for effective and (time-) efficient exercise therapy protocols has led us to a specific form of lumbar extension training. Each training session consists of no more than 5 to 10 minutes training of the isolated lumbar extensors on a special training device. Arguments for this study ------------------------ For a number of years, we have experience with high-intensive, isolated training of the lumbar extensors in military personnel with nonspecific LBP, by using a special training device. In this a sports medicine approach is followed, partly according to established exercise protocols \[[@B3],[@B4]\], in which three key principles are emphasized: (1) isolation of the lumbar extensors through fixation of the pelvis and thighs; (2) training in the individual\'s full range of motion; (3) avoiding \'sticking points\' in the training load -- i.e. points in the range of motion in which a relatively high resistance is experienced -- by tuning the load curve of the weight stack to the individual\'s strength curve. In individual cases, we observed satisfying to sometimes excellent results in terms of pain relief and functional restoration, when giving a training stimulus of no more than 5 to 10 minutes (1 to 2 training sessions) per week. These findings, however, need to be confirmed in a randomized controlled trial. Four main reasons led to the choice of doing research on the efficacy of this sports medicine approach for our working population with LBP. First, recent systematic reviews indicate that exercise therapy is a successful approach for the restoration of chronic and recurrent LBP, at least in the short term \[[@B2],[@B3]\]. However, higher quality studies generally show a lack of treatment specificity of different exercise modalities, e.g. aerobic exercises, strength and endurance reconditioning or mobilizing exercises \[[@B4],[@B5]\]. Moreover, controversy remains regarding the impact of a training stimulus, in terms of intensity, duration and frequency, on the reduction of LBP. Different explanations for this lack of specificity are given in the literature, such as non-specific, more centrally induced training effects, e.g. a shift in pain perception \[[@B5]\], or large heterogeneity in the chosen study populations \[[@B6]\]. If, indeed, no specific dimension or type of exercise therapy is superior to one another in producing optimal therapeutic outcomes, other aspects are more relevant when introducing an intervention program, such as: treatment affinity, expectation and compliance of patient and provider, costs, facilities, and personnel capacity. From this perspective, back strength and endurance training in CLBP patients with the use of training devices is an interesting concept for our military population. RNLA personnel are, from their very first initial military education, used to participate in physical exercise programs, including progressive resistance training on exercise machines. The RNLA is well equipped with an extensive line of modern fitness devices on all major military locations throughout the country, including state-of-the-art lower back machines. Moreover, protocolized treatment sessions with our training device take no longer than 5 to 10 minutes once or twice a week from both patient and provider, compared to (on an average) 30 minutes in regular treatment sessions. We expect this time efficiency to be highly appreciated by our personnel, who work in a typical military culture of \"running into extremes\": relatively quiet (maintenance) periods on the military base are interspersed with extremely busy periods shortly before and during out-of-area operations. For several target groups, longstanding and time-consuming rehabilitation programs are out of the question. For instance, recruits who drop out of their initial training because of (back) injuries, need to return as quick as possible to prevent a stagnation in their military career. For soldiers standing by for military operations, everything revolves around the mission when being commanded to be prepared within the next weeks. A second reason for our approach is that the majority of studies on LBP management consist of multimodal interventions, which include physical, behavioral, educational and/or ergonomic elements. To obtain a better view on the (relative) efficacy of either of these concepts, unimodal intervention programs like ours need to be evaluated \[[@B7],[@B8]\]. Besides, we strongly believe that exercise as the primary entrance for restoring back function has a wide span of treatment effects, including improvements for cognitive and/pr behavioral variables. Although exercise has a primary goal of improving functioning of targeted tissues, successful completion of exercise protocols in the presence of chronic pain may for example lead to a reduction in pain-related fears. As standardized exercise on a training machine is based on measured performance (number of kilograms and repetitions), patients are continuously given numerical feedback regarding their increasing physical capacities \[[@B9]\]. An increased awareness of improving physical capacities may draw their attention away from pain and suffering. Third, the choice for a particular intervention approach depends in many cases on the stage and severity of the back problem, the extent to which psychosocial aspects are involved, and the needs and preferences of the patient. For instance, behavioral therapy is mainly focused on issues that are prevalent in chronic patients, such as low feelings of self-control or fear of movement/(re-)injury. Since the population of the present study, military employees of the RNLA, is a working population with mostly short-term, intermittent and moderately severe LBP, we chose to apply a more physical approach. As we have seen in our previous research (see the next paragraph), this links well with the health perceptions of our target population, in which perceived health problems were not severe and much more focused on physical than on mental aspects. Fourth, the efficacy of isolated extension training in chronic back patients has been studied by several other research groups as well \[[@B10]\]. Although promising results were reported regarding lumbar strength improvements and pain relief, several methodological shortcomings hinder solid interpretation of these findings. Most problems encountered were a small sample size, lack of randomization, lack of long-term follow-up results, variation in study populations (e.g. healthy volunteers, employees receiving worker\'s compensation), and inadequate or missing control groups \[[@B7],[@B10]-[@B14]\]. In a review on lumbar extension training with MedX-equipment in LBP patients, Miltner et al \[[@B8]\] conclude that more controlled studies are needed \"to delineate further the role of isolated lumbar extension exercise for the treatment of LBP and to test the efficacy compared to other methods of care.\" Earlier research on our minimal intervention approach ----------------------------------------------------- Especially in recent years, we have scientifically studied the potential of our sports medicine approach. In two previous trials we compared the efficacy of a high-intensive, progressive resistance training program of the isolated lumbar extensors, with a low-intensive, non-progressive program of the same extend, in a group of workers with nonspecific LBP. Total intervention time of both \'minimal intervention programs\' was limited to 14 sessions of 5 to 10 minutes, over a period of 12 weeks (1^st^trial) or 8 weeks (2^nd^trial). In the first trial, we were unable to demonstrate that either of the two training programs was superior in alleviating back complaints \[[@B15]\]. However, the magnitude of the improvements in back function found in this study were in line with those reported in other studies, which used more extended (multimodal) exercise programs. Therefore, it would be interesting to compare the efficacy of our minimal intervention program with the usual care RNLA personnel with nonspecific LBP. Moreover, the results of our first trial indicated that some individuals with LBP might benefit more from an aggressive approach, showing a trend towards a higher improvement rate (self-assessed percentage decrease in complaints) directly after the \'minimal intervention\' treatment, as well as a higher compliance to the treatment and a higher willingness to participate in physical exercise on the longer term. In the present multicenter study, we aim at identifying relevant subgroups of patients that show higher success rate due to this training approach. Methods ======= Study design and population --------------------------- In a randomized, single-blinded multicenter trial, we evaluate the efficacy of progressive, isolated resistance training of the lumbar extensor muscles, compared to the usual care. The trial started in April 2002; data will be collected till the end of 2005. The source population (n = 23,000) consists of military employees of the RNLA. Our in- and exclusion criteria are listed in table [1](#T1){ref-type="table"}. Recruitment of participants takes place during regular office hours of the military general practitioner. A brief outline of the study design is presented in Figure [1](#F1){ref-type="fig"}. Study sites ----------- Almost every military location in the Netherlands has a health center, in which general practitioners, dentists and physiotherapists give primary care to military personnel of the RNLA. For the present study we selected five military health centers on the basis of: (1) representing a major part of the total military population; (2) holding at least two full-time physiotherapists, and (3) the willingness to unconditionally participate in the project. The selected military health centers include the following locations: • Amersfoort, in the middle-east of the Netherlands: approximately 15% younger soldiers on stand-by for military operations abroad (18--25 years), 25% military instructors (35+ years), 10% staff personnel (40+ years), and 46% civilian workers from supporting units (40+ years); • Den Haag, in the west of the Netherlands: approximately 90% older staff personnel (40+ years) at office work; • Oirschot, in the south of the Netherlands: approximately 75% younger soldiers in their initial military education or on stand-by for military operations abroad (18--25 years), 25% military instructors and staff personnel (35+ years); • Schaarsbergen, in the south-east of the Netherlands: approximately 65% younger soldiers in their initial military education or on stand-by for military operations abroad (18--25 years), and 30% staff personnel (35+ years); • Seedorf, in the north-west of Germany (part of the 1 German/Netherlands Corps): approximately 65% younger soldiers on stand-by for military operations abroad (18--25 years), 20% staff personnel (35+ years), and 10% civilian workers from supporting units (40+ years). Study population ---------------- ### Recruitment, enrollment, and randomization All general practitioners at each of the selected study centers identify potential subjects from among their clinic\'s patients, according to the aforementioned criteria. Each subject is submitted to regular history taking and physical examination by the physician; checklists with standard elements for LBP have been provided to all physicians. If eligible, patients are informed about the study. All relevant information from the intake is written down in a referral; a visit to one of the physiotherapists of the center is planned within the next days. At the first visit to the physiotherapist, patients receive further information about the trial. A pre-assessment of the isometric back strength is taken. A written explanation of one of the questionnaires, in which patients have to choose the three most disabled daily activities due to back pain, is given them to take home, which allows them to make a well-considered choice at the next visit. Written informed consent is obtained from all patients who are willing and eligible to participate. At the second visit, participants undergo a baseline measurement consisting of a compound questionnaire and an isometric back strength test. At the third visit, patients are randomized into either the back strengthening group or usual care group. Directly after randomization, the first treatment session starts. Patients are allowed to withdraw from the study at any time, although the importance of full compliance of every participant is emphasized. Randomization is done by means of a computer-generated table of random numbers per study center, using a block size of ten. Prestratification is applied for the duration of the back complaints, with a cut-off point of 12 weeks, suspecting duration of complaints to influence the individual response to the exercise program. One intervention group receives a back-strengthening program; the other group receives a usual care program. The randomization is concealed, which means that the treating physiotherapist obtains the allocated treatment by means of a computer software program, by entering the name and military registration number of the patient. The study protocol was approved by the Medical Ethics Committee of the Netherlands Central Military Hospital. Study interventions ------------------- ### Back strengthening program Subjects allocated to the back-strengthening program (BS) undergo a 10-week, progressive resistance-training program of the isolated lumbar extensor muscle groups. The program includes 14 training sessions (2 days per week) and 3 isometric back strength tests (in week 1, 5, and 10). The initial training load is set at approximately 35% of the maximal isometric back extension strength of the participant, measured at baseline. The goal of every training session is to perform 15 to 20 repetitions (reps) on the lower back machine, equivalent to approximately 50% and 70% of the one-repetition maximum (1 RM) respectively. If the subject is able to perform a higher number of reps, 2 1/2 kg weight is added in the next training session. Vice versa, if the participant is unable to perform the minimal number of reps, the subsequent training load is lowered with 2 1/2 kg. This training protocol is partly based on existing protocols \[[@B5],[@B18]\], and partly on our own experiences. A comprehensive training protocol can be obtained from the authors. Training sessions are carried out on a Total Trunk Rehab (Technogym Inc, Italy). This lower back machine is equipped with a knee-lock system and a thigh-restraining belt to immobilize both hips and thighs, allowing the participant only to move the isolated lower back. All training sessions are conducted as much as possible by the same physiotherapist. The physiotherapist pays special attention to the execution of the training in terms of pace and movement. The flexion and extension of the lower back has to be executed in the full range of motion of the participant, and movements have to be slow and controlled (moving in two seconds from maximal flexion to maximal extension when lifting the weight stack, and returning from maximal extension to maximal flexion in four seconds when lowering the weight stack). During this movement, emphasis is put on the hollowing and flattening of the lumbar lordosis. Every training session is preceded by a 5-minute all-body warming-up on an arm/leg ergometer (Schwinn Airdyne Pro, Balans Inc., Nieuwegein, The Netherlands). The weight load used and the number of reps completed during each training session are recorded. ### Usual care program Subjects allocated to the usual care program (UC) receive regular physical therapy for their lower back for at most 10 weeks, or earlier when the patient indicates to be free of complaints. Based on the physiotherapist\'s judgment, this could include \'hands-on\' treatment (e.g. passive mobilizing and pain-cushioning techniques, manual therapy) and/or \'hands-off\' treatment (e.g. exercise therapy, individual education and instruction on the back function). In the RNLA, active therapy forms are favored. To increase the contrast between both intervention programs, physiotherapists are not allowed to use the lower back machine in their usual care. Patients are not allowed to undergo co-treatment beside the interventions programs during the treatment period, nor exercise on equipment that mimics the specific components of the lower back machine. Therapeutic activities in every therapy session as well as the number of sessions are written down on a form. Outcome measurements -------------------- In this study we have chosen primary outcome measures that are most relevant to the patient and clinician: self-assessed degree of complaints and degree of handicap in daily activities due to LBP. In addition, our secondary measurements focus on several other LBP-related areas, like kinesiophobia or mental health perception. We have included some potential prognostic factors into our measurements, i.e. characteristics of the patient that possibly influence the effect of the intervention: job characteristics, overall health, physical activity, patient satisfaction with the allocated treatment, and attitudes and beliefs of the physiotherapist. This trial is mainly focused on the efficacy of our minimal intervention strategy, and not on unraveling the physiological or psychological working mechanisms of isolated lumbar extension training on LBP. Nevertheless, by including potential prognostic variables, starting points for further research into the \'black box\' of this type of intervention could be identified. ### Baseline characteristics The following demographic variables are registered during the intake: age, time since first episode of LBP, pain radiation, treatment history, and work absenteeism due to LBP in the last year. Moreover, the status of the patient before treatment, in terms of job aspects, overall health and the degree of physical activity, is assessed using a compound questionnaire. Several job characteristics, i.e. content, relation with superior and colleagues, conflicts, and physical aspects of the job, are measured using subscales of a validated Dutch version of the Job Content Questionnaire \[[@B16]\]. Physical aspects of former jobs are assessed using one item of the Dutch Musculoskeletal Questionnaire \[[@B17]\]. Overall health is assessed with one item from the MOS 36-item Short Form Health Survey \[[@B18]\]: \"What do you think, in general, of your health?\" (1 = bad, 2 = moderate, 3 = good, 4 = very good, 5 = excellent). The degree of physical activity is measured with the Short Questionnaire to Assess Health Enhancing Physical Activity \[[@B19]\], a validated and fairly reliable 5-item questionnaire. To assess the attitudes and beliefs of the participating physiotherapists about the relationship between low back pain and function before the start of the study, we use the Pain Attitudes and Beliefs Scale for Physiotherapists \[[@B20]\]. These attitudes and beliefs are believed to influence the physiotherapist\'s commitment to a certain treatment approach. ### Primary outcome measures Global perceived effect \[[@B21]\] is measured by self-assessment on a 7-point scale (1 = completely recovered, 2 = much improved, 3 = slightly improved, 4 = no change, 5 = slightly worsened, 6 = much worsened, 7 = vastly worsened). We defined scores of 1 and 2 as a clinically important change. Patient-specific functional status \[[@B22]\] is measured by a questionnaire following a patient-specific approach. At baseline, individual patients select three main complaints, i.e. frequent activities which they perceive as important in their daily life, but which are hampered by their back pain. Patients rate the severity of these three complaints on a 100 mm visual analogue scale at each test moment. The responsiveness of the questionnaire is fairly good, with an area under the ROC-curve of 0.82, and with mediate correlations with the Roland Disability Questionnaire (r = 0.69--0.75) and the Visual Analogue Scale (r = 0.70--0.80) \[[@B21]\]. Low-back specific functional status is measured by the validated Dutch version of the Roland Disability Questionnaire \[[@B23],[@B24]\], a widely used 24-item scale that reflects the functional disability due to LBP. Test-retest reliability of this scale is considered good for three weeks (r = 0.83) and six months (r = 0.72) respectively \[[@B25]\]. ### Secondary outcome measures Fear of movement or re-injury is measured by the Tampa Scale for Kinesiophobia \[[@B26]\], a 17-item scale to obtain a score for the extent to which a chronic back patient fears (new) physical damage due to physical activity. The Dutch version of this questionnaire has been found sufficient reliable and valid \[[@B27],[@B28]\]. Mental health is measured by the Dutch translation of the 12-item General Health Questionnaire \[[@B29],[@B30]\], assessing problems concerning psychological distress, like depression, sleep deprivation, stress coping, and self confidence. Test-retest reliability of the scale is high in a general population, with reported Cronbach\'s alpha coefficients between 0.86 and 0.90 \[[@B30]\]. Social health is measured by a subscale of the Impact on Participation and Autonomy Questionnaire \[[@B31]\], focusing on the influence of the disability on social relationships; Cronbach\'s alpha for this factor is 0.87. Overall work status is measured by a 4-level item (1 = \"currently working full duties and/or able to do all of my regular home duties\", 2 = \"able to do all work and/or home duties but it causes extra pain\", 3 = \"on restricted duties at work and/or need help with some of my home duties\", 4 = \"off work and/or need help with most of my home duties\"). Individual back extension strength progression was evaluated using repeated isometric measurements on the lower back training and testing machine. A detailed description of these measurements can be find elsewhere \[[@B15]\]. Patient satisfaction is measured at the end of the treatment program by two 3-level items and one 5-level item, in which the degree of satisfaction with the allocated treatment is assessed: (a) \"Were you satisfied with the allocated treatment at the start of the program?\" (b) \"Has your opinion about the treatment changed during the program?\" (c) \"How satisfied are you now about the treatment that was given to you?\" Short- and long-term follow-up ------------------------------ Beside a baseline measurement, follow-up data are gathered at two short-term intervals and two long-term intervals. Short-term follow-up measurements are at 5 and 10 weeks after randomization. Long-term follow-up measurements are at 6 months and one year after the end of the intervention, respectively. At every test moment, participants fill in a compound questionnaire on a stand-alone PC, and they undergo an isometric back strength measurement on the lower back machine. The content of the questionnaire varies per test moment: parts of the questionnaire referring to potential prognostic variables (job characteristics, overall health, and physical activity) are only displayed at baseline, and at 6 and 12 months of follow-up. Figure [1](#F1){ref-type="fig"} presents a flow chart of the different phases of the study. Blinding -------- Double blinding or placebo control is virtually impossible in trials that involve treatment modalities like the ones used in this study, since both patient and provider are inevitably aware of the content of the treatment. Because our physiotherapists are aware of which treatment they provide, there is always the possibility that they may inadvertently convey different expectations to the patients in each treatment program. This could enhance non-specific effects in either of the two groups. Therefore, as mentioned, beliefs and attitude of the physiotherapists towards back treatment in general are evaluated at baseline, as well as at the end of the study period. Another limitation of trials comparing a relatively new treatment to the usual care is the risk of a potential nocebo effect; i.e. patients might feel disappointed after being allocated to the standard therapy. To minimize this effect, both programs are introduced to the patient as potentially equally effective treatments in restoring back function, with the relative efficacy of both programs as the main focus of the study. Moreover, at baseline patients are informed about the opportunity they have to continue with a treatment modality of their choice (e.g. our training device) after finishing the treatment period. Patient satisfaction with the allocated treatment is measured directly after the treatment period. Other efforts to achieve a certain level of blinding within patients, physiotherapists, and data researchers, are: • low back strength training and measurement are done as much as possible by two different physiotherapists; • an independent data manager collects data from all study locations, and recodes patients and locations to unique codes before handing the database to the researchers. Statistics ---------- ### Sample size estimates We attempt to enroll 200 patients at the four military health centers, i.e. 100 patients per treatment group. According to power calculations (α = 0.05 and 1-β = 80%), this sample size is sufficient to detect a 20% difference in our primary outcome measure Global Perceived Effect, between the BS and UC program. In our beliefs, a 20% difference reflects a clinical relevant change in health status. ### Data analysis Statistical analysis will be performed according to the intention-to-treat principle; i.e. patients will be analyzed in the treatment group to which they were randomly allocated. In addition, a per-protocol analysis will be performed, in which only patients with no major protocol deviations will be analyzed. Comparing these analyses with the results of the intention-to-treat analysis will indicate, to what extend protocol deviations and lack of compliance might have biased the results. In our analyses, we will compare the size of the effect, if any, of isolated back strengthening and of usual care for the low back on our primary and secondary outcome variables. Further analysis will determine whether several potential prognostic variables will influence the magnitude of the treatment responses, and if subsets of patients can be distinguished that can be indicated as good-/bad-responders to our specific back training. Demographic and clinical characteristics, as well as baseline outcome measures will be summarized by descriptive statistics. Longitudinal multilevel analyses will be used to examine differences in all continuous outcome measures at 5 and 10 weeks after randomization, and 6 and 12 months of follow-up. Supervision of the centers -------------------------- In order to obtain full commitment from the participating military health centers, as well as to make sure that every center uses the study protocols in the same way, we organize several feedback and feedforward sessions with all participating physiotherapists. In these sessions, we explain different aspects of the trial design, give instructions on the test and training protocols, and answer remaining questions. After these sessions, we install the required equipment (test/training machine, soft- and hardware) at the four locations. Each center has a practice period of about 8 weeks to become familiar with the instruments, logistics and protocols, by means of ad hoc training and test sessions with non-participating (regular) back patients. Our researchers join these sessions and, if necessary, give correcting feedback during and after a test or training. After this practice period, each location officially starts with the study. Once every two weeks, our researchers visit the centers to monitor progress in recruitment and data handling, and to observe the execution of test and training sessions. Discussion ========== This article describes the rationale and design of a multicenter randomized controlled trial in which the efficacy of a specific type of lumbar extension training and usual care are compared in patients with nonspecific LBP longer than 4 weeks. A substantial number of trials have been conducted that included lumbar extension training in low back pain patients \[[@B4],[@B7],[@B11],[@B12],[@B32],[@B33]\]. So far, only the study by Risch et al \[[@B11]\] has emphasized a minimal intervention approach comparable to ours, which was, however, conducted in a non-randomized study design. Our population at risk can be seen as a selected population of mainly male employees, who work in a dynamic organization with a strong culture of physical fitness. We realize that this selection might limit the external validity of the outcomes of this study. However, results of the trial may be extrapolated to other working populations with a more-than-average degree of physical straining job activities, e.g. policemen and firemen on duty or construction workers. Besides, despite the \"fit-and-healthy\" image that soldiers have in our society, a large health survey among a cross-section of male military personnel of 30--40 years showed no favorable scores on several cardiovascular and fitness parameters in comparison to other populations of Dutch men \[[@B34],[@B35]\]. In this perspective, the RNLA -- with over 30,000 military and civilian employees a major professional organization in the Netherlands -- seems to be a good reflection of Dutch society in general with regard to general health parameters. Moreover, for reasons of homogeneity it might be even an advantage to only have a study population of working men with (probably) moderate low back trouble in this study. We hope with this trial to give greater insight to caregivers within and outside the RNLA on treatment options for workers in the sub-acute or chronic phase of their LBP. If, for instance, our minimal intervention approach is equally or more effective than the usual care, medical decision makers may consider implementing this treatment modality in the daily practice of the physiotherapist, by weighing the costs (e.g. price and depreciation costs of materials) and benefits (e.g. reduction of treatment time). List of abbreviations ===================== RNLA = Royal Netherlands Army RCT = randomized controlled trial LBP = low back pain BS = Back Strength intervention UC = Usual Care intervention rep = repetition on back extension machine Competing interests =================== The authors 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/1471-2474/5/40/prepub> Acknowledgements ================ The authors gratefully acknowledge all participating military general practitioners and physiotherapists for their work on location. We would like to thank Peter de Putter and Wim Veenman for their efforts in the development and maintenance of the ALBATROS software program, as well as in the management of all data. Finally we would like to thank Colonel René Roelofs, MD, Surgeon General RNLA, and Colonel Cees IJzerman, MD, Director Occupational Health & Safety Service RNLA, for authorizing this multicenter study. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Flow chart of the different phases of the multicenter trial ::: ![](1471-2474-5-40-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### In- and exclusion criteria of the study ::: ----------------------------------------------------------------------------------------------------------- Inclusion criteria: •military employees of the RNLA between the age of 18 and 54 years\ •at least 4 weeks of continuous or recurrent (at least 3 times a week) episodes of LBP pain localized\ •between posterior iliac crests and angulus inferior scapulae\ •availability to visit the local military health center 2 times a week during 10 consecutive weeks, with\ •no more than two sessions of absence due to job-related activities (e.g. military exercise, course,\ leave)\ •willingness to abandon other treatment interventions for the lower back during the intervention\ period\ •signed informed consent Exclusion criteria •spinal surgery in the last 2 years\ •specific treatment for LBP in the last 4 weeks (e.g. physiotherapy, manual therapy)\ •severe LBP which hinders in performing maximal isometric strength efforts\ •specific LBP, defined as herniated disc, ankylosing spondylitis, spondylolisthesis or other relevant\ •neurological diseases ----------------------------------------------------------------------------------------------------------- :::	PubMed Central	2024-06-05T03:55:49.830703	2004-11-9	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533884/", "journal": "BMC Musculoskelet Disord. 2004 Nov 9; 5:40", "authors": [ { "first": "Pieter H", "last": "Helmhout" }, { "first": "Chris C", "last": "Harts" }, { "first": "J Bart", "last": "Staal" }, { "first": "Rob A", "last": "de Bie" } ] }
PMC533885	Background ========== The global AIDS epidemic has proceeded relentlessly for ≈ 24 years with no promising prophylactic intervention in sight. In 2003 there were 5 million new HIV infections, and 3 million AIDS deaths \[[@B1]\]. To date the number of individuals living with HIV-1 infection/AIDS has reached 40 million, and ≈ 30 million people have already died from AIDS since the beginning of the pandemic \[[@B1],[@B2]\]. Most new infections have been acquired by the mucosal route, heterosexual transmission playing the major (≈ 80%) role. Although the incidence of transmission per unprotected coital act is estimated to be low (0.0001 -- 0.004), but strikingly increased when acutely infected individuals are involved \[[@B3],[@B4]\], the cumulative effect is overwhelming. Anti-HIV-1 vaccines applicable to global immunization programs are not expected to become available for many years. Thus, other prevention strategies are urgently needed. This includes educational efforts and application of mechanical and/or chemical barrier methods. The latter correspond to microbicides, i.e. topical formulations designed to block HIV-1 infection (and possibly transmission of other sexually transmitted diseases) when applied vaginally (and possibly rectally) before intercourse \[[@B3],[@B5]-[@B7]\]. Conceptually, it is preferred that the active ingredient(s) of microbicide formulations (1) block virus entry into susceptible cells by preventing HIV-1 binding to the cellular receptor CD4, the coreceptors CXCR4/CCR5 and to receptors on dendritic/migratory cells (capturing and transmitting virus to cells which are directly involved in virus replication), respectively \[[@B3],[@B8]-[@B11]\], and/or (2) are virucidal. The formulations must not adversely affect the target tissues, and should not cause them to become more susceptible to infection after microbicide removal \[[@B12],[@B13]\]. Treatment with anti-retroviral drugs has decreased mortality from AIDS in industrialized countries but has had a minimal effect so far in developing countries \[[@B14]\]. To avoid a similar dichotomy with respect to microbicides, they should be designed and selected to become affordable and widely accessible, while shortening the time between research and development and their marketing and distribution as much as possible. This would be facilitated if mass manufactured products with established safety records were to be found to have anti-HIV-1 activity. Qualifying candidates to be considered for microbicide development may possibly be discovered by screening pharmaceutical excipients (= \"inactive\" ingredients of pharmaceutical dosage forms) and foods, respectively, for anti-viral properties. This approach has already led to the discovery of cellulose acetate 1,2-benzenedicarboxylate (used for coating of enteric tablets and capsules) as a promising candidate microbicide \[[@B15]-[@B19]\]. Here we report the outcome of screening fruit juices neutralized to pH ≈ 7 to discount nonspecific effects caused by acidity. Methods ======= Reagents -------- Pomegranate juices (PJ) were purchased in local New York City stores; their origin is given in parentheses: PJ1 (Madeira Enterprises Inc., Madeira, CA); PJ2 was prepared from fresh ripe pomegranates in our laboratory; PJ3 (Sadaf^®^; Sadaf^®^Foods, Los Angeles, CA; additional ingredients: fructose, citric acid); PJ4 (Cortas Canning & Refrigeration Co. S.A.L., Beirut, Lebanon); PJ5 (Kradjian, Import & Wholesale Distribution, Glendale, CA. Product of Iran); PJ6 (R.W. Knudsen ; Just Pomegranate; Knudsen & Sons, Inc., Chico, CA); PJ7 (Aromaproduct Ltd., Product of Georgia; distributed by Tamani, Inc., New York, NY). Starches used were: PURE-DENT^®^B815 Corn Starch NF, PURE-DENT^®^B816 Corn Starch USP, Spress^®^B825 Pregelatinized corn starch NF, Spress^®^B820 Pregelatinized corn starch NF, INSTANT PURE-COTE™ B792 Food starch-modified, INSCOSITY™ B656 Food starch-modified (Grain Processing Corporation, Muscatine, IN); PURITY^®^21 corn starch NF and PURITY^®^826 corn starch NF (National Starch and Chemical Company, Bridgewater, NJ); Remyline AX-DR Waxy rice starch and Remy DR native rice starch, medium grind (A&B Ingredients, Fairfield, NJ); ARGO^®^corn starch (Best Foods Division, CPC International Inc., Engelwood Cliffs, NJ); STALEY^®^pure food powder starch (Tate & Lyle, Decatur, IL); STARCH 1500 pregelatinized starch NF (Colorcon, West Point, PA). The following polymers were used: polyethylene glycols (PEG) 1000 NF, 1500 NF and 8000 NF; and hydroxypropyl methylcellulose, 50 cps, USP (Spectrum, New Brunswick, NJ); Carbopol 974P-NF (B. F. Goodrich Co., Cleveland, OH); Carbophil, Noveon AA1 (Noveon, Inc., Cleveland OH); and Pharmaburst B2 (SPI Pharma, New Castle, DE). Fattibase was from Paddock Laboratories, Inc., Minneapolis, MN. Recombinant proteins employed were: HIV-1 IIIB gp120, biotinyl-HIV-1 IIIB gp120, CD4, and biotinyl-CD4 (ImmunoDiagnostics, Inc., Woburn, MA); HIV-1 IIIB BaL gp120 and FLSC (a full length single chain protein consisting of BaL gp120 linked with the D1D2 domains of CD4 by a 20 amino acid linker) (produced in transfected 293T cells \[[@B20]\]). Phycoerythrin (PE)-labeled streptavidin was from R & D Systems, Minneapolis, MN. Biotinylated Galanthus nivalislectin was from EY Laboratories, Inc. San Mateo, CA. Rabbit antibodies to synthetic peptides from gp120 (residue numbering as in reference \[[@B21]\]) were prepared as described \[[@B21]\]. Monoclonal antibodies (mAb) 588D, specific for the CD4 binding site, and 9284, specific for the gp120 V3 loop, were from Dr. S. Zolla-Pazner and NEN Research Products, Du Pont, Boston, MA, respectively. A \"generic\" version of the nonnucleoside HIV-1 reverse transcriptase inhibitor TMC-120 \[[@B22]\] was synthesized by Albany Molecular Research, Inc., Albany, NY, and used in control experiments at a final 5 μM concentration. Pelletted, 1000-fold concentrates of HIV-1 IIIB (6.8 × 10^10^virus particles/ml) and BaL (2.47 × 10^10^virus particles/ml) were from Advanced Biotechnologies, Inc., Columbia, MD. Primary HIV-1 isolates, MT-2 cells, HeLa-CD4-LTR-β-gal and U373-MAGI-CCR5E cells (both contributed by Dr. Michael Emerman) and Cf2Th/synCCR5 cells (contributed by Dr. Tajib Mirzabekov and Dr. Joseph Sodroski) were obtained from the AIDS Research and Reference Reagent Program operated by McKesson BioServices Corporation, Rockville, MD. CEMx174 5.25M7 cells, transduced with an HIV-1 long terminal repeat (LTR)-green fluorescent protein and luciferase reporter construct, expressing CD4 and CXCR4 and CCR5 coreceptors \[[@B23]\], were obtained from Dr. Cecilia Cheng-Mayer. The cells were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), 1 μg/ml puromycin and 200 μg/ml G418. These cells are suitable for titration of both X4 and R5 HIV-1 isolates and for determining the effectiveness of anti-HIV-1 drugs with reliable reproducibility. This is impossible to accomplish by using peripheral blood mononuclear cells (PBMCs) because of their variations in susceptibility to HIV-1 infection among cells derived from distinct individuals \[[@B24]-[@B26]\]. PBMCs were isolated from HIV-1 negative donors as described \[[@B27]\]. Formulations ------------ In attempts to separate gp120-CD4 binding inhibitory activity from most other ingredients of PJ, 200 mg of distinct starch preparations were added per ml of PJ1. After mixing for 1 h at 20°C, excess juice was decanted, and the pellets resuspended in 1 ml of distilled water. Based on results of enzyme linked immunosorbent assays (ELISA), PURITY^®^21 corn starch, NF grade (S21) was selected for further studies, and the corresponding PJ complex was designated as PJ-S21. PJ-S21 was freeze dried and used to prepare the following formulations: PEG suppositories (50% PJ-S21, 45% PEG 1000, 5% PEG 1500); Fattibase suppositories (50% PJ-S21, 50% Fattibase); and mucoadhesive instantly dispersible tablets (50% PJ-S21, 20% HPMC, 20% Pharmaburst, 7.5% Carbopol 974P and 2.5% Carbophil). Enzyme linked immunosorbent assays (ELISA) ------------------------------------------ Inhibition of infection by HIV-1 IIIB and BaL, respectively, was determined relying on a β-galactosidase readout system \[[@B18]\]. The enzyme was quantitated with a Galacto-Light Plus System chemiluminescence reporter assay (Applied Biosystems, Foster City, CA) using a Microlight ML 2250 luminometer (Dynatech Laboratories, Inc., Chantilly, VA). To measure virucidal activity, virus was separated from excess inactivating agent by centrifugation and/or precipitation with PEG 8000 \[[@B18],[@B19]\]. Serial dilutions of the treated virus were assayed for infectivity as described above. Dose response curves (i.e. luminescence vs. dilution) for treated and control viruses were obtained, and the percentages of virus inactivation were calculated \[[@B19]\]. To determine inhibition of infection by primary HIV-1 strains, CEMx174 5.25 M7 cells were incubated with 100 × TCID~50~of primary HIV-1 strains in the absence or presence of PJ-S21 at graded concentrations for 3 days at 37°C. The experiments were done in triplicate. Infection was quantitated by measuring luciferase activity \[[@B23]\] using a kit from Promega (Madison, WI) in an Ultra 384 luminometer (Tecan, Research Triangle Park, NC). CD4-HIV-1 gp120 binding and its inhibition were measured by ELISA. Wells of 96-well polystyrene plates (Immulon II, Dynatech Laboratories, Inc., Chantilly, VA) were coated with 100 ng/well of either gp120 IIIB or gp120 BaL, and post-coated as described \[[@B16]\]. Dilutions of PJs and of PJ-S21, respectively, in 0.14 M NaCl, 0.01 M Tris, 0.02% sodium merthiolate, pH 7.0 (TS) containing 100 μg/ml bovine serum albumin (BSA) were added to the wells for 1 h at 37°C. The wells were washed 5 × with TS. Biotinyl-CD4 (1 μg/ml) in TS-1% gelatin was added to the wells for 5 h at 37°C. After washing 1 × with TS-0.1% Tween 20 and 5 × with TS, horseradish peroxidase (HRP)-streptavidin (0.625 μg/ml; Amersham, Arlington Heights, IL) in TS-2% gelatin-0.05% Tween 20 was added. After 30 min at 37°C, the wells were washed 4 × with TS-0.1% Tween 20 and 2 × with TS. Bound HRP was detected using a kit from Kirkegaard and Perry Laboratories Inc. (Gaithersburg, MD) and the absorbance (A) read at 450 nm. A in the absence of inhibitors was 1.0 to 1.5, and 0 to 0.005 in the absence of biotinyl-CD4. In an alternative assay, CD4 (500 ng/ml) was mixed with biotinyl-gp120 (1 μg/ml) in the presence or absence of inhibitors for 30 min at 20°C. Serial dilutions of the mixtures were added to wells coated with the anti-CD4 mAb OKT 4 (Ortho-Clinical Diagnostics, Rochester, NY) and captured biotinyl-gp120 was detected with HRP-streptavidin as described above. To measure binding to gp120 of antibodies to gp120 peptides, the respective rabbit antisera were diluted 50-fold in a mixture of FBS and goat serum (9:1) containing 0.1% Tween 20 (pH 8.0) and added to gp120 wells. Bound IgG was detected with HRP labeled anti-rabbit IgG (Sigma, St. Louis, MO; 1 μg/ml in TS-10% goat serum-0.1% Tween 20). A cell-based ELISA was used to measure the blocking of CCR5 binding sites on HIV-1 BaL gp120 by PJ and PJ-S21, respectively \[[@B20]\]. Briefly, FLSC (125 ng/ml) in the absence or presence of graded amounts of inhibitors was added to Cf2Th/synCCR5 cells fixed with 5% formaldehyde in wells of 96-well plates. After 1 h at 37°C, bound FLSC was detected with mAb M-T441 (125 ng/ml; Ancell, Bayport, MN) specific for the CD4 D2 domain, followed sequentially by biotinylated anti-mouse IgG and HRP-streptavidin. Results ======= Anti-HIV-1 activity of pomegranate juice ---------------------------------------- Serial twofold dilutions of juices \[apple, black cherry, blueberry, coconut milk, cranberry, elderberry, grape (red), grapefruit, honey, lemon, lime, pineapple, pomegranate and red beet (10% reconstituted dry powder)\] were assayed for inhibition of infection by HIV-1 IIIB of cells expressing the CD4 and CXCR4 receptors and coreceptors. Most juices (4-fold diluted) had no inhibitory activity, except blueberry, cranberry, grape and lime juice, respectively \[endpoints for 50% inhibition of infection (ED~50~) between 1/16 and 1/64\]. Consistently, PJs from distinct geographical areas had the highest inhibitory activity (Fig. [1](#F1){ref-type="fig"}; blue shaded area). Since HIV-1 viruses utilizing CCR5 as coreceptor (=R5 viruses) are predominantly transmitted sexually \[[@B3],[@B28]\], it was important to test whether PJ can inhibit not only infection by HIV-1 IIIB, a virus utilizing CXCR4 as coreceptor (=X4 virus), but also infection by an R5 virus, HIV-1 BaL. Results in Fig. [1](#F1){ref-type="fig"} (red shaded area) show that infection by the latter virus is also inhibited, albeit less effectively than that by HIV-1 IIIB. Blocking virus entry is a primary target for microbicide development \[[@B3],[@B8]-[@B11]\]. Therefore, it was of interest to determine whether or not PJ inhibited the binding of the HIV-1 envelope glycoprotein gp120 to CD4, the common receptor for both X4 and R5 viruses. Pretreatment of both gp120 IIIB and BaL by PJ inhibited subsequent binding of soluble labeled CD4 (Fig. [2](#F2){ref-type="fig"}). This suggested that one or more PJ ingredients bound strongly or irreversibly to the CD4 binding site on gp120. These results, obtained in an ELISA using gp120 immobilized on polystyrene plates, were confirmed in another assay in which both gp120 and CD4 were in soluble form (data not shown). In reverse experiments, pretreatment of CD4 with PJ failed to block subsequent gp120 binding. Other juices having anti-HIV-1 activity (blueberry, cranberry, grape and lime) failed to block gp120-CD4 binding. To delineate sites on gp120 blocked by the PJ inhibitor(s), the inhibitory effect of PJ on binding to gp120 IIIB of antibodies to peptides derived from the amino acid sequence of gp120 was studied. The binding of antibodies to peptides (102--126), (303--338), (306--338), (361--392), (386--417), (391--425), (411--445) and (477--508) was significantly (≥ 50%) inhibited (Fig. [3](#F3){ref-type="fig"}). The binding to gp120 IIIB of monoclonal antibodies 9284 and 588D, specific for the gp120 V3 loop (residues 303 -- 338) and the CD4 binding site, respectively \[[@B29],[@B30]\] was each inhibited by 97%. Some of the relevant peptides contain residues involved in CD4 binding \[[@B31]-[@B33]\] while all discerned peptides include residues involved in coreceptor binding \[[@B34]-[@B39]\]. The locations of the peptides and of residues involved in receptor/coreceptor binding on the X-ray crystallographic structure of gp120 are shown in Fig. [4](#F4){ref-type="fig"}. These results suggest that the PJ inhibitor(s) may also block gp120-coreceptor binding. This will be addressed subsequently. Separation of anti-HIV-1 inhibitor(s) from pomegranate juice ------------------------------------------------------------ PJ is intensely colored. Therefore, it cannot be directly formulated into a microbicide since it would stain clothing, which is unacceptable. Attempts were made to separate or isolate the active ingredient(s) from PJ. After striving intermittently for over four years to accomplish this, it was discovered that the inhibitor(s) of gp120-CD4 binding can be adsorbed effectively (≥ 99%) onto a selected brand of corn starch (Fig. [5](#F5){ref-type="fig"}), resulting in a nearly colorless product, designated as PJ-S21. PJ-S21, suspended in water or unbuffered 0.14 M NaCl had a pH of 3.2, compatible with the acidic vaginal environment in which it would remain stabile after application (see below). Inhibitors of gp120-CD4 binding could be eluted from PJ-S21 by extraction with ethanol/acetone 6:4. Drying of the extract followed by gravimetry indicated that the extract contained 3.17 mg solids per gram of PJ-S21. PJ-S21, to the same extent as the original PJ, inhibited the binding of gp120 IIIB-CD4 complexes to cells expressing CXCR4, as determined by flow cytometry (Fig. [6](#F6){ref-type="fig"}). Similarly, binding of a gp120 BaL-CD4 fusion protein to cells expressing CCR5 was blocked by PJ and PJ-S21, as detemined by a cell based ELISA \[[@B20]\]; (Fig. [7](#F7){ref-type="fig"}). Thus, PJ-S21 is an inhibitor of both X4 and R5 virus binding to the cellular receptor CD4 and coreceptors CXCR4/CCR5. PJ-S21 also inhibited gp120 binding to PBMCs as determined by flow cytometry (Fig. [8](#F8){ref-type="fig"}). To confirm that PJ-S21 functions as a virus entry inhibitor, the complex was added to cells at time intervals before and after infection of cells by HIV-1 IIIB and BaL, respectively. Results shown in Fig. [9](#F9){ref-type="fig"} demonstrate that PJ-S21 interferes with early steps of the virus replicative cycle. To be considered as a topical microbicide, PJ-S21 must be formulated to withstand storage in a tropical environment. Accelerated thermal stability studies revealed that a water suspension of PJ-S21 maintained only 4, 11, and 33%, respectively, of its original activity (measured by inhibition of gp120-CD4 binding) when stored for 30 min at 60°C, and one week at 50°C or 40°C. On the other hand, a dried PJ-S21 powder remained fully active after storage at 50°C for 12 weeks (the longest time used in the evaluation). Consequently, anhydrous formulations should be preferred for further development. Three such formulations were prepared: two kinds of suppositories, melting at 37°C, and a tablet (for compositions see Methods section). The inhibitory activity of PJ-S21 was fully preserved after 12 weeks storage at 50°C within tablets, and at 30°C within the suppositories (the highest temperature considered to prevent melting). Data showing the inhibition of infection by HIV-1 IIIB and BaL respectively, by PJ-S21 and its formulations (except the tablets which also contain anti-HIV-1 inhibitors other than PJ-S21, i.e. Carbopol 974P \[[@B18]\]) are summarized in Fig. [10](#F10){ref-type="fig"}. Their inhibitory activities against HIV-1 IIIB and BaL were similar, unlike the inhibitory activities of the original PJs (Fig. [1](#F1){ref-type="fig"}). These formulations were also virucidal, albeit at concentrations higher than those sufficient for inhibition of infection. These experiments also revealed that PJ-S21 was not cytotoxic under the experimental conditions used. The inhibitory/virucidal activities were maintained in the presence of seminal fluid (SF) at a 1:1 (w/w) ratio of SF to PJ-S21; (data not shown). A microbicide can be considered potentially successful, only if it has antiviral activity against primary virus isolates belonging to distinct virus clades and phenotypes. PJ-S21 meets this requirement since it inhibited infection by primary HIV-1 strains of all clades tested having R5 and X4R5 (= dual-tropic) phenotypes (Table [1](#T1){ref-type="table"}). Discussion ========== Pomegranates have been venerated for millennia for their medicinal properties and considered sacred by many of the world\'s major religions. In deference to pomegranates, the British Medical Association and several British Royal Colleges feature the pomegranate in their coat of arms. The Royal College of Physicians of London adopted the pomegranate in their coat of arms by the middle of the 16^th^Century \[[@B40]\]. The best known literary reference to the contraceptive power of pomegranate seeds is classical Greek mythology. Persephone (IIερσεφονη) had eaten six pomegranate kernels (from which juice is derived) while in the Underworld and for that many months the land remained infertile during the Fall and Winter (Fig. [11](#F11){ref-type="fig"}). Ironically, this report shows that pomegranate juice contains HIV-1 entry inhibitors targeted to the virus envelope corresponding to a class of anti-retroviral drugs still scarce in development \[[@B41]\]. PJ contains several ingredients \[[@B42],[@B43]\] which, isolated from natural products other than PJ, were reported to have anti-HIV activity, for example: caffeic acid \[[@B44]\], ursolic acid \[[@B45]\], catechin and quercetin \[[@B46],[@B47]\]. However, these compounds, in purified form, obtained commercially, did not block (at 200 μg/ml) gp120-CD4 binding as measured by the ELISA described above and did not adsorb to corn starch, unlike the entry inhibitor(s) from PJ. In fact, the supernatant after treatment of PJ with starch, and removal of the entry inhibitors, retained anti-HIV-1 activity and also inhibited infection by herpes virus type 1, unlike the HIV-1 entry inhibitors which adsorbed onto starch. Thus, the antiviral activities in the supernatant appeared to be non-specific, and probably similar to those of extracts from pomegranate rind \[[@B48],[@B49]\], and were not characterized further. Additional information \[[@B50]-[@B53]\] has revealed that the findings apply to crude extracts from pomegranate rind prepared at elevated temperatures under conditions which destroy the HIV-1 entry inhibitor described here. The inhibitor(s) interfering with gp120 binding to CD4 (Fig. [2](#F2){ref-type="fig"} and [5](#F5){ref-type="fig"}) blocked additional sites on gp120 (Fig. [3](#F3){ref-type="fig"}) involved in interaction with the CXCR4/CCR5 coreceptors (Fig. [4](#F4){ref-type="fig"}, [6](#F6){ref-type="fig"} and [7](#F7){ref-type="fig"}). This was not completely expected and can be explained either by the presence of multiple inhibitors with distinct or overlapping specificities in PJ-S21 or by induction of gp120 conformational changes \[[@B54]\] resulting in blockade of both CD4 and CXCR4/CCR5 binding sites on gp120. Similar effects have been noticed for other small molecule inhibitors \[[@B55]\]. Simultaneous blocking of more than a single site on HIV-1 involved in virus entry is expected to increase the effectiveness of candidate microbicides \[[@B11]\]. The target sites for the inhibitor(s) are likely to be located within the protein moiety of gp120 since binding of labeled Galanthus nivalislectin (specific for terminal mannose residues \[[@B56]\]; and other lectins to gp120 oligosaccharides was not diminished in the presence of PJ or PJ-S21 (data not shown). Blocking of CD4 binding sites on HIV-1 gp120 by monoclonal antibodies or a CD4-IgG2 recombinant protein has been shown to be sufficient to inhibit HIV-1 infection of human cervical tissue ex vivo\[[@B11]\] and in preventing virus transmission to macaque monkeys when applied vaginally \[[@B57]\]. Therefore, it seems likely that PJ-S21 will be similarly effective, an expectation which remains to be confirmed. The application of PJ-S21 as a topical anti-HIV-1 microbicide requires reasonable uniformity among batches produced at distinct times and locations. Similarities in gp120-CD4 binding inhibitory activity among distinct freshly prepared and commercial juices stored for unknown periods (Fig. [2](#F2){ref-type="fig"}) suggest that this should be feasible. Pasteurization of juice for 30 seconds at 85°C resulted in complete loss of inhibitory activity. A commercial PJ concentrate exposed to 61°C, and two other concentrates, presumably prepared by evaporation at elevated temperatures, had no or drastically diminished activity. The gp120-CD4 inhibitory activity from PJ3 (juice with fructose and citric acid added), failed to bind to starch. Separate experiments revealed that these compounds interfere with inhibitor binding to corn starch. Therefore, PJs intended for production of the PJ-S21 complex must be sterilized by filtration and be free of additives. Particular attention must be devoted to the selection of starch, a pharmaceutical excipient generally used in vaginal formulations \[[@B58]\], for effective binding of the virus entry inhibitors from PJ. Among a dozen starches tested, the best results have been obtained with S21. With other brands, the adsorption of the inhibitors was either incomplete or their binding did not result in a complex having activity in the ELISA measuring gp120-CD4 binding inhibition (ARGO^®^corn starch), presumably, because of irreversible binding of the PJ inhibitors. Interestingly, there are only a few references available regarding the use of starch as an adsorbent for different compounds: flavors \[[@B59],[@B60]\], dyes \[[@B61]-[@B63]\], low-molecular mass saccharides \[[@B64]\], lipids \[[@B65],[@B66]\], proteins \[[@B67]\] and iodine \[[@B68]\]. The intended dose of PJ-S21 for vaginal application is 1.0 to 1.5 g, (= 3.17 -- 4.76 mg solids from PJ adsorbed onto starch) i.e. ≥ 100-fold higher than the dose needed for blocking HIV-1 infection in vitro(Fig. [10](#F10){ref-type="fig"}, Table [1](#T1){ref-type="table"}), and thus expected to meet requirements for likely in vivoprotection against vaginal challenge \[[@B69]\]. This quantity of PJ-S21 is produced from 5 to 7.5 ml of PJ, i.e. ≤ 5% of a single (150 ml) serving of juice, attesting to the safety, feasibility and economy of this proposed candidate topical microbicide. In an alternative approach to formulation development, PJ-S21 can be incorporated into a water dispersible film (similar to the widely available \"breath control\" strips) or into water dispersible sponges \[[@B70]\] which are converted into a gel following topical application \[[@B19]\]. Each of the above formulations would meet the following requirements: (1) minimization of waste disposal problems associated with the use of applicators needed for delivery of microbicidal gels/creams; (2) simplicity; (3) small packaging and discretion related to purchase, portability and storage; (4) low production costs; (5) amenability to industrial mass production at multiple sites globally and (6) potential application as rectal microbicides. Furthermore, it would remain possible to produce for local use PJ-S21 based gel formulations with a limited shelf life, avoiding the costs of producing dry PJ-S21 powders via appropriate low temperature drying processes. Whichever of these formulations is selected, adequate quality control will be needed to assure uniform anti-HIV-1 activity of the final product(s) and to establish reproducible conditions for manufacture. Conclusions =========== PJ-S21 can be classified as an AAAA candidate microbicide: Acceptable; Accessible; Affordable; and Accelerative in transition from development to marketing. Thus, PJ-S21 would be expected to circumvent some problems associated with antiretroviral drugs and possibly some of the other candidate microbicides, i.e. uncertainty related to potential side effects, investment and time needed to establish inexpensive large scale production, and monopoly of supply. Abbreviations used ================== AIDS, acquired immunodeficiency syndrome; BSA, bovine serum albumin; ED~50(90)~, effective dose(s) for 50% (90%) inhibition of infection; ELISA, enzyme linked immunosorbent assays; FBS, fetal bovine serum; FLSC, a full length single chain protein consisting of BaL gp120 linked with the D1D2 domains of CD4 by a 20 amino acid linker; HIV-1, human immunodeficiency virus type 1; HRP, horseradish peroxidase; LTR, long terminal repeat; PBMCs, peripheral blood mononuclear cells; PBS, phosphate buffered saline; PEG, polyethylene glycols; PJ, pomegranate juice; S21, PURITY^®^21 corn starch NF grade; SF, seminal fluid. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= ARN developed the concepts representing the basis of the manuscript and designed most experiments. NS contributed to the development of experimental techniques and carried out experiments other than infectivity assays. YYL did all the tissue culture work and viral infectivity assays. AKD did all the molecular modeling studies and contributed to the development of cell based enzyme linked immunosorbent assays. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/4/41/prepub> Acknowledgements ================ We thank Dr. Shibo Jiang and Dr. Hong Lu for carrying out all experiments with primary HIV-1 isolates and for analysis of the resulting data; Dr. Qian Zhao for experiments with the full length single chain protein consisting of HIV-1 BaL gp120 linked with the D1D2 domains of CD4 (FLSC); Veronica L. Kuhlemann for assistance and editorial help in preparing the manuscript and for production of all graphs; and Ruth A. Croson-Lowney for flow cytometry. Financial support for this research was provided by the Marilyn M. Simpson Charitable Trust, the Glickenhaus Foundation, and an NIH grant PO1HD41761. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Inhibition of HIV-1 infection of HeLa-CD4-LTR-β-gal and U373-MAGI-CCR5E cells, respectively, by pomegranate juice (PJ).Blue shaded area = HIV-1 IIIB; red shaded area = HIV-1 BaL. Four distinct PJs (PJ1 to PJ4) were tested. Infection was monitored by measuring β-galactosidase. ::: ![](1471-2334-4-41-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Inhibition of CD4 binding to recombinant gp120 IIIB and BaL, respectively, by pomegranate juice (PJ).Recombinant gp120 coated wells were incubated with dilutions of the PJ for 1 h at 37°C. After removal of the juice, and washing the wells, biotinyl-CD4 was added, and its binding to the wells was measured by ELISA. ::: ![](1471-2334-4-41-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Inhibition by pomegranate juice (PJ) of binding to gp120 of antibodies to synthetic peptides from the gp120 sequence.Wells of polystyrene plates coated with gp120 IIIB were incubated with 4-fold diluted PJ for 1 h at 37°C. After removal of PJ, the wells were washed, and 50-fold diluted anti-peptide antisera \[21\] were added. Bound IgG was quantitated by ELISA. PJ was not added to control wells. Decreases of absorbance, as compared to the respective control wells, are plotted. ::: ![](1471-2334-4-41-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Location on the gp120 structure of segments corresponding to anti-peptide antibodies whose attachment to gp120 is inhibited by ≥ 50 % in the presence of pomegranate juice (red) and of amino acid residues involved in CD4 and CXCR4/CCR5 coreceptor binding, respectively.Green, portions of the structure corresponding to anti-peptide antibodies whose attachment to gp120 is not significantly inhibited by PJ. The CD4 domains and the antigen-binding fragment of a neutralizing antibody were excised from the structure of the gp120-CD4-antibody complex \[31\] (1gc1 retrieved from the Protein Data Bank (pdb) \[http://www.rcsb.org/pdb/). The V3 loop, generated by homology modeling, was added to the gp120 structure as described \[16\]. The figure was generated by Molscript \[71\] and Raster3D \[72,73\]. The locations of gp120 variable loops (V1 -- V5) and of the N- and C-termini of the sequence are indicated. ::: ![](1471-2334-4-41-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Adsorption onto corn starch of gp120-CD4 binding inhibitor(s) from pomegranate juice (PJ).Corn starch (PURITY^®^21, NF grade; 200 mg/ml) was added to PJ prefiltered to remove particulates. After mixing for 1 h at ≈20°C, the starch was allowed to settle and the supernatant fluid was removed by aspiration. The pellets, resuspended (200 mg/ml) in phosphate buffered saline, and the supernatant fluids were tested at serial dilutions for inhibition of CD4 binding to gp120 IIIB as described in the legend for Fig. 2. The inhibitory activity of the resuspended pellet against gp120 BaL-CD4 binding was then confirmed. Control starch did not inhibit gp120-CD4 binding. ::: ![](1471-2334-4-41-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Inhibition by pomegranate juice (PJ) and PJ-S21, respectively, of gp120 IIIB-CD4 complex binding to cells expressing CXCR4 coreceptors.HIV-1 IIIB gp120 (5 μg) and biotinyl-CD4 (2.5 μg) were added to 100 μl phosphate buffered saline (PBS) containing 100 μg/ml bovine serum albumin (BSA) (PBS-BSA) and PJ (final 3-fold dilution) or PJ-S21 (67 mg; corresponding to 212 μg solids from PJ adsorbed onto starch). After 1 h at 20°C, the respective mixtures were added to 10^6^MT-2 cells. After 30 min, the cells were washed 3 times with PBS-BSA and PE-streptavidin (a fluorescent label specific for biotin; 0.1 μg) was added. After 20 min, the cells were washed and fixed by 1% formaldehyde in PBS. Flow cytometry analysis was performed in a FACSCalibur flow cytometer (Becton Dickinson Immunocytometric Systems, San Jose, CA). The median relative fluorescence values for cells exposed to gp120-CD4; gp120-CD4 + PJ; gp120-CD4 + PJ-S21; and control cells were: 13.7; 4.0; 4.3; and 2.1, respectively. ::: ![](1471-2334-4-41-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Inhibition by pomegranate juice (PJ) and PJ-S21, respectively of FLSC binding to CCR5 expressing Cf2Th/synCCR5 cells.FLSC is a chimeric recombinant protein consisting of gp120 BaL linked with D1D2 domains of CD4. The inhibitory effect was quantitated using a cell-based ELISA \[20\]. The starting concentration of PJ-S21 was 200 mg/ml, corresponding to 634 μg/ml solids adsorbed onto starch from PJ. ::: ![](1471-2334-4-41-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Inhibition by PJ-S21 of biotinyl-gp120 IIIB binding to peripheral blood mononuclear cells (PBMCs).HIV-1 IIIB biotinyl-gp120 (5 μg) was added to 100 μl of PBS-BSA containing graded quantities of PJ-S21. After 1 h at 20°C, the respective mixtures were added to 10^6^PBMCs. After 30 min the cells were washed 3× with PBS-BSA and PE-streptavidin (0.1 μg was added). Subsequently the procedures described in the legend to Fig. 6 were used. The median relative fluorescence values for control cells and cells exposed to biotinyl-gp120 in the absence and presence of PJ-S21 (100, 6.25 and 3.12 mg/ml) were 4.1, 81.31, 12.2, 35.2 and 50.0 respectively. 100 mg of PJ-S21 corresponds to ≈ 320 μg solids adsorbed from PJ onto starch. ::: ![](1471-2334-4-41-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Inhibition of HIV-1 IIIB or BaL replication depends on the time of PJ-S21 addition pre- or post-infection.For comparison, the inhibition of infection by the nonnucleoside reverse transcriptase inhibitor TMC-120, added to cells at distinct intervals after HIV-1, was determined (dotted lines). Virus infection was measured by quantitation of β-galactosidase. ::: ![](1471-2334-4-41-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### HIV-1 inhibitory and virucidal activity of PJ-S21 and its formulations.Inhibition of infection by HIV-1 IIIB and BaL, respectively, was determined as described in the legend for Fig. 1. To measure virucidal activity, the respective viruses were mixed with graded quantities of PJ-S21 for 5 min at 37°C. After low speed centrifugation, the viruses were separated by precipitation with PEG 8000 and centrifugation. The resuspended pellets and control untreated viruses were serially diluted, and the dilutions assayed for infectivity. The concentration range given on the abscissa corresponds to 0.31 -- 1,268 μg solids adsorbed from PJ to starch. ::: ![](1471-2334-4-41-10) ::: ::: {#F11 .fig} Figure 11 ::: {.caption} ###### Persephone (IIερσεφονη) holding a pomegranate.In Greek mythology she had eaten seeds of the fruit and consequently was condemned to remain in Hades, the Underworld, for six months of every year. Derived from a painting of Dante Gabriel Rossetti in the Collection of the Tate Gallery in London. ::: ![](1471-2334-4-41-11) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Inhibitory activity of PJ-S21 on infection by primary HIV-1 strains ::: Primary strain Subtype, Coreceptor use ED~50~\* mg/ml ED~90~\* mg/ml ---------------- ------------------------- ---------------- ---------------- 92RW008 A, R5 0.50 ± 0.05 2.76 ± 0.28 94UG103 A, X4R5 1.42 ± 0.54 3.42 ± 0.98 92US657 B, R5 0.62 ± 0.11 2.86 ± 0.33 93IN101 C, R5 3.56 ± 1.10 8.87 ± 2.55 93MW959 C, R5 1.02 ± 0.19 3.54 ± 0.90 92UG001 D, X4R5 0.62 ± 0.17 2.94 ± 0.85 93THA051 E, X4R5 0.86 ± 0.01 4.09 ± 0.08 93BR020 F, X4R5 4.25 ± 0.78 8.31 ± 1.04 RU570 G, R5 0.42 ± 0.09 1.54 ± 0.16 BCF02 Group O, R5 0.59 ± 0.29 3.92 ± 0.27 \* ED~50(90)~= effective dose(s) of PJ-S21 for 50% (90%) inhibition of infection. One gram of PJ-S21 contains approximately 3.2 mg of the inhibitors adsorbed to starch from pomegranate juice. :::	PubMed Central	2024-06-05T03:55:49.833447	2004-10-14	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533885/", "journal": "BMC Infect Dis. 2004 Oct 14; 4:41", "authors": [ { "first": "A Robert", "last": "Neurath" }, { "first": "Nathan", "last": "Strick" }, { "first": "Yun-Yao", "last": "Li" }, { "first": "Asim K", "last": "Debnath" } ] }
PMC533886	Background ========== Mental fatigue with reduced capacity for attention, concentration, and learning, as well as subsequent disturbance of short-term memory, is a common symptom in diseases with general or patchy neuroinflammation, such as multiple sclerosis (MS) and neurodegenerative diseases, such as Ahlzheimer\'s and Parkinson\'s diseases \[[@B1]-[@B6]\]. The mental fatigue often appears prior to other more prominent mental, cognitive, or physical symptoms from the nervous system in these diseases. Mental fatigue is also common during the rehabilitation after meningitis or encephalitis (postinfectious mental fatigue), stroke or brain trauma (posttraumatic mental fatigue), being especially troublesome when major neurological symptoms have disappeared and the patient is on his way back to work. According to the International Classification of Diseases, 10th revision (ICD-10), mental fatigue is covered by the diagnoses \"mild cognitive disorder\" or \"neurasthenia\" and according to the Diagnostic and Statistical Manual of Mental Disorders, 4th edition \[[@B7]\], mental fatigue is included in the group of \"mild neurocognitive disorders\". According to the diagnostic classification by Lindqvist and Malmgren \[[@B8]\], mental fatigue is one of the symptoms of the \"astheno-emotional syndrome\". Although mental fatigue is not exactly the same as depression, where the patient has a feeling of not being able to do anything, there are overlaps and both disorders have behavioral manifestations such as reduction in motivation that would appear similar in animal models, where affective state is either irrelevant or difficult to assess. Even the \"sickness behavior\" \[[@B9]\] contains a component of fatigue. Mental fatigue is also prominent after sleep deprivation. In addition to the fatigue itself, the patient with mental fatigue often suffers from loudness and light sensitivity, irritability, affect lability, stress intolerance, and headaches \[[@B8]\]. Mental fatigue appears as a decreased ability to intake and process information over time. Mental exhaustion becomes pronounced when cognitive tasks have to be performed for longer time periods with no breaks (cognitive loading). Often, the symptoms are absent or mild in a relaxed and stress-free environment. To explore the possible cellular neurobiology of mental fatigue, we start by looking at some components important for information intake and processing within the central nervous system, namely glutamate neurotransmission, and focus on the clearance of extracellular glutamate (\[Glu\]~ec~). Glutamate neurotransmission is indispensable for information intake and processing within the central nervous system ==================================================================================================================== Glutamate neurotransmission is crucial in information intake and information processing within the brain \[see \[[@B10]\]\]. Glutamate transmission is also indispensable for long-term potential (LTP) formation, the cellular correlate to memory formation \[see \[[@B11]\]\]. In brain, the \[Glu\]~ec~has to be maintained at approximately 1--3 μM in order to assure a high precision (high signal-to-noise ratio) at normal glutamate neurotransmission \[[@B12]\] and also, to avoid excitotoxic actions of glutamate on neurons. The clearance of glutamate from the extracellular space is achieved by high-affinity, sodium (Na^+^)-dependent electrogenic uptake transporters. The glutamate aspartate transporter (GLAST) and glutamate transporter 1 (GLT-1) are most abundantly located on astrocytes surrounding synapses of glutamate-bearing neurons \[[@B13]\]. In fact GLAST and GLT-1 have different expression patterns. GLAST is the major transporter for glutamate uptake during development while expression of GLT-1 increases with the maturation of the nervous system. Glutamate transporter 1 expression seems to follow the formation and maturation of synapses and especially synaptic activity \[[@B14]\]. Even more convincing for the role of astroglia in keeping the \[Glu\]~ec~low, it has been demonstrated with knockout techniques in rats that loss of GLT-1 or GLAST produces elevated \[Glu\]~ec~and neurodegeneration characteristic of excitotoxicity, while the loss of neuronal glutamate transporter does not elevate \[Glu\]~ec~\[[@B15]\]. Regulation of astroglial glutamate transporter capacity -- role of proinflammatory cytokines ============================================================================================ A large number of factors have been shown to affect the activity and expression of the glutamate transporters GLT-1 and GLAST. For example, GLT-1 is stimulated by phosphorylation by protein kinase C (PKC), while GLAST is inhibited by PKC at a non-PKC consensus site \[[@B16]\]. The synthesis of GLT-1 has been shown to be stimulated by factors acting via receptor tyrosine kinases and pathways dependent on phosphatidylinositol-3-kinase (PI3K) and the nuclear transcription factor NFκB. One mechanism of regulation of GLT-1 is related to formation of cysteine bridges. Glutamate transporter 1 contains cysteines that are sensitive to oxidative formation of cysteine bridges. Oxidative species such as hydrogen peroxide can readily oxidize the functional sulfhydryl groups of cysteines, to form disulfide bridges which exert an inhibitory effect towards glutamate transports \[[@B17]\]. Examples of factors or altered conditions that impair astroglial glutamate transport are arachidonic acid, lactic acid, cytokines, and leukotrines, nitric oxide (NO), β-amyloid protein, peroxynitrate, and glucocorticoids. The altered conditions could be disturbed energy metabolism with lowering of adenosine triphosphate (ATP) levels or lowering of pH. Notable is the finding that many of these substances or conditions also decrease astroglial gap junction communication and even disintegrate the BBB, thus impairing the astroglial support of the glutamate neurotransmission \[for references, see \[[@B18]\]\]. Proinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 have since long been known to impair astroglial glutamate uptake even if the mechanisms are not fully understood. The inhibitory function of TNF-α was established as early as the 1990s, when TNF-α was shown to inhibit astroglial glutamate uptake \[[@B19]\]. Hu and coworkers \[[@B20]\] reported a dose-dependent inhibition of astrocyte glutamate uptake by a mechanism involving nitric oxide (NO). In a study from 2001, Liao and Chen \[[@B21]\] demonstrated that TNF-α potentiates glutamate-mediated oxidative stress, which results in a decrease in glutamate transporter activity. Recently, Wang and coworkers \[[@B22]\] showed a reduced expression of GLT-1 and GLAST, and also, an impaired glutamate transport in human primary astrocytes, by TNF-α. The nuclear factor NFκB has been suggested to be involved in this regulation \[[@B23]\]. Even IL-1β and IL-6 have been shown to impair astroglial glutamate uptake capacity by involvement of oxidative stress or NO \[[@B20],[@B24],[@B25]\]. Even dysregulation of the blood brain barrier (BBB) is seen early in neuroinflammation, and parallels the release of proinflammatory cytokines \[[@B26]-[@B28]\]. Mechanisms for disruption of the BBB in neuroinflammation are incompletely understood, but appear to involve direct effects of cytokines on endothelial regulation of BBB components. Exposure of endothelium to TNF-α interrupts the BBB by disorganizing cell-cell junctions. Furthermore, TNF-α has been shown to depress calcium (Ca^2+^) signaling between BBB endothelial cells by reducing gap junction coupling and inhibiting triggered ATP release \[[@B29]\]. Could glutamate neurotransmission be dynamically regulated by extracellular glutamate levels? ============================================================================================= As stated above, already when the \[Glu\]~ec~exceeds some 3--5 μM, the efficiency of the glutamate signaling is considered to be reduced \[[@B12]\]. There is prolonged postsynaptic and adjacent glial receptor activation \[[@B30]\], with less precision (with a decreased signal-to-noise ratio) in the glutamatergic transmission. As a consequence, the information taken into the brain will be less distinct. In addition, activation of astroglial networks, with induction of Ca^2+^oscillations, both within and between the gap junction-coupled astroglial syncytia \[[@B31]-[@B33]\], and with subsequent astroglial glutamate release \[[@B34]\] could increase the excitability level in neighboring neuronal circuits. The overall result may be that more, and larger, neuronal circuits would be activated over time \[[@B35],[@B36]\]. This conclusion is further supported by studies demonstrating that inhibition of GLT-1 could facilitate hippocampal neurotransmission \[[@B37]\] and lead to increased neuronal excitability, as seen in for example hepatic encephalopathy \[[@B38]\]. Increased \[Glu\]~ec~would also lead to astroglial cell swelling, with a resulting decrease in the extracellular space volume, and locally further increased \[Glu\]~ec~\[[@B39]-[@B42]\]. The astroglial swelling would give rise to relative depolarization of the astroglial cell membrane, with a further decreased astroglial glutamate uptake capacity, and in addition, a decreased capacity of the astrocytes to remove \[K^+^\]~ec~\[[@B43],[@B44]\]. Even moderately increased (up to 8--10 mM) \[K^+^\]~ec~levels have been shown in experimental systems to inhibit glutamate release \[[@B45]\]. Recent data indicate a dynamic and fine-tuning regulation of the glutamatergic transmission. One mechanism by which neurons regulate excitatory transmission is by altering the number and composition of glutamate receptors at the postsynaptic plasma membrane. This has been shown for the NMDA receptor in experimental systems and could have prominent importance for dynamic processes as learning and memory \[[@B46]\]. Of great importance in this context are also studies where stimulation of metabotropic glutamate receptors (mGluR3 and mGluR5) have been shown to critically and differentially modulate the expression of glutamate transporters \[[@B47]\] thus creating a substrate for a fine-tuning of the glutamate neurotransmission. Even the proinflammatory cytokine IL-1β could act as a regulator of glutamate transmission, as it was shown recently that this cytokine inhibits glutamate release and reduces LTP as a consequence of the formation of reactive oxygen species \[[@B11]\]. Furthermore, in states of decreased astroglial glutamate uptake capacity, even astroglial glucose uptake, and consequently the supply of metabolic substrates to the neurons, has been reported to decrease \[[@B48]-[@B50]\] and there may be relative energy insufficiency at the cellular level in neuronal circuits. In addition, glutamate release from the presynaptic terminals could decrease due to factors such as a decreased glutamine supply of the neurons. Experimental investigations in the rat and monkey have demonstrated a feedback loop from the left basal frontal cortex, with an inhibitory influence on the locus coeruleus in the brain stem \[[@B51]\]. If this loop also exists in humans, a slight increase in the neuronal firing due to slightly elevated \[Glu\]~ec~in the basal frontal cortex could lead to a decrease in the noradrenaline and serotonin (5-HT) release in the cerebral cortex, which would also decrease glucogenolysis \[[@B52],[@B53]\] and, furthermore, impair metabolic substrates for cortical neurons. Thus, it might be that glutamate neurotransmission could be regulated by changing astroglial glutamate transporter capacity, and thus, increases in \[Glu\]~ec~levels could be one factor to impair glutamate transmission. Proinflammatory cytokines and neuroinflammatory and degenerative diseases, major depression, sickness behavior, and sleep deprivation ===================================================================================================================================== There is an extensive literature on inflammatory response with microglial activation and the production of proinflammatory cytokines (TNF-α, IL-1β and IL-6) in neuroinflammatory/infectious and neurodegenerative diseases as well as in stroke and trauma \[[@B5],[@B54]\]. The inflammatory activation starts early in some neurodegenerative disease such as Alzheimer\'s and Parkinson\'s diseases, being prominent for long time in these diseases and also in neuroinflammatory diseases, in meningitis, encephalitis and in trauma or stroke \[see \[[@B54]\]\]. Several groups have also described enhanced production of proinflammatory cytokines in major depression \[see \[[@B55]\]\] and sickness behavior \[[@B9],[@B56],[@B57]\]. This is interesting as there are overlaps between mental fatigue and these disorders. Furthermore, proinflammatory cytokines are activated in sleep deprivation \[[@B58]\], a state where mental fatigue is often prominent. In states of anxiety and stress, often experienced as secondary to mental fatigue, increased glucocorticoid levels have been demonstrated. Interestingly, long-term increases in glucocorticoids have been demonstrated to result in the production of both TNF-α and IL-1β \[[@B59]\]. Could mental fatigue be the consequence of a dysfunction in a specific brain region? ==================================================================================== In the search for pathophysiological correlates to fatigue in MS, Roelcke and co-workers \[[@B60]\] demonstrated reduced glucose metabolism in the frontal cortex and basal ganglia in MS patients with fatigue. A hypotheses by Chaudhuri and Behan \[[@B6]\] also focused on basal ganglia as one part of the brain crucial for mental fatigue to appear. Using patients with chronic fatigue syndrome, which is not however exactly the same as mental fatigue, studies have revealed prefrontal and temporal cortices, anterior cingulate and cerebellum as regions possibly involved in fatigue \[[@B61]\]. Interestingly these later studies also pointed at a possible connection between glutamate transmission and fatigue. Even if the mental fatigue is not the central problem in attention deficit hyperactivity disorder (ADHD), some of the symptoms in this disorder is similar to the symptom complex associated with mental fatigue, and there is some support for glutamate being involved in the disorder and its treatment \[[@B62]\] and also, at least hypothetically, a deficient astroglial metabolism due to decreased noradrenaline and serotonin levels \[[@B63]\]. Until now there is no evidence for a specific brain region being affected in mental fatigue. On the contrary, it seems that mental fatigue could appear from disturbances of different neuronal systems. We will therefore present a hypothesis (figure [1](#F1){ref-type="fig"}) where the functional disturbance of mental fatigue at the cellular level is coupled to the fine-tuning of the glutamate neurotransmission. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Schematic drawing of cellular regulation of extracellular glutamate concentrations (\[Glu\]~ec~) in normal brain function (left), and in the presence of the proinflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 (right). Possible pathophysiology underlying mental fatigue at the cellular level is outlined below. [To the left:]{.underline} Two neuronal cell bodies with processes (white) make contact with each other through a synapse (center). Astrocytic (pink) processes encapsulate the synapse and cover also the abluminal side of the blood vessel wall (right). The endothelial cells covering the luminal (blood) side of the vessel wall and the astrocytic processes make up the blood brain barrier (BBB). An oligodendroglial cell (bluish), with its myelin encapsulating the axon, and a microglial cell (yellow) are seen. The astrocytes, with their high-affinity glutamate transporters, are the main site for keeping \[Glu\]~ec~low. Even neurons express glutamate transporters, as do oligodendroglial cells, and endothelial cells at their abluminal side. [To the right:]{.underline} TNF-α, IL-1β and IL-6 attenuate astroglial glutamate uptake transport and disintegrate the BBB, allowing glutamate from the blood to enter the brain. The overall result is slightly increased \[Glu\]~ec~. Tumor necrosis factor-alfa also decreases oligodendroglial cell glutamate uptake \[78\], while microglial glutamate uptake has been demonstrated to increase (Persson, M., Hansson, E., and Rönnbäck, L, to be published), though not to levels to compensate for the decreased astroglial glutamate uptake capacity. Due to increased \[Glu\]~ec~, astroglial swelling is shown. [Below:]{.underline} Hypothetic cellular events underlying mental fatigue. Slightly increased \[Glu\]~ec~could make the glutamate neurotransmission less distinct (decrease the signal-to-noise ratio). At the cellular level, there would be astroglial swelling, which in turn would decrease the local extracellular (ec) volume and, as a consequence, lead to further increased \[Glu\]~ec~. Astroglial swelling also depolarizes the astroglial cell membrane, which further attenuates the electrogenic glutamate uptake and, in addition, the astroglial K^+^uptake capacity. As a consequence, even \[K^+^\]~ec~may rise. The increased \[K^+^\]~ec~, together with decreased glutamine production and reduced glucose uptake concomitant with the decreased glutamate uptake, could lead to decreased presynaptic glutamate release and thereby decreased glutamate transmission, which, according to our hypothesis, is one cellular correlate to mental fatigue/exhaustion. Increased extracellular glutamate levels in the prefrontal region could lead to inhibition of the brain stem nuclei locus coeruleus (LC) and raphe nuclei and thereby inhibit noradrenaline (NA) and serotonin (5-HT) release in the cerebral cortex resulting in decreased astroglial metabolism and neuronal metabolic supply. Increased neuronal excitability may be part of the loudness and light sensitivity often accompanying the mental fatigue. In addition, the decrease in noradrenaline and serotonin release might be part of decreased attention and the appearance of depression often accompanying the mental fatigue. ::: ![](1742-2094-1-22-1) ::: Mental fatigue -- a stereotypical reaction upon brain function disturbance -- a hypothesis focusing on impaired glutamate neurotransmission (figure [1](#F1){ref-type="fig"}) ============================================================================================================================================================================= It may be that mental fatigue is a stereotypical reaction to disturbance of \"higher\" brain functions. The brain, with its billions of specialized neurons and supporting glial cells, works as a \"whole\" organ. Every disturbance of brain homeostasis, no matter where the anatomical localization is, would therefore attenuate brain capacity for information processing and, as a consequence, information intake. One way to diminish information intake and processing at the cellular level would be to impair glutamate neurotransmission by attenuating the glial support and especially diminishing the astroglial capacity to clear \[Glu\]~ec~. The initial consequence would be slightly increased \[Glu\]~ec~, with less precision in glutamate transmission. This would disintegrate the \"filter\", which normally selects information and prevents it from reaching the cerebral cortex. We can take the sound from a low-frequency fan as an example. This sound is normally sorted out after hearing it for a while. If this sound is handled with less precision by auditory recognition systems, it will continually be recognized by brain centers as \"new\" information and be processed in the cerebral cortex as long as the sound is on. The \"filter\" that normally restrains already recognized information from reaching higher brain centers, has been \"opened\". From a physiological point of view, it seems appropriate that the individual, and not the brain at the synaptic level, should determine which information should reach, and be processed by, the cerebral cortex. The decreased attention, increased loudness and light sensitivity, and irritability could be physiological ways of avoiding overstimulation of higher cortical centers. In case the individuals cannot protect themselves from too much sensory stimulation, the filter\'s opening leads to overstimulation of the cerebral cortex. Here, the final shutdown of the glutamate transmission could be one mechanism underlying mental exhaustion (figure [1](#F1){ref-type="fig"}). In line with these theoretical proposals, increased \[Glu\]~ec~has in fact been demonstrated in MS, meningitis, and encephalitis, Alzheimer\'s disease, ischemia and traumatic brain injury \[[@B64]-[@B69]\]. Furthermore, it has been shown in experimental studies that even extracellular K^+^is involved in the post-traumatic hyperexcitability, and a recent study has proposed that the larger extracellular K^+^increase evoked by neuronal activity is a consequence rather than the primary mechanism underlying post-traumatic hyperexcitability \[[@B70]\]. The theory also involves the possibility of a disturbed noradrenaline/serotonin turnover in the cerebral cortex due to a slight hyperexcitability in the frontal cortex. Interestingly, increased \[Glu\]~ec~in the prefrontal cortex has been reported by Bossuet and coworkers \[[@B67]\] in asymptomatic simian immunodeficiency virus (SIV)mac251-infected macaques without major brain involvement, being consistent with our theory at least in this set of animal experiments. If valid even in humans, a disturbed noradrenaline/serotonin turnover in the cerebral cortex could be coupled to the disturbed attention and depression often occurring in addition to the mental fatigue \[see \[[@B71]-[@B73]\]\]. Testing of the hypothesis ========================= It is not possible at present to ultimately prove whether or not the altered neuronal-glial interactions in glutamatergic transmission induced by proinflammatory cytokines could serve as a model to explain cellular mechanisms underlying mental fatigue. Brain imaging techniques able to determine and follow \[Glu\]~ec~and \[K^+^\]~ec~over time would be important to use in humans suffering from mental fatigue. Today, this is not possible for technical reasons. Instead, we must use experimental systems to learn about glial cell biology and neuron-glia-neuron signaling and interactions, and thus test specific parts of the hypothesis. Neuroactive substances produced by, or altered conditions related to, the production of proinflammatory cytokines could be evaluated with regard to their effects on astroglial support of glutamate transmission, and especially glutamate transport capacity. The role of the intact astroglial network in higher brain functions (cognition and behavior) could be studied in animal models. Effects of astroglial dysfunction with regard to glutamate transport capacity would be of special interest. Even clinical studies with different treatment strategies could be important in casting some light on the accuracy of the hypothesis. Of utmost importance in all such studies would be test batteries making it possible to objectify and even quantify the degree of mental fatigue. Why do the symptoms persist in some patients? ============================================= Normally, mental fatigue and the associated symptoms disappear when the brain dysfunction is over. In some patients, the symptoms persist. We have at present no explanation for this, but if our hypothesis is correct, there could be a genetic failure preventing astroglial glutamate transporters from upregulating. Another explanation for why the symptoms persist could be that the pathological stimulation by brain plasticity creates new neuronal networks \[[@B18],[@B36]\]. Aspects of treatment ==================== Providing information about mental fatigue, its cause and the prognosis, is of utmost importance for breaking the vicious circle, which comes with the risk for secondary anxiety and depression. Furthermore, it is important for the patient to imagine and learn how much sensory stimulation they can tolerate prior to feeling too exhausted. Due to recent results on changes in cell signaling and neuronal plasticity \[[@B18],[@B36]\], it may be important to identify the symptoms and treat them as early as possible to avoid formation of new and functionally disturbing neuronal circuits due to overstimulation of neuronal-glial units. If our hypothesis is correct, it may be possible to further improve the symptoms by suppressing the production of proinflammatory cytokines and, thereby, restoring the normal astroglial glutamate uptake. In this context, xanthine derivatives may be of use \[[@B74]\]. Another substance, worth considering, may be minocycline, a synthetic tetracycline derivative that has been shown to attenuate microglial activation and, consequently, the production of proinflammatory cytokines \[[@B75]\]. During recent years substances, which enhances glutamate uptake have been identified. Nicergoline \[[@B76]\], different growth factors including pituitary adenylate cyclase-activating polypeptide (PACAP) \[[@B77]\], some low molecular weight factors \[[@B23]\] as well as metabotropic glutamate agonists \[[@B47]\] have all been able to stimulate glutamate transport in experimental systems and could be of interest in the pharmacotherapy of mental fatigue. Interestingly, even AMPA receptor modulators have been demonstrated as cognitive enhancers \[[@B10]\]. List of abbreviations used ========================== ADHD attention deficit hyperactivity disorder AMPA alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate ATP adenosine triphosphate BBB blood brain barrier Ca^2+^calcium Ec extracellular GLAST glutamate aspartate transporter GLT-1 glutamate transporter-1 \[Glu\]~ec~extracellular glutamate concentration 5-HT 5-hydroxytryptamine ICD-10 International Classification of Diseases, 10^th^revision IL-1/-6 interleukin-1/-6 K^+^potassium \[K^+^\]~ec~extracellular potassium concentration LC locus coeruleus LTP long term potential MS multiple sclerosis Na^+^sodium NA noradrenaline NFκB nuclear transcription factor kappaB NMDA N-methyl-D-aspartate NO nitric oxide PACAP pituitary adenylate cyclase-activating polypeptide PI3K phosphatidylinositol-3-kinase PKC protein kinase C Siv mac simian immunodeficiency virus macaques TNF-α tumor necrosis factor alpha Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= Equal contributions by both authors. Acknowledgments =============== This work, performed in the authors\' laboratories, was supported by the Swedish Research Council (grant No. 21X-13015; 21BL-14586), Swedish Council for Working Life and Social Research, Edith Jacobsson Foundation, Rune and Ulla Amlöv Foundation for Neurological and Rheumatological Research, and John and Brit Wennerström Foundation for Neurological Research. The authors are grateful to Eva Kraft, Göteborg, Sweden, for drawing Figure [1](#F1){ref-type="fig"}.	PubMed Central	2024-06-05T03:55:49.836553	2004-11-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC533886/", "journal": "J Neuroinflammation. 2004 Nov 4; 1:22", "authors": [ { "first": "Lars", "last": "Rönnbäck" }, { "first": "Elisabeth", "last": "Hansson" } ] }
PMC534089	Background ========== Paraneoplastic syndromes (PNS) are signs or symptoms attributable to tissue damage at sites that are remote from a primary malignancy or their metastases. PNS involving virtually every level of the neuro-muscular system have been described \[[@B1]-[@B3]\]. Typically, the paraneoplastic syndromes causing neurologic disorders precede the diagnosis of the neoplasm and are often the main reasons that medical attention is sought \[[@B2],[@B3]\]. Recent attention has thus centered on discovering novel serum or cerebrospinal fluid markers that can specifically identify not only the presence of a malignancy, but the type of malignancy involved if present. A wide variety of anti-neuronal antibodies have been associated with the many PNS-associated neurologic disorders \[[@B2]\]. These antibodies have varying degrees of sensitivity and specificity for the underlying types of malignancies. However, in the right setting, the presence of a given auto-antibody in combination with specific signs and symptoms, may reasonably predict the primary site and tumor type. The vast majority of tumors associated with PNS-neurological disorders are malignant \[[@B2]\]. Notable exceptions include the identification of antibodies to voltage gated potassium channels (VGKC) in patients with thymomas \[[@B4]\]. We describe in this report the finding of a common anti-neuronal antibody (anti-Ri) in a patient with a benign neoplasm: mature cystic teratoma of the ovary, and whose neurologic symptoms, short-term memory deficits, was apparently associated with her tumor. Clinical history ================ A 33-year-old nulligravid female with no significant past medical history presented to her physician with complaints of a short-term memory loss of approximately three months\' duration. This included an inability to remember details about 24-hour old events. There had been no major socio-economic or personal changes in her life over this period. A detailed neurologic examination was notable only for her presenting complaint. Routine laboratory work-up, including a lumbar puncture were all within normal limits. A physical examination revealed a large right adnexal mass, which upon ultrasonographic assessment showed internal features suggestive of a malignancy. A panel of serum paraneoplastic autoantibodies was then requested, including anti-Hu, anti-Yo, anti-Ri, anti-Tr and anti-Ma1/2. All were normal with the exception of IgG anti-Ri, measured at 1:15,360 by an indirect immunofluorescence method. An extensive diagnostic work-up failed to reveal any malignancies. The patient subsequently underwent a right salpingo-oophorectomy, and her adnexal mass was diagnosed as a benign mature cystic teratoma of the ovary. Almost immediately following her surgery, the patient expressed a subjective improvement in her symptoms. Within a month, the serum anti-Ri had decreased to 1:256, and a detailed neurologic examination revealed resolution of her symptoms. She has not experienced any relapse in her symptoms in the 1 year since her surgery. Discussion ========== In 1988 and 1991, Budde-Steffen at al \[[@B5]\] and Luque et al \[[@B6]\] described a subpopulation of patients with opsoclonus and a history of breast cancer in whose serum and CSF were identified an antibody that reacted against 55 kD and 80 kD proteins that were designated Anti-Ri (also known as ANNA-2). It has since been shown that these antigens are highly conserved but widely distributed central nervous system neuronal proteins which are encoded by the Nova-1and Nova-2genes and which may play a role in neuronal maturation and homeostasis \[[@B7]-[@B9]\]. As those seminal reports indicate, Anti-Ri was initially associated with opsoclonus/myoclonus and cerebellar ataxia symptomatology in patients with breast and gynecologic cancers. They have however subsequently been associated with a wide variety of malignancies that have included lung, gastric and bladder carcinomas \[[@B10]\]. Indeed, in one study, 32% and 36% of cancers associated with anti-Ri in patients with suspected PNS were breast and lung carcinomas respectively \[[@B10]\]. The spectrum of associated neurologic symptoms has also expanded considerably and now includes vertigo, muscle weakness, dysarthria, dysphagia, dementia, deafness, myelopathy, opthalmoplegia, encephalomyelitis, rigidity, nausea, myelopathy, sensorimotor neuropathy \[[@B10]\]. What these cases illustrate is that with a few exceptions \[[@B4]\], the vast majority of tumors associated with PNS-neurological disorders are malignant. Our case thus highlights the potential association between a benign neoplasm and the presence of these antibodies and neuronal symptomatology. Is it possible that the presence of the autoantibody and the ovarian tumor are completely fortuitous?. Given the temporal relationship between the resolution of her symptoms and the sharp decrease in her anti-Ri titer following her surgery, we believe this is unlikely. However, the precise mechanistic basis for this association as well as potential influence of outside factors remains to be elucidated. It should also be noted that high titers of Anti-Ri have been identified in patients with a history of ovarian cancer but without any evidence of a PNS, suggesting caution in assessing the specificity of this auto-antibody for PNS. In summary, we describe in this report an association of a common anti-neuronal antibody (anti-Ri) in a patient with a benign neoplasm, mature cystic teratoma of the ovary, and whose neurologic symptoms, short-term memory deficits, was apparently associated with her tumor. Although, Anti-Ri antibodies are typically associated with malignancies, this case illustrates the potential association between benign tumors and this autoantibody. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= Dr Fadare wrote the initial version of the manuscript. Dr Hart managed the patient, provided clinical information, and revised the manuscript. Acknowledgements ================ Written permission was obtained from the patient to report this case	PubMed Central	2024-06-05T03:55:49.839061	2004-11-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534089/", "journal": "Int Semin Surg Oncol. 2004 Nov 10; 1:11", "authors": [ { "first": "Oluwole", "last": "Fadare" }, { "first": "Hassan J", "last": "Hart" } ] }
PMC534090	Introduction ============ This paper identifies the major challenges faced and the lessons learned in addressing them by the collaborative research groups involved in developing and conducting a large, multi-national case-control study of acute leukemia among children in areas of the former Soviet Union (FSU) that were most heavily exposed to radioactive fallout as a result of the April 1986 accident in reactor vessel \#4 of the Chernobyl Nuclear Power Plant. In this accident, a variety of radioisotopes including iodine (^131^I), cesium (^137^Cs, ^134^Cs), and strontium (^90^Sr), were released from the damaged reactor vessel contaminating soil, vegetation, and groundwater \[[@B1]\]. Fallout from the Chernobyl accident contaminated large portions of Eastern Europe, the then Union of Soviet Socialist Republics (USSR) and more distant regions. Areas of the FSU, including the now independent republics of Belarus, Russia, and Ukraine, were among the most heavily contaminated. The intent of the research project was to examine acute leukemias without specific regard to national boundaries, while recognizing the requirement to bring together investigators from these three republics in a common effort. Acute external exposures to ionizing radiation have been etiologically linked with observed increases in the risk of all types of leukemia, except chronic lymphocytic leukemia; the risk is greatest for acute myeloid leukemia \[[@B2]-[@B12]\]. The association between exposure to ionizing radiation from the Chernobyl accident and the occurrence of leukemia has been summarized in a recent review \[[@B10]\] which highlights mixed results from published studies to date (see the review article for a comprehensive overview of published studies). Accounting for differences between the studies in the methodologies used to assess the radiological exposures, the procedures for identifying childhood malignancies and in the lengths of follow up, these authors concluded that there is not strong evidence demonstrating increases in childhood or adult leukemia from Chernobyl exposures \[[@B10]\]. Nonetheless, because the link between high dose ionizing radiation exposure and the development of leukemias is widely established, and children are felt to represent a uniquely susceptible population, this issue remains of high scientific and public heath interest. The study described herein focused on this most susceptible subgroup. Collaborative studies are challenging at best; international consortia efforts are even more so. The study of border-crossing disasters such as that in Chernobyl need be investigated as a single scientific challenge to take advantage of standardizing the investigative process across national boundaries, and to afford adequate sample size and the associated statistical power to reach meaningful conclusions. This paper will not recount the events of the Chernobyl disaster or specific results of the investigation. Rather, it will share the lessons learned while conducting a collaborative, multinational study for the benefit of other investigators. Lesson \#1: Developing a basis for collaboration ------------------------------------------------ One of our first challenges was to establish an understanding of and a presence in each of the affected areas of the FSU. Multinational/multi-site studies are labor intensive and difficult to coordinate from distant locations. Therefore, the identification of a strong, on-site research team is essential. Each group must be treated as a full-member of the larger study team and be fully engaged. The local team is primarily responsible for study implementation. It is imperative that local teams with relevant expertise be formed. Our teams employed physicians, epidemiologists, statisticians, and dosimetrists. Local teams participated in study design, as well as study management in the field and troubleshooting problems. This required teams that were extremely knowledgeable about geopolitical boundaries, location of cities/small villages, governmental structures, and the health care delivery system. The latter area was particularly important given that the entire study hinged on the identification of acute leukemias and pair matched healthy controls. The health care systems in Belarus, the Russian Federation, and Ukraine have been adapted from the FSU system. Each Oblast (an Oblast is a large administrative unit -- equivalent to a state in the United States) has 1--2, and in rare cases 4, large cancer treatment and diagnostic facilities (termed oncodispensaries). There are separate facilities for childhood (i.e., \<16 years of age at time of diagnosis) and adult patients. In addition to providing nearly all cancer-related medical treatment, oncodispensaries house records of all reported cancer patients within the Oblast. These treatment centers contained critical source records vital to the success of our study. On-site research teams knew how to identify cases (e.g., review of oncodispensary records), where to identify controls (e.g., polyclinics), and what agency permission was needed to obtain access to these data. This would have been impossible for an \"outsider\" to do. The identification of committed and skilled collaborators from each of the Republics facilitated project coordination and monitoring from outside the FSU. However, not surprisingly, there have been some difficulties related to language and culture. Although English language proficiency is progressing rapidly within the FSU, many of the individual researchers were unable to effectively communicate via English. Moreover, the collaborators based in the USA were even more limited in their Russian language fluency. This issue of language was addressed early in this collaboration through a consensus that English would be used as the common language, which was endorsed by our FSU collaborators. Certain investigators were well trained in English and the Consortium relied on simultaneous interpretation for initial meetings of collaborators, and then switched to having an interpreter present to provide general and personal assistance as needed. All printed materials, such as study protocols and survey instruments were produced in dual language versions (Russian and English) and verified via back translation. Since the late 1990s, key project staff members in the USA and each FSU Republic maintained frequent linkages through e-mail communications at intervals between site visits and periodic project reviews. However, it should be noted that prior to the late 1990s, e-mail was scarce in the FSU and somewhat unreliable. International communications were first established via facsimile transmissions then evolved to an electronic platform (e-mail) as Internet coverage in the FSU expanded. Lesson \#2: Infrastructure Development -------------------------------------- This study was supported by the International Consortium for Research on the Health Effects of Radiation (ICRHER), based in the United States. (A series of articles summarizing two research conferences appears in a special supplement of the Stem Cells journal \[[@B13]\].) Our research objective was to examine the relationship between exposure to chronic low doses of ionizing radiation and the incidence of acute leukemias among children in the 3 FSU republics without specific regard to national boundaries (see figure [1](#F1){ref-type="fig"}), while recognizing the obligation to bring investigators from the three republics together with each other and those in the USA in a common effort. Individuals in the 3 republics exposed during childhood to radiation from Chernobyl were felt to represent a uniquely susceptible group and an appropriate population for a retrospective study. The ICRHER was incorporated in June 1993. It was born of the insight and enthusiasm of one man, the late Admiral Elmo Zumwalt, Jr., United State Navy (retired), who was concerned about environmental exposures and cancer risk; particularly, how such exposures might affect military personnel. Admiral Zumwalt was also concerned about the humanitarian aspects of accidental exposure to toxic agents, including ionizing radiation. The Chernobyl reactor accident provided an opportunity to pursue both these interests. Initially, the ICRHER (the \"Consortium\") was comprised of collaborative groups at institutions in the United States of America (USA) and FSU republics, specifically, the Baylor College of Medicine, Houston, Texas; the Research Center for Radiation Medicine, Kiev, Ukraine; the Fred Hutchinson Cancer Research Center (FHCRC), Seattle, Washington; the National Center for Hematology, Moscow, Russia; Hadassah Medical Organization, Jerusalem and Haifa, Israel; and the National Marrow Donor Program, Minneapolis, Minnesota. The Bridgeport Hospital/Yale University and the Research Institute of Radiation Medicine and Endocrinology, Minsk, Belarus, joined the Consortium later. The final research team for the multinational Leukemia case-control study includes the FHCRC in collaboration with the Medical Radiological Research Center in Russia and two groups from the Roswell Park Cancer Institute (one working with the Research Institute of Radiation Medicine and Endocrinology, in Minsk, Belarus and another with the National University \"Kiev-Mohyla Academy\", Kiev, Ukraine). The Consortium helped to bring structure and organization in a variety of areas, from assembling the principal investigators to creating working groups to empanelling external advisory boards. A corporate entity or central office was formed to coordinate these activities, and to manage common logistic requirements, to draw up contracts for U.S. and Israeli institutions, to support the FSU collaborators, and to provide an administrative office of record. Satellite offices were established in Kiev, Minsk, Moscow, Obninsk, Bryansk and Jerusalem to provide administrative, communication and logistical support to the research activities in the FSU and Israel. FSU offices, as will be seen later, were critical to the success of this study. Given the changes in leadership and make up of the ICRHER since its inception, it was imperative to maintain flexibility and responsiveness to change. Modifications in research partners may occur for many reasons. Therefore, it is critical that some type of \"corporate entity\" exist to aid in these transitions through detailed documentation and to provide continuity at the executive/advisory level. Consortium investigators developed a pilot study to test the feasibility of initiating a full-scale epidemiologic investigation, and to determine whether needed collaborations could be established. The primary goals were to assess radiation dose, to identify individuals at risk for radiation-related illness (including émigrés to Israel), to develop data collection instruments and common study protocols, to elucidate mechanisms of radiation damage, and to establish core support through the central office. The original group invested considerable time in reviewing exposure data, the populations involved, and possible health outcomes. Following these critical discussions, a decision was made to launch a pilot investigation of acute leukemia. This study would prove challenging, but it was facilitated by the knowledge and experiences gleaned from the feasibility study. Lesson \#3: Study Design ------------------------ Based on the feasibility and pilot studies, a consensus decision was made to begin a multinational leukemia case-control study in Belarus, the Russian Federation, and the Ukraine, including the Israeli component (the Israeli studies concluded in August 2000). The focus has been on the most radiosensitive malignancy in the most radiosensitive at-risk age group: occurrence of acute leukemia in those 0 to \<6 years at the time of the accident \[[@B2],[@B10]\]. Initially, the aim of the Consortium was to investigate the occurrence of acute leukemia among individuals who were 0 to age 20 years at the time of the Chernobyl accident. The original population was selected due to early indications of high radiation dose exposures and easily achievable sample size estimates based on power calculations. When doses were not found to be as high as predicted, the sample size requirements became exceedingly large. Therefore, the researchers decided to narrow their focus to individuals aged 0 to \<6 years at the time of the accident, since they represented a much more radiosensitive subpopulation. Hence, there is the need for well-designed feasibility studies to provide critical information in study planning and decision-making. The final study population included 421 confirmed cases of acute leukemia and 842 population controls pair-matched to cases on age, gender, and type of residence. All participants were in uteroto \<6 years of age at the time of the Chernobyl accident in April 1986. ### Case identification Case identification was often difficult, particularly when compared to studies conducted in the U.S. that typically rely on either population-based cancer registries or hospital-based rapid case identification. Many regions studied did not have an up-to-date, population-based cancer registry. And, since nearly all medical records in the FSU exist in paper form, exhaustive manual reviews of hard copy records were often required. This task was somewhat simplified by the local custom of referring cancer cases to regional oncodispensaries, thereby, restricting record reviews to a finite number of facilities. Moreover, it was also necessary to review death files to ascertain cases not previously brought to medical attention. Trained physicians reviewed the records at the oncodispensaries in Belarus, the Russian Federation, and the Ukraine. Records were retrieved for all potential patients (i.e., date of birth, date of diagnosis, and residence location). After verifying eligibility, trained interviewers contacted parents of these patients and scheduled an interview. ### Control selection Control selection was equally vexing. In the US, researchers typically use neighborhood controls, random digit telephone dialing methods, and/or computerized databases \[[@B14]-[@B16]\]. In the FSU, control selection began by meeting with district health officials to obtain permission to review polyclinic records. Considerable time was then spent reading through racks of paper files and recording information for potential participants. Interviews were completed with two controls pair-matched to each case based upon age at diagnosis, sex, region/district of residence, and type of settlement. The research teams visited the polyclinic and randomly selected 20 potential controls fitting the inclusion criteria. Interviews were generally scheduled and completed for the first two potential controls on the list. Other names on the list were contacted and scheduled for interviews as needed. Over 90% of potential controls contacted by the research teams agreed to participate. These high rates of case and control participation may be attributed to the dedication and resourcefulness and commitment of the research teams and the use of modest incentives (food baskets). ### Interview As formidable as the above tasks were, they paled in comparison to actual data collection. During the feasibility study, investigator meetings were held in the US to develop common data collection instruments. These instruments were structured to address issues of cultural and language compatibility for use in the three Republics. While Russian was adopted as the standard language for data collection, study instruments contained both Russian and English text. Instruments were back translated into English to ensure accuracy of translation. Next, a team of interviewers needed to be recruited and trained. We relied almost exclusively on recruiting physicians who were familiar with the disease process, were credible representatives, were respected by study participants, and who could be depended on to provide accurate and verifiable data. The mix of urban areas and rural settlements presented logistical challenges in terms of tracking participants, arranging appointments, and completing face-to-face interviews. To overcome this challenge, some interviewers mailed letters to introduce the project, and to request that the participant contact the interviewer. Others traveled to local communities to personally discuss participation and to schedule interviews. Still others used local contacts to identify participant places of employment for either telephone or direct contact. Traveling to interview study participants was another challenge. In contrast to conducting research in the US where there are widespread and reliable telecommunication systems, well-developed highway systems, and accurate maps, FSU travel was more problematic. For example, not all roads to small villages in the FSU are paved and most are limited to a single lane in either direction. Roads can be particularly treacherous in the winter and rainy seasons. Maps do not always show the precise location of small villages. Villages have neither street signs nor house numbers. Further, the collapse of the Soviet Union resulted in changes in the names of many streets, settlements, and villages. This arduous process of locating and interviewing controls took considerable effort and time. Where interviewers in the U.S. are generally out and back in the same day, FSU colleagues were out in the field for days at a time and would be fortunate if they could complete a handful of interviews each day. These differences in operations must also be factored in to the overall cost of the study. Finally, local residents tended to be wary of strangers and special introductions were often necessary to gain entry. One cannot overstate the importance of having a well-trained and dedicated on-site research team that is familiar with cultural norms and local \"maps\". The two-hour, face-to-face interviews were generally completed with the mothers of cases and controls. Interview items were developed by US and FSU scientists and included sections addressing demographics, general health status, maternal and paternal occupational history, and a detailed questionnaire for obtaining information necessary for developing estimates of individualized internal and external radiation exposures, including questions regarding consumption of locally produced milk, meats, and vegetables; residence history and type of housing structure; use of protective measures immediately after the accident; and time spent outdoors. Lesson \#4: Working Groups -------------------------- Those involved in multinational or multi-site investigations may consider the creation of Working Groups to monitor the various components of the study. As has been utilized in other international studies \[[@B17]\], the lead investigators could separate the overall study into its basic components and organize working groups that represent all collaborating teams. For example, we established a leukemia diagnostic working group comprised of hematologic morphologists and hematologists from representative ICRHER institutions, and chaired by a leading pediatric hematologist who was not associated with the Consortium. Members completed blinded reviews of bone marrow pathology slides, or other information (e.g., clinical histories, laboratory data) for cases without slides, and then assigned a histopathologic diagnosis based upon group consensus. A subset of cases was randomly selected for repeat review to assess consistency. Results of this review process affirmed the accuracy of acute leukemia diagnoses made within these areas of the FSU \[[@B18]\]. A common methodology to assess individual absorbed radiation doses and corresponding uncertainties was developed and tested by the dosimetry intercomparison working group (DIWG) for all subjects based on interview data and available exposure data. The interview collected detailed exposure data for each subject from the time of the Chernobyl accident until the reference date. It should be noted that interview data alone were not sufficient to determine individual dose estimations. For retrospective dose estimation specialized radioecological data were necessary. These resources provided information concerning local soil types, food contamination with different radionuclides, dates of radioactive cloud arrivals to each local community, etcetera. These data were collected by the members of DIWG based on information published within the FSU. Dosimetrists are a good example of the specialized personnel that need to be a full-time, on-site presence. They have critical knowledge of the local area as well as access to primary data essential in quantifying exposure. Individual dose estimates reflected local conditions (e.g., contamination levels, soil type, soil to milk transfer coefficients) at each location where a particular subject lived during the appropriate time interval. The subject\'s residence history and other important personal information, such as milk consumption and food sources, were collected during the standardized interviews by trained examiners. Fieldwork was performed jointly by physicians and dosimetrists. A Data Analysis Working Group was essential for oversight of the analytic phase. Composition included representatives from each site with a strong chair to maintain focus and momentum. Also, a Data Audit Working Group was established at study inception to conduct periodic reviews to assure adherence to study protocols. And finally, a centralized Data Coordination Office served as a repository of the common dataset and oversaw periodic computerized checks for quality and completeness. Conclusions =========== Our experiences in the organization and successful implementation of a multinational, retrospective study of acute leukemia in regions impacted by the Chernobyl disaster have been highlighted. Issues identified during the implementation of our multi-national epidemiology study, along with strategies for resolution are summarized in table [1](#T1){ref-type="table"}. While trained research teams within each Republic were responsible for collecting data, we relied on a distinctive series of working groups of collaborators from participating institutions to coordinate various aspects of the study such as case confirmation, data quality, dosimetry, and data analyses. This allowed all project teams to remain interconnected and equally involved while utilizing the unique expertise of various collaborators. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Potential issues regarding the implementation of multi-national epidemiology studies ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Challenge: Resolution strategy: -------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------- •Language •Dual language versions (Russian & English) for all printed materials; use of interpreters •Geographic distance between collaborators •e-mail accounts for key collaborators; site visits, progress meetings •Limited experience with epidemiology •Mandatory training workshops for interviewers; audits to assure compliance with protocols •Subject ascertainment •Cases identified through manual record reviews at oncodispensaries and childhood oncology centers; controls identified from manual review of raion medical records •Limited comprehensive cancer registry data •Manual records review at oncodispensaries, childhood cancer centers and mortality files •Lack of telephone to contact participants •Mailed letters of introduction; field trips to communities •Locations of study participants •Field trips for data collection; assistance of local residents •Radiologic contamination data in multiple locations •Visits to multiple Institutes & offices; contacts of collaborators •Adequate communications •All research sites provided immediate Internet access •Timely compensation for local investigators •Direct pay facilitated by USA agencies (e.g., Civilian Research and Development Foundation) •Common research protocols and joint methodology for individual radiation assessment •Periodic meetings of all USA/FSU investigators to promote personal relationships and scientific value of combined data •Data collation and analysis •Establish Data Coordination Office in the Former Soviet Union\ •Transfer all data electronically •Data access and archives •Access by mutually-agreeable policy\ •Transfer data to USA institution for permanent storage •Multidisciplinary international study •Highly cooperative, joint international consortium with working groups ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ::: The breakup of the former Soviet Union in 1991 created national autonomy in Belarus, the Russian Federation, and Ukraine. Although the study area for this project included selected regions of the FSU, the intent was to study acute leukemia without regard to specific national boundaries, while recognizing the requirement to bring together investigators from these three Republics in a common effort. Proprietary concerns and country-specific restrictions on the sharing of scientific information were thoroughly addressed to gain agreement and to facilitate the pooling of analytic information. The Consortium played an integral role in providing infrastructure support for this project through the appointment of project support administrators at each research site to oversee communications, equipment procurement/maintenance, and compensation. The fiscal aspects of supporting research are unique to each country. A careful examination of collaborating scientific institutions and the financial regulations of each participating country prior to setting up any support mechanisms is critical, and may result in country-specific arrangements. Essential computer, laboratory, and communication equipment was supplied. Computer software, which was compatible across the three research settings, was installed and upgraded periodically. Equipment was segregated and secured to insure exclusive use by project staff. It should be emphasized that this investigation represents the largest retrospective study examining the relationship between Chernobyl radiation exposures and risk of acute leukemia conducted to date and the only research effort to bring together data from the most exposed areas into a single study; results of the multinational case-control study are presented in a separate paper \[[@B19]\]. The conduct of multinational epidemiologic studies presents numerous challenges \[[@B17],[@B20]\], including issues such as language, physical infrastructure, telephone coverage, and road conditions, as well as geographic distances and issues of participant ascertainment. However, as demonstrated by our experiences, these challenges can be effectively overcome through attention to organization, communication, and quality assurance. Moreover, these challenges are greatly offset by unique opportunities to yield information of great significance to science and society. List of Abbreviations ===================== FSU, former Soviet Union USSR, Union of Soviet Socialist Republics USA, United States of America ICRHER, International Consortium for Research on the Health Effects of Radiation; the \"Consortium\" FHCRC, Fred Hutchinson Cancer Research Center DIWG, dosimetry intercomparison working group Competing Interests =================== The author(s) declare that they have no competing interests. Author contributions ==================== MCM and AMM were responsible for the study concept. MCM and AMM drafted the manuscript; KMM, PLM, RCM, VFS and RWD provided critical review and input. MCM, KMM, PLM, RCM, VFS, RWD and AMM participated in interpretation, as well as in data acquisition efforts. All authors read and approved this manuscript. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Regions surrounding the Chernobyl Nuclear Power PlantShading identifies areas included in the International Consortium for Research on the Health Effects of Radiation (ICRHER) study of acute childhood leukemia: Gomel & Mogilev Oblasts in Belarus; Cherkassy, Chernigov, Rivno, & Zhitomir Oblasts in Ukraine; and Bryansk Oblast, Russian Federation. Solid lines identify boundaries between countries/republics. Shaded square in center of figure identifies location of Chernobyl Nuclear Power Plant in northern Ukraine. ::: ![](1476-069X-3-12-1) ::: Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/content/backmatter/1476-069X-3-12-b1.pdf> Acknowledgments =============== This work was supported by Grant \# N00014-94-1-0049 issued to Georgetown University from the Office of Naval Research in support of the International Consortium for Research on the Health Effects of Radiation. The contents are solely the responsibility of the authors and do not necessarily represent the views of the Office of Naval Research, Georgetown University or the International Consortium for Research on the Health Effects of Radiation. The collegiality and enthusiastic support, as well as critical feedback on earlier drafts of this paper, received from ICRHER members and others, are gratefully acknowledged.	PubMed Central	2024-06-05T03:55:49.840006	2004-11-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534090/", "journal": "Environ Health. 2004 Nov 8; 3:12", "authors": [ { "first": "Martin C", "last": "Mahoney" }, { "first": "Kirsten B", "last": "Moysich" }, { "first": "Philip L", "last": "McCarthy" }, { "first": "Richard C", "last": "McDonald" }, { "first": "Valery F", "last": "Stepanenko" }, { "first": "Robert W", "last": "Day" }, { "first": "Arthur M", "last": "Michalek" } ] }
PMC534091	Background ========== There are 9.6 M cancer survivors in the US today, and 61% are age 65 or older \[[@B1]\]. Over the next 50 years, the number of elderly cancer survivors is expected to double \[[@B2]\]. Unfortunately, these individuals are at greater risk for other cancers, cardiovascular disease, osteoporosis, diabetes, and functional decline \[[@B1]-[@B5]\]. While all elderly are at increased risk for functional decline \[[@B6]\], those diagnosed with cancer are even more vulnerable and may experience functional losses that threaten independent living \[[@B7]-[@B10]\]. Adherence to healthful lifestyle behaviors may be one way that older cancer survivors can maintain or regain higher levels of physical functioning. Yet, there are little data to support this premise. Methods ======= We explored associations between lifestyle factors and physical functioning among elderly cancer survivors who were screened for Project LEAD (Leading the Way in Exercise And Diet), a home-based, diet and exercise intervention trial \[[@B11]\]. Briefly, individuals (age 60+) who were no more than 18 months beyond a diagnosis of early stage (I-II) breast or prostate cancer were identified through cancer registries, private practices, and self-referral. Patients were mailed a letter of invitation, consent forms, a postage-paid return envelope, and a brief survey. The purpose of the survey was to screen-out individuals already practicing healthful behaviors \[i.e., those engaging in regular, vigorous exercise or consuming a low fat diet with 5 or more daily servings of fruits and vegetables (F&V)\] and individuals unlikely to benefit from the telephone counseling-mailed material intervention \[i.e., those who were mentally incompetent, severely hearing impaired, or who had conditions that precluded unsupervised exercise or a high F&V diet (i.e., scheduled hip or knee replacement, walker or wheelchair use, recent stroke or heart attack, angina, congestive heart failure, chronic obstructive pulmonary disease, end-stage renal failure, and/or warfarin-use)\]. The survey incorporated validated scales to assess dietary fat (Block Dietary Fat Screener) \[[@B12]\], and F&V consumption (5 A Day items) \[[@B13]\]. Given space constraints of the survey and our focus on vigorous exercise, one item was used to assess exercise (\"On average, do you do continuous vigorous exercise for at least 20 minutes, 3 or more times per week?\") \[[@B14],[@B15]\]. The SF-36 Physical Function Subscale (SF-36 PFS) also was modified slightly to omit item \#2 (\"Does your health limit you in moderate activities\") and scores were scaled 0--100 with the assumption that the average value for the missing item was the same as that for the remaining items \[[@B16]\]. T-tests and chi-square analyses were performed to determine if respondents differed from non-respondents on age, time since diagnosis, race and gender, and to test for differences between male and female respondents. Pearson correlation coefficients indicated associations between F&V intake and percent dietary fat, and physical function scores. T-tests were used to explore associations between exercise and function. Linear regression analyses permitted an examination of associations between physical function and health behaviors controlling for several likely confounders including gender, age, race, time since diagnosis and concurrent health behaviors. Given the homogeneity in stage at diagnosis, stage information was omitted from this analysis. Results and Discussion ====================== Between August 2000 and May 2003, 2,431 cases were identified and physician approval for contact was granted for 2,034 cases. Incomplete address information existed for 24 of these cases, yielding 2,010 viable posted surveys, of which 688 complete surveys were returned (34% response rate). Characteristics of respondents and non-respondents are provided in Table [1](#T1){ref-type="table"}. Respondents were significantly more likely than non-respondents to be younger, white, male, and more proximal to their date of diagnosis. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographic characteristics of respondents and non-respondents ::: Characteristic Respondents (n = 688) Non-Respondents (n = 1322) P-value ----------------------------------------------------- ----------------------- ---------------------------- --------- [Age (years)]{.underline} Mean (sd) 71.4 (5.0) 73.0 (5.9) \<.0001 Range 60 -- 94 64 -- 96 [Race \[% (N)\]]{.underline} White 83.4% (574) 75.0% (991) \<.0001 African American 12.4% (85) 21.0% (278) Other/Unknown 4.2% (29) 4.0% (53) [Gender \[% (N)\]]{.underline} Female 41.6% (286) 52.7% (696) \<.0001 Male 58.4% (402) 47.4% (626) [Time Elapsed Since Diagnosed (months)]{.underline} Mean (sd) 10.8(4.9) 11.3 (5.8) .046 Distribution \[%/(N)\] 0 -- 3 months post-diagnosis 9.2% (63) 11.4% (151) \>3 -- 6 months post-diagnosis 8.9% (61) 10.1% (134) \>6 -- 9 months post-diagnosis 24.0% (165) 17.4% (230) \>9 -- 12 months post-diagnosis 21.8% (150) 20.7% (274) \>12 -- 15 months post-diagnosis 18.0% (124) 15.9% (210) 15+ months post-diagnosis 18.2% (125) 24.4% (323) ::: Data regarding physical function and lifestyle behaviors are provided in Table [2](#T2){ref-type="table"}. In bivariate analyses, elders with prostate cancer (men) have significantly higher SF-36 PFS scores than those with breast cancer (women). A majority of all respondents adhere to a low fat diet, while a minority pursue regular vigorous exercise or eat 5 or more daily servings of F&Vs. Men are significantly more likely than women to exercise and to consume 5 or more daily servings of F&Vs. However, female cancer survivors are more likely to report low fat diets. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Physical function and health behaviors of survey respondents (N = 688) ::: ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Variable Total Sample (N = 688) Women with Breast Cancer (N = 286) Men with Prostate Cancer (N = 402) p-value --------------------------------------------------------------------------------- ------------------------ ------------------------------------ ------------------------------------ --------- [Physical Function]{.underline} \[SF-36 -- 9 items scaled 0--100, mean (sd)\] 74.3\ 68.3\ 78.5\ \<.0001 (25.3) (25.5) (24.3) [Exercise]{.underline} (% who \"vigorously exercise for at least 20 minutes, 3 or more times a week\") 44.6% 36.7% 50.2% .001 [% of Energy (Calories) from Fat]{.underline} mean (sd) 29.1\ 25.6\ 31.7\ .0003 (21.8) (16.2) (24.7) \% consuming \<30% of Energy from Fat 61.1% 65.8% 57.6% .031 [Fruit and Vegetable Consumption]{.underline} mean number of servings (sd) 4.1\ 3.7\ 4.4\ .0005 (2.9) (2.3) (3.2) \% consuming 5+ servings/day 20.4% 15.4% 24.1% .005 ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: Modest correlations were obtained between the indicators of exercise and diet, with positive agreement noted between F&V intake and exercise (Pearson ρ = .12/p = .003) as well as F&V intake and dietary fat (ρ = .42/p \< .0001). An inverse association was noted between dietary fat and exercise (ρ = -.08/p = .05). In bivariate associations, all three behaviors were significantly associated with SF-36 physical functioning scores (p \< .05): F&V intake (ρ = .09), dietary fat (ρ = -.10), and vigorous exercise (ρ = .37). In multivariable linear regression analyses, with the SF-36 PFS score serving as the dependent variable and controlling for age, race, gender, time since diagnosis, and concurrent health behaviors, the simultaneous associations of the three indicators remained statistically significant (independent) and yielded the following estimates: (1) a 0.2 increase in the SF-36 PFS score with each reported 1% decrease in percent energy from fat (p \< .0001); (2) a 0.9 increase in the SF-36 PFS score for each reported serving of F&V/day (p = .0049); and (3) a 15.4 increase in the SF-36 PFS score with a positive response for regular vigorous exercise (p \< .0001). In comparing these cross-sectional data on lifestyle behaviors of older cancer survivors to normative data reported on healthy elders, as well as to previous data reported on general populations of breast and prostate cancer survivors (where a dearth of data have been reported on older cancer survivors per se), we find both differences and similarities. Like data that exist on healthy elders (age 65+) responding to the 2000 Behavioral Risk Factor Surveillance System (BRFSS) survey \[[@B17]\], or findings of a previous study of 978 breast and prostate cancer survivors \[[@B18]\], a minority of the respondents to this survey report consuming 5 or more daily servings of F&Vs. However, the percentage of our respondents who reportedly achieved 5 A Day guidelines was lower than that reported by these two previous studies (20.4% as compared to 34.4% and 42%, respectively) \[[@B17],[@B18]\]. In contrast, a majority of survivors in both this study (61.1%) and the previous study on cancer survivors (69%) report adherence to a low fat diet \[[@B18]\], whereas higher mean intakes of fat 32--33% were noted among a general population of elders (age 60+) responding to the National Health and Nutrition Examination Survey, Phase I (1988--1991) \[[@B19]\]. Like the previous study on breast and prostate cancer survivors (where 58% reported routine exercise of moderate intensity or greater compared to 47% within the general population) \[[@B18],[@B20]\], a greater proportion of these elderly cancer survivors report routine vigorous exercise when compared to BRFSS data on the general population (i.e., 44.6% vs. 11%) \[[@B20]\]. Findings may differ due to differences in survey instruments, populations and varying amounts of respondent bias, however at least for dietary fat and exercise, data largely support the prevalent finding that cancer survivors tend to report healthier lifestyle behaviors \[[@B21]\]. The fact, that we did not see this trend in F&V consumption may be due to non-penetrance of the 5 A Day message among the survivor population, or may be the result of our population being significantly older than that reported in previous studies -- a population where proportionally more individuals may be edentulous or suffering from G.I. intolerances that serve as barriers to F&V consumption. Our data on physical function, however, indicate higher levels within our sample (74.3 ± 25.3) when compared to U.S. age-matched norms (69.4 ± 26.3) \[[@B22]\], despite previous findings, which suggest decreased physical functioning among older cancer survivors \[[@B7]-[@B10]\]. A potential explanation for the higher physical function scores exhibited within our sample may relate to the higher prevalence of healthful lifestyle practices which in turn may increase physical function. This links back to the primary thrust of this study, which was to determine evidence for a link between functional status and health behavior. Our results suggest that regular vigorous exercise and a diet rich in F&Vs and low in fat is associated with higher levels of physical functioning among elderly recently diagnosed with cancer. Although the association between exercise and improved function has been reported repeatedly in other studies among elders \[[@B6],[@B23]\], this is the first study to show this association exclusively among elderly cancer survivors. Further, the fact that regular vigorous activity is associated with a 15.4 point differential in functional status, accounts for a magnitude of effect that is comparable to 0.61 standard deviations (sd), and far exceeds the 0.22 sd noted in a previous study by Baker et al. as being both statistically and clinically significant \[[@B7]\]. Furthermore, only one study to date has reported the link between diet composition and physical function. Ortega et al. found that diets low in fat and high in F&Vs were associated with higher levels of physical functioning among a sample of upper socio-economic, elderly male Spaniards at risk for cardiovascular disease \[[@B24]\]. This previous report however did not include defined estimates, so it is difficult to draw exact comparisons regarding the magnitude of dietary change associated with a given difference in SF-36 PFS score. Nonetheless, our data suggest that modest gains in physical functioning may be achieved with dietary changes that are both feasible and realistic \[i.e., a 0.9 increase in PFS score with each additional serving of F&V or a 0.2 increase in PFS score with each 1% decrease in percent energy from fat (roughly equivalent to a half a teaspoon of butter, margarine or oil for most caloric intakes)\]. Albeit, multiple servings of F&Vs (roughly 5 per day) or substantial decreases in fat (approximately 20% of total Calories) would be necessary to achieve clinical significance if dietary changes were pursued in isolation, however if taken together, as in the pursuit of a healthier overall diet, it is conceivable that appreciable functional improvement could occur. While the relationship between exercise and physical function is strong and mechanistically more direct, low fat diets (with lower amounts of saturated and trans fats) and increased F&Vs also provide theoretically viable, yet not fully elucidated pathways, to increased function \[[@B25]\]. It must be noted, however that our findings differ somewhat from those of a recently reported study of cancer survivors by Blanchard et al. \[[@B26]\], who found a significant association between Health Related Quality of Life (HRQOL) and exercise, but not between HRQOL and F&V intake. Differences between studies with regard to the sample (e.g. our sample included 688 elderly breast and prostate cancer survivors whereas the sample of Blanchard et al. \[[@B26]\] was comprised of 316 breast, prostate and colorectal cancer survivors of all ages), and survey items (e.g., we used validated items taken from the National 5 a Day trials \[[@B13]\], whereas Blanchard et al. \[[@B26]\] used one composite item) may account for the discrepancy in findings. Accordingly, and for the purposes of designing effective interventions to improve physical function in older cancer survivors, further research is necessary to corroborate associations between diet and physical function (if any) and to clarify responsible mechanisms. The primary limitations of this study relate to respondent bias and cross-sectional design. Our response rate of 34% is indeed less than the 59% we have achieved in previous mailed surveys among similar populations \[[@B18]\]. Given that this survey was linked to accrual efforts for a yearlong behavioral intervention trial \[[@B11]\], a response rate in this range was anticipated. Indeed, our response rate is similar to those of 35--50% cited by Martin Brown, PhD (Chief of the National Cancer Institute\'s Health Services and Economics Branch) in a published interview regarding survey responses rates among cancer patients \[[@B27]\]. In addition, in attempting to control for factors that differed between respondents and non-respondents, we acknowledge that there likely exist other important factors that were not included (e.g. items that our survey did not ascertain such as socio-economic status), or the fact that we were unable to assess and subsequently control for lifestyle behaviors among those who did not respond -- non-respondents whose lifestyle behaviors may have placed them at risk (i.e., sedentary, poor diets) and less inclined to respond, or conversely those already adhering to healthy lifestyle behaviors and less compelled to participate. Another limitation of our study was the use of abbreviated scales or items to obtain health-related data. As an example, the single item used to capture vigorous exercise may have led to inflated rates of reporting due to the absence of a response category for moderate exercise. Finally, our study was cross-sectional, and cause and effect possibly are confounded. Conclusions =========== Findings of this study support the recent American Cancer Society diet and exercise guidelines for survivors \[[@B25]\], which call for a physically active lifestyle and a plant-based diet. However, more research is needed to confirm associations between lifestyle factors and physical function, especially among broader populations of survivors (i.e., other types of cancer, other age groups, and among short-term versus long-term survivors). Ultimately, randomized controlled trials enrolling of older persons with cancer are needed to determine the potential benefit of diet and exercise interventions in reorienting the trajectory of functional decline. List of Abbreviations ===================== F&V: Fruits and Vegetables SF-36: Short Form-36 Health Survey PFS: Physical Function Subscale Competing Interests =================== The author(s) declare that they have no competing interests. Authors\' Contributions ======================= WDW, ECC, MCM, CFP and HJC conceived of the study, and participated in its design. DCS participation in the coordination of the study, as well as data cleaning and interpretation of findings. RS performed the statistical analysis. All authors read and approved the final manuscript. Acknowledgements ================ Sources of Financial Support: NIH\# P60-AG11268, R01-CA81191 (WDW, ECC, DCS, RS), R01-CA106919 (WDW, ECC, MCM, CFP, DCS, RS, HJC), P20-NR07795 (ECC), and the Mary Duke Biddle Foundation. We thank Heather MacDonald, Rebecca Tesh and Diane Parham for their dedication and hard work. Finally, we appreciate the advice of our advisory committee members (Drs. Steven Clinton, Charles Evans, Linda Fried, Charles Poole and Alfred Siu).	PubMed Central	2024-06-05T03:55:49.842797	2004-10-29	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534091/", "journal": "Int J Behav Nutr Phys Act. 2004 Oct 29; 1:16", "authors": [ { "first": "Wendy", "last": "Demark-Wahnefried" }, { "first": "Elizabeth C", "last": "Clipp" }, { "first": "Miriam C", "last": "Morey" }, { "first": "Carl F", "last": "Pieper" }, { "first": "Richard", "last": "Sloane" }, { "first": "Denise Clutter", "last": "Snyder" }, { "first": "Harvey J", "last": "Cohen" } ] }
PMC534092	Background ========== During HIV-1 infection, an increase in DC-SIGN and CD40 has been reported, as has a decrease in the expression of CD80 and CD86 in dendritic cells (DCs) of lymphoid tissue \[[@B1]\]. Although some suggest that HIV-1 infection reduces the production of IL-12 by DCs,\[[@B2]\] others have shown that DCs derived from HIV-1-infected individuals express both IL-12 and IL-10 at levels similar to those in non-infected individuals\[[@B3]\] While these studies have explored the effects of wild-type HIV-1 on DC functions, the possible effects of HIV-1-derived lentiviral vectors (LVs) on DC functions have not been well characterized \[[@B1]\]. LVs are useful gene transfer tools that can efficiently target many types of cells including DCs. As important immune modulating cells for immunotherapy and vaccine applications, DCs play critical roles in activating the host immune response. DCs can capture, process, and present foreign antigens, migrate to lymphoid-rich tissues, and stimulate antigen-specific immune responses \[[@B4]\]. DCs present a variety of signals to stimulate T cells and initiate immune response; these signals involve multiple signaling mediators, including MHC molecules harboring antigenic peptides (signal 1), the co-stimulatory molecules CD80, CD86, and ICAM-1 (signal 2), and cytokines such as IL-12, IL-4, and IL-10 (signal 3) \[[@B5]\]. Engagement between DCs and T cells not only stimulates T-cell proliferation, but also polarizes differentiation of naïve T helper (Th) cells into IFN-γ-producing Th1 or IL-4-producing Th2 effector cells \[[@B6],[@B7]\]. Production of IL-12 by DCs early in an immune response is critical for polarization of CD4^+^T cells toward Th1 function, which is essential for the clearance of intracellular pathogens. IL10, on the other hand, suppresses IL-12 production from DCs and diminishes the commitment of Th1 differentiation. Besides cytokine signaling, there is accumulating evidence that co-stimulatory molecules and adhesion molecules such as CD80, CD86, and ICAM-1 not only engage in T-cell stimulation, but also direct the differentiation of naive T cells \[[@B8]-[@B10]\]. Efficient gene transfer into DCs without cytotoxicity has always been difficult \[[@B11],[@B12]\]. LVs transduce DCs at high efficiencies with little to no cytotoxicity, and the transduced DCs retain their immature phenotype, are able to respond to maturation signals, and maintain immunostimulatory potential in both autologous and allogeneic settings \[[@B13]-[@B16]\]. In this study, we carefully analyzed cellular response to LV transduction by evaluating changes in DC phenotypes using monocyte-derived DCs prepared from more than 40 blood donors. We investigated the function of DCs to polarize naive T cells to Th effectors after LV infection. Our results demonstrated altered DC functions after LV gene transfer. Most importantly, we illustrated effective modulation of DC immunity by LV expression of different cytokines or siRNA molecules. Materials and Methods ===================== Generation of monocyte-derived dendritic cells ---------------------------------------------- Peripheral blood mononuclear cells (PBMCs) from healthy donors (Civitan Blood Center, Gainesville, FL) were isolated from buffy coats by gradient density centrifugation in Ficoll-Hypaque (Sigma-Aldrich, St. Louis, MO) as previously described \[[@B17]\]. DCs were prepared according to the method of Thurner et al. \[[@B18]\], with the following modifications: On Day 0, five million PBMCs per well were seeded into twelve-well culture plates with serum-free AIM-V medium (Invitrogen Corp. Carlsbad, CA). The PBMCs were incubated at 37°C for 1 hr and the non-adherent cells were gently washed off; the remaining adherent monocytic cells were further cultured in AIM-V medium until Day 1. The culture medium was removed with care not to disturb the loosely adherent cells, and 1 ml per well of new AIM-V medium containing 560 u/ml of recombinant human GM-CSF (Research Diagnostic Inc., Flanders, NJ) and 25 ng/ml of IL-4 (R&D Systems, Minneapolis, MN) was added and the cells were cultured at 37°C and 5% CO~2~. On Day 3, 1 ml of fresh AIM-V medium containing 560 u/ml of GM-CSF and 25 ng/ml of IL-4 was added to the culture. On Day 5, the non-adherent cells were harvested by gentle pipetting. After washing, the DCs were frozen for later use or used immediately. Lentiviral vector construction and preparation ---------------------------------------------- MLV and LVs were constructed as described previously \[[@B19],[@B20]\]. The self-inactivating pTYF vectors expressing CD80, CD86, GM-CSF, and IL-12 genes under the EF1α promoter control were constructed by inserting cDNAs that have been previously functionally characterized \[[@B21]-[@B23]\]. The cDNA of ICAM-1 was derived from pGEM-T-ICAM-1 kindly provided by Dr. Eric Long. The cDNAs of Flt3L, CD40L, and IL-7 were amplified by RT-PCR using the primers listed below with a modified eukaryotic translation initiation codon (CCACC-AUG): Flt3L sense 5\'-TTT CTA GAC CAC CAT GAC AGT GCT GGC GCC AG-3\' and antisense 5\'-AAG GAT CCT CAG TGC TCC ACA AGC AG-3\'; CD40L sense 5\'-TTT CTA GAC CAC CAT GAT CGA AAC ATA CAA C-3\' and antisense 5\'-TTG AAT TCT TAT GTT CAG AGT TTG AGT AAG CC-3\'; IL-7 sense 5\'AAG CGG CCG CCA CCA TGT TCC ATG TTT CTT-3\' and antisense 5\'-TTC TCG AGT TAT CAG TGT TCT TTA GTG CCC ATC-3\'. The LVs were produced and concentrated as described previously \[[@B20]\]. Lentiviral siRNA vectors were generated as previously described, using four oligonucleotides. IL-10i\#1: sense 5\'-GAT CCC CAG CCA TGA GTG AGT TTG ACT TCA AGA GAG TCA AAC TCA CTC ATG GCT TTT TTG GAA A-3\' and antisense 5\'-AGC TTT TCC AAA AAA GCC ATG AGT GAG TTT GAC TCT CTT GAA GTC AAA CTC ACT CAT GGC TGG G-3\'; IL10i\#2: sense 5\'-GAT CCC CGG GTT ACC TGG GTT GCC AAT TCA AGA GAT TGG CAA CCC AGG TAA CCC TTT TTG GAA A-3\' and antisense 5\'-AGC TTT TCC AAA AAG GGT TAC CTG GGT TGC CAA TCT CTT GAA TTG GCA ACC CAG GTA ACC CGG G-3\' \[[@B24]\]. Lentiviral transduction of immature DCs and DC maturation --------------------------------------------------------- We plated Day-5 immature DCs at 5 × 10^5^per well in a 24-well plate containing 200 μl of medium supplemented with GM-CSF (560 u/ml) and IL-4 (25 ng/ml). DC infection was carried out by adding concentrated LVs to the cells at a multiplicity of infection (MOI) of 50--100 (\~10^5^--10^6^transducing units/ng of p24) as previously described \[[@B25]\]. The infected cells were incubated at 37°C for 2 hr with gentle shaking every 30 min, then 1 ml of DC medium was added and the culture was incubated with the viral vectors for an additional 12 hr. DC maturation was induced by adding lipopolysaccharide (LPS) at a final concentration of 80 ng/ml and TNF-α at a final concentration of 20 u/ml and incubated for 24 hr. To collect mature DCs, the cells were treated with AIM-V medium containing 2 mM EDTA at 37°C for 20 min, and washed three times with PBS. Antibody staining and flow cytometry ------------------------------------ For analysis of cell-surface marker expression by flow cytometry, we incubated DCs for 10 min with normal mouse serum and then 30 min with fluorochrome-conjugated anti-human monoclonal antibodies. In different experiments, these antibodies included HLA-ABC (Tu149, mouse IgG2a, FITC-labeled, Caltag Laboratories, Burlingame, CA); HLA-DR (TU36, mouse IgG2b, FITC-labeled, Caltag Laboratories); CD1a (HI49, mouse IgG1k, APC-labeled, Becton Dickinson Pharmigen, San Diego, CA); CD80 (L307.4, mouse IgG1k, Cychrome-labeled, Becton Dickinson);CD86 (RMMP-2, Rat IgG2a, FITC-labeled, Caltag Laboratories); ICAM-1 (15.2, FITC-labeled, Calbiochem); DC-SIGN (eB-h209, rat IgG2a, APC-labeled, eBioscience, San Diego, CA); CD11c (Bly-6, mouse IgG1, PE-labeled, BD Pharmigen); CD40 (5C3, mouse IgG1, Cy-chrome-labeled, Becton Dickinson); CD123 (mouse IgG1, PE-labeled, BD Pharmigen); and CD83 (HB15e, mouse IgG1, R-PE-labeled, Becton Dickinson). We included the corresponding isotype control antibody in each staining condition. After two washes, the cells were resuspended and fixed in 1% paraformaldehyde in PBS and analyzed using a FACSCalibur flow cytometer and the CELLQUEST program (Becton Dickinson). The live cells were gated by forward- and side-light scatter characteristics and the percentage of positive cells and the mean fluorescence intensity (MFI) of the population were determined. FACS sort of lacZ-positive cells -------------------------------- The lentiviral siRNA vector-transduced cells co-expressing nuclear lacZ gene were separated from un-transduced cells by staining with fluorescent LacZ substrate and sorted by FACS. To label the lacZ-positive cells, we resuspended cells in 100 ul medium and added 100 ul of FDG (fluorescein di-beta-D-galactopyranoside) working solution (2 mM) which was diluted from a 10 × stock FDG solution (20 mM). The stock solution was made by dissolving 5 mg FDG (MW 657, Molecular Probe, Eugene, OR) in a 1:1 mixture of DMSO/ethanol and mixing with ice-cold ddH~2~O to make an 8:1:1 ddH~2~O/DMSO/ethanol solution. The cells were incubated in 37°C water bath for 1--1.5 min, and diluted with 10-fold volume of cold medium and kept on ice until FACS sorting. Preparation of naïve CD4+ T cells --------------------------------- The CD4^+^T cells from PBMCs were collected by negative selection, using a CD4^+^T cell isolation Rosette cocktail (StemCell Technologies, Vancouver, BC) according to the manufacturer\'s instructions. Briefly, we centrifuged 45 ml of buffy coat (approximately 5 × 10^8^PBMCs) in a sterile 200-ml centrifuge tube with 2.25 ml of the CD4^+^T cell-enrichment Rosette cocktail at 25°C for 25 min. Thereafter, 45 ml of PBS containing 2% FBS was added to dilute the buffy coat. After gentle mixing, we layered 30 ml of the diluted buffy coat on top of 15 ml of Ficoll Hypaque in a 50-ml centrifuge tube and centrifuged for 25 min at 1,200 g. Non-rosetting cells were harvested at the Ficoll interface and washed twice with PBS (2% FBS), counted, and cryopreserved in aliquots in liquid nitrogen for future use. The purity of the isolated CD4^+^T cells was consistently above 95%. The CD4^+^CD45RA naïve T cells were purified based on negative selection of CD45RO^-^cells using the MACS (Miltenyi Biotec, Auburn, CA) magnetic affinity column according to the manufacturer\'s instructions. In vitroinduction of Th functions and intracellular cytokine staining ----------------------------------------------------------------------- The in vitroDC:T cell co-culture method was modified based on Caron et al\[[@B26]\]. Briefly, we co-cultured purified naïve CD4 T cells with allogeneic mature DCs at different ratios (20:1 to 10:1) in serum-free AIM-V media. On day 5, 50 u/ml of rhIL-2 was added and the culture was expanded and replenished with fresh AIM-V medium containing rhIL-2 every other day for up to 3 weeks. After day 12, we washed the quiescent Th cells and re-stimulated them with PMA (10 ng/ml or 0.0162 μM) and ionomycin (1 μg/ml, Sigma-Aldrich) for 5 hr, adding Brefeldin A (1.5 μg/ml) during the last 2.5 hr of culture. We then fixed, permeablized, and stained the cells with FITC-labeled anti-IFN-γ and PE-labeled anti-IL-4 mAb (Pharmigen, San Diego, CA). The cells were analyzed in a FACSCalibur flow cytometer (BD Biosciences, San Diego, CA). DC-mediated mixed lymphocyte reaction ------------------------------------- We co-cultured serial dilutions of DCs, from 10,000 cells per well to 313 cells per well, with 1 × 10^5^allogeneic CD4 T cells in a 96-well U-bottomed plate in a total volume of 200 μl for 5 days. The proliferation of T cells was monitored by adding 20 μl of the CellTiter96 solution to each well according to the manufacturer\'s instruction (Promega). The cells were further cultured for 4 hr before reading the OD~490~value using a microplate reader (EL808, BIO-TEK Instrument Inc.,Winooski, VT). Results ======= LVs altered surface marker expression in peripheral blood monocyte-derived DCs ------------------------------------------------------------------------------ To investigate the effects of lentiviral vectors (LVs) on DCs, we transduced monocyte-derived DCs with LVs encoding different reporter genes. The efficiency of LV transduction of DCs is illustrated by a reporter gene assay (Fig. [1A](#F1){ref-type="fig"}). The DCs were derived from healthy donors\' PBMCs, and on day 5 (d5) of culture, the immature DCs (imDC) were infected with LV-PLAP (encoding placenta alkaline phosphatase). Analysis of PLAP activity on day 7 demonstrated transduction efficiency of \> 80% (Fig [1B](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### LV transduction of DCs and analysis of surface marker expression.PBMC-derived DCs were infected with LV on Day 5 (d5) and after maturation, co-cultured with naïve CD4 T cells for 1--2 weeks before intracellular cytokine staining (ICCS) and flow cytometry (FACS) analysis. The d5 DCs were transduced by LV-PLAP and 48 hr later, analyzed by PLAP enzyme assay. (B) FACS analysis of DC surface markers after viral transduction. The d5 DCs were transduced with LV carrying PLAP or Cre as reporter gene, MLV vectors, empty LV, or no vector controls (mock) and treated with TNF-α plus LPS. The cell surface markers were stained with antibodies and analyzed by FACS. The numbers represent mean fluorescence index (MFI) and the results are representatives of six experiments. ::: ![](1742-4690-1-37-1) ::: ### DC functions through surface receptor signaling To see if LVs affected DC surface marker expression, we examined the expression profile of surface molecules on DCs by antibody immunostaining. We transduced PBMC-derived imDC with mock (control 293 supernatants) vectors, empty LV particles, LV, and MLV carrying a reporter gene. After induction of maturation with LPS plus TNF-α, we harvested the DCs for antibody staining and FACS. The results are shown in Fig. [1C](#F1){ref-type="fig"} and summarized in Table [1](#T1){ref-type="table"}. Among the surface molecules tested, CD1a, CD80, CD86, ICAM-1, and DC-SIGN were down-regulated after LV transduction, but not after transduction with empty LV or MLV. The same result was obtained using different preparations of LVs carrying either PLAP or Cre as the reporter gene. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Surface marker profile of DCs transduced with LVs or MLV vectors. ::: Geometrical Mean Fluorescence ± SD ---------------- ------------------------------------ -------------- ------------------- ------------- Surface Marker Mock Empty LV LV MLV CD11c 48.8 ± 3.2 47.2 ± 1.3 52.3 ± 2.3 55.3 ± 1.1 CD123 13.0 ± 0.4 13.4 ± 0.8 14.9 ± 0.6 15.7 ± 0.1 CD1a 27.3 ± 1.1 27.6 ± 2.9 21.5 ± 0.2\* 31.0 ± 0.3 CD40 8.6 ± 0.1 8.9 ± 0.6 8.6 ± 0.1 9.0 ± 0.3 ICAM-1 462.6 ± 57.5 376.5 ± 30.1 179.5 ± 3.4\\\* 498.5 ± 6.9 CD62L 3.3 ± 0.1 3.2 ± 0.03 3.7 ± 0.1 3.3 ± 0.4 CD80 (B7-1) 9.9 ± 0.9 10.6 ± 0.7 9.3 ± 0.2\* 11.3 ± 0.4 CD83 5.8 ± 0.3 5.8 ± 0.1 6.4 ± 0.01 6.0 ± 0.3 CD86 (B7-2) 39.6 ± 3.5 39.6 ± 2.5 31.4 ± 0.4\* 47.3 ± 1.5 DC-SIGN 62.7 ± 4.5 55.7 ± 0.4 50.6 ± 1.5\* 68.6 ± 4.1 HLA-ABC 13.9 ± 1.3 15.8 ± 1.0 14.6 ± 0.3 17.2 ± 0.9 HLA-DR 31.5 ± 0.8 28.6 ± 2.2 26.9 ± 0.4 33.2 ± 1.7 Results are presented as geometrical mean fluorescence after FACS. Asterisks (\) denote significance of difference by Student t-test (\P \< 0.05, \\P \< 0.01, \\\P \< 0.001). ::: LV transduction impaired DC-mediated Th1 immunity ------------------------------------------------- It has been reported that retroviral infection induces up-regulation of Th2 cytokines including IL-10 and impairs DC maturation \[[@B27],[@B28]\]. Because HIV causes immune suppression and the preceding results showed that LV infection altered the surface marker expression profile of DCs, we suspected that LV infection might also affect DC activation of T cells. To test this, we set up an in vitroimmunity assay using co-culture of human DCs and naïve T cells. We generated DCs from PBMCs and infected the d5 DCs with LV carrying a reporter gene. To characterize the function of DCs, we purified naïve CD4^+^T cells from healthy donors\' blood and co-cultured the T cells with allogeneic monocyte-derived DCs treated with TNFα and LPS to induce maturation, as illustrated in Fig. [2](#F2){ref-type="fig"}. The co-cultured T cells were allowed to expand and rest for more than 7 days after DC priming. To analyze Th response, on days 7 and 9 we reactivated the resting T cells with ionomycin and PMA, and subjected the T cells to ICCS using antibodies against IFN-γ and IL-4. We found that the IFN-γ-producing Th1 cell populations were dramatically reduced when incubated with DCs transduced with LVs, from 72% (day 7) and 75% (day 9) for the control to 27% (day 7) and 22% (day 9) for the LV-transduced DCs. The Th2 populations remained essentially unchanged (Fig. [2](#F2){ref-type="fig"}). In naïve T cells the Th1 response is regulated by the \"master transcription regulators\" T-bet and GATA-3.\[[@B29]\] Analysis of T-bet and GATA-3 expression in T cells after coculture with LV-transduced DCs showed decreased expression of both T-bet and GATA-3 RNA, and the relative T-bet expression correlated with the Th differentiation according to ICCS of T cells after 8 days of co-culture (data not shown). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Impaired Th1 response induced by LV-transduced DCs.We analyzed T helper function by using DC:T cell co-culture and IL-4 and IFN-γICCS. Immature DCs were infected with mock (293T supernatants) or LV on d5 and treated with LPS and TNFα. The DCs were harvested and co-cultured with naïve CD4+ T cells at a DC:T cell ratio of 1:10. On Day 7 amd 9 after co-culture, the cells were re-stimulated and the T helper cell populations were examined by INF-γ and IL-4 antibody ICCS as described in the Materials and Methods. The percentages of cell populations are indicated in the FACS quadrants. The results are representative of four independent experiments. ::: ![](1742-4690-1-37-2) ::: Up-regulation of CD80 and CD86 expression did not restore DC functions ---------------------------------------------------------------------- Because T cell co-stimulatory molecules are important mediators of DC functions, the down-regulation of CD80 and CD86 in DCs after LV transduction might contribute to the observed Th1 impairment. To examine this possibility, CD80 and CD86 were up-regulated in DCs using LVs encoding these two genes to see if the impaired Th1 response could be corrected. The LVs encoding human CD80 and CD86 were constructed as shown in Fig. [3A](#F3){ref-type="fig"}. The functions of these CD80 and CD86 genes have been previously demonstrated in an in vivostudy \[[@B21]\]. DCs were transduced with LVs expressing a reporter, CD80 or CD86 gene, and then treated with LPS and TNF-α 12 hr later. Thirty-six hours after LV transduction, we analyzed the transduced DCs for CD80 and CD86 expression by FACS, using anti-CD80 and anti-CD86 antibodies. The results were consistent with our earlier findings; CD80 expression was reduced from 41% to 35% after LV-PLAP infection, while CD86 expression was reduced from 61% to 49% (Fig. [3B](#F3){ref-type="fig"}). Their expression was up-regulated after transduction with LVs encoding CD80 and CD86; the expression of CD80 was up-regulated from 35% to 44%, and the expression of CD86 from 49% to 76%. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### LV modification of DC immune functions.(A) Diagram of LV constructs containinging different immune modulatory genes. (B) Up-regulation of T cell costimulators in DCs transduced with LV-CD80 or LV-CD86. Immature DCs were transduced with mock, LV-PLAP, LV-CD80, or LV-CD86 for 12 hr, induced to mature, and analyzed 24 hr later using anti-CD80 and anti-CD86 antibodies. The mean fluorescence intensity and percentage of positive cells are shown. (C) Th1/Th2 assay of DCs with up-regulated CD80 or CD86. The T-cell activation function of DCs was analyzed by DC:T cell co-culture. ICCS and FACS for T helper function were performed 8 days after co-culture. The percentages of different T-cell populations are shown. (D) Th1/Th2 assay of DCs co-transduced with different LV immune modulatory genes. DCs were transduced with LV (LV-PLAP), and co-transduced with LVs encoding different immune modulatory genes, including IL-12, CD40L, IFN-γ, FL, GM-CSF, and ICAM-1, or incubated with soluble IFN-γ. DCs were then treated with TNF-α and LPS and co-cultured with naïve CD4 T cells. The T cells were analyzed for IL-4 and IFN-γ expression by ICCS and FACS 9 days after co-culture. The percentages of different T cell populations are shown in the quadrants. The results are representative of six independent experiments. ::: ![](1742-4690-1-37-3) ::: To see if the up-regulation of the T-cell co-stimulatory molecules in DCs could restore the Th1 response, we co-cultured naïve CD4 T cells with DCs transduced with mock, LV-PLAP, LV-PLAP plus LV-CD80, or LV-PLAP plus LV-CD86. After 8 days, the T cells were reactivated and analyzed by ICCS and FACS using anti-IL-4 and anti-IFN-γ antibodies as described earlier. We found that after LV transduction the Th1 population was reduced from 24% to 13%. Moreover, this impairment could not be corrected by the up-regulation of CD80 and CD86 in DCs (from 13% to 12% and 13%, respectively, Fig. [3C](#F3){ref-type="fig"}). Modification of DC immunity by LVs encoding immune modulatory genes ------------------------------------------------------------------- Cytokine signaling is important in DC-mediated Th differentiation; for examples, IL-12 is critical to Th1 development, and Flt3-ligand (FL) has been shown to enhance IL-12 production in DCs \[[@B30]\]. To overcome the impaired Th1 response after LV transduction, we investigated whether modification of the local cytokine environment in the DC:T cell synapse could promote a Th1 response. LVs expressing different cytokine and receptor genes, including FL, GM-CSF, IL-12 (a bi-cistronic IL-12A and IL-12B construct), CD40L, IFN-γ, and ICAM1 were constructed (Fig. [3A](#F3){ref-type="fig"}). Expression or function of these different immune modulatory genes has been previously demonstrated. \[[@B21]-[@B23]\] DCs were transduced with LVs carrying reporter gene PLAP either alone or co-transduced with different immune modulatory genes. As positive control, we treated DCs with soluble IFN-γ before maturation and DC:T-cell co-culture. The Th function of the LV-transduced DCs was analyzed by DC:T cell co-culture followed by ICCS and FACS analysis of IFN-γ and IL-4, as described earlier. The results showed that LV transduction alone reduced IFN-γ-producing Th1 cell population as found above, from 8.16% to 3.46%. However, co-transduction with LV encoding IL-12 enhanced Th1 response from 3.46% to 9.38%, while co-transduction with LV encoding IFN-γ increased such response from 3.46% to 13.08%, an increase that was similar to that produced by soluble IFN-γ (Fig. [3D](#F3){ref-type="fig"}). LVs expressing FL, GM-CSF, CD40L, or ICAM-1, on the other hand, exhibited no significant effect. Modulation of DC function by LVs expressing siRNA targeting IL-10 ----------------------------------------------------------------- IL-10 is a critical immune modulatory gene and modulation of IL-10 gene expression may alter DC function. To test this, we constructed LVs encoding siRNA targeting IL-10. We chose two regions in the IL-10 mRNA as the siRNA target sites (Fig. [4A](#F4){ref-type="fig"}). The siRNA expression was driven by a human H1 polIII promoter that was cloned into LVs as previously reported.\[[@B24]\] The LV-siRNA vector also carries a nlacZ reporter gene convenient for vector titer determination and for the identification of transduced cells. To demonstrate the siRNA effects, we transduced B cells with IL-10-siRNA LVs or a control siRNA LV targeting GFP gene, and after transduction, the B cells were expanded and the lacZ-positive cells were FACS-sorted using fluorescent substrate FDG. The expression of IL-10 was quantified by ICCS and FACS using anti-IL-10 Ab. The result demonstrated IL-10 suppression in the lacZ-positive B cells that were transduced with LVs expressing the two IL-10 specific siRNAs but not the non-specific siRNA targeting GFP gene (Fig. [4B](#F4){ref-type="fig"}). ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Modification of DCs by LV-siRNA targeting IL-10.(A) LV-siRNA targeting IL-10. LV siRNAs targeting two different sites of IL-10 mRNA were illustrated. The predicted hairpin siRNA structure is shown. (B) Illustration of efficient down-regulation of IL-10 in B lymphocytes after LV IL-10 siRNA transduction. Epstein-Barr virus (EBV) transformed B cells were transduced with LV siRNA targeting IL-10 (\#1 and \#2) or GFP gene. The siRNA LVs also carry a lacZ reporter gene which could be labeled with fluorescein di-b-D-galactopyranoside (FDG) to separate the transduced from un-transduced cells by FACS sort. (C) Immature DCs were transduced with mock, empty LVs, LV-nlacZ, or LV-nlacZ plus LV-siIL-10 \#1 or \#2, treated with LPS, and analyzed by ICCS and FACS using anti-IL-10 antibody. (D) Enhanced Th1 response by DCs transduced with LV-siRNA targeting IL-10. DCs were transduced with LVs and either co-transduced with LV-siRNA targeting IL-10 (LV-IL10i\#2) or GFP (GFPi) or treated with soluble IFN-γ as controls, and the DCs were then assayed for T-cell activation function by DC:T cell co-culture. The T cells were fully rested before reactivation with PMA and ionomycin after 10 days of co-culture. The numbers shown in the FACS quadrants are percentages of the total gated cell population. Results are representative of three independent experiments. ::: ![](1742-4690-1-37-4) ::: The effect of the IL-10 LV siRNAs was then examined in DCs by co-transduction using a reporter LV and the IL-10 LV-siRNAs. The transduced DCs were then treated with LPS and analyzed for IL-10 expression as described above. Again, the empty LV had no effect and LV transduction alone up-regulated IL-10 expression. However, co-transduction with LV-siRNA targeting IL-10 down-regulated IL-10 expression (Fig. [4C](#F4){ref-type="fig"}); the low level of IL-10 expression in DCs was expected as the DC culture was derived and maintained in GM-CSF and IL-4 supplemented media. To examine whether co-transduction of DCs with LVs expressing the IL-10 siRNA could promote a Th1 response, we transduced DCs with LV alone or together with either an LV-siRNA (\#2) or a control LV-siRNA (GFPi). For positive control, we incubated DCs with soluble IFN-γ as previously described. After the DCs were co-cultured with naïve T cells for 10 days, the T cells were reactivated and analyzed for Th functions by ICCS to determine intracellular expression of IFN-γ and IL-4. The results clearly demonstrated that the IL-10 LV-siRNA vector, but not the GFPi LV-siRNA vector, enhanced Th1 response at levels comparable to that of the positive control (DCs treated with soluble IFN-γ, Fig. [4D](#F4){ref-type="fig"}). Discussion ========== Although HIV-1 is an immunopathogen in humans, HIV-1 derived vectors do not contain viral genes and have been rendered replication-defective. In this study, we found that LV transduction of DCs resulted in altered DC surface marker phenotypes. These changes in DC phenotypes led to suppressed function in mediating the Th1 immunity. DCs transduced by LVs did not lose the capacity to stimulate allogeneic T-cell proliferation, as reported by others \[[@B13],[@B14],[@B16]\]. However, in the DC:T cell co-culture functional assay, we showed that after LV transduction, DCs had significantly reduced ability to polarize naïve CD4+ T cells to differentiate into Th1 effectors. The changed gene-expression profile of DCs after LV transduction correlates with Th1 suppression. As demonstrated here, DC-mediated immunity requires antigen presentation, T cell co-stimulation, and cytokine production, all of which were down-regulated upon LV infection. These results are consistent with a recent study demonstrating cultured immature DCs and DCs from 6 of 10 HIV-1 patients display reduced maturation function and diminished MLR in DC:T cell coculture \[[@B28]\]. Cytokines have critical roles in shaping up the immune response \[[@B31],[@B32]\]. We have detected up-regulation of IL-10 in HUVEC, B cells and DCs after LV infection suggested possible immune suppression by LVs (data not shown). Earlier work has shown that IL-10 inhibits the expression of IL-12 and co-stimulatory molecules in DCs,\[[@B32]\] a finding that correlates with its ability to inhibit the primary T-cell response and induce a stage of anergy in allo- or peptide-antigen-activated T cells \[[@B33]\]. IL-10 has also been shown to down-regulate ICAM-1 in human melanoma cells \[[@B34]\]. Here we showed that LV transduction of DCs, led to down-regulation of CD80, CD86, and ICAM-1. Many of these immune regulatory genes are activated through transcriptional factor NF-κB. Using cDNA microarray analysis, we detected reduced NF-κB expression in DCs after LV infection (not shown), suggesting that LV infection may trigger a cascade of immune suppression through down-regulation of the NF-κB signaling pathway. It has been reported that HIV-1 Tat up-regulates IL-10 as a result of intranuclear translocation of NF-κB and activation of the protein kinases ERK1 and ERK2 \[[@B35]\]. However, the LVs used in this study do not carry a tat gene. The fact that empty LV particles did not induce the same effects as did intact LVs, suggests that Tat or other virion-associated proteins do not play a role. Thus, it is plausible that events after retroviral attachment and fusion, such as reverse transcription and integration, might trigger the observed cellular response. It would be interesting to see if such immune suppression also occurs in vivo*following LV gene transfer. DCs, during their interaction with T cells, provide multiple signals to polarize naïve T cells. These signals include the co-stimulatory molecules CD80, CD86, and ICAM-1, which are considered \"signal 2\" for T-cell stimulation. The roles of these co-stimulatory molecules on Th differentiation remain controversial. Many studies have shown that ICAM-1 promotes Th1 commitment \[[@B36]\]. CD80 and CD86 have been reported to polarize CD4^+^T cells toward the Th2 subset through engagement with CD28 \[[@B37]-[@B39]\]. However, CD80 could also interact with CTLA-4 to induce Th1 polarization \[[@B40]\]. Moreover, CD86 has been reported to be a Th1-driving factor \[[@B41]\]. Further studies are needed to address the roles of co-stimulatory molecules in the development of DC and T-cell immunity. Nevertheless, the down-regulation of T cell co-stimulatory molecules in DCs after LV transduction could potentially have an impact on the DC-mediated Th1 response. The analysis of surface-marker expression profile also revealed down-regulation of CD1a and DC-SIGN in DCs after LV transduction. CD1a is a nonpolymorphic histocompatibility antigen associated, like MHC class I molecules, with beta-2-microglobulin, and is responsible for the presentation of lipid antigens. DC-SIGN (DC-specific, ICAM-3 grabbing nonintegrin) is a 44-kDa type I membrane protein with an external mannose-binding, C-type lectin domain \[[@B42]\]. It has been postulated that DC-SIGN interacts with ICAM-3 on T cells to allow sufficient DC-T cell adhesion and, in addition, that DC-SIGN is a new member of the co-stimulatory molecule family \[[@B5],[@B43]\]. With these characteristics, the down-regulation of CD1a and DC-SIGN might also contribute to the impaired Th1 function of DCs. Polarization of naïve Th cells into Th1 cells is critical for the induction of cellular immunity against intracellular pathogens and cancer cells. The observed impairment of the Th1 response by LV-transduced DCs raises a potential issue with LV-based immunotherapy. We illustrated that co-transduction with LV encoding IL-12 or IFN-γ, but not CD80, CD86, or ICAM-1, in DCs effectively restored Th1 immunity. In addition, co-transduction with LVs expressing small interfering RNA targeting IL-10 could also promote DC-mediated Th1 immunity. In a step toward future generation of vaccines, LVs encoding IL-12 and IL-10-siRNA as potent Th1 adjuvants may be used to enhance the cellular immune response in the prime-and-boost vaccination regimen. In summary, our study has addressed an important immune suppression effect of LVs and presented a solution that is important for future LV-based DC immunotherapy applications. List of abbreviations used ========================== HIV-1 human immunodeficiency virus type 1 LV lentiviral vector DC dendritic cell Th T helper MLV murine leukemia virus RT-PCR reverse transcription-polymerase chain reaction FACS fluorescence activated cell sorter ICCS intracellular cytokine staining IL interleukin PLAP placenta alkaline phosphatase siRNA small interfering RNA PBMC peripheral blood mononuclear cells MOI multiplicity of infection LPS lipopolysaccharide TNFα tumor necrosis factor alpha INF-γ interferon-gamma Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= The study was conceived by LJC; XC and LJC participated in designing and coordinating the study; JH carried out some of the lentiviral constructions, siRNA designs and participated in result discussion; XC performed the statistical analysis; LJC and XC carried out detailed analysis of the results and XC drafted and LJC finalized the manuscript. All authors read and approved the final manuscript. Acknowledgment ============== We thank F. Higashikawa, W. Chou and B. Lo for technical assistance, M. Chen and N. Benson for assistance in flow cytometry, and Dr. E. Long for the plasmid pGEM-T-ICAM-1. We are grateful to the blood donors of Life South. This work was supported by an NIH grant.	PubMed Central	2024-06-05T03:55:49.844683	2004-11-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534092/", "journal": "Retrovirology. 2004 Nov 1; 1:37", "authors": [ { "first": "Xiaochuan", "last": "Chen" }, { "first": "Jin", "last": "He" }, { "first": "Lung-Ji", "last": "Chang" } ] }
PMC534093	Introduction ============ During the past decades many methods and factors have been proposed as good prognostic tools or markers for cardiovascular risk stratification. However, even with the most sophisticated stress testing procedures the prediction capability still remains imperfect \[[@B1]\]. Previous studies have shown that atherosclerosis of the descending aorta detected by transesophageal echocardiography (TEE) is a good marker of coexisting coronary artery disease \[[@B2]-[@B9]\]. Cohen et al. have demonstrated, that in patients with brain infarction, \"complex\" plaques (with ulcerated surface, mobile parts and thrombi) are powerful predictors of future cardiovascular events \[[@B10]\]. The aim of this study was to evaluate whether the presence of atherosclerosis on the descending aorta observed by routine scanning during TEE has any prognostic impact in predicting cardiovascular events such as cardiac death, myocardial infarction or fatal stroke. Therefore, we conducted a prospective study; collecting and analyzing the data of 238 consecutive patients referred to the echo lab for transesophageal echocardiography. Methods ======= Patient selection ----------------- During the year 1998, 238 consecutive patients (135 males, 103 females, and mean age 58 ± 11 years) were studied with transesophageal echocardiography at the 2^nd^Department of Medicine and Cardiology Center, University of Sciences, Szeged, Hungary. The patients underwent TEE examination for the following reasons: TIA or suspected cerebral embolism (n = 100), coronary flow reserve evaluation (n = 71), evaluation of the native or artificial mitral valve (n = 23), suspected endocarditis (n = 15), evaluation of the aortic valve (n = 13), suspected aortic dissection (n = 6), atrial septal defect or patent foramen ovale (n = 6), others (n = 4). Thirty patients had suffered a previous myocardial infarction. The patients were followed-up to a period of at least 2 years, median 31 ± 9 months. Transthoracic and transesophageal echocardiography -------------------------------------------------- All patients had a transthoracic echocardiographic examination to assess the global and regional left ventricular function. The ejection fraction was calculated using the area-length single plane method \[[@B11]\]. Left ventricular function was considered to be depressed in case of ejection fraction ≤40%. The TEE examination was carried out according to recommendations of the Mayo clinic procedure \[[@B12]\]. Two-dimensional echocardiograms were obtained by using commercially available imaging system (ATL-HDI) with a biplane transducer. Echocardiographic images were recorded on videotape for subsequent playback and analysis. The atherosclerotic lesions of the descending aorta were graded according to the modified scoring system originally proposed by Fazio et al \[[@B2]\]: Grade 0 -- no sign of atherosclerosis Grade 1 -- intimal thickening Grade 2 -- plaque \< 5 mm Grade 3 -- plaque \> 5 mm and/or \"complex\" plaque with ulcerated or mobile parts. Significant atherosclerosis was considered in case of grade 2 or 3 (fig [1](#F1){ref-type="fig"} and [additional files 1](#S1){ref-type="supplementary-material"}, [2](#S2){ref-type="supplementary-material"}, [3](#S3){ref-type="supplementary-material"}, [4](#S4){ref-type="supplementary-material"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Grading of the transesophageally detected aortic lesions in the descending aorta. Grade 1: intimal thickening (left upper panel); Grade 2: small plaque indicated by arrow right upper panel; Grade 3: lower panels. On the lower panels: left a huge, multiple plaque, right: an ulcerated plaque with mobile part. ::: ![](1476-7120-2-21-1) ::: Follow up data -------------- Follow-up data were obtained from at least one of four sources: 1) review of the patient\'s hospital record; 2) personal communication with the patient\'s physician and review of the patient\'s chart; 3) a telephone interview with the patient conducted by trained personnel; or 4) a staff physician visiting the patients at regular intervals in the outpatient clinic. Follow-up data were obtained in all patients. The outcome events were: cardiac related death, fatal stroke, non-fatal myocardial infarction and revascularization either by percutaneous transluminal coronary angioplasty (PTCA) or by coronary artery by-pass grafting (CABG). Myocardial infarction was documented by a consistent history, EKG changes and cardiac enzyme level elevations and confirmed by hospital chart or hospital discharge letter review. In the next step, patients with revascularization were censored and the remaining patients were considered for the analysis of hard events (cardiac death, myocardial infarction, and fatal stroke). All-cause mortality (cardiovascular death plus death of other causes) was also analyzed. Statistical analysis -------------------- Values are expressed as mean ± Standard Deviation. Continuous variables have been compared by the means of Student\'s t test (two-tailed). Statistical analysis of discrete variables has been performed with chi-square test; a Fisher\'s exact test has been used when appropriate. The individual effect of certain variables on infarction-free survival has been evaluated with the use of the Cox proportional hazard model with univariate and multivariate analysis and with the Kaplan-Meier method. The patients were stratified into two subgroups: patients with and without cardiac events. The examined variables were age, sex, risk factors (arterial hypertension, diabetes, hypercholesterolemia), previous myocardial infarction, ejection fraction, and significant atherosclerosis of the descending aorta. A p value \<0.05 was considered statistically significant. Results ======= Transesophageal echocardiography -------------------------------- The examination was successful and complete in all patients, without any side effect. Significant atherosclerosis of the descending aorta was observed in 102 patients, (grade 3 in 16, and grade 2 in 86 patients) whereas 136 patients had only mild intimal thickening (n = 46) or normal endocardial surface (n = 90). Follow-up data -------------- There were 57 events in the follow-up period: cardiac related death: n = 14, fatal stroke: n = 4, non-fatal myocardial infarction: n = 5 and coronary artery revascularization: n = 34. Ten patients died of non-cardiovascular causes and the cause of the death of 2 patients was undetermined. Cardiovascular events and TEE findings -------------------------------------- The number of events was significantly higher in the 102 patients with (n = 34) as in patients the 136 patients without significant atherosclerosis (n = 23, p \< 0.01). The results of univariate and multivariate analysis are shown in table [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Univariate analysis -- all events (cardiac death + nonfatal AMI + stroke + revascularization) ::: Variable p value -------------- ------------- Aorta plaque 0,0033 Previous MI 0,0074 Male gender 0,0088 Hypertension 0,0758 Age 0,1687 Cholesterol 0,1154 Diabetes 0,2954 LV EF 0,7810 LV EF = Left ventricular ejection fraction; MI = myocardial infarction ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Multivariate analysis -- all events (cardiac death + nonfatal AMI + stroke + revascularization) ::: 95% CI -------------- ------------- -------- ------------ ----------- Variable p value HR Lower Upper Aorta plaque \<0,01 2,1 1,2 3,5 Male gender \<0,05 0,49 0,3 0,9 HR = Hazard Ratio; CI = Confidence Interval ::: The frequency of events was in close correlation with the severity of the atherosclerosis of the descending aorta (fig [2](#F2){ref-type="fig"}). Fifty percent of the patients with grade 3 experienced cardiovascular events. There was no significant difference when all causes of death were considered between subjects with aortic lesions or free of atherosclerosis of the descending aorta (5% vs 1%, p = ns). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### A Kaplan-Meier curve showing the association between the severity of the transesophageally detected aortic plaques and the long term survival including all events. It can be clearly seen that more severe the atherosclerosis on the descending aorta was, higher the probability of future cardiovascular events. ::: ![](1476-7120-2-21-2) ::: Spontaneous cardiovascular events and TEE findings -------------------------------------------------- Patients with early (\<3 months, n = 29) or late (\>3 months, n = 5) revascularization have been censored. Nine events have occurred in the 122 patients with TEE score ≤1 and 14 in the 82 patients with TEE score ≥2 (7% vs 17%, p \< 0.05) (fig [3](#F3){ref-type="fig"}). The results of univariate analysis are shown in table [3](#T3){ref-type="table"}. Impaired left ventricular function (EF ≤ 40%), significant atherosclerosis of the descending aorta, and age were predictive for future cardiovascular events. By multivariate analysis, only left ventricular function with EF ≤ 40% (HR 3.0, 95% CI 1.3--7.1) and TEE atherosclerotic plaque ≥2 (HR 2.4, 95% CI 1.0--5.5) predicted cardiovascular events (table [4](#T4){ref-type="table"}). Similarly to the entire group, in patients with no revascularization, the more severe the atherosclerosis on the descending aorta was, higher the probability of future cardiovascular events. There was no significant difference when all causes of death were considered between subjects with aortic lesions or free of atherosclerosis of the descending aorta (5% vs 1%, p = ns). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### A Kaplan-Meier curve survival showing a better outcome of patients without transesophageally detected aortic plaques. ::: ![](1476-7120-2-21-3) ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Univariate analysis -- Hard events (cardiac death + nonfatal AMI + stroke) ::: Variable p value -------------- ------------- LV EF 0,0069 Aorta plaque 0,0283 Age 0,0440 Cholesterol 0,5642 Diabetes 0,9161 Hypertension 0,3212 Previous MI 0,6053 Male gender 0,9880 LV EF = Left ventricular ejection fraction; MI = myocardial infarction ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Multivariate analysis -- Hard events (cardiac death + nonfatal AMI + stroke) ::: 95% CI -------------- ------------- -------- ------------ ----------- Variable p value HR Lower Upper LV EF \<0,01 3,0 1,3 7,1 Aorta plaque =0,04 2,4 1,0 5,5 HR = Hazard Ratio; CI = Confidence Interval ::: Discussion ========== Atherosclerosis of the descending aorta observed during transesophageal echocardiography is a useful predictor of future cardiovascular events. Comparison with previous studies -------------------------------- Our data are in keeping up with previous findings, showing that patients with atherosclerosis of the thoracic aorta have higher probability of coexisting coronary artery disease \[[@B2]-[@B8]\]. In those series the positive predictive value of TEE varied between 64% and 95%, whereas the negative predictive value was consistently high (between 82 and 99%.), indicating that in the absence of echocardiographically assessed atherosclerotic plaque in the thoracic aorta the probability of coronary artery disease is unlikely. Furthermore, Khoury et al have demonstrated, that atherosclerotic plaques in patients with coronary artery disease were found predominantly in the descending aorta (in 93%) and in the aortic arch (in 80%), whereas the ascending aorta was the least involved (in 37%) \[[@B9]\]. Atherosclerosis is a complex polygenic, multifactorial vascular disorder associated with many differing and changing metabolic, anatomic and clinical manifestations \[[@B13]\]. The presence of atherosclerotic plaque in the thoracic aorta, as shown by chest x-ray, has been shown in previous studies to be correlated with an increased risk of cardiovascular death \[[@B14],[@B15]\]. However, several studies have also demonstrated that the generation of acute coronary syndromes is not necessarily related to plaque severity rather to its morphology and complexity. From histopathologic and vascular biologic studies \[[@B13],[@B16]\] plaque composition and vulnerability (type of lesion) rather than degree of stenosis (size of lesion) have emerged as crucial factors leading to sudden rupture of the plaque surface, usually with thrombosis superimposed, which underlies the great majority of infarctions. Angiographic studies also suggest that the most frequent situation giving rise to infarction is the occlusion of previously noncritical stenoses \[[@B17]\], which are more prevalent than the possibly more dangerous severe stenoses \[[@B18]\]. Taken together, these studies suggest that in two of three infarctions the culprit lesions had only mild to moderate stenosis on initial evaluation in a substantial number of patients. This is again consistent with our finding that significant coronary artery disease is in close relationship with the atherosclerosis of the aorta but more severe the atherosclerosis is, higher the probability of spontaneous cardiovascular events. Clinical implications --------------------- One of the most important first steps in stratifying risk among patients with proven or suspected coronary artery disease is the identification of patients at high risk for coronary or vascular events during the course of the next few months or years. To date, left ventricular dysfunction, the number of diseased vessels, and the severity of myocardial ischemia have emerged as important determinants of survival \[[@B19]\]. Our data suggest that atherosclerosis of the descending aorta observed by a simple, routine transesophageal echocardiographic examination can be an additional prognostic marker in identifying patients for higher risk for cardiovascular events. When patients are referred to transesophageal testing for whatever reason, a semiquantitative description of atherosclerotic burden of the descending aorta should be always included in the prognostic stratification. Study limitations ----------------- This study has several limitations. The study population was highly heterogeneous, reflecting the garden variety of patients referred to the echo lab for transesophageal testing: patients with known or suspected coronary artery disease or cerebrovascular disease coexist with patients with congenital or acquired valvular disease. Therefore, our findings cannot be directly translated into the general population, and further prospective studies are needed at this point to evaluate the prognostic value of transesophageally detected aortic pathology in more sharply defined clinical subsets. Future directions ----------------- The morphological characterization of atherosclerotic plaque has not been performed in our study, neither in terms of plaque content \[[@B20]\] nor of plaque geometry \[[@B21]\]. Ultrasonic tissue characterization technology can be applied for a more accurate and quantitative description of echocardiographic plaque structure and profile \[[@B22]\]. Both these criteria have documented the prognostic impact in the carotid artery \[[@B23]\]. Thereby, the prognostic value of ultrasonic assessment of aortic atherosclerosis can certainly be further improved with more quantitative, albeit more technologically demanding, image analysis. It is however important, that even a semiquantitative, subjective and extremely simple assessment of atherosclerosis from transesophageal images applied on an extremely heterogeneous population yields powerful prognostic stratification, even when hard prognostic end-points are considered. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Grade I. atherosclerosis of the descending aorta. Intimal thickening. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Small plaque on the descending aorta, corresponding to Grade 2. atherosclerosis. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 Grade 3. atherosclerosis, with large, multiple plaques. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 4 Grade 3. atherosclerosis, plaque with mobile parts. ::: ::: {.caption} ###### Click here for file :::	PubMed Central	2024-06-05T03:55:49.847939	2004-10-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534093/", "journal": "Cardiovasc Ultrasound. 2004 Oct 22; 2:21", "authors": [ { "first": "Albert", "last": "Varga" }, { "first": "Noemi", "last": "Gruber" }, { "first": "Tamás", "last": "Forster" }, { "first": "Györgyi", "last": "Piros" }, { "first": "Kálmán", "last": "Havasi" }, { "first": "Éva", "last": "Jebelovszki" }, { "first": "Miklos", "last": "Csanády" } ] }
PMC534094	Background ========== Self-completed postal questionnaires are an economical and simple method of data collection for both research studies and audit activity. They are cheaper than telephone or personal interviews \[[@B1]\] and may be particularly useful in medical research as the response rate to sensitive questions may be greater than from other methods of data collection \[[@B2]\]. However, questionnaire based studies are subject to non-response bias \[[@B3]\]. If there are differential response rates from certain groups in the sample population, then generalisability of results to the target population is questionable. The lower the response rate, the more open to criticism the conclusions drawn from the research will be. High response rates are important not only because they reduce the risk of non-response bias but also because they increase the precision of parameter estimates \[[@B4]\]. Ensuring a high response rate to initial mailings has the additional benefit of reducing the costs associated with re-mailing or other methods of follow-up such as telephone interviews. Strategies for increasing response rates can be summarised as falling into five broad categories; the covering letter (personalisation, use of an appeal etc), incentives (cash or other reward), contact (pre-notification and follow-up), mailing (return envelopes, type of outgoing postage etc) and questionnaire (length, format, colour etc) \[[@B5]\]. Pre-notification, follow-up, university sponsorship, cash incentives, first class postage, freepost return and questionnaire colour have all been shown to increase response rates \[[@B4]\]. A Cochrane review \[[@B3],[@B6]\] reported similar findings, with monetary incentives and recorded delivery having the greatest effect on response (response rates doubled) and a range of other strategies significantly increasing the odds of response. However, individual studies demonstrated wide variability, were conducted across a variety of disciplines (psychology, medicine, business etc) and in a variety of countries; their generalisability is, therefore, questionable. An individual\'s decision to reply to a questionnaire is bound up in the content of the questionnaire, its relevance to the individual and who is perceived as benefiting from completion \[[@B3]\]. Questionnaires asking about personal experience may be treated differently to those asking for opinion, and information requested by a retail company may be perceived as less important than that sought by a charity, academic institution or health provider. Much of the research relating to the effectiveness of specific interventions on response rates in general population surveys was conducted before 1990 and from a market or sociological perspective. More recent work has been conducted on the effects of incentives on increasing response rates in populations of medical professionals. A lottery style incentive (prize of a weekend break) was reported to generate a small increase in response rates from General Practitioners (GP, Family physicians) in Quebec \[[@B7]\] and cash incentives also increased response rates in a GP population \[[@B8]\]. It is not clear, however, whether results from these professional groups can be generalised to general population surveys. Studies sampling members of the public tend to confirm the more general findings of the Cochrane review \[[@B6]\], which included surveys of professionals as well as the public, that financial incentives have a positive effect on response and even a small financial incentive (\$1 -- \$2) increased response rates to a health related survey \[[@B9],[@B10]\]. However general population surveys often require large samples and the cost of offering even a small individual monetary reward may be prohibitive. The Cochrane Review \[[@B6]\] identified 45 trials (44,708 participants) of non-monetary incentives (e.g. key ring, offer of entry into a lottery, offer of study results) and found that the odds of response were approximately a fifth higher when using such incentives. However, their meta-analysis did not discriminate between the type of non-monetary incentive, the population (i.e. professional versus public) or the subject matter (i.e. health versus non-health). Of the nine studies identified that utilised some form of lottery incentive \[[@B6]\], only one demonstrated a significant benefit \[[@B11]\]. The remaining eight studies had non-significant results although five of these suggested a trend towards an improved response \[[@B12]-[@B16]\], two tended to suggest a detrimental effect \[[@B17],[@B18]\] and one showed no effect \[[@B19]\]. Evidence on the use of lottery style incentives is therefore conflicting. One of the studies that showed no overall benefit in including a lottery incentive \[[@B13]\] demonstrated a significant difference in favour of the lottery group (24.4% versus 18.5%) with respect to responses to a first mailing. Although this benefit was not maintained after a reminder, such a result could reduce the cost of undertaking surveys. Three of the five \[[@B13],[@B15],[@B16],[@B18],[@B19]\] evaluations of a lottery incentive using a general population sample, indicated small but non-significant increases in response. The three studies that demonstrated a tendency towards increased response rates had offered entry into a draw for a restaurant meal to the value of \$100 (1985) \[[@B15]\], \$50--\$200 (1988) \[[@B16]\], and a range of prizes including a television, \$100 and a trip to Las Vegas (1989) \[[@B13]\]. There is, therefore, some evidence to support the use of lottery incentives in increasing questionnaire response rates. However, studies focussing on health related issues are limited to only two that used a general population sample \[[@B15],[@B19]\] and three that sampled patients \[[@B12],[@B14],[@B17]\]. The wide range of incentives used, study settings and subjects mean that it is difficult for medical researchers to determine the applicability of this evidence to community based surveys in the UK. Determining the community prevalence of disease is important for needs assessment, service planning and determining the potential economic implications of new treatments. Studies aiming to precisely estimate the prevalence of disease usually require large samples and often use postal questionnaires. A large community mailing to determine the prevalence of Irritable Bowel Syndrome (IBS) \[[@B20]\] provided an opportunity to embed a randomised controlled trial to investigate the effect of including an incentive on response and completion rates. Medical research rarely, unless sponsored by the pharmaceutical industry, has the capacity to pay a cash incentive for all returned questionnaires. This study therefore aimed to determine the effect of including a low-cost lottery style incentive (returned questionnaires being entered into a draw for a prize) on the response and completion rate of a health questionnaire survey. A secondary objective was to combine data from this study with that from previously published studies based in a health environment \[[@B15],[@B19]\] using patient/general population responders to provide a more precise estimate of effect. Methods ======= Location -------- This study was undertaken in Birmingham, a large city in the West Midlands region of the United Kingdom. Ethical approval was obtained from North Birmingham and West Birmingham Research Ethics Committees prior to commencement of the study. Participants ------------ Eight general practices in the North and West Birmingham areas were recruited to participate in a health survey designed to determine the prevalence of IBS in the community \[[@B20]\]. All practices in these areas were invited to participate in this survey and practices were randomly selected from those practices who had expressed an interest in participation, after stratification for deprivation scores. To provide a sample with representation from all socio-economic groups, practices were selected from each of the four quartiles of the Townsend scores. Townsend scores are calculated from small area statistics collected during the decennial census (most recent in 2001) and provide an indicator of deprivation. Practices were allocated a Townsend score based on their location (postcode). All patients aged 18 and over were eligible for inclusion. The only exclusion criteria were patients for whom the general practitioner indicated that mailing would be inappropriate; this typically included patients known to be terminally ill or patients unable to complete the questionnaire e.g. those with severe learning disability. Questionnaires returned by the postal service as \'not at this address\' were removed from the sampling frame (denominator) and response rates calculated as the number returned divided by this denominator. Trial size ---------- Estimates were based on the prevalence study within which this trial was embedded and indicated that 8,000 patients should be mailed. This provided 4,000 in each arm of the RCT, sufficient to demonstrate a 4% difference in response rates with 90% power at the 5% significance level, assuming a 60% response rate. Randomisation ------------- To minimise contamination (two or more individuals in a household receiving a questionnaire, but not all receiving the lottery incentive) only one person per domestic address was selected. Practice registers were utilised to generate randomly ordered lists of addresses and then one individual aged 18 or over was randomly selected from each address until the required number of patients had been identified. The prevalence survey, within which this trial was embedded, aimed to recruit a stratified random sample, comprising 1,150 patients from each participating practice to ensure sufficient cases were included from each of the Townsend quartiles. Where the practice was unable to generate 1,150 patients (due to smaller list sizes) the maximum number of patients available was included. Randomisation was performed on a 50:50 basis within each practice, to control for practice effects, using a computerised simple random number sequence. Intervention ------------ All patients selected received a health questionnaire. Questionnaires had three sections; section one requested personal and demographic details in addition to details of personal and family medical history; section two was the SF12 \[[@B21]\], a validated generic quality of life measure; section three was a self completed questionnaire version of the ROME II criteria \[[@B22]\] (to confirm diagnosis of IBS). A covering letter, sent in the joint names of the University and the relevant general practice, explained that the practice was participating in a research project to find out about the number of patients affected by certain conditions and the ways in which ill health affects people\'s quality of life. All patients received a reply paid envelope (addressed to the research team at the University of Birmingham) with the questionnaire and were informed that return of a blank questionnaire would indicate the wish not to be involved and they would not be contacted further. The intervention group received an identical questionnaire to the control group. The covering letter was also identical apart from the addition of a paragraph explaining that all returned questionnaires would be entered into a draw for a prize of £100 of \"High Street shopping vouchers\". This letter stressed that entry into the draw was dependent on return rather than completion of a questionnaire, as this was deemed to be more ethically responsible. In addition to the letter, a flyer printed on brightly coloured paper (yellow) was included for intervention patients, highlighting the fact that returned questionnaires would be entered into a draw. All non-responders were re-mailed after 4 weeks. Again, the intervention group received the additional paragraph in the covering letter and an additional flyer. All follow-up mailings included a copy of the questionnaire and a reply paid return envelope. Data handlers were not blinded to the intervention status of responders, but this was not considered to be a source of bias as response rates were the primary outcome. Mailings took place in the period January to July 2001. Mailings were conducted by practice and within each practice, control and intervention patients were mailed on the same day. Outcomes -------- The principal outcome was the overall response rate. Response rates to initial and follow-up mailings, and numbers of blank responses were also compared between groups. Analysis -------- Analysis was on an intention to treat basis. Response rates were compared between the two arms of the trial using chi-squared tests. Predictors of response were identified by logistic regression using backward elimination. Variables entered into the starting model included randomisation arm, age, sex, Townsend score derived from patient postcodes, practice and all two-way interactions with the randomisation arm. Two recent systematic reviews of strategies to influence response rate were identified \[[@B3],[@B4]\] and from these, all studies of non-professional groups using lottery-style incentives in a health environment were identified and full papers obtained. Data from these was combined with those of this study and a meta-analysis undertaken (Rev Man software). Results ======= Eight thousand six hundred and forty five patients were included in the trial; 4,325 were randomised to the lottery arm and 4,320 to the control group and a 50:50 split was maintained within each practice. The trial profile is shown in Figure [1](#F1){ref-type="fig"}. Two hundred and sixty questionnaires were returned by the Royal Mail as \'not at address\' and were not included in the analysis. The proportion of these returned questionnaires were comparable between trial arms (121 (2.8%) in the intervention arm and 139 (3.2%) in the control arm). Baseline characteristics of patients were similar between the two randomised groups (Table [1](#T1){ref-type="table"}). Four thousand and twelve individuals responded to the initial mailing; 1996 (47.5%) in the intervention arm and 2017 (48.2%) in the control group (χ^2^= 0.5, p = 0.48). A further 1197 replied to the reminder mailing; 616 in the intervention arm and 581 in the control arm giving an overall response of 2612/4204 (62.1%) in the intervention arm and 2598/4181 (62.1%) in the control arm (χ^2^= 0, p = 0.99). The numbers of questionnaires returned blank was similar for both groups, 197 (4.7%) in the intervention and 217 (5.2%) in the control arm (χ^2^= 1.1, p = 0.29). No objections to the use of an incentive or requests for exclusion from the prize draw were made to the research team or participating general practices. Response rates varied by practice from 37% to 77% (Table [2](#T2){ref-type="table"}). Practice, patient age, patient sex and Townsend score were identified as predictive of response, with response rates increasing with age ((OR per 1 year change in age) (95% CI) = 1.02 (1.016, 1.024)) (i.e. if the odds of responding at age 40 are 1.0 (1:1), then odds of responding at age 45 are 1.1 (1:1.1)). Lower response rates were associated with being male (OR = 0.53 (0.48, 0.58)) and living in an area with a higher Townsend score (more deprived) (OR per 1 point change in deprivation score= 0.93 (0.92, 0.95) (Table [3](#T3){ref-type="table"}). The small number of practices, and limited number of explanatory variables associated with each practice, meant that whilst practice was predictive of response it was not possible to explore the practice characteristics related to this. Randomisation arm and its interaction with other terms were not identified as significant predictors of response rate. This indicates that practice, patient age, sex and Townsend score affected response rates whereas the lottery incentive did not. Given the lack of difference between groups, no economic evaluation was conducted. The lottery incentive presented additional costs in terms of production and inclusion of flyers (\<£0.01 per individual) and provision of the pre-specified prize (£100 overall), and there was therefore a net disbenefit to the use of the incentive. Two previously published relevant studies were identified \[[@B15],[@B19]\]. Results of the meta-analysis are provided in Figure [2](#F2){ref-type="fig"}. Discussion ========== This RCT using a large community sample suggests that using a lottery draw style incentive for £100 of High Street vouchers has no effect on overall response rates to a postal questionnaire asking about health and medical history. Whilst it is possible that the locality of the study (Birmingham, UK) is not typical, the range of practices and Townsend scores of the sample, suggest this is unlikely. It is also possible that a larger incentive or a cash prize, rather than vouchers, may influence response rates. However the value of the incentive offered was comparable to, or greater than, previous studies. The only published study which has shown a significant benefit of a prize draw was of professionals \[[@B11]\] and we, therefore, believe that the findings of this large trial are likely to be generalisable to health related surveys of the general public. There was no difference between the groups in the numbers of questionnaires returned blank indicating that the incentive did not just encourage people to return incomplete questionnaires. There was also no difference in the numbers replying to the first mailing. Had the incentive increased numbers replying to the initial mailing, even if it failed to increase response overall, it may have proved cost-effective in terms of reducing the costs of follow-up mailings. Our results indicate that previous research suggesting increased response to initial mailings \[[@B13]\] is not generalisable. The questionnaire used in this RCT was designed to determine disease prevalence. It is possible that such mailings over-inflate prevalence estimates, as individuals responding may be encouraged to do so because of their vested interest as an individual experiencing disease. Inclusion of an incentive not related to disease status may encourage response from a wider range of the population and may provide more accurate estimates of prevalence. However, this study does not suggest that future health surveys would gain any response benefit from the inclusion of a prize draw incentive. Indeed the addition of the data from this study to that of the two previously published studies based in a health environment \[[@B15],[@B19]\] using patient/general population responders provides further evidence of a lack of effect (Figure [2](#F2){ref-type="fig"}). Future work in this area may best be conducted using qualitative methodologies to explore factors related to response in community surveys. Conclusions =========== A lottery draw style incentive for £100 of High Street vouchers does not affect response rates to a postal health survey, when used in a general population sample. On the basis of this large RCT we would not recommend utilising similar incentives in general population health research. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' Contributions ======================= SW and LR conceived the idea and designed the study. PB contributed to the study design and collected and validated the data. AR completed the analyses. LR wrote the first draft of the manuscript, all other authors made significant contributions to the writing of the manuscript and approved the final version. SW is the guarantor. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/4/30/prepub> Acknowledgements ================ This paper was written on behalf of the Birmingham IBS Research Group. Members of the Birmingham IBS Research Group comprise the authors of this paper in addition to Dr. Sheila Greenfield (Medical Sociologist), Dr. Tina Maddison (Specialist Registrar in Public Health), Ms. Charlotte Mann (Project Officer), Ms Elisabeth Rawson (Research Associate and Hypnotherapist), Dr. Sukhdev Singh (Gastroenterologist and Senior Lecturer), and Dr. Kate Thomas (GP and Senior Lecturer). The introduction to this paper has been informed by the comprehensive Cochrane review that has been completed by Edward et al. \[[@B3],[@B6]\] This project has benefited from discussion of the research idea with all members of the Birmingham IBS Research Group. We are also grateful for the help and support provided by the eight practices in the Birmingham area that collaborated with this study. Our particular thanks go to Najam Mughal from Birmingham Health Authority for her assistance and support during this study. The prevalence survey was funded by a grant from the NHS Office (West Midlands). During the period this work was completed, Lesley Roberts was funded by a NHSE (West Midlands) New Blood Fellowship. Sue Wilson is funded by a Department of Health NHS R&D Primary Care Career Scientist Award Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Questionnaire response rates ::: ![](1472-6963-4-30-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Meta-analysis of lottery studies using health related general population surveys ::: ![](1472-6963-4-30-2) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Baseline characteristics of patients by randomisation arm ::: Characteristic Lottery Control ------------------ -------------- -------------------- -------------------- Age (years) mean (sd) 48.5 (18.8) 48.4 (18.8) range 18--100 18--98 n 4323 4316 Sex (male) % 47.8 48.2 n 4325 4320 Townsend score\* mean (sd) 1.06 (3.6) 1.06 (3.7) median (IQR) 1.9 (-2.25 to 4.3) 1.9 (-2.11 to 4.3) n 4199 4189 Townsend scores are calculated at enumeration district level and standardised to England and Wales (Range -7.6 to 11.8). Positive scores indicate greater deprivation than the national average. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Response rates ::: Lottery Control Total --- --------- --------- ------- ----- ----- ------ ------ 1 365 558 65.4 359 557 64.5 64.9 4 394 555 71.0 390 547 71.3 71.1 5 333 522 63.8 335 519 64.6 64.2 8 423 553 76.5 426 556 76.6 76.6 3 329 538 61.2 318 547 58.1 59.6 2 371 558 66.5 355 553 64.2 65.4 6 137 367 37.3 132 353 37.4 37.4 7 259 553 46.8 283 549 51.5 49.2 ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Factors related to response. ::: Variable β (SE) P value Odds ratio (95% confidence interval) ----------------------------------------- -------------- ---------- -------------------------------------- Intercept -0.36 (0.06) \<0.0001 \- Age (per increase of 1 year) 0.02 (0.001) \<0.0001 1.02 (1.02, 1.02) Male -0.64 (0.05) \<0.0001 0.53 (0.48, 0.58) Townsend score (per increase of 1 unit) -0.07 (0.01) \<0.0001 0.93 (0.92, 0.95) Practice 3 -0.22 (0.07) \<0.0001 0.80 (0.69, 0.92) Practice 6 -0.90 (0.09) \<0.0001 0.41 (0.34, 0.48) Practice 7 -0.67 (0.07) \<0.0001 0.51 (0.45, 0.59) Variables considered were randomisation arm, age, sex, Townsend score, practice and 2 way interactions with randomisation arm. :::	PubMed Central	2024-06-05T03:55:49.849295	2004-11-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534094/", "journal": "BMC Health Serv Res. 2004 Nov 8; 4:30", "authors": [ { "first": "LM", "last": "Roberts" }, { "first": "S", "last": "Wilson" }, { "first": "A", "last": "Roalfe" }, { "first": "P", "last": "Bridge" } ] }
PMC534095	Background ========== We are interested in understanding how Specificity proteins (Sp) govern extracellular matrix deposition during bone formation. The Sp proteins are a family of transcription factors with three zinc-fingers that bind GC-boxes and assist the further binding of the multiprotein complex TFIID promoting the initiation of gene transcription \[[@B1]-[@B4]\]. GC-boxes have the consensus sequence GGGCGGG. Sp1 and Sp3 are associated with chondrocytic differentiation by regulating the alpha1(II) procollagen gene (COL2A1), a chondrocytic marker. Sp1 has been shown to be an activator and Sp3 a repressor of COL2A1 transcription \[[@B5]\]. Despite the ubiquitous expression of Sp3, Sp3 null mice suffer from specific skeletal defects associated with a delay in osteoblast differentiation, having reduced ossification and impaired bone formation and show a decrease in the expression of osteocalcin, an osteoblast specific gene \[[@B6]\]. Sp7, also known as Osterix, is required for osteoblast differentiation since in Sp7 null mice bone formation does not occur \[[@B7]\]. Previously, we cloned two isoforms of human Sp7 that are generated from distinct promoters upstream of exon 1a and exon 1b \[[@B8]\]. We identified another human Sp gene that is closely related to Sp7 and named it Sp8. Recently, Sp8 null mice have been described \[[@B9],[@B10]\]. Note that the mouse mBtd gene \[[@B10]\] is Sp8 and not a specific homologue of the Drosophilagene buttonhead (Btd) \[[@B11]\]. However, Btd is a member of the Sp family \[[@B12]\]. Sp8 null mice have a well-defined phenotype, having severe truncation of all limbs and no tail. Their malformed brains have defective closure of the anterior and posterior neuropores that leads to exencephaly and spina bifida. Sp8 is required for proper maintenance and maturation of the apical ectodermal ridge, a signalling centre, which forms at the limb bud apex and governs the outgrowth of the limb during development. Here we describe the sequence of two isoforms of human Sp8. Results ======= In our search for additional Sp transcription factors, we performed a TblastN search of the human genome with the sequence of Sp7 \[[@B8]\]. The most closely related sequences were found on genomic DNA BAC clones CTA-324D18 and CTB-86D3 that were in the draft stage (GenBank accession Nos. AC005251 and AC005060 Genome Sequencing Center, Washington University, USA). This new gene was named Sp8 since it is closely related to Sp7. Human Sp8 cDNAs --------------- Utilising the sequences of the two BAC clones, PCR primers were designed to amplify probable cDNAs. Two different cDNA clones were obtained by RT-PCR from the human MG-63 osteosarcoma cell line and sequenced (Fig. [1](#F1){ref-type="fig"}). Successful amplification and sequencing required methods designed to overcome problematic GC-rich regions. These transcript variants differed at their 5\' termini. Clone 1 comprises exon 1a and 2, and together they encode the long protein isoform of Sp8 (Sp8L)(Fig. [1](#F1){ref-type="fig"})(GenBank accession No. AY167047). Clone 2 comprises the untranslated exon 1b and exon 2. Exon 1b contains an in-frame stop codon and no initiation methionine codon. Together they encode the short protein isoform (Sp8S)(Fig. [1](#F1){ref-type="fig"})(GenBank accession No. AY167048). The first ATG codon of Sp8L is in excellent sequence consensus for an initiation methionine having a guanosine residue at positions -3 and +4, and that of Sp8S is in good sequence consensus having a guanosine at -3 \[[@B13]\]. The ORF is GC rich (70%). Another Sp8 cDNA clone was isolated from the osteoblasts of a patient with osteoporosis. This has a glycine-165 deletion; having lost one of the five sequential GGC codons (nucleotides 569--583) (Fig. [1](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Human Sp8 cDNA sequences encompassing the ORF and translation. (A) Sequence encoding exon 1a encoding the start of Sp8L protein isoform. (B) Sequence encoding the untranslated exon 1b encoding the Sp8S protein isoform. (C) Sequence encoding the common exon 2. The atg/methionine start codon for the long protein isoform is shown in bold and coloured red and that of the short protein isoform coloured pink. The ORF stop codon, tga, is shown in bold and coloured red and is indicated by a red asterisk. An in-frame stop codon, tag, in the 5\'UTR of exon 1b is shown in bold and coloured red. The exon/exon boundaries were determined by comparison with the sequence of genomic DNA clone CTA-324D18. The nucleotides underlined and shown in bold type indicate the location of the exon/exon boundaries. The loss of a GGC (glycine) codon found in an additional Sp8 clone is shown in green and bolded. ::: ![](1471-2164-5-86-1) ::: Human Sp8 gene -------------- After the completion of the human genome the location and structure of the human Sp8 gene was identified by mapping the sequences of Sp8L and Sp8S cDNA clones. The human Sp8 gene maps to 7p21.3 between the multidrug resistance protein (P-glycoprotein) and ribosomal protein L23 genes and is separated by 0.65 Mbp from the related Sp4 gene by seven genes encoding two ribosomal proteins, four putative genes of unknown function and a gene similar to argininosuccinate synthetase. The Sp8 and Sp4 genes are linked to the homeobox gene cluster HOX A, which is 5.6 Mbp away. The human Sp8 gene spans 4.6 kb and has three exons (Fig. [2](#F2){ref-type="fig"}). Exons 1a and 1b encode different transcript variants that are likely to be initiated from distinct promoters. The first two exons are separated by a 733 bp intron 1a, followed by a shorter, 152 bp intron 1b. All splice donor/acceptor sites contained consensus GT/AG dinucleotides. The gene possesses a CpG island upstream of exon 1a (438 bp, 63% GC). Computer analysis of the immediate human Sp8 promoter preceding exon 1a contains a number of RNA polymerase II promoter elements including a TATA-box, CCAAT-box and two GC-boxes that are conserved in rodents and fish. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Gene structure of human Sp8. The human genomic sequences corresponding to the Sp8 cDNAs are located on clones CTA-324D18 and CTB-86D3. The location of a CpG island is shown in green. The first exon encoding the start of the Sp8L transcript is labelled 1a and that of Sp8S, 1b. The ORF is indicated by closed boxes. The sequences around the initiation methionine codons (underlined) are shown with those nucleotides corresponding to Kozak consensus sequence coloured red. The location of the TGA stop codon and a potential polyadenylation signal, AATAAA are also shown. The sizes of the exons and introns, in base pairs, are indicated. ::: ![](1471-2164-5-86-2) ::: Comparison of the human Sp8 protein with those of other species --------------------------------------------------------------- The ORF of Sp8L clone 1 encodes a 508-residue protein with a 50,500 Da molecular mass and an isoelectric point 9.02. The first ATG codon of clone 2 corresponds to the second ATG of clone 1, thereby omitting the first 18 residues of the ORF, encoding a 490-residue protein with a 48,674 Da molecular mass and an isoelectric point 9.10. The human Sp8 protein belongs to the Sp/Krüppel like factor (KLF) family of proteins that are characterised by three Cys2-His2 zinc-fingers \[[@B14]\]. The zinc-fingers are located towards the COOH terminus and are involved in binding DNA in a sequence specific manner. However, the Sp proteins are distinguished from the KLF family by an amino-terminal hydrophobic domain called the Sp-motif with the consensus sequence PLALLA and a buttonhead box (BTD) with the consensus sequence CxCP(N/Y)C prior to the zinc finger domain. Database searches identified complete Sp8 genes and protein sequences from other vertebrate species. The chimpanzee gene is located on chromosome 7 (clone RP43-37F3); the mouse gene at 12F2 (BAC clone RP23-161L22); the rat gene at 6q33 on BAC clone CH230-1K24 and the zebrafish within BAC clone CH211-180P9. A comparison of the protein sequences shows that Sp8 is well conserved through vertebrate evolution (Fig. [3](#F3){ref-type="fig"}). The human and chimpanzee proteins are very similar with one conservative substitution and an additional glycine residue. The mouse Sp8 protein \[[@B9],[@B10],[@B15]\] has 97% identity and the zebrafish 79% identity to human Sp8. An amphibian Sp8 EST from Xenopus laeviswas identified by our database search (accession No. BI313193). The mammalian Sp8 proteins differ from that of the zebrafish by the insertion of two poly-alanine and a poly-glycine region in the amino-half of the protein. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Sequence comparison of the human Sp8 protein with those from other vertebrate species. The species are man, Homo sapiens, Hs; mouse, Mus musculus, Mm; chimpanzee, Pan troglodytes, Pt; rat Rattus norvegicus, Rn and Zebrafish, Danio rerio, Dr. The location of the exon/exon boundaries are underlined in black on the protein sequences. The methionine residues at the start of translation of Sp8S are underlined in red. The positions of the Sp, BTD-box and three zinc-finger domains found in most Sp protein family members are indicated. The conserved residues are shown by (\), strongly conserved residues by (:) and weakly conserved residues by (.). Stop codons are indicated by a hash. Residues are colour coded: basic, DE, blue; acidic,\_KR, pink; polar, CGHNQSTY, green and hydrophobic, AFILMPVW, red. ::: ![](1471-2164-5-86-3) ::: Database searches also showed that homologues of the vertebrate Sp8 genes are found in insects, but not in nematodes, yeasts and higher plants (Fig. [4](#F4){ref-type="fig"}). The fruit fly, Drosophila melanogaster, D-Sp1 gene is a homologue of Sp8 and not, as the name implies, Sp1 nor Sp4 as previously stated \[[@B16]\]. The red flour beetle, Tribolium castaneum, Sp8 protein \[[@B17]\] has 43% identity and 80% similarity to human Sp8. However, D-Sp1 differs from the vertebrate and beetle Sp8 proteins possessing a much longer, glutamine and histidine-rich carboxy-terminus. The possession of Sp, BTD and zinc-fingers domains in a protein is characteristic of Sp proteins in general; these domains in these three Sp8 proteins are very highly conserved. Outside these domains, a motif of unknown function is located towards the amino-terminal with the sequence GKGFHPWKKS and is unique to Sp8 proteins. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Sequence comparison of the human Sp8 protein with those from other invertebrate species; red flour beetle, Tribolium castaneum, TcSp8 and fruit fly, Drosophila melanogasterSp8, DmD-Sp1 proteins. The locations of the exon/exon boundaries are underlined in black on the protein sequences ([GE]{.underline} and [S]{.underline}). The methionine residues at the start of translation for the short protein isoforms are underlined in red ([M]{.underline}). The positions of the Sp, BTD-box and three zinc-fingers motifs are shown. Stop codons are indicated by a hash. ::: ![](1471-2164-5-86-4) ::: By comparison with other Sp proteins, the Sp8 protein can be divided up into 5 domains. The amino-terminal domain A (residues 1--53) is highly conserved in Sp8 proteins from other species. Two alpha-helices, residues 3--7 and 15--27, are predicted in this domain; whereas the rest of the protein appears to lack any secondary structure except for the zinc-finger domain. Domain A is also found in the Sp7 proteins. Domain B, residues 54--167, is a low-complexity region, having poly- serine, alanine and glycine stretches that are not present in fish Sp8 proteins. Domain C, residues 168--373, is similar to the same region of Sp7 and Sp6 that has been shown to be involved in transcriptional activation \[[@B7]\] and contains a basic region (residues 360--371) that probably contributes to a nuclear localization signal and a BTD-box, a highly conserved region that is present in all Sp family members. The BTD-box was first described as a 10-amino-acid region in the Drosophila zygotic gene, buttonhead \[[@B11]\]. Domain D, residues 374--456 contains three classical zinc finger structures (residues 374--456) of the Cys2-His2 type where the conserved cysteine residues in two short beta-sheets and the histidine residues in an alpha-helix tetrahedrally co-ordinate a zinc ion \[[@B18]\]. Between the fingers are five-residue linker sequences with the consensus sequence TGE+x. The negatively charged carboxy-terminal domain E, residues 457--508, contains a glycine-rich region (458--475). Tissue distribution of Sp8 mRNA ------------------------------- The expression of Sp8 in human and murine adult tissues was examined by reverse transcriptase PCR using primers located in the 3\'UTR. Human Sp8 mRNA expression was detected in a wide range of tissues at a low level, being found in heart, brain, placenta, liver, pancreas, prostate, testis, ovary and colon, but was below the limit of detection in lung, skeletal muscle, kidney, spleen, thymus, small intestine and peripheral blood leukocytes (Fig. [5A](#F5){ref-type="fig"}). Murine Sp8 mRNA expression, relative to the expression of beta-actin mRNA, was detected in all tissues examined, with the highest levels being found in brain and prostate and the lowest in spleen (Fig. [5B](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Expression of Sp8 mRNA in human and mouse adult tissues determined by RT-PCR using primers located in the 3\'UTR. A. Ethidium bromide stained agarose gel of PCR products from human tissues. The lanes are: skeletal muscle, muscle; small intestine, intestine; peripheral blood leukocytes, leukocyte; negative control, -c and 100 bp ladder, m. The expression of beta-actin mRNA, a housekeeping gene, in the same samples is shown below. B. RT real time PCR of murine Sp8 mRNA expression normalised to that of beta-actin. ::: ![](1471-2164-5-86-5) ::: Expression of human Sp8 mRNA in osteoblast-like cells ----------------------------------------------------- The expression of both transcript variants mRNAs encoding Sp8L and Sp8S in osteoblast-like cells was examined by reverse transcriptase PCR using primers specific for each isoform (Fig. [6](#F6){ref-type="fig"}). The Sp8L was most abundant in the osteosarcoma cell lines HOS and MG63 and was present in adult and craniofacial osteoblasts, but was below the limit of detection in foetal osteoblasts and chondrocytes. The Sp8S was most abundant also in the osteosarcoma cell lines HOS and MG63 and was present in craniofacial and foetal osteoblasts, but was below the limit of detection in adult osteoblasts and chondrocytes. Runx2, a transcription factor important for osteoblast differentiation, and beta-actin, a housekeeping gene, were expressed in all the osteoblast-like cells. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Expression of Sp8 mRNAs in human osteoblast-like cells by reverse transcriptase PCR. Amplicons were run on an ethidium bromide stained agarose gel. The amplicons were: Sp8 long protein isoform, Sp8L; Sp8 short protein isoform, Sp8S; Runx2/Cbfa1, Runx2 and the housekeeping gene beta-actin. The sizes of the amplicons are indicated. The lanes are: primary adult osteoblasts, AO; HOS osteosarcoma cell line, HS; MG63 osteosarcoma cell line, MG; primary craniofacial osteoblasts, CF; primary foetal osteoblasts, FO; primary adult chondrocytes, CH; negative control, (-) and 100 bp markers, m. ::: ![](1471-2164-5-86-6) ::: BMP-2 regulation of Sp8 mRNA expression in the murine cell line in C3H10T1/2 ---------------------------------------------------------------------------- BMPs are potent secreted factors that promote osteoblast differentiation during development and bone remodelling. Since Sp8 was found in human osteosarcoma cell lines and is important in skeletal development \[[@B9],[@B10]\] we examined the regulation of Sp8 by BMP-2 in the murine pluripotent embryonic cell line, C3H10T1/2 that can be induced to form a chondrogenic/osteoblastic phenotype by treatment with BMPs \[[@B19]-[@B21]\]. Sp8 mRNA expression, normalised to that of beta-actin, was measured by RT-real time PCR and was induced by 100 ng/mL BMP-2 after one day and remained upregulated at 20 days (Fig. [7](#F7){ref-type="fig"}). Data normalised to the housekeeping gene G3PDH gave similar results (data not shown) indicating that Sp8 was upregulated and that beta-actin was not down-regulated. ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Time course of effect of 100 ng/mL BMP-2 treatment on C3H10T1/2 cells. Murine Sp8 and beta-actin mRNA expression was determined by RT real time PCR. The relative levels of expression in treated (•) and untreated cells (◆) were examined after 0, 7, 14 and 20 days treatment. Significant increases in Sp8 expression were seen at 7, 14 and 20 days (p \< 0.05). ::: ![](1471-2164-5-86-7) ::: Discussion ========== In the human genome three Sp gene pairs had been described previously; being maximally separated by 3.2 Mbp, transcribed in opposite directions, being orientated 5\' to 5\' manner and colocalized with a specific HOX gene clusters \[[@B8]\]. Sp3 and Sp5 are located on chromosome 2q31.1; Sp1 and Sp7 on 12q13.13 and Sp2-Sp6 on 17q21.3-q22. A search for a partner to Sp4 located on 7q21.3-q22 revealed a putative eighth Sp gene most similar to Sp7 suggesting that Sp8, like Sp7, may play a role also in skeletal development. Consequently, we used RT-PCR to amplify Sp8 cDNAs from a human osteosarcoma cell line and isolated two transcript variants clones that encode a full-length protein (long isoform) Sp8L and an amino-terminal truncated protein (short isoform) Sp8S, with 508 and 490 residues respectively. We found a glycine-165 deletion mutation in an additional cDNA clone isolated from a patient with osteoporosis. The length of this poly-glycine region is conserved in primates and rodents, but is absent in fish. We speculate that this Sp8 mutation, and other mutations yet to be discovered, may play a role in susceptibility to osteoporosis, and since Sp8 plays an important role in neuropore closure be a candidate gene for spina bifida \[[@B9]\]. The sequence of our Sp8L clone is similar to that of two other human clones, but the other two clones lack 170 and 128 bp of the low-complexity, B domain (GenBank accession Nos. BAB71297 and AAH38669). These deletions may be caused by incomplete reverse transcriptase reactions occurring during cDNA library synthesis, presumably because the high GC content (81%) generates strong secondary structure in the mRNA in this region. These deletion-carrying clones are unlikely to be generated by the presence of cryptic intron(s) since no suitable donor or acceptor splice sites are present in the genomic DNA sequence. One of the deletion-carrying clones, AAH38669, possesses a polymorphism, cac\>cgc, at nucleotide 1263 that results in a His\>Arg mutation at residue 448. His-448 is located in the third zinc- finger and is conserved in all Sp proteins and, by homology with Sp1, is likely to contact DNA \[[@B18]\]. This mutated protein would be expected to be deleterious, having reduced affinity for GC-box binding sites in promoter regions. We found that in man Sp8 has two transcript variants utilising two different first exons. Sequence analyses of mouse and zebrafish genomic and EST data support this gene structure for Sp8 in vertebrates and not a gene structure with two untranslated 5\' exons that has been suggested for murine Sp8 \[[@B10]\]. Interestingly, the drosophila D-Sp1 gene, a Sp8 homologue, also has two transcript variants, encoding long and short protein isoforms (accession No. AE003448) \[[@B22]\]. Both Sp8 and Sp7 have a similar exon structure, being three-exon genes with two transcript variants with different first exons \[[@B8]\]. The long protein isoforms use translated first exons that encodes seven residues and the short protein isoforms use untranslated first exons; overall, the proteins have 39.5% identity and 65.9% similarity. Both the Sp8S and Sp7S proteins lack an 18-residue amino-terminus thereby disrupting a hydrophobic region termed the Sp domain that is conserved in other Sp proteins. The conservation of the two protein isoforms through evolution suggests that they have differing roles. Although the function of this hydrophobic region is unknown, other zinc-finger transcription proteins often have a conserved protein-protein interaction domain at their amino-terminus (e.g. BTB or kelch domains). This indicates that the Sp motif may also be involved in a protein-protein interaction and that the short protein isoforms do not have this protein interaction domain. Amino-terminal spliced variants expressed from separate promoters are a feature of other important transcription factors that regulate skeletal development. An oestrogen regulated protein in osteoblasts, KLF10 is another member of the Sp/Krüppel-like factor family that has two amino-terminal variant isoforms generated in a similar way to those of Sp8. These isoforms are named the TGFβ-inducible early gene (TIEG1) and the early growth response gene-alpha \[[@B23]-[@B25]\]. Runx2 has two major amino-terminal isoforms that exert different functions during the process of osteoblast differentiation; the Runx2-type-I isoform is widely expressed in osteoprogenitor cells and active osteoblasts, whereas the Runx2-type-II isoform is restricted to cells lining mineralised bones \[[@B26]\] and BMP-2 preferentially upregulates the Runx2-type-II isoform \[[@B27]\]. Sp8 does not initiate BMP-mediated signalling during apical ectodermal ridge formation, but may function downstream of the BMP receptor-1a in the signalling cascade \[[@B9],[@B10]\]. The signalling events downstream of the BMP receptor that result in tissue-specific gene expression and skeletal development have been only partially elucidated. We found that Sp8 expression was induced by 100 ng/mL BMP-2 only after 24 hours in C3H10T1/2 cells. BMP-2 has been shown to induce several other transcription factors that promote differentiation such as Runx2/Cbfa1, Sp7/osterix and ZNF450 in addition to the negative regulator Id1 \[[@B7],[@B28]-[@B30]\]. Induction of these genes in C3H10T1/2 cells occurs within 4 hours, preceding that of Sp8, suggesting that Sp8 is not directly regulated by BMP signalling and that it may be induced by one or more of the BMP-early-induced genes. The Sp8 gene has a wider phylogenetic distribution than Sp7, being found in coelomates, whereas Sp7 is limited to vertebrates. In view of the close similarities between human Sp8 and Sp7 in gene structure and amino acid sequence we speculate that they evolved from an ancestral Sp8 gene during a duplication of a Sp/Hox gene cluster \[[@B31]\]. The Sp8 gene has retained its function in regulating appendage/limb growth \[[@B9],[@B10],[@B12],[@B17]\] and that Sp7 has subsequently evolved a novel function, namely, regulating cartilage/bone formation \[[@B7]\]. Conclusions =========== In humans, there are two transcript variants of Sp8 that utilise different first exons encoding long and short protein isoforms. These two isoforms are conserved being found also in the zebrafish suggesting that they have distinct functions. Sp8 is upregulated by BMP-2 in the murine pluripotent cell line C3H10T1/2 and may play a role in mesenchymal differentiation. Methods ======= Cell culture and RNA extraction ------------------------------- Two cell lines derived from osteosarcomas, HOS and MG-63, were cultured at 37°C in 5% CO~2~using Dulbecco\'s Modified Eagle\'s Medium, containing 4 mM L-glutamine, 4500 mg glucose/L, 1500mg bicarbonate/L (Invitrogen, UK) with the addition of 10% foetal bovine serum, 10 μM ascorbic acid, 100 IU/mL penicillin and 50 μg/ml streptomycin. Primary human osteoblasts were isolated from trabecular bone of femoral heads taken during total hip arthroplasty and cultured as previously described \[[@B32]\]. Primary human craniofacial osteoblasts were obtained from paediatric skull and cultured as previously described \[[@B33]\]. Human primary foetal osteoblasts were obtained and cultured as previously described \[[@B34]\]. Human primary articular chondrocytes were obtained from isolated femoral heads and cultured as previously described \[[@B35]\]. The murine cell line C3H10T1/2 was cultured in Dulbecco\'s modified Eagle\'s medium supplemented with 50 U/mL penicillin, 50 μg/mL streptomycin (Invitrogen) and 10% new born bovine serum (Sigma) at 37°C under 5% CO~2~in a humidified incubator. Prior to BMP-2 treatment, cells were seeded at a density of approximately 40,000 cells/cm^2^, left for 24 hours, then treated with fresh media with or without 100 ng/mL human recombinant BMP-2 (Wyeth Corporation) and the media replaced every three days. Total RNA was extracted from cells using guanidine thiocyanate and treated with DNase-I to remove any contaminating genomic DNA (SV Total RNA isolation system, Promega, UK). The concentration and purity of eluted RNA was determined spectrophotometrically and the quality of the RNA was verified by non-denaturating agarose gel electrophoresis. For Sp8 cloning, total RNA was reverse transcribed with an oligo-dT primer using ThermoScript, an AMV RNase H- reverse transcriptase at an elevated temperature of 60°C (Invitrogen, UK). Molecular cloning of human Sp8 ------------------------------ The human Sp7 protein sequence \[[@B8]\] was used to search the human genome sequence and the EST database to identify closely related genes. PCR primers designed from the sequence of genomic DNA clones CTA-324D18, CTB-86D3 and EST sequence from Image clone 2721342 (accession No. AW207154) were used to clone two transcript variants spliced isoforms of the human Sp8 cDNA by PCR from MG-63 osteosarcoma cell line cDNA. The PCR primers were: Sp8L forward, ATTGTATTGCACACCTCTAAAAAAAACA; Sp8S forward, GCGTGGTGCTTGCTCCC and common reverse, GCGTCACTCTAGGCCGTTG (Helena Biosciences, France). The cDNAs were amplified by PCR with an annealing temperature of 60°C using the Advantage-GC Advantage kit (Clontech, UK) with the addition of 0.5 M GC-Melt since the DNA sequence of Sp8 is GC-rich. PCR products were excised from agarose gels stained with ethidium bromide and eluted from the agarose using a DNA extraction kit (Qiagen, UK). The PCR products were cloned into the T-A vector pCR4-TOPO (Invitrogen, The Netherlands). Transformed colonies were picked and vectors containing inserts were extracted using the Wizard Plus SV minipreps DNA purification system (Promega, UK) and sequenced in both directions using ThermoFidelase 2 (Fidelity Systems Inc., USA). Tissue and cellular distribution of human Sp8 mRNA by reverse transcriptase PCR ------------------------------------------------------------------------------- Human cDNA was analysed for the relative expression of the Sp8, Runx2/Cbfa1 and beta-actin mRNA by PCR. Sixteen adult tissue cDNAs (BD Clontech, UK) were generated from polyA^+^selected RNA and reverse transcribed using an oligo-dT primer. Total RNA from cell type cDNAs were reversed transcribed using random hexamer primers using an AMV RNase H- reverse transcriptase (ThermoScript, Invitrogen, UK) at 60°C. Approximately 4.0 ng of cDNA from each tissue, and cDNA derived from 50 ng of total RNA from each cell type were amplified by PCR using Taq Gold polymerase. Tissue master mixes were divided into gene specific mixes with the addition of PCR primers to a final concentration of 200 μM. The primers were: Sp8L forward primer, GCAACTTCACTTCTAGGG[GAAGA]{.underline}(exon 1a/2); Sp8S forward primer, TGGGGGTGCCAG[GAAGAAC]{.underline}(exon 1b/2) and a common reverse primer, AGCTGTCCGAGAGGGAGGA (exon 2), producing a 118 and 112 bp amplicons respectively; Sp8 3\'UTR, TTAGTCCGGCCGTCAATTGT and TGGTATTTAAACTACAGCCTCGTCTGA, producing a 151 bp amplicon; Runx2, AGAAGAGCCAGGCAG[GTGCT]{.underline}*(exon 6/7) and TTCGTGGGTTGGAGAAGCG (exon 7), producing a 102 bp amplicon, as measure of the sum of all Runx2 isoforms, and beta-actin, GGCCACGGCTGCTTC and GTTGGCGTACAGGTCTTTGC, producing a 208 bp amplicon. The amplification conditions were; a 10 min hot start to activate the polymerase followed by 50 cycles of 95°C for 15 sec and 60°C for 1 min. Amplification specificity was confirmed by direct sequencing of the amplicons. Expression of murine Sp8 mRNA by real time PCR ---------------------------------------------- Utilising a CAS-1200 robotic precision liquid handling system, PCR was carried out on a Rotor Gene 3000 (Corbett Research, Australia) using a SYBR Green I double-stranded DNA binding dye assay. Copy DNA derived from 200 ng of total RNA from each sample was amplified by PCR using Taq Gold polymerase using PCR primers to a final concentration of 50 nM. The primers were: mouse Sp8 3\'UTR, CCATTCAGCTCTGGCTAGGTCTT and GATTCCCGTTCGCAGAACTC producing a 67 bp amplicon. Beta-actin mRNA was used as a control gene as previously described \[[@B36]\]. The amplification conditions were; a 10 min hot start to activate the polymerase followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. The number of cycles required for the fluorescence to become significantly higher than background fluorescence (termed cycle threshold \[C~t~\]) was used as a measure of abundance. A comparative C~t~method was used to determine gene expression. Expression levels in each cDNA sample were normalised to the expression levels of the control gene (ΔC~t~). The ratios of Sp8 mRNA/control gene RNA from each cDNA were standardised to that of the untreated cells on day 0 that was taken as 100% (ΔΔC~t~). The formula E^-ΔΔCt^was used to calculate relative expression levels where E is the efficiency of the PCR per cycle. Statistically significant changes in gene expression were determined using the t-test on data from three replicate experiments. The amplification specificity was confirmed by melting curve analysis and agarose gel electrophoresis. Authors\' contributions ======================= AJE conceived of the study, and participated in its design and coordination. MAM, JEG and AJE carried out the cell culture work, MAM and AJE carried out the expression studies and MAM and AJE carried out the gene cloning. All authors participated in writing the manuscript, read, and approved the final manuscript. Acknowledgements ================ We thank the Advanced Biotechnology Centre for DNA sequencing (Charing Cross Campus, Imperial College, London), Drs Bruce C. Knight, Chris Murphy and Colin Scotchford for providing primary foetal osteoblasts, adult chondrocytes and the osteosarcomas cell lines and June Edgar for critically reading the manuscript.	PubMed Central	2024-06-05T03:55:49.851693	2004-11-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534095/", "journal": "BMC Genomics. 2004 Nov 8; 5:86", "authors": [ { "first": "Maria-athina", "last": "Milona" }, { "first": "Julie E", "last": "Gough" }, { "first": "Alasdair J", "last": "Edgar" } ] }
PMC534096	Background ========== Why a design article? --------------------- Publishing the design of the trial has several advantages. It may prevent publication bias \[[@B1]\]. A study producing positive results seems more likely to be published than a study that reports no or negative results \[[@B2],[@B3]\]. Also, the study can be included in systematic reviews because data can be retrieved from the researcher \[[@B2]\]. Publishing the design of a study before the results are available provides an opportunity to reflect critically on the design of the study, irrespective of the results. Also, a design article provides detailed information about the intervention within the trial to care givers. The lumbosacral radicular syndrome (LRS) is a complex of symptoms related to the lumbosacral nerve roots. The LRS is a disorder with radiating pain in the leg below the knee in one or more lumbar or sacral dermatomes, and can be accompanied by phenomena associated with nerve root tension or neurological deficits (i.e. sensory deficits in the leg, decreased muscle strength in the leg, decreased reflexes, urinary problems) \[[@B4]-[@B7]\]. A prolapsed disc is a frequent cause of LRS, but other causes include spinal or lateral recess stenosis, tumours and radiculitis \[[@B4],[@B5],[@B7],[@B8]\]. The incidence of LRS in the Netherlands is estimated at 5 per 1000 persons a year \[[@B8]\]. Most patients seeking medical care in the Netherlands will first visit a general practitioner (GP), who is regarded as the \'gatekeeper\' of the health care system. The majority of health problems presented to GPs are treated by the GPs themselves and they are responsible for most referrals to (para) medical specialists. In 1996 the Dutch College of General Practitioners published their clinical guideline for LRS \[[@B5]\]. There is consensus that treatment of LRS in the first six to eight weeks should be conservative. The exact content of the conservative treatment is yet not clear \[[@B9]\]. Since the study of Vroomen et al. \[[@B10]\] bed rest is not regarded a treatment option for LRS anymore. Primarily, treatment consists of adequate pain medication, giving information about the natural course of LRS, which in general is favourable, and stimulating to continue the normal daily activities of the patient. GPs in the Netherlands largely comply with the recommendations stated in the clinical guideline regarding management in patients with LRS \[[@B11]\]. However, they deviated regarding the referral to physical therapy (PT), almost half of patients with LRS were referred, whereas this was not recommended in the guideline. No specific patients characteristics could be found for the prescription of physical therapy. So, in general practice referral to PT in patients with LRS is common. However, there is a lack of knowledge of the effectiveness of PT in LRS. Therefore, the aim of this article is to present the design of a randomised clinical trial of conservative treatment (general practitioners and physical therapy) in patients with acute LRS. Methods/Design ============== Aim --- The LRS trial aims to assess the effectiveness of PTs management added to GPs management compared to GPs management only in patients with acute LRS. We will use a multicentre, randomised clinical trial as study design. Figure [1](#F1){ref-type="fig"} shows the flow chart of the proposed design of the LRS trial. The procedures and design of this study are approved by the Erasmus Medical Center Ethics Committee. Study population ---------------- Participating GPs in and around Rotterdam, the Netherlands, will invite patients with suspected acute LRS to participate in the trial. GPs will invite patients from May 2003 till November 2004 if they have radiating (pain) complaints in the leg below the knee; duration of the (pain)complaints is less than 6 weeks, the age is above 18 years and they present one of the following symptoms: more pain on coughing, sneezing or straining, decreased muscle strength in the leg, sensory deficits in the leg, decreased reflex activity in the leg or a positive straight leg raising test. Patients will receive a letter of information about the LRS trial from their GP. Patients\' name and telephone number will be faxed to the research institute. Subsequently, a researcher (PL) will screen eligible patients by telephone and make an appointment to check inclusion and exclusion criteria, to complete the informed consent procedure and to perform the baseline measurement. Figure [2](#F2){ref-type="fig"} shows the criteria that must be fulfilled to participate in the LRS trial. A research assistant will check these criteria during patients first visit. The informed consent procedure is completed when patients meet the criteria, are willing to participate and give their written consent. Hereafter, the baseline measurement will take place. Randomisation ------------- Randomisation will take place after baseline measurement by the research assistant. We use a concealed randomisation procedure using a computer generated randomisation list developed by an independent person. Patients\' specific and unique trial number will be typed in a special developed database (i.e. not editable for research assistant and a second randomisation action using the same trial number is not possible) and the random allocation will appear on screen. In order to prevent unequal treatment group sizes, block randomisation will be used with blocks of 10 patients \[[@B12]\]. This means that after every 10th patient the number of patients allocated to both treatment groups is equal. Towards every randomised patient will be explained that the management of their complaint by his or her GP will be continued. Patients who are allocated to physical therapy will be shown a list of participating physical therapists of which he or she can make a choice. The research assistant makes the first appointment with the physical therapist most easily accessible by the patient. Blinding -------- For obvious reasons GPs and PTs are not blinded for treatment allocation. But they are not involved with treatment effect measurements. The patients cannot be blinded because of the ethical reasons as stated by the Medical Ethical Committee. The researcher is involved in the statistical analysis, but the analysis and interpretation of the findings will be audited and verified by an independent and not involved statistician. In this trial the primary outcome measurement and most of the secondary outcome measurements will be scored by the patients. Studies from Ostelo et al \[[@B13]\] and Scholten-Peeters et al \[[@B14]\] mentioned that in this type of study patients are blinded to a certain extent because they are unaware of the exact content of both treatments or may be called naive to the content of the treatment not received. Other more or less similar designed trials from Vroomen et al \[[@B10]\] and Hofstee et al \[[@B15]\] reported that it is not possible to blind participating patients for allocated treatment. Therefore, we think it is important to know any treatment preference of the patients at baseline. Supplementary analysis may show to what extent this effects the scores on outcome measurements of the patients. GP intervention --------------- All patients will be treated by the GP according to their clinical guideline (see Figure [3](#F3){ref-type="fig"}). GPs will give information and advice about LRS. If necessary they prescribe adequate pain medication. We asked the GPs not to refer patients to paramedical specialists (i.e. manual therapist, physical therapist, exercise therapist, etc.). Referral to PT is based on randomisation and performed by the research assistant. PT intervention --------------- PT treatment will imply information and advice about LRS and exercise therapy. Passive modalities such as massage, manipulation techniques or applying applications (e.g. ultra sound or current waves) are not allowed in the PT treatment. This PT treatment protocol was accomplished in a consensus meeting with participating PTs. The PT will report what kind of information/ advice and what type of exercise the patient receives in each session. Both GP and PT intervention will be restricted to a maximum of 9 treatments/ consultations in the first 6 weeks after randomisation. Theoretical background ---------------------- In the Netherlands, PTs are mainly taught the biomechanic model \[[@B13]\]. This model focuses on somatic issues; it assumes a causal relation between tissue damage and pain. PT could be of additional value in the management of patients with LRS because PTs are \'the experts\' in treating musculoskeletal disorders with exercises and advice/ information. The pain reported by a patient is used as guidance to determine the intensity of the exercises and the advice about resuming normal daily activities and work. This study assumes that focussing on (pain) complaints with exercises and advice is the optimal PT treatment in the acute phase (0 to 6 weeks) of LRS. It is possible that patients may suffer from a fear of movement because of pain \[[@B16]\]. Good advice/ information will reassure these patients and exercises will show them that movement is possible. So, the secondary treatment goal of the PT is to decrease the possibly present fear of movement in these patients. Sample size ----------- This trial attempts to enrol 182 patients with LRS, 91 patients in both treatment groups. This sample size is regarded sufficient to detect a difference of 20% (with a α of 0.05 and a power of 80%) in the primary outcome (GPE) between the two treatment groups. A difference of 20% is considered to be clinically relevant \[[@B17]\]. Measurements ------------ Figure [4](#F4){ref-type="fig"} shows the outcome measures and the points of time they are collected. At baseline we will collect patients characteristics such as gender, date of birth, height and body weight. In standardised history taking there will be established whether patients are familiar with LRS in the past, report more pain in the leg on coughing/ sneezing or straining, on sitting, standing, walking and lying down, and if patients report a decreased muscle strength and sensory deficits in the leg. The physical examination consists of the straight leg raising test, the crossed straight leg raising test, test of Bragard, finger-floor distance, standing on toes and heels, knee tendon reflex, ankle tendon reflex, strength of m. extensor hallicus longus, sensory tests (touch, sharp and blunt) in the dermatomes L5/ S1 in the feet. Primary outcome measure ----------------------- The primary outcome measure is the Global Perceived Effect (GPE), measured on a 7 points scale ranging from 1 = completely recovered to 7 = vastly worsened. It is regarded a clinical relevant outcome measure and is regarded valid and responsive to measure the patients\' perceived benefit \[[@B18]-[@B20]\]. Secondary outcome measures -------------------------- Pain severity of the leg and the back will be scored on a 11 points Visual Analogue Scale (VAS) ranging from 0 = no pain to 10 = unbearable pain. Reliability, validity and responsiveness of the VAS have been shown \[[@B21]-[@B23]\]. The functional status will be measured with the Roland Morris Disability Questionnaire (RDQ) for sciatica \[[@B24]\]. The scoring of the RDQ is achieved by counting the number of positive responses: a patient individual score can vary from 0 (no disability) to 24 (severe disability). The RDQ has proved to be a valid instrument and appears to be responsive for clinical relevant changes \[[@B20],[@B25]-[@B28]\]. Health status will be measured by the 36-item short form (SF-36) \[[@B29]\] and the Euroqol (EQ-5D) instrument \[[@B30],[@B31]\]. Validity and responsiveness on both SF-36 \[[@B32]-[@B34]\] and EQ-5D \[[@B35]-[@B37]\] proved to be sufficient. Fear of movement will be measured by the Tampa scale for kinesiophobia (TSK) \[[@B38],[@B39]\]. The TSK consists of 17 items; each rated on a 4-point likert scale. The TSK has been shown to be a valid and responsive instrument \[[@B40],[@B41]\]. Costs will be calculated and include LRS related sickness absence from work, medical consumption (i.e. medication use, additional therapies, visits to health care providers), out-of-pocket expenses and paid help. Patients\' treatment preference will be evaluated at baseline and at 4 follow-up measurements. Statistical analysis -------------------- Baseline comparability will be investigated by descriptive statistics to examine whether randomisation was successful. If necessary, adjustments for baseline variables will be performed in the analysis. Group differences and 95% confidence intervals will be calculated for all outcome measures. The statistical analysis will be performed according tot the intention-to-treat principle, analysing the patients in the treatment group to which they were randomly allocated. Between group differences will be calculated using the Student t-test for continuous variables or Chi-Square for dichotomous variables. In addition a per-protocol analysis will be performed, analysing only those patients with no serious protocol deviations. Comparing the results of the intention-to-treat and the per-protocol analysis will indicate if and to what extent protocol deviations might have biased the results. Multivariate regression analysis will be conducted to examine the influence of baseline variables on outcome. Discussion ========== This article introduces a design of a RCT to evaluate the additional effectiveness of PTs management added to GPs management in patients with LRS. The study is designed in a way that GP and PT treatment is transparent (according a guideline and a consensus meeting) and not a complete \"black box\". The results of this trial will contribute to the decision of the GP regarding referral of patients with LRS to PT. The inclusion of patients will run until the end of the year 2004. The follow-up measurements will be completed in the end of the year 2005. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2474/5/39/prepub> Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Flow chart of the LRS trial. ::: ![](1471-2474-5-39-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Selection criteria for trial eligibility. ::: ![](1471-2474-5-39-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Summary of the clinical guideline \'Lumbosacral radicular syndrome\' of the Dutch College of General Practitioners (1996). ::: ![](1471-2474-5-39-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Data collection and outcome measures. ::: ![](1471-2474-5-39-4) :::	PubMed Central	2024-06-05T03:55:49.854309	2004-11-9	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534096/", "journal": "BMC Musculoskelet Disord. 2004 Nov 9; 5:39", "authors": [ { "first": "Pim AJ", "last": "Luijsterburg" }, { "first": "Arianne P", "last": "Verhagen" }, { "first": "Raymond WJG", "last": "Ostelo" }, { "first": "Hans JMM", "last": "van den Hoogen" }, { "first": "Wilco C", "last": "Peul" }, { "first": "Cees JJ", "last": "Avezaat" }, { "first": "Bart W", "last": "Koes" } ] }
PMC534097	Background ========== One of the promises of comparative genomics is to annotate previously undetectable functional signals in genomic sequence, by identifying and characterising evolutionarily conserved elements. A principled way to extract such signals is by fitting the data to probabilistic models of the molecular evolutionary process. The logic runs as follows: suppose there are various kinds of conserved element x, y, z\... (e.g. exons, bits of RNA, promoters, etc) that mightexplain an observed sequence homology. For each of these scenarios, we can construct a probabilistic model M~x~, M~y~, M~z~\... and compare the likelihood of the observed data under each of these models. The model with the best fit indicates the type of functional element present in the sequence. A groundbreaking example of how this probabilistic approach can be used is the QRNA program, designed as a comparative RNA gene predictor \[[@B1]\]. The three types of element considered by QRNA are noncoding RNA (called RNA), protein-coding exons (called CODfor codon), and unidentified DNA homology (called OTHfor other). The former (RNA) was modeled using a Pairwise Stochastic Context-Free Grammar (Pair SCFG); the latter two (CODand OTH) using Pairwise Hidden Markov Models (Pair HMMs). The noncoding RNA predictions generated a high yield of experimental hits, and offered an information-theoretic glimpse into a modern-day RNA world \[[@B2]\]. It is natural to consider how such an approach might be applied to a pairwise comparison where the evolutionary \"distance\" between the two sequences can vary. One approach, analogous to the BLOSUM series of BLAST matrices for proteins \[[@B3]\], is to partition a set of training alignments into an ad hocnumber of bins based on the percentage sequence identity. Alignments in the same bin (i.e. having comparable sequence identity) then represent pairs of sequences at approximately equivalent distances. For example, the BLOSUM62 substitution matrix was estimated from pairwise alignments with at least 62% identity. This sort of approach is used by the RIBOSUM basepair substitution matrices developed for RSEARCH \[[@B4]\], recent versions of QRNA, and the stemloc program in the author\'s DART software package. An alternative approach, analogous to the PAM series of BLAST matrices \[[@B5]\], is to treat the \"distance\" as a timemeasurement, by postulating an underlying evolutionary stochastic process or continuous-time Markov chainwhose mutation rate parameters are constant over time (called stationarityin stochastic process theory). This evolutionary rate approach uses fewer parameters -- and makes fuller use of the data -- than the dividing-into-bins approach, since it postulates an infinitesimal generator for all time-scales of the process. For the PAM series, this generator takes the form of an instantaneous substitution rate matrix; for a primary-sequence model, the generator is a conditionally-normalized Pair HMM or transducer\[[@B6]\]; for an RNA secondary-structure model, we will see that the generator is a Pair SCFG; and so on. Furthermore, the evolutionary rate model is supremely compatible with likelihood-based phylogenetic methods \[[@B7]\]. It\'s therefore worth considering such evolutionary rate-based models, although (since they\'re trickier to analyse mathematically) they\'re less suited to quick software prototyping than the \"bin-by-percent-ID\" approach. With this in mind, we can consider the evolutionary rate-based equivalents of the three pairwise grammars used in QRNA. The OTHmodel, for noncoding DNA sequence, is a Pair HMM with affine gaps; the closest evolutionary equivalent is the \"long indel\" model \[[@B8],[@B9]\]. The long indel model incorporates multi-residue indels and single-residue (point) substitutions; it is based on the TKF91 model, which only allows single-residue indels \[[@B10]\]. In contrast, the current best evolutionary versions of the COD\[[@B11]\] and RNA\[[@B12]\] models do not attempt to model indels, changes in exon/intron structure or changes in RNA secondary structure. These are deficiencies which must eventually be addressed; ultimately they will limit the usefulness of the models. For example, the lack of a treatment of indels means that these models can only be used on a pre-generated alignment; they cannot, by themselves, be used to align sequences. In this report we present a simple but improved model of RNA structure evolution, called the TKF91 Structure Tree (Figure [1](#F1){ref-type="fig"}). This model allows not just covariant point substitutions of nucleotides, but also covariant insertions and deletions of bases, base-pairs, whole stems and multi-stem structures (Figure [2](#F2){ref-type="fig"}). Although we have not, in this paper, applied the Structure Tree to multiple alignment, or adapted it to include \"long indels\", the similarity to existing models \[[@B8],[@B13]\] suggests very natural forms for such adaptations of our model. Furthermore, the TKF91 Structure Tree is algebraically tractable, yielding SCFG-based scoring schemes for simultaneous RNA alignment and structure prediction (from which alignment algorithms naturally follow). To our knowledge, this is the first such model for the evolution of RNA structureto be described within an evolutionary rate framework. A computer program for simultaneous pairwise alignment and secondary structure prediction using the TKF91 Structure Tree has been developed in C++. The potential of the model for RNA sequence alignment has been demonstrated by testing the pairwise aligner on four functional elements from the RFAM database \[[@B14]\]: the purine riboswitch, the nanos translational control element, the U2 splicing factor and the bacterial nuclear RNase P gene. The TKF91 Structure Tree is a very simple evolutionary model lacking some \"obvious\" features, such as natural selection to favour the thermodynamically stable overlap of π-orbitals between adjacent stacked bases in RNA double helices. The fact that the model appears to work reasonably well, despite the exclusion of such features, suggests that very simple models of RNA evolution may turn out to be sufficient to uncover a surprisingly large proportion of RNA sequence homology. Methods ======= We begin by reviewing the TKF91 model \[[@B10]\]. This model describes the evolution of a single sequence under the action of two kinds of mutation event: (i) point substitution events, which act on a single residue only; and (ii) single-residue indel events, which insert or delete a single residue. The rates of both types of event are independent of the neighboring sequence. The TKF91 model, as defined by Thorne et al, is time-reversible. This has the implication, called the pulley principleby Felsenstein, that the position of an ancestral node in a phylogenetic tree can be slid around like a pulley without changing the likelihood of the observed data \[[@B7]\]. Aligning a pair of observed present-day sequences is therefore identical to aligning an ancestral sequence with its descendant, and we can talk about ancestor-descendant alignment without loss of generality. The TKF91 model can be analysed algebraically \[[@B10]\], and the probability distribution function (PDF) over ancestor-descendant alignments can be expressed as a Pair HMM \[[@B13]\] and extended to multiple sequences (using a \"Multiple HMM\") \[[@B13]\]. While it is straightforward to define a more general \"long indel\" model allowing multi-residue deletions and insertions \[[@B8]\], the only Pair HMMs for this general model that have been described to date are approximations, inspired by the form of the TKF91 model: so far there is no exact Pair HMM solution of the long indel model \[[@B8],[@B9],[@B15],[@B16]\]. In this paper, we will not be considering such long-indel models. Definition of the TKF91 model ----------------------------- The state of the TKF91 process is described by a TKF91 link sequence: a permanent immortal linkat the left end of the sequence, followed by zero or more mortal links. Over time, mortal links can be deleted, and new mortal links can be inserted to the right of either immortal or mortal links. This can be treated as a birth-death process (λ~0~, μ~0~) with constant immigration (λ~0~), where \"births\" are identified with single-link insertions occuring to the immediate right of the parent mortal link. and \"immigration\" with insertions immediately right of the immortal link. A further site-independent labeling is introduced on mortal links using the singlet nucleotide alphabet, Ω = {A, C, G, U}. Each site\'s alphabet label evolves as an independent four-state reversible continuous-time Markov chain(RCTMC) with substitution rate R~0~(i, j) from state ito state j. Labels for newly inserted mortal links are drawn from the equilibrium distribution p~0~(i) of this substitution process. By reading off the labels of mortal links, the state of the TKF91 process can be equated to a sequence in Ω\. Analysis of the TKF91 model --------------------------- The following functions of (λ~n~, μ~n~) arise in analyses of equilibrium and transition probabilities in the TKF91 model \[[@B10]\]. Here tis a time parameter. ![](1471-2105-5-166-i1.gif) Here exp(R~n~t) ≡ exp(A) is the exponential of the matrix with elements A~ij~= R~n~(i, j)t. The meaning of the above functions is as follows. α~n~is the probability of non-deletion; β~n~, γ~n~are the probabilities of insertion, following (respectively) an insertion and a deletion; κ~n~is the probability of continuing the ancestral sequence; and M~n~(i, j) is the conditional substitution probability from ito j. Note (1 - γ~n~)κ~n~(1 - α~n~) = β~n~(delete → delete and insert → insert transition probabilities are equal). Note also lim~t→∞~β~n~= κ~n~. The equilibrium probability distribution over sequences in the TKF91 model is a geometric distribution with parameter κ~0~. The residues at individual positions of the sequence are independently, identically distributed at equilibrium and are sampled from the equilibrum distribution of the point substitution process. The TKF91 singlet grammar is shown in Figure [3](#F3){ref-type="fig"}. The TKF91 pair grammar is shown in Figure [4](#F4){ref-type="fig"}. Note that two alternate sets of rule probabilities, jointly and conditionally normalised, can be read off from Figure [4](#F4){ref-type="fig"}: the conditional probabilities can be read off from column P(d\\|a), while the joint probabilities can be found by multiplying the expressions in columns P(a) and P(d\\|a) to obtain P(a, d). Extending the TKF91 model ------------------------- Various extensions to TKF91 have been proposed \[[@B8],[@B9],[@B15]\]. The most tractable kind of extension changes the meaning of a \"link\" but leaves the indel process on links intact \[[@B15]\]. Our RNA model is one such extension, allowing two different kinds of TKF91 model that can be mutually nested to form loop and stem regions. Consider the following extension to the TKF91 model, which we call the TKF91 Structure Tree, and which is shown in Figures [1](#F1){ref-type="fig"} and [2](#F2){ref-type="fig"}. This model uses the fact that an RNA secondary structure (excluding pseudoknots, kissing loops and other \"tertiary\" interactions) can be identified with a tree. The state of our stochastic process can thus be described by a rooted tree: every node in this tree is either a singlet, paired, loopor stemnode. The tree can be broken into overlapping loop sequencesand stem sequences, which correspond to strands of unpaired RNA (loops) or double helices of basepaired RNA (stems). Loops are allowed to contain unpaired nucleotides, and can also serve as a branching-off point for nested stems. Stems, on the other hand, are allowed to contain paired nucleotides, and are terminated by a loop (this reflects the smallest unit of RNA structure, which is a stem terminated by a loop). The tree is rooted by a loop sequence. The above description will now be made more precise. Definition of the TKF91 Structure Tree -------------------------------------- The state of the TKF91 Structure Tree is described by a rooted tree where each node has degree ≤ 3. There are four basic kinds of node in the tree: singlet, paired, loop and stem. Singlet and paired nodes correspond to observable nucleotides. Singlet nodes (labeled from Ω) represent independently evolving nucleotides, as in TKF91. Paired nodes (labeled from Ω^2^) represent covariant basepairs. Loop and stem nodes determine the tree structure (Figure [1](#F1){ref-type="fig"}). Loop nodes (labeled L), of which the root node is one, are present at the beginning of loop sequences, which contain singlet and stem nodes and are written horizontally. Stem nodes (labeled S) are present at the beginning of stem sequences, which contain paired nodes, are terminated by a loop node, and are written vertically. The set of loop and stem node labels is written Φ. The full set of node labels is Ω ∪ Ω^2^∪ Φ. Φ = {L, S} Ω = {A, C, G, U} Ω^2^= {AA, AC, AG, AU, CA, CC, CG, CU, GA, GC, GG, GU, UA, UC, UG, UU} ### Loop sequences A loop sequence is very similar to a TKF91 link sequence: as with TKF91, we have a leftmost immortal loop linkfollowed by zero or more mortal loop links. The mortal links are inserted and deleted with rates λ~1~and μ~1~, in the style of TKF91. Each link is also a node in the Structure Tree. Links are labeled from Ω ∪ Φ: the immortal loop link is labeled L, while the mortal loop links are labeled from {A, C, G, U, S}. As with the TKF91 model, the alphabet labeling of each mortal link evolves as an independent five-state RCTMC with substitution rate R~1~(i, j) from ito jand equilibrium probability p~1~(i) of being in state i, plus the additional restriction that R~1~(X, S) = R~1~(S, X) = 0 for all X∈ Ω: in other words, embedded stems can\'t interconvert with singlet nucleotides. See step 1 → 2 of Figure [2](#F2){ref-type="fig"} for examples of single-nucleotide substitution in loop sequences, and steps 3 → 4 and 4 → 5 for single-nucleotide insertion and deletion. The S-labeled links possess an independently evolving embedded stemsequence that can be considered to \"nest\" inside the loop sequence. If the S-link is deleted, then the embedded stem (and all its children) is deleted with it. Conversely, when a new S-link is inserted, it is inserted with a complete subtree that is sampled from the equilibrium distribution over Structure Trees. See steps 6 → 7 and 7 → 8 of Figure [2](#F2){ref-type="fig"} for examples of substructure insertion and deletion. Since a loop sequence is effectively a TKF91 sequence with a special \"fifth nucleotide\" character representing an embedded stem (the Slink), it obeys the same statistics as a TKF91 sequence. In particular, the probability distribution over loop lengths at equilibrium is a geometric distribution with parameter κ~1~. ### Stem sequences A stem sequence is also derived from a TKF91 link sequence. Unlike the TKF91 link sequence or the loop sequence, however, it is written vertically (rather than horizontally) in Figure [1](#F1){ref-type="fig"}. It consists of a topmost immortal stem link, zero or more mortal stem links, and a bottommost, terminating looplink. Again, each link is also a node in the Structure Tree. Links are labeled from Ω^2^∪ Φ: the immortal stem link is labeled S(this is the node in the parent loop sequence), the mortal links are labeled with the paired nucleotide alphabet Ω^2^(each with an independent sixteen-state RCTMC modeling covariant pair substitution along RNA stems, with substitution rate matrix R~2~(i, j) and equilibrium p~2~(i)), and the terminating loop link is labeled L. The mortal stem links experience TKF91-style insertion and deletion with rates λ~2~and μ~2~(although, in the diagrammatic form of Figure [1](#F1){ref-type="fig"}, newly inserted links are placed immediately under their parent link, rather than immediately to the right). The terminating loop link Ldoes not contribute to insertion or deletion (so is effectively immortal but inert) but possesses an independently evolving loop sequence. See step 2 → 3 of Figure [2](#F2){ref-type="fig"} for examples of covariant basepair substitution in stem sequences, and step 5 → 6 for covariant basepair insertion and deletion. Note that the immortal stem link, S, is only immortal from the point of view of the stem sequence beneath it. The Sis itself a mortal link in a parent loop sequence, and may be deleted as that sequence evolves. In this event, the loop link Lwill also be deleted, along with all its children (step 7 → 8, Figure [2](#F2){ref-type="fig"}). Thus, the only truly immortal link is the loop node at the root of the Structure Tree, which has no parents to deal death from above. As with the loop sequence, a stem sequence is effectively a TKF91 sequence with minor modifications, and it obeys the same statistics as a TKF91 sequence. The probability distribution over stem lengths at equilibrium is a geometric distribution with parameter κ~2~. Analysis of the TKF91 Structure Tree ------------------------------------ Figure [5](#F5){ref-type="fig"} shows the SCFG for generating the TKF91 Structure Tree at equilibrium. There are two nonterminals, Φ, and four terminals, Ω. Figure [6](#F6){ref-type="fig"} shows the pair stochastic context-free grammar for an ancestor and descendant sequence separated by evolutionary time t. Again, conditional and joint probabilities can both be read from the figure. Nonterminals are Φ~1234~; terminals are Ω~a~for the ancestor and Ω~d~for the descendant. Φ~1234~= {L~1~, L~2~, L~3~, L~4~, S~1~, S~2~, S~3~, S~4~} Ω~a~= {A~a~, C~a~, G~a~, U~a~} Ω~d~= {A~d~, C~d~, G~d~, U~d~} Dynamic programming alignment of sequences to these grammars has the typical complexity for single-sequence \[[@B17]\] and pairwise \[[@B18]\] SCFGs. That is, for Figure [5](#F5){ref-type="fig"}, the time complexity is O(L^3^) and the memory complexity O(L^2^), while for Figure [6](#F6){ref-type="fig"}, the time complexity is O(L^3^M^3^) and the memory complexity O(L^2^M^2^), where Land Mare sequence lengths. This is also the complexity of the single-sequence and two-sequence Sankoff algorithm \[[@B19]\], for which SCFGs may be regarded as a probabilistic scoring scheme. The time and memory complexity may be reduced by the use of \"banding\" techniques \[[@B20],[@B21]\], that restrict the dynamic programming computation to the (typically) highest-scoring central diagonal band of the dynamic programming matrix, or by more flexible constraints on the DP iteration \[[@B18]\]. Grammar transformations ----------------------- We now describe some transformations of Figures [5](#F5){ref-type="fig"},[6](#F6){ref-type="fig"} performed before implementing the grammar parsers. ### Null cycles The presence in a grammar of \"null cycles\" -- sequences of production rules which cause no net change -- complicates the parsing algorithms for that grammar. Generally, null cycles are avoided by programmers designing SCFGs or HMMs for sequence analysis \[[@B17]\]. However, in the grammars derived automatically for the TKF91 Structure Tree, null cycles arise naturally due to the possibility of zero-length loop or stem sequences in the model. There are several classes of null cycle in the grammars for the Structure Tree model, shown in Table [1](#T1){ref-type="table"}. ### Degeneracies As well as null cycles, there are other undesirable degeneracies in the Structure Tree grammars. Grammatical degeneracy occurs when more than one parse has the same meaning, so parses are degeneraterather than unique. Most stochastic grammars useful for bioinformatics are degenerate in the sense that there are always many folds or alignments consistent with the observed sequence data; this sort of degeneracy is technically called ambiguity. We are more concerned with other forms of degeneracy, such as structural degeneracy(multiple parses denote a single pattern of basepairing) and alignment degeneracy(multiple parses denote a single alignment). TKF91, in effect, skirts alignment degeneracy by assigning meaning to the ordering of deletions and insertions in an alignment, but alignment degeneracies arise in the Structure Tree model because there are multiple ways to delete and insert things (e.g. deleting a whole stem, versus deleting all its elements individually). There are also structural degeneracies arising from \"silent\" (i.e. non-emitting) loops or stems. In addition to the null cycles described above, these include (for the singlet grammar) the undesirable \"loop bifurcation\" L→ LLand the \"silent bulge\" S→ S(a null cycle). A full list of degeneracies for the singlet and pair grammars is shown in Table [1](#T1){ref-type="table"}. ### Prevention of zero-length stems The null cycles all involve zero-length stems and can be broken (NB not marginalised; the likelihood is discarded) by adding extra nonterminals ![](1471-2105-5-166-i2.gif) before the corresponding S~k~, copying all outgoing rules except the nonemitting S~k~→ L~k~. This also removes the loop bifurcations, but leaves silent bulges of the form S~k~→ ![](1471-2105-5-166-i2.gif). The silent bulges can be removed by adding nonterminals ![](1471-2105-5-166-i3.gif) before L~k~, copying all outgoing rules except L~k~→ ε, changing ![](1471-2105-5-166-i4.gif) to ![](1471-2105-5-166-i5.gif) so as to prevent escape from ![](1471-2105-5-166-i3.gif) without an unpaired emission, and adding new rules of the form ![](1471-2105-5-166-i6.gif) to allow escape if there is a genuine bifurcation. A more careful analysis, marginalising null cycles and silent bulges rather than simply ignoring them, is almost certainly possible. ### Transformation to canonical form Figures [7](#F7){ref-type="fig"} and [8](#F8){ref-type="fig"} show the singlet and pairwise grammars with null cycles and silent bulges removed, in the canonical form used by the DART software package \[[@B18]\]. As well as the new sets of nonterminals described above (Φ\' for singlet, ![](1471-2105-5-166-i7.gif) for pair) the grammar includes nonterminals dedicated to bifurcations (![](1471-2105-5-166-i8.gif) for singlet, ![](1471-2105-5-166-i9.gif) for pair) and emissions (![](1471-2105-5-166-i10.gif) for singlet, ![](1471-2105-5-166-i11.gif) for pair). The separation of the nonterminals into null, bifurcation and singlet/pair emission sets puts the grammar in the form understood by the DART library \[[@B18]\]. The full nonterminal alphabets are Ψ for singlet states and Ψ~1234~for pair states. ![](1471-2105-5-166-i12.gif) The asymptotic complexity of the dynamic programming recursions implied by these grammars is unchanged by the transformation to DART form. For Figure [7](#F7){ref-type="fig"}, the time complexity is O(L^3^) and the memory complexity O(L^2^), while for Figure [8](#F8){ref-type="fig"}, the time complexity is O(L^3^M^3^) and the memory complexity O(L^2^M^2^), where Land Mare sequence lengths. Again, the complexity may be reduced by the use of \"banding\" \[[@B20],[@B21]\] or other \[[@B18]\] constraints. Parameterisation of the TKF91 Structure Tree -------------------------------------------- The Expectation Maximisation (EM) algorithm is often used for training BLOSUM-like models, e.g. estimating emission and transition probabilities for Pair HMMs \[[@B17]\] or Pair SCFGs \[[@B1]\]. It is also useful for training evolutionary rate models, which have roughly the same number of parameters and can make use of larger training sets (since the training data don\'t have to be \"binned\" by percent identity). The EM algorithm for the TKF91 Structure Tree can be separated into two parts, one for the substitution process and one for the indel process. Earlier work \[[@B22]\] showed how to estimate the maximum-likelihood substitution rate matrix R~n~using the EM algorithm, given the following sufficient statistics: ![](1471-2105-5-166-i13.gif), the expected number of insertions of state d; ![](1471-2105-5-166-i14.gif), the expected number of aligned sites with ancestral state aand descendant state d. A forthcoming paper describes how to estimate the maximum-likelihood indel rates λ~n~, μ~n~for a TKF91 model using the EM algorithm, given the following sufficient statistics: ![](1471-2105-5-166-i15.gif), the expected number of deleted links not followed by an insertion; ![](1471-2105-5-166-i16.gif), the expected number of surviving links not followed by an insertion; ![](1471-2105-5-166-i17.gif), the expected number of deleted links followed by an insertion; ![](1471-2105-5-166-i18.gif), the expected number of surviving links followed by an insertion. We can calculate all the above update statistics simultaneously from data (the E-step) using a constrained version of the Inside-Outside algorithm \[[@B18]\] for the grammar in Figure [8](#F8){ref-type="fig"}, as follows. Assume the joint normalisation, P(d, a), and suppose that ![](1471-2105-5-166-i19.gif) is the posterior expectation of the number of times rule mof Figure [8](#F8){ref-type="fig"} was applied, as returned by the Inside-Outside algorithm. For emit rules, let ![](1471-2105-5-166-i20.gif) be the expected number of times rule mwas used to emit the specific nonterminals X, Y\... ∈ Ω. Then ![](1471-2105-5-166-i21.gif) The terms in parentheses are to be omitted if the conditional normalisation, P(d\\|a), is used. The relationship between the expected insert and match usage ![](1471-2105-5-166-i22.gif), ![](1471-2105-5-166-i14.gif) and the expected start, wait and transition usage ![](1471-2105-5-166-i23.gif) of the previous work \[[@B22]\] is as follows ![](1471-2105-5-166-i24.gif) where ![](1471-2105-5-166-i25.gif) is defined as in the previous work \[[@B22]\] ![](1471-2105-5-166-i26.gif) Results ======= The pairwise aligner for the TKF91 Structure Tree is distributed as part of the DART package at the following URL: <http://www.biowiki.org/> The aligner is based on the Stochastic Context-Free Grammars (SCFGs) shown in Figures [7](#F7){ref-type="fig"} and [8](#F8){ref-type="fig"}, as explained in the Methods section. The specific implementation uses a general Pair SCFG dynamic programming (DP) engine with accelerating heuristics, to be described in a later paper (manuscript in preparation). To test the performance of the model at aligning and predicting structure of RNA sequence, we considered pairs of RNA sequences from four different families, with varying degrees of homology at the level of secondary structure. The four families were the purine riboswitch (Figure [9](#F9){ref-type="fig"}), the nanos translational control element (TCE) from Drosophila(Figure [10](#F10){ref-type="fig"}), the U2 spliceosomal factor (Figure [11](#F11){ref-type="fig"}) and bacterial nuclear RNase P (Figure [12](#F12){ref-type="fig"}). For a given family, denote the two sequences in the family by A, B. The following computations were performed: (1A), (1B)For each of the two sequences (A, B) taken individually, the secondary structure was predicted without the aid of comparative information from the other sequence, using the single-sequence SCFG of Figure [7](#F7){ref-type="fig"}. (2)The two sequences (A, B) were then aligned using the TKF91 model, without making use of any model of RNA structure, using the Pair HMM of Figure [4](#F4){ref-type="fig"}. (3)Finally, the two sequences (A, B) were aligned using the TKF91 Structure Tree model introduced in this paper, using the Pair SCFG of Figure [8](#F8){ref-type="fig"}. These computations allow a comparison between the TKF91 model, the single-sequence SCFG of Figure [7](#F7){ref-type="fig"} and the TKF Structure Tree. The results, including structure and alignment predictions, are illustrated in a compact visual representation that we call a \"fold/alignment dotplot\". The key to interpreting the fold/alignment dotplot is shown in Figure [13](#F13){ref-type="fig"}. The subregions labeled a-f have the following meaning: (a)This triangular dotplot illustrates the single-sequence structure prediction for sequence Aof computation (1A). The pixel color at co-ordinates (x, y) represents the posterior probability that residues xand yof Aare base-paired, in the absence of any information from sequence B. (b)This triangular dotplot illustrates the single-sequence structure prediction for sequence Bof computation (1B). The pixel color at co-ordinates (x, y) represents the posterior probability that residues xand yof Bare base-paired, in the absence of any information from sequence A. (c)This rectangular dotplot illustrates the structure-free pairwise alignment of computation (2). The pixel color at co-ordinates (x, y) represents the posterior probability that residue xof Ais homologous to residue yof B, in the absence of any structural information from the two sequences. (d)This triangular dotplot illustrates the comparative structure prediction for sequence Aof computation (3). The pixel color at co-ordinates (x, y) represents the marginal posterior probability that residues xand yof Aare base-paired, summed over all alignments to sequence B. (e)This triangular dotplot illustrates the comparative structure prediction for sequence Bof computation (3). The pixel color at co-ordinates (x, y) represents the marginal posterior probability that residues xand yof Bare base-paired, summed over all alignments to sequence A. (f)This rectangular dotplot illustrates the structural pairwise alignment of computation (3). The pixel color at co-ordinates (x, y) represents the marginal posterior probability that residue xof Bis homologous to residue yof A, summed over all secondary structures of sequences Aand B. Note that the orientation of this plot is flipped (reflected about the diagonal axis) relative to (c). In addition, the \"true\" (published) structures and alignments are overlaid on the computational results as blue squares (or blue dots, on the larger images). The rate parameters used for the TKF91 Structure Tree were obtained by maximum likelihood training from a random selection of structurally-annotated RFAM alignments, as follows: λ~1~= 0.027, μ~1~= 0.03; λ~2~= 0.007, μ~2~= 0.01; p~1~(S) = 0.01. The substitution rate parameters were taken from the PFOLD program \[[@B12]\]. The evolutionary \"time\" between the two sequences was set to 1 in each case. In the case of the RNase P and U2 genes, the DP algorithms were constrained to a band along the main diagonal of the DP matrix; this constraint was imposed due to limited memory. No such constraint was imposed for the purine riboswitch computations. The posterior probabilities of folding and alignment (dotplots a-f) obtained by DP on these three classes of element are shown in Figure [14](#F14){ref-type="fig"} (for the purine riboswitches), Figure [15](#F15){ref-type="fig"} (for the nanos TCEs), Figure [16](#F16){ref-type="fig"} (for the U2 snRNAs) and Figure [17](#F17){ref-type="fig"} (for the bacterial nuclear RNase P genes). In all cases, the Pair SCFG sharply resolves the most probable stems in the sequences; for the nanos, U2 and RNase P sequences, it also resolves the pairwise alignment. Purine riboswitch ----------------- The purine riboswitches are a class of cis-acting regulatory elements that specifically bind adenine or guanine and are involved in the post-translational regulation of purine transport and biosynthesis \[[@B23]\]. Figure [9](#F9){ref-type="fig"} shows the alignment of the two riboswitch sequences, from Bacillus haloduransand Streptococcus pneumoniae, which was taken from the RFAM database \[[@B14]\]. The two secondary structures of this pair are exactly identical, although the primary sequences are considerably diverged. Figure [14](#F14){ref-type="fig"} shows the posterior dotplots for the purine riboswitches. This is an easy case for the model, with a strong signal and few gaps. The TKF91 Structure Tree grammar (Figure [8](#F8){ref-type="fig"}) is able to identify all stems correctly, with some slight uncertainty over the alignment. The primary-sequence TKF91 grammar (Figure [4](#F4){ref-type="fig"}) is similarly able to find the correct alignment, although the singlet folding grammar (Figure [7](#F7){ref-type="fig"}) has difficulty resolving the stems (note that this grammar does not model basepair stacking effects). Nanos translational control element ----------------------------------- The translational control element (TCE) is a regulatory sequence from the 3\' untranslated region of the Drosophilananos gene, involved in post-translational degradation and transport of nanos mRNA, which localises to the posterior of oocytes and other cell lines \[[@B24]\]. Figure [10](#F10){ref-type="fig"} shows the alignment of the two TCE sequences, from Drosophila virilisand Drosophila melanogaster, which was curated by hand from the description by Gavis et al\[[@B24]\]. The two secondary structures of this pair share the same overall bifurcating-stem structure, but with some changes in stem length. Figure [15](#F15){ref-type="fig"} shows the posterior dotplots for the nanos TCEs. This time the TKF91 Structure Tree grammar (Figure [8](#F8){ref-type="fig"}) does considerably better than the primary-sequence TKF91 grammar (Figure [4](#F4){ref-type="fig"}) at finding the correct alignment, probably due to the gaps at the end (the TKF91 grammar in Figure [4](#F4){ref-type="fig"} is effectively a global aligner with linear gaps, so that the alignments it produces tend to form a continuous line from corner to corner of the DP matrix, without major discontinuities, as can be seen in region (c) of Figure [15](#F15){ref-type="fig"}). Again, the Structure Tree does much better than the singlet folding grammar (Figure [7](#F7){ref-type="fig"}) at distinguishing real stems from background noise, since it is able to use covariation of basepaired residues as a clue. U2 snRNA -------- The U2 small nuclear RNA recognizes and binds the branch point region of introns in pre-mRNA \[[@B25]\]. Figure [11](#F11){ref-type="fig"} shows the alignment of the two splicing factors, from Tetrahymena thermophilaand Leptomonas collosoma, was taken from the RFAM database \[[@B14]\]. The secondary structures of the two sequences are quite similar, but the LeptomonasU2 has a deletion of roughly 35 bp that eliminates an entire stem (stems 4--6 on Figure [11](#F11){ref-type="fig"}). Figure [16](#F16){ref-type="fig"} shows the posterior dotplots for the U2 snRNAs. As before, the Structure Tree\'s stem predictions (regions (d) and (e), above the main diagonal of Figure [16](#F16){ref-type="fig"}) are far more specific than the singlet grammar\'s predictions (regions (a) and (b), below the main diagonal). The primary-sequence TKF91 grammar (Figure [4](#F4){ref-type="fig"}) is, again, hampered by its global alignment and linear gap penalty, and the alignment in region (c) is stretched and also uncertain. However, the Pair SCFG (Figure [8](#F8){ref-type="fig"}) manages to identify the 35-bp deletion and correctly finds stem 4 of Figure [11](#F11){ref-type="fig"}, though stems 5--6 have a lower probability (when predicting the structure of this deleted region, the Pair SCFG is unable to use covariation and must rely on basepairing information alone). Bacterial nuclear RNase P ------------------------- Nuclear RNase P is a class of endoribonuclease ribozyme involved in the production of mature 5\' ends of transfer RNAs during tRNA biosynthesis \[[@B26]\]. Figure [12](#F12){ref-type="fig"} shows the alignment of the two ribozyme sequences, from Pichia canadensisand Clavispora opuntiae, which was taken from the RFAM database \[[@B14]\]. The secondary structures of the two sequences are quite different, with major change in stem length and deletion of whole stem structures, characteristic of this gene family (stems 0--2 and 8--9 of Figure [12](#F12){ref-type="fig"}). Figure [17](#F17){ref-type="fig"} shows the posterior dotplots for the RNase P genes. This family is one of the most mutable in RFAM, and the TKF91 Structure Tree performs poorly on this case. Both the Pair HMM (Figure [4](#F4){ref-type="fig"}; region (c) of Figure [17](#F17){ref-type="fig"}) and the Pair SCFG (Figure [8](#F8){ref-type="fig"}; region (f) of Figure [17](#F17){ref-type="fig"}) get the alignment almost entirely wrong, except for a region toward the 3\' end that doesn\'t contain any stems (the region just before stem 8 of Figure [12](#F12){ref-type="fig"}). As a consequence, the Pair SCFG also fails to predict any stems correctly; the singlet SCFG (Figure [7](#F7){ref-type="fig"}) does no better. Region (f) of Figure [17](#F17){ref-type="fig"} displays the continuous-line alignment from corner-to-corner, that is characteristic of global aligners with linear gaps: unlike the case of the U2 alignment, the structural signal here is insufficient to compensate for the indel-modeling deficiencies of the TKF91 Structure Tree. The log-odds score of the \"true\" alignment (Figure [12](#F12){ref-type="fig"}) under the Structure Tree model is highly negative (-547 bits), suggesting that the model is poorly adapted for this example. Compare this with the scores for the previous examples: Figure [9](#F9){ref-type="fig"} scored 2 bits, Figure [10](#F10){ref-type="fig"} scored -82 bits and Figure [11](#F11){ref-type="fig"} scored 35 bits. The low score for the nanos TCEs (Figure [10](#F10){ref-type="fig"}) was due primarily to the deletions in the outermost stem; the score rose to -5 bits with judicious trimming and careful choice of the \"time\" parameter. Discussion ========== We have described a reversible continuous-time Markov chain, called the \"TKF91 Structure Tree\", that describes both (i) covariant substitutions and indels in RNA sequence contingent upon a particular secondary structure, and (ii) changes in the underlying RNA secondary structure, corresponding to gain and loss of substructures. A pairwise alignment algorithm based on the model has been implemented in C++ and tested on four homologous pairs of RNA functional element from RFAM \[[@B14]\]. As with the TKF91 model on which the TKF91 Structure Tree is based \[[@B10]\], it should be possible, systematically, to design corresponding algorithms for multiple sequences, using either exhaustive dynamic programming \[[@B6],[@B27]\] or Markov Chain Monte Carlo \[[@B13]\]. It should be noted that the present implementation of the TKF91 Structure Tree is not designed to be a direct competitor to programs like FOLDALIGN \[[@B20]\], DYNALIGN \[[@B21]\] or CARNAC \[[@B28]\]. Such pairwise alignment programs are optimized for criteria like alignment accuracy and sensitivity. The TKF91 Structure Tree, on the other hand, was designed as an expository evolutionary model, ultimately aimed at phylogenetic analysis of multiple RNA sequences in an evolutionary likelihood context. The pairwise alignment program reported in this paper was implemented to demonstrate the potential of this evolutionary model, rather than for use as a practical alignment tool. The author\'s STEMLOC program, which is similarly based on Pair SCFGs, has been optimized for practical applications (preferring short-term performance advantages over long-term design considerations) and may be freely downloaded from <http://www.biowiki.org/>. The results of our tests on pairwise alignments from RFAM reveal the strengths and weaknesses of our model. When RNA structure is very strongly conserved and indels are few, as with the purine riboswitches selected for this comparison (Figure [14](#F14){ref-type="fig"}), the TKF91 Structure Tree performs well at both structure prediction and alignment. On such alignments, the model is expected to be similar to PFOLD \[[@B12]\], which uses an SCFG and an evolutionary substitution model but lacks an evolutionary treatment of gaps. When the alignment has numerous indels in loops and stems, as in the selected nanos TCEs (15), or even minor rearrangements of structure, as in the selected U2 splice factors (16), the Structure Tree still seems to work well. However, beyond a certain level of structural change, as in the selected RNase P alignment (17), the model performs poorly and leaves considerable room for improvement. In view of the room for improvement, we can identify a number of weaknesses of the TKF91 Structure Tree that could be improved in future models: • Sources of degeneracy such as zero-length stems and loops were removed \"by hand\" from the Pair SCFG (Table [1](#T1){ref-type="table"}). These degeneracies could have beeen specifically excluded from the evolutionary model, but with the apparent cost of making an exact solution much harder to find. One might expect the nondegenerate grammars of Figures [7](#F7){ref-type="fig"} and [8](#F8){ref-type="fig"} to approximate the transition probabilities of such a nondegenerate model. • Indel rates for whole stems/multistems are same as for unpaired residues. In nature, stem gain and loss is much slower than unpaired residue insertion/deletion, since the former is a structural change while the latter is not. • Multiple-residue indel events, and hence affine gap penalties, are not allowed. Again, the poor performance on the RNase P alignment may in part be due to this: the alignment generated has many small gaps scattered throughout, whereas the \"true\" alignment has fewer, longer gaps. This is a characteristic artefact of using a point indel model (linear gap penalty) where a multi-residue indel model (affine penalty) would be more appropriate. • Stems cannot be deleted without deleting all their \"children\" as well (i.e. all stems nested inside). Empirical inspection of alignments in RFAM, however, reveals many structures where an outer stem has been deleted or truncated, while inner stems are preserved. Again, perhaps an affine gap penalty for covariant indels (i.e. indels in stems) would address this. Alternatively, one might contrive some kind of \"ragged-end\" local alignment model, e.g. by embedding the finite TKF91 Structure Tree in an infinite, unobserved Structure Tree (c.f. \[[@B8]\]), though this may not be the ideal way to model such effects. • The equilibrium distribution over structures is highly simplified. For example, there is currently no modeling of fine-scale energetics such as basepair stacking propensities due to π-orbital conjugation. Mathematically, the complexities of modeling such effects are somewhat similar to those involved in modeling nearest-neighbor substitution biases in DNA (such as methylation-induced CpG deamination). Since recent progress has been made with such models \[[@B29],[@B30]\] we might eventually expect inclusion of stacking effects in models of covariant RNA substitution, as well. • Bulges cannot be inserted into stems, except via the following awkward mechanism: the insertion of a bulge into a stem requires the pre-existence of a null S→ L→ Stransition, where the Lis empty. To fix this, Lnodes could be allowed in stem sequences, just as Snodes are allowed in loop sequences (in fact, one should probably introduce \"left\" and \"right\" L-nodes, corresponding to left & right bulges). However, this would increase the potential for degeneracies. • We have assumed that all stems and loops evolve at the same rate, whereas empirical inspection of RFAM of suggests otherwise. It is known that the analogous assumption in proteins (that all sites evolve at the same rate) can skew phylogenetic distance estimation \[[@B31]\], and perhaps a similar correction to the discretized gamma priors used in proteins could be applied here \[[@B32]\]. • There is no special treatment of structural features such as triloops, tetraloops, triple-A platforms, U-turns and the like. Such features are often observed to be evolutionary conserved \[[@B33],[@B34]\] and seem likely to be involved in intermolecular interactions \[[@B35],[@B36]\]. It would be relatively easy to incorporate such features into the TKF91 Structure Tree, as special classes of L- or S-branch. • While the lengths of stem sequences are geometrically distributed in the TKF91 Structure Tree, due to their roots in the TKF91 model, empirical observations of real RNA structures suggest that real stem lengths follow a fairly tight length distribution. Such approximations in modeling stem lengths could conceivably contribute to poorer performance of the model. (In practise, we have not observed unnaturally long stems in the output of the TKF91 Structure Tree aligner, but the existence of a long, perfect inverted repeat in the sequence could conceivably bring out this problem.) Despite these drawbacks, the results of our preliminary benchmark suggest that the TKF91 Structure Tree may be useful for aligning (at least the better-conserved) RNA functional elements. Given the recent growth of RNA sequence and structure databases such as RFAM \[[@B14]\] and SCOR \[[@B34]\], it would be interesting to carry out a broad-scale, empirical study of the mutations of RNA structures. This could then be used as a starting point for systematically designing and benchmarking an improved evolutionary model for RNA. In the meantime, the results presented here suggest new ways of designing evolutionary grammars that recognise higher-level structural change as well as point substitutions and indels, offering new ways of using high-throughput comparative sequencing to interpret the contents of genomes. Acknowledgements ================ This manuscript has evolved over the course of discussions with Sam Griffiths-Jones, Alex Bateman, Sean Eddy, Elena Rivas, Eric Westhof, Bjarne Knudsen, Kushal Chakrabarti, Jotun Hein, Gerton Lunter and David Haussler. The author thanks two anonymous reviewers for helpful criticism. The work was partly developed during a workshop in Benasque, Spain organised by Elena Rivas and Eric Westhof in 2003. The work was partially supported by grants from the UK\'s EPSRC (code HAMJW) and MRC (code HAMKA). Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### A TKF91 Structure Tree, and the corresponding RNA secondary structure (inset). ::: ![](1471-2105-5-166-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### An example mutation path for a TKF91 Structure Tree, illustrating the instantaneous transitions of the process. In each figure (N), gray arrows indicate the sites of past mutations in step (N- 1) → (N), while black arrows indicate the sites of upcoming mutations in step (N) → (N+ 1). The types of mutation are as follows. (1) → (2): Point substitution of unpaired nucleotides in loop sequences. (2) → (3): Point and/or covariant substitution of paired nucleotides in stem sequences. (3) → (4): Insertions of single unpaired nucleotides in loop sequences. (4) → (5): Deletions of single unpaired nucleotides in loop sequences. (5) → (6): Insertion and deletion of paired nucleotides in stem sequences. (6) → (7): Insertion of stems into loop sequences. (7) → (8): Deletion of stems from loop sequences. ::: ![](1471-2105-5-166-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### An SCFG with nonterminals {L} and terminals Ω for generating the equilibrium distribution over TKF91 sequences. Here X∈ Ω is a generic terminal symbol. ::: ![](1471-2105-5-166-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### A Pair SCFG with nonterminals {L~1~, L~2~} and terminals Ω~a~∪ Ω~d~for generating pairwise alignments of TKF91 sequences for an ancestor and a descendant. Here X, Y∈ Ω are generic terminal symbols. ::: ![](1471-2105-5-166-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### An SCFG with nonterminals Φ and terminals Ω for generating the equilibrium distribution over Structure Trees. Here W, X∈ Ω are generic terminal symbols. ::: ![](1471-2105-5-166-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### A Pair SCFG with nonterminals Φ~1234~and terminals Ω~a~∪ Ω~d~for generating pairwise alignments of Structure Trees for an ancestor and a descendant. Here W, X, Y, Z∈ Ω are generic terminal symbols. ::: ![](1471-2105-5-166-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### A DART-form SCFG with nonterminals Ψ and terminals Ω for generating ancestral Structure Trees. Here W, X, Y, Z∈ Ω are generic terminal symbols. ::: ![](1471-2105-5-166-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### A DART-form Pair SCFG with nonterminals Ψ~1234~and terminals Ω~a~∪ Ω~d~for generating pairwise alignments of Structure Trees for an ancestor and a descendant. Here W, X, Y, Z∈ Ω are generic terminal symbols. ::: ![](1471-2105-5-166-8) ::: ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Alignment of purine riboswitches from Bacillus halodurans(AP001509.1) and Streptococcus pneumoniae(AE007476.1). ::: ![](1471-2105-5-166-9) ::: ::: {#F10 .fig} Figure 10 ::: {.caption} ###### Alignment of nanos TCEs from Drosophila virilis(DVU24695) and Drosophila melanogaster(DRONANOS). ::: ![](1471-2105-5-166-10) ::: ::: {#F11 .fig} Figure 11 ::: {.caption} ###### Alignment of U2 splicing factors from Tetrahymena thermophila(X63784.1) and Leptomonas collosoma(X56455.1). ::: ![](1471-2105-5-166-11) ::: ::: {#F12 .fig} Figure 12 ::: {.caption} ###### Alignment of nuclear RNase P genes from Pichia canadensis(AF186219.1) and Clavispora opuntiae(AF186216.1). ::: ![](1471-2105-5-166-12) ::: ::: {#F13 .fig} Figure 13 ::: {.caption} ###### Fold-alignment dotplot key. These plots compare separate and integrated methods for RNA alignment and folding. Regions (a) and (b) use the single-sequence SCFG of Figure 7; region (c) use the TKF91 pair HMM of Figure 4; and regions (d), (e) and (f) use the pair SCFG of Figure 8, which is based on the TKF91 Structure Tree (see Results section). ::: ![](1471-2105-5-166-13) ::: ::: {#F14 .fig} Figure 14 ::: {.caption} ###### Fold-alignment dotplot of purine riboswitches (see Figure 13 for key). From a wide range of potential stems (faint red diagonal lines, plots a-b), the Pair SCFG clearly resolves the three strongest (sharp white diagonal lines, plots d-e). In this case, the primary sequence alignment is relatively clear (plot c) and so little alignment clarity is gained by including structural information (plot f). ::: ![](1471-2105-5-166-14) ::: ::: {#F15 .fig} Figure 15 ::: {.caption} ###### Fold-alignment dotplot of nanos translational control elements (see Figure 13 for key). From a range of potential stems (faint red diagonal lines, plots a-b), the Pair SCFG resolves the three true stems sharply using the comparative signal (white diagonal lines, plots d-e). Some uncertainty in the primary sequence alignment (parallel blurred red lines, plot c) is resolved by including structural information, including a deletion in the outermost stem (broken diagonal line, plot f). ::: ![](1471-2105-5-166-15) ::: ::: {#F16 .fig} Figure 16 ::: {.caption} ###### Fold-alignment dotplot of U2 splicing factors (see Figure 13 for key). From a range of potential stems (faint red diagonal lines, plots a-b), the Pair SCFG resolves the true stems sharply using the comparative signal (white diagonal lines, plots d-e). The primary sequence alignment is highly uncertain (blurred red lines, plot c) but this uncertainty, including the deletion of a whole stem, is resolved by including structural information (broken diagonal line, plot f). ::: ![](1471-2105-5-166-16) ::: ::: {#F17 .fig} Figure 17 ::: {.caption} ###### Fold-alignment dotplot of nuclear RNase P genes (see Figure 13 for key). From a wide range of potential stems (faint red diagonal lines, plots a-b), the Pair SCFG resolves several sharply using the comparative signal (sharp white diagonal lines, plots d-e). Uncertainty in the primary sequence alignment (wide red lines, plot c) ?? is also resolved by including structural information (arrow white line, plot f). ::: ![](1471-2105-5-166-17) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Classes of degeneracy in the Structure Tree grammars. Permutations and combinations of these cycles are also possible. ::: Degeneracy Figure Nonterminal sequence Rule sequence Comment ------------------ -------- ------------------------ --------------- ---------------------------------------------- Null cycle 5 L→ S→ L* 2,3,5 6 L~1~→ \... → L~1~ 4,7,11 via S~1~L~1~ 5,32,30 via S~4~L~1~ L~2~→ \... → L~2~ 17,27,25 L~1~→ L~2~\... 6,27,25 \...L~2~→ L~1~ 15,11,7 via S~1~L~1~ 16,32,30 via S~4~L~1~ L~3~→ S~3~→ L~3~ 24,25,27 L~4~→ S~4~→ L~4~ 29,30,32 Loop bifurcation 5 L→ LL 2,5 6 L~1~→ L~1~L~1~ 4,11 L~2~→ L~1~L~1~ 15,11 L~3~→ L~3~L~3~ 24,27 L~4~→ L~4~L~4~ 29,32 Silent bulge 5 S→ L→ S 5,3,2 Cyclic permutation of L→ S→ L 6 S~2~→ L~1~→ S~1~ 22,4,7 S~1~→ L~1~→ S~1~ 11,4,7 Cyclic permutation of L~1~→ S~1~→ L~1~ S~3~→ L~3~→ S~3~ 27,24,25 Cyclic permutation of L~3~→ S~3~→ L~3~ S~4~→ L~4~→ S~4~ 32,29,30 Cyclic permutation of L~4~→ S~4~→ L~4~ :::	PubMed Central	2024-06-05T03:55:49.856509	2004-10-26	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534097/", "journal": "BMC Bioinformatics. 2004 Oct 26; 5:166", "authors": [ { "first": "Ian", "last": "Holmes" } ] }
PMC534098	Background ========== The discovery of novel transcription factor binding sites in regulatory sequences of genes has been an important scientific challenge for some years now. Computational approaches to this problem have come in two flavors. One class of methods looks for overrepresented motifs in sequences that are believed to contain several binding sites for the same factor (such as promoters of co-regulated genes) \[[@B1]-[@B6]\]. The second class of methods identifies motifs that are significantly conserved in orthologous sequences, e.g., promoters of the same gene in different species \[[@B7],[@B8]\]. These two approaches have been applied to their respective kinds of data sets, with moderate success. However, with new genomes being sequenced regularly, motif-finding applications today often present heterogeneoussequence data that includes promoters/enhancers of multiple co-regulated genes in one species, as well as their orthologs in other species. This paper presents a probabilistic algorithm, called \"PhyME\" (Phylogenetic Motif Elicitation), for ab-initiodetection of binding site motifs in such heterogeneous sequences. PhyME integrates two different axes of information in evaluating a candidate motif\'s significance. One axis is that of overrepresentation, which depends on the number of occurrences of the motif in each species. The other axis is the level of conservationof each motif instance across the species. A real motif that is not sufficiently significant along any one axis may turn out to be significant when both axes are considered simultaneously, leading to increased sensitivity and specificity of the integrated approach. Given the regulatory regions of potentially co-regulated genes along with their orthologs from other species, PhyME uses an Expectation-Maximization (E-M) algorithm to search for the motif that best explains the data. When evaluating a motif, its orthologous occurrences are assumed related to each other by a probabilistic model of evolution that takes into account the varying phylogenetic distances among the species. (The species may be related by any user-specified phylogenetic tree.) Each E-M iteration scales linearly with the total length of the input sequences and also with the number of species. The algorithm can also handle cases where the heterogeneous data is incomplete, i.e., where the orthologous regulatory regions are missing from some species. This capability makes it particularly suitable for applications that include data from incomplete genomes, or where orthology information is incomplete. An important feature of PhyME is that it allows motifs to occur in (evolutionarily) conserved as well as unconserved regions in orthologous promoters, treating the two kinds of occurrences differently when scoring a motif. It does not require each binding site occurrence in one promoter to have an orthologous occurrence in any or all other species. As a result, PhyME affords some flexibility in terms of the evolutionary distances spanned by the input sequences. For instance, using a distantly related ortholog will help pinpoint motifs located in conserved regions but will not hamper the discovery of motifs absent from that ortholog. Comparison with previous work ----------------------------- Traditionally, motif finding algorithms have treated input sequences as being independently generated, and searched for statistically overrepresented motifs in them. These algorithms \[[@B1]-[@B6]\] do not have the notion of sequence orthology built into them, and are therefore typically run on sequences from the same species. PhyME has an obvious advantage over them, since it takes motif conservation into account. (Henceforth, conservationof motifs will be assumed to mean conservation across species.) Another class of motif-finding methods take as input sets of orthologous sequences, either aligned \[[@B8]\] or unaligned \[[@B7]\] and search for well-conserved motifs. These methods however, unlike PhyME, do not exploit the other important aspect of a motif\'s significance -- that of overrepresentation. Some algorithms \[[@B9],[@B10]\] take as input a heterogeneous pool of co-regulated and orthologous promoters, and find overrepresented motifs after treating all sequences (including orthologous ones) as independent. However, this \"homogenizing\" strategy has its disadvantage, since it treats orthologous (and hence, directly related) motif occurrences as statistically independent observations. PhyME, on the other hand, respects the distinction between orthologous and co-regulated motif occurrences. There are algorithms that attempt to handle the two axes of information by a two-step approach. For instance, Cliften et al. \[[@B11]\] and Kellis et al. \[[@B12]\] find a set of highly conserved motifs (in yeast promoters) in the first step, and then extract overrepresented ones from this set, in a second step. The algorithm CompareProspector \[[@B13]\] takes a Gibbs-sampling approach to find overrepresented motifs but biases the search in regions conserved across species. Conversely, one may identify overrepresented motifs in the first step, and then isolate evolutionarily conserved ones among these \[[@B14]\]. In either case, a motif that is relatively weak by either criterion alone, but strong when considering both, may be missed out. PhyME\'s integrated approach to the heterogeneous data problem addresses this issue. Admittedly, the methods of Cliften et al. and Kellis et al. have a broader range of applications, since these are genome-wide searches for motifs. A recent algorithm called orthoMEME (Prakash et al. \[[@B15]\]) tackles the heterogeneous data problem by using Expectation-Maximization to search the space of motifs and the space of motif alignments (orthology) simultaneously. Each motif occurrence is assumed to have an orthologous copy in the other species, that could be located anywhere in the corresponding promoter. This is in contrast to PhyME\'s approach, where orthologous motif occurrences are restricted to pre-aligned regions of the promoters. This restriction comes with the advantage that PhyME scales better with the number of species than does orthoMEME. This is a significant advantage in practice, since the orthoMEME implementation is able to handle only two species data, whereas we have experimented with PhyME on orthologs from up to six species. Moreover, PhyME also allows non-conserved occurrences (those residing outside aligned regions), or occurrences that are conserved in some species and missing in others. Requiring that all motif occurrences come in orthologous sets may be justified for very closely related species, but for more diverged pairs of species (e.g., D. melanogasterand D. pseudoobscura) the promoters are known to have a mix of conserved and unconserved binding sites \[[@B16]\]. PhyME therefore gains an advantage by looking at both kinds of occurrences. However, orthoMEME\'s phylogenetic model is more powerful than that of PhyME and can handle a greater range of motif variation than PhyME can. Our approach is most similar to the algorithm PhyloGibbs (Siddharthan et al. \[[@B17]\]), the main differences being that PhyloGibbs (i) uses a Gibbs-sampling approach and (ii) assumes a star topology for the phylogeny, whereas PhyME uses an E-M approach and can handle arbitrary tree topologies. Thus PhyME has a broader domain of applicability in terms of the phylogenetic relationships among input sequences. It may therefore be preferable over PhyloGibbs when the phylogeny is far removed from a star, e.g., in a scenario where a pair of close species is included along with another pair of closely-related species, but the two pairs are greatly diverged from each other. On the other hand, an advantage of using PhyloGibbs is that multiple motifs (for different transcription factors) may be searched in parallel. The algorithm EMnEM (Moses et al. \[[@B18]\]) uses E-M and a phylogenetic model to find motifs, much like PhyME does, except that the former assumes that the input sequences are completely aligned. This assumption may be unsuitable for species at relatively large evolutionary distances, e.g., human and mouse, or D. melanogasterand D. pseudoobscura. Therefore, PhyME can handle a broader range of species divergence in its input. Another important difference between EMnEM and PhyME is the probabilistic model that each uses to model evolution. While EMnEM is implemented to use the Jukes-Cantor model \[[@B19]\], PhyME uses a more realistic model that incorporates binding site specificities. Thus, in calculating the joint likelihood of aligned motif occurrences, the EMnEM implementation does not use the fact that the effective mutation probability of an ancestral base to some base βdepends on the fitness of a binding site with βat that position. The evolutionary model used in PhyME reflects this dependence, and the incorporation of the model into an Expectation-Maximization framework is one of the main technical contributions of our work. The Results section includes a preliminary comparison of PhyME\'s performance with that of EMnEM, orthoMEME and PhyloGibbs, on real data. The algorithm PhyloCon (Wang and Stormo \[[@B20]\]) extends the greedy algorithm of CONSENSUS (Hertz et al. \[[@B2]\]) to incorporate multiple species data. However, it treats all orthologous sequences uniformly, ignoring the fact that different species may be at different relative distances from each other. As such, it may be more suitable to use PhyME in cases where the phylogeny is far removed from a star topology of uniform branch length. Also, the PhyloCon algorithm proceeds by first identifying several local multiple alignments in orthologous sequences and then searching for common patterns (motifs) among these multiple alignments. As a result, it may miss motif occurrences that are not well-conserved (or are completely missing) in orthologous sequences. An advantage of PhyloCon is that it does not require the motif length to be input, and instead reports motifs of varying lengths. Results ======= In this section, we first present the new algorithm, and then describe its evaluation on synthetic data, as well as biological data sets from various organisms. Algorithm --------- Suppose that the input includes ndifferent promoters (e.g., from co-regulated genes), and for each promoter there are sequences for Kspecies. (Kmay be different for different promoters.) PhyME requires that there be one designated \"reference species\" σ~r~in the input, for which there is sequence data corresponding to each of the npromoters. We shall describe PhyME\'s algorithm for the special case n= 1, though allowing multiple motif instances in this one sequence. The extension to n\> 1 is trivial, and omitted here for simplicity. Thus, the input consists of a set of sequences S= {S~1~, S~2~,\..., S~K~}, where S~i~is the orthologous sequence from species i, and one of the S~i~\'s comes from species σ~r~. The input also includes the motif length l, and the phylogenetic tree Ψ over the Kspecies, with neutral point mutation rates (probabilities) along each branch. The output is a position weight matrix (PWM) representing the discovered motif, and its score. PhyME first partially aligns the input sequences and identifies contiguous regions (\"blocks\") in each ![](1471-2105-5-170-i1.gif) that are highly conserved in ![](1471-2105-5-170-i2.gif). It then inputs all the sequences, along with the locations of the conserved blocks, to the core motif-finding algorithm. ### Alignment of sequences In this pre-processing step, PhyME computes the regions of high local similarity between ![](1471-2105-5-170-i2.gif) and each of the other S~i~. The assumption is that such regions are of common evolutionary origin, and any sequence outside them is independently evolved. PhyME runs the LAGAN alignment program of Brudno et al. \[[@B21]\] on each sequence pair (![](1471-2105-5-170-i2.gif), S~i~\\|i≠ σ~r~), and extracts all ungapped aligned blocks of a certain minimum size (of the order of the motif\'s length) and percent identity, to serve as the blocks of common origin. This is illustrated in Figure [1a](#F1){ref-type="fig"}, which shows orthologous promoters from three species, σ~1~(the reference species), σ~2~and σ~3~. An example of a block is region BC in σ~1~, aligned with region UV in σ~3~. Note how blocks can overlap in the reference species (BC overlaps with KL). The input is now reorganized into two kinds of sequences: 1\. The sequence from the reference species, with aligned blocks of the other species \"hanging off\" it. (In Figure [1b](#F1){ref-type="fig"}, this is shown as the sequence AJ, with blocks MN, OP, QR of σ~2~and blocks UV, WX of σ~3~aligned with corresponding blocks in σ~1~.) Thus, any position in this sequence either has a single base from the reference species, or has an alignment of bases from multiple species, one of which is the reference species. This entire construct is called the \"reference sequence\". 2\. Any subsequence not from the reference species, and bracketed by blocks on both sides. (e.g., regions NO, PQ, VW in Figure [1b](#F1){ref-type="fig"}.) The terminal sequences in the non-reference species, which are to the left of the left-most block and to the right of the right-most block, may be optionally included, as per the user\'s specification. PhyME fits the parameters of a probabilistic model on the reference and bracketed sequences simultaneously, and the desired motif comes out as a by-product of this training procedure, which is described next. ### Hidden Markov Model The probabilistic process that is assumed to generate sequences is described by a very simple Hidden Markov Model(HMM). For the moment, let us assume that the sequence Sbeing generated is entirely from one species, with no aligned positions. The HMM parameters include a \"motif weight matrix\" W~m~of length l, and a \"background weight matrix\" W~b~of length 1. (The (k, j)^th^entry of a weight matrix is the probability of emitting the base jat position kof the sequence being sampled from the matrix.) At each step, the generative process of the HMM chooses either W~m~or W~b~according to their transition probabilitiesp~m~= pand p~b~= 1 - prespectively, where pis a model parameter. A sequence is then sampled from the chosen weight matrix, and appended at the end of the sequence Screated so far. The process then proceeds to the next step. It stops when the length of Sreaches its known length L. The series of motifs chosen in the successive steps of the process is called a \"parse\" of the sequence. The model parameters θ, which include W~m~, W~b~, and p, associate a well-defined probability Pr(S, T\\|θ) with each parse Tof the sequence S. The probability that Swas generated by an HMM with parameters θis then given by Pr(S\\|θ) = Σ~T~Pr(S, T\\|θ). Let Pr(S\\|θ~b~) be the probability of generating Sby using only W~b~. For a given θ, we define ![](1471-2105-5-170-i3.gif) This log-likelihood ratio is the function optimized by PhyME -- the parameters W~m~and pare trained so as to maximize F(S, θ). (The background weight matrix W~b~is not trained during this maximization, rather it is pre-computed from S, or optionally from specified background sequence, by measuring nucleotide frequencies.) The value of the objective function for a set of independent sequences is the sum of its values for the sequences taken separately. This additive property allows easy extension of the parameter training procedure to the general case of multiple sequences (n\> 1). The objective function maximized then is ![](1471-2105-5-170-i4.gif), the set Snow including, for each of the ninput promoters, the \"reference sequence\" as well as all \"bracketed sequences\" (defined earlier) as separate elements. An important aspect of computing F(S, θ) is the subsequence probabilityPr(s\\|W). This is the probability of generating a subsequence sof length l, (length of W), when sampling from weight matrix W; so ![](1471-2105-5-170-i5.gif), where s= s~1~s~2~\... s~l~, and W~kj~is the probability of sampling base jat the k^th^position of W. This formula applies when subsequence shas a single base at each position. However, we need to adapt this formula to the case where one or more positions in subsequence smay be an alignment of orthologous bases from multiple species. In this general case, we can write sas ψ~1~ψ~2~\... ψ~l~, where each ψ~k~is either a single base, or an alignment of orthologous bases at a single position of the reference sequence. The subsequence probability Pr(s\\|W) can then be computed as ![](1471-2105-5-170-i6.gif), where Pr~e~(ψ\\|W, k) denotes the probability of observing ψat position kwhen sampling from W. Let the vector ψ= (s~1~, s~2~, \... s~K~), where s~σ~is the nucleotide from species σin the single-base alignment ψ. If the s~σ~were independent, we could write ![](1471-2105-5-170-i7.gif). However, the s~σ~\'s occur in an alignment (ψ), meaning that this assumption of independence is obviously untenable. Thus we need an expression for Pr~e~(ψ\\|W, k) that explicitly takes the phylogenetic relationships among the species (given by the phylogenetic tree Ψ) into account. We present such an expression in the next section, and we shall thereafter return to the topic of maximizing the function F(S, θ). ### Evolutionary model This section describes the probabilistic evolutionary model that PhyME uses to incorporate phylogenetic relationships in the computation of the term Pr~e~(ψ\\|W, k) mentioned above. It was first proposed in Sinha et al. \[[@B22]\] to model binding site evolution, and applied successfully on the two fly genomes. The model makes the crucial assumption that all positions in a binding site evolve independently, at equal rates, and the probability of fixation of a mutation α→ βat position kis proportional to the weight matrix entry of βat that position. If we further assume, for simplicity of exposition, that the phylogenetic tree Ψ has a star topology, then the model assumptions give us (from Sinha et al. \[[@B22]\]; also see Methods.) ![](1471-2105-5-170-i8.gif) where s~σ~is the nucleotide from species σin alignment ψ, δ~xy~= 1 if x= yand 0 otherwise, and μ~σ~is the neutral mutation probability between the ancestor and the species σ. For the position k, one \"creates\" a base αin the ancestor with frequency W~kσ~, and each such base is either passed unchanged to the species σ(probability 1 - μ~σ~) or mutated in species σwith probability μ~σ~and a new base selected with a frequency defined again by W. In the general case, when Ψ does not have a star topology, Formula (1) can be written in a recursive manner. (See Methods.) ### Expectation Maximization The function F(S, θ) that is maximized by PhyME measures how much more likely it is that Swas generated using the motif weight matrix, than without it. Naturally, a PWM that maximizes this score is the motif that best explains the data. PhyME tries to find such a motif by training the parameters (W~m~, p) of the HMM, using the Baum-Welch algorithm \[[@B23]\], which iteratively converges to a locally optimum θusing Expectation Maximization (E-M). Let ![](1471-2105-5-170-i9.gif), for i∈ {m, b} be the expected number of times the HMM plants motif W~i~in generating the sequence(s), the expectation being over all parses. Similarly, let ![](1471-2105-5-170-i10.gif) be the expected number of times that the nucleotide alignment ψis sampled at the k^th^position of the motif W~m~. ![](1471-2105-5-170-i9.gif) and ![](1471-2105-5-170-i10.gif) are expected values of hidden variablesof the HMM. These averages are computed during the \"E-step\" in each iteration, using dynamic programming (the Forward-Backward algorithm, \[[@B23]\]). In the \"M-step\", two kinds of updates are made, using the values of ![](1471-2105-5-170-i9.gif), ![](1471-2105-5-170-i10.gif) computed in the E-step. The parameter pis updated according to ![](1471-2105-5-170-i11.gif). The motif weight matrix W~m~is updated by solving, for each column kof the matrix, the following set of five simultaneous equations, in variables u~β~(β∈ Σ) and λ. ![](1471-2105-5-170-i12.gif) ![](1471-2105-5-170-i13.gif) The derivation of the update formulas is somewhat involved, and is described in the Methods section. The equations are solved using Newton\'s method, and the solution value of u~β~is used to update the (k, β)^th^entry of W~m~, according to W~mkβ~= e^uβ^. Newton\'s method involves computation of the first and second partial derivatives of log Pr~e~(ψ\\|W~m,\ k~), as described in Methods. In practice, we found that Newton\'s method always converges from a single initial condition, and the convergence almost always happens within 3--5 iterations. The time complexity of (each E-M iteration in) PhyME is O(LKl), where Lis the length of the sequences, Kis the number of species, and lis the length of the motif desired. (See Methods for details.) Results on synthetic data ------------------------- We first present the results of running PhyME on synthetic data. The experimental framework is largely borrowed from Wang and Stormo \[[@B20]\]. In each experiment, 5 \"ancestral\" sequences, each of length 600 bp, are created at random, and 20 \"binding sites\", each of length 8, are \"planted\" at randomly chosen locations in these sequences. The sites are chosen such that the weight matrix formed by them has a relative entropy of R. Each ancestral sequence is then \"evolved\" by point mutations to create Kadditional \"orthologous\" copies, assuming a star topology (with Kleaves) and a common \"background mutation rate\" μ~b~along each branch. (No insertions or deletions were included in this simulated evolution, for simplicity.) The motif instances are subjected to a common \"motif mutation rate\" μ~m~, which is the probability of mutation of any position in a motif. The ancestral set of sequences is then removed and the remaining Korthologous sets are input to the motif discovery algorithm, with one arbitrarily designated the reference species. The algorithm is made to report 3 different motifs, thereby making some allowance for false positives, especially when Ris low. For each reported motif, its 20 best occurrences in the reference species are compared with the planted occurrences, to give a score ranging between 0 and 1. (1 represents the best possible performance; see the Methods section for details.) The score for the best of the 3 reported motifs is the \"performance score\" of the algorithm. The three algorithms being compared are PhyME, MEME \[[@B1]\], and GIBBS (Wadsworth Gibbs sampler) \[[@B24]\]. PhyME was run with an evolutionary tree with a star topology, the mutation rate along each branch being μ~b~. MEME and GIBBS were run on the entire data set pooled together, ignoring the orthology of sequences. Figure [2](#F2){ref-type="fig"} shows the effect of varying Kon performance scores of the algorithms. Note that the performance of PhyME, while similar to MEME and GIBBS for K= 1, improves relative to them as Kincreases. The absolute performance score of GIBBS (and of MEME, to some extent) deteriorates with increasing K. With more orthologous sequences, conserved stretches of background sequence may distract the algorithm from the motif occurrences. PhyME, with the additional knowledge of orthology, is able to pick out the motif better. Figure [3](#F3){ref-type="fig"} shows the effect of varying the mutation rates. The background mutation rate μ~b~was varied from 0.2 to 0.5 and the motif mutation rate μ~m~was varied between 0.1 and μ~b~- 0.1). As per expectation, the performance of each algorithm improved with decreasing μ~m~(for a fixed μ~b~). Interestingly, as μ~b~decreases, the performance of PhyME for μ~m~= 0.1 first improves and then falls down. The initial improvement is because the alignment step is able to find more conserved blocks with diminishing background mutation rate. However, when the latter approaches the motif mutation rate, the distinction (in cross-species conservation) between motif and background becomes weaker, hence performance goes down. In another set of experiments, we examined the effect of the alignment step on the performance. Sequences were created with K= 2, μ~b~= 0.3, μ~m~= 0.1 and R= 12. After the alignment step of PhyME (in which the entire sequence was aligned as one conserved block), we artificially \"unpaired\" some number nof the planted orthologous motif occurrences, i.e., the alignment was modified so that these npairs fell outside conserved blocks. This was followed by the usual motif-finding step, and the entire procedure was repeated for various values of n. We find a gradual degradation in performance as PhyME moves from maximum utilization of motif orthology (n= 0, no unpaired motifs) to minimum (n= 20, no motif pairs considered orthologous). (Figure [4](#F4){ref-type="fig"}.) We also evaluated the effect of mis-estimates of the neutral mutation rates on performance. PhyME was run on random sequences created with experiment parameters K= 3, μ~b~= 0.3, μ~m~= 0.1 and R∈ {11, 12, 13}, and in different runs, the value of μ~b~input to PhyME ranged from 0.1 (underestimate) to 0.5 (overestimate). We observed that underestimates of μ~b~resulted in significantly greater performance degradation than overestimates of equal magnitude. (Data not shown.) For instance, using μ~b~= 0.4 instead of the true value of 0.3 made no difference to the performance, whereas using μ~b~= 0.2 resulted in 15 -- 50% decline. Results on biological data -------------------------- In the following sections, we present results of running PhyME on real data sets from yeast, fly and human. The results are compared to MEME (run by pooling orthologous sequences together), orthoMEME \[[@B15]\], PhyloGibbs \[[@B17]\], and EMnEM \[[@B18]\]. The latter three programs address the heterogeneous data problem directly, just as PhyME does. Another program that solves the same problem is PhyloCon \[[@B20]\]. PhyloCon was not evaluated in our study because we did not have a clear method to post-process its output to extract a specified number of top-scoring motifs that are non-redundant. (Our evaluations described below use the top three motifs reported by each program.) ### Yeast data sets We first present some examples in yeast, where sequence data from four species, S. cerevisiae, S. mikatae, S. kudriavzeviiand S. bayanuswas used. We performed motif-finding (with PhyME, MEME, orthoMEME, PhyloGibbs and EMnEM) on some regulons from the SCPD \[[@B25]\] database, which catalogues sets of co-regulated genes. For each regulon, the top ηmotif occurrences in S. cerevisiaereported by the algorithm (ηbeing the number of known sites in S. cerevisiae) were examined for \"matches\" to the known weight matrix for that regulon\'s motif. (See Methods for details.) The number of matches was the performance score of the algorithm. We counted matches to the weight matrix, rather than to known sites, so that a reported motif occurrence that is very similar to the known motif (but not annotated as a site by SCPD) will not be counted as a false positive. Each algorithm reported 3 motifs (with ηoccurrences for each motif), and the results are for the best scoring motif, thereby making some allowance for false positives, such as simple repeats. Even though PhyME is implemented to handle arbitrary phylogenies, for efficiency it was run with a phylogenetic tree with a star topology, having S. cerevisiaeat the center and the mutation rate along the branches of S. mikatae, S. kudriavzevii, and S. bayanusbeing (0.25, 0.3, 0.35) respectively. These values are based on average substitution rate per base in the corresponding pairs of species. (A more accurate tree can be derived from the work of Kellis et al. \[[@B12]\].) For multiple species data, MEME was run by pooling all sequences together. OrthoMEME was run only for the case K= 2 (i.e., on sequence from the two species S. cerevisiaeand S. mikatae), since its current implementation can only handle two species data. The other four programs were run for K= 1, 2, 3, 4, in separate executions. See Methods for details on how orthoMEME, PhyloGibbs and EMnEM were run. Figure [5](#F5){ref-type="fig"} plots the performance scores for regulons RAP1, MIG1, CAR1, PHO4 and MCM1, which show interesting results. Note how the performance of PhyME improves with Kfor RAP1 and MIG1. For CAR1, both PhyME and MEME improve from K= 1 to K= 2, and then deteriorate for higher K, but PhyME at K= 3 is still better than at K= 1. For PHO4, PhyME\'s performance first goes up (for K= 2) and then dips below the K= 1 level, whereas MEME shows best performance at K= 4. For MCM1, PhyME scores well (over 90% matches) for K∈ {1,3,4}, whereas MEME\'s performance degrades for K\> 1. Thus, these examples illustrate that PhyME\'s approach can lead to improved motif discovery in multiple species data, and also that there may be situations when more orthologous sequences distract either algorithm from the true motif. For regulons CSRE, GCN4, MATα2, URS1H, REB1, and PDR3, the performance score was high (typically over 80% matches) and largely invariant of the number of species. Figure [5](#F5){ref-type="fig"} also reports the scores of PhyloGibbs. This program has similar scores as PhyME on CAR1, PHO4 and MCM1. (It did not execute to completion for K= 3, 4 in MCM1.) PhyME has better scores on MIG1 and RAP1, though PhyloGibbs\' scores on RAP1 with a different choice of parameters (\"-G 0.7\", see Methods) were similar to PhyME. (Data not shown.) We also report the scores of EMnEM in Figure [5](#F5){ref-type="fig"}. (The program did not execute to completion in CAR1.) This program performs well for K= 2 (comparable to the best scores in the data sets RAP1, PHO4 and MCM1). For K= 3, 4 also, EMnEM scores are comparable to PhyloGibbs. PhyME typically performs better than EMnEM (with K= 3, 4) for RAP1, MIG1, MCM1, and comparably for PHO4. We find the scores of orthoMEME, as reported in Figure [5](#F5){ref-type="fig"}, to be lower than those of PhyME (for K= 2). However, we observed that in all five regulons reported, orthoMEME reported fewer than ηoccurrences in S. cerevisiaeper motif. This is because orthoMEME was run in the \"zoops\" mode (zero or one motif occurrence per sequence), since the \"tcm\" mode (any number of occurrences per promoter) does not perform well. Thus, with the total number of predictions being fewer than η, orthoMEME\'s scores are expected to be lower than other programs even for the same level of specificity. We suggest caution in comparing PhyME\'s scores directly with those of the other programs, since we lacked expertise in choosing optimal parameter settings for them. This is particularly true for EMnEM, which has several parameters for modeling the evolution of motifs, and we lacked experience in setting these parameters optimally. We clearly have more expertise at using PhyME than the other programs, and this makes the comparisons biased. Our goal in these experiments was to provide some examples of how multiple species data can be exploited to improve performance, rather than assessing the different motif finding programs available. A proper comparative assessment of these programs has to address several challenges not addressed here. Such a task was undertaken for several motif finding programs, in the work of Tompa et al. (unpublished). A similar assessment of the motif-finding programs in the context of the heterogeneous data problem is an important topic for future work. ### Fly data sets Next, we present results from fruitfly, where data from two species, D. melanogasterand D. pseudoobscura, is available. Nine different enhancers were chosen -- enhancers eve1, eve2, eve5, ftz3\', gtposterior, hairy2, hairy34, and run1have binding sites for the Krtranscription factor, and btdheadhas Bcdsites. Well-defined weight matrices are available for both Krand Bcd\[[@B26]\]. For each enhancer, the top ηmotif occurrences (in D. melanogaster) reported by the algorithm (ηbeing the number of \"strong\" occurrences of the known weight matrix in D. melanogaster-- see Methods) were examined, and the number of matches was the performance score of the algorithm. Six different motif-finding strategies were tested separately -- (i) MEME\_1 (MEME on single species) (ii) MEME\_2 (MEME on both species pooled together), (iii) PhyloGibbs, (iv) orthoMEME, (v) EMnEM and (vi) PHYME (PhyME on both species, with μ= 0.5). Each strategy was required to report occurrences only in D. melanogaster. (See Methods for details of how orthoMEME, PhyloGibbs and EMnEM were run.) Figure [6](#F6){ref-type="fig"} compares the scores of the different strategies for all nine enhancers. Note that either PHYME or MEME\_2 performs better than MEME\_1 for seven of the nine enhancers, and worse only for one (ftz3\'), thereby making the case for using two species data. Moreover, on btdhead, gtposteriorand hairy2, PhyME performs significantly better than MEME\_2, demonstrating the advantage of using orthology information. Similarly, we find PhyME to perform better than PhyloGibbs on gtposterior, hairy2and hairy34. EMnEM performs well in these data sets, scoring comparably to PhyME or PhyloGibbs, except on btdhead, eve2, and gtposterior, where both PhyME and PhyloGibbs perform better, and hairy2, where PhyME alone performs better. OrthoMEME was run in the \"tcm\" mode, since the \"zoops\" mode is not appropriate here, with several putative sites to be found in each promoter. However, orthoMEME tends not to perform well in the \"tcm\" mode in general, and in our tests also, its scores were poor on most of the enhancers. We thus find that PhyME is preferable to orthoMEME for cases where we expect several motif occurrences per sequence. As in the yeast data sets, the comparison of scores between PhyME and the other programs should be interpreted with caution, since we lacked expertise in choosing optimal parameter settings for the other programs. ### Human data sets Finally, we present results of running PhyME on two data sets from human, where orthology with mouse and rat was utilized. These data sets were chosen because all of 15 different motif-finding programs tested in an assessment project (Tompa et al., unpublished) failed to report the correct motif in them. The first set corresponds to the transcription factor SP1, a zinc-finger protein. The heterogeneous sequence data included 35 human promoters (of length 2 Kbp each), of which four have orthologous promoters from mouse and rat, 20 from mouse only, 4 from rat only, and 7 have no orthologs. Each of the human promoters is known to have at least one functional Sp1 binding site, with a total of 76 known sites overall. Figure [7a](#F7){ref-type="fig"} shows a \"sequence logo\" representation \[[@B27]\] of an alignment of these known sites. PhyME was run to find motifs of length 7, using the phylogenetic tree shown in Figure [7c](#F7){ref-type="fig"}. The second motif reported by PhyME (Figure [7b](#F7){ref-type="fig"}) is almost identical to the known SP1 weight matrix. The top 27 instances (in human promoters) of this motif included 16 that overlapped with known binding sites. We also ran MEME on the heterogeneous data set (pooling orthologous sequences together), and the second motif reported was a good match to SP1. However, of its 41 instances reported in human promoters, only 9 were overlapping with known sites. Moreover, when MEME was run on human promoters alone, none of the top three motifs matched the SP1 motif. Thus, PhyME showed a clear performance improvement over MEME, both in the single species run, and when the orthologous sequences were pooled together. The second data set used in our tests corresponds to the leucine zipper transcription factor c-Jun. The heterogeneous data set included 500 bp promoters for 11 human genes targeted by c-Jun, as well as orthologs from mouse and rat for 3 genes, from mouse only for 5 genes, and from rat only for the remaining three genes. PhyME was run exactly as in the previous data set. The known binding sites of c-Jun (in human) were aligned to produce a weight matrix that is shown in Figure [8a](#F8){ref-type="fig"}. The third ranked motif reported by PhyME is shown in Figure [8b](#F8){ref-type="fig"}, and we can see that its last five positions are similar to the first five positions of the known weight matrix. Of the top 13 instances of the discovered motif, 4 overlap with known binding sites of c-Jun, whereas a maximum of 9 could have been obtained. (Of the 11 known sites, 9 are in the 500 bp upstream regions used in our analysis.) We also ran MEME on the heterogeneous data set (using the pooling strategy), and none of the three best motifs reported by MEME matched the c-Jun motif. Thus, both the human data sets tested demonstrate how PhyME can improve motif discovery in typical motif finding scenarios by exploiting heterogeneous sequence data properly. Discussion ========== Issues in algorithm design -------------------------- ### Alignment step In the alignment step, PhyME extracts blocks of high sequence similarity between the reference species and each of the other species. Motif occurrences in such locally conserved regions are deemed orthologous, an assumption well-justified by traditional interpretations of sequence alignment. Conversely, all orthologous motif occurrences are assumed to be aligned in such blocks. This assumption is not always true since there may be orthologous motif occurrences not aligned by the alignment program, but it heavily constrains the space of orthologous motif occurrences, implying greater efficiency of the search algorithm. Moreover, the assumption does not mean that \"true\" orthologous occurrences in unaligned regions are ignored -- they are merely treated as independent occurrences. Our experiments on synthetic data (see Results) demonstrate that the performance is not very sensitive to the correct alignment of all orthologous motif pairs. The blocks computed in the alignment step have to be with respect to the reference species, but the alignment itself need not be done in a pairwise manner. A multiple alignment of all sequences may be computed (e.g., with M-LAGAN \[[@B21]\], using the input phylogenetic tree Ψ) and blocks between ![](1471-2105-5-170-i2.gif) and each of the other S~i~may then be extracted. (The alignment step is implemented as a separate tool in PhyME, making it easy to switch to such alternative schemes.) Furthermore, the implementation may be modified in the future to drop the requirement of a reference species, since this requirement is not crucial to the motif finding step of PhyME. For instance, the alignment step may utilize the \"Threaded Block Alignment\" (TBA) program of Blanchette et al. \[[@B28]\], which completely circumvents the notion of a reference species in multiple alignments. Once the blocks of high sequence conservation have been identified, a possible strategy is to restrict attention to motif occurrences in these blocks, assuming that all functional binding sites must be evolutionarily conserved. However, this assumption is not true even for as closely related species as D. melanogasterand D. pseudoobscura, separated by about 25--30 Myrs. An empirical study \[[@B16]\] on these two species revealed that a good fraction (35--40%) of occurrences of relevant motifs occur outside of locally conserved contexts, and should therefore be taken into account when discovering motifs. ### Motif Finding In the probabilistic process that is assumed to generate sequences, the transition probability does not depend on the previous choice(s) made during the process, meaning that the HMM is of zeroth order, nor on the position in the sequence, meaning that any information about spatial distribution of motifs is ignored. The model, unlike that of MEME, does not fragment the sequence into all l-length words to be treated independently. Rather, it parses the sequence into a series of non-overlapping occurrences of the motif and background. The evolutionary model described by Formula 1 applies only to phylogenies having a star topology. The general case of arbitrary tree topology is described in Methods. In Formula 1, if μ~σ~is small (as for very closely related species), then finding different bases in orthologous positions has low probability Pr~e~(ψ\\|W, k), even if their frequency in Wis the same. This mirrors the intuition that mutations in locally conserved regions of closely related species are evidence against a binding site residing there. For largely diverged species (i.e., if μ~σ~\~1, ∀ σ), Pr~e~(ψ\\|W, k) reduces to the product of the individual bases\' probabilities. It is worth emphasizing here that the weight matrix Wbeing searched by the algorithm is assumed to be unchanged over the entire phylogenetic tree (including the ancestor). The neutral mutation rates (probabilities) along each branch of Ψ are input by the user and not trained during E-M. Training them on input data may cause overfitting, producing values that are largely inconsistent with the known evolutionary distances. The work of Moses et al. \[[@B18]\] studies this issue, and finds that it is more important to use correct phylogenetic relationships, e.g., an appropriate evolutionary tree, than to use accurate mutation rates. Note that the evolutionary model used by PhyME comes into play only in Equations 2 as the term Pr~e~(ψ\\|W~m~, k). Other models of evolution, e.g., F81 \[[@B29]\], can be incorporated into PhyME by simply using the appropriate formulation of this term, as long as the derivatives of log Pr~e~(ψ\\|W~m~, k) can be computed efficiently. Conclusions =========== We have developed a new algorithm, PhyME, that detects motifs in heterogeneous sequence data by integrating two important aspects of a motif\'s significance -- overrepresentation and cross-species comparison -- into one probabilistic score. We have evaluated different aspects of the algorithm on synthetic data, and demonstrated on some biological data sets that the new approach improves motif detection. Methods ======= The evolutionary model ---------------------- The evolutionary model makes the following assumptions: (i) Nucleotides in an aligned position are evolved from a common ancestor. (ii) The weight matrix applies to the common ancestor and to all descendants, a reasonable assumption given the propensity of DNA binding domains of proteins to evolve slower than cis-regulatory modules. (iii) All positions evolve independently, at equal rates, and the probability of fixation of a mutation α→ βat position kis proportional to the weight matrix entry of βat that position. Suppose we are given a phylogenetic tree Ψ, with the species {σ~1~, σ~2~, \.... σ~K~} at the leaves. Let the vector ψ= (s~1~, s~2~, \... s~K~), where s~σ~is the nucleotide from species σin the (single position) alignment ψ. The term Pr~e~(ψ\\|W, k) denotes the probability of observing ψat position kwhen sampling from weight matrix W. For each node jof the tree, except the root, let μ~j~be the probability of a base in the parent species of jhaving mutated (under neutral evolution) in species j. Also, let ψ~j~be the vector formed by elements of ψthat correspond to leaf nodes descended from node j. Let C(j) denote the set of children of node jand let rbe the root of the tree. Then, we can write (using the model assumptions): ![](1471-2105-5-170-i14.gif) where f(ψ~j~, α) denotes the probability of observing ψ~j~given that the base at the parent of jis α. For a leaf node σ, this can be written as ![](1471-2105-5-170-i15.gif), from the model assumptions. (δ~ij~= 1 if i= j, and 0 otherwise.) For an internal node j(except root r), the expression is : ![](1471-2105-5-170-i16.gif) For the special case where Ψ has a star topology, Equation 4 reduces to Equation 1. Training parameters in a HMM ---------------------------- Given a sequence Sand a set of position weight matrices {W~i~}, the objective function to be maximized is F(S, θ) = log(Pr(S\\|θ)/Pr(S\\|θ~b~)), where Pr(S\\|θ) is the probability of generating the sequence Susing the parameters θ, and θ~b~represents the parameter values that only allow the background motif W~b~to be used by the HMM. The sequence Scan be written as ψ~1~ψ~2~\... ψ~L~, where each ψ~i~is either a single base or an alignment of orthologous bases at a single position. θincludes the weight matrices W~i~and their transition probabilities p~i~. Since Pr(S\\|θ~b~) depends only on W~b~, which is assumed constant, we shall outline how to maximize log Pr(S\\|θ), following the description in \[[@B23]\]. A parse of the sequence Sin terms of the W~i~(i.e., the series of motifs chosen in the successive steps of the generative probabilistic process) is denoted by T. We thus have ![](1471-2105-5-170-i17.gif) The maximization is iterative, with the t^th^iteration computing a model θ^t+\ 1^that improves the objective function from the current model θ^t^. In classical E-M fashion, let us define a function Q(θ\\|θ^t^) as ![](1471-2105-5-170-i18.gif) It is easily shown that log Pr(S\\|θ) - log Pr(S\\|θ^t^) ≥ Q(θ\\|θ^t^) - Q(θ^t^\\|θ^t^). Thus, if we maximize Q(θ\\|θ^t^) over all θ, we shall always improve upon log Pr(S\\|θ^t^), or remain there if the local maximum has been reached. Let A~i~(T, S) be the number of times W~i~occurs in the parse Tof S. Also, let E~ikψ~(T, S) denote the number of times that the alignment ψis emitted (sampled) at the k^th^position of the matrix W~i~, in parse Tof S. Let l~i~denote the length of W~i~. Then we have ![](1471-2105-5-170-i19.gif) which gives us ![](1471-2105-5-170-i20.gif) Note that the only the first term in this expression depends on p~i~, and only the second term depends on W~i~. Hence, we maximize each of these terms independently, with respect to the appropriate free parameters. We first maximize the term ![](1471-2105-5-170-i21.gif) Note that ![](1471-2105-5-170-i22.gif) is the average number of occurrences of W~i~in Sover all parses T. Thus the term to maximize is ![](1471-2105-5-170-i23.gif), and this is maximized when ![](1471-2105-5-170-i24.gif) Next, we maximize the second term: ![](1471-2105-5-170-i25.gif) Again, note that ![](1471-2105-5-170-i26.gif) is the average number of times that the alignment ψis sampled at the k^th^position of the matrix W~i~while generating S, the average being over all parses T. Thus, the term to maximize is ![](1471-2105-5-170-i27.gif). We first note that in our case, there is a single weight matrix W~m~to be trained. Hence, we need to maximize Qwith respect to W~m~. We can do this maximization with respect to each column kindependently. Let W~kβ~denote the (k, β)^th^entry of W~m~. Thus, for each k= 1 \... l, we need to maximize Qwith respect to W~kβ~(β∈ Σ), with the constraint Σ~β~W~kβ~= 1. Using Lagrangian multiplier λ, the objective function becomes Q+ λ(Σ~β~W~kβ~- 1). Transforming to log variables u~β~= log W~kβ~to ensure that the W~kβ~remain positive during optimization, we then have the following necessary conditions for optimality (in addition to the constraint ![](1471-2105-5-170-i28.gif)) : ![](1471-2105-5-170-i29.gif) We therefore have a system of five equations (including the constraint) in the variables u~β~(∀β∈ Σ and λ. Denoting the vector of these five variables by u, we solve this system of equations using Newton\'s iterative method. Let us write the above system of equations as F(u) = 0, where F(u) = \[\[f~β~\], f~λ~\], with f~β~being the left side of Equation 9, and ![](1471-2105-5-170-i30.gif). Newton\'s method uses the update relation: Δu= -(J(u))^-1^F(u) where Δuis the change in uin the current iteration and Jis the Jacobian matrix of F. The important terms in the computation of Fand Jare the first and second partial derivatives of log Pr~e~(ψ\\|W~m,\ k~) with respect to the u~β~variables. For this purpose, we need to compute Pr~e~(ψ\\|W~m,\ k~) and its first and second partial derivatives. Computation of Pr~e~(ψ\\|W~m,\ k~) uses the formulas 4 and 5. The partial derivatives can be computed recursively (over the tree Ψ) by using the chain rule of differentiation. These recursive computations are implemented in a bottom-up manner, so as to avoid redundant computations. Newton\'s method uses Fand Jto iteratively compute new values of u, until convergence. The Jacobian matrix Jin our case is not positive definite, hence Newton\'s method is not guaranteed to converge. However, in practice, we found the method to always converge from a single initial seed. Upon convergence, the log variables u~β~are transformed back to W~kβ~= e^uβ^. The procedure is repeated for each k= 1 \... l, and W~m~is then updated with the new values. This update, along with that given by Equation 8, is used iteratively to improve F(S\\|θ) until the local maximum is reached, as indicated by a very small change in its value. Time complexity --------------- The E-step computes ![](1471-2105-5-170-i31.gif), ![](1471-2105-5-170-i32.gif) and ![](1471-2105-5-170-i33.gif), for k= 1 ... l, ∀ ψ. The Forward-Backward algorithm is run once, in O(LKl) time, where Lis the total length of the input sequences, Kis the number of species, and lis the length of the motif W~m~. (This time complexity assumes that nodes in the phylogenetic tree have a fixed maximum degree.) Thereafter, ![](1471-2105-5-170-i31.gif), ![](1471-2105-5-170-i32.gif) are computed in O(L) time, and all the ![](1471-2105-5-170-i33.gif) are computed in one scan of the input, expending O(Ll) time. The M-step runs Newton\'s method to solve a system of equations, once for each column of W~m~. Each run of Newton\'s method goes through a small number (3--5) of iterations. Each iteration computes the first and second partial derivatives of log Pr~e~(ψ\\|W~m,\ k~) Each of these derivatives can be computed in O(K) time, where Kis the number of species (since \\|ψ\\| ≤ K) Hence, Fand Jcan be computed in O(LK) time, where Lis the total length of the sequence. Hence, Newton\'s method takes O(LK) time, and is run ltimes, for an overall time complexity of O(LKl) for the M-step. Thus, the running time of (each E-M iteration in) PhyME scales linearly with the length of the sequences, the length of the motif desired, and the number of species. Implementation details ---------------------- PhyME is implemented in C++ for Linux, and the source code will be made freely available at <http://edsc.rockefeller.edu/cgi-bin/phyme/download.pl>. The current implementation runs in a few minutes (on a workstation) for typical applications with total sequence length \~10000 bp, 2--4 species, and motif length of \~10. PhyME uses the LAGAN alignment tool of Brudno et al. \[[@B21]\] for the alignment step. After alignment, the ungapped blocks extracted are required to be at least 10 bp long, and have at least 70% identity. PhyME is implemented to handle an arbitrary phylogenetic tree Ψ relating the input species. The E-M algorithm is guaranteed to converge only to a local optimum. To address this problem, the motif-finding step is executed a fixed number of times, each time using a randomly chosen substring of the input sequence as the \"seed\" to initialize W~m~, and truncating the E-M procedure after a small number of iterations. The seed with greatest score F(S, θ) among these runs is then used to run the E-M to convergence and the trained motif is reported, along with all its instances with posterior probability above a certain threshold. To find more motifs, PhyME masks out the central base of each of these instances. Optionally, the user may specify nsites, the expected number of occurrences of each of the desired motifs. In such a case, PhyME turns off training of the parameter p, and uses a fixed value computed from nsites. Similarly, an option maxsites specifies the maximum number of occurrences expected. PhyME considers occurrences on both strands by introducing a new weight matrix W~r~, and an associated transition probability p~r~, in the HMM parameters. The weight matrix is constrained to be the reverse complement of W~m~. The model has a fixed bias of planting the motif in one orientation versus the other, and this bias is trained from the data. PhyME also has the option of capturing local correlations in background nucleotide composition. To implement a κ^th^order Markov background, PhyME uses a special background weight matrix that is of length 1 but uses the knowledge of the previous κbases generated to determine the emission probabilities of the next base. Performance score in experiments with synthetic data ---------------------------------------------------- We use the following score for measuring the performance of a motif-finding algorithm on synthetic data. Let S= {S~1~, S~2~, \... S~n~} be the set of ninput sequences. For any motif m, let I~mi~be the set of positions in sequence S~i~that are occupied by an occurrence of m. We know the occurrences of the planted motif m^k^, and are evaluating the motif m^r^reported by an algorithm. The performance score Φ is defined as follows: ![](1471-2105-5-170-i34.gif) In other words, it is the number of positions, over all sequences, where occurrences of the known and reported motifs overlap, divided by the total number of positions at which the known orthe reported motif occurs. Note that if the reported occurrences exactly concur with the known occurrences, the score is 1, and when the reported and known occurrences have no position in common, it is 0. Details of experiments with biological data sets ------------------------------------------------ ### Yeast The genes regulated by each transcription factor are listed in SCPD. For each such \"regulon\", the known sites and the known weight matrix were extracted from SCPD. Also, 800 bp long upstream sequences of the genes in each regulon were extracted (for S. cerevisiae) from the RSA-Tools web site \[[@B30]\]. Orthologous promoters in the other yeast species were obtained from Cliften et al. \[[@B11]\]. Let ηbe the number of known binding sites in S. cerevisiae. The input to the motif finding algorithm consisted of the sequences from S. cerevisiaeand their orthologs from one or more of the other species, depending on K. (In addition to S. cerevisiae, we used S. mikataefor K= 2, S. mikataeand S. kudriavzeviifor K= 3, and S. mikatae, S. kudriavzeviiand S. bayanusfor K= 4.) The length of the motif was also input to each program. Each algorithm was made to report 3 motifs, and for each motif, the top ηreported occurrences in S. cerevisiaewere examined. For each such occurrence, the logarithm of the probability of sampling it from the known weight matrix was computed, and a z-score of this logarithm was obtained. If the z-score was above 3, the occurrence was called a \"match\". To allow for slight offsets in the reported motif, each reported occurrence was padded with 3 bp of its context, on either side. PhyME was run with the maxsitesoption set to η, and MEME was run with the same option set to ηK. We also experimented with running MEME with the nsitesparameter set to ηK. OrthoMEME was run with a zeroth order Markov background, in the \"zoops\" mode, with expected number of sites between 0.8\*η(\"minsites\") and 1.2\*η(\"maxsites\"). PhyloGibbs was run with mutation probability 0.7 (\"-G 0.3\") for all species, and was asked to report three motifs (three \"colors\") each with 1.5 × ηoccurrences (\"-I\") initially. A 3^rd^order Markov background (\"-N 3\") trained on the full complement of yeast promoters was used, as with PhyME and MEME. The \"loose align\" option (\"-D 1\") and the \"stop after anneal\" option (\"-X\") were used. These options were suggested by an author of PhyloGibbs (Rahul Siddharthan, personal communication). We experimented with a different value for the mutation probability (\"-G 0.7\"), with no improvement, except in the RAP1 regulon. EMnEM was run with default parameters, the motif length being input through the \"-w\" parameter. Phylogenetic trees were derived from each input promoter, using the fastDNAML software of Olsen et al. \[[@B31]\]. The alignments were done using the MLAGAN program of Brudno et al. \[[@B21]\]. In separate runs, we also tried non-default values of the parameters \"-p\" (relative rate of motif to background; default 0.5, also tried 0.25) and \"-m\" (evolutionary model; default Jukes-Cantor, also tried F81). The expected number of instances of each motif per sequence (\"-e\") was set to η/nand η/n+ 1 in separate runs, where nis the number of input promoters. For each data set, and for each value of K, we took the best scoring choice of parameters. This was done to give some advantage to EMnEM, since we lacked expertise in choosing optimal parameter values. ### Fly The locations of cis-regulatory modules involved in body-patterning of the early embryo in D. melanogasterwere obtained from \[[@B26]\], and their sequences were extracted from BDGP \[[@B32]\]. The evaluation procedure was identical to that in yeast, with the following difference. Since there is no complete list of verified sites in the enhancers, we first scanned the sequences (in D. melanogaster) with the known weight matrix, and counted matches, by the same measure as above. This count was the value of ηused in the experiment. An extra complication in the fly data is caused by the fact that each enhancer typically contains sites for multiple transcription factors. We restricted our tests to the factors Kr and Bcd, because their weight matrices are of better quality than others. Moreover, for each enhancer, we chose to test with the transcription factor with most putative sites (matches to its weight matrix). OrthoMEME was run as in the yeast data sets (see above), except that the \"tcm\" mode was used now. PhyloGibbs was also run as in the yeast data sets, except that we used a mutation probability of 0.5 (\"-G 0.5\"), and a 2^nd^order Markov background (\"-N 2\"), trained on non-coding regions in fly. We also experimented with a higher value of the mutation probability, and tried specifying the initial number of occurrences per motif (\"-I\") differently, with no clear improvement. EMnEM was run with the Jukes-Cantor evolutionary model (\"-m 0\") and with the relative rate of motif to background (\"-p\") set to 0.5 and 0.25 in separate runs. The expected number of motifs was set to ηand 1.5 × ηin separate runs. The best performing choice of parameters was used for each data set. ### Human The genes comprising each regulon were obtained from TRANSFAC \[[@B33]\]. Mouse and rat orthology information for human genes was obtained from Homologene \[[@B34]\]. Human, mouse and rat promoters were obtained from the UCSC Genome Browser \[[@B35]\]. Authors\' contributions ======================= All authors participated in initial discussions leading to the key idea of using Expectation-Maximization and a phylogenetic model to search in a weight-matrix space. SS designed the algorithm details, derived the E-M calculations, implemented and tested the program, and drafted the manuscript. All authors contributed to, read and approved the final manuscript. Acknowledgments =============== This material is based upon work supported in part by the National Science Foundation under grant DBI-0218798, in part by the National Institutes of Health under grant R01 HG02602, and in part by a Keck Foundation fellowship. We are very grateful to Amol Prakash for experiments with orthoMEME, and to Rahul Siddharthan for help in running PhyloGibbs. Several useful discussions on the topic with Eric Siggia are also acknowledged. An anonymous referee, who suggested several useful changes to the manuscript, is also thanked. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Orthologous promoters and blocks of sequence conservation. Shaded areas represent ungapped aligned blocks. σ~1~is the reference species. (a) Alignment of input sequences and extraction of blocks. (b) Reorganization of input sequences. ::: ![](1471-2105-5-170-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effect of varying the number of species (K) on motif-finding performance. The x-axis is the relative entropy (R) of the planted motif. Each point is an average over 10 experiments with synthetic data. (μ~b~= 0.3, μ~m~= 0.1.) ::: ![](1471-2105-5-170-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effect of varying background and motif mutation rates (μ~b~and μ~m~respectively) on motif-finding performance. Each point is an average over 10 experiments with synthetic data. (K= 3, R= 12.) ::: ![](1471-2105-5-170-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effect of the alignment step on motif-finding performance. The x-axis shows how many of the orthologous pairs of planted motifs are artificially unpaired in the alignment step. Each solid line represents a separate experiment. The squares plot the average score over eight experiments. ::: ![](1471-2105-5-170-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Effect of multiple species information on motif-discovery in the regulons RAP1, MIG1, CAR1, PHO4 and MCM1 in yeast. The y-axis plots the number of matches to the known motif, among the top ηreported occurrences, where ηis the number of known sites, plotted as \"KNOWN\" . Only matches in S. cerevisiaeare considered. ::: ![](1471-2105-5-170-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Comparison of PhyME to 1 species and 2 species MEME, and to PhyloGibbs and EMnEM, for fly enhancers. The parenthetical number next to an enhancer name is the number of strong occurrences of the known weight matrix, in the D. melanogastersequence. ::: ![](1471-2105-5-170-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Results on the human SP1 regulon. (a) The known motif. (b) Motif reported by PhyME, using mouse and rat orthologs. (c) The phylogenetic tree used by PhyME. ::: ![](1471-2105-5-170-7) ::: ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Results on the human c-Jun regulon. (a) The known motif. (b) Motif reported by PhyME, using mouse and rat orthologs. ::: ![](1471-2105-5-170-8) :::	PubMed Central	2024-06-05T03:55:49.861242	2004-10-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534098/", "journal": "BMC Bioinformatics. 2004 Oct 28; 5:170", "authors": [ { "first": "Saurabh", "last": "Sinha" }, { "first": "Mathieu", "last": "Blanchette" }, { "first": "Martin", "last": "Tompa" } ] }
PMC534099	Background ========== Many of the hopes for achieving clinical benefits of genomic medicine will hinge on the ability to develop an efficient specimen conduit from clinic to laboratory. Quantitative gene expression studies have created unprecedented tissue collection and handling challenges. In particular, the rapid degeneration of RNA, and possible perturbation of expression following excision place a high premium on prompt stabilization of tissue samples intended for expression analysis. This can be accomplished by sending a dedicated trained technologist outfitted with the necessary specialized equipment, such as liquid nitrogen, into the clinical environment. Alternatively, clinicians can be enabled to process the specimens directly in the course of patient care and send them in some stable form by unrushed and routine means for centralized processing. The latter is greatly preferred when patients are physically dispersed, and becomes essential in a multi-institutional setting. High throughput quantification of RNA expression in solid tissues has become a commonplace modality for genome-wide discovery of mechanisms of disease. Typically, groups of samples classified into comparison groups are used as a training set for expression pattern discovery, followed by validation in a fresh challenge set of annotated cases. The likelihood of success is highly dependent on the accuracy of classification within the training set, and ability to control random variables introduced during tissue processing and analytical measurement of RNA abundance. Efforts to standardize RNA quantification include sharing of information regarding probe design and use \[[@B1]\], or centralized design and production of analytical reagents and platforms by commercial entities using good manufacturing procedures (GMP). Flash freezing, either by immersion in liquid nitrogen or on dry ice, is the most common means of stabilizing tissue samples intended for RNA analysis. Local access to the necessary materials and expense of cold shipping and/or storage limit these collection capabilities in most clinical settings. An additional disadvantage of frozen storage is that homogenization of frozen tissue must be accomplished rapidly to avoid the rapid RNA degeneration that occurs during thawing of a previously frozen sample. Room temperature immersion of fresh tissue samples in aqueous sulfate salt solutions (such as ammonium sulfate) at controlled pH precipitates degenerative RNAses \[[@B2]\] and other solubilized proteins, thereby preserving the tissue with intact RNA \[[@B3]\]. Tissues preserved in this manner are compatible with most RNA isolation protocols, and may be archivally stored for extended periods at -60°C. A commercial preparation of this preservative, RNALater (Ambion), is increasingly being used by individual investigators and cooperative groups \[[@B4]\] for collection of human tissues. There have been promising reports of microarray-based RNA expression studies using RNALater-preserved tissues \[[@B5]-[@B10]\]. Solid tissues stored for a week in RNALater at room temperature give comparable RNA yields, and specific gene RNA abundance as with frozen tissue\[[@B8]\]. RNA yields are not affected substantially by storage at room temperature compared to 4°C, for storage intervals up to 3 months \[[@B11]\]. RNALater preserved tissues and cell suspensions are suitable starting points for RNA quantification by quantitative RT-PCR \[[@B11]\] and expression microarray hybridization \[[@B12]\]. One shortcoming of the prior work is that the potential changes contributed by RNALater use have not been precisely measured relative to random processing effects. We studied the effects of differences between storage conditions on gene expression as measured by expression array. Duplicate uterine myometrial tissue samples from three women were processed under each of 4 fixed storage conditions -- fresh, frozen, 24 hours RNA-later and 72 hours RNA-later. The 24 labeled cRNA samples (Figure [1](#F1){ref-type="fig"}) were hybridized to HG-U133A Affymetrix microarrays. Then, for each microarray a data matrix was generated of 22,283 probe sets (genes) by quantitative expression levels in each RNA sample, and the effect of subject source, tissue processing, and replicates (Table [1](#T1){ref-type="table"}) determined by ANOVA. Subset analysis by gene functional class was then performed to determine if storage condition has a specific effect on particular groups of genes. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Experimental design.Tissue aliquots from 3 women were aliquoted, in duplicate, into four storage groups before RNA isolation and microarray hybridization. ANOVA design elements including fixed (storage group), random (woman, duplicate processing), and random interactive (woman × storage) effects as listed in Table 1. ::: ![](1471-2164-5-88-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Variability Sources in ANOVA Model (See Figure 1). Mixed ANOVA Model:Xij = u + ai + Bj + Eij where Xij is the observation (LN intensity), ai is the tissue storage effect, Bj is the individual variability effect and Eij is the noise term. ::: Variability Source Type df ---------------------------------------------- ---------- -------- Tissue Storage Fixed 3 Woman, Individual Variation Random 2 Interaction (Woman × Storage) Random 6 Replication (RNA isolation, chip Processing) Random 12 ::: We found no systematic bias in measured quantitative level of gene expression by processing method, indicating that short term storage in RNALater is a valid alternative to traditional frozen storage. Results ======= Of the 22,283 genes, 14,639 did not have absolutely null expression across all 24 samples. We fit the mixed model ANOVA from their log values and recorded this F statistic. The permutation distribution was used to assess the significance of F statistics calculated for each gene in the dataset. In this approach all 13,824 or (4!)^3^possible ways of permuting 4 pairs of replicate samples within each subject were considered. For each of these, the F statistics were computed for each gene. To control the overall error rate, the distributions of the maximum F statistics over the genes were used. That is, for each gene, the p-value is the proportion of permutations with the maximum F statistics over all genes greater or equal to the observed value for a particular gene. A test declaring as significant any genes with p \< 0.05 then guarantees that the chance of any false positives being selected is \< 5%. Similar analyses were performed replacing the distribution of the maximum F statistic with the distribution of the F statistics at the 95^th^percentile and then at the 90^th^percentile. After closer examination of the 387 genes in the 5% tail, we noted that most were exhibiting expression values below 100 for all 24 samples. In fact, within a storage condition, 2 out of 3 patients exhibited null expression while the third patient showed expression values other than null but less than 100 for at least one of their replicates. Therefore, as an additional analysis, any expression values less than 100 were recoded as 100. Genes that showed expression levels of 100 across all 24 samples, and therefore lacked variability, were then removed from the analysis. This resulted in 7,853 genes for which there was at least one sample with expression level greater than 100 across all 24 samples. Patient subjects provided the greatest sources of variation in the mixed model ANOVA, with replicates and processing method the least (Figure [2](#F2){ref-type="fig"}). The magnitude of variation conferred by processing method (24 hours RNALater vs 72 hours RNALater vs. fresh vs frozen) was similar to the variability seen within replicates. This is strong evidence that those individual expression profiles characteristic of the source tissue (woman) are unlikely to be obscured by the small amount of variation introduced by the processing method chosen. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Sources of variation in the mixed ANOVA model. Distribution of Mean Squares Errors by variation source are plotted for those genes with at least one tissue showing expression at a level above LN(100) (7853 genes). Note that individual women emerge as the dominant source of variation. Variation contributed by tissue storage is of approximately the same magnitude as that seen between duplicate samples within the same storage group. Boxes encompass inner quartiles, horizontal line represents the median or the second quartile, and whiskers delimit 1.5 times the interquartile range. Because of the very large number of data points, outliers were suppressed in this summary plot. ::: ![](1471-2164-5-88-2) ::: The distribution of ANOVA test statistics on those 7,853 genes where at least one sample of 24 had expression at a level exceeding 100 is compared in Figure [3](#F3){ref-type="fig"} to those seen in the randomly permuted dataset. In the actual dataset, the maximum observed F statistic was 25.52; the observed F statistic at the 95th percentile was 3.51; and the observed F statistic at the 90^th^percentile was 2.58. Corresponding p-values were 0.94, 0.55 and 0.51, respectively. The values of test statistics seen at the 95% level in a randomly permuted dataset (Figure [3](#F3){ref-type="fig"}, thin solid line) are greater than those of the observed dataset (Figure [3](#F3){ref-type="fig"}, thick solid line). This indicates that the model variation contributed by processing method is of the same magnitude as that seen randomly. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Distribution of actual test statistics vs. randomly permuted background.Distribution of ANOVA F-statistics from the model shown in Figure 1 were calculated for the observed (FStat Observed) and permuted datasets. The maximum, 95th percentile, and 90th percentile F-Statistics in the permuted dataset provide an index of the distribution of test results expected for a random sample. The values of test statistics seen at the 95% level in a randomly permuted dataset are greater than those of the observed dataset. Genes were included if at least one tissue showed expression above LN(100) (7853 genes). ::: ![](1471-2164-5-88-3) ::: Subset analysis of the test statistic according to gene functional class (Table [2](#T2){ref-type="table"}) showed that the frequency of \"outlier\" ANOVA results within each functional class is overall no greater than expected by chance. Test statistic distribution was compiled by functional annotation for those 7853 genes which had at least one sample with detectable expression above a level of 100. Nine separate classification schemas containing a total of 127 functional classes were studied. Probe sets were rank ordered by decreasing ANOVA test statistic, and enrichment in the nominal p = 0.05 tail (F\>3.51, containing top 387 of 7853 expressed) plotted by functional class (circles) and schema (box plot) (Figure [4](#F4){ref-type="fig"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Test statistic results by functional class within 9 annotation schema. Amongst 9 schema, a total of 127 functional classes (\"Classes\") contained expressed genes (\"Total Genes\"), and of these, 102 classes had a sufficient number of expressed genes (\>50, \"Class\>50\") to enumerate (\"Genes F\>3.51\") and calculate fractional representation (\"%\>3.51\", also Figure 3) in the nominal p = 0.05 tail at a test statistic cutoff of 3.51. 7853 genes with expression in at least one tissue \>LN(100) were included. ::: Code Schema Classes Class\>50 TotalGenes Genes F\>3.51 \%\>3.51 ------ ------------------------------------- --------- ----------- ------------ --------------- ---------- 1 Biological Process (GO) 22 14 3591 197 5.5 2 Cellular Role (Proteome) 14 13 1775 92 5.2 3 Cellular Component (GO) 15 12 2845 154 5.4 4 Molecular Localization (Proteome) 9 10 1788 93 5.2 5 Organismal Role (Proteome) 17 12 1524 64 4.2 6 Biochemical Function (Proteome) 16 14 2727 150 5.5 7 Subcellular localization (Proteome) 8 7 2047 116 5.7 8 Molecular Function (GO) 21 15 4473 239 5.3 9 Pathways (GenMAPP) 6 5 756 33 4.4 ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Proportion of functional gene classes in which the test statistic falls within the nominal p \< 0.05 tail.Nine publically available gene functional annotation schemas (listed in Table 2), each composed of multiple functional gene classes, were used to determine if specific functional subsets of expressed genes were more likely to show significant change in RNA detection between tissue treatments. Percentage of individual gene classes with test statistics above the nominal p = 0.05 level (F Statistic \>3.51 in the dataset of 7853 expressed genes) are plotted on the Y axis for each of 9 classification schema (X axis). Results for functional classes of genes with a minimum of 50 available expressed genes are shown as individual data points (circles) the distribution of which is summarized for each schema by the superimposed notch plot. There are only two high outliers (arrows) amongst the 113 gene classes shown. These are a messenger RNA splicing factors in the Biological Process (GO) schema (Schema 1), and translation factor in the Pathways (GenMAPP) schema (Schema 9). This frequency of 2/113 outliers is no greater than expected by chance, (\>5). ::: ![](1471-2164-5-88-4) ::: Discussion ========== Storage of fresh whole human tissues for up to 72 hours at room temperature in RNALater does not introduce quantitative bias into RNA expression determinations with the Affymetrix U133A array. Several differing standards justify this conclusion. First, by construction of a test model (Figure [1](#F1){ref-type="fig"}) incorporating both random reproducibility estimates (replicate determinations) and between-sample differences it has been possible to demonstrate that the magnitude of variation introduced by RNALater processing is equivalent to that seen within routine repeat specimens in a common processing group (Figure [2](#F2){ref-type="fig"}). Second, the extent of result variation conferred by RNALater processing is not statistically significant when measured against the randomly permuted dataset (Figure [3](#F3){ref-type="fig"}). This is an important element in evaluation of large datasets in which small numbers of individual variables may randomly demonstrate extreme values of the test statistic. Lastly, there is no evidence that specific functional subgroups of genes have aberrant behavior in this regard (Figure [4](#F4){ref-type="fig"}). Uterine myometrium was selected for these experiments because its components (myocytes, fibroblasts, vascular elements) are evenly intermingled throughout the myometrial compartment, lending itself to physical subdivision into equivalent aliquots. This would not be possible with more complex tissues in which differing cell types are distributed asymmetrically within the specimen. Despite the equivalency of subdivided fractions that underwent varying storage treatments, it must be noted that this is a hormonally responsive tissue whose expression patterns would be expected to differ between individual women as a function of monthly changes in circulating sex hormones. We did not control for hormonal factors or indication for hysterectomy (prolapse or fibroids) but selected patients randomly. It comes as no surprise that expression differences between women, irrespective of processing method, emerged as the dominant source of inter-sample variation. This was anticipated in constructing the model, by assigning the subject source of specimens as a random variable which could be measured against the fixed processing effects. It is likely that if a larger number of women had been included in the study, the observed biologic variation attributable to subject would have been even greater. Since our goal was to compare magnitude of variation contributed by subjects to that conferred by processing method, we achieved a balanced design by having comparable degrees of freedom for those two variables. There are several critical procedural elements that must be highlighted for successful preservation of solid tissues in aqueous sulfate salt solutions such as RNALater. These reagents enter the tissue through passive diffusion, a process which follows simple physical principles. The distance between the tissue surface, which is exposed to preservative, and the innermost regions of the fragment should be minimized. We did this by cutting the tissues into 2 mm thick slices, thereby reducing the diffusion distance to 1 mm or less. Clumping of multiple fragments into a mass that excludes preservative may obviate the benefits of fine division. This can be avoided either by gentle agitation or placement in a sufficiently large container that individual pieces are likely to disperse. Results reported here are for tissues stored at room temperature (23--25°C). Storage under cooler conditions (4°C) as recommend by the manufacturer of RNALater were not directly evaluated in this experiment because it was our intent to mimic storage interval and conditions commonly encountered when sending a specimen by express courier to a centralized processing facility. Storage at temperatures substantially higher than 25°C, especially before the preservative has had an opportunity to penetrate the tissue, should be avoided. Conclusions =========== Split samples of fresh human tissue yield quantitatively similar RNA expression profiles whether processed fresh, frozen, or following 24--72 hour storage in RNALater. Formal statistical analysis shows patient source is the predominant source of variation between samples, with processing method contributing a random level of variation comparable to that seen in split duplicates (replicates). Subset analysis by functional gene category did not identify a specific class of genes which responded differently by processing method. Use of nontoxic ambient environment tissue preservatives makes it practical to engage practicing clinicians directly in decentralized sample collection for high throughput expression analysis in a central location. Tissue handling closely resembles that used by clinicians to prepare specimens for routine pathology analysis. Upon receipt in a centralized facility, the samples can either be immediately homogenized or archived at -60°C. Methods ======= Tissue handling and storage --------------------------- Normal fresh uterine myometrial tissues were collected randomly from three women undergoing hysterectomy for benign uterine disease. For each hysterectomy, a single 4 to 8 gram tissue fragment was subdivided into eight aliquots composed of thin slices measuring no more than 2 mm in thickness. Replicate aliquots were immediately triaged into one of four storage conditions prior to homogenization: 1)immediate homogenization; 2)flash frozen in liquid nitrogen and storage for 48 hours at -80°C; 3)24 hour immersion in RNALater at room temperature with gentle agitation; or 4)72 hours immersion in RNALater at room temperature with gentle agitation. RNA isolation ------------- Tissue was solubilized in Trizol reagent (Gibco BRL, Grand Island, NY), and RNA isolated according to the manufacturers instructions. In brief, the aqueous phase was resolved by addition of chloroform, and RNA precipitated from the aqueous phase by addition of isopropyl alcohol. Pelleted RNA was washed with 70% ethanol, dried, and resuspended in water. Quality of total RNA was assessed by running a non-denaturing 1% agarose tris-acetate buffer which confirmed the integrity of 18S and 28S ribosomal bands for all 24 total RNA preparations. Microarray chip hybridization and data normalization ---------------------------------------------------- Double-stranded cDNA was generated from 8 μg total RNA using the Superscript Choice System (Life Technologies) with T7-(dT)24 oligomer. cDNA was purified by phenol/chloroform extraction and ethanol precipitation. Biotin-labeled cRNA was prepared using the Enzo BioArray HighYield RNA Transcript labeling kit (Affymetrix). Unincorporated NTPs were removed from the biotinylated cRNA using an RNeasy kit (Qiagen). 10 μg of quality, fragmented cRNA was hybridized to each Affymetrix HG-U133A arrays containing probe sets representing approximately 22,000 genes. Array hybridization, washing was done according to the manufacturer\'s protocol (Affymetrix, GeneChip^®^Expression Analysis Technical Manual) and all arrays were scanned under a low PMT (Photo Multiplier Tube) of 570 nm. Global scaling to a target value of 75 was applied to normalize all the arrays so they were comparable (Affymetrix Microarray Analysis Suite MAS5.0). The Affymetrix average-difference expression data and the P/A calls were used in the analysis. Those probe sets determined to have no detectible signal above background mismatch hybridization (Call of \"Absent\") were assigned a nominal value of 1 to facilitate future log transformations. Probesets having at least one tissue with detectable expression (call of \"present\") and an average difference above either 1 or 100 were selected to define subsets of 14639 permissively or 7853 stringently expressed genes, respectively. Further analysis was performed using the natural log transformed data of these probe subsets. Data files for all specimens processed are deposited online at the Gene Expression Omnibus at the National Center for Biotechnology Information \[[@B13]\]. Biostatistical analysis ----------------------- For this two factor study, a mixed model analysis of variance (ANOVA)was used, regarding storage condition as a fixed factor with four levels and subject as a random factor with three levels. The analysis of variance calculations for sums of squares in the mixed model ANOVA are identical to those for the fixed ANOVA model. Similarly, the degrees of freedom and mean squares are exactly the same. The mixed ANOVA model departs from the fixed ANOVA model only in the expected mean squares and the consequence choice of the appropriate test statistic. The mixed model also included a random storage by subject interaction. Replicate samples enabled us to estimate the replication error in the model. To test for the presence of storage main effects for each gene we divided the mean square for storage by the mean square for the interaction effect between storage and subject \[[@B14]\]. The ANOVA test statistic was calculated using C++. Functional annotation of probesets on the U133A chip, were downloaded from the Netaffx^tm^download center \[[@B1]\]. The March, 2003 version matches individual probesets with functional annotations (Table [2](#T2){ref-type="table"}) from public domain databases including: the Gene Microarray Pathway Profiler, Gene Ontology Consortium, Proteome BioKnowledge Library, and Kyoto Encyclopedia of Genes and Chromosomes. Within each schema (comprised of many functional classes of genes), each gene is assigned to a primary functional class. Each probe set may be represented in several different schemas. Individual functional classes with at least 50 probesets represented within the U133A array were plotted by schema to show fractional representation within the nominal 0.05 tail (Figure [4](#F4){ref-type="fig"}). This provides a rapid and intuitive manner to identify functional classes of genes biased towards high test statistics in the ANOVA model. Authors\' contributions ======================= GM and JW conceived and designed the research plan and participated in all aspects of data collection and analysis. DF participated in data analysis and interpretation. DN and DZ performed the statistical analysis. CL and HB performed the RNA isolations, chip hybridizations, and data collation. Acknowledgments =============== The authors wish to thank Dr. John Palma for discussions and input during data analysis	PubMed Central	2024-06-05T03:55:49.867053	2004-11-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534099/", "journal": "BMC Genomics. 2004 Nov 10; 5:88", "authors": [ { "first": "George L", "last": "Mutter" }, { "first": "David", "last": "Zahrieh" }, { "first": "Chunmei", "last": "Liu" }, { "first": "Donna", "last": "Neuberg" }, { "first": "David", "last": "Finkelstein" }, { "first": "Heather E", "last": "Baker" }, { "first": "Janet A", "last": "Warrington" } ] }
PMC534100	Background ========== Although it is clear that elevated concentrations of endogenous glucocorticoids can cause apoptosis in the thymus \[[@B1]-[@B3]\], the role of normal concentrations of glucocorticoids in thymic homeostasis remains controversial \[[@B4]-[@B7]\]. Results reported by Ashwell and colleagues suggest glucocorticoids are essential at very low concentrations for early development and survival of thymocytes and that glucocorticoids can alter the sensitivity of more mature thymocytes to positive selection, thereby influencing the T cell receptor repertoire \[[@B5]\]. In addition, there is convincing evidence that corticosterone is produced in the thymus and that it acts locally to affect thymocyte development \[[@B8],[@B9]\]. Therefore, it was surprising when normal cellular development (including repertoire) was observed until the time of birth in glucocorticoid receptor knockout mice \[[@B10]\]. This raised serious questions about the necessity of glucocorticoids as a required or permissive agent in thymic development. It has also been suggested that glucocorticoids play a role in homeostasis in the adult thymus by inducing death by neglect of thymocytes that are neither positively nor negatively selected. This idea has been based on the observation that the predominant cell type subjected to death by neglect, CD4^+^CD8^+^non-mature thymocytes, is most susceptible to elevated concentrations of glucocorticoids \[[@B11]\]. Recent results indicate that overexpression of glucocorticoid receptors (GR) in developing and mature T cells leads to decreased cell number in the thymus and a decreased number of T cells in the periphery in adult mice. In addition, decreased expression of GR is associated with increased cell number in the thymus \[[@B4]\]. However, results obtained with knockout or transgenic mice have been contradictory \[[@B4],[@B7],[@B12]-[@B14]\]. For example, one group using transgenic mice that express anti-sense GR mRNA in the thymus found increased thymus cellularity \[[@B4]\], whereas another group using a cre-lox conditional knockout system to eliminate glucocorticoid receptor in cells that express CD4 (including double positive cells) reported no increase in cellularity \[[@B14]\]. Both groups verified that the expected decrease in sensitivity to high concentrations of glucocorticoids occurred in the transgenic mice. Until the basis for such differences can be determined, it seems reasonable to use an alternate approach that does not alter the glucocorticoid receptor (except by natural mechanisms relating to glucocorticoid concentration). In addition, transgenic approaches cannot provide the concentration-response information for corticosterone that would be needed to distinguish normal physiological effects and stress-related effects. A small number of studies have been reported in which systemic glucocorticoid concentrations were reduced by adrenalectomy, leading to increased numbers of cells in the thymus \[[@B15]-[@B17]\]. However, this observation has not been universal, with one report indicating no increase in thymus cellularity in adrenalectomized mice \[[@B18]\]. Thus, confirmation of an adrenalectomy-induced increase in thymus cellularity would be useful. Even if confirmation is obtained, it would still be possible that an adrenal product other than corticosterone was responsible for increased cell number in the thymus. However, if corticosterone was the major regulator of thymus homeostasis, restoring corticosterone to physiological levels in adrenalectomized mice should return thymus cell number and subpopulation ratios to normal values. Therefore, this approach was used in the present study to determine the role of systemic corticosterone in thymus homeostasis. The study was designed so the results would also indicate whether thymus-derived corticosterone is sufficient to permit normal maintenance of number of cells in each major subpopulation in the thymus. If physiological (adrenal-derived) concentrations of corticosterone are important in the induction of death by neglect of thymocytes, it would seem likely that any increase in cell number in the thymus of adrenalectomized mice would be explained mostly by an increase of CD4^+^CD8^+^cells, which are the predominant cell type subjected to death by neglect \[[@B11]\]. In addition, it has also been proposed that physiological concentrations of corticosterone increase the sensitivity of thymocytes to negative selection. Preventing this would presumably cause fewer single positive thymocytes to die, thus increasing the percentages of these cells in the thymus. It might also be expected that immature single positive thymocytes (CD3 low, CD4^-^CD8^+^) would be increased, as these cells have been reported to be particularly sensitive to glucocorticoids \[[@B19]\]. If physiological (non-stress) concentrations of corticosterone contribute to the induction of death by neglect or negative selection, it would be expected that sub-physiological concentrations of corticosterone would decrease apoptosis in the thymus. Failure to observe these changes in mice with sub-physiological concentrations of corticosterone would suggest either that thymus-derived corticosterone \[[@B8]\] is sufficient to compensate for loss of systemic (adrenal-derived) corticosterone or that corticosterone is not directly involved in these processes under physiological, non-stress conditions. The studies described here were designed to directly test these predictions and thus to indirectly evaluate the role of endogenous glucocorticoids in death by neglect in the thymus. In addition, this study was designed to distinguish the relative contributions of systemic (mostly adrenal-derived) glucocorticoids and those produced in the thymus \[[@B8]\]. The use of a dose-response approach permitted identification of the point on the corticosterone concentration vs. thymocyte subpopulation plot that corresponds to a physiological corticosterone concentration, and it permitted identification of a distinction between the effects of sub-physiological concentrations of corticosterone and stress-inducible concentrations. Results and discussion ====================== Adrenalectomy increases cell number and alters subpopulation percentages in the thymus, and this effect is inhibited 24 hr after restoration of corticosterone -------------------------------------------------------------------------------------------------------------------------------------------------------------- The results shown in Figure [1](#F1){ref-type="fig"} demonstrate that adrenalectomy significantly increases the total number of cells in the thymus as well as the number of CD4^+^CD8^-^and CD4^+^CD8^+^cells. The number of CD4^-^CD8^+^and CD4^-^CD8^-^cells was also greater in adrenalectomized mice than in the naive control group, but the difference was not significant. This indicates that the number of CD4^+^CD8^-^and CD4^+^CD8^+^cells is diminished to a greater extent than CD4^-^CD8^+^and CD4^-^CD8^-^cells by normal, physiological concentrations of corticosterone. Results for adrenalectomized mice were comparable whether a placebo pellet was implanted or not, indicating that non-adrenal-derived stress mediators induced by pellet implantation did not affect cell number. Timed release corticosterone pellets were used to restore corticosterone in adrenalectomized mice, and 0.5 and 1.5 mg pellets had only minimal effects on any subpopulation 24 hours after implantation of pellets (Figure [1](#F1){ref-type="fig"}). Pellets containing 2.5 mg of corticosterone returned the number of CD4^+^CD8^-^and CD4^+^CD8^+^cells to near normal values. A pellet containing 5.0 mg of corticosterone significantly decreased the number of CD4^-^CD8^-^and CD4^+^CD8^+^cells as compared to the naive control group, suggesting that CD4^-^CD8^-^and CD4^+^CD8^+^are more sensitive than CD4^+^CD8^-^and CD4^-^CD8^+^cells to stress-inducible corticosterone concentrations. The greater sensitivity of CD4^+^CD8^+^cells as compared to the other subpopulations at high corticosterone concentrations has prompted an assumption that these cells are more sensitive to physiological (unstressed) concentrations of corticosterone. However, this was not supported by the results presented here. The results in Figure [1](#F1){ref-type="fig"} do indicate that CD4^+^CD8^+^cells increase in number to a greater extent than CD4^-^CD8^+^or CD4^-^CD8^-^cells in ADX mice, but CD4^+^CD8^-^cells increase in number proportionally more than all these other subpopulations in mice with sub-physiological concentrations of corticosterone (ADX groups). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Effect of adrenalectomy and restoration of various concentrations of corticosterone on the number of nucleated cells in the thymus 24 hours after implantation of corticosterone pelletsTreatment groups were: Naive, untreated; ADX-N, adrenalectomized naive; ADX-P, adrenalectomized with placebo pellet implanted; other groups, adrenalectomized with corticosterone pellets of the indicated size implanted. The thymus was evaluated 24 hours after pellet implantation (\~3--4 weeks after ADX). Group size was 5--12. Values shown are mean ± S.E. obtained by normalizing groups to the mean value for the Naive group (defined as 100%). Results shown are pooled from two independent experiments, and groups significantly different from the naive group (by ANOVA followed by Dunnett\'s test) are shown by \* (p\< 0.05) or \\ (p\< 0.01). ::: ![](1471-2172-5-24-1) ::: The changes in subpopulation percentages (as opposed to cell numbers, which are shown in Figure [1](#F1){ref-type="fig"}) in the thymus 24 hours after implantation of pellets are shown in Figure [2](#F2){ref-type="fig"}. These results confirm the greater sensitivity of CD4^+^CD8^+^cells than the other subpopulations to stress-inducible concentrations of corticosterone (in mice with a 5 mg pellet). The greater sensitivity of CD4^-^CD8^-^cells than CD4^-^CD8^+^and CD4^+^CD8^-^cells noted in Figure [1](#F1){ref-type="fig"}, was also evident in Figure [2](#F2){ref-type="fig"} in terms of a lesser increase in percentage of the former cell type as compared to the latter cell types in mice treated with a 5 mg pellet. However, it should be emphasized that all subpopulations decreased in absolute number in these mice, so these percentage values do not reflect increases in the number of cells in these subpopulations but increases relative to CD4^+^CD8^+^cells, which are the most abundant and were diminished to the greatest extent. No significant change in subpopulation percentages was caused by adrenalectomy with or without a placebo pellet. However, it should be noted that the results shown in Figure [1](#F1){ref-type="fig"} indicate that the number of CD4^+^CD8^+^and CD4^+^CD8^-^cells increased to a greater extent than the number of CD4^-^CD8^-^or CD4^-^CD8^+^cells. This did not result in a substantial change in percentages (Figure [2](#F2){ref-type="fig"}), because the cells that increased the most (CD4^+^CD8^+^cells) account for over 90% of the total thymocyte population, and changes in this subpopulation were reflected in the denominator (total cell number) of the equation that determines the percentage of cells in each population. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effect of adrenalectomy and restoration of various concentrations of corticosterone on the percentage of cells in the 4 major subpopulations in the thymus 24 hours after implantation of corticosterone pelletsData from the experiments noted in Figure 1 were analyzed for changes in the percentage of each cell type as compared to the total number of nucleated cells per thymus. The values did not differ significantly between experiments for any subpopulation, so the data were pooled (without normalizing). Groups significantly different from the naive group (by ANOVA followed by Dunnett\'s test) are shown by \* (p\< 0.05) or \\ (p\< 0.01). ::: ![](1471-2172-5-24-2) ::: Adrenalectomy increases cell number and alters subpopulation percentages in the thymus, and this effect is inhibited 72 hr after restoration of corticosterone -------------------------------------------------------------------------------------------------------------------------------------------------------------- Changes in the thymus were first evaluated 24 hours after implantation of pellets to allow determination of the role of apoptosis in loss of cells at a time point at which cell numbers were still decreasing. To determine if greater or different effects were evident after a longer period of corticosterone exposure, thymuses were evaluated 72 hours after implantation of pellets. As shown in Figure [3](#F3){ref-type="fig"}, the effects on cell number and on the number of cells in most subpopulations were greater than observed after 24 hours (Figure [1](#F1){ref-type="fig"}). For example, mice with a 2.5 mg corticosterone pellet had significantly fewer total thymocytes and significantly fewer cells in all but one of the major subpopulations than the naive group, whereas such decreases were only observed in the group with a 5 mg pellet after 24 hours (Figure [1](#F1){ref-type="fig"}). Similarly, the changes in subpopulation percentages were more pronounced after 72 hours (Figure [4](#F4){ref-type="fig"}) than after 24 hours (Figure [2](#F2){ref-type="fig"}). Some of the increases in cell number in adrenalectomized naive mice (ADX-N) or adrenalectomized mice with a placebo pellet implanted (ADX-P) compared to naive control that were observed in Figure [1](#F1){ref-type="fig"} were not significant in the 72 hour experiment (Figure [3](#F3){ref-type="fig"}). It should be noted that these groups were essentially equivalent in all experiments, because adrenalectomy occurred 3 weeks before analysis in all mice. Evaluation of pooled, normalized data from 4 independent experiments indicates that compared to naive mice (100 ± 4.3%), ADX-N (143 ± 5.2%) and ADX-P (144 ± 8.3%) groups had significantly more total thymocytes. The mean number of nucleated cells per thymus in the naive (non-adrenalectomized) groups was 8.1 × 10^7^in these 4 experiments. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effect of adrenalectomy and restoration of various concentrations of corticosterone on the number of nucleated cells in the thymus 72 hours after implantation of corticosterone pelletsTreatment groups were: Naive, untreated; ADX-N, adrenalectomized naive; ADX-P, adrenalectomized with placebo pellet implanted; other groups, adrenalectomized with corticosterone pellets of the indicated size implanted. The thymus was evaluated 72 hours after pellet implantation (\~3--4 weeks after ADX). Group size was 5--12. Values shown are mean ± S.E. obtained by normalizing groups to the mean value for the Naive group (defined as 100%). Results shown are pooled from two independent experiments, and groups significantly different from the naive group (by ANOVA followed by Dunnett\'s test) are shown by \* (p\< 0.05) or \\ (p\< 0.01). ::: ![](1471-2172-5-24-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effect of adrenalectomy and restoration of various concentrations of corticosterone on the percentage of cells in the 4 major subpopulations in the thymus 72 hours after implantation of corticosterone pelletsData from the experiments noted in Figure 3 were analyzed for changes in the percentage of each cell type as compared to the total number of nucleated cells per thymus. Groups significantly different from the naive group (by ANOVA followed by Dunnett\'s test) are shown by \* (p\< 0.05) or \\ (p\< 0.01). ::: ![](1471-2172-5-24-4) ::: Serum corticosterone concentrations indicate that corticosterone replacement with pellets yields appropriate corticosterone concentrations and that sub-physiological corticosterone concentrations (in ADX mice) are not sufficient to maintain thymus homeostasis ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- The serum corticosterone concentration measured in naive mice was 185 ng/ml (Figure [5A](#F5){ref-type="fig"}). Normal corticosterone values in female B6C3F1 mice vary from less than 100 ng/ml to approximately 300 ng/ml in a circadian pattern \[[@B20]\]. Adrenalectomized mice had barely detectable concentrations of corticosterone in the serum, and corticosterone pellets containing increasing amounts of corticosterone produced increasing corticosterone concentrations in the serum. These results were obtained 24 hours after implantation of pellets. These serum corticosterone concentrations range from values that can be found in normal (unstressed) mice at certain times of the day \[[@B20]\] (observed in groups treated with 0.5 and 1.5 mg pellets) to values that are comparable to those measured in mice exposed to moderately or highly stressful conditions \[[@B20],[@B21]\] (observed in mice treated with 2.5 or 5.0 mg pellets). Non-linear curve fitting was used to extrapolate the size of pellet that would be required to produce the same corticosterone concentration measured in naive mice. The extrapolated value (1.75 mg) is shown in Figure [5B](#F5){ref-type="fig"}. Thus, the effects of adrenalectomy on the thymus should not quite be reversed by a 1.5 mg pellet and should be more than reversed by a 2.5 mg pellet. The results shown in Figure [3](#F3){ref-type="fig"} are reasonably consistent with this expectation. It seems appropriate to examine the 72-hour data in this regard (Figure [3](#F3){ref-type="fig"}), because this exposure period most likely represents the time required to achieve the maximum effects of corticosterone on the thymus. The slight differences from expected effects for some subpopulations may be related to the fact that pellets do not reproduce the circadian changes in corticosterone that occur in normal animals. These changes likely contribute to thymus homeostasis, and restoring corticosterone to a constant concentration may not mimic this effect precisely. Nevertheless, these results clearly demonstrate that corticosterone alone is able to reverse the effects of adrenalectomy and that this reversal occurs at concentrations within physiological levels. This suggests that corticosterone is the only adrenal product required to regulate thymocyte number. More importantly, these results conclusively demonstrate that corticosterone produced in the thymus is not sufficient to maintain normal thymocyte numbers. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Serum corticosterone concentration 24 hours after implantation of corticosterone pelletsMice from one of the experiments noted in Figures 1 and 2 were bled prior to removal of the thymus, and serum corticosterone concentrations were determined by radioimmunoassay. In panel A, values shown are means ± SE (n = 5 mice/group), and values significantly different from the naive control are indicated by \* (p\< 0.05) or \\ (p\< 0.01). The treatments are described in the legend for Figure 1. In panel B, non-linear regression was used to determine a best-fit line through points from 0 (ADX-N) to 5 mg corticosterone pellets (open squares). The extrapolated pellet size required to produce the same corticosterone concentration shown for Naive mice in panel A is indicated by an open circle. Dotted lines indicate the 95% confidence interval for the regression line. The R-squared value for this relationship was 0.987. ::: ![](1471-2172-5-24-5) ::: Differential sensitivity of thymic subpopulations as indicated by linear regression analysis -------------------------------------------------------------------------------------------- It is clear that thymic subpopulations differ in their sensitivity to high (stress-inducible) corticosterone concentrations. However, linear regression analysis (Figure [6](#F6){ref-type="fig"}) indicates that the sensitivities of the various cell populations are not as different as might be expected on the basis of the decreased percentage of CD4^+^CD8^+^cells and the increased percentages of the other subpopulations. The results demonstrate that increasing concentrations of corticosterone affect CD4^-^CD8^-^and CD4^+^CD8^+^similarly, but the slope for CD4^+^CD8^+^cells is significantly greater (by the method of Zar as implemented by Prism software) than for CD4^-^CD8^-^cells, indicating slightly greater sensitivity to high concentrations of corticosterone. The effect of increasing concentrations of corticosterone on CD4^+^CD8^-^cells was significantly less than the effect on CD4^+^CD8^+^cells with regard to the slopes of the respective lines. The slope for CD4^-^CD8^+^cells was less than the slope for all other subpopulations, suggesting a lower sensitivity of these cells to corticosterone across the whole range of concentrations. However, the decrease for CD4^+^CD8^-^cells and CD4^-^CD8^+^cells relative to naive control was essentially the same in mice treated with a 5 mg pellet, suggesting that the difference in sensitivity is minimal as the corticosterone concentration increases. Although non-linear models would likely have given better correlation coefficients than linear ones for some of these data, linear models facilitate comparison. In addition, the Runs Test was conducted for all linear regression analyses, and the non-linear component was not significant for any of these data. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Linear regression analysis of corticosterone pellet size (mg) and the number of cells per thymus for each of the 4 major subpopulationsThe thymus was evaluated 24 hours after pellet implantation (\~3--4 weeks after ADX), and all groups shown were ADX. Group size was 5--12. Values shown are mean ± S.E. obtained by normalizing groups to the mean value for the Naive group (defined as 100%). Results shown are pooled from two independent experiments, and statistically significant differences in the slope and intercept of each pair-wise combination are described in the text. ::: ![](1471-2172-5-24-6) ::: Role of changes in the rate of apoptosis in decreased and increased cell number in the thymus of ADX mice with or without a corticosterone pellet ------------------------------------------------------------------------------------------------------------------------------------------------- There are several mechanisms by which corticosterone might act to alter the number of cells in each subpopulation in the thymus. Although induction of apoptosis is generally regarded to be the major mechanism \[[@B1]-[@B3],[@B22]\], altering differentiation or proliferation of thymocytes, altering the development of pro-thymocytes in the bone marrow, or altering pro-thymocyte or thymocyte trafficking to or from the thymus are all possible mechanisms. To determine if changes in apoptosis may play a role in the effects noted in this study, apoptosis was evaluated using two criteria: cell size (indicated by forward scatter) and TUNEL labelling for DNA fragmentation. As shown in Figure [7](#F7){ref-type="fig"}, the results demonstrate that the percentage of apoptotic cells in the thymus was decreased in mice that had sub-physiological concentrations of corticosterone in the serum (ADX-naive, ADX-placebo, and ADX mice with a 0.5 mg corticosterone pellet). The percentage of apoptotic cells was substantially increased in mice with a high (stress-inducible) concentration of corticosterone in the serum (caused by a 5 mg pellet). ::: {#F7 .fig} Figure 7 ::: {.caption} ###### Apoptosis in the thymus in response to sub-physiological and stress-inducible concentrations of corticosteroneApoptosis was evaluated using the TUNEL technique with fluorescein-labelled dUTP. The cells were also surface labelled with fluorescent-labelled antibodies to CD4 and CD8. Values shown represent means ± SE for groups of 5 mice. The values in the upper panel represent the percentage of apoptotic cells in the thymus. The other values represent the percentage of apoptotic cells that are within each of the major 4 subpopulations in the thymus. Groups significantly different from the naive group (by ANOVA followed by Dunnett\'s test) are shown by \* (p\< 0.05) or \\ (p\< 0.01). ::: ![](1471-2172-5-24-7) ::: Initially, changes in the percentage of apoptotic cells in the various subpopulations did not seem entirely consistent with the changes in the percentages of each subpopulation in the thymus (compare Figure [2](#F2){ref-type="fig"} and Figure [7](#F7){ref-type="fig"}). Three-color flow cytometry allowed determination of the percentage of apoptotic cells in each subpopulation. In naive mice, most apoptotic cells were CD4^+^CD8^+^, as expected, with a substantial percentage in the CD4^-^CD8^-^category and lesser percentages of the mature single positive categories. The percentages of apoptosis in all populations increased in mice treated with a 5.0 mg corticosterone pellet, except CD4^+^CD8^+^cells, for which the percentage decreased. This almost certainly reflects the fact that this subpopulation was depleted by 24 hours of elevated corticosterone concentrations (Figure [1](#F1){ref-type="fig"}), and at least some of the remaining CD4^+^CD8^+^cells were likely glucocorticoid resistant \[[@B23]\]. However, it should also be noted that an increase in apoptosis was only observed in mice treated with a 5 mg pellet, not in mice treated with a 0.5 mg pellet. In contrast, a 0.5 mg pellet diminished the significant increase in cell numbers caused by adrenalectomy (Figure [3](#F3){ref-type="fig"}). This suggests the possibility that mechanisms other than apoptosis may also be involved glucocorticoid-mediated homeostasis in the thymus. The large percentage of CD4^-^CD8^-^cells in the apoptotic population was not entirely unexpected, because there is a developmental checkpoint that can lead to death in CD4^-^CD8^-^cells that do not productively rearrange a TCR β chain \[[@B24]\]. In the human thymus, the percentage of apoptotic CD4^-^CD8^-^is lower than we noted (13% in humans vs. 34% in the present study) \[[@B25]\]. However, the human thymuses in that study were obtained from newborns, and the differences could thus reflect age as well as species differences. Effect of ADX with or without corticosterone pellets on immature single positive cells in the thymus ---------------------------------------------------------------------------------------------------- Single positive CD8 cells that express low levels of CD3 (and TCR) are apparently the immediate precursors for CD4^+^CD8^+^cells in the thymus \[[@B24]\]. One report indicates that these cells are more susceptible than mature single positive cells to glucocorticoids \[[@B19]\]. Results shown in Figure [8](#F8){ref-type="fig"} are consistent, at least in part, with this finding. The increase in the percentage of this subpopulation in mice treated with a 5 mg pellet (2.4 fold) was substantially less than the increase in mature single positive cells (\~5-fold) (Figure [2](#F2){ref-type="fig"}). In addition, the number of immature CD8^+^cells was decreased to a greater extent than for mature single positive cells at this time point (Figure [1](#F1){ref-type="fig"}). This suggests that immature single positive cells are more sensitive to high levels of corticosterone than mature single positive cells, though they are apparently less sensitive than CD4^+^CD8^+^and CD4^-^CD8^-^cells. The number of immature single positive cells did not increase significantly in ADX mice that did not receive a corticosterone pellet, indicating that homeostasis of this cell type is not affected by physiological levels of corticosterone. This further illustrates that it is not appropriate to infer the effects of physiological concentrations of glucocorticoids on various cells types on the basis of the action of pharmacological concentrations of glucocorticoids. The percentage of immature single positive cells in the thymus of untreated mice in our study was comparable to values noted by others \[[@B19],[@B26]\]. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### Effect of sub-physiological and stress-inducible concentrations of corticosterone on immature single positive (CD8^+^) thymocytesThe histograms at the top of this figure illustrate the region containing cells that express low levels of CD3 (the larger purple peak on the histogram to the left, in which the isotope control is indicated by a green line), and the subsequent analysis of those cells for CD4 and CD8. The cells in the lower right quadrant (outlined with bold lines) are CD3^low^CD8^+^, immature single positive cells. The values shown in the graphs are means ± SE for groups of 5 mice representing either the percentage of immature single positive cells in the thymus or the total number of these cells in the thymus (obtained by multiplying the percentage by the total thymus cell number for each mouse). Groups significantly different from the naive group (by ANOVA followed by Dunnett\'s test) are shown by \\ (p\< 0.01). ::: ![](1471-2172-5-24-8) ::: Relationship of present results and results from other studies -------------------------------------------------------------- The findings reported here are consistent with some, but not all, results from other laboratories. The study most relevant to the present one involved the use of transgenic mice that express glucocorticoid receptor at higher than normal or lower than normal levels \[[@B4]\]. Comparing changes in the number of thymocytes in various subpopulations in mice expressing twice the normal level of glucocorticoid receptor in the thymus with our results at moderately elevated corticosterone concentrations (2.5 mg pellet, figure [3](#F3){ref-type="fig"}) indicates some similarities and some differences. In both studies, the total number of thymocytes was decreased significantly. However, expression of higher levels of glucocorticoid receptor caused significant suppression of cell numbers for CD4^+^CD8^+^, CD4^-^CD8^-^, and CD4^+^CD8^-^cells, but not CD4^-^CD8^+^cells \[[@B4]\]. In contrast, the 2.5 mg pellet in adrenalectomized mice (a situation that should be analogous to higher levels of glucocorticoid receptors with normal corticosterone concentrations) caused significant decreases in cell number in CD4^+^CD8^+^, CD4^-^CD8^+^, and CD4^-^CD8^-^cells, but not in CD4^+^CD8^-^cells (Figure [3](#F3){ref-type="fig"}). In mice expressing lower than normal levels of glucocorticoid receptor in the thymus (due to incorporation of anti-sense DNA under the control of the lck promoter) \[[@B4]\], the pattern of change was very similar to that which we observed in adrenalectomized mice (with no corticosterone pellet). In both cases, the significant increases in cell number were noted only for the CD4^+^CD8^-^and CD4^+^CD8^+^subpopulations. Cell number in the other two major subpopulations was increased slightly, but not significantly. The overall relationships between these results might be explained by a report indicating that normal expression of glucocorticoid receptor in the thymus is a very dynamic process, with substantial changes in expression in different subpopulations of cells \[[@B19]\]. In addition, the evidence suggests that sensitivity of the various subpopulations to glucocorticoids is not always strictly dependent on the amount of glucocorticoid receptor expressed. Thus, other factors that change during the development of T cells play an important role in sensitivity to glucocorticoids. Causing excess production of glucocorticoid receptor in all cells of the thymus (as in transgenic mice with an extra glucocorticoid receptor gene, transcribed in all thymocytes under the control of the lck promoter) \[[@B4]\] would not be likely to produce the same differences in glucocorticoid receptor levels among cellular subpopulations in the thymus as noted in normal animals (in which glucocorticoid receptor levels vary in different subpopulations). A portion of the glucocorticoid receptor production would be subject to the normal dynamic regulatory process, but a portion of production (the portion under the control of the lck promoter) would not. This may explain the differences in the results obtained using transgenic mice with elevated levels of glucocorticoid receptor \[[@B4]\] as compared to our results using elevated corticosterone concentrations. However, expression of glucocorticoid receptor anti-sense RNA in the thymus in a uniform manner \[[@B4]\] seems to have produced similar results as decreased corticosterone concentrations (in adrenalectomized animals) (Figs. [1](#F1){ref-type="fig"},[2](#F2){ref-type="fig"},[3](#F3){ref-type="fig"},[4](#F4){ref-type="fig"},). This may reflect the fact that the action of anti-sense RNA in a particular cell type would be expected to be proportional to the amount of glucocorticoid receptor expressed in that cell. Thus, the normal differences between subpopulations with regard to glucocorticoid sensitivity might be retained. Thus, it is not surprising that the results with anti-sense glucocorticoid receptor transgenic mice are comparable to those for adrenalectomized mice in our study with regard to the differential increase in cell number for different subpopulations. It is not clear why no increase in thymus cellularity or changes in subpopulations were noted by other investigators using a conditional knockout system to produce mice in which the thymus contains little glucocorticoid receptor \[[@B14]\]. However, glucocorticoid receptor knockout mice can express portions of the glucocorticoid receptor, which may have unexpected functions \[[@B27]\]. Such contradictory findings with transgenic approaches have been common in this field of research (see Introduction), indicating a useful role for classical pharmacological approaches such as those in the present study. The relationship between the results reported here and results from studies on the interactions between glucocorticoids and self-antigen in positive selection is not clear. A recent study indicates that activation through the TCR down regulates SRG3, a protein that associates with the glucocorticoid receptor and increases sensitivity to glucocorticoids \[[@B28]\]. This has been proposed as an explanation for the decreased sensitivity of mature single positive thymocytes as compared to non-mature double positive thymocytes to high concentrations of glucocorticoids. However, as already noted, this pattern did not seem to apply when comparing the effects of sub-physiological and physiological concentrations of glucocorticoids, i.e., the CD4^+^CD8^-^cells increased to a greater extent than CD4^+^CD8^+^cells in mice with sub-physiological corticosterone concentrations. This suggests that the observed changes in SRG3 may not account for differences in sensitivity of cells in different subpopulations to physiological concentrations of corticosterone. This leaves open the question of what does mediate those differences and the role (if any) of TCR signalling. It would be of interest to explore this with TCR transgenic mice. One study in which adrenalectomy has been used to evaluate the effects of glucocorticoids on cellular subpopulations in the thymus yielded different results than those reported here. In that study, there was no increase in total cell number in the thymus, and there were no changes in subpopulation percentages in adrenalectomized mice two weeks after adrenalectomy \[[@B18]\]. The basis for the difference in this result and the results of other studies, which indicate increased numbers of thymocytes in adrenalectomized mice or rats \[[@B15]-[@B17]\] is not clear. In a study in which one of the authors of the present report (E. L. P.) was involved, there was a greater increase in cell number in the thymus in adrenalectomized mice than in the present study \[[@B16]\]. Perhaps because the overall increase in cell number was greater, the increases in all subpopulations were significant \[[@B16]\]. The age and housing conditions (and resulting environmental stress levels) of the control group probably plays an important role in this regard, and it would be very difficult to assure precisely the same conditions for the control group in every case. Nevertheless, the results reported here along with results from most other adrenalectomy studies and results from one study using transgenic mice \[[@B4]\] indicate that physiological (non-stress) concentrations of corticosterone normally decrease the number of cells in the thymus. Conclusions =========== The results presented here do not directly demonstrate the extent to which corticosterone contributes to death by neglect or the extent to which it contributes to the death of negatively selected thymocytes. The fact that both CD4^+^CD8^+^and CD4^+^CD8^-^cells are increased in number in ADX mice is consistent with the idea that death by neglect of CD4^+^CD8^+^cells is mediated by corticosterone. The increase in CD4^+^CD8^-^cells could be explained by the failure of CD4^+^CD8^+^cells to die before reaching maturity, as they would have done in the presence of corticosterone. The observation that CD4^-^CD8^+^cells are not increased to the same extent as CD4^+^CD8^-^cells is exactly what would have been predicted if sub-physiological concentrations of corticosterone allow survival of cells that would normally die by neglect. Whereas maturation of CD4^+^CD8^+^cells to CD4^-^CD8^+^cells requires MCH class I-dependent signals, maturation to CD4^+^CD8^-^status can be MHC-independent and apparently occurs by default \[[@B29]\]. Thus, cells that are not selected positively may preferentially mature to CD4^+^CD8^-^cells before dying by corticosterone-mediated apoptosis. However, sub-physiological concentrations of corticosterone apparently allow these non-selected cells to survive. Methods ======= Animals and animal care ----------------------- Female C57BL/6 × C3H F1 (B6C3F1) mice were used in this study. Normal, adrenalectomized (ADX), and sham adrenalectomized mice were purchased from Charles River Labs (Wilmington, MA). The mice were allowed to recover from shipping stress for at least two weeks before use in experiments, and surgery was performed approximately one week before shipping. Thus, mice were evaluated at least three weeks after surgery at an age of 8--12 weeks. Mice were maintained on a 12 hour light/dark cycle, with free access to lab chow and water, except that ADX mice were given water with 0.9% sodium chloride. Sentinel mice housed periodically in the same room as the mice used in this study were negative for common adventitious agents and pathogens of mice. Animal care and use was in accord with the regulation of LSU Health Sciences Center and the NIH Guide for Care and Use of Laboratory Animals. The animal facility in which the mice were maintained is approved by the American Association for Accreditation of Laboratory Animal Care. Implantation of timed release corticosterone pellets ---------------------------------------------------- Timed-release corticosterone pellets and placebo pellets were purchased from Innovative Research of America (Sarasota, FL). The pellets are designed to yield constant blood levels of corticosterone for 3 weeks. Pellets were implanted subcutaneously in the scapular area of mice that were anesthetized with sodium pentobarbital (55 mg/kg) and inhalation of methoxyflurane, as described in a previous study \[[@B2]\]. The incision was closed with a surgical staple. The entire process was conducted aseptically. Mice were allowed to recover on a heating pad prior to being returned to their home cages. Parameters were evaluated after 24 hours in two experiments and after 72 hours in a third experiment. Preparation of cells and flow cytometry --------------------------------------- In two experiments, mice were euthanized by CO~2~inhalation and the thymus was removed for analysis. In one experiment mice were euthanized by decapitation, trunk blood was obtained and allowed to clot, and serum was isolated after centrifugation. The serum was used to determine corticosterone concentration, using a radioimmunoassay kit (Diagnostic Products Corporation, Los Angeles, CA) as described previously \[[@B30],[@B31]\]. The thymus was then removed from each mouse. Single cell suspensions were prepared in 3 ml of RPMI 1640 by pressing the organs between the frosted ends of sterile glass microscope slides, as in previous studies \[[@B1],[@B2]\]. After centrifugation, the cells were resuspended in 3 ml of RPMI 1640, 20 μl samples were taken, diluted in 10 ml of Isoton II isotonic buffered saline (Coulter Corp., Miami, FL), and counted using an electronic cell counter (Coulter Model Z1). Cells were adjusted to 2 × 10^7^per ml, and 50 μl was added to the wells of a 96-well V-bottom microplate. Antibodies diluted in 50 μl of FACS buffer (phosphate buffered saline without calcium and magnesium plus 0.1% bovine serum albumin and 0.1% sodium azide) were added to appropriate wells. In each experiment in which multiple antibodies were used, controls included cells labeled with each antibody singly, cells labeled with each isotype control antibody singly, cells labeled with all isotype control antibodies together, and unlabeled cells. The following antibodies were used: anti-CD4 (GK1.5) labeled with phycoerythrin (PE), anti-CD8a labeled with Cychrome, and anti-CD3 labeled with fluorescein isothiocyanate (FITC). These antibodies and matching isotype controls were obtained from BD Pharmingen. Titration of the antibodies indicated that a 1:8 dilution of anti-CD4 and anti-CD8 and a 1:5 dilution of anti-CD3 were appropriate for this study. After labeling for 30 min at 4°C, the cells were washed, fixed with 1% paraformaldehyde (EM Sciences, Ft. Washington, PA), washed again, and resuspended in FACS buffer. Samples were diluted in Isoton II (0.4 ml) for analysis. Cells were analyzed using a FACScan flow cytometer (Becton-Dickinson). A gate was set using forward scatter and side scatter to exclude debris, erythrocytes, and clumps of cells. All cells within this gate were then analyzed for CD3, CD4, and CD8. In some experiments, cells were labeled to detect DNA fragmentation instead of CD3. Cells were first labeled with anti-CD4 (phycoerythrin) and anti-CD8 (cychrome) as described above, then the cells were fixed with 4% paraformaldehyde (in phosphate buffered saline). A terminal dUTP nick end labeling (TUNEL) kit from Boehringer-Mannheim (Indianapolis, IN) with fluorescein-labeled dUTP was used to label apoptotic cells. Flow cytometry was used to identify apoptotic cells by two criteria. Cells that were small (as indicated by forward scatter) and labeled with fluorescein (indicating DNA fragmentation) were regarded to be apoptotic. Statistical analysis -------------------- Values significantly different from the naive (untreated) control group were determined by analysis of variance (ANOVA) followed by Dunnett\'s post hoc test. Statistical analysis, linear regression, and non-linear regression analysis were performed using Prism 4.0 software (GraphPad, Inc., San Diego, CA). Comparison of slope or intercept of pairs of regression lines was done using the method of Zar \[[@B32]\] as implemented by Prism software. Authors\' contributions ======================= The authors contributed approximately equally to conceiving, designing, and conducting these experiments. S.B.P. wrote the manuscript, and E.L.P. revised it. Acknowledgements ================ This work was supported by NIH grants R01ES09158 and R01AA009505 (to S.B.P.).	PubMed Central	2024-06-05T03:55:49.869546	2004-11-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534100/", "journal": "BMC Immunol. 2004 Nov 2; 5:24", "authors": [ { "first": "Stephen B", "last": "Pruett" }, { "first": "Eric L", "last": "Padgett" } ] }
PMC534101	Background ========== Leishmaniasis is endemic in several parts of the world, with a global prevalence of over 12 million cases and 1.5--2 million new cases emerging every year \[[@B1]\]. The infection is caused by protozoan parasites of the genus Leishmania, transmitted through the bite of the sand fly vector. Several Leishmaniaspecies are able to cause a wide spectrum of clinical manifestations, ranging from the mild cutaneous form, the disfiguring mucosal form and the life-threatening visceral form, also known as kala-azar. In Brazil, Leishmania (L.) braziliensiscauses cutaneous and mucosal disease, L. amazonensiscauses cutaneous and, sporadically, visceral disease, while L. chagasiis exclusively associated with visceral disease. The clinical outcome of infection thus not only depends on the species involved, but also on the patient\'s immunocompetence. In recent years, a protective immune response against intracellular pathogens, such as Leishmania, Listeriaand mycobacteria, has been defined as type 1 (Th1), whereas protection against extracellular pathogens, such as helminths, requires a type 2 (Th2) response. The murine model of experimental leishmaniasis has been instrumental for the elaboration of the Th1/Th2 paradigm, inasmuch as the preferential action of Th1 (IFN-γ, IL-12, TNF-α) or Th2 cytokines (IL-4, IL-5, IL-10) results in cure or progression of the disease, respectively \[[@B2],[@B3]\]. In human leishmaniasis, this Th1/Th2 dichotomy is much less explicit for in vitro or ex vivo cytokine production. However, striking differences in cellular (lymphoproliferation and IFN-γ production) and humoral (total and anti-LeishmaniaIgG) immune response can be observed in different clinical forms of the disease. Our group has recently shown that patients with localized cutaneous leishmaniasis (LCL) display a diminished Th1 response during the early phase of disease, which is reverted after treatment \[[@B4]\]. In mucosal leishmaniasis (ML), on the other hand, an exacerbated Th1 response with increased IFN-γ and TNF-α levels, is believed to provoke tissue destruction \[[@B5]\]. In patients with visceral leishmaniasis (VL), characterized by immunosuppression and absence of IFN-γ production \[[@B6]\], we were able to show the beneficial effect IFN-γ in vivo\[[@B7]\]. Although zinc deficiency has been shown to lead to a selective Th1 deficiency in human volunteers \[[@B8]\], only few data are available on the role of trace elements in human leishmaniasis, being restricted to Old World LCL, showing increased serum copper and decreased serum zinc in Turkish LCL patients infected by L. major\[[@B9]\]. In this study, we investigated if Zn and Cu levels differ in different clinical forms of the disease, and if these trace metals might be correlated to anti-parasite immune response. Methods ======= Blood samples (10 ml heparinized tubes, Vacutainer) from 21 patients and 15 healthy controls (mostly patient\'s relatives) were obtained in an outpatient clinic in the district of Corte de Pedra, (Bahia state, Northeast of Brazil). This rural area has a low socio-economic status and a high incidence of infection with Leishmania braziliensisand, sporadically, Leishmania amazonensis. During a one year period, 14 patients with LCL (single lesion with less than 4 weeks of duration) were selected and treated (20 mg/kg of Sb (Glucantime) i.v. during 20 days). Of those, only 7 patients returned to draw blood after 3 months of treatment, but all patients cured during follow-up. Seven patients with ML were selected after several rounds of unsuccessful treatment and severe disease progression. Blood samples from 10 patients (at diagnosis, before treatment) with VL were obtained from two different urban areas (Salvador-Bahia and Teresina-Piaui). Ten healthy urban controls were recruited among students and laboratory staff (Salvador-Bahia). Diagnosis was confirmed by Montenegro skin test, serology, direct culture of parasites from lesions \[[@B4]\] or bone marrow aspiration for VL \[[@B7]\]. This study was approved by the Ethics Committee of the University Hospital Edgard Santos, Salvador. Cu and Zn were quantified by atomic absorption spectrophotometry (Varian 220) using an air/acetylene flame. One ml of plasma was diluted tenfold with 0.05 % Triton X-100, 1 % HNO3, sonicated for 10 min and analyzed in triplicate. All reagents used were analytical grade (Merck). Due precautions were taken to avoid external and internal (hemolysis) trace metal contamination. In order to investigate the influence of trace metals, antibodies and other endogenous plasma components, such as cytokines, on the ex vivocellular immune response, we used a recently described model using whole blood and live Leishmaniapromastigotes \[[@B10]\], closely mimicking the early in vivoevents following a sandfly bite. Whole blood was diluted tenfold in culture medium (RPMI supplemented with L-glutamine and gentamycin, all from Gibco-BRL) and stimulated with L. amazonensispromastigotes (10^5^/mL). Buffy coats from normal blood donors were used to obtain large quantities of cells required for in vitroexperiments to examine the effect of exogenous trace metals upon cytokine production. Mononuclear cells were separated by density gradient centrifugation (Ficoll-Paque, Pharmacia, Uppsala, Sweden) and cultured in complete culture medium (supplemented with 10 % fetal calf serum, Gibco-BRL). Cu and Zn concentrations were below the detection limit in RPMI and less than 2 μM in complete culture medium. Supernatants were collected after 72 h of culture and frozen in aliquots for cytokine determination. Lymphoproliferation was quantified by measuring \[H\]-thymidine incorporation after 120 h of culture. IFN-γ, TGF-β1, TNF-α and IL-5 in plasma or culture supernatants were quantified using commercial ELISA kits (DuoSet, R&Dsystems). All results are expressed as mean ± SEM. Statistical evaluation of data was performed using GraphPad Prism software: Mann-Whitney test for comparing patients and control groups, Spearman rank test for correlation and Wilcoxon signed rank test for comparing in vitrotreatments; a p-value \<0.05 was considered significant. Results ======= A significant decrease in plasma Zn was observed for both LCL and ML patients, as compared to controls from the same endemic area (0.80 ± 0.04 and 0.77 ± 0.05 vs. 1.01 ± 0.06 μg/mL, p \< 0.01, Figure [1A](#F1){ref-type="fig"}), and in VL patients, as compared to urban controls (0.55 ± 0.08 vs. 0.83+/-0.03 μg/mL, p \< 0.001, Figure [1A](#F1){ref-type="fig"}). Zn deficiency (plasma Zn \<0.65 μg/mL), however, was observed only in VL (7/10) and ML (1/7) patients. As shown in Fig. [1B](#F1){ref-type="fig"}, plasma Cu was significantly increased in LCL (1.32 ± 0.10 vs. 1.01 ± 0.05 μg/mL, p \< 0.001) and VL (1.42 ± 0.13 vs. 0.72 ± 0.06 μg/mL, p \< 0.001), but not in ML (1.04 ± 0.05 vs. 1.01 ± 0.05 μg/ml). After three months of treatment, plasma Zn increased and Cu decreased in LCL patients, resulting in values indistinguishable from endemic controls. Although within normal physiological ranges \[[@B11]\], Cu and Zn levels were significantly increased in healthy controls from the endemic area, as compared to urban controls (p \< 0.01, Fig, [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}). Cu/Zn ratios, however, were similar in both control groups (p = 0.12), but significantly increased in all three patient groups (Fig. [1C](#F1){ref-type="fig"}), reaching a three-fold molar excess of Cu to Zn in VL patients. To determine if these observations reflect possible changes in the patients\' immune status, we quantified humoral and cellular anti-Leishmaniaimmune response ex vivoand correlated them to trace element levels. When comparing patient groups, a stepwise increase in anti-LeishmaniaIgG and Cu/Zn ratio can be observed, with ML\<LCL\<VL (Table [I](#T1){ref-type="table"} and Fig. [1](#F1){ref-type="fig"}). In addition, a highly significant correlation between plasma Cu, but not Zn or Cu/Zn ratio, and anti-LeishmaniaIgG was observed across patient groups (Fig. [2](#F2){ref-type="fig"}, Spearman r = 0.65, p = 0.0028). Since lymphoproliferation and IFN-γ production were virtually absent in VL, correlation with trace element levels across patient groups could not be calculated, but an inverse order was observed, with ML\>LCL\>\>VL (Table [I](#T1){ref-type="table"}) as compared to Cu/Zn ratio with ML\<LCL\<\<VL (Fig. [1](#F1){ref-type="fig"}), confirming reciprocal regulation of Th1/cellular immune response and Th2/humoral immune response. However, we found a significant negative correlation between plasma Cu and ex vivoIFN-γ production (Spearman r = -0.86, p = 0.024) in untreated patients only, whereas no significant correlation was observed for Leishmania-or mitogen-induced lymphoproliferation, TGF-β1, TNF-α and IL-5 levels in plasma or culture supernatants (not shown). To verify the hypothesis that increased Cu levels might down-regulate IFN-γ production, we added Cu (10 μM, corresponding to the increase of plasma Cu observed ex vivo in LCL patients) to mitogen-or anti-CD3-stimulated in vitrocultures from healthy controls. As shown in Figure [3](#F3){ref-type="fig"}, 10 μM of CuCl~2~significantly decreased anti-CD3-induced IFN-γ production (40.5 ± 9.3 % inhibition, p \< 0.05). Interestingly, the addition of physiological concentrations of Zn (10--30 μM) to ex vivoor in vitrocultures did not revert the apparently inhibitory effect of endogenous or exogenous Cu on IFN-γ secretion. (not shown). Discussion ========== Although plasma Zn was significantly decreased in all three patient groups, plain Zn deficiency was only observed in seven VL patients and one ML patient, being absent in LCL patient and in both control groups. In parallel, plasma Cu in VL patients increased to levels which have been shown to be toxic in vitro\[[@B12]\]. In addition, a highly significant positive correlation between plasma Cu and parasite-specific IgG across patient groups suggests that the trace element might interfere in anti-Leishmaniaimmune response, e.g. by leading to a non-protective Th2/humoral immune response, known to be exacerbated in visceral leishmaniasis. Increased plasma Cu cannot be considered as a mere marker of inflammation, since it was not observed in ML, a chronic inflammatory condition characterized by high production of pro-inflammatory cytokines, such as TNF-α and IFN-γ \[[@B5]\]. Absence of correlation between TNF-α and trace metal levels underscores the specificity of the inhibitory effect of Cu upon IFN-γ production, which might in fact be the upstream event to an increased humoral anti-Leishmaniaresponse. Since our in vitrofindings indicate Zn as a monocyte/macrophage activator \[[@B13]\], the significant decrease in Zn in ML patients might be responsible for the inability of the patients to clear the parasite, in spite of high IFN-γ levels and several rounds of treatment. LCL patients represent an intermediate group between ML and VL, displaying a detectable, but variable humoral and cellular immune response with production of both Th1 and Th2 cytokines, undergoing a shift towards the Th1/cellular pole after successful treatment \[[@B4]\]. We observed a reciprocal association between Cu/Zn levels and humoral and cellular immune response between the three patient groups (Table [I](#T1){ref-type="table"} and Fig. [1](#F1){ref-type="fig"}), as well as a complete reversal of increased Cu/Zn ratios after treatment in LCL patients. Taken together, these data indicate that Cu/Zn imbalance might serve as a marker for decreased Th1 response and immunodeficiency in leishmaniasis, being more pronounced in its most severe and possibly fatal visceral form. Unfortunately, no clinical follow-up was possible in VL patients, but mortality, mostly due to co-infections, and therapeutic failure occur in 6,1 % of the cases \[[@B14],[@B15]\]. It is tempting to speculate that environmental exposure to copper might increase susceptibility to Leishmaniaand other intracellular pathogens, such as Listeriaand mycobacteria, e.g. by directly interfering with cytokine production as previously shown \[[@B16]\]. Increased plasma Cu in endemic controls might reflect the use of Cu as a fungicide in cocoa plantations in the Corte de Pedra area. It should be stated that spouses and relatives were preferentially chosen as endemic controls, since environmental exposure and nutritional status are far more important determinants for Cu and Zn levels than sex or age \[[@B11]\]. In addition, (epi)genetic factors related to copper homeostasis might render normal individuals more susceptible to copper toxicity \[[@B17]\]. Thus, increased Cu levels and decreased Zn levels might be a cause, rather than a consequence of LCL. On the other hand, absence of Cu increase, linked to uncontrolled IFN-γ production, might underlie evolution towards ML. Long-term follow-up of treated patients and comparison with other endemic areas might learn if trace metal levels have predictive value for clinical evolution of and/or susceptibility to leishmaniasis. In addition, we propose that trace metal levels should be taken into account in vaccine strategies for leishmaniasis, because of the importance of Zn in a protective Th1 response \[[@B8]\] and because of the possibly deleterious effect of Cu described in this study. Two recent large-scale vaccination trials for cutaneous and visceral leishmaniasis \[[@B18],[@B19]\] were carried out in regions where Zn deficiency is prevalent, namely Iran and Sudan, which might have contributed in part to the low protection rate observed in both trials. Administration of Zn in vivo has been shown to down-regulate increased Cu levels in patients with Wilson disease and to revert its toxicity \[[@B20]\], suggesting that systemic administration of Zn might be beneficial in addition to its direct immunostimulatory effect \[[@B8]\]. A recent report \[[@B21]\] demonstrated the safety and efficiency of oral Zn in Old World cutaneous leishmaniasis, a mild and self-healing form of the disease, with patients displaying normal Zn levels. Our results strongly suggest that zinc therapy should be considered in the mucosal and visceral forms of leishmaniasis, associated with high morbidity and mortality, as well as frequent failure of antimonial therapy. Conclusions =========== Zn deficiency in visceral and mucosal leishmaniasis indicate possible therapeutic administration of Zn in these severe forms of leishmaniasis. Plasma Cu positively correlates to (non-protective) humoral immune response across patient groups, and reciprocally, increased Cu levels decreased in vitro(protective) IFN-γ production, implying that environmentally or genetically determined increases in Cu levels might augment susceptibility to infection with intracellular pathogens. Our data indicate that Cu/Zn imbalance can be a useful marker for immune dysfunction in leishmaniasis and suggest that trace metals are implicated in both humoral and cellular anti-Leishmaniaimmune response, which should inspire future strategies for therapy and immunoprophylaxis of human leishmaniasis. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JVW designed the scientific project, analyzed the data and wrote the paper. GS was responsible for sample processing and data collection. AD selected LCL and ML patients, as well as controls from the endemic area. CHC selected VL patients. AFS did the trace metal analysis. AB and EMC supervised the field work and reviewed the paper. MBN analyzed the data and reviewed the paper. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/4/50/prepub> Acknowledgements ================ We thank the patients, their families, the field workers and the medical staff from the Corte de Pedra outpatient clinic; André Bafica, Cristiano Bahia, Daniele Decanine, George Soares, Jorge Tolentino, Maria da Purificaçao Silva and Silvia Cardoso for practical assistance; Dr Maria das Graças Korner for advice on trace element analysis; and Dr. Claudia Brodskyn, Dr. Mitermayer dos Reis and Dr. Roque Almeida for critical reading of the manuscript. Financial support: NIH (TMRC grant AI-30639), CNPq (Brazilian National Research Council) and PRONEX. EMC, AB and MBN are CNPq investigators. GS received a PIBIC-UFBA and CAPES fellowship. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Plasma levels of Zn and Cu in controls and patients. Bars represent mean (+/-SEM) Zn (A) and Cu (B) plasma levels, and plasma Cu/Zn ratios (C) of urban (U) and endemic (E) controls; and patients with mucosal leishmaniasis (ML), localized cutaneous leishmaniasis before (LCL0) and after 3 months of treatment (LCL3), and visceral leishmaniasis (VL). \p \< 0.05, \\p \< 0.01, \\\p \< .001 for LCL0 and ML vs. E and for VL vs. U (Mann-Whitney test). ::: ![](1471-2334-4-50-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### *Correlation between plasma Cu and anti-LeishmaniaIgG. Plasma levels of Cu and anti-LeishmaniaIgG were positively correlated in patients with localized cutaneous leishmaniasis before treatment (LCL), mucosal leishmaniasis (ML) and visceral leishmaniasis (VL) (Spearman r = 0.65, p = 0.0028). ::: ![](1471-2334-4-50-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### In vitro effects of exogenous Cu upon IFN-γ production in healthy controls. Mononuclear cells from normal donors were incubated for 48 h in the absence or presence of anti-CD3 (200 ng/ml), Cu (10 μM CuCl^2^) or both. Bars represent mean (+/-SEM) IFN-γ concentration in supernatants from six different donors. Unstimulated cells or cells stimulated with Cu alone did not produce IFN-γ. \p \< 0.05 vs. anti-CD3-stimulated cells (Wilcoxon signed rank test). ::: ![](1471-2334-4-50-3) ::: ::: {#T1 .table-wrap} Table I ::: {.caption} ###### Characteristics and immune parameters of patient groups and controls ::: Group (n) Age Male/Female *Anti-LeishmaniaIgG (O.D.)* Lympho-proliferation (cpm)^b^ IFN-γ (pg/ml)^b^ --------------------------- ------------- ----------------- --------------------------------- ----------------------------------- ---------------------- Urban controls (10) 24.9 (2.0) 7/3 \< cut-off \< 100 \< 20 Endemic controls (15) 28.7 (8.8) 3/12 \< cut-off^a^ ND \< 20 LCL0 (14) 28.4 (12.7) 10/4 0.178 (0.073) 2737 (1178) 612 (207) LCL3 (7) 28.5 (11.2) 5/2 0.074 (0.021) ND 685 (348) ML (7) 44.4 (11.9) 5/2 0.016 (0.005) \> 5000 \> 2000 VL (10) 18.4 (3.8) 6/4 0.188 (0.072) \< 100 \< 20 Results are expressed as mean (SEM). ND: not done. LCL0 and LCL3, localized cutaneous leishmaniasis at 0 and 3 months of treatment, ML mucosal leishmaniasis, VL visceral leishmaniasis. ^a^One endemic control displayed positive serology and was discarded from analysis. ^b^Following stimulation of tenfold diluted whole blood with live Leishmaniapromastigotes. :::	PubMed Central	2024-06-05T03:55:49.872918	2004-11-17	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534101/", "journal": "BMC Infect Dis. 2004 Nov 17; 4:50", "authors": [ { "first": "Johan", "last": "Van Weyenbergh" }, { "first": "Gisélia", "last": "Santana" }, { "first": "Argemiro", "last": "D'Oliveira" }, { "first": "Anibal F", "last": "Santos" }, { "first": "Carlos H", "last": "Costa" }, { "first": "Edgar M", "last": "Carvalho" }, { "first": "Aldina", "last": "Barral" }, { "first": "Manoel", "last": "Barral-Netto" } ] }
PMC534102	Background ========== The organisms in a log phase, actively multiplying culture of Mycobacterium tuberculosisall grow well on plates and are estimated as colony forming units (cfu). However, cultures that have been grown undisturbed in the depths of liquid medium for 100 days contain a majority population which grows in liquid medium but is not able to form colonies on solid medium \[[@B1],[@B2]\]. Since the bacilli in such cultures are hardly multiplying and have an uptake of \[^3^H\] uridine of only 15% of log phase cultures \[[@B1]\], they may be considered as dormant. A smaller population, that could grow on plates as well as in broth, possibly the survivors of the log phase bacilli, has also been demonstrated in these cultures. At the end of a 10-month mouse experiment on vaccines, we questioned whether the same two populations would be found, particularly in view of the evidence that there are similarities in gene expression patterns following adaptation to micro-aerophilic conditions in stationary cultures and exposure to NO in macrophages \[[@B3]\]. We therefore estimated populations present in the chronic infections in the organs of the 10-month mice both by conventional plate counts and by counts of the probable number of viable organisms obtained from serial dilutions in liquid medium. Similar counts were also set up, as a control, in a short-term acute infection in mice. Methods ======= Culture media and bacteria -------------------------- The media used were 7H9 liquid medium with 10% albumin, dextrose, catalase supplement and 0.05% Tween 80, and 7H11 agar medium with 10% oleic acid, albumin, dextrose, catalase supplement (Becton Dickinson, Oxford, UK). They were made selective by the addition of 100 μg carbenicillin, 200 U polymyxin B, 20 μg trimethoprim and 10 μg amphotericin B per ml (Mast, Bootle, UK) \[[@B4]\]. The two experiments both used 6-week, female Balbc mice, which were infected, with a mouse passaged H37Rv strain of M. tuberculosissuspended in 0.1% gelatin. The spleens and lungs were obtained at sterile autopsy and were homogenised as described elsewhere \[[@B5]\] in 5 ml water. From this suspension 100 μl amountsfrom the neat suspension and from serial 10-fold dilutions in 1 ml, were inoculated onto duplicate thirds of selective 7H11 medium plates. The number of colonies was counted after 3--4 weeks incubation at 37°C to give the plate count. A negative plate therefore had \<25 cfu / organ. For the broth counts, serial 10-fold dilutions of the organ homogenate were made, in triplicate, in 1 ml amounts to 9 ml amounts of selective 7H9 broth with 0.05% Tween 80 in plastic 28 ml screw-capped bottle. In the acute infection, 10 serial dilution were set up, so as to obtain 30 tubes in all, while in the chronic infection 6 serial dilutions were set up yielding 18 tubes in all. These were incubated at 37°C and examined at 3 and 6 weeks and finally at 9 weeks for the characteristic growth of M. tuberculosis, namely a clear supernatant in undisturbed cultures with an upwards swirl of white growth on shaking,. Probable numbers of bacilli (pnb) per organ were obtained from a table of densities of organisms estimated by the dilution method \[[@B6]\]. Samples of the positive growth from 18 broth cultures were plated out on 7H11 medium. Acute infection experiment -------------------------- Each of 6 mice was infected by the intravenous route with 200 μl of a suspension of a containing 2.6 × 10^6^cfu of the H37Rv strain. Plate and broth counts were carried out 7 days later. Chronic infection experiment ---------------------------- Each of 88 mice was infected by the intra-peritoneal route with 200 μl of a suspension containing 3.1 × 10^3^cfu of the H37Rv strain. Mice in our experiments are usually housed in an isolator, connected by a tunnel port to a Class 1 safety cabinet through which air from the environment is sucked. The intra-peritoneal route was chosen so that mice could be kept throughout the experiment in the isolator to prevent cross infection with mouse pathogens during exposure to the outside air in the Class 1 cabinet. One day after infection, samples of 6 mice yielded scanty or no colonies in plate counts of spleens and lungs. After a further 4 weeks these organ counts in 6 mice had risen to 2.24 × 10^4^in spleens and 1.15 × 10^4^in lungs. The mice were then divided into 6 experimental groups, 4 of which were vaccinated with various DNA vaccines over a 4-week period and 2 were unvaccinated controls. At 12 weeks after infection only 9 of 36 lungs and 18 of 36 spleens yielded positive growth on plates. At 10 months after infection, the 36 remaining mice were sacrificed, and plate and broth counts were set up on all, using dilutions estimated from a sample of 4 mice sacrificed 3 weeks earlier. Statistics ---------- The results of the plate and broth counts were examined by 2-way analysis of variance using the Stata package, release 8 (Stata Corp., College Station, TX) Results ======= As the experimental vaccines appeared to have only small effects, which will be reported elsewhere, in the chronic infection model, the results in all 36 mice at 10 months are considered together. A typical broth count is shown diagrammatically in Table [1](#T1){ref-type="table"}. Note that there were never any sporadic positive tubes in the no growth zones (inoculated in the example with 10^-4^or 10^-5^dilutions) of any set of broth cultures. The results in the 6 mice in the acute infection experiment and in 27 of the 36 mice in the chronic infection experiment that had assessable numbers of bacilli estimated as cfu by the plate method and as pnb by the broth method are set out in Table [2](#T2){ref-type="table"}. In the analysis of variance of the acute infection counts, neither the variation between individual mice nor the difference between the counting methods was statistically significant. However, in the chronic infection, the broth counts were higher than the plate counts. In the spleens, the mean broth count was 5.290 log~10~cfu / organ and the plate count was 3.858 log~10~cfu /spleen. The difference between these counts is 1.432 log~10~cfu / spleen (27-fold) so that, on the assumption that all bacilli that grew on plates also grew in broth, the bacilli capable of growing on plates comprised 3.7% (100/27) of the total count. In the lungs the mean broth count was 5.523 log~10~cfu /lungs, about 73-fold higher than the plate count for the lungs. Thus the bacilli able to grow on plates comprised about 1.4% (100/73) of the total. The differences between individual mice were significant (p = 0.01) and highly significant between the counting methods (p \< 0.001). In the remaining 9 mice no colonies were obtained on the neat dilution plates in either the lungs alone or in both lungs and spleen (Table [3](#T3){ref-type="table"}). However, broth counts were obtainable from both organs in all 9 mice, though their mean values were appreciably lower than those in Table [1](#T1){ref-type="table"}. Where a comparison could be made between counting methods in the spleens of the 7 mice with colonies in plate counts, the means of the broth counts (4.347 log~10~cfu / ml) were 19-fold higher than the corresponding plate counts (3.079 log~10~cfu / ml, giving 5.3% of the total), in approximate agreement with the 27-fold (3.7%) estimate of the difference between broth and plate counts obtained from Table [1](#T1){ref-type="table"}. The following changes occurred in the broth counts during incubation. The counts between the 3-week and the 6-week readings increased on average in the lungs of each acute mice by 2.3 tubes and by 4.7 tubes in the spleens, and in each of the chronic mice by 2.0 tubes in lungs and 1.6 tubes in spleens. Thereafter, the increase from 6 weeks to 9 weeks was 1.5 tubes in the lungs of acute mice and 0.83 tubes in their spleens, while the increases in the lungs of chronic mice were 1.3 tubes and 1.0 tube in the spleens. Since an increase of 1 tube indicates a rise of about log~10~0.8, that is about 12%, in the count, it is evident that counts increased during incubation, rapidly between 3 and 6 weeks and slowly between 6 and 9 weeks. Discussion ========== The chronic infection experiment was unusual in that the intraperitoneal infection in vaccinated mice led to trapping of the bacilli in the peritoneal cavity, so that few bacilli reached the organs, and thus the plate counts were sometimes negative, with a count of less than 25 cfu / organ. Variation, considerably greater than after intravenous or airborne infection, was also evident, the SD of the 4-week spleen counts, expressed as log10 cfu/organ, being 0.51 as compared to 0.23 for intravenous infection \[[@B5]\]. Evidence that growth in the broth cultures was M. tuberculosiswas provided by the growth of typical colonies on 7H11 plates. That it was not due to sporadic contamination was shown by the usual complete absence of contamination in selective media, by a clear supernatant in the unshaken cultures and by the absence of any growth in the \"no growth\" zones of the broth cultures. In log phase cultures (Hu Y-M, personal communication) and in the acute infection of mice, similar estimates of viable organisms were obtained in plate and broth counts However, our best estimate indicated that the bacilli in the chronic infections that would grow on plates was about 3.7% of the total population in spleens and 1.4% in lungs. This can be compared to the findings on a culture in 7H9 broth grown undisturbed for 100 days in the depths of liquid medium with caps screwed tightly on. In such a 100-day culture, population A, capable of growth in broth but not on plates, was estimated by broth dilution counts to be 7.60 log~10~pnb / ml, while a smaller population B, that grew on plates, was estimated from parallel plate counts as 5.85 log~10~cfu / ml.\[[@B1]\] Thus, population A was 1.75% of the total population. The corresponding estimates for a 30-day static culture were population A = 9.983 log10 pnb / ml and population B = 8.013 log10 cfu /ml, so that population B comprised 1.07% of the total population (Hu Y-M, personal communication). These estimates suggest that the extent to which bacilli have gone into ill-defined dormancy might be measured as the proportion of a total bacterial population that can grow on plates. The lower this proportion, the greater the overall degree of \"dormancy\". Whatever, the theoretical significance of this simple technique for measuring dormancy, there is a practical implication for those undertaking long-term mouse experiments. Some end such an experiment with plate counts and others with culture in liquid medium. Those using only plate counts may be seriously underestimating the residual populations. It is also clear that there is much work to be done in seeing how various experimental conditions, such as the duration of the infection and the immune state of the mice, affect the ratio between broth and plate counts. Those exploring the development of new drugs need to know how these two populations respond to current anti-tuberculosis drugs and to novel drugs. Competing interests =================== The author(s) declare that they have no competing interests. Authors contributions ===================== All three authors took part in the running of the experiments with JD contributing the most. DAM contributed the concept of parallel broth and plate counts. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2334/4/51/prepub> Figures and Tables ================== ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Example of a broth count from a chronic infection mouse ::: Tubes -------- ------- ---- ---- 10^-5^ 0 0 0 10^-4^ 0 0 0 10^-3^ 0 \+ 0 10^-2^ \+ \+ \+ 10^-1^ \+ \+ \+ 10^0^ \+ \+ \+ The mean fertile level (X) = no. of fertile cultures (10) / no. of cultures at each level (3). K = 0.760 from Table \[6\]. Log pnb = X-K + log5 (organ in 5 ml) = 3.272 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Plate and broth counts of M. tuberculosisin spleen and lungs of mice with acute and chronic infections ::: Infection Mouse organ Type of count\* Mean count SD 95% confidence limits ------------------- ------------- ----------------- ------------ ------- ----------------------- ------- Acute (6 mice) Spleen Plate 7.070 0.214 6.845 7.295 Broth 6.663 0.771 5.854 7.472 Lungs Plate 6.703 0.095 6.603 6.803 Broth 6.609 0.421 6.167 7.050 Chronic (27 mice) Spleen Plate 3.858 0.882 3.509 4.207 Broth 5.290 0.820 4.966 5.614 Lungs Plate 3.662 1.462 3.084 4.240 Broth 5.523 0.939 5.152 5.894 \* Plate counts are log~10~cfu/organ. Broth counts are log~10~probable number of bacilli/organ ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Plate and broth counts of M. tuberculosisin spleen and lungs of mice with chronic infections, when plate counts had no colonies ::: Organ with no colonies in plate counts\* No. of mice Organ Type of count^†^ Mean count SD 95% confidence limits ------------------------------------------ ------------- -------- ------------------ ------------ ------- ----------------------- -------- Spleen & lungs 2 Spleen Broth 3.728 0.708 -2.638 10.094 Lungs Broth 4.247 1.428 -8.580 17.073 Lungs only 7 Spleen Plate 3.079 0.960 2.192 3.968 Broth 4.347 0.823 3.586 5.108 Lungs Broth 4.202 0.624 3.625 4.778 \* A count of \<1.40 log~10~cfu/organ ^†^Plate counts are log~10~cfu/organ: broth counts are log~10~probable number of bacilli/organ :::	PubMed Central	2024-06-05T03:55:49.875016	2004-11-17	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534102/", "journal": "BMC Infect Dis. 2004 Nov 17; 4:51", "authors": [ { "first": "Jasvir", "last": "Dhillon" }, { "first": "Douglas B", "last": "Lowrie" }, { "first": "Denis A", "last": "Mitchison" } ] }
PMC534103	Background ========== Lipoprotein lipase (LPL) hydrolyses triglycerides (TG) in circulating lipoproteins \[[@B1],[@B2]\]. This is a necessary first step in catabolism of the TG-rich lipoproteins as evidenced by the massive hypertriglyceridemia in patients with genetic deficiency of the enzyme \[[@B3]\]. Fine-tuned regulation of LPL activity is an important aspect of energy homeostasis \[[@B4]\]. Patients on chronic hemodialysis (HD) often have reduced LPL activity and derangements of lipoprotein profiles \[[@B5]-[@B7]\]. During HD, conventional heparin is commonly used as anticoagulant and this releases LPL from its binding sites at the vascular endothelium into the circulation. It has been suggested that repeated heparinisation may induce release and subsequent degradation of LPL that exceeds the rate of enzyme synthesis and thereby causes a depletion of LPL stores \[[@B8]-[@B11]\]. In a previous study we followed the LPL activity and the TG changes during a dialysis-session using conventional heparin as anticoagulant \[[@B12]\]. There was a peak of LPL activity accompanied by a reduction in TG during the first hour, followed by a decrease in LPL activity to a stable plateau during the remaining session while TG increased towards and beyond the original baseline. When compared to a group of healthy control subjects, the peak LPL activity was only about 50 % in the HD-patients while the plateau activities were comparable. Our interpretation was that the functional pool of LPL, represented by the initial peak, was impaired in HD-patients, while the production of lipase molecules, reflected by the plateau, was only marginally reduced. In recent years, conventional heparin has increasingly been replaced by various low molecular weight (LMW) heparins. A major argument is the ease of administration \[[@B13],[@B14]\]. A single injection of a LMW heparin can often replace a primed infusion of conventional heparin. The increase in plasma LPL activity is less pronounced after LMW compared to conventional heparin \[[@B15]\], and it has been suggested that this causes less disturbance of lipoprotein metabolism \[[@B10]\] although this conclusion has been questioned \[[@B16]\]. Direct studies of the lipase-heparin interaction have shown that a heparin decasaccharide is enough to fill the heparin-binding grove on the lipase molecule \[[@B17],[@B18]\]. Decasaccharides fall in the middle or lower size range in preparations of LMW heparins \[[@B19]\]. Several lines of evidence indicate that also in biological systems, decasaccharides are sufficiently long to exert full effect on LPL. Chevreuil et al. found that on a weight basis, decasaccharides released more LPL from perfused rat hearts than conventional heparin did \[[@B20]\]. Several groups have reported that LMW heparins or decasaccharides release LPL from tissues in vitro or from cultured cells as efficiently as or even more efficiently than conventional heparin does. It is therefore unlikely that the lower plasma LPL activities after LMW heparin are due to less release of the lipase. More likely, LMW retards clearance of the lipase by the liver less efficiently than conventional heparin does. Two studies have directly demonstrated such a difference in liver perfusion experiments \[[@B20],[@B21]\]. In a recent study we infused a LMW heparin (dalteparin) for eight hours to healthy volunteers to explore the influence on LPL activity and TG response \[[@B22]\]. The peak LPL activity was only about 30%, and the subsequent plateau activity only about 40%, compared to the activities observed during a similar infusion with conventional heparin. A bolus of conventional heparin given when the LPL activity had levelled off to a plateau brought out about the same amount of activity irrespective of if the subjects had been infused with dalteparin or conventional heparin. We concluded that dalteparin and conventional heparin had reduced the peripheral stores of LPL to a similar extent and that the difference in plasma levels of LPL activity was due to a more rapid hepatic clearance of the LPL-dalteparin complex. There was a tendency towards a more pronounced increase in TG after dalteparin compared to conventional heparin, indicating that lipoprotein metabolism might be more, rather than less, disturbed by the use of LMW heparin. As HD-patients are increasingly subjected to repeated treatment with LMW heparin during dialysis we have now followed LPL activity and TG during a dialysis-session with dalteparin using the same regime as in our previous study with conventional heparin, i.e. a primed infusion \[[@B12]\]. Methods ======= Subjects and protocol --------------------- The study design was based on the protocol used in a previous investigation in which nine HD-patients were studied during a dialysis-session using conventional heparin as anticoagulant \[[@B12]\]. The present study, on the same HD-patients, was performed three months later with a LMW heparin (dalteparin, Pharmacia, Stockholm, Sweden) as anticoagulant. In all other respects the dialysis regime, as well as medication and diet recommendations, was kept unchanged. The median age was 73 years and the median BMI was 24.7. The diagnoses were diabetes nephropathy (BE), chronic pyelonephritis (AJ), nephrosclerosis (HB), polycystic kidney disease (MK, CH), chronic glomerulonephritis (RH, RS, KL) and in one patient the origin was unclear (BV, not biopsied). The patients had been on maintenance hemodialysis for 5--38 months and were treated with bicarbonate hemodialysis either two (RS, HB) or three (MK, BE, KL, AJ, RH, CH, BV) times a week, depending on residual renal function. All dialyses were performed with hemophan dialysers (GFS+16, GAMBRO, Lund, Sweden) and Biosol dialysis solution (Pharmalink, Stockholm, Sweden). A central dialysis catheter was used as dialysis-access in five of the patients and an arteriovenous-fistula/graft in four. The patients were treated with antihypertensive drugs (ACE-inhibitors, beta-blockers, calcium channel inhibitors), diuretics, sodium bicarbonate and phosphate-binding drugs. The diabetic patient was non-insulin dependent and was treated only by diet recommendations. One patient (RS), having a rejecting renal transplant, was treated with low doses of corticosteroids and cyclosporine. No one was treated with lipid lowering drugs. The experiments were carried out after an overnight fast, and 48--96 hours had passed since the previous hemodialysis. A loading dose of 40 IU dalteparin per kg body weight was given, followed by a continuous infusion of 1000 IU/hour, in accordance with the manufacturer\'s recommendations. Blood samples were drawn before start and then regularly at 15, 30, 60, 120, 180 and 240 minutes. According to existing routines, the patients had a combined breakfast/lunch, containing 25 g fat, about two hours after the dialysis was started. The ethical committee approved the study and informed consent was obtained from all patients prior to participation. Handling of samples and assay methods ------------------------------------- Blood samples for measurement of LPL activity were collected in heparinized tubes. They were immediately chilled in ice water and centrifuged in a cooling centrifuge within 15 minutes. The plasma was frozen at -20°C and then stored at -70°C until analyses. LPL activity was measured as described \[[@B23]\] using an emulsion containing a trace amount of \[^3^H\]-oleic acid-labelled triolein, 100 mg soybean TG and 10 mg egg yolk phospholipids per mL, prepared by Fresenius-Kabi, Uppsala, Sweden. Hepatic lipase was inhibited by pre-incubation of the plasma samples with immunoglobulins from a rabbit antiserum to human hepatic lipase. The assay medium contained a relatively high concentration of heparin, and possible differences in the heparin concentration or type in the sample would not affect the activity. All assays were made in triplicate and the mean value was used. A standard sample of human post-heparin plasma was run on each assay day and the value was used to calibrate for between-assay-variations. LPL protein mass was determined with an enzyme-linked immunosorbent assay, as previously described \[[@B24]\], using immunoaffinity-purified chicken antibodies raised against bovine LPL for capture and the monoclonal antibody 5D2, also raised against bovine LPL, for detection (a gift of Dr. J. Brunzell. Seattle, Washington, USA). Blood samples for lipid determination were drawn in tubes without anticoagulant, immediately chilled in ice water, centrifuged and frozen as described above. Total cholesterol, HDL-cholesterol and TG were determined by routine methods on a multianalyzer (Vitros 950 IRC; Johnson & Johnson, Clinical Diagnostics Inc, New York, NY, USA). Baseline LDL-cholesterol levels were calculated using the Friedewald formula \[[@B25]\]. Antifactor Xa activity was determined using a chromogenic substrate (COACUTE^®^, Chromogenix AB, Mölndal, Sweden). Statistics ---------- The values are expressed in terms of median and range and were examined for significant differences by paired Wilcoxon signed-rank test. Simple linear regression and the Spearman rank correlation test were used to evaluate relationships between variables. Two-tailed P values below 0.05 were considered to be statistically significant. Results ======= Baseline data ------------- Table [1](#T1){ref-type="table"} gives baseline data for the HD-patients at the time of the dialysis-session with dalteparin. There were no significant differences compared to the values at the time of the dialysis with conventional heparin three months earlier \[[@B12]\]. There was no significant difference between the ultrafiltration rates during the two dialyses. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Baseline data for the HD-patients at the time for the dialysis-session with dalteparin. Median values for the dialyses with dalteparin (D) and conventional heparin (H), respectively. There were no statistically significant differences between values on the two occasions. ::: HD-patients Gender Age (years) Dbw\* (kg) Ultra-filtration(L) BMI (kg/m^2^) TG (mmol/L) Cholesterol (mmol/L) HDL (mmol/L) LDL (mmol/L) ------------- ---------- ------------- ------------ --------------------- --------------- ------------- ---------------------- -------------- -------------- MK F 53 71.5 3.0 24.7 3.41 4.5 0.65 2.3 BE F 64 81 2.2 34.2 2.55 5.4 1.24 3.1 KL F 79 76 2.8 28.3 2.14 6.3 0.76 4.5 CH M 55 62.5 3.5 19.5 1.21 3.6 0.99 2.1 BV M 70 77.5 1.3 26.2 1.47 4.6 0.99 2.9 RS M 73 65 2.7 23.3 2.86 7.1 0.96 4.9 RH M 78 62 2.7 21.5 1.09 4.6 1.26 2.9 AJ M 78 73 3.0 26.5 2.84 5.0 0.74 2.9 HB M 90 70 3.0 24.2 1.38 4.4 1.30 2.5 median D 73 71.5 2.8 24.7 2.14 4.6 0.99 2.9 median H 73 70.5 2.2 24.6 1.84 5.6 1.09 3.7 \Dry body weight ::: Anticoagulation effect ---------------------- During the dialysis with dalteparin, the anti-Factor Xa activity was between 0.52 and 0.87 IU/mL. The target value recommended by the manufacturer is 0.5--1.0 IU/mL. Hence, the plasma dalteparin concentration remained well within the range for effective anticoagulation throughout the dialysis-session. LPL activity and mass --------------------- The LPL-activity rose rapidly when dalteparin was administered. The highest value was at 15 minutes, median 15 mU/mL (range 9--32). The activity remained high at 30 minutes, but then decreased so that at 120 minutes the median was only 9 mU/mL (range 5--15) (Fig [1](#F1){ref-type="fig"}). The activity then remained essentially unchanged to the end of the dialysis at 240 minutes (6 mU/mL, range 5--11). The shape of the curve resembled that during the earlier dialysis with conventional heparin (Fig [1](#F1){ref-type="fig"} inset), but the values were much lower during the dialysis with dalteparin (p \< 0.01). The area under the curve (AUC) for LPL activity during the peak period (0--180 minutes) was 1774 mU/mL × minutes (range 1116--3001). This is only 27% of the AUC observed during the earlier study with conventional heparin \[[@B12]\] (p \< 0.01). For the plateau period (180--240 minutes) the AUC was 390 mU/mL × minutes (range 308--618) which is 36% of the corresponding AUC during the dialysis with conventional heparin (p \< 0.01). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Plasma LPL activities during infusion of dalteparin.The figure shows individual curves for the nine subjects in the present study. The inset compares median values from the present study (□) with those from three earlier studies where the same protocol for infusion was used. Control subjects given conventional heparin \[23\] (●), control subjects given dalteparin \[22\] (○), HD patients given conventional heparin \[12\] (■). ::: ![](1471-2369-5-17-1) ::: The inset in Fig. [1](#F1){ref-type="fig"} compares the median values for LPL activities in the present study to the LPL activities observed in earlier studies with age and gender matched healthy subjects given conventional heparin \[[@B23]\], or dalteparin \[[@B22]\], and with HD patients given conventional heparin \[[@B12]\]. The values during dalteparin infusion were lower in both controls and in HD patients. Values in HD patients were lower than in controls both with dalteparin and with conventional heparin. Thus, the highest values were for controls given conventional heparin and the lowest values were those in the present study with dalteparin in HD patients. The differences were remarkably large. These studies have been carried out on separate occasions over several years, but duplicate samples had been saved frozen. To check the consistency of the values, the duplicate samples from the 0, 15 and 30 min time points were thawed and assayed for LPL activity and mass. There was good agreement between the activities from the earlier and the repeated assay for all four studies (Fig [2A](#F2){ref-type="fig"}). Hence, the large differences between the LPL activities registered for controls and HD patients and for the two different heparin preparations were real. As an additional test of the consistency of the data, we plotted the increase in LPL activity and LPL mass from basal (before heparin) at the 30 min time points (Fig [2B](#F2){ref-type="fig"}). The basis for this is that previous studies have indicated that heparin releases mainly the active form of the lipase \[[@B26]\]. For regression analysis, changes in LPL mass of less than 100 ng/mL were excluded because heparin may release some inactive LPL \[[@B26]\] and because of the uncertainty in calculating small differences between pre- and post-heparin mass. The analysis returned a slope of 0.46 ± 0.05 mU/ng (r = 0.94, p \< 0.001), consistent with the expected specific activity of human LPL (around 0,4 mU/ng under our assay conditions). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Evaluation of the consistency of data from four separate studies by repeated assay of frozen samples.Samples had been obtained and treated as described in the methods section and then stored frozen at -70°C in three earlier studies of plasma LPL during infusion of conventional heparin or dalteparin in control subjects or in dialysis patients \[12,22,23\] and the present study. Samples from the 0, 15 and 30 min time points were thawed and assayed for LPL activity and mass. Panel A shows the LPL activities at 15 and 30 min recorded on the second assay, as a function of the value recorded on the original assay. Regression analysis gave a slope indicating that the repeated value was 113 % of the original (r = 0.94, p \< 0.0001). Panel B shows the increase of LPL activity over the baseline samples plotted against the increase of LPL mass for the 30 min samples. For this, values from the second assay were used (LPL mass was not determined in some of the earlier studies). A regression analysis, excluding samples for which the increase in LPL mass was less than 100 μg/mL, returned a slope of 0.46 ± 0.05 mU/ng LPL (r = 0.94, p \< 0.001). Same symbols as in Fig 1. ::: ![](1471-2369-5-17-2) ::: Triglycerides ------------- TG remained essentially unchanged for the first two hours and then increased. The change from 2.14 mmol/L (range 1.09--3.41) at the start, to 2.59 mmol/L (range 1.49--5.04) at the end of the dialysis represents a 21 % increase (p \< 0.01). Compared to the values during the earlier dialysis with conventional heparin, there was no statistically significant difference at the start of dialysis, but from 30 minutes and through the remaining session TG values were significantly higher during the dialysis with dalteparin (p \< 0.05) (Fig [3](#F3){ref-type="fig"}). There was no drop in TG at 30 to 60 min like that found using conventional heparin. There was no correlation between LPL activity and changes in TG during any of the dialysis-sessions. To further illustrate the changes in TG concentrations we have set the baseline value for each individual to 100% and calculated the changes from this. Median values for the changes are plotted in Figure [4](#F4){ref-type="fig"} which reinforces the conclusion that there was no significant decrease of TG during the first two hours during the dialysis with dalteparin, in contrast to the marked drop observed during dialysis with conventional heparin \[[@B12]\], and during infusion of either conventional heparin \[[@B23]\] or dalteparin in control subjects \[[@B22]\]. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Plasma TG concentrations during infusion of dalteparin.The figure shows individual curves for the nine subjects in the present study. The inset compares median values from the present study (□) with those from three earlier studies where the same protocol for infusion was used. Control subjects given conventional heparin (●), control subjects given dalteparin (○), HD patients given conventional heparin (■). P \< 0.05 for dalteparin compared to conventional heparin given to HD patients. ::: ![](1471-2369-5-17-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Changes in plasma TG concentrations during infusion of dalteparin or conventional heparin to HD patients or matched controls.For this, the basal TG concentration for each individual was set to 100% and the concentrations observed at the subsequent time points were calculated relative to this. The figure shows median values from the present study (□) and from three earlier studies where the same protocol for infusion was used. Control subjects given conventional heparin (●), control subjects given dalteparin (○), HD patients given conventional heparin (■). ::: ![](1471-2369-5-17-4) ::: Cholesterol ----------- Total cholesterol increased from 4.6 mmol/L (range 3.6--7.1) at start, to 6.1 mmol/L (range 3.8--8.4) at the end (32%, p \< 0.05). This differs from the earlier dialysis with conventional heparin, when total cholesterol did not change from baseline \[[@B12]\]. HDL-cholesterol did not change from baseline. Again, this differs from the dialysis with conventional heparin when HDL-cholesterol increased from 1.09 mmol/L (range 0.67--2.06) at start to 1.19 mmol/L (range 0.67--2.31) at the end (p \< 0.05). No correlation was found between LPL activity and total or HDL-cholesterol changes during any of the dialysis-sessions. The median LDL-cholesterol, calculated by the Friedewald formula, was 2.9 mmol/L (range 2.1--4.9) before the dialysis and increased to 3.75 (2.1--5.6) at 240 min. This corresponds to an increase of 29% (p \< 0.05). Discussion ========== This study shows the same pattern of plasma LPL activity in HD patients given dalteparin as observed in previous studies with control subjects given dalteparin \[[@B22]\] or conventional heparin \[[@B23]\] and in HD patients given conventional heparin \[[@B12]\]. The novel aspect is how remarkably low the plasma LPL activities were in the HD patients. This prompted us to re-analyze frozen samples from the earlier studies to rule out the possibility of assay variations as a confounding factor. The increase in LPL mass was also low in the HD patients given dalteparin, and corresponded well to the increase of LPL activity. This further supports the conclusion that infusion of dalteparin to HD patients resulted in low levels of LPL in the circulating blood, compared to what was seen when conventional heparin was infused \[[@B12]\] or when controls were given dalteparin or conventional heparin \[[@B22],[@B23]\]. There are several earlier studies that show that administration of LMW heparin results in lower plasma levels of LPL than conventional heparin \[[@B27]-[@B30]\]. LMW heparin preparations differ considerably in their molecular characteristics and caution should be exercised when extrapolating from one preparation to another. The comparisons to our earlier study with conventional heparin \[[@B12]\] are based on clinically relevant doses during HD, as recommended from manufacturer\'s and clinical guidelines, not on a molecule-for-molecule basis. In a study with dalteparin Persson et al. found that the early LPL activity was only about half as high compared to values observed after conventional heparin \[[@B27],[@B29]\]. This is similar to the difference between the AUCs for the early peak of plasma LPL activity observed here and in an earlier study with conventional heparin in HD patients \[[@B12]\]. The LPL activities were lower in HD patients than observed in controls given dalteparin \[[@B22]\]. A similar difference was earlier found for infusion of conventional heparin in HD patients \[[@B12]\] compared to controls \[[@B23]\]. One possible explanation is that the depletion of LPL stores during the dialysis-sessions is not fully restored between the sessions. Arnadottir et al. have, however, found that the amount of LPL released by a bolus of heparin is restored within 24 hours \[[@B30]\]. In rats, it takes about four hours after a single bolus of LMW heparin before the LPL stores are replenished \[[@B31]\] and chylomicron catabolism occurs at normal rate \[[@B32]\]. A more likely explanation is that the kidney dysfunction as such causes an impairment of the LPL system \[[@B33]\]. Yet another possibility, that has not been explored, is that the kidney itself makes an important contribution to overall LPL stores. This is the case in mink, where kidney has a higher LPL activity than any other tissue, and in mice \[[@B34],[@B35]\]. Administration of heparin causes a temporary derangement of lipoprotein metabolism. In our studies with control subjects given conventional heparin or dalteparin the TG concentration decreased after heparin and then gradually increased again so that at the end of the study period TG exceeded the baseline level (see inset in Fig [3](#F3){ref-type="fig"}). This probably reflects that LPL first becomes more available for lipoprotein catabolism as it circulates in blood but then becomes less available when the lipase is removed from blood by the liver and the tissues stores become depleted. This is in accord with observations in animal experiments. Chevreuil et al. found that the clearance of injected radioactively labeled chylomicron TG was dramatically increased five minutes after rats had been given conventional heparin or LMW heparin \[[@B32]\]. This was associated with an increased appearance of label in plasma FFA, supporting the view that the rate of lipolysis was increased. In contrast, injection of chylomicrons one hour after the heparins resulted in substantially slower clearance compared to saline-treated controls. Appearance of label in plasma FFA was also decreased, suggesting that impaired lipolysis was responsible, at least in part, for the impeded chylomicron clearance. The decrease of plasma TG was small and statistically not significant in the HD patients given dalteparin. This may, at least in part, be explained by the relatively low plasma LPL activities. In addition, there are reports that in patients with nephrosis there are inhibitors of LPL in the circulating blood and that VLDL isolated from such patients are lipolyzed slowly by LPL \[[@B36]\]. On the other hand, the rise of TG from two hours was pronounced in the HD patients. This indicates that the LPL stores were depleted in these patients even though the plasma LPL activities were low. This is in accord with results from animal experiments. Chevreuil et al. injected decasaccharides to rats \[[@B20]\]. This resulted in only a small and short-lived increase of LPL activity in blood. Nonetheless, the decasaccharides had apparently removed most of the functional LPL from peripheral tissues. The LPL activity that could be released from isolated hearts by single-pass perfusion with heparin (\"functional LPL\") was decreased by 75% one hour after the rats had been injected with decasaccharides. The catabolism of chylomicron TG by perfused hearts was delayed to a similar extent. The clearance of labeled chylomicrons injected to rats was markedly delayed from 30 minutes to 2 hours after a decasaccharide injection. After one hour, the fractional catabolic rate was only one-third of the control value. All of this suggests that dalteparin causes a profound depletion of functional LPL even though the plasma levels of LPL activity are relatively low. Conclusions =========== The peak level of LPL in plasma after injection of dalteparin is less than half of that after conventional heparin. This was shown here for a group of HD patients, but a similar difference has earlier been observed for healthy controls. The peak level of LPL is lower in HD patients than in controls both after dalteparin and after conventional heparin. This is probably a consequence of the kidney disease but the detailed mechanism is not known. These two effects compound to an almost ten-fold difference in peak LPL activity comparing the present HD patients given dalteparin to a group of healthy controls given conventional heparin. Analysis of LPL activity and mass in frozen samples from our earlier studies ruled out the possibility of assay variation as a confounding factor. Prior in vitro*studies of the LPL-heparin interaction, and animal experiments, indicate that decasaccharides, or longer heparins, release the enzyme efficiently from its binding sites at the vascular endothelium. The difference in plasma levels of LPL is probably due mainly to a difference in how much the respective heparin retards the uptake of LPL by the liver. Immediately after heparin, plasma LPL activity is high and catabolism of TG-rich lipoproteins is accelerated. This acceleration was not evident during infusion of dalteparin to HD-patients. Then follows a period when tissue stores of LPL are depleted and lipoprotein metabolism is retarded. This depletion was at least as marked after dalteparin as after conventional heparin, despite the lower plasma LPL levels. In the present study, the TG level increased significantly more after dalteparin than after conventional heparin. Our results indicate that LMW heparins disturb the LPL system as much or more than conventional heparin does. Abbreviations ============= AUC -- area under the curve; HD -- hemodialysis; HDL -- high density lipoprotein; LMW heparin -- low molecular weight heparin; LPL -- lipoprotein lipase; TG -- triglycerides; VLDL -- very low density lipoprotein Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= BN participated in the design of the study, carried out the patient studies, assembled the data, did the statistical analyses, and participated in writing of the manuscript; BS and GO conceived of the study and coordinated the work. TO participated in the design of the study and in writing 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/1471-2369/5/17/prepub> Acknowledgements ================ We thank Ann-Sofie Jacobsson for technical assistance and Ann-Sofie Lindgren for blood sampling and subject contact. The study was supported by grants 03X-727 and 13X-12203 from the Swedish Research Council and by grants from the Department of Public Health and Clinical Medicine, Umeå University, and the Department of Internal Medicine, University Hospital of Northern Sweden, Umeå.	PubMed Central	2024-06-05T03:55:49.876590	2004-11-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534103/", "journal": "BMC Nephrol. 2004 Nov 3; 5:17", "authors": [ { "first": "Birgit", "last": "Näsström" }, { "first": "Bernd", "last": "Stegmayr" }, { "first": "Gunilla", "last": "Olivecrona" }, { "first": "Thomas", "last": "Olivecrona" } ] }
PMC534104	Background ========== Despite some promising early results, gene therapy does not, as yet, live up to expectation \[[@B1]\]. The main stumbling block remains gene delivery, and all advances in the control of gene expression and selection of therapeutic genes are hampered by inefficient gene transfection. Hence the development of a safe and effective method of gene delivery in vivois of utmost importance if gene therapy is to move from the experimental to the clinical stage. Electroporation is currently receiving much attention as a way to increase drug and DNA delivery \[[@B2]-[@B5]\]. Electroporation has long been used as an effective in vitrogene delivery system in both prokaryotes and eukaryotic cells. Electroporation is a physical means of importing small molecules and macromolecules into cells via increased cell membrane permeability. Electroporation combined with chemotherapeutic drugs bleomycin and cisplatin (electrochemotherapy) has shown to be very promising antitumor therapy. It was tested on many different tumor types on the preclinical level demonstrating high antitumor effectiveness resulting in tumor cures at very low chemotherapeutic doses. It was also tested in clinical trials for the treatment of accessible cutaneous tumors of different histological types in cancer patients resulting in up to 100% objective responses \[[@B4],[@B6],[@B7]\]. Recent studies demonstrated the feasibility of electrogene therapy using a range of therapeutic genes for the treatment of experimental tumors \[[@B4],[@B8]-[@B14]\]. However, the transfection efficiency of electroporation-assisted DNA delivery is still low compared to viral methods and there is a clear need to optimize this approach \[[@B5],[@B14],[@B15]\]. In studies designed to determine transfection efficiency in tumors, tissue homogenates, tissue sections or measurement of fluorescence in whole-tumor specimens using fluorescence stereomicroscope have been employed. Different plasmids have also been used to analyze transfection efficiencies, such as those encoding the green fluorescence protein (GFP), β-galactosidase or luciferase \[[@B5],[@B14],[@B15]\], making direct comparisons difficult. In order to optimize treatment, knowledge about spatial and time dependency of gene expression following delivery is of utmost importance in order to improve gene delivery. This information is also important for the timing of gene therapy with other cancer treatment modalities. Intravital microscopy of tumors growing in dorsal skin fold window chambers is a useful method for monitoring gene transfection, since it allows non-invasive dynamic monitoring of gene expression in tumors in a live animal \[[@B16],[@B17]\]. So far, only one study has used this technique to monitor the activity of an adenovirus in infecting a mammary cell carcinoma over the course of several days \[[@B16]\]. In the present study, we used intravital microscopy to monitor real time spatial distribution of gene transfection using the GFP reporter gene in rat P22 tumors. We followed time dependency of transfection efficiency; compared liposome-DNA complexes and electroporation assisted DNA delivery using two different sets of electrical parameters. Methods ======= Animals and tumors ------------------ All animal experiments were carried out in accordance with the UK animals (Scientific Procedures) Act 1986, following the UKCCCR guidelines and with approval from the local Ethical Review Committee of the Gray Cancer Institute. Early generations of the P22 transplanted rat carcinosarcoma were used in experiments. Donor tumors were grown subcutaneously in the left flank of 8--10 week old male BD9 rats \[[@B18]\]. Surgery ------- Surgery was carried out under general anesthesia using intraperitoneal injection of fluanisone (10 mg/kg), fentanyl citrate (0.315 mg/kg) (\'Hypnorm\', Janssen Animal Health, UK) and midazolam (2 mg/kg) (\'Hypnovel\', Roche Products Ltd., UK) as described previously \[[@B18]\]. Briefly, animals were kept warm using heating pads throughout the surgical procedure and aseptic technique was used throughout. Window chambers, consisting of a double-sided aluminum frame, holding two parallel glass windows approximately 200 μm apart, were surgically implanted into the dorsal skin of male BD9 rats, weighing approximately 200 g. Surgery involved removal of the epidermal and dermal layers of both skin layers of a dorsal skin flap, except for the deepest fascia layer on each side, and then securing the two sides of the chamber to the skin using stainless steel screws and sutures. The two fascia layers moved freely between the two glass windows, following these procedures. Early generation subcutaneous transplants of the P22 rat carcinosarcoma were used as donor tumors, when they reached approximately 0.5 cm in diameter. A tumor fragment (\~0.5 mm diameter) was placed onto one of the fascia layers within the window chamber on the day of surgery. Animals were given an intraperitoneal injection of a few milliliters dextrose-saline, immediately following surgery and kept on a warmed pad until recovery from anesthesia. Subsequently, animals were kept in a warm room (30--34°C) until the day of experiment. Three animals were used for each experimental group. Study design ------------ Transfection was carried out 7 to 14 days following surgery, when tumors measured 3--4 mm in diameter, using a plasmid encoding the green fluorescent protein (GFP; p-EGFP-N1, Clontech, Basingstoke, UK). Animals were anaesthetized with Hypnorm and midazolam. The window on the tumor side was removed and 40 μg of DNA alone or encapsulated in lipofectin (30 μl; Life Technologies, Paisley, UK) vesicles was carefully placed on the tumor surface in a total volume of 75 μl in phosphate buffered solution \[[@B5]\]. One minute thereafter, electroporation was carried out by application of 8 square electric pulses generated by an electroporator (built in-house). Pulses were delivered by two flat, parallel stainless steel electrodes (two stainless steel strips: length 15 mm, width 4 mm with rounded corners) 3 mm apart that were placed at the diametrically opposed edges of the tumor. Two different electroporation protocols were used: EP1-- amplitude 180 V (voltage/distance ratio 600 V/cm); pulse length 5 ms; repetition frequency 1 Hz and EP2-- amplitude 390 V (voltage/distance ratio 1300 V/cm); pulse length 0.1 ms; repetition frequency 1 Hz. Immediately after the procedure, the glass window was replaced and GFP fluorescence monitored at selected time points for 6 days. Intravital microscopy --------------------- Intravital microscopy was carried out using an inverted Nikon Diaphot 200 fluorescence microscope, with a stage modified in-house for holding rats. Animals were anaesthetized with Hypnorm and midazolam and placed on the stage, in such a way that the window chamber was located centrally above the objectives using location screws. Rectal temperature was maintained between 34--37°C throughout the experiment, using a thermostatically controlled heating pad beneath the rat and an infrared overhead lamp for maintenance of tumor temperature. Tumor preparations were alternatively viewed at each time point under transmitted visible light for measurement of tumor diameter and under fluorescence epi-illumination using a 100 W mercury arc lamp, for visualization of GFP fluorescence (excitation filter of 450--490 nm, emission filter of 520 nm) using x1.6, x4, x10 and x20 objective. Prior to transfection, two different regions of interest (ROI) using x10 objective were selected, one in the center of the tumor and one in the tumor periphery. Tumor preparations were monitored at each time point for 15 s using transmitted or epi-illumination. Data analysis ------------- Observations were recorded in digital format, using a Sony DSR-30P digital videocassette recorder, for off-line analysis. Multiple frames (typically 10) were captured onto computer and the images averaged for the analysis of the fluorescence intensity using the Visilog Image Processing package (Noesis, France). Increase in fluorescence intensity in the tumors was determined by subtracting the values of fluorescence intensity of non-transfected granulation tissue from the values of tumors and normalizing them to the values of non-transfected tissue. The calculations were performed for each animal at all time points. Statistical analysis -------------------- Statistical analysis was carried out using SigmaStat Statistical software (Systat Software GmbH, Erkrath, Germany). Data were tested for normality using Kolmogorov-Smirnov test and differences between the groups were tested for significance using Holm-Sidak method, after one-way repeated measures analysis of variance was performed. A value of p \< 0.05 for the comparisons was considered to represent a significant difference between groups. Results and discussion ====================== Three different types of non-viral transfection methods were compared: DNA alone, liposome-DNA complex and electroporation-assisted DNA delivery, using two different sets of electric pulse parameters. Transfection of GFP was monitored using intravital microscopy on syngeneic P22 rat carcinosarcoma tumors growing in the dorsal skin flap window chamber of BD9 rats (Figure [1](#F1){ref-type="fig"}). The transfection efficiency was evaluated by monitoring real time spatial distribution and time dependence of GFP fluorescence. Among the tested transfection methods, electroporation-assisted gene delivery was the most effective method for transfection of tumors growing in the window chamber. Two different sets of electric pulse parameters were tested; EP2 resulted in up to 17%, while EP1 in up to 22% increase in fluorescence intensity in the tumors. EP2 has previously been proven to be effective in electrochemotherapy of accessible cutaneous tumors in patients with histologically different types of tumors \[[@B6],[@B7]\], and also for electrogenetherapy \[[@B8]\]. On the other hand, EP1 electric pulses have already been shown to be effective for gene delivery of solid tumors of different histology and origin (rat, mouse and human) \[[@B5]\]. Transfection efficiency using either DNA alone or liposome-DNA complex was lower (7% and 12%, respectively) compared to electroporation-assisted gene delivery. Spatial distribution of transfected cells differed between the two sets of electric pulses parameters. When using EP1 electric pulse parameters, cells expressing GFP were more spread out through the whole tumor, compared to EP2 conditions, where cells expressing GFP were limited to the areas that were close to the positioning of the electrodes (Figure [2](#F2){ref-type="fig"}). Electroporation of the cell membrane is a physical phenomenon that occurs above a certain threshold of induced transmembrane potential and is dependent on electric pulse parameters such as pulse length, pulse amplitude, number and pulses sequence \[[@B4]\]. According to the current knowledge at a single cell level, DNA electro-transfer is a process involving attachment of DNA to the electropermeabilized side of the cell facing the cathode, aggregation of DNA and its translocation to the cytoplasmic side of the cell \[[@B19]\]. Therefore, in the case of EP2, the conditions suitable for DNA electro-transfer (above the threshold level) in vivoappeared to be obtained only in the vicinity of the electrodes, whereas in the case of EP1 a larger area of the tumor was prone to DNA transfer. Effectiveness of electroporation-assisted gene delivery was also evident by lack of transfection efficiency in the granular tissue surrounding the tumor that was not exposed to electric pulses, but to DNA only (Figure [2](#F2){ref-type="fig"}). In addition, more homogenous distribution of transfection efficiency using EP1 conditions compared to EP2 conditions could be due to the electrophoresis of the DNA caused by longer duration of the EP1 pulses. Functional GFP was formed within 5 h after transfection regardless of the transfection method used (Figures [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}). Detectable green fluorescence is the end result of a series of events, including transfer of the DNA encoding GFP into the cell, evasion of intracellular nucleases, transfer to the nucleus, transcription and translation, and finally, folding of the protein into a functional protein\'s fluorophore. Topical administration of DNA to the tumors in the window chamber (40 μg of DNA) resulted in very low transfection efficiency, the increase in fluorescence intensity in the tumors was up to 7% at day 2 post-transfection, and remained at this level up to day 6. Administration of liposome-DNA complexes resulted in increased transfection efficiency in the tumors compared to DNA alone (P = 0.032). The increase was up to 12% with the similar level of GFP expression on day 6 post-transfection as after the administration of DNA only (Figure [3](#F3){ref-type="fig"}). Similar results were obtained in our previous study on dense cell suspensions and solid subcutaneous P22 tumors where liposome-DNA complexes resulted in significantly higher GFP transfection efficiency compared to DNA injection only \[[@B5]\]. These results are in accordance with several preclinical and clinical studies demonstrating efficient gene-transfer to solid tumors using liposome-DNA complexes \[[@B20],[@B21]\]. The highest increase in fluorescence intensity was obtained with electroporation-assisted gene delivery using EP1 in the present study. The fluorescence intensity increased to 22% at day 2 post-transfection and then remained at this level over the observation period of 6 days. Electroporation assisted gene delivery with EP2 was less effective than with EP1, although it was better than liposome-DNA complex alone. These results are in accordance with our previous study, where electroporation assisted gene delivery using either of the electric pulse parameters yielded higher transfection efficiency compared to lipofectin-enhanced method. The transfection efficiencies that were obtained in the present study were much higher compared to the transfection efficiency that we obtained in our previous study on P22 solid tumors growing subcutaneously in SCID mice \[[@B5]\]. The possible reason for this is the absence of the skin overlying the tumors in the window chamber. In subcutaneously growing tumors, the presence of the stratum corneum of the skin causes the electric field intensity to drop substantially, especially in the centre of the tumor, compared to the electric field intensity in the skin \[[@B22],[@B23]\]. The absence of this effect in the window tumors may account for the increased transfection efficiency observed in the current study, as predicted from theoretical analyses \[[@B22],[@B23]\]. It is worth noting that the fluorescence level remained approximately constant throughout the observation period for all transfection methods. This means that, as the tumors grew during the 6-day observation period (tumor diameter increased approximately 2-fold), plasmid DNA appeared to be present in the progeny cells. In addition, intravital microscopy demonstrated that application of electric pulses to the tumors did not induce major cell damage as no effect on the tumor growth was observed compared to untreated controls. Six days post transfection there was no difference in increase in tumor diameter between the tumors that were transfected with DNA alone (2.2 fold), liposome-DNA complexes (2.3 fold), and electroporation-assisted DNA delivery using EP1 (2.4 fold) or EP2 (2.2 fold). Conclusions =========== In conclusion, this study showed that intravital microscopy is useful for monitoring the spatial and temporal efficacy of electroporation methods for gene transfection in animal tumor models. As such, it will be valuable for the evaluation of new methods of optimizing gene delivery. Electroporation-assisted gene delivery using EP1 was found to result in early onset and homogenous distribution of gene expression in the P22 tumor model. Further improvement in transfection efficiency may be gained by optimizing electric pulse parameters. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= MC conceived the study, participated in its design and experiments, and drafted the manuscript. IW participated in design of the study and experiments. GUD participated in design of the study and critically revised the draft. GMT participated in design of the study and coordination, and critically revised the draft. GS helped to draft the manuscript and participated in analysis and interpretation of the data. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2407/4/81/prepub> Acknowledgements ================ This work was supported by Cancer Research UK and the Ministry of Education, Science and Sport of the Republic of Slovenia. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Expression of GFP following electroporation-assisted gene delivery using EP1GFP expression was monitored in the rat P22 tumor growing in rat dorsal flap window chamber following electroporation-assisted gene delivery using EP1 (8 electric pulses, 600 V/cm, 5 ms, 1 Hz). a.image taken under visible light condition on day 0 (x4 objective), b.image taken under fluorescence epi-illumination on day 0 before administration of DNA and electroporation (x4 objective), c.image taken under fluorescence epi-illumination on day 2 (x4 objective); d.image taken under fluorescence epi-illumination on day 2 (x20 objective). ::: ![](1471-2407-4-81-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Comparison of transfection efficiency between tumour center and periphery following two different electroporation protocolsGFP expression in rat P22 tumor growing in rat dorsal flap window chamber 2 days after two different electroporation conditions: EP1: 8 electric pulses, 600 V/cm, 5 ms, 1 Hz (a, c); EP2: 8 electric pulses, 1300 V/cm, 0.1 ms, 1 Hz (b, d). Comparison of transfection efficiency in the ROI\'s of tumor center (a, b) and periphery (c, d) (x10 objective). Arrows: edge of the tumor. ::: ![](1471-2407-4-81-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Time dependence of transfection efficiency comparing different transfer methodsDNA or liposome-DNA complexes were administered to the top of the tumor growing in window chamber. Electric pulses were applied to the tumor immediately after the addition of DNA. Two sets of electric pulse parameters were used: EP1 (8 electric pulses, 600 V/cm, 5 ms, 1 Hz) and EP2 (8 electric pulses, 1300 V/cm, 0.1 ms, 1 Hz). Average of three animals/group +/- standard error of the mean. ::: ![](1471-2407-4-81-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Representative images of time related transfection efficiency of electroporation-assisted gene delivery using EP1 conditionsExample images taken under visible light condition (a) and under fluorescence epi-illumination (b) on day 0 before administration of DNA and electroporation. Example images of transfection efficiency in tumors at 5 h (c), 2 (d), 4 (e) and 6 days post-transfection (f). (x10 objective). ::: ![](1471-2407-4-81-4) :::	PubMed Central	2024-06-05T03:55:49.879313	2004-11-16	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534104/", "journal": "BMC Cancer. 2004 Nov 16; 4:81", "authors": [ { "first": "Maja", "last": "Cemazar" }, { "first": "Ian", "last": "Wilson" }, { "first": "Gabi U", "last": "Dachs" }, { "first": "Gillian M", "last": "Tozer" }, { "first": "Gregor", "last": "Sersa" } ] }
PMC534105	Background ========== The growth of a fetus in an undernourished intrauterine environment is considered to result in adaptive fetal programming or metabolic imprinting with pathophysiologic consequences later in life \[[@B1]-[@B4]\]. It is contended that low birth weight at term (\<2500 g), a surrogate for impaired gestational environment, is uncommon in industrialized societies, and deprivations that existed before the second world war no longer apply to pregnancies at present \[[@B5],[@B6]\]. In reality, the United States birth data for year 2002 show a prevalence of 7.8% low birth weight, with blacks showing almost twice the rate of whites \[[@B7]\]. Studies world-wide, regardless of socio-economic background, have linked low birth weight to increased risk of insulin resistance, dyslipidemia, hypertension, coronary heart disease, and type 2 diabetes \[[@B8]-[@B13]\], although some studies have found a weak or no associations in this regard \[[@B5],[@B14]-[@B16]\]. Several studies including our own have examined the association between low birth weight and selected cardiovascular risk factor variables in childhood and adolescence \[[@B17]-[@B27]\]. However, information is scant on data linking low birth weight to longitudinal changes of cardiovascular risk factor variables measured simultaneously and serially from childhood to adolescence. As part of the Bogalusa Heart Study, a biracial (black-white) community-based investigation of evolution of cardiovascular risk since childhood \[[@B28]\], the present analysis examines the relationship between low birth weight and the longitudinal trends of adiposity, blood pressure, lipids and lipoproteins, and measures of glucose homeostasis from childhood to adolescence. Methods ======= Study cohorts ------------- Between 1973 and 1996, 7 cross-sectional surveys of children and adolescents were conducted in the community (65% white, 35% black) of Bogalusa, LA. This panel design, based on repeated cross-sectional examinations performed every 3 to 4 years, resulted in serial observations required for the longitudinal analysis. For the present report, two sets of data were merged as described previously \[[@B21]\]: 1) singleton new born cohort participants (n = 233) whose weights were measured at birth as part of the initial examination during 1973--1974; and 2) singletons (n = 1213) aged 7--11 years who participated in 1987--1988 cross-sectional survey and whose birth weight records were obtained from the Office of Vital Statistics in New Orleans in 1991. Of those with birth weight data (n = 1436), 1329 subjects participated in 2 to 7 surveys of children and adolescents. Exclusion of those with missing data (n = 170), congenital heart disease (n = 11), and diabetes (n = 7) resulted in 1141 eligible subjects (57% white, 47% female). Low birth weight and control groups were selected from the eligible cohort, according to birth weight percentile cut points \[[@B29]\]. Subjects (n = 123) who had birth weight below the race-specific 10^th^percentile (whites: \<2749 g; blacks: \<2438 g) were categorized as low birth weight group; those (n = 296) in the upper normal range of 50--75^th^percentile (whites: 3402 -- 3770 g; blacks: 3133 g -- 3487 g) as control group. Birth weights above the 75^th^percentile were not included in the control group because of the u-shaped associations between birth weight and risk factors or disease \[[@B3],[@B30]\]. Race-specific percentile, rather than World Health Organization (WHO) criterion for low birth weight (\<2500 g) was used to define low birth weight because of black-white differences in birth weight distribution \[[@B31],[@B32]\]. General examination ------------------- Identical protocols were used by trained examiners across all surveys \[[@B33]\]. Subjects were instructed to fast for 12 hours prior to screening, and compliance was determined by interview on the morning of examination. Anthropometric and blood pressure measurements were made in replicate and mean values were used. Height and weight were measured 2 times; subscapular skinfold thickness 3 times. Body mass index (BMI) calculated as weight in kg divided by the square of the height in meters was used as a measure of overall adiposity; subscapular skinfold for truncal fatness. The reproducibility in terms of intraclass (intra-observer) correlation coefficient was greater than 0.99 for weight and height, and greater than 0.97 for subscapular skinfold. Blood pressure levels were measured in 6 replicates by 2 randomly assigned nurses on the right arm of subjects in a relaxed, sitting position. Systolic and diastolic blood pressures were recorded at the first, fourth, and fifth Korotkoff phases using mercury sphygmomanometer. For this analysis fourth phase was used for diastolic blood pressure because in our experience the fourth phase is more reliably measured in children and more predictive of adult hypertension \[[@B34]\]. Laboratory analyses ------------------- From 1973 to 1986 cholesterol and triglyceride levels in serum were measured using chemical procedures on Technicon Autoanalyzer II (Technician Instrument Corp., Tarrytown, NY). Since then these measurements were done using enzymatic procedures on Abbott VP instrument (Abbott laboratories, North Chicago, IL). Serum lipoprotein cholesterols were analyzed by a combination of heparin-calcium precipitation and agar-agarose gel electrophoresis procedures \[[@B35]\]. Both chemical and enzymatic procedures met the performance requirement of the Lipid Standardization Program of the Centers for Disease Control and Prevention, Atlanta, GA. The laboratory has been monitored for precision and accuracy by the agency\'s surveillance program since 1973. For example, the average bias in levels of total cholesterol on CDC control samples ranged from -0.1 to -1.6 mg/dL between different cross-sectional surveys, with no consistent pattern over time within or between surveys. The intraclass correlation coefficients between the blind duplicate (10% random sample) values ranged from 0.87 to 0.99 for total cholesterol; 0.88 to 0.99 for triglycerides; 0.86 to 0.98 for LDL cholesterol; and 0.86 to 0.98 for HDL cholesterol. Plasma immunoreactive insulin levels were measured by a commercial radioimmunoassay kit (Phadebas, Pharmacia Diagnostics, Piscataway, NJ). Plasma glucose was measured by a glucose oxidase method either using a Beckman glucose analyzer (Beckman Instruments, Fullerton, CA) or as part of a multichemistry (SMA20) profile. The intraclass correlation coefficient between blind duplicate values ranged from 0.94 to 0.98 for insulin and 0.86 to 0.98 for glucose. An index of insulin resistance was calculated according to the homeostasis assessment model formula \[[@B36]\]: HOMA-IR=fasting insulin (μu/mL) × fasting glucose (mmol/L) ÷ 22.5. Statistical analyses -------------------- For test of significance glucose and insulin were logarithmically transformed to approach normality. The average of multiple measurements for subjects within age groups 4 to 11 and 12 to 18 years corresponding to childhood and adolescence periods was used to calculate mean levels of risk variables by birth weight status and age groups. Mean levels of risk variables within each age group were compared between low birth weight and control group by a general linear model, adjusting for age, race, and gender. The longitudinal rates of change in risk variables was assessed by the generalized estimation equation (GEE) method \[[@B37]\] with age as predictor, adjusting for race and gender. Independent association of low birth weight with longitudinal trends of risk variables from childhood to adolescence was assessed by multivariate analysis (GEE). The model included birth weight (low vs control) and risk variables as applicable along with age, age^2^, race and gender and their interaction with birth weight (low vs control). A backward stepwise method was used to remove nonsignificant terms. Results ======= Mean levels of cardiovascular risk variables during childhood (ages 4--11 years) and adolescence (ages 12--18 years) periods are shown in table [1](#T1){ref-type="table"} by birth weight groups. Of the risk variables adjusted for age, race, and gender, levels of HDL cholesterol were significantly lower and LDL cholesterol higher among low birth weight group vs control group during childhood. During adolescence, only glucose levels were significantly higher among low birth weight group vs control group. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Levels (mean ± SD) of risk variables during childhood and adolescence by birth weight. The Bogalusa Heart Study ::: Variable Childhood (4--11 years) Adolescence (12--18 years) ------------------------- ------------------------- ---------------------------- --------------- ------------- BMI (kg/m^2^) 16.7 ± 2.6 17.5 ± 2.9 21.6 ± 5.2 22.7 ± 5.0 Subsc. Skinfold (mm) 8.2 ± 5.1 8.3 ± 6.4 15.9 ± 10.0 16.0 ± 10.7 Syst. BP (mm Hg) 96.4 ± 9.0 98.6 ± 8.1 108.8 ± 9.1 105.2 ± 8.7 Diast. BP (mm Hg) 57.2 ± 10.2 58.7 ± 7.9 66.1 ± 8.0 66.4 ± 7.4 Triglycerides (mg/dL) 62.6 ± 23.2 52.8 ± 20.3 87.1 ± 29.0 83.2 ± 35.3 HDL cholesterol (mg/dL) 44.3 ± 22.6^a^ 54.7 ± 17.4 49.9 ± 12.8 51.2 ± 11.5 LDL cholesterol (mg/dL) 76.0 ± 35.4 ^a^ 68.6 ± 37.6 99.5 ± 24.7 98.4 ± 24.3 Glucose (mg/dL) 79.7 ± 8.1 80.9 ± 9.7 85.4 ± 8.2^b^ 81.6 ± 7.4 Insulin (μu/mL) 8.5 ± 5.7 7.4 ± 4.6 14.8 ± 7.5 13.2 ± 8.6 HOMA-IR 1.7 ± 1.2 1.6 ± 1.0 3.0 ± 2.4 2.7 ± 2.0 Difference between groups (adjusted for age, race, and gender), a: p = 0.05; b: p = 0.02 HOMA-IR: homeostasis model assessment index of insulin resistance ::: Longitudinal rates of change in cardiovascular risk variables from childhood to adolescence, adjusted for race and gender, are presented in table [2](#T2){ref-type="table"} by birth weight groups. The rate of increase in BMI was significantly lower in low birth weight group compared with control group, while the rate of increase in subscapular skinfold remained similar between the groups. With respect to blood pressure, the rate of increase in systolic blood pressure was significantly higher in low birth weight group than control group. Of the measures of glucose homeostasis, rate of increase of glucose was marginally significant in low birth weight group vs control group. Low birth weight was associated with significantly higher rate of increase in LDL cholesterol; and no significant trends in HDL cholesterol and triglycerides. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Rates of change in risk variables from childhood to adolescence by birth weight. The Bogalusa Heart Study ::: Variable Low Birth Weight Control p-value --------------------------- ------------------ --------- --------- BMI (kg/m^2^/y) 0.60^†^ 0.71 0.03 Subsc. Skinfold (mm/y) 0.91 1.12 0.27 Syst. BP (mmHg/y) 1.70 1.30 0.02 Diast. BP (mm Hg/y) 1.21 1.02 0.26 Triglycerides (mg/dL/y) 2.28 2.92 0.80 HDL cholesterol (mg/dL/y) -0.53 -0.91 0.24 LDL cholesterol (mg/dL/y) 0.80 0.64 0.05 Glucose (mg/dL/y) 0.50 0.11 0.07 Insulin (μu/mL/y) 0.79 0.67 0.70 HOMA-IR 0.18 0.14 0.44 ^†^Regression slope with respect to age in years (y) adjusted for race and gender (generalized equation estimation method). HOMA-IR: homeostatis model assessment index of insulin resistance. ::: In a multivariate analysis, low birth weight was retained as an independent predictor variable for adverse longitudinal trends in systolic blood pressure, triglycerides, and glucose (marginal) from childhood to adolescence, regardless of race or gender (table [3](#T3){ref-type="table"}). Further, there was a significant interaction between low birth weight and age in this regard, denoting that these variables increased to a greater extent in the low birth weight group than in the control group as individuals became older. An analysis of this data set using the WHO criterion for low birth weight (\<2500 g) showed that only 36 subjects were reclassified as having normal birth weight, and the results were essentially the same (data not shown). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Independent association of low birth weight with longitudinal trends of systolic blood pressure, triglycerides and glucose from childhood to adolescence ::: Independent Variables Retained Syst. BP Triglycerides Glucose -------------------------------- ---------- --------------- --------- --------- ------- --------- Birth Weight (low vs control) 3.84 0.02 48.6 0.08 15.20 0.07 Gender (male vs female) \-- \-- \-- \-- 4.31 \<0.001 Age 0.39 0.40 12.21 0.01 6.60 \<0.001 Age^2^ 0.06 0.002 -0.44 0.04 -0.31 \<0.001 Insulin 0.11 0.03 1.16 \<0.001 0.22 0.02 BMI 0.34 \<0.001 \-- \-- \-- \-- Birth weight × age 0.45 0.004 12.71 0.03 0.10 0.07 Birth weight × age^2^ \-- \-- 0.55 0.02 2.65 0.10 ^†^GEE regression coefficient. The model included birth weight (low vs control) along with age, age^2^, race, and gender, and their interaction with birth weight; and risk variable as applicable. ::: Discussion ========== Information is sparse at a community level in the U.S. general population regarding the effects of low birth weight on the longitudinal trends in C-V risk factor variables measured serially and concurrently from childhood to adolescence. The present community-based study demonstrates the adverse effects of low birth weight on the longitudinal (developmental) trends in systolic blood pressure, triglycerides, and glucose during childhood and adolescence, regardless of race or gender. These observations are in accord with the emerging evidence supporting the concept of intrauterine imprinting and its pathophysiologic consequences enunciated by the fetal origin or thrifty phenotype hypothesis \[[@B3]\]. Many, but not all, previous studies in children and adolescents have found adverse associations between birth weight and levels of cardiovascular risk factor variables \[[@B16]-[@B27]\]. In this study, the magnitude of differences in mean levels of cardiovascular risk factor variables between low birth weight and control groups during childhood and adolescence periods were small and nonsignificant for most of the study variables, except for the adverse levels HDL cholesterol and LDL cholesterol in childhood and glucose in adolescence among the low birth weight group. However, in a multivariate analysis of the serial data, the independent adverse effects of low birth weight on the longitudinal trends of systolic blood pressure, triglycerides, and glucose were discernable in the study cohort. Of note, the observed adverse trends associated with low birth weight vs control group were influenced by age in that the differences became greater in magnitude as the children got older. Earlier studies have reported that the inverse associations between birth weight and levels of cardiovascular risk factor variables became stronger with increasing age \[[@B26],[@B38]\]. Whether the potentiating effect of increasing age on low birth weight -- risk variable relationship reflects the interaction between fetal programming related to intrauterine malnutrition and the increasing burden with age of unhealthy life-style behaviors including overnutrition and sedentary life style is not clear. In this context, it should be noted that although the rate of yearly increase in BMI, which also includes muscle mass, was significantly lower in low birth weight group, the rate of increase in subscapular skinfold, a measure of truncal fat, remained similar to that of control group. This suggests a gaining of truncal fat, in relative term, over muscle mass in the low birth weight group. Although observational studies like the present one can not establish causality, several putative mechanisms link low birth weight to adverse trends in risk factor variables. It has been suggested that insulin resistance may be one mechanism by which intrauterine events may program disease risk \[[@B39]\]. Undernutrition in utero is known to cause permanent impairment in growth, structure and function of muscle \[[@B39],[@B40]\], fat \[[@B41],[@B42]\], endocrine pancreas \[[@B2],[@B43]\], liver \[[@B30]\], renal nephrons \[[@B44],[@B45]\] and vasculature \[[@B46]\] due to biologic programming, resulting in insulin resistance/glucose intolerance, hypertension, and dyslipidemia. Further, it has been suggested that intrauterine programming of the hypothalamic-pituitary-adrenal axis may be a functional mechanism underlying the link between low birth weight and above disorders \[[@B47]\], known as components of insulin resistance or metabolic syndrome \[[@B48]\]. This study has certain limitations. The lack of information on the duration of gestation precluded us from adjusting the birth weight for gestational age, a potential confounder. However, earlier studies have found that adverse effects of low birth weight on cardiovascular risk factor variables were independent of gestation period \[[@B10],[@B17],[@B49]\]. Further, it has been pointed out that inclusion of preterm births could actually underestimate these associations \[[@B28]\]. This study also lacks measurements of glucose tolerance and insulin action and secretion used in etiologic studies. Instead, we used the glucose homeostasis measures that are relatively easily measured and applicable at a population level. Conclusions =========== Low birth is characterized by adverse developmental trends in metabolic and hemodynamic variables during childhood and adolescence, especially as the children get older. These observations in conjunction with earlier findings support the view that low birth weight, albeit a crude marker of prenatal growth and physiological environment, is a potential early risk factor for the emergence of metabolic and hemodynamic disorders and related diseases \[[@B1],[@B50]\]. Abbreviations ============= BMI, body mass index; LDL, low-density lipoprotein; HDL, high-density lipoprotein Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= MGF participated in study design, data analysis and manuscript preparation. SRS and GSB contributed to study concept and design, data collection, acquisition of funding and manuscript preparation. JX was involved in measurements of laboratory variables. 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/1471-2431/4/22/prepub> Acknowledgements ================ Supported by NIH grants AG-16592 from the National Institute on Aging; HL-38844 from the National Heart, Blood and Lung Institute; and HD-043820 from the National Institute of child Health and Human Development. The Bogalusa Heart Study is a joint effort of many investigators and staff members whose contribution is gratefully acknowledged. We especially thank the Bogalusa, LA school system and the participants and their parents.	PubMed Central	2024-06-05T03:55:49.881032	2004-11-3	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534105/", "journal": "BMC Pediatr. 2004 Nov 3; 4:22", "authors": [ { "first": "Maria G", "last": "Frontini" }, { "first": "Sathanur R", "last": "Srinivasan" }, { "first": "Jihua", "last": "Xu" }, { "first": "Gerald S", "last": "Berenson" } ] }
PMC534106	Background ========== Research on pain in infants has progressed considerably over the past twenty years. The nature and frequency of procedural pain in the neonatal intensive care unit (NICU) is now understood \[[@B1]\], many measures have been developed for assessment of acute pain in the NICU \[[@B2]\] and many pain interventions have now been evaluated \[[@B3]\]. However, much less is known about the pain experienced by neonates who are at risk for neurological impairment (NI), as most studies of neonatal pain have either excluded this group or have not examined data specific to them within larger data sets. We do know that this group represents approximately 10% of infants admitted to the Neonatal Intensive Care Unit \[[@B4]\] and that they experience more painful procedures in the NICU during the first days of life than infants who are not at risk for NI \[[@B5]\]. It also appears this vulnerable group may be particularly susceptible to potential long-term negative consequences of pain because of their neurological fragility, concomitant illnesses, and repeated exposure to painful stimuli \[[@B6]\]. The crucial first step of pain management is pain assessment. Without a valid and reliable approach to assessing pain, and the demonstrated efficacy of interventions for pain, decisions about pain management may not improve care. However, even with valid, reliable pain assessment tools, the characteristics of healthcare providers may affect ratings provided by them. These characteristics can include the healthcare providers\' views of pain interventions \[[@B7]\], lack of awareness of advances in pain management \[[@B8]\], and use of pain cues that are not reliable \[[@B9]\]. The present study was designed to move beyond self-report of beliefs to examine whether healthcare professionals\' judgments of pain in neonates are affected by their perception that a neonate has mild, moderate or severe risk for neurological impairment. Research in this area is only emerging, but has important implications for how healthcare professionals deliver care to this vulnerable population. In a previous study using questionnaires, we found that caregivers of children with severe cognitive impairment view the pain of children with more severe impairment as reduced \[[@B10]\]. A second study using the same questionnaire with healthcare professionals and students revealed a similar pattern of beliefs \[[@B11]\]. Most recently, we adapted that questionnaire to assess the beliefs of healthcare professionals\' regarding the pain of infants with varying degrees of risk for neurological impairment and again found that those who took part believed that the degree of pain experienced decreases as risk for neurological impairment increases \[[@B12]\]. These studies suggest that those who manage the pain of infants at risk for, or children with, intellectual deficits believe that pain is less for those at greater risk or who have greater impairment. These results may explain why we also found infants at risk for neurological impairment receive less pain treatment in the NICU \[[@B13]\]. However, to extrapolate from questionnaires to clinical behaviour can be problematic. Thus, the current study was designed as a step to linking these two sets of results. Specifically, we felt it was important to know if professionals\' beliefs about pain in this group influence their assessment of infants\' pain, which could lead to those infants\' being provided with less pain treatment. As with any experimental study, the circumstances could not completely replicate those in a clinical setting. For example, the participants would not have access to physiological data or to the infants\' recent history of pain. However, we hypothesized that the participants in this study would rate the pain lower for infants described as having greater risk for neurological impairment, corresponding with the beliefs expressed in our three previous studies. Methods ======= Participants ------------ One hundred and one healthcare professionals, with at least one year of experience working with infants with neurological impairment in the NICU, were recruited from two tertiary level university affiliated NICU settings in central and eastern Canada. They were recruited through information provided by the research nurses and notices posted within their centers. Each participant was paid a small honorarium for their participation, and all provided informed consent. The study was approved by each health centre\'s Research Ethics Board. Materials ========= Demographic information ----------------------- Participants completed a questionnaire that requested information regarding their age, gender, education and work experience. Video clips ----------- Nine video clips were viewed by each participant. The 30 second video clips depicted term and preterm neonates of Caucasian descent experiencing a heel stick and squeezing for blood collection. Video clips were of the infants\' faces only, with most lying on their sides and all bundled. Audio was not included. Prior to each videoclip, a verbal description of the neonate suggesting he/she was at mild, moderate or severe risk for neurological impairment was provided to the participant. These descriptions had been previously recorded on audio tape by a researcher to ensure each participant was read the description in an identical fashion. Descriptions were counterbalanced such that each videoclip was described for some participants as having either mild risk, moderate risk, or severe risk within each of two orders of presentation. Thus, each participant viewed three infants that were described as having mild, moderate or severe risk for neurological impairment, but the level of risk, and the order in which the clips were presented varied. Examples of the descriptions provided to participants are shown in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Sample descriptions of infants viewed on videotape provided ::: Mild Risk Moderate Risk Severe Risk ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Brianna is 6 days old and has been treated for neonatal jaundice. She will make a complete recovery. Otherwise she is healthy. Samuel was born 4 weeks prematurely and was mildly asphyxiated at birth because the cord was wrapped around his neck during delivery. An MRI shows a small area of damage in the brain. Matthew was born with a serious metabolic condition which caused significant brain damage. He will likely not survive past 2 years of age. Jason was born prematurely and is gaining weight slowly. He is now one month old. He suffered a unilateral Grade I bleed in his brain and will likely have no permanent damage from that. Matthew was born with a serious metabolic condition which caused moderate brain damage. With the aid of a special diet, he will develop fairly well but will likely have significant learning disabilities. Samuel was born 4 weeks prematurely and was severely asphyxiated at birth because the cord was wrapped tightly around his neck at delivery. An MRI shows extensive damage throughout the brain. ::: Ratings of pain and distress ---------------------------- Participants rated the pain of infants shown on videotape from 0 (no pain) to 10 (extreme pain). They also rated each infant\'s pain using a Faces Pain Scale (0 = no pain, 6 = extreme pain). These measures were chosen because they are easy to use and were feasible for this experimental task. Although many validated measures of neonatal pain exist, these are multidimensional in nature. As such, they require the person using them to have access to information regarding the infant\'s physiological status, something we were unable to provide in the context of this task. The 7 face scale \[[@B14]\], was included to allow a check of the validity of the 0--10 pain rating, since the latter is not commonly used in clinical neonatal settings. The Faces Pain Scale is also not typically used in a neonatal setting, but research indicates most adults find it easy to use \[[@B15]\], making it a useful check of participants\' 0--10 pain ratings. Preliminary analyses indicated there was a significant relationship, similar to results for reliability computed for other sets of observational pain tools used in pediatric research \[[@B16]\], between 0--10 pain ratings and Faces Pain Scale ratings for infants at mild ([r]{.underline} = .74), moderate (.[r]{.underline} = .56) and severe risk for neurological impairment ([r]{.underline} = .60). Ratings of cuddling and calm ---------------------------- Participants provided a rating of how effective they believed a behavioural intervention (i.e. cuddling) would be for minimizing the procedural pain for each infant (0 = no effect, 10 = very effective) and of how long (seconds) they believed it would take each infant to calm without intervention. Ratings of risk for neurological impairment ------------------------------------------- To ensure that the descriptions provided were valid depictions of infants at each level of risk and were understood and accepted by participants, participants were asked to rate the level of risk they believed each infant had for future neurological impairment (0 = no risk, 10 = certain impairment). Procedure --------- Participants took part in small groups of 5 to 6 professionals that were randomly assigned to one of the two orders of presentation. They completed the demographic questionnaire and the rating tasks were explained to them. They were then shown the nine video clips. After each videoclip was viewed, the participants were provided with time to make their independent ratings for that videoclip before the next was shown. Participants were not permitted to interact with each other until all tapes had been viewed and rated. After ratings were complete, they were debriefed regarding the purpose of the study and a discussion of their experience was facilitated by the research assistant. Preliminary analyses -------------------- ### Exclusions due to missing data Preliminary analyses indicated that two participants were missing more than 10% of ratings. As per an a priori decision as to how to handle missing data, their data were excluded from further analyses. The remaining 99 participants were missing 0% ([n]{.underline} = 88) to 7% ([n]{.underline} = 1) of responses ([M]{.underline} = 0.2, [SD]{.underline} = 0.6). ### Exclusions due to presentation order effects A 2 (group) X 6 (rating) Repeated Measures Analysis of Variance (RM ANOVA) was conducted on the six ratings provided by participants who viewed the tapes in the two orders to determine whether order of presentation had affected ratings. This analysis revealed a significant effect of order of presentation \[[F]{.underline}(1,97) = 4.4, [p]{.underline} = .04). A more detailed look at the data using stem and leaf plots revealed 4 participants in one group had extreme scores for ratings of Time to Calm ([M]{.underline} = 192.8, [SD]{.underline} = 39.2) relative to the other participants in that group ([M]{.underline} = 37.4, [SD]{.underline} = 28.7). The data of these four participants were removed. A second RM ANOVA revealed no significant effect due order of presentation of the video clips \[[F]{.underline}(1,93) = 1.7, [p]{.underline} = .20). Thus, 95 professionals formed the final sample for the study. ### Manipulation check To determine if the descriptions provided to the participants were effective in leading them to believe the infants viewed had mild, moderate or severe risk for neurological impairment, each participant\'s mean rating for degree of risk for future impairment for the infants they were told had a mild (3), moderate (3) or severe risk (3) for impairment were computed. These ratings were then compared using a RM ANOVA. There was a significant difference in the ratings provided to those clips described as having mild ([M]{.underline} = 3.8, [SD]{.underline} = 2.8), moderate ([M]{.underline} = 4.9, [SD]{.underline} = 2.0) and severe risk ([M]{.underline} = 6.1, [SD]{.underline} = 2.8; [F]{.underline}(2, 93) = 13.9, [p]{.underline} \< .001). Post-hoc paired t-tests indicated these differences were significant between infants described as having mild and moderate risk ([t]{.underline}((94) = -3.2, [p]{.underline} = .002), mild and severe risk ([t]{.underline}((94) = -4.8, [p]{.underline} \< .001), and moderate and severe risk ([t]{.underline}((94) = -5.0, [p]{.underline} \< .001), Thus, participants believed the infants had different levels of risk when they provided ratings for the video clips. Statistical procedures ---------------------- The data were analyzed using SPSS Version 10.0.7 \[[@B17]\]. Power computations were completed using Sample Power 1.2 \[[@B18]\] or based on tables prepared by Stevens \[[@B19]\]. Alpha was set at .05 for all tests and Bonferroni corrections were applied to sets of post hoc matched sample t-tests to maintain alpha at .05 for each set. Because the corrected [p]{.underline} values varied with the number of tests in each set, raw [p]{.underline} values are reported. Wilks Lambda was used to test significance for all RM ANOVA\'s. There was .80 power or greater to detect medium size effects using repeated measures analyses with 3 to 5 levels of factors and greater than .99 power to detect medium size differences in means using matched sample t-tests. Power was .86 to detect a significant medium size correlation between ratings and years of experience. ### Descriptive statistics Descriptive statistics were generated for the demographic characteristics of the participants (Table [2](#T2){ref-type="table"}) and for the ratings provided for each set of video clips (Table [3](#T3){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Characteristics of the participants (N = 95) ::: Characteristic n \% ------------------------- ---- ---- Site Toronto 46 48 Halifax 49 52 Profession Nurse 50 53 Physician 19 20 Respiratory Therapist 17 18 Occupational Therapist 2 2 Physiotherapist 2 2 Psychologist 1 1 Other Clinician 4 4 Gender Female 82 86 Age 20--30 years 19 20 31--35 years 15 16 36--40 years 32 34 41--45 years 11 12 46 years or more 18 19 Note. Percentages rounded. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Mean ratings given to infants described as at risk for mild, moderate or severe risk for neurological impairment (N = 95) ::: Rating Mild Risk Moderate Risk Severe Risk ----------------------------------- ----------- --------------- ------------- ------ ------ ------ M SD M SD M SD Pain (0--10) 6.3 1.7 6.6 1.7 6.2 1.6 Faces pain Scale (0--6) 4.7 1.0 4.8 0.8 4.8 1.0 Distress (0--10) 6.1 1.7 6.5 1.5 6.3 1.6 Effectiveness of Cuddling (0--10) 7.0 2.1 7.1 1.9 6.5 2.2 Time to calm (Seconds) 35.2 34.8 34.9 28.3 32.8 31.8 ::: ### Effect of risk for neurological impairment on ratings To compare the ratings provided for the 9 video clips, a 3 (level of risk) X 5 (rating type) RM ANOVA was conducted on the scores of the 95 participants. This was followed by 5 one-way RM ANOVA\'s on each rating (0--10 pain rating, Faces Pain Scale rating, distress rating, effectiveness of cuddling rating, time to calm) and matched sample t-tests on ratings when the one-way ANOVA was significant. ### Effect of participants\' characteristics on ratings The effect of participants\' characteristics on ratings was examined using Mixed Measures ANOVA\'s on the five ratings at three levels of risk. The first three included Gender, Age, and Site (Toronto, Halifax) as between-subjects effects. The fourth included three levels of profession (i.e. staff nurse, physician, respiratory therapist) as the between-subjects effect. Other professionals were not included due to small numbers. The relation between the participants\' years of experience in a neonatal setting and their ratings were investigated using Pearson Correlations. Results ======= Participants ------------ The characteristics of the participants are displayed in Table [2](#T2){ref-type="table"}. The majority were nurses and the number of years experience in a neonatal setting ranged from 1.5 to 36 years ([M]{.underline} = 11.8, [SD]{.underline} = 7.7). The 50 nurses included staff nurses ([n]{.underline} = 34), advanced practice nurses ([n]{.underline} = 9) and nurse managers/educators ([n]{.underline} = 7). The physicians specialized in neonatology ([n]{.underline} = 10), neurology ([n]{.underline} = 4), pediatrics ([n]{.underline} = 3) and other specialties ([n]{.underline} = 2). Six of the 19 physician participants were residents or fellows. Additional professions are listed in Table [2](#T2){ref-type="table"}. Effect of risk for neurological impairment on ratings ----------------------------------------------------- The mean ratings provided for video clips of infants described as having mild, moderate or severe risk for neurological impairment are depicted in Table [3](#T3){ref-type="table"}. The RM ANOVA on the five ratings revealed a nonsignificant effect of Level of Risk \[[F]{.underline} (2,93) = 0.6, [p]{.underline} \> .05\], a significant effect of Rating \[[F]{.underline} (4,91) = 91.6, [p]{.underline} \< .001\] and a significant interaction between the two \[[F]{.underline} (8,87) = 3.4 [p]{.underline} = .002\]. Thus, there was no overall effect due to the level of risk described, but level of risk described did affect some ratings. One-way RM ANOVA\'s revealed Level of Risk had a marginal effect on participants\' ratings of pain on the 0--10 scale \[[F]{.underline} (2,93) = 2.9, [p]{.underline} = .06\] and a significant effect on ratings of the perceived effectiveness of cuddling \[[F]{.underline} (2,93) = 4.4, [p]{.underline} = .02\], but nonsignificant effects on Faces Pain Scale ratings \[[F]{.underline} (2,93) = 0.3, [p]{.underline} = .70\], distress \[[F]{.underline} (2,93) = 2.2, [p]{.underline} = .12\] and time to calm \[[F]{.underline} (2,93) = 0.4, [p]{.underline} = .65\]. As Table [3](#T3){ref-type="table"} shows, there was a slight tendency for participants to rate pain lower for infants who were described as having greater risk for impairment. Participants did believe cuddling would be less effective when risk for neurological impairment was greater. Ratings of the effectiveness of cuddling were significantly lower for infants described as at high risk than they were for those described as at mild risk \[[t]{.underline}(94) = 2.5, [p]{.underline} = .01\] or moderate risk \[[t]{.underline}(94) = 3.0, [p]{.underline} = .004\]. The difference in ratings between those described as at mild or moderate risk were nonsignificant \[[t]{.underline}(95) = -0.2, [p]{.underline} = .77\]. Thus, participants believed that beyond a moderate level of risk, the effectiveness of cuddling dropped significantly. In summary, participants did not view the pain of the infants as varying due to level of risk for neurological impairment. Nor did they perceive the distress or time to calm after pain as differing between groups of infants described as having mild, moderate or severe risk for neurological impairment. However, they did perceive that cuddling would be less effective as an intervention for infants with high risk, than for those with mild or moderate risk of neurological impairment. Effect of participants\' characteristics ---------------------------------------- ### Site, gender and age Three Mixed Measures ANOVA\'s were used to examine the effect of participants\' characteristics on the five ratings ratings. The first result indicates a nonsignificant main effect of Site \[[F]{.underline}(1,93) = 0.7, [p]{.underline} = .39\], the second revealed a nonsignificant main effect of Gender \[[F]{.underline}(1,93) = 0.9, [p]{.underline} = .34\], and the third indicated Age also did not significantly effect ratings on the five measures \[[F]{.underline}(4,90) = 0.2, [p]{.underline} = .95\]. Thus, participants\' ratings did not vary due to their institution, gender or age. ### Profession To examine the effect of participants\' profession on their ratings, three groups were included in a Mixed Measures ANOVA: staff nurses (n = 34), physicians (n = 11), and respiratory therapists (n = 17). Residents and Fellows, Nurse Managers and Educators and Specialists, and other professionals were not included due to small numbers in those groups. The analysis revealed a significant main effect of Rating Scale \[[F]{.underline}(4,56) = 3.9, [p]{.underline} = .001\]. However, the main effect of Level of Risk was nonsignificant and the main effect of Profession only approached significance \[[F]{.underline}(2,59) = 2.7, [p]{.underline} = .07\]. Participants\' ratings were not affected by their professional background. The interaction between Rating and Level of Risk was significant \[[F]{.underline}(8,52) = 36.6, [p]{.underline} \< .001\], but all other interactions were nonsignificant. Games-Howell post-hoc comparisons revealed a significant difference in the ratings provided by staff nurses and physicians ([p]{.underline} = .004) and a difference between respiratory therapists and physicians that approached significance ([p]{.underline} = .06). As shown in Table [4](#T4){ref-type="table"}, Nurses\' ratings did not appear to differ greatly due to level of risk for impairment, while Physicians\' showed a tendency to rate all aspects of the experience higher as level of risk increased, and respiratory therapists tended to provide lower ratings as the infants\' level of described risk for neurological impairment increased. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Mean pain, distress, effectiveness of cuddling and time to calm scores given to infants described as at risk for mild, moderate or severe risk for neurological impairment by physicians and other clinicians ::: Rating Level of Risk Staff Nurses (n = 34) Physicians (n = 11) Respiratory Therapists (n = 17) ---------------------------------------- --------------- ----------------------- --------------------- --------------------------------- ------------------ ----------------- ------------------ [M]{.underline} [SD]{.underline} [M]{.underline} [SD]{.underline} [M]{.underline} [SD]{.underline} 0 -- 10 Pain rating Mild 6.4 1.6 4.8 2.3 6.9 1.3 Moderate 6.9 1.5 5.6 2.4 6.9 1.8 Severe 6.4 1.7 5.8 2.1 5.8 1.6 Faces pain rating (0--6) Mild 4.6 1.0 3.7 0.8 5.3 0.6 Moderate 4.9 0.7 4.8 0.6 4.9 1.1 Severe 4.9 1.0 5.4 0.6 4.3 1.3 0 -- 10 Distress rating Mild 6.4 1.6 4.3 2.1 6.9 1.3 Moderate 6.8 1.3 5.6 2.2 6.8 1.9 Severe 6.4 1.6 6.3 1.9 5.4 1.6 0--10 Effectiveness of cuddling rating Mild 6.4 1.6 4.3 2.1 6.9 1.3 Moderate 7.2 2.0 6.9 1.9 6.5 2.4 Severe 6.6 2.3 5.8 2.6 4.9 2.1 Time to calm estimate (seconds) Mild 40.0 36.1 14.9 15.5 46.8 43.8 Moderate 39.8 28.9 18.4 13.8 42.2 35.4 Severe 40.5 40.3 23.3 15.7 31.3 33.0 ::: ### Professional experience Eighty-nine participants provided information regarding their amount of professional experience. Correlations indicated that years of experience were not correlated significantly with any of the five ratings provided after corrections for multiple tests. Thus, the importance of an infants\' level of risk for neurological impairment was neither greater nor less as experience in this setting increased. Discussion ========== Overall, the professionals in this study did not rate the pain of neonates differently when provided with information indicating those infants had mild, moderate or severe risk for neurological impairment. The professionals\' perception of the infants\' level of risk also did not affect their ratings of the infants\' distress, or their belief in how long the infant would take to calm after pain without intervention. Professionals did perceive that cuddling would be significantly less effective for infants at high risk for neurological impairment than for infants with mild or moderate impairment. However, this effect was not large, and, although it was statistically significant, it may be spurious. Further research should examine whether beliefs regarding pain experience in this group and beliefs regarding the effectiveness of cuddling and other nonpharmacological interventions are truly independent. These results are inconsistent with the results of our previous questionnaire study indicating professionals, with similar levels of experience in neonatal intensive care settings, perceive the pain experience of infants as reduced as their level of risk for neurological impairment increases \[[@B12]\]. There are several possible reasons for these discrepant results. The professionals who participated in this study were asked to rate the risk for neurological impairment of each infant they viewed on videotape. Asking them to do this may have alerted them to the purpose of the study and elicited efforts on their behalf to provide ratings that were unbiased. However, their ratings of the perceived effectiveness of cuddling did vary by level of risk for impairment, suggesting attempts to appear unbiased do not fully explain the results found. In our previous studies, questionnaires elicited beliefs about the pain experience of infants and children with varying levels of risk relative tothe pain experience of those without risk \[[@B10]-[@B12]\]. In contrast, no infants in the current study were described as having no risk for neurological impairment. This was because the infants\' appearance made it apparent that they were not healthy full-term infants. It may be that the comparative nature of the questions in the previous studies made the possibility of differences in pain experience due to neurological risk more salient to participants. Thus, the pain ratings provided here did not differ among levels of risk, but had ratings of healthy infants been included in the task, they may have differed significantly from them. It is also possible that the beliefs expressed by professionals in our previous study \[[@B12]\] do not moderate professionals\' behaviour in relation to pain assessment for specific infants, as was found here. A discordance between expressed beliefs and behaviour, in regard to pediatric pain management, has been reported elsewhere \[[@B20],[@B21]\]. Thus, the professionals here may hold similar beliefs to the professionals in our previous study, but these beliefs did not alter their behaviour when asked to judge pain in a specific infant based on observable behaviour. This interpretation is supported by the current results because no differences were found due to level of risk for ratings that the professionals could base on behaviour they observed on the video clips: pain, distress, time to calm. In contrast, professionals\' judgments of the effectiveness of cuddling were influenced by the descriptions of the infants\' level of risk for neurological impairment. This may be because there was no visual information to base this rating upon, so professionals used the descriptions of risk provided, presumably in light of their previous experience with these groups in the neonatal setting. The finding that pain ratings did not vary due to level of risk for neurological impairment raises questions about our previous study that revealed infants at risk for neurological impairment receive less pain treatment in the NICU \[[@B13]\]. When a group is provided less medication for pain, it is typically assumed that this is because their pain was judged as less. However, it is possible that professionals hold beliefs about pain treatment that directly impact upon treatment decisions, irregardless of pain assessment. For example, they may hold beliefs about the appropriateness of medication for specific groups that are unrelated to beliefs about the amount of pain that group experiences. In support of this perspective, research indicates that nurses hold negative attitudes towards pharmacological treatment for pain \[[@B7]\] and that steps to improve pain assessment do not necessarily result in changes in pain management \[[@B22]\]. Further research is needed to reconcile the current results with beliefs that risk for neurological impairment does affect pain experience expressed by a similar group of professionals in our previous survey \[[@B12]\] and the results of our study indicating procedural pain is not treated as frequently for infants in the NICU who have greater risk for neurological impairment \[[@B13]\]. If this reflects a disconnect between pain beliefs related to assessment and those related to treatment for infants at risk for neurological impairment, then educational interventions aimed at improving care through changes in pain assessment may be ineffective. In that case, other avenues to changing professionals\' pain management for this group should be explored. Another finding in this study warrants discussion. Professionals\' judgments of the effectiveness of cuddling decreased with increasing risk for neurological impairment, despite their having judged pain as similar in intensity. This result is similar to a finding by Fanurik et al. \[[@B23]\]. They found nurses, but not physicians, responding to vignettes of children undergoing painful procedures, indicated nonpharmacological interventions would be less appropriate as level of cognitive impairment increased. The same professionals\' ratings of the pain intensity experienced by the children in that study did not differ due to perceived level of cognitive impairment. The current results, along with those of Fanurik\'s group \[[@B23]\], raise the question of whether professionals perceive the pain experienced by those at risk for or with neurological impairment as similar in intensity, but differing in quality from those at lesser risk. Because the current study elicited ratings only of the intensity of pain and distress and professionals were not asked about the nature of the pain the infants experienced, the results cannot confirm this possible explanation, as data regarding pain quality was not collected. However, professionals in our survey study differentiated between physiological aspects of pain and internal and external responses to pain, such as emotional reaction, behavioural reaction and communication of pain \[[@B12]\]. They also believed the experience of infants at greater risk was more reduced along the latter aspects that are more psychological in nature. Caregivers\' have expressed similar beliefs, and also perceived the behaviour of children with more severe impairment is more closely related to their physiological pain experience \[[@B10]\]. From this finding, we could suggest that there is a belief, on the part of professionals and caregivers, that the pain behaviour of those at greater risk for, or with, neurological impairment is more reflexive in nature. We could further speculate that the underlying rationale may be that they are seen as less able to interpret their pain, both cognitively and emotionally, due to their neurological impairment. However, we would need to conduct further research to substantiate this rationale. If professionals and caregivers do believe pain behaviour is more reflexive, and that pain experience is more physiologically based when a child has neurological impairment, it could explain the current results regarding the effectiveness of cuddling. Professionals viewing the video clips may have perceived the behavioural responses of the infants with different levels of risk for impairment as being similar in intensity. Nonetheless, they may have interpreted the behaviour of those with more risk as more of a reflexive response to a physiological insult, while they saw the behaviour of those with lesser risk as reflecting a more multidimensional pain experience incorporating both physical and psychological suffering. Thus, we could again speculate that they may have felt cuddling, an intervention that would address physical and psychological aspects of pain, would be more effective for the less impaired groups. This phenomenon would not be novel or unique. For most of recorded history, there has been a belief that cognitive interpretation of pain was necessary for pain to result in long-term negative consequences. This belief was often the justification for poorer pain management for both children and infants \[[@B24]\]. Although this belief is fading in regard to children and infants in general, it is still held in relation to those who are most severely at risk for, or have neurological impairment, and are perceived as least capable of interpreting their pain. Alternatively, this belief may be based on the actual experience of professionals in this study, that it is more difficult to calm an infant at risk for neurological impairment. This experience may also be an accurate perception of the difficulty infants at greater risk for impairment may have in responding to behavioural interventions because of their reduced ability to organize behavioural state and biobehavioural responses. Further research should examine these areas of speculation to specifically determine whether the perception that a behavioural intervention will be less effective for infants at greater risk for neurological impairment does reflect professionals\' direct experience with this group or their understanding of how the pain experience may be affected by neurological impairment that may affect pain interpretation. The current study has several limitations. Professionals were asked to rate the pain experience of infants receiving heel sticks from videotape. Although this may approximate the real situation in a NICU setting, it is not identical. In a NICU setting, professionals would have rich information from the environment, previous contact with an infant, physiological data, and medical records that guide their assessment of pain. They would also view this infant within the context of all other infants in the unit. Professionals here were also asked only to provide ratings of pain intensity. As the results suggest, this is only one dimension of pain and may not be the dimension that plays the largest role in their judgments regarding pain in a clinical setting. The professionals here were experienced in the types of pain experienced in the NICU and may have held a priori beliefs about the painfulness of this procedure that moderated their judgments. Research suggests professionals\' beliefs regarding the painfulness of a procedure play a large role in their assessments of children\'s pain \[[@B7],[@B9],[@B25]\]. Conclusions =========== The current study indicates professionals\' perception of the pain intensity of infants does not differ due to their understanding of the infants\' level of risk for neurological impairment. Professionals also view cuddling as less effective for infants at greater risk for neurological impairment. Further research is needed to examine the reasoning behind the judgments made by healthcare professionals and to clarify why they might view an intervention as less effective for infants with greater risk of neurological impairment, despite having rated their pain intensity as similar to that of infants at lesser risk. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= The study was conceived and designed by BS, PM & LB with assistance from all remaining authors. The study was conducted under the supervision of PM, BS, KO, AO. Statistical analyses were conducted by LB, with assistance from PM, BS and JB. Interpretation of results were conducted by LB, PM, BS, JB, CC, LF, KB and AO. The manuscript was prepared by LB, and edited by PM and BS, with review and assistance from all remaining authors. 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/1471-2431/4/23/prepub> Acknowledgments =============== We would like to acknowledge the research nurses who participated in the collection and management of this data, including Kim Caddell, Kim Dionne, and Anne Jack. The assistance of Celeste Johnston, Patricia McKeever and Sharyn Gibbons in carrying out this project is also appreciated. Lynn Breau is supported by a Postdoctoral Fellowship from AstraZeneca Canada, Inc. and is a CIHR Strategic Training Fellow in the Canadian Child Health Clinician Scientist Program which is supported by the SickKids Foundation, BC Research Institute for Children & Women\'s Health and the Child Clinician Scientist Program of the Canadian Institutes for Health Research. Funding for this project is gratefully acknowledged from the Canadian Institutes of Health Research (MOP-37884).	PubMed Central	2024-06-05T03:55:49.883129	2004-11-12	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534106/", "journal": "BMC Pediatr. 2004 Nov 12; 4:23", "authors": [ { "first": "Lynn M", "last": "Breau" }, { "first": "Patrick J", "last": "McGrath" }, { "first": "Bonnie", "last": "Stevens" }, { "first": "Joseph", "last": "Beyene" }, { "first": "Carol S", "last": "Camfield" }, { "first": "G Allen", "last": "Finley" }, { "first": "Linda", "last": "Franck" }, { "first": "Alexandra", "last": "Howlett" }, { "first": "Karel", "last": "O'Brien" }, { "first": "Arne", "last": "Ohlsson" } ] }
PMC534107	Background ========== Suicide is an important public health problem that is among the leading causes of death in Western countries \[[@B1]\]. Over the last years, the relationship between suicide and mental disorders has been the focus of several studies and has generated important debate \[[@B2]\]. This relationship has been investigated by different strategies, but particularly by the psychological autopsy method \[[@B3]\], which is generally considered the method of choice to retrieve postmortem information on psychopathology. The psychological autopsy procedure entails the retrospective psychiatric assessment of the deceased by variable methodologies, but generally by means of proxy-based interviews. This procedure is also frequently completed by having access to medical and other relevant dossiers from the subject on whom the psychological autopsy is carried out \[[@B4],[@B5]\]. It is well established that psychopathology is an important predictor of suicide completion \[[@B6]\], but there is considerable variability between studies in rates of total and specific psychiatric disorders \[[@B7]\]. One of the most consistent findings in suicidology is the excess of male suicides observed in most countries \[[@B8]\], with a few notable and important exceptions, such as China \[[@B1],[@B9]\]. Geographic origin is another important source of variation \[[@B1]\]. However, the possibility that clinical and other behavioural factors could at least partly mediate gender and geographic differences in suicide rates has been little explored. The aim of this study was to carry out quantitative syntheses of overall and specific psychiatric diagnoses found in suicide studies and to explore possible gender and geographical differences in the distribution of psychiatric disorders among suicide completers. Methods ======= Study identification -------------------- To identify studies for this review, the National Library of Medicine (NLM) PubMed database was searched up to December 2002 using English language and human study limits. The Medical Subject Heading (MeSH) terms \"suicide AND psychological autopsy\", \"suicide AND psychopathology\", \"suicide AND (postmortem diagnoses OR postmortem diagnosis)\", and \"(mental disorders/\epidemiology) AND prevalence AND ((suicide/\statistics & numerical data) NOT suicide attempts)\" were used. Finally, in order to find other articles not obtained through electronic searches, reference lists from original studies as well as from not independent studies were screened. Study selection --------------- The inclusion criteria for considering articles for this review were as follow. Studies had to: 1) be original, 2) be published in English, 3) contain information on diagnostic distribution, 4) include suicide completers unselected according to specific mental disorders, 5) use of a psychological autopsy method, which for the purpose of this review was considered as the process of reconstructing psychiatric diagnoses based either on interviews with informants (regardless of the specific diagnostic instrument methodology) or on review of multiple official records that contained interviews with informants such as general practitioners, other professionals and relatives or friends, 6) use of standard diagnostic criteria (any versions of the Diagnostic and Statistical Manual of Mental Disorders, the International Classification of Diseases or the Research Diagnostic Criteria). Studies were excluded if: 1) their sample was not independent from that investigated in another study (see below for criteria on which one was included), 2) they were reports on suicide in one specific diagnostic category and 3) if diagnoses were simply extracted from medical records without review of multiple sources of information. A single reviewer (G.A.L.) made a prior screening to identify and select articles. When titles and abstracts were deemed adequate or when they remained too obscure to reach a verdict, full texts were retrieved for further evaluation in conformity with the inclusion and exclusion criteria. Study assessment ---------------- A total of 23 variables were defined, three of which relate to demographic information, four other concern the method of diagnosis, and 16 evaluate the presence of psychiatric diagnoses. To obtain the latter 16 variables (shown in table 1; see [additional file](#S1){ref-type="supplementary-material"}), every diagnostic term used in the original studies was categorised into one of the 16 pre-defined groups. So diagnoses such as \"intermittent depressive disorder\" or \"neurotic depression\" reported in some studies were coded under \"depressive disorders\' variable and diagnoses such as \"alcohol use\", \"alcohol misuse\" and \"alcohol abuse\" were coded as \"alcohol problems\". All substances noted as other than alcohol were coded under \"other substances problems\". These two variables were then recoded as \"any substance problems\". The same was achieved with the \"depressive disorders\" and \"bipolar disorders\" which were recoded as \"any affective disorders\". Disorders labelled as \"other\" or as a subset of various disorders without further specification were left aside. For all studies the most specific diagnosis was considered. That is, when the authors broke down general diagnosis such as \"affective disorder\" into \"depressive disorders\" and \"bipolar disorders\", only these more specific diagnoses were noted and accounted for in our study. When two studies or more were carried on the same population, the study with the largest sample and the most informative report was consistently selected. When multiple diagnoses and principal diagnoses (those deemed by the investigators as more related to the suicide) were reported, preference was given to the former. In four cases, secondary diagnoses were added to principal diagnoses to obtain multiple diagnoses \[[@B10]-[@B13]\]. Studies for which controls were selected among psychiatric in-patients or matched to suicides by mental diagnosis, only suicide cases were included in our analysis \[[@B12],[@B14]\]. In the study by Graham and Burvill \[[@B15]\], controls were older suicide completers, and so they were included in our suicide group. In the study by Hawton et al. \[[@B10]\], only diagnoses for suicides obtained by means of an interview were included. In three case-control studies \[[@B16]-[@B18]\], not all suicide cases were matched to a control. In these cases, we considered the full suicide sample in the descriptive analyses, but only the control-matched suicides in the quantitative analyses. Statistical analysis -------------------- Descriptive analyses and homogeneity tests were carried out before pooling the data. In order to determine the risks of having had a disorder, suicides and controls were recorded in 2 × 2 tables. These data were then stratified by the 16 outcome variables and Mantel-Haenszel Weighted Odds Ratios (OR) and 95% confidence intervals (95% CI) were estimated. Gender differences were also explored by means of Odds Ratios. Major disorders were then compared between the different demographic areas by means of χ^2^to assess variations in the diagnostic distribution across these demographic areas. All statistical analyses were carried out using Epi Info 6, version 6.04d (C.D.C., U.S.A.; W.H.O., Geneva, Switzerland). Results ======= A total of 152 studies were initially identified. After selection according to inclusion/exclusion criteria, 27 studies were included in this review. The most common reasons for exclusion were that a) no diagnostic distribution was provided (n = 46) \[[@B6],[@B19]-[@B63]\], b) samples were pre-selected according to a psychiatric disorder (n = 30) \[[@B64]-[@B93]\], c) there was another report on the same sample that either included more subjects or was more informative (n = 29) \[[@B3],[@B94]-[@B121]\]. Four other studies were about non-completers \[[@B122]-[@B125]\]. Another was not in English \[[@B126]\], and others reported only on one type of disorder \[[@B127],[@B128]\], and therefore, they were all excluded. Additional 14 studies \[[@B7],[@B129]-[@B141]\] were excluded because the diagnostic criteria were either unspecified or not standard. The studies by Rich et al. \[[@B99]\] and by Foster et al \[[@B142]\] were not independent from, respectively, Rich et al. \[[@B143]\] and Foster et al. \[[@B144]\]. Although non-independent, these studies provided information of different quality, and thus, were included in our review. Accordingly, Rich et al. \[[@B99]\] and Foster et al. \[[@B142]\] were considered, respectively in the gender difference analysis and the case-control comparisons, whereas the study by Rich et al. \[[@B143]\] and Foster et al. \[[@B144]\] were considered for the descriptive analysis. Methodological assessment ------------------------- Among the 27 studies that were retained, 52% (14/27) were case-control studies. Eighty-one percent (22/27) of the studies were published after 1990. Sixty-seven percent of the studies (18/27) used DSM diagnostic criteria, whereas only 22% (6/27) and 11% (3/27) used the ICD and RDC diagnostic criteria respectively. Multiple diagnoses were investigated in 63% (17/27) of the studies, whereas principal diagnoses only were given for the other 10 studies. A description of the demographic and methodological features of these 27 studies is shown in table [2](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 2 ::: {.caption} ###### Description of the 27 studies included in this meta-analysis ::: Study Year Origin Diagnostic criteria Methods Number of diagnoses n Suicide With a Dx (%) n Control with a Dx (%) Matched ----------------------------- ----------- ----------- --------------------- --------------------------------- --------------------- ----------- --------------- ----------- --------------- ------------------------------------------------------------------------------------- Appleby et al.\[151\]\* 1999 England ICD-10 Official records and interviews Multiple 84 76 (90%) 64 17 (27%) Living ± 5 year and sex Apter et al.\[145\]\* 1993 Israel DSM-III Official records and interviews Principal 43 35 (81%) Asgard U.\[147\]\* 1990 Sweden RDC Official records and interviews Principal 104 99 (95%) Cavanagh et al.\[14\] 1999 Scotland DSM-III Official records and interviews Principal 45 44 (98%) Cheng et al.\[16\]\* 1995 Taiwan DSM-III-R Official records and interviews Multiple 116 114 (98%) 226 130 (58%) Living ± 5 years, sex, area of residence Conwell et al.\[156\]\* 1996 USA DSM-III-R Official records and interviews Multiple 141 127 (90%) Foster et al.\[142,144\] 1997/1999 Ireland DSM-III-R Official records and interviews Multiple 118 106 (90%) 117 30 (26%) List of deceased\'s GP Age, gender, marital status Harwood et al.\[17\]\* 2001 England ICD-10 Official records and interviews Multiple 100 93 (93%) 54 N/A Natural deaths Age and sex Hawton et al.\[10\] 2002 England ICD-10 Official records and interviews Multiple 42 38 (90%) 84 6 (7%) Living nurses ± 10 years, specialty and seniority Henriksson et al.\[11\] 1993 Finland DSM-III-R Official records and interviews Multiple 229 225 (98%) Houston et al.\[12\] 2001 England ICD-10 Official records and interviews Multiple 47 40 (85%) Lesage et al.\[150\] 1994 Canada DSM-III-R Official records and interviews Multiple 75 69 (92%) 75 N/A Living Neighbourhood, age, marital status and occupation Phillips et al.\[9\]\* 2002 China DSM-IV Interviews with informants Principal 519 325 (63%) 536 93 (17%) Accidental deaths Geographical areas Rich et al.\[143\] 1986 USA DSM-III Official records and interviews Multiple 283 258 (91%) Runeson B.\[153\] 1989 Sweden DSM-III-R Official records and interviews Principal 58 57 (98%) Shaffer et al.\[18\]\* 1996 USA DSM-III Official records and interviews Multiple 119 108 (91%) Shaffi et al.\[13\]\* 1988 USA DSM-III Official records and interviews Multiple 21 20 (95%) 21 11 (52%) Living friends Sex, age, race, education, religion, income, and father\'s education Vijayakumar et al.\[159\]\* 1999 India DSM-III-R Official records and interviews Principal 100 88 (88%) 100 14 (14%) Living SES, sex and ± 2 years Waern et al.\[154\]\* 2002 Sweden DSM-IV Official records and interviews Multiple 85 82 (96%) 153 28 (18%) Living Sex, ± 2 years Boardman et al.\[152\] 1999 England ICD-10 Multiple official records Multiple 212 151 (71%) 212 40 (19%) Unnatural deaths ± 5 years and sex Cantor et al.\[157\] 1989 Australia DSM-III-R Multiple official records Principal 47 41 (87%) Groholt et al.\[149\]\* 1997 Norway DSM-III-R Multiple official records Multiple 121 90 (74%) Thacore et al.\[158\] 2000 Australia ICD-9 Multiple official records Principal 75 46 (65%) Graham et al.\[15\] 1992 Australia DSM-III Multiple official records Multiple 136 120 (88%) Brent et al.\[148\] 1999 USA DSM-III Interviews with informants Multiple 140 115 (82%) 131 32 (24%) Living Age, race, gender, country and SES Cerel et al.\[155\] 2000 USA RDC Interviews with informants Multiple 15 13 (87%) 201 70 (35%) Non-suicide bereaved family Arato et al.\[146\]\* 1987 Hungary RDC Interviews with informants Principal 200 162 (81%) \* Based on axis I disorders only. N/A -- information not available or not clear ::: Demographic features -------------------- A total of 3275 suicides were included in our study with a mean number of 121 (standard deviation (SD) 103) suicides per study. There were 11 studies where diagnoses were given by gender for a subtotal of 933 males and 462 females \[[@B10],[@B11],[@B18],[@B99],[@B144]-[@B150]\] There were 14 studies \[[@B10]-[@B12],[@B14],[@B17],[@B142],[@B145]-[@B147],[@B149],[@B151]-[@B154]\] carried out in Europe, including one in Israel \[[@B145]\]. These 14 European studies comprised a total of 1488 suicides. Seven studies were from North America \[[@B13],[@B18],[@B143],[@B148],[@B150],[@B155],[@B156]\] with 794 suicides, three others were from Australia \[[@B15],[@B157],[@B158]\] with 258 suicides and, finally, three were from Asia \[[@B9],[@B16],[@B159]\]. with 735 suicides. Diagnostic distribution ----------------------- The mean percentage of suicides with a psychiatric diagnosis was 87.3 % (SD 10.0 %). However, only 14 of the 27 studies reported both axes I and II disorders (see table [2](#T1){ref-type="table"}). The remaining 13 studies only assessed axis I diagnoses. The mean percentage of controls with a diagnosis was, as expected, lower (34.9 % SD 25.1 %). As a comparison, among studies not included because the diagnostic criteria were not specified or not standard, the mean percentage of suicides with a diagnosis was not statistically different from that of the studies included in this review (78.7% SD 21.0%, χ^2^: 2.27, p = 0.13). On average, 43.2% (SD 18.5%) of suicide cases were diagnosed with any affective disorders (including depressive and bipolar disorders) and 25.7% (SD 14.8%) with other substance problems. In these groups, respectively, depressive disorders and alcohol problems were the most frequent. Finally, personality disorders represented 16.2% (SD 8.6%) of the suicide diagnoses and psychotic disorders, including schizophrenia accounted for 9.2% (SD 10.2%). The samples from the 14 case-control studies were found homogeneous for the 16 outcome variables according to a homogeneity test (results not shown), allowing us to pool the individual studies and determine overall risks. Table 1 (see [additional file](#S1){ref-type="supplementary-material"}) shows that, with the exception of organic disorders and adjustment disorders, suicide cases had a higher risk of being diagnosed than controls with each of the diagnoses considered. Of these diagnoses, the risks for psychotic disorders were the highest (OR = 15.38; 95% CI: 3.53--97.82) followed by the variable \"at least one psychiatric disorder\" (OR = 10.50; 95% CI: 9.60--13.56). The risk for schizophrenia was also particularly high (OR = 5.56; 95% CI: 3.12--10.24). This is due to the fact that there were only 15 control subjects altogether diagnosed with schizophrenia and two with psychotic disorders. Statistically significant differences were found when male and female suicide cases were compared (see table [3](#T2){ref-type="table"}). However, gender-based comparisons should be considered cautiously as, when available, demographic information indicated that female suicides included in the studies reviewed tended to be older than males (table [5](#T4){ref-type="table"}). Nevertheless, even considering this potential limitation, the results are interesting. The risks for alcohol (OR = 2.19; 95% CI: 1.63--2.95), other substance problems (OR = 2.02; 95% CI: 1.32--3.10), and any substance problems (OR = 3.58; 95% CI: 2.78--4.61), personality disorders (OR = 2.01; 95% CI: 1.38--2.95) or childhood disorders (OR = 4.95; 95% CI: 2.69--9.31) were greater in male as opposed to female suicides. On the other hand, the risks of having depressive disorders (OR = 0.53; 95% CI: 0.42--0.68) or any affective disorders (OR = 0.66; 95% CI: 0.53--0.83) were lower in males. ::: {#T2 .table-wrap} Table 3 ::: {.caption} ###### Odds Ratios for major outcome variables across sexes ::: Disorders n for females n for males OR (95% CI) χ^2^ p-value -------------------------------------------- --------------- ------------- ------------------- -------- --------- Any psychiatric disorders 398 801 0.98 (0.70--1.36) 0.02 0.881 Schizophrenia 17 44 1.30 (0.71--2.39) 0.79 0.373 Other psychotic disorders or psychosis NOS 15 40 1.33 (0.71--2.56) 0.88 0.347 Somatoform, anxiety and neurotic disorders 33 83 1.27 (0.85--1.97) 1.24 0.265 Bipolar disorders 26 43 0.81 (0.48--1.38) 0.68 0.409 Organic disorders 6 15 1.24 (0.45--3.60) 0.20 0.656 Adjustment disorders 31 64 1.02 (0.64--1.64) 0.01 0.917 Disorders more likely if male Alcohol problems 73 272 2.19 (1.63--2.95) 29.57 0.000 Other substances problems 32 122 2.02 (1.32--3.10) 11.89 0.001 Any substances problems 110 436 3.58 (2.78--4.61) 110.18 0.000 Personality disorders 41 153 2.01 (1.38--2.95) 14.60 0.000 Childhood disorders 13 117 4.95 (2.69--9.31) 34.57 0.000 Disorders more likely if female Depressive disorders 199 268 0.53 (0.42--0.68) 28.56 0.000 Any affective disorders 272 454 0.66 (0.53--0.83) 12.91 0.000 Other disorders 16 12 0.36 (0.16--0.82) 7.44 0.006 ::: ::: {#T4 .table-wrap} Table 5 ::: {.caption} ###### Descriptive analysis of the age and sex of subjects ::: Age (mean ± SD) n \[Studies\] -------------------- --------------------- -------------------------------------- All regions ♂ 28.5 ± 12.8 880 \[11,18,143,145,148-150,158\] ♀ 34.5 ± 17.8 333 \[11,18,143,147-149,158\] Both sexes\* 41.6 ± 17.8 794 \[11,14,17,149,151,154,157,158\] American Studies ♂ 26.0 ± 12.3 491 \[18,143,148,150\] ♀ 27.3 ± 18.9 127 \[18,143,148\] Both sexes\* N/A N/A European Studies ♂ 27.2 ± 15.4 314 \[11,145,149\] ♀ 37.9 ± 18.9 191 \[11,147,149\] Both sexes\* 42.3 ± 20.8 672 \[11,14,15,17,151,154\] Australian Studies ♂ 42.5 491 \[158\] ♀ 45.7 15 \[158\] Both sexes\* 39.5 ± 5.2 122 \[157,158\] Asian Studies ♂ N/A N/A ♀ N/A N/A Both sexes\* N/A N/A N/A -- information not available \* Both sexes refers to studies in which information on age by sex was not provided, and thus, only mean age for the whole sample was available. ::: Analysing the data according to geographic areas, the diagnostic distribution of the key diagnoses found in suicides differed significantly between world regions (see table [4](#T3){ref-type="table"}), but as mentioned above, potential age-related biases may apply (table [5](#T4){ref-type="table"}). The American suicides were more often diagnosed with a psychiatric disorder than suicides in the other regions of the world; 89.7 % (SD 4.2 %) of the American suicides had at least one diagnosis, whereas 88.8 % (SD 8.9 %) of the European suicides, 83.0 % (SD 18.4 %) of the Asian suicides and 78.9 % (SD 15.3 %) of the Australian suicides had at least one psychiatric diagnosis. ::: {#T3 .table-wrap} Table 4 ::: {.caption} ###### Diagnostic distribution across different regions of the world ::: European (%) North American (%) Australian (%) Asian (%) χ^2^ -------------------------------------------------------------- -------------- -------------------- ---------------- ------------ ----------- Affective disorders 753 (48.5) 390 (33.6) 71 (32.7) 335 (51.3) 11.3\* Substances-related disorders 390 (18.6) 573 (40.1) 106 (24.1) 135 (26.7) 12.1\* Schizophrenia and other psychotic disorders or psychosis NOS 125 (7.5) 42 (4.2) 29 (24.3) 53 (8.4) 24.1\* Personality disorders 197 (16.8) 75 (13.4) 75 (17.7) 20 (17.7) 1.2^n.s.^ At least one Diagnosis 1298 (88.8) 710 (89.7) 207 (78.9) 527 (83.0) 6.4^n.s.^ \* Significant at p ≤ 0.01 ^n.s.^Non significant ::: Discussion ========== Total psychopathology --------------------- Since the first psychological autopsy studies by Robins et al. \[[@B139]\] in North America and by Barraclough et al. \[[@B7]\] in Europe, a relatively small number of studies have been carried out. These original studies were descriptive in nature, and only more recently case-control studies have been performed. The data from these studies have consistently suggested a clear relationship between mental disorders and suicide. Here we systematically reviewed these studies and pooled their results whenever possible. Our results show that, on average, 87.3 % of the subjects who committed suicide had a mental disorder. On the other hand, an average of 14.0 % of these subjects was not diagnosed with a psychiatric disorder. A possible explanation is that a diagnosis failed to be detected due to various methodological shortcomings. This possibility is concrete, as psychological autopsy studies rely on informants and/or available medical information to generate diagnostic data. In some cases, the informant has little information on the last weeks or months of life of the subject. Therefore, it is possible that the overall rate of psychopathology observed is still underestimated. This is consistent with findings from recent studies by our group focusing on suicides without an axis I diagnosis \[[@B160]\]. Specific diagnoses ------------------ This review confirms the overall impression from individual studies that affective, substance-related, personality and psychotic disorders account for most of the diagnoses among suicides. The two single most common diagnostic categories among suicide completers were any affective disorders (diagnosed in 43.2 % of suicide cases), and any substance disorders (present in 25.7 % of suicide cases). Recent studies on comorbidity indicate that suicide completers are more likely to have more than one psychiatric diagnosis \[[@B142],[@B161]\]. In a comparison with matched community controls, Foster et al. \[[@B142]\] found a significant increase in suicide risk in the presence of Axis I-Axis II comorbidity (OR = 346.0, p \< 0.0001). Our group \[[@B161]\], investigating male completers and controls from the general population, found that suicide cases had an average of 2.36 diagnoses and that comorbidity in completers tended to be of three different patterns, according to mean number of diagnoses (range 1.19 -- 4.05) and presence of impulsive-aggressive behaviours. Thus, it would have been interesting to assess overall levels of comorbidity in this review, as well as to investigate what is the amount of overlap between the different diagnoses investigated. However, very little, if any, information about comorbidity was present in the original studies reviewed and this information was impossible to retrieve from the published data. Gender differences ------------------ The investigation of gender differences in rates of psychopathology associated to suicide should be regarded in light of the methodological limitations of this review, which are primarily related to difficulties in comparing studies carried out using different methodological procedures, diagnostic instruments and criteria, in addition to potential differences in sample characteristics, including age distribution. However, given the important effect that gender seems to have as a suicide risk moderator and the relative lack of appropriate investigation focusing on gender differences in suicide completion, the observed differences in rates of psychopathology in male and female suicides are interesting and should be considered for validation in future studies. Our results indicate that the risk of substance-related disorders, personality disorders and childhood disorders are significantly higher in male suicides, whereas, the risk of affective disorders, specifically, depressive disorders, are greater in female suicides. On average, any substance problems represented 41.8 % (SD 21.1 %) of the male diagnoses and 24.0 % (SD 16.5 %) of the female diagnoses (χ^2^7.29 p = 0.007), whereas affective disorders represented 59.4 % (SD 13.9 %) of the female diagnoses and 47.4 % (SD 12.7 %) of the male diagnoses (χ^2^2.88 p = 0.089). Although there has been much discussion on possible factors that could help explain gender differences in suicide rates, most of the studies have primarily focused on psychosocial and demographic risk factors. There is very little data on the possible role of psychiatric and/or behavioural characteristics, which may also mediate gender differences in suicide risk. This study suggests that the underlying psychiatric morbidity may be different in male and female suicide completers. An important question that follows is whether or not the differences found in this study between male and female suicides are the consequence of gender differences in the prevalence of psychiatric disorders in the general population. Although possible, it is unlikely that differences in population rates of psychiatric disorders could explain the different distribution of psychiatric disorders observed in this study, as the gender-specific risks found were not consistently reflecting gender-differences observed in prevalence rates (for instance, schizophrenia and psychotic disorders) and they were not always in the same direction (for instance, personality disorders). An interesting finding of this study was precisely the absence of gender differences in schizophrenia. This is not necessarily inconsistent with suggestions that most of the suicide cases in schizophrenia are males \[[@B162]-[@B164]\], as our findings basically indicate that there are no relative differences between genders in the proportion of suicide cases that are diagnosed with schizophrenia. However, our findings are inconsistent with the common generalization that schizophrenics tend to commit suicide early in the course of the disease because, given gender-differences in the age at onset in schizophrenia \[[@B165]\], with males more likely to have the onset at younger ages, one would expect a considerably higher proportion of schizophrenia among male completers, even if the age distribution in our sample suggests that women in general seemed older than men. In summary, despite the potential methodological limitations discussed above, our results in gender differences in clinical correlates of suicide are interesting and should be further investigated. Geographic differences ---------------------- We also found differences in rates of psychiatric disorders in studies from different geographic origins. This finding may indicate social and cultural factors influencing how one views and interprets suicide and cultural biases towards or against specific diagnoses. Alternatively, as discussed for gender-based comparisons, demographic (age, rural vs. urban samples, socioeconomic and educational level, etc.) differences between the samples could explain some of these results. In view of that, similar limitations, as those for the analysis of gender differences, apply to the analysis of geographical differences in rates of psychopathology associated to suicide (see table [5](#T4){ref-type="table"}). American women seem younger than in any other region, Australian women and men appear older than those in the other regions, and no Asian studies provide age means for their sample. In spite of these limitations, our review suggests that, although psychopathology mediates suicide worldwide, there seem to be differences across different parts of the world in the relative proportion of the specific psychiatric disorders found among suicide completers. As mentioned above, these differences may be attributed to variance in psychological autopsy methodologies between countries, or yet, to important differences in the prevalence of psychiatric disorders. Although it is possible that methodological differences between studies play a certain role explaining some of the differences found, it is unlikely that they accounted for all differences found as the studies included in these regional comparisons used similar methods and diagnostic criteria, whereas the differences found were substantial. It is not likely either that diversity between countries in prevalence of psychiatric disorders account for all the observed regional differences, as for some of these disorders, such as schizophrenia, it is thought that there is little variation in prevalence rates between different populations \[[@B166]\]. Thus, the geographical differences observed in the relative proportion of psychiatric disorders among suicide completers is an interesting issue that should be further explored. Most limitations of this study are common to all quantitative systematic reviews. In particular to this study, one should take into account that the quantitative review was carried out with studies that, although published in a relatively short period of time (from 1986 to 2002), have variation in diagnostic criteria used and have different methodological rigor. Moreover, it is possible that between-study variation in the distribution of a series of demographic variables could have had an impact on our findings. We chose not to control for these methodological differences as given the diverse sources of possible variation, doing so would have considerably limited the number of studies included in the review. Therefore, we opted to be more inclusive and consider the results of this review as preliminary and providing information to be further investigated. Over the course of this study, a report on another meta-analysis of psychological autopsy studies was published. This study, by Cavanagh et al. \[[@B167]\], reviewed the literature on psychological autopsies and yielded similar overall results. However, there are differences between the study by Cavanagh et al \[[@B167]\] and ours, both in methodology and major aims. While they identified studies through a larger number of library databases, they included only studies up to June 2000. Moreover, they did not investigate risks attributed to specific diagnostic categories, but rather risks attributed to mental health disorders, presence of an affective disorder and comorbidity. They also investigated the role of a few social variables and did not carry out analyses exploring a possible gender and geographic difference in relative rates of psychopathology. Conclusions =========== Our study carried out a systematic review of psychological autopsy studies of suicide and indicates that overall, 87.3% of suicide cases have a history of psychiatric disorders. We also found that male suicides have a different psychiatric profile than female suicide cases and that the relative proportion of psychiatric disorders in suicide completers tends to vary according to geographical region. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= GAL carried out the search, extraction of data, analysis and drafted the manuscript. CK helped with the design of the review, and the statistical analysis. GT conceived the study and participated in the design and coordination. 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/1471-244X/4/37/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Table 1 -- Mantel-Haenszel Weighed Odds Ratio. This table gives Mantel-Haenszel Weighed Odds Ratio for the 14 case-control studies included in this meta-analysis for the 16 variables of psychiatric disorders. ::: ::: {.caption} ###### Click here for file ::: Acknowledgment ============== This study was partly funded by CIHR grant MOP-38078 and a NARSAD grant. GT is a CIHR scholar.	PubMed Central	2024-06-05T03:55:49.886776	2004-11-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534107/", "journal": "BMC Psychiatry. 2004 Nov 4; 4:37", "authors": [ { "first": "Geneviève", "last": "Arsenault-Lapierre" }, { "first": "Caroline", "last": "Kim" }, { "first": "Gustavo", "last": "Turecki" } ] }
PMC534108	Background ========== With the recent emergence of severe acute respiratory syndrome (SARS), a newly discovered infectious disease, the importance of primary infection control measures have been highlighted \[[@B1],[@B2]\]. Routine handwashing with soap and water has been cited by the World Health Organization (WHO) as being \"the most important hygiene measure in preventing the spread of infection\" \[[@B3]\]. This statement has been reiterated by both the United States Centers for Disease Control (CDC) and Health Canada, in reference to reducing the transmission of SARS, the influenza virus, and other infectious pathogens \[[@B3]-[@B5]\]. The epidemiological evidence supporting the effectiveness of this basic measure in healthcare settings dates back to the mid-nineteenth century \[[@B6],[@B7]\]. Ignaz Semmelweis, a Hungarian obstetrician, implemented routine handwashing with chlorinated lime by maternity ward staff, as a mechanism to reduce the incidence of puerperal fever \[[@B6],[@B7]\]. This simple routine elicited dramatic results, reducing the mortality rate from 13--18 percent to 2 percent \[[@B6],[@B7]\]. These findings have been replicated numerous times in hospital environments- underlining the magnitude of routine handwashing \[[@B8],[@B9]\]. The elementary school environment is also negatively impacted by outbreaks of disease causing microorganisms \[[@B10],[@B11]\]. These occasional outbreaks result in increased student and teacher absenteeism, increased healthcare expenditures, and an overall decline in the children\'s learning environment \[[@B11]\]. The United States CDC has estimated that the average school-aged child missed approximately one week annually due to illness-related absenteeism in 1995 \[[@B12]\]. Despite the scientifically proven evidence of the effectiveness of handwashing, and the increasing promotion of proper hand hygiene techniques, observational studies in school settings have indicated that handwashing practices are often lacking \[[@B13],[@B14]\]. Guinan et al. (1997) reported that proper handwashing compliance, with soap and water, in school-aged children ranged from 8 to 28 percent. Reported reasons for the observed inadequacy in compliance included insufficient time during the day, and the use of substandard washing facilities in hard to access locations of the school environment \[[@B13],[@B14]\]. In attempts to overcome the obstacles of routine handwashing in school environments, antimicrobial rinse-free hand sanitizers are being used as an alternative hand hygiene technique. The concern is that such programs may be carried out in the absence of evidence of effectiveness in the school environment. Thus, it is timely to review the evidence currently available for the effectiveness of antimicrobial rinse-free hand sanitizer programs in reducing absenteeism due to communicable illness. The aim of this systematic review was to determine whether antimicrobial rinse-free hand sanitizer interventions are effective in preventing illness-related absenteeism in elementary school children. Methods ======= A detailed written protocol was prepared and reviewed in advance (complete protocol can be obtained from the corresponding author). Search strategy --------------- A comprehensive search was conducted to identify all relevant studies regardless of publication status. Six electronic databases were searched for studies published in any language. The databases included: Biological Abstracts (1990-May 2003), CINAHL (1982--2003), the Cochrane Controlled Trials Register (1981--2003), EMBASE (1980-May 2003), HealthSTAR (1975-May 2003), and MEDLINE (1966-May 2003). A detailed search strategy was developed for use in MEDLINE and an iterative process was completed to refine the MEDLINE search for each database. Descriptions of the database search strategies are presented in Appendix 1 (see Additional File [1](#S1){ref-type="supplementary-material"}). OVID served as the primary search interface, and the SDI feature was used to monitor newly posted citations, the most recent date September 30, 2004. Due to the low occurrence of studies in this subject area, no filters were used to identify specific study types or reviews. The reference lists of all relevant articles were reviewed for additional studies. A letter was sent to all corresponding authors of the articles identified by hand-search, excluding two newly eligible citations posted between May 2003 and September 2004 \[[@B15],[@B16]\], or by searching bibliographic databases. Additionally, contact experts and the industrial companies that manufactured the antimicrobial hand gels used in the included trials (GOJO Industries and Woodward Laboratories, Inc.) were contacted in attempts to identify other eligible trials. A detailed list of contacts is provided in Appendix 2 (see Additional File [2](#S2){ref-type="supplementary-material"}). Finally, conference proceedings for the American Journal of Infection Control (2000--2004) and recently published issues of the American Journal of Infection Control (February 2003 to August 2004) were searched by hand. Eligibility criteria -------------------- Studies were evaluated for inclusion on the basis of four criteria: target population, intervention, outcome, and study design. The target population of interest consisted of elementary school children between 4 and 12 years of age (including senior kindergarten and grades 1 through 8). The interventions of interest were those that administered antimicrobial rinse-free hand hygiene programs compared with no intervention or placebo treatment arm in a school setting. The outcome of interest was the comparison of the number of absences due to communicable illnesses in children who received the antimicrobial rinse-free hand hygiene intervention with the number of such absences in those who received a placebo or no intervention. We included cluster randomized controlled trials (RCTs) and cluster non-randomized controlled trials regardless of publication status. Study selection --------------- All relevant citations, titles and abstracts, were imported into a reference database where duplicates were manually removed. Priority in downloading was given to MEDLINE. Reviews were excluded, but the bibliographies from such articles were examined for relevant studies. The screening was completed in an unblinded manner; there is inconclusive evidence that blinding introduced bias into the process \[[@B17]\]. One individual (EM) independently screened the titles and abstracts of each citation and identified all citations for full review. One reviewer was deemed appropriate as it was thought that the reviewer would error on the side of caution. Hard copies of all potentially relevant citations were retrieved, and two reviewers (EM, NLS) independently assessed each article using the aforementioned eligibility criteria, excluding the newly published citations in which eligibility was assessed by EM \[[@B15],[@B16]\]. Disagreements were discussed and a final decision was made by means of open consensus. A pilot test assessing the eligibility criteria on a sample of articles was not performed. Recent studies by both Juni et al. 2002 and Moher et al. 2003 indicate that the exclusion of trials in languages other than English (LOE) does not bias measures of effectiveness- however, both are cautionary, advocating language inclusive search strategies \[[@B18],[@B19]\]. Due to limited fiscal resources, an English language restriction was applied at this level, however the number of citations in LOE that met eligibility criteria will be noted. Data abstraction ---------------- Two reviewers (EM, NLS) independently abstracted data from all studies meeting the eligibility criteria, excluding one abstract where EM independently abstracted pertinent information \[[@B15]\], using pre-printed data collection forms presented in Appendix 3 (see Additional File [3](#S3){ref-type="supplementary-material"}). Information pertaining to the descriptive details of the study (e.g., year published, language of publication, publication status), design (e.g., randomized controlled trial), population (e.g., age, grade level), intervention (e.g., type of antimicrobial rinse-free hand sanitizer, inclusion of an educational component), and primary outcome(s) (e.g., absences due to illness) were collected. Adverse advents were not considered due to the relatively benign nature of the intervention. Reviewers resolved differences by means of open consensus. For the case of crossover study designs, data from the both arms of the study was abstracted. A pilot test assessing the data collection form on a sample of articles was not performed. Quality assessment ------------------ Two reviewers (EM, NLS) independently assessed the quality of each of the included studies, excluding the abstract by Thompson (2004) previously mentioned \[[@B15]\], using the previously validated 3-item Jadad scale, which assesses the quality of the report in terms of randomization generation, double blinding, and withdrawals and drop-outs by intervention group \[[@B20]\]. Studies were not given a quantitative score; rather this was used as a qualitative tool. (Due to the nature of the intervention, not all items apply). Additionally, if trials were randomized, allocation concealment was assessed and qualitatively evaluated as adequate, inadequate or unclear \[[@B21]\]. Disagreements were resolved through open consensus. A pilot study applying the quality assessment criteria on a subset of studies was not completed. Data analysis ------------- Data synthesis and analysis was performed in accordance with the Cochrane Reviewers\' Handbook \[[@B22]\]. Firstly, data was qualitatively synthesized to examine the overall pattern of studies with respect to study design, population, intervention, and outcome characteristics. Sources of clinical and statistical heterogeneity were identified and results were examined. All data was abstracted as reported. The primary outcome, frequency of absences due to communicable illness was analyzed. Percent relative differences were presented along with 95 percent confidence intervals as the estimate of intervention effectiveness in the four studies which calculated rate and risk ratios as the measure of association \[[@B10],[@B27],[@B31],[@B41]\]. Percent relative differences and 95 percent confidence intervals were calculated enabling the results to be compared between studies, without altering the measure of association reported in the studies (rate and risk ratios). In the studies where data could not be abstracted, measures of association were reported \[[@B15],[@B16]\]. The validity of performing a quantitative synthesis was considered, however based on a qualitative inspection of heterogeneity and estimates of intervention effectiveness this was not deemed appropriate. Thus, sensitivity and subgroup analyses were not performed, and publication bias was not assessed quantitatively. Results ======= Description of studies ---------------------- A flow diagram of the search results is illustrated in Figure [1](#F1){ref-type="fig"}. From the searches of the electronic databases, a total of 211 citations were identified, of which 70 were duplicates, resulting in the identification of 141 unique citations. In all, 18 potentially relevant trials were retrieved from the searches of the relevant databases. Hand-searching of the reference lists of relevant articles and conference proceedings resulted in a further 7 trials, which were also reviewed for consideration. Thus, 25 studies were determined potentially relevant \[[@B10],[@B23]-[@B46]\]. Using both titles and abstracts, no trials meeting the inclusion criteria in LOE were found during the study selection phase. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Flow diagram outlining the results of literature search and review of studies retrieved ::: ![](1471-2458-4-50-1) ::: After review of the full text of these studies, 21 were excluded for the following reasons: no outcomes of interest (n = 1), inappropriate population (n = 1), inappropriate interventions (n = 9), inappropriate study design (n = 3), irrelevant subject matter (n = 5), and review (n = 2). Thus, a total of 4 trials fulfilled the inclusion criteria \[[@B10],[@B27],[@B31],[@B41]\]. However, during the time between manuscript submission and revision, 2 additional citations were deemed to be eligible \[[@B15],[@B16]\], bringing the total to n = 6 eligible studies, one of which was a published manuscript \[[@B16]\], and the other a published abstract \[[@B15]\]. Of the 6 remaining studies, 2 were crossover studies \[[@B16],[@B27]\], 1 was a placebo-controlled cluster randomized controlled trial (RCT) \[[@B41]\], 2 were cluster non-randomized controlled trials (NRCT) \[[@B10],[@B31]\], and the published abstract was a cluster trial, however randomization was unclear \[[@B15]\]. McNemars\'s test was used to assess observer agreement, chi-square = 2.00; df = 1, p = 0.1573; and there was 92 percent agreement between the two reviewers with respect to study relevance for the initial four trials included \[[@B10],[@B27],[@B31],[@B41]\]. All of the relevant trials are described in Table [1](#T1){ref-type="table"}, and an assessment of their quality of reporting is presented in Table [2](#T2){ref-type="table"} (the study by Thompson 2004 was excluded as only the abstract was available). Our overall agreement was 89 percent with respect to data collection for the five included studies outlined in Table [1](#T1){ref-type="table"}. For quality abstraction, percent agreement was 80 percent. When examining items relating to blinding and assessment of withdrawals and dropouts, observer agreement was 100 and 80 percent, respectively. Kappa statistics were not calculated as sample size was insufficient. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of studies included in the systematic review, demographics and descriptive statistics ::: --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Author(s), Year, and Country Source of Funding Study Population Definition Illness-related Absenteeism Unit of analysis Study Duration Intervention Arm Control Arm ------------------------------------------------------ -------------------------------------------------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------- ------------------------------------------------------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- -------------------------------------------------------------------------------------------------------- Cluster Randomized Controlled Trials (RCTs) White ^41^2001 United States Industry (Woodward Laboratories, Inc.) 1 private and 2 public elementary schools grades K-6; 72 initial classes (n = 1626 students) \\ • GI or respiratory-related\ Grouped by classroom 5 weeks • Education: presentation and video describing germs and proper handwashing techniques (1 hr session)\ • Education: presentation and video describing germs and proper handwashing techniques (1 hr session)\ [Target group]{.underline}:\ • Parents reported events • Large involvement of both parents and school staff\ • Large involvement of both parents and school staff\ • 16 classes, n = 388 students\ • Each child received 1-oz bottle of SAB (CleanHands^®^) (alcohol-free) instant hand sanitizer\ • Each child received 1-oz bottle of placebo formulation\ [Control group]{.underline}:\ • Instructed to use spray under teacher supervision to supplement handwashing • Instructed to use spray under teacher supervision to supplement handwashing • 16 classes, n = 381 students Crossover* Dyer ^27^2000 United States Industry (Woodward Laboratories, Inc.) Private elementary school (K-6); 2 classrooms per grade level, n = 30 students per classroom\ • GI (symptoms including vomiting, abdominal pain, and diarrhea)\ Grouped by classroom 10 weeks (4 weeks first arm, 2 week washout period, 4 weeks second arm) • Education: presentation and video describing germs and proper handwashing techniques (1 hr session)\ • Education: presentation and video describing germs and proper handwashing techniques (1 hr session)\ [Target group]{.underline}:\ • Respiratory-related (symptoms included cough, sneezing, sinus trouble, bronchitis, fever alone, pink eye, headache, mononucleosis, and acute exacerbation of asthma)\ • Each child received 1-oz bottle of SAB (CleanHands^®^) (alcohol-free) instant hand sanitizer\ • Instructed to wash hands as usual • Children grades K-6\ • Parents reported events • Instructed to use spray under teacher supervision to supplement handwashing • 1 classroom per grade level\ • n = 210 students\ [Control group]{.underline}:\ • Children grades K-6\ • 1 classroom per grade level\ • n = 210 students Morton ^16^2004 United States Maine Administrative School District \#35 in Eliot, and South Berwick, Maine; Erie Scientific donated AlcoSCRUB^®^ 1 elementary school in northern New England, grades K-3; 17 classrooms and n = 285 students eligible\ • GI (symptoms including influenza, diarrhea, nausea, or vomiting (with or without fever))\ Grouped 100 days (46 day first arm, 1 week washout period, 47 day second arm) • Education: guardians provided with study information and a contact number for the school nurse; additionally, monthly updates were provided\ • Education: information was presented by school nurse about proper handwashing\ PHASE 1:\ • Respiratory-related (symptoms included nasal congestion, cough, or sore throat (with or without fever))\ • Education: students received a carefully planned education program; 45 minute \"Germ Unit\", Glo Germ™ presentation\ • Instructed to wash hands as usual [Target group]{.underline}:\ • Parents reported events • Reinforcement: 1^st^week: daily reminders given to students, after 1^st^week reinforcement given weekly and after holidays; each classroom visited twice by school nurse during two arms\ • 9 classrooms\ • AlcoSCRUB^®^dispensers were furnished in each classroom, located near the classroom entrance at a height that was accessible to all students\ [Control group]{.underline}:\ • Gel use was monitored, and reinforcement was given to classes with low use • 8 classrooms\ • PHASE 2:\ • reversed Cluster Non-randomized Controlled Trials (NRCTs) Hammond ^31^2000 United States GOJO Industries, Inc. 18 public elementary schools grades K-6 in 6 school districts \\ • Infectious process such as cold, flu, and gastroenteritis (common infectious illnesses such as pink eye, abscesses, and skin infections were not included)\ Grouped by school and grouped by classroom 10 months • Each test classroom was equipped with a dispenser of PURELL instant alcohol-based hand sanitizer; also placed in other locations around the school\ • No intervention [Target group]{.underline}:\ • No information regarding reporting of events • Reinforcement by study co-ordinators every 3 months • 5 school districts: District 1 (Ohio; K-5; n = 1440 students), District 2 (Ohio; 2,3; n = 266 students), District 3 (Delaware; 3,4; n = 110 students), District 4 (Tennessee; K-6; n = 680 students), District 5 (California; K-5; n = 579 students)\ [Control group]{.underline}:\ • 5 school districts: District 1 (K-5; n = 1136 students), District 2 (2,3; n = 552 students), District 3 (3,4; n = 113 students), District 4 (K-6; n = 592 students), District 5 (K-5; n = 612 students) Guinan ^10^2002 United States GOJO Industries, Inc. 5 elementary schools; 4 schools had 4 classrooms, 1 school had 2 classrooms (coed and single sex schools)\ • Infectious process such as cold, flu, and gastroenteritis\ Grouped by classroom 3 months • Education: presentation and video describing germs and proper handwashing techniques (1 hr session)\ • No intervention [Target group]{.underline}:\ • Children and parents reported events\ • Each test classroom was equipped with a dispenser of an alcohol-based hand sanitizer • Children grades K-3\ • Had to be 5 days between episodes to count • 4 schools with 2 classrooms, 1 school with 1 classroom\ • n = 145 students\ [Control group]{.underline}:\ • Children grades K-3\ • 4 schools with 2 classrooms, 1 school with 1 classroom\ • n = 145 students Cluster Controlled Trials-randomization unclear* Thompson ^15^2004 United States (abstract only) Not available 5 grade two classrooms, and 1 one/two combination classroom, n = 138 children\ • Illness = cold, flu, conjunctivitis, and gastrointestinal symptoms\ n/a n/a • Age appropriate interactive learning session\ n/a [Target group]{.underline}:\ • Teachers recorded absences • Alcohol-based hand sanitizer installed in intervention classrooms • 3 classrooms\ [Control group]{.underline}:\ • 3 classrooms --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- \* Not including drop-outs or withdrawals † n/a = not available ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Quality assessment of trials meeting inclusion criteria ::: ---------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ---------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Author(s), Year, and Country Quality Assessment Allocation Concealment Additional Comments Cluster RCT White ^41^2001 United States Study was described as randomized however did not explain method of randomization; participants and study-coordinators were blinded; description of withdrawals and dropouts provided: of the 72 initial classes (1626 students), 32 classes (16 target and 16 control; 769 students) participated (remainder dropped from analysis) Unclear Sample size calculation not defined; statistical methods unclear; parents required to sign detailed informed consent form; study reviewed and approved by the two school boards; soap and handwashing not monitored; clustering not accounted for Crossover Dyer ^27^2000 United States Study was not formally randomized; neither participants or study-coordinators were blinded; description of withdrawals and dropouts provided: no exclusions from the population were necessary Not Relevant Sample size calculation not defined; statistical methods unclear; no parental consent form; study not approved by a formal university institutional review board (approved by school board of education); limited SES diversity; soap and handwashing not monitored; clustering not accounted for Morton ^16^2004 United States Study was described as randomized however did not explain method of randomization; neither participants or study-coordinators were blinded; description of withdrawals and dropouts provided: of the 17 initial classes (285 students), 17 classes ((253 students, 120 girls and 133 boys), non-consent = 22 children, adverse-events = 10 children) Unclear Sample size calculation not defined; data not in a format which could be easily extracted; study approved by Board of Education, and the Institutional Review Board at the state\'s largest hospital; a consent form was sent to all parents and guardians; clustering not accounted for Cluster NRCTs Hammond ^31^2000 United States Study was not formally randomized; neither participants or study-coordinators were blinded; description withdrawals and dropouts provided: 1 school district did not comply with protocol; 25/3080 students did not participate or complete the protocol (in each case, data was not used for results) Not Relevant Sample size calculation not defined; no parental consent form; formal review not mentioned; clustering not accounted for Guinan ^10^2002 United States Study was not formally randomized; participants and study-coordinators were not blinded; description withdrawals and dropouts not provided Not Relevant Sample size calculation not defined; statistical methods unclear; no parental consent form; formal review not mentioned (approved by each school); limited SES diversity (high SES); performed in peak absenteeism season; clustering not accounted for ---------------------------------- ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ ---------------------------- --------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: All six trials were conducted in the United States, 4 with reported industrial sponsorship, and were published between 2000 to 2004. The trials varied in size (range = 138 to 6080 students; range = 1 to 18 schools), and geographic locations (Pennsylvania \[[@B10]\], California \[[@B27],[@B37]\], Ohio/Tennessee/Delaware/California \[[@B31]\], New England \[[@B16]\]). One of the studies assessed only private schools \[[@B28]\]; another study assessed both private and public schools \[[@B41]\], three assessed only public schools \[[@B10],[@B16],[@B31]\], while one\'s type of school assessed was not available \[[@B15]\]. Additionally, there was considerable variation in the type of school included both within and between trials: Christian private school, public elementary schools, same-sex schools and co-ed schools. The duration of the studies ranged from 5 weeks to 10 months, the longest trial being that of the RCT \[[@B41]\]. The trials also varied with respect to the intervention administered. White et al. (2001) and Dyer et al. (2000), provided each student with alcohol-free instant hand sanitizer \[[@B27],[@B41]\], whilst Hammond et al. (2000), Guinan et al. (2002), Morton et al. (2004), and Thompson et al. (2004) provided each class with alcohol-based instant hand-sanitizer dispensers \[[@B10],[@B15],[@B16],[@B31]\]. Education was concurrently provided for both the control and intervention arms in two studies \[[@B16],[@B27],[@B41]\], education on germs and hygiene was provided only to the intervention arm in one study \[[@B10]\], one study did not provide any education however study reinforcement was provided for teaching staff \[[@B31]\], and one study provided education to the intervention arm but as only the abstract was available for this study it was unclear if the control arm received any education \[[@B15]\]. Methodological quality ---------------------- The quality of reporting of the 5 trials that were examined in detail was low. Only one study was described as being randomized and double-blinded, however, it failed to describe a detailed and appropriate method of randomization and allocation concealment was unclear \[[@B41]\]. Four of the five studies, as previously mentioned, discussed withdrawals and dropouts however the description was quite basic and detailed flow-diagrams outlining the passage of participants through the trial were not supplied \[[@B16],[@B27],[@B31],[@B41]\]. White et al. (2001) reported a significant number of dropouts (857 of 1626 students did not complete the study) however no explanations were offered \[[@B41]\]. Four studies received industrial sponsorship either by GOJO industries or Woodward Laboratories. In addition, two studies received financial support from another external source \[[@B16],[@B41]\]. Other characteristics of poor quality reporting included: no sample size calculation defined for all five studies, and the statistical methods were vague. No studies took into consideration clustering when analyzing their results. Our overall agreement for all items of quality was greater than 80 percent; again observer agreement statistics were not calculated as the sample size was insufficient. Primary outcome --------------- All six studies varied in their definition of communicable illness-related absenteeism, refer to Table [1](#T1){ref-type="table"}. Out of the five studies with published manuscripts, four of the studies reported the estimate of intervention effectiveness in terms of risk/rate ratios, subsequently calculating percent relative effect, and one reported and odds ratio for a pair-matched study; results are reported in Table [3](#T3){ref-type="table"}, Table [4](#T4){ref-type="table"}, and Table [5](#T5){ref-type="table"}. The percent relative effect measures the decreased rate of the occurrence of absenteeism when the rate ratio is the measure of association or the decreased risk of absenteeism when the relative risk is the measure of association. Tests of significance were completed in all five of the studies using chi-squared tests or t tests however no confidence intervals were calculated. Two of the studies used rate ratios as the measure of association \[[@B10],[@B31]\], two studies used relative risks as the measure of association \[[@B27],[@B41]\], and the other used the odds ratio \[[@B16]\]. Table [3](#T3){ref-type="table"}, [4](#T4){ref-type="table"} and [5](#T5){ref-type="table"} distinguish between the measures of association used. All studies found a statistically significant effect of the antimicrobial rinse-free hand gel interventions. In the study by Hammond et al. (2000), the experimental group had a 20% (95% CI = 19--21%) reduction in absences due to communicable illness, the experimental group in the trial completed by Guinan et al. (2002) had a 49% (95% CI = 42--56%) reduction, White et al. (2001) demonstrated a 33% (95% CI = 17--45%) reduction in the experimental group, Dyer et al. (2000) had a 34 % (95% CI = 10--50%) reduction in absences due to communicable illness in the experimental group in the first phase and a 56 % (95% CI = 31--72%) in the second phase, and Morton et al. (2004) reported a significant odds ratio for McNemar\'s test (chi-square = 7.787; p = 0.0053). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Absences due to communicable illness, person-time incidence rates and percent relative effects of a non-alcoholic rinse-free hand sanitizer ::: Intervention Control ------------------- --------- ------------ ------------------ ------------- ------------ ------------- ------------- White et al. ^41^ 770 153 (9615) 1.59 222 (9459) 2.35 33 (17, 45) Dyer et al. ^27^ Phase 1 420 70 (4136) 1.69 105 (4120) 2.55 34 (10, 50) Phase 2 420 28 (4156) 0.674 63 (4140) 1.52 56 (31, 72) \* Percent relative effect = (1- intervention rate/control rate)\100 95 percent confidence interval = (1--95% UCL of Rate Ratio)\100 to (1--95% LCL of Rate Ratio)\100 ::: ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Absences due to communicable illness, cumulative incidence rates and percent relative effects of an alcoholic rinse-free hand sanitizer ::: Intervention* Control --------------------- ------ ------------------ ------------- ------------- ------ ------------- Hammond et al. ^31^ 6080 7441.5 (3075) 2.42 9066 (3005) 3.02 20 (19, 21) Guinan et al. ^10^ 290 140 (145) 0.97 277 (145) 1.91 49 (42, 56) \* Percent relative effect = (1- intervention risk/control risk) \100 95 percent confidence interval = (1--95% UCL of Risk Ratio)\100 to (1--95% LCL of Risk Ratio)\100 ::: ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Measures of association reported for studies in which no data could be extracted ::: ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Trials* No. of students Raw data reported Measures of association, and statistical tests reported -------------------- --------------------- ------------------------------------------------------------------------------------------------------ ----------------------------------------------------------------------------------------------------------------------------------------------- Thompson ^15^ 138 days absent per student in intervention group = 2.30 days absent per student in control group = 3.20 • Overall reduction in absenteeism due to illness was 28 percent for children using alcohol hand rub Morton et al. ^16^ 253 N = 211 absent overall\ • McNemar\'s test for dichotomous variables with paired subjects, was used to assess strength of intervention: chi-square = 7.787; p = .0053\ n = 42 never absent due to illness\ • Odds of being ill decreased by 43 percent with use of AlcoSCRUB^®^ n = 103 ill regardless of participation in the control or the AlcoSCRUB^®^group\ n = 69 ill in control group\ n = 39 ill in AlcoSCRUB^®^group ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- ::: Discussion ========== Many studies have examined the importance of preventing the transmission of infectious diseases in the school environment, one such studied completed by Cramer et al. 1999, indicated this item to be of great concern for the parent\'s of school-aged children \[[@B47]\]. The most common infections transmitted in school environments are respiratory (influenza, pharyngitis etc) and diarrheal illnesses (i.e., Norwalk virus). Most of the infections occur at a constant low level but occasionally outbreaks do occur resulting in increased absenteeism and involvement of public health authorities. Since hands are the primary mechanism of transmission of these illnesses, proper hand hygiene techniques have been endorsed as the first defence at reducing the risk of transmission \[[@B1]-[@B5],[@B8],[@B10],[@B28]\]. In health care settings, the routine use of antimicrobial alcohol based hand gels has been endorsed as an alternative to handwashing when hands are not visibly soiled \[[@B48]-[@B50]\]. Effectiveness in the hospital setting has not been easy to document given the relative low incidence of documented infections that can be specifically related to nosocomial transmission relative to the high number of handwashing opportunities in specific environments such as intensive care unit settings. Can the evidence from these six trials reported here be used to promote this type program in elementary schools at the present time? This systematic review of antimicrobial rinse-free hand sanitizers for prevention of illness-related absenteeism in elementary school children is the first review, of the author\'s awareness, to assess this issue. Although randomized controlled trials are the study design least likely to provide biased estimates of effect, due to the nature of school-based interventions, the inclusion of both randomized and non-randomized cluster controlled trials was required \[[@B51]\]. Of the six studies that met our inclusion criteria, three were non-randomized cluster controlled trials. Recent evidence indicates that non-randomized designs overestimate the effect of an intervention, thus the magnitude of the results should be interpreted with caution \[[@B52]\]. Four of the six studies used an alcohol-based product, the other two using a benzalkonium chloride based disinfectant. The FDA in the United States has indicated that insufficient data exits to classify the latter compounds as safe and effective to use as antiseptic handwashes. They are also adversely affected in the presence of organic material such as food residues, which may be an issue in schools \[[@B53]\]. Four studies were industry sponsored, and five were flawed due to the lack of sample size calculations. The five studies included were of low quality and methodologically weak. The only blinded randomized study using a placebo incorporated in this review was reported to be randomized and double-blinded, however, a description of the randomization technique was not discussed in the report and allocation concealment was unclear \[[@B41]\]. Additionally, this study suffered from a large proportion of withdrawals and drop-outs, thus the results had to be cautiously interpreted. Current studies have indicated that poor quality studies are associated with exaggerated treatment effects \[[@B52]\]. Although all studies reported statistically significant effects of the antimicrobial rinse-free hand gel in the experimental group, the aforementioned evidence suggests the reader should interpret these results cautiously. Thus, a clear delineation of the effectiveness of the intervention cannot be resolved from this review. Several limitations were encountered when completing this review, the major one being the scarcity of high quality studies. Additionally, although content experts, primary authors and industrial companies were contacted, no grey literature was found. The possible existence of unpublished non-significant trials should not be discounted. The validity of performing a quantitative synthesis was considered, however based on a qualitative inspection of heterogeneity and estimates of intervention effectiveness this was not deemed appropriate. Sources of heterogeneity included study designs, population characteristics, intervention characteristics, case definition and primary outcome measure. Thus, sensitivity and subgroup analyses were not performed, and publication bias was not assessed quantitatively. Another limitation was the fact that one reviewer was used to do the broad screen of articles and review the two citations identified between September 2003 and the present time. This may have biased the results; however, it is believed that this reviewer would overestimate the citations to be included. Conclusions =========== In wake of the recent worldwide emergence of Severe Acute Respiratory Syndrome (SARS), the importance of proper hand hygiene has been brought to the spotlight. Comprehensive hand hygiene programs with occasional reinforcement are an inexpensive intervention, which potentially can work for a broad population, with minimal adverse effects. Future research should concentrate on developing study protocols that are scientifically sound with regards to randomization generation, blinding, allocation concealment and other factors that will minimize or avoid bias. Hand hygiene programs are the most important infection control measure in the school environment and have potentially large public health and economic implications therefore their design, implementation, and analysis should be carried out with the rigour. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= EM conceived and designed the study as part of a graduate course in systematic reviews, reviewed trials for inclusions, abstracted data, participated in data analysis, and drafted and revised the manuscript. NLS participated in initial study design, reviewed trials for inclusion, abstracted data, participated in data analysis, and revised the manuscript. Both EM and NLS agreed upon the final revision. Appendices ========== Appendix 1 -- Syntax for searches Appendix 2 -- List of corresponding authors, content experts and industrial companies contacted Appendix 3 -- Data collection form Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/4/50/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional file 1 Additional file 1 - Syntax for searches ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional file 2 Additional file 2 - List of corresponding authors, content experts and industrial companies contacted ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional file 3 Additional file 3 - Data collection form ::: ::: {.caption} ###### Click here for file :::	PubMed Central	2024-06-05T03:55:49.891199	2004-11-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534108/", "journal": "BMC Public Health. 2004 Nov 1; 4:50", "authors": [ { "first": "Emily", "last": "Meadows" }, { "first": "Nicole", "last": "Le Saux" } ] }
PMC534109	Background ========== It is known that assessment of bronchial responsiveness incorporating measurements of forced expiration can be problematic because of limited co-operation and fatigue and dizziness due to repeated forced expiratory manoeuvres. In addition, a deep inspiration, as it is required during an FEV~1~(Forced Expiratory Volume in 1 sec) procedure, causes transient bronchodilatation particularly in normals during challenge with pharmaceutical substances, resulting in interpretation difficulties \[[@B1]\]. Determining bronchial reactivity using a technique which measures airways resistance is less influenced by inspiratory and expiratory efforts. Furthermore, it is more sensitive to small changes in bronchoconstriction \[[@B2]\] and hence more suitable for studies in normal subjects, in whom the response to bronchoconstrictors is, limited \[[@B3]\]. The interrupter method has been shown to be a simple and non-invasive technique of measuring airway mechanics in children or patients with limited co-operation \[[@B4]\]. It is also suitable for diagnostic purposes in the detection and exclusion of asthma \[[@B5]\] and in obtaining valid rhinomanometric measurements in various groups of patients \[[@B6]\]. An official statement by the ATS (American Thoracic Society) on methacholine provocation indicated that the interrupter method may be useful in testing patients who cannot perform acceptable spirometry manoeuvres but its use should be restricted to laboratories with expertise in their application and interpretation \[[@B7]\]. Furthermore, concerns have been raised about pressure equilibration during flow interruption \[[@B8]\] and when small increases in resistance are used as provocation thresholds, the repeatability of the method was found unacceptably low and unsuited for clinical and research purposes \[[@B9]\]. In addition, the studies performed so far with this technique were done on too small numbers of subjects to allow firm scientific conclusions. We hypothesized that, since normals present with lower levels of airway obstruction during challenge, the interrupter technique in this case might be suitable and comparable with the reference PD~20~FEV~1~method, and therefore clinically useful. Methods ======= Subjects -------- The study was conducted in a tertiary referral centre for respiratory disease and 198 subjects were initially enrolled. All subjects were healthy with a negative history and physical examination, normal blood counts, chemistries, chest radiography and spirometry. One individual from this sample reacted to the diluent control solution (0.6%), defined as a resistance difference of \> 30% baseline \[[@B9]\] and was excluded together with non evaluable data from 27 subjects. The final data of 170 normals were finally included, consisting of 100 (59%) males and 70 (41%) females. Predicted values for spirometry were obtained according to the European Community Coal and Steel (ECCS) \[[@B10]\]. All participants were given detailed information of the purpose of the study, which was approved by the hospital ethics committee, and signed a consent form. They were asked to come in the next morning, avoiding all factors listed in the ATS guidelines \[[@B7]\] that might cause a false negative test. Methods ------- Routine spirometry was performed according to standardized guidelines \[[@B11]\]. Interrupter resistance was measured at end interruption (Rint, EI) using the technique by Phagoo et al.\[[@B12]\], who showed that the Rint, EI reflected changes in lung mechanics more sensitively, than interrupter resistance measured at mid or begining of interruption. The Rint, EI is calculated from the airway opening pressure (time function) signal airway opening pressure Pao(t) as follows: based on the assumption that, during a brief (100 ms) airflow interruption there is equilibration between alveolar pressure (PA) and Pao, the Rint, EI is obtained by dividing the change in pressure by the immediately preceding flow. In this study we used the alternative method of opening the interrupter \[[@B13]\], which calculates Rint, EI from the Pao signal using a calibration resistance. The airflow interruptions were performed using the Bronchoscreen system (Jaeger, Würzburg, Germany) \[[@B8]\], a computerized apparatus with a combined nebulizer-shutter head, which allows the changes in resistance of the respiratory system Rint, EI) to be measured with each breath. During quiet breathing, the opening interrupter Rint, EI was calculated. The seated subject (with noseclip in place and the cheeks partly supported by a rubber mouthpiece) breathed in a relaxed manner (in order to avoid glottic artifacts) ambient air to get accustomed to the apparatus. The shutter closed within 15 ms. The time of complete airflow interruption was 100 ms. It was triggered 150 ms after the onset of expiration. The dead space of the apparatus was 0.35 L. The pressure transducer (Honeywell 142 PC 01G; Chesham, Bucks, UK) was connected via a side port directly to the mouthpiece at a distance of 18 cm from the airway opening. Rint, EI was calculated by the formula: Rint, EI = (PA/Pm) × Rref, where PA is the end interruption mouth pressure, Pm the pressure generated during free flow and Rref is a fixed serial resistance. The triggering volume was determined by integrating the signal from a low resistance Lilly Pneumotach, which had a linearity of ± 2% at a flow below 12 L/s. Before each challenge the interrupter was calibrated. A vent produced an airflow of 105 L/min, which was led through the shutter and a calibrating resistance (0.10 kPa/L/s) and the determined Rint,EI had to be within ± 10% of the reference resistance. The above method has been found valid in the presence of mild to moderate bronchoconstriction \[[@B14]\], conditions that are normally met during bronchial challenge. Bronchial responsiveness was measured by a rapid methacholine provocation dosimetric test, as previously described by our group with the same apparatus but using histamine instead \[[@B15]\]. Briefly, 1% methacholine in saline (Lofarma, Italy) was inhaled in doubling doses starting from 200 μg, until FEV~1~had fallen = 20% compared with FEV~1~after an initial saline inhalation. The bronchial aerosol provocation system (APS Jaeger, Wurzburg, Germany) was used in this procedure. The nebulizer was calibrated to draw 5 μL of solution per automatic actuation lasting 0.6 seconds. The 100 μL of aerosol bolus had a mass median aerodynamic diameter of 1.9 μm with 80% of the droplets being less than 5.5 μm at a set pressure of 1.6 bar (22.8 psi). The subjects inhaled methacholine by slow inspiratory capacity manoeuvres guided by the green colour of light emission diodes (so that inspiratory flow was \<0.5l/s) and the response was assessed 1 min after each inhaled dose. Data was assessed by using two different estimates: 1) provocation dose which increases Rint, EI by 100% (PD~100~Rint, EI), calculated by interpolation from the last two points of the cumulative semilogarithmic dose-response diagram, and 2) the percent slope (dose-response ratio-DRR) of a line extending from the origin to the last point of the curve (DRR) \[[@B16]\]. Plateau response was defined as difference in Rint, EI \<40% after the delivery of three consecutive doubling doses and/or DRR\<40% after a total cumulative dose of 4 mg or a PD~100~Rint, EI \>4 mg. The DRR data were analyzed from the whole sample. The 10 day reproducibility of the PD~100~Rint, EI was investigated by randomly asking 39 subjects to come again after one week for a second examination. During the second visit we compared Rint, EI with FEV~1~as measurements of response to provocation, the latter determined 30 s after the assessment of Rint, EI \[[@B9],[@B17]\]. At least two technically correct forced expiratory manoeuvres with an FEV~1~variation within ± 5% were received and the highest value was used for calculating the dose producing a 20% fall in FEV~1~(PD~20~FEV~1~). Statistical analysis -------------------- Regression analysis and correlation, Shapiro-Wilk test for normality and the non-parametric Mann-Whitney-U-/Wilcoxon Rank Sum test with normal approximation and the x^2^test, were used for statistical analysis. The relative duplicate error was used to assess test-retest reproducibility of the PD~100~Rint, EI (assuming a normal distribution), defined as a standard deviation of the differences divided by the v2 after log transformation (approximates coefficient of variation)\[[@B18]\]. Agreement between PD100Rint, EI and PD~20~FEV~1~was defined and calculated according to Bland and Altman \[[@B19]\]. Normal bronchial responsiveness was defined at a cut-off level of \> 0.8 mg methacholine \[[@B20]\], while negative non-response reactions were those \> 2.0 mg (8.8 μmoL)\[[@B21]\]. Results ======= Subjects\' anthropometric data and baseline spirometry are shown in Table [1](#T1){ref-type="table"}. Mean values of vital capacity (VC), FEV~1~and maximal expiratory flow when 50% of the forced vital capacity (FVC) remains to be exhaled (Vmax~50~), were higher in males by 16.3%, 14, 7% and 2.5% than in females. While Rint, EI was higher in females, possibly reflecting smaller airway size, but these differences were not statistically significant. The distribution of PD~100~Rint, EI is shown in Figure [1](#F1){ref-type="fig"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Characteristics of the study population stratified by gender. ::: Variables Men Women ------------------------------------- ----------------- ---------------- n = 100 n = 70 Age, mean (range) yr 38 (18--60) 35 (18--55) Height, mean (cm), SEM 174 (0.78) 160 (0.71) Weight, mean (kg), SEM 79 (0.9) 63 (0.9) Rint, EI, mean (kPa/l/s), SEM 0.24 (0.069) 0.29 (0.074) VCin, mean (%pred), (range) 111.5 (83--144) 95.2 (76--129) FEV~1~, mean(%pred), (range) 107.9 (75--125) 93.2 (78--110) FEV~1~% mean (range) 83 (77--92) 82 (75--90) Vmax~50~, mean (%pred), (range) 83.5 (70--155) 81 (65--145) *Abbreviations: VC in: inspiratory vital capacity, FEV~1~: forced expiratory volume in 1 sec, FEV~1~%: ratio of forced expiratory volume in 1 sec to forced vital capacity, Vmax~50~: maximum flow at 50% of forced vital capacity, Rint, EI:Interrupter Resistance at End Interruption ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Analysis of the distribution of PD~100~Rint, EI (threshold dose) in males and females. Values \>4 mg are derived by extrapolation. ::: ![](1471-2466-4-11-1) ::: Twenty one males and ten females (18%) of our subjects exhibited bronchial hyperesponsiveness. These values were normally distributed (W = 0.93, p = 0.12), with no gender related difference (x^2^= 1.48, p = 0.22, odds = 1.79). Furthermore, 5 of these 31 subjects (3 men and 2 women, 3% of total) were found to show moderate bronchial hyperesponsiveness (PD~100~Rint, EI \< 0.4 mg or \< 1.66 μmol methacholine), as frequently found in current symptomatic asthmatics \[[@B21]\]. No correlation was found of PD~100~Rint, EI to baseline post-saline Rint, EI and the respective DRR. Subjects with negative reactions (\> 8.8 μmol) showed DRRs that were ten times lesser compared to those with BHR (mean ± SD = 67.52 ± 10.66 vs 690 ± 390, p \< 0.001). Plateau response was exhibited by 66 (38%) of the subjects, (36 males) without gender related statistical difference (x^2^= 0.81, p= 0.36). They had DRRs that were 2.5 times smaller compared to the subjects with normal but measurable reactions (30.1 ± 9.8 vs. 75.0 ± 49.9, p = 0.024). PD100Rint, EI was found reproducible with a duplicate error of 8.3% or 0.65 doubling doses (within 140 μg). A close correlation was found between PD~100~Rint, EI and PD~20~FEV~1~(r = 0.76, 95% CI 0.53-0.88) with relatively narrow limits of agreement (Figure [2](#F2){ref-type="fig"}.) Stratification of data according to BHR status is shown in Table [2](#T2){ref-type="table"}. The interrupter method showed DRRs that were more reactive in comparison to the respective DRRs of FEV~1~(approximately seven-fold). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Bland and Altman plot of the differences between two methods against their mean value. The limits of agreement (![](1471-2466-4-11-i1.gif) -2s and ![](1471-2466-4-11-i1.gif) +2s) are -0.334 mg (-1.39 μmol) and 0.306 mg (1.27 μmol) respectively. The 95% confidence intervals are -0.364 to -0.303 mg and 0.275 to 0.336 mg, respectively. ::: ![](1471-2466-4-11-2) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Comparison of the methods described in the text in terms of threshold dose (sensitivity) and dose-response ratio (reactivity), stratified according to BHR status. NS: p value not statistically significance between the two methods. The greater reactivity of the interrupter method is shown. ::: Methods (× ± SD)* ------------------------------------------------------------ ------------- ---------------------------- --------------- Subjects showing BHR PD~100~Rint, EI (mgs) 0.57 ± 0.20 Dose-response ratio (%/mg) 690 ± 390 PD~20~FEV~1~(mgs) 0.72 ± 0.66 Dose-response ratio (%mg) 98 ± 90 NS P \< 0.05 Subjects with normal measurable reactions (\> 0.8 mgs) PD~100~Rint, EI (mgs) 3.42 ± 3.10 Dose-response ratio (%/mg) 74.86 ± 49.87 PD~20~FEV~1~(mgs) 3.13 ± 2.65 Dose-response ratio (%/mg) 20 ± 3.82 NS P \< 0.05 *Abbreviations*: BHR :Bronchial Hyperresponsiveness, Rint, EI:Interrupter Resistance at End Interruption, PD~20~FEV~1~:Dose Producing a 20% fall in FEV~1~. ::: Discussion ========== In this study we have shown that the interrupter technique, and specifically PD~100~Rint, EI, is comparable to the conventional PD~20~FEV~1~method for evaluation of BHR in a large sample of normal subjects. We have also found that this technique has a specificity of 81.2% for normal subjects and its dose response ratio is 7-fold more sensitive than the conventional FEV~1~method. It is known that specific airway conductance is four times more sensitive than FEV~1~as a measure of response to provocation but the use of a body plethysmograph makes assessment of bronchial challenge expensive and time consuming. The present methodology is particularly useful in children and in the elderly, since it is non-invasive, sensitive to changes in airway calibre and requires no subject co-operation. The opening interrupter technique offers the additional advantage of simplicity and ease of application, being particularly useful in subjects unable to perform forced manoeuvres. The Rint, EI was measured during expiration above forced residual capacity (FRC), because resistance hardly changes above this level and since subjects performed relaxed tidal flow maoeuvres, measurements were not affected by variations in breathing. Furthermore, we did not correct Rint, EI by lung volumes because the variability formed in the FRC can reduce the benefit of standardization of Rint, EI and the correlation between respiratory resistance and FRC is not significant over the limited FRC range of healthy subjects \[[@B21]\]. Problems in repeatability have been reported when one uses provocative concentration causing a 30% increase in Rint, EI (PC~30~Rint, EI), so we assessed the PD~100~Rint, EI threshold, which is above the 95 % confidence interval at one tail direction (1.96SD) observed in our normal sample at baseline (Table [1](#T1){ref-type="table"}). Furthermore, although a correlation of the PC~40~Rint, EI with the classical provocative concentration causing a 20% fall in FEV~1~(PC~20~FEV~1~) has been reported (17), data on agreement are presented for the first time in this study. If a cut-off value is set at 0,4 mg PD~100~Rint, EI methacholine, which defines severe and moderate hyperresponsiveness compatible with asthma \[[@B22]\] then 3% of the studied normal population was found to be in this area. This is similar to the percentage found by Malo et al. \[[@B23]\] working with PC~20~FEV~1\ as~well as to 2.5%, which represents the proportion of subjects beyond the 2 SD of the mean on one side of a normal distribution. If the PD~100~Rint, EI threshold is set at 0,8 mg methacholine \[[@B18]\], which includes mild BHR, then 18% of the total subjects studied had some degree of hypersensitivity. Contradictory results have been previously reported regarding the clinical significance of asymptomatic BHR. Some studies, using even stricter definitions of BHR, showed that asymptomatic BHR is of no significance \[[@B24]\]. In contrast other studies have reported an increased rate of decline of lung function in an asymptomatic population with BHR \[[@B25]\]. A plateau with a low maximal response was exhibited by 38% of the subjects, thus representing the least reactive part of the sample. Seppala et al.\[[@B26]\], using a method incorporating a deep inhalation (FEV~1~) showed that 50% of normals had no calculable PC~20~FEV~1~. In this study, by direct comparison of two methods, one involving a deep inhalation, the relative effects of maximal expiratory manoeuvres on airway calibre can be indirectly assessed. The data in Table [2](#T2){ref-type="table"} show that while threshold doses between the two methods are essentially similar and not statistically significant, there is a significant difference in the DRRs between the two methods, being more pronounced in normal subjects showing BHR. Recently, Sundblad et al \[[@B27]\] reported a significant correlation between dose response slopes of FEV~1~and airway conductance in a large sample of subjects but not all were normals. It is known that in bronchial challenge the dose-response curve is expressed mainly by the threshold dose indicating hyperresponsiveness and the rate and magnitude of the response (hyperreactivity, DRR). The less reactive DRRs of the FEV~1~method lend support to the perturbed actomyosin equilibrium hypothesis recently described, in that with stretching there is a decrease in myosin duty cycle and the magnitude of the contractile response becomes functionally disengaged from the level of the contractile stimulus \[[@B28]\]. Furthemore, since there is indirect evidence of a lack of airway inflammation or remodelling that could prevent smooth muscle from stretching, our data are in agreement with those of Kolnaar et al. \[[@B24]\]. The fact that airway elastic recoil decreases (increase in hysteresis) when smooth muscle is contracted \[[@B1]\], explains the greater difference in DRRs exhibited by subjects showing BHR (the prevailing distending force of the lung allows the airway to dilate more after deep inhalation). No correlation was found in this study between BHR and baseline airway calibre, although Malo et al.\[[@B23]\] found a weak correlation by using a more sensitive parameter i.e. the PC~6~FEV~1~. The limits of agreement between PD~100~Rint, EI and the classical PD~20~FEV~1~were found relatively small at -1.39 μmol and 1.27 μmol respectively. This implies that this method may be used as an alternative to FEV~1~during provocation, as it is simple and easy to perform and requires no patient co-operation. Gender differences in BHR were explored because of the smaller lung size in women. Our data are in agreement with recent studies \[[@B29]\] that in non smoking women, lung size has no effect on bronchial sensitivity. Since RintL was found more sensitive than RintEI, a study comparing the two methods with the classical method would be interesting \[[@B30]\]. Conclusions =========== In summary, the interrupter technique as an index of response to provocation has been shown to be useful to assess bronchial responsiveness in normal subjects, when maximal efforts cannot be performed. We recommend threshold doses of 100% baseline, because they show reliable agreement with the classical PD20FEV1 method. List of abbreviations ===================== ATS:American Thoracic Society, BHR:Bronchial Hyperresponsiveness, DRR:Dose Response Ratio, FEV~1:~Forced Expiratory Volume in 1 sec, FVC:Forced Vital Capacity, FRC:Functional Residual Capacity, PA:Alveolar Pressure, Pao: airway opening Pressure, PC~20~FEV~1~:Provocative Concentration causing a 20% fall in FEV~1~, PC~30~Rint, EI:Provocative Concentration causing a 30% increase in Rint, EI, PD~100~Rint, EI: Provocation dose which increases Rint, EI by 100%, PD~20~FEV~1~:Provocation Dose producing a 20% fall in FEV~1~, Rint, EI:Interrupter Resistance at End Interruption, VC:Vital Capacity, Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= PP, IK, AT, SA, DB were involved with the study conception. PP, AT, SA performed the interrupter technique and collected the data. PP did the statistical analysis. PP, AT, DB prepared 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/1471-2466/4/11/prepub> Acknowledgement =============== The authors thank Professor J.Milic-Emily for his review of the manuscript and his valuable advice	PubMed Central	2024-06-05T03:55:49.896234	2004-11-12	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534109/", "journal": "BMC Pulm Med. 2004 Nov 12; 4:11", "authors": [ { "first": "Panagiotis", "last": "Panagou" }, { "first": "Ioannis", "last": "Kottakis" }, { "first": "Argyris", "last": "Tzouvelekis" }, { "first": "Stavros", "last": "Anevlavis" }, { "first": "Demosthenes", "last": "Bouros" } ] }
PMC534110	Background ========== Dupuytren\'s disease (DD) is a fibro-proliferative disorder of the palmar fascia (PF) characterized by the formation of fibrous nodules and cords \[[@B1]\]. The disease results in digital contractures, leading to loss of hand function. Surgical excision of the diseased PF is currently the principal form of management since the lack of a clear etiology has precluded the development of other effective and rational forms of treatment. Since Baron Guillaume Dupuytren\'s classical description of the disease in 1831, multiple clinical associations have been described, however, no clear molecular mechanism for the disease has been established \[[@B2]\]. Histochemical studies of DD have demonstrated the presence of myofibroblasts \[[@B3]\], increased production of type III collagen \[[@B4]-[@B7]\], and alterations in other extra-cellular matrix proteins including various fibronectin isoforms \[[@B8]-[@B14]\]. These biological features are characteristic of abnormal growth factor regulation, specifically fibrogenic cytokines such as transforming growth factor-beta (TGF-β). Several studies have documented TGF-β expression in DD palmar fascia using RT-PCR \[[@B15]\], in-situhybridization \[[@B16]\], and immunohistochemistry \[[@B16]-[@B18]\], while others have shown that TGF-β can stimulate cell proliferation \[[@B18]-[@B20]\] and promote myofibroblast differentiation in vitro\[[@B21]\]. As a result of these and other studies it has been suggested that an aberrant TGF-β activity may be involved in the pathogenesis of DD. In this study, we chose to focus on TGF-β~2~and its effects on collagen contraction in vitrousing a three-dimensional fibroblast populated collagen lattice (FPCL) contraction assay and DD patient-matched disease and control primary cell cultures. Previous reports have examined the role of TGF-β in DD by comparing disease fibroblasts to \'control\' fibroblasts obtained from transverse carpal ligament material obtained from patients undergoing carpal tunnel release (CTR). By contrast, the control fibroblast cultures used in this study were established from unaffected PF tissue from the same patient, thus providing us with unique patient- and tissue- matched control cultures. The observed phenotypic differences between patient/tissue-matched control and disease FPCL cultures, specifically elevated collagen contraction activity, and β-catenin and fibronectin (Fn) expression in disease cells \[[@B22]-[@B24]\], raises the intriguing possibility that pro-fibrotic factors, such as TGF-β~2~, may be regulating these disease-associated events in vitro, since TGF-βs are known to promote fibroblast mediated collagen contraction \[[@B21],[@B25],[@B26]\] and up-regulate collagen, Fn and β-catenin \[[@B20],[@B27]-[@B30]\]. As described in detail below, we have found that exogenous TGF-β~2~could significantly stimulate \'normal\' and disease FPCL contraction in a dose-dependent manner. While neutralizing anti-TGF-β~2~antibodies completely blocked exogenous TGF-β~2~stimulated FPCL contraction they had no effect on the enhanced basal collagen contraction activity of disease FPCL cultures. Methods ======= Patient samples and primary cell cultures ----------------------------------------- Our study protocol was cleared through the UWO Ethics Committee for Research Involving Human Subjects. Areas of diseased fascia and uninvolved normal (control) PF tissue were collected during surgery. DD explant cultures were initially cultured in starter media consisting of α-MEM (Gibco, Invitrogen Corporation) supplemented with 20% fetal bovine serum (FBS, Clontech Laboratories, Palo Alto, CA), and antibiotics (Penicillin G and streptomycin sulfate) and fungizone (Gibco, Invitrogen Corporation) as previously described \[[@B23]\]. Established primary culture lines were maintained in α-MEM + 10% FBS + antibiotics + fungizone. Culture flasks were incubated at 37°C in a humidified chamber with 5% CO~2~. Medium was changed every 4--5 days and the cells sub-cultured using 0.05% Trypsin-EDTA (Gibco, Invitrogen Corporation, Grand Island, NY) when confluent. Fibroblast Populated Collagen Lattice (FPCL) contraction assay -------------------------------------------------------------- Collagen contraction was carried out using patient-matched disease (D) and control (C) primary cultures (passages 2 -- 6) established from patient-matched DD lesions and uninvolved palmar fascia (control). Collagen lattices were prepared by mixing cell suspensions with a neutralized solution of collagen type I matrix (8 parts Vitrogen100 collagen type I, 2.9 mg/ml, Collagen Corp, Santa Clara, CA, USA + 1 part 10 × α-MEM + 1 part HEPES buffer, pH 9.0). The cell-collagen concentrations were adjusted with sterile phosphate buffered solution (PBS) to attain a final collagen concentration of 2.0 mg/ml and a final cell concentration of 8.6 × 10^4^cells/ml of matrix. The cell-collagen mixture was then aliquoted into 24 well culture dishes (0.5 ml/well) that were pre-treated with a PBS solution containing 2% (w/v) bovine serum albumin (BSA). Following FPCL polymerization (1 hr, 37°C) culture medium (0.5 ml) consisting of α-MEM + 10% fetal bovine serum (FBS) was added atop each lattice. After 2 days of culture the attached FPCLs were mechanically released from the sides of the culture plates. Digital images of the contracting FPCL were captured at various time points over a 5-day assay period using a conventional flatbed scanner. Collagen lattice areas were then quantified using the Image J program \[[@B31]\]. Each assay was done in quadruplicate. TGF-β~2~and neutralizing anti-TGF-β~2~antibody treatments --------------------------------------------------------- Commercially available human recombinant TGF-β~2~(expressed in NSO murine myeloma cells) was acid activated in a solution of 4N HCl + 0.1% (w/v) BSA according to the manufacturer\'s instructions (Product \# T 2815, Sigma, St. Louis, MO). Acid activated human recombinant TGF-β~2~was then aliquoted and frozen for extended storage at -70°C. The indicated concentrations of activated human recombinant TGF-β~2~were added to complete culture media immediately following FPCL polymerization. Neutralizing anti-TGF-β~2~antibodies (R&D Systems, Minneapolis, MN) were added to complete culture media either immediately following FPCL polymerization or added to the cell suspension prior to mixing with the neutralized Vitrogen collagen type I solution. Control FPCL cultures were treated with appropriate carrier solutions. Cell proliferation assay ------------------------ FPCL cultures were assayed using a commercially available CellTiter 96^®^A~queous~One Solution cell proliferation assay according to the manufacturer\'s instructions (Promega, Madison, WI, USA). This cell proliferation assay is a colorimetric method that uses a tetrazolium compound 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) in combination with a stable electron coupling reagent PES (phenazine ethosulfate). This produces a chemically stable MTS tetrazolium compound that can be bio-reduced by cells to form a soluble colored formazan product \[[@B32],[@B33]\]. Briefly, cells from patient-matched primary cultures were cultured as stressed-relaxed FPCLs as described above. Each primary cell line was plated as quadruplicate FPCL cultures for each of the indicated time points with 0.5 ml of culture media ± TGF-β~2~(1 ng/ml) atop each FPCL. At each of the designated contracting time points 100μl of CellTiter 96^®^A~queous~One solution reagent was added to the FPCL cultures and incubated for 3 hours (37°C, 5% CO~2~atmosphere, humidified chamber). FPCL media was then collected and aliquoted into a 96-well culture plate for absorbance reading at 450 and 650 nm (background reference λ) using a 96-well BIO-RAD microplate reader. Media was also collected from FPCLs containing no cells (negative control) for background absorbance readings. The assay was conducted over 5 days during the course of FPCL contraction. A standard curve was also generated to calculate relative cell numbers per FPCL (range of 4 × 10^3^--10^5^cells/FPCL). Statistical analysis -------------------- Student two-tailed ttest was used to compare data between two groups. Values were expressed as mean ± standard deviation of the mean (SDM). Pvalues \< 0.05 were considered statistically significant. Results ======= TGF-β~2~stimulates FPCL contraction ----------------------------------- We first examined the basal collagen contraction activity of three independent patient-matched early passage control and disease primary FPCL cultures. As shown in Figure [1](#F1){ref-type="fig"}, disease FPCL cultures contracted collagen faster and to a greater degree when compared to patient-matched control FPCL cultures. This is in agreement with previous results that show distinct phenotypic differences between control and disease primary DD cultures \[[@B22],[@B24]\], including enhanced collagen contraction by disease FPCL cultures \[[@B23]\]. Next, we explored the role of exogenous TGF-β~2~on FPCL contraction. Initial experiments showed a typical dose-dependent response for TGF-β~2~stimulated FPCL contraction (Fig. [2](#F2){ref-type="fig"}). Because maximum collagen contraction was achieved in response to 1 ng/ml of TGF-β~2~, all subsequent experiments used this dose. As shown in Figure [3a](#F3){ref-type="fig"}, we observed enhanced contraction rates and total collagen contraction for both control and disease cells treated with exogenous (1 ng/ml) TGF-β~2~. This enhanced collagen contraction activity observed in disease FPCL cultures was not due to differences in cell proliferation/viability between control or disease FPCL cultures. In fact, TGF-β~2~appears to exert a significant pro-apoptotic effect on relaxed disease FPCL cultures that is absent in the \'control\' FPCL cultures (Fig [3b](#F3){ref-type="fig"}). Thus, the amount of collagen contraction exerted by disease FPCL cultures is more pronounced if one also considers the changes in cell viability over the course of FPCL contraction. Neutralizing anti-TGF-β~2~antibodies block TGF-β~2~stimulated FPCL contraction but do not alter basal FPCL contraction ---------------------------------------------------------------------------------------------------------------------- Exogenous TGF-β~2~stimulated collagen contraction in both control and disease FPCL cultures was inhibited in a dose-dependent manner by neutralizing anti-TGF-β~2~antibodies. As shown in Figure [4](#F4){ref-type="fig"}, lower concentrations of neutralizing antibodies (100 ng/ml) partially inhibited FPCL contraction, while higher concentrations of neutralizing antibodies (1000 ng/ml) completely inhibited TGF-β~2~stimulated FPCL contraction, thus confirming the ligand-dependent nature of this response in vitro. We also examined the effect of neutralizing anti-TGF-β~2~antibodies on the basal collagen contraction activity of primary disease and control FPCL cultures. As shown in Figure [5](#F5){ref-type="fig"}, high concentrations (1000 ng/ml) of neutralizing anti-TGF-β~2~antibodies had no effect on the basal levels of collagen contraction observed in either control or disease FPCL cultures, suggesting that there is little or no endogenous active TGF-β~2~that could account for the FPCL contraction in vitro. To preclude that more localized interactions between endogenously produced TGF-β~2~and its cell-surface receptors may account for the observed increase in disease FPCL contraction, we also pre-incubated the cells with neutralizing anti-TGF-β~2~antibodies prior to forming FPCL. Regardless, the blocking antibodies had no effect on the basal levels of FPCL contraction (data not shown). Discussion ========== Members of the TGF-β family are potent fibrogenic factors that play an important role in the patho-physiology of numerous fibro-proliferative disorders, including DD \[[@B34]\]. The study presented here focused on the effect of TGF-β~2~on collagen contraction using primary FPCL cultures derived from patient-matched disease and control unaffected PF tissue. Here, we found that disease FPCL cultures contracted collagen faster and to a greater degree than patient-matched control FPCL cultures. Although neutralizing anti-TGF-β~2~antibodies effectively blocked exogenous TGF-β~2~stimulated FPCL contraction for both control and disease cultures, the same neutralizing antibodies had no effect on the basal collagen contraction activity of either disease or control FPCL cultures, suggesting that enhanced disease FPCL contraction is not due to elevated levels of endogenous active TGF-β~2~. These results, however, do not exclude the possibility that there may be differences in the levels of latent TGF-β~2~produced by these cell cultures that may be subsequently activated in vivo. Recent work by Kuhn et. al. (2002) reported reduced DD FPCL contraction in response to tamoxifen, which was associated with a decreased TGF-β~2~production \[[@B35]\]. However, the TGF-β~2~assayed in this study required an acid-activation step, suggesting that TGF-β~2~produced by these cells is largely in its latent, non-activate form. Thus, it is possible that TGF-β~2~produced by DD fibroblasts and/or other resident cell types in vivomay be important to disease cord contraction, provided appropriate regulators of latent TGF-β activation are also present and active. However, it does not explain the enhanced basal FPCL contraction rates of disease cell cultures, suggesting that other signalling factors may be regulating this disease cell function in vitro. It is interesting to note that both TGF-β and β-catenin signalling pathways show some degree of \'cross-talk\' \[[@B29],[@B30],[@B36]-[@B38]\], suggesting that β-catenin plays an important role in TGF-β signalling. The degree and significance of signalling \'cross-talk\' between β-catenin and TGF-β in the context of DD is unknown and needs further examination. Although previous DD studies have used different \'control\' primary fibroblast as \'disease-free\' controls, namely fibroblasts derived from transverse carpal ligament material obtained from patients undergoing carpal tunnel release (CTR), they have the disadvantage of being of different anatomical origin than PF derived \'control\' fibroblast cultures. This is an important consideration given that fibroblasts exhibit functional heterogeneity depending on their origin \[[@B39]-[@B41]\]. For example, human fibroblasts that express the cell surface antigen Thy-1 are capable of TGF-β~1~stimulated myofibroblast differentiation, while Thy-1 negative fibroblasts appear to be only capable of lipofibroblast differentiation \[[@B42]\]. Phenotypic differences have also been attributed to fibroblasts of different dermal origins \[[@B43]\]. For example, Chipev and colleagues showed that TGF-β~1~had a pro-apoptotic effect on non-palmoplantar (keloid) fibroblasts and an anti-apoptotic effect on palmoplantar fibroblasts. Similar to what we have observed for detached or contracting DD FPCL cultures, they showed that TGF-β~1~treated (in the presence of serum) keloid FPCL cultures underwent the most extensive apoptosis response upon mechanical release compared to dermal fibroblasts from different body sites \[[@B43]\]. Perhaps the similar myofibroblast phenotype attributed to both DD and keloid fibroblast cultures also dictates a similar apoptotic fate to relaxed FPCL cultures in response to TGF-βs. Although the loss of tension in these cultures triggered a mostly uniform loss in cell viability across all groups, unlike disease FPCL cultures TGF-β~2~did not appear to have any significant pro-apoptotic effect on relaxed \'control\' FPCL cultures. In light of these and other findings, it appears that the patient-matched control fibroblast cultures employed in these studies may truly represent a suitable \'control\' phenotype, with the added advantage of having the same PF origins as the disease cell cultures. While this does not exclude the possibility that the control cultures may harbour some residual disease cells, this is not supported by the distinct phenotypic differences we have observed between these two types of patient-matched PF cultures in the current and previous studies. In these earlier studies, we reported elevated levels of β-catenin and fibronectin isoforms in disease FPCL cultures \[[@B22]-[@B24]\], as well as enhanced disease FPCL contraction rates which we have subsequently confirmed in these studies. This together with the distinctive pro-apoptotic affects of TGF-β~2~on disease FPCL cultures described here, further support the notion that the patient/tissue-matched \'control\' cultures have a non-disease phenotype that is suitable for these types of investigations. Although the \'synthetic\' myofibroblast features of the disease cells described in previous studies are known to be stimulated by TGF-β \[[@B20],[@B21],[@B25]-[@B30],[@B35]\], our results suggests that endogenous TGF-β~2~does not play a role in regulating these phenotypic differences in vitro. Nevertheless, aberrant expression of various TGF-β signalling components have been previously shown to trigger this \'synthetic\' myofibroblast phenotype in other fibro-proliferative disorders, specifically keloids and burn hypertrophic scarring \[[@B34],[@B44],[@B45]\], that can to some extent be inhibited by neutralizing anti-TGF-β~2~antibodies \[[@B46],[@B47]\]. Hopefully future studies will unravel the extent of these phenotypic differences between these patient/tissue-matched control and disease FPCL cultures with respect to pro-fibrotic factors like TGF-β~2~, tension and other important intersecting signaling pathways. Conclusions =========== Primary disease FPCL cultures contract collagen faster and to a greater extent than control PF-matched FPCL cultures. While neutralizing anti-TGF-β~2~antibodies can block exogenous TGF-β~2~stimulated collagen contraction for both control and disease FPCL cultures, it had no effect on the basal contraction rates of either control or disease FPCL cultures. We, therefore, conclude that the enhanced collagen contraction activity of disease FPCL cultures is not due to differences in the levels of endogenous active TGF-β~2~. List of abbreviations ===================== DD, Dupuytren\'s disease; PF, palmar fascia; CTR, carpal tunnel release; TGF-β, transforming growth factor-beta; FPCL, fibroblast populated collagen matrix; PBS, phosphate buffered saline; RT, room temperature; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; α-MEM, alpha-minimal essential medium; FBS, fetal bovine serum; Fn, fibronectin; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt; PES, phenazine ethosulfate; RT-PCR, reverse transcriptase polymerase chain reaction; SDM, standard deviation of the mean. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= RT and YW carried out the experiments. RT drafted the manuscript. JCH and BSG designed and coordinated the research, and reviewed and edited 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/1471-2474/5/41/prepub> Acknowledgements ================ This study was supported by grants from the Canadian Institutes of Health Research, Plastic Surgery Education Fund, American Society for Surgery of the Hand, and the Lawson Health Research Institute\'s Internal Research Fund. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Collagen contraction of primary control and disease FPCL cultures. Digital images of contracting FPCL cultures were captured and analyzed using Image J software to quantify collagen contraction. The mean FPCL surface area (mm^2^) was plotted over time with each data point representing the mean ± the standard deviation of the mean (SDM) of three independent patient-matched cell lines. Experiments were repeated in quadruplicate. ::: ![](1471-2474-5-41-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### TGF-β~2~dose response. The dose response curve (upper panel) shows the accumulated FPCL contraction (%) over 5 days. The degree of TGF-β~2~stimulated contraction was measured over a large concentration range (\>5 orders of magnitude). The plotted data shows a typical dose-response relationship, with maximal response elicited by 1 ng/ml of TGF-β~2~. Representative images of contracting FPCL after 5 days are shown for increasing concentrations of TGF-β~2~(lower panel). ::: ![](1471-2474-5-41-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### FPCL contraction in response to exogenous TGF-β~2~. Contracting FPCL cultures (± 1 ng/ml TGF-β~2~) were analyzed using Image J software to quantify collagen contraction. (A) The plotted data points represent the mean surface area ± SDM for three independent patient-matched primary FPCL cultures. Experiments were repeated in quadruplicate. (B) Cell proliferation/viability assays were performed on contracting FPCL cultures. The plotted bar graph represents the mean cell number ± SDM for the indicated time points for one representative patient-matched disease and control primary culture. Significant differences between groups are indicated by the P-values. The stars denote significance differences (P\< 0.05) between the same treatment groups of different time points (white star -- Day 1 vs. Day 3, black star -- Day 1 vs. Day 5). ::: ![](1471-2474-5-41-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Neutralizing anti-TGF-β~2~antibodies block TGF-β~2~stimulated FPCL contraction. (A) Control and (B) disease FPCL cultures (± 1 ng/ml TGF-β~2~) were treated with the indicated concentrations of neutralizing anti-TGF-β~2~antibodies. FPCL contraction was analyzed and plotted as the mean surface area ± SDM for quadruplicate cultures per treatment. ::: ![](1471-2474-5-41-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Neutralizing anti-TGF-β~2~antibodies do not inhibit basal collagen contraction activity of control or disease FPCL cultures. Disease and control FPCL cultures were incubated in the absence or presence of the indicated concentrations of neutralizing anti-TGF-β~2~antibodies. Measurements of the contracting FPCL were plotted as the mean surface area ± SDM for quadruplicate cultures per treatment. ::: ![](1471-2474-5-41-5) :::	PubMed Central	2024-06-05T03:55:49.898125	2004-11-12	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534110/", "journal": "BMC Musculoskelet Disord. 2004 Nov 12; 5:41", "authors": [ { "first": "Raymond", "last": "Tse" }, { "first": "Jeffrey", "last": "Howard" }, { "first": "Yan", "last": "Wu" }, { "first": "Bing Siang", "last": "Gan" } ] }
PMC534111	Background ========== Myasthenia gravis (MG) is an acquired, neuromuscular, autoimmune disease that presents clinically with weakness and fatigue of the skeletal muscles. The disorder is characterized by a decrease of the number of acetylcholine receptors in the neuromuscular plates, due to an autoimmune process mediated by antibodies directed against the alpha-subunit of the nicotine receptor of the acetylcholine \[[@B1]\]. The disease is twice as common in women as in men and frequently affects young women in the second and third decades of life, overlapping with the childbearing years \[[@B2],[@B3]\]. Generally, during pregnancy in one third of patients the disease exacerbates, whereas in two thirds it remains clinically unchanged \[[@B4]-[@B8]\]. Of the women who experience worsening, it usually occurs during the first trimester. Signs and symptoms of MG in pregnant women tend to improve during the second and third trimesters coinciding with the physiological immunosuppression which normally takes place in that period. Complete remission can occur in some patients \[[@B4]-[@B8]\]. Papazian \[[@B9]\] reported a 21% incidence of transient neonatal myasthenia gravis (TNMG) on infants born to mothers with MG. In this report 67% of infants developed TNMG within the first few hours after birth and within the first 24 hours of life in 78% of neonates \[[@B9]\]. Onset of TNMG beyond 3 days after birth has not been reported. Two clinical forms of TNMG have been described: typical (71%) and atypical (29%). Clinical features of the atypical form include the presence of arthrogriposis multiplex congenita (AMC) in the fetus or newborn \[[@B10]\]. The severity of AMC in the infant is variable and does not co-relate with neither the severity of maternal MG during pregnancy, or if it is the first or subsequent pregnancies \[[@B10]\]. In anti acetylcholine-receptor (anti-AchR) antibody-associated AMC, fetal or neonatal death is common. The possible mechanisms could be crossing of maternal antibodies through the placenta with consequent blockage of the function of the fetal isoform of the AchR leading to fetal paralysis causing AMC. In the typical form of TNMG the usual clinical findings include poor sucking and generalized hypotonia \[[@B11]\]. Other reported clinical manifestations are week cry (60% to 70%), facial diplegia or paresis (37 to 60%), swallowing and sucking difficulties (50 to 71%), and mild respiratory distress \[[@B9]-[@B11]\]. Ptosis (15%) and ophthalmoparesis (8%) are less common. Respiratory distress requiring assisted mechanical ventilation can occur in severe cases (29%) \[[@B9],[@B10],[@B12]\]. Response to an oral or parenteral anticholinesterase agent is usually very good. Complete recovery is expected in less than 2 months in 90% of patients and by 4 months of age in the remaining 10% \[[@B9],[@B10],[@B12]\]. It is not clear why only some babies develop TNMG, but the ability of the mother\'s serum antibodies to bind to the fetal isoform of the AchR in newborn may be a contributing factor \[[@B12]\]. The purpose of our present study was to report on the clinical course, delivery and neonatal outcome of pregnant women with the diagnosis of myasthenia gravis, followed in our perinatal center. Methods ======= Patient population ------------------ From January 1,1996 to December 31, 2003, 18 pregnant women with MG were treated during pregnancy and delivered at the National Institute of Perinatology, a tertiary referral center in Mexico City, Mexico. The clinical course of the disease during pregnancy, labor and post-partum period was reviewed, as well as the neonatal period in the 17 infants born to MG mothers. All clinical data was ascertained after reviewing and collecting data from the patient\'s medical records. The diagnosis of myasthenia gravis was made on clinical grounds and confirmed by positive edrophonium chloride and electromyography tests \[[@B13],[@B14]\]. Transient neonatal myasthenia gravis was diagnosed on the bases of clinical signs of generalized hypotonia, sucking disturbances, weak cry and respiratory difficulties. Criteria for defining clinical improvement or deterioration ----------------------------------------------------------- After reviewing the medical records of patients the following criteria was used to define clinical change of MG during pregnancy: a) the first was the type and dosage of medications that the patient received before, during and after pregnancy. Data was collected on the type and doses of medications administered to the patient during the 3 periods, b) the second parameter was the stage of the disease according the Osserman\'s classification before, during and after pregnancy. The criteria for improvement, unchanged or worsening of MG during pregnancy were the following: 1) Remission: those patients that presented a total disappearance of the symptoms (Osserman\'s stage 0) and who did not require any specific medication, 2) Improvement: patients who had clinical improvement of the symptoms and decrease of the dosage of the medications that they received before pregnancy by 30% or more, 3) No change: patients with no clinical change in their symptoms (According to Osserman\'s classification) and same doses of medications compared with before pregnancy. 4) Deterioration: patients who had a deterioration of the disease (worsening of the Osserman\'s stage) and an increase in the dosages of medications compared with before the pregnancy, or the need for immunosuppressant drugs such as azathioprine and/or prednisone. The Osserman\'s classification used in this study was the one used by the Myasthenia Gravis Foundation of America: grade I: any ocular muscle weakness; grade II: mild weakness affecting other than ocular muscles; III: moderate weakness affecting other than ocular muscles; IV Severe weakness affecting other than ocular muscles; and grade V: Defined by tracheal intubation, with or without mechanical ventilation, except when employed in routine postoperative management \[[@B15]\]. ### Patient follow-up In the first two trimesters all patients were seen in the clinic once a month, every 15 days between 32 and 36 weeks, and weekly after 36 weeks of gestation. During every visit the dosage of the medications, and the Osserman\'s stage were reviewed. All patients were seen by a team of obstetricians and clinical neurologists. Statistical analysis -------------------- Descriptive statistics was used to compute the results. Results ======= During the study period 18 pregnant women with MG were seen at the hospital and had the medical records available for review. The mean ± SD maternal age was 27.4 ± 4.0 years. Before pregnancy 3 patients (17%) were in remission (Osserman\'s stage 0) and 15 patients (83%) were classified as Osserman\'s stage II. All the patients were clinically stable before pregnancy. Of the 15 patients with in stage II, 13 (86%) used pyridostigmine, one used pyridostigmine plus steroids (7%), and one used pyridostigmine, azathioprine and steroids. Thymectomy was performed in 17 patients (94%) before the pregnancy. The mean length of time from the start of symptoms to thymectomy was 24.0 months (range: 1--168 months). Other clinical conditions were also diagnosed in 5 patients (28%) before pregnancy: 3 (17%) had impaired glucose tolerance and 2 autoimmune thyroiditis (11%). Serum antibodies against the human acetylcholine receptor assayed by standard RIA were positive in 14 patients (77%). The patients became pregnant at a mean of 2 years post-thymectomy. In our center we prefer to do the thymectomy first and then when the patient is stable we recommend the pregnancy. This is not a generalized protocol in many centers but the majority of our patients were in good conditions before the pregnancy. During pregnancy 9 patients (50%) did not change the clinical status compared with before pregnancy, 2 (11%) had improvement and 7 (39%) had worsening of the MG. Of the seven patients who deteriorated, one did so in the first trimester and six in the second trimester. Only one patient experienced a myasthenic crisis during pregnancy. During pregnancy 11 patients (61%) received pyridostigmine, one patient (6%) received pyridostigmine plus steroids and another (6%) received pyridostigmine, steroids, and azathioprine. The pregnancy in two patients (11%) was complicated by eclampsy; one woman was diagnosed with chorioamnionitis and another with thrombocytopenia. Pregnancy duration was 37.5 ± 3.0 weeks (range 29--41 weeks). The clinical characteristics of patients is shown in Table [1](#T1){ref-type="table"}. Table [2](#T2){ref-type="table"} shows the delivery mode and neonatal outcome of our patients. Eight patients were delivered by caesarian section. The other ten were delivered by vaginal delivery (one forceps assisted), one of these products was a stillbirth. Seventeen infants were born at mean ± SD gestational age of 37.5 ± 3.0 weeks and a mean birth weight of 2710 ± 73 g. Only one infant presented with transient neonatal myasthenia gravis. This baby presented with sucking difficulties, which resolved spontaneously by day 7 and did not require any specific treatment. The patient with myasthenic crisis delivered by spontaneous vaginal delivery at 37 weeks. The weight of the newborn was 2800 g without complications. No congenital anomalies were identified in any of the newborns. Discussion ========== Myasthenia gravis is not rare among women of reproductive age, the reported incidence ranges from 1:10,000 to 1:50,000 \[[@B3]\]. Literature describing the clinical course of pregnant myasthenic women mostly consists of single case reports and case series \[[@B6]-[@B8]\]. Generally it has been assumed that pregnancy is associated with physiological immunosuppression. There is evidence, as yet unexplained, that polymorphonuclear leukocyte chemotaxis and adherence functions are depressed beginning in the second trimester and continuing during the rest of the pregnancy. It is possible that these depressed leukocyte functions of pregnant women account in part for the improvement observed in some autoimmune diseases. It may also explain the increased susceptibility to certain infections \[[@B17]\]. On the other hand it is well known that some diseases could exacerbate during the pregnancy. This has been reported for example in patients with systemic lupus erythematosus and myasthenia gravis \[[@B16],[@B17]\]. The clinical course of myasthenia gravis in pregnancy is considered to be unpredictable. It has been reported that: a) approximately one third of patients remain the same, one third improve, and the remaining one third worsens, b) the course in one pregnancy does not predict the course in subsequent pregnancies, c) exacerbations occur equally in all three trimesters and 4) therapeutic termination does not demonstrate a consistent benefit in cases of first trimester exacerbation \[[@B4],[@B6]-[@B9],[@B18]\]. Schlezinger \[[@B4]\] described the course of MG during pregnancy in 22 myasthenic women with a total of 33 pregnancies. He showed that in one third of the pregnant woman an exacerbation occurred, whereas two thirds showed no change or a remission occurred. In his series the exacerbation usually occurred during the first trimester, with minor clinical changes during the second and third trimesters \[[@B4]\]. Djelmis et al \[[@B8]\] reviewed their experience with 69 pregnancies among 65 women with MG managed over a 28 year-period. 24.6% of patients showed an improvement during the pregnancy, 44.9% did not change and 30.4% suffered exacerbations. In Djelmis et al \[[@B8]\] report the deterioration was observed in the last 4 weeks of pregnancy and in the puerperium. In another study Mitchell et al \[[@B6]\] reported the clinical course of 11 cases of pregnant myasthenia gravis patients. 27.2 % had improvement and 72.7% deteriorated during pregnancy. The deterioration was observed in the third trimester in all patients. One of their patients suffered respiratory failure. They concluded that there were no predictive factors to identify the mother at risk of exacerbation during pregnancy and the risk of neonatal myasthenia gravis. Batochi et al \[[@B7]\] evaluated the course of 47 women who became pregnant after the onset of MG. During pregnancy 42% had no change, 39% improved and 19% got worse. In the experience of Batochi et al \[[@B7]\] the clinical worsening was more frequent in the second trimester and two patients developed respiratory failure. He concluded that the course of the myasthenia gravis during gestation is highly variable and unpredictable and can change in subsequent pregnancies. Recently Picone at al \[[@B18]\] in a series of 12 patients showed worsening in 42% of patients during pregnancy. Our study showed a frequency of worsening of 33%, being an intermediate frequency compared with the reported frequencies of 15 to 55%. The majority of our patients showed that the worsening occurred in the second trimester as in the Batochi et al \[[@B7]\] series. In the series of Osserman \[[@B4]\] and Djlemis et al \[[@B8]\] the worsening was observed in the third trimester. It is clear from these reports that the clinical course of the disease during pregnancy is highly variable, and difficult to predict. In our study 8 patients had a cesarean section for delivery (47%) and 9 (53%) delivered vaginally (one by forceps extraction). In one patient the pregnancy ended in a stillbirth. Djelmis et al \[[@B8]\] reported vaginal deliver in 82%, Batochi et al \[[@B7]\] in 70%, Mitchel et al \[[@B6]\] in 90% and Picone at al \[[@B18]\] in 58%. Our study showed a rate of cesarean section of 47%, similar to the rate of 42% reported by Picone et al \[[@B18]\]. In a recent study Hoff et al \[[@B19]\] reported the results of a retrospective cohort in Norway. The study population consisted of 127 births to mothers with MG compared with a reference group of 1.9 million births to mothers without MG. They showed that women with MG had a higher rate of complications at delivery, and in particular the risk of preterm rupture of amniotic membranes was three times higher in the MG group compared with the reference group. The rate of interventions during birth was raised and cesarean sections doubled. They concluded that MG is associated with an increased risk of complications during delivery, leading to a higher need for surgical interventions. Regarding the newborns, our study showed that their weight is lower compared with other studies \[[@B6],[@B7]\]. This may be explained by racial differences between our population and the population reported by others. The incidence of TNMG has been reported between 9 and 30% \[[@B6],[@B7]\] Typical clinical findings in the typical form of TNMG are poor sucking and generalized hypotonia. Other manifestations are swallowing and sucking difficulties and mild respiratory distress. Response to oral or parenteral anticholinesterase agents is usually very good. Complete recovery is expected in less than 2 months in 90% of patients and in the remaining 10% by 4 months \[[@B9],[@B10]\], \[[@B12],[@B13]\]. Only 5% of our patients presented with TNMG, which is less than the rate reported by others. The reason for this lower rate is unexplained but it could be due to genetic variation as suggested by others \[[@B9]\]. In conclusion the present literature in pregnant patients with myasthenia gravis is somewhat limited. It consists mostly of case reports and case series. Our study adds to the body of literature showing that about third of our patients deteriorated during pregnancy, which was observed in the second trimester. Our cesarean section rate was high and the rate of TNMG was relatively low. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= JFTZ. Has made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. LHR. Has made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. VS. Has made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data. BE. Has been involved in drafting the article or revising it critically for important intellectual content ODS. Has been involved in drafting the article or revising it critically for important intellectual content 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/1471-2474/5/42/prepub> Acknowledgments =============== FUNSALUD and CONACYT (SNI) funded Dr. Tellez-Zenteno\'s postdoctoral fellowship. Figures and Tables ================== ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Patient characteristics ::: Variable Mean ± SD, median (range), \# of patients (%) ------------------------------------------------------ --------------------------------------------------- Maternal variables Maternal Age (years) 27.4 ± 4.04 Length of time from diagnosis to thymectomy (months) 24 (1--168) Period of time from thymectomy to pregnancy (months) 24 (6--36) Duration of pregnancy (weeks) 37.5 ± 3.0 (29 to 41) Previous thymectomy (%) 17 (94%) Clinical course of disease during pregnancy No change 9 (50%) Improvement 2 (11%) Deterioration 7 (39%) ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Delivery and neonatal outcome ::: Variable Mean ± SD, \# of patients (%) -------------------------------------- ----------------------------------- Type of delivery (n = 18) Vaginal (without Forceps)\* 9 (50%) Vaginal (Forceps) 1 (6%) Caesarian 8 (44%) Neonatal outcome (n = 17) Birth weight (g) 2710 ± 73 Neonatal transient myasthenia gravis 1 (6%) \*One vaginal delivery was a stillbirth. :::	PubMed Central	2024-06-05T03:55:49.899858	2004-11-16	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534111/", "journal": "BMC Musculoskelet Disord. 2004 Nov 16; 5:42", "authors": [ { "first": "José F", "last": "Téllez-Zenteno" }, { "first": "Lizbeth", "last": "Hernández-Ronquillo" }, { "first": "Vicente", "last": "Salinas" }, { "first": "Bruno", "last": "Estanol" }, { "first": "Orlando", "last": "da Silva" } ] }
PMC534112	Background ========== The use of recombinant proteins as drugs has deeply modified the therapeutic approach to many severe diseases. However, a variety of practical problems limits the use of biotechnological protein drugs. Stability of the active proteins, need for parenteral administration, and high costs of the final purified materials are among the most significant drawbacks. A way of circumventing these issues is represented by the direct administration of recombinant bacteria, acting simultaneously as cell factory and delivery system for pharmacologically active proteins. This approach has been already extensively experimented for the mucosal delivery of vaccine antigens \[[@B1],[@B2]\]. In recent years, the local delivery of therapeutic antibodies \[[@B3],[@B4]\], adjuvant cytokines \[[@B5],[@B6]\], and anti-inflammatory cytokines \[[@B7]-[@B9]\] has been successfully attempted with food-grade bacteria (e.g., Lactococcus lactis, Streptococcus gordonii), although limited to the therapy of localised pathologies (e.g., inflammatory bowel diseases, IBD, in the gastro-intestinal tract). Among anti-inflammatory strategies, both at systemic and local level, the use of the IL-1 receptor antagonist (IL-1Ra) has received vast attention. IL-1 is a family of cytokines highly active in the modulation of immune amplification and inflammation. The IL-1 family includes two agonist proteins, IL-1α and IL-1β, and one antagonist protein, IL-1Ra. IL-1β is a very potent immunostimulatory and inflammatory cytokine, responsible for initiating and amplifying the host response to invasion. If not properly controlled, IL-1 can cause fever, acute inflammation, tissue destruction, organ failure, and eventually shock and death (reviewed in \[[@B10]\]). IL-1Ra inhibits IL-1 by acting as a competitive receptor antagonist with no detectable agonist activity, thus representing a natural powerful mechanism to control IL-1-dependent responses and avoid pathological derangement (reviewed in \[[@B11],[@B12]\]). In experimental animal models, IL-1Ra has demonstrated excellent therapeutic effects against acute and chronic inflammatory pathologies, being also effective at high doses in prolonging survival in endotoxic shock \[[@B11]-[@B17]\]. In human trials, IL-1Ra has been administered to patients with septic shock, rheumatoid arthritis, graft-versus-host disease, and multiple sclerosis (reviewed in \[[@B11],[@B12],[@B16]\]). While only a modest benefit was achieved in patients with septic shock \[[@B11],[@B12],[@B16],[@B18]\], IL-1Ra had a clear beneficial effect in reducing joint destruction in rheumatoid arthritis \[[@B11],[@B12],[@B19]-[@B21]\]. From the clinical experience with purified recombinant IL-1Ra it became clear that most of the problems of variability of efficacy were due to difficulties in adequate timing and dosage of the drug \[[@B12]\]. To overcome these problems, gene therapy with adenoviral vectors carrying the IL-1Ra gene has been attempted in experimental animals, yielding promising results in models of type 1 diabetes and ischemic brain damage \[[@B22],[@B23]\]. The clinical application of the gene therapy approach may however meet with difficulties for safety reasons, besides the problems of controlling drug release, concentration, and localisation. Based on previous experience of using recombinant bacteria as in vivocell factory, here we describe a novel system of local delivery of IL-1Ra, able to achieve systemic effects. The system exploits the ability of certain bacteria (such as Bacillus subtilis) to undergo autolysis in non-permissive conditions (as it occurs in the mammalian intestine) thereby releasing the cytoplasmic proteins. Intra-colon instillation of B. subtilisexpressing recombinant human mature IL-1Ra induces significant serum levels of the recombinant protein in rats and rabbits, and prevents the inflammatory effects of systemic IL-1. Intra-peritoneal administration of recombinant B. subtilisin the mouse could inhibit LPS-induced shock and death. Further experimental evidence with a B. subtilisstrain producing human IL-1β demonstrates that this delivery system can be generalised to other recombinant proteins. Results ======= The ability of B. subtilisto generate spores by autolysis of the cell wall, thereby releasing cytoplasmic proteins, was exploited as system for delivery of proteins in vivo. Following a sporulation signal (e.g., nutrient depletion) bacteria undergo an autolytic process with release of most of their cellular components and formation of a highly resistant spore containing DNA and few essential proteins (Figure [1A](#F1){ref-type="fig"}). As already shown in a previous study \[[@B24]\], in vitrosporulation of B. subtilisengineered for endocellular expression of human IL-1Ra (strain pSM539) caused the release of large amounts of intact and active recombinant protein within a few hours after the sporulation signal (Figure [1B](#F1){ref-type="fig"}). The in vitrorelease of intracellular recombinant IL-1Ra was equally evident in pSM539 Spo^+^(normally sporulating) and Spo^-^bacteria, i.e.genetically modified cells which, in response to the sporulation signal, start the autolysis process but are unable to form a complete spore thus undergoing complete cell destruction (Figure [1C](#F1){ref-type="fig"}). Both Spo^+^and Spo^-^strains of pSM539 were used in subsequent in vivoexperiments with identical results. The IL-1Ra recovered after B. subtilisautolysis in vitroretained full biological activity, with a specific activity of 1.1 × 10^6^inhibitory units (IU)/mg vs.0.9 × 10^6^IU/mg of reference standard IL-1Ra \[[@B24]\]. To assess whether the recombinant protein could also be released in vivoby engineered B. subtilis, the bacterial strain engineered with IL-1Ra was administered intra-peritoneally in the mouse, in the small and large intestine of rats, and in the rabbit distal colon. The presence of human IL-1Ra was assessed at different times after administration, both locally and in the serum of treated animals, by Western blotting, ELISA, and BIAcore analysis (Table [1](#T1){ref-type="table"}). Intact human IL-1Ra was found locally at high levels 3 hours after administration of recombinant bacteria and persisted for several hours. In the serum, intact human IL-1Ra could be found at measurable levels when B. subtiliswas administered intra-peritoneally (2/2 mice) or in the colon (5/9 rats, 27/31 rabbits), but not when bacteria were administered in the small intestine (0/5 rats). The delivery of IL-1Ra at the intestinal mucosal level was examined. Live cells of the IL-1Ra-producing pSM539 strain were instilled in the rat distal colon, a non-permissive environment that does not allow B. subtilisvegetative life \[[@B26]\]. As a control, animals received equal numbers of the pSM214 strain, i.e. B. subtiliscells transformed with the β-lactamase-expressing control plasmid pSM214. Data in Figure [2](#F2){ref-type="fig"} show that the presence of intact IL-1Ra can be measured both locally (in the intestinal washings) and in serum for several hours after intra-colonic inoculum of the IL-1Ra-producing B. subtilisstrain pSM539, whereas serum of animals receiving B. subtilispSM214 remained negative. As a control, pSM539 bacteria delivered in the small intestine released detectable amounts of IL-1Ra locally, but no IL-1Ra could be found at the serum level (data not shown; Table [1](#T1){ref-type="table"}). The serum pharmacokinetic parameters of IL-1Ra released by recombinant B. subtilis, as compared to the purified protein administered intra-colonically, show a few differences (Table [2](#T2){ref-type="table"}). The C~max~was higher and the T~max~quicker for the purified protein, as compared to IL-1Ra released from intra-colonically administered B. subtilis. On the other hand, the AUC/dose was almost identical. Administration of control bacteria pSM214 intra-colonically together with the purified protein did not significantly change the pharmacokinetics parameters of IL-1Ra, except for a slight decrease of the total dose absorbed, indicating that the physical presence of bacteria has little effect on IL-1Ra absorption. It is concluded that engineered B. subtilisdelivered intra-colonically releases, conceivably by autolysis, the cytoplasmic recombinant protein, which is subsequently absorbed and can be detected intact at measurable levels in the bloodstream. To verify that IL-1Ra found in serum after B. subtilisadministration at the mucosal level is functional, its ability to inhibit IL-1 was evaluated both in vitroand in vivo. In vitro, activity of standard IL-1β was assessed with the classical co-stimulation assay on thymocytes of LPS-unresponsive C3H/HeJ mice. Inhibition by IL-1Ra was evaluated as capacity to decrease IL-1-induced thymocyte proliferation. The presence of biologically active IL-1Ra was measured as inhibition of IL-1β-induced thymocyte proliferation by the IL-1Ra-containing serum of rabbits administered pSM539 intra-colonically. The presence and amount of IL-1Ra was measured by ELISA in the serum of pSM539-treated rabbits and of control pSM214-treated or untreated animals. As shown in Figure [3](#F3){ref-type="fig"}, IL-1Ra-containing serum from pSM539-treated rabbit (used at dilutions containing from 0.1 to 10 ng/ml IL-1Ra) was as effective in inhibiting IL-1β activity as the same concentrations of standard purified recombinant IL-1Ra (Figure [3](#F3){ref-type="fig"}, left), whereas the same dilutions of serum from pSM214-treated or from untreated animals (devoid of IL-1Ra) did not possess any IL-1-inhibiting activity (Figure [3](#F3){ref-type="fig"}, left and right panels). To confirm that the IL-1β-inhibiting activity observed in sera of pSM539 treated animals is indeed due to IL-1Ra, data in the Figure [3](#F3){ref-type="fig"} (right) show that the inhibitory capacity of pSM539 serum is significantly decreased or abolished by an antiserum against human IL-1Ra. Thus, it can be concluded that the IL-1Ra present in serum after intra-colonic administration of pSM539 is biologically active. The in vivoefficacy of IL-1Ra released by intra-colonic pSM539 was evaluated in antagonising the effects of parenterally administered IL-1 \[[@B27]\]. As shown in Figure [4](#F4){ref-type="fig"} (upper left), the increase in body temperature induced in rabbits by i.v. administration of 75 ng/kg human IL-1β was significantly reduced by preventive intra-colonic treatment with 2 × 10^9^cells of B. subtilispSM539. The reduction of IL-1β-induced fever was more pronounced with lower doses of IL-1β (90% reduction of peak fever induced by 50 ng/kg IL-1β), but it was still highly significant when fever was induced by 100 ng/kg IL-1β (\>60% reduction of peak fever) (data not shown). To confirm these data, the effect of intra-colonic treatment with IL-1Ra-producing pSM539 was evaluated on other inflammation-related parameters induced by IL-1β, i.e., granulocytosis and decrease of blood glucose and iron concentrations. As shown in Figure [4](#F4){ref-type="fig"} (upper right), the increase in circulating PMN induced in rats by IL-1β i.p. was abrogated by previous intra-colonic administration of IL-1Ra-producing pSM539 but not by the control strain pSM214. Likewise, the IL-1β-induced decrease in the blood levels of iron (Figure [4](#F4){ref-type="fig"}, lower left) and glucose (Figure [4](#F4){ref-type="fig"}, lower right) was still evident in animals administered control pSM214 bacteria but was significantly reduced by intra-colonic instillation of IL-1Ra-producing pSM539 bacteria. It is inferred that human recombinant IL-1Ra delivered in vivoby intra-colonic administration of engineered B. subtilisis biologically active and able to counteract the systemic inflammatory effects of IL-1. That IL-1Ra delivered by B. subtiliscan have an anti-inflammatory protective effect in vivowas shown in a model of shock and death induced by bacterial endotoxin (LPS) in the mouse (Figure [5](#F5){ref-type="fig"}), an acute syndrome in which IL-1β plays a major role \[[@B14]-[@B16],[@B25]\]. LPS-sensitive C3H/HeOuJ mice receiving recombinant pSM539 bacteria intra-peritoneally 24 hours before administration of a lethal dose of bacterial LPS could survive significantly longer than mice administered the control pSM214 bacteria or PBS, in agreement with previous data on the efficacy of IL-1Ra in inhibiting LPS-induced shock \[[@B13]-[@B15]\]. To validate the concept of delivery of bioactive recombinant proteins via the colonic mucosa by means of recombinant B. subtilis, another B. subtilisstrain was constructed (pSM261, engineered for production of human mature IL-1β) and administered in vivoto rabbits. As shown in Figure [6](#F6){ref-type="fig"}, the intra-colonic administration of 1 × 10^9^live cells of B. subtilispSM261 induced a significant increase in body temperature, superimposable to that caused by intra-colonic instillation of purified recombinant IL-1β. Furthermore, in agreement with the systemic effects of massive doses of IL-1β administered parenterally \[[@B25],[@B28]\], intra-colonic administration of pSM261 caused shock and death in 9/14 animals (64%). It is concluded that the mucosal delivery of engineered B. subtilisin the large intestine is a suitable system for attaining significant blood levels of bioactive recombinant proteins and systemic effectiveness. Discussion ========== The use of live bacteria is very common in particular in vaccinology, where attenuated or mutant bacteria have been employed for decades as antigen carriers. The advantage of live bacteria relies on their capacity of colonising the host and enter the host organs/tissues with the same modalities as their virulent counterparts, thus eliciting the relevant immune response and immune memory, at variance with killed bacteria or purified bacterial components. Thus, attenuated strains of Salmonella, Listeria monocytogenes, Mycobacterium tuberculosis, Vibrio choleraeare being developed and used as vaccine carriers \[[@B29]-[@B31]\]. A further development in the use of live bacteria as antigen carriers in vaccination exploits the technologies of genetic engineering for introducing multiple antigens from different micro-organisms into a single non-virulent bacterial carrier (e.g., food-grade lactic acid bacteria), with the possibility of including T- and B-stimulating epitopes from different antigens, and also to engineering into the same carrier adjuvant sequences derived for instance from an immunostimulating cytokine \[[@B30]-[@B34]\]. Among bacterial systems developed for antigen delivery in vaccination, some strains of non-pathogenic, food-grade or GRAS (generally regarded as safe) bacteria have been examined for the topical delivery of pharmacologically active protein drugs, after cell engineering with the DNA coding for the protein of interest. This is the case of Lactococcus lactisand of Streptococcus gordonii, which have been engineered to produce recombinant antibodies, adjuvant and anti-inflammatory cytokines, and used to deliver these proteins locally at the mucosal surface after oral administration \[[@B3]-[@B9]\]. The goal of these delivery approaches was that of making the recombinant proteins available for therapy of local pathologies or for local effects: antibodies for passive immunotherapy of local infections \[[@B3],[@B4]\], cytokines as adjuvants for mucosal vaccines \[[@B5],[@B6]\], inhibitory cytokines for anti-inflammatory therapy of localised chronic inflammatory diseases (IBD-like pathologies) \[[@B7]-[@B9]\]. Although undoubtely promising and susceptible of vast applications, the method of mucosal delivery of therapeutic protein through recombinant bacteria acting as cell factories needs further and deeper investigation. This should include the central issue of safety and contained/controlled release of recombinant micro-organisms \[[@B8]\], the problem of assessing the mucosal permanence of bacteria (extent and duration of colonisation depending on the changes in the mucosal environment in different conditons of health and nutrition) and the extent of protein release, and the issue of pharmacodynamics of the delivered protein in particular for its systemic effects, beyond the boundaries of the local delivery environment. The delivery system proposed here is not based on the permanence/colonisation capacity of bacteria in the host mucosal surfaces, but it relies on the capacity of sporulating bacteria of releasing intracellular proteins in non-permissive environments. B. subtiliscells engineered to produce human IL-1Ra were able to release the recombinant protein (intact and biologically active) following a sporulation signal in vitro\[[@B24]\]. This observation could be repeated in vivo, when recombinant B. subtiliscells were inoculated in the intestine of rats or rabbits (a non-permissive environment that does not allow the vegetative life of B. subtilis). The recombinant protein could be detected locally shortly after administration of bacteria and persisted at measurable levels for several hours. Release and recovery of recombinant IL-1Ra was much more abundant and consistent in the large intestine as compared to the small intestine. Most interestingly, the recombinant protein released from sporulating bacteria delivered in the large intestine was absorbed in the bloodstream at detectable levels, whereas no circulating IL-1Ra could be found after bacterial delivery in the small intestine. IL-1Ra present in the blood was intact, as judged by its molecular mass in Western blotting, and retained full IL-1-inhibiting activity, as judged by its capacity of dose-dependent neutralisation of IL-1β in vitro. The passage of an intact protein from the intestinal lumen to the bloodstream is not a new concept. Indeed, transcytosis has been extensively described in intestinal epithelial cells, and allows transport of intact proteins and macromolecules from the intestinal lumen to the circulation through an endocytic non-degradative pathway in physiological conditions of integrity of the intestinal mucosal barrier \[[@B35]-[@B39]\]. This mechanism of transcytotic transport, quantitatively scarce as compared to the degradative pathway of protein absorption, may have a role in physio-pathological passage of antigens, allergens, and toxins. Delivery of IL-1Ra through engineered sporulating bacteria apparently had some pharmacokinetics advantages as compared to the purified protein. Whereas the absorption into the bloodstream was quick after administration of the purified protein (T~max~at 60 min), IL-1Ra released from intra-colonically administered B. subtilishad a much slower kinetics of absorption (T~max~200 min), as expected by the fact that the protein must be released from bacteria before being absorbed. Furthermore, although the C~max~was decreased for B. subtilisIL-1Ra (136 ng/ml vs.482 ng/ml for the purified protein; only partially attributable to the higher dosage of the purified protein), the AUC/dose were almost identical. Thus, IL-1Ra delivered intra-colonically by B. subtilisis absorbed into the bloodstream at a slower and more constant rate than the purified protein delivered in the same site, which is absorbed quickly into the bloodstream and rapidly disappears thereafter. Thus, it appears that bacteria do not undergo sporulation all at the same time (which would result in a rapidly appearing and disappearing peak of protein), but release the protein constantly from the moment of administration for about 8 h. This would allow a controlled and sustained circulating level of the protein, thus a more favourable pharmacodynamic profile, with a single administration. The protein selected for in vivodelivery with B. subtilisis the IL-1 receptor antagonist IL-1Ra, a competitive non-activating ligand of the IL-1 receptor with IL-1 inhibitory activity \[[@B11],[@B12]\]. IL-1 is a potent inflammatory cytokine which, in pathological conditions, is responsible of chronicisation of inflammation, tissue destruction, organ failure, hypotensive shock \[[@B10]\]. Anti-IL-1 strategies have been attempted in acute an chronic inflammatory diseases with the use of recombinant IL-1Ra protein \[[@B11],[@B12]\]. The poor outcome of clinical trials in septic shock has highlighted the problems of a therapy based on the systemic administration of a purified recombinant protein, whose efficacy is hampered by its rapid pharmacokinetics \[[@B11],[@B12],[@B18]\]. At present, experimentation of therapeutic IL-1Ra is being targeted to slowly progressive chronic diseases with defined organ/tissue targets (e.g., rheumatoid arthritis) \[[@B40],[@B41]\]. To achieve sustained IL-1Ra levels, gene therapy approaches have been attempted with promising results in animal models of experimental arthritis, ischemic brain damage, autoimmune diabetes \[[@B19]-[@B23]\]. However, the risk remains of side effects due to the uncontrolled inhibition of the physiologically important IL-1 activity. Indeed, a precise balance between between IL-1β and IL-1Ra should be maintained for achieving proper tissue homeostasis, as shown for the intestinal mucosa \[[@B42]\]. The drug delivery strategy here described merges the well-known approach of vaccination with live bacteria with that of gene therapy. The delivery of pharmacologically active proteins by live sporulating bacteria, as described here, presents a series of advantages over other similar approaches. At variance with conventional gene therapy, the gene coding for the drug protein is introduced in a bacterial carrier rather than in host cells, a situation that would allow a complete control of its permanence in the body. In a previous study, intragastric or vaginal administration of Streptococcus gordoniiengineered to release human IL-1Ra resulted in a prolonged local delivery of the protein, consequent to the capacity of S. gordoniito colonise the mucosal surfaces \[[@B9]\]. Mucosal delivery of IL-1Ra (by intragastric administration of engineered S. gordonii) also had a local therapeutic effect in a model of ulcerative colitis \[[@B9]\]. The delivery system with sporulating bacteria described here differs from that with S. gordonii, as it causes rapid local release of the recombinant protein (e.g.in the large intestine, where IL-1Ra peaks at 4 h and decreases towards background at 24 h), followed by absorption into the bloodstream. In preliminary experiments in the mouse, IL-1Ra-expressing bacteria were also administered intragastrically or subcutaneously. This achieved appearance of human IL-1Ra in the serum, and systemic effects of inhibition of LPS-induced shock and death (data not shown). This is a new finding, that opens the possibility of exploiting localised bacterial administration (e.g.at mucosal sites) for systemic drug delivery. The amount of protein released at the mucosal site directly correlates with the number of administered bacteria, since the internal body environment does not sustain bacterial replication but induces sporulation. This allows an exact control of the dose of drug delivered and, based on the pharmacokinetics parameters, of the blood levels that can be reached. The same result could not be easily obtained with S. gordonii, as amount and timing of protein release may be influenced by variation of the colonisation capacity depending on variations of environmental conditions of the host tissues. A problem that should be faced when using recombinant bacteria in vivofor therapy or vaccination is that of safety and contained release of genetically modified organisms (GMO). The use of suicidal genes or the deletion of genes vital for survival outside the host organism have been explored with very promising results \[[@B8],[@B43]\]. The bacterial system proposed here can be modified in the sporulation mechanism for the control of its survival. In preliminary experiments, the recombinant B. subtilispSM539 strain was engineered in order to inactivate a gene involved in sporulation control. As a consequence, in response to in vitrosporulation signals (adverse environmental conditions) the mutated Spo^-^strain could regularly initiate the sporulation process, undergoing cell autolysis and release of the cytoplasmic proteins (including the recombinant IL-1Ra), but it was incapable of eventual spore formation and further survival. Likewise, release of the recombinant protein from Spo^-^in vivowas comparable to that of Spo^+^bacteria, but spores could never be recovered from intestinal lavage and faeces (data not shown). This suggests that the system can be optimised to full biological containment and environmental safety without altering its delivery properties. Conclusions =========== The novel system of protein drug delivery here proposed links some of the advantages of gene therapy (endogenous production of the relevant protein, targeted delivery) to the possibility of controlled release in terms of timing and protein amount. Exploitation of the mechanism of bacterial autolysis in non-permissive environments allows release of intracellular proteins, including the known amount of the pharmacologically active recombinant protein drug. The release is persistent for several hours, allowing to maintain more constant protein levels in the bloodstream. The system is simple, cheap, and can be developed to full environmental safety (i.e., avoiding the risk of release of genetically modified bacteria in the environment). The concept that pharmacologically active proteins released at the colonic mucosal surface can be absorbed and reach the circulation intact and retaining full activity (validated with two proteins with opposite effects, IL-1Ra and IL-1β) opens promising avenues to the use of local delivery for the therapy of systemic diseases. Methods ======= Bacterial strains ----------------- Engineered B. subtilisstrains were constructed as previously described in detail \[[@B44],[@B45]\]. Briefly, cDNA coding for mature human IL-1Ra (encompassing the mutation N91\>R), and cDNA coding for mature human IL-1β were cloned between EcoRI and HindIII in pSM214, a B. subtilisplasmid which promotes the synthesis of recombinant products intracellularly, to obtain recombinant plasmids pSM539 (carrying the cDNA for IL-1Ra) and pSM261 (carrying the cDNA for IL-1β). Plasmids were used to transform the B. subtilisSMS118 strain. The pSM539-harbouring B. subtilisstrain SMS118(pSM539) could produce 1.0--2.0 mg IL-1Ra/10^9^cells/0.35--0.49 g (wet weight), after conventional culture overnight in 1 liter flasks. The SMS118(pSM261) strain in the same culture conditions produced 0.15--0.25 mg IL-1β/10^9^cells/0.35--0.49 g. As negative control, B. subtilisstrain SMS118 was transformed with the pSM214 plasmid, which contains the gene of β-lactamase (conferring resistance to penicillin). All strains were leu^-^, pyrDI, npr^-^, apr^-^. Sporulation-defective (Spo^-^) strains were constructed by mutation in the srfAgene, as previously described \[[@B46]\] and were kindly provided by Dr. G. Grandi (Chiron S.r.l., Siena, Italy). Bacterial preparations ---------------------- Bacteria were grown in LB medium containing 5 mg/l chloramphenicol for 7 h at 37°C under shaking and harvested by centrifugation (3,000 × g, 20 min, 4°C). For sporulation supernatant preparation, 0.25 g wet weight of bacteria (corresponding to 0.5--0.7 × 10^9^cells) were suspended in Difco sporulating medium (bacto beef extract 3 g/l, peptone 5 g/l, NaOH 0.25 mM, MgSO~4~10 mM, KCl 0.1%, MnCl~2~0.1 mM, Ca(NO~3~)~2~1 mM, FeSO~4~1 mM, pH 6.8) without chloramphenicol and incubated at 35°C with shaking. Aliquots of sporulation supernatant were harvested by centrifugation (14,000 × g, 5 min) at different time points. Following sporulation signals, both Spo^+^and Spo^-^bacteria initiate the autolysis process, which ends in cell autolysis with release of cytoplasmic content. However, whereas in Spo^+^bacteria there is formation of a spore with preservation of strain survival, Spo^-^bacteria are unable to form a spore thus undergoing complete cell destruction (Figure [1A](#F1){ref-type="fig"}). Upon sporulation signals, both Spo^+^and Spo^-^bacteria released 100% of intracellular recombinant products in a time-dependent fashion, with maximal relaease between 2 an 8 h (Figure [1C](#F1){ref-type="fig"}) \[[@B24]\]. SDS-PAGE analysis ----------------- Protein samples were run on 13.5% mini SDS-PAGE according to Lämmli \[[@B47]\] and stained with Coomassie R-250. The gel was subjected to laser scanning on a Molecular Dynamics Personal Densitometer, and the densitometric analysis was made using Image Quant software (Molecular Dynamics, Sunnyvale, CA). Animals ------- Experimental animals were: female C3H/HeOuJ mice of 10--12 weeks of age (20--25 g) (for all in vivoexperiments), female C3H/HeJ mice of 5--8 weeks of age (for thymocyte proliferation), female Sprague-Dawley rats (around 300 g), and female New Zealand rabbits (1.9--2.5 kg). All animals were purchased from Charles River Italia (Calco, Italy) and were housed in standard cages at 22 ± 1°C with 12 h light-12 h dark cycle. Animals received standard diet and tap water ad libitum. In vivoadministration of B. subtilis ---------------------------------------- Bacteria were harvested and resuspended in LB medium or sterile PBS. Mice received a single intra-peritoneal injection of 0.2 ml of bacterial suspension in PBS. Rats were fasted overnight before the surgical procedure and maintained under urethane anaesthesia throughout. Bacteria (in LB medium diluted 1:1 in PBS) were instilled in the small intestine (duodenum) with a 22 1/2 G needle, in a volume of 1--10 ml. Two surgical ligatures were applied, one at the beginning of the duodenum immediately below the needle entry puncture (to avoid exit of instilled bacteria), and another one near the ileocecal valve, to limit to the small intestine the transit of bacteria. Intra-colon instillation was performed again with a 22 1/2 G needle in the ceacum immediately below the ileo-caecal valve, in a volume of 5--10 ml. Two surgical ligature were applied just below the needle entry point and at the colon terminal region, to avoid loss of bacteria. Animals were sacrificed by exanguination at different times after treatment, to collect blood and intestinal washings. For intra-colonic administration of bacteria in rabbits, animals were fasted overnight prior to treatment, then lightly restrained in conventional stocks and maintained conscious throughout the experiment. A rounded-tip urethral catheter (Rüsh, Germany) was carefully inserted 10 cm into the distal colon via the anal route and 2 ml of B. subtilissuspension were administered. Serum samples were prepared from blood collected from the rabbit marginal ear vein at different times (0--8 h) after intra-colonic administration of bacteria. In some experiments, animals were sacrificed, to collect the large intestine content (saline washing). Protocols of animal experimentation were reviewed by the institutional ethical board for adherence to ethical guidelines for animal research conduct (Italian D. L.vo 27/01/1992 n. 116 and corresponding EU directive 86/609; policy of refinement, reduction and replacement towards the use of animals for scientific procedures 99/167/EC -- Council Decision of 25/1/99), and previously authorised by the Italian Ministry of Health. Detection of human IL-1Ra in animal samples ------------------------------------------- Western blotting: samples were subjected to reducing 15% mini SDS-PAGE and analysed by Western blotting using a polyclonal rabbit serum anti-human IL-1Ra and a goat anti-rabbit IgG secondary antibody conjugated with horseradish peroxidase, as described in detail elsewhere \[[@B48]\]. Serum samples were filtered on Microcon 100 (MWCO 100,000; Amicon, Beverly, MA) before analysis. ELISA measurement: samples were subjected to quantitative determination of human IL-1Ra using a specific ELISA (Amersham, Little Chalfont, UK), following the manufacturer\'s instructions. The lower detection limit was 20 pg/ml. Purified human recombinant IL-1Ra was used as standard. Serum samples were filtered on Microcon 100 (Amicon) before analysis. Biosensor measurement: detection of IL-1Ra in serum samples and intestinal washings was confirmed with the biosensor BIAcore™ system (Pharmacia Biosensor AB, Uppsala, Sweden), which allows real time biospecific interaction analysis by means of the optical phenomenon of surface plasmon resonance, as previously described in detail \[[@B49]\]. The lower detection limit for human IL-1Ra was 2 pg/ml. IL-1-induced thymocyte proliferation ------------------------------------ The classical assay of co-stimulation of murine thymocyte proliferation was used to evaluate the bioactivity of IL-1 and IL-1Ra. Briefly, thymocytes from 5--8 week-old C3H/HeJ mice (preferentially used because of their LPS unresponsiveness) were cultured at 6 × 10^5^cells/well of Cluster^96^plates (Costar, Cambridge, MA) in 0.2 ml of RPMI-1640 medium (Life Technologies, Paisley, Scotland) supplemented with 2 mM L-glutamine, 25 mM HEPES buffer, 50 μg/ml gentamycin sulfate, 1.25 × 10^-5^M 2-ME (all from Sigma Chemical Co.), 5% fetal bovine serum (Hyclone, Logan, UT) for 72 h in moist air with 5% CO~2~\[[@B50]\]. The biological activity of IL-1β was assessed as co-stimulation of thymocyte proliferation, by adding to the culture wells a selected amount of human recombinant IL-1β (30--300 pg/ml) \[[@B51]\] and a suboptimal concentration of purified PHA (1.5 μg/ml; Murex Diagnostics, Dartford, UK). Cells were then pulsed for 18 h with 18.5 kBq/well of \[^3^H\]TdR (sp. act. 185 GBq/mmol; Amersham) and their proliferation was measured as radiolabel incorporation with a β-counter. The biological activity of IL-1Ra was evaluated as inhibition of IL-1β-dependent thymocyte proliferation. To this end, cells were stimulated to proliferate (with IL-1β and PHA) in the presence of increasing concentrations of human recombinant IL-1Ra \[[@B51]\] or serial dilutions of serum from rabbits receiving pSM214 or pSM539 intra-colonically, or from untreated rabbits. The concentration of IL-1Ra in serum of pSM539-treated rabbits was determined by ELISA and serum was added to the cultures after appropriate dilution. Control sera from pSM214-treated or untreated rabbits were used at the same dilutions as IL-1Ra-containing serum. Cell proliferation was then evaluated as radiolabel incorporation as described above. To assess that the effect of IL-1Ra-containing serum was indeed due to IL-1Ra, a polyclonal rabbit antibody against human IL-1Ra \[[@B48]\] was added to the cultures at a dilution of 1:300, i.e.the dilution previously found to inhibit 50% of the activity of 10 ng/ml IL-1Ra in the thymocyte assay (not shown). IL-1-induced fever ------------------ Rabbits were lightly restrained in conventional stocks throughout the experiment, and accustomed to the stocks over a period of 2 h, to minimise variations in body temperature. Body temperature was measured by means of a cutaneous thermistor probe (TM-54/S and TMN/S; LSI-Lastem, Settala Premenugo, Italy) placed between the left posterior paw and the abdomen and allowed to stabilise for 2 min. B. subtilissuspensions (2 × 10^9^live cells/rabbit) were instilled in the distal colon 1 h before i.v. administration of 50--100 ng/ml highly purified LPS-free human recombinant IL-1β in pyrogen-free saline through the marginal ear vein. Temperature was recorded every 20 min for 3 h starting from IL-1β administration. In experiments with IL-1β-producing strain pSM261, rabbits received an intra-colonic administration of 1 × 10^9^pSM214 (control) or pSM261 (IL-1β) bacteria, or 250 μg purified human IL-1β. Temperature was recorded up to 22 h after treatment. IL-1-induced neutrophilia, hypoferremia, hypoglycemia ----------------------------------------------------- Live cells of B. subtilisstrains pSM214 and pSM539 were instilled in the distal colon (1 × 10^9^cells/kg), 2 h before administration of IL-1β. Blood samples were drawn 2, 4, 6, 8 and 24 h after intra-peritoneal inoculum of 0.1 μg/kg human recombinant IL-1β. The number of circulating neutrophils was assessed by flow cytometry. The plasma iron concentration was determined colorimetrically with a commercially available kit (Fe; Boehringer Mannheim, Mannheim, Germany). Hypoferremia (60--75% decrease of plasma iron level) was evident from 4 to 24 h after IL-1β inoculum. The blood glucose concentration was measured in serum samples by the glucose/glucose oxidase/peroxidase method with commercially available kits (Glucose GOD Perid; Boehringer Mannheim) or by biosensor detection with devices for diagnostic monitoring (Roche Diagnostics, Milano, Italy). Overlapping results were obtained in rats and rabbits. LPS-induced shock in the mouse ------------------------------ LPS-sensitive C3H/HeOuJ mice received an intra-peritoneal inoculum of 0.5 ml PBS alone or containing bacterial suspensions (control pSM214, IL-1Ra-producing pSM539; 3 × 10^6^bacteria/mouse), 24 h before i.p. administration of 15--20 mg/kg of LPS (from E. coli055:B5; Sigma Chemical Co., St. Louis, MO). LPS inoculum was delayed to 24 h after bacteria administration to avoid interference of pre-inoculum. In fact, preliminary experiments showed that intra-peritoneal inoculum of PBS decreased significantly LPS toxicity when administered at shorter times before LPS (data not shown). Mice were observed for 7 days after LPS administration and deaths recorded. Statistical analysis -------------------- Results are presented as mean ± SEM. Statistical significance was assesed by two-tailed Student\'s ttest. Comparison of survival curves was performed by the χ^2^test. Calculation of percentiles was performed by survival analysis. All calculations were performed with the Stratgraphics Plus 5 programme (Manugistics, Inc., Rockville, MD). List of abbreviations ===================== IL, interleukin; IL-1, interleukin-1; IL-1Ra, interleukin-1 receptor antagonist; IBD, inflammatory bowel disease; LPS, bacterial lipopolysaccharide, AUC, area under the curve; PMN, polymorphonuclear leukocytes; GRAS, generally regarded as safe; GMO, genetically modified organisms. Authors\' contributions ======================= SP carried out the in vivoand pharmacokinetics studies in rats and rabbits, and performed the statistical analysis. PB designed and performed the bioactivity studies. PR designed and performed the microbiological and biochemical work. DB coordinated the bioactivity studies, organised the data, and wrote the manuscript. AT designed and coordinated the entire study. All authors read and approved the final manuscript. Acknowledgments =============== This work was supported by the Commission of the European Union (contract no. QLK4-2001-00147), a research grant from AIRC (Associazione Italiana Ricerca sul Cancro), Milano, Italy, and the FIRB project \"NIRAM\" of the Italian MIUR. The authors are particularly indebted to Giovanni Maurizi (Consorzio Biolaq, L\'Aquila, Italy) for his seminal contribution to this work. The support of Cinzia D\'Ettorre (Dompé SpA, L\'Aquila, Italy) for BIAcore analysis is gratefully acknowledged. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Release of IL-1Ra from recombinant B. subtilis. A. Schematic representation of the life cycle of B. subtilisshowing the mechanism of recombinant IL-1Ra release upon sporulation (autolysis with spore formation, as in Spo^+^strain), and destructive autolysis (with cell lysis without spore formation, as in Spo^-^strain). B. Presence of recombinant IL-1Ra in sporulation supernatant of pSM539 Spo^+^strain, assessed by ELISA (mean ± SEM). IL-1Ra amount in sporulation supernatants was also quantitatively evaluated by laser scanning densitometry. The amount of IL-1Ra released in vitroby 0.5--0.7 × 10^9^bacteria (0.25 g wet weight) 3 h after the sporulation signal was 1.152 mg in 9.6 mg total released proteins (i.e., 12% of the total protein in the sporulation supernatant). Overlapping results were obtained with Spo^-^pSM539 cells (data not shown). C. SDS-PAGE analysis of wild-type B. subtilis(lane 1) and of recombinant B. subtilispSM539 Spo^-^strain expressing IL-1Ra (lane 2). Asterisk indicates the migration position of human mature IL-1Ra. The protein identity was confirmed in Western blotting. Identical results were obtained with the Spo^+^strain (data not shown). ::: ![](1472-6750-4-27-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Presence of intact IL-1Ra in serum after intra-colonic administration of IL-1Ra-producing B. subtilis. Left: Human IL-1Ra in colon washings and in serum of rats receiving a single intra-colonic instillation of 3 × 10^8^live cells of B. subtilispSM214 (β-lactamase control) or pSM539 (producing IL-1Ra). The presence of IL-1Ra was assessed by Western blotting on samples of colon washings (upper panel) and serum (lower panel) taken at different time points. Samples at time 0 were from animals receiving pSM539 immediately before sampling. Results at time 0 from rats treated with pSM214 and untreated rats were also negative (data not shown). An aliquot of standard human recombinant IL-1Ra (7--14 ng) was included as reference in each gel. Right: Presence of human IL-1Ra in serum of rabbits receiving an intra-colonic instillation of 2 × 10^9^live cells of B. subtilispSM539 (producing IL-1Ra; ●) or pSM214 (β-lactamase control;○). IL-1Ra concentration was assessed by ELISA. Data are the mean ± SEM of values from 3 rabbits/group in one representative experiment. Overlapping results were obtained with sera of rats after intra-colonic administration of recombinant B. subtilis, and by BIAcore analysis of human IL-1Ra presence in rat and rabbit serum samples (data not shown). Statistical significance: pSM539 vs. pSM214 p \< 0.01 at every time. ::: ![](1472-6750-4-27-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Serum IL-1Ra from intra-colonic B. subtilisinhibits IL-1-induced thymocyte proliferation in vitro. Co-stimulation of murine thymocyte proliferation by IL-1β was assessed in cultures of C3H/HeJ thymocytes stimulated with a suboptimal concentration of PHA (1.5 μg/ml) in the absence (zero control; empty column) or in the presence of IL-1β (solid column). Left panel: Serum samples were taken from rabbits administered intra-colonically 3 h earlier with B. subtilispSM539 (producing IL-1Ra; ●) or pSM214 (β-lactamase control; ○), and assayed by ELISA for the presence of IL-1Ra (1.99 μg/ml in the pSM539 serum; 0.00 μg/ml in the pSM214 serum). Inhibition of IL-1β activity (30 pg/ml) was assessed as decrease of thymocyte proliferation in the presence of different dilutions of the pSM539 serum (1:200, 1:2,000, 1:20,000; corresponding to 10, 1, and 0.1 ng/ml IL-1Ra) and compared to the same concentrations of human recombinant IL-1Ra (■). Right panel: Thymocyte proliferation to IL-1β (300 pg/ml) was evaluated in the presence of rabbit serum taken 6 h after intra-colon administration of pSM539 (●) or serum from the same rabbit taken before treatment (□). Serum from pSM539-treated rabbit contained 1.35 μg IL-1Ra/ml (tested by ELISA). Serum was added at dilutions (1:450, 1:1,350, 1:4,050) containing 3, 1, and 0.3 ng/ml IL-1Ra, either alone (●) or in the presence of a polyclonal antibody against human IL-1Ra (diluted 1:300; ◇). Untreated rabbit serum (negative for IL-1Ra in ELISA) was used as control at the same dilutions as pSM539 serum. Results are presented as mean ± SEM of triplicate cultures within single representative experiments. Statistical significance: p \< 0.01 pSM539 serum (at 1, 3, 10 ng IL-1Ra/ml) vs. IL-1β alone and corresponding serum controls (pSM214, untreated, + anti-IL-1Ra). ::: ![](1472-6750-4-27-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Inhibition of IL-1 systemic effects in vivoby intra-colonic administration of IL-1Ra-producing B. subtilis. Upper left: Increase in body temperature in rabbits treated with human recombinant IL-1β (75 ng/kg i.v.) 1 h after a intra-colonic instillation of 2 × 10^9^live cells of B. subtilispSM539 (producing IL-1Ra; ●) or pSM214 (β-lactamase control; ○). Data are the mean ± SEM of values from 3 rabbits/group. Difference was statistically significant with p \< 0.02 at 40 and 60 min. Upper right: Granulocytosis induced by human recombinant IL-1β in rats. Animals were administered intra-colonically with 1 ml/kg saline (![](1472-6750-4-27-i1.gif)) or with the same volume of saline containing 1 × 10^9^live cells/kg of pSM214 (□) or pSM539 (■). After 2 h animals received 100 ng/kg human recombinant IL-1β i.p. A control group received saline i.p. (![](1472-6750-4-27-i2.gif)) instead of IL-1β. All animals were bled 2 h later, and the number of circulating PMN was evaluated cytofluorimetrically. PMN numbers are expressed as percent blood leukocytes and reported as the mean ± SEM of values in 3 rats/group. Lower left: Hypoferremia induced by human recombinant IL-1β in rabbits. Animals received two intra-colonic instillations of 1 ml/kg saline alone (![](1472-6750-4-27-i1.gif)) or containing 2 × 10^9^live cells of B. subtilispSM539 (producing IL-1Ra; ■) or pSM214 (β-lactamase control; □), 3 h before and 10 min after administration of IL-1β (100 ng/kg i.p.). Control rabbits administered intra-colonically with saline received an i.p. inoculum of saline instead of IL-1β (![](1472-6750-4-27-i2.gif)). Serum iron levels were measured 8 h later. Data are the mean ± SEM of values of 3--25 rabbits, tested in five separate experiments and representative of results obtained at different times after IL-1β inoculum (4--24 h). Lower right: Hypoglycemia induced by human recombinant IL-1β in rats. Animals were administered intra-colonically with 1 ml/kg saline (![](1472-6750-4-27-i1.gif)) or with the same volume of saline containing 1 × 10^9^live cells/kg of pSM214 (□) or pSM539 (■). After 2 h animals received 100 ng/kg human recombinant IL-1β i.p. A control group received saline i.p. (![](1472-6750-4-27-i2.gif)) instead of IL-1β. All animals were bled 2 h later, and serum samples were assayed for glucose concentration. Data are reported as the mean ± SEM of values in 3 rats/group. ::: ![](1472-6750-4-27-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### IL-1Ra-producing B. subtilisprotects mice from endotoxic shock. LPS-sensitive C3H/HeOuJ mice (29--52 mice/group tested in four separate experiments) received 3 × 10^6^pSM539 bacteria (engineered with human mature IL-1Ra; ●) i.p. 24 h before administration of LPS (15--20 mg/kg). Mice were observed for 96 h and deaths recorded. Control mice received either the B. subtilisstrain pSM214 (β-lactamase control;) or PBS (×) 24 h before LPS administration. Statistical analysis of survival curves (χ^2^) indicated a significant increase in survival of mice receiving pSM539 as compared to controls receiving PBS or receiving pSM214. At 16 h difference was statistically significant at p \< 0.05 vs. pSM214 but not significant vs. PBS (+). From 24 to 40 h, difference was statistically significant only vs. pSM214 at p \< 0.01 (\#). From 44 h on, difference was statistically significant vs. both controls at p \< 0.01 (\). Comparison of survival percentiles showed a statistically significant difference (p \< 0.01) between pSM539 and control groups pSM214 and PBS at 75% and 50% percentiles. Survival of control mice receiving pSM214 was never statistically different from that of mice receiving PBS (p \> 0.2). ::: ![](1472-6750-4-27-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Intra-colonic administration of IL-1β-producing B. subtilisprovokes fever and shock in rabbits. Increase in body temperature in rabbits receiving a single intra-colonic instillation of 1 × 10^9^live cells of B. subtilispSM261 (producing IL-1β; ●) or pSM214 (β-lactamase control; ○). As positive control, rabbits received a single intra-colonic administration of 250 μg purified human recombinant IL-1β (■). Data are the mean ± SEM of values of 5 rabbits within a single representative experiment. In the group treated with IL-1β-producing pSM261 bacteria, 2/5 animals died within 30 h of hypotensive shock. From 0.5 to 3 h after treatment, body temperature of pSM216-treated rabbits was significantly higher than that of animals receiving control pSM214 (§p \< 0.05;,\ p \< 0.01). ::: ![](1472-6750-4-27-6) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Local and systemic IL-1Ra after administration of IL-1Ra-producing B. subtilis ::: *Delivery of B. subtilispSM539* Detection of IL-1Ra -------- ------------------------------------- ------------------------- ------------------ -------------------------- Mouse peritoneal cavity 0 h \- \- 3 h ++ \+ 6 h \+ \- 24 h \- \- Rat small intestine 0 h \- \- 3 h ++ \- 4 h \+ (B: 149.4 μg) \- 6 h +/- \- 8 h +/- (B: 16.5 μg) \- 24 h \- n.t. large intestine 0 h \- \- 2 h n.t. ++ (B: 0.6--1.3 μg/ml) 3 h +++ n.t. 4 h +++ ++ (B: 0.5--1.9 μg/ml) 6 h ++ \+ 8 h ++ \+ (B: 0.1--0.4 μg/ml) 24 h \+ n.t. Rabbit large intestine 0 h \- \- (B/E: 0 μg/ml) 0.5 h n.t. \+ (B: 0.2--0.4 μg/ml) 1 h n.t. ++ (B/E: 0.2--1.2 μg/ml) 2 h n.t. ++ (B/E: 0.6--1.6 μg/ml) 3 h n.t. ++ (B: 0.6--2.0 μg/ml) 4 h ++ ++ (B/E: 0.4--2.1 μg/ml) 6 h n.t. ++ (B/E: 0.3--0.9 μg/ml) 8 h ++ ++ (E: 0.6--2.4 μg/ml) 24 h n.t. \+ (B: 0.3 μg/ml) Mice, rats and rabbits were administered live cells of B. subtilisstrain pSM539 (engineered to produce human mature IL-1Ra; 1 × 10^8^-2 × 10^9^cells/kg) into the peritoneal cavity, the small intestine, or the large intestine. Animals were sacrificed at different time points after bacterial inoculum, and samples of serum and intestinal washings were taken. The presence of human IL-1Ra was assessed in all samples by Western blotting. Semi-quantitative analysis was performed after laser scanning densitometry in comparison to different amounts of standard human recombinant IL-1Ra, and scored as follows: -, no detection; +/- very faint and/or inconsistent detection; + consistently positive; ++ abundant detection; +++ very high levels. Western blotting analysis revealed that IL-1Ra recovered from serum and intestinal washings had the same molecular mass as the standard IL-1Ra. No proteolytic fragments or larger aggregates could be detected. For some samples, quantitative assessment of IL-1Ra was performed by ELISA (E) or by BIAcore (B) analysis. Assessed animals were 2--16/group/time. n.t., not tested. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Pharmacokinetic analysis of IL-1Ra in rabbit serum after administration of IL-1Ra-producing B. subtilis ::: Intra-colon instillation IL-1Ra (mg/kg) C~max~(μg/ml) T~max~(min) AUC (0--6 h)/dose (g × h)/ml ------------------------------ -------------------- ------------------- ----------------- ---------------------------------- IL-1Ra 1.00 0.482 60 1.34 IL-1Ra + pSM241 1.00 0.372 60 1.05 pSM539 0.44 0.136 200 1.10 Groups of four rabbits received an intra-colonic instillation of highly purified human recombinant IL-1Ra (1.0 mg/kg) alone or admixed with control pSM214 bacteria (4 × 10^8^cells/kg), or of IL-1Ra-producing pSM539 bacteria (4 × 10^8^cells/kg). Serum samples were taken just before treatment (time zero) and after 1, 2, 4, and 6 h from intra-colonic instillation. The presence of human IL-1Ra in serum samples was determined by ELISA. :::	PubMed Central	2024-06-05T03:55:49.901535	2004-10-30	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534112/", "journal": "BMC Biotechnol. 2004 Oct 30; 4:27", "authors": [ { "first": "Stefano", "last": "Porzio" }, { "first": "Paola", "last": "Bossù" }, { "first": "Paolo", "last": "Ruggiero" }, { "first": "Diana", "last": "Boraschi" }, { "first": "Aldo", "last": "Tagliabue" } ] }
PMC534113	Background ========== The impact of infertility on the psychological well being of couples involved has been the object of increasing attention in recent years. It cannot be denied that infertility is a deeply distressing experience for many couples \[[@B1]\]. In latter part of twentieth century, psychogenic cause was an accepted topic in infertility until when diagnostic abilities improved \[[@B2]\]. Edelmann et al (1985) found that infertility has a significant effect on psychological factors. Some authors have paid attention to the fact that health problems, loss of self-esteem, feeling akin to mourning, threat, sexual distress, depression, guilt, anxiety, frustration, emotional distress and marital problems are all associated with infertility \[[@B3]\]. We aimed to examine prevalence and severity of anxiety/depression in relation to duration/cause of infertility in Iranian infertile women. Several studies have demonstrated anxiety has a detrimental effect on fertility \[[@B4]\] and that reduction of anxiety increases pregnancy rate \[[@B5],[@B6]\]. Other researches failed to support a relationship between anxiety and infertility \[[@B7]\] Lapane et al (1995) indicated that depression could play an important role in the pathogenesis of infertility \[[@B8],[@B9]\]. Infertility sometimes is accompanied by existential crises and emotional tensions such as anxiety, interpersonal problems, and suppressed anger, unsatisfactory in interpersonal, frustration, inferiority feeling, depression, rejected feeling and unconscious guilt feeling. Those couples with a history of failure in Assisted Reproductive Technique (ART) have shown personality maladjustment \[[@B10]\]. Overall percentage of psychological problem in infertile couples ranges between 25 and 60% \[[@B11]\]. One study has demonstrated that 74.6% patients reported changes in their mood \[[@B12]\]. Psychological difficulties of infertile patients are complex and influenced by a number of factors such as gender differences, cause and length of infertility. Freeman et al (1987) found that half of their sample of infertile couples described infertility as the most upsetting experience of their lives, whereas 80% of the sample reported by Mahlstedtet et al (1987) described their experience of infertility to be either stressful or very stressful \[[@B1]\]. Duration of infertility increases of stress \[[@B13]\]. Depression and anxiety were improved in infertile women as their age and duration of infertility increased \[[@B14]\]. Long lasting infertility and unsuccessful treatment cycles intensifies stress and psychopathologic problems especially depression \[[@B15],[@B16]\]. Although, some studies showed that there is no relation between duration of infertility and depression or psychological factors \[[@B17]\]. Another study showed those who had 2--3 years infertility had more depression / anxiety than those who had this problem for a year or more than 6 years. Peak of depression could be seen during third year of infertility. After six years there will be a reduction in psychological symptoms in women. During first three years, infertility is accompanied by signs such as anxiety, depression, loss of self-esteem, impotence and maladjustment of marital status. After 3 years, optimistic attitude would change to despair and at last there will be some emotional changes to adopt a child or live without one, thereafter. Those who have social support, positive personal characteristics, and have a satisfactory life with their spouse show no signs of anxiety/depression \[[@B18]\]. Since most literature on psychological aspects of infertility is from developed countries it was thought that a study from a developing country with a different culture might contribute to existing knowledge on the topic. We aimed to examine prevalence and severity of anxiety/depression in relation to duration of infertility in Iranian infertile women. The results of this study, which included counseling and couple-therapy, are being prepared for infertile couples. Methods ======= The subjects were 370 infertile women who were referred to Vali-e-Asr Reproductive Health-Research Center between January 2001 and January 2002 for treatment of their infertility problems. In this group mean (± SD) age was 28 (± 5.37) and mean (± SD) duration of infertility was 6.36 (± 4.18). Among them 293 (79.2%) women were housewives and the others were working outside. A gynecologist evaluated patients and then they were visited by a psychologist and were informed of the study purposes. Diagnosis of male factor is based on WHO criteria 1999. Menstrual history and mid-luteal progesterone level considers ovulatory factor, diagnosis of endometriosis is done by lapascopy, tubal factor is diagnosed by HSG and cervical facto is detected in Post Coital Test. After obtaining oral consent from each patient, data were collected using BDI \[[@B19],[@B20]\], and Cattle \[[@B21]\]. inventories: Beck Depression Inventory (BDI) ------------------------------- The test used was a translated and validated Persian version of Beck\'s depression Inventory. A full 21-items BDI was administered. This scale is a widely used measure for intensity of depression. Each item describes a specific behavioral manifestation of depression. Scores on each item can range from 0, indicating no depressive symptomatology, to 3, indicating a severe level of symptomatology. Total scale scores can thus range from 0 to 63. Scores of 17 or above it indicates of a clinically significant depression. The classification of depression scores involves: 1\. 0--16 (without depression) 2\. 17--27 (mild depression) 3\. 28--34 (moderate depression) 4\. 35--63 (severe depression) Cattle Inventory ---------------- The Cattle inventory is a 40 items self-report measure of anxiety. This test was a translated and validated Iranian version of Cattle\'s Inventory. Scores can range from 0 to 80, with scores of 28 or above demonstrate anxiety. Classification of anxiety scores involves: 1\. 0--27 (without anxiety) 2\. 28--40 (moderate anxiety) 3\. 41--49 (neurotic anxiety) 4\. 50--80 (severe anxiety) For each patient the following data were recorded: age, cause and duration of infertility, education and job. Data were analyzed by using statistical SPSS. The relationship between continuous and binary explanatory variables with Beck and Cattle scores were assessed using spearman\'s rho and unpaired t-test, respectively. In addition, the relationship between categorical responses and explanatory variables were evaluated using chi-square test. For descriptive purposes, we presented frequency tables. Results ======= Three hundred seventy infertile women were considered in this cross-sectional study. The results of Beck and Cattle inventories showed that 40.8% and 86.8% of these women had depression and anxiety symptoms, respectively (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Prevalence of depression and anxiety ::: Depression (Beck) Frequency Percent Anxiety (Cattle) Frequency Percent ------------------- ----------- --------- ------------------ ----------- --------- Normal 219 59.2% Normal 49 13.2% Mild 96 25.9% Moderate 141 38.1% Moderate 37 10% Neurotic 117 31.6% Severe 18 4.9% Severe 63 17% Total 370 100% Total 370 100% ::: Mean scores of depression and anxiety by age groups are shown in figure [1](#F1){ref-type="fig"}. Depression and anxiety were more severe in 21--25 years and under 20 years respectively, although there was no significant difference between age groups. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Mean score of depression/anxiety by age groups ::: ![](1472-6874-4-9-1) ::: In next step, we compared the prevalence of depression (depression group is consisted of mild, moderate and severe depressive women) in all categories of infertility causes. Chi^2^statistic for this 2 × 9 table (chi-square = 20.643 P = 0.014) showed that prevalence of depression is not equal in these categories. Same analysis for the prevalence of anxiety (anxiety group is consisted of moderate, neurotic and severe anxietic women) in different groups of infertility causes showed no significant difference between them (chi-square = 7.491 P = 0.485) (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Frequency of depression and anxiety by cause of infertility ::: Causes of Infertility Percent of Depression Percent of Anxiety ------------------------------ --------------------------- ------------------------ Oligo-astheno-terato spermia 24.6% 86.2% Azospermia 31.6% 80.7% Ovulatory 48.0% 85.7% Endometriosis 20.0% 80.0% Uterus 52.2% 82.6% Tubal 50.0% 90.5% Habitual abortion 33.3% 100% Unexplained 56.5% 95.7% Male & Female (Both) 49.0% 93.9% P-value 0.014 0.485 chi-square = 20.643 ::: The correlation between Beck and Cattle scores based on Spearman\'s Rho was 0.707 (P \< 0.001), which shows a significant relation between depression and/or anxiety scores. Then, we checked correlation coefficient between Beck and Cattle scores with quantitative variables like age, education (in years) and especially the duration of infertility. Duration of infertility showed a significant relation with both Beck (r = 0.15, P = 0.004) and Cattle (r = 0.157, P = 0.002) scores. Based on duration of infertility 31(29.2%), 51 (42.1%), 30 (46.8%) and 39 (49.3%) patients had depression in different groups, but there was no significant relationship between duration of infertility and depression (P-value = 0.106) (Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Frequency and rate of depression and anxiety based on infertility duration. ::: 1--3 years 4--6 years 7--9 years \>10 years All ---------------------------------- -------------- ---------------- ---------------- ---------------- ---------------- --------- ------ ---- ------ ----- ------ Depression stage (P = 0.106) Normal 75 70 70 57.8 34 53.8 40 50.6 219 59.2 Mild 22 20.5 34 28.1 16 25 24 30.3 96 25. Moderate 5 4.6 12 9.9 8 12.5 12 15.1 37 10. Severe 4 3.7 5 4.1 6 9.3 3 3.7 18 4.8 Anxiety Stage (P = 0.048) Normal 24 22.6 11 9 6 9.3 8 10.1 49 13.3 Moderate 41 38.6 51 42.1 24 37.5 25 31.6 141 38.1 Neurotic 29 27.3 36 29.7 24 37.5 28 35.4 117 31.6 Severe 12 11.3 23 19 10 15.6 18 22.7 63 16.9 ::: Also 82 (77.3%), 110 (90.9%), 58 (90.6%) and 71 (89.8%) of patients had different stages of anxiety and there was a significant relation between anxiety and duration of infertility (P-value = 0.048) (Table [3](#T3){ref-type="table"}). Educational level had a significant and negative relation with these two scores, but age showed no significant effect on depression and/ or anxiety (Table [4](#T4){ref-type="table"}). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Correlation between Beck and/or Cattle scores with duration of infertility, women\'s age and women\'s education ::: Beck scores Cattle scores ----------------------------------- ----------------- ------------------- -------- ------- Duration of Infertility 0.150 0.004 0.157 0.002 Women\'s Age -0.052 0.315 -0.044 0.395 Women\'s Education (in years) -0.319 \< 0.001 -0.156 0.003 ::: Finally, we tested the relation between depression and/or anxiety scores with women\'s job. Results of unpaired t-test showed significant difference between depression scores of housewives and employees (t = 9.179, P = 0.003). Anxiety and depression were observed more in homemakers comparing to outside employees though we found no significant effect of women\'s job on Cattle scores (t = 2.943, P = 0.087) (Table [5](#T5){ref-type="table"}). ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Relation between depression and/or anxiety scores and women\'s job ::: Women\'s job Mean SD P-value ------------ ------------------ ---------- --------- ------------- Beck Housewife 16.3549 10.2261 \0.003 Employee* 12.4286 9.7014 Cattle Housewife 6.3857 2.084 \\0.087 Employee 5.9091 2.4878 \t = 9.179 \\*t = 2.943 ::: Discussion ========== Patients participating in this study were from different geographical areas in Iran. The finding of this study provides information about frequency and severity of anxiety/depression in order to duration of infertility in childless women. The prevalence of psychiatric morbidity specially depression and/or anxiety in infertile patients have been assessed in several countries, for example Jones et al (1993) found that, there was mild to moderate depression in 28.3% of infertile women, moderate to severe depression in 7.2% and 1.2% had most severe depression based on BDI \[[@B7]\]. Another study showed that 67% of infertile women suffered from anxiety \[[@B1]\] and the same studied by Oddens et al (1999) reported that 24.9% had depressive disorders \[[@B22]\]. Anxiety were investigated in 130 infertile women in China, the results showed that different levels of mental pressure were found in 83.8% of infertile women, and moderate or severe types in 25% \[[@B23]\]. There was depression and/or anxiety disorder in 33% (Hong Kong), in 32 % (Scotland) of infertile women \[[@B16],[@B22]\]. The overall percentage of depression disorder in infertile women ranges between 24 and 36% and also anxiety disorder ranges between 67 and 84%. Our study showed 40.8% depression and 86.8% anxiety in infertile women. Consistent with our research, Iranian infertile women show higher rates of anxiety and/or depression than the other countries. In Islamic and eastern countries such as Iran, family status especially childbearing is very important and valuable. Having a child stabilizes family and increases marital satisfaction. In our culture and society, negative attitudes to infertility are so throbbing. Having a child is psychologically or effectively, a vital factor for women, and the absence of children may cause marital problems such as divorce or even second marriage especially in Islamic societies which it is possible for men to marry with more than one woman. Intervention of relatives especially husband\'s family, negative attitude and behavior of surroundings (family, friends, neighbors, etc.) causes psychological problems for infertile women. Generally infertile women experience negative social consequences including marital instability, stigmatization and abuse. Infertility can have a serious effect on both psychological well being and social status of women in our country. The most common age for depression and/or anxiety in our study was 21--25 years. In this study, anxiety and/or depression had negative correlation with education. In other words, with the raise of education level, anxiety and/or depression decrease. Results of different studies about relationship of age and education with anxiety and/or depression were not similar. Age and education level have no significant relationship with depression and/or anxiety \[[@B24]\]. Another study showed that there was positive correlation between them \[[@B18]\]. In such closed societies as some parts of our country, education and job may be the lone gate leading women to joyful aspects of their life other than maternity. This is why education plays a considerable role in decreasing their depression/anxiety. Having a job may reduce stress from In Vitro fertilization (IVF) \[[@B25]\]. In our study, anxiety and/or depression were observed more in housewives (vs. outside employees). It seems being at work outside home decreases psychological signs of anxiety and depression. Based on previous researches \[[@B26]-[@B28]\], infertile women showed higher rates of psychiatric symptoms than their partners, especially in female and unexplained factors. Women are necessarily more deeply involved in treatment procedures and it is normal for them to be more affected. One of the characteristics of infertile couples is that women are habitually more affected by the situation of infertility than men \[[@B29]\]. Based on our study depression is more common in \"unexplained cause\" group comparing to other causes and anxiety is more common in \"endometriosis\" group comparing to other causes, our results is similar to other studies \[[@B2],[@B3],[@B6],[@B9],[@B13]\]. Anxiety and/or depression increases with duration of infertility \[[@B30]\]. A study demonstrated that women who had experienced infertility for a long or medium range of time presented a significantly lower state of anxiety \[[@B31]\] and there was a trend of decreasing psychological stress with lengthening of infertility time. Based on depression scales, infertile patients who had infertility for an intermediate to a long time showed less symptoms than those who are in their first stage of their problem \[[@B32]\] but other studies showed that psychological distress in infertile women increase with time \[[@B15]\] and depression peaks between the second and third year of infertility and does not return to normal range until after 6 years of infertility \[[@B18]\]. In our study, women with lower stages of depression and anxiety can be seen during 1--3 years of infertility, but during 4 through 6 years after infertility diagnosis their signs become more prominent, especially severe depression has the most common frequency during 7--9 years. It was shown that the first three years (1--3) anxiety and/or depression is in its lowest limit and after 4 to 9 years it becomes worse. It seems that our results are completely different comparing to other countries. It may show that having a child is very important for our people, especially our women, therefore women show higher and longer emotional reactions and psychiatric symptoms lasts longer in comparison to other countries. In conclusion it can be suggested that psychological interventions especially in 4--9 years of infertility may prevent the surge in depression/anxiety and could presumably lead to increased pregnancy rates. Authors\' contribution ====================== FM contributed to design and management of the study. NA contributed to study design and data gathering. The initial idea of this study was by MMA. NK contributed in design, writing of the manuscript and analysis of this research. FZ contributed in design, writing of the manuscript and analysis of this research. MS contributed in edition and revision of the article. MJ contributed in edition and revision of the article. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6874/4/9/prepub> Acknowledgement =============== Here by we appreciate all our colleagues in Vali-e Asr reproductive Health Research Center specially Mrs. Masoumi and Miss Bagheri for their aids in data collection.	PubMed Central	2024-06-05T03:55:49.906322	2004-11-6	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534113/", "journal": "BMC Womens Health. 2004 Nov 6; 4:9", "authors": [ { "first": "Fatemeh", "last": "Ramezanzadeh" }, { "first": "Malek Mansour", "last": "Aghssa" }, { "first": "Nasrin", "last": "Abedinia" }, { "first": "Farid", "last": "Zayeri" }, { "first": "Navid", "last": "Khanafshar" }, { "first": "Mamak", "last": "Shariat" }, { "first": "Mina", "last": "Jafarabadi" } ] }
PMC534114	Background ========== Ligand stimulation of the epidermal growth factor receptor (EGFR) and the three other members of the EGFR family of receptor tyrosine kinases (ErbB2, ErbB3 and ErbB4) results in tyrosine autophosphorylation, recruitment of signaling proteins, and activation of distinct complement of signaling pathways that regulate a great variety of cellular responses \[[@B1],[@B2]\]. One of the signaling pathways that is activated by the EGFR is the phosphatidylinositol-3 kinase (PI-3 kinase)/Akt anti-apoptic signaling pathway \[[@B3]\]. The PI-3 kinase holoenzyme consists of a regulatory subunit (p85) and a catalytic p110 subunit. The regulatory subunit contains two SH2 domains that bind specifically to pYXXM motifs in a variety of cellular proteins, including receptor tyrosine kinases such as the PDGF (platelet-derived growth factor) receptor, and c-kit and docking proteins such as IRS (insulin receptor substrate) 1, IRS2 and Gab1. Although the cytoplasmic domain of the EGFR does not contain any canonical p85 binding motifs, EGF stimulation leads to PI-3 kinase activation by an indirect mechanism. It has been proposed that the PI-3 kinase is activated in response to EGF stimulation as a consequence of formation of EGFR/ErbB3 heterodimers \[[@B4]\]. Unlike EGFR, the cytoplasmic region of ErbB3 contains at least six pYXXM motifs \[[@B5],[@B6]\]. Indeed, EGF stimulation of cells co-expressing the EGFR and ErbB3 results in recruitment and activation of PI-3 kinase by tyrosine phosphorylated ErbB3 \[[@B4]\]. EGF stimulation of PI-3 kinase may also be mediated by the docking protein Gab1 (Grb2-associated binder-1). EGF stimulation leads to tyrosine phosphorylation of Gab1 enabling recruitment and activation of PI-3 kinase by the three canonical pYXXM motifs on Gab1 \[[@B7]\]. Gab1 was originally identified as a Grb2 binding protein, and was shown to be tyrosine phosphorylated in response to treatment with a variety of growth factors \[[@B7]-[@B9]\]. Gab1 contains a number of tyrosine residues that could serve as potential binding sites for the SH2 domain containing proteins Grb2, PI-3 kinase, and the protein tyrosine phosphatase Shp2 \[[@B10]\]. While there have been reports that Gab1 binds directly to the EGFR via an 83-amino acid stretch termed the Met-binding-domain or MBD \[[@B9]\], the majority of Gab1 is believed to be indirectly associated with the EGFR via the adaptor protein Grb2, which binds to a proline rich region on Gab1 via its C-terminal SH3 domain \[[@B10]-[@B13]\]. Cells over-expressing a mutant Gab1 protein containing tyrosine to phenylalanine mutations at the three p85 binding sites have been shown to be defective in EGF-induced JNK activation, and treatment of cells over-expressing wild type Gab1 with PI-3 kinase inhibitors interferes with MAPK signaling in response to EGF treatment, thus revealing a link between Gab1 and PI-3 kinase in EGF-induced mitogenic signaling \[[@B9],[@B14]\]. Furthermore, the PI-3 kinase product phosphatidylinositol (3,4,5) triphosphate (PIP3) has been shown to bind to the pleckstrin homology (PH) domain of Gab1 resulting in membrane-association of Gab1, suggesting a positive feedback loop in which PI-3 kinase acts as both an upstream regulator and a downstream effector of Gab1 signaling via the EGFR \[[@B9]\]. Gab1 thus acts as a docking protein facilitating the recruitment of a multi-protein signaling complex including the EGFR, p85 and Shp2 in response to EGF treatment. Although the role of the Shp2 protein in the control of EGFR/Gab1 interactions is not well understood, several studies have suggested that Gab1-associated Shp2 may influence EGF-induced PI-3 kinase signaling. Previous work has shown that Gab1 is not a global substrate of Shp2, as complex formation between Gab1 and Shp2 does not reduce the total EGF-induced tyrosine phosphorylation levels of Gab1 \[[@B15]\]. However there have been several reports suggesting that Shp2 may specifically de-phosphorylate the tyrosine phosphorylation sites on Gab1 that bind to p85, thus terminating recruitment of PI-3 kinase and EGF-induced activation of the PI-3 kinase pathway \[[@B16]-[@B18]\]. It has been shown that cells devoid of Shp2 show an increase in PI-3 kinase activity, as well as elevated and sustained levels of Akt activation in response to EGF treatment \[[@B18]\]. It was reported that treatment of cells with PI-3 kinase inhibitors or with the phosphatidylinositol (3,4,5) triphosphate (PIP3) phosphatase PTEN interferes with the association between Gab1 and Shp2 in response to EGF treatment, suggesting PI-3 kinase may be required for Shp2 recruitment of Gab1 following EGF stimulation \[[@B14]\]. However, the mechanism for this postulated recruitment is unknown. In the experiments presented here we utilized fibroblasts isolated from Gab1-/- mouse embryos in order to examine the role of Gab1 in EGF-mediated activation of the PI-3 kinase/Akt cell survival pathway. We also address the question of whether ErbB3 recruitment of PI-3 kinase is dependent on or independent of Gab1. Our results demonstrate a clear requirement for Gab1 in recruitment and activation of PI-3 kinase in response to EGF stimulation. Additionally, we show that while Shp2 does not mediate global dephosphorylation of Gab1, it does appear to negatively regulate the EGF-induced activation of PI-3 kinase through an undefined mechanism. Finally we demonstrate that ErbB3 is capable of recruiting PI-3 kinase in the absence of Gab1, but Gab1 functions as the major mediator of PI-3 kinase activation in response to EGF stimulation. Results and Discussion ====================== Previous studies have suggested that ErbB3 and Gab1 can function as links between EGFR and PI-3 kinase. In this report we use MEFs derived from Gab1 -/- embryos \[[@B19]\] to explore the contribution of Gab1 and ErbB3 to EGF stimulation of PI-3 kinase and Akt in these cells. Gab1 is essential for EGF stimulation of PI-3 kinase and Akt ------------------------------------------------------------ MEFs derived from Gab1-/- or wild type (WT) embryos were stimulated with EGF and assayed for Gab1 tyrosine phosphorylation, for activation of PI-3 kinase and for Akt stimulation. As shown in Figure [1A](#F1){ref-type="fig"}, the endogenous Gab1 present in WT MEFs is tyrosine phosphorylated in response to EGF treatment. As shown in Figure [1B](#F1){ref-type="fig"}, Gab1 -/- MEFs displayed very low levels of EGF-induced PI-3 kinase activity relative to cells expressing Gab1. We did observe an approximate 2-fold increase in this low-level basal PI-3 kinase activity in Gab1 -/- MEFs, which represents a Gab1-independent signaling pathway. Gab1 -/- and WT MEFs were additionally stimulated with EGF and the activation of Akt was analyzed by immunoblotting with antibodies which recognize specifically the activated form of Akt. As shown in Figure [1C](#F1){ref-type="fig"} (top and middle panels), Gab1 -/- cells display no activation of Akt in response to EGF, while WT MEFs show EGF-stimulated Akt activation within two minutes of EGF treatment. The cDNA encoding the wild type murine Gab1 sequence was cloned into a retroviral vector, and the virus was used to infect Gab1 -/- MEFs. Stable cell lines were selected for co-transduction of a puromycin resistance gene and pools of selected cells were used for further analysis. As shown in Figure [1A](#F1){ref-type="fig"}, the ectopic Gab1 protein was expressed at slightly lower levels in the Gab1 -/- MEFs relative to endogenous Gab1 expression seen in the wild type MEFs. Quantitation by densitometry reveals Gab1 expression in wild type MEFs to be 1.4-fold higher than ectopic Gab1 expression in Gab1 -/- MEFs. Treatment with EGF induced tyrosine phosphorylation of the exogenous Gab1 protein expressed in the Gab1 -/- MEFs at levels similar to endogenous Gab1 in wild type MEFs. As shown in Figure [1B](#F1){ref-type="fig"}, expression of exogenous Gab1 in the Gab1 -/- MEFs results in Gab1-associated PI-3 kinase activity that is augmented following EGF treatment. The low level of EGF-induced PI-3 kinase activity observed in the Gab1 -/- cells may be due to signaling via an alternate, Gab1-independent mechanism. These cells were additionally treated with EGF over a period of time and the activation of Akt was assayed by immunoblotting with antibodies specific for the Ser473 phosphorylated form of Akt. The experiment presented in Fig [1C](#F1){ref-type="fig"} shows that ectopic expression of Gab1 in the Gab1 deficient cells rescues EGF-induced Akt activation to levels similar to those observed in EGF-treated wild type MEFs. Taken together, these results demonstrate that Gab1 is required for EGF-stimulation of PI-3 kinase and Akt. The canonical p85 binding sites on Gab1 are essential for PI-3 kinase and Akt activation in response to EGF stimulation ----------------------------------------------------------------------------------------------------------------------- The cDNA encoding a mutant Gab1 protein, containing tyrosine to phenylalanine point mutations at the three binding sites for the p85 regulatory subunit of PI-3 kinase (Y446F/Y472F/Y589F) (Gab1^F446/472/589^), was cloned into a retroviral vector and used to generate pools of stable MEF cell lines as described above. We first assayed the cells for Gab1 expression, and for the ability of the mutant Gab1^F446/472/589^protein to become tyrosine phosphorylated in response to EGF treatment. As shown in Figure [2A](#F2){ref-type="fig"}, both wild type Gab1 and Gab1^F446/472/589^undergo tyrosine phosphorylation in response to EGF treatment (upper left panel). Quantitation of multiple experiments by densitometry reproducibly demonstrates that Gab1^F446/472/589^is tyrosine phosphorylated following EGF treatment to similar levels when normalized for Gab1 expression levels. We next subjected lysates from unstimulated or EGF stimulated cells to immunoprecipitation with anti-Gab1 antibodies followed by immunoblotting with anti-p85 antibodies. As has been demonstrated previously \[[@B20]\], wild type Gab1 readily coimmunoprecipitated p85 following EGF treatment, while the Gab1^F446/472/589^mutant protein failed to show an association with p85, confirming that the Gab1^F446/472/589^protein is deficient in p85 binding. Similar levels of Gab1 expression in these cells were confirmed by reprobing the Gab1 phosphotyrosine blot with anti-Gab1 antibodies (Figure [2A](#F2){ref-type="fig"}, bottom left panel). Additionally, total cell lysates of all Gab1 expressing cell lines described in this study were subjected to anti-Gab1 immunoblotting, providing independent evidence for similar levels of Gab1 expression across all cell lines (Figure [2A](#F2){ref-type="fig"}, right panel). Because the substrates of Shp2 are for the most part unknown, we were additionally interested in examining the state of EGFR tyrosine phosphorylation following treatment with EGF in order to determine if the failure of Gab1 to bind p85, and potentially recruit Shp2, would influence levels of EGFR autophosphorylation. However, stimulation with EGF for varying time intervals revealed no significant differences in the levels of autophosphorylation of EGFR in cells expressing wild type Gab1 versus the Gab1^F446/472/589^mutant (Figure [2B](#F2){ref-type="fig"}). A linear representation of the EGF-induced EGFR tyrosine phosphorylation following normalization for EGFR expression levels is shown in Figure [2B](#F2){ref-type="fig"} (bottom). These results are consistent with our finding that p85 binding to Gab1 does not influence the recruitment of Shp2 to the Gab1 signaling complex, and are inconsistent with the conclusion that Gab1 mediates a PI-3 kinase-dependent recruitment of Shp2 \[[@B14]\]. We next explored the role of Gab1 in EGF-induced activation of the PI-3 kinase/Akt cell survival pathway utilizing the Gab1^F446/472/589^expressing cells. We first assayed the Gab1-associated PI-3 kinase activity directly through a PI-3 kinase assay. As shown in Figure [2C](#F2){ref-type="fig"}, immunoprecipitation of wild type Gab1 following EGF treatment brings down associated PI-3 kinase activity. However immunoprecipitation of Gab1^F446/472/589^is not associated with significant levels of PI-3 kinase in the presence or absence of EGF stimulation. In order to assay the effects of EGF stimulation on signaling downstream of PI-3 kinase, Gab1 -/- MEFs expressing no Gab1, wild type Gab1 or Gab1^F446/472/589^were treated with EGF over varying times and cell lysates were immunoblotted for serine-phosphorylated Akt. Mutation of the p85 binding sites on Gab1 essentially eliminated all EGF-induced Akt activation relative to cells expressing wild type Gab1 (Figure [2D](#F2){ref-type="fig"}). The binding of p85 is absolutely required for Gab1-mediated activation of PI-3 kinase and Akt following EGF treatment \[[@B9]\]. Previous work has indicated that treatment of cells with PI-3 kinase inhibitors reduces levels of EGF-induced complex formation between Gab1 and the protein tyrosine phosphatase Shp2 \[[@B14]\]. This finding suggests a role for Gab1 in the PI-3 kinase-dependent recruitment of Shp2 following EGF stimulation. We have examined the possibility that mutation of the p85 binding sites on Gab1, which prevents PI-3 kinase activation, altered EGF-induced recruitment of Shp2 as compared to the recruitment of Shp2 by wild type Gab1. For this purpose, lysates from unstimulated or EGF-stimulated cells were subjected to immunoprecipitation with anti-Gab1 antibodies following immunoblotting with anti-Shp2 antibodies. The experiment presented in Fig [2A](#F2){ref-type="fig"} shows that mutation of the p85 binding sites on Gab1 did not affect recruitment of Shp2 by Gab1 following EGF stimulation (Figure [2A](#F2){ref-type="fig"}, fourth panel from the top). As has been previously observed \[[@B20]\], we noted a low level of basal association between Shp2 and both Gab1 and Gab1^F446/472/589^, which may be due to incomplete growth factor starvation prior to EGF stimulation in these experiments. Expression of Gab1 mutant protein deficient in Shp2 binding rescues EGF-induced PI-3 kinase/Akt activation in Gab1 -/- MEFs --------------------------------------------------------------------------------------------------------------------------- To assess the role of the protein tyrosine phosphatase Shp2 in Gab1-mediated signaling induced by EGF, two Gab1 mutants were generated and expressed in pools of Gab1 -/- MEFs. The first contained tyrosine to phenylalanine point mutations at the two binding sites for the Shp2 protein tyrosine phosphatase (Y627F/Y659F) (designated Gab1^F627/659^) and the second contained mutations at the Shp2 binding sites as well as at the three PI-3 kinase binding sites described above (designated Gab1^F446/472/589/627/659^). We first assayed the ability of the mutant Gab1 proteins to become tyrosine phosphorylated in response to EGF. As shown in Figure [3A](#F3){ref-type="fig"} (top panel) both wild type Gab1 protein and the Gab1^F627/659^protein readily undergo tyrosine phosphorylation when stimulated with EGF. Quantitation following densitometry indicates that Gab1^F627/659^is reproducibly tyrosine phosphorylated to levels approximately 1.5-fold higher than Gab1. This result suggests that Gab1 may be a substrate of Shp2, and that blocking Shp2 binding thereby increases EGF-induced Gab1 tyrosine phosphorylation. The Gab1^F446/472/589/627/659^mutant reproducibly displayed lower levels of tyrosine phosphorylation following treatment with EGF suggesting that these five tyrosines are the main phosphorylation sites on Gab1. Immunoprecipitation of cell lysates with anti-Gab1 antibodies followed by immunoblotting with anti-Shp2 antibodies demonstrates that wild type Gab1 forms a complex with Shp2 following EGF treatment, while the Gab1^F627/659^mutant proteins fail to show an association with Shp2 thus confirming that phosphorylation of Tyr627 and 659 is required for Shp2 binding (Fig [3A](#F3){ref-type="fig"}). The basal interaction we observed between Gab1 and Shp2 in the absence of EGF stimulation (Figure [2A](#F2){ref-type="fig"}) is absent in the Gab1^F627/659^mutant, even following prolonged exposures of the western blot. We did not detect a change in the tyrosine phosphorylation of EGFR in cells expressing Gab1 proteins that are deficient in recruitment of Shp2. The experiment presented in Fig [3B](#F3){ref-type="fig"} shows cells stimulated with EGF over varying periods of time and cell extracts assayed for levels of EGFR tyrosine autophosphorylation. As has been previously reported \[[@B15],[@B18],[@B20]\], recruitment of Shp2 by Gab1 does not alter the magnitude or kinetics of tyrosine autophosphorylation of EGFR (Figure [3B](#F3){ref-type="fig"}, left panels). Levels of EGFR autophosphorylation are represented linearly following quantitation by densitometry and normalization for protein expression levels (Figure [3B](#F3){ref-type="fig"}, bottom). Previous work with Shp2 -/- cells demonstrated an elevated and sustained activation of PI-3 kinase and Akt in response to EGF treatment, and it was proposed that Shp2 may act to dephosphorylate Gab1 at one or both of the p85 binding sites \[[@B18]\]. We utilized the Gab1 proteins deficient in Shp2 binding to assay more directly the role of the Shp2-Gab1 complex in mediating activation of PI-3 kinase and Akt in response to EGF stimulation. As shown in Figure [3C](#F3){ref-type="fig"}, immunoprecipitation of Gab1^F627/659^brings down 1.6-fold higher basal levels of PI-3 kinase activity relative to wild type Gab1 as assayed by PIP3 production. Importantly, Gab1 mutants defective for Shp2 binding show approximately 2-fold higher Gab1-associated PI-3 kinase activity in response to EGF treatment. Consistent with these findings, previous studies have demonstrated that cells transiently over-expressing Gab1^F627/659^bound more p85 \[[@B18]\]. In both Gab1 and Gab1^F627/659^expressing cells the Gab1-associated PI-3 kinase activity is augmented by EGF treatment. As expected, the additional mutation of the p85 binding sites eliminates Gab1-associated PI-3 kinase activity. In order to assay the effects of EGF stimulation on signaling downstream of PI-3 kinase, cells were treated with EGF for varying periods of time and cell lysates were assayed for Akt activation by immunoblotting with P-Ser473 Akt antibodies. Interestingly, cells expressing the Gab1^F627/659^protein reproducibly showed activation of Akt with significantly sustained kinetics relative to cells expressing wild type Gab1 (Figure [3D](#F3){ref-type="fig"}, left panels). As expected, the additional mutation of the p85 binding sites (Gab1^F446/472/589/627/659^) limited Akt activation to levels similar to those observed in the Gab1 -/- cells, confirming the requirement for PI-3 kinase association with Gab1 to induce EGF-mediated activation of the Akt pathway. Taken together, these results suggest a role for Shp2 in negatively regulating the EGF induced activation of the PI-3 kinase pathway via Gab1, possibly by dephosphorylating Gab1 at p85 binding sites. Expression of ErbB3 in Gab1 -/- MEFs enhances activation of the PI-3 kinase signaling pathway --------------------------------------------------------------------------------------------- As described above, PI-3 kinase is recruited to the EGFR via the adaptor protein Gab1. The results presented here demonstrate that Gab1 is required for EGF-induced activation of the PI-3 kinase pathway via the EGFR, presumably because this receptor does not contain binding sites for the p85 regulatory subunit of PI-3 kinase. The catalytically inactive ErbB3 receptor, however, contains at least six binding sites for p85 \[[@B5]\], and thus may bypass the requirement for Gab1 in response to EGF by heterodimerizing with the catalytically active EGFR. In order to test this hypothesis, retroviral vectors were used to introduce either the Gab1 or ErbB3 genes into Gab1 -/- MEFs that endogenously express the EGFR but not ErbB3, and pools of stable cell lines were selected for further analysis. We first assayed the ability of the endogenous EGFR to be tyrosine autophosphorylated in response to EGF, as well as the ability of the exogenous ErbB3 receptor to be tyrosine phosphorylated in response to stimulation with either EGF or neuregulin (NRG). The experiment presented in Fig [4A](#F4){ref-type="fig"} shows that all cell lines exhibit EGFR autophosphorylation in response to EGF treatment (Figure [4A](#F4){ref-type="fig"}, upper left panel), while only cells expressing the ectopically introduced ErbB3 protein show ErbB3 tyrosine phosphorylation in response to EGF stimulation. Interestingly, ErbB3 reproducibly shows constitutive low-level tyrosine phosphorylation that is augmented only 1.3-fold in response to EGF treatment. Cells expressing ErbB3 show tyrosine phosphorylation in response to treatment with NRG (Figure [4A](#F4){ref-type="fig"}, upper and middle right panels). Expression of EGFR and ErbB3 in the appropriate cell lines was confirmed by immunoblotting with antibodies specific for EGFR and ErbB3, respectively (Figure [4A](#F4){ref-type="fig"}, lower panels). The apparent decrease in EGFR expression in cells co-expressing EGFR and ErbB3 following EGF treatment was not observed in repetitions of this experiment, and is likely due to a stripping anomaly. Additionally, we demonstrated that Gab1 -/- MEFs that express wild type Gab1 display Gab1 tyrosine phosphorylation in response to EGF treatment, while Gab1 -/- control cells or those expressing ErbB3 do not show Gab1 phosphorylation (Figure [4B](#F4){ref-type="fig"}, upper panel). In order to test the ability of ErbB3 to rescue the EGF-induced activation of the PI-3 kinase/Akt signaling pathway in Gab1 -/- MEFs, we first assayed these cells for EGF-induced PI-3 kinase activity. Cells were either left unstimulated or were stimulated with EGF and cell lysates were immunoprecipitated with anti-phosphotyrosine antibody. Phosphotyrosine-associated PI-3 kinase activity was then assayed by analysis of PIP3 production. As shown in Figure [4C](#F4){ref-type="fig"}, both cells expressing wild type Gab1 and ErbB3 show PI-3 kinase activity, while Gab1 -/- cells do not. Interestingly, EGF induces PI-3 kinase activity to a greater degree in cells expressing Gab1 relative to cells expressing ErbB3. In order to assay the effects of EGF stimulation on signaling downstream of PI-3 kinase, cells were treated with EGF over varying periods of time and assayed for the presence of Ser473-phosphorylated Akt. Treatment of cells expressing either wild type Gab1 or ErbB3 with EGF induced rapid activation of Akt, although cells expressing wild type Gab1 reproducibly displaed higher levels of phosphorylated Akt with significantly sustained kinetics relative to Gab1 -/- cells expressing ErbB3 (Figure [4D](#F4){ref-type="fig"}, left panels). Equal loading and expression levels of Akt were confirmed by immunoblotting (Figure [4D](#F4){ref-type="fig"}, right panels). Cells expressing ErbB3 displayed activation of Akt in response to treatment with NRG at levels similar to or greater than that seen in Gab1-expressing cells following EGF treatment, with activation reproducibly observed at longer time points (Figure [4E](#F4){ref-type="fig"}, left panels). Again, equal loading and expression levels of Akt were confirmed by immunoblotting (Figure [4E](#F4){ref-type="fig"}, right panels). The phosphorylation of AKT in cells expressing Gab1, which shows modest enhancement following treatment with NRG, may be attributed to alternate signaling pathways including those mediated by ErbB2 and ErbB4. Involvement of these receptors was not explored in this study. Taken together, these results demonstrate that ErbB3 can partially compensate for Gab1 deficiency in EGF-induced activation of the PI-3 kinase/Akt signaling pathway, although Gab1-mediated activation appears to be more robust, and likely represents the primary mechanism by which EGF stimulates PI-3 kinase and Akt. While ErbB3 is relatively ineffective at mediating EGF-stimulation of PI-3 kinase activation, it is an efficient mediator of PI-3 kinase stimulation in response to NRG stimulation. Thus, EGF and NRG can stimulate PI-3 kinase activation in normal and transfected cells by means of Gab1 and ErbB3, respectively. Conclusions =========== The results presented here demonstrate an absolute requirement for Gab1 in EGF-induced activation of the PI-3 kinase/Akt signaling pathway. Using this approach we demonstrated a strict requirement for association between Gab1 and p85 in EGF-induced PI-3 kinase activation, suggesting that Gab1 indeed provides an essential link between the EGFR and PI-3 kinase. Additionally, p85 binding does not play a significant role in Shp2 recruitment to the EGFR-Gab1 signaling complex, in contrast to previous studies \[[@B14]\]. We further demonstrated that the Gab1-Shp2 complex is responsible for the negative regulation of the strength and duration of PI-3 kinase/Akt signaling in response to EGF previously observed in Shp2 -/- cells. ErbB3 expression can bypass the requirement for Gab1 in EGFR signaling and can partially rescue EGF-induced activation of the PI-3 kinase/Akt cell survival pathway. This alternate pathway to PI-3 kinase activation may provide cells with a means of controlling either the strength or duration of PI-3 kinase signaling through differential expression of Gab1 and ErbB3, since the ErbB3-mediated response appears to be weaker. Thus Gab1 plays an essential role in bringing together a multi-protein signaling complex in response to EGF that modulates a critical aspect of cellular survival. Methods ======= Expression constructs --------------------- Expression vectors for wild type Gab1 and for Gab1Δp85 (Y446F/Y472F/Y589F) were previously described \[[@B21]\]. The cDNAs of Gab1 (pcDNA3-Gab1-WT), Gab1^F446/472/589^(pcDNA3-Gab1-3YF), and ErbB3 (pcDNA3-ErbB3) were subcloned into the mammalian retroviral vector pBabe containing a gene for puromycin resistance. The Gab1^F627/659^and Gab1^F446/472/589/627/659^mutants were generated by site-directed mutagenesis (Strategene) carried out on pBabe-Gab1-WT (to generate Gab1^F627/659^) or pBabe-Gab1^F446/472/589^(to generate Gab1^F446/472/589/627/659^) according to the manufacturers\' specifications. Cell lines and culture ---------------------- Gab1-deficient (Gab1 -/-) mouse embryonic fibroblasts (MEFs) were obtained from Walter Birchmeier \[[@B22]\]. Wild type or Gab1-/- MEFs were maintained in DMEM supplemented with 10% fetal bovine serum (Gibco), 2 mM L-glutamine (Gibco), and 100 μg each of penicillin and streptomycin. The retroviral vectors described above (pBabe, pBabe-Gab1, pBabe-Gab1-Gab1^F446/472/589^, pBabe-Gab1-Gab1^F627/659^, pBabe-Gab1-Gab1^F446/472/589/627/659^, and pBabe-ErbB3) were used to transfect the amphitropic retroviral packaging cell line GPG (obtained from Joan Brugge) and high-titer viral stocks were used to infect Gab1 -/- MEFs. Cells were selected in medium supplemented with puromycin and pools of selected cells were used for further experiments. Prior to stimulation with EGF, cells were starved in serum-free medium. Immunoprecipitation and immunoblotting -------------------------------------- Cells were grown on 15-cm plates as described above to approximately 80% confluence and then starved overnight in DMEM without serum. Cells were left unstimulated or were stimulated with recombinant human EGF (Invitrogen) as indicated. Stimulations were halted by the addition of ice-cold PBS. Cells were washed in PBS and lysed in buffer containing 1% Triton X-100 as previously described \[[@B23]\]. For Gab1 immunoprecipitations, a mixture of polyclonal antibodies directed against both the N- and C-termini of Gab1 and cross-linked to Protein A Sepharose (Zymed) was incubated with cell extracts for 2--4 hours at 4°C. For p85 immunoprecipitations, a mixture of polyclonal antibodies directed against the N-terminal SH2 domain of p85 and the full-length p85 protein (Upstate) was incubated with cell extracts for 2--4 hours at 4°C. For EGFR immunoprecipitations, a polyclonal antibody directed against the C-terminus of the EGFR was incubated with cell extracts overnight at 4°C. For ErbB3 immunoprecipitations, a mixture of polyclonal antibodies directed against both the N- and C-termini of ErbB3 was incubated with cell extracts overnight at 4°C. Protein A Sepharose was added to immunoprecipitates (except anti-Gab1) and incubated for 1 hour at 4°C. Immunoprecipitates were then washed in buffer containing 0.1% Triton X-100, separated by SDS/PAGE, and transferred to nitrocellulose membranes (Bio-Rad). For Akt immunoblotting, total cell extracts were separated directly by SDS/PAGE and transferred to nitrocellulose membranes. Membranes were blocked for 1 hour or overnight in 5% BSA/TBS and immunoblotted as indicated. The anti-phosphotyrosine blotting was carried out with antibody 4G10 (Upstate). Anti-Gab1, anti-EGFR and anti-ErbB3 blotting was performed with the indicated polyclonal antibodies (Upstate). Anti-Shp2 blotting was carried out with polyclonal antibody (Santa Cruz). Anti-phosphoSer473-Akt and anti-Akt blotting were performed with the respective polyclonal antibodies (Cell Signaling Technology). Proteins were visualized by incubation with Enhanced Chemiluminescence (Amersham Pharmacia) according to the manufacturer\'s specifications. PI-3 kinase assay ----------------- The PI-3 kinase assay was performed essentially as previously described \[[@B22]-[@B24]\]. Briefly, cells were serum-starved for 24 hours and stimulated with EGF as indicated, and cell extracts were prepared as described above. The lysates were immunoprecipitated using anti-Gab1 polyclonal antibodies or the monoclonal anti-phosphotyrosine antibody PY20 (Santa Cruz) for 2 hours at 4°C. Protein A Sepharose was incubated with immunoprecipitates for 1 hour at 4°C. The immunoprecipitates were washed three times with Buffer 1 (1X PBS, 1% NP-40), twice with Buffer 2 (0.5 M LiCl, 0.1 M Tris pH 7.5), twice with TNE (10 mM Tris pH 7.5, 100 mM NaCl, 1 mM EDTA pH 8.0), and twice with Buffer 4 (20 mM Hepes pH 7.5, 50 mM NaCl, 5 mM EDTA pH 8.0, 0.03% NP-40, 30 mM Tatrasodium Pyrophosphate (Sigma)). L-α-Phosphatidylinositol (Sigma) was added (10 μl of a sonicated solution at 10 mg/ml in 20 mM HEPES pH 7.5) and the reaction was initiated by the addition of 50 μl of the kinase buffer (20 mM Tris pH 7.5, 75 mM NaCl, 10 mM MgCl~2~10 μM ATP, 100 mM adenosine, 10 μCi \[γ-^32^P\] ATP) per sample. Samples were incubated at 30°C for 15 minutes, and the reaction was stopped by addition of 100 μl 1N HCl. Samples were extracted by addition of 200 μl CHCl~3~/CH~3~OH (1:1). The samples were vortexed and centrifuged, and the lower organic phases containing phospholipids were dried at 27°C for two hours. Samples were resuspended in 10 μl of PI-4-P standard (0.5 ml CHCl~3~, 0.5 ml CH~3~OH, 2.5 μl HCl, 1 mg L-α-Phosphatidylinositol 4-monophosphate (Sigma)) and subjected to thin layer chromatography (TLC plates -- VWR) in CHCl~3~/CH~3~OH/NH~4~OH/H~2~O (45:35:7:3). The TLC plates were exposed in a PhosphorImager cassette for four days. Authors\' contributions ======================= BL carried on the initial biochemical experiments before leaving the laboratory. DM continued this study and carried out the biochemical studies, the PI-3 kinase assays, and drafted the manuscript. IL participated in the PI-3 kinase assays. JS initiated and supervised the project. All authors read and approved the final manuscript. Acknowledgements ================ This work was supported in part by a James Hudson Brown-Alexander B. Coxe Postdoctoral Fellowship (to D.M.). Joseph Schlessinger is supported by NIH grant R01-AR051448 and by funds from the Ludwig Institute for Cancer Research. MEFs from Gab1-/- were kindly provided by Walter Birchmeier. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Expression of wild type Gab1 rescues EGF-induced PI-3 kinase and Akt activation in Gab1 deficient MEFs. A.The indicated cell lines were serum-starved for 24 hours and stimulated with 10 ng/ml EGF for five minutes at 37°C. Cell extracts were prepared and analyzed for Gab1 tyrosine phosphorylation and Gab1 expression. Both WT MEFs and cells expressing exogenous Gab1 show Gab1 tyrosine phosphorylation in response to EGF treatment. B.The indicated cell lines were serum-starved for 24 hours and stimulated with 100 ng/ml EGF for five minutes at 37°C. Cell extracts were prepared and phosphotyrosine immunoprecipitates were analyzed for PI-3 kinase activity. Gab1 -/- cells fail to show phosphotyrosine-associated PI-3 kinase activity, while cells expressing exogenous Gab1 display phosphotyrosine-associated PI-3 kinase activity that is augmented by EGF treatment. C.The indicated cell lines were serum-starved for 24 hours and stimulated with 1 ng/ml EGF for varying periods of time at 37°C. Cell extracts were prepared and analyzed for activation of Akt by using antibodies that specifically recognize the serine473 phosphorylated form of Akt. Membranes were subsequently stripped and immunoblotted for Akt to confirm equal loading. Ectopic expression of Gab1 in Gab1 -/- MEFs rescues the EGF-induced activation of Akt. ::: ![](1741-7007-2-24-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Expression of a Gab1 mutant protein deficient in p85 binding fails to rescue EGF-induced PI-3 kinase/Akt activation in Gab1 deficient MEFs. A.The indicated cell lines were serum-starved for 24 hours and stimulated with 10 ng/ml EGF for five minutes at 37°C. Cell extracts were prepared and analyzed for Gab1 tyrosine phosphorylation, Gab1 co-immunoprecipitation with p85, p85 expression, Gab1 co-immunoprecipitation with Shp2, and Gab1 expression. Both Gab1 and Gab1^F446/472/589^are tyrosine phosphorylated in response to EGF treatment, and both form a stable complex with Shp2. However, Gab1^F446/472/589^fails to associate with the p85 subunit of PI-3 kinase. Additionally total cell lysates from the indicated cell lines were immunoblotted with anti-Gab1 antibodies, providing independent evidence that Gab1 is expressed at approximately equal levels in all cell lines. B.The indicated cell lines were serum-starved for 24 hours and stimulated with 10 ng/ml EGF for varying periods of time at 37°C. Cell extracts were prepared and analyzed for EGFR tyrosine phosphorylation. All of the cell lines examined show similar kinetics of EGF-induced EGFR activation. Immunoblots were quantitated by densitometry, normalized for EGFR expression and represented linearly. Diamonds = Gab1 -/-, squares = Gab1, triangles = Gab1^F446/472/589^. C.The indicated cell lines were serum-starved for 24 hours and stimulated with 100 ng/ml EGF for five minutes at 37°C. Cell extracts were prepared and Gab1 immunoprecipitates were analyzed for PI-3 kinase activity. Ectopic expression of wild type Gab1 restored EGF-induced PI-3 kinase activity, while expression of Gab1^F446/472/589^fails to rescue PI-3 kinase activity in response to EGF treatment. D.The indicated cell lines were serum-starved for 24 hours and stimulated with 1 ng/ml EGF for varying periods of time at 37°C. Cell extracts were prepared and analyzed for activation of Akt by using antibodies that specifically recognize the serine473-phosphorylated form of Akt. Membranes were subsequently stripped and immunoblotted for Akt to confirm equal loading. Ectopic expression of Gab1 in Gab1 -/- MEFs rescues activation of Akt in response to EGF treatment, while expression of Gab1^F446/472/589^fails to rescue the EGF-induced Akt activation. ::: ![](1741-7007-2-24-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Expression of a Gab1 mutant protein deficient in Shp2 binding enhances EGF-induced activation of the PI-3 kinase/Akt signaling pathway. A.The indicated cell lines were serum-starved for 24 hours and stimulated with 10 ng/ml EGF for five minutes at 37°C. Cell extracts were prepared and analyzed for Gab1 tyrosine phosphorylation, Gab1-Shp2 co-immunoprecipitation and Gab1 expression. Gab1^F627/659^becomes tyrosine phosphorylated in response to EGF treatment to levels approximately 1.5-fold higher than Gab1 as determined by densitometry, while Gab1^F446/472/589/627/659^does not show EGF-induced tyrosine phosphorylation in this assay. Wild type Gab1 forms a stable complex with Shp2 in response to EGF treatment, while Gab1^F627/659^and Gab1^F446/472/589/627/659^do not. B.The indicated cell lines were serum-starved for 24 hours and stimulated with 10 ng/ml EGF for varying periods of time at 37°C. Cell extracts were prepared and analyzed for EGFR tyrosine phosphorylation and EGFR expression. All of the cell lines examined show similar kinetics of EGF-induced EGFR activation. Immunoblots were quantitated by densitometry, normalized for EGFR expression, and represented linearly. Diamonds = Gab1 -/-, squares = Gab1, triangles = Gab1^F627/659^, circles = Gab1^F446/472/589/627/659^. C.The indicated cell lines were serum-starved for 24 hours and stimulated with 100 ng/ml EGF for five minutes at 37°C. Cell extracts were prepared and Gab1 immunoprecipitates were analyzed for PI-3 kinase activity. Cells expressing exogenous Gab1^F627/659^display enhanced PI-3 kinase activity relative to cells expressing wild type Gab1. Expression of exogenous Gab1^F446/472/589/627/659^fails to rescue EGF-induced PI-3 kinase activity in Gab1 deficient MEFs. D.The indicated cell lines were serum-starved for 24 hours and stimulated with 1 ng/ml EGF for varying periods of time at 37°C. Cell extracts were prepared and analyzed for activation of Akt by using antibodies that specifically recognize the serine473-phosphorylated form of Akt. Membranes were subsequently stripped and immunoblotted for Akt to confirm equal loading. Cells expressing exogenous Gab1^F627/659^display enhanced activation of Akt with sustained kinetics relative to cells expressing wild type Gab1. Expression of exogenous Gab1^F446/472/589/627/659^fails to rescue EGF-induced Akt activation in Gab1 deficient MEFs. ::: ![](1741-7007-2-24-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Expression of ErbB3 in Gab1-deficient MEFs partially rescues EGF-induced activation of the PI-3 kinase/Akt signaling pathway. A.The indicated cell lines were serum-starved for 24 hours and stimulated with 10 ng/ml EGF for five minutes at 37°C. Cell extracts were analyzed for EGFR tyrosine phosphorylation and EGFR expression, and for ErbB3 tyrosine phosphorylation and ErbB3 expression. Cells were additionally stimulated with 10 ng/ml NRG and cell extracts analyzed for ErbB3 tyrosine phosphorylation. The endogenous EGFR is tyrosine phosphorylated in response to EGF in all cell lines. ErbB3 exhibits weak constitutive tyrosine phosphorylation that is enhanced by NRG treatment, but is not significantly enhanced by treatment with EGF. Selected bands were quantitated by densitometry to determine relative increase in growth factor-induced tyrosine phosphorylation. B.The indicated cell lines were serum-starved for 24 hours and stimulated with 10 ng/ml EGF for five minutes at 37°C. Cell extracts were analyzed for Gab1 tyrosine phosphorylation and Gab1 expression. Cells rescued with wild type Gab1 exhibit Gab1 tyrosine phosphorylation in response to EGF treatment, while Gab1 -/- cells and Gab1-deficient cells expressing ErbB3 do not. C.The indicated cell lines were serum-starved for 24 hours and stimulated with 100 ng/ml EGF for five minutes at 37°C. Cell extracts were prepared and phosphotyrosine immunoprecipitates were analyzed for PI-3 kinase activity. Expression of wild type Gab1 in Gab1-deficient MEFs rescues the EGF-induced PI-3 kinase activity. Gab1 deficient MEFs exogenously expressing ErbB3 exhibit PI-3 kinase activity that is largely EGF-independent. D.The indicated cell lines were serum-starved for 24 hours and stimulated with 1 ng/ml EGF for varying periods of time at 37°. Cell extracts were analyzed for activation of Akt by using antibodies that specifically recognize the phosphorylated form of Akt. Membranes were subsequently stripped and immunoblotted for Akt to confirm equal loading. Ectopic expression of ErbB3 in Gab1 deficient cells results in a partial rescue of EGF-induced Akt activation relative to cells expressing wild type Gab1. E.The indicated cell lines were serum-starved for 24 hours and stimulated with 1 ng/ml NRG for varying periods of time at 37°C. Cell extracts were analyzed for activation of Akt by using antibodies that specifically recognize the phosphorylated form of Akt. Membranes were subsequently stripped and immunoblotted for Akt to confirm equal loading. Treatment of Gab1-deficient cells exogenously expressing ErbB3 with NRG results in a robust and sustained activation of Akt, while cells expressing exogenous Gab1 do not exhibit Akt activation in response to NRG treatment. ::: ![](1741-7007-2-24-4) :::	PubMed Central	2024-06-05T03:55:49.909356	2004-11-18	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534114/", "journal": "BMC Biol. 2004 Nov 18; 2:24", "authors": [ { "first": "Dawn R", "last": "Mattoon" }, { "first": "Betty", "last": "Lamothe" }, { "first": "Irit", "last": "Lax" }, { "first": "Joseph", "last": "Schlessinger" } ] }
PMC534115	Background ========== The immune system is composed of a multiplicity of individual cell types that derive from a relatively small number of immuno-hematopoietic progenitors that undergo complex developmental and exposure-driven differentiation and activation. Cell-type specific gene expression is driven to a large measure by complex transcriptional regulation that orchestrates differential expression of a wide variety of genes necessary to accomplish immune effector functions. A number of specific transcription factors (TFs) which regulate gene expression in immune system cell types have been identified, largely through gene knockout experiments and isolation of protein complexes that bind to regulatory regions of target genes. Examples include PU.1/Ets, Ikaros, E2A, EBF, PAX5, GATA3, NFAT, cMYB, and OCT-2 \[[@B1]-[@B4]\]. These proteins bind to clusters of cis-regulatory elements in multiple diverse combinations to give rise to specific patterns of gene expression \[[@B5]\]. However, the layout of regulatory and coding regions is not known for most genes that are preferentially expressed in lymphocytes and immune tissues (see, for examples, \[[@B6]-[@B11]\]). Based on the nearly completed nucleotide sequences of the mouse and human genomes (<http://genome.ucsc.edu>\[[@B12]\]; <http://www.ensembl.org>\[[@B13]\]), we have sought to expand our knowledge of the structure and function of compartment-specific genes, and in particular, to find clusters of cis-elements that bind TFs and regulate gene expression during biological processes. DNA sequences of both coding regions and non-coding regions which harbor cis-elements that govern expression, are phylogenetically conserved \[[@B14]-[@B16]\]. This conservation of functionally important regions of DNA underpins current methods of identifying putative regulatory regions by comparative sequence analysis. In practice, finding relevant clusters of cis-elements is difficult and computationally intensive. High-throughput gene expression profiling provides a powerful approach to the investigation of relative transcriptional activity as a function of biological differentiation across a variety of cells and tissues. Published examples that probe a wide variety of distinct, differentiated materials include the Human Gene Expression (HuGE) Index database <http://www.hugeindex.org>\[[@B17]\] and the GNF (Genomics Institute of the Novartis Research Foundation; <http://web.gnf.org/>) database of human and mouse gene expression \[[@B18]\]. These resources provide access to patterns of expression of a significant fraction (15--25%) of all mouse and human genes in several dozen tissues and cell types. We have created a large database locally, which has permit investigators from our campus to profile gene expression in mouse tissues and cell types specific to their interests \[[@B19]\]. To do this, we used the Incyte Mouse GEM1 microarray, an 8638 element spotted cDNA gene expression platform and a universal reference design that employed poly A+ mRNA was prepared from whole day-1 postnatal mouse. Two channel Cy3-Cy5 microarray hybridization technology was used to identify relative strength of signals from each element of the array for a specific tissue. From this database, we identified 360 cDNAs on the microarray that exhibit preferential expression in immune tissues such as lymph nodes, thymus, and activated T-cells relative to most other types of tissues. We identified 333 genes that encode these sequences and have grouped them by biological functions and by patterns of expression. Cis-element clusters that are conserved in pairs of orthologs are strong predictors of regulatory regions within mammalian genes \[[@B15],[@B20],[@B21]\]. We have used this method to identify putative regulatory modules, which are clusters of conserved cis-regulatory elements that occur in coordinately regulated genes of the immune system and may play a role in controlling their expression during development or mature cell function. Several of the modules identified through this approach contain cis-elements whose biological relevance has been experimentally validated in previous studies. Other computationally identified modules from this immunomic database have not been studied in detail, but the results and a tool to analyze them further, are provided at the website <http://cismols.cchmc.org>\[[@B22]\]. Taken together these data provide valuable guidance to the design of experiments that seek to identify regulatory modules in genes with specific patterns of expression. Results ======= Selection of set of immune genes -------------------------------- Our goal is to identify genes, which are essential for the differentiation, maintenance, and function of the immune system, and their associated regulatory elements. Polymorphisms or mutation in these might underlie well-known variation among individuals in effectiveness of their immune response. Mouse immune genes were identified from our gene expression database constructed using the 8638 element microarray and probed with mRNA prepared from 65 normal adult and fetal tissues. We chose to select relevant genes by collecting those expressed above a threshold value rather than by statistical analysis of variance. Given the small number of replicates and the large number of comparisons being made, we would not have enough statistical power to detect differentially expressed genes by using traditional statistical tests with appropriate specificity. In addition, with the reduced specificity of statistical tests, the biologically non-significant, but somewhat reproducible differences in gene expression will obscure changes that are of biologically significant magnitude, but vary from replicate to replicate. Expression of genes is not discontinuous from tissue to tissue, but varies quantitatively over a wide range. The threshold to distinguish expressed from non-expressed genes was set to identify the hundred or so most highly expressed gene in each relevant tissue. Genes were considered to be \"immune genes\" if they were more highly expressed in one or more of 6 immune tissues (lymph nodes from normal and antigen stimulated mice, thymus, activated T-cells, spleen, peripheral blood mononuclear cells) than in most other normal adult and fetal mouse tissues. 680 genes were identified where the amount of cDNA hybridized from one or more immune tissues was 3 or more times greater than hybridization of cDNAs from the reference whole mouse (Figure [1A](#F1){ref-type="fig"}). To increase specificity, the 680 genes were then filtered to remove those with 2-fold or greater expression in normal brain, spinal cord, heart, kidney, pancreas or stomach. These tissues were chosen because they do not play a role in the immune response, contain very few cells of the immune system, and should not express immune-specific genes. By contrast, no effort was made to remove genes expressed in the intestinal tract, lung, or fetus where cells of the immune system might be expected. The resulting set of 483 genes was examined by hierarchical cluster analysis. Spleen and peripheral blood mononuclear cells were noted to express genes encoding proteins of immature erythroid cells and polymorphonuclear leukocytes. To remove these, the set was restricted to genes that were expressed 2 fold or greater in at least one of stimulated and unstimulated lymph nodes, activated T cells, or thymus. The end result is a set of 360 expressed sequences, which we call \"immune\" genes (Figure [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}). 265 of the expressed sequences were linked to specific genes and gene symbols, using the Mouse Genome Database (MGD) <http://informatics.jax.org>\[[@B23]\] and NCBI-LocusLink \[[@B24]\]. The remainders were analyzed using MouseBLAST and BLAT \[[@B12]\] to find sequence homologies with known genes. An additional 78 sequences could be linked to specific genes, 9 (seven occurring twice and one occurring thrice) of these were redundant, so that a total of 333 previously known unique genes were represented by the 360 expressed sequences. 292 of these genes were assigned a probable function, using criteria described in Methods. 5 sequences were repetitive elements and 12 sequences could not be linked to a known gene or function. Gene symbols, names, functions, and extensive additional annotations are provided in the supplementary materials ([Additional file 1](#S1){ref-type="supplementary-material"}). Human orthologs of these mouse immune genes were sought by sequence homology. Where found, pairs of mouse-human orthologs were annotated with regard to function and were analyzed for phylogenetically conserved regulatory regions. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Expression profiles of sequences across tissues:Hierarchical tree clustering of genes and tissues was carried out using Pearson correlation and the log of the average of the relative expression ratio for each gene, as measured in replicate arrays. Sequences with similar expression patterns across all tissues are clustered together in the resulting trees, the closeness of the sequences in sub trees is a measure of how closely correlated their expression is. (A) Hierarchical tree clustering of genes across 65 normal adult and fetal tissues. 680 sequences were identified that were highly expressed in thymus, unstimulated and stimulated lymph nodes, spleen, peripheral blood mononuclear cells, and in vitroactivated T-cells. To increase specificity, 320 sequences were removed because they were also highly expressed in one or more non-lymphoid tissues, as described in the text. The pattern of expression of the remaining 360 \"immune genes\" across tissues is shown. (B) Hierarchical tree clustering of 360 immune genes across 18 normal adult and fetal tissues. There are 3 major groups of tissues that show clusters of highly expressed \"immune genes\" These include the 6 immune tissues, various segments of adult intestine, and fetal day 16.5 lung and intestine. Less prominent clusters are seen in adult lung and liver. Genes in these clusters are described in the text. While the band of high expression extends across all genes for the 6 immune tissues, relative expression of each gene within the immune tissues shows distinct patterning. ::: ![](1471-2164-5-82-1) ::: Hierarchical clustering of genes and tissues -------------------------------------------- Hierarchical tree clustering of the 360 sequences and 65 normal adult and fetal tissues was carried out by Pearson correlation using the log of the average of the relative expression ratio for each gene as measured in replicate arrays (Figure [1A](#F1){ref-type="fig"}). While the band of high expression extends across the 6 immune tissues, relative expression of each gene within the immune tissues shows distinct patterning (Figure [1B](#F1){ref-type="fig"}). For intestinal and fetal tissues, the areas of high expression are localized and do not include the majority of the immune genes. Function of the genes expressed in these tissues will be described. Function of the immune genes ---------------------------- A putative function could be assigned to 298 expressed sequences ([Additional file 1](#S1){ref-type="supplementary-material"}) based on one or more known functional annotation or sequence analysis-based structural classifiers. This annotation is independent of pattern of expression and gives an overview of the types of functions carried out by immune genes. Six functional groups derived from these annotations are shown in Table [1](#T1){ref-type="table"}. The HGNC <http://www.gene.ucl.ac.uk/nomenclature>\[[@B25]\] and MGI <http://informatics.jax.org>\[[@B23]\] approved gene symbols are used in the table, although many of these genes are better known by their aliases as provided in supplementary materials. Table [1](#T1){ref-type="table"} shows 59 genes that have functions associated with defense-immune or defense response (immune is a subcategory of defense in GO annotations). Defense-immune genes were more directly related to antigen recognition and receptor signaling of T- and B-lymphocytes than defense genes, although the separation of defense and immune is somewhat arbitrary. 47 genes in Table [1](#T1){ref-type="table"} are involved in cell signaling, 14 in apoptosis, 8 in chemotaxis, and 6 in lysosomes. Additional lists of genes grouped by function and shown in the supplementary materials include 39 in transcription, 23 in DNA replication/cell cycle control, 20 in protein synthesis, 13 in transport, and 10 in adhesion. Smaller groups of genes that are important in function of the immune system include protein trafficking and degradation, and maintenance of the cytoskeleton. Functions carried out by some of the genes that are highly expressed in immune tissues are common to cells and tissues that are actively proliferating and synthesizing proteins. These include, for example, genes involved in DNA synthesis and the cell cycle such as the minichromosome maintenance proteins, Mcm2through Mcm7; the DNA polymerases and primase, Pola2, Cdc6, Prim1; the processivity factor Pcna, and cyclin E1, Ccne1. They play a role in regulation of chromosomal replication in many types of cells \[[@B26]\]. In the immune tissues, high expression of these genes is characteristic of activated T-cells, which are proliferating. Similarly, other immune genes are involved in protein synthesis and are not specific to the immune system. Twelve immune genes encode ribosomal proteins. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Six sets of genes that are highly expressed in immune tissues, grouped by function. Gene symbol and GenBank accession number identify genes ::: Defense -- Immune -- 38 Genes Defense -- 21 Genes Signal -- 47 Genes ----------------------------------- --------------------------- --------------------------- ---------------- ----------------- ------------ 1190001G19Rik NM\_026875 5830443L24Rik BC031475 1200013B08Rik NM\_028773 A630096C01Rik BB629669 Arl6ip2 BC006934 2410118I19Rik AK004869 AI789751 AI789751 Bst1 NM\_009763 2610207I05Rik AK011909 B2m NM\_009735 C1qg NM\_007574 Adcy7 NM\_007406 BB219290 NM\_145141 C1s NM\_144938 AI325941 BF181435 Btla-interim BM240873 Camp NM\_009921 Arhh AK017885 Cd14 NM\_009841 Daf1 NM\_010016 Cd37 NM\_007645 Cd79b NM\_008339 Gbp2 NM\_010260 Cd53 NM\_007651 Cd86 BC013807 Gzmb NM\_013542 Cd97 NM\_011925 Cxcl9 NM\_008599 Klra24-pending AA288274 Clecsf12 NM\_020008 Fcgr3 NM\_010188 Klrd1 NM\_010654 Clecsf5 NM\_021364 Gp49a NM\_008147 Ncf2 NM\_010877 Clk3 AF033565 H2-Aa NM\_023145 Ncf4 NM\_008677 Coro1a NM\_009898 H2-Ab1 NM\_010379 Oas2 NM\_145227 D530020C15Rik BC027196 H2-DMa NM\_010386 Oasl2 NM\_011854 Dgkz BC014860 H2-Eb1 NM\_010382 Ocil-pending NM\_053109 Dok2 NM\_010071 H2-K U47328 Prg NM\_011157 E430019B13Rik AA881918 H2-L M34961 Tnfrsf13b AK004668 G431001E03Rik AA387272 H2-Oa NM\_008206 Tnfrsf4 NM\_011659 Gnb2-rs1 NM\_008143 H2-Ob NM\_010389 Tnfrsf9 NM\_011612 Gpcr25 NM\_008152 H2-Q7 NM\_010394 Zbp1 AA175243 Gprk6 NM\_011938 Igh-4 L36938 Hck NM\_010407 Igj BC006026 Apoptosis -- 14 Genes Iigp-pending NM\_021792 Igl AK008551 Il2rg NM\_013563 Igsf7 AF251705 5630400E15Rik AK017464 Il4ra NM\_010557 Lst1 AF000427 AI447904 BF179348 Jak1 BC031297 Ly86 NM\_010745 Axud1 BC029720 Lck BC011474 Mpa2 NM\_008620 Biklk BC010510 Lcp2 BC006948 Mpeg1 L20315 Birc2 NM\_007464 Lyn BC031547 Ms4a1 NM\_007641 Casp4 NM\_007609 Lypla1 BF160555 Ms4a4b NM\_021718 Dnase1l3 NM\_007870 Map3k1 AF117340 Ms4a6c NM\_028595 Ian4 NM\_031247 Map4k1 BC005433 Sema4d NM\_013660 Ifi203 AA174447 Mbc2 BC011482 Tactile-pending NM\_032465 Ripk3 NM\_019955 P2y5 AK011967 Tcrd AI530748 Scotin-pending NM\_025858 Pilra AJ400844 Tcrg NM\_011558 Stk17b NM\_133810 Pip5k2a AK012196 Tlr1 NM\_030682 Stk4 W77521 Ptpn2 NM\_008977 Trygn16 M97158 Trp53inp1 NM\_021897 Ptpn8 NM\_008979 Ptprc NM\_011210 Lysosomes -- 6 Genes Chemotaxis -- 8 Genes Ptprcap NM\_016933 Rac2 NM\_009008 Acp5 AA002801 Ccl19 NM\_011888 Stat1 NM\_009283 Ctsl NM\_009984 Ccl22 NM\_009137 Stat3 BC003806 Ctss NM\_021281 Ccl4 NM\_013652 Stat4 NM\_011487 Ctsz NM\_022325 Ccl6 NM\_009139 Stk10 NM\_009288 Man1a NM\_008548 Ccr2 NM\_009915 Syk NM\_011518 Man2b1 NM\_010764 Cxcl13 NM\_018866 Tln NM\_011602 Cxcr4 NM\_009911 S100a8 NM\_013650 ::: There are sets of genes that work together to produce the cellular and humoral immune responses. For example, molecules of the major histocompatibility complex present foreign peptides to T cells. They are encoded by genes such H2-Aa, H2-Ab1, H2-DMa, H2-Eb1, H2-K, H2-L, H2-Oa, H2-Ob, H2-Q7, and B2m(Table [1](#T1){ref-type="table"}, Defense -- Immune). Signal transduction pathways are abundant and play critical roles in the function of lymphocytes. They link the recognition of antigens or chemokines by receptors on the cell surface to the transcription of genes required for cell division and new protein synthesis. This process of lymphocyte activation requires an intracellular signaling cascade with participation of protein kinases, G-proteins, and products of cleavage of membrane phospholipids \[[@B27]-[@B29]\] (Table [1](#T1){ref-type="table"}, Signal). Janus kinases, encoded by genes such as Jak1, phosphorylate both signal transducers and activators of transcription (Stat1, Stat3, and Stat4) as part of the lymphocytes\' response to cytokines. The product of Rac2is a G protein that participates in the cascade of kinases leading to activation of TFs. Chemokines are a family of small proteins that activate cells such as lymphocytes as part of the host response to infection. Genes that encode the chemokines (Ccl4, Ccl6, Ccl19, Ccl22, Cxcl13) and chemokine receptors (Cxcr4and Ccr2) (Table [1](#T1){ref-type="table"}, Chemotaxis) are highly expressed in immune tissues. Twenty-one sequences representing 19 known genes were highly expressed in gastrointestinal tissue (Figure [1B](#F1){ref-type="fig"}). Of these, 5 were classified as \"Defense -- Immune\", including B2m, H2-Q7, Tcrg, Tlr1, and H2-K. Of the 51 genes expressed in fetal tissues (Figure [1B](#F1){ref-type="fig"}), 46 are annotated. Sixteen genes functioned in protein synthesis and 13 in cell cycle/DNA synthesis. No \"Defense\" or \"Defense-Immune\" genes were highly expressed in fetal tissues. Genes expressed in fetal tissues reflect active growth and proliferation of cells. In immune tissues, these same genes are particularly well expressed in activated T-cells and thymus, where cell proliferation is occurring. Cis-regulatory elements of MHC class I genes -------------------------------------------- Regulatory modules predicted by comparative analyses of DNA sequences must be validated by genomic footprinting and other biochemical techniques, which prove that the predicted TFbinding sites are biologically relevant. Because extensive data are available, we compared the structure of the promoter elements of the H2-Kand HLA-Agenes (MHC class I) as predicted by computational and biochemical studies. Experimentally identified, conserved, regulatory elements within the promoter of MHC class I genes include: an enhancer A element (two NFKB sites), an interferon-stimulated response element (ISRE), site α (cAMP-response element), enhancer B (inverted CCAAT), CCAAT, and TATA elements \[[@B30]\]. Computationally predicted arrangements of conserved cis-elements in the promoters of H2-Kand its human ortholog, HLA-A, are shown in Figure [2](#F2){ref-type="fig"}. FASTA sequences and corresponding coordinates of the regions used in the analysis are given in [Additional file 10](#S10){ref-type="supplementary-material"}. The predicted arrangements are in close agreement with results of genomic footprinting, electrophoretic mobility shift assays, and other techniques. For HLA-A, computationally identified binding sites previously found by biochemical analyses include: IRFF, CREB, ECAT, PCAT, TBPF and two NFKB. The enhancer-A element of the MHC class I promoter encompasses two NFKB binding sites and plays an important role in the constitutive and cytokine-induced expression of MHC class I genes. Our IRFF site is the reported ISRE and can bind interferon regulatory factor 1 to activate MHC class I transcription. Site α of the MHC Class I promoter corresponds to our CREB binding site and plays an important role in regulation of expression of Class I genes. Our PCAT and ECAT sites include sequences consistent with the CCAAT site and our TBPF is a TATA binding site, as reported in the MHC class I promoter immediately upstream of the transcription start site \[[@B30]\]. Computational analysis identifies additional potential binding sites that have not yet been tested for biological relevance. These include families IKRS, WHZF, EKLF, EGRF, and AHRR. Several of these may play a specific role in the immune system. For instance, the IKRS family of sites bind Ikaros zinc finger transcription factors, which are regulators of lymphocyte differentiation; the WHZ family of TFs includes members that are critical to the proper expression of genes during development of the thymus \[[@B31]\]; the EGR family of zinc finger transcription factors is induced as a consequence of activation of the mitogen-activated protein kinase (MAPK) signaling pathway during positive selection in the thymus \[[@B32]\]; and AhR is known to effect immunosuppression by inducing bone marrow stromal cells to deliver a death signal to lymphocytes \[[@B33]\]. We conclude that computational analyses both identify previously reported TF binding sites and predict phylogenetically conserved sites that should be examined for biological relevance in future biochemical studies. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### *Computationally predicted clusters of cis-elements in the promoter region of mouse H2-Kand its human ortholog HLA-A**: The ATG of human HLA-Ais at position 10,001 while that of mouse H2-Kis at 10,463. Thus, the region represented relative to ATG is -305 to +97 (human) and -653 to -293 (mouse). Additionally, these regions correspond to chr 6: 30,015,866--30,016,268 (+) of the Human Genome July 2003 Assembly and chr17: 33,638,839--33,639,199 (-) of the Mouse October 2003 Assembly <http://genome.ucsc.edu>. Families of transcription factor binding sites and the relative positions of the sites in the nucleotide sequences of the two genes are represented as different colored bars stretching across the ortholog gene pair. ::: ![](1471-2164-5-82-2) ::: Cis-Regulatory elements in genes grouped by patterns of expression ------------------------------------------------------------------ Locally developed programs, TraFaC and CisMols, were used to identify and display putative regulatory modules in genes grouped by patterns of expression. The algorithms use a moving 200 bp window to scan regions of DNA for specific sequences characteristic of TF binding sites (Figure [3](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Example of a CisMols display of location and composition of clusters of cis-elements that are putative regulatory modules.The genes are those with high expression in thymus. The algorithm used by TraFac and CisMols to display regulatory modules uses a moving 200 bp window to scan regions of DNA for specific sequences characteristic of TF binding sites (cis-elements). Clusters of these cis-elements are not generally distributed evenly across a segment of DNA, but are highly localized to specific segments which are likely to play a role in regulation of gene expression. Because the scanning window is limited to 200 bp and the scan changes the frame of sequences within the window, a regulatory module that contains multiple cis-elements may not be displayed as one list of multiple elements, but rather as a list of several modules of different composition and arrangement within one small segment of DNA. Each colored cube indicates a cluster of 3 or more cis-elements with at least one \"lymphoid element\". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI\'s RefSeq database). The legend in the lower left half indicates the composition of each of the modules and the genes that share them. In the lower right hand panel is the Trafac image of one of the cis-element dense region with multiple shared modules of Arid1agene. ::: ![](1471-2164-5-82-3) ::: Cluster analysis of genome wide expression data from microarrays permits the grouping together of genes with similar patterns of expression across cells, tissues or experimental conditions. Clustering of genes by patterns of expression was first applied on a large scale to yeast \[[@B34]\], where control of important variables like genotype, phase of cell cycle, and growth conditions permits precise identification of coordinately regulated genes. Clustering has also been used to catalog mammalian genes that are differentially expressed in normal and malignant immune cells \[[@B35],[@B36]\]. While yeast genes with similar patterns of expression have been found to share regulatory elements \[[@B37]\], identification of such elements in clustered genes of mammals is complex and not very successful \[[@B38],[@B39]\]. Conservation of functionally important regions of DNA underpins current methods of identifying putative regulatory regions by computational analysis of nucleotide sequences \[[@B14]-[@B16]\]. Using K-means clustering in GeneSpring (Version 4.2.1), 160 genes, which had been annotated using SOURCE \[[@B40]\] early in our studies, were divided into distinct sets based on similarity of expression patterns across 15 tissues. Tissues were given equal weight, the number of clusters was set at 20, and similarity was measured by standard correlation. For technical reasons, GeneSpring did not assign 4 genes to clusters. The cluster sets are shown in [Additional file 2](#S2){ref-type="supplementary-material"}. ### K-cluster set 15 K-cluster set 15 contained 14 genes. While these genes had similarities in patterns of expression across a group of 15 tissues, their most prominent shared characteristic was preferential expression in thymus. They were diverse in function. For instance, the group comprised transcription factors Ets1and Tcf12, chromatin matrix associated protein Smarcf1(recently renamed Arid1a), the ATP-binding cassette transporter Abcg1(transports peptides during antigen processing), the 2\'-5\'-oligoadenylate synthetase Oasl2that is induced by interferon, and the histocompatibility antigen H2-Kthat plays a role in antigen presentation and processing. Sequences of both the mouse gene and its human ortholog were available for seven genes (Abcg1, Ctsl, Man2b1, Sgpl1, Arid1a, Tcf12, and Zfp162). The 3 kb upstream regions of all 7 genes were compared to identify modules of shared cis-elements. The search criteria were limited by (1) requiring modules to contain at least 3 TF binding sites, one of which is a lymphoid element (see this list of lymphoid elements in Methods), (2) to be evolutionarily conserved, that is, to occur within the phylogenetic footprints in the aligned mouse-human orthologs, and (3) to be located within 3 kb upstream and 100 bp downstream of the first bp of exon 1 (transcription start). Examples of modules of cis-elements are shown in Table [2](#T2){ref-type="table"}. Arid1a, Abcg1and Sgpl1are most similar to one another. They also have the most similar patterns of expression across tissues, when clustered in hierarchical trees. One module, AP2F EGRF MAZF SP1F ZBPF, contains 5 cis-elements within a 200 bp window and is present within 3 kb upstream of transcription start in Arid1a, Abcg1 and Sgpl1. The conserved modules containing multiple transcription factor binding sites (Table [2](#T2){ref-type="table"} and Figures [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}; [Additional file 11](#S11){ref-type="supplementary-material"} gives fasta sequences, list of binding sites and coordinates) are likely to play a role in regulation of expression of these genes, but this hypothesis must be experimentally verified. Ctsl, Man2b1, Zfp162 and Tcf12did not share modules (within upstream 3 kb region and having at least one \"lymphoid element\") with the other genes. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Examples of modules of shared cis-elements in K-cluster, set 15 genes. All elements of a module are within a 200 bp window and are present in both the human and mouse orthologs. Modules are located within 3-kb upstream of the transcription start site. ::: Genes* -------------------------- ----------- ---- ---- ---- ---- ---- ---- AP2F EGRF MAZF SP1F ZBPF \+ \+ \+ \- \- \- \- AP2F EGRF SP1F ZBPF \+ \+ \+ \- \- \- \- AP2F MAZF SP1F ZBPF \+ \+ \+ \- \- \- \- AP2F HESF MAZF SP1F \+ \+ \+ \- \- \- \- MAZF SP1F ZBPF \+ \+ \+ \- \- \- \- AP2F MAZF SP1F \+ \+ \+ \- \- \- \- AP2F SP1F ZBPF \+ \+ \- \- \- \- \- EGRF MAZF ZBPF \+ \+ \+ \- \- \- \- ETSF SP1F ZBPF \+ \+ \+ \- \- \- \- AP2F EGRF ETSF SP1F ZBPF \+ \+ \+ \- \- \- \- AP2F MAZF MZF1 SP1F ZBPF \+ \+ \- \- \- \- \- AP2F MAZF MZF1 SP1F \+ \+ \- \- \- \- \- AP2F MZF1 SP1F ZBPF \+ \+ \- \- \- \- \- ETSF MAZF SP1F \+ \- \+ \- \- \- \- EGRF ETSF ZBPF \+ \- \+ \- \- \- \- ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Clusters of TF binding sites immediately upstream of the transcription start site in 3 genes of cluster set 15 (co-expressed genes with high expression in thymus): The upper panels compare the location of TF binding sites surrounding and upstream regions of transcription start site (based on the corresponding mRNA annotations from NCBI\'s RefSeq database) of human and mouse Arid1a, Abcg1and Zfp162genes in GenomeTrafac database <http://genometrafac.cchmc.org>. The bottom panels list the gene descriptions and each of the binding sites in their order of occurrence from distal (top) to proximal (bottom) to exon 1 of the human gene, which is on the left within each panel. Binding sites in bold are known \"lymphoid elements\". The first nucleotide of exon 1 is at bp 40,001. Each of the colored bars represents a class of TF binding sites and connects homologous binding sites in genes of the two species. The orthologous genes may differ in the number and location of specific TF\'s binding sites. The corresponding coordinates of the regions on human (NCBI Build 35, May 2004) genome assembly are: chr1: 26,706,590--26,706,986 (+), chr21: 42,512,113 42,512,317 (+) and chr11: 64,302,720--64,302,924 (-) for human ARID1A, ABCG1and SF1respectively. Coordinates in the mouse genome assemblies (Build 33 Mouse Assembly, May 2004) are: chr4: 132,206,952--132,207,348 (-), chr19: 6,151,958--6,152,162 (+) and chr17: 29,663,342--29,663,546 (+) for Arid1a, Abcg1and Zfp162respectively. ::: ![](1471-2164-5-82-4) ::: Figure [4](#F4){ref-type="fig"} shows the computationally predicted arrangement of cis-elements immediately upstream of the transcription start site (promoters) of specific individual genes: Arid1a, Abcg1, and Zfp162. Elements were required to be within 500 bp of transcription start to be shown in Figure [4](#F4){ref-type="fig"}, which focuses on sequence conservation in classical promoters of pairs of orthologs and does not require that elements be shared with other genes. Modules in Table [2](#T2){ref-type="table"} were within 3 kb of transcription start, which could include both classical promoters and upstream enhancers, and were shared by more than one pair of orthologs. A number of the elements of modules listed in Table [2](#T2){ref-type="table"} are also present in the predicted promoters. For example, MAZF and SP1F are also present in the promoters of Arid1a, Abcg1and Zfp162. ### K-cluster Set 7 K-Cluster set 7 includes 19 genes. As a group the genes were better expressed in stimulated lymph nodes and activated T-cells than in the other tissues. Expression was characteristically low in peripheral blood mononuclear cells and in other non-immune adult and fetal tissues. Among the genes in set 7 are the integral surface membrane protein CD72found on B-cells, the transcription regulators Irf5and Icsbp1, the tyrosine kinases Hck, Stk10, and Lynthat are a part of the intracellular signaling cascade, the mitogen activated protein kinases Map3k1and Map4k1that participate in the very earliest steps of induction of new gene expression after lymphocytes are exposed to antigen, and the ATP-binding cassette transporters Abca7and Tap1of the type that transport peptides during antigen processing. Other less well-characterized genes in these sets may have functions similar to the genes that are better annotated. Sequences of 11 genes from Set 7 and their human orthologs were examined for the presence of clusters of TF binding sites, at least one of which is a lymphoid element, as defined in Methods. The 11 genes shared relatively few clusters of TF binding sites. There were 7 clusters shared by 3 genes. The largest cluster contained 6 elements, AP2F CDEF EGRF SP1F ZBPF ZF5F and was shared by Irf5and Stk10. There were 21 clusters shared by 2 genes and containing 3 to 6 TF binding sites. The composition and location of these are shown in images from CisMols (Additional files [3](#S3){ref-type="supplementary-material"},[4](#S4){ref-type="supplementary-material"},[5](#S5){ref-type="supplementary-material"},[6](#S6){ref-type="supplementary-material"},[7](#S7){ref-type="supplementary-material"},[8](#S8){ref-type="supplementary-material"} and [9](#S9){ref-type="supplementary-material"}). ### Highly expressed genes In addition to searching for potential regulatory regions within sets of genes clustered by similarities of patterns of expression across sets of tissues and within regions immediately upstream of exon 1, we also sought to identify genes characterized by high expression in specific immune tissues. It is not known whether clustering by pattern of expression across tissues and/or grouping by high expression in specific tissues (or neither) will be a useful way to group genes for computational identification of regulatory elements and regulatory regions. It is clear, however, that although modules of cis-elements that regulate expression of genes in tissues can occur at many different locations relative to a gene\'s promoter, at least some regulatory elements are located within promoter regions and this is the region we have searched most intensively for conservation of known TF binding sites. For the purposes of this analysis, we defined genes that were highly expressed based on their normalized expression being at least 4 times higher in an individual immune tissue relative to their median signal across the entire database. High expression in a single tissue does not preclude significant expression in other tissues, so high expression is not synonymous with unique expression. We examined highly expressed mouse genes and their human orthologs for the presence of clusters of TF binding sites, with the additional constraint that at least one of the cis elements present in the cluster was a lymphoid element, as defined in Methods. Grouped by tissue, suitable paired mouse/human orthologs were: activated T-cells, 17 genes: Ctsz, Kpnb1, Tnfrsf9, Tnfrsf4, Myc, Mcm2, Mcm5, Mcm6, Mcm7, Gzmb, Ncf4, Gapd, Ccl4, Pcna, Rpl13, Cd86, Icsbp1; thymus, 7 genes: Satb1, Hdac7a, Sgpl1, Abca1, Prss16, Abcg1, C1qg; stimulated lymph node, 4 genes, Stk10, Irf5, Cxcl9, Tnfrsf1. Identical analyses of 6 genes highly expressed in skeletal muscle (Ckm, Myf6, Aldo1, Myog, Dmd, Chrm3) and 8 in liver (G6pc, Cyp7a1, Proc, Ttr, Aldo2, Ins2, Igf1, Pah) served as negative controls, i.e. not tissues that play a critical role in lymphocyte differentiation or the immune response. The MCM family and Mycare involved in replication of DNA and chromosomes. The TNF and TNFR families of genes encode receptors and ligands that couple directly to signaling pathways for cell proliferation, survival and differentiation \[[@B41]\]. Prss16encodes a thymus specific protease which is specifically expressed by epithelial cells in the thymic cortex and plays a role in T-cell development and, perhaps, in susceptibility to autoimmunity \[[@B42]\]. Hdac7aencodes a histone deacetylase. Members of the Hdacfamily of genes modify histones and play a role in the regulation of expression of genes such as those functioning in the cell cycle, apoptosis, and transcription \[[@B43]\]. Cxcl9 is an inflammatory chemokine induced by interferon. Its promoter contains binding sites for CREB, STAT1, and NFKB \[[@B44]\]. The results of the above approach are shown in Table [3](#T3){ref-type="table"}, which lists examples of putative computationally identified regulatory modules of immune genes and the cis-elements that they contain. When modules of genes highly expressed in thymus, stimulated lymph nodes, or activated T-cells were compared with one another and to modules of genes expressed in muscle and liver, it is clear that the composition (cis-elements) of modules are not unique to a specific gene. However there is some evidence of unique arrangements of elements within modules. There are also cis-elements that are not commonly shared. For example, the individual cis-elements HESF, HAML, MYT1 and P53F were not found in modules of genes other than those highly expressed in thymus. Likewise, E2FF was only present in modules of activated T-cells, but clearly does not play a unique role in the immune system. Members of the E2F family of TFs are key participants in cell proliferation, apoptosis, and differentiation \[[@B45]\]. E2FF is found in promoters of Mcm2, Mcm5, Mcm6, Mcm7, and Myc. These genes are highly expressed in proliferating cells generally, an example of which is the activated T-cell. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Examples of modules of shared cis-elements found in highly expressed genes in 3 immune tissues. Elements were clustered with at least 2 other cis-elements within a 200 bp window, indicating the presence of a putative regulatory module which contained at least 3 transcription factor binding sites, one of which was required to be a lymphoid element. All are located within 3 kb upstream and 100 bp downstream of the first bp of exon 1. The modules were present in the mouse and human orthologs of at least 2 genes from sets of genes that were highly expressed in thymus, stimulated lymph nodes, or activated T-cells. The number of genes for which orthologs were available: thymus, 7; lymph node, 4; activated T-cells, 17. ::: Thymus Stimulated Lymph Node Activated T-cell ------------------------------- ------------------------------- ------------------------------- MAZF SP1F ZBPF AP2F CDEF EGRF SP1F ZBPF ZF5F MAZF SP1F ZBPF EGRF MAZF SP1F ZBPF LHXF NKXH OCT1 RBIT CREB SP1F ZBPF ETSF SP1F ZBPF EGRF ETSF NFKB E2FF MAZF SP1F AP2F EGRF HESF MAZF SP1F ZBPF GATA HOXF NKXH ECAT PCAT SP1F ZBPF ETSF MAZF SP1F STAT ZBPF LHXF NKXH OCT1 ETSF MAZF MZF1 AP2F EGRF ETSF SP1F ZBPF NKXH OCT1 RBIT EGRF SP1F ZBPF EGRF MAZF P53F SP1F IKRS MAZF NFKB GATA HAML MYT1 E2FF EBOX ETSF MAZF SP1F ZF5F BCL6 CREB E2FF STAT HOXF LEFF LHXF OCT1 MAZF MZF1 NFKB PAX5 ::: Regulatory modules, which have been proved biologically to regulate expression of genes, contain multiple TF binding sites, much as is shown in Figures [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"} and [4](#F4){ref-type="fig"}. Examples of modules of shared cis-elements (i.e., within a 200 bp window) in highly expressed genes are listed in Table [3](#T3){ref-type="table"}. For example, of the modules highly expressed in thymus SP1F MAZF ZBPF was present in paired orthologs of Abca1, C1qg, Abcg1and Sgpl1; module AP2F EGRF HESF MAZF SP1F ZBPF was present in Sgpl1and Abcg1. Of the modules highly expressed in stimulated lymph nodes, AP2F CDEF EGRF SP1F ZBPF ZF5F was present in Stk10and Irf5; module GATA HOXF NKXH was present in Stk10and Irf5. Of the modules highly expressed in activated T-cells, E2FF EBOX ETSF MAZF SP1F ZF5F was present in Kpnb1and Mcm6; module BCL6 CREB E2FF STAT was present in Icsbp1and Tnfrsf4. Discussion ========== Individual differentiated biological states can be characterized by gene expression profiling. Large-scale comparisons of profiles of cells, tissues, and developmental stages have the potential to identify a wealth of coordinately regulated groups of genes that reflect the interplay of their functional relationships and transcriptional control mechanisms. We have built a database comprised of the mRNA expression profiles of 65 normal adult and fetal C57BL/6J mouse tissues using the Incyte Mouse GEM1, 8638 element, clone set. Using microarray analysis, 680 sequences were identified that were highly expressed in one or more of 6 immune tissues. Many were also expressed in certain other tissues. Some of these other tissues were organs such as heart, kidney, and brain which do not normally contain lymphocytes in large numbers and do not play a role in the immune response. Others, such as intestine and lung, interface with the external environment, contain significant numbers of lymphocytes, and can mount an immune response. The 680 expressed sequences were filtered to remove 320 that were expressed in \"non-immune\" brain or heart or kidney. This resulted in a list of 360 expressed sequences called \"immune genes\" that were less broadly expressed in tissues without immune function than were the 680. Mutations and polymorphisms in both the 680 expressed sequences and the 360 immune genes have a significant chance of specifically affecting immune function. We predict this will be more common with changes in the 360 immune genes. We tested this by comparing reports of disease causing mutations in the 360 immune genes with those reported for the 320 genes that were more broadly expressed (Online Mendelian Inheritance in Man). Of the 360 mouse immune genes, 32 had an ortholog with gene symbol in OMIM and17 had annotations that described a function clearly linked to development or function of the immune system. Mutations in 2 (LCP2and PARVG) cause severe immunodeficiency disease. Examples of other diseases caused by mutations in these 32 genes were B- and T-cell malignancies, autoimmune disorders, and reduced viral or bacterial resistance. Of the 320 genes removed from the list of 680, 37 had orthologs with gene symbols listed in OMIM. 4 genes were expressed in lymphocytes and mutation in one, Bruton\'s tyrosine kinase, causes agammaglobulinemia. Mutations in other genes caused disorders of coagulation, red cells, or granulocytes, rather than the immune system. We conclude that the list of 360 immune genes includes a higher percentage of genes preferentially expressed in immunocompetent tissues and with more specific immune-related functions than does the full list of 680 sequences expressed in immune tissues, but also with expression in non-immune tissues. The 360 immune genes represent a portion of the complete set of genes that encode proteins and processes necessary for the differentiation, maintenance, and function of the immune system. These genes are functionally diverse and represent both ubiquitous and specialized cellular processes. 10 or more genes are in specific functional clusters that carry out general processes such as DNA and chromosomal replication, cell cycle regulation, transcription, and translation. Other genes are in functional clusters that carry out specialized functions, largely restricted to immune tissues. These include genes that encode proteins involved in antigen recognition and transport, chemokine synthesis, chemokine recognition, and the intracellular signaling cascade necessary to initiate transcription and new protein synthesis in lymphocytes, as part of the host response to antigen. Functional annotation of these genes is a work in progress. While probable functions have been assigned to most of the expressed sequences and the genes that encode them, using information shown in the [Additional file 1](#S1){ref-type="supplementary-material"}, there is much work to be done. Most functional annotations are based on the sharing of presently known protein domains and sequence homologies and provide general clues to the role a gene or protein may play in cells that participate in the immune response to antigen. A more precise understanding will come about as new laboratory data are correlated with studies of the expression of specific immune genes, their coordination with expression of other genes, and the structure and function of their products. For several reasons, the \"immune genes\" that we have identified are not all of the genes that are expressed in immune tissues: (1) the Incyte set of 8638 genes probably contains representative cDNAs from 25% or less of all mouse genes; (2) genes that are essential to immune function, but are expressed at similar levels in immune and other tissues will not be included in the immune set; and (3) a gene with a very low level of expression will be missed, if cDNA made from its RNA is not present in sufficient quantity to give a signal on the microarray. Genes may be included or excluded in error because of the large number of genes screened for expression with a limited number of replicates. Incyte cDNA microarrays are no longer manufactured and no Incyte arrays or public databases are available to check expression of our immune genes in other species. There are two relevant publicly available Novartis gene expression databases (Genomics Institute of the Novartis Research Foundation, \[[@B18]\]), which can be accessed. One uses Affymetrix chip U74Av2 and a set of 90 mouse tissues and cell lines and the second uses Affymetrix HG U133 and 158 human tissues and cells. Relating Affymetrix probes to Incyte cDNA probes is complex and the Novartis tissue sets do not contain the same tissues we have used. However, our immune genes, when expressed on the Novartis arrays, are generally clustered in tissues of the immunohematopoietic system, the gastrointestinal tract, and lung. These types of publicly available databases will permit identification and functional annotation of new immune genes with consequent availability of larger sets of coordinately regulated genes for searches of conserved regulatory modules. Using comparative genomics-based, cis-element analyses (<http://trafac.cchmc.org>\[[@B15]\] and <http://genometrafac.cchmc.org>\[[@B46]\]), we identified compositionally similar clusters of cis-elements in upstream regions of mouse/human orthologs of several immune genes. There was an excellent agreement between the computationally predicted and experimentally determined arrangements of cis-elements in the promoters of the mouse H2-Kand human HLA-Agenes. Analyses of other immune genes identified a wealth of potential immune system-specific regulatory modules. For example (Table [2](#T2){ref-type="table"}), Arid1a, Abcg1, and Sgpl1are members of a K-clustered set of immune genes and share a phylogenetically conserved module of 5 cis-elements: AP2F EGRF MAZF SP1F and ZBPF, all within a 200 bp interval. Other examples of clustered TF binding sites that could be within regulatory modules of genes highly expressed in specific tissues are given in RESULTS. Striking examples of putative modules include the 6 cis-element module AP2F EGRF HESF MAZF SP1F ZBPF in genes highly expressed in thymus; the 6 cis-element module E2FF EBOX ETSF MAZF SP1F ZFSF in genes highly expressed in activated T-cells; and the 6 element module AP2F CDEF EGRF SP1F ZBPF ZF5F in genes highly expressed in stimulated lymph nodes (Table [3](#T3){ref-type="table"}). Putative regulatory modules are not distributed randomly across an entire segment of DNA, but are highly clustered within distinct short segments that are the computationally identified promoters and enhancers (Figure [3](#F3){ref-type="fig"}). Because of the nature of the scanning algorithm with its 200 bp window, variations of multiple modules may occur within one segment. These phenomena are more easily understood by examining Figure [3](#F3){ref-type="fig"}. Our data support the hypothesis that (1) regulatory modules of genes are highly clustered in a few sites that can be computationally identified, (2) modules in different genes may share cis-elements that bind TFs, and (3) certain combinations of TF binding sites are phylogenetically conserved and appear to be reused across genes when specific patterns of expression are required. Cis-elements from the same family have a high probability of interacting with similar groups of transcription factors, although they will not necessarily be in the same position relative to the transcription start site. We have identified genes and putative regulatory modules that play a role in the differentiation, maintenance, and function of the immune system. These results serve to advance both our understanding of normal gene and immune system function and also to identify genes and regulatory regions whose mutation or polymorphic variation lead to immunologic disease. Methods ======= C57BL/6J mice from The Jackson Laboratory were the source of normal adult and fetal tissues. The complete panel of tissues for microarray analyses by our group has been described \[[@B47]\]. Peripheral blood mononuclear cells were separated from whole blood on Ficoll/Hypaque gradients; unstimulated lymph nodes, spleen, and thymus were each collected from unimmunized mice and pooled separately; \"stimulated\" lymph nodes were collected from mice 10 days after they were immunized with hen egg-white lysozyme (HEL) in complete Freund\'s adjuvant; activated T cells were prepared by enriching T cells from peripheral blood and treating them with anti-CD3 and anti-CD28. Except for activated T-cells and pancreatic islet cells, all cells and tissues were collected in duplicate. 128 preparations of poly (A)-RNA were made from 65 different tissues, checked for quality, and quantified as previously described \[[@B19],[@B47]\]. Microarray analyses were carried out using Incyte mouse GEM1 cDNA arrays (Incyte Genomics, Palo Alto, CA), as described previously for our group \[[@B19],[@B47]\]. Relative abundance of probes was calculated as the ratio of the sample value against the value from the labeled whole mouse reference cDNA for each gene on each array. Data analyses were carried out with GeneSpring version 4.2.1 (Silicon Genetics) software, including filtering, K-means and hierarchical clustering. A list of all tissues in the full set of 65 normal adult and fetal tissues is provided in [Additional file 12](#S12){ref-type="supplementary-material"}. Our analyses focused on comparison gene expression in 18 tissues that were selected to represent a variety of adult and fetal tissues (Figure [1B](#F1){ref-type="fig"}), most with immunological function. 6 of the 18 tissues were the \"immune tissues\" -- unstimulated and stimulated lymph nodes, spleen, peripheral blood mononuclear cells, activated T-cells, and thymus. The remaining 12 tissues of the 18 tissue set were: fetal day 16.5 intestine and lung; adult duodenum, jejunum, ileum, proximal and distal colon; adult lung and liver, and joint synovium from normal adult mice and mice with acute and chronic arthritis. All pertinent microarray data are available through the Children\'s Hospital Research Foundation expression database web server <http://genet.chmcc.org> within the ExpressionDB folders of the Incyte Mouse GEM1 chip genome. Genes on the Incyte array were identified by NCBI GenBank accession and systematic numbers and by gene symbol, where available. For those sequences that could not be assigned a gene symbol, sequence homologies to known mouse genes were sought using MouseBLAST \[[@B23]\], BLAT \[[@B12]\], MGD \[[@B23]\], and LocusLink \[[@B24]\]. BLAST comparisons of the human and mouse confirmed Ensembl predictions of human orthologs of mouse genes. Identity of genes was confirmed by BLAST comparison of the GenBank sequences from NCBI <http://www.ncbi.nlm.nih.gov>\[[@B48]\] with Ensembl \[[@B13]\] sequences. When downloading the genomic sequences with flanking sequences, it was important to have an mRNA that contained exon 1, so the site of initiation of transcription was correctly identified. Presence of an upstream exon 1 in an isoform would lead to re-defining of the promoter and intronic regions. Criteria for presence of exon 1 included: comparison of the number and location of exons in orthologous genes, alignment of transcripts of the gene as reported by different databases, and alignment of the 5\' end of the transcript with the putative start site and signals in the gene. In cases where we encountered multiple high scoring transcript hits against the genome, we manually looked into the alignments to rule out the occurrence of pseudogenes that frequently lacked introns when compared to the \"true\" genes. Additional information about sequences of both the transcript and the gene was obtained from UCSC Golden Path \[[@B12]\]. Confirmation of the presence of exon 1 in orthologs was particularly important because of the need to locate the start site of transcription. Computational prediction of exons is error prone. DNA sequences of genes were downloaded to include at least 10,000 flanking base pairs upstream and downstream of the first and last exons respectively. The November 2002 and April 2003 assemblies of human and the February 2002 and February 2003 assemblies of mouse genome were used for this purpose depending upon their availability at the time of our analyses ([Additional files 10](#S10){ref-type="supplementary-material"} and [11](#S11){ref-type="supplementary-material"} list relevant FASTA sequences and genomic coordinates). The GO and MGI databases were searched for annotations of the immune genes, using Stanford SOURCE <http://source.stanford.edu>\[[@B40]\]. For genes not found or incompletely annotated, manual annotation was done using criteria similar to the Gene Ontology (GO) \[[@B49]\], Mouse Genome Informatics (MGI) \[[@B23]\], and LocusLink classifications <http://www.ncbi.nlm.nih.gov/LocusLink>\[[@B50]\]. A function was assigned if the encoded protein contained distinctive InterPro functional domains, or sequence similarity to paralogs previously annotated, or sequence similarity to functionally characterized SwissProt/TrEMBL proteins. Using the information about structure and function, the authors simplified annotations and grouped genes by major functions, such as antigen binding and processing (defense -- immune function), transcription, protein synthesis, apoptosis, cell division. Highly detailed annotations are provided in the supplementary materials ([Additional file 1](#S1){ref-type="supplementary-material"}). To identify putative consensus cis-acting regulatory sequences in genes that were coordinately regulated, we first selected groups of genes based on their expression patterns in different immune tissues. The complete genomic sequences (with flanking upstream and downstream regions of 40 kb) of the selected genes and their orthologs were extracted from the Ensembl/UCSC human and mouse databases \[[@B12],[@B13]\]. Where available the NCBI-RefSeq mRNAs were used as references for downloading the genomic sequences with upstream and downstream gene flanking regions of 40 kb. The transcription start site was thus at 40,000 in the downloaded sequences used in comparative genomic analysis for identification of potential regulatory clusters using Trafac server \[[@B15]\]. Repeat elements were masked using the RepeatMasker <http://ftp.genome.washington.edu>\[[@B51]\]. Conserved clusters of regulatory elements in the evolutionarily conserved non-coding regions of mouse and human orthologs were displayed using the TraFaC <http://trafac.cchmc.org>\[[@B15]\] or GenomeTraFaC <http://genometrafac.cchmc.org>\[[@B46]\] servers which integrate results from MatInspector Professional (Version 4.1, 2004; 356 individual matrices in 138 families) <http://www.genomatix.de>\[[@B52]\] and Advanced PipMaker (chaining option) <http://pipmaker.bx.psu.edu/cgi-bin/pipmaker?advanced>\[[@B53]\] programs. We compared conserved putative cis-regulatory regions of each of the different groups of genes from mouse and human to identify known TF binding sites. The CisMols analyzer <http://cismols.cchmc.org>\[[@B22]\] permits selection of TFs that must be present in clusters of TFs that constitute a putative regulatory module. To convey specificity to the search for modules relevant to regulation of gene expression in immune tissues, we required the presence of one or more of the following TFs, which we call \"lymphoid elements\". They have been reported to play a role in some aspect of lymphoid biology (see for example, \[[@B1]-[@B3]\]: BCL6, CMYB, CREB, EGRF, ETSF, GATA, IKRS, IRFF, MZF1, NFAT, NFKB, OCT1 (site also binds OCT2), PAX5, SP1F, VMYB, and WHZF. ECAT and PCAT were also included because of their frequent occurrence in promoters at the start of transcription. The search was limited to a region 3 kb upstream and 100 bp downstream of the start site of exon 1 (based on the NCBI-RefSeq mRNA annotations). This is where the promoter and associated regulatory elements would be expected, given that additional regulatory elements (enhancers/silencers) are almost certain to be located elsewhere. Images of the CisMols analyses of genes to identify regulatory elements are also provided in supplementary materials ([Additional files 3](#S3){ref-type="supplementary-material"} to [9](#S9){ref-type="supplementary-material"}). One example is shown in Figure [3](#F3){ref-type="fig"}. Authors\' contributions ======================= JJH and BJA were primarily responsible for the design, coordination and conduct of the study. AGJ and AG were responsible for regulatory region analyses and software development. AGJ was responsible for ortholog analysis and novel ortholog assignments. JJH, BJA and AGJ drafted the manuscript and figures and JJH, BJA and AGJ contributed editorial revisions. SK, SW and CE were responsible for generating, quality assurance, and initial assembly of the gene chip data. JDK provided purified lymphoid cells, read the manuscript and provided comments and discussion. All authors read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 List and annotation of 360 expressed sequences (\"immune genes\"). ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 10 FASTA sequences and the corresponding coordinates on the human and mouse genome assemblies (May 2004) of the promoter regions used in the analysis and displayed in figures 2 and 4. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Using K-means clustering in GeneSpring (Version 4.2.1), 160 annotated genes, were divided into distinct sets based on similarity of expression patterns across 15 tissues. Tissues were given equal weight, the number of clusters was set at 20, and similarity was measured by standard correlation. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 11 FASTA sequences and the corresponding coordinates on the human and mouse genome assemblies (May 2004) of the promoter regions used in the analysis and displayed in figures 2 and 4. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 3 CisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one \"lymphoid element\". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI\'s RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 4 CisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one \"lymphoid element\". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI\'s RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 5 CisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one \"lymphoid element\". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI\'s RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 6 CisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one \"lymphoid element\". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI\'s RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 7 CisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one \"lymphoid element\". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI\'s RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 8 CisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one \"lymphoid element\". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI\'s RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 9 CisMols display of location and composition of clusters of cis-elements that are putative regulatory modules for the genes in various groups (test and control). Each colored cube indicates a cluster of 3 or more cis-elements with at least one \"lymphoid element\". The region searched is upstream 3 kb and downstream 100 bp of transcription start site (as defined by the respective mRNAs from NCBI\'s RefSeq database). The legend in the lower left half of the figure indicates the composition of each of the modules and the genes that share them. ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 12 Tissue lists used in the generation of microarray profile data. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This work was supported in part by an award from the Howard Hughes Medical Institute to the University of Cincinnati for the development of Bioinformatics Core Resources, and by grants NIEHS U01 ES11038 and ES06096 Mouse Centers Genomics Consortium, Center for Environmental Genetics, NCI Mouse Models of Human Cancer Consortium and the National Library of Medicine G08 LM007853 IAIMS. We thank Amy Sherman of Incyte Genomics, Andrew Conway of Silicon Genetics, Paul Spellman and Rodney DeKoter for valuable discussion.	PubMed Central	2024-06-05T03:55:49.912122	2004-10-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534115/", "journal": "BMC Genomics. 2004 Oct 25; 5:82", "authors": [ { "first": "John J", "last": "Hutton" }, { "first": "Anil G", "last": "Jegga" }, { "first": "Sue", "last": "Kong" }, { "first": "Ashima", "last": "Gupta" }, { "first": "Catherine", "last": "Ebert" }, { "first": "Sarah", "last": "Williams" }, { "first": "Jonathan D", "last": "Katz" }, { "first": "Bruce J", "last": "Aronow" } ] }
PMC534613	Background ========== The human uterine cervix undergoes biochemical changes resulting in softening, effacement and dilatation during pregnancy and labor. This remodeling, or ripening, is a prerequisite for parturition \[[@B1]\]. It is characterized by inflammatory events, such as extravasation of neutrophils and macrophages \[[@B2],[@B3]\] and an increased cervical level of pro-inflammatory cytokines such as interleukin (IL)-8 \[[@B3],[@B4]\]. Progesterone, essential for the maintenance of pregnancy, and glucocorticoids have anti-inflammatory properties \[[@B5]\]. Placental production of progesterone and adrenal synthesis of glucocorticoids \[[@B6]\] increase markedly during human pregnancy. The antiprogestin (RU486), successful for labor induction at term in humans \[[@B7]\], also has anti-glucocorticoid properties. Progesterone and cortisol regulate the human placental corticotropin-releasing hormone (CRH) gene \[[@B8]\]. Placental CRH, synthesized in abundance in the human placental syncytiotrophoblasts and trophoblasts \[[@B9]\], has been proposed to be a key regulator for human parturition trough interactions with adrenal steroids and estrogen \[[@B10]\]. Among the group of structurally related, ligand-inducible nuclear steroid receptor transcription factor proteins, GR was the first that was cloned and sequenced \[[@B11]\]. GR and the progesterone receptor (PR) share structural similarities, and they interact with the same hormone responsive elements \[[@B12]\]. The GRα and GRβ isoforms are derived from the same gene, and GRα is the major form found in human cells and tissues \[[@B13]\]. NFκB is a key pro-inflammatory regulator. GR and NFκB are both inducible transcription factors with diametrically opposed functions in inflammatory responses. A mutual negative direct and indirect cross talk between GR and NFκB has been well described in previous studies \[[@B5],[@B14]\]. Prostaglandin E~2~(PGE~2~) is widely implicated for cervical ripening in clinical practice \[[@B15]\]. Among the COX enzymes, regulating prostaglandin synthesis, the COX-1 form is constitutively expressed, whereas COX-2 is inducible and particularly involved in inflammatory events \[[@B16]\]. The COX enzymes are down regulated by cortisol in human decidua, myometrium and cervix \[[@B17]\]. Platelet-activating factor (PAF) is a lipid pro-inflammatory mediator, involved in several reproductive processes, i.e. parturition \[[@B18]\]. PAF is synthesized by some leukocytes, blood platelets and vascular endothelial cell \[[@B19]\]. The PAF receptor (PAF-R) is a G-coupled membrane receptor with an estrogen responsive element within its promoter region, enabling regulation by estrogens \[[@B20]\]. The activation of PAF-R is associated with cytoskeletal remodeling and expression of pro-inflammatory modulators, such as COX-2, IL-6 and IL-8 \[[@B21]\]. Thus, PAF-R and COX enzymes have been widely demonstrated as factors involved in the events promoting and proceeding parturition, yet their cell origin in the human uterine cervix remains to be clarified. Nitric oxide (NO) is synthesized intracellularly from the amino acid L-arginine through the activity of specific synthase enzymes (NOS) \[[@B22]\]. The inducible form, iNOS, present in e.g. macrophages, regulates the level of NO in response to various inflammatory stimuli, including proinflammatory cytokines and lipopolysaccharides. NO stimulates PGE~2~release from human cervical tissue explants \[[@B23]\], and is a powerful regulator of COX-2 thereby increasing local PGE~2~concentrations in inflammatory tissues \[[@B24],[@B25]\]. NO donors do induce cervical ripening in human pregnancy in the first trimester \[[@B26],[@B27]\], at term \[[@B28]\] and in non-pregnant women \[[@B29]\]. Besides, treatment with the NO donor isosorbide-5-mononitrate stimulates production of e.g. COX-2 and PGE~2~in human cervix \[[@B27]\]. The action of NO on cervical ripening appears to be accomplished by effects on connective tissue and smooth muscle cells in a similar way as previously been shown for prostaglandins \[[@B27]\]. Our hypothesis is that glucocorticoids exert a direct receptor mediated effect in the human cervix uteri, and that a changed biological response to glucocorticoids could be a mechanism behind the events resulting in cervical ripening at parturition. Since NFκB has opposed functions in inflammatory responses as compared to GR, we presume that NFκB could also be a regulator of the inflammatory events leading to cervical ripening. These inflammatory events could be mediated via factors such as the PAF-R, iNOS and/or COX enzymes. Methods ======= Study patients -------------- All women gave their informed consent and the Local Ethics Committee of the Karolinska Hospital approved the study. All were healthy, had uncomplicated pregnancies and were without medication prior to parturition. The non-pregnant (NP) women were hysterectomised due to benign disorders not involving the cervix. The women in the term pregnant (TP) group all had unripe cervices with a Bishop score \<5 points and none of them were in labor. Biopsies were obtained during elective caesarean sections before onset of labor. The biopsies from the post partal (PP) women were taken after a normal vaginal delivery. For the immunohistochemistry study of GR and NFκB, cervical biopsies were obtained from one para and eleven primipara TP women (n = 12) with a mean age (range) of 33 (28--38) years, and a mean gestational age of 38 (37 to 40) weeks. The women of the PP group (n = 14) were all primipara and had a mean age of 31 (22--37) years, and a gestational age of 40 (39 to 42) weeks. The NP control group (n = 8) had a mean age of 43 (32--49) years, and a mean parity of II (I-III). The cervical samples available for the immunohistochemistry studies of COX-1, COX-2 and PAF-R were TP (n = 8), PP (n = 10) and NP group (n = 6). Biopsies for RNA preparations were not available for the RT-PCR study from all subjects, NP (n = 5), TP (n = 6) and PP (n = 5). The women in the NP group were significantly older than those of the other two groups. Since hysterectomies in young women are uncommon most biopsies in the NP group are from women in the middle of their 40s, but they were all menstruating regularly and did not receive any medication. Tissue collection ----------------- Cervical biopsies were obtained transvaginally (for the TP and PP groups) from the anterior cervical lip at the 12 o\'clock position, from 10--20 mm depth. The tissue samples from the hysterectomies were obtained directly after the uterus was removed during operation. The same physician (YSV) collected all the samples. The biopsies were immersion-fixed in 4% phosphate buffered formaldehyde at 4°C overnight, stored at 4°C in 70% ethanol and thereafter embedded in paraffin. From the biopsies that were large enough, a small piece was cut off prior to fixation, and frozen in -70°C until RNA preparation. RNA preparation and reverse transcription ----------------------------------------- Total RNA from frozen cervical tissue samples was purified with the SV Total RNA isolation system (Promega, Madison, WI) according to a procedure recommended by manufacturer. One microgram of total RNA from each sample was reverse transcribed at 42°C for 45 min in a final volume of 40 μl with a reaction mixture containing 50 mmol/l Tris-HCl (pH 8.3), 75 mmol/l KCl, 3 mmol/l MgCl~2~, 7.5 mmol/l dithiothretiol, 0.5 mmol dNTPs, 1 μg random hexameters, and 400 U of Moloney murine leukemia virus reverse transcriptase (Gibco-BRL, Paisley, UK). RT-PCR ------ Oligonucleotide primers for the GRα gene were as follows \[[@B30]\]: 5\'-CCT AAG GAC GGT CTG AAG AGC-3\' and 5\'-GCC AAG TCT TGG CCC TCT AT-3\', corresponding to nucleotides 2158-2178 and 2616-2635 of the human GRα cDNA (GenBank accession No X03225). Oligonucleotide primers for the GRβ gene were as follows \[[@B30]\]: 5\'-CCT AAG GAC GGT CTG AAG AGC-3\' and 5\'-CCA CGT ATC CTA AAA GGG CAC-3\', corresponding to nucleotides 2158-2178 and 2503-2523 of the human GRβ cDNA (GenBank accession No X03348). Oligonucleotide primers for the COX-1 gene were as follows: 5\'-TGC CCA GCT CCT GGC CCG CCG CTT-3\' and 5\'-GTG CAT CAA CAC AGG CGC CTC TTC-3\', corresponding to nucleotides 568-591 and 871-847 of the human COX-1 cDNA \[[@B31]\]. Oligonucleotide primers for the COX-2 gene were as follows: 5\'-TTC AAA TGA GAT TGT GGG AAA ATT GCT-3\' and 5\'-AGA TCA TCT CTG CCT GAG TAT CTT-3\', corresponding to nucleotides 574-601 and 878-854 of the human COX-2 cDNA \[[@B32]\]. The predicted size of the PCR products was 477 bp for GRα, 366 bp for GRβ, 304 bp for COX-1 and 305 bp for COX-2. For PCR, the cDNAs corresponding to 50 ng RNA were added to 10 μl of HotStarTaq^®^master mix (Quiagen GmbH, Hilden, Germany) containing 2.5 μM of each oligonucleotide primer in a final volume of 20 μl. The reaction mixture was overlaid with mineral oil. After an initial incubation for 15 min at 95°C, the samples were subjected to 33 (GRα, COX-1 and COX-2) and 40 (GRβ) cycles of 30 s at 94°C, 40 s at 60°C, and 60 s at 72°C with a final extension step at 72°C for 10 min in the DNA Thermal Cycler 480 (Perkin-Elmer, Norwalk, CT). The amount of PCR product for GRα increased linearly up to 36 cycles and for COX-1 and COX-2 it increased linearly up to 38 cycles (data not shown). Quantitative measurement of GRβ mRNA would require larger amounts of cervical RNA than were available, since GRβ showed very low expression with a visible band only after 40 cycles. To standardize the quantification method, an endogenous 18S rRNA was used as an internal standard. The 18S rRNA primers and Competimers™ (modified at their 3\' ends to block extension by DNA polymerase) were obtained from Ambion (Quantum RNA 18S Internal Standards; Ambion Austin, TX). The standard, and the GR and COX mRNAs were amplified in parallel and under the same conditions. A mixture of 18S primers and Competimers™ (1:9) was used to modulate amplification efficiency of 18S rRNA to the same linear range as GRα, COX-1 and COX-2 when amplified under the same conditions. The predicted size of the PCR product for 18S was 489 bp. The PCR products were run on 2% agarose gel and stained with ethidium bromide. Bands were captured and analyzed using ChemiDoc Gel Documentation System (Bio-Rad Laboratories, Hercules, CA). The levels of GR and COX PCR products were normalized against the 18S product. The RT-PCRs were repeated twice. Immunohistochemistry -------------------- Paraffin sections (5 μm) were used and a standard immunohistochemical technique (avidin-biotin-peroxidase) was carried out as described before \[[@B33]\] to visualize GR, NFκB, COX-1, COX-2, PAF-R and iNOS. After the tissues were dewaxed and rehydrated, an antigen retrieval procedure was performed. The sections were pre-treated by heating in a microwave oven at 700 W in 0.01 M sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide. All tissue sections were exposed to a non-immune block with normal goat serum. A polyclonal rabbit anti-human antibody was used for the detection of GR (ABR PAI-511A, Affinity Bioreagents, Inc., Golden, CO, USA). This antibody recognizes both GRα and GRβ. The tissue sections were incubated over night in +4°C with the primary antibody diluted 1:1000. A polyclonal rabbit anti-human antibody (DB033 Delta Biolabs, Cambell, CA, USA) was used for the NFκB immunostaining. The tissue sections were incubated with the primary antibody diluted 1:500 over night at +4°C. The primary antibodies were replaced with non-immune rabbit IgG in negative controls. Polyclonal goat anti-human (sc-1752 and sc-1745, Santa Cruz) antibodies were used (diluted 1:100) for the COX-1 and COX-2 respectively. The incubation with primary antibody was 1 hr at 37°C for both COX-1 and COX-2. A polyclonal goat anti-human (sc-8741, Santa Cruz) antibody was used for the PAF-R immunostaining. The tissue sections were incubated with the primary antibody diluted 1:100 over night at +4°C. Replacing the primary antibody with non-immune goat IgG was used as negative control. A monoclonal mouse anti-iNOS antibody was used for detection of iNOS (N32020, Transduction Laboratories, Lexington, KY, USA). It recognizes the C-terminal domain of iNOS. The antibody was diluted 1:50 and incubated for 70 minutes in room temperature. Replacing the primary antibody with non-immune mouse IgG was used as negative control. Image analysis -------------- A microscope and CCD video camera connected to a computer with an image analysis program (Leica Imaging System Ltd., Cambridge, UK) was used to assess quantitative values from GR and NFκB immunohistochemistry. The quantification of nuclear immunostaining was performed on the digitized images of systematic randomly selected fields of stroma and squamous epithelium. Ten fields were analyzed separately in each section of tissue, using the color-discrimination software. Positive staining is presented as a ratio of the area of positively stained nuclei (brown) to the total area of cell nuclei (brown and blue). Manual scoring -------------- Two observers blinded to the identity of the slides, performed all the assessments. The staining was evaluated semi-quantitatively using a grading system. The staining intensity was graded on a scale of (0) no staining, (1) very faint, (2) faint, (3) moderate and (4) strong staining. Statistics ---------- Statistical calculations for the data from the relative quantification of RT-PCR products, the immunohistochemistry results by image analysis and manual scoring were performed by ANOVA on Ranks (Kruskal-Wallis\' test) and significances were evaluated by Dunn\'s test. Values with different letter designations are significantly different (p \< 0.05). Results ======= GR -- By immunohistochemistry the GR protein was localized to the nuclei of cervical stroma (S), squamous epithelium (SQ), glandular epithelium (GE) and vascular endothelium (Figure [1a,1b,1c,1d,1e,1f,1g,1h,1i](#F1){ref-type="fig"}) in samples from the NP (left column), TP (middle column) and PP (right column) groups. By image analysis the GR levels were determined in SQ and stroma (Figure [2](#F2){ref-type="fig"}). The stromal cells include vascular epithelium and the leukocytes within the stroma. Blood cells within vessels (V) are excluded from the image analysis. Strong immunostaining was present in SQ, particularly in the basal and parabasal cell layers (Figure [1a,1b,1c](#F1){ref-type="fig"}). There was a significant decrease in immunostaining of the PP group as compared to the TP group, both in stroma and SQ (Figure [2](#F2){ref-type="fig"}). The stromal GR immunostaining was increased in the TP group as compared to the NP and PP groups (Figure [2](#F2){ref-type="fig"}). It was noted in all groups that GE (Figure [1g](#F1){ref-type="fig"} and [1i](#F1){ref-type="fig"}), vascular endothelium (Figure [1d,1e](#F1){ref-type="fig"} and [1f](#F1){ref-type="fig"}) and some intravascular and perivascular leukocytes (as identified by their morphology, black arrowheads, Figure [1d](#F1){ref-type="fig"} and [1f](#F1){ref-type="fig"}) stained positive for GR, while some leukocytes were negative (white arrowheads, Figure [1d](#F1){ref-type="fig"} and [1f](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Immunostaining of GR in the non-pregnant (NP) (left column, a, d, g), term pregnant (TP) (middle column, b, e, h) and postpartum (PP) (right column, c, f, i) groups. GR protein (brown staining) is present in the nuclei of cervical stroma (S), squamous epithelium (SQ), vessels (V) and glandular epithelium (GE) in all three groups. Some leukocytes, identified by their morphology, were positive for GR (black arrowheads) while others were negative (white arrowheads) (dand f). A negative control where the primary antibody was replaced by rabbit IgG is shown in h. ::: ![](1477-7827-2-74-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### GR levels, as assessed by image analysis of immunohistochemistry results, in cervical squamous epithelium (SQ) and stroma in samples from TP (n = 12) and PP (n = 14) as compared to NP (n = 8) women. Box and whisker plots represent the median value with 50% of all data falling within the box. The \"whiskers\" extend to the 5^th^and 95^th^percentiles. Boxes with different letter designations are significantly different, p \< 0.05. ::: ![](1477-7827-2-74-2) ::: NFκB ---- Positive NFκB immunostaining in cervix uteri was present in stroma, GE, SQ and vascular endothelium in NP (left column), TP (middle column) and PP (right column) groups (Figure [3a,3b,3c,3d,3e,3f,3g,3h,3i,3j,3k,3l](#F3){ref-type="fig"}). Strong positive staining for NFκB was also seen in neuronal ganglions (G, Figure [3h](#F3){ref-type="fig"}) and in smooth muscle cells/activated fibroblasts, both around blood vessels (Figure [3j](#F3){ref-type="fig"}) and within the stroma (Figure [3l](#F3){ref-type="fig"}). In GE the nuclear and cytoplasmatic NFκB immunostaining was increased in the PP group as compared to the NP group (Figure [4](#F4){ref-type="fig"}). No changes in NFκB staining were observed in SQ (Figure [4](#F4){ref-type="fig"}). The stroma displayed an increased nuclear immunostaining, but unchanged cytoplasmatic staining, in the PP group as compared to the other groups (Figure [5](#F5){ref-type="fig"}). Vascular endothelium showed an increased nuclear but unchanged cytoplasmatic staining, in the PP group as compared to the NP group (Figure [5](#F5){ref-type="fig"}). In all groups some leukocytes (black arrowhead, Figure [3l](#F3){ref-type="fig"}), stained positive for NFκB. As for image analysis of GR, the manual scoring of NFκB in stromal cells could include leukocytes within the stroma, but not blood cells within vessels. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Immunostaining of NFκB protein in stroma (S), glandular epithelium (GE), squamous epithelium (SQ) and vessels (V) in cervical biopsies from the NP (left column, a, d, g, j), TP (middle column, b, e, h) and PP (right column, c, f, i, l) groups. Positive nuclear and cytoplasmic immunostaining is also observed in neuronal ganglions (G) (h). Some leukocytes, identified by their morphology, display positive NFκB staining (l, black arrowhead). A negative control where the primary antibody is replaced by rabbit IgG is shown in k. ::: ![](1477-7827-2-74-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### NFκB protein levels, as assessed by manual scoring of immunohistochemistry results, in nuclei and cytoplasma of glandular epithelium (GE) and squamous epithelium (SQ) in cervical samples from TP (n = 10) and PP (n = 10) as compared to NP (n = 8) women. Box and whisker plots represent the median value with 50% of all data falling within the box. The \"whiskers\" extend to the 5^th^and 95^th^percentiles. Boxes with different letter designations are significantly different, p \< 0.05. ::: ![](1477-7827-2-74-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### NFκB protein levels, as assessed by manual scoring, in nuclei and cytoplasma of stroma and vascular endothelium in cervical samples from TP (n = 10) and PP (n = 10) as compared to NP (n = 8) women. Box and whisker plots represent the median value with 50% of all data falling within the box. The \"whiskers\" extend to the 5^th^and 95^th^percentiles. Boxes with different letter designations are significantly different, p \< 0.05. ::: ![](1477-7827-2-74-5) ::: COX-1 ----- Immunostaining for COX-1 (Figure [6a,6b,6c](#F6){ref-type="fig"}) was found in platelets (not shown), some leukocytes (not shown), vessel endothelium (Figure [6a,6c](#F6){ref-type="fig"}), stroma (Figure [6a,6b,6c](#F6){ref-type="fig"}), neuronal ganglion (not shown), SQ (Figure [6c](#F6){ref-type="fig"}) and GE (not shown). Manual scoring was performed and there was a significant increase of COX-1 staining in the stroma of the TP and PP groups as compared to the NP group (Figure [7](#F7){ref-type="fig"}, top). No differences were found in staining of the SQ, GE and endothelium between the three groups (data not shown). ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Immunostaining of COX-1 (a-c), COX-2 (d-i), PAF-R (j-n) and iNOS (p, r), in cervices from women in the NP (left column), TP (middle column) and PP (right column) group. A representative negative control for COX-1, COX-2 and PAF-R immunohistochemistry (primary antibody replaced by goat IgG, same secondary antibody) is shown in o, the negative control for iNOS is shown in q(primary antibody replaced by mouse IgG). Abbreviations: S = stroma, GE = glandular epithelium, SQ = squamous epithelium, V = vessel and G = ganglion cells. ::: ![](1477-7827-2-74-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### COX-1 (top) and COX-2 (middle, bottom) protein levels, as assessed by manual scoring of stroma (top, middle) and glandular epithelium (bottom) in cervical samples from the TP (n = 8), PP (n = 9) and NP (n = 6) groups. Box and whisker plots represent the median value with 50% of all data falling within the box. The \"whiskers\" extend to the 5^th^and 95^th^percentiles. Boxes with different letter designations are significantly different, p \< 0.05. ::: ![](1477-7827-2-74-7) ::: COX-2 ----- Immunostaining of COX-2 (Figure [6d,6e,6f,6g,6h,6i](#F6){ref-type="fig"}) was found in the stroma, GE and smooth muscle cells/activated fibroblasts, both around vessels and arranged as bundles within the stroma. The intensity of COX-2 staining was overall less than that of COX-1, except in GE where the immunostaining was almost maximal in all samples of the TP and PP groups (Figure [6h,6i](#F6){ref-type="fig"}). There was an increased immunostaining in the stroma of the PP group and in GE from the TP and PP groups, as compared to the NP group (Figure [7](#F7){ref-type="fig"} middle and bottom, respectively). PAF-R ----- Immunostaining of PAF-R was found in stroma, neuronal ganglion (G) and GE (Figure [6j,6k,6l,6m,6n](#F6){ref-type="fig"}). The immunostaining in the stroma was higher in the TP group (Figure [6k](#F6){ref-type="fig"}) compared with NP (Figure [6j](#F6){ref-type="fig"}) and PP groups (Figure [6l](#F6){ref-type="fig"}) (Figure [8](#F8){ref-type="fig"} top). The immunostaining in GE was increased in the PP group (Figure [6l](#F6){ref-type="fig"}) as compared to the TP group (Figure [6k](#F6){ref-type="fig"}) (Figure [8](#F8){ref-type="fig"} bottom). A negative control representative for the goat-derived antibodies, where goat IgG replaced the primary antibody, is shown in Figure [6o](#F6){ref-type="fig"}. ::: {#F8 .fig} Figure 8 ::: {.caption} ###### PAF-R protein levels, as assessed by manual scoring, in stroma (top) and glandular epithelium (bottom) in cervical samples from the TP (n = 11), PP (n = 13) and NP (n = 9) groups. Box and whisker plots represent the median value with 50% of all data falling within the box. The \"whiskers\" extend to the 5^th^and 95^th^percentiles. Boxes with different letter designations are significantly different, p \< 0.05. ::: ![](1477-7827-2-74-8) ::: iNOS ---- Faint positive staining was found in SQ, stroma and GE (Figure [6p,6r](#F6){ref-type="fig"}). There were no differences in immunostaining of iNOS found between the groups (data not shown). A negative control where the primary antibody was replaced by mouse IgG is shown in Figure [6q](#F6){ref-type="fig"}. GRα mRNA -------- The GRα mRNA level was determined by RT-PCR (Figure [9](#F9){ref-type="fig"}). GRα mRNA levels were not significantly different between the three study groups, but there was a tendency of an increased level in the TP group as compared to the other two groups (Figure [9a](#F9){ref-type="fig"}). When the relative GRα mRNA level in the NP group was defined to 100%, the TP group showed 120% and the PP group 90% of that level. ::: {#F9 .fig} Figure 9 ::: {.caption} ###### Images of representative RT-PCR gels for a: GRα, b: GRβ and c: COX-1 (upper band) and COX-2 (middle band) in the human cervix in the non-pregnant (NP; n = 5), term pregnant (TP; n = 6) and postpartum (PP; n = 5) groups. The gels are stained with ethidium bromide. a: GRαmRNA(upper band) and 18S mRNA (lower band). b: 18S mRNA (upper band) and GRβmRNA(lower band). c: The 18S mRNA (bottom band) and the COX-1 (upper band) and COX-2 (middle band) PCR products. ::: ![](1477-7827-2-74-9) ::: GRβ mRNA -------- Very low levels of GRβ mRNA were present in 4 out of 6 samples from the TP group, and in 2 out of 5 samples in the NP and PP groups (Figure [9b](#F9){ref-type="fig"}). COX-1 mRNA ---------- There was no difference in the COX-1 mRNA level between the groups, as assessed by RT-PCR (Figure [9c](#F9){ref-type="fig"}) (Figure [10](#F10){ref-type="fig"}, top). ::: {#F10 .fig} Figure 10 ::: {.caption} ###### COX-1 (top) and COX-2 (bottom) mRNA levels in the human cervix from the NP (n = 5), the TP (n = 6) and the PP (n = 5) groups as determined by RT-PCR. Intensities of the PCR product bands were normalized against the internal 18S standard. Box and whisker plots represent the median value with 50% of all data falling within the box. The whiskers extend to the 5th and 95th percentiles. Group medians with different letter superscripts are significantly different (P \< 0.05). ::: ![](1477-7827-2-74-10) ::: COX-2 mRNA ---------- The COX-2 mRNA level was the highest in the PP group (Figure [9c](#F9){ref-type="fig"}) (Figure [10](#F10){ref-type="fig"}, bottom), but there was a great variation between the individual PP patients (Figure [9c](#F9){ref-type="fig"}), probably due to the different amount of glands present in the biopsies. Discussion ========== This study is, to our knowledge, the first reporting GR expression in human cervix uteri during pregnancy and at parturition. In a recent study on human endometrium \[[@B34]\], GR protein was observed in stromal cells and within leukocytes invading the stroma. Pujols et al. \[[@B13]\] reported that the expression of GRα mRNA is 400-fold higher than GRβ mRNA expression in human tissues. The GRα protein was found in all cells and specimens in their study, while GRβ was not detected in any specimen. In our study, GRα mRNA was much more abundant than GRβ mRNA. Therefore, we conclude that GRα is the major receptor type observed in our study, although GRβ could contribute to the GR protein level in the TP group. Glucocorticoids exert their anti-inflammatory effects primarily by inhibiting the expression of cytokines e.g. IL-8, as demonstrated in human fibroblasts \[[@B35]\], tumour necrosis factor (TNF)-α \[[@B36]\], colony-stimulating factor (CSF) and macrophage stimulating factor (M-CSF) \[[@B5]\]. The majority of these pro-inflammatory genes have no glucocorticoid responsive elements (GRE) in their promoter regions that could explain the effect of glucocorticoids, but many of them contain sites for the transcription factors activating protein (AP)-1 and NFκB \[[@B37]\]. In the review by McKay and Cidlowski \[\[[@B5]\], and references therein\] it is obvious though, after comparing the genes transcriptionally regulated by NFκB (e.g. IL-8, iNOS, COX-2) and genes repressed by GR (e.g. IL-8, iNOS, COX-2, GR, CRH, fibronectin, and metalloproteinases (MMPs)), that NFκB and GR have diametrically opposed functions. The oppositely regulated IL-8, iNOS and COX-2 have all proven to be important for cervical ripening and labor induction \[[@B4],[@B17],[@B38]\]. Maternal plasma levels of cortisol increase until term \[[@B6]\], but their diurnal variation is maintained \[[@B39]\] although more long-term variations have been suggested \[[@B40]\]. At parturition, maternal plasma levels of placental CRH \[[@B10],[@B41]\], pituitary adrenocorticotropic hormone (ACTH) and adrenal cortisol \[[@B6]\] increase exponentially. Our data shows that the GR protein was present in cervical stroma, SQ and vascular endothelium in samples from non-pregnant, term pregnant and postpartum women. The decrease in GR levels in stroma and SQ at parturition, as compared to term, could be interpreted in terms of a GR mediated glucocorticoid anti-inflammatory activity during pregnancy that is ended at parturition. The NFκB protein was present in cervices from non-pregnant, term pregnant and postpartum women. This protein, ubiquitously expressed in a variety of cell types, is found in the cytoplasm in its inactive form, but translocates into the nucleus upon activation \[[@B14]\]. Therefore, the increase in nuclear NFκB levels in cervical stroma, GE and vascular endothelium in the PP group suggests an activation of NFκB at parturition. Thus, our present results of decreased GR and increased NFκB levels at parturition are in agreement with the reports of their opposed effects on the activity of several inflammatory genes \[\[[@B5]\], and references therein\] and the idea of cervical ripening being an inflammatory reaction \[[@B42]\]. Polymorphonuclear leukocytes and macrophages migrate from blood vessels and accumulate in the cervix uteri before parturition \[[@B2]\]. GR and NFκB were identified by immunohistochemistry in morphologically recognized leukocytes in the samples from all groups in this study. GR-positive leukocytes have also been observed in the endometrium of non-pregnant women and the decidua of early pregnant women \[[@B43]\]. Cortisol is a potent inhibitor of COX-2 in myometrium, decidua and cervix \[\[[@B17]\], and references therein\]. We found the constitutive COX-1 protein to be present at higher levels in the stroma at term and postpartum, whereas COX-1 mRNA was unchanged. Inducible COX-2 mRNA was increased at parturition as compared to term, and the COX-2 protein was increased in stroma postpartum and in GE at term and postpartum as compared to the NP group. Our observations suggest, that prostaglandin synthesis occur especially in the GE, where a highly intensive COX-2 immunostaining was observed at term and postpartum. This is in agreement with a recent report on COX-2 mRNA in the pregnant baboon cervix \[[@B44]\]. This would also explain the large variation in COX-2 mRNA levels found in the present study. The RNA is prepared from homogenates of cervical biopsies, and the amount of glands present in the biopsies varies from none to plenty. Our results indicate that prostaglandin synthesis could be regulated predominantly via COX-2 in cervical GE at parturition. The stromal increase in COX enzymes could be due to the influx of macrophages and leukocytes before parturition \[[@B2]\], since leukocytes are an abundant source of PGE~2~in the human body \[[@B45]\]. We could not exclude that COX-1 and COX-2 are present in the stromal cells, thereby adding another cervical source of prostaglandin synthesis, but it seems likely that the enzymes are mainly expressed in the invading leukocytes. Further, our data together with previous findings, suggest suppression not only by progesterone \[[@B46]\], but also by cortisol \[\[[@B17]\], and references therein\] of prostaglandin synthesis in the human uterine cervix during pregnancy and a release of this suppression at parturition. Since the COX-2 promoter contains NFκB binding sites \[[@B47]\], the activation of COX-2 could be regulated via NFκB. Nitric oxide stimulates PGE~2~release from human cervical tissue explants \[[@B23]\]. Nitric oxide donors induce cervical ripening in term pregnancy in humans \[[@B28]\]. Treatment with the NO donor isosorbid-5-mononitrate stimulates the synthesis of COX-2 and PGE~2~in human uterine cervix \[[@B27]\]. The immunostaining of iNOS in the present study did not differ between the groups, and thus did not vary due to pregnancy or parturition. This is not in agreement with previous studies on human cervix \[[@B38],[@B48]\]. Tschugguel et al. found increased iNOS immunostaining with an antibody from Transduction laboratories (no number stated), but not on the mRNA level using RT-PCR, indicating a post-transcriptional regulation of iNOS \[[@B38]\]. Ledingham et al. \[[@B48]\] found increased cervical immunostaining and stronger bands on Western blot in term pregnancy as compared to the non-pregnant state, using an antibody from Transduction laboratories (39120, clone 54). We also used a monoclonal iNOS antibody from Transduction laboratories, but a different clone (32020, clone 6), which could explain the different results. We found some staining in GE, while Ledingham et al. state no staining of the glands \[[@B48]\]. Platelet activating factor (PAF), like prostaglandins derived from the arachidonic acid precursor, is a multifactorial pro-inflammatory mediator, which has been implicated in parturition. Local application of PAF in rats induced cervical ripening \[[@B49]\], whereas a PAF-R antagonist prolonged parturition \[[@B50]\]. PAF-R has been identified in human cervical fibroblasts in vitro \[[@B51]\]. We show, for the first time, presence of the PAF-R protein in the human uterine cervix in vivo. Stromal PAF-R immunostaining was most pronounced at term, and decreased after parturition. PAF-R immunostaining was, like for COX-2, further increased in GE postpartum. PAF increases the expression of pro-inflammatory cytokines e.g. IL-8, and this effect can be abolished using a PAF-R antagonist (WEB2170) \[[@B18],[@B51]\]. Furthermore, the COX-2 promoter contains NFκB binding sites \[[@B47]\], and the PAF stimulated COX-2 induction is NFκB dependent \[[@B51]\], indicating that the PAF-R could activate NFκB and thereby induce COX-2. PAF also increases expression of MMP-1 \[[@B51]\], which has been shown to effectuate collagen degradation and cervical ripening \[[@B1],[@B52]\]. If the process of cervical ripening is disturbed, either resulting in a preterm delivery or to a prolonged delivery time, possibly ended by a cesarean section, it will lead to increased risks for both the mother and the child. Preterm delivery is the leading factor causing neonatal mortality and morbidity \[[@B54]\]. An increased knowledge of the factors regulating the cervical ripening process will give tools for developing pharmaceuticals that can regulate cervical ripening. Conclusions =========== We have demonstrated that the human uterine cervix is a potential target organ for glucocorticoids during pregnancy. The higher GR protein levels in cervical stroma and SQ before parturition may reflect a GR mediated anti-inflammatory effect of cortisol during pregnancy, with a subsequent decline of this activity at parturition. The concomitant increase in nuclear NFκB levels in the cervix suggests activation of this transcription factor at parturition. NFκB activity promotes pro-inflammatory events and could be responsible, at least in part, for the observed increase in COX-1, COX-2 and PAF-R levels. Acknowledgements ================ The present study received financial support from the Swedish Research Council (grant 7189, GE), The Swedish Society of Medicine (LS), Sigurd and Elsa Goljes mine (DS) and Karolinska Institutet.	PubMed Central	2024-06-05T03:55:49.918137	2004-10-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534613/", "journal": "Reprod Biol Endocrinol. 2004 Oct 22; 2:74", "authors": [ { "first": "Ylva", "last": "Stjernholm-Vladic" }, { "first": "Denis", "last": "Stygar" }, { "first": "Christopher", "last": "Mansson" }, { "first": "Britt", "last": "Masironi" }, { "first": "Sonja", "last": "Akerberg" }, { "first": "Hong", "last": "Wang" }, { "first": "Gunvor", "last": "Ekman-Ordeberg" }, { "first": "Lena", "last": "Sahlin" } ] }
PMC534783	Introduction ============ In 1998 Fire and coll. coined the term RNA interference (RNAi) referring to the phenomenon of post-translational silencing of gene expression that occurs in response to the introduction of double-stranded RNA (dsRNA) into a cell \[[@B1]\]. This phenomenon can result in highly specific suppression of gene expression. RNAi technology is rapidly spreading in research laboratories worldwide, as it is associated with a number of practical and theoretic advantages over preexisting methods of suppressing gene expression (Table [1](#T1){ref-type="table"}). RNAi promises to revolutionize key areas of medical research, as demonstrated by the preliminary findings obtained in the fields of cancer, infectious diseases and neurodegenerative disorders. In this review the principles underlying this phenomenon as well as the technical challenges encountered while using RNAi for research purposes are discussed. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Comparison between different methods for gene silencing. ::: --------------------------------------------------------------------------------------------------------------------------- Method Advantages Drawbacks ----------------------------- ------------------------------------------ -------------------------------------------------- RNA interference Specific\ Knock-down (not knock-out)\ Relatively easy Needs transfection Anti-sense DNA Easy\ Variable efficiency\ Inexpensive Variable specificity\ Needs transfection Dominant negative mutants Stable suppression\ Needs transfection\ Specific protein domains can be targeted Variable/unexpected effect Knock-out animal Complete gene silencing Labor intensive, expensive\ Lethal mutants may prevent embryonic development Small molecule inhibitors Easy delivery Variable specificity\ Labor intensive development --------------------------------------------------------------------------------------------------------------------------- ::: The physiology of RNAi ====================== Introduction of long dsRNA into a mammalian cell triggers a vigorous nonspecific shutdown of transcription and translation, in part due to activation of dsRNA-dependent protein kinase-R (PKR) \[[@B2]\]. Activated PKR phosphorylates the translation initiation factor EIF2: this effect, in association with activation of Rnase-L and induction of interferon production, halts protein synthesis and promotes apoptosis. Overall, this is believed to represent an antiviral defense mechanism \[[@B3]\]. Owing to this phenomenon, initial observations of RNAi induced by long dsRNA in plants \[[@B4]\] and the nematode Caenorhabditis elegans \[[@B1]\] were at first applied to mammalian cells with little success. In a breakthrough experience reported by Elbashir et al., it was discovered that dsRNAs 21--23 nucleotides long -- termed small interfering RNAs (siRNAs) -- could suppress mammalian gene expression in a highly specific manner \[[@B5]\], pointing the way to gene silencing in mammalian cells. RNAi is a highly conserved mechanism throughout taxonomical species \[[@B6]\]. In addition to have an antiviral activity, RNAi is also believed to suppress the expression of potentially harmful segments of the genome, such as transposons, which might otherwise destabilize the genome by acting as insertional mutagens \[[@B7]\]. Though its mechanisms are not fully elucidated, RNAi represents the result of a multistep process (Figure [1](#F1){ref-type="fig"}). Upon entering the cell, long dsRNAs are first processed by the RNAse III enzyme Dicer \[[@B8]\]. This functional dimer contains helicase, dsRNA binding, and PAZ (named after piwi, argonaute, and zwille proteins) domains. Whereas the former two domains are important for dsRNA unwinding and mediation of protein-RNA interactions, the function of the PAZ domain species, is not completely elucidated \[[@B9],[@B10]\]. Dicer produces 21--23 nucleotide dsRNA fragments with two nucleotide 3\' end overhangs, i.e. siRNAs. Recently it has been suggested that Dicer has functions other than dsRNA cleavage that are required for siRNA-mediated RNAi in mammals \[[@B11]\]. RNAi is mediated by the RNA-induced silencing complex (RISC) which, guided by siRNA, recognizes mRNA containing a sequence homologous to the siRNA and cleaves the mRNA at a site located approximately in the middle of the homologous region \[[@B9]\]. Thus, gene expression is specifically inactivated at a post-transcriptional level. In C. elegans, Dicer has been shown to interact with rde proteins. The rde proteins bind to long dsRNA and are believed to present the long dsRNA to Dicer for processing \[[@B12]\]. Mutants displaying a high degree of resistance to RNAi have been reported to possess mutations at rde-1 and rde-4 loci \[[@B13]\]. Given the highly conserved nature of these enzymes, similar mutations may be of significance in mammalian cells. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Mechanism of RNA interference (RNAi). The appearance of double stranded (ds) RNA within a cell (e.g. as a consequence of viral infection) triggers a complex response, which includes among other phenomena (e.g. interferon production and its consequences) a cascade of molecular events known as RNAi. During RNAi, the cellular enzyme Dicer binds to the dsRNA and cleaves it into short pieces of \~ 20 nucleotide pairs in length known as small interfering RNA (siRNA). These RNA pairs bind to the cellular enzyme called RNA-induced silencing complex (RISC) that uses one strand of the siRNA to bind to single stranded RNA molecules (i.e. mRNA) of complementary sequence. The nuclease activity of RISC then degrades the mRNA, thus silencing expression of the viral gene. Similarly, the genetic machinery of cells is believe to utilize RNAi to control the expression of endogenous mRNA, thus adding a new layer of post-transciptional regulation. RNAi can be exploited in the experimental settings to knock down target genes of interest with a high specific and relatively easy technology (see text for more details). ::: ![](1479-5876-2-39-1) ::: Besides gene silencing, RNAi might be involved in other phenomena of gene regulation. DNA/RNA interactions are known to influence DNA methylation. It appears that RNAi can also function on this level by methylating cytosines as well as CpG sequences more classically associated with methylation. If the target sequence shares homology with a promoter, transcriptional silencing may occur via methylation. Moreover, RNA appears to interact with chromatin domains, which may ultimately direct DNA methylation. Studies of C. elegans have shown that RNAi can spread among cells through mechanisms that may not hinge upon siRNA \[[@B14]\]. The systemic RNA interference-deficient (sid) locus, sid-1, encodes a conserved protein with a signal peptide sequence and 11 putative transmembrane domains, suggesting that the sid-1 protein may act as a channel for long dsRNA, siRNA, or a currently undiscovered RNAi-related signal. Sid-1 mutants retain cell-autonomous RNAi but fail to show spreading of RNAi. It remains unclear whether this systemic RNAi occurs in mammals, although a strong similarity is reported between sid-1 and predicted human and mouse proteins. siRNA synthesis and delivery strategies ======================================= Several strategies for inducing siRNA-mediated gene silencing have been developed, each of them presenting specific advantages and disadvantages (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Comparison between siRNA delivery methods. ::: --------------------------------------------------------------------------------------------------------------------------------------------------------- Method Advantages Drawbacks ----------------------------------- ------------------------------------------------------------------------- ------------------------------------------- Chemical or enzymatic synthesis Rapid\ Transient RNAi\ Enzymatic: no need to test individual siRNA\ Needs transfection\ Chemical: high purity Enzymatic: variable purity & specificity\ Chemical: expensive DNA plasmid vector or casette Less expensive\ Labor intensive\ Stable RNAi Needs transfection Viral vector Stable RNAi\ Labor intensive\ May be effective in cells resistant to transfection with dsRNA/plasmids Potential biohazard --------------------------------------------------------------------------------------------------------------------------------------------------------- ::: Synthesis, purification, and annealing of siRNAs by industrial chemical processes \[[@B15]\] is becoming increasingly popular. This method is rapid and purity is generally high. This may be the best approach for initial \"proof of principle\" experiments. In vitro siRNA synthesis is an alternative and relies upon the T7-phage polymerase \[[@B16]\]. This polymerase produces individual siRNA sense and antisense strands that -- once annealed -- form siRNAs. Extra nucleotides required by the T7 promoter are removed by RNase digestion and cleaning steps. Otherwise, recombinant Rnase-III can be used to cleave long dsRNAs to produce multiple siRNAs \[[@B17]\]. Although technically easy, this approach presents the drawback of the generation of non-specific siRNAs. siRNAs can be produced by polymerase-III promoter-based DNA plasmids or expression cassettes \[[@B18]\]. These constructs produce small inverted repeats, separated by a spacer of three to nine nucleotides, termed short hairpin RNAs (shRNAs), which are processed by Dicer into siRNAs \[[@B19]\]. Transcription begins at a specific initiation sequence, determined by the promoter used. In addition to a defined initiation sequence, the U6 polymerase-III promoter terminates with TTTT or TTTTT \[[@B20]\]. The products are shRNAs that contain a series of uridines at the 3\' end, a feature that seems to favor RNAi \[[@B21]\]. Suppression of gene expression by RNAi is generally a transient phenomenon \[[@B22]\]. Gene expression usually recovers after 96 to 120 hours or 3 to 5 cell divisions after transfection, which is likely due to dilution rather than degradation of siRNAs. However, by introducing plasmids which express siRNA and a selection gene, stable RNAi can be sustained as long as two months after transfection \[[@B23]\]. Interest is growing in the use of viral vector-mediated RNAi. Adenoviral and retroviral vectors have been reported to produce siRNAs in vivo \[[@B24],[@B25]\] and stable RNAi is obtained using this method, though in the absence of a selective pressure \[[@B26],[@B27]\]. Virus-mediated RNAi may circumvent some of the problems associated with cells that are generally refractory to RNAi, such as non-transformed primary cells \[[@B28]\]. At present, the question of whether functional RNAi will continue in all progeny of a cell with stable vector integration remains unanswered. Designing RNAi experiments ========================== Several crucial considerations should be beard in mind while designing RNAi experiments. The below examples regard RNAi experiments performed with chemically synthesized siRNA. 1\. The first step is to design a suitable siRNA sequence. A growing number of libraries of validated siRNAs directed toward some frequently targeted genes are available. However, if the gene of interest has not been targeted using siRNA before, a novel siRNA must be developed. In mammalian cells RNAi is mediated by 21- to 23-nucleotide siRNAs containing symmetrical two nucleotide 3\' overhangs. Given a siRNA sequence alone, it is not currently possible to predict the degree of gene knockdown produced by a particular siRNA. Nevertheless, several observations have been made that can be taken into account to increase the probability of producing an effective siRNA. The chief variable is the gene target site. Generally, it is recommended that a target site located at least 100--200 nucleotides from the AUG initiation codon is chosen. Targets within 50--100 nucleotides of the termination codon should instead be avoided. The 5\' and 3\' untranslated region (UTR) should also be avoided, since associated regulatory proteins might compromise RNAi. This is just a general recommendation, as some siRNAs targeting the 3\' UTR have also been shown to induce RNAi \[[@B29]\]. Numerous on-line design tools will produce a list of suitable gene target sites. It is important to ensure that the sequence is specific to the target gene by performing a BLAST search in order to avoid cross reaction with unwanted genes. As an example, Biocomputing at the Whitehead Institute for Biomedical Research -- a nonprofit independent research and educational institution affiliated with the Massachusetts Institute of Technology -- is one of several organizations that has developed a freely available web-based siRNA design tool. 2\. The structural characteristics of the siRNA molecules are another crucial aspect to be considered while designing RNAi experiments. SiRNA of 21 nucleotides with 3\'-d(TT) or (UU) overhangs are considered the most effective \[[@B30]\]. Despite the fact that nucleotide-protein steric interactions contribute to the relationship between siRNA length and activity, the reason for this relationship is not completely elucidated. For optimal siRNA secondary structure, the GC ratio should ideally be between 45 and 55%, and multiple identical nucleotides in series, particularly poly(C) and poly(G), should be avoided to determine any requirements for modification, such as fluorophore labeling to allow for siRNA tracking and quantification of transfection efficiency. 3\. To induce RNAi, siRNA must be transfected into the cells of interest. Several transfection reagents exist, the most commonly used being liposomal or amine-based. In some cases electroporation may be used, but cell toxicity can be high with this technique \[[@B31]\]. Cell lines show varying responses to different transfection reagents, and it may be necessary to try more than one reagent or approach. Transfection efficiency is optimized by titrating cell density, transfection time, and the ratio of siRNA-to-transfection reagent. The cell passage number and antibiotic use can also affect the efficiency of transfection. 4\. Recently, experimental design features have been suggested to guarantee the rigor of RNAi experiments \[[@B32]\]. Due to the high specificity of RNAi, a siRNA with a one-nucleotide sequence mismatch can serve as a negative control. If this approach is used, absence of homology with other targets should be confirmed at the design stage. It is important to remember that mismatched siRNAs could target mutant gene sequences. Therefore loss of functional target gene silencing should be demonstrated to validate this approach. Alternatives include sequences that preset no homology to any known gene. Some investigators have suggested that scrambled siRNA is not sufficiently homologous to the target sequence to function as an adequate control; therefore, they propose a combination of mismatched and scrambled controls \[[@B32]\]. A more challenging functional control is to demonstrate the \"rescue\" of the target gene function following artificial overexpression of the target gene. Transfection of a plasmid expressing the gene sequence to which a siRNA is targeted results in production of mRNA that would also be targeted by the siRNA. This problem can be overcome using plasmids containing silent mutations. This approach takes advantage of degeneracy of gene coding, i.e., amino acids are represented by more than one three-nucleotide codon sequence. Rescue is achieved by expression of a protein identical to the native protein from a nucleotide sequence that differs from the native nucleotide sequence to which the siRNA is targeted. Alternatively, siRNAs directed to the 3\'-UTR can be used. Many researchers use more than one siRNA, with each targeted to different areas of the gene sequence. A consistent RNAi response using different siRNAs with a variety of targets within the gene sequence of interest would increase confidence in experimental results. Dose-respons characteristics should be determined and the lowest effective concentration of siRNA be used to avoid nonspecific effects. 5\. The effect of RNAi should be quantified at both the mRNA and the protein level. The knockdown of a protein should be probably evaluated after mRNA reduction has been proved: in fact, a reduction in protein levels not accompanied by a decrease in mRNA might indicate that other mechanisms are at work, such as RNAi mediated by microRNA. Northern blot analysis is considered by many to be the gold standard. Real-time reverse transcriptase polymerase chain reaction, incorporating internal controls to quantify \"housekeeping\" gene transcript levels, can also be used \[[@B33]\]. Protein knockdown can be confirmed by Western blot analysis, immunofluorescence, flow cytometry and phenotypic and/or functional assays. Although RNAi generally occurs within 24 h of transfection, both onset and duration of RNAi depend on the turnover rate of the protein of interest, as well as the rate of dilution and longevity of the siRNAs. The duration of gene silencing can also be modified by factors such as the concentration of serum in the culture medium, which affects cell-cycle rate. It is therefore necessary to determine the time course of any silencing observed under specific conditions using the modalities discussed. Conclusions =========== RNAi is now commonly used in biological and biomedical research to study the effect of blocking expression of a given gene. As the effect is rarely complete, it is generally termed a \"knock-down\" to distinguish it from the \"knock-out\" achieved by deletion of the gene. Although significant advances have been made as compared to previous methods, RNAi has its own limitations. Not every sequence works, most researchers reporting a success rate of about one in three. Moreover, although the effects are generally believed to be highly sequence-specific, some doubts remain as to whether or not some of the observed effects are \"off target.\" Some residual activation of the interferon system has been reported, as well as degradation of closely related, but non-identical, mRNAs. Nevertheless, RNAi remains the most promising functional genomics tool recently developed. DNA microarray technology has now enabled the level of expression of every gene in the genome to be determined under any condition \[[@B34],[@B35]\]. This has led to a huge accumulation of data on genes whose expression is significantly altered in several diseases. To take an example, large databases have been established of genes that are pathologically regulated in cancer. In some cases this has resulted in the identification of key genes involved in tumor development and provided important new therapeutic targets. However, in most cases the pattern of gene expression is far too complex to allow for identification of the relatively small number of misexpressed genes that are involved in causing or maintaining the disease rather than the much larger number that are innocent bystanders. The ability of RNAi to provide relatively easy ablation of gene expression has opened up the possibility of using collections of siRNAs to analyse the significance of hundreds or thousands of different genes whose expression is known to be upregulated in a disease, given an appropriate tissue culture model of that disease. Perhaps more important still is the possibility of using genome-wide collections of siRNAs, whether synthetic or in viral vectors, as screening tools. Two main avenues of research can rely upon RNAi libraries. First, in a high-throughput manner each gene in the genome is knocked-down one at a time and the cells or organism scored for a desired outcome, such as death of a cultured cancer cell but not a normal cell. Due to the very large numbers of assays required to screen all 35--50,000 genes in the human genome, this approach is highly labor-intensive and time consuming. The other approach is to use large pools of RNAi viral vectors and apply a selective pressure that only cells with the desired change in behavior can survive. The identity of the genes knocked-down in the surviving cells can then be identified by sequencing the RNA interference vectors that they carry. Both approaches show consider-able promise in identifying novel genes that may make important therapeutic targets for inhibition either by conventional drug discovery methods or, more intriguingly, by RNA interference itself. Acknowledgements ================ We truly thank Ms. Marta Briarava for her help with graphics.	PubMed Central	2024-06-05T03:55:49.921507	2004-11-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534783/", "journal": "J Transl Med. 2004 Nov 22; 2:39", "authors": [ { "first": "Simone", "last": "Mocellin" }, { "first": "Maurizio", "last": "Provenzano" } ] }
PMC534784	Background ========== Lipoprotein lipase (LPL) hydrolyses triglycerides in chylomicrons and VLDL and thereby makes fatty acids available for cellular uptake and use in metabolic processes \[[@B1],[@B2]\]. Relatively high levels of LPL mRNA are found in adipose tissue, heart, red skeletal muscle and lactating mammary gland \[[@B3],[@B4]\]. Parenchymal cells, such as adipocytes and myocytes, synthesize and secrete the enzyme, which is then transferred to the endothelium and anchored to the oligosaccharide chains of heparan sulfate proteoglycans (HSPG) \[[@B1],[@B2]\]. There is continuous recycling of the enzyme between the luminal and abluminal side of the endothelial cells, and perhaps to other extracellular sites in the tissue \[[@B1],[@B5],[@B6]\]. It is not known how the extracellular enzyme is turned over. One possibility is that it is transported with blood to the liver and degraded there \[[@B7]\]. LPL activity in the circulating blood is normally low and most of the LPL protein in blood is catalytically inactive \[[@B7]-[@B10]\]. Release of lipase from extrahepatic tissues into blood has been demonstrated \[[@B11],[@B12]\]. Model studies with labeled LPL have demonstrated uptake and degradation of both active and inactive LPL in the liver \[[@B13]-[@B15]\]. Heparin releases LPL from its endothelial binding sites into the circulating blood. The uptake in the liver is retarded, but not abolished \[[@B13],[@B14]\]. This has been taken as evidence that there are both heparin-sensitive and heparin-insensitive binding sites in the liver. An implication is that the high lipase activity in blood after heparin injection is due to release from peripheral tissues combined with retarded uptake in the liver. Studies in rats and in human subjects indicate that the net effect of heparin is an accelerated transport of LPL to the liver \[[@B16],[@B17]\]. If this hypothesis is correct, LPL mass and activity should increase in the liver after injection of heparin, in contrast to the decrease that occurs in extrahepatic tissues \[[@B6]\]. To test these concepts we have followed LPL activity and mass in liver after injection of heparin, and we have used immunofluorescence to explore if heparin changes the pattern of where in the liver LPL binds. Results ======= Amount and distribution of LPL in liver --------------------------------------- LPL activity in rat liver was 26 ± 1 mU/g (Table [1](#T1){ref-type="table"}), similar to the activity reported by Peterson et al \[[@B15]\]. This is low compared to the activities in adipose tissue (around 1600 mU/g in fed rats \[[@B6]\]) and heart (around 1100 mU/g \[[@B18]\]). LPL mass was 120 ng/g. The relation between LPL activity and mass in plasma was similar to that in liver; activity was 8 mU/ml and mass was 29 ng/ml (Table [1](#T1){ref-type="table"}). The specific activity of the enzyme in plasma increased to around 1.2 after injection of heparin. This indicates that most of the LPL in plasma or liver before heparin was inactive, in accord with studies on LPL in human plasma \[[@B7]-[@B10]\]. To study the distribution of endogenous LPL in rat liver we used affinity-purified chicken antibodies raised against bovine LPL. These antibodies have previously been used for ELISA of LPL in rat tissues \[[@B19]\]. There was faint immunofluorescence in a granular pattern (green) over hepatocytes, and stronger staining over scattered cells (Figure [1](#F1){ref-type="fig"}). Some of these reacted positively with the ED2 antibodies indicating that they were Kupffer cells. Sections treated with pre-immune IgG instead of anti-LPL (inset in Fig [1](#F1){ref-type="fig"}), as well as sections where the second antibody was omitted, showed no immunofluorescence. Effects of heparin ------------------ LPL activity and mass in plasma increased many-fold after heparin injection (Table [1](#T1){ref-type="table"}). The highest value was at 15 min, but even after 60 min the activity was still more then 50-fold higher than in normal plasma. In liver the level of LPL activity had increased already two min after heparin injection and it was about 10-fold increased both at 15 and 60 min compared to time 0. The amount of LPL protein in the liver, measured by the ELISA, had increased about two-fold at two min, 3.5-fold at 15 min and five-fold after 60 min. These data give direct evidence that after injection of heparin some of the LPL released into plasma was taken up by the liver. Hepatic lipase (HL) was also measured in the livers (Table [1](#T1){ref-type="table"}). As expected, but in sharp contrast to what was found for LPL, the HL activity decreased after heparin. Already after two min the activity had decreased by two thirds. This reflects the release of HL into the circulating blood. The activity in liver remained constant at 15 min and had begun to increase again after 60 min. The pattern of immunofluorescence for LPL after heparin was similar to that before heparin, with faint staining over most cells and more intense staining over scattered cells, some of which were ED2 positive (not shown). To explore the origin of the LPL released into plasma and taken up by the liver we measured LPL activity in heart and adipose tissue before and 20 min after injection of heparin (Table [2](#T2){ref-type="table"}). Data for plasma and liver in fed rats were similar to those in the experiment in Table [1](#T1){ref-type="table"}. In fasted rats, plasma post-heparin LPL activity was less than half of that in fed rats in accord with previous studies \[[@B18],[@B20]\]. The LPL activity in liver increased after heparin, in concert with the results in Table [1](#T1){ref-type="table"}. There was no statistically significant difference of the LPL activity in liver between fed and fasted rats either before or after heparin. In adipose tissue the LPL activity was about 5-fold higher in fed compared to fasted rats. Heparin caused a washout of 45 % of the LPL activity from epididymal and 65% from perirenal adipose tissue (p \< 0.01). Values for LPL activity in heart were somewhat higher in fasted than in fed rats, but this did not reach statistical significance (p = 0.16). Heparin caused no significant decrease of heart LPL in fed rats, but a highly significant (p \< 0.01) washout in the fasted rats, about 40%. Injection of labelled bovine LPL -------------------------------- The immunostaining of endogeneous LPL was faint and not suitable for detailed analysis or quantitation. The reason is that the only reagents available were chicken antibodies raised against bovine LPL. To further explore the hepatic binding and uptake of the enzyme we therefore injected bovine LPL. A trace amount of ^125^I-labeled LPL was included so that we could quantitate the uptake/metabolism. Values are given in Table [3](#T3){ref-type="table"} for the times at which localization of the lipase was studied by immunofluorescence. These values agree with an earlier study when more complete time curves were obtained \[[@B13]\]. For active LPL earlier studies have shown that at short times after injection about half of the lipase locates in extrahepatic tissues and about half in the liver \[[@B13],[@B21],[@B22]\]. In the present study 33 % of the radioactivity was in liver after two min (Table [3](#T3){ref-type="table"}). This increased to 52 % after 15 min and then decreased again to 20 % after 60 min. Heparin slows down the clearance of LPL from the blood \[[@B13],[@B21]\]. Fifteen min after injection of the labeled lipase, about half is still in the circulating blood \[[@B13]\]. In our experiments 30 % was in the liver at this time (Table [3](#T3){ref-type="table"}). Hence, 60 % or more of the removal from plasma had taken place in the liver. After 60 min radioactivity in the liver had decreased to 23 % of the injected dose (Table [3](#T3){ref-type="table"}). Earlier studies have demonstrated that acid soluble breakdown products of the labeled lipase appear in blood \[[@B13]\] and in the perfusion fluid in experiments with isolated livers \[[@B14]\]. Hence, the decrease of label in liver at longer times probably occurred through degradation of the lipase. For inactive lipase earlier studies have shown that only a minor fraction locates in extrahepatic tissues, and more than 70% of the uptake occurs in the liver \[[@B13]\]. In the present study, 50 % of the radioactivity was in the liver 15 min after injection, while 9 % was in the blood. After 60 min the radioactivity in the liver had decreased to 19 % of the injected dose. To visualize the injected bovine LPL we used rabbit antibodies. These antibodies did not inhibit endogenous LPL in rat post-heparin plasma or in extracts of adipose tissue. No immuno-reaction was seen in sections from control rats not injected with LPL (inset in Figure [2E](#F2){ref-type="fig"}). Likewise, there was no immunofluorescence in sections treated with non-immune rabbit IgG instead of anti-LPL, or when the second antibody was omitted. Two min after injection of the active lipase, intense immuno-staining was seen along sinusoids (Figure [2A](#F2){ref-type="fig"}). This staining was strongest in the periportal areas. There was little staining outside the sinusoids. Occasionally a few fluorescent dots were seen in hepatocytes, possibly representing endocytic vesicles. At 15 min after injection there was still staining over the sinusoids (Figure [2C](#F2){ref-type="fig"}), but most of the staining was now associated with hepatocytes and the number of granulae seen in hepatocytes had increased, indicating that the lipase had been internalized in vesicles (Figure [3A](#F3){ref-type="fig"}). Some cells, localized predominantly around the portal area, had many fluorescent dots. This staining was mainly granular in contrast to the more continuous staining over sinusoids at this time. Double staining at 15 min after injection of LPL demonstrated that some of these cells were ED2-positive (Figure [3A](#F3){ref-type="fig"}). At 60 min little or no staining remained at sinusoidal surfaces and the total staining had decreased (Figure [2E](#F2){ref-type="fig"}), but there were still grains in cells close to the portal area. This was probably enzyme that had been taken up in intracellular vesicles and had not yet become degraded. To get more detailed information about the binding sites in liver we used electron microscopy. For this, bovine ^125^I-labeled LPL was injected and 10 min later the livers were fixed by perfusion. The enzyme was visualized as silver grains by means of autoradiography (Figure [4A](#F4){ref-type="fig"}). Counting of silver grains in the sections showed that about 55% of the lipase was within spaces of Disse, mostly associated with hepatocytes. Twenty-five to 30% was on the luminal side of endothelial cells. Only about 15% was inside hepatocytes and other cell types, probably Kupffer cells. The pattern of distribution was quite different for the inactive lipase. At the first time, two min, there was intense staining for LPL over scattered cells concentrated to the portal regions but very little staining over sinusoids or hepatocytes (not shown). Double staining with the ED2 antibody showed that the intensively LPL-positive cells were Kupffer cells. After 15 min the immunofluorescence had changed to a more punctuate pattern that still co-localized with Kupffer cells (Figure [5C](#F5){ref-type="fig"}). This indicated that the lipase had been internalized in vesicles. There was some immunostaining over other cells, presumably hepatocytes, but this was much weaker than the staining over Kupffer cells (Figure [5A](#F5){ref-type="fig"} and [5C](#F5){ref-type="fig"}). After 60 min the intensity of the staining had decreased, but the pattern with more intense staining over Kupffer cells and much weaker staining over other cells remained (not shown). Hence, there was no indication that the inactive lipase first bound to one type of cell and then transferred to another type for uptake. Electron microscopic autoradiography of sections from livers of rats ten min after injection the inactive LPL, confirmed that the labelled lipase was mostly associated to sinusoidal cells that morphologically seemed to resemble Kupffer cells. Heparin markedly changed the pattern of localization for the active lipase. The initial (two min after injection) staining along the sinusoids was much weaker than in sections from rats that had not received heparin. The staining was generally more intense at 15 min compared to at two min after injection of the lipase (Figure [2D](#F2){ref-type="fig"}). This is in accord with the radioactivity data that showed that more LPL had been taken up (Table [2](#T2){ref-type="table"}). Compared to the pattern without heparin, the staining was spread throughout the liver parenchyma rather than concentrated in the portal areas (compare Figure [2A,2C](#F2){ref-type="fig"} and [2E](#F2){ref-type="fig"} with [2B,2D](#F2){ref-type="fig"} and [2F](#F2){ref-type="fig"}). There was more staining associated with scattered cells in the portal areas than in sections from rats that had not received heparin (Figure [2B](#F2){ref-type="fig"}). These cells were ED2-positive (not shown). Already at 15 min the LPL-staining had taken on a granular character both in the ED2-positive cells and in hepatocytes (Figure [3](#F3){ref-type="fig"}). At 60 min the intensity of staining had decreased (Figure [2F](#F2){ref-type="fig"}). The ED2 positive cells still dominated the picture but there was also granular staining over hepatocytes. More staining remained compared to the same time point without heparin (compare Figure [2F](#F2){ref-type="fig"} and [2E](#F2){ref-type="fig"}). Electron microscopic autoradiography showed that when heparin was injected ten min after active LPL there was a strong reduction in the amount of LPL in the spaces of Disse and on endothelial cells, while the radioactivity found in hepatocytes and Kupffer cells remained (data not shown). Heparin had no marked effect on the distribution of the inactive LPL (Figure [5](#F5){ref-type="fig"}). Most of the staining co-localized with staining for ED2 positive Kupffer cells (Figure [5D](#F5){ref-type="fig"}). Discussion ========== This study shows that after injection of heparin, LPL activity and mass in liver increases several-fold, in concert with the hypothesis that heparin causes accelerated transport of LPL to the liver. In other parts of the body LPL is attached to HSPG \[[@B1],[@B2]\]. Heparin competes efficiently with these binding sites. The rapid extraction of LPL by the liver in the presence of heparin implies that some other type of binding site must be present there. Members of the LDL receptor (LDL-R) family bind both the active and the inactive form of the lipase \[[@B23]\] and two recent studies indicate that LRP is involved in hepatic uptake of LPL \[[@B24],[@B25]\]. The binding of active LPL to LRP is, however, strongly impeded by heparin \[[@B26]\]. Therefore, it is unlikely that the heparin-resistant binding of active LPL is mediated by LRP or some other receptor of the LDL-R family. Heparin markedly decreased the binding of LPL along the sinusoids. This presumably reflects that binding to HSPG was competed by heparin. Staining associated with Kupffer-like cells increased. This may be the same sites as those that bind inactive LPL. Another possibility is that the LPL-heparin complexes were recognized and taken up as such. There is evidence for binding and uptake of heparin by Kupffer cells \[[@B27]\]. Most of the binding was, however, to hepatocytes even in the presence of heparin. Our data are qualitative, based on the immunolocalization. For more accurate quantitation one should label the lipase with a non-degradable label like ^125^I-tyramine cellobiose and isolate the different cell types. It has been suggested that LPL and HL bind, at least in part, to the same sites in the liver \[[@B24],[@B28]\]. It is, however, unlikely that HL shares the heparin-insensitive sites. The response of the two enzymes to heparin was very different. HL activity decreased by 60% within two min after heparin injection, reflecting release of the lipase into blood. In contrast, LPL activity in the liver increased, reflecting binding to the heparin-insensitive sites. Earlier studies have shown that there are also heparin-sensitive sites that bind LPL in liver \[[@B13]\]. Wallinder et al perfused livers in situ with heparin 15 min after injection of ^125^I-LPL to rats and found that about 10% of the lipase that had bound in the liver could be released \[[@B13]\]. Vilaró et al perfused isolated livers with ^125^I-LPL in a recirculating system for 10 min. After wash the perfusion was then continued in single pass mode with a heparin-containing medium. About 50% of the LPL that had bound in the liver reappeared in the perfusion medium within four min \[[@B14],[@B29]\]. At least some of these heparin-sensitive sites are likely HSPG, and they are probably the main sites that mediate the initial capture of LPL from blood. This binding may correspond to the decoration of the sinusoids seen by immunofluorescence at the earliest time after injection of the lipase. HSPG are present on virtually all cells in the body, including hepatocytes and endothelial cells in the liver \[[@B30],[@B31]\]. Vilaró et al studied binding and uptake of LPL in cultured hepatocytes \[[@B32]\]. The enzyme was concentrated at the tips of the microvilli, a site where also HSPG are highly abundant \[[@B33],[@B34]\]. Immunofluorescence now showed that at short times after injection the lipase located along the sinusoids, and electron microscopy showed that most of the lipase was in the spaces of Disse. The immunofluorescence was strongest in the portal areas, indicating that the lipase was extracted soon after it entered the liver. Most of the staining was over hepatocytes. With time the immunofluorescence shifted to a granular pattern, indicating that the enzyme had been internalized. The internalization may have occurred with lipase bound to HSPG, as demonstrated with cultured fibroblasts and with hepatocytes \[[@B32],[@B35],[@B36]\] and/or with lipase bound to LRP as suggested by several authors \[[@B24],[@B25],[@B35],[@B37]\]. In fibroblasts, both these pathways contribute to LPL internalization \[[@B38]\]. The lipase may well recycle as demonstrated by Heeren et al in experiments with cultured hepatocytes \[[@B39],[@B40]\]. The immunostaining gradually faded with time indicating that the lipase was degraded, in accord with previous studies, and with the decrease of ^125^I-radioactivity in the liver observed in the present study. Inactive LPL, as prepared here, was taken up in Kupffer cells. Most of the LPL in plasma is inactive \[[@B8],[@B10]\] and there is inactive LPL also in the tissues \[[@B19]\]. The nature and metabolic significance of this inactive LPL is not clear. Western blot analysis indicates that it is full-length LPL \[[@B7]\]. On heparin-agarose it elutes in the position expected for monomeric LPL \[[@B7]\]. Gel filtration of plasma indicates that it is associated with the lipoproteins \[[@B8]\]. The turnover of inactive LPL does not appear to be much influenced by heparin \[[@B6],[@B8],[@B13]\]. Earlier studies had shown that injected, inactive LPL is rapidly bound and degraded in the liver, both in vivo\[[@B13]\] and on perfusion through an isolated liver \[[@B14]\]. In these studies the inactive lipase was produced by complete denaturation in 6 M guanidinium chloride. In the present study the inactive lipase was gently prepared by incubation in rat plasma at 45°C. This probably results in dissociation to inactive but still folded monomers \[[@B41],[@B42]\]. Our preliminary experiments had shown that under these conditions the enzyme slowly lost its catalytic activity. After 90 min, the time used here, less than 5 % of the activity remained. In terms of clearance rate and tissue distribution, the present preparation behaved as the fully denatured lipase used in previous studies. Whether any of these lipase preparations faithfully reproduces the metabolic behaviour of the inactive lipase present in plasma is not clear. A recent study has defined several conformational states of the LPL molecule, and the kinetics of conformational transitions \[[@B42]\]. Before heparin, the ratio of LPL activity to LPL mass was about 0.2 mU/ng in liver and 0.3 mU/ng in blood (Table [1](#T1){ref-type="table"}). After heparin, the specific activity for LPL in plasma increased to about 1.2, indicating that heparin released mainly or almost exclusively the active form of the lipase as has been shown to be the case in humans \[[@B7]\]. In the liver, however, the ratio stayed well below one. At the 60 min time point LPL mass had increased by about 500 ng/g, but LPL activity had only increased by about 240 mU. This indicates that the lipase loses catalytic activity after it is taken up in the liver, but is degraded more slowly. These observations are in accord with earlier studies. Chajek-Shaul et al. perfused rat livers with LPL-containing media and found that the enzyme lost its catalytic activity soon after binding/uptake in liver \[[@B43]\]. Wallinder et al compared the uptake and degradation of ^125^I-labeled LPL in liver to that for asialofetuin, which is taken up by the galactose receptor \[[@B13]\]. The half-life for asialofetuin was about 15 min, whereas that for the lipase was longer, about one hour. To explore the source of LPL released into plasma and taken up by the liver we measured LPL activity in adipose tissue and heart before and after injection of heparin. In fed rats there was a large decrease of LPL activity in adipose tissue, in accord with a previous study \[[@B6]\]. From these data and the tissue weights at least 7000 mU LPL activity was washed out from white adipose tissue during the first 20 min after heparin. To this should be added an unknown amount of LPL washed out from other tissues. During the same time the LPL activity increased by the 1600 mU in the liver and about 5100 mU in blood. These data indicate that the dominant source of LPL released to plasma in fed rats is the white adipose tissue. Post-heparin LPL activity was lower in fasted than in fed rats, less than half, in accord with previous studies \[[@B18],[@B20]\]. LPL activity in adipose tissue is suppressed during fasting, and we did not find any significant loss of activity after heparin. Hence, the adipose tissue releases much less LPL activity into plasma in fasted than in fed rats. The main contributors are presumably heart and skeletal muscle. We observed a large washout of LPL activity from heart in the fasted rats, about 40%. In other studies we have noted a similar washout from the Soleusmuscle. Kuwajima et al perfused some of the rat hindlimb muscles (gastrocnemius, soleus and plantaris) with heparin in situ and observed a large release in fasted but not in fed rats \[[@B20]\]. These data suggest that in fasted rats, the main source of LPL released into plasma by heparin are skeletal muscles and heart. LPL in plasma is bound to lipoproteins and it has been suggested that the lipase serves as a ligand for binding and uptake of lipoproteins in the liver. Chevreuil et al injected doubly labelled chylomicrons to rats shortly after heparin \[[@B44]\]. The results showed accelerated lipolysis of the triglyceride moiety of the chylomicrons, as expected. In addition, clearance of chylomicron remnants, as traced by retinyl esters, was greatly accelerated. Together with the present results this suggests that after heparin, the large increase of LPL in blood may accelerate the hepatic uptake of some lipoproteins. Conclusions =========== • In the liver, the active form of LPL initially binds to sinusoidal surfaces but then transfers to and is taken up mainly in hepatocytes • An inactive form of LPL, presumably monomers, was mainly taken up in Kupffer cells • Heparin retards the uptake of active LPL in liver, but there are heparin insensitive binding sites for LPL both on hepatocytes and on Kupffer cells • Release of LPL into blood by heparin results in accelerated transport of the lipase to the liver • The observation that rat liver contained substantial amounts of LPL, most of which was inactive, is in accord with the hypothesis that one route for turnover for endothelial LPL is transport to and degradation in the liver • The observations that most of the LPL in blood is inactive, that injected inactive bovine LPL located to Kupffer cells, and that the immunostaining for endogenous LPL was more intense over Kupffer cells than over hepatocytes suggest that a substantial fraction of the transport from extrahepatic tissues occurs with LPL that has lost its activity. • The main source of LPL released into plasma and taken up by the liver in fed rats is the adipose tissue, whereas in fasted rats the main sources are heart and skeletal muscles. Methods ======= Animals ------- Male Sprague-Dawley rats (Moellegard Breeding centre, Denmark) weighing 180--220 g were used. They were kept on a standard pellet diet in a 12-hour light cycle. In order not to disturb blood circulation or the metabolic functions of the liver, we performed all experiments on unanaesthetized rats. They were killed through decapitation at the time of tissue removal. Injections were made in the tail vein. Mean liver weight for the rats was about 9 g. In some of the rats we dissected out all visible adipose tissue. The mean total weight was 14 g, including fibrous tissue removed with the subcutaneous adipose tissue. To correct values for LPL mass/activity in the liver we used an estimated figure of 3% for the amount of blood plasma remaining in the liver after exsanguination. This was based on earlier experiments with ^125^I-albumin and Cr^51^-labeled red blood cells \[[@B45],[@B46]\]. The local Animals Care Committee in Umeå approved all animal procedures. Materials --------- Vectashield mounting medium was from Vector Laboratories, Burlingame, CA. Tissue-Tec OCT compound was purchased from Sakura Finetek Europe BV, Zoeterwoude, The Netherlands. Microscope slides and cover slips were from Menzel -- Gläser, Germany. Plasma, used in the preparation of catalytically inactive LPL, was taken from fasted rats with EDTA as anticoagulant. Heparin was obtained from Leo Pharma AB, Malmö, Sweden. The dose given was 500 IU/kg body weight. Lipase and antibody preparations -------------------------------- LPL was purified from bovine milk as previously described \[[@B47]\] and was labeled with ^125^I using the lactoperoxidase/glucose oxidase method \[[@B13]\]. The labeled LPL was separated from damaged protein and free iodine by chromatography on heparin-Sepharose using a gradient of NaCl. The labeled preparations were stored at -70°C in the presence of 2 mg BSA per ml. The specific activity of the labeled LPL was approximately 10 000 cpm/ng. Inactive LPL for the electron microscopy study was prepared by dissociation in guanidinium hydrochloride as described \[[@B13]\]. To find suitable conditions to prepare inactive LPL for the immunofluorescence experiments, we diluted bovine LPL in rat plasma to the concentration we would later use in the in vivoexperiments and incubated this at different temperatures. On incubation at 37°C the LPL activity remained essentially stable for one hour. At higher temperatures the lipase became unstable. Based on these results we decided to use 45°C for gentle inactivation of LPL aimed to prevent aggregation of the enzyme. Chromatography on heparin-Sepharose of active and inactivated LPL showed, as expected \[[@B19]\], that the active form of LPL eluted around 1 M NaCl, while the inactive form(s) eluted earlier in the salt gradient. After 30 min at 45°C most of the lipase eluted early in the gradient. Only about 20 % remained in the form with high heparin affinity. At 60 min this form had been reduced to 8 % and after 90 min it had virtually disappeared. From this we decided to use incubation at 45°C for 90 min to transform LPL to the inactive (presumably monomeric) form with low affinity for heparin. A trace amount of ^125^I-labeled LPL was included in the preparation, to enable us to follow the distribution and metabolism of the injected material. Each rat received about 40 μg lipase protein, except in the electron microscopy studies where only a trace amount of the labeled lipase was injected. Antibodies against bovine LPL were raised in a chicken (chicken no 225) and IgG were isolated from egg yolks as previously described \[[@B48]\]. Antibodies against bovine LPL were also raised in a rabbit and IgG were isolated on a Protein A-Sepharose column. Both the chicken and the rabbit antibodies were affinity purified on LPL-Sepharose. They were eluted with 0.2 M glycine at pH 2.7, and 50 mM diethylamine at pH 12, respectively, and immediately dialysed against 10 mM Tris/HCl, pH 7.4. Monoclonal antibody 5D2 to LPL was a kind gift from Dr. J. Brunzell, Seattle. A mouse monoclonal antibody (ED2) against a surface antigen expressed on rat Kupffer cells was obtained from Becton Dickinson, San Diego, CA. Goat anti-rabbit IgG labeled with Alexa Fluor 488, goat anti-chicken IgG labeled with Alexa Fluor 488, and goat anti-mouse IgG labeled with Alexa Fluor 546 were from Molecular Probes, Leiden, The Netherlands. Goat IgG, used for control sections, was from Sigma, St.Louis, MO. Preparation of tissue for immunofluorescence studies and confocal microscopy ---------------------------------------------------------------------------- Small pieces of liver were mounted in Tissue -- Tec OCT and snap frozen in propane chilled with liquid nitrogen. The tissue pieces were then stored at -70°C until sectioning. Cryosections were fixed for 10 min in 4 % paraformaldehyde. After rinsing, the sections were blocked in 5 % goat serum for 10 min and then incubated overnight with the primary antibody. All these procedures were made at room temperature. Incubation with the secondary antibody was then for 30 min at 37°C. The sections were rinsed in 0.01 M phosphate 0.15 M NaCl at pH 7.4 and mounted in Vectashield medium (Vector laboratories, Burlingame, CA). The immunostained samples were analyzed by confocal laser scanning microscopy (Leica SP2 or Nikon Eclipse E 800). To avoid potential signal crossover the two fluorophores were sequentially scanned. Data were collected with sequential laser excitation to eliminate bleed through and with confocal parameters such as pinhole size set to minimize the thickness of the optical sections. The images were digitally optimized using the Adobe Photoshop software. Electron microscopy ------------------- For autoradiographic studies, ^125^I-labeled LPL was injected to rats and 10 min later, the livers were perfused with 2% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for 10 min. After fixation the livers were washed in 0.1 M phosphate buffer, cut in small pieces, dehydrated through graded acetone solutions and embedded in Spurr resin. Postfixation with 1% osmium tetroxide was not performed due to its known effect of fading latent images in autoradiography. Ultrathin sections, 70 nm thick, were collected over Formvar-carbonated copper grids. These sections were coated with a monolayer of Ilford L4 nuclear emulsion, diluted 1:4 with distilled water, by means of a tungsten wire loop following the \"loop interference\" technique. After an exposure of 8 months, the silver grains were revealed with Phenidon development. Sections were stained with uranyl acetate and lead citrate and examined in a Hitachi MT 800 electron microscope at 75 kV. Quantitative analysis was performed by counting the number of silver grains per area in each experimental condition. The area analyzed was about 25 × 10^4^μm^2^. Lipase assays ------------- The activities of LPL and of hepatic lipase were determined as described \[[@B49]\]. In the assay for LPL the substrate was an emulsion of soybean triglycerides and a trace amount ^3^H oleic acid-labeled triolein in egg yolk phospholipids. Hepatic lipase (HL) was inhibited by incubation of the samples on ice for two h with rabbit anti-HL IgG. In the HL assay, LPL is inactivated by 1 M NaCl. Both assays were run at 25°C for 30 min. All determinations were carried out in triplicate. The activities are expressed in mU/ml plasma. One mU corresponds to 1 nmol of fatty acid released / min. All determinations were carried out in triplicates. Plasma samples were stored frozen at -70°C before the analysis. Tissue samples were rinsed in cold 0.9 % NaCl, blotted dry, weighed and then immediately frozen in liquid nitrogen in 9 volumes of buffer at pH 8.2 containing per ml: 1 mg BSA, 10 mg Triton X-100, 1 mg SDS, 5 IE heparin, and protease inhibitor Complete Mini (Roche) 1 tablet / 50 ml buffer. They were stored at -70°C and later thawed and homogenized with a Polytron homogenizer (PT-MR 3000; Kinematica AG, Littau, Switzerland). The homogenates were centrifuged for 15 min at 3000 rpm in a Beckman Microfuge and the supernatants were used for the assays. For assay of LPL in liver and post-heparin plasma, the activity of hepatic lipase was suppressed by incubating the extract with an excess of anti-HL immunoglobulins before assay. Detergent containing extraction buffers are needed to solubilize and stabilize active and inactive forms of LPL efficiently \[[@B19],[@B50]\], but the detergents may interfere with the assay. Bergö and Olivecrona \[[@B19]\] used the same assay conditions as in the present study and found that the assay system tolerated at least 10 μl of the detergent buffer without any decrease in LPL action. We have repeated these studies in the context of the present experiments. With extracts from adipose tissue, the assay system showed good linearity between the amount of extract added and the lipase activity displayed, but with extracts from heart, kidney or liver there was a definite nonlinearity. Our interpretation is that other tissue proteins, solubilized by the detergents, interfere. We have therefore used a small volume of tissue extract (usually 2 μL), to stay within, or close to, the linear range of the assay. To explore the recovery of LPL activity in liver extracts as prepared here, we added purified bovine LPL to the homogenate which was then treated and assayed as the other samples. The recovery of the added bovine LPL was complete within experimental error. LPL protein mass was measured by an ELISA, using chicken antibodies for capture and the monoclonal 5D2 antibody coupled to peroxidase for detection \[[@B19]\]. Bovine LPL was used as standard. List of abbreviations ===================== LRP -- low density lipoprotein receptor-related protein, LPL -- lipoprotein lipase, HSPG -- heparan sulphate proteoglycan, HL -- hepatic lipase, ELISA -- enzyme-linked immunoassay, BSA -- bovine serum albumin, SDS -- sodium dodecyl sulphate, VLDL -- very low density lipoprotein Authors\' contributions ======================= LN carried out the immunolocalization studies, C L-I carried out the electron microscopic studies, SV participated in the design of the study and supervised the electron microscopy, JG carried out the studies on wash-out of LPL from tissues after heparin, TO conceived of the study, participated in its design and drafted the manuscript. GO participated in the design of the study and coordinated the work. All authors read and approved the final manuscript. Acknowledgements ================ This study was supported by grants from the Swedish Medical Research Council 03X-727 and 13X-12203. We thank Dr Inga Hägerstrand, Umeå University Hospital, for help with liver morphology, David Bellido, Scientific and Technical Services of the University of Barcelona, for technical help with autoradiography, Ann-Sofie Jacobsson for carrying out all lipase assays, Åsa Lundsten for preparing the antibodies Solveigh Nilsson for preparing the bovine lipase, and Toralph Ruge for labeling it with ^125^I. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Detection of endogenous LPL in rat liver by immunofluorescence. Tissue sections were double-stained with chicken anti-LPL IgG (detected with Alexa 488-labeled goat anti-chicken antibodies (green)), and the monoclonal anti-Kupffer cell antibody ED2 (detected with Alexa 546-labeled goat anti-mouse antibodies (red)). All panels show sections from livers of rats that did not receive heparin. The magnification in panels A and B was × 20-fold whereas it was × 60 + zooming in panel C. Panel A shows staining only for LPL. Panel B is the same area as in A but with staining also for ED2. Panel C shows both stainings. The inset in panel A shows a control section with non-immune chicken IgG instead of anti-LPL. ::: ![](1472-6793-4-13-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Distribution of bovine LPL in livers at different times after injection, and the effect of heparin. Tissue sections were stained with the rabbit polyclonal anti-LPL IgG and then with goat anti-rabbit IgG labelled with Alexa fluor 488 (green). Panels A, C and E are from rats that did not receive heparin. Panels B, D and F are from rats that had been given heparin five min before the injection of active bovine LPL. Panels A and B are two min, panels C and D are 15 min, and panels E and F are 60 min after injection of the lipase. The inset in panel E shows a section from the liver of a rat that was not injected with bovine LPL. The magnification was × 20. ::: ![](1472-6793-4-13-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Higher magnification of sections from the same experiment as in Figure 2Both sections are 15 min after injection of active bovine LPL. The rat in panel A did not receive heparin; the rat in panel B had been given heparin five min before LPL. Green colour represents staining of LPL by the rabbit polyclonal antibody. Red colour represents staining of Kupffer cells by the ED2 antibody. The magnification was × 60 + zooming. ::: ![](1472-6793-4-13-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Ultrastructural localization of injected bovine LPL^125^I-labeled active (upper panel) or inactive (lower panel) LPL was injected. Ten min later the rats were killed and sections of their livers were processed for autoradiography as detailed in the methods section. H, hepatocytes, EC, endothelial cell, KC, Kupffer cell. Bar: 2 μm. ::: ![](1472-6793-4-13-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Distribution of injected, inactive bovine LPL in livers, and the effect of heparinAll sections are 15 min after injection of inactive bovine LPL. The sections in panels A and C are from rats that did not receive heparin; the sections in panel B and D are from rats that had been given heparin five min before the lipase. Green colour represents staining of LPL by the rabbit polyclonal antibody. Red colour represents staining of Kupffer cells by the ED2 antibody. Panels C and D show both stainings. The magnification was × 20 in panels A and B; × 60 + zooming in panels C and D. ::: ![](1472-6793-4-13-5) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Lipases in plasma and liver after injection of heparin ::: Time min Plasma Liver ------------ ---------- --------- ----- ------ --- ----- ------ --- ------ ----- --- ---- ----- --- ---- ------ --- ------ ----- --- ---- 0 8 ± 2 29 ± 1 0.28 ± 0.06 26 ± 1 119 ± 12 0.22 ± 0.02 450 ± 21 2 844 ± 35 746 ± 62 1.17 ± 0.11 116 ± 21 243 ± 19 0.46 ± 0.04 181 ± 22 15 1161 ± 146 1047 ± 190 1.18 ± 0.11 245 ± 30 397 ± 37 0.62 ± 0.05 181 ± 23 60 485 ± 124 502 ± 118 1.24 ± 0.15 265 ± 27 624 ± 66 0.45 ± 0.06 255 ± 17 Rats were injected with heparin. After the indicated times the rats were killed, blood samples were taken from the heart and livers were removed and processed for determination of LPL activity and mass as described in Material and Methods. The values have been corrected for the contribution of blood remaining in the liver, and are means ± SEM, n = 6. ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Effect of heparin injection on tissue LPL activities ::: Treatment LPL activity mU/g ----------------- --------------------- ------------- ------------- ------------- ------------- Fed rats Before heparin 1.9 ± 0.2 28 ± 8 999 ± 111 1700 ± 138 1094 ± 195 After heparin 1057 ± 59^a^ 173 ± 20^a^ 868 ± 67 938 ± 70^a^ 383 ± 73^a^ Fasted rats Before heparin 2.5 ± 0.3 54 ± 12 1199 ± 63 345 ± 48^b^ 188 ± 49^b^ After heparin 374 ± 76^a,b^ 189 ± 21^a^ 729 ± 70^a^ 357 ± 56^b^ 263 ± 28 The fasted rats had been deprived of food overnight (16 -- 18 hours). The values have been corrected for the contribution of blood remaining in the liver, and are means ± SEM, n = 5. The difference is statistically significant (p \< 0.05) ^a^when comparing the effect of heparin,^b^when comparing the effect of nutritional state. ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Clearance of ^125^I-labeled LPL from blood and uptake in liverin the presence and absence of heparin ::: Time after injection (min) % of injected ^125^I-labeled LPL ------------------------------ ------------------------------------ ------------ ------------ Blood Liver Liver Active LPL 2 32.6 ± 3.1 13.9 ± 0.6 15 8.5 ± 13.6 51.5 ± 2.8 30.4 ± 1.4 60 6.7 ± 5.5 18.2 ± 1.7 22.8 ± 0.4 Inactive LPL 15 9.4 ± 0.7 50.0 ± 5.5 60 6.2 ± 1.0 18.8 ± 3.1 Rats were injected with active or inactive ^125^I-labeled LPL either directly, or five min after intravenous injection of heparin. After the indicated times the rats were killed, blood samples were taken from the heart and livers were removed. The values have been corrected for the contribution of blood remaining in the liver, and are means ± SEM, n = 5. :::	PubMed Central	2024-06-05T03:55:49.923779	2004-11-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534784/", "journal": "BMC Physiol. 2004 Nov 15; 4:13", "authors": [ { "first": "Lucyna", "last": "Neuger" }, { "first": "Senén", "last": "Vilaró" }, { "first": "Carmen", "last": "Lopez-Iglesias" }, { "first": "Jitendra", "last": "Gupta" }, { "first": "Thomas", "last": "Olivecrona" }, { "first": "Gunilla", "last": "Olivecrona" } ] }
PMC534785	Review ====== Introduction ------------ Chronic disease affects approximately 18% of children \[[@B1]\]. Although cure is not possible, survival rates have improved substantially for many conditions (e.g. cancer \[[@B2]\] and cystic fibrosis \[[@B3]\]). Many diseases require daily self-management and restrict children\'s physical and social activities. Consequently questions are increasingly raised about the quality of life (QOL) of children with chronic disease. Efforts to measure child QOL have proved complex but a number of generic and disease-specific measures have been reported \[[@B4]\]. Generic measures are designed to assess and compare health status in patients with different diseases and may provide valuable information for comparing outcomes between sick and healthy populations. They are generally well validated and reliable but are often not recommended for work involving evaluation of randomised controlled trials (RCTs), as they lack sensitivity to detect small but clinically significant changes in QOL over time or due to treatment for specific diseases \[[@B5]\]. Disease specific measures are more suitable for evaluation of clinical trials designed to assess a particular treatment. These measures include items that are likely to be affected by the specific disease or treatment and are therefore more responsive to clinically significant changes. The quality of measures must be evaluated according to performance characteristics. Guidelines suggest good measures of QOL are reliable and valid for the group of patients for whom they are used, include a form for self-report wherever possible, are brief and developmentally appropriate, and allow completion by proxy \[[@B4]\]. There is little evidence that QOL measures are routinely used in clinic practice \[[@B6]\] or clinical trials \[[@B7]\], despite the fact that the aim in many trials is to improve QOL. In both child \[[@B8]\] and adult work \[[@B9]\], few trials include measures of QOL, and amongst those, non-standardised measures continue to be used. QOL is also frequently insufficiently analyzed, reported or discussed in the study report or subsequent publications \[[@B5]\], despite the increasing emphasis in clinical practice and research to use patient centered outcomes and child perspectives \[[@B10]\]. We report a systematic review drawing on established methodologies \[[@B11]\] to determine first, the extent to which QOL measures are used in paediatric clinical trials and RCTs, and second, the quality of QOL measures currently used. Method ====== Search Strategy --------------- The following databases were searched: MEDLINE 1966 to Nov Week 2 2003, CINAHL 1982 to December Week 1 2003, EMB Reviews: Cochrane Central Register of Controlled Trials, Your Journals at OVID, EMB reviews: ACP Journal Club 1991 to July / August 2003, EMB reviews: Database of Abstracts and Reviews of Effects 3^rd^quarter 1993, AMED (Allied & Contemporary Medicine) 1985 to December 2003, British Nursing Index (BNI) 1985 -- October 2003, EMBASE, PSYCHINFO 1872--2003. Text word and thesaurus searches were used to minimise the chance of missing relevant articles. The following keywords were searched: • child, childhood, children, adolescent, infant, pediatric, paediatric, • quality of life, QOL, • clinical trial, randomised controlled trial Searches were restricted to English language papers. Search engines were used to search the Internet with keywords and Boolean logic. Additional references from articles identified through these searches were also pursued. Inclusion Criteria ------------------ These included: 1\) Children and adolescents up to the age of 20 years, 2\) RCT, formal cross-over trial, or studies evaluating one or more active drug treatment with or without placebo, 3\) Standardised QOL measure (For these purposes we drew on a previous review \[[@B4]\] and defined minimal psychometric criteria to include some preliminary reliability and validity data), 4\) Articles published in English between January 1994 and December 2003. Exclusion Criteria ------------------ 1\) Samples including both adults and children. 2\) Comparison of surgical treatment, pain control, palliative medication, or psychological/homeopathic intervention. 3\) Outcomes evaluated in terms of medical data only, non-standardised measures of QOL or standardised psychological measures including symptom checklists, measures of self-esteem, or coping. Procedure --------- Abstracts were reviewed for relevance and full articles obtained where appropriate. A summary sheet was developed and both authors independently reviewed papers to ensure reliability. Data extracted by reviewers was second coded and compared and any discrepancies were resolved through discussion. Results ======= Of the 917 records retrieved from the databases, initial inspection suggested that 27 abstracts met the inclusion criteria. On reading the full articles, nine failed to meet inclusion criteria. The resulting 18 articles were included in the review \[[@B12]-[@B29]\]. Study characteristics (summarised in Table 1 \[see [Additional file 1](#S1){ref-type="supplementary-material"}\]) ----------------------------------------------------------------------------------------------------------------- • Disease:QOL was most frequently included in trials in atopic diseases (Asthma = 4, Rhinitis = 4, Dermatitis = 2 and Eczema = 1). Single studies were identified in Cystic fibrosis, Otis media, Amblyopia, Diabetes, Obesity associated with a brain tumour, Idiopathic short stature, and Congenital agranulocytosis. • Location:Seven studies were conducted in the U.S.A, 4 in the U.K, 3 in the Netherlands, one in Taiwan, and one in Israel. Two studies were multi-national. • Child\'s age:Three studies recruited children across a broad age range (1 to 18 years), 2 focused on pre-school children (1--5 years), 4 on pre-school and middle childhood (2--10 years), 2 on middle childhood (6--12 years), 6 on middle childhood and adolescence (5--18 years), and one on adolescents alone (12--17 years). • Sample size:Sample size ranged from 19 \[[@B29]\] to 689 \[[@B15]\]. Power calculations were reported in six studies. • Design and trial aim:We identified 11 RCTs, 2 cross-over studies, and 5 studies comparing two or more active treatments without placebo or control group. Of the 11 RCTs, 1 was multi-national, 7 multi centre, and 3 single centre studies. Of the 7 non RCTs, 1 was multi-national, 2 were multi centre, and 4 single centre. Nine articles involved comparisons of two or more treatment and the remainder involved comparison of treatments with placebo. • Blinding:Seven RCTs reported blinding procedures. • Parent and caregiver QOL:Fifteen studies measured the impact of the disease on the child\'s QOL. Three included assessment of the caregivers QOL. • Respondent for child QOL:Of the 15 studies focusing on child QOL, 10 were based on child, and 3 on parent reports. In two studies both children and parents reported the child\'s QOL and in one of these clinicians also rated child QOL \[[@B28]\]. Quality of QOL measures (Table 2 \[see [Additional file 2](#S2){ref-type="supplementary-material"}\]) ----------------------------------------------------------------------------------------------------- • Generic or disease specific:In total, 12 disease specific and two generic measures were used \[[@B30]-[@B38]\]. The four asthma trials involved three different measures of asthma specific QOL. The four perennial rhinitis trials used two different measures of rhinitis specific quality of life, and the two atopic dermatitis trials and one atopic eczema trial used two different dermatology specific measures of QOL. In two studies authors had developed their own disease specific measure \[[@B25],[@B29]\]. • Quality of measure:We assessed quality of measures based on minimal accepted criteria \[[@B4]\] whereby measures should be brief, allow proxy and self report and include reliability and validity data and age appropriate versions. Although all measures included some preliminary psychometric data, only two measures fulfilled all of these criteria \[[@B36],[@B38]\]. Three measures fulfilled four criteria but lacked age appropriate versions. The remaining measures fulfilled three or less criteria. Discussion ========== Despite extensive searches we identified only 18 published reports of paediatric trials including standardised QOL measures. This undoubtedly represents a very small percentage of paediatric trials and supports previous findings that QOL data is seldom reported in paediatric clinical trials \[[@B8]\]. Asthma and rhinitis were most frequently studied, perhaps because there is higher incidence for these conditions in children compared to other conditions such as cancer and cystic fibrosis \[[@B39]\]. Further explanations include the non-life threatening nature of these conditions as well as the availability of disease specific measures compared to rarer illnesses. In considering why there are relatively few trials including QOL measures, it is important to take into account the aims and purpose of the trial \[[@B40],[@B41]\]. The aim of most trials is to assess the impact of treatment on clinical variables, with QOL viewed to be of secondary importance if at all. It is not necessarily appropriate that QOL measures are included in all trials. Where QOL assessment is appropriate however, inclusion of a QOL measure must be hypothesis driven and an integral part of the clinical development programme rather than an added afterthought \[[@B5]\]. Quality of measures ------------------- Where QOL was measured, disease specific measures were most often used (N = 12) as is normally recommended for use in clinical trials. Only two trials included measures that satisfactorily fulfilled accepted criteria \[[@B4]\]. Typically, information about measures included some reliability data although a third of studies failed to provide information about the validity of the scale. Most measures were brief and contained less than 30 items but many lacked age appropriate versions or parallel versions for child and proxy raters. Selection of a measure of QOL is dependent on the psychometric properties of the instrument, as well as clinical and demographic variables characteristic of the sample. However, psychometric properties depend upon samples for which the scale has been validated. Hence it is important to ensure measures are used with clinical populations where psychometric data are available. There are some grounds for assuming that QOL changes during childhood, and therefore satisfactory measures target specific age groups \[[@B42]\]. There are difficulties identifying single measures that are appropriate across a wide age range and only half of measures identified in this review included age appropriate versions. It is also generally recommended that ratings of QOL should be made by children themselves whenever possible \[[@B43]\]. In cases of younger children proxy reports are necessary but there are questions about the relationship between child and parent report \[[@B4]\]. It is therefore positive that most (73.3%) studies obtained ratings from children with only four relying on parents alone to provide proxy ratings. CONSORT \[[@B44]\] guidelines recommend methods of reporting RCTs, but do not adequately deal with the issues concerning QOL assessment and psychometric validity. It is essential that trial developers select appropriate measures and are aware of the problems associated with QOL assessment. Barriers to inclusion of QOL measures ------------------------------------- Objections to inclusion of QOL measures in trials involve anticipated increased costs, extra time needed to gain patient and parent consent, and lack of sophistication of currently available measures \[[@B8]\]. A major restriction to inclusion of QOL assessment in clinical trials remains limitations in currently available measures, especially for less prevalent chronic conditions. However, it is only through including measures that we will learn more and be able to develop a second generation of measures that do show more sophisticated properties. A second problem is that disease specific measures may simply not be available for rare conditions. Attempts to develop such measures are promising and in this review instruments for ambylopia \[[@B25]\] and agranulocytosis \[[@B29]\] had been developed. In order to facilitate collection of QOL data from children with chronic illness, reliable and valid measures are increasingly required \[[@B46]\]. Other methodological limitations in current work include the lack of power calculations. Where the aim of the trial includes QOL assessment, power calculations must be performed and are an essential element of clinical trial design. In cases where measurement of QOL is a secondary endpoint, sample size calculations are rare and difficult to establish. However attempts should be made to hypothesise expected changes in QOL scores in relation to the agreed sample size prior to the trial \[[@B5]\]. Conclusion ========== This review supports previous findings of limited use of QOL measures in paediatric cancer trials \[[@B9]\] and extends this to include a number of conditions other than cancer. QOL assessment is most common in trials where the aim is to compare the impact of treatment on clinical variables and is largely limited to common non-life threatening conditions. The measurement of QOL provides valuable information about the psychological and social impact of treatment on children especially where no differences in survival rates are anticipated. For this reason, the inclusion of QOL measurement in paediatric trials is becoming increasingly valued and mandatory \[[@B47],[@B48]\]. There are still questions concerning selection of QOL measures and how best to report findings \[[@B49]\], but our review provides useful information for trial developers regarding the availability and quality of QOL measures. Author\'s contributions ======================= Both authors were responsible for planning, conducting and reporting this work and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Table 1: Study characteristics ::: ::: {.caption} ###### Click here for file ::: ::: {.caption} ###### Additional File 2 Table 2: Quality of life measures ::: ::: {.caption} ###### Click here for file :::	PubMed Central	2024-06-05T03:55:49.927780	2004-11-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534785/", "journal": "Health Qual Life Outcomes. 2004 Nov 22; 2:66", "authors": [ { "first": "Sally-Ann", "last": "Clarke" }, { "first": "Christine", "last": "Eiser" } ] }
PMC534786	Background ========== The Menopause Rating Scale (MRS) was initially developed in the early 1990s \[[@B1],[@B2]\] to measure the severity of age-/menopause-related complaints by rating a profile of symptoms. The validation of the MRS began some years ago \[[@B2]-[@B6]\] with the objectives (1) to enable comparisons of the symptoms of aging between groups of women under different conditions, (2) to compare severity of symptoms over time, and (3) to measure changes pre- and post-treatment \[[@B4]-[@B6]\]. Development and standardization of the scale were published elsewhere \[[@B2]\]. In brief, the standardization of this scale was performed on the basis of a representative sample of 500 German women aged 45--60 years in 1996. A factorial analysis was applied to establish the raw scale of complaints or symptoms. Statistical methods were used to identify the dimensions of the scale. Finally, three dimensions of symptoms/complaints were identified: a psychological, a somatic-vegetative, and a urogenital factor that explained 59 % of the total variance \[[@B2]\]. This is indicative for a high efficiency of a scale with only 11 items -- compared to other international scales. Reference values for the severity of symptoms or complaints were calculated based on a population sample \[[@B2]\]. The scale consisting of 11 items is self-completed by the woman. A 5-point rating scale permits to describe the perceived severity of complaints of each item (severity 0 \[no complaints\]\...4 scoring points \[very severe symptoms\]) by checking the appropriate box. The composite scores for each of the dimensions (sub-scales) are based on adding up the scores of the items of the respective dimensions. The composite score (total score) is the sum of the dimension scores. Details as how to apply and evaluate the scale were published \[[@B8],[@B9]\] and can be also obtained from the website <http://www.menopause-rating-scale.info>. The scale was defined as a menopause-specifc, health-related quality of life scale (HRQoL), because the profil of complaints in this scale importantly determines the HRQoL of women in this age span. Moreover, a good correlation between the results obtained with the MRS scale and the generic QoL scale was observed \[[@B6]\]. The MRS scale became internationally well accepted as far as the usage many countries is concerned. The first translation was into English \[[@B7]\]. Other translations followed \[[@B8]\], i.e. taking international methodological recommendations \[[@B10]-[@B12]\] into consideration. Currently, the following versions are available: Brazilian, English, French, German, Indonesian, Italian, Mexican/Argentine, Spanish, Swedish, and Turkish language. These versions are available in a published form, and can be downloaded in PDF-format from the internet (see reference 8 and <http://www.menopause-rating-scale.info>). Like in other health-related QoL scales, it is a challenge to satisfy the demands of a clinical utility and outcomes sensitivity. A comprehensive overview regarding conventional psychometric requirements of test reliability and validity were recently published elsewhere \[[@B9]\]. It is the aim of this paper is to share methodological information about the capacity of the scale to assess changes after hormone treatment since no methodologically detailed work in this regard was published before. Methods ======= A multicenter, open post-marketing study was conducted with a product for hormone therapy (CLIMEN^®^= 2 mg estradiol valerate/2 mg estradiol valerate + 1 mg cyproterone acetate) using the MRS scale as outcome measure under routine conditions of office-based gynaecologists. The study was described in detail elsewhere \[[@B6]\]. In brief, 1801 gynaecologists from all parts of Germany participated in the study on a voluntary basis. 10,904 women who required hormone treatment were included. The median age was 49 years. Beside others, the MRS scale was documented before therapy and 6 months after starting the hormone treatment. A specific problem was the transformation of an older MRS version into the advanced, relatively broad validated current version of MRS. The old version of the MRS was read by the physician and the patient answered to which extend she perceived suffering from a specific symptom, and if yes to which extend. The new scale is self-completed by the woman without interaction with the physician. The symptoms itself are the same in both scales. Nevertheless, this is a methodological limitation. We, however, are not interested in the absolute score values but relative changes after treatment compared to before. In addition, the scoring system of the old version was adjusted into the new coding system using a linear transformation. Additionally, one question of the old version was split into two questions -- as recommended for the current version of the MRS (see later discussion on limitations of the study). The statistical analyses were performed with the commercial statistical package SAS 8.2. Results ======= Altogether, data of 9311 women were available for most of our analysis. However, the sample size varied slightly depending on the variables used because we had also missing information in a few variables. The mean age was 49.8 years (SD 6.4). About half of the participating women were still perimenopausal (51.9%) or were already in the postmenopausal phase (48.1%). The mean body mass index was not eye-catching with 24.7 (SD 3.7). The improvement of the health-related quality of life (HRQoL) -- measured with the MRS scale -- is described in Table [1](#T1){ref-type="table"}. The means and SD of the scoring points of the total scale (and the three subscales) can be seen at baseline (before therapy) and after six months of hormone treatment. Significant declines of the mean scores were observed after treatment indicating an improvement of HRQoL altogether and in the three subscales of the MRS. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Means (SD) of MRS scores at baseline (before therapy) and at end of observation (after therapy. Improvement of scores after therapy by absolute difference in scoring points. ^§^Total scale and for each subscale. ::: n Scores before Scores after Absolute change Percent (%) change *P\\* ---------------------------- ------- --------------- -------------- ----------------- -------------------- ----------- Total scale 9311 11.0 (8.2) 1.7 (3.2) 9.3 (7.4) 36.1 (20.6) \<0.0001 Psychological subscale 9311 4.5 (4.1) 0.7 (1.7) 3.8 (3.7) 34.5 (27.1) \<0.0001 Somatic subscale 9311 4.2 (3.2) 0.5 (1.2) 3.6 (3.0) 37.3 (23.1) \<0.0001 Urogenital subscale** 9311 2.3 (2.6) 0.5 (1.1) 1.8 (2.3) 24.5 (25.3) \<0.0001 ^§^Summary score \"before therapy\" minus \"after therapy\" \* Percent (%) change compared with the change before treatment: pre-treatment score minus post-treatment divided by pre-treatment score multiplied by 100 (%) \\ Paired t-test for dependent samples: significance of the absolute difference ::: Apart from the comparison of means, we calculated the relative improvement compared with the situation before therapy (baseline) to better understand the magnitude of change after therapy (Table [1](#T1){ref-type="table"}), i.e. in absolute and relative terms. There was not much difference in relative improvement (%) among subscales (all highly significant): In average, the scores improved by one third after six months hormone treatment. The scale is able to measure an improvement in patients starting with \"no/little complaints\" (total score = 0--4), \"mild\" (5--8), \"moderate\"(9--15), and \"severe\" (16 + points) before therapy (= baseline). This is presented in Table [2](#T2){ref-type="table"}: The more severe the complaints were before treatment the better the effect regarding relative improvement of symptoms measured by the MRS, which gives evidence for the clinical utility of the MRS as outcome measure. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Relative change of MRS scoring points as percent of the baseline total score: Mean values (SD) of the relative change (% improvement of the complaints) in four categories of severity at baseline. ::: Severity of complaints at baseline --------------------- ------------------------------------ ----------------------- ----------------------- ----------------------- No/little (0--4) Mild (5--8) Moderate (9--15) Severe (16+) Means (SD) (n = 2460) Means (SD) (n = 1693) Means (SD) (n = 2592) Means (SD) (n = 2566) Total score 10.8 (10.6) 32.2 (9.8) 43.9 (11.8) 55.1 (13.8) Psychological score 6.0 (14.7) 27.6 (21.5) 43.7 (20.6) 57.1 (17.9) Somatic score 13.8 (17.3) 34.4 (18.5) 44.1 (16.9) 54.8 (15.9) Urogenital score 5.7 (13.9) 17.0 (20.6) 27.5 (23.6) 44.4 (22.6) ::: It is interesting to compare the HRQoL before and after hormone treatment with the norm values of MRS obtained in an average population of aging women, i.e. not patients as in our post-marketing study. To this end, we compared only the MRS total scores in patients with the average female population (Table [3](#T3){ref-type="table"}). It became evident in this crude and simple comparison, that the severely deteriorated distribution of complaints in the patient group before therapy -- compared with the normal population -- improved after therapy remarkable, i.e., at least as far as the total score of the MRS is concerned. The three subscales showed a similar tendency towards the better. The extremely high proportion of patients without complaints immediately after therapy could be due to a selection problem in this post-marketing study and the application of the physician-administered version of the MRS (see discussion). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Level of complaints in the population in percent (%) compared with patients of the Climen treatment study: Frequency distribution before/after therapy compared with population norm values\* ::: Frequency in patients: Percent (%)in four categories of severity --------------------- -------- -------------------------------------------------------------------- ---------- Total score No or little (-4) 48 26.4 86.8 Mild (5--8) 25 18.2 8.4 Moderate (9--15) 20 27.8 4.0 Severe (16+) 8 27.6 0.8 Psychological score No or little (-1) 48 30.7 82.5 Mild (2--3) 23 17.0 10.4 Moderate (4--6) 20 22.2 5.5 Severe (7+) 9 30.1 1.6 Somatic score No or little (-2) 53 35.7 93.0 Mild (3--4) 24 22.7 5.1 Moderate (5--7) 15 25.5 1.6 Severe (8+) 8 16.1 0.3 Sexual score No or little (0) 64 37.3 76.8 Mild (1) 13 13.3 11.6 Moderate (2--3) 16 22.2 8.7 Severe (4+) 7 27.3 2.9 \* The population data came from the standardization of the MRS scale \[2,3\] ::: The treating gynaecologist (who also applied the MRS scale) assessed individually the efficiency of the hormone treatment in the above mentioned intervention study. The gynaecologist\'s expert opinion regarding treatment efficiency was categorized into two categories for the purpose of this analysis: successful(very effective and effective) and not successful(little, no, or negative effects). This alternative variable was then used for the comparison with the alternative \"success-variable\" based on MRS (total score only): \"successful\" (5 and more points reduction after therapy compared with baseline test) and \"not successful\" (less than 5 scoring points reduction after therapy compared with baseline test). The prediction of the expert opinion of the treating gynaecologist with the MRS data seems to be good: sensitivity (correct prediction of a positive assessment by the physician) 70.8% and specificity (correct prediction of a negative assessment by the physician) 73.5% (Table [4](#T4){ref-type="table"}). ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Prediction of a positive assessment by the physician concerning \"successful treatment\" by means of the MRS scale (total score).\"Not successful\" was defined for the MRS as: less than 5 scoring points improvement at the end of the HRT treatment compared with \"before treatment\". ::: MRS: not successful MRS: successful Total ------------------------ --------------------- ----------------- ------- Doctor: not successful 311 112 423 Doctor: successful 2570 6227 8797 Total 2881 6339 9220 Sensitivity: 70.8% Specificity: 73.5 ::: Discussion ========== The MRS scale was developed (a) to assess symptoms of aging/menopause (independent from those that are disease-related) or HRQoL between groups of women under different conditions, (b) to evaluate the severity of symptoms over time, and (c) to measure changes pre- and post hormone replacement therapy. The aim of this paper was to empirically demonstrate that the latter claim is evident. Reliability and validity are important to show the usefulness of the scale as a clinical utility in monitoring treatment effects -- once all other methodological requirements are successfully demonstrated before. Reliabilitymeasures (internal consistency and test-retest stability) were found to be good across countries \[[@B9]\]. Regarding validityit was shown that the internal structure of the MRS across countries was sufficiently similar to conclude that the scale really measures the same phenomenon \[[@B9]\]. The comparison with another scale for aging women -- although not a validated HRQoL scale (Kupperman) -- showed sufficiently good correlations of the total score, which is compatible with the notion of a good criterion-oriented validity. The same is true for the comparison with the generic quality-of-life scale SF36 where also high correlation coefficients have been shown \[[@B3]-[@B5]\]. Another fact in favor of the scale is that it was translated into 10 languages so already \[[@B7]-[@B9]\]. Having the above-mentioned psychometric data available, a point was reached to critically evaluate the capacity of the scale to reliably measure health-related effects of hormone treatment independent from the severity of complaints and -- in addition -- to the comparison of treatment effects measured by the MRS scale and the subjective assessment by the treating physician. To this end, many clinicians use the term \"validity\" and mean high utility for clinical work or research. The only hormone treatment study with the MRS scale as outcome measure in women during menopausal transition we could get data for methodological analysis was the above described postmarketing study. We hope to repeat/confirm this analysis with data of a more stringently designed clinical trial. But even on the basis of a methodologically weak dataset, in absence of other data, we got re-assuring methodological information about the MRS scale. It is a well-established experience that women with menopausal complaints respond to hormone therapy with a marked improvement of the HRQoL. This is what the MRS scale should be able to detect. We saw that the increased mean MRS total score at baseline (before treatment) markedly decreased after 6 months under treatment indicating a significant improvement of complaints & HRQoL. This was also the case for the mean scores of the three subscales. These data cannot disentangle the effect of treatment and \"natural variation\" of complaints over time. This however was not the point: It was not the intention of this paper to evaluate effectiveness of hormone therapy in an uncontrolled post-marketing study. The absolute improvement of symptoms during treatment was 9.3 points of the MRS total score on average. This is equivalent to 36% of the baseline score, and similar also for all three subscales. In other words, the MRS scale was shown to be successful in detecting treatment effects. The impressive magnitude of the therapy-related improvement of HRQoL should be obviously discussed in the context of selection of women with complaints susceptible for this kind of treatment by the participating gynaecologists. Another critical remark is that we cannot comment as to what extend the MRS scale is able to measure true or placebo treatment effects. But this is more a question concerning efficacy and the study draw any conclusions in this regard by definition of the study design. To answer the question whether the sensitivity of the MRS scale is good enough to detect even treatment-related changes in women with only little or mild symptoms as compared with severe ones, the analysis was stratified. An improvement of complaints/QoL was seen in an increasing degree in patients with little, mild, moderate and severe symptoms at baseline. The relative improvement increased with the degree of severity of symptoms at baseline, which is consistent with the general expectation. It seems to be important to underscore: The MRS scale seems to detect also a positive treatment effect in women with little complaints -- although to a lesser degree. Moreover, we showed the capacity of the MRS scale to determine therapeutic efficiency with another approach: a face-value-comparison with norm values of the population \[[@B2],[@B3]\]. The level of complaints in patients before therapy expressed a higher degree of severity (higher MRS total score). After 6 months of hormone treatment the frequency distribution of patients with a certain severity of complaints returned towards a similar distribution as observed in the general population. The extreme proportion of patients with no/ little complaints after therapy should be again seen in the context of apparent patient selection (patients were not only treated because of their symptoms but also for other indications such as prevention) and/or effects of the interaction of patients with the treating physician (who also administered the MRS. Thus, we cannot exclude that such a biases have inflated the impression of a \"too positive therapy efficiency\". But we do not intent to draw conclusions about therapeutic efficiency anyway. It is another way to look at therapeutic efficiency with the assistance of the MRS scale. Although this indicates at least that comparisons with norm values could be helpful for interpreting results of intervention studies, we are not recommending formal statistical testing of differences between patient groups and the reference values of the population: Patients are usually too different from the general population, a difference hard to adjust for. It is just a visual comparison (as in Table [3](#T3){ref-type="table"}) to get a crude idea for the interpretation of results. The MRS scale was also tested whether it predicts the therapeutic assessment of the treating physician. At face value, the individually assessed efficiency of hormone treatment by the treating gynaecologists was comparable with the assessment by the MRS scale, i.e. using a simple dichotomization of the treatment effect in \"successful\" and \"not successful\" for both the subjective opinion of the physician and the result of the MRS scale: The sensitivity (correct prediction of a positive assessment by the physician) was 70.8% and specificity (correct prediction of a negative assessment by the physician) 73.5%. In other words, the MRS scale fits well with the subjective assessment of the treatment effect estimated by the physician. However, conclusions have to be drawn very carefully because of a possibly inherent bias that may have inflated the positive result: The subjective assessment of \"success\" by the treating physician was obviously not as independent from the assessment by the MRS scale as desirable because the physician applied the scale to the patient. Even without being able to recall the result of the MRS six month ago or to calculate and compare the total score of both administrations, the interaction with the patients is likely to have introduced this bias in the direction of a higher compatibility between both assessments. Although the result may too positive compared with a blinded, really independent assessment, it permits to generate the working hypothesis of a sufficiently good prediction of the therapeutic effect by means of the MRS scale. This needs to be confirmed with better data, i.e. data from a blinded, independent comparison, i.e. with the currently used, self-administered MRS scale. The aim of this exercise was only to demonstrate that the MRS scale may well predict the clinical opinion about efficiency of hormone therapy, what was not empirically shown before. We recommend the MRS as standardized/validated \"objective\" scale for use in clinical studies, although some aspects discussed above need confirmation in a new study. Moreover, since the scale is already broadly used at the international level, it is important to sensitise users about some lacking information or weak evidence. The limitations of this study should be shortly summarized. First of all, this study was performed in a dataset where an earlier version of the MRS scale was used, i.e. the scale was not self-administered but completed in an interview of the physician with the patient. This could have influenced the magnitude of the absolute scores of the total and sub-scales. As far as pre-/post-treatment changes are concerned, the magnitude of the absolute changes may have been more influenced than the relative changes of the HRQoL assessment of the patients as discussed in this paper. Another problem along the same line is that we had to transform the old coding system into the new one. This was done with a simple linear transformation and is not likely to have introduced any bias. Another limitation is that this is the first study we are aware of for this kind of assessment of the validity to measure therapeutic intervention. It is not likely that the main conclusions of the study are materially biased. However, the results should be cautiously used (e.g., for planning clinical trials or outcomes studies) as long as not confirmed with data obtained with the currently used self-administered MRS scale without potential influence of the physician. It can be assumed that a new study with the currently recommended MRS scale -- in the sense of \"patient-reported-outcome\" -- would demonstrate positive results but to a lesser degree. Conclusions =========== The MRS scale showed some evidence for its ability to measure treatment effects on quality of life across the full range of severity of complaints in aging women. This however needs confirmation in other and better-designed clinical studies. Competing interests =================== The authors FS and SG are employees of the company that produces HRT products. We cannot however see any conflict of interest as far as the methodological aspects of the validation of the MRS scale are concerned. Authors\' contributions ======================= LAJH: responsible for the collection and evaluation of the data, and involved in writing of the paper. DMT: responsible for building the database of this publication, responsible for the statistical evaluation, and contributed to writing of the paper. FS: responsible for the post-marketing study and designing this paper, contributed to the manuscript. SG: responsible for designing and overseeing the post-marketing study of Climen, contributed to writing and revising of the paper JS: responsible for the field work of the post-marketing study, setting up the initial database, and for the preparation of the subset of data used for this publication. HPGS: Major responsibility in developing the MRS scale, contributed to writing/revision of the manuscript.	PubMed Central	2024-06-05T03:55:49.929086	2004-11-22	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534786/", "journal": "Health Qual Life Outcomes. 2004 Nov 22; 2:67", "authors": [ { "first": "Lothar AJ", "last": "Heinemann" }, { "first": "Thai", "last": "DoMinh" }, { "first": "Frank", "last": "Strelow" }, { "first": "Silvia", "last": "Gerbsch" }, { "first": "Jörg", "last": "Schnitker" }, { "first": "Hermann PG", "last": "Schneider" } ] }
PMC534787	Background ========== Postal surveys are routinely used to obtain information from patients and groups within the general population, over a range of topics. Postal surveys are a cost-efficient method compared with intensive methods such as face-to-face interviews and capable of obtaining systematically, information on many thousands of people. A key quality component of postal surveys relates to the number of people sampled, and the proportion returning a completed useable questionnaire \[[@B1]\]. Lower response rates can reduce the statistical power of the study, and mask statistically significant relationships, which \'truly\' exist within the population studied. Responders may also be different to non-responders. This can introduce bias into the survey findings, if the decision to respond (or not) relates to the outcome being analysed within the survey, thereby reducing generalisability to the initial reference population \[[@B2]\]. Many studies conclude that non-responders in surveys and other epidemiological studies can differ to responders with respect to a range of specific health, lifestyle and social variables. Non-responders have been found to differ with respect to their sex, age, race, social class, home circumstances, education, and healthy lifestyle behaviours \[[@B3]-[@B7]\]. They can also differ in terms of existing health and healthcare utilisation \[[@B8]-[@B11]\] with differences extending through to higher rates of mortality \[[@B12]\] compared with responders. However, it can be difficult to make clear conclusions about the characteristics of non-responders in surveys and other types of studies, as factors such as the purpose of the study and the way in which it was carried out, will no-doubt have some effect. Furthermore, differences have not always been found between responders and non-responders, at least in terms of what factors were assessed, and nonresponse will not always affect estimates of prevalence \[[@B13]-[@B15]\]. Nevertheless, nonresponse bias should always be considered a possibility \[[@B16],[@B17]\]. Moreover, there is evidence that in general, response rates to postal questionnaires are falling, \[[@B18]\], making this topic worthy of continued investigation. There is however, no agreed level of acceptable response in postal surveys \[[@B19]-[@B21]\]. A systematic review \[[@B22]\] found a number of factors associated with postal questionnaires can influence the likelihood of response. This included providing incentives; questionnaire length and appearance, method of delivery, method for return, if any pre-warning/contact was given, the content and layout of the questions, through to the origin/sponsor of the questionnaire and how it was communicated. The review found that monetary incentives, recorded delivery and using an \'interesting\' questionnaire were the three strongest influences on response rates. Statistical heterogeneity was found for all of these factors, thus limiting the extent that pooling of results was viable. Moreover, only a third of studies were from medical/epidemiological/healthcare journals and with no distinction between studies of patients or of staff and subgroup analyses were absent. As such, it is difficult to generalise the findings from the review to particular groups of patients in a healthcare setting. In the current study, we carried out a randomised controlled trial to examine the effect of a pre-warning letter on response rates to a postal questionnaire. The questionnaire was sent to patients who had previously been referred to a community based exercise referral scheme because of a sedentary lifestyle and sought information on the quality of the service offered. An earlier study \[[@B23]\] of a similar population suggested poor response rates could be a problem. With a limited budget, we were unable to offer financial incentives, as suggested in the review by Edwards et al \[[@B22]\], but wanted to explore the suggestion that pre-warning letters might increase final response. Methods ======= The sample consisted of patients who had been referred to a community based exercise referral scheme during the past 12 months, identified from the service register. Patients were referred by a primary care practitioner because of concerns about their sedentary behaviour and its impact on their health. The questionnaire formed part of a project examining the relationship between patient service-expectations and service outcomes. We examined the effect of a pre-warning letter, posted to patients seven-days before sending the main questionnaire, compared with no pre-warning letter. The pre-warning letter was printed on one-side of letter-headed paper, and informed the respondent that a survey would be sent to them within the next few days. It informed them about the purpose of the survey and the importance of it being completed and returned. The main questionnaire was sent with a covering letter and a pre-paid business franked addressed envelope for its return \[[@B24]\]. A standard reminder letter was sent to all non-responders seven days after posting the questionnaire, and after a further seven days, persistent non-responders were sent a further copy of the questionnaire, with a standard letter and return envelope. Randomisation was done using computer generated random numbers, and stratifying by age and sex. Participants remained unaware as to group allocation. The primary outcome was the final response rate to postal questionnaires after sending all reminder letters. This was calculated at least 6 weeks after sending the initial questionnaire, to allow for late responders. Differences in proportions between groups were examined using Pearson\'s chi-square test and logistic regression to adjust for age and sex with 95% confidence intervals (95% CI). To observe a difference of at least 10% between trial arms required 752 participants, based on a return rate of 60% in the control groups, with 80% power. Approval for the study was received in advance from the local research ethics committee and the research governance committee. Results ======= The number of patients referred to the exercise referral scheme in the past year with complete name and address information was 627. Their mean age was 48 years (SD 13, range 18 to 78) and 69.2% (434/627) were women. Randomisation allocated 49.0% (307/627) of patients to receive a pre-warning letter and 51.0% (320/627) no pre-warning letter (Figure [1](#F1){ref-type="fig"}). The two groups were balanced with respect to sex (66.8% female in the pre-warning group compared with 71.6% in the control group) and age (mean 48.7 years, SD 13.3, in the pre-warning group compared with mean 47.6 years, SD 13.9 in the control group). The final response rate to the postal survey, after completing two stages of follow-up was 25.4% (159/627). In the pre-warning group, the response rate was 30.0% (92/307) compared with 20.9% (67/320) in the control group (χ^2^6.75, p = 0.009) (Figure [2](#F2){ref-type="fig"}). Thus giving a difference between the two groups of 9.1% (95% CI for risk difference, 2.2% to 15.8%) (Figure [2](#F2){ref-type="fig"}). In a logistic regression model, the pre-warning letter increased the odds of returning the questionnaire by 1.61 (95% confidence interval 1.12, 2.32) and this was not altered after adjusting for age (in years) or sex (OR^adj\ age\ sex^1.60, 95% CI 1.11, 2.30). Conclusions =========== Sending a pre-warning letter seven days in advance of mailing out a postal questionnaire had a modest impact on increasing final response rates by almost 10%, with a relative 43% increase compared with sending no pre-warning letter. Patients in our study were selected on the basis of a previous referral to a community based exercise referral scheme, and the questionnaire sent to them sought information about their perceptions of the service quality. An earlier trial examining the impact of this service on a similar group of patients, in terms of increasing physical activity, had achieved average response rates of 60% \[[@B23]\]. The much lower than expected response rate in the current study could have been influenced by the topic and purpose of the questionnaire along with the layout of the questionnaire \[[@B22]\]. Our findings are consistent with the evidence from a systematic review \[[@B22]\] that pre-contact can lead to an increased odds of response by as much as 50%. Our study confirms that this benefit extends to groups of sedentary patients known in advance, to be reluctant to reply. Pre-warning letters are simple to administer and relatively low cost compared with more labour intensive methods to increase response rates, such as telephone reminders or face-to-face visits. Moreover, this intervention method does not require any additional information or administrative systems over and above those required for sending the main questionnaire. Give that the response rate in the intervention group still remained low, at 30%, we may need to consider alternative approaches to postal questionnaires to obtain information from this group of patients, particularly if non-response could be associated with the outcomes examined within the survey -- in this case, the patient experience of the exercise referral service. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= RAH conceived the study design, drafted the main protocol, carried out randomisation, analysed the results and wrote the first draft of the paper. DC was responsible for completing the study, for data entry, for assisting with data analysis and significant comments on the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6963/4/31/prepub> Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Flow diagram of the randomised controlled trial. ::: ![](1472-6963-4-31-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effect of pre-warning on final questionnaire response rates. ::: ![](1472-6963-4-31-2) :::	PubMed Central	2024-06-05T03:55:49.931845	2004-11-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534787/", "journal": "BMC Health Serv Res. 2004 Nov 10; 4:31", "authors": [ { "first": "Roger A", "last": "Harrison" }, { "first": "Don", "last": "Cock" } ] }
PMC534788	Background ========== Endometriosis is a condition that affects 10% of women of reproductive age \[[@B1]\]. In the condition, endometrial glands and/or stroma from the eutopic endometrium lodge and survive at ectopic sites such as the fallopian tubes, ovaries and peritoneal cavity. The most accepted theory as to how these cells migrate to ectopic areas is Sampson\'s retrograde menstrual transport theory, where endometrial cells are shed through the fallopian tubes \[[@B2]\]. However, what is not known is how these foreign cells continue to survive in their new ectopic location. Ubiquitin is a 76 amino acid protein \[[@B3]\] that is involved in the degradation of short lived, regulatory or misfolded proteins, thus maintaining cellular homeostasis \[[@B4]\]. Ubiquitin tags these proteins to be taken to the proteasome and in some instances also to the lysosomic machinery to prevent damage to cells. This is the most cited function of ubiquitin, although ubiquitin also shares a very similar sequence homology to the B-cell lymphoma athanogene 1 (BAG-1), which has known anti-apoptotic properties. To our knowledge, the hypothesis that ubiquitin may mediate anti-apoptosis within the endometrium has never been examined and consequently has not been investigated in the area of ectopic endometrial cell survival in endometriosis \[[@B5]\]. Accumulating evidence suggests that dysregulated apoptotic machinery in endometriosis has a role in its pathogenesis, but no consensus exists as to which pathway/s this is mediated by \[[@B6]\]. Transformed ectopic endometrial cells are able to survive due to a number of factors, such as down regulation of soluble Fas concentrations and a lack of cell-surface Fas expression on T-cells \[[@B7]\]. Therefore, this study was undertaken to determine the expression of ubiquitin within endometriosis during the menstrual cycle and to establish whether it is related to a decrease in the incidence of apoptosis in this tissue, thus potentially promoting ectopic endometrial cell survival. Methods ======= Tissues ------- Approval to conduct this study was granted by the Human Research Ethics Committee of the Western Sydney Area Health Service. Archived formalin fixed, paraffin embedded tissues from 14 women with endometriosis and 55 women undergoing laparoscopy for non-endometrial pathologies such as leiomyomata and benign ovarian cyst were studied. Endometriotic implants (n = 20) and endometrial tissues (n = 59) were obtained from the women above, respectively. All tissues were observed from the Department of Pathology at Westmead Hospital between January 2000 and July 2003. Mean ages for endometriosis and control groups were 42.5 ± 2.69 and 36.4 ± 1.17 years, respectively. Women with histological evidence of malignancy, necrosis, active inflammation or hormonal treatment were excluded from the study. Control subjects had normal, regular menstrual cycles and endometrial samples were collected from the proliferative and secretory phases of the cycle. Sectioning ---------- Sections (4 μm) of endometriosis and control endometrial tissue blocks were cut on a microtome (Microtome, Stauffenberg, Germany), placed on superfrost slides (Menzel-Glaser, Braunscheig, Germany) and air-dried at room temperature for 24 hours. Localisation of ubiquitin ------------------------- De-waxing was performed with two changes of histolene and absolute ethanol through a single change of 95% (v/v) and 70% (v/v) ethanol and a final change of tap water. All incubations were of 5 min duration. Following the suppression of endogenous peroxidase activity for 10 min with a blocking agent (DAKO Pty Ltd, Botany, Australia) sections were incubated with normal goat serum for 60 min to prevent non-specific binding. A polyclonal rabbit anti-ubiquitin primary antibody (DAKO Pty Ltd, Botany, Australia) prepared at a titre of 1:100 was added for a further 60 min at 37°C. Slides were then washed three times in tris-buffered saline (TBS: 50 mM Tris HCl, 150 mM NaCl, pH 7.5; 5 min) before the biotinylated goat anti-rabbit IgG secondary antibody (DAKO Pty Ltd, Botany, Australia) prepared at 1:200 was applied (30 min). A horseradish peroxidase avidin biotinylated complex kit (HRP-ABC) and diaminobenzidine (DAB) were used according to the manufacturer\'s instructions (DAKO Pty Ltd, Botany, Australia) to detect the secondary antibody. All washes used TBS except after DAB administration, where water was used. Sections were counterstained with haematoxylin and eosin (H&E) and cover slips were attached using a DAKO aqueous based mounting medium. Negative controls were obtained through the omission of the primary antibody to ubiquitin (that showed the absence of specific staining). Ubiquitin has increased expression in the normal endometrium in both the late proliferative and late secretory phase. In this study, late secretory phase tissue was used as a positive control \[[@B8]\]. Currently, no single assay exists that detects apoptosis with high specificity and sensitivity \[[@B9]\]. Thus the TUNEL technique used in this study was used in conjunction with the classical method of H&E staining (which detects nuclear shape changes during the early stages of apoptosis) as positive strand break detection alone (TUNEL) may overestimate the true occurrence of cell death within a given cell population. A presence of DNA strand breaks, for example, may not correlate with nuclear segmentation or may be detected during the late lytic stage of apoptosis, where most cells are no longer viable. TUNEL labelling --------------- Sections were deparaffinized and rehydrated with histolene and a graded series of alcohols (absolute, 95%, 90%, 80% and 70%) for 5 min each. This was followed by a 20 min incubation of sections with proteinase K \[15 μg/ml in 10 mM Tris/HCl, pH 7.5\] at room temperature. Sections were then rinsed twice in phosphate-buffered saline (PBS) and reacted with 50 μl of the TUNEL reaction mixture (Roche Diagnostics, Castle Hill, Australia) for 60 min in a dark, humidified chamber at room temperature. The sections were then rinsed three times in PBS and incubated for a further 30 min with 50 μl of the Converter-POD (Roche Diagnostics, Castle Hill, Australia) followed by 10 min with DAB. This procedure ensures the detection of TUNEL labelled cells. Finally, sections are counterstained and mounted as described for ubiquitin staining. For positive controls, sections were treated with DNAse 1 to induce DNA strand breaks or peroxidase blocking solution was excluded. Negative controls were achieved by omitting terminal deoxynucleotidyl transferase (TdT; Roche Diagnostics, Castle Hill, Australia). H&E stains ---------- The Department of Pathology, Westmead Hospital, kindly provided the H&E slides used for this study. The basis for this procedure is to identify cells with cell blebbing, nuclear condensation and cell shrinkage, which are characteristic features of apoptosis. Scoring of ubiquitin sections ----------------------------- The ubiquitin staining in the normal and endometriotic tissue was calculated using a semi-quantitative method to determine the average intensity scores of the protein in the nucleus of glands and the stroma using an Optimas Image Analysis program (Silver Spring, USA). Five randomly selected fields were viewed and evaluated with a grading of 0, 1, 2 or 3 (negative, weak, moderate, strong) according to a scale created by Watanabe and colleagues \[[@B10]\]. Scoring of TUNEL sections ------------------------- TUNEL apoptotic cell numbers were determined by counting darkly labelled cells in five randomly selected fields at X400 and expressed as the apoptotic cell mean/field according to Meresman and colleagues \[[@B11]\]. Scoring of H&E sections ----------------------- Ten randomly chosen fields at X600 magnification were used to determine the number of apoptotic stromal and glandular cells according to a modified grading scale used by Meresman and colleagues \[[@B12]\]: (-) \< 3 apoptotic cells/field; (+) \>3 apoptotic cells/field. This modified grading system was used to make statistical analysis more accurate, as there were only a few samples that exhibited \> 8 apoptotic cells/field. Also, apoptotic cells were identified by their characteristic morphological features in H&E-stained late secretory endometrial sections. These included cell shrinkage and chromatin margination or chromatin condensation with formation of apoptotic bodies \[[@B12]\]. Apoptotic bodies were identified by the presence of one, or several of the following features \[[@B13],[@B14]\]: 1\) Single round mass of condensed strongly eosinophilic cytoplasm with a clump of strongly basophilic, homogenous chromatin. 2\) Cytoplasm with more than one piece of condensed chromatin. 3\) Condensed chromatin fragments without cytoplasm. Statistical analysis -------------------- All data is expressed as mean ± SEM. Comparisons between experimental groups were performed using the independent t-test and Mann-Whitney U test for parametric and non-parametric analysis, respectively. A P-value less than 0.05 was considered a significant difference between groups. Results ======= Immunohistochemistry of ubiquitin in the control endometrium and endometriotic implants --------------------------------------------------------------------------------------- Analysis of the effect of the menstrual cycle phase on glandular epithelial cell ubiquitin expression showed a greater level of ubiquitin during the proliferative phase than the secretory phase in controls (P = 0.032; Figure [1A](#F1){ref-type="fig"}, [3A,3D,3G,3J](#F3){ref-type="fig"}, and [3M](#F3){ref-type="fig"}) in contrast to ectopic glandular cells of endometriosis tissues where ubiquitin is higher during the secretory phase (P = 0.022; Figure [1A](#F1){ref-type="fig"}, [4A](#F4){ref-type="fig"} and [4B](#F4){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### A. Ubiquitin grades for glandular cells of control endometrium and endometriotic implants during the menstrual cycle. Bars represent mean ± SEM; p \< 0.05. P = proliferative and S = secretory. B Ubiquitin grades for stromal cells of control endometrium and endometriotic implants during the menstrual cycle.Bars represent mean ± SEM; p \< 0.05. P = proliferative and S = secretory. ::: ![](1477-7827-2-69-1) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Immunohistochemical staining of control endometrium.Panels A, D,G,J and M are ubiquitin stained sections where brown cells are ubiquitin labelled (X400). Panels B, E, H, K and N are H&E stained sections where □ are apoptotic cells (X600). Panels C, F, I, L and O are TUNEL stained sections where ▼ are TUNEL positive cells.; Menstrual cycle phases: MP = mid proliferative; LP = late proliferative; ES = early secretory; MS = mid secretory and LS = late secretory. ::: ![](1477-7827-2-69-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Immunohistochemical staining of endometriotic implants.Panels A and B are ubiquitin stained sections where brown cells are ubiquitin labelled (X400). Panels C and D are H&E stained sections where □ are apoptotic cells (X600). Panel E and F are TUNEL stained sections where ▼ are TUNEL positive cells. Menstrual cycle phases: P = proliferative and S = secretory. ::: ![](1477-7827-2-69-4) ::: Furthermore, the effect of the menstrual cycle phase on stromal cell ubiquitin expression, showed a similar level of ubiquitin between the proliferative and secretory phase of controls (P \> 0.05) in contrast to ectopic stromal cells of endometriosis tissues where ubiquitin is higher during the secretory phase (P = 0.020; Figure [1B](#F1){ref-type="fig"} and [4B](#F4){ref-type="fig"}). Detection of apoptotic cells in the control endometrium and endometriotic implants ---------------------------------------------------------------------------------- A significantly higher level of apoptosis was observed using H&E in ectopic glandular cells of patients with endometriosis in comparison to controls in the proliferative phase (2.79 ± 0.43 vs 1.90 ± 0.15 respectively; P = 0.04; Figure [2A](#F2){ref-type="fig"}, [3B,3E](#F3){ref-type="fig"} and [4C](#F4){ref-type="fig"}) whereas no significant difference was seen during the secretory phase (1.29 ± 0.23 vs 1.64 ± 0.19 respectively; P \> 0.05; Figure [2A](#F2){ref-type="fig"}, [3H,3K,3N](#F3){ref-type="fig"} and [4D](#F4){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### A. Apoptotic grades for glandular cells of control endometrium and endometriotic implants during the menstrual cycle using H&E.Data are expressed as the apoptotic cell mean/field. Bars represent mean ± SEM; \p \< 0.05. Menstrual cycle phases: P = proliferative and S = secretory. B. Apoptotic grades for glandular cells of control endometrium and endometriotic implants during the menstrual cycle using TUNELData are expressed as the apoptotic cell mean/field. Bars represent mean ± SEM; Menstrual cycle phases: P = proliferative and S = secretory. C. Apoptotic grades for stromal cells of control endometrium and endometriotic implants during the menstrual cycle using H&E.Data are expressed as the apoptotic cell mean/field. Bars represent mean ± SEM; \p \< 0.05. Menstrual cycle phases: P = proliferative and S = secretory. D. Apoptotic grades for stromal cells of control endometrium and endometriotic implants during the menstrual cycle using TUNEL.Data are expressed as the apoptotic cell mean/field. Bars represent mean ± SEM; Menstrual cycle phases: P = proliferative and S = secretory. ::: ![](1477-7827-2-69-2) ::: Similar levels of apoptosis were observed within proliferative phase ectopic stromal cells of patients with endometriosis and controls using the H&E method (4.57 ± 0.85 vs 5.08 ± 0.50 respectively; P \> 0.05; Figure [2C](#F2){ref-type="fig"}, [3B,3E](#F3){ref-type="fig"} and [4C](#F4){ref-type="fig"}). In contrast, a considerably lower level of apoptosis was observed using H&E in secretory phase ectopic stromal cells of patients with endometriosis than in controls (2.73 ± 0.63 vs 4.17 ± 0.33 respectively; P = 0.03; Figure [2C](#F2){ref-type="fig"}, [3H,3K,3N](#F3){ref-type="fig"} and [4D](#F4){ref-type="fig"}). Apoptotic cells are seen using the TUNEL technique (Figure [3C,3F,3I,3L,3O](#F3){ref-type="fig"}, [4E](#F4){ref-type="fig"} and [4F](#F4){ref-type="fig"}). However no statistically significant difference in levels of cell death were observed for both ectopic glands of endometriosis patients and controls in either the proliferative (1.73 ± 0.88 vs 1.51 ± 0.38 respectively; P \> 0.05; Figure [2B](#F2){ref-type="fig"}, [3C,3F](#F3){ref-type="fig"} and [4E](#F4){ref-type="fig"}) or secretory phase (1.05 ± 0.48 vs 0.70 ± 0.19 respectively; P \> 0.05; Figure [2B](#F2){ref-type="fig"}. [3I,3L,3O](#F3){ref-type="fig"} and [4F](#F4){ref-type="fig"}). Similar levels of apoptosis were also observed in ectopic stromal cells of endometriosis patients and controls using the TUNEL technique in both the proliferative (4.53 ± 0.93 vs 3.59 ± 0.85 respectively; P \> 0.05; Figure [2D](#F2){ref-type="fig"}, [3C,3F](#F3){ref-type="fig"} and [4E](#F4){ref-type="fig"}) and secretory phase (1.83 ± 0.43 vs 2.08 ± 0.52 respectively; P \> 0.05; Figure [2D](#F2){ref-type="fig"}, [3I,3L,3O](#F3){ref-type="fig"} and [4F](#F4){ref-type="fig"}). Discussion ========== Ubiquitin is implicated in the removal of short lived, regulatory or misfolded proteins but is also widely known to play a part in DNA repair and removal of virus budding \[[@B4]\]. The expression of ubiquitin during the proliferative phase of control tissues is likely to be modulated by increasing oestrogen since there is an increasing oestrogen level in response to FSH during folliculogenesis. Thus ubiquitin may play a role in supporting the developing endometrium should implantation occur, as its maximal expression correlates to day 14 of the menstrual cycle. In addition, Bebington and colleagues have previously shown an increase in ubiquitin levels during the late secretory phase as an indication that the protein may take part in the differentiation of the endometrium \[[@B8]\]. In this study, apoptosis was found to be present in endometriotic tissue with varying intensity. A previous study of endometriosis has also shown this result \[[@B15]\] but the possible biological relationship of ubiquitin to this condition has not previously been elucidated. Our data shows that the expression of ubiquitin is increased during the secretory phase of the menstrual cycle in both glands (Figure [1A](#F1){ref-type="fig"}) and stroma (Figure [1B](#F1){ref-type="fig"}) in endometriosis tissues compared to controls. In addition, the level of apoptosis observed within the glands was greater during the proliferative phase (Figure [2A](#F2){ref-type="fig"}), in contrast with a significant decrease in apoptosis in ectopic stromal cells during the secretory phase (Figure [2C](#F2){ref-type="fig"}). This study suggests that increased levels of ubiquitin within ectopic endometrial cells may allow their continued survival, through a yet to be established pathway. The up-regulation of ubiquitin in human endometriotic tissue may facilitate ectopic endometrial cell survival, particularly allowing those of stromal origin to grow, survive and evade T-cell mediated disposal. This finding is of particular interest because ubiquitin has primarily been attributed to the removal of aberrant proteins but seems, from our data, to be also associated with cell survival. This duality in ubiquitin\'s role may be attributed to the type of lysine residue linkage that occurs during polyubiquitination. Studies by Deng and colleagues \[[@B16]\] have shown that if ubiquitin proteins are linked to each other through lysine 48, the target protein (in this case proteins on ectopic endometrial cells), will be directed to the proteasome for degradation. However, if the linkage occurs through lysine 63, the target protein associates with other proteins, aiding in its survival. Our data is consistent with the hypothesis that ubiquitin has a protective effect on ectopic endometrial cells, particularly those of stromal origin, as shown by the increased level of ubiquitin with an associated decrease in apoptosis during the secretory phase. However, a different mechanism may apply to glandular endometriosis, where despite a greater ubiquitin expression during the secretory phase, no significant association was found with ectopic glandular cell survival. This suggests that an insufficient ubiquitin tagging during the proliferative phase may cause ectopic glandular cells to undergo apoptosis. Other factors that may explain ectopic endometrial cell survival include the down-regulation of apoptotic receptors \[[@B15]\], failure of immune cells to recognise and eliminate ectopic cells \[[@B17]\] and an increase in cytokine levels and growth factors in the peritoneal fluid of women with endometriosis \[[@B18]\]. The significant increase in age of patients with endometriosis may have an unknown effect on the level of apoptosis as older women may have increased incidence of endometrial cell death due to increased longevity. However, this age difference is consistent with another study that also shows a higher age range in women with the condition \[[@B19]\]. Our results suggest that ubiquitin is important for endometrial function throughout the menstrual cycle where it may play an important role in the regeneration of the endometrium \[[@B5]\]. Also, a loss of ubiquitin regulation in the ectopic environment may have an important role in the pathogenesis of endometriosis. In this respect, it is interesting that ubiquitin has recently been shown to exert an immunomodulatory effect in other tissues \[[@B20]\]\[[@B21]\]. Endometriotic implants may survive at ectopic locations due to a combination of factors, including protection from apoptosis and immune attack. Conclusions =========== In conclusion, this study demonstrates for the first time that ubiquitin is expressed in endometriotic cells. Furthermore, the up-regulation of ubiquitin expression may contribute preferentially to the development of these ectopic endometrial lesions due to their reduced sensitivity to apoptosis. Authors\' contributions ======================= RI was responsible for carrying out this study as part of her honours degree. CB provided RI with training in the techniques used. SF and CM were responsible for the conception and design of the study, research funding, and for supervision of RI\'s work. SF, CB and CM read and approved the final manuscript. Acknowledgements ================ This work was supported by a Sesqui NSSS Grant (K9671 U3023) from the University of Sydney (SDF). We are grateful to Dr Billous and Dr Jaworski from the Department of Pathology at Westmead Hospital for providing us with archived endometriosis tissues. We also thank Dr Karen Bythe from the Westmead Millenium Institute for assisting us with statistical analysis.	PubMed Central	2024-06-05T03:55:49.933061	2004-9-25	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534788/", "journal": "Reprod Biol Endocrinol. 2004 Sep 25; 2:69", "authors": [ { "first": "Romina S", "last": "Ilad" }, { "first": "Steven D", "last": "Fleming" }, { "first": "Catherine R", "last": "Bebington" }, { "first": "Christopher R", "last": "Murphy" } ] }
PMC534789	Background ========== The tumor suppressor protein p53 protects organisms from malignancy by either inducing programmed cell death or by arresting the cell cycle in response to cellular stress. The intracellular concentration of p53 is tightly regulated at the posttranslational level and the protein is very unstable under physiological conditions. Upon stress, p53 is stabilized and can act as a potent transcription factor that activates a plethora of downstream target genes \[[@B1],[@B2]\]. The p53 target genes can be grouped into classes according to their effect on a cell. One class is represented by p21^CIP^, a cyclin dependent kinase inhibitor that is a potent inhibitor of the cell cycle. Another class of p53 target genes, of which bax is the most known representative, mediates p53-induced apoptosis. Other p53 target genes prevent the process of angiogenesis \[[@B2]\]. Not surprisingly, p53 is inactivated in a wide variety of human cancers \[[@B1],[@B3]\]. Most mutations found in cancers are mis-sense mutations mapping to the central core domain of p53, which confers sequence-specific DNA binding activity to the protein. These mutations can cause destabilization of the core domain and loss of the DNA binding function. Thus, most mutant p53 proteins lack the ability to bind the DNA control elements of their target genes and fail to activate their expression. As a consequence, cells lacking functional p53 are unable to arrest the cell cycle or to undergo apoptosis in response to genotoxic stress. Since lack of p53 function plays such a central role in cancer development and in resistance to treatment, there has been much interest in the search of means and molecules to reactivate mutant forms of p53 \[[@B4]-[@B9]\]. A report by Foster et al. \[[@B7]\] generated special interest since it reported the discovery of a class of small molecules that was able to stabilize the p53 core domain. Not only were these compounds reported to stabilize the active conformation of wild type p53 but they were also shown to stabilize mutant p53 forms and enable them to activate transcription of p53 target genes. While the initial screening was conducted by an in vitroassay, activity of these compounds was subsequently confirmed in cell culture experiments and in a xenograft tumor mouse model \[[@B7]\]. One of their compounds, termed CP-31398, was reported to increase reporter gene activation by mutant p53 proteins about tenfold in the human p53-null lung carcinoma cell line H1299. We tested CP-31398 in a yeast cell-based assay and in human tissue culture cells. We could not detect any reactivation of mutant p53 in these cellular assays. Our results are in agreement with, and provide support to the results obtained by Rippin et al. \[[@B10]\], which indicate that CP-31398 intercalates with DNA rather than binding p53. Results ======= The yeast Saccharomyces cerevisiaedoes not contain p53 homologous proteins. However, it has been demonstrated that p53 expressed in yeast can function as a potent transcriptional activator of artificial genes bearing its specific recognition sequence \[[@B11]\]. To test different mutant forms of p53 and the potential effect of various molecules on the activity of such mutants, we constructed a yeast strain carrying an integrated bi-directional reporter gene construct in which a single p53 binding site from the human p21^CIP1^promoter \[[@B12]\] was inserted between the divergent HIS3and lacZgenes (figure [1A](#F1){ref-type="fig"}). The p53-dependent expression of the yeast marker gene HIS3allows growth selection on media lacking histidine and containing 3-amino-triazole (3-AT), which is a competitive inhibitor of the HIS3gene product. The p53-dependent activation of this reporter gene is convenient for library screening, while expression of the bacterial lacZgene allows verification and quantitation of the transcriptional activity of the various p53 forms and putative modulators. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Human p53 protein activates transcription from a reporter construct in Saccharomyces cerevisiae. (A) Schematic representation of our yeast reporter construct integrated into our yeast strain. The black circle represents a single p53 responsive element from the human p21 promoter. (B) β-galactosidase assay to measure activation of the lacZreporter gene. Wild type p53 and the indicated point mutant variants were transformed into the p53 responsive reporter strain and β-galactosidase activity in solution was determined. The activity of wild type p53 was arbitrarily set to 100%. p53R282W and p53V173A showed about 40% of activation compared to wild type p53. No activation of the reporter gene was detected in yeast cells containing the other point mutant variants. Average and standard deviation were determined from three independent experiments. (C) Growth on selective plates containing 20 mM 3-AT depends on expression of the HIS3reporter gene and correlates with the activation of the lacZreporter gene. Control plates consist of standard drop-out plates lacking the corresponding growth marker without 3-AT. Growth under selective conditions was dependent on activation of the p53 dependent reporter gene. ::: ![](1477-5751-3-5-1) ::: Transformation of this strain with an episomal plasmid expressing human wild type p53 led to activation of the integrated lacZand HIS3reporter genes, which resulted in increased β-galactosidase activity (figure [1B](#F1){ref-type="fig"}) and cell growth on plates lacking histidine and containing 20 mM 3-AT (figure [1C](#F1){ref-type="fig"}). In contrast, expression of three mutant forms of p53 \[[@B1]\] with point mutations in their DNA-binding domain that completely abolish sequence-specific DNA-binding activity (p53R175H, p53R248W, p53R273H) did not activate transcription of the reporter genes (figure [1B](#F1){ref-type="fig"} and [1C](#F1){ref-type="fig"}, and data not shown). Expression of mutant forms that retain some DNA-binding activity in vitroand in mammalian cells \[[@B13]\] led to reduction of reporter gene expression compared to wild type p53 (figure [1B](#F1){ref-type="fig"}). All p53 variants were expressed to comparable levels, as verified by western blot analysis (data not shown). Thus, the results of these transcriptional assays, taken together with published results of experiments performed in mammalian cells, indicate that the relative transcriptional activity of wild type p53 and the tested derivatives is comparable in yeast and in human cells. Since lack of p53 function plays such a central role in cancer development and in resistance to chemotherapeutic treatment, many efforts have been directed towards trying to reactivate mutant forms of p53 \[[@B4]-[@B9],[@B14]\]. The report by Foster et al. \[[@B7]\] generated special interest since it presented the discovery of a small molecule (CP-31398) that was able to stabilize the core domain of p53 in vitro. In addition, this compound was reported to enable some otherwise silent p53 mutants to activate transcription from target gene promoters in cell culture experiments. We tested the effect of CP-31398 on human p53 activity in our p53-responsive yeast strain. Yeast cells expressing either wild type p53 or the mutant p53R173A were grown in media containing increasing concentrations of CP-31398. Activation of transcription of the p53-dependent reporter gene was assessed by measuring β-galactosidase activity in extracts from these cells (figure [2](#F2){ref-type="fig"}). No significant difference in lacZreporter gene expression was observed between untreated cells and cells that were incubated with increasing concentrations of the compound. Very high concentrations of CP-31398 (500 μg/ml) reduced reporter gene activity, both in the case of wild type p53 expression and in the case of p5R173A expression. Results of growth assays on selective plates to indirectly measure HIS3expression paralleled our data from the lacZexperiments (data not shown). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Treatment with the p53 stabilizing compound CP-31398 shows no effect on reporter gene activity in yeast. Yeast cells expressing wild type p53 (lanes 1--5), p53V173A (lanes 6--10) or empty vector (-, lanes 11--15, white bars) were incubated with the concentrations of CP-31398 indicated (0--500 μg/ml) and expression of β-galactosidase was determined. β-galactosidase activity of wild type p53 without CP-31398 treatment was arbitrarily set to 100%. Yeast cells were treated with CP-31398 for 16 hours. ::: ![](1477-5751-3-5-2) ::: Since these negative results regarding the lack of expected effects of CP-31398 on p53 could be due to our assay system in yeast, we tested CP-31398 in experiments with human tissue culture cells. We transfected the human p53-null H1299 lung carcinoma cell line that was also used for some of the experiments described by Foster et al. \[[@B7]\] with plasmid DNA expressing either human wild type p53 or the p53R173A mutant together with a reporter plasmid carrying a p53-responsive luciferase gene \[[@B12]\]. When we treated these cells with CP-31398 in concentrations that were shown to be effective by Foster et al. (5--20 μg/ml and higher concentrations), reporter gene signals decreased and massive cell death was observed (figure [3A](#F3){ref-type="fig"} and data not shown). Lower concentrations that showed no obvious toxicity to the cells had no significant effect on reporter gene activity. We observed very similar effects when we performed corresponding experiments in the osteosarcoma cell line Saos-2 (p53 null cell line) (data not shown). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Treatment of H1299 lung carcinoma cells with CP-31398 provokes massive cell death and p53 independent decline of luciferase reporter gene activity. (A) H1299 cells were transfected with expression constructs for wild type p53 (lanes 1--3) and p53V173A (lanes 4--6). All the samples were cotransfected with a p53-responsive luciferase reporter (p21 luciferase, containing a single p53 responsive p53 binding site from the human p21 promoter, termed WWP-luc, see material and methods) and a constitutive reference β-galactosidase construct (CMV-lacZ) for normalization. These cells were subsequently incubated with 0, 10, 15 μg/ml CP-31398 respectively and relative luciferase activities were determined. (B) H1299 cells were transfected with an expression construct for the synthetic activator GAL4-VP16. All samples were cotransfected with a gal4p responsive luciferase reporter (UAS~G~luciferase) and a reference β-galactosidase plasmid (CMV-lacZ) for normalization. The control cells were transfected with CMV-lacZand UAS~G~luciferase only. These cells were subsequently incubated with 0, 10 and 15 μg/ml CP-31398 and relative luciferase activities were determined. The cells were treated with CP-31398 for 16 hours. ::: ![](1477-5751-3-5-3) ::: Cell death and decreased reporter gene activity was not dependent on the expression of p53 since treatment with CP-31398 of the same cell lines expressing the unrelated activator GAL4-VP16 co-transfected with the respective reporter construct caused similar toxicity and lower reporter gene activity (figure [3B](#F3){ref-type="fig"}). We next tested whether CP-31398 might have an effect on endogenous wild type p53 in the human cell line HeLa. These cells express wild type p53 protein, but p53 levels are low because of the presence of the viral HPV E6 protein, which targets p53 for degradation \[[@B15]\]. We transfected HeLa cells with the same p53-dependent luciferase reporter construct that was used with the other cell lines and treated the cells with increasing concentrations of CP-31398 (figure [4](#F4){ref-type="fig"}). To our surprise, there was a strong increase in reporter gene activation. When we expressed additional human wild type p53 from a transfection plasmid, the signal increased even more (data not shown). In contrast to the previous effect on other cell lines described above, we did not observe any significant cell death in the case of HeLa. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Treatment of HeLa cervical carcinoma cells with CP-31398 leads to p53 dependent induction of the luciferase reporter. HeLa cells were transfected with a p53 responsive reporter gene (WWP-luc) and a reference β-galactosidase plasmid (CMV-lacZ) for normalization. Control cells were transfected with CMV-lacZalone. The cells were subsequently incubated with CP-31398 (0--10 μg/ml) and relative luciferase activities were determined. Cells were treated for 16 hours. ::: ![](1477-5751-3-5-4) ::: We subjected extracts from HeLa cells treated with CP-31398 to western blot analysis. The p53 signals correlated with increasing CP-31398 concentrations, whereas the actin control signals did not (figure [5A](#F5){ref-type="fig"}). These results are consistent with a classical response to genotoxic stress by compounds causing stabilization of p53 \[[@B16]\]. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Western Blot analysis of HeLa cells treated with CP-31398 and daunorubicin. (A) HeLa cells were treated with increasing concentrations of CP-31398 and protein extracts were subjected to SDS-PAGE and subsequent detection with an anti-p53 antibody (DO-1). (B) HeLa cells were treated with the established p53 inducing agent daunorubicin. Protein extracts were subjected to SDS-PAGE and subsequent detection with an anti-p53 antibody (DO-1). Expression of actin is detected as a loading control in experiments 5A and B. ::: ![](1477-5751-3-5-5) ::: We also measured changes in p53 levels in HeLa cells after treatment with increasing concentration of daunorubicin, a known anticancer agent that is highly cytotoxic by a number of proposed mechanisms -- intercalation into DNA among them \[[@B17]\]. We found, as expected, that daunorubicin treatment led to a progressive stabilization of p53 in HeLa cells comparable to the response when cells were treated with CP-31398 (figure [5B](#F5){ref-type="fig"}). Discussion ========== We assessed the proposed p53 stabilizing action of CP-31398 in yeast cells and in human cells. CP-31398, a compound isolated in an antibody-based in vitroscreen, was reported to stabilize the p53 DNA-binding core domain and to reactivate mutant p53 in vivo\[[@B7]\]. We were unable to detect any effect of CP-31398 on p53-dependent reporter gene activation by a mutant form of human p53 neither in human cells nor in yeast cells. In our hands, CP-31398 did not stabilize mutant p53 proteins so as to show differences in activation of p53-dependent reporter genes in yeast and in mammalian cells. In addition, concentrations that were shown to be effective in cell culture by Foster et al. \[[@B7]\] led to extensive cell death. Most importantly, such cell death was independent of p53 expression. The p53 protein expressed within yeast cells functions as a potent transcriptional activator. Reconstitution of transcriptional activation by p53 in a heterologous, yet cellular system such as a yeast cell should be suitable to assess DNA-binding and transcriptional activation activity regardless of posttranslational modifications and other influences that are inevitable when p53 is studied in the context of its regulatory network in mammalian cells. It has been proposed that such posttranslational modifications like acetylation and phosphorylation activate the latent DNA binding activity of p53 by allosteric mechanisms \[[@B18]\]. However, more recent in vivoand in vitrostudies question whether DNA binding itself is regulated at all and suggest that induction of p53 activity primarily occurs at the level of increasing protein concentration within the nucleus \[[@B16],[@B19],[@B20]\]. The evident p53 activity in yeast cells, in which the proposed mammalian-specific p53 modifying enzymes are missing, seems to be more readily consistent with the conclusions of such studies. With our system in yeast, we should be able to detect stabilization of the p53 core domain as long as this leads to increased binding of p53 to its specific DNA recognition sequence and subsequent activation of reporter gene expression. Therefore, our yeast system provides a convenient means to screen compound libraries for identifying molecules that can reactivate mutant p53 proteins in a cellular environment. Thanks to the easy genetic malleability of yeast and the lack of endogenous p53-related pathways, cellular screens with this organism should allow not only identification of compounds that can permeate cellular membranes and be active in an intracellular environment but also rapid exclusion of molecules that are not specific for the chosen target. In contrast to the results obtained with the exogenous expression of wild type p53 in yeast cells or with the H1299 and Saos-2 human cells, we observed a strong increase in wild type p53-dependent reporter gene activation in HeLa cells. These cells showed no apparent cell death after treatment with CP-31398. Wang et al. \[[@B21]\] reported stabilization of wild type p53 and an increase in p53 levels in other cell lines. These observations are consistent with the results we obtained in HeLa cells. These authors also reported that ubiquitination and degradation of wild type p53 is blocked by CP-31398. This effect seems to be specific to mdm2-mediated p53 degradation since HPV (human papilloma virus) E6-mediated degradation of p53 was unaffected. We do not know why we do not see any stabilization of exogenous p53 in H1299 or Saos-2 cells, but it is possible that unspecific toxicity induced by CP-31398 masks the increasing p53-dependent reporter signal. While these results indicate that CP-31398 might stabilize wild type p53, they do not explain the mechanism. Direct interaction and stabilization of p53 is not excluded. However, other explanations seem plausible. Stabilization of the core domain structure by CP-31398 as proposed in the original article should presumably have no effect on p53 protein levels. But p53 levels increase after treatment with CP-31398. Such a response is in line with a classical stabilisation of p53 after genotoxic stress. In contrast, Wang et al. reported that no serine 15 or 20 phosphorylation was detected in their cells after treatment with CP-31398. Interaction with mdm2 was unaffected, but p53 degradation was nevertheless blocked \[[@B21]\]. Therefore, it remains unclear by which mechanism CP-31398 stabilizes p53; it seems unlikely that core domain stability and DNA binding are influenced by CP-31398 directly. It is interesting to note that CP-31398 can intercalate into DNA as reported by Rippin et al. \[[@B10]\]. This intercalation is probably toxic to the cell and likely induces a classical p53 response, similar to the known p53 inducer daunorubicin. Our results strongly suggest a classical p53 stabilization through reduced degradation due to genotoxic effects caused by CP-31398. In fact, wild type p53 levels changed quite dramatically in HeLa cells, which are resitant to the apoptotic effects of p53, whereas the other human cell lines did not survive the treatment, probably because they underwent apoptosis in response to CP-31398 \[[@B22]\]. In support to this interpretation, our control substance daunorubicin showed very similar and expected results as those obtained with CP-31398. Conclusions =========== In contrast to the results reported by Foster et al. \[[@B7]\], we did not detect any stimulation of mutant p53 activity in vivoby CP-31398, a potential anti-cancer compound. Concentrations of CP-31398 that were reported to be active in the published work were highly toxic to human cells in our experiments. The results of our in vivoexperiments are in agreement with the recently published biochemical analysis of CP-31398, which shows that this molecule does not bind p53 as previously claimed, but rather intercalates into DNA. Methods ======= Yeast strains ------------- The yeast strain used in our experiments is a derivative of the S. cerevisiaestrain JPY5 \[[@B23]\] (MAT![](1477-5751-3-5-i1.gif)ura3-52 his3Δ200 leu2Δ1 trp1Δ63 lys2Δ385). The p53 responsive yeast strain was constructed by integration of the reporter construct described in the result section and in figure [1A](#F1){ref-type="fig"} into the HIS3locus by homologous recombination. The integrating p21 reporter plasmid was linearized with AflIIthat cuts in the 3\' untranslated region (3\'UTR) of the S. cerevisiae HIS3gene. Yeast growth and manipulations ------------------------------ Yeast genetic techniques and media were as described in \[[@B24]\]. For selection of plasmids, dropout media containing all except the specified amino acids were used. Yeast transformation was performed by the lithium acetate procedure \[[@B25]\]. Recombinant plasmids -------------------- All p53 forms tested in yeast were expressed from the vector pGAD424 (Clontech, Inc). Wild type p53 was subcloned from a mammalian expression vector with primers containing HinDIIIrestriction sites by polymerase chain reaction (PCR). The PCR product was introduced into the HinDIIIsites of pGAD424, removing the GAL4AD ORF from pGAD424. All the point mutant p53 variants were generated by assembled PCR with mismatched primer pairs and subsequent cloning into pGAD424 analogous to wild type p53. The yeast reporter plasmid was derived from pDE96 (yeast integrating plasmid, bi-directional HIS3, lacZ) \[[@B26]\] by introduction of a hybridised double stranded oligo containing the p53 responsive element from the p21^CIP1^promoter (p21\_sense\_SalI 5\'-TCG AGC CGT CAG GAA CAT GTC CCA ACA TGT TGA GCT G-3\' and p21\_anti\_XbaI 5\'-CTA GCA GCT CAA CAT GTT GGG ACA TGT TCC TGA CGG C-3\') into the XbaIand SalIsites of the vector backbone. The plasmid WWP-luc is described in \[[@B12]\]. The mammalian p53 expression plasmids were constructed by subcloning the HinDIIIp53 fragments from the yeast expression vectors into the GAL4expression plasmid pSCETV-GAL4(1-93)RV, this resulted in p53 expression under the control of the CMV promoter. The mammalian GAL4 dependent reporter Gal5-luc contains five GAL4 responsive binding sites in front of the luciferase cassette \[[@B27]\]. Gal4-VP16 is described elsewhere \[[@B28]\]. Yeast β-galactosidase assay --------------------------- Yeast β-galactosidase assays in solution using permeabilized cells were performed as described in \[[@B24]\]. Activity was normalized to the number of cells assayed. Mammalian cell culture ---------------------- Cells were obtained from ATCC (American Type Culture Collection, Manassas, Virginia, USA) and cultured according to the recommendations of ATCC. Transient transfection and luciferase assays -------------------------------------------- We used Polyfect^®^transfection reagent (Qiagen, Inc) according to manufacturers recommendations for transfection of all cell lines. Cells for luciferase assays and western blotting were harvested by scraping 48 hours after transfection and subjected to three freeze thaw cycles in 100 mM potassium phosphate pH 7.8 1 mM dithiothreitol buffer. Supernatants were clarified by centrifugation (5 min, 13000 rpm) and resuspended in 100 μl extraction buffer. 10 μl of extract was mixed with 100 μl luciferase assay solution (Promega) and analyzed in a luminometer (EG&G Berthold Lumat LB 9507). β-galactosidase assays were performed according to standard methods using 50 μl of the extract and luciferase units were normalized according to β-galactosidase values. All measurements were performed from at least two independent transfections experiments. Western blot analysis and antibodies ------------------------------------ Protein extracts were prepared as described above. Proteins were separated by SDS-PAGE, electrophoretically transferred to nitrocellulose membranes, and western blotting was performed according to standard procedures. Anti-p53 antibody DO-1 (Santa Cruz Biotechnology, Inc) reacts with an amino terminal epitope mapping between amino acid residues 11--25 of wild type and mutant p53. Anti-actin antibody (I-19; Santa Cruz Biotechnology, Inc) is an affinity purified goat polyclonal antibody raised against a peptide mapping to the carboxy terminus of human actin. Authors\' contributions ======================= All experimental work was carried out by ST. AB conceived of the study and participated in its design and coordination. Both authors read and approved the final manuscript. Acknowledgements ================ We thank Dr. R. Eckner for providing the p53 expression plasmid and the p53 mammalian reporter plasmid WWP-luc, and Drs. W. Schaffner and M. Noll for stimulating discussions. This study was supported in part by the Commission of Technology and Innovation (CTI) of the Swiss Government.	PubMed Central	2024-06-05T03:55:49.934653	2004-11-17	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534789/", "journal": "J Negat Results Biomed. 2004 Nov 17; 3:5", "authors": [ { "first": "Stefan", "last": "Tanner" }, { "first": "Alcide", "last": "Barberis" } ] }
PMC534790	Background ========== Working in a burn unit has been described as a stressful occupation \[[@B1]\]. Every nurse who cares for a burn victim knows that stress is a part of working in this field. Some authors have emphasized that these nurses experience dealing with self-inflicted burns, uncooperative patients, inter-staff conflicts and dying patients on a daily basis \[[@B2]\]. Unresolved job stress may results in emotional withdrawal and burnout \[[@B1]\]. Professional burnout has been defined as a syndrome manifested by emotional exhaustion, depersonalization, and reduced personal accomplishment \[[@B3]\]. Nurses who have worked in burn centers of Tehran have experienced burnout in comparison to nurses working in other areas. The main researcher\'s previous study of burnout and coping in burn centers of Tehran indicated that the majority of nurses had been experienced high levels of burnout \[unpublished thesis\]. The consequences of professional burnout for nurses are serious. It results in emotional withdrawal or indifference; reduces the limits of nurses\' activity and their contact with patients \[[@B4]\]. Burnout results in a poor quality and quantity of nursing care and has negative effects on the most areas of personal, interpersonal and organizational performance \[[@B5]\]. While no health-care professional is immune to these pressures, there is evidence that suggests that areas of nursing particularly those areas we think of as critical care environments such as burn units, are often the most vulnerable to stress, and in need of much support \[[@B6],[@B7]\]. Nurses in burn centers of Tehran are also vulnerable to burnout because these centers have many problems. The managers of the burn centers have not the authority for recruiting new nurses. Moreover, self-management of burn centers in Tehran, poverty of most of the burn victims and lack of supportive organizations, resulted in financial problems in burn centers. These in turn have resulted in intense staff shortages, a heavy workload, and low pay. These factors, in addition to inherent characteristics of burn centers have put nurses under a huge pressure and many times they have indicated that they haven\'t any motivation to work and they wish to leave burn centers as soon as possible. Lewis et al. had the same idea and concluded that the scope and intensity of problems nurses encounter in burn units indicate that they need psychiatric consultation \[[@B2]\]. However, regarding emotional, attitudinal, psychosomatic, behavioral and organizational responses of these nurses to burnout, it is vital to identify the factors that involve in their perception of burnout. Some authors also referred to these factors in burn centers \[[@B1]\] and other units or populations \[[@B8],[@B9]\]. Nurses in burn centers of Tehran also pointed implicitly or explicitly to some factors that have played a role in their perceived stress and altered their responses to burnout. The challenge for managers and nurse executives of burn centers is to understand the intervening factors and their impacts on these burn nurses\' responses to burnout. As a result they can develop and promote delivery systems that support positive adaptation to stressors in burn centers, retain nurses and facilitate quality nursing care. Methods ======= In order to understand nurses\' perceptions of factors modifying their responses to job stress and burnout, qualitative research adapted from the grounded theory method was chosen \[[@B10]\]. Grounded theory --------------- The value of using a qualitative research method such as grounded theory is embedded in the subjective and often emotional nature of care, stress and coping. As a descriptive study, the qualitative paradigm, with its emphasis on understanding factors modifying nurses\' responses to job stress and burnout from the view point of practicing nurses themselves seemed logical. Grounded theory is a theory that is derived from data, systematically gathered and analyzed through the research process \[[@B10]\] The aim of grounded theory is to generate rather than verify theory \[[@B11]\]. The researcher\'s purpose in using grounded theory is to explain a phenomenon from within the social situation itself and to identify the inherent processes operating therein \[[@B12]\]. In effect, grounded theory is guided by simultaneous analysis. Both analysis and data collection inform each other. The analysis process is systematic and ends when new data no longer generate new insights. This has been also described as \' category saturation\' \[[@B13],[@B14]\]. Pilot study ----------- Five clinical nursing instructors participated in the pilot study. They were faculties of School of Nursing in Iran University of Medical Sciences (IUMS) and had been supervising nursing students in burn centers of Tehran for many years. Their age ranged from 40--48 years and had been working in burn centers for 7--14.5 years. The aim of pilot study was using the experiences of nursing instructors in the original study and reducing the informant and researcher bias in the interviews and participant observations \[[@B15]\]. The results of the study indicated that the staff of burn units felt drained, they haven\'t had any motivation or desire to care and they had been working purely for their pay. The findings were strongly indicative of the symptoms of burnout. It revealed that their behavior was representative of an indication of their professional dilemma. The pilot study also indicated that social support, patients\' cooperation/motivation and the nurses\' unique characteristics had been modified to alter the nurses\' responses to burnout. Analysis of data from the original study was conducted keeping these findings in mind. Conduct of study ---------------- The research proposal was approved by the ethics committee of IUMS. Then permission was granted from the managers of two burn centers and their nursing administrators. Further permission and written consent was obtained from all who participated in this study. Data collection and sample -------------------------- Following ethical approval, data was collected through tape- recorded, unstructured interviews. Initially data was collected in one center and analyzed. Then data gathering was initiated in the second center. There were 19 informants from the first center and 14 from the second center that participated (except 5 participants in pilot study). From this sample, 25 were nurses in different levels and positions and 8 were other members of burn team. The nurses\' sample included 8 staff nurses, 8 licensed practical nurses, 2 nurses\' aids, 3 head nurses, 2 supervisors, and 2 nursing administrators. Since nursing staff pointed to some issues concerning the burn team, the researcher interviewed one physician, one social worker, 2 physiotherapists, and 4 patients in the process of theoretical sampling. Criterion for recruiting nursing participants was at least one year of experience in the burn center. Patients were selected according to their desire as well as their physical and psychological stability. Selecting patients occurred by consultation with head nurses. Participants were 11 males and 22 females. The nursing staff participated were 19 females and 6 males. Twelve of the nursing staff had been working 2--3 shifts in burn centers or other hospitals as well as working in other jobs due to financial needs. Other demographic information of the nursing staff is displayed in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Demographic information of nursing staff. ::: Demographic item Mean Range SD ---------------------------------- ---------- ----------- -------- Age of participants 39 23--52 ± 9.5 Years of experience 17.5 1--29.5 ± 10.5 Length of time at study hospital 12.85 1--29.5 ± 10.9 ::: Samples were recruited from all units of both centers. The first purposeful sample included 6 of nursing staff. Theoretical sampling was used after emerging the tentative theory. The basis for theoretical sampling was the questions which emerged during data analysis. At this stage the researcher interviewed nursing administrators and other members of the burn team. Theoretical sampling helped in verifying nursing staff\'s responses and credibility of categories and resulted in more conceptual density. No new data were emerged in the last two interviews; therefore data gathering by interviews were terminated. Interview process ----------------- Interviews were conducted in a private place with mutual agreement of the interviewer and interviewees. All interviews were completed by the main researcher. The duration of interviews ranged between 30--165 minutes. All the interviews were tape recorded except one. Some notes were taken during dialogues. Unstructured interviews were conducted using a topic guide which has been drawn up by the researchers initial review of the literature related to the concepts of the subject of the study. This topic guide included the structure, process and outcome of care \[[@B16]\]. The following grand tour question guided the study:\" please tell me about the nursing care in your unit\". Subsequent questions were based on the participants\' responses and demands of the emerging theory. Interviews were terminated when data redundancy occurred. Participant observation ----------------------- In each center after the termination of interviews, participant observations were performed in all wards at morning, evening and night. 14 sessions of observation in the first center and 7 in the second center occurred. For this purpose the researcher informed the nursing administrators of her program. By selecting all the wards in all shifts there was no need for theoretical sampling in this stage (place and time) \[[@B10]\], but theoretical sampling of different situations were made in each ward or dressing room based on the questions which emerged during the interviews and observations. Descriptive, focused and selective observations were occurred in a non- linear fashion. Theoretical sampling occurred during focused and selective observations. Some of the questions which guided the theoretical sampling were,\" is there any difference between nursing cares received by different patients?\", \"is nursing care different in a large or small ward?\" Prolonged engagement of the researcher in the field reduced the obtrusiveness. The level of participation varied from complete observation to participation in some activities. Some informal interview also occurred during the observations. Immediately after each session of observation field notes were completed systematically. Analysis of the field notes helped in determining contextual conditions and explaining variations in the nurses\' responses in each context. This led to proposition of several hypotheses. Data analysis ------------- Data collection, analysis and interpretation occurred simultaneously, in keeping with grounded theory methodology \[[@B10]\]. After each interview the transcript was manually transcribed by the main researcher onto a personal computer, providing an opportunity for identifying themes as the tape was transcribed(for the purpose of this paper, quotes from the participants were translated verbatim). Following transcription, a print- out was obtained and the tape replayed making notes onto the transcripts. Notes included comments about tone of voice, recurrent themes and the researcher\'s own initial thought and feelings about the nature and significance of the data. Field notes of each session of observations were also typed in double space and were analyzed. The transcripts were re- read and codes assigned to recurrent themes. This is known as \"open coding\", whereby the data are examined word by word and line by line \[[@B10]\], and codes were freely generated, often reflecting the words of the respondents themselves. For example the code \"head nurse support\" was given to the response:\" relationship is heartfelt, perhaps I do many extra things because she is positive, she is supportive, she gives me motivation\". The codes similar in meaning grouped in the same categories. Analytical tools include asking questions and making comparisons helped in finding the properties of each concept \[[@B10]\]. In axial coding, categories were related to their subcategories; coding was occurred around the axis of a category, linking categories at the level of properties and dimensions \[[@B10]\]. In this stage the structures of care were related to the processes. For example it indicated that which group of factors has contributed to the nurses\' distancing from patients. The process of integrating and refining the theory occurred in selective coding \[[@B10]\]. In this stage the core category\" emergence of negative trends: nurses\' responses to burnout\" was identified. Selective sampling of literature related to job stress and burnout was very helpful. The core category linked other main categories (emotional, attitudinal, psychosomatic, behavioral, and organizational responses) and their subcategories. For the purpose of this article, the main categories and their subcategories are displayed in Table [2](#T2){ref-type="table"}. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Emergence of negative trends: nurses\' responses to burnout. ::: Main categories Subcategories ------------------------------ -------------------------- Emotional responses Personal desperation Professional desperation Attitudinal responses Depersonalization Negativity Psychosomatic responses Physical attrition Psychological attrition Behavioral responses Intolerance Justification Organizational responses Perfunctory care Declining performance ::: Data trustworthiness -------------------- The researchers accepted the perspective of Guba and Lincoln. They translated internal validity into credibility, external validity into transferability, reliability into dependability, and objectivity into confirm ability \[[@B17]\]. Credibility enhanced by the researchers\' describing and interpreting her experiences. For this purpose the researcher kept a field journal in which she noted the content and the process of interactions, including reactions to various events. This journal became the record of relationships and provided material for reflection. Prolonged engagement and persistent observation helped to data credibility. In this way the process of data collection and analysis took 8 months. Data triangulation and method triangulation confirmed credibility \[[@B15]\]. Maximum variation sampling, participant observation and using published literature met this criterion. Furthermore, once the description of the phenomenon was complete, it was returned for verification to 4 participants of each center and they validated the descriptions. The original context described adequately, so that a judgment of transferability can be made by readers. The process of the study was audited for meeting dependability \[[@B18]\]. In doing so, student\'s supervisors and two other experts reviewed the process of the study and they arrived at a same conclusion. Confirm ability requires one to show the way in which interpretations have been arrived at via the inquiry. In this study, confirm ability was established, because credibility, transferability and dependability were achieved \[[@B17]\]. The signposts indicating research decisions and influences were present throughout the study and the entire study functioned as an inquiry audit. Results ======= The findings related to factors which involved in the nurses\' responses to burnout are presented in this article. Analysis and interpretation of data indicated that personal characteristics and social support have involved in the nurses\' responses. These factors are presented in Table [3](#T3){ref-type="table"}. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Factors involve in nurses\' responses to burnout. ::: ------------------------------ ------------------------------- Personal characteristics Nurses\' characteristics Patients\' characteristics Social support Head nurse support Nursing administrator support Peer support ------------------------------ ------------------------------- ::: Nurses\' characteristics ------------------------ Data from interviews and participant observations indicated that special personal characteristics and personality traits have involved in the nurses\' emotional, attitudinal, behavioral, and organizational responses to burnout. Personal characteristics such as conscience, religious beliefs, personal philosophy, commitment, a sense of responsibility, and altruism facilitated caring behaviors. Nurses with these characteristics were more patient and empathetic. They were more cooperative and rarely justified their faults by fatigue, workload or staff shortage. Conscience, commitment, and religious beliefs such as fear of divine requital were the most prominent traits that modified the responses to burnout. One of participants stated:\" God knows. I always feel it\'s me there on the bed. Sometimes a patient calls and I ignore, but I tell myself, what I expected if I were on this bed? I fear god and say to myself, his authority is great and whatever I do, I will see the reflection of my doings\". Some of the participants pointed to interest and love in caring of burn victims. One of participants stated:\" these patients are different from other ones. I have worked more than 18 years in general hospitals, I haven\'t worked more than 7 years in burn centers, but I think that was a blessing in disguise, I am glad because clinical work for a burn patient means love, means everything, believe me. I don\'t care the managers\' behaviors, workload, nursing shortage and other deficiencies, because I love burn victims. Believe me\". Many of the nursing staff, distanced from patients, they had immoral beliefs and demonstrated humiliation and reproach in their behaviors. They related these attitudes to fatigue, micro and macro conditions in burn centers, and loss of motivation; but participant observation indicated that, this isn\'t the case for all the nurses. Nurses, who had been known as good nurses, were very calm and intimate with their patients and focused on the patients\' needs. The researcher wrote in one of her field notes:\" she is very calm and speaks with compassion. She makes jokes and patients are relaxed with her. She follows the principles and procedures more strictly than others\". Nurses in all levels were under pressure of workload, low pay, staff shortage, environmental conditions of burn units and other structural inhibitors, but as the excerpts of interviews indicated, appraisal of these inhibitors was different in the presence of specific personal characteristics. Data indicated that in some instances, when there were a number of inhibitors and they were long stay, even positive personal characteristics couldn\'t work. This was often the case in infectious dressing rooms and busy wards. The worst kinds of treating patients were seen in these places. It seemed that the inhibitory factors which are persistent and too frequent interact with personal characteristics and finally overcome the positive characteristics. One of participants stated:\" patients expect to receive care, expect a friendly encounter which does not happen. To tell the truth, some days I am excessively distressed and tired that I don\'t have the patience to answer the patient\' questions and concerns. I emotionally can not do what I really want to do on a daily basis for the patients. The reason is persistent day and night problems occurring with this job\". This process is displayed in Table [4](#T4){ref-type="table"}. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Interrelationships between personal characteristics, inhibitory factors and caring behavior in burnout. ::: Frequency and intensity of inhibitory factors Positive personal characteristics Caring behavior --------------------------------------------------- --------------------------------------- --------------------- High Defeat Deteriorates Low Overcome Improves ::: Patients\' characteristics -------------------------- Data strongly indicated that nurses\' appraisal of the patients\' characteristics have influenced some aspects of their attitudinal and behavioral responses. When the appraisal was positive, the relationships improved, and when it was negative, relationships deteriorated. Positive appraisal occurred often when the patients were cooperative and motivated for recovery and in cases where they had an advantage of high socio- economic class, cultural and educational levels, or whenever they stimulated the nurses\' senses of compassion and pettiness. Negative appraisal occurred often when the patients were from lower socio- cultural levels, addicts or there was a possibility of having acquired immune deficiency syndrome (AIDS) or hepatitis. The first group was treated kindly and more respectfully. The use of humiliating words and reproach towards them diminished and as a result the aggressive behavior and physical withdrawal less occurred. One of participants stated:\" \...I am more supportive and compassionate towards children, those who are very alone, who have no one to love and care for them, those who have committed self-inflicted burns, a woman whose husband caused her to burn herself and doesn\'t have any one to support her. In many occasions I have even paid them to buy juice from outside the hospital. I feel that these patients are needy\". The second group was treated very unethical. They encountered a humiliating, reproaching, and aggressive behavior. One of participants stated:\" \...he fights when I am dressing him, he pulls his hand and leg, he isn\'t cooperative, and he has no class. They drain all my energy to the point that I don\'t want to talk to them. I think they are mentally retarded. They keep still when I shout at them just the way that the children act. I tell them I\'ll pull your ear, and I\'ll beat you up. This is the behavior that has worked with them \". Moreover patients with extensive burns, whose survival was an improbable event, not only were badly treated, but also were sometimes ignored and received poor care. In other words, they received only those treatments which had been ordered by physicians to prevent being reprimanded by supervisors. One of the participants justified herself and stated:\" if you want the truth, a patient with 90% burns can not benefit from tetracycline ointment, but it\'s there in his order, I prefer to spend my time with a patient who has a better chance of surviving. Right or wrong I don\'t apply the ointment, because I can spend that time for a patient who will survive\". We can conclude that burnout has made nurses to modify their caring behaviors to fit the different type of patients they care for. Interaction between nurses and patients\' characteristics --------------------------------------------------------- As described later, nurse\'s and patient\'s characteristics modified the nurse\'s responses to burnout and altered caring behaviors. More analysis and interpreting of data indicated that interaction between these two variables resulted in a more powerful combination that alters responses to burnout and identifies the kind of caring behavior. This process is displayed in table [5](#T5){ref-type="table"}. It is important to mention that the meaning of patient\'s characteristics in this study is the nurse\'s appraisal or perception of these characteristics and nurse is clinical nursing staff in different levels. ::: {#T5 .table-wrap} Table 5 ::: {.caption} ###### Interrelationships between nurses and patient\'s characteristics and caring behavior in burnout. ::: Nurse\'s characteristics Patient\'s characteristics ------------------------------ ----------------------------------------------------------------------- ---------------------------------------------------------------------------- + - + Naturally good behavior Relatively good behavior related to ethical aspects- sometimes non-ethical - Relatively good behavior related to inhibition of non-ethical aspects Bad behavior related to the opportunity for emerging non-ethical aspects ::: Table [5](#T5){ref-type="table"} indicates that when both nurse\'s characteristics and her/ his appraisal of patient\'s characteristics are positive, then the nurse\'s caring behavior is naturally effective and efficient. In this case, patient is treated respectfully, there is an empathetic behavior, and nurses spend more time with her/his clients to value their emotional needs. When the nurse\'s characteristics are positive and her/ his perception or appraisal of the patient\'s characteristics is negative the nurse doesn\'t have a natural empathetic behavior. She thinks that she has to be good and behave well because of her beliefs; therefore she demonstrates a good behavior. Sometimes when the patient has been perceived as having a negative outburst, from the nurse\'s point of view, she/he has a very negative attitude towards the patient. This will cause ethical issues and misbehavior by the nurse. The good behavior occurs when the nurse\'s characteristics are negative but the appraisal of the patient\'s characteristics is positive. In this case, the patient\'s characteristics do not permit for emergence of negative characteristics of the nurse; therefore an ethical/respectful and caring behavior will result. At times when nurse\'s characteristics are negative and her/his perception of the patient\'s characteristics is also negative, non-ethical behaviors find a good opportunity to emerge. In this situation the patient encounters the worst behavior. Humiliation is intense, physical withdrawal is often seen and aggressive behavior is routine. One of participants stated: \"I take care of some patients with love and conscience and take care of the other patients only with conscience and some nurses doesn\'t have love at all, I take care of silent, calm and lonely patients better, I perform routine care for the other\". Another participant also stated:\" it seems I give positive energy to the patients to whom I am more interested and take care of them with more love. I\'ve seen that they respond better to therapeutic measures\". Therefore interaction between nurses and patients\' characteristics has a very powerful effect on the nurses\' responses to burnout and determines their nature of caring behavior. Social support -------------- Data from interviews and participant observations strongly suggested that social support influences the nurses\' responses to burnout. Supportive behavior of head nurses, nursing administrators and coworkers modified the nurses\' responses. Among these, head nurse\'s the support was the most effective factor. Nurses believed that they do not have any motivation or desire to perform well when they are not supported well. One of participants stated:\" we have a very close loving relationship with the head nurses who are supportive. In that situation I do many things for her. Her supportive attitude and caring/positive attitude helps me a lot. Do you understand?\" another participant believed that he couldn\'t endure if the nursing administrator weren\'t supportive. He stated:\" I have seen that she is doing a good job. I have been so stressed at times that I have thought of quitting. The nursing administrator has changed my mind during those stressful moments by being caring, loving and supportive and I have decided to stay in spite of low pay and benefits.\" Support from peers also modified the nurses\' responses. They could tolerate more with the support of their coworkers. One of participants stated:\" by god, I like every single one of them. It seems like we live together seven hours a day (major part of our day). We are so familiar with each other\'s character and behavior patterns. You may not believe this, these relationships has been very helpful and caused us to have a very strong unit. We care for each other in times of weakness, illness or pressure\". It is worthy of mention that the effect of social support on the nurses\' responses to burnout was not as powerful as the nurses\' and patients\' characteristics. Social support influenced the emotional and in some cases the attitudinal responses, but it didn\'t have enough power to alter the organizational and behavioral responses, therefore it didn\'t change caring behaviors significantly. Moreover, there were not any data indicative of the modifying effect of the factors proposed in this article on the psycho- somatic responses to burnout. Discussion ========== Findings of this study indicated that nurses\' and patients\' characteristics and social support modified the nurses\' responses to burnout. These factors altered the nurses\' perceptions of the inhibitory factors; in other words they could have a more positive appraisal and this in turn modified their responses. Lazarus and Folkman (1988) proposed that the initiative of behavioral manifestations is a transactional appraisal which is important for the person\'s wellbeing and betrays the confrontation as noxious, useful, threatening or requiring struggle \[[@B19]\]. Moreover Lazarus (1976) believed that personal variables including values, beliefs, commitment and a sense of control over the environment are modifying factors that influence a person\'s cognitive appraisal \[[@B20]\]. In this study, conscience, religious beliefs and commitment were the most prominent characteristics that modified the nurses\' responses to burnout. Nurses with positive characteristics had a non- threatening evaluation of their confrontations, therefore they cared better for their patients. Garrett and MC Daniel (2001) in their study concluded that a nurse\'s perception of the environment is more a function of personality than education or experience \[[@B8]\]. Conscience and commitment were of the most prominent characteristics that modified nurses\' responses to burnout, and were related to the caring behavior. Focusing on caring attitudes, Roach (1987) also proposed that caring behavior in nursing is manifested through the five \'C \' attributes: compassion, competence, confidence, conscience and commitment \[[@B21]\]. Stuart and Sundeen (1987) referred commitment as representative of whatever important for the person. It includes decisions the person considers as necessary in his life and it can direct people to (or far from) the conditions which could be threatening, noxious or probably useful \[[@B22]\]. Participants pointed mostly to religious beliefs as a modifying factor. Stuart and Sundeen (1987) also concluded that spiritual beliefs can essentially reduce stress and influence on the persons\' potential coping capabilities \[[@B22]\]. In this study, when inhibitory factors were persistent and frequent, even positive personal characteristics defeated. Selye (1976) concluded that the influence of stressors on a person depends on the number of stressors that must be confronted in a time, the duration of confrontation and existence of previous experience with the same stressors \[[@B23]\]. This happened more in infectious dressing rooms, where nurses had the closest longest contact with the bare bodies of burn victims. There were only one dressing room with 18--27 patients per each day for dressing and some of nurses had been there for more than 28 years. Patients with different characteristics treated differently. Nurses changed their caring behavior with different patients. It seemed that they didn\'t have enough emotional and physical energy and motivation for caring for all patients; but some patients stimulated their emotions and gave them the needed positive energy for caring. In other words, patients\' characteristics could both reduce and intensify the nurses\' responses to burnout. When these characteristics appraised as positive, caring behaviors improved, and when it was negative, the worst kind of behaviors occurred. Maslach (1982) in congruence with this conclusion believed that there is little evidence that caring is a uniform state \[[@B24]\], and Benner and Wrubel (1984) concluded that it\'s not clear that this is because the caring affect is depleted, or the nurse\'s personal needs for emotional protection take precedence over the human caring for others. They also believed that physical exhaustion may reduce the nurse\'s ability to continue to provide care \[[@B25]\]. Patients with extensive burns received the worst kind or care. Nurses stated that caring for these patients is futile. Data implied that caring for these patients have been incongruous with the nurses\' values. Meltzer and Huckabay (2004) in their study of the relationship between critical care nurses\' perceptions of futile care and its effect on burnout, concluded that feeling of emotional exhaustion in these nurses was highly influenced by the frequency with which nurses were involved in life- sustaining interventions that conflicted with the nurses\' values and standards in term of what the nurses thought are ethically appropriate and could result in improvement in a patient\'s condition and outcome \[[@B26]\]. It was the interaction between nurses\' and patients\' characteristics that identified the caring behavior. Some authors have found according to their clinical practice that nurses have the ability to adjust their approach and their style of interaction with different patients. These authors proposed that they not only alter the nature of dialogue and the tone of voice to meet each patient\'s needs, but also adjust their affective response. They pointed that delineation of these behaviors would be a significant contribution, yet to date these styles of care have not been explored \[[@B27]\]. In this study some patients received a natural care, but others faced with an ethical and in some instances a non- ethical care. Natural care occurred spontaneously and without thinking, but ethical care happened thoughtfully by the mediation of the patients and/ or nurses positive characteristics. Typically non- ethical cares was thoughtful, but in this case both the nurses and patients characteristics were negative and the appraisal was too threatening. In her discussion of caring, Noddings (1984) also distinguished between natural caring and ethical caring. According to Noddings, natural caring comes from a remembrance of being cared for, whereas ethical caring \" is an active relation between my actual self and a vision of my ideal self as one- caring and cared- for\" \[[@B28]\]. Other authors also proposed that nurses may care naturally or they may care out of a desire to be a good nurse \[[@B29]\]. Social support from head nurses, nursing administrators and peers made nurses to endure and tolerate in the face of problems. Lazarus (1976) proposed that the resources reducing the potential harm could be found in the environment and essentially in others who indicated one can rely on them \[[@B20]\]. Social support didn\'t have enough power to modify behavioral and organizational responses and it could only change emotional and some aspects of attitudinal responses. Therefore caring behaviors didn\'t change. Garrett and MC Daniel (2001) also concluded that other people in the work setting like supervisors and peers might limit depersonalization and emotional exhaustion \[[@B8]\]. Conclusions =========== This study as a part of more extensive research (PhD dissertation) identified the most important factors that intervened in the nurses\' responses to burnout in burn centers of Tehran. Nurses had responded to burnout. These responses included emotional, attitudinal, psychosomatic, behavioral and organizational. This part of study indicated that the nurses and patients characteristics and interaction between these two factors had a very powerful effect on the responses and determined the kind of caring behavior. Moreover social support from managers (e.g. head nurses and nursing administrators) and peers modified some of the nurses\' responses to burnout. The influence of positive personal characteristics, especially conscience, religious beliefs, philosophy, commitment, a sense of responsibility and altruism on the nurses\' responses to burnout, the finding that, long lasting and persistent problems in the work setting can deteriorate even the personal characteristics, and regarding the numerous problems in burn centers of Tehran, there is an urgent need for helping the nurses. We suggest that due to the intense staff shortage in these centers, the managers try to keep these nurses. The only way they can do this is by using stress reduction programs. Data strongly indicated that these nurses need to rest periodically to preserve energy and to refresh their morale. Moreover, giving importance to moral and ethical aspects of care by managers could be helpful and motivating. Changing burn patients\' inherent characteristics and their other characteristics such as poverty and socio- cultural level is not a possible alternative. Promoting nurses\' morale is possible and must be done promptly if we want our burn survivors receive at least an ethical effective care. Social support made the problems tolerable. Therefore we recommend nurse executives in burn centers of Tehran promote a work environment that help to decrease the perception of pressures and increase perceptions of social support. The best solution is of course eliminating the micro and macro conditions which overshadow the nurses\' responses and caring behaviors. This grounded theory created several hypotheses in this stage. Nurses with specific traits were more resistant to burnout and were more caring. It is suggestive of conducting a quantitative research to test the relationship between personality traits, burnout and caring behaviors. Differences in caring behavior for different patients were related to the influence of burnout on the nurses\' personality traits and appraisal of patients\' characteristics. It was a very new finding that needs to be investigated in more detail. The dramatic effect of social support on the nurse\' perceptions of pressures in burn centers of Tehran is suggesting of identifying the relationship between social support and the level of burnout the nurses experience in these burn centers. However the nature of a qualitative research like this, limits it\'s generalizability; therefore we suggest conducting more qualitative research in other critical care units to support these findings. Application of the findings of this study and conducting the suggested studies would help the managers of burn centers to enhance an environment conductive to morale, promote natural and ethical care, refresh the nurses\' positive emotions and facilitate a supportive environment for their nurses. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= FR initiated and designed the research, collected and analyzed the data and wrote the paper. FO was the main supervisor, helped in analysis, and revised and edited the drafts. MN was co- supervisor and revised the drafts. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6955/3/6/prepub> Acknowledgements ================ The authors thank Iran University of Medical Sciences for its financial support and Mrs. Minoo Maasoumi- the nursing administrator of Los Angeles Unified School District- for copy editing of this paper.	PubMed Central	2024-06-05T03:55:49.936754	2004-11-13	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534790/", "journal": "BMC Nurs. 2004 Nov 13; 3:6", "authors": [ { "first": "Forough", "last": "Rafii" }, { "first": "Fatemeh", "last": "Oskouie" }, { "first": "Mansoure", "last": "Nikravesh" } ] }
PMC534791	Background ========== In Finland, as in most industrialised countries mortality from cardiovascular diseases has shown decreasing trends since around 1970 \[[@B1],[@B2]\]. The Mini-Finland survey from the years 1979--80 revealed that the angina pectoris symptom (i.e. reported chest pain under physical strain) may already appear in both sexes at the age of 30, though it was not until the age of 65 that it becomes more common among men compared to women \[[@B3]\]. The typical symptoms of coronary heart disease (CHD), chest pain and breathlessness, are well-known. They are considered quite dramatic, and thus can be fairly reliably mapped by a survey \[[@B4]\]. However, coronary heart disease patients also have other complaints in respect of their health, for example fatigue and sleep problems \[[@B5]\]. It has also been estimated that 17% to 27% of patients with coronary artery disease have major depression and a significantly larger percentage has subsyndromal symptoms of depression \[[@B6]\]. The diagnosis of CHD is usually based on medical examinations or register data and not on what people by themselves experience. However, people act and use health services according to what they experience to suffer from and what they experience to limit their ability to work. Since CHD patients have many other symptoms than the traditional and well-known, it is important to know what the spectrum of symptoms and complaints among working-aged people with self-reported coronary heart disease is in relation to functional capacity. Methods ======= Design ------ The Health and Social Support study (HeSSup) is a prospective etiological follow-up study on the psychosocial health of the Finnish working-aged population. The HeSSup population consisted of a random sample of 39,563 individuals drawn from the Finnish Population Register in three age groups: 30--34, 40--44, and 50--54. The survey was carried out by postal questionnaire. Forms were returned by 15,477 individuals (approximately 5,000 in each age group), a response rate of 38.9% (37.6% in 30--34, 37.9% in 40--44, and 41.1% in 50--54). The sample was subjected to a thorough analysis of non-response \[[@B7]\]. The analysis was made using the official statistics of the Finnish population for the corresponding age groups in 1998 to assess whether the study population adequately represented the Finnish population. Diagnosed epilepsy and diagnosed hypertension were selected to represent chronic diseases. The major reasons for refuse were the length of the questionnaire and above all suspicion of the purpose behind the request for written consent. Less educated, divorced, widowed, unemployed and those on disability pension were least willing to participate. Differences in physical conditions between the study participants and the whole population were, however, small. It was also noted that people suffering from hypertension returned the questionnaire somewhat less readily than others. Material and methods -------------------- The subjects were asked whether or not a doctor had told them that they had angina pectoris or had had myocardial infarction. The perceived state of health was determined according to Likert\'s five-step scale (good, quite good, fair, rather poor and poor). In order to avoid small frequencies in the analyses this was modified to a three-step scale (good, fair, poor). The appearance of dyspnoea was categorised into four degrees of difficulty according to a widely used cardiovascular survey method \[[@B4]\]. Persons who suffered from dyspnoea when walking uphill or upstairs comprised the group of mild symptoms. Those out of breath when walking on level ground at normal speed with other people of the same age comprised the intermediate group, those who had to stop walking on level ground due to breathlessness comprised the group of difficult dyspnoea and those who became breathless even while standing still or while washing or dressing themselves, comprised the group of extremely difficult symptoms. Participants were also asked whether or not they had experienced daily or weekly chest pain during anger or other kind of emotion, palpitation and perspiration without physical exercise, flushing, trembling of hands and voice, and jerking of muscles. Irregular heartbeats daily or weekly were asked, too. Depression was estimated by Beck\'s \[[@B8]\] depression scale ranging from 0 to 63. The normal score on this scale is below 10. In mild depression the scores are between 10 and 19 \[[@B9]\]. It was also asked how well and how many hours a day the participants had usually slept and how often they had felt day-time sleepiness, which when occurring daily or almost daily has been proved to be associated with depression, insomnia and breath interruptions during sleep \[[@B10]\]. Analyses -------- In interpretation of results the coronary heart disease (CHD) patients were divided into two groups. The first comprised coronary patients not having had heart infarction (angina pectoris group) and the second patients having had heart infarction (infarction group). In order to have the best available comparison groups, four randomly selected age and sex matched controls for comparison were selected for every patient. Thus there were altogether 740 controls for the angina pectoris group and 536 controls for the infarction group. Stroke was ruled out in the control groups, but otherwise there were no differences between the CHD groups and their respective controls. The associations, odds ratio (OR) with 95% confidence intervals (CI), between symptoms and coronary heart disease with and without heart infarction, were computed by multivariate logistic regression analysis. The analyses were made using the SAS System for Windows, release 8.2/2000. Results ======= The data comprised 319 patients: 185 coronary heart disease patients who had not experienced heart infarction (55.1% were men) and 134 patients who had (78.4% were men) (Table [1](#T1){ref-type="table"}). Most of the CHD patients were in the oldest age group, and almost 90% of those who had had a heart infarction were in the age group 50--54. In all age groups the prevalence of self-reported CHD was higher among men than among women (Table [2](#T2){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The coronary heart disease patients studied according to age and gender ::: Angina pectoris group Myocardial infarction group -------- ----------------------- -------- ----------------------------- -------- -------- -------- -------- ----- 30--34 16 14 30 16 4 3 7 5 40--44 20 28 48 26 3 9 12 9 50--54 47 60 107 58 22 93 115 86 Total 83 102 185 100 29 105 134 100 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Prevalence of self-reported coronary heart disease according to age and gender ::: Angina pectoris Myocardial infarction ----------- --------------------- --------------------------- ----- ----- Age group \% \% \% \% 30--34 0.5 0.7 0.1 0.2 40--44 0.7 1.4 0.1 0.4 50--54 1.7 2.8 0.7 4.0 ::: Perceived state of health ------------------------- State of health was perceived as good or quite good by 37.3% in the angina group and by 24.6% in the infarction group. The corresponding figures in the control groups were 76.0% and 67.4%, the differences being statistically significant (p \< 0.001). State of health was perceived as poor or rather poor by 28.1% in the angina group and by 32.8% in the infarction group. Symptoms and complaints ----------------------- At least mild breathlessness occurred in two thirds of the angina group and three fourths of the infarction group (Table [3](#T3){ref-type="table"}). Difficult or extremely difficult breathlessness was reported by 20.2% in the angina group and by 27.8% in the infarction group. The corresponding figures in the control groups were 1.4% and 4.0%, the differences also being statistically significant (p \< 0.001). Chest pain during anger or any kind of emotion, palpitation and perspiration without physical exercise, irregular heart beats, and jerking of muscles were all both daily and weekly statistically significantly more common among CHD patients than among controls. Almost daily CHD patients reported more daytime sleepiness and trembling of hands and voice than controls. CHD patients also slept more poorly than controls, and sleeping hours ≤ 6 in a day was more common among then than among controls. CHD patients scored higher on the depression scale than the controls, the average score being 10.2 (95% CI 9.0--11.4) in the angina group and 5.8 (95% CI 5.4--6.2) in the control group. In the infarction group the average score was 9.7 (95% CI 8.4--11.0). The corresponding figure in the control group was 5.9 (95% CI 5.4--6.5). In both coronary heart disease groups at least mild depression was twice as common as among controls. Flushing was the only complaint, which was not statistically significantly more common among CHD patients than among controls. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Occurrence (%) of symptoms and complaints in coronary heart disease (CHD) patients and the control population ::: Angina pectoris Controls Myocardial infarction Controls -------------------------------------------------- ----------------- ---------- --------- ----------------------- ---------- --------- At least mild dyspnoea (Rose and Blackburn 1968) 66.5 33.2 \<0.001 75.4 35.3 \<0.001 Chest pain during anger or emotion Almost daily 12.2 1.0 \<0.001 16.3 0.8 \<0.001 Weekly 12.8 2.5 \<0.001 16.3 3.3 \<0.001 Palpitation without physical exercise Almost daily 14.1 3.4 \<0.001 20.8 3.5 \<0.001 Weekly 15.3 5.2 \<0.001 16.9 5.4 \<0.001 Perspiration without physical exercise Almost daily 22.7 9.7 \<0.001 26.0 11.0 \<0.001 Weekly 18.2 10.2 0.003 16.8 8.3 0.001 Irregular heart beats Almost daily 15.7 3.5 \<0.001 24.0 3.7 \<0.001 Weekly 12.4 6.1 0.004 14.0 4.4 \<0.001 Depression (Beck ≥ 10) 41.6 20.4 \<0.001 43.3 21.0 \<0.001 Sleeping hours ≤ 6 in a day 16.8 10.3 0.015 19.4 10.1 0.003 Poor sleep usually 25.0 15.4 0.002 32.1 13.0 \<0.001 Daytime sleepiness Almost daily 33.2 11.2 \<0.001 32.1 14.9 \<0.001 Flushing Almost daily 11.1 7.0 0.066 10.9 7.6 0.228 Weekly 12.8 7.1 0.014 9.3 8.0 0.624 Trembling of hands Almost daily 13.8 2.3 \<0.001 9.2 3.3 0.004 Weekly 8.3 4.7 0.053 10.7 4.0 0.003 Trembling of voice Almost daily 3.3 1.1 0.030 5.3 1.2 0.002 Weekly 3.3 1.2 0.049 3.8 2.3 0.341 Jerking of muscles Almost daily 13.7 2.7 \<0.001 12.3 4.4 0.001 Weekly 11.5 2.7 \<0.001 9.2 3.8 0.011 ::: ORs of reported symptoms ------------------------ In the multivariate logistic regression analysis chest pain during anger or other kind of emotion and dyspnoea were the symptoms most associated with CHD (Table [4](#T4){ref-type="table"}). Irregular heart beats and perspiration without physical exercise were also strongly associated with heart infarction, but not with CHD without heart infarction. On the other hand, jerking of muscles was strongly associated with CHD without heart infarction, but not with heart infarction. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Age- and sex-matched ORs with 95% CI in the multivariate logistic regression analysis for reported symptoms of coronary heart disease (CHD) without and with heart infarction. All of these symptoms were in the same model. Statistically significant associations are bolded. ::: Angina pectoris Myocardial infarction ------------------------------------------ ----------------------- ----------------------- Dyspnoea 3.81 (2.16--6.72) 2.33 (1.21--4.50) Chest pain during anger or emotion\* 3.61 (1.68--7.77) 4.12 (1.72--9.84) Palpitation without physical exercise\* 1.19 (0.55--2.59) 1.39 (0.57--3.39) Irregular heart beats\* 1.46 (0.69--3.08) 3.12 (1.28--7.60) Perspiration without physical exercise\* 1.44 (0.87--2.38) 2.19 (1.15--4.14) Flushing\* 1.11 (0.62--1.97) 2.02 (0.89--4.59) Trembling of hands\* 1.36 (0.58--2.93) 1.01 (0.37--2.67) Trembling of voice\* 2.58 (0.85--7.87) 1.17 (0.29--4.69) Jerking of muscles\* 2.44 (1.20--4.96) 1.37 (0.55--3.41) Depression (Beck ≥ 10) 1.57 (0.99--2.48) 1.01 (0.52--1.98) Poor sleep 1.30 (0.74--2.27) 1.39 (0.70--2.77) Daytime sleepiness 1.24 (0.76--2.01) 1.41 (0.78--2.56) Sleeping hours ≤ 6 in a day 1.21 (0.64--2.26) 1.08 (0.50--2.33) \* almost daily or weekly ::: Discussion ========== The principal finding in this study was that many working-aged coronary heart disease patients experience unpleasant symptoms such as dyspnoea, chest pain during anger or emotion, irregular heart beats, perspiration without physical exercise, and jerking of muscles. In addition, the frequency of most of the self-reported symptoms among the study population is higher also in respect of those symptoms, which would not be expected at least among those CHD patients whose disease is in good balance. Working-aged CHD patients may be regarded as a special group compared with the main part of CHD patients who are already by age entitled to a pension. It is likely that working-aged CHD patients have experienced the most widespread and intense exposure to risk factors, which thus has caused them this disease among the first ones within their age group. As working-aged they are wished, however, to return back to normal life and work as soon as possible. According to our study they still have a lot of symptoms concerning their every day life, which harm their recovery and rehabilitation. It is also noteworthy that many of the working aged CHD patients are still in working life. Although chest pain and dyspnoea do not prevent them to work at customer service, many of the symptoms such as trembling of hands interfere their normal jobs while appearing mostly in rest. Trembling of hands and voice are also very irritating symptoms, and they may be considered shaming. Thus they may interfere social life and reduce the quality of life. The study material may be considered representative of the Finnish working-aged population, although the response rate was only 39%. Careful non-response analysis indicated that respondents and non-respondents were comparable in respect of the most important demographic variables \[[@B7]\]. It is possible that CHD patients respond to the questionnaire more actively than other people. On the other hand, there are certainly those among CHD patients who neglect their disease and are not willing to respond. However, we do not know for sure whether there is an over or under estimation of the associations, but we can presume that these two factors compensate each other. Moreover, it is unlikely that the principal association studied, i.e. the association between CHD and appearance of symptoms would be a substantially different one in non-participants. The findings reflect the respondents\' own conception of their symptoms. The own conception of symptoms is important, since according to findings from a 3 years\' follow-up of 4,000 men, self-reported coronary heart disease predicts very strongly a new coronary event \[[@B11]\]. In addition, the presence of anginal symptoms may be an important independent correlate of prognosis in patients with CHD \[[@B12]\]. Our method to determine the existence of CHD is based on the patients\' report on whether a doctor had told them that they suffered from this particular disease. Thus, we cannot know for sure the accuracy of the information reported. Nowadays, CHD is rare among young people \[[@B1],[@B2]\]. In our data there still is some. It is possible that there is a combination of several risk factors in the background. There are validated instruments, such as the generic The Short Form 36 Health Survey \[[@B13]\] (SF-36), the Nottingham Health Profile \[[@B14]\], and the Seattle Angina Questionnaire \[[@B15]\], to investigate health-related quality of life. Our method to examine the subject was to ask about complaints and symptoms. Most of these questions have been successfully used in cardiovascular surveys \[[@B4]\] and in the Finnish Twin Cohort Study \[[@B16]\]. In addition, mood was estimated according the Beck\'s depression scale \[[@B8]\]. The occurrence of dyspnoea and chest pain even during anger or other kind of emotion may be considered a finding that was expected. In an American study on the care of coronary heart patients at the emergency department the most frequently reported symptom was chest pain (70% among men and 71% among women) and dyspnoea (30% men and 29% among women) \[[@B17]\]. The typical chest pain is also a symptom more predictive of an acute coronary attack in working-aged than in older patients \[[@B18]\]. It was suggested as far back as 1987 that palpitations are not an independent risk factor for increased cardiac morbidity or mortality \[[@B19]\]. However, according to a British study those experiencing palpitations at work and while asleep were more likely to have a cardiac cause for their palpitations \[[@B20]\]. Our finding was that working-aged CHD patients report palpitations more often than the control population. The high occurrence of trembling of hands and voice, and jerking of muscles may be considered an unexpected finding. Most CHD patients use beta-blocking medicines, which in addition to protecting the heart muscle also reduce the adrenergic stimulation and thus relief the symptom of trembling. Concerning depression our findings support those of previous studies. One out of four of symptomatic coronary heart disease patients have namely been found to have a probable depressive disorder, but none of them had previously been identified as suffering from depression or been treated for this reason \[[@B21]\]. In primary care it is of vital importance to notice symptoms of this illness, since continuing depression has been found to be associated with increased risk of mortality among CHD patients following hospital discharge \[[@B22]\]. It has also been verified that depression is common after coronary heart disease events such as bypass grafting, coronary angioplasty, myocardial infarction and myocardial ischaemia \[[@B23]\]. In our study about 40% of CHD patients had at least minor depression compared with 20% among controls. The high depression rates are probably due to our method to diagnose depression at ≥ 10 points in Beck\'s inventory scale. Thus we do not think there are any selection bias, since the controls were randomly selected. Furthermore, it is not probable that depressed people respond to our questionnaire more readily than people not suffering from low mood. It was also of noteworthy that daytime sleepiness was connected with coronary heart disease. A cross-sectional study of 5,419 Finnish adult men found a higher prevalence of diagnosed myocardial infarction among those who slept more than nine hours, whilst those sleeping less than six hours per night had more symptomatic coronary disease \[[@B24]\]. In a Swedish study concerning working-aged women poor sleep was associated with an increase in spasmodic chest pain and irregular heart beat \[[@B25]\], whereas in men an association between difficulties falling asleep and CHD mortality has been found \[[@B26]\]. From previous research we know that despite having survived a life-threatening clinical event, CHD patients appear to have continued adverse behaviours such as smoking, being obese and having frequent hangovers more than the control population \[[@B27]\]. The follow-up of our cohort will show in what extent the symptoms we found are indicators of increased risk of CHD among working-aged people and to what extent the symptoms are result of CHD and its care. In both cases particular attention should be paid to these aspects in primary care. Conclusions =========== According to the present findings many working-aged people with self-reported coronary heart disease perceive their state of health as poor or rather poor. They suffer from a wide range of symptoms limiting their every day life. It is noteworthy that many of these symptoms are not only irritating, but constitute a threat to health. The health related quality of life is poor among working-aged coronary heart disease patients. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= MPTS drafted the manuscript; SBS participated in drafting of the manuscript; MJK participated in the design of the study and the statistical analyses; LHS participated in the statistical analyses; KJM conceived of the study, and participated in its design and co-ordination. All authors have read and approved the final manuscript.	PubMed Central	2024-06-05T03:55:49.940969	2004-11-8	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534791/", "journal": "Health Qual Life Outcomes. 2004 Nov 8; 2:60", "authors": [ { "first": "Markku PT", "last": "Sumanen" }, { "first": "Sakari B", "last": "Suominen" }, { "first": "Markku J", "last": "Koskenvuo" }, { "first": "Lauri H", "last": "Sillanmäki" }, { "first": "Kari J", "last": "Mattila" } ] }
PMC534792	Background ========== Recent studies of disease burden have demonstrated the importance of psychological disorders. For instance, depression was the fourth leading cause of disease burden, accounting for 4.4% of total disability adjusted life years in the world in 2000 \[[@B1]\]. The 12-item General Health Questionnaire (GHQ-12) has been widely used in many countries for detecting psychological morbidity. Some major national studies such as the British Household Panel Survey (BHPS) also employ this instrument \[[@B2]\]. Calibration of this instrument may therefore contribute significantly to a large community of researchers. While the longer versions of the GHQ are normally considered multidimensional, the GHQ-12 is often regarded as measuring only a single dimension of psychological health. For example, Corti \[[@B3]\] analyzed the GHQ-12 data in the BHPS and maintained that the high Cronbach\'s alpha value indicated the unidimensionality of this instrument. However, several authors suggested that the GHQ-12 contained two or three clinically meaningful factors. Using principal component analysis, Politi et al. \[[@B4]\] identified two factors: general dysphoria and social dysfunction. Andrich and van Schoubroeck \[[@B5]\] suggested that the positively worded items formed one factor and the negatively worded items formed another. Graetz \[[@B6]\], Martin \[[@B7]\] and Worsely and Gribbin \[[@B8]\] proposed three different 3-factor models. In a multi-centre study, although considerable between-centre variation was found, the final solution tended to have either two or three factors \[[@B9]\]. Using confirmatory factor analysis (CFA) to analyze the BHPS data, Cheung \[[@B10]\] compared various models and found that the 3-factor model proposed by Graetz \[[@B6]\] gave the best fit. The factors are anxiety and depression (4 items), social dysfunction (6 items), and loss of confidence (2 items). In a study of employees in New Zealand, Kalliath et al \[[@B11]\] also employed CFA to compare various models. They also found that Graetz\'s 3-factor model gave better goodness-of-fit than the others. However, they maintained that none of the models they examined gave a sufficient level of goodness-of-fit. Hence they modified the instrument to propose a short (8-item) version of GHQ. In a study of college students and young adolescents in Australia, French and Tait \[[@B12]\] found that Graetz\'s model not only fitted the data better than other models, but also satisfactorily achieved some fit indices targets such as Comparative Fit Index \> 0.95. In a study of a rural population in Australia \[[@B13]\], the model of Worsely and Gribbin fitted best and that of Graetz was second best. While the structure of the GHQ-12 has been studied using factor analysis methods, the construct validity and usefulness of those resulting factors are not often tested. The question is whether the additional information provided by the 2 or 3 factors, if they exist, is clinically useful. In other words, will multiple scores be more useful than a total single score in helping us to understand respondents\' health status? The purpose of this study was therefore two-fold. First, we aimed to compare the previously proposed models of the GHQ-12 in an oriental population and identify the best-fitting one. It was not our objective to assess their absolute level of fit or to derive new model or version of the GHQ. Second, we aimed to assess whether the factors identified relate to clinical and health-related quality of life variables in different ways. Methods ======= Subjects and study design ------------------------- A consecutive sample of outpatients with anxiety disorders and/or depressive disorders was recruited from a psychiatric clinic at a tertiary hospital in Singapore. Inclusion criteria were the presence of any anxiety disorder and/or major depressive disorder, literacy in English or Chinese, and completion of an informed consent form. Patients with organic brain syndrome or psychosis were excluded. During routine consultation visits, diagnoses of recruited patients were ascertained by a psychiatrist using DSM-IV criteria and the severity of their psychiatric disorders was assessed using a Clinical Global Impression (CGI) scale, which ranges from 1 (very mild) to 5 (very severe). Patients were then given a questionnaire containing the General Health Questionnaire (GHQ-12) \[[@B14]\], the Beck Anxiety Inventory (BAI) \[[@B15]\], and the Short Form-36 Health Survey (SF-36) \[[@B16]\] for self-completion. Identical English and Chinese questionnaires were prepared for subjects to select according to their preference. A research assistant checked returned questionnaires for completeness. Instruments ----------- The General Health Questionnaire (GHQ-12) consists of 12 items, each assessing the severity of a mental problem over the past few weeks using a 4-point scale (from 0 to 3). The score was used to generate a total score ranging from 0 to 36, with higher scores indicating worse conditions \[[@B14]\]. The Chinese version of GHQ-12 used in this study had been validated \[[@B17],[@B18]\]. A previous study of the 60- and 30-item versions of English and Chinese GHQ yielded comparable scale scores, suggesting equivalence for the two language versions \[[@B19]\]. The Beck Anxiety Inventory (BAI) is a valid and reliable self-report checklist for anxiety symptoms \[[@B15]\]. This instrument consists of 21 items, each describing an anxiety symptom for a respondent to assess how much he or she has been bothered by the symptom over the past week on a 4-point scale. Responses to all items are summed up to a total score ranging from 0 to 63, with higher scores indicating more severe anxiety. A Chinese BAI was developed by the authors using forward- and back-translation procedures, and refined after a pilot study of subjects with anxiety disorders \[[@B20]\]. The Short Form 36 Health Survey (SF-36) \[[@B16]\] is a 36-item questionnaire assessing functional health-related quality of life (HRQoL) in 8 domains: physical functioning, role limitations due to physical problems, bodily pain, general health, vitality, social functioning, role limitations due to emotional problems, and mental health. The instrument yields each domain a score ranging from 0 to 100, with higher scores indicating better HRQoL. The validity and reliability of SF-36 have been extensively documented \[[@B21]\]. In Singapore, both the UK English \[[@B16]\] and Chinese (Hong Kong) \[[@B22]\] versions of SF-36 have been validated \[[@B23],[@B24]\] and these two language versions appear to be equivalent \[[@B25]\]. Statistical analysis -------------------- Various factor structures of the GHQ-12 were tested by confirmatory factor analysis. Model I was unidimensional. Model IIA contained 2 factors: General Dysphoria and Social Dysfunction \[[@B4]\]. Model IIB also contained 2 factors: positively worded items forming one factor and negatively worded items forming another \[[@B5]\]. Model IIIA contained 3 factors: Cope, Stress and Depress, identified by Martin \[[@B7]\]. Model IIIB was the 3-factor model proposed by Graetz \[[@B6]\]: Anxiety and Depression, Social dysfunction, and Loss of Confidence. Model IIIC was also a 3-factor model: Anhedonia-Sleep disturbance, Social Performance and Loss of Confidence \[[@B8]\]. In the confirmatory factor analysis the number of factors and the relationship between factors and observed GHQ-12 items were pre-specified according to the models. The loading of an item on a factor within a model was estimated using the maximum likelihood method. Methodologists have emphasized that it is desirable to use different indicators to examine a model\'s goodness-of-fit \[[@B26]\]. The fit of the six models was assessed by three measures. The Akaike\'s Information Criterion (AIC) penalizes the maximum log likelihood of a model according to its number of parameters. A model with a lower AIC is more plausible than one with a higher AIC. Instead of showing relative fitness, the Comparative Fit Index (CFI) assesses the fit of a model itself. The values range between 0 and 1. A CFI larger than 0.90 indicates an acceptable model. (Hu and Bentler \[[@B27]\] suggested that a CFI value above 0.95 indicates an acceptable model. In a later section we will discuss the more stringent cutoff.) The Root Mean Square of Approximation (RMSEA) assesses a model\'s amount of error. An RMSEA value larger than 0.08 indicates too much error. The best-fitting model was examined in detail. The Kruskal-Wallis test was used to compare the GHQ-12 overall and factor scores of patients with different diagnosis. Pearson\'s correlation coefficient (r) was used to assess the association between GHQ-12 scores and various variables, namely Beck Anxiety Inventory, Clinical Global Impression and SF-36 scores. The Fisher\'s Z transformation was used to produce 95% confidence interval. Results and Discussion ====================== A total of 120 participants (63 man and 57 women) were included in the analysis (Table [1](#T1){ref-type="table"}). Most (90%) respondents were Chinese; the mean (SD) age was 43.1 (12.7). Sixty six percent of the participants chose to administer an English version of the questionnaire. The mean scores of clinical and HRQoL data reported by the respondents in both gender were shown in Table [1](#T1){ref-type="table"}. Men tended to have less anxiety, better clinical global impression, and higher SF-36 scores. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Mean (SD) clinical and SF-36 health-related quality of life values by gender ::: Clinical or psychological data Men (N= 63) Women (N= 57) ^(a)^ -------------------------------- --------------- ----------------------- Beck Anxiety Inventory 20.65 (13.48) 21.89 (13.59) Clinical Global Impression 2.76 (0.84) 3.02 (0.82) Physical Functioning 76.50 (17.88) 73.72 (17.97) Physical Problems 51.06 (43.57) 43.42 (42.13) Bodily Pain 62.06 (24.89) 53.46 (24.05) General Health 49.48 (21.14) 49.21 (20.07) Vitality 45.40 (18.15) 41.26 (19.94) Social Functioning 56.15 (23.75) 50.22 (26.88) Emotional Problems 39.15 (42.56) 26.32 (41.18) Mental Health 51.05 (17.65) 46.67 (19.11) \(a) N = 56 for Clinical Global Impression scale due to a missing value. ::: Table [2](#T2){ref-type="table"} shows goodness-of-fit statistics for the 1-, 2- and 3-factor models. The 3-factor model (IIIB) proposed by Graetz (1991) was the best in terms of all three fit statistics. It gave the lowest AIC and RMSEA and highest CFI. Its CFI was 0.935. All six models produced RMSEA\'s which exceeded 0.08. The one-dimensional model (Model I) had the highest AIC, highest RMSEA and lowest CFI. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Goodness-of-fit of six confirmatory factor analysis models (N = 120) ^(a),(b)^ ::: Statistics Model I (1 factor) Model IIA (2 factors) Model IIB (2 factors) Model IIIA (3 factors) Model IIIB (3 factors) Model IIIC (3 factors) ------------ -------------------- ----------------------- ----------------------- ------------------------ ------------------------ ------------------------ AIC 69.529 29.220 29.956 51.611 21.075 48.956 CFI 0.888 0.927 0.925 0.908 0.935 0.910 RMSEA 0.139 0.115 0.115 0.130 0.109 0.128 \(a) AIC: Akaike\'s Information Criterion; CFI: Comparative Fit Index; RMSEA: Root Mean-Square Error of Approximation \(b) See text for details about the models. ::: Figure [1](#F1){ref-type="fig"} displays the standardized factor loadings and between-factor correlation of model IIIB. The factor loadings ranged between 0.72 and 0.90. The three factors were strongly correlated. The correlation between factor 1 (Anxiety and Depression) and factor 2 (Social Dysfunction) was 0.89. The correlation between factor 2 and factor 3 (Loss of Confidence) was 0.83. That between factor 1 and 3 was 0.90. These strong correlations suggest that even if there were in fact three factors, in practice it may be very difficult to discern them. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Standardised factor loadings and between-factor correlations of Graetz\'s model \[6\]. Boxes represent GHQ-12 items; ellipses represent factors. One-way and two-way arrows indicate factor loadings and between-factor correlations, respectively. ::: ![](1477-7525-2-63-1) ::: Having established that Graetz\'s 3-factor model fitted the data better than the other models, we calculated the factor scores as unweighted sums of the items concerned. From figure [1](#F1){ref-type="fig"} we could see that the loadings on each factor did not vary substantially. Hence we chose to use unweighted sums for simplicity. Table [3](#T3){ref-type="table"} shows the mean (SD) factor scores and the overall GHQ-12 score by clinical diagnosis. Some patients had multiple diagnoses; we categorized them into one of three major clinical diagnoses. The three factor scores and the overall GHQ-12 scores behaved in fairly similar ways. All four scores were significantly different between patients with and without depression; none was significantly different between patients with and without general anxiety disorder. Patients with panic disorder had lower scores on the factor Loss of Confidence (difference = 0.68; P = 0.043). The SD of the two diagnosis groups pooled was about 1.75; the between group difference was therefore approximately about 0.4 SD. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Comparison of mean (SD) values of GHQ-12 scores by clinical diagnosis. ::: Diagnosis N Overall GHQ-12 score Anxiety and depression (Factor 1) Social dysfunction (Factor 2) Loss of confidence (Factor 3) -------------------------- ---- ---------------------- ----------------------------------- ------------------------------- ------------------------------- Depression Yes 60 32.15 (9.18) 11.28 (3.10) 15.73 (4.74) 5.13 (1.83) No (Other diagnosis) 60 27.43 (6.74) 9.48 (2.86) 13.73 (3.06) 4.22 (1.62) P-value ^(a)^ 0.002 0.002 0.010 0.005 General anxiety disorder Yes 47 30.02 (8.10) 10.68 (2.99) 14.64 (3.96) 4.70 (1.82) No (Other diagnosis) 73 29.64 (8.58) 10.19 (3.18) 14.79 (4.20) 4.66 (1.77) P-value 0.712 0.410 0.987 0.993 Panic disorder Yes 54 28.48 (7.88) 9.96 (3.08) 14.22 (3.71) 4.30 (1.69) No (Other diagnosis) 66 30.86 (8.64) 10.73 (3.10) 15.15 (4.37) 4.98 (1.80) P-value 0.158 0.179 0.244 0.043 \(a) Kruskal-Wallis test. ::: Table [4](#T4){ref-type="table"} presents the results of the correlation of 3 factors of Graetz\'s model and BAI, Clinical Global Impression Score, and SF-36 scales. The 3 factors were correlated with the 10 clinical and HRQoL variables to very similar degree. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Pearson\'s correlation coefficients (95% confidence intervals) between GHQ-12 scores and clinical and health-related quality of life variables ::: Clinical/HRQoL scales Overall GHQ-12 score Anxiety and depression (Factor I) Social dysfunction (Factor II) Loss of confidence (Factor III) ---------------------------- ------------------------ ----------------------------------- -------------------------------- --------------------------------- Beck Anxiety Inventory 0.69 (0.58 to 0.77) 0.68 (0.57 to 0.77) 0.62 (0.50 to 0.72) 0.63 (0.50 to 0.72) Clinical Global Impression 0.49 (0.34 to 0.61) 0.45 (0.29 to 0.58) 0.47 (0.31 to 0.60) 0.43 (0.27 to 0.56) Physical functioning -0.17 (-0.34 to 0.01) -0.18 (-0.35 to 0.00) -0.16 (-0.33 to 0.02) -0.12 (-0.29 to 0.06) Role physical -0.63 (-0.73 to -0.51) -0.60 (-0.71 to -0.48) -0.61 (-0.71 to -0.48) -0.51 (-0.63 to -0.36) Bodily pain -0.52 (-0.68 to -0.44) -0.57 (-0.68 to -0.44) -0.43 (-0.56 to -0.27) -0.46 (-0.59 to -0.31) General health -0.57 (-0.68 to -0.44) -0.57 (-0.68 to -0.43) -0.52 (-0.64 to -0.38) -0.50 (-0.62 to -0.35) Vitality -0.71 (-0.79 to -0.61) -0.73 (-0.80 to -0.63) -0.63 (-0.73 to -0.51) -0.62 (-0.72 to -0.50) Social functioning -0.65 (-0.74 to -0.54) -0.62 (-0.72 to -0.50) -0.60 (-0.70 to -0.47) -0.59 (-0.70 to -0.46) Role emotional -0.62 (-0.72 to -0.50) -0.63 (-0.73 to -0.51) -0.55 (-0.66 to -0.41) -0.55 (-0.66 to -0.41) Mental health -0.67 (-0.76 to -0.56) -0.67 (-0.76 to -0.56) -0.60 (-0.70 to -0.47) -0.61 (-0.71 to -0.49) ::: Several previous confirmatory factor analyses found that the 3-factor model of Graetz gave better fit to survey data from Australia \[[@B12]\], Britain \[[@B10]\] and New Zealand \[[@B11]\]. In this study we examined the issue in an Asian population in Singapore, whose members are mainly ethnic Chinese. All three goodness-of-fit indices employed, namely AIC, CFI and RMSEA, agreed that the 3-factor model of Graetz out-performed the other five models. The CFI value was 0.935. Conventionally, a CFI of 0.90 or larger is taken as evidence of sufficient fit. A more stringent criterion of CFI larger than 0.95 has recently been proposed and debated \[[@B27],[@B28]\]. The RMSEA also indicated that even the best-fitting model did not fit well, using the cut-off of 0.08 as a criterion. However, our aim is to compare the models rather than to modify the instrument. So for our purpose it is the comparison of the goodness-of-fit of the six models that matters, not the absolute values of the fit indices. We consider the \"correctness\" and \"usefulness\" of a model two fairly separate issues. Although the goodness-of-fit of Graetz\'s model was limited, we proceeded to examine the factor scores in relation to external criteria in order to reach a conclusion about the usefulness of the model. The one-dimensional model was the worst according to all three goodness-of-fit indices. The three factors in the model proposed by Graetz were found to be strongly correlated with each other, with correlation coefficients in the neighborhood of 0.8 to 0.9. Such strong correlations suggest that even if there were indeed three different factors, in practice it is quite difficult to differentiate them. The study of French and Tait \[[@B12]\] also showed strong correlation between the factors, which led the authors to recommend that it may be prudent to use the overall score rather than overinterpret the factors within the GHQ-12. We examined the three factor scores and the overall GHQ-12 score in relation to clinical diagnoses. The four scores behaved in fairly similar ways. Although the Loss of Confidence scale was significantly different between patients with and without panic disorder while the other three scales did not show significant differences between the two groups of patients, the difference was only about 0.4 SD. This is smaller than a recommended threshold (0.5 SD) corresponding to minimal clinically important differences for health states questionnaires \[[@B29]\]. We also examined the association between the three GHQ scores and the Beck Anxiety Inventory, a clinical impression score, and the 8 scales of the SF-36. The three factors were associated with the clinical and HRQoL variables to similar degrees. Two limitations of the study should be noted. Firstly, the sample size was somewhat small for confirmatory factor analysis. Secondly, the participants were clinical cases. This homogeneity might have made it more difficult to detect variations in GHQ-12 scores. We believe that the question about the relative plausibility of various factor models have been sufficiently answered by this and several previous studies \[[@B10]-[@B12]\]. Nevertheless, future studies of non-clinical participants based on larger sample sizes will be helpful to further assess the practical usefulness of the factors of the GHQ-12. Conclusions =========== Several studies, including the present one, have found that Graetz\'s 3-factor model of the GHQ-12 is more plausible than other models. However, the factors were strongly correlated and difficult to discern. Our analysis of the three GHQ scores in relation to clinical variables and aspects of health-related quality of life did not appear to be more informative than analysis of a single overall GHQ-12 score. As such, from a pragmatic point of view we consider it acceptable to use this instrument as a one-dimensional measure. Unless one has specific questions that are best answered by a subset of the three factors, there is no need to consider the multi-dimensionality. Authors\' contribution ====================== FG carried out the confirmatory factor analysis, interpreted the findings, and drafted part of the manuscript. NL designed the study, participated in the development of the statistical framework and interpreted the findings. JT participated in the study design, discussion of the statistical framework, and the interpretation of findings. CF participated in the study design and carried out the data collection and clinical assessments. SCL participated in the study design and discussion and interpretation of findings. YBC conceived of the study, developed the statistical framework, carried out part of the statistical analysis, and drafted part of the manuscript. All authors read and approved the final manuscript.	PubMed Central	2024-06-05T03:55:49.943610	2004-11-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534792/", "journal": "Health Qual Life Outcomes. 2004 Nov 11; 2:63", "authors": [ { "first": "Fei", "last": "Gao" }, { "first": "Nan", "last": "Luo" }, { "first": "Julian", "last": "Thumboo" }, { "first": "Calvin", "last": "Fones" }, { "first": "Shu-Chuen", "last": "Li" }, { "first": "Yin-Bun", "last": "Cheung" } ] }
PMC534793	Background ========== An increase in the prevalence of asthma and atopy during the last two decades is documented for both children \[[@B1]\] and adults \[[@B2]\]. The International Study of Asthma and Allergies in Childhood (ISAAC) has demonstrated a large variation in the prevalence of asthma symptoms in children throughout the world \[[@B3]\]. An ISAAC-study on prevalence of childhood allergic diseases in Scandinavia and Eastern Europe has shown that the prevalence among Swedish children 13--14 years of age is 15% for asthma/wheezing, 17% for eczema and 26% for rhino-conjunctivitis \[[@B4]\]. It is well known that there are more individuals with perceived hypersensitivity than individuals with verified/doctor-diagnosed allergy. This is especially true when it comes to perceived food hypersensitivity with up to tenfold higher figures versus verified food allergy \[[@B5],[@B6]\]. Prevalence figures for food hypersensitivity vary considerably (1--25%) with regard to study design, population of subjects \[[@B7]-[@B9]\] and country \[[@B7],[@B8]\]. Research has demonstrated that allergic diseases have a negative impact on individuals\' health-related quality of life (HRQL) and a number of studies describe HRQL-deteriorations in children and adults with asthma \[[@B10]-[@B12]\], eczema \[[@B13]-[@B15]\] and rhinitis \[[@B16]-[@B18]\]. Studies have also shown that allergy-associated physical and organ-related measures and tests do not always correlate with HRQL-scores \[[@B11],[@B12]\]. Adverse reactions to food constitute an important part of allergy-associated problems, especially among children. Still, studies on food allergy and HRQL are scarce. However, parental perception of physical and psychosocial functioning, measured with the Children\'s Health Questionnaire (CHQ-PF50), has shown that childhood food allergy has a significant emotional impact on the parent and limits the family activities \[[@B19]\]. It has also been documented that, from a parental perspective, children with peanut allergy have significantly more disruption in their daily life compared with children with rheumatological disease, due to their children\'s risk of death \[[@B20]\]. Furthermore, peanut allergic children have been shown to report more anxiety about eating and more fear of an adverse event compared with children with diabetes mellitus \[[@B21]\]. Previous studies have shown that adolescent males have a higher prevalence of atopy than adolescent females \[[@B22]\] although there is a female dominance in self-reported allergic diseases \[[@B23]\]. Furthermore, the prevalence of asthma has been found to vary with age and sex, showing a male predominance before puberty that changes into a higher prevalence in females in adolescence \[[@B24],[@B25]\]. Research in the allergy field is mainly focused on individuals with verified allergy and their suffering from these conditions, i.e. leaving out those with self-reported allergy-like conditions but with no doctor-diagnosis. Still, one can presume that perceived allergy could have an impact on the health-related quality of life (HRQL) and involve suffering, regardless of verifiable diagnosis. In order to obtain information about the extent to which adolescent females and males themselves experience allergy-like conditions and the impact of these conditions on their everyday life, the present study aimed to investigate the magnitude of self-reported allergy-like conditions in adolescence and to evaluate their HRQL. Special focus was put on food hypersensitivity as a specific allergy-like condition and on gender differences. Methods ======= Subjects and procedure ---------------------- The present study involved adolescents at the senior level of the nine-year compulsory school and at the upper secondary school in a municipality in the south of Stockholm, Sweden. The socio-demographic distribution of the inhabitants in this municipality was slightly above in socio-economics and slightly below in number of immigrants compared with the country as a whole. A total of 2064 adolescents were registered in the schools at the relevant levels, all with the Swedish language as their school language. One week prior to starting the study (May 2003), an information letter outlining the purpose of the investigation, including an assurance of confidentiality and voluntary participation, was distributed to both the adolescents (n = 2064) and their parents. In connection with lessons at school, the teachers distributed questionnaires to the 1488 adolescents who were present at school. The instructions given by the teachers and the administration of the questionnaires were standardized. Two questionnaires were distributed together in one envelope, with the HRQL-questionnaire (se below) at the top. The adolescents themselves completed the questionnaires during that particular lesson. No other support was offered than the possibility to ask the teacher clarifying questions regarding the wording. After completion of the questionnaires, or in case of renouncing participation, each adolescent put the questionnaires into the envelope, sealed it, and handed it over to the teacher, who forwarded the envelopes to one of the authors (BM). The discrepancy between the number of registered adolescents (n = 2064) and the number, who were actually present when the data was collected (n = 1488), was partly due to the fact that it was close to the summer holiday and graduation and some adolescents attended activities outside school. The school records confirmed their absences. As 37 adolescents had not properly filled in the questionnaires, those were excluded from the study. In total 1451 questionnaires (97%) remained for data analysis. Age ranged between 13 and 21 years (mean 16.2 years), with 99% of the adolescents between 14 and 20 years. In total 696 females and 716 males had reported their gender. For 39 adolescents the gender was not reported. The terminology used in this study is according to ISAAC (The International Study of Asthma and Allergies in Childhood) \[[@B3]\] and EAACI (The European Academy of Allergology and Clinical Immunology) position paper \[[@B26]\] (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Terminology and definitions^1^ ::: Allergy-like conditions ----------------------------------------------------------------------------------------------------------- ----------------------------------------------------------- ----------------------------------------------------------------------- --------------------------------------------------------------------------------------- Hypersensitivity Allergic diseases Asthma/wheezing Eczema/rash Rhino-conjunctivitis Self-reported hypersensitivity, allergy or intolerance to food or other defined environmental substances. Self-reported asthma, wheezing or whistling in the chest. Self-reported recurrent eczema or itchy rash for at least six months. Self-reported sneezing, runny nose, blocked nose or itchy-watery eyes without a cold. ^1^Terminology according to ISAAC \[3\] and EAACI position paper \[26\]. ::: Questionnaires -------------- A study-specific questionnaire was used to evaluate the magnitude of allergy-like conditions during the past twelve months and to evaluate the frequency of allergy testing. The questionnaire, devised by the authors, was based on relevant literature on similar subjects \[[@B3]\]. Prior to the data collection, a pilot test of the questionnaire was performed with fourteen adolescents, who were not included in the present study, and subsequently minor lexical adjustments were made. Some of the questions included were as follows: \"Are you allergic or hypersensitive to any of the following?\" (Possible answers: furred animal, pollen, dust/mite, food, nickel, other substances: Yes/No.) \"If you are allergic or hypersensitive to any food items, what reactions or symptoms do you perceive? (Possible answers: not allergic or hypersensitive, eczema, rash, eye-nose-symptoms, itchy mouth, breathing difficulties, vomiting-diarrhoea-stomach ache, allergic chock, other).\" \"Have you had asthma, wheezing or whistling in the chest [in the past 12 months]{.underline}? (Yes/No)\" \"[In the past 12 months]{.underline}, have you had recurrent eczema or itchy rash for at least 6 months? (Yes/No)\" \"[In the past 12 months]{.underline}, have you had a problem with sneezing, or a runny, or a blocked nose when you [did not]{.underline} have a cold? (Yes/No)\" \"[In the past 12 months]{.underline}, have you had a problem with itchy-watery eyes when you [did not]{.underline} have a cold? (Yes/No)\" The questions concerning allergic diseases were taken verbatim from the ISAAC study \[[@B3]\]. The generic instrument Medical Outcome Trust Short Form 36 Health Survey (SF-36) was used to measure HRQL. SF-36 is a well-validated and reliable measure of HRQL in adults and adolescents from the age of 14, and normative data are available for the Swedish population \[[@B27]\]. The SF-36 consists of 36 items, which refer to eight health scales related to daily life activities. Four of these health scales represent the physical dimension and the remaining four health scales represent the mental dimension of the HRQL concept. The scale scores range from 0 to 100, with 100 representing the highest level of functioning and well being \[[@B27],[@B28]\]. The footnote of Table [4](#T4){ref-type="table"} conveys a summary of the contents in the SF-36 health scales. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Comparison of SF-36 scores between adolescents with and without allergy-like conditions and between females and males with allergy-like conditions ::: Allergy-like conditions No such conditions Females with allergy-like conditions Males with allergy-like conditions ---------------------------------- ------------------------- -------------------- ------ -------------------------------------- ------------------------------------ ------ ------ ------ ------ ----------- N = 931 N = 520 N = 501 N = 412 SF-36 health scales^1^ Mean SD Mean SD p-value Mean SD Mean SD p-value Physical dimension Physical functioning (PF) 91.1 16.4 91.7 19.1 NS 91.1 13.8 91.2 18.8 NS Role functioning-physical (RP) 75.7 31.3 83.2 27.2 \<0.001 73.6 31.5 78.4 30.4 \<0.05 Bodily pain (BP) 71.3 23.6 81.2 20.3 \<0.001 67.9 24.1 75.3 22.4 \<0.001 General health (GH) 69.6 20.5 80.8 16.7 \<0.001 66.3 21.1 73.5 19.2 \<0.001 Mental dimension Vitality (VT) 53.0 21.0 62.2 21.0 \<0.001 50.2 20.5 56.4 21.2 \<0.001 Social functioning (SF) 81.3 21.6 86.9 18.9 \<0.001 79.1 22.0 84.1 20.8 \<0.001 Role functioning-emotional (RE) 65.4 40.3 75.4 36.8 \<0.001 58.2 42.1 73.7 36.3 \<0.001 Mental health (MH) 67.3 19.6 74.9 19.1 \<0.001 62.7 20.2 72.8 17.4 \<0.001 ^1^Summary of contents of SF-36 health scales:Physical dimension: PF = Ability to perform daily physical activities, e. g. walking, running, lifting, and other moderate physical efforts; RP = Extent to which physical health limits work/daily activities; BP = Intensity of pain and its interference with normal activities; GH = Personal evaluation of general health status, presently and in the future. Mental dimension: VT = Personal evaluation of energy, tiredness, etc; SF = Extent to which physical health or emotional problems interfere with normal social activities; RE = Extent to which emotional problems limit work/daily activities; MH = Personal evaluation of mental health, including anxiety, depression, and general positive and negative affects \[27, 28\]. ::: Statistical analyses -------------------- For statistical analyses the SPSS 11.0 program was used. SF-36 data was processed by means of an SPSS program provided by the HRQL-group at the University of Gothenburg, Sweden \[[@B27]\]. Internal consistency of the SF-36 health scales was tested by means of Cronbach\'s alpha and in this study ranged between 0.72 and 0.91, with the exception of the scale for social functioning (SF) showing 0.61. To test differences in proportions between groups, the Chi-square test was used. The Student\'s t-test and, when appropriate, the one-way analysis of variance (ANOVA) were used to assess differences in means between groups. A p-value \<0.05 was considered to be statistically significant. Ethical approval ---------------- This study was approved by the Director of School Administration in Tyresö municipality and by the research ethics committee at Huddinge University Hospital. Results ======= Allergy-like conditions ----------------------- As shown in Table [2](#T2){ref-type="table"}, a total of 931 adolescents (64%) reported that they suffered from some kind of allergy-like condition, i.e. either hypersensitivity to defined substances (46%) or allergic diseases (51%) (definitions given in Table [1](#T1){ref-type="table"}). In particular, 19% of the whole group (24% of the females and 14% of the males) reported that they reacted to some food (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Self-reported allergy-like conditions among the 1451 adolescents ::: ------------------------------------------- ------------- ---------- --------- ------------ --------- ---------- ------------- Total Females Males N = 1451^1^ N = 696 N = 716 p-values N (%) N (%) N (%) Allergy-like conditions, totals 931 (64) 501 (72) 412 (58) \<0.001 Hypersensitivity to defined substances 663 (46) 364 (52) 282 (39) \<0.001 pollen 335 (23) 161 (23) 162 (23) NS food 271 (19) 165 (24) 98 (14) \<0.001 furred animal 234 (16) 117 (17) 110 (15) NS dust/mite 194 (13) 114 (16) 75 (11) \<0.001 nickel 169 (12) 132 (19) 32 (5) \<0.001 other substances^2^ 94 (7) 62 (9) 31 (4) \<0.001 Allergic diseases 739 (51) 408 (59) 319 (45) \<0.001 asthma/wheezing 231 (16) 156 (22) 72 (10) \<0.001 eczema/rash 286 (20) 178 (26) 105 (15) \<0.001 rhino-conjunctivitis 546 (38) 287 (41) 248 (35) \<0.05 ------------------------------------------- ------------- ---------- --------- ------------ --------- ---------- ------------- ^1^Gender unknown n = 39. ^2^Offending substances reported: insects (n = 15), drugs (n = 14), detergents (n = 9), mildew (n = 8), perfume (n = 8) and smoke (n = 5). Substances reported for less than five persons each are not specified. ::: The adolescents suffered to a large extent from complex allergy-like conditions, i.e. hypersensitivity to multiple offending substances and/or allergic diseases. Fifty per cent of those with hypersensitivity (n = 334/663) reported more than one kind of offending substance and 35% of those who reported allergic diseases (n = 260/739) reported more than one type of disease. Fifty-one per cent of those with allergy-like conditions (n = 471/931) reported both hypersensitivity and allergic disease. Significantly more females than males reported allergy-like conditions (Table [2](#T2){ref-type="table"}). In addition, hypersensitivity to more than one type of offending substance was more frequently reported by females than by males (55% and 43%, respectively, p \< 0.01). Females reported also to a greater extent more than one kind of allergic disease (40% and 29%, respectively, p \< 0.001). Out of the 931 adolescents with allergy-like conditions, 404 (43%) reported that they had been tested for allergy by means of blood test or skin prick test (results not shown). For the majority (n = 324) of these adolescents with self-reported allergy testing, the tests had been performed during their school age years. Sixty-one per cent of those tested (n = 246/404) reported that the test results verified allergy. This figure corresponds to a 17% prevalence of self-reported verified allergy within the whole population of 1451 adolescents. Food hypersensitivity --------------------- In the group of 271 adolescents reporting food hypersensitivity, 139 (51%) reported allergy test results that verified some kind of allergy, albeit not necessarily food allergy (results not shown). The most common food-induced symptoms were OAS (Oral Allergy Syndrome)-like symptoms (Table [3](#T3){ref-type="table"}), i.e. itching and swelling of the lips and oral cavity, reported by 52% of the adolescents, similarly reported by females and males. Significantly more females than males reported food-induced symptoms from the skin (p \< 0.001) and from the gastro-intestinal tract (p \< 0.05). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Food-induced symptoms, hypersensitivity to other substances besides food and allergic diseases among adolescents with food hypersensitivity ::: Adolescents with food hypersensitivity Total Females Males -------------------------------------------- --------- ----------- --------- ----------- -------- ----------- ------------ Food-induced symptoms 271 (100) 165 (100) 98 (100) OAS^2^-like symptoms 140 (52) 83 (50) 55 (56) NS skin symptoms 81 (30) 62 (38) 16 (16) \<0.001 gastro-intestinal symptoms 76 (28) 55 (33) 20 (20) \<0.05 breathing difficulties 67 (25) 38 (23) 29 (30) NS eye/nose symptoms 35 (13) 20 (12) 15 (15) NS anaphylaxis 32 (12) 21 (13) 11 (11) NS Hypersensitivity to defined substances 208 (77) 128 (78) 72 (74) NS pollen 141 (52) 79 (48) 56 (57) NS furred animals 125 (46) 67 (41) 53 (54) \<0.05 dust/mite 90 (33) 56 (34) 33 (34) NS nickel 64 (24) 54 (33) 7 (7) \<0.001 other substances^3^ 26 (10) 20 (12) 6 (6) NS Allergic diseases 210 (78) 135 (82) 70 (71) \<0.05 asthma/wheezing 88 (33) 61 (37) 24 (25) \<0.05 eczema/rash 93 (34) 66 (40) 25 (26) \<0.05 rhino-conjunctivitis 167 (62) 103 (62) 58 (59) NS ^1^Gender unknown n = 8. ^2^OAS = Oral Allergy Syndrome ^3^The substances reported for at least three persons each were: mildew (n = 4), drugs (n = 3), insects (n = 3) and perfume (n = 3). ::: Offending food items reported were: nuts (39%), fruit and berries (35%), peanut (32%), almond (22%), tomato (19%), carrot (16%), lactose (12%), vegetables (10%), shellfish (9%), soy (7%), milk (7%), fish (5%) and egg (5%). Substances reported for less than five per cent of the 271 adolescents are not specified. For two food items there were significant gender differences. The offending food items fruit and berries were more commonly reported by females (44% and 24% respectively, p \< 0.001) and peanut was more commonly reported by males (43% and 27% respectively, p \< 0.01). A total of 63 adolescents reported food as the only offending substance. However, the majority of the 271 adolescents who reported food hypersensitivity suffered from complex allergy-like conditions that included additional offending substances besides food (77%) as well as allergic diseases (78%). In this group of food hypersensitive adolescents there were significantly more males than females who reported hypersensitivity to furred animals and as regards hypersensitivity to nickel the result was reverse (Table [3](#T3){ref-type="table"}). Allergic diseases such as asthma/wheezing and eczema/rash were also significantly more often reported by the females (Table [3](#T3){ref-type="table"}). Health-related quality of life ------------------------------ Adolescents with allergy-like conditions scored significantly lower on seven of the eight SF-36 health scales compared with adolescents without such conditions (Table [4](#T4){ref-type="table"}). The adolescents with allergy-like conditions scored similar on the health scales whether they had reported verified allergy or not (results not shown). Moreover, Table [4](#T4){ref-type="table"} also demonstrates gender differences among the adolescents with allergy-like conditions. Females reported significantly lower SF-36 scores in seven of the eight health scales. When comparing females with and females without allergy-like conditions, the former group scored significantly lower on all eight scales (Figure [1a](#F1){ref-type="fig"}). For the males, statistically significant differences were seen for all health scales, except for physical functioning (PF) and role functioning-physical (RP) (Figure [1b](#F1){ref-type="fig"}). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Comparison of SF-36 scores between: a) females with allergy-like conditions and females with no such conditions, and b) males with allergy-like conditions and males with no such conditions. (Physical dimension:PF, physical functioning; RP, role functioning-physical; BP, bodily pain; GH, general health. Mental dimension:VT, vitality; SF, social functioning; SE, role functioning-emotional; MH, mental health.) ::: ![](1477-7525-2-65-1) ::: Figure [2](#F2){ref-type="fig"} shows comparisons between food hypersensitive adolescents, females and males, with or without other allergy-like conditions. Females with food hypersensitivity scored significantly lower on three health scales (BP, GH and SF) compared with females with other allergy-like conditions (Figure [2a](#F2){ref-type="fig"}). A corresponding comparison for the males showed no HRQL-deterioration for the food hypersensitive males (Figure [2b](#F2){ref-type="fig"}). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Comparison of SF-36 scores between: a) females with food hypersensitivity and females with other allergy-like conditions, b) males with food hypersensitivity and males with other allergy-like conditions, and c) adolescents with food hypersensitivity with or without allergic diseases. (Physical dimension:PF, physical functioning; RP, role functioning-physical; BP, bodily pain; GH, general health. Mental dimension:VT, vitality; SF, social functioning; SE, role functioning-emotional; MH, mental health.) ::: ![](1477-7525-2-65-2) ::: When comparing food hypersensitive adolescents who reported allergic diseases (i.e. asthma/wheezing, eczema/rash and rhino-conjunctivitis) (n = 210/271) to those food hypersensitive adolescents who did not report such conditions (n = 61/271), the groups showed a similar pattern as regards the mental dimension scales of the SF-36, i.e. no statistically significant differences were found. As regards the physical dimension scales, the food hypersensitive adolescents who also reported allergic diseases scored significantly lower on BP (bodily pain) and GH (general health) (Figure [2c](#F2){ref-type="fig"}). A comparison within the whole group of adolescents with allergy-like conditions (n = 931), i.e. between those who reported only hypersensitivity to any defined environmental substances (A), those who reported only allergic diseases (B), and those who reported both (C), showed that the three groups scored similar on the four health scales representing the mental dimension of the SF-36. As regards the physical dimension, adolescents with allergic diseases only (B) or in combination with hypersensitivity (C) scored lower on the health scales for bodily pain (BP) and general health (GH) compared with those with only hypersensitivity to defined substances (A) (Figure [3](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Comparison of SF-36 scores between adolescents with (A) only hypersensitivity to defined substances, (B) only allergic diseases and (C) both (p \< 0.05: BP A \> B and C; GH A \> C). (Physical dimension:PF, physical functioning; RP, role functioning-physical; BP, bodily pain; GH, general health. Mental dimension:VT, vitality; SF, social functioning; SE, role functioning-emotional; MH, mental health.) ::: ![](1477-7525-2-65-3) ::: Discussion ========== The present study focuses on self-reported allergy-like conditions among adolescents, in particular food hypersensitivity. The results show that as many as 64% of the adolescents reported allergy-like conditions, of which nearly one third reported food hypersensitivity. In most cases the allergy-like conditions were complex, i.e. included hypersensitivity to multiple offending substances and/or allergic diseases. Compared with the prevalence for Swedish children 13--14 years of age, shown in an ISAAC-study \[[@B4]\], the adolescents (13--21 years) in the present study reported about the same prevalence of asthma/wheezing (15% and 16%, respectively) and of eczema (17% and 20%, respectively), but a higher prevalence of rhino-conjunctivitis (26% and 38%, respectively). The differences in prevalence of rhino-conjunctivitis between these studies might to some extent be explained by the fact that different age groups were sampled. Furthermore, the referred ISAAC-study was performed at least six years earlier than the present one. During this time span the allergy problem has been a growing concern among both children and adults and the rates may have risen \[[@B1],[@B2]\]. The adolescents with allergy-like conditions generally showed significantly lower HRQL than adolescents without such conditions. Previous studies have shown that doctor-diagnosed asthma, eczema, and rhinitis have negative impacts on HRQL \[[@B10]-[@B18]\]. In the present study we have shown that those who reported that they suffered from these allergic diseases scored low on SF-36, regardless whether the diseases were verified by medical expertise or not. Furthermore, also the adolescents who reported hypersensitivity without having such diseases, scored low on SF-36, especially on the scales concerning mental health and emotional-social functioning. This is in accordance with previous findings in children with doctor-diagnosed peanut allergy \[[@B20],[@B21]\]. Living with constant vigilance, uncertainties and risks of adverse reactions, is likely to influence HRQL in adolescents in a negative way. The question of co-morbidity has been discussed \[[@B29]\], as it is not always possible to grasp what component(s) of a complex allergy-like condition affects the HRQL. Furthermore, one has to consider the possibility that the HRQL-deteriorations in these adolescents may not be a direct effect of their allergy-like conditions, but related to an overall poorer general state of health. The presence of poor social, mental or somatic health may increase the perception of allergy-like conditions. Still, irrespective of what the underlying causes are, it is evident that adolescents who experience allergy-like conditions also experience HRQL-deterioration. In most of the comparisons between different subgroups of adolescents with and without allergy-like conditions, the SF-36 health scale for physical functioning (PF) showed no significant difference. This is noteworthy, as the physical parameter often is in focus when health-care professionals assess an individual\'s state of health. However, it has been previously shown that in patients with asthma/wheezing, the link between lung function and HRQL is weak \[[@B11],[@B12]\] and the results of the present study indicate that the link between physical parameters and the HRQL may be weak also in other kinds of allergy-like conditions. Hypersensitivity may perhaps be considered as a practical, emotional and psychosocial health problem -- not primarily a physical. It has been shown, however, that HRQL-deterioration among peanut allergic children is related to anxiety and fear for adverse reactions \[[@B20],[@B21]\]. Moreover, the adolescents with a non-severe chronic allergic disease may be well adapted to the disease, physiologically and/or psychologically, so that the disease as such has no significant impact on their physical quality of life. In the present study we show that in adolescence, significantly more females than males experienced not just asthma/wheezing but also eczema/rash, rhino-conjunctivitis, and hypersensitivity to food, dust/mite and nickel. The females presented more complex allergy-like conditions compared with the males. In addition, females with allergy-like conditions showed more severe HRQL-deterioration compared with the males. It is known that female gender among adults implies a larger report of burden of health problems in general \[[@B30]\] and SF-36 Swedish normative data show that females 15--19 years of age score lower compared with males in this age group \[[@B27]\]. Thus, the gender differences with respect to allergy-like conditions were in accordance with a known pattern within the health-and-gender-field. Biological, hormonal and socio-cultural explanations to gender differences in asthma and allergy have been discussed \[[@B24],[@B31],[@B32]\] as well as possible gender biased diagnostic practices \[[@B33]-[@B35]\] such as underdiagnosis of females due to gender differences in disease severity. Further research in this area is needed so that health-care professionals, school personnel, relatives and friends can improve their care and support given to both females and males suffering from allergy-like conditions. Seventeen per cent of the adolescents, of whom the most part were tested during school age, reported positive allergy test results. It can be assumed that there were some additional adolescents with doctor-diagnosed allergy but without verifying test results. Thus, the total of adolescents doctor-diagnosed as having allergy may well be more than 17%. However, in the present study the focus was on self-reported (perceived or verified) allergy-like conditions. The mean SF-36 scores did not differ whether the adolescents reported objectively verified allergy or not. The lack of difference in HRQL was not surprising as a diagnostic test that verifies allergy says nothing about the individuals experience or the severity of the allergic condition. Food hypersensitivity --------------------- Most food allergies are something that children outgrow \[[@B36]\] and adverse reactions to food should consequently be a problem that decreases with age. The prevalence of food allergy is estimated to 5--8% in children and 1--2% in adults \[[@B37]\]. However, figures of perceived reactions to food may well be over 20% \[[@B6]\]. In the present study, 19% of the adolescents did report adverse reactions to food. This high figure may be explained by the fact that the reactions were self-perceived and not necessarily doctor-diagnosed and that the figure includes all kinds of food hypersensitivity regardless of the mechanisms behind the adverse reactions. The existing diagnostic methods for food hypersensitivity are not sufficient and the underlying mechanisms of perceived food hypersensitivity are not always known \[[@B38]\]. Nevertheless, it is noteworthy that almost every fifth adolescent perceive herself or himself as food hypersensitive and subsequently avoid certain food items. Food items constituted a considerable part of the offending substances reported in this study and up to 41% of all adolescents with hypersensitivity specified at least one food item as an offending substance. Professional counselling and diagnostic procedures may to some extent be able to help the adolescents to reduce their food avoidance. Yet, this kind of perceived allergy-like condition -- regardless of what the underlying mechanisms were -- was evidently associated with HRQL-deterioration and the adolescents\' experiences deserve sincere attention. Previous research show that food allergy in a child implies disruption in daily life and HRQL-deterioration for both the child and the family \[[@B19]-[@B21]\]. A pattern emerged in this group of food hypersensitive adolescents, showing a great deal of hypersensitivity to pollen, rhino-conjunctivitis and OAS-like symptoms, which is in accordance with the well known cross-reactivity of pollen and food allergens \[[@B39]\]. Significantly more males than females reported additional hypersensitivity to furred animals. To our knowledge this has not been reported before but may be associated with the higher atopy prevalence among males \[[@B22]\]. The results also pointed to a possible gender difference in what offending food items females and males, respectively, experience. Further research is needed to elucidate such a phenomenon. More than half of the adolescents with food hypersensitivity reported positive allergy tests, but as a consequence of how the questions in the survey were asked, it is not known if they were verified as allergic specifically to food. However, a vast majority of the food hypersensitive adolescents did undoubtedly suffer from complex allergy or allergy-like conditions, which included multiple types of hypersensitivity and/or allergic diseases. This should require competence in health-care when trying to tackle the adolescents\' multifaceted problems, including food hypersensitivity. Limitations ----------- The extensive number of adolescents who participated in the present survey, together with a very high answer rate, makes the results reliable. However, the sample of adolescents used in this study may limit the generalizability as the socio-demographic distribution in this particular municipality was slightly above in socio-economics and slightly below in number of immigrants compared with the country as a whole. Additional studies are warranted to confirm the results. The results of the present study emanate exclusively from adolescents\' statements. There is always a risk that the respondents involved do not correctly remember things that were asked for in a questionnaire. However, our main interest was in adolescents\' experiences of allergy-like conditions during the past twelve months and daily functioning during the past four weeks. Remembering correctly over a longer period of time was only of importance in questions about allergy testing. It seems likely that the adolescents would remember such events as skin prick tests or blood tests during their school age, although tests carried out in early childhood might have been unknown or forgotten. The magnitude of allergic diseases was measured by means of ISAAC-questions. The ISAAC questionnaire, which asks for events during the past 12 months, has been used in many countries all over the world and for many years. Its results constitute basis for international comparisons and it can be considered well validated. However, hypersensitivity items are not included in the ISAAC questionnaire. The questions in the present study about hypersensitivity were developed specifically for the present study and a pilot test was performed. Only lexical adjustments were needed. It could be discussed if the fact that the questionnaires asked for events during two distinct periods: 12 months (allergy-like conditions/ISAAC-questions) and 4 weeks (daily functioning/SF-36) might have biased the present results. It is also possible that an allergy-specific HRQL measure instrument would give another picture of the physical scale in relation to allergy-like conditions. Conclusions =========== The prevalence of self-reported allergy-like conditions among adolescents was high -- 64%. Significantly more females than males reported allergy-like conditions and females with allergy-like conditions showed more severe HRQL-deterioration compared with males with such conditions. The results indicate a need to consider not merely physical consequences but also the psychosocial quality of life impact of allergy-like conditions among both females and males. Further research is needed to elucidate the reasons behind the gender differences in this area. Most adolescents suffered from complex allergy-like conditions that included multiple types of hypersensitivity and/or allergic diseases. Food items constituted a considerable part of the offending substances reported. When attending to a young individual who suffers from an allergy-like condition, the whole syndrome should be in focus -- not only one specific offending substance, or one specific hypersensitivity or allergic disease. A team approach accompanied by an understanding of how allergy-like conditions impair the quality of life may improve the management of the adolescent\'s health problems, both in health-care services and in schools. Authors\' contributions ======================= BM and GN conceived of the study. All authors made substantial contributions to conception, planning and design. BM carried out the acquisition, analysis and interpretation of data. BM drafted the manuscript. GN and SA have been involved in revising it critically for important intellectual content. All authors read and approved the final manuscript. Acknowledgements ================ We thank Mette Kjörstad at the Tyresö municipality, the teachers and pupils in Tyresö schools, and Centre for Allergy Research, Karolinska Institutet, for making this study possible. The authors are grateful to Ms Ellinor Larsen, University of Karlstad, for revising the English language.	PubMed Central	2024-06-05T03:55:49.946882	2004-11-19	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534793/", "journal": "Health Qual Life Outcomes. 2004 Nov 19; 2:65", "authors": [ { "first": "Birgitta", "last": "Marklund" }, { "first": "Staffan", "last": "Ahlstedt" }, { "first": "Gun", "last": "Nordström" } ] }
PMC534794	Background ========== Clove is the dried flower bud of Syzygium aromaticum(L.) Merr. & Perry. (Family: Myrtaceae) an evergreen tree 10--20 m in height indigenous to India, Indonesia, Zanzibar, Mauritius and Sri Lanka \[[@B1]\]. It is one of the most important drugs used in indigenous medicine in India, especially in Unani medicine. Clove is reported as aphrodisiac \[[@B2]\], stomachic \[[@B3],[@B4]\], carminative, antispasmodic \[[@B5],[@B6]\]. It is reported to be useful in conceiving in high doses and act as a contraceptive in low doses \[[@B7]\] and useful in cataract \[[@B8]\]. Clove is also reported to have anticarcinogenic property \[[@B9]\]. It inhibits platelet aggregation and alters arachidonic acid metabolism in human platelets \[[@B10]\]. It posseses antiviral activity against Herpes simplex\[[@B11]\]. Phytochemical studies indicate that the clove contains free eugenol, eugenol acetate, caryophyllene, sesquetrepene ester \[[@B12]\], phenyl propanoid \[[@B13]\], β caryophyllene \[[@B14]\], eugenol and acetyle eugenol \[[@B15]\]. Eugenol, the major constituent, inhibits lipid peroxidation and maintains activities of enzyme superoxide dismutase, catalase, glutathione peroxidase-6 phosphate dehydrogenase \[[@B16]\], and has also been reported to have vasodilatory \[[@B17]\] and smooth muscle relaxant property \[[@B18]\]. Phyto chemical study of the test drug that was carried out according to the methods described by Jenkin et al \[[@B19]\], showed that it contains alkaloids, amino acids, flavonoids, proteins, sterols, reducing sugar, tannins and phenols. However, clove, or its known compound had not been scientifically studied for their effect on sexual function. Earlier we carried out a preliminary study of hydro alcoholic extract (50%) of clove using only mounting frequency and mating performance as the marker for sexual function in normal male mice, and the results from our study demonstrated the aphrodisiac activity and safety from short term toxicity of the test drug \[[@B20]\]. The present study thus, is aimed to investigate the aphrodisiac effect of 50% ethanolic extract of clove in detail, using multiple parameters along with its probable gastric ulceration and adverse effects in sexually normal male albino rats. The doses used in the study were selected according to the Freirich \[[@B21]\], multiplying the Unani clinical doses reported in standard Unani text \[[@B2]\] by the conversion factor of 7. Methods ======= Plant material and extraction ----------------------------- The authenticated dried flower bud of S. aromaticum(clove) was procured from the market (Delhi, India). A voucher (S721) sample was kept for further reference. The clove was crushed to coarse powder and sieved through No. 20 mesh size. The extraction was carried out by mixing the powdered clove with 1:3 w/v in 50% ethanol v/v by Soxhlet apparatus for 6 h. The extract was filtered and the solvent from the filtrate was removed by rotary evaporator under reduced pressure and low temperature. The yield of extract was 10.40% w/w in terms of dried starting material. It was yellowish and of pleasant smell. The extract was preserved in a refrigerator. Chemicals used -------------- Sildenafil citrate was purchased from Zydus Cadila, (Ahmadabad, India). Other drugs used were ethinyl oestradiol (Infar Limited, Calcutta, India), progesterone (Sun Pharmaceutical Industries Limited, Mumbai, India) and 5% xylocane ointment (Astra IDL Limited, Bangalore, India) Animals ------- Twelve weeks old male and female albino rats of wistar strain weighing 350--400 g and 225--275 g respectively, were used for the study. They were housed singly in separate standard cages and maintained under standard laboratory conditions (temperature 24--28°C, relative humidity 60--70%, 12 h light-dark cycle) with free access to solid pellet diet (Gold Mohar, Lipton-India) and water ad libitumthroughout the study except during the experiment. The study design was approved by the ethical committee of the Department for animal care and use. Drug preparation ---------------- Since clove in Unani medicine is orally administered, therefore, the extract of clove was suspended in distilled water using Tween 80 (1%) for oral administration. Sildenafil citrate and ethinyl oestradiol were also suspended in distilled water using Tween 80 (1%) separately, for oral use. Progesterone was dissolved in olive oil for subcutaneous injection. All the drug solutions were prepared just before administration. Mating behaviour test --------------------- The test was carried out by the methods of Dewsbury and Davis Jr \[[@B22]\] and Szechtman et al \[[@B23]\], modified by Amin et al \[[@B24]\]. Healthy and sexually experienced male albino rats (350--400 g) that were showing brisk sexual activity were selected for the study. They were divided into 5 groups of 6 animals each and kept singly in separate cages during the experiment. Group 1 represented the control group, which received 10 ml/kg of distilled water orally. Groups 2--4 received suspension of the extract of clove orally at the doses of 100, 250 and 500 mg/kg, respectively, daily for 7 days at 18:00 h. Group 5 served as standard and given suspension of sildenafil citrate orally at the dose of 5 mg/kg, 1 h prior to the commencement of the experiment. Since the male animals should not be tested in unfamiliar circumstances the animals were brought to the laboratory and exposed to dim light (in 1 w fluorescent tube in a laboratory of 14\' × 14\') at the stipulated time of testing daily for 6 days before the experiment. The female animals were artificially brought into oestrus (heat) \[[@B25]\] by the Szechtman et al method (as the female rats allow mating only during the estrus phase) They were administered suspension of ethinyl oestradiol orally at the dose of 100 μg/animal 48 h prior to the pairing plus progesterone injected subcutaneously, at the dose of 1 mg/animal 6 h before the experiment. The receptivity of the female animals was confirmed before the test by exposing them to male animals, other than the control, test and standard animals. The most receptive females were selected for the study. The experiment was carried out on the 7^th^day after commencement of the treatment of the male animals. The experiment was conducted at 20:00 h in the same laboratory and under the light of same intensity. The receptive female animals were introduced into the cages of male animals with 1 female to 1 male. The observation for mating behaviour was immediately commenced and continued for first 2 mating series. The test was terminated if the male failed to evince sexual interest. If the female did not show receptivity she was replaced by another artificially warmed female. The occurrence of events and phases of mating were called out to be recorded on audio-cassette as soon as they appeared. Their disappearance was also called out and recorded. Later, the frequencies and phases were determined from cassette transcriptions: number of mounts before ejaculation or Mounting Frequency (MF), number of intromission before ejaculation or Intromission Frequency (IF), time from the introduction of female into the cage of the male upto the first mount or Mounting Latency (ML), time from the introduction of the female up to the first intromission by the male or Intromission Latency (IL), time from the first intromission of a series upto the ejaculation or Ejaculatory Latency (EL), and time from the first ejaculation upto the next intromission by the male or Post Ejaculatory Interval (PEI). In the second mating series only the EL was recorded. The values for the observed parameters of the control, test and standard animals were statistically analysed by using one-way analysis of variance (ANOVA) method. Test for libido --------------- The test was carried out by the method of Davidson \[[@B26]\], modified by Amin et al \[[@B24]\]. Sexually experienced male albino rats were divided into 5 groups of 6 animals each and kept singly in separate cages during the experiment. Group 1 represented the control group, which received 10 ml/kg of distilled water orally. Groups 2--4 received suspension of the extract orally at the doses of 100, 250 and 500 mg/kg, respectively, once a day in the evening (18:00 h) for 7 days. Group 5 served as standard and given suspension of sildenafil citrate orally at the dose of 5 mg/kg, 1 h prior to the commencement of the experiment. The female rats were made receptive by hormonal treatment and all the animals were accustomed to the testing condition as previously mentioned in mating behaviour test. The animals were observed for the Mounting Frequency (MF) on the evening of 7^th^day at 20:00 h. The penis was exposed by retracting the sheath and 5% xylocaine ointment was applied 30, 15 and 5 min before starting observations. Each animal was placed individually in a cage and the receptive female rat was placed in the same cage. The number of mountings was noted. The animals were also observed for intromission and ejaculation. The MF in control, test and standard animals was statistically analysed by employing one-way analysis of variance (ANOVA) method. Test for potency ---------------- The effect of the test drug was studied according to the methods described by Hart and Haugen \[[@B27]\] and Hart \[[@B28]\], modified by Amin et al \[[@B24]\]. The male animals were divided into 5 groups of 6 animals each and kept singly in separate cages during the experiment. Group 1 represented the control group, which received 10 ml/kg of distilled water orally. Groups 2--4 received suspension of the test drug orally at the doses of 100, 250 and 500 mg/kg, respectively, daily for 7 days. Group 5 received a suspension of sildenafil citrate orally at the dose of 5 mg/kg, 1 h before the commencement of the experiment. On the 8^th^day, the test for penile reflexes was carried out by placing the animal on its back in a glass cylinder partial restraint. The preputial sheath was pushed behind the glans by means of thumb and index finger and held in this manner for a period of 15 min. Such stimulation elicits a cluster of genital reflexes. The following components were recorded: Erections (E), Quick Flips (QF), and Long Flips (LF). The frequency of these parameters observed in control, test and standard groups was statistically analysed by using one-way analysis of variance (ANOVA) method. Test for ulcerogenecity ----------------------- The male animals (350--400 g) were divided into 4 groups of 6 animals each. Group 1 represented the control group, which received 10 ml/kg of distilled water. Groups 2--3 received suspension of the extract orally at the doses of 100, 250 and 500 mg/kg, respectively, daily for 7 days. After the treatment, on 8^th^day all the animals were killed and the stomach was then incised along the greater curvature and washed carefully with physiological saline. Any gastric lesions were observed immediately using a magnifying glass. The number of erosions per stomach was assessed for severity, according to the score of Cioli et al \[[@B29]\] : (o) absence of lesion, vasodilation or up to 3 pin point ulcers; (1) more than 3 pin point ulcers, (2) from 1 to 5 small ulcers (\< 2 mm); (3) more than 5 small ulcers (\< 2 mm), (4) 1 or more giant ulcers. Evaluation of gastric damage was carried out by two observers, who followed the same evaluation criteria. Adverse effects --------------- All treated rats were observed at least once daily for any overt sign of toxicity (salivation, rhinorrhoea, lachrymation, ptosis, writhing, convulsions and tremors), stress (erection of fur and exophalmia) and changes in behaviour (such as spontaneous movement in the cage, climbing, cleaning of face). In addition food and water intake were noted. Statistical analysis -------------------- The significance of difference between the means was determined by one-way analysis of variance (ANOVA) with post-hoc\'t\' test. P value \<0.05 was considered as significant. Results ======= The data obtained with the mating behaviour test indicated that the clove-extract at the dose of 500 mg/kg increased the Mounting Frequency (MF) (P \< 0.0001), Intromission Frequency (IF) (P \< 0.001) ; Ejaculatory latency in first series (EL~1~) (P \< 0.0001) and decreased Mounting Latency (ML) (P \< 0.0001), Intromission Latency (IL) (P \< 0.01) in a significant manner. The dose 250 mg / kg of the extract significantly increased the MF (P \< 0.001), IF (P \< 0.05) and significantly reduced the ML (P \< 0.001), EL~1~(P \< 0.05); Post Ejaculatory Interval (PEI) (P \< 0.001) and did not significantly alter the IL (P \< 0.61), Ejaculatory Latency in second series (EL~2~) (P \< 0.41). Whereas the dose of the test drug at 100 mg/kg significantly increased the MF (P \< 0.05), PEI (P \< 0.05) but did not significantly affect the IF (P \< 0.61), ML (P \< 0.61), IL (P \< 0.61); EL~1~(P \< 0.61); EL~2~(P \< 0.81). However, the standard drug increased the MF (P \< 0.0001), IF (P \< 0.0001); EL~1~(P \< 0.0001), EL~2~(P \< 0.0001); PEI (P \< 0.0001) as well as decreased ML ((P \< 0.0001) and IL (P \< 0.0001) in a highly significant manner (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of 50% ethanolic extract of clove (S. aromaticum) on mating behaviour in male rats ::: Mean Frequency ± SEM ------------------------------------------------------ ---------------------- ------------------- ------------------------ ------------------------ ------------------------------ Parameters Control (10 ml/kg) Clove (100 mg/kg) Clove (250 mg/kg) Clove (500 mg/kg) Sildenafil citrate (5 mg/kg) Mounting Frequency (MF) 11.50 ± 1.22 13.20 ± 1.47\* 19.00 ± 1.10\\\* 31.80 ± 2.32\\\\ 48.70 ± 2.34\\\\ Intromission Frequency (IF) 5.50 ± 1.22 5.83 ± 1.17 NS 6.67 ± 0.81\* 8.17 ± 1.47\\\* 24.70 ± 0.81\\\\ Mounting Latency (ML, in sec) 35.30 ± 1.51 36.70 ± 1.21 NS 31.30 ± 1.63\\\* 24.30 ± 1.63\\\\ 11.70 ± 1.37\\\\ Intromission Latency (IL, in sec) 40.00 ± 5.29 41.30 ± 3.89 NS 38.50 ± 3.89 NS 21.00 ± 3.83\\ 15.00 ± 0.89\\\\ Ejaculatory Latency in first series (EL~1,~in sec) 198.00 ± 0.98 201.00 ± 13.00 NS 208.00 ± 13.70\* 233.50 ± 12.00\\\\ 344.50 ± 12.00\\\\ Ejaculatory Latency in second series (EL~2~, in sec) 297.33 ± 8.10 295.50 ± 11.70 NS 311.33 ± 9.67 NS 343.16 ± 7.70\\\\ 398.16 ± 13.50\\\\ Post Ejaculatory Interval (PEI, in sec) 364.00 ± 12.22 343.16 ± 7.70\* 309.33 ± 10.90\\\\ 217.33 ± 8.96\\\\ 99.00 ± 5.68\\\\ Tabular values are mean ± SEM, n = 6 (number of animals in each group); significant difference from control, NS: Not significant. \* P \< 0.05, \\ P \< 0.01; \\\* P \< 0.001, \\\\ P \< 0.0001. ::: The test for libido showed that the extract at the dose of 500 mg/kg increased the Mounting Frequency (MF) in a significant manner (P \< 0.001). The extract at the doses of 100 mg/kg and 250 mg/kg did not significantly alter the MF(P \< 0.24, P \< 0.75 respectively). The standard drug strikingly increased the MF (P \< 0.0001). Intromission and Ejaculation were found absent in control, test and standard groups (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Effect of 50% ethanolic extract of clove (S. aromaticum) on Mounting Frequency (test for libido) in male rats ::: Mean Frequency ± SEM ----------------------------- ---------------------- --------------- ---------------- ---------------- ------------------ Mounting Frequency (MF) 6.17 ± 0.98 6.33 ± 0.81NS 7.17 ± 1.72 NS 11.20 ± 1.17\* 23.00 ± 2.17\\ Intromission Frequency (IF) Nil Nil Nil Nil Nil Ejaculation (EJ) Absent Absent Absent Absent Absent Tabular values are mean ± SEM, n = 6 n (number of animals in each group); significant difference from control, NS: Not significant. \P \< 0.001,\\* P \< 0.0001 ::: The test for potency exhibited that the higher dose (500 mg/kg) of the test drug significantly increased the frequency of Erections (E) (P \< 0.0001), Quick Flips (QF) (P \< 0.0001), Long Flips (LF) (P \< 0.0001) as well as the aggregate of these penile reflexes (TPR) (P \< 0.0001). The extract at the dose of 250 mg/kg significantly increased the E (P \< 0.01), LF (P \< 0.05) and TPR (P \< 0.05) but comparatively less than the higher dose of the extract and standard drug, and did not significantly increase the QF (P \< 0.10). whereas, the test drug at the dose of 100 mg/kg did not alter the E (P \< 0.78), QF (P \< 0.78); LF (P \< 0.44) and TPR (P \< 0.44) in a significant manner (Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Effect of 50% ethanolic extract of clove (S. aromaticum) on Penile reflexes (test for potency) ::: Mean Frequency ± SEM ----------------------------- ---------------------- ----------------- ------------------ -------------------- -------------------- Erections (E) 7.67 ± 1.63 8.33 ± 1.21 NS 10.50 ± 1.76\\ 13.80 ± 0.98\\\* 19.00 ± 2.64\\\* Quick Flips (QF) 5.17 ± 0.75 5.33 ± 1.21 NS 6.00 ± 1.22 NS 9.67 ± 1.37\\\* 17.30 ± 4.13\\\* Long Flips (LF) 2.17 ± 1.17 3.17 ± 1.50 NS 4.00 ± 1.55\* 7.50 ± 1.22\\\* 12.00 ± 2.26\\\* Total Penile Reflexes (TPR) 15.01 ± 3.55 16.83 ± 7.21 NS 20.50 ± 4.53\* 30.97 ± 3.50\\\* 48.30 ± 9.03\\\* Tabular values are mean ± SEM, n = 6 (number of animals in each group); significant difference from control, NS: Not significant. \* P \< 0.05, \\ P \< 0.01; \\\* P \< 0.0001. ::: Seven days treatment of low and high doses of the extract caused no significant ulceration in gastric mucosa of albino rats. Moreover, there were neither treatment related defects nor overt clinical signs of toxicity, stress or changes in behaviour and appearance evident. The food and water intake of all test drug treated rats remained similar to those of the control group. Discussion ========== In the present study, clove (S. aromaticum) was tested in animal experimentation for its effect on sexual behaviour, and sildenafil citrate was used as the standard referent. The study showed that the 50% ethanolic extract of clove possesses significant sexual function enhancing activity as observed in sexual behaviour tests. Mating behaviour test revealed that the test drug significantly increased the Mounting Frequency (MF) and Intromission Frequency (IF) as compared to control but less than that of the standard drug. The (MF) and (IF) are considered as the indices of both libido and potency. So, this is an indication that the test drug possesses a sexual function improving effect. The premature ejaculation is one of the important causes of sexual dysfunction, so the assessment of Ejaculatory Latency in first series (EL~1~) and in second series (EL~2~) was studied. The test drug significantly increased the EL~1~and EL~2~as compared to control animals, whereas a highly significant increase was observed with the standard drug. The test drug was found to produce a significant reduction in the Mounting Latency (ML) and Intromission Latency (IL) as compared to control while a highly significant decrease was found in ML of animals treated with sildenafil citrate. This is also an evidence of the sexual function improving effect of the clove. The Post Ejaculatory Interval (PEI) is considered as an index of potency and libido, and also a parameter of the rate of recovery from exhaustion after first series of mating. PEI was found significantly decreased with clove-extract and also with the standard drug. The test drug decreased PEI either by enhancing the potency and libido or by producing lesser exhaustion in the first series of mating or by both. The effect of the test drug on libido was also evaluated by testing the MF after genital anaesthetization which does away with the reinforcing effect of genital sensation thus affording the study of pure libido or intrinsic sexual desire. During the experiment the extract produced a significant increase in the MF of sexually normal male rats whereas the efficacy of the standard drug, as expected, was found to be highly significant. The MF was much reduced in control, test and standard animals in comparison with the MF of corresponding groups in mating behaviour test where the penis had not been anaesthetized. None of the control, test and standard animals were observed to show Intromission or Ejaculation because their occurrence depends upon local genital sensation which was obstructed due to anaesthetization. The effect on potency was also evaluated by testing the effect of the drug on the frequency of penile reflexes namely Erections (E), Quick Flips (QF), and Long Flips (LF). The test drug significantly increased the frequency of all the components of penile reflexes (E, QF, & LF) in the test animals as compared to control group but comparatively lesser than the standard drug. The aggregate of penile reflexes (TPR) was also found increased in the animals treated with the extract and sildenafil citrate. Therefore, the experiment revealed that the test drug produced a marked increase in potency. The spices are reported to produce an increase in gastric acid secretion by a cholinergic mechanism \[[@B30]\], and so, their use for sexual invigoration may cause gastric ulceration and other adverse effects. Therefore, ulcerogenic and other adverse effects of the extract were also evaluated. The results of this evaluation were negative. This suggests that the short term use of clove for this purpose is apparently safe. The results of the present study clearly proved that the clove is endowed with sexual function improving activity. This is in consonance with our earlier study showing sexual function improving effect of the test drug in male mice. However, the established drug i.e. sildenafil citrate exhibited, as expected, tremendous activity. With regard to the mechanism of the test drug, it is difficult to interprete the mechanism involved in potentiation of sexual function. The drugs induced changes in neurotransmitter levels or their action at cellular levels could change sexual behaviour \[[@B31]\]. Hence, the increased sexual function could be due to the nervous stimulant action of the test drug \[[@B32]\]. Further, phyto chemical study of the extract indicated that it contains sterols and phenol. Thus, the resultant aphrodisiac effectivity of the test drug might also be attributed to sterols or phenolic compounds. Moreover, research should be aimed at isolating the active principle(s) responsible for aphrodisiac activity and the mechanism by which the drug enhances sexual function. In addition, to discover the applied effective concentration or dosages of the extract, more studies are also required. Conclusions =========== The present results indicated that the 50% ethanolic extract of clove possesses potent aphrodisiac activity in normal male albino rats without any gastric ulceration and adverse effects and provided scientific evidence in favour of the claims made in Unani medicine that the clove is clinically useful as sexual invigorator in males. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= T-Supervised the design and coordinatrion of the study. SA-Practically conducted the design of the study. AL-Participated and performed the statistical analysis IA-Participated in the drafting 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/1472-6882/4/17/prepub> Acknowledgments =============== The authors are thankful to the Department of Ilmul Advia of Aligarh Muslim University, Aligarh, India for providing all the facilities to carry out the study and Dr. K.M.Y. Amin (In-charge, Pharmacology Section) and Prof. S. H. Afaq (In-charge, Pharmacognosy Section) for their valuable suggestions.	PubMed Central	2024-06-05T03:55:49.951881	2004-11-5	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534794/", "journal": "BMC Complement Altern Med. 2004 Nov 5; 4:17", "authors": [ { "first": "Shamshad", "last": "Ahmad" }, { "first": "Abdul", "last": "Latif" }, { "first": "Iqbal Ahmad", "last": "Qasmi" } ] }
PMC534795	Introduction ============ Bedside echocardiography can bring important anatomical and hemodynamic information for the management of critically ill patients, and is often required in hospitalized patients for the assessment of left ventricular function. Standard echocardiographic equipments, while optimal, have large size and sometimes are difficult to maneuver in the emergency room or intensive care units. Recently, hand-carried ultrasound (HCU) devices have been demonstrated to broaden the versatility in ultrasound application. Due to their portability and low cost, HCU acts like a stethoscope, providing information beyond physical examination at the point-of-care \[[@B1],[@B2]\]. Although it has been shown to improve the detection of cardiovascular abnormalities over the physical examination, its role in the assessment of hospitalized patients is not completely established \[[@B3],[@B4]\]. This study was undertaken to compare the findings of the bedside evaluation using HCU to the comprehensive echocardiography (CE), in cardiology inpatient setting. Methods ======= Patients -------- We studied 44 consecutive hospitalized patients with cardiovascular disorders. Patients were included in the study when their referring physicians asked for a bedside evaluation with conventional echocardiography. In all patients, we performed the echocardiography with both HCU and CE within a maximal interval of 24 hours. The clinical characteristics of patient population are shown in Table [1](#T1){ref-type="table"}. Among these patients, 61% were in the cardiac ward, 27% in the emergency room, and 12% in the intensive care unit. This study was approved by our Institutional Ethical Committee and informed consent was obtained from all participants or their legal representatives. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Clinical characteristics ::: Variables ----------------------------------------- ---------- Age (years) 54 ± 18 Male gender 25 (57%) Cardiomyopathy 16 (36%) Acute coronary syndrome 10 (23%) Postoperative of cardiac surgery 9 (21%) Valvulopathy 5 (11%) Cardiogenic shock 3 (7%) Total atrioventricular conduction block 1 (2%) Data are mean ± SD or number (%) of patients. ::: Study Protocol -------------- All patients underwent two echocardiographic evaluations. First, HCU was performed with the portable device OptiGo (Philips Medical Systems, Andover, Massachusetts, USA) and, consecutively, by a commercially available system (HDI 5000, Philips Medical Systems, Bothell, Washington, USA) equipped with a 4-2 MHz transducer and second-harmonic imaging. HCU was performed by one same cardiologist with level 2-training in the performance and interpretation of echocardiography according to the specifications of the American Society of Echocardiography \[[@B5]\], after a period of instructions about the HCU settings. CE was performed by one experienced echocardiographer with level-3 training \[[@B5]\], and was considered the gold standard. Each investigator was blinded to the results of the other examination. The final echocardiographic report was based on the results of CE. Imaging Analysis ---------------- OptiGo is equipped with a 2.5 MHz phased-array transducer and operates on a rechargeable Lithium ion battery, which facilitates its use at bedside. HCU was performed using two-dimensional imaging, color Doppler flow mapping, and simple caliper measurements. Images were frozen and scrolled for review and the measurements were performed on-line. CE evaluation included two-dimensional with second-harmonic imaging, M-mode, and both spectral and color Doppler flow mapping. Images were recorded on videotape or digitalized. The aorta, left atrium, left ventricular end-diastolic and end-systolic diameters, as well as interventricular septal and posterior wall thickness were measured according to the recommendations of the American Society of Echocardiography \[[@B6]\]. The left ventricular ejection fraction (LVEF) was visually estimated, and a normal ventricular function was defined as LVEF ≥ 55%. According to the segmental wall motion analysis, patients were divided as those with segmental wall motion abnormality (WMA) and those without WMA. Pericardial effusions were classified as mild, moderate, or large effusion. Valve structure and function were analyzed. Dysfunctions were classified into mild, moderate, or severe degree according to the qualitative evaluation by HCU, and using both qualitative and quantitative parameters by CE \[[@B7]\]. A significant valvular regurgitation was defined in our study as those of moderate or severe degree \[[@B8]\]. On the other hand, non-significant valvular regurgitation was defined as those of no, trace, or mild degree. The estimation of transvalvar gradients and valvular areas in patients with prosthetic and stenotic valves, as well as the estimation of pulmonary artery pressure, were performed only by CE \[[@B7]\]. The intraobserver variability of CE findings was assessed in 15 randomly assigned patients, with analysis made at least 4 weeks apart. The interobserver agreement between the experienced echocardiographer and the level 2-trained cardiologist was assessed by the analysis of 15 CE recorded examinations. Statistical analysis -------------------- Continuous data are expressed as mean ± one standard deviation (SD) and categorical data as proportions. Comparisons between groups for continuous variables were made using Student ttest. Chi-square and Fisher Exact tests were used for categorical variables. Agreement between HCU and CE results were assessed by the Kappa statistics. Interobserver and interobserver variability was determined by intra-class correlation and linear regression. A two-tailed p value \<0.05 was considered significant. Results ======= Imaging analysis was feasible with HCU and CE in all patients. The percentage of patients with good, regular and bad image quality using CE were 80%, 18% and 2%, respectively. HCU had a lower percentage of patients with good image quality (59%), and a higher percentage of patients with regular (27%) and bad (14%) image quality (p \< 0.05 versus CE), as shown in Figure [1](#F1){ref-type="fig"}. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Image quality by hand-carried ultrasound and comprehensive echocardiography. Image quality obtained by hand-carried ultrasound device (solid bars) and by comprehensive echocardiography with second-harmonic imaging (open bars). Comprehensive echocardiography had a higher percentage of patients with good quality than hand-carried ultrasound. \* p \< 0.05 between groups. ::: ![](1476-7120-2-24-1) ::: Determination of cardiac chamber dimensions and left ventricular function ------------------------------------------------------------------------- There were no significant differences in cardiac chamber dimensions obtained by HCU and CE, except for the posterior wall thickness, which was lower when assessed by HCU (Table [2](#T2){ref-type="table"}). Among the 44 studied patients, 24 (55%) had some degree of left ventricular dysfunction and 20 (45%) had normal left ventricular function. There was no difference between the LVEF estimated by CE and by HCU (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Cardiac chamber measurements obtained by comprehensive echocardiography (CE) and by hand-carried ultrasound (HCU) ::: Variables CE HCU ------------------ ------------- ------------- Aorta (mm) 28.7 ± 4.1 27.8 ± 4.2 Left atrium (mm) 45.5 ± 7.7 44.0 ± 7.5 LVED (mm) 57.1 ± 11.2 54.9 ± 10.7 LVES (mm) 44.4 ± 15.5 42.8 ± 10.7 IVST (mm) 10.0 ± 2.4 9.4 ± 2.4 PWT (mm) 9.6 ± 1.8 8.7 ± 1.7\* LVEF (%) 47 ± 16 44 ± 15 Data are mean ± SD. IVST = interventricular septal thickness; LVED = left ventricular end-diastolic diameter; LFEF = left ventricular ejection fraction; LVES = left ventricular end-systolic diameter; PWT = posterior wall thickness. \* p \< 0.05 compared to CE. ::: The analysis of segmental wall motion was not possible by HCU in two patients, due to poor endocardial border delineation, and was deemed feasible in all patients using CE. In the remaining 42 patients, HCU correctly identified eight of the 11 patients with WMA by CE, and failed to identify three of them. These three false-negative results occurred in patients with bad image quality by HCU. On the other hand, among the 31 patients without WMA by CE, HCU correctly identified 27 patients, and had four false-positive results. Among these four cases, three patients had global left ventricular dysfunction and one had asynchronic movement of the interventricular septum. The agreement between HCU and CE for the detection of WMA was 83% (Kappa = 0.58; p \< 0.001). CE identified nine patients with pericardial effusion, one patient with a moderate effusion and the remaining eight patients with mild effusions. HCU correctly detected and classified pericardial effusion in seven patients, and failed to diagnose two patients with mild effusions, both localized posterior to the left ventricle. Analysis of valvular dysfunction -------------------------------- Significant mitral valve regurgitation was diagnosed by CE in 16 patients, and by HCU in 13 of them. The agreement between HCU and CE for detection of this abnormality was 93% (Kappa = 0.85; p \< 0.001). Significant aortic valve regurgitation was detected in three patients by CE and in two by HCU, and the agreement between the two techniques was 98% (Kappa = 0.89; p \< 0.001). Significant tricuspid regurgitation was detected by CE in 16 patients and by HCU in 12 of them. However, there was one additional case misdiagnosed by HCU as of significant degree. The agreement between HCU and CE for the detection of significant tricuspid regurgitation was 88% (Kappa = 0.74; p \< 0.001). Four patients (9%) had aortic valve stenosis, two cases of moderate, one of mild, and the other one of severe degree. Qualitative analysis of valve structure by HCU was capable to identify aortic stenosis in two of these patients, but the severity of these lesions was not determined. There were six (14%) patients with prosthetic valves and eight (18%) patients with pulmonary hypertension, in which estimation of transvalvar gradients or valvular areas were performed only by CE. A complete evaluation of these patients was not possible using HCU due to its technical limitations in determining hemodynamic parameters. Intra and interobserver variability ----------------------------------- The intraobserver agreement for detection of pericardial effusion, WMA, and significant valvular dysfunction was 100%, and the interobserver agreement was 91%. There was an excellent correlation between LVEF estimated in the first and second evaluation by the same observer (r = 0.91; p \< 0.05). The correlation between LVEF estimated by the experienced echocardiographer and the cardiologist with level 2-training in echocardiography was r = 0.88 (p \< 0.05). Discussion ========== The present study describes the value of bedside evaluation of cardiac patients using HCU in comparison to CE. Our results demonstrate that HCU may be used for the assessment of cardiac chamber dimensions, estimation of left ventricular function, and detection of WMA. Moreover, we found a good agreement between HCU and CE for detecting significant valvular regurgitation. However, HCU presents important limitations regarding the assessment of prosthetic and stenotic valves, as well as for the evaluation of patients with pulmonary hypertension, due to the lack of spectral Doppler. Bedside echocardiography is a frequently used diagnostic tool in the cardiology inpatient setting and can affect the patient management, direct further diagnostic work-up, and modify therapeutic decisions. The recent development of portable ultrasound devices has the potential to allow quick and easy-to-use echocardiography at the point-of-care, although its value in hospitalized patients was not completely defined. In patients with cardiovascular disorders, HCU has been shown to increase the diagnostic accuracy over physical examination when performed by cardiologists with level-2 training in echocardiography \[[@B1]\]. In the present study we confirmed the usefulness of these portable devices for estimating cardiac chamber dimensions and left ventricular function, which are frequent indications for bedside echocardiographic examination. We also demonstrated a good agreement between CE and HCU for detecting WMA. We would like to emphasize that CE with second-harmonic imaging was chosen as the gold standard for evaluation of segmental wall motion, since it has already been proven that this imaging modality ameliorates the endocardial border delineation \[[@B9],[@B10]\]. Another point to be noted is that our population included a high proportion of patients with left ventricular global dysfunction, in whom detection of segmental abnormality can be somehow challenging by non-experienced observers. In at least three of the four false-positive results, the presence of global myocardial impairment due to cardiomyopathy could lead to a confounding diagnosis of segmental impairment. In the false-negative cases, the use of second-harmonic modality improved the quality of imaging, allowing for a better visualization of endocardial thickening and detection of segmental left ventricular dysfunction. Our results are in accordance with recently published data demonstrating that HCU was highly concordant with clinical diagnosis of acute coronary syndrome based on the analysis of wall motion by these portable devices \[[@B11]\]. However, when considering the full spectrum of abnormalities in hospitalized patients, previous reports demonstrated that hand-carried bedside echocardiography failed to quantify valvular regurgitation and also missed findings relevant to clinical questions in a significant number of patients. These studies concluded that HCU falls far short of standard echocardiography in evaluation of critically ill patients \[[@B3],[@B12]\]. We do believe that the lack of spectral Doppler in HCU is an important limitation for evaluation of cardiac patients, since this technique provides unique hemodynamic information, especially in patients with prosthetic or stenotic valves, and pulmonary hypertension. In our study population, these clinical conditions occurred in a considerable number of cases, since we included patients in the pre and postoperative period of valvular surgery. Limitations ----------- In the present study, we did not evaluate the effect of HCU on patient management. Agreement between CE and HCU for detection of WMA was analyzed in 42 patients, since two patients were initially excluded because of inadequate acoustic window. The specific issue of agreement between HCU and CE according to image quality was not addressed in the present study because of the limited number of patients in each group. Conclusions and clinical implications ===================================== We concluded that HCU is useful for bedside assessment of left ventricular global and segmental left ventricular function as well as for evaluation of significant valvular regurgitation. However, it has limitations regarding hemodynamic assessment, which is an important issue in the cardiology inpatient setting. Therefore, we emphasize that bedside echocardiography using HCU should be performed by cardiologist with at least level 2-training in echocardiography, and always complemented with CE when hemodynamic evaluation is necessary. Competing Interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= JMT and WMJ designed the study, did the selection and recruitment of the patients, and wrote the text. RRM and JMC performed the echocardiograms and participated in the subsequent analysis of data. JLA and JFR analyzed the statistics and revised this manuscript.	PubMed Central	2024-06-05T03:55:49.953866	2004-11-17	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534795/", "journal": "Cardiovasc Ultrasound. 2004 Nov 17; 2:24", "authors": [ { "first": "Jeane M", "last": "Tsutsui" }, { "first": "Raquel R", "last": "Maciel" }, { "first": "Joicely M", "last": "Costa" }, { "first": "Jose L", "last": "Andrade" }, { "first": "Jose F", "last": "Ramires" }, { "first": "Wilson", "last": "Mathias" } ] }
PMC534796	Background ========== Nocardiosis is a rare localized or systemic infection caused by several species of the genus Nocardia. This genus consists of strictly aerobic, Gram-positive, variably acid-fast, filamentous bacteria with a tendency to fragment into bacillary and coccoid forms \[[@B1]\]. N. asteroides, N. farcinica, N. nova(included in the N. asteroidescomplex) and N. brasiliensisare the species most often involved in human disease \[[@B1],[@B2]\]. N. brasiliensishas been recovered from the soil in many tropical and subtropical areas but rarely in temperate areas. Traumatic inoculation of N. brasiliensisinto the skin is the most typical mode of acquisition of the infection due to this organism \[[@B1],[@B2]\]. Herein, we describe a case of a man, who had an accident at work and 1 week later developed lymphocutaneous infection caused by N. brasiliensis. Case Presentation ================= A previously healthy 32-year-old man was referred to the emergency department of orthopedics with traumatic injuries of the index, middle and ring fingers of the left hand. The injury happened at work while he was operating a machine of cotton elaboration. On admission, routine laboratory investigations showed only elevated white blood cell count (12,400/mm^3^), while red blood cell count, haemoglobin, chemistry and urine analysis were within normal limits. Radiography of the bones of his left hand revealed fractures of the nail bones of the middle and the ring finger. Surgical debridement of the damaged soft tissue was undertaken with amputation of these two nail bones. The patient was hospitalized and intravenous therapy with ceforanide (1 g/12 h), ofloxacin (200 mg/12 h) and metronidazole (500 mg/8 h) was initiated. Four days after his admission the injury of the hand became tender, erythematous, swelling and began to drain. The purulent material expressed from the hand was sent for culture. Three days later the lesion worsened and was complicated with lymphangitis. The patient was noted to be febrile (38.5°C) without any other systemic symptoms. Physical examination revealed multiple erythematous subcutaneous nodules along the lymphatics extending up the patient\'s left forearm. These nodules were tender and painful. There was no regional lymphadenopathy. Debridement of the lesions was performed and the tissue was submitted for bacterial and fungal cultures. The Gram-stained smear showed polymorphonuclear leucocytes and Gram-positive fine, branching filaments, partially acid-fast, with a tendency to fragment into coccoid and bacillary forms. Laboratory tests showed: white blood cell count (18,000/mm^3^), absolute neutrophil count, 12,780/mm^3^, erythrocyte sedimentation rate, 75 mm/h; and C-reactive protein, 14.2 mg/dl (normal \< 0.8 mg/dl). After 5 days of incubation cultures of the pus and the tissue on Columbia blood agar grew white colonies adherent to the agar, rough with a velvety surface, having a characteristic mouldy odor (Figure [1](#F1){ref-type="fig"}). Colonial characteristics, physiological properties and biochemical tests performed identified the isolate as Nocardia brasiliensis(Table [1](#T1){ref-type="table"}). Susceptibility to the antibiotics by the determination of the MICs using the E-test method (AB Biodisk, Solna, Sweden), showed that the isolate was sensitive to trimethoprim-sulfamethoxazole, amoxicillin-clavulanic acid, gentamicin, tobramycin, amikacin, and minocycline, intermediate to ciprofloxacin and resistant to ampicillin, second and third generation cephalosporins, erythromycin, clindamycin, ofloxacin and pefloxacin. The patient\'s antimicrobial therapy was changed to intravenous trimethoprim-sulfamethoxazole (160/800 mg b.i.d). The patient responded to therapy. Following 2 weeks of treatment the patient improved and all laboratory tests returned to normal. He was discharged 3 weeks after his admission on oral trimethoprim-sulfamethoxazole (160/800 mg b.i.d). The antibiotic therapy was continued for a total of 3 months. His hand and arm lesions were healing well and 6 months later revealed complete resolution of the infection without signs of recurrence. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Rough chalky-white colonies of Nocardia brasiliensisgrown on Columbia blood agar ::: ![](1476-0711-3-24-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Physiological characteristics and biochemical reactions of our Nocardia brasiliensisisolate ::: Test or characteristic Reaction of our strain ----------------------------------- ---------------------------- Decomposition of: Adenine \- Casein \+ Tyrosine \+ Xanthine \- API 20C AUX assimilation results: Glucose \+ Glycerol \+ Galactose \+ N-acetyl-D-glucosamine \+ Inositol \+ Adonitol \- Trehalose \+ Equivalent growth at 35°/45°C \- Lysozyme broth \+ Production of arylsulfatase (7d) \- Gelatin liquefaction (7d) \+ Sensitivity to: Gentamicin \+ Tobramycin \+ Amikacin \+ Erythromycin \- ::: Discussion ========== In the United States, there are an estimated 500--1,000 new cases of nocardiosis each year \[[@B3]\]. On the basis of epidemiological surveys conducted in France and Italy, the annual estimated incidence of human nocardiosis is 150--250 and 90--130 cases, respectively \[[@B4],[@B5]\]. N. brasiliensisaccounts for only about 7--14% of the reported cases \[[@B3]\]. The incidence of nocardiosis in Greece is still unknown because the number of nocardial infections are not reported to the public health authorities. Only one case of N. brasiliensislymphocutaneous syndrome has been previously described in the same geographic area \[[@B6]\]. N. brasiliensisalthough rarely implicated in pulmonary and disseminated infections in immunocompromised patients, has been most commonly associated with cutaneous infections \[[@B7]\]. Nocardiaenters the skin after traumatic inoculation injuries, varying from contaminated abrasions and puncture wounds to insect and animal bites. The most common resulting skin lesions are on the upper and lower extremities \[[@B7]\]. Cutaneous manifestations include: (i) mycetoma, (ii) lymphocutaneous (sporotrichoid) infection, (iii) superficial skin infection, and (iv) disseminated infection with cutaneous involvement. The present case is consistent with the classical presentation of lymphocutaneous infection with a primary lesion at the site of injury on the hand and an ascending lymphangitis involving the forearm. The inoculation probably occurred from the cotton that had been contaminated by Nocardiaand entered the wound after the accident. The lymphocutaneous syndrome can be caused by a wide variety of microorganisms. The most common causative agents of the syndrome in addition to Sporothrix schenkiiand Nocardia brasiliensis, include Mycobacterium marinumand Leishmaniaspecies. Less common causes are Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Blastomyces dermatitidis, Pseudoallescheria boydii, other species of Mycobacterium, Streptococcus pyogenes, Staphylococcus aureusand viruses as cowpox virus and herpes simplex \[[@B8]\]. A history of a traumatic wound contaminated with soil and the relatively brief incubation period (less than 2 weeks) suggest nocardiosis. Diagnosis of nocardial infection can be established by cultural isolation of the microorganism. Identification to the species level can be successfully performed either by conventional biochemical methods or by molecular techniques \[[@B1],[@B9]\]. Trimethoprim-sulfamethoxazole combination is recognized the drug of choice for nocardiosis \[[@B10]\]. Primary lymphocutaneous nocardiosis may be curable after a course of 2 to 4 months, although several studies report clinical cures of cutaneous nocardiosis caused by N. brasiliensisafter only 2 to 3 weeks of therapy. In patients with sulfa intolerance or those who fail therapy with trimethoprim-sulfamethoxazole, alternative therapy must be based on sensitivity testing. Minocycline, tetracycline, amikacin and amoxicillin-clavulanic acid have been successfully used \[[@B11]\]. Although rare, lymphocutaneous nocardiosis must be considered, diagnosed with appropriate cultures and adequately treated, in order to prevent progression to dissemination of the primary skin disease. Acknowledgements ================ Written consent was obtained from the patient for publication of the study.	PubMed Central	2024-06-05T03:55:49.955177	2004-11-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534796/", "journal": "Ann Clin Microbiol Antimicrob. 2004 Nov 15; 3:24", "authors": [ { "first": "Sofia", "last": "Maraki" }, { "first": "Stavros", "last": "Chochlidakis" }, { "first": "Eleni", "last": "Nioti" }, { "first": "Yannis", "last": "Tselentis" } ] }
PMC534797	Background ========== The literature from English-speaking countries indicates that teaching communication skills is effective in improving learners\' communication skills with patients \[[@B1]\]. However, the evidence from non-English speaking countries is sparse \[[@B1]\]. In addition, the conceptual frameworks for communication skills teaching are based on research evidence from English-speaking countries \[[@B2]\]. There is an ongoing debate about whether the principles and methods for teaching communication skills developed in English-speaking countries could be applied to other places with different languages and cultures \[[@B2]-[@B4]\]. Teaching communication skills is gaining popularity and proliferating for Japanese health professional students \[[@B5]\]. Yoshida et al. conducted a controlled study to examine the effects of such training with 16 Japanese dental students and had a positive result \[[@B6]\]. A few reports have been published on Japanese medical students \[[@B7]-[@B9]\]. However, to our best knowledge, no controlled studies for communication skills teaching have been conducted for that population. In many traditional medical schools in Japan, communication skills teaching is limited in time and scope, and isolated from other formal curricula. Thus it is important to know whether such type of training make a difference, at least in the short run. This should also be of interest to educators elsewhere who similarly work in settings where there is not enough formal curricular time for communication skills teaching. The objective of this study was to evaluate the impact of a short, intensive small group seminar, which was based on Western educational principles, on Japanese medical students\' communication skills with patients. Methods ======= Participants ------------ Medical schools in Japan last six years with the last two years consisting of clerkships. Before the fifth-year, Japanese students typically have few direct interactions with patients. Throughout the academic year 2001--2002, a total of 105 fifth-year students from the Nagoya University School of Medicine rotated through the various clinical services of the Nagoya University Hospital. Students divided themselves into 18 groups of 5 to 7, but the sequential order of rotations is set by the medical school officials. Educational intervention ------------------------ As part of a 1-week clerkship rotation at the Department of General Medicine, students participated in a two-day, small group seminar on the medical interview and communication skills. Typically either or both of two of the authors (KM, NB) facilitated the seminar. Both facilitators had had an experience in learning and teaching the medical interview and communication skills in the United States. The seminar utilized learner-centered, skills-oriented, experiential, and interactive learning methods. To guide the teaching of communication skills, we created a conceptual model for patient-physician communication referring to 3 existing models \[[@B2],[@B10],[@B11]\]. Although our main teaching focus is on communication process skills, we also addressed the content aspects of the medical interview (e.g., discussion of differential diagnosis). The learning activities during the seminar are summarized in Figure [1](#F1){ref-type="fig"}. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Learning activities during a two-day seminar on medical interviewing and communication skills. ::: ![](1472-6920-4-24-1) ::: Outcome measures ---------------- In September 2001, half way through the academic year, a five-station objective structured clinical examination (OSCE) was conducted for all fifth-year students. The primary purpose of the OSCE was to provide trainees with the opportunity to receive feedback on their clinical skills from the faculty in a safe and structured environment. One OSCE station focused on the medical interview. Students engaged in a 5-minute interaction with a standardized patient presenting with cough. A total of 10 fourth-year students were trained to serve as standardized patients in a series of 3 small group sessions, each lasting 60 minutes \[[@B12]\]. During the interview, the fifth-year students were observed by faculty and evaluated for both station-specific and general communication skills on the pre-defined rating scale. The faculty gave students a 3-minute feedback immediately after the encounter. Standardized patients did not give feedback. All interactions were videotaped and subsequently reviewed by faculty members to provide students with additional written feedback. Placed at the mid point of the academic year, the OSCE provided us with the opportunity to evaluate the short-term effectiveness of the small group seminar on students\' communication skills. We reviewed the videotapes of 52 students who had attended the seminar (the experimental group) and 53 students who had not at the time of the OSCE (the control group). The group assignment was based on the sequential order of clinical rotations, arbitrarily set by the medical school officials. The time intervals between the seminar and the OSCE ranged from 1 week to 5 months. Students were asked to provide informed consent using a form that had been approved by the Institutional Review Board at the Nagoya University Hospital. The interview rating form was created by one of the authors (KM) and includes 16 essential communication skills items. They are grouped into 6 communication tasks that should be accomplished during the initial 5 minutes of an encounter (establish initial rapport, survey patient\'s reason(s) for the visit, determine the patient\'s chief concern, elicit and understand the patient\'s perspective, manage flow -- provide the structure for the interview, and use of relationship building skills). The performance was rated on a 4-point scale labelled as good, satisfactory, insufficient and poor. The skills items were selected for their association with improved patient outcomes. They were derived from evidence-based communication assessment tools (i.e., the Calgary-Cambridge observation guide, the SEGUE framework, and the checklist developed by the investigators of the Macy Initiative in Health Communication) \[[@B2],[@B10],[@B11]\]. These instruments are based on the same conceptual models for patient-physician communication we referred to during our teaching seminar. Two staff members (KK, HW) were trained to serve as raters. The tapes were independently reviewed and scored using the students\' communication skills rating scale. Ten arbitrarily selected videotapes of students\' role-plays of a doctor-patient encounter during the small group seminar were used to ensure accuracy and inter-rater reliability. At the time of the research the raters primarily worked outside the University and did not participate in the teaching seminars. Thus they were blinded to the students\' group assignments. From the 105 students who attended the OSCE, 2 did not return the consent form, 5 did not give permission for the video review, for 1 the videotape quality was too poor to be analyzed. Thus, a total of 97 videotapes were available for the analysis. A skill item was considered \'acceptable\' if both raters scored the students\' performance as \'good\' or \'satisfactory.\' It was labeled as \'unacceptable\' if both raters scored the performance as \'insufficient\' or \'poor.\' When these two raters disagreed over the judgment about the students\' performance (e.g., one rater scored the performance of a skill item as \'acceptable\' and the other scored the performance of the same item as \'unacceptable\'), a communication educator and researcher (KA) served as the tiebreaker. The overall disagreement rate between the two raters (KK, HW) was 21%. The raters disagreed more often on inviting the patient to tell the story chronologically (41%), actively responding to the patient\'s concerns and nonverbal cues (34%), and being attentive and empathic nonverbally (31%). Statistical analysis -------------------- We compared baseline characteristics of the two groups using t-tests for a continuous variable (age) and chi-square tests for categorical variables. To evaluate the effect of the educational intervention, the proportion of students with \'acceptable\' performance was compared with those whose performance was unacceptable using chi-square tests for all 16 skills items. All statistical analyses were done by JS using Stat View Version 5.0 (SAS Institute Inc. North Carolina). Results ======= Student characteristics including gender did not differ between the groups except that more students in the control group engaged in self study as a preparation for the OSCE (p \< .05) (Table [1](#T1){ref-type="table"}). There were trends that more students in the control group took an elective on communication skills at the 4^th^year and were interested in a future generalist career. For both groups combined, the mean age was 23.5 years and 38 % were women. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Baseline characteristics of the students ::: Intervention Group (N = 47) Control Group (N = 50) P-value ---------------------------------------------------------------- ----------------------------- ------------------------ --------- Mean age (SD) 23.6 (1.5) 23.4 (1.5) 0.48 Women 36% (N = 17) 40% (N = 20) 0.70 Did a self-study preparing for OSCE 34% (N = 16) 56% (N = 28) 0.03 Took an elective on communication in medicine at the 4^th^year 43% (N = 20) 54% (N = 27) 0.26 Interested in becoming a generalist 13% (N = 6) 26% (N = 13) 0.10 ::: The proportions of students who were judged to have performed as \'acceptable\' for each of the 16 items are shown in Table [2](#T2){ref-type="table"}. There was a statistically significant difference for one particular skill related to understanding patient\'s perspectives: \"exploring how the illness or problem affected the patient\'s life\" (53% in the intervention group vs. 30% in the control group, p = .02). No significant differences were observed for the other 15 skills, although there was a trend favouring the intervention in the skill for \"asking the patient about ideas concerning the illness or problem (60% vs. 40%, p = .054) and one of the relationship building skills: \"being attentive and empathic nonverbally (87% vs. 72%, p = .064).\" ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Student performance of the skill judged as \'acceptable\' ::: Communication Tasks and Related Skills Intervention Group (N = 47) Control Group (N = 50) P-value ------------------------------------------------------------ ----------------------------- ------------------------ --------- Establish Initial Rapport Greet patient and obtain patient\'s name 92% 94% 0.43 Introduce self and clarify the role 100% 98% 1.0 Survey Patient\'s Reason(s) for the Visit Allow the patient to complete his/her opening statement 9% 6% 0.71 Invite the patient to tell the story chronologically 49% 46% 0.77 Actively listen, using verbal and nonverbal techniques 66% 58% 0.42 Summarize. Check for understanding. Invite more questions? 70% 60% 0.29 Determine the Patient\'s Chief Concern Ask closed-questions that are non-leading, one at a time 100% 100% 1.0 Define the concern completely 96% 94% 1.0 Elicit and Understand the Patient\'s Perspective Explore contextual factors (e.g., job, family, hobbies) 66 % 62% 0.69 Ask the patient\'s ideas about the illness or problems 60% 40% 0.054 Explore how the problem affects the patient\'s life 53% 30.0% 0.02 Manage Flow -- Provide the Structure to the Interview Summarize periodically throughout the interview 81% 76% 0.56 Use signposting 40% 30% 0.28 Use of Relationship Building Skills Be attentive and empathic nonverbally 87% 72% 0.064 Actively respond to patient\'s concerns and nonverbal cues 38% 40% 0.8637 Use appropriate language 100% 100% 1.0 ::: Discussion ========== A short, intensive small group seminar on medical interviewing appeared to have had an impact on some specific skills, pertaining to \"eliciting and understanding the patient\'s perspectives.\" It did not seemed to have improved the skills associated with the other tasks: establishing initial rapport, surveying the patient\'s reason(s) for the visit, determining the patient\'s chief concern, and managing flow -- providing the structure for the interview, and the skills for building relationships. There are several strengths of our study. First, this is one of the few empirical, controlled studies from a non-English speaking country. Even though the students were not strictly randomized into intervention and control groups, the assignment occurred arbitrarily by the administration, without regard to students\' preferences or interests in medical interviewing. Thus, it is unlikely that the higher scores in the intervention group are attributable to self-selection. Although there was a significant difference between the groups in proportions of students who did a self-study for the OSCE, which might have caused the results of no difference in most of the skills, the other characteristics such as age and gender were similarly distributed (Table [1](#T1){ref-type="table"}). Second, interventions and evaluations were guided by the conceptual framework, modelled after the 3 widely used theoretical models that are based on rigorous, empirical research in the field of patient-physician communication \[[@B2],[@B10],[@B11]\]. Third, the communication skills evaluation instrument was matched with the competencies taught in the small group sessions \[[@B13]\]. By carefully delineating and defining specific communication skills that should be addressed in the teaching session and by evaluating the effect of the teaching intervention on these individual skills, we sought to examine whether some skills were more teachable than others in such a brief, small group sessions. Our study also has weaknesses that should be addressed. First, our teaching method was based on the research findings in Western world, and this is based on the untested assumption that these findings are equally valid in Japan. There is evidence that patient-physician communication patterns in Japan are different from those in the West. Previous research by Ohtaki and colleagues compared patient-physician communication patterns in Japan and the USA \[[@B14]\]. It included 20 outpatient consultations of four physicians in Japan and 20 outpatient consultations of five physicians in the USA. Japanese physicians spent less time on social talk than the USA counterparts (5% vs. 12%). Japanese patient-physician encounters included more pauses than those in the USA (30% vs. 8.2% of the total consultation length). There is a need for more empirical studies linking physicians\' communication skills to patient outcomes specifically for Japanese population. Second, our assessment of students\' communication skills was based on observations of a single, five-minute OSCE station. The reliability of which as a measure of communication skills is known to be low \[[@B15]\]. Third, because we assessed the students\' skills at only one time, we could not assess the change in students\' performance before and after the intervention. Fourth, the use of junior students as standardized patients may have influenced the performance of the examinees. The accuracy of student-standardized-patients\' (student-SPs\') portrayal would be a critical issue especially when the OSCE is used to grade students. Although we did not objectively investigate the consistencies of the portrayal by student-SPs, our examinees rated highly the fidelity of student-SPs, i.e., the degree to which they were acting as if they were real patients (mean score, 3.9 on a 5-point Likert scale) \[[@B12]\]. Fifth, our study might have only shown that the intervention was effective in improving students\' skills for eliciting \'expert\' observations of patient perspectives, not actual patient perspectives. We did not ask student-SPs whether examinees elicited their perspectives. Rather, we judged examinees\' ability to elicit patient perspectives through their \'observable\' behaviours from the experts\' point of view. The role of student-SPs in evaluating fellow students\' communication skills, particularly skills for eliciting patient perspectives should be addressed in future studies. Finally, the statistically significant difference observed for only 1 skill among a total of 16 skills could be due to chance alone. It is certainly possible that our intervention was too weak to influence any of the 16 communication skills. One can hypothesize the reasons why the intervention appeared to make a difference to some communication skills competencies but not to others. One could speculate that the competencies that were not influenced by the intervention were either very easy in general or too difficult to acquire in such a short teaching session. For example, the skills for establishing initial rapport (greet patient and obtain the patient\'s name, introduce self and clarify roles) and skills for determining the patient\'s chief concern (ask closed-ended questions that are non-leading and one at a time, define the concern completely) may be already present from the outset or so easy to acquire that a self-study just before the OSCE would make no differences in scores between the groups regardless of the intervention. On the other hand, the skills for surveying the patient\'s reason(s) for the visit, which requires being open at the beginning of the interview, may be too difficult for students to demonstrate, with or without the intervention. In particular, only 9% in the intervention group and 6% in the control group demonstrated an acceptable performance for the skills of allowing patients to complete their opening statements. These very low scores may also indicate that during small group sessions, we did not emphasize enough the importance of not interrupting patients at the beginning of the interview. Another explanation is that \'content\' skills (i.e., what we communicate) are easier for students to acquire than \'process\' skills (i.e., how we communicate). Kurtz at al. noted that the skills for understanding patient\'s perspectives, which our intervention made a difference, are actually \'content\' skills, not \'process\' skills \[[@B16]\]. One could argue that the intervention was just too short to influence other \'process\' skills. These interesting hypotheses should require further investigations. Conclusions =========== The results of this study suggest that a short, intensive small group seminar for Japanese medical students may have had an impact on specific communication skills, namely, skills for exploring how the illness or problem affected the patient\'s life, asking the patient about ideas concerning the illness or problem, and being attentive and empathic nonverbally at least in the short term. Further studies should be done to confirm this preliminary finding and to clarify the skills for which educational interventions could make a difference. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= KM contributed to the conception and design of the study, design and implementation of the educational intervention, interpretation of the data, and drafting of the manuscript. KK and KW contributed to the collection of the data and reviewing of the manuscript. KA contributed to the collection of the data and reviewing of the manuscript. JS contributed to the conception and design of the study, analysis and interpretation of the data and reviewing of the manuscript. NB contributed to the conception and design of the study, design and implementation of the educational intervention, interpretation of the data, and reviewing of the manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1472-6920/4/24/prepub> Acknowledgements ================ We thank students who participated in the study. We thank Elizabeth Kachur, Ph.D. in New York for useful comments on earlier drafts.	PubMed Central	2024-06-05T03:55:49.956465	2004-11-18	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534797/", "journal": "BMC Med Educ. 2004 Nov 18; 4:24", "authors": [ { "first": "Kei", "last": "Mukohara" }, { "first": "Kazuya", "last": "Kitamura" }, { "first": "Hideki", "last": "Wakabayashi" }, { "first": "Keiko", "last": "Abe" }, { "first": "Juichi", "last": "Sato" }, { "first": "Nobutaro", "last": "Ban" } ] }
PMC534798	Background ========== Conjugated fatty acid (CFA) is the general term of positional and geometric isomers of polyunsaturated fatty acids with conjugated double bonds. It has been reported that conjugated linoleic acid (CLA), the CFA form of linoleic acid, has favorable physiological effects, such as anti-atherosclerosis, anti-obesity, anti-tumor, and anti-hypertension \[[@B1]-[@B9]\]. There are also other types of CFA in some plant seed oils. Punicic acid (9cis, 11trans, 13cis-conjugated linolenic acid; 9c, 11t, 13c-CLNA) is contained about 72% in pomegranate seed oil \[[@B10]\]. α-Eleostearic acid (9cis, 11trans, 13trans-CLNA) is contained in bitter gourd oil and tung seed oil about 60% and 70%, respectively \[[@B10],[@B11]\]. Catalpa seed oil also contains catalpic acid (9trans, 11trans, 13cis-CLNA) about 31% and pot marigold seed oil contains calendic acid (8trans, 10trans, 12cis-CLNA) about 33% \[[@B10]\]. There are some studies showing that mixtures of CLNA isomers, prepared by alkaline isomerization of α-linolenic acid or plant seed oil, have some physiological functions including body fat reduction and anti-tumor activity \[[@B12],[@B13]\]. In addition, purified α-eleostearic acid (9c, 11t, 13t-CLNA) and α-eleostearic acid rich bitter gourd seed oil also reveal anti-carcinogenesis in vitro and in vivo \[[@B10],[@B11],[@B14],[@B15]\]. However, there are few studies evaluated the physiological function of punicic acid (9c, 11t, 13c-CLNA) \[[@B10],[@B16]\]. Previously, we reported the hypolipidemic effect of purified punicic acid in human liver derived HepG2 cells \[[@B17]\]. In the present study, we investigated the effects of pomegranate seed oil rich in 9c, 11t, 13c-CLNA on lipid metabolism in Otsuka Long-Evans Tokushima fatty (OLETF) rats. OLETF rats develop a syndrome with multiple metabolic and hormonal disorders that shares many features with human obesity \[[@B18]-[@B21]\]. OLETF rats have hyperphagia, because they lack receptors for cholecystokinin, and become obese, developing hyperlipidemia, diabetes, and hypertension. To clarify the physiological function of 9c, 11t, 13c-CLNA, we measured hepatic enzyme activities in relation to lipid metabolism and fatty acid composition in plasma of these obese, hyperlipidemic rats. Results and Discussion ====================== In comparison with their progenitor Long-Evans Tokushima Otsuka (LETO) rats, OLETF rats had increased body weight gain with enhanced food intake during 2 weeks feeding period (Table [1](#T1){ref-type="table"}). In OLETF rats, food intake was not different between the groups. There was also no significant difference between groups in the relative liver weights of LETO and OLETF rats. Food efficiency, however, was higher in 9c, 11t, 13c-CLNA group than in other two groups. Chin et al. previously reported that CLA is a growth factor for rats as shown by enhanced weight gain and improved feed efficiency \[[@B22]\]. Thus, we consider that 9c, 11t, 13c-CLNA may have some growth promotional function. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Effect of 9c, 11t, 13c-CLNA on body weight, relative liver weight, food intake, and food efficiency ::: LETO OLETF -------------------------------------- --------------- --------------- --------------- Body weight (g) Initial 223 ± 3^a^ 266 ± 12^b^ 265 ± 8^b^ Final 282 ± 5^a^ 357 ± 15^b^ 369 ± 11^b^ Gain 59.4 ± 2.7^a^ 91.3 ± 3.6^b^ 104 ± 6^b^ Relative liver weight (g/100 g BW) 3.12 ± 0.09 3.40 ± 0.11 3.35 ± 0.06 Food intake (g) 17.8 ± 0.4^a^ 25.8 ± 1.1^b^ 26.2 ± 1.1^b^ Food efficiency (g BW gain/g intake) 25.7 ± 1.0^a^ 27.3 ± 0.6^b^ 30.4 ± 1.1^b^ ^a,b^Different superscript letters show significant difference at P\< 0.05. ::: The effect of dietary 9c, 11t, 13c-CLNA on the accumulation of abdominal white adipose tissue (WAT) was investigated (Figure [1](#F1){ref-type="fig"}). After 2 weeks feeding period, OLETF rats developed marked abdominal obesity. Compared with LETO rats, the control diet increased perirenal, epididymal, and omental WAT weights of OLETF rats to 2.6-, 1.5-, and 2.1-fold, respectively. There was no significant effect of 9c, 11t, 13c-CLNA on the accumulation of abdominal WAT in OLETF rats. However, 2 weeks feeding of the diet supplemented with 5% pomegranate seed oil resulted in a significant reduction of omental WAT weight (by 27%) compared with the feeding of control diet in OLETF rats (unpublished data). These results suggested that 2 weeks feeding of 1% pomegranate seed oil diet might not be enough to reveal anti-obese effect of 9c, 11t, 13c-CLNA. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Effect of 9c, 11t, 13c-CLNA on abdominal white adipose tissue weight in LETO and OLETF rats. Rats were fed a control or 9c, 11t, 13c-CLNA diet for 2 weeks. Values are expressed as mean ± SE for 6 rats. ^a,b^Different letters show significant differences at P\< 0.05. Omen, omental; Peri, perirenal; Epi, epididymal. ::: ![](1476-511X-3-24-1) ::: After the 2 weeks feeding period, OLETF rats revealed hyperlipidemia. Serum triacylglycerol, phospholipids, and cholesterol levels of OLETF rats fed the control diet were significantly higher than those of LETO rats fed the control diet (Figure [2](#F2){ref-type="fig"}). However, feeding of 9c, 11t, 13c-CLNA did not affect to serum lipid levels in OLETF rats. Although the present results showing that dietary 1% pomegranate seed oil rich in 9c, 11t, 13c-CLNA could not alleviate hyperlipidemia in OLETF rats, our previous report indicated that purified 9c, 11t, 13c-CLNA suppressed the secretion of apolipoprotein B100 from human liver derived HepG2 cells \[[@B17]\]. Further studies are needed to elucidate the effect of purified 9c, 11t, 13c-CLNA on the pathogenesis of hyperlipidemia in OLETF rats. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Effect of 9c, 11t, 13c-CLNA on serum lipid levels in LETO and OLETF rats. Rats were fed a control or 9c, 11t, 13c-CLNA diet for 2 weeks. Values are expressed as mean ± SE for 6 rats. ^a,b^Different letters show significant differences at P\< 0.05. TG, triacylglycerol; PL, phospholipids; Chol, cholesterol. ::: ![](1476-511X-3-24-2) ::: Next, we investigated the effect of dietary 9c, 11t, 13c-CLNA on the distribution of lipids to the liver. There was no significant difference in relative liver weight between control and 9c, 11t, 13c-CLNA group in OLETF rats. Previous reports indicated that CLA feeding resulted in the development of hepatomegaly and fatty liver in mice \[[@B23]-[@B25]\], and a mixture of CLNA also induced hepatic lipid accumulation in rat \[[@B13]\]. In the present study, the triacylglycerol concentration in OLETF rats was significantly higher than that in LETO rats, and the triacylglycerol accumulation in the liver of OLETF rats was markedly alleviated by the 9c, 11t, 13c-CLNA diet (Figure [3](#F3){ref-type="fig"}). There was no significant difference in liver phospholipids and cholesterol levels among groups in LETO and OLETF rats. These results suggest that 9c, 11t, 13c-CLNA has a preventive effect against the triacylglycerol accumulation in the liver. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Effect of 9c, 11t, 13c-CLNA on hepatic lipid levels in LETO and OLETF rats. Rats were fed a control or 9c, 11t, 13c-CLNA diet for 2 weeks. Values are expressed as mean ± SE for 6 rats. ^a,b^Different letters show significant differences at P\< 0.05. TG, triacylglycerol; PL, phospholipids; Chol, cholesterol. ::: ![](1476-511X-3-24-3) ::: To further investigate the regulation of hepatic lipid metabolism, we analyzed the effect of dietary 9c, 11t, 13c-CLNA on the activities of enzymes related to fatty acid synthesis and fatty acid β-oxidation. As shown in Figure [4A](#F4){ref-type="fig"}, the activities of glucose-6-phosphate dehydrogenase (G6PDH) and malic enzyme (ME), key enzymes of NADPH production, and fatty acid synthase (FAS), a key enzyme of fatty acid synthesis, were markedly increased in OLETF rats fed the control diet compared with LETO rats. There was no significant effect of dietary 9c, 11t, 13c-CLNA on these enzyme activities in OLETF rats. The activities of carnitine palmitoyltransferase (CPT), a key enzyme of fatty acid β-oxidation, and peroxisomal β-oxidation were not different between OLETF and LETO rats, and 9c, 11t, 13c-CLNA diet did not affect on these activities in OLETF rats (Figure [4B](#F4){ref-type="fig"}). Koba et al. previously reported that a mixture of CLNA isomers, prepared by alkaline isomerization, enhanced hepatic mitochondrial and peroxisomal β-oxidation compared with linoleic acid, α-linolenic acid, and CLA \[[@B13]\]. Thus, we consider that the effect of 9c, 11t, 13c-CLNA on the fatty acid β-oxidation is weak compared with those of other CLNA isomers. In addition, the alleviation of hepatic triacylglycerol accumulation by 9c, 11t, 13c-CLNA could not be attributed to the regulation of enzyme activities related to the fatty acid synthesis and fatty acid β-oxidation. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Effect of 9c, 11t, 13c-CLNA on activities of enzymes related to lipid metabolism, (A) G6PDH, ME, FAS (B) CPT, peroxisomal β-oxidation, in the liver of LETO and OLETF rats. Rats were fed a control or 9c, 11t, 13c-CLNA diet for 2 weeks. Values are expressed as mean ± SE for 6 rats. ^a,b^Different letters show significant differences at P\< 0.05. ::: ![](1476-511X-3-24-4) ::: To gain insight into the effect of dietary 9c, 11t, 13c-CLNA on lipid metabolism, we analyzed fatty acid composition in serum triacylglycerol. As shown in Table [2](#T2){ref-type="table"}, saturated fatty acid (SFA) levels were lower and monounsaturated fatty acid (MUFA) levels were higher in OLETF rats fed the control diet than those in LETO rats. Feeding of 9c, 11t, 13c-CLNA significantly reduced MUFA levels in plasma triacylglycerol of OLETF rats. It has been recognized that MUFAs are the major fatty acid form in fat depots \[[@B26]\]. Alterations in the ratio of SFA to MUFA have been implicated in various disease states including cardiovascular disease, obesity, and diabetes \[[@B27]-[@B29]\]. Therefore, the ratio of SFA to MUFA is of physiological importance in normal and disease states. A key enzyme involved in the cellular synthesis of MUFA from SFA is the membrane-bound stearoyl-CoA desaturase (SCD), which inserts a cis-double bond in the delta-9 position of fatty acid substrates. Previous reports indicated that 10t, 12c-CLA, an active isomer of anti-obese effect of CLA, suppresses delta-9 desaturation and SCD activity in vitro and in vivo \[[@B30]-[@B32]\]. In the present study, the index of delta-9 desaturation, ratio of oleic acid (18:1) versus stearic acid (18:0), was higher in obese, hyperlipidemic OLETF rats compared with in lean LETO rats, and it was significantly decreased by dietary 9c, 11t, 13c-CLNA in OLETF rats. As far as we know, this is the first study showing that 9c, 11t, 13c-CLNA also suppresses delta-9 desaturation in vivo. We suggest that the alleviation of hepatic triacylglycerol accumulation by dietary 9c, 11t, 13c-CLNA was, at least in part, attributable to the suppression of delta-9 desaturation in OLETF rats. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Effect of 9c, 11t, 13c-CLNA on fatty acid composition in serum triacylglycerol. ::: LETO OLETF -------------------- ------------------ ------------------ ------------------ \% 14:0 2.28 ± 0.23^a^ 1.05 ± 0.15^b^ 1.10 ± 0.13^b^ 16:0 36.8 ± 0.7^a^ 28.1 ± 0.7^b^ 41.2 ± 2.7^a^ 16:1 0.492 ± 0.066^a^ 3.83 ± 0.25^b^ 2.39 ± 0.20^c^ 18:0 5.58 ± 0.63^a^ 3.29 ± 0.26^b^ 4.04 ± 0.32^b^ 18:1 11.7 ± 0.7^a^ 24.5 ± 1.4^b^ 20.4 ± 1.4^c^ 18:2 35.5 ± 0.7^a^ 33.2 ± 0.9^a^ 27.8 ± 1.4^b^ 20:4 7.74 ± 0.76^a^ 4.80 ± 0.40^b^ 3.12 ± 0.44^c^ Desaturation index Δ9 desaturation 2.18 ± 0.24^a^ 7.73 ± 0.83^b^ 5.34 ± 0.75^c^ Δ6 desaturation 0.219 ± 0.024^a^ 0.144 ± 0.009^b^ 0.110 ± 0.011^b^ ^a,b,c^Different superscript letters show significant difference at P\< 0.05. ::: Conclusions =========== Dietary pomegranate seed oil rich in 9c, 11t, 13c-CLNA alleviates hepatic triacylglycerol accumulation in obese, hyperlipidemic OLETF rats. The mechanism of this effect could not be attributed to the regulation of enzyme activity related to fatty acid synthesis and fatty acid β-oxidation. However, suppression of delta-9 desaturation by dietary 9c, 11t, 13c-CLNA may be, at least in part, involved this effect. Materials and Methods ===================== Animals and diets ----------------- All aspects of the experiment were conducted according to the guidelines provided by the ethical committee of experimental animal care at Saga University. Five weeks old male OLETF rats and LETO rats, the progenitor of OLETF rats, were provided by Tokusima Research Institute (Otsuka Pharmaceutical, Tokushima, Japan). Rats were housed individually in metal cages in temperature-controlled room (24°C) under a 12-hour light/dark cycle. After a 1-week adaptation period, OLETF rats were assigned to two groups (six rats each) that were fed with a semisynthetic diet supplemented with 10% safflower oil (the control group) or a semisynthetic diet supplemented with 9% safflower oil and 1% pomegranate seed oil rich in 9cis, 11trans, 13cis-CLNA (the 9c, 11t, 13c-CLNA group). LETO rats were fed the same diet as the OLETF rats in the control group. The pomegranate seed oil rich in 9c, 11t, 13c-CLNA (69.0%) was prepared by Kaneka Co. (Hyogo, Japan). The semisynthetic diet were prepared according to recommendations of the AIN-76 \[[@B33]\] and contained (in weight %): casein, 20; fat, 10; cornstarch, 15; vitamin mixture (AIN-76™), 1; mineral mixture (AIN-76™), 3.5; DL-methionine, 0.3; choline bitartrate, 0.2; cellulose, 5; and sucrose, 45. The rats received different diets for 2 weeks and were killed by aortic exsanguinations under diethyl ether anesthesia. Liver and abdominal (perirenal, epididymal, and omental) WATs were also excised for analysis. Analysis of lipids ------------------ Serum was separated by centrifuging the blood. Triacylglycerol, cholesterol, and phospholipids in serum were measured using enzyme assay kits from Wako Pure Chemicals (Tokyo, Japan). Liver lipids were extracted and purified according to the method of Folch et al \[[@B34]\]. The concentrations of triacylglycerol, cholesterol, and phospholipids were measured according to the methods of Fletcher \[[@B35]\], Sperry and Webb \[[@B36]\], and Bartlett \[[@B37]\]. Measurement of fatty acid composition in plasma was carried out as previously described \[[@B38],[@B39]\]. Preparation of liver subcellular fractions ------------------------------------------ A piece of liver was homogenized in 6 volumes of a 0.25 M sucrose solution that contained 1 mM EDTA in a 10 mM tris Tris-HCL buffer (pH 7.4). Fractions of mitochondria, microsomes, and cytosol were obtained as previously described\[[@B40]\]. The protein concentration was determined according to the method of Lowry et al \[[@B41]\], with bovine serum albumin used as the standard. Assays of enzyme activity ------------------------- The enzyme activities of ME (EC 1.1.1.40) \[[@B42]\], G6PDH (EC1.1.1.49) \[[@B43]\], FAS (EC 2.3.1.85) \[[@B44]\] in the liver cytosol fraction, mitochondrial CPT (EC2.3.1.23) \[[@B45]\] and peroxisomal β-oxidation \[[@B46]\] were determined as described. Statistical analysis -------------------- All values are expressed as means ± SE. Data were analyzed by one-way ANOVA, and all differences were inspected by Duncan\'s new multiple-range test \[[@B47]\]. Differences were considered to be significant at P\<0.05. List of abbreviations ===================== CFA, conjugated fatty acid; CLA, conjugated linoleic acid; CLNA, conjugated linolenic acid; OLETF rat, Otsuka Long-Evans Tokushima fatty rat; LETO rat, Long-Evans Tokushima Otsuka rat; WAT, white adipose tissue; G6PDH, glucose-6-phosphate dehydrogenase; ME, malic enzyme; FAS, fatty acid synthase; CPT, carnitine palmitoyltransferase; SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; SCD, stearoyl-CoA desaturase Acknowledgement =============== We thank Satoko Ikegami and Masanori Ushijima for technical assistance. This work was supported by a research grant from the Japanese Ministry of Education, Science and Culture.	PubMed Central	2024-06-05T03:55:49.958692	2004-11-9	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534798/", "journal": "Lipids Health Dis. 2004 Nov 9; 3:24", "authors": [ { "first": "Keisuke", "last": "Arao" }, { "first": "Yu-Ming", "last": "Wang" }, { "first": "Nao", "last": "Inoue" }, { "first": "Junichi", "last": "Hirata" }, { "first": "Jae-Young", "last": "Cha" }, { "first": "Koji", "last": "Nagao" }, { "first": "Teruyoshi", "last": "Yanagita" } ] }
PMC534799	Review ====== Genomes in the thousands ------------------------ At the date of October 30^th^, 2004 the GOLD (<http://www.genomesonline.org/>) site provided links to 1205 ongoing or completed genome programmes, most of which from prokaryotic organisms. More than 40,000 pages are indexed in the WWW Browser Engine Google for the keyword \"cell factory\". Early in 1999 the European Union launched a research programme on the cell factory[(]{.underline}<http://europa.eu.int/comm/research/rtdinf21/en/key/03.html>), stating that « The concept of the \"bio-product\" is as old as the knowledge involved in the making of bread, beer, wine or cheese. However, recent techniques and knowledge in molecular biology and genetics mean that living cells -- from bacteria to man -- are now becoming real \"factories\". In vast fermentation vats, engineers can direct and control natural metabolism in order to produce all sorts of substances with a high added value: proteins, amino acids, alcohols, citric acid, solvents and even bio-plastics. This industrial mastery of the mechanisms of life opens up revolutionary perspectives in the development of new kinds of medicines, foodstuffs with specific nutritional properties, and biodegradable biochemical products » \[[@B1]\]. Taken together these pieces of information show that exploration of the potential of microbes as industrial tools is shifting from its former status of traditional biotechnology assets to new high technology devices, meant to perform highly specific tasks, with the highest possible yields and security, and genomics as the background support. We shall not here review the use of microbes in traditional production (bread, beer, cheese and wine have been invented since the origin of the Neolithic, and perhaps even earlier \[[@B2]\]) but, rather, see how the coupling between knowledge of bacterial genome sequences and new genomics techniques such as expression profiling and biotechnology processes have interacted recently. The numbers of works in the domain is growing exponentially (it counts certainly in the thousands), and we shall therefore restrict our choice to leads that may be used for further reading. In order to limit the scope of this already extensive study, we shall also restrict this review to cells using the standard genetic code, not considering the extremely interesting attempts to reprogramme the code (for a review see \[[@B3]\]). There is also interesting work developed in vitro, that allows variations on the nature of the building blocks of macromolecules, which will lead to fascinating new aspects of the microbial (artificial) cell factory. We shall not consider this further here (see for example \[[@B4]-[@B6]\]). General versus particulars -------------------------- ### Individual genes, from genomics to metagenomics Genomes are most often viewed as bags of genes. While this is not our personal view -- we see genomes as highly organized set of genes, certainly not randomly distributed along the chromosomes \[[@B7]\], this is certainly a limited, but sufficient view for a large area of biotechnology applications. When reading papers emphasizing the biotechnological potential of bacteria, it is not uncommon to witness sentences like that one: « The completion of the \<name of species\>genome sequencing project resulted in the discovery of a number of new genes with potential interest for biotechnological applications ». Indeed, many biotechnological procedures still rest (and will rest) on the isolation of individual genes, or series of genes involved in the biosynthesis of a specific compound (this is illustrated in the complex case of coenzyme B12 biosynthesis in Escherichia coli, for example \[[@B8]\]). Remarkably, it has become a practical fact that it is now much easier (and often significantly less costly) to sequence a genome in its entirety than to isolate a gene of interest and then submit it to mutagenesis for improvement. There are already so many examples of this situation that they cannot be all given here. Many proteases, amylases, lipases and other enzymes of general biotechnological interest (in particular in agro-food industry) are side-products of genes isolated from a variety of genomes \[[@B9]-[@B11]\]. As a vivid and lively illustration of the potential of genome programs in the domain of complex molecules such as antibiotics, genes involved in non-ribosomal protein synthesis are continuously collected from genomes, sometimes in an unforeseen way. This was the case, for example with the genome of the entomopathogen Photorhabus luminescens\[[@B12]\], that possesses a variety of such highly complex « megasynthases » \[[@B13]\], when it was assumed that most would be present in Gram positives, Streptomycesspecies in particular \[[@B13]-[@B16]\], and certainly not in enterobacteria. With this simple gene family expanding exponentially in parallel with the genome programs trend, we need a focused resource to keep track of important developments: the NRPS/PKS database provides us with an updated resource that tries to keep trace of these interesting by-products of genomics certainly promised to a bright future in the domain of chemistry of fine chemicals \[[@B17]\]. The genome concept for identifying new genes of biotechnological interest has now been expanded to that of a « metagenome », formed of communities of organisms (often non-cultivatable) in a given environment \[[@B18],[@B19]\]. This revived the interest for biotechnology of fine chemicals \[[@B20]\], that was proposed for a long time, but remained of limited use. Gene prospecting has already been used to extract interesting variants of genes coding for interesting enzyme activities \[[@B21]\]. There are voices, however, that go against that particular trend, emphasising that the variety provided by artificial means will be much larger than that conceivably produced during evolution \[[@B22]\]. However, although chemistry is extremely efficient, some steps, in particular associated with the chirality of molecules, are costly in terms of the process and its yield. In contrast, chirality is an in-built property of life. We can therefore safely speculate that we shall witness in the near future the use of metagenomics for the revival of biotechnology processes in solving expensive bottlenecks in chemical industrial processes. However interesting, these « single-gene » approaches remain conceptually very limited, they only explore the surface of what could be provided by the knowledge of genomes. Furthermore, they often aim at the preparation of a single enzyme, that is meant to be used in a process that does not make use of the adaptation and maintenance potential of living organisms. In short, the cell is not used for what it is in reality, a factory. This is however dramatically changed with the advent of genomics as we shall now see. Functional genomics ------------------- Progresses in genome sequencing were followed by attempts to better understand how a cell behaves as a whole. The knowledge of complete genome sequences permitted scientists to set up expression profiling techniques that play an ever increasing role in biotechnology \[[@B23]\]. Indeed the corresponding knowledge can be used, when the genome of a bacterium used in industry is known, to improve its behaviour, stability, yield in production or security \[[@B24]\]. Many metabolic engineering strategies now use genome-wide methodologies such as DNA sequencing, transcription profiling and global analysis of metabolites. These techniques allow the identification of genetic differences and provide insight into their cellular effects. Inverse metabolic engineering endeavours to map differences between strains with different degree of a certain desired phenotype and subsequent identification of factors conferring that phenotype. Briefly reviewed, expression profiling can be divided into three major branches that each have a particular outcome, and gives a specific knowledge on the organism. ### The transcriptome With all genes known from a cell it has been possible to create DNA arrays sampling a subfamily or all genes on a variety of physical supports. These arrays can subsequently been used to monitor the level of expression of each gene in a particular condition. While this transcriptome approach is widely used, its interpretation is still a matter of research \[[@B25],[@B26]\] but is continuously improving \[[@B26],[@B27]\]. Indeed the very fact that an experiment has, embedded in the data, a collective behaviour is until now rarely used as such, while multifactorial analysis techniques would certainly provide new insights \[[@B28]\]. However transcriptome expression profiling has already had considerable impact in biotechnology. A case in point is improvement of lysine production, despite the fact that this amino acid has previously been manufactured using bacteria for more than 40 years \[[@B29]\]. ### The proteome The second level of expression profiling is, of course, that of the direct access to the gene products, the proteins. Two-dimensional gel electrophoresis has been developed for thirty years, with considerable success, but it is still extremely limited by the lack of reproducibility of 2-D gel patterns \[[@B30]\]. Other methods try to by-pass the 2-D gel step by direct coupling of high-performance mass spectrometry instrumentation with highly efficient chromatographic and electrophoretic separations \[[@B31]\]. While it is a method of choice for qualitative studies, the latter however are usually difficult to use when one wishes to compare the outcome of several experiments. 2D-gel electrophoresis appears therefore to have still a bright future in the domain. Proteomic studies are complementary to transcriptome analysis \[[@B10],[@B32]-[@B35]\], because translation efficiency is variable \[[@B36],[@B37]\], and because mRNA stability can also vary \[[@B38],[@B39]\]. They are just beginning to demonstrate their importance in the study of complexes that organize the cell factory \[[@B40]\]. ### The metabolome Fermentation processes often aim at producing a given metabolite. The major problem facing industry in this domain is to improve the production yield, often for products that do not have a very high added-value (as opposed to proteins used in medicine, for example), in a background that has already been improved by generations of mutational improvement. Furthermore, many metabolites have to be as pure as possible, trying to prevent contamination by side-products that may be toxic \[[@B41]\]. It is therefore of importance to be able to analyse the whole metabolite set of cultures growing in a variety of conditions, and to relate it to gene expression \[[@B42],[@B43]\], so that educated guesses may be explored for improvement of the processes of interest \[[@B44]\]. While there is currently no efficient large-scale way to systematically monitor metabolites in cells (Nuclear Magnetic Resonance, for example, is limited by its poor sensitivity to those metabolites that are at a high concentration in the cell and Mass Spectrometry needs preliminary purification steps to sort out the zoo of molecules generated in a cell) \"metabolomics\" is one of the most fashionable \"omics\" at present \[[@B45],[@B46]\]. It has already been used efficiently in the case of focused production, such as synthesis of antibiotics \[[@B47]\]. There is little doubt that this domain will expand considerably in the near future \[[@B45]\]. Gene expression and genome organisation --------------------------------------- The traditional way for biotechnology to improve its processes was to select mutants having interesting properties (in terms of stability, resistance to foreign agents such as viruses, and of course metabolite or biomass production). This required long and tedious procedures where relevant features were usually gradually improving \[[@B48]\]. However these slow changes had a remarkable, although unobtrusive, consequence. Rather than involving isolated mutations, in many cases a coordinate set of mutations was improving the quality of production. Unfortunately, in the absence of direct access to the genome sequence, it was not possible either to identify or to tell those which were important and those which were dispensable. Furthermore, even when the sequence is known it is far from being straightforward to tell, from the differences observed with the parent strains, what are the important ones. Genomics, with all its \"omics\" complements, nevertheless completely changed the picture, and it is now possible to optimise production knowingly, using molecular targets that are directly extracted from knowledge of the genome. This has been applied for example in the case of the much studied Corynebacterium glutamicum\[[@B49]\]. Further progress is certainly possible. It is important to try to understand whether genomes are simply random collection of genes, or whether they show rules, that might be exploited for using cells as factories. Remarkably, at least in bacteria, the organisation of the genome reflects some kind of optimisation of gene expression \[[@B50]\]. Genes do not work in isolation, and their products, even in bacteria, are likely to be compartmentalised. The study of the landscape of all neighborhoods of a gene (proximity in the chromosome, codon usage bias, phylogeny of its products, electric charge, amino acid composition, participation in complexes, and even a neighborhood benefiting from the expertise of other scientists, such as the co-occurrence of gene names in a same article -- \"in biblio\") provides a systemic view that must be used to optimise the behavior of the cell \[[@B51]\]. While this has not yet, to our knowledge, be taken into consideration for improvement of production by industrial strains, it is more than likely that this will be performed in the near future (in fact it is likely that optimisation of the global properties of gene or gene islands text has already been used for the industrial production of proteins, but because protection by patenting is difficult, if this has been done the corresponding know-how is likely to be protected by secrecy). Regulation of gene expression is also of major interest. It must be understood however that this feature of life is evolving much more rapidly than catalytic or structural components of the cell. One should therefore be cautious in extrapolating knowledge from an organism to another one. Theoretical studies, associated to validation experiments have now begun to decipher the rules that govern regulation of gene expression, and it is certainly already possible to construct subtle regulation systems \[[@B52]\], that are much more sophisticated than the ubiquitous on/off systems using positive or negative control of transcription \[[@B53]-[@B56]\]. Among the recent discoveries that will play a considerable role in genome-mediated control of gene expression is that of riboswitches \[[@B57]\]. This mode of control seems to be ubiquitous, but significantly different between Gram negatives and Gram positives (where it appears to be more widely spread). It is still early to have an exact idea of the impact on industrial processes, but the very fact that many coenzymes (vitamins) biosynthetic pathways are controlled using riboswitches warrants further exploration. In the same way quorum-sensing has much to say for the control of gene expression at high cell density \[[@B58]\]. Until now this general control process -- which is still under investigation -- has not been explicitly used to control production in cell factories. It seems likely that, once deciphered in its details, it will be a parameter introduced in large-scale productions. The recent serendipitous discovery that borate was involved in the construction of the mediator AI-2 (autoinducer-2) demonstrates however that unexpected features should always be considered as a possibility when a process does not go entirely as planned \[[@B59]\]. Model cell factories -------------------- Many bacteria have been used as cell factories. In most cases this was to produce small molecules (in particular antibiotics, vitamins and amino acids). These bacteria were usually the result of continuous improvement using standard mutagenesis/screening techniques of bacteria isolated in the wild. Streptomyces species, for example, account for a large number of antibiotics production. Streptomyces lividans\[[@B60]\], Corynebacterium glutamicum\[[@B61]\], Bacillus subtilis\[[@B24]\], Escherichia coli\[[@B62]\], Zymomonas mobilis\[[@B63]\], to give a few names, are used not only as models but also as large-scale production factories. Perhaps the largest scale production fermenters (often 150 m^3^) are now growing Xanthomonas campestris, used as a supplement not only in food, but also in dentrifrice, housekeeping products and even in painting, to prevent it from making drops \[[@B64]\]. In the past decades even larger fermenters were used to produce biomass or ethanol, a trend that was abandoned with cheap oil prices, but that will most probably resume its older importance as the price of oil rises sharply. Among those, most bacteria were chosen for their industrial purpose, as a prime intention. As a consequence, until the advent of genome programs, they were only known for their physiological and physico-chemical properties in fermenters, with limited knowledge of their genetic properties. This was such an inconvenience that, very early on, industry explored the usability of the universal model E. colias a ubiquitous cell factory. This trend was particularly emphasized as soon as genetic engineering techniques were developed, as early as in the early eighties, with the construction of new vectors for expressing foreign proteins at will (e.g. \[[@B65]\]). Mid-eighties many proteins of medical interest were produced using E. colias the factory. This is still so today, with only little shift to the use of other bacterial species as factories. This was initially limited to high added-value products, allowing for very expensive purification steps and compliance to very tight regulations. In quite a few cases however E. coliand sometimes other model bacteria, in which appropriate genes were introduced either on plasmids or in the chromosome, was highly efficient in producing low cost metabolites \[[@B66]\]. Escherichia coliis even used in the production of amino acids, in industrial quantities, a production that was initially reserved to specific mutants of species that had been slowly improved over the years. GRAS organisms, such as AT-rich Gram positives, such as B. subtilis, are much more difficult to use, except for biomass production, or secreted proteins, because heterologous protein expression is difficult there. This has been understood after the genome was deciphered, as a consequence of the poorly versatile control of translation initiation (lack of ribosomal S1 protein in particular \[[@B67]\]), as well as of the large number of proteases harbored by the organism \[[@B68]\]. Taken together these observations suggest the rational choice of a new organism that would play the role of a ubiquitous cell factory. This organism should have several properties. It should be non pathogenic, and its envelope should not trigger inflammation reactions in animals (Man included). It should be easily transformable and allow recombination with linear DNA, with as little matches needed for recombination as possible. It should grow fast at temperature compatible with the size of fermenters (metabolic activity heats up the medium), and it should reach high cell density. More specialized views, adapted to specific productions will also be considered at some point, but it will be interesting to witness the choice of new model bacteria in the new era of the cell-factory. Conclusions =========== Bacteria have been used as factories for a long time. A first step to rationalize this approach has been met with the first genetic engineering of E. coli, producing heterologous proteins. As we now sequence a new genome every third day or so, it is clear that we will be able soon to understand the core of bacterial life, and probably be able to choose new models, better suited to the goals of industry. However we must always remember that life is full of surprises, even in the best explored domains: who would have thought that E. colicommunicates with its kins using the boron atom? Discovery cannot be planned, and the most surprising observations, that have the most considerable consequences in terms of applications of research, come from studies that are totally academic in nature (who would have thought that the discovery of RNAi would have come from the study of variagation in petunia flowers?). One should not mix up domains: discovery first, and this needs a considerable degree of freedom of choice in the topics explored, and then, naturally, one can think of applications of research. Constructing the best of bacterial cell factory would be such a goal. List of abbreviations ===================== GRAS: Generally Recognized As Safe RNAi: RNA interference Authors\' conflict of interest ============================== The author declares that he has no competing interests. Acknowledgements ================ This work, meant to explore an unexpected neighbourhood of genes, that of industrial interest, benefited from support from the BlastSets Program (ACI IMPBIO), [BioSapiens]{.underline}<http://www.biosapiens.info/> (LSHG-CT-2003-503265), from the European Union and [BIOSUPPORT]{.underline}<http://bioinfo.hku.hk/>, from the government of Hong Kong. Discussions related to the topic reviewed here are developed in the [Stanislas Noria]{.underline}<http://www.pasteur.fr/recherche/unites/REG/causeries/causeries.html> group.	PubMed Central	2024-06-05T03:55:49.960573	2004-11-10	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534799/", "journal": "Microb Cell Fact. 2004 Nov 10; 3:13", "authors": [ { "first": "Antoine", "last": "Danchin" } ] }
PMC534800	Review ====== Types of spider silk -------------------- Spiders and silks always go together. Currently there are over 34,000 described species of spiders, all of which have a varied tool kit of task-specific silks with divergent mechanical properties \[[@B1]-[@B6]\]. Although some spiders may use silk sparingly, most make rather elaborate nests, traps and cocoons typically using more than one type of silk (Figure [1](#F1){ref-type="fig"}), which are produced by a wide and diverse range of glands, ducts and spigots. Among the various spider silks the major ampullate (MA) silk, which forms the primary dragline, is extremely tough. MA silk reveals a tensile strength that is comparable to Kevlar (4 × 10^9^N/m^2^) coupled with a reasonable viscoelasticity (dragline 35 %, Kevlar 5 %). Spiders use dragline silk as a strong yet flexible structural element in the web, providing a framework to which other silks are attached, and as a life line when a spider is dropping off to escape an enemy. Minor ampullate (MI) silk, used for structural reinforcement in construction of the web, has a similar high tensile strength in comparison to major ampullate silk but has little elasticity \[[@B7],[@B8]\]. Due to the low elasticity of MI silk it is irreversibly deforming when stretched. An orb web\'s capture spiral, in part composed of viscid silk formed by the flagelliform gland, which is therefore named flagelliform silk, is stretchy and can triple in length before breaking, but provides only half the tensile strength of major ampullate silk \[[@B9]\]. The combination of strength and stretchiness gives the capture spiral a toughness (energy to break) greater than elastin, tendon, silkworm silk, bone, synthetic rubber, Kevlar, and high-tensile steel. Composition and structural architecture of spider silks ------------------------------------------------------- Spider silks are protein polymers that display extraordinary physical properties \[[@B1]-[@B4],[@B8]\], but there is only limited information on the composition of the various silks produced by different spiders. Among the different types of spider silks, draglines from the golden orb weaver Nephila clavipesand the garden cross spider Araneus diadematusare most intensely studied. Dragline silks are generally composed of two major proteins \[[@B5],[@B10]-[@B13]\] and it remains unclear whether additional proteins play a significant role in silk assembly and the final silk structure. The two major protein components of draglines from Nephila clavipesare termed MaSp1 and MaSp2 ([M]{.underline}ajor [a]{.underline}mpullate [S]{.underline}pidroins) and from Araneus diadematusADF-3 and ADF-4 ([A]{.underline}raneus [D]{.underline}iadematus [F]{.underline}ibroin). The dragline silk proteins have apparent molecular masses between 180 kDa and 720 kDa depending on the conditions of analysis \[[@B14]-[@B16]\]. It is assumed that, based on amino acid composition, within the dragline fiber the molecular ratio between MaSp1 and MaSp2 and between ADF-4 and ADF-3 is approximately 3 to 2 \[[@B10],[@B11],[@B17]\]. Based on DNA analysis it could be shown that all spider silk proteins are chains of iterated peptide motifs (so called repeating units) (Figure [2](#F2){ref-type="fig"}). The small peptide motifs can be grouped into four major categories: GPGXX (with X often representing Q), alanine-rich stretches (A~n~or (GA)~n~), GGX, and spacers (Figure [2A](#F2){ref-type="fig"}). A fifth category is represented by non-repetitive (NR) regions at the amino- and carboxyl termini of the proteins (Figure [2](#F2){ref-type="fig"}), often representing polypeptide chains of 100 amino acids and more \[[@B7],[@B10],[@B11],[@B18]-[@B22]\]. So far the largest sequence information could be obtained for flagelliform silk from Nephila clavipes(Figure [2B](#F2){ref-type="fig"}). This flagelliform silk protein is translated from a \~15.5 kb mRNA transcript originating from a 30 kb Flaglocus \[[@B9],[@B23]\]. The coding sequence is divided into 13 exons. The NR amino-terminal region is split between exons 1 and 2. All of the other exons are found to encode exactly one repeating unit, built from the described motifs (Figure [2B](#F2){ref-type="fig"}). The final exon 13 in addition includes the NR carboxyl-terminal region. On the basis of several studies, the major categories of peptide motifs in spider silk proteins have been assigned structural roles \[[@B24]-[@B28]\]. The GPGXX motif has been suggested to be involved in a β-turn spiral, probably providing elasticity, based on structures of comparable proteins \[[@B29]-[@B32]\]. If elasticity is due to GPGXX β-spirals, then this motif should be found in the more elastic silks. Flagelliform silks, which show the highest elasticity with more than 200 %, consist of contiguous repeats of this motif for at least 43 times in each repeating unit (Figure [2B](#F2){ref-type="fig"}). The only non-flagelliform silk proteins with GPGXX motifs are MA proteins MaSp2, ADF-3, and ADF-4, which also display some viscoelasticity. In accordance to the lower elasticity of dragline silk in comparison to flagelliform silk the number of tandemly arrayed repeats depicts at most 9 concatenated GPGXX motifs before interruption by another motif \[[@B1],[@B21]\]. Alanine-rich motifs contain typically 6--9 alanine residues and have been found to form crystalline β-sheet stacks leading to tensile strength \[[@B6],[@B24],[@B25],[@B12]\]. The MA and MI silks are both very strong, and at least one protein in each silk (there are always pairs) contains the A~n~or (GA)~n~motif. Interestingly, this motif is not found in flagelliform silks. A glycine-rich 3~1~-helix is adopted by the GGX motif forming an amorphous matrix that connects crystalline regions and that provides elasticity \[[@B26],[@B33],[@B34]\]. The postulated GGX motif is widely distributed and this motif can be found in MA, MI and flagelliform silks (Figure [2A](#F2){ref-type="fig"}). Several groups have suggested that the motifs GPGXX and GGX might be involved in forming an amorphous matrix, which would provide the elasticity of the fiber. The spacers contain charged groups and separate the iterated peptide motifs into clusters. Non-repetitive termini are common to all sequenced MA, MI and flagelliform silks belonging to the Araneoidea family with highly conserved carboxyl-terminal sequences \[[@B19],[@B35],[@B36]\]. The structural impact of the spacer and terminal regions is so far undetermined \[[@B37]\]. Recent findings on single NR-regions of ADF-3 and ADF-4 (without additional repeating units) revealed a secondary structure comprising α-helices as determined by Circular Dichroism and they seem to retain this structural feature in proteins that additionally contain repeating units \[[@B36]\]. It can be speculated that the α-helical NR carboxyl-termini might play a crucial role during assembly of the silk fiber \[[@B19],[@B36],[@B38]\]. Natural spider silk assembly ---------------------------- Silk assembly in vivois a remarkable process. For instance, dragline silk proteins are stored at concentrations up to 50 % (w/v) in the respective glands \[[@B39]\]. This highly concentrated protein solution forms the silk dope (spinning solution), which displays properties of a liquid crystal \[[@B40]-[@B42]\]. Therein, the polyalanine motifs are thought to adopt an α-helical conformation, while the glycine-rich motifs form either β-turns or random coil conformation \[[@B39],[@B43],[@B44]\]. Thread assembly is initiated during a passage of the silk dope through the spinning duct accompanied by extraction of water, sodium and chloride \[[@B45],[@B46]\]. Simultaneous secretion of potassium and hydrogen ions into the lumen of the duct lowering the pH from 6.9 to 6.3 is thought to initiate partly unfolding of the proteins by disrupting their water shell and altering coulombic forces \[[@B42],[@B45]-[@B48]\]. The silk proteins are thought to extend somewhat, align and get packed much closer in the extensional flow-field of the draw-down taper found in the distal part of the duct. As the hydrophobic polyalanine segments of the silk proteins align and are drawn closer together by extensional flow, they are exposed to an increasingly hydrophobic environment, which might trigger their conversion from an α-helical to a β-pleated structure resulting in the formation of numerous interchain hydrogen bonds. The latter would act as multifunctional crosslinks at nodes between the more mobile glycine-rich segments. Thus the assembly of the thread can be seen as a liquid-crystalline phase transition involving separation into polymer-rich and solvent-rich phases \[[@B47]\]. While some aspects of spider silk assembly have been unraveled, the contribution of the individual silk proteins to the assembly process still needs to be resolved in more detail. Comparative studies of the two major dragline silk proteins of Araneus diadematus, ADF-3 and ADF-4, revealed that, although their amino acid sequences are rather similar \[[@B5]\], they display remarkably different solubility and assembly characteristics: While ADF-3 is soluble even at high concentrations \[[@B49]\], ADF-4 is virtually insoluble and self-assembles into filamentous structures under specific conditions \[[@B50]\]. At a closer look, the different solubilities of ADF-3 and ADF-4 could be explained by the hydrophobicities of the two proteins. The hydrophilic ADF-3 interacts favourably with the aqueous solvent and thus remains soluble under most conditions. In contrast, the more hydrophobic ADF-4 favours interactions with other protein molecules and thus tends to aggregate. Interestingly, all pairs of dragline silk proteins from different spider species display a common distinct distribution of hydrophobicity. In direct comparison, MaSp1 / ADF-4 proteins generally display relatively high hydrophobicity, while the corresponding MaSp2 / ADF-3 partner protein is more hydrophilic \[[@B50]\]. Applications for spider silks ----------------------------- Laboratory-scale production of spider silk would initiate a new generation of ecological materials. Spider silk is for instance a promising tool with broad usability in medical devices. In the middle ages spider webs were used as wound dressing -- some reports are even dated back to ancient Greek and Roman cultures. Silkworm silk does not cause allergic reactions and it is thought that spider silk behaves similarly \[[@B51]\]. The unmatched toughness of spider silk would allow to improve several medical products such as wound closure systems, band-aids, and extremely thin sutures for neurosurgery. Additionally, spider silks can be further used in artificial ligaments and tendons for durable implants. High performance fibers built from spider silks can be employed in several technical and industrial applications. In addition to specialty ropes and fishing nets, spider silk can be utilized for parachutes, ballistic applications (body armor), sporting goods, textiles, and lightweight constructions for airplanes \[[@B52],[@B53]\]. Therefore, one day industrially produced spider silk could out-compete man-made fibers. Production of recombinant spider silk proteins ---------------------------------------------- Recombinant production of spider silk proteins has been complicated by the highly repetitive nature of the underlying genes, by their high gc-content, by the length of the constructs, and by the specific codon usage of spiders. In first studies, in vitrotranslation of mRNA from excised major ampullate glands of Nephila clavipeswas performed using tRNA from E. coli, but translation was discontinuous \[[@B14],[@B54]\]. In the era of recombinant proteins and genetic engineering one would envisage to easily produce spider silk proteins (mainly from draglines) in microbes or cell culture. Unfortunately, no dragline silk gene has been cloned in its entirety and only sequence data from the 3\' end of partial cDNA clones of dragline genes from Nephila clavipesand Araneus diadematusand other spiders have been reported \[[@B10],[@B11],[@B20]-[@B22]\]. Therefore, all recent studies used partial cDNA constructs of dragline silk genes to produce recombinant silk proteins in E. coli\[[@B55]\], in MAC-T (bovine) and BHK (hamster) cells \[[@B49]\], or in insect cell lines from Spodoptera frugiperdausing the baculovirus expression system \[[@B50]\]. The most promising expression system seems to be the baculovirus system, since it was possible to efficiently produce dragline silk components at a high yield. Designing of synthetic spider silk proteins ------------------------------------------- Cloning strategies for designing genes for bacterial, yeast or plant expression have been developed to produce recombinant silk-like proteins closely resembling natural dragline \[[@B29],[@B36],[@B56]-[@B62]\] or flagelliform silk proteins \[[@B63]\]. Since gene manipulation and amplification of spider silks is difficult by PCR due to the repetitive nature of silk genes, cloning strategies involved engineering of synthetic DNA modules. These modules were optimized for the codon usage adapted by the corresponding expression host. The use of synthetic modules constructed from small size oligonucleotides repeats has allowed control over primary gene and protein sequence and final protein size. Tobacco, potatoes, the yeast Pichia pastorisand mainly E. colihave been utilized as expression hosts for synthetic genes yielding proteins with up to 150 kDa \[[@B29],[@B56]-[@B62]\]. Unfortunately, expression levels from the synthetic genes have been low and mostly the recombinant silk proteins represented only up to 5% of the total protein in the cell \[[@B13]\]. Although once production levels of up to 1000 mg/l of cell culture have been reported \[[@B57]\], large losses in yield are encountered during purification due to precipitation and non-specific interactions. For the microbial expression systems, yields of purified proteins have been generally in the 10 to 40 mg/l range (\>90% purity) \[\[[@B55],[@B56],[@B59],[@B60]\], summarized in \[[@B13]\]\]. In a recent study, genes coding for spider silk-like proteins were generated using a cloning strategy, which was based on a combination of synthetic DNA modules and PCR-amplified authentic gene sequences (Figure [3](#F3){ref-type="fig"}) \[[@B36]\]. This approach was in contrast to previous protein designs, which did not include the carboxyl-terminal NR-regions that are found in all dragline silks. The dragline silk proteins ADF-3 and ADF-4 \[[@B10]\] from the garden spider Araneus diadematuswere chosen as templates for the synthetic constructs. A seamless cloning strategy \[[@B64]\] allowed controlled combination of different synthetic DNA modules as well as authentic gene fragments. A cloning vector was designed comprising a cloning cassette with a spacer acting as placeholder for synthetic genes \[[@B36]\] (Figure [3B](#F3){ref-type="fig"}). To mimic the repetitive sequence of ADF-3, two modules have been designed. The sequence of one module, termed A, was derived from the poly-alanine containing consensus sequence of ADF-3 (Figure [3A](#F3){ref-type="fig"}). The sequence of a second module termed Q contained four repeats of the GPGQQ motif. In a first cloning step the spacer region of the cloning vector was replaced by one of the synthesized DNA modules. Subsequently two modules could be joined in a site-directed way. To study different length repeat units, one or two Q modules were combined with one A module to obtain (AQ) (Figure [3B](#F3){ref-type="fig"}) or (QAQ) (Figure [3C](#F3){ref-type="fig"}). The complementary 3\'-single strand extensions gg(sense) and cc(antisense) were used for connecting two modules (Figure [3B](#F3){ref-type="fig"}). Thus the DNA sequence required to link two modules was confined to a glycine codon (ggn). Glycine is naturally abundant in spider silk proteins (\~30%), therefore modules could be designed to match authentic amino acid sequences. Since the arrangement of the cloning cassette\'s elements remained unchanged upon cloning, repeat units could be multimerized to generate synthetic genes coding for the repetitive proteins (rep-proteins) (AQ)~12~and (QAQ)~8~(Figure [3C](#F3){ref-type="fig"}). The repetitive part of ADF-4 is generally composed of a single conserved repeat unit displaying only slight variations. These variations were combined and one consensus module termed C has been designed (Figure [3A](#F3){ref-type="fig"}), which was multimerized to obtain the rep-protein C~16~(Figure [3C](#F3){ref-type="fig"}). ADF-3 and ADF-4 both display NR-regions at their carboxyl termini, comprising 124 and 109 amino acids respectively. Gene sequences coding for these regions were amplified by PCR, and codons problematic for bacterial expression were changed to more suitable codons by site directed mutagenesis. In the described system, all synthetic genes could be combined with the appropriate authentic NR-regions. Additionally NR3 and NR4 could be expressed individually. All constructs could be purified by a heat step followed by an ammonium sulfate precipitation \[[@B36]\], which has been employed in previous studied for purifying spider silk proteins \[[@B35],[@B62]\]. Based on this protein engineering approach, which combines synthetic repetitive sequences with authentic NR-regions, proteins closely resembling authentic silk proteins could be produced at high yields. Bacterial production in Erlenmeyer flasks yielded similar protein amounts for all constructs. Yields of individual preparations ranged from 10 to 30 mg of purified protein per liter of culture medium. Fermentation of cells increased the yield of purified protein to 140 and 360 mg/l (purity \>95%). Therefore, the established bacterial expression system provides the basis for cost-efficient large scale production of spider silk-like proteins. Assembly of recombinant spider silks ------------------------------------ One important feature of spider silk proteins is their storage at high protein concentrations (up to 50% w/v) in the dope without apparent aggregation or assembly. However, spider silk proteins can rapidly assemble into highly stable fibers when needed. The determination of solubility and self-assembly of recombinant spider silk proteins is therefore important to create commercially available silk fibers. For instance, pH-shifts are involved in natural silk assembly, but the exact function of acidification during spider silk assembly has not yet been determined. It seems likely that negatively charged groups are protonated reducing the net charge of spider silk proteins. Phosphoryl groups of phosphorylated amino acid residues, which have been detected in dragline silk \[[@B65]\], display pK~A~-values \[[@B66]\] near the range of the pH-shift observed during the spinning process and thus could be involved in triggering silk assembly. Therefore, changes in pH can be used to initiate assembly of recombinant spider silk proteins \[[@B36]\]. Interestingly the intracellular pH 6.3 of Sf9cells (derived from the fall armyworm Spodoptera frugiperda) used for producing ADF-4 corresponds to the pH in the spinning dope prior to silk thread assembly \[[@B50]\]. These pH conditions, among additional factors, might be involved in initiating aggregation of ADF-4 within the cytosol of Sf9cells \[[@B50]\]. Surprisingly, investigating the aggregates in adf-4expressing cells revealed filaments that coiled throughout the cytoplasm, whereby most of the cells contained only one or few filaments. In contrast, Sf9cells infected with control viruses or the adf-3encoding virus never produced such filaments. The diameter of the ADF-4 filaments (0.2 -- 1.5 μm) was in the range of native dragline silks (1.0 -- 4.0 μm), but length of the filaments formed in the Sf9cells seemed to be constrained by the volume of the cells, making them too short for mechanical force measurements. Strikingly, the purified ADF-4 filaments (Figure [4B](#F4){ref-type="fig"}) showed a similar morphology and chemical stability in comparison to natural dragline silk threads of Araneus diadematus\[[@B50]\]. Phosphate, like other lyotropic ions, is known to increase the surface tension of water, promoting hydrophobic interactions \[[@B67]\]. In the case of spider silk proteins it is likely that the addition of phosphate initiates interactions between the hydrophobic poly-alanine motifs, causing the aggregation of the proteins. Accordingly aggregation of polyalanine-rich proteins is pronounced in comparison to synthetic silks which contained one third less poly-alanine motifs \[[@B36]\]. Strikingly, recombinant spider silk proteins are highly soluble in most aqueous solutions, but form nanometer-sized fibers upon addition of methanol, phosphate or other suitable ions (Figure [4A](#F4){ref-type="fig"}). Artificial spinning of spider silks ----------------------------------- A remaining critical step concerning commercial production of silk fibers is the successful spinning of recombinant proteins into fibers resembling the natural silks in their microstructure and in their mechanical properties, which are outstanding by any measure. Besides the chemical parameters discussed above, several mechanical parameters play important roles in generating silk. To draw silk under natural spinning conditions, spiders attach their dragline to an object with glue from the piriform glands, before drawing the silk out by moving away or by descending and using their weight to draw the silk. It is common practice to take advantage of the drawing process by the forced silking of captive animals to collect silk for experiments. Analysis of the differences between naturally and forcibly spun dragline silk provided evidence for discrepancies in their material properties \[[@B44],[@B68],[@B69]\]. Forced spinning under spinning speeds ranging from 0.1 to 400 mm/s and temperatures ranging from 5 to 40°C revealed dramatic differences in strain at breaking, breaking energy, initial Young\'s modulus and point of yielding \[[@B70]\]. Therefore, in case of spinning recombinant silk proteins in vitroseveral aspects have to be taken into account to gain materials with expected properties. Several attempts are reported in the literature and even more have been performed to wet-spin recombinant spider silk proteins. In a first attempt, microfabricated spinnerets were constructed using silicon microfabrication methods \[[@B71]-[@B73]\]. These spinnerets allowed for the production of meters of silk fibers from solutions containing as little as 10 mg of protein. First the spinneret was validated and tested by producing fibers from dissolved silk from the silkworm Bombyx mori\[[@B71]\], before solubilized dragline silk from Nephila clavipeswas wet-spun \[[@B72]\]. The diameters and mechanical properties of the regenerated silkworm silks converged the native silk ones. However, the wet-spun spider silks exhibited diameters of about 40 μm compared to the natural fiber diameter of 2.5 to 4.0 μm with mechanical properties that did not match the natural ones \[[@B72]\]. Other attempts of wet-spinning revealed fiber diameters of approximately 10 -- 60 μm \[[@B49],[@B74]\]. These fibers were subjected to either single or double postspinning draw, first in 70 to 80 % methanol (single and double draw) and then in water (double draw only) to increase their mechanical properties. Fibers subjected to higher draw ratios displayed greater toughness, tenacity, and modulus values \[[@B49]\]. However, even the best values obtained by such technique were in the range of the regenerated Nephilafibers \[[@B72]\], but lower than the reported values for natural dragline silks \[[@B2]\]. Perspectives ============ Engineering of precision fibers ------------------------------- The future objective might not be to prepare identical copies of natural silk fibers, but rather to capture its key structural and functional features in designs that could be useful for engineering applications (Figure [5](#F5){ref-type="fig"}). Using \"protein engineering\" based on knowledge achieved from investigations of the natural silks, artificial proteins can be designed that allow bacterial synthesis at high yields \[[@B75]\]. The soluble synthetic silk would be able to assemble into protein fibers with desired properties, which includes the possibility to specifically functionalize the fiber surface by chemical cross-linking with biologically or chemically active groups. Such protein fibers could be optimized by additional protein engineering in order to gain fibers that allow the formation of interconnected nano- or micrometer-scale networks, which are capable of various biological, chemical or physical processes (e.g. enzymatic reactions, chemical catalysis, electronic signal propagation, etc.) (Figure [5](#F5){ref-type="fig"}). The main strategy would be to modify the monomeric proteins either with the desired functionality prior to assembly into fibers, or to incorporate a reactive group that will subsequently permit the conjunction with desired functionality after the fibers have assembled. Protein fibers could for instance be covalently linked with external units through chemically specific amino acid side chains (e.g. SH-groups of cysteines) \[[@B76]-[@B78]\]. Since the physical and chemical properties of bio-polymers and their assembly processes depend on the amino acid composition of the underlying polypeptide, engineering \"synthetic\" proteins with specific structural features will create a new class of fibrous proteins. However, to design new biomaterials based on spider silk, all properties of the underlying proteins have to be analyzed and in the best case successfully mimicked \[[@B35]\]. Therefore, the crucial design features of both the feedstock of the dope and the spinning process have to be closely adopted, which would allow for managing the commercial production of new materials. Acknowledgements ================ The work is supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. I would like to thank Bettina Richter for scanning electron microscopy and Daniel Huemmerich for atomic force microscopy. Further I acknowledge Daniel Hümmerich and Christian Ackerschott for critical comments on the manuscript and the members of the fiberlab for inspiring discussions. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### *Scanning electron microscopy of major and minor ampullate and flagelliform silks collected from the garden cross spider Araneus diadematus.*Silk harvested from a web was placed on Thermanox plastic cover slips (Nalgene Nunc). Samples were vacuum coated with a gold layer and analyzed with a JSM-5900LV (JEOL Ltd.) at 20 kV. ::: ![](1475-2859-3-14-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Composition of spider silks.*(A) Structural motifs occurring within spider silks. X indicates a residue that may vary within or between proteins. The spacer represents non-repetitive but conserved regions that disrupt the glycine-rich repeats. More details on the motifs can be found in the text. (B) The sequenced cDNAs of adf-3and adf-4code for the shown amino acid motifs and represent approximately 1/6^th^of the entire dragline silks. Both comprise a non-repetitive (NR) carboxyl-terminal region and a large repeat unit based on three major peptide motifs as visualized. The amino-terminal region is so far unresolved for any dragline silk. The predicted flagelliform silk protein is organized into non-repetitive (NR) amino-terminal and carboxyl-terminal regions that flank a repetitive region made up of 11 iterations of a repeating unit. Each unit contents approximately 440 amino acids. Three types of sub-repeats are present in an ensemble with the predominant unit being GPGXX. ::: ![](1475-2859-3-14-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Cloning strategy for constructing synthetic spider silk genes.(A) Amino acid sequences of designed silk modules were derived from dragline silk proteins ADF-3 and ADF-4 and back translated into nucleotide sequences using the bacterial codon usage. (B) The cloning cassette comprised restriction sites required for module insertion and multimerization. During gene construction a spacer region was replaced by modules and module multimers. The first codon of each module (ggn) determined the \"linking\" amino acid glycine. (C) Single modules were connected resulting in controlled assembly of synthetic genes. To study different length repeat units, one or two Q modules were combined with one A module to obtain (AQ) or (QAQ). These repeat units were multimerized to generate synthetic genes coding for the repetitive proteins (rep-proteins) (AQ)~n~and (QAQ)~n~. The repetitive part of ADF-4 is generally composed of a single consensus module termed C, which was multimerized to obtain the rep-protein C~n~. Additionally, carboxyl-terminal non-repetitive (NR)-regions from the natural genes were amplified by PCR and optionally linked with the synthetic genes \[50\]. ::: ![](1475-2859-3-14-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Morphology of self-assembled fibers of recombinant spider silk proteins.Images of nanofibers of the synthetic protein C~16~assembled in vitro(A) and of ADF-4 fibers assembled in insect cells (B) were obtained by atomic force microscopy (AFM). The left images depict the height information, the right images the deflection. The average height of the C~16~-nanofibers is 2 -- 4 nm, the height of the visualized ADF-4 fiber is 0.7 μm. ::: ![](1475-2859-3-14-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Engineering of a synthetic silk protein.*The future objective of silk engineering might not be to prepare identical copies of natural silks, but rather to capture its key structural and functional features in designs. The soluble synthetic silk should be able to assemble into protein fibers with desired properties, which includes the possibility to specifically functionalize the fiber surface e.g. by chemical cross-linking with biologically or chemically active groups. Synthetic silk engineering could be accomplished by assembling modules either originating from authentic genes, mimicking silk motifs or being entirely synthetic with defined functionality. The follow-up strategy would be to modify the resulting monomeric soluble protein either with the desired functionality prior to assembly into fibers, or to incorporate a reactive group that will subsequently permit the conjunction with desired functionality after the fibers have assembled. ::: ![](1475-2859-3-14-5) :::	PubMed Central	2024-06-05T03:55:49.963245	2004-11-16	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534800/", "journal": "Microb Cell Fact. 2004 Nov 16; 3:14", "authors": [ { "first": "Thomas", "last": "Scheibel" } ] }
PMC534801	Introduction ============ Plasmodium vivaxis the most widely distributed human parasite, with an estimated burden of 70--80 million cases annually \[[@B1]\]. In some parts of the world (Asia, South America), it is the most prevalent form of the four human malarial parasites. Although it causes a less severe disease than Plasmodium falciparum, being rarely lethal, P. vivaxaffects the working capacity of the population and the lack of efficient drug distribution favors the onset of drug resistant strains \[[@B2],[@B3]\]. Imported malaria is an increasing health problem in Western Europe, where about 6,500 cases are reported annually in Germany, France, Italy and the United Kingdom \[[@B4]\]. Although P. falciparuminfections account for the majority of cases (64%), P. vivaxis responsible for an additional 23% \[[@B4]\]. Presence in this area of residual anopheline populations susceptible to P. vivaxinfection represents a permanent risk for the occurrence of P. vivaxindigenous malaria cases, as recently occurred in central Italy \[[@B5],[@B6]\]. Since 1970, malaria had been eradicated in central Asia, except for some residual foci in two countries belonging to the Newly Independent States (NIS), i. e. Azerbaijan and Tajikistan (WHO, Regional Office for Europe, unpublished document). At the beginning of the 1990s, the situation changed dramatically due to the re-emergence of malaria in the NIS area and especially in Tajikistan, where at present an epidemic is still in progress \[[@B7],[@B8]\]. In these countries, the existing state of the primary health care system is extremely precarious, especially in rural areas and in small villages. Malaria is a common disease, which can easily re-establish itself when a lack of control occurs. In comparison with P. falciparum, molecular studies of the genetic diversity and dissemination of P. vivaxare scanty. Recently, 33 polymorphic tandem repeats (TRs) of P. vivaxand a P. vivaxpolymorphic microsatellite have been identified and shown to be useful in population studies \[[@B9],[@B10]\]. The merozoite surface protein 3α (MSP3-α) gene also seems to be a good candidate for studying the genetic diversity of P. vivaxpopulations, since PCR-RFLP products indicate the presence of up to 13 alleles \[[@B11],[@B12]\]. However, the circumsporozoite protein (CSP) and merozoite surface protein 1 (MSP-1) genes still remain the most studied molecular markers in genetic epidemiological surveys carried out in P. vivaxendemic areas. In the frame of a malaria research project funded by the European Commission, a molecular study was undertaken in Azerbaijan, aimed at collecting information on the genetic make-up of P. vivaxnatural populations present in this endemic country. For this purpose the extent of polymorphism of CSP and MSP-1 genes were analysed in parasite isolates from five localities of central Azerbaijan by using PCR amplification and sequencing. Materials and Methods ===================== Study area and samples collection --------------------------------- Azerbaijan covers an area of 29.540 Km^2^, with a populations of approximatively 2,5 millions. The climate is typically continental with an average temperature between 12 and 15°C and a rainfall between 200 and 600 mm per year. Climatic and agro-ecological conditions of this area make the environment favourable to mosquito vectors breeding. The major malaria vector is Anopheles sacharovithat breeds preferably in lakes, swamps, irrigation canals and pools. Although it prefers well-oxygenated water, it is known to tolerate moderate salty water. Other anopheline species found in this area are A. maculipennis, A. subalpinus, A. superpictusand A. hyrcanus, all of which are considered secondary vectors of malaria transmission \[[@B13]\]. Malaria transmission occurs in Azerbaijan mainly from June to October. In the last years, number of malaria cases showed a negative trend, accounting for 610 cases in 2000, 418 cases in 2001, 203 cases in 2002. During summer 2002, a malaria epidemiological survey was performed in central Azerbaijan, in the frame of a Malaria Surveillance Programme launched in year 2001 by the Ministry of Health in collaboration with WHO. Active case detection was carried out in five districts included in previously identified sentinel sites, namely Mingaçevir, Beylagan, Imisli, Saatli and Sabirabad. A map of Azerbaijan with the study area is shown in Figure [1](#F1){ref-type="fig"}. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Map of central Azerbaijan showing localities (underlined names on the map) included in the present study. ::: ![](1475-2875-3-40-1) ::: All individuals who visited the district health centres or were found in villages with a history of recent fever and no history of travel in the past few months were considered. In this context, a total of 36 infected individuals with positives blood smears at the microscopic examination, collected between August and September 2002, were selected for the genetic study. The age of patients ranged from 6 to 78 years and parasitaemia varied from 288 to 12,800 parasites/μl. For the molecular analysis, a blood sample of about 1 ml was taken from by venipuncture before drug treatment was given. Patients or the guardians of children were informed about the study. According to the international rules for research involving human subjects, any information which would identify a participant was removed in order to keep each sample processed anonymous. Number of samples for each district is shown in Table [1](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Geographic origin of Azerbaijan isolates with the corresponding MSP-1 and CSP characteristics identified in the present study. ::: ISOLATE NAMES MSP-1 CSP ------------------- ------------- --------- --- ------- --- Bey1 Belem 21 G VK210 4 Bey2 Belem 21 G \" 4 Bey4 Sal1 \- O \" 5 Bey7 Belem 21 G \" 8 Bey14 recombinant 19 S \" 2 Im3 Belem 21 G \" 4 Im5 Belem 21 G \" 4 Im8 Sal1 \- H \" 1 Im9 Belem 21 G \" 4 Im10 Belem 21 F \" 4 Im11 Belem 21 G \" 4 Im12 Belem 21 C \" 4 Im14 Belem 21 D \" 4 Im15 Belem 21 G \" 4 Min1 Belem 21 G \" 4 Min3 Belem 21 G \" 4 Min6 Sal1 \- L \" 3 Min7 recombinant 19 S \" 2 Min8 Sal1 \- I \" 2 Min9 Belem 21 G \" 4 Min10 Sal1 \- P \" 6 Sat2 Belem 21 G \" 4 Sat3 Belem 21 F \" 4 Sat5 recombinant 18 U \" 5 Sat7 recombinant 18 U \" 5 Sat11 recombinant 12 T \" 5 Sab1 Belem 21 G \" 7 Sab2 Belem 21 A \" 4 Sab4 Belem 21 B \" 4 Sab6 Sal1 \- M \" 4 Sab7 Belem 21 G \" 4 Sab8 Belem 21 G \" 4 Sab10 Sal1 \- M \" 4 Sab12 Belem 21 G \" 4 Sab13 Sal1 \- R \" 4 Sab15 Sal1 \- Q \" 4 ::: DNA preparation --------------- Plasmodial DNA was extracted from 200 μl of each infected blood sample using QIAamp DNA blood kit following the manufacturer\'s instructions (Qiagen, CA). Circumsporozoite (CSP) marker analysis -------------------------------------- The CSP gene was amplified for the most part of samples using PV5 and PV6 primers \[[@B14]\]. Samples that did not provide good PCR products with this set of primers were processed a second time by using CSP-A2 \[[@B15]\] as forward primer and PV6bis (5\'-CACAGGTTACACTGCATGGAGT-3\') as original reverse primer. PCR amplification was performed in a reaction mixture of 50 μl containing the parasite DNA, 1x reaction buffer, 2.5 mM MgC1~2~, 80 μM of each deoxynucleotide triphosphate, 6 pmol of each primer and 1.3 U of Taqpolymerase (Promega, Madison, USA). The PCR programme was: denaturation at 94°C for five minutes; 34 cycles of one minute at 94°C, one minute at 54°C and two minutes at 72°C. The PCR products were separated using electrophoresis on a 1.5 % NuSieve gel and the band of interest was cut out and purified using the QIAquick PCR purification Kit (Qiagen). The purified product was sequenced in both directions using an ABI-PRISM 373 sequencer. Nucleotide or amino acid sequences were aligned first using the CLUSTAL X programme \[[@B16]\] with manual editing and adjustments made using the MUST package \[[@B17]\]. The ExPASy Molecular Biology Server <http://us.expasy.org/> was used to convert nucleotide sequences into amino acid sequences. The GenBank accession numbers of the eight sub-types of VK210 type are from AY792359 to AY 792366. Merozoite surface protein 1 (MSP-1) marker analyses --------------------------------------------------- A portion of the MSP-1 gene (the region encompassing the interspecies conserved blocks ICB5 and ICB6) was amplified using a nested PCR with, respectively, the two outer primers A5 and A6 \[[@B18]\] and the two inner primers MSP1N1 forward and MSP1N2 reverse \[[@B6]\]. The first round of amplification was performed in a reaction mixture of 50 μl containing parasite DNA, 1x reaction buffer, 2.5 mM MgC1~2~, 200 μM of each deoxynucleotide triphosphate, 30 pmol of each primer and 2.5 U of Taqpolymerase (Promega, Madison, USA). For the second round, 10 μl of the first amplification product was added to a fresh PCR mixture with 30 pmoles of each inner primer. The thermal profile was: denaturation at 94°C for five minutes; 35 cycles of 94°C for one minute, 60°C for one minute and 75°C for three minutes. All nested-PCR products were purified by Microcon-PCR (Millipore), following the manufacturer\'s instructions and sequenced in both directions at the MWG Biotech. The results were analysed by means of Omiga 2.0 (ACCELRYS, Cambrige) and Mega 2 (S. Kumar, K. Tamura, M. Nei and Pennsylvania State University) computer programmes. The GenBank accession numbers of the 36 nucleotide sequences from P. vivaxisolates are from AY789657 to AY789692. Distance analyses ----------------- The aligned nucleotide sequences of CSP were converted to a distance matrix (% of differences) using the Net algorithm of the MUST package \[[@B17]\]. The dendrogram was generated using the neighbour-joining method \[[@B19]\]. Bootstrap proportions were used to assess the robustness of the tree with 1,000 bootstrap replications \[[@B20]\]. MSP-1 and CSP data were analysed using the Cavalli-Sforza distance \[[@B21]\] from Genetics v.4.01 package. The dendrogramme was generated using the neighbour-joining method \[[@B19]\] Results ======= CSP marker ---------- CSP sequences obtained from 36 Azerbaijan P. vivaxisolates were found to belong to the VK210 type \[[@B22]\]. The isolates tested displayed variations in the peptide repeat motifs GDRA(A/D)GQPA with different alternations of non-synonymous codons GCT or GAT, respectively, coding for alanine (A) and aspartic acid (D) (Figure [2](#F2){ref-type="fig"}). All our sequence types had the same three repeat units (GDRAAGQPA) at the 3\' end, identical to that of the VK210 type. Furthermore four non-synonymous mutations were found, one being the RDRADGQPA variant (sequence named in the present study as sub-type 1), already described in North Korean and Chinese isolates \[[@B23]\]. In summary, eight different sub-types of VK 210 were observed (Figure [2](#F2){ref-type="fig"} and Figure [4](#F4){ref-type="fig"}). Among all 36 azeri isolates analysed, 24 isolates were found to have identical sequence (sub-type 4, Table [1](#T1){ref-type="table"} and Figure [4](#F4){ref-type="fig"}). In particular, the Beylagan (n = 5) and Mingaçevir (n = 7) isolates appeared the most diversified since they displayed four and five different sub-types respectively. The Imishli (n = 9), Saatli (n = 5) and Sabirabad (n = 10) isolates only showed two different sub-types each-one. Figure [4](#F4){ref-type="fig"} clearly shows that the genetic diversity of CSP is relatively small inside the Azerbaijan isolates when compared to the South Korean and Chinese isolates. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Amino acid sequence alignment of eight CSP sub-types found from 36 Azerbaijan P. vivaxisolates with that of VK210 type (Accession No. M28745). ^a^Imi 8; ^b^Bey 14, Min 7, 8; ^c^Min 6; ^d^Bey 1, 2, Imi 3, 5, 9, 10, 11, 12, 14, 15, Min 1, 3, 9, Sat 2, 3 Sab 2, 4, 6, 7, 8, 10, 12, 13, 15; ^e^Bey 4, Sat 5, 7, 11; ^f^Min 10; ^g^Sab 1; ^h^Bey 7. ::: ![](1475-2875-3-40-2) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Distance tree (built with the neighbor-joining method) inferred from 443 nucleotide positions and 264 variable sites of CSP gene. Numbers on the branches indicate bootstrap proportions (1000 replicates); only bootstrap values above 70 % are displayed on the tree. ::: ![](1475-2875-3-40-4) ::: MSP-1 marker ------------ The majority of Azerbaijan isolates (Table [1](#T1){ref-type="table"}) belong to either the Belem (22 isolates, all with the same poly-Q region of 21 repeats) or the Sal I (9 isolates) types already described \[[@B24]\]. Only 5 P. vivaxisolates were identified as recombinant types. Isolate Satl1 (sequence named in the present study as sub-type T) could be ascribed to the type 3a (accession no. D85252, \[[@B25]\]), while isolates Bey 14 and Min7 (sub-type S) and isolates Sat5 and Sat7 (sub-type U) seem to be the result of recombinant events between the recombinant type 3a and Sal I. All the three recombinant types showed a different number of poly-Q repeats (Table [1](#T1){ref-type="table"}). In addition to these sources of diversity, nucleotide substitutions could be observed, allowing the identification of 17 sub-types (Table [1](#T1){ref-type="table"} and Figure [3](#F3){ref-type="fig"}). The Imisli and Sabirabad districts appeared to be less diversified, accounting for five different genotypes for 9 isolates and six genotypes for 10 isolates, respectively. Finally, Saatli district was found to have the greatest variability, with four different genotypes for 5 isolates. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Amino acid sequence alignment of seventeen MSP-1 sub-types found from 36 Azerbaijan P. vivaxisolates compared with that of MSPlBelem (Accession No. M60807), MSPlSal1 (Accession No. M75674) and recombinant type 3a (D85252). Classification of Azeri isolates according to the different types is shown in Table 1. ::: ![](1475-2875-3-40-3) ::: Combined analysis between the two markers ----------------------------------------- By combining the results of genotyping obtained by CSP and MSP-1, 19 P. vivaxsub-types (Figure [5](#F5){ref-type="fig"}) were identified as circulating in the central region of Azerbaijan. The sub-type named G/4 with the greatest representation (n = 14 isolates), was detected in all districts investigated. Genotypes identified as M/4 and U/5 were observed twice in the districts of Sabirabad and Saatli, respectively. Genotype F/4 was detected once in Imisli and Saatli districts, as was for genotype S/2, detected once in Beylagan and Mingacevir districts. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Neighbour-joining tree from the MSP-1 and CSP data (results in parenthesis) reflecting the relationships between the Azerbaijan P. vivaxisolates. ::: ![](1475-2875-3-40-5) ::: Discussion and conclusions ========================== For CSP, the main variations already reported in the literature consist of two variant sequences, VK210 and VK247 that show a variable number of repeat units, GDRA(A/D)GQPAA and ANGAGNQPG, respectively, with some variant positions within the sequence \[[@B22],[@B23]\]. These two variants have a worldwide distribution, and locally their distribution have been also correlated with climatic gradients or with the Anophelines vector specificity \[[@B26]\]. Studies carried out in some Asian endemic countries, i.e. South and North Korea, China, the Philippines, the Solomon Islands and Thailand \[[@B27],[@B15],[@B28],[@B12]\] suggest that CSP has a limited value as a molecular marker for genetic variability when used alone. In the present study, all the analysed P. vivaxisolates from Azerbaijan were found to belong to the type VK210 and they constitute a group of eight CSP sub-types that closely linked in the dendrogramme shown in Figure [4](#F4){ref-type="fig"}. Differently from what reported by other Authors \[[@B28]\] who showed that the CSP sequence analysis allows detecting the geographic origin of plasmodial isolates, our results did not support its use for tracking the geographic origin of Azeri isolates since we dealt with a limited number of samples studied. No genotype association with particular sampling districts was observed since, for example, the most common sub-type identified (G/4) was present in all five districts. Our results confirmed that MSP-1 is a good polymorphic marker. In particular, the region of the gene known to be highly polymorphic and discriminative between the Belem and Sal I types was analysed \[[@B23]\]. A variable poly-glutamine (poly-Q) region is characteristic of the Belem type and represents the principal source of genetic diversity of this marker. Moreover, a poli-Q region is the recombination site between the two types Belem and Sal I and, as shown in the literature, interallelic recombinations between the two types are frequent \[[@B25]\]. The total genetic diversity observed when including the nucleotide substitutions is relatively important taking into account the low endemicity of studied area. Similar results were observed in Southeastern Iran and in Thailand \[[@B29],[@B12]\], low endemic countries for P. vivaxmalaria as well, where the authors detected the two types Belem and Sal I, together with several recombinant types. In particular, in the study carried out in Iran by Zakeri et al., the analysis of MSP1 genetic diversity on a total of 16 plasmodial isolates leaded to the identification of 14 genetic sub-types. It is worth noting that such a high level of diversity is probably due to the small sample size. Our results show 17 genetic sub-types on a total of 36 isolates analysed and a quite high MSP-1 polymorphism also in Azerbaijan. As suggested in other studies \[[@B12]\] and also reported by Zakeri et al., it is possible to speculate that the observed genetic diversity could be also explained considering the studied area, i.e. central Azerbaijan, as transit road of the country and also of neighboring Asian country, where the circulating P. vivaxpopulations show considerable MSP-1 genetic diversity. However, further studies aimed at collecting more information about people moving within the whole country and to closer countries are needed to verify the above hypothesis. The combined analysis of CSP and MSP1 sequence polymorphism has led to the identification of a total of 19 P. vivaxsub-types, confirming that the simultaneous use of more than one genetic marker in this kind of study enhances the knowledge of genetic diversity existing in the parasite populations. The results of the current study show the circulation of multiple plasmodial clones in the studied area thus leading to the conclusion that malaria surveillance activities must be maintained in Azerbaijan in order to avoid serious disease outbreaks in the future. The understanding of the polymorphism extent in surface antigens as CSP and MSP-1 and the resulting genetic diversity in P. vivaxfield populations could help in implementing malaria control activities being a crucial step for the development of a malaria vaccine. Authors\' contributions ======================= S. Mammadov, N. Aliyev, E. Gasimov were involved in field collection of blood samples and microscopy examinations. M.C. Leclerc and A. Cligny did the CSP sequence analysis and M. Menegon did the MSP-1 sequence analysis. M.C. Leclerc and J.L. Noyer did the distance analyses. M.C. Leclerc wrote the report with major contributions of C Severini, M Menegon and G. Majori. G. Majori coordinated the field activities carried out in Azerbaijan. C. Severini, as scientific coordinator of the VIVAXNIS project mentioned below, got the financial support. Acknowledgements ================ We are grateful to N. G. Eyvazov (MOH/RCHE, Baku) and F. Abdullayev (WHO/EURO, Baku) for their valuable support to our research activities in Azerbaijan. We thank F. Severini (ISS, Rome) for the figures editing and N. Billote (CIRAD, Montpellier) for advises improving the manuscript. The work was supported by a grant from the European Commission, INCO-Copernicus 2 project contract ICA2-CT-2000-10046 (acronym VIVAXNIS).	PubMed Central	2024-06-05T03:55:49.965854	2004-11-9	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534801/", "journal": "Malar J. 2004 Nov 9; 3:40", "authors": [ { "first": "Marie Claude", "last": "Leclerc" }, { "first": "Michela", "last": "Menegon" }, { "first": "Alexandra", "last": "Cligny" }, { "first": "Jean Louis", "last": "Noyer" }, { "first": "Suleyman", "last": "Mammadov" }, { "first": "Namig", "last": "Aliyev" }, { "first": "Elkhan", "last": "Gasimov" }, { "first": "Giancarlo", "last": "Majori" }, { "first": "Carlo", "last": "Severini" } ] }
PMC534802	Background ========== It is estimated that 15 -- 40% of chronic low back pain patients have pain arising from the lumbar zygapophysial joints (ZJ)\[[@B1],[@B2]\]. Previous studies have indicated that historical and physical examination findings cannot predict results from diagnostic ZJ blocks \[[@B1]-[@B6]\]. A specific sequence of injections of local anesthetic under fluoroscopic guidance into the joint space or targeting the medial branches of the dorsal rami, are reference standards for diagnosis. For the ZJ to be considered the sole source of pain, there must be a total or near total pain ablation for a time consistent with the known properties of the anesthetic agent\[[@B7],[@B8]\]. In a recent study by Revel M et al (1998), the authors found that a cluster of seven items, (hereinafter called Revel\'s criteria) were shown to be of value in predicting a 75% reduction of pain following a single intra-articular anesthetic injection into the ZJs. The items in the cluster are: age over 65 years, pain well relieved by recumbency, no exacerbation of pain with: coughing and sneezing, forward flexion, extension, rising from flexion and the extension-rotation test\[[@B9]\]. These authors suggested two clinical strategies: Strategy 1 consists of five or more items being true. Strategy 2 is the same except that one of the true statements must be \'pain well relieved by recumbency\'. Estimated sensitivities and specificities for strategies 1 and 2 were 100/92%, and 66/80% respectively. A subsequent study of 200 consecutive patients using double blocks failed to confirm these findings\[[@B6]\]. Because prognosis for acute low back is good, invasive and expensive and invasive diagnostic testing cannot be justified. However, persistent disabling pain needs more intensive investigation in order to determine appropriate management strategies. Back pain of ZJ origin may be treated using intra-articular steroid injection\[[@B7],[@B10]\]., or radio frequency neurotomy \[[@B11]-[@B14]\]. However, the low prevalence of the condition dictates that a clinical selection process capable of identifying patients unlikely to respond to the initial screening diagnostic block is desirable. Patients with a low probability of a positive anesthetic response need not be subjected to the screening block and the tissue origin of pain should be sought elsewhere. Revel\'s criteria are currently the only documented clinical means by which response to an initial screening block may be predicted, but it remains a provisional finding only, until further research confirms the previous findings. The current study objectives were to estimate the predictive value of the two clinical strategies of Revel et al (1998), using similar measurement parameters (75% reduction in pain VAS after ZJ block), and apply alternative analytic methods to explore any potential utility of the variables used. Methods ======= Design ------ This study utilized a prospective, blinded, concurrent, reference standard-related validity design with intra-articular ZJ or medial branch blocks as the reference standard against which clinical variables were compared. Local Institutional Review Board approval was granted at the beginning of the study. A 75% or more reduction in pain following ZJ block on pain VAS was designated as reference standard A, the same standard used by Revel et al (1998). Based on pain drawings and patient self-report, complete abolition of the patient\'s primary or dominant pain was designated as reference standard B. Dominant pain location was acquired by pain drawing and direct questioning, and documented prior to clinical examination and diagnostic injection in a prospective manner. Post injection dominant pain location was acquired by reference to pain drawings and direct questioning also. Patients -------- Patients with low back pain with or without lower extremity symptoms, referred to a private radiology practice in New Orleans, USA specializing in the diagnosis of spinal pain, were invited to participate in the study. Patients receiving ZJ blocks were either referred specifically for that procedure or had the procedure included in their radiology examination based on pre-injection clinical evaluation by the injectionist (CA). Between May 2001 and October 2002, physical therapists attended the clinic in blocks that ranged from 4 to 8 weeks (ML) and examined patients. Normal scheduling was not affected by the presence of the visiting therapists, so patients were consecutive during these periods. All patients had undergone imaging studies prior to referral from a variety of medical and paramedical practitioners. Some were self-referred. Patients were excluded from the study if they were unwilling to participate, were too frail to tolerate a physical examination, or were deemed by any member of the clinic team to be unable to comprehend the study procedure. Prior to the formal clinical examination, clinic staff recorded basic demographic and medical data. Measurements ------------ ### Pain 100 mm visual analog scales (VAS) scales for current, best and worst pain. A current pain VAS was repeated after the clinical examination and following ZJ blocks. The 23-point Roland-Morris Disability Questionnaire \[[@B15]\] was completed to evaluate disability, and psychosocial distress estimated using the Zung Depression Index\[[@B16]\], the Modified Somatic Perception Questionnaire\[[@B17]\] and the Distress Risk Assessment Method (DRAM) \[[@B18]\]. The physical examination ------------------------ History taking and a structured physical examination were carried out by a physical therapist with 30 years of clinical experience as a manipulative therapist (ML). Some patients were examined by a physical therapist with 17 years experience (SBY). The clinical examination occupied 30 to 60 minutes and included many tests besides those necessary for the current analysis, as part of a larger project. Inconclusive findings or incomplete examinations were documented. The physical examination included a visual assessment of range of motion, recording anatomical location of dominant pain, nerve tension tests, key muscle strength tests, tendon reflex tests, light touch sensitivity, a McKenzie\[[@B19]\] styled examination and where possible, Waddell\'s tests for signs of inappropriate pain behaviour\[[@B20]\]. The data for Revel\'s criteria\[[@B9]\]were obtained in a prospective and systematic manner using standardized language and terminology. The four physical tests that form part of the criteria are depicted in Figure [1](#F1){ref-type="fig"}. (see file Figure [1](#F1){ref-type="fig"} Revels criteria.png) Radiology examination --------------------- Prior to ZJ blocks, the radiologist reviewed case notes and imaging studies, and conducted a physical examination that guided the type of diagnostic procedure to be employed and the target structures. Intra-articular ZJ joint injection or MBB using standard technique\[[@B5]\] was carried out by an interventional radiologist (CA) with 20 years experience, or by an injectionist under his guidance. Patient pain responses to injections were recorded as 0.5 cc Lidocaine 2% was slowly injected into the target joint or at medial branch targets. Pain intensity 100 mm VAS\'s were recorded 30 to 45 minutes post procedure, then hourly in a pain journal for eight hours post-injection. Reference standards A and B were evaluated. A positive anesthetic response was recorded if reduction or abolition of pain lasted the known duration of lidocaine, about one and a half hours. Where appropriate and possible, positive responders were rescheduled for confirmatory blocks using bupivacaine 0.75%. A ZJ source of pain was confirmed if a confirmatory block was positive and relief of pain lasted for at least four hours. Some patients received ZJ blocks and sacroiliac joint injections during the same session. If the combined block was positive, the patient was scheduled to return for confirmatory blocks to identify which structure was responsible for the effect. If the combined block produced less than 75% reduction in pain, a negative ZJ block was recorded. Blinding -------- Physical therapists conducting the clinical examination were blinded to the results of previous imaging studies and diagnostic injections, the Roland, Zung and MSPQ questionnaires. The injectionist was blinded to the results of the physical therapy examination and diagnostic conclusions. Data analysis ------------- Basic statistical values for demographic variables and regression analyses were calculated using statistics software (Minitab version 13.31 © Minitab Inc 2000). Differences between included and excluded patients were evaluated with the student\'s t, chi square, and Kruskal-Wallis tests where appropriate. Significance for differences was set at p \< 0.05. Calculations of sensitivity, specificity, predictive values and likelihood ratios with 95% confidence intervals were performed using Confidence Interval Analysis software © Bryant T.N. 2000\[[@B21]\]. Results ======= Initial ZJ blocks were carried out on 151 chronic low back pain patients. Thirty-four patients were excluded from analysis as they received another intervention in the same procedure session and did not return for differentiating and confirmatory blocks. One case was excluded through incomplete data on Revel\'s criteria. Following ZJ block, 27 of 116 patients satisfied reference standard A. Data required for determination of Reference standard B were missing for five of the 116 cases. Eighteen of these 111 patients satisfied reference standard B. Table [1](#T1){ref-type="table"} contains demographic and other descriptive characteristics with comparisons between included and excluded patients. Included patients had longer time off work than excluded patients (mean 55 versus 99 weeks) but otherwise had similar characteristics. In specifically evaluating Revel\'s criteria against reference standard A, logistic regression failed to achieve significance as a model (n = 108, p = 0.46). Two variables; \"absence of pain with coughing and sneezing\" and \"no exacerbation of pain rising from flexion\", showed a trend towards significance as predictors within the model (p = 0.07). Using Revel\'s criteria within a logistic regression with reference standard B as the response variable, a strong trend towards significance was reached (n = 100, p = 0.06). One component variable (age over 65) individually reached significance within the model: (p = 0.004, odds ratio 16.1 with 95% confidence intervals of 2.4 and 107.8). In the whole sample 12.5% were aged over 65 years whereas of the 19 positive responders six were over 65 years (31.6%). The same patients satisfied both strategies of Revel et al. Estimated sensitivity, specificity, predictive values and positive likelihood ratios for both strategies of Revel et al are presented in Table [2](#T2){ref-type="table"}. With respect to reference standards A & B, sensitivities are low at 11 and 17%, specificities are high at 91 and 93% and likelihood ratios are 1.2 and 2.5 respectively. Of the 27 patients satisfying reference standard A, 13 (48%) returned for confirmatory ZJ blocks. Three of these reported 75% or more reduction in pain following the confirmatory block. None of the three patients with confirmed ZJ pain satisfied Revel\'s criteria. Discussion ========== The current data produces results that are in stark contrast to those of Revel et al (1998) with sensitivity low and specificity high. However, \'no pain with cough and sneeze\' and \'no exacerbation of pain rising from flexion\' approached statistical significance in relation to a 75% reduction pain after ZJ block (reference standard A). These variables are in line with Revel\'s results. Age over 65 is associated with reference standard B (abolition of primary pain). Likelihood ratios for the criteria are lower in the current data also. Some of the differences in results may be explained by a number of factors: 1\. Revel et al\'s study was a placebo controlled design whereas we did not routinely utilize a similar or equivalent control. 2\. The patients in Revel et al\'s sample were older (mean 58 versus 43 years), and had a shorter duration of symptoms (mean 78 versus 160 weeks). 3\. It is also possible that the high level of standardization when acquiring the criteria data in the current study, and the prospective methodology might have contributed to the differences in results. Following anaesthetic blocks, patients frequently state that the pain prompting consultation is abolished, yet a post-procedure pain VAS still registers more than 0/100. In this study reference standard B was evaluated as an alternative to the usual standard, so that pain in areas above the lumbar spine or pain directly attributable to the needle insertion site do not result in inappropriate post procedure VAS scores. In the interests of developing more precise instruments documenting results of diagnostic blocks, we propose that in future studies, the VAS scale should specifically refer to the primary pain complaint for which the diagnostic block is undertaken. Based on the current data, Revel\'s criteria are not suitable as a screening test to select patients suitable or unsuitable for an initial screening ZJ block. Such a screening device should have high sensitivity to ensure that most patients likely to respond are included in the initial diagnostic block. Our study suggests that, at best, Revel\'s criteria, might identify a small subset (11%) of patients likely to respond to a screening block (specificity 93%). Conclusions =========== Neither strategy utilizing Revel\'s criteria is suitable as a clinical screening device for selection of chronic LBP patients for initial diagnostic ZJ blocks. In contrast to Revel\'s findings, the current data demonstrated low sensitivity and high specificity for these clinical criteria. The high specificity of the criteria reported in this study relates to a single uncontrolled screening block. Consequently these criteria can not considered diagnostic of painful lumbar ZJ. Only placebo controlled or double ZJ blocks are able to diagnose this source of low back pain. Competing Interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= ML conceived the design of the study, examined all but 10 patients, carried out data analysis and prepared the manuscript BÖ assisted in project design and manuscript preparation CNA assisted in project design, provided facilities and conducted fluoroscopically guided injections BMcD carried out data analysis and assisted in manuscript preparation. All authors have read and approved the final manuscript. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2474/5/43/prepub> Acknowledgements ================ This projected was supported by grants from The International Spine Injection Society, The New Zealand Society of Physiotherapists Scholarship Fund and the New Zealand Manipulative Therapists Education Trust Fund.Thanks to Sharon B Young for examining 10 patients. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### The four physical examination components of Revel\'s criteria for lumbar zygapophysial joint pain: standing flexion, returning from standing flexion, standing extension, the extension rotation test. ::: ![](1471-2474-5-43-1) ::: ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Basic demographic data results for chronic LBP patients receiving screening ZJ blocks ::: All patients (n = 151) Excluded patients (n = 35) Included patients (n = 116) Significance -------------------------------------- ---------------------------- -------------------------------- --------------------------------- ------------------ -------- ------ --------------- -------- ------- --------- Variable Mean Median STD Mean Median STD Mean Median STD p value Age (years) 44.3 43.0 13.2 48.03 48.0 14.3 43.2 41.5 12.6 0.09 Duration of current symptoms (weeks) 147.1 91.0 170.2 103.7 74.0 93.5 160.2 104.0 186.2 0.08 Time off work (weeks) 89.5 (n = 69) 74.0 82.4 55.4 (n = 15) 56.0 41.3 99.0 (n = 54) 82.0 88.6 0.04\* VAS (today) 55.9 61.0 24.6 50.8 53.0 27.0 57.4 62.0 23.7 0.24 VAS (at best) 30.3 27.0 22.5 24.6 15.0 22.5 32.0 30.5 22.3 0.06 VAS (at worst) 85.8 89.0 13.3 80.6 87.0 18.4 87.4 90.0 10.9 0.07 Roland Morris Questionnaire 17.8 19.0 4.8 18.0 19.0 4.6 17.7 19.0 4.8 0.68 Zung Depression Index 29.7 29.0 11.6 26.1 26.0 11.8 30.7 30.0 11.4 0.06 MSPQ Questionnaire 9.7 9.0 6.8 8.8 8.0 6.4 9.9 9.0 7.0 0.49 \% Male 53.0 47.1 54.7 0.55 \% Smoker 34.4 35.3 34.2 0.98 \% Off work 50.0 44.1 51.7 0.33 \% Previous spinal surgery 28.8 17.7 28.2 0.14 \% traumatic onset 69.5 73.5 68.4 0.78 Notes: 1. p value refers to comparisons between included and excluded patients \* significant at p \< 0.05 ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Diagnostic value of clinical examination variables in relation to single anaesthetic zygapophysial joint block ::: ------------------------------------------------------ -------------------- --------------------- --------------------- -------------------- Reference Standard A (75% reduction in Pain VAS) N = 116^1^ Contingency table Injection result Positive Negative Criteria satisfied 3 8 Criteria not satisfied 24 81 % Sn^3^(95 CI^8^) % Sp^4^(95 CI) % PPV^5^(95 CI) % NPV^6^(95 CI) + LR^7^(95 CI) 11.1 (3.9,28.1) 91.0 (83.3,95.4) 27.3 (9.8,56.6) 77.1 (68.2,84.1) 1.2 (0.3,4.3) Reference Standard B (Abolition of Primary Pain) N = 111^2^ Contingency table Injection result Positive Negative Criteria satisfied 3 6 Criteria not satisfied 15 84 % Sn(95 CI) % Sp(95 CI) % PPV(95 CI) % NPV(95 CI) + LR(95 CI) 16.7 (5.8,39.2) 93.3 (86.2,96.9) 33.3 (12.1,64.6) 84.9 (76.5,90.6) 2.5 (0.7,9.1) ------------------------------------------------------ -------------------- --------------------- --------------------- -------------------- Notes: ^1^n = 116. One case with missing data items. Unable to determine satisfaction of criteria ^2^n = 111. Cases with available data on abolition of primary pain ^3^Sn = sensitivity, Sp^4^= specificity, PPV^5^= positive predictive value, NPV^6^= negative predictive value, +LR^7^= positive likelihood ratio, 95 CI^8^= 95% confidence interval :::	PubMed Central	2024-06-05T03:55:49.967606	2004-11-16	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534802/", "journal": "BMC Musculoskelet Disord. 2004 Nov 16; 5:43", "authors": [ { "first": "Mark", "last": "Laslett" }, { "first": "Birgitta", "last": "Öberg" }, { "first": "Charles N", "last": "Aprill" }, { "first": "Barry", "last": "McDonald" } ] }
PMC534803	Background ========== Chronic asthma is driven and maintained by the persistence of a subset of chronically activated memory T lymphocytes. The development of allergen-specific CD4^+^T-helper 2 (Th2) immunoresponses is responsible for the cellular and molecular events underlying the initiation and progression of allergic asthma \[[@B1],[@B2]\]. The Th2 lymphocyte, therefore, is potentially an important target cell for therapy in allergic asthma. Dendritic cells (DC) are well defined as antigen presenting cells (APC) able to initiate and regulate T cell responses \[[@B3]\]. Besides skewing T-cell responses into Th1 or Th2 responses \[[@B4]\], DC have been shown to mediate the induction of antigen-specific regulatory T (Treg) cells, like CD4^+^Th3 cells and CD4^+^T regulatory 1 (Tr1) cells \[[@B5],[@B6]\]. Macrophages (Mφ), however, can also serve as APC and play a pivotal role in controlling and directing immune responses \[[@B7],[@B8]\]. To exert these functions, Mφ express MHC-II molecules and secrete a variety of mediators. By secreting pro-inflammatory cytokines, such as IL-1, IL-6 and TNF-α, Mφ can trigger immune responses against microbial pathogens \[[@B8],[@B9]\]. Moreover, by releasing IL-12 Mφ can specifically skew immune responses towards Th1 responses \[[@B10]-[@B12]\]. Although Mφ favor the induction of Th1 responses \[[@B13],[@B14]\], it has also been demonstrated that Mφ can induce differentiation of Th2 lymphocytes \[[@B15],[@B16]\]. Similar to DC, Mφ are nowadays thought to be capable of suppressing immune responses by secreting anti-inflammatory mediators, such as PGE~2~, TGF-β and IL-10 \[[@B7],[@B9],[@B17]\]. In the lung, alveolar Mφ participate in the maintenance of immunological homeostasis. By secreting pro-inflammatory cytokines and chemokines they direct the recruitment and activation of inflammatory cells, while they also play a key role in dampening immune responses against non-pathogenic antigens \[[@B9],[@B18]\]. Alveolar Mφ have been shown to suppress T-lymphocyte proliferation in vitro\[[@B19],[@B20]\] and APC-function of DC in vitroand in vivo\[[@B21]\]. Additionally, several studies have demonstrated that Mφ induce tolerance against inhaled allergens, likely at the level of allergen-specific T lymphocytes \[[@B22]-[@B24]\]. Interestingly, selective elimination of alveolar Mφ potentiated IgE Ab production in response to inhaled allergen, indicating a key role for alveolar Mφ in tolerance against allergen inhalation \[[@B25]\]. Moreover, we \[[@B26]\] and others \[[@B12],[@B27]\] demonstrated that treatment with allergen-loaded Mφ effectively suppresses allergen-induced airway manifestations of asthma. In vitrostudies demonstrated that allergen-specific T cells induced IL-10 production by OVA-loaded Mφ (OVA-Mφ), suggesting that the immunosuppressive effects of OVA-Mφ might be mediated by IL-10 \[[@B26]\]. In this study, we investigated whether stimulation with toll like receptor 4 (TLR4)-ligand LPS \[[@B28]\] and the TLR9-ligand immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) \[[@B29]\] increases the IL-10 production by allergen-loaded Mφ and, thereby, can potentiate their immunosuppressive effects. Subsequently, using Mφ isolated from IL-10^-/-^mice, we examined whether Mφ-derived IL-10 is crucial in the suppression of allergen-induced allergic airway inflammation. Methods ======= Animals ------- Animal care and use were performed in accordance with the guidelines of the Dutch Committee of Animal Experiments. Specific pathogen-free (according to the Federation of European Laboratory Animal Science Associations \[[@B30]\]) male BALB/c mice (6 wk old) were purchased from Charles River (Maastricht, The Netherlands). The mice were housed in macrolon cages in a laminar flow cabinet. Breeding pairs of IL10^-/-^BALB/c mice were kindly provided by DNAX (Palo Alto, CA) and were housed in macrolon cages with filter top. All mice were provided with food and water ad libitum. Materials --------- OVA (chicken egg albumin, grade V) and purified LPS from Escherichia coli0111:B4 were purchased from Sigma-Aldrich (St. Louis, MO). CpG-containing phosphorothioate ISS-ODN and control phosphorothioate mutated oligodeoxynucleotide were synthesized by Isogen Bioscience BV (Maarsen, The Netherlands). The ISS-ODN used had the sequence 5\'-TGACTGTGAA-CGTTCGAGATGA-3\' and the mutated-ODN had the sequence 5\'-TGACTGTGAA-GGTTAGAGATGA-3\' \[[@B31]\]. Loading and stimulation of Mφ ----------------------------- Peritoneal Mφ were isolated from naïve BALB/c mice as described previously \[[@B26]\]. For in vitroexperiments, Mφ were plated in triplicate wells of a 96-well round-bottomed plate (Greiner Bio-One GmbH, Kremsmuenster, Austria) at 1 × 10^5^Mφ/well in RPMI 1640 enriched with 2% FCS, penicillin/streptomycin (all GIBCO BRL) and 50 μM β-mercaptoethanol (Sigma-Aldrich). Mφ were loaded with 2 mg/mL OVA and stimulated with different concentrations of LPS, ISS-ODN or mutated-ODN, for 20 h at 37°C and 5% CO~2~. Subsequently, supernatants were harvested and the amount of IL-10 was determined using an IL-10-specific sandwich ELISA. Stimulation with 10 μg/mL LPS or 3 μg/mL ISS-ODN triggered the highest IL-10 production by Mφ. For in vivostudies, 1 × 10^7^Mφ/mL were loaded with 2 mg/mL OVA and were stimulated with 10 μg/mL LPS or 3 μg/mL ISS-ODN. After incubation for 3 h at 37°C and 5% CO~2~, the Mφ were extensively washed (3 times with 50 mL saline) to remove all residual soluble OVA, LPS, and ISS-ODN. Sensitization, treatment and challenge -------------------------------------- Mice were sensitized to OVA by active sensitization with 7 i.p. injections of 10 μg OVA in 0.5 mL pyrogen-free saline on alternate days \[[@B32]\]. Treatment was performed 17 days after the last sensitization by administration (i.v.) of 3 × 10^5^Mφ in 50 μl saline. As an additional control group, mice were i.v. injected with 50 μL saline (sham treatment). One week after treatment, mice were exposed to OVA (2 mg/mL saline) aerosol challenges for 5 min on 8 consecutive days. Determination of OVA-specific IgE levels in serum ------------------------------------------------- Mice were sacrificed and were bled by cardiac puncture. Subsequently, serum was collected and stored at -70°C until analysis. OVA-specific IgE in serum was measured as described \[[@B33]\]. A reference standard was obtained by i.p. immunization of mice with OVA and arbitrarily assigned a value of 1000 experimental units/mL (EU/mL). The detection level of the IgE ELISA was 0.5 U/mL for IgE. Analysis of the cellular composition in the bronchoalveolar lavage fluid ------------------------------------------------------------------------ Bronchoalveolar lavage (BAL) was performed immediately after bleeding of the mice by lavage of the airways through a tracheal cannula with 1 mL saline containing 2 μg/mL aprotinine (Roche Diagnostics) and 5% BSA (Sigma-Aldrich). Cytokines in the supernatant of this first mL of the BAL fluid (BALF) were determined by ELISA. Subsequently, mice were lavaged 4 times with 1 mL saline. The cells in the BALF were pooled in cold PBS (including those from the first mL) and subsequently differentiated into mononuclear cells (monocytes, Mφ and lymphocytes), eosinophils, and neutrophils as described previously \[[@B33]\]. Cytokine ELISAs --------------- IL-5, IL-10, IL-12p70 ELISAs (all BD PharMingen) were performed according to the manufacturer\'s instructions. The detection limit of the IL-5 ELISA was 10 pg/mL, of the IL-10 ELISA 15 pg/mL, and of the IL-12p70 ELISA 62.5 pg/mL. Statistical analysis -------------------- All data are expressed as mean ± standard error of mean (SEM). Statistical analysis on BALF cell counts was performed using the non-parametric Mann-Whitney Utest (2-tailed). For ELISA, results were statistical analyzed using a Student\'s ttest (2-tailed, homoscedastic). Results were considered statistically significant at the P\< .05 level. Results ======= IL-10 production by Mφ is increased by LPS and ISS-ODN ------------------------------------------------------ In vitrostudies suggest that the immunosuppressive effects of OVA-Mφ could be mediated by Mφ-derived IL-10 \[[@B26]\]. To further enhance these immunosuppressive effects we attempted to increase the IL-10 levels produced by Mφ. Since Mφ express TLR4 and TLR9 \[[@B28],[@B29]\], we tested whether activation of these receptors (using LPS and ISS-ODN, respectively) would increase the IL-10 production by peritoneal Mφ. As Figure [1](#F1){ref-type="fig"} shows, stimulation with LPS or ISS-ODN highly increased the IL-10 production by OVA-Mφ in vitro, while control mutated oligodeoxynucleotide did not. The IL-10 levels produced by OVA-Mφ increased 2.5-fold upon stimulation with LPS and 4.5-fold upon stimulation with ISS-ODN. IL-12p70 was not detectable in any of these cultures (data not shown). ::: {#F1 .fig} Figure 1 ::: {.caption} ###### LPS and immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) enhance the IL-10 production by OVA-Mφ. 1 × 10^5^ Mφ/well were loaded with 2 mg/ml OVA and stimulated with either 10 μg/ml LPS or 3 μg/ml ISS-ODN for 20 h. As a control, Mφ were stimulated with mutated-ODN (M-ODN, 3 μg/ml). One of four representative experiments is shown. ::: ![](1465-9921-5-21-1) ::: Increased production of IL-10 potentiates the suppressive effects of OVA-Mφ --------------------------------------------------------------------------- To examine the in vivoeffect of the increased production of IL-10 by OVA-Mφ, peritoneal Mφ were isolated and subsequently loaded with OVA and stimulated with LPS (10 μg/mL) or ISS-ODN (3 μg/mL) for 3 h. The stimulated and OVA-loaded Mφ were administered (i.v.) to OVA-sensitized mice. In sham-treated mice, OVA-inhalation challenge strongly increased OVA-specific IgE Ab in serum (Figure [2](#F2){ref-type="fig"}). Treatment with OVA-Mφ that were not stimulated or stimulated with LPS caused no significant suppression in the up-regulation of serum OVA-specific IgE (Figure [2](#F2){ref-type="fig"}). In contrast, ISS-ODN-stimulated OVA-Mφ significantly suppressed (60%, P\< .05) the up-regulation of serum OVA-specific IgE (Figure [2](#F2){ref-type="fig"}). OVA-specific IgG2a levels in serum of sham-treated mice were also increased upon OVA-inhalation challenge. However, these levels were not affected upon treatment with OVA-Mφ or stimulated OVA-Mφ (data not shown). ::: {#F2 .fig} Figure 2 ::: {.caption} ###### OVA-specific IgE levels in serum are significantly suppressed upon treatment with ISS-ODN-stimulated and OVA-loaded Mφ. OVA-sensitized BALB/c mice were treated (i.v.) with saline (sham), OVA-Mφ, ISS-ODN-stimulated OVA-Mφ (ISS-ODN/OVA-Mφ), or LPS-stimulated OVA-Mφ (LPS/OVA-Mφ). Subsequently, these mice were challenged by OVA inhalation. Serum OVA-specific IgE levels were measured prior to and after challenge. Values are expressed as the mean ± SEM (n = 6 to 8). \*P\< .05 compared with sham-treated and OVA-challenged mice. ^†^P\< .05 compared with mice treated with OVA-Mφ and that were OVA-challenged. ::: ![](1465-9921-5-21-2) ::: The BALF of mice, sensitized and challenged with OVA, contained high numbers of eosinophils (Figure [3A](#F3){ref-type="fig"}). OVA-Mφ partially suppressed (30%, not significant) the influx of eosinophils into the BALF. Ex vivostimulation of OVA-Mφ with LPS further enhanced the suppression of airway eosinophilia (60%, P\< .05), compared with sham-treated mice (Figure [3A](#F3){ref-type="fig"}). OVA-Mφ stimulated with ISS-ODN effectively suppressed the airway eosinophilia. The number of eosinophils in the BALF were significantly (P\< .01) suppressed by 85% compared to sham-treated mice and by 79% compared to mice treated with OVA-Mφ (Figure [3A](#F3){ref-type="fig"}). ::: {#F3 .fig} Figure 3 ::: {.caption} ###### ISS-ODN-stimulated and OVA-loaded Mφ (ISS-ODN/OVA-Mφ) significantly suppress airway eosinophilia and IL-5 levels in the bronchoalveolar lavage fluid. The number of eosinophils (eo), neutrophils (neutro) and mononuclear cells (MNC) in the BALF (A), and IL-5 levels in the BALF (B) after OVA inhalation challenge. Values are expressed as the mean ± SEM (n = 6 to 8). \*P\< .01 and ^†^P\< .05 compared with sham-treated mice. ^‡^P\< .01 and ^§^P\< .05 compared with mice treated with OVA-Mφ. ::: ![](1465-9921-5-21-3) ::: The BALF of sham-treated mice contained high levels of the Th2 cytokine IL-5 (Figure [3B](#F3){ref-type="fig"}), that correlates with the numbers of eosinophils. Treatment with OVA-Mφ or LPS-stimulated OVA-Mφ reduced the IL-5 levels in the BALF by 50% (p = 0.07 and p = 0.06, respectively), compared to sham-treated mice (Figure [3B](#F3){ref-type="fig"}). ISS-ODN-stimulated OVA-Mφ, significantly reduced (P\< .01) the IL-5 levels in the BALF by 80%, compared to sham-treated mice. IL-10 was not detectable in the BALF of any of the mice (data not shown). Since ISS-ODN-stimulated OVA-Mφ produced the highest levels of IL-10 and most strongly suppressed OVA-induced airway inflammation, we used these Mφ to further analyze the underlying mechanism of immunosuppression by allergen-loaded Mφ. IL-10 produced by OVA-Mφ suppress OVA-induced airway inflammation ----------------------------------------------------------------- To prove that IL-10 produced by OVA-Mφ indeed mediates the observed immunosuppressive effects, we isolated peritoneal Mφ from IL-10^-/-^BALB/c mice. After loading with OVA and stimulation with ISS-ODN ex vivo, the IL-10^-/-^Mφ were administered (i.v.) to OVA-sensitized BALB/c mice. Serum OVA-specific IgE levels of allergic mice that were treated with ISS-ODN-stimulated IL-10^-/-^Mφ were as high as that of sham-treated mice (Figure [4](#F4){ref-type="fig"}). However, the up-regulation of serum OVA-specific IgE levels was partially affected by ISS-ODN-stimulated IL-10^-/-^OVA-Mφ (Figure [4](#F4){ref-type="fig"}). The serum OVA-specific IgE levels were approximately 50% suppressed compared with unloaded ISS-ODN-stimulated IL-10^-/-^Mφ. Still, these IgE levels were 45% higher (P\< .05) than in mice treated with ISS-ODN-stimulated OVA-Mφ. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### The suppression of OVA-specific IgE in serum by ISS-ODN-stimulated and OVA-loaded Mφ is partially mediated by IL-10. Sensitized mice were treated (i.v.) with saline (sham), ISS-ODN-stimulated OVA-Mφ (ISS-ODN/OVA-Mφ), ISS-ODN-stimulated IL-10^-/-^Mφ (ISS-ODN/IL10^-/-^Mφ), or ISS-ODN-stimulated IL-10^-/-^OVA-Mφ (ISS-ODN/IL10^-/-^OVA-Mφ). Serum OVA-specific IgE levels were measured prior to and after OVA challenge. Values are expressed as the mean ± SEM (n = 6 to 8 per group). \*P\< .05 compared with sham-treated and OVA-challenged mice. ^†^P\< .05 compared with mice treated with ISS-ODN/IL10^-/-^OVA-Mφ and that were OVA-challenged. ::: ![](1465-9921-5-21-4) ::: Importantly, after OVA-inhalation challenge, treatment with ISS-ODN-stimulated IL-10^-/-^OVA-Mφ did not suppress airway eosinophilia (Figure [5A](#F5){ref-type="fig"}). Unloaded IL-10^-/-^Mφ that were stimulated with ISS-ODN had also no effect on airway eosinophilia, while immunotherapy with ISS-ODN-stimulated OVA-Mφ suppressed the influx of eosinophils by 88% (P\< .05), compared to sham-treated mice (Figure [5A](#F5){ref-type="fig"}). ::: {#F5 .fig} Figure 5 ::: {.caption} ###### IL-10 is crucial in the suppression of airway eosinophilia and IL-5 levels in the bronchoalveolar lavage fluid by ISS-ODN-stimulated and OVA-loaded Mφ (ISS-ODN/OVA-Mφ). (A) Number of eosinophils (eo), neutrophils (neutro) and mononuclear cells (MNC) in the BALF after OVA challenge. (B) Levels of IL-5 in the BALF after OVA challenge. Values are expressed as the mean ± SEM (n = 6 to 8 per group). \*P\< .05 compared with sham-treated mice. ^†^P\< .05 compared with mice treated with ISS-ODN/IL10^-/-^OVA-Mφ. ::: ![](1465-9921-5-21-5) ::: The BALF of sham-treated mice, sensitized and challenged with OVA, contained high levels of IL-5 (Figure [5B](#F5){ref-type="fig"}). Treatment with ISS-ODN-stimulated IL-10^-/-^OVA-Mφ as well as with ISS-ODN-stimulated IL-10^-/-^Mφ had no effect on the IL-5 levels in the BALF (Figure [5B](#F5){ref-type="fig"}). In contrast, these IL-5 levels were significantly reduced by 78% (P\< .05) upon treatment with ISS-ODN-stimulated OVA-Mφ (Figure [5B](#F5){ref-type="fig"}). Together, IL-10 produced by OVA-Mφ mediated the anti-inflammatory effects of allergen-loaded Mφ on allergen-induced airway eosinophilia and IL-5 production. Discussion ========== Previously, we showed that allergen-loaded Mφ partially suppress allergen-induced airway manifestations in mice \[[@B26]\]. Here, we demonstrated that the anti-inflammatory effects of allergen-loaded Mφ are IL-10 dependent and that both the IL-10 production and the immunosuppressive effects can be potentiated by stimulation of ISS-ODN. Stimulation with ISS-ODN, a ligand for TLR9 \[[@B29]\], readily increased the IL-10-production by peritoneal Mφ that were loaded with OVA. In contrast, these Mφ produced no detectable IL-12p70. We (data not shown) and others \[[@B34]\] confirmed these data using the Mφ-like cell line RAW264.7. In our mouse model of allergic asthma, stimulation of OVA-Mφ with ISS-ODN significantly potentiated their immunosuppressive effects. The suppression of the OVA-induced serum OVA-specific IgE levels, airway eosinophilia and IL-5 levels in the BALF was enhanced. Measuring the enhanced pause (Penh) in response to inhalation of different doses of methacholine (data not shown), we confirmed our previous finding that OVA-Mφ significantly suppressed OVA-induced airway hyperresponsiveness to methacholine \[[@B26]\]. As potentiation of the IL-10 production by Mφ (using LPS or ISS-ODN) did not further suppress the allergen-induced airway hyperresponsiveness (data not shown), it can be speculated that the mechanisms underlying the suppression of airway hyperresponsiveness and of allergic airway inflammation, at least in part, differ. However, we would like to stress that Penh values may not correlate with changes in pulmonary resistance \[[@B35]\]. Compared to ISS-ODN, stimulation with LPS showed an intermediate capacity to enhance the immunosuppressive effects of OVA-Mφ. OVA-Mφ produced higher levels of IL-10 upon stimulation with ISS-ODN compared to stimulation with LPS, suggesting a correlation between the levels of IL-10 produced by the Mφ and the extent of suppression of allergen-induced airway inflammation. As LPS and ISS-ODN trigger signaling via different intracellular pathways \[[@B36]\], we hypothesized that stimulation of Mφ with a combination of LPS and ISS-ODN could further increase the production of IL-10. However, the levels of IL-10 produced by OVA-Mφ stimulated with LPS and ISS-ODN were as high as when stimulated with ISS-ODN only (data not shown). Implying that stimulation of Mφ with ISS-ODN results in maximal production of IL-10. Using Mφ isolated from IL-10^-/-^BALB/c mice, we demonstrated that Mφ-derived IL-10 is crucial in the suppression of airway eosinophilia and IL-5 levels in the BALF, while the suppression of serum IgE is partially IL-10 dependent. Although a lack of IL-10 production could up-regulate the MHC class II and B7 expression in Mφ of IL-10^-/-^mice, as increased IL-10 levels could down-regulate these molecules \[[@B37],[@B38]\], the shift in expression of these molecules will most probably not be the underlying mechanism of suppression of airway eosinophilia and Th2 cytokines, because low levels of MHC class II or B7 itself do not result in suppressive functions. Furthermore, we can not exclude that there are, at present unknown, developmental changes in Mφ derived from IL-10-deficient mice, that may affect their capacity to suppress an allergic inflammatory response. The observation that the suppression of IgE is partially IL-10 independent suggests that the suppression of serum IgE levels is only slightly correlated to Th2-cytokine levels. This is in agreement with the finding that memory IgE responses are inferior mediated by Th2 cytokines \[[@B39]\]. These data indicate that a second, IL-10 independent, suppressive pathway has to be induced by OVA-Mφ that causes a further suppression of serum IgE levels. Mφ can reside for long period of time in tissue, including the lung \[[@B40]\]. By secreting the immunosuppressive cytokine IL-10 the Mφ could, allergen-independently, suppress allergen-induced airway inflammation. In the past, IL-10 has been shown to suppress allergen-induced airway manifestations of asthma in the mouse \[[@B41]-[@B43]\]. However, we found that, after i.v. treatment, the ISS-ODN-stimulated OVA-Mφ specifically migrate to the spleen of OVA-sensitized mice. Subsequently, at this site, an allergen-specific and long-lasting immunosuppressive response is induced (preliminary results by Vissers JLM et al). These results demonstrate that the IL-10 produced by the Mφ is not directly responsible for the suppression of allergic inflammation in the lungs, but that an allergen-specific suppressive T-cell subset is induced in the spleen. This hypothesis is supported by the finding that IL-10 production by OVA-Mφ, upon recognition of OVA-specific T cells in vitro, is dependent on MHC class II/TCR interaction \[[@B26]\]. Direct targeting of OVA to alveolar Mφ, for instance by intratracheal treatment with OVA-loaded liposomes, could demonstrate whether Mφ in the lung can directly induce suppression of OVA-induced airway inflammation. However, we (data not shown) and others \[[@B44]\] found that alveolar Mφ from OVA-sensitized mice do not produce IL-10 upon stimulation with LPS or ISS-ODN. Although we were not able to test the suppressive capacity of OVA-loaded alveolar Mφ in our mouse model, targeting of allergens to alveolar Mφ could still be promising to induce immunosuppressive effects in humans, because alveolar Mφ from patients with allergic asthma produce IL-10 \[[@B45],[@B46]\]. In this study and in our previous study \[[@B26]\], we observed no indications for an increased Th1 response upon immunotherapy with OVA-Mφ that could counteract the Th2 response. In contrast, others demonstrated that allergen-loaded Mφ, stimulated with IFN-γ ex vivo, promote a switch from Th2 cells to Th1 cells \[[@B12],[@B27]\]. This dissimilarity can mainly be explained by the difference in cytokines which are produced by the Mφ used. IFN-γ-stimulated Mφ produce IL-12 upon allergen-specific interaction with T cells \[[@B12]\], while our Mφ produce IL-10 upon antigen recognition \[[@B26]\]. Likely, IL-12 will favor skewing towards Th1 \[[@B4],[@B11]\], whereas IL-10 will act as a suppressive cytokine. In our model, allergen-loaded Mφ will, most probably, induce Treg cells via secretion of IL-10. Antigen-induced Treg cells are typically induced in microenvironments with APCs presenting antigens and local high levels of IL-10 \[[@B6]\]. This T-cell subset plays a pivotal role in the maintenance of T-cell tolerance against foreign-antigens. They exhibit their suppressive activity by secreting the suppressive cytokine IL-10 (Tr1 cells) or TGF-β (Th3 cells) \[[@B47]\]. By using CD4^+^T lymphocytes, ex vivotransduced to express IL-10, it was shown that allergen-specific lymphocytes can suppress allergen-induced asthma manifestations via production of IL-10 \[[@B43]\]. Recently, Akbari and colleagues found that pulmonary dendritic cells from mice exposed to respiratory allergen produced IL-10 and, thereby, induced allergen-specific Tr1 cells \[[@B5],[@B48]\]. Furthermore, treatment of mice with killed Mycobacterium vaccaeinduced allergen-specific Treg cells that produced IL-10 and TGF-β \[[@B49]\]. In agreement with our study, these studies indicate a pivotal role for IL-10 in limiting allergen-induced asthma manifestations. Conclusions =========== Here, we demonstrated, in a mouse model of allergic airway inflammation, that treatment with allergen-loaded Mφ suppress asthma manifestations in an IL-10-dependent manner. Importantly, the IL-10 production and anti-inflammatory effects of allergen-loaded Mφ can be potentiated by stimulation with ISS-ODN. Further detailed analysis of the mechanisms underlying this Mφ-based immunotherapy may lead to the development of new strategies to induce tolerance against allergen-specific Th2 responses in allergic diseases, including asthma. Authors\' contributions ======================= JLMV carried out the allergic model, subsequent analysis, writing and preparation of the manuscript. BCAMvE, PVJ and GAH assisted with the allergic model. AJMvO participated in the direction of the study as well as writing and preparing the manuscript. All authors read and approved the final manuscript. Acknowledgments =============== This study was supported by research grants 3.2.00.48 (JLMV) and 3.2.99.23 (PVJ) from The Dutch Asthma Foundation and by Stichting Astma Bestrijding (BCAMvE). We thank Dr. D. Rennick (DNAX) for providing us the breeding pairs of IL10^-/-^BALB/c mice. We thank Dr. Nanne Bloksma for stimulating discussions and Dr. Sue McKay for critical reading of the manuscript.	PubMed Central	2024-06-05T03:55:49.970205	2004-11-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534803/", "journal": "Respir Res. 2004 Nov 11; 5(1):21", "authors": [ { "first": "Joost LM", "last": "Vissers" }, { "first": "Betty CAM", "last": "van Esch" }, { "first": "Prescilla V", "last": "Jeurink" }, { "first": "Gerard A", "last": "Hofman" }, { "first": "Antoon JM", "last": "van Oosterhout" } ] }
PMC534804	Background ========== Management of acute myocardial infarction (AMI) has underwent considerable changes in the last two decades and the management of patients with AMI has become more established \[[@B1],[@B2]\]. In patients with ST-segment elevation myocardial infarction (STEMI), primary angioplasty is becoming first-choice therapy \[[@B2]-[@B4]\]. However, because of the low availability of such treatment, even in developed countries, most patients with STEMI are reperfused with intravenous thrombolysis. Many patients do not receive a reperfusion therapy at all \[[@B5],[@B6]\]. Data from large clinical trials indicate that after successful thrombolysis more than 50% of patients have a significant residual stenosis and about 30% of patients have spontaneous or inducible ischemia \[[@B7]-[@B9]\]. In this group reocclusion of the infarct-related artery is a potential threat, as it is associated with recurrent ischemia or recurrent infarction \[[@B10],[@B11]\]. Therefore, early risk assessment is of great importance, especially in patients treated with thrombolysis and patients who did not receive reperfusion therapy. This risk assessment should be followed by an effective treatment strategy in order to prevent recurrent cardiac events and deterioration of left ventricular function \[[@B12],[@B13]\]. As these recurrent events are mainly due to the presence of an unstable residual stenosis of the infarct-related coronary artery, an effective therapy should include an invasive procedure like angioplasty to optimize coronary flow to the infarct area in high risk patients \[[@B11]\]. Trials have been performed to study the effect of routine angioplasty on clinical outcome early after AMI treated with thrombolysis \[[@B14]-[@B20]\]. Most of these studies failed to show improvement of clinical outcome with a standard invasive approach. Possible explanations for these results are the unselected approach, and the high risk profile of balloon angioplasty in the early days, when abciximab and stents were not available. Because angioplasty always carries an inherent risk, it remains important to select those patients after a recent myocardial infarction, who will actually benefit from angioplasty of the infarct-related artery. To date, non-invasive risk stratification after AMI has mainly focused on exercise stress testing. The inability to exercise, the low diagnostic accuracy and resting ECG abnormalities, however, remain important limitations in the detection of ischemia \[[@B21]\]. Several post-infarction observational studies investigated viability as prognosticator and showed that the presence of viability in the infarct area was highly predictive for future coronary events like recurrent ischemia, recurrent infarction, left ventricular failure, and death \[[@B22]-[@B31]\]. Viable tissue is potentially jeopardized by an unstable residual stenosis in the infarct-related coronary artery. In a meta-analysis of non-randomized data by Iskandrian, the impact of revascularization on clinical outcome in patients with viability after AMI was studied \[[@B32]\]. A significant decrease in future cardiac events was observed in the patients with viability who were revascularized. In contrast, the outcome in patients without viability in the infarct area did not change by an invasive strategy. In this context it should be noted that other post-infarction studies have shown viability to be associated with an improved prognosis \[[@B33]-[@B36]\]. Two studies demonstrated that especially patients with severe LV dysfunction and viability show a better survival than patients with LV dysfunction but without viability \[[@B34],[@B36]\]. The reason for this paradox is not quite understood. However, it can be argued that patients with viability have a potential of recovery of LV function (spontaneous or by revascularization), thereby improving their survival \[[@B37]\]. Thus, on one hand viability may improve survival by recovery of function especially in patients with moderate to severe LV dysfunction, on the other hand viability is related to a worse prognosis by increased risk of recurrent ischemic events. Based on the aforementioned assumptions, we initiated a clinical trial to investigate the benefits of percutaneous coronary intervention (PCI) of the infarct-related artery in patients with viability detected in the early, subacute phase of myocardial infarction. To demonstrate viability, we use low-dose dobutamine echocardiography (LDDE). This test can safely be performed 48 hours after acute MI \[[@B23],[@B27],[@B30],[@B31]\]. It is a well validated bedside test with a diagnostic accuracy of about 80%, which is comparable to scintigraphical techniques (SPECT/PET) \[[@B38]\]. Coronary stenting will be performed with concomitant use of abciximab. After stenting, oral clopidogrel is given in a standard way. With this approach the lowest possible periprocedural event rate will be attained, with a low rate of target vessel revascularization (TVR) \[[@B39]-[@B43]\]. We hypothesize that, in order to prevent future cardiac events, PCI is only useful in patients with viability in the infarct zone early after myocardial infarction. Methods ======= Patient Selection ----------------- Patients admitted to any of the participating centers with an acute or recent myocardial infarction, who are not treated by direct or rescue angioplasty, and who are stable during the first 48 hours after the acute event, are screened for the study. Patients \< 80 years of age are considered suitable for the study when they have definite myocardial infarction, as demonstrated by an significant rise in creatine kinase-MB levels (twice the upper limit of normal: ULN), 1 mm ST segment elevation in two or more standard leads or 2 mm ST segment elevation in two contiguous chest leads, and/or the development of Q waves. The criteria for exclusion are: viability testing technically not possible (poor echo-window), contraindications for dobutamine echocardiography (arrhythmia), and coronary angiography (severe diabetic nephropathy or known contrast-allergy), serious life-threatening non-cardiac illness, ECG abnormalities making the evaluation of the ST segment impossible (left bundle branch block, pacemaker), and an unreliable follow-up. The study complies with the Declaration of Helsinki and all ethics committees of the participating centers have approved the protocol. All eligible patients provide written informed consent. Study design ------------ The study is a prospective, multicenter, randomized, controlled clinical trial. In the VIAMI-trial, patients who are admitted to the hospital with an acute myocardial infarction and who did not undergo primary or rescue PCI, are evaluated by LDDE within 3 days of admission. Patients with unequivocal signs of viability in the infarct-area are randomized to an invasive or a conservative treatment strategy. In the invasive strategy patients undergo coronary angiography with the intention to perform PCI with stenting of the infarct-related coronary artery. In the conservative group an ischemia-guided approach is adopted with stress testing before discharge from the hospital. When the test is highly suggestive for ischemia, coronary angiography will be performed. If revascularization is performed, this will be scored as a secondary endpoint. Patients without viability will serve as a registry group with long-term follow-up (Fig [1](#F1){ref-type="fig"}). These patients are assigned to the conservative group in order to prevent physicians\' bias during the trial. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Flow chart ::: ![](1468-6708-5-11-1) ::: The primary endpoint is the composite of death from any cause, recurrent infarction, and unstable angina. The secondary endpoints are need for revascularization, the occurrence of angina pectoris (CCS classification), and the incidence of heart failure (NYHA classification). Left ventricular function is also evaluated as determined by echocardiography at 3 months, 6 months, and 1 year follow-up. A reinfarction is diagnosed if there is an increase in the total creatine kinase and MB isoenzyme activity (2 times ULN) and either a history of ischemic chest discomfort, or electrocardiographic changes indicative for transmural ischemia or necrosis. For the diagnosis of unstable angina, the patient must be hospitalized with ischemic chest pain or discomfort occurring at rest or with minimal exertion. In addition, the need for intravenous medical intervention and/or objective evidence of myocardial ischemia is required. For extensive description of the end points definitions, see Table [I](#T1){ref-type="table"}. ::: {#T1 .table-wrap} Table I ::: {.caption} ###### Primary end point definitions ::: ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- Definition of reinfarction 1\. Reinfarction during hospitalization for index infarct and not related to revascularization procedures\ - Either ischemic type of chest discomfort or new electrocardiographic changes indicative for transmural ischemia or necrosis with an increase in the total creatine kinase and MB isoenzyme activity. The activity of CK-MB has to be at least 2 times the upper limit of normal and more than 50% above the previous baseline value. 2\. Reinfarction discharge for index infarct and not related to revascularization procedures\ - Either a history of ischemic chest discomfort, usually lasting \> 20 minutes, or classic electrocardiographic changes indicative for transmural ischemia with an increase in the total creatine kinase and MB isoenzyme activity of at least 2 times the upper limit of normal.\ - New abnormal Q-waves (amplitude ≥ 1/3 of total QRS amplitude and ≥ 0.04 seconds) in ≥ 2 contiguous leads. 3\. Periprocedural reinfarction during revascularization after index infarct\ PCI\ - Classic electrocardiographic changes indicative for transmural ischemia with an increase in the total creatine kinase and MB isoenzyme activity of at least 2 times the upper limit of normal.\ - New abnormal Q-waves (amplitude ≥ 1/3 of total QRS amplitude and ≥ 0.04 seconds) in ≥ 2 contiguous leads. CABG \- Classic electrocardiographic changes indicative for transmural ischemia with an increase in the total creatine kinase and MB isoenzyme activity of at least 5 times the upper limit of normal.\ - New abnormal Q-waves (amplitude ≥ 1/3 of total QRS amplitude and ≥ 0.04 seconds) in ≥ 2 contiguous leads. Definition of unstable angina \- Ischemic type of chest discomfort at rest or with minimal exertion, with a duration of at least 15 minutes. The presenting symptoms must represent a change from the patients\' usual anginal pattern.\ - Either a need for intravenous medical intervention and/or objective evidence of myocardial ischemia (dynamic ST changes in ≥ 2 contiguous electrocardiographic leads, or an abnormal elevation of Troponin-T or Troponin-I without a significant rise of CK MB isoenzyme activity) ---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- PCI, percutaneous coronary intervention; CABG, coronary artery bypass grafting; CK-MB, creatine kinase myocardial enzyme ::: A clinical endpoints and safety monitoring committee will continuously check and monitor all events in a blinded manner. Reinfarction after revascularization will be scored as described in Table [I](#T1){ref-type="table"}. Major and minor bleeding complication in the invasive treated group are defined according to the criteria of the Thrombolysis in Myocardial Infarction trials \[[@B44]\]. All patients will be treated with aspirine, beta blockers, angiotensin-converting-enzyme inhibitors and statins as accepted in international guidelines \[[@B2],[@B4]\]. Low-dose dobutamine echocardiography ------------------------------------ If the inclusion criteria are met and none of the criteria for exclusion, viability testing by LDDE is performed within 3 days of admission. Preferably, beta-blockers are withdrawn 24 hours before the test \[[@B23],[@B27],[@B30],[@B31]\]. Discontinuation of beta-blockers seems to to enhance LDDE accuracy \[[@B45],[@B46]\]. Before the administration of dobutamine, a baseline echocardiogram is obtained according to the guidelines of the American Society of Echocardiography \[[@B47]\]. Five standard views are obtained: the parasternal long-axis and short-axis view and the apical two, three- and four-chamber view. A 16-segment model is used in which the apex is divided in 4 segments. Segmental wall motion and thickening is scored according to a 4-point scale: 1 = normal, 2 = hypokinetic, 3 = akinetic, and 4 = dyskinetic. Left ventricular volumes and ejection fraction are measured by use of the modified Simpson\'s rule algorithm from orthogonal apical long-axis projections as recommended by the American Society of Echocardiography. Dobutamine is administrated intravenously at doses of 5, 10, and 15 μg/kg/min, for 5 minutes at each dose. When a 10% increase in heart rate is not achieved with 15 μg/kg/min, a 5-minute infusion with 20 μg/kg/min can be used as the final stage of the procedure. Viability is defined as the improvement of wall motion abnormalities in two or more segments of the infarct zone. Changes from hypokinesia to normokinesia and from dyskinesia or akinesia to hypo- or normokinesia are considered an improvement in wall motion abnormality. Dyskinesia changing to akinesia is not considered as an improvement. Patients are continuously monitored by a 12 lead ECG and blood pressure is recorded at the end of each stage. All views are recorded at rest and during dobutamine on VHS videotape. All videotapes are sent to the core-lab (VU University medical center) and will be analyzed by 2 experienced observers. A third observer is used in case of disagreement to reach consensus. For subsequent off-line analysis, the echocardiographic images will be digitized from VHS videotape and transferred to a working station (Enconcert^®^by Philips). The images will be displayed side-by-side in a quadscreen format to facilitate the comparison of images. Coronary angiography and angioplasty ------------------------------------ Angiography and angioplasty will be performed as soon as possible after randomization. Coronary angiography will be performed according to standard protocols. To determine the severity of culprit lesions, quantitative coronary arteriography (QCA) will be performed to measure percent diameter stenosis, reference diameter and cross-sectional area stenosis. The degree of stenosis will be determined in the view in which the stenosis is most severe. When feasible, PCI will be performed (with or without etc) when there is a significant (≥ 50%) stenosis or occlusion of the infarct-related coronary artery, with the intention to perform primary stenting of the infarct-related artery, with concomitant use of abciximab, according to the EPILOG protocol \[[@B39]\] After stenting, all patients recieve oral clopidogrel in a standard way. In case of severe 3-vessel disease or significant left main stem stenosis, where PCI is judged to be a high risk, coronary artery bypass grafting will be considered. Follow-up --------- Patients will be followed for a period of three years. Follow-up data will be obtained of all patients during visits at the outpatient clinic in the first year, and by telephone interview in the second and third year of follow-up. Left ventricular function (volumes and ejection fraction) is determined by echocardiography at 3 months, 6 months, and 1 year of follow-up. Statistical design ------------------ The VIAMI-trial is conducted to investigate the differences in clinical outcome between an invasive and a conservative strategy in patients with demonstrated viability in the infarct-area. The expected event rate in the viability positive group is estimated to be 35 percent. To demonstrate with a power of 80% (α = 0.05, two-sided) that PCI leads to a 50% reduction in event rate in the invasive group compared to the conservative group, 200 patients will be needed in each group. Therefore, we intend to randomize a total of 400 patients in this trial, with interim analysis after 200 included patients. The formal stopping rules for the study are the following: If one of the treatment strategies appears significant superior at interim analysis (P ≤ 0.01), the study will be stopped. Statistical analysis -------------------- Baseline descriptive data will be presented as mean ± standard deviations (SD). Differences in clinical and echocardiographic variables will be assessed by unpaired Student\'s ttest. Differences between proportions will be assessed by chi-square analysis; a Fisher\'s exact test will be used when appropriate. Event-free survival curves are computed with the Kaplan-Meier method, and the differences between these curves are tested with a Mantel-Cox log-rank test. A primary endpoint analysis is planned at 30 days, 6 months, and 1 year of follow-up. Subgroup analyses are planned to determine whether the treatment effect is more or less pronounced in certain subgroups. The data will be subgrouped by sex, age, diabetes, anterior infarction, time from onset of symptoms to treatment, and the use of thrombolytics. All analyses will be performed on an intention-to-treat basis. Also, the outcome per-protocol will be evaluated. Such an analysis seems worthwhile, as it will reflect the true influence of PCI on post-thrombolytic ischemic events. Current status -------------- Enrollment of patients started April 1, 2001. Recently, an interim analysis was performed after the inclusion of 200 patients, having a 30 day follow-up. The Clinical Event Committee recommended continuation of the trial. Conclusion ========== The VIAMI-trial is the first multicenter, randomized, controlled clinical trial, investigating the clinical benefits of percutaneous catheter intervention of the infarct-related artery in patients with demonstrated viability in the early, subacute phase of myocardial infarction. Competing interests =================== The authors declare that they have no competing interests. Authors\' contribution ====================== RBvL recruited and analysed all data and drafted the manuscript. GV have been involved in drafting the article and revised it critically. OK and JGFB revised the manuscript critically. CAV and FCV have given final approval of the version to be publiced.	PubMed Central	2024-06-05T03:55:49.971989	2004-11-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534804/", "journal": "Curr Control Trials Cardiovasc Med. 2004 Nov 11; 5(1):11", "authors": [ { "first": "Ramon B", "last": "van Loon" }, { "first": "Gerrit", "last": "Veen" }, { "first": "Otto", "last": "Kamp" }, { "first": "Jean GF", "last": "Bronzwaer" }, { "first": "Cees A", "last": "Visser" }, { "first": "Frans C", "last": "Visser" } ] }
PMC534805	Background ========== The mechanism of action of homeopathic remedies has baffled practitioners and scientists for two centuries. A widely accepted premise of those doing research in this field is that if remedies are more than placebos, then the process of making remedies by alternating dilution and succussion must alter the solvent, encoding in it a \"memory\" or \"information\" that biological systems can detect. Experiments attempting to measure or document solvent alteration through direct study of the physical and chemical properties of remedies have so far failed to yield any independently replicated positive effects. Proton nuclear magnetic resonance (^1^H-NMR, or simply NMR) is among the techniques that have been used to look for differences between remedies and control samples. The term \"NMR\" encompasses both solvent mobility studies (results are given as a pair of relaxation times denoted T~1~and T~2~) and analytical studies. In analytical studies, also called spectroscopy, the results are displayed as a graph or spectrum plotting concentration against a variable called chemical shift. There are also more complex applications of NMR such as imaging and two-dimensional NMR that are not relevant to the study of discrete remedy samples. The chemical shift of a proton in a molecule in a sample reflects the (time-averaged) amount of magnetic shielding provided by the electrons making up the covalent or hydrogen bond(s) in which the proton participates, with greater electron density generally correlating with lower chemical shift numbers. Chemical shifts are measured in units of parts per million (ppm) deviation from a reference shift. Recent literature reviews by Baumgärtner \[[@B1]\] and by Becker-Witt et al.\[[@B2]\] identified 18 published articles on the use of NMR to study remedies, of which 9 projects used NMR spectroscopy \[[@B3]-[@B11]\]. Of these nine, eight \[[@B3]-[@B10]\] reported finding differences between remedies and controls when focusing on the relative height, chemical shift, or width of one or more of the peaks due to H\'s in the solvent. However Aabel and coworkers\' recent study \[[@B11]\] found no differences between remedies and controls. It should be noted that all studies had methodological weaknesses according to the criteria developed by Becker-Witt et al.\[[@B2]\] One hypothesis to explain how UD remedies might be different from controls states that remedies contain long-lived stable clusters of solvent molecules that are not present in controls \[[@B12]-[@B14]\]. If this hypothesis is correct, then the H\'s making up H-bonds in the stable clusters would experience a bonding environment different from that of the ambient solvent, and they should generate a separate signal on the NMR spectrum. There would be one discrete signal or peak for each symmetry-distinct H in the cluster, along with the expected large peak at 4.8 ppm for the ambient water. \"Unexplained\" discrete peaks have never been reported in NMR spectroscopy studies of remedies, but we wondered if this might be because cluster concentrations are very small and the methodology has been insufficiently sensitive to detect them. A technical but relevant point must be introduced here. One version of the \"cluster theory\" posits that once a solvent molecule becomes part of a stable cluster it will stay in that cluster indefinitely. We call such a cluster \"non-dynamic\". A trickier but more believable hypothesis is that solvent molecules cycle back and forth between the ambient solvent and the clusters. If only one molecule at a time leaves a cluster and it is replaced quickly by another solvent molecule, then the pattern or geometry of the cluster could be sustained without indefinitely tying up any individual solvent molecules. Exchanges of surface hydrogens could also occur rapidly without losing the cluster geometry. A cluster whose components exchange with the ambient solvent would be called \"dynamic\". The key concept is a parameter called \"dwell time\", which is the average time a molecule spends in a cluster. If the dwell time is shorter than 10^-3^sec or so, NMR will not be able to \"see\" it because the chemical shift will be the average over some milliseconds, and the discrete signals will blend back in with the ambient water signal. Dwell times are considerably shorter than 10^-3^sec for a variety of processes, such as ion solvation \[[@B15]-[@B17]\] and protein association \[[@B18]-[@B22]\]. Therefore NMR spectroscopy is a good method for testing the cluster hypothesis only if, as part of the hypothesis, we postulate that clusters are non-dynamic or that dwell times are on the order of several milliseconds or longer. The project described in this article used a high-sensitivity NMR method to test this possibility, i.e., do remedies made in water contain low concentrations of long-lived non-dynamic regions of structured H-bonding? A good starting point was to quantify the detection limits of previous studies and of the present study. Without a detection limit a negative finding is hard to interpret. The methodology of Aabel et al.\[[@B11]\], which did not include individual shimming of tubes and collected the standard 16 scans per tube, might be expected to have a detection cutoff around 1 mM on a 500 MHz magnet. That is, peaks representing concentrations of H smaller than 0.001 mol/L would be lost in the noise. This project undertook to improve the detection limit by conducting individual shimming, increasing the number of scans collected, and most importantly, utilizing a high sensitivity method called presaturation. With presaturation, the H\'s contributing to the 4.8 ppm peak are excited in advance in a manner which causes their contributions to nearly cancel each other out rather than combine into a huge peak. As a result the receiver gain can be increased and signals that would otherwise be overwhelmed become detectable. By combining these techniques, peaks belonging to H-containing compounds at concentrations as low as 5 μM were consistently detected. Assuming unsplit peaks, this means that if a remedy contained a population of a particular stable cluster species at a concentration of 5 μM, the method would be able to detect it. Assuming peaks are split as doublets, the cutoff rises to 10 μM. Although the H\'s on an H~2~O molecule are normally NMR-equivalent, they need not be equivalent if the H~2~O is embedded in a fixed stable cluster. Then each H of an H~2~O could split the other\'s peak, yielding doublets. Protons on distinct H~2~O units are far enough apart that their coupling constants can be expected to be too small to generate further peak splitting. Based on this reasoning we assume doublets as the norm for a hypothetical stable cluster, and we take 10 μM as the detection cutoff. For perspective, at 10 μM, we could detect structuring if just 1 in every 10^7^protons were involved in a fixed H-bond. Methods ======= Chemicals --------- Chemicals including 100.0% D~2~O, 100.0%-d DMSO-d6, and 98% 1,4-cyclohexanedione (C~6~H~8~O~2~) were obtained from Sigma-Aldrich \[[@B23]\]. Distilled deionized water was either obtained from Sigma-Aldrich or prepared on site via a purifier capable of producing 18 MΩ·cm water. Ultra-pure water was kept in tightly closed polypropylene bottles. Measurements at the point of use found that the water had conductivity no greater than 2 μS (or 0.5 MΩ·cm). The rise in conductivity occurs within minutes as ultra-pure water picks up CO~2~from exposure to air and ions from exposure to glass, and we considered it to be unavoidable. Remedies -------- Two animal (Sepiaand Lachesis), two plant (Ignatiaand Lycopodium) and two mineral remedies (Natrum Muriaticumand Argentum Nitricum), all of which occur commonly in clinical homeopathy, were used for the project. Commercial remedies were obtained from Helios Pharmacy \[[@B24]\] and from Washington Homeopathic \[[@B25]\]. Both pharmacies are widely considered to make quality products that give good clinical results. Helios remedies are regarded by many to be among the best homeopathic remedies in the world. Each pharmacy provided all six remedies at the 12 C, 30 C, 200 C, 1 M, and 10 M potencies. Staff at both pharmacies made the 12 C and 30 C remedies via the Hahnemann process in washed and rinsed vials using distilled deionized water at each dilution, starting from a standard mother tincture (MT). For the 200 C potencies, a 195 C potency in ethanol-water \"off the shelf\" was used as the starting point for five serial dilutions and succussions in deionized distilled water. Likewise, 1 M and 10 M potencies were derived from \"library\" 995 C and 9995 C potencies made in ethanol-water. It was a consensus among homeopathic practitioners that despite the switch from ethanol-water to water, this was a valid way of generating 200 C and higher potency remedies. Upon receipt, commercial remedies were stored in the bottles in which they were sent, at room temperature, in a box in a dark cupboard. Each pharmacy also provided an unsuccussed water control. We also made our own mineral remedies up to the 12 C potency starting from MT\'s consisting of a hand-made 1 M NaCl solution and a stock bottle of 0.1 N AgNO~3~. To make a remedy series, twelve 12 mL capped borosilicate glass vials were labeled 1 C through 12 C, and to each was added 4 mL water. Two drops of MT were added to the first and it was succussed, then 2 drops of the 1 C were added to the next vial and it was succussed, and so on. Transfers were via sterilized Pasteur pipettes. Vials and pipettes were not re-used. Succussion consisted of 120 strokes of forcefully pounding the closed vials held in the fist against a rubber mouse pad on a counter top. Succussed water to be used as a control was made the same way. Over the course of the project, seven series of Nat Murpotencies and six of Arg Nitrpotencies were made. Remedies were made less than 24 hours in advance of their scheduled testing times. They were placed into NMR tubes and readied for analysis within an hour of being produced. The tubes generally waited overnight in a light-resistant foil-wrapped container at room temperature before undergoing analysis. NMR Methodology --------------- To prepare a sample for analysis, a borosilicate glass NMR tube rated for 500 MHz (Wilmad Lab Glass \[[@B26]\]) was primed with 50 to 90 μL of locking agent (either D~2~O or DMSO-d6) and 20 μL of a dilute water solution containing a known concentration of a marker molecule (markers tried were acetone (CH~3~COCH~3~) and 1,4-cyclohexanedione (C~6~H~8~O~2~)). The remedy or control sample was then added to fill the tube to the 700 μL mark, followed by gentle tilting and turning to mix. The marker served several purposes: it provided a reference line for zeroing the chemical shift scale, it provided a reference peak for comparing concentrations, and its sharpness and shape gave feedback about the accuracy of the shimming process. By carefully varying the marker concentration we also determined the method\'s limits of detection. \[C~6~H~8~O~2~was considered an \"ideal\" marker in that it met all of these criteria: it is available cheaply at high purity; it dissolves in water without altering pH and does not evaporate over time; its ^1^H-NMR spectrum has a single unsplit line; its unique peak occurs at a distinctive location far from the water peak and is not easily confused with other common peaks (2.77 ppm); and it will not normally occur as a contaminant or from other sources, so one can be sure its concentration is exactly what one intends.\] Proton NMR spectra were obtained at the Department of Chemistry Instrumentation Facility at M.I.T. (\"DCIF\"). All spectra reported here were obtained on a Varian INOVA 500 tuned to 499.759 MHz and equipped with an inverse probe. Tubes were run with the temperature clamped at either 20°C or 21°C and were not spun. Shimming was done manually for Z1 through Z7 and for first and second order XY magnets, using the lock signal. Presaturation used the standard presat pulse sequence (satdly = 1.5 sec), with the optimal presat frequency being determined to within 0.1 Hz via an array method. Locking, shimming and presat frequency optimization were repeated each time a tube was inserted. Between 128 and 200 transients were collected for each sample. Start-to-finish time for each tube was typically around 35 minutes. Spectrum analyses were done via the standard Varian programs running on an SGI workstation provided by DCIF. Randomization ------------- Randomization and blinding are among the recommendations in Ref. \[[@B2]\] for how to conduct high quality studies on homeopathy. We did not conduct strict randomization, but we did intentionally \"mix up\" the samples in some respects. We labeled all NMR tubes at the outset and we deliberately changed around which tubes were used for controls and for remedies, in case tube-specific effects were to occur. On most days when we were analyzing remedies we included at least one control, and we always ran the control neither first nor last, in case a time-related trend or drift were to influence the results. Our plan was this: if any signals appeared which seemed to be occurring significantly more often in remedies than in controls, the studies would be followed up with strictly randomized, blinded trials to verify those particular signals. If no significant differences were found, then strict randomization could not alter the outcome, and the follow-up step would be unnecessary. Results ======= We analyzed a total of 57 remedy samples, 5 succussed water controls, and 41 unaltered water controls. Of this total, 28 samples were non-commercial (i.e. made on site) Nat Murand Arg Nitrremedies of potency 6 C to 12 C. Because the details of the protocol evolved over the course of the project, it could be argued that this total represents the combination of many different experiments. Realizing this, we made a point toward the end of the project, of applying what we considered to be our best methodology to screen a set of what were arguably the best remedies. Specifically, we screened 18 Helios remedies, namely the 12 C, 30 C and 10 M potencies of the six MT\'s. The screening protocol consistently used 70 μL of D~2~O for locking and concentrations of 12.5 μM or less of C~6~H~8~O~2~as marker. These 18 trials are included in the 57 for the purposes of discussion. Spectra can be conveniently split into those obtained during 2002 (listed by sample and date in Table 1, Additional File [1](#S1){ref-type="supplementary-material"}) and the Helios screening run (listed in Table 2, Additional File [1](#S1){ref-type="supplementary-material"}). Sensitivity ----------- As indicated above, when marker concentration was 5 μM of H or greater (e.g. 0.62 μM of C~6~H~8~O~2~), a peak was always seen above noise, using the 3σ criterion \[[@B27]\]. This included four samples where the concentration was just 5 μM. A peak was seen in two of three samples where the concentration was 4 μM and in neither of two samples where it was 3 μM. Thus 5 μM was taken as the detection cutoff for this methodology. Expected and unexpected signals ------------------------------- Expected signals that were seen in all spectra included the large water signal peaking at 4.81 ppm, which dominated the spectrum despite presaturation. The water signal is so dominating that small peaks between approximately 4.4 and 5.2 ppm could be \"lost\" in it, and this interval must be viewed as an inaccessible region of the spectrum for our methodology. Marker peaks for CH~3~COCH~3~and C~6~H~8~O~2~were observed respectively at 2.22 \[[@B28]\] and 2.77 ppm. ^13^C satellites were seen as expected when marker concentrations were high enough and are not listed separately in Table 3, Additional File [1](#S1){ref-type="supplementary-material"}. When DMSO-d6 was the locking agent, a residual DMSO-d5 quintuplet centered at 2.68 ppm was always present. The focus of this project was to look for discrete peaks other than these expected peaks. Combining the 103 spectra, there were 35 positions where a discrete signal occurred that was not among these expected signals. These positions are listed in Table 3, Additional File [1](#S1){ref-type="supplementary-material"}. Signals that had the same structure (i.e. singlet, doublet, etc.) and occurred at the same position (within ± 0.01 ppm) in multiple spectra were assumed to have the same genesis. (This assumption could be challenged, but it would not affect our overall conclusions.) We therefore examined each of the 35 \"unexpected\" positions to see if we could offer an explanation for why signals occurred there. Artifacts --------- Fifteen of the 35 signals were classified as artifacts, i.e. machine-generated spectrum \"glitches\" that were unrelated to the sample. A signal was dismissed as artifact if it could not be phased consistent with the marker peak. Artifacts were further classified as either \"consistent\" or \"intermittent\". The consistent artifacts occurred in more than half of all samples and throughout the eighteen months when our data was collected. Consistent artifacts were seen at 3.60, 8.17, and 11.53 ppm. Signals at positions 8.17 and 11.53 were explained as mid-spectrum artifact and as a \"reflection\" of the water signal respectively (Δ = 8.17 - 4.81 = 3.36, reflection across midpoint is at 8.17 + Δ = 11.53), but no rationale for the 3.60-ppm artifact was identified. We labeled the consistent artifacts as C1, C2, and C3. Intermittent artifacts were labeled R1 through R12 and are listed in Table 3, Additional File [1](#S1){ref-type="supplementary-material"}. These artifacts tended to be small, often little more than bumps or wiggles in the baseline. With the marker peak phased to go up (i.e. wholly above the baseline), artifacts might be entirely below the baseline (downgoing), entirely above (upgoing), or both (biphasic). An artifact that occurred repeatedly could have different behaviors in different spectra. Table 3, Additional File [1](#S1){ref-type="supplementary-material"} indicates whether each artifact was downgoing (d), biphasic (b), or upgoing (u), or whether more than one behavior was seen. The criterion for classification as artifact was that a signal was downgoing or biphasic in at least some of the spectra where it was seen. Half of the intermittent artifacts were seen in only one spectrum each. Those that occurred repeatedly demonstrated a \"burst\" pattern in the sense that they were present for spectra collected on a single day or during an interval of weeks or months, but were not seen at other times. For instance, the artifact R5 occurred only on 4-Jun-03 and was seen in every spectrum obtained that day (the control as well as the remedies: Table 2, Additional File [1](#S1){ref-type="supplementary-material"}). Contaminants ------------ Signals that were consistently upgoing could be artifacts or they could be measuring something actually present in the sample. Among such signals, some were evidence of contamination by small organic molecules. Seven signals were positively identified, and an eighth was given a probable assignment. Zacharias \[[@B29],[@B30]\] raised the issue that some amount of contamination was probably unavoidable in remedies, and that it could affect the outcome of studies of remedies. In our spectra signals representing contamination by small organic molecules were frequently seen. Non-commercial remedies and controls often contained acetate (CH~3~COO^-^, 1.90 ppm \[[@B28],[@B31]\]) or formate (HCOO^-^, 8.44 ppm \[[@B28]\]) ions at concentrations ranging from barely detectable (1.7 μM for acetate, 5 μM for formate) to 30 μM. Long soaking of the NMR tubes in water between uses and exercising extra care to avoid hand contact with the remedies decreased but did not eliminate the occurrence of these two contaminants. Lactate (CH~3~CHOHCOO^-^, 1.32 ppm \[[@B31]\]) occurred at barely detectable levels in 3 non-commercial samples. The fact that its signal is a doublet with the characteristic 5 Hz coupling constant assists with the identification, and improves one\'s confidence that one is seeing a real signal and not just noise that happens to occur at the chemical shift of lactate. The α hydrogen signal of lactate, expected around 4.2 ppm, is a quartet and would be predicted to have 1/4 the height of the methyl peak. The quartet would have been a nice confirmation of the lactate identification, but it could not be seen above noise. One signal, an upgoing singlet at 1.28 ppm, occurred in all but one spectrum run using DMSO-d6 between April and November 2002, and did not occur in spectra run using D~2~O or outside this time interval. We pegged it as coming from a contaminant in the DMSO-d6, which was not present in different bottles of DMSO-d6 that were used before April and after November. Its base was broader by a factor of four than the other singlets seen, and despite its trace size we could often make out that it had a symmetric stepped shape like a ziggurat. A good guess, which fits all of these facts, is that it comes from ethylmethylsulfoxide-d7 (CD~3~SOCD~2~CD~2~H), which could plausibly be introduced during the manufacture of DMSO-d6 (CD~3~SOCD~3~). We did not find anywhere listed the chemical shift of ethylmethylsulfoxide, but the ethyl\'s methyl group of the very similar molecule ethylmethylketone resonates at 1.26 ppm \[[@B28]\] in D~2~O. We gave it the identifier \'X\' in Table 3. A DMSO-d6 control run on 1-Aug-02 yielded two peaks in the vicinity of 1.3 ppm, one of which may have been \'X\'. For the 18 Helios remedies screened with the C~6~H~8~O~2~protocol, results are given in Table 2, Additional File [1](#S1){ref-type="supplementary-material"}. All but one had measurable quantities of ethanol, with concentrations typically around 300 μM but in one case as high as 3.6 mM (range 116 -- 3632 μM, median 310 μM). The ethanol signal, a triplet at 1.17 ppm paired with a quartet at 3.65 ppm, was unmistakable. The ratio between the peak areas of ethanol\'s methyl triplet at 1.17 ppm and its methylene quartet at 3.65 ppm is theoretically 3:2. In Table 2, Additional File [1](#S1){ref-type="supplementary-material"}the range of ratios is 1.37 to 1.66. The proximity of the variably-shaped artifact near 3.60 ppm sometimes interfered with accurate determination of the area of the methylene signal, and ethanol concentrations were taken to be 1/3 of the 1.17-ppm peak area. Repeat measurements of a single sample showed that the concentration figures obtained this way have an experimental error of 5 -- 10 %. The 18 Helios remedies of Table 2, Additional File [1](#S1){ref-type="supplementary-material"} all contained CH~3~COO^-^(range 22 -- 214 μM, median 55 μM) and HCOO^-^(range 8 -- 75 μM, median 44 μM). None contained detectable lactate, but we sometimes saw acetone or methanol (CH~3~OH, 3.34 ppm \[[@B28]\]). Six of the 18 contained detectable acetone (range 3 -- 21 μM, median 5 μM) and a different but overlapping set of six held detectable methanol (range 2 -- 10 μM, median 4 μM). Because the samples prepared on site never contained detectable ethanol, methanol, or acetone (excepting the added acetone markers), and all samples were analyzed by the same procedure, it is safe to conclude that the Helios samples came with these contaminants. While we admit that our procedures and lab technique were apparently introducing some extraneous acetate and formate, the Helios remedies\' levels of these ions typically ran higher than the levels seen in remedies prepared on site (medians of 55 and 44 versus a maximum of about 30 for on site remedies). We deduce that the source of these contaminants in Helios remedies was at least partially from the remedies themselves, i.e. most or all of the Helios remedies came with some acetate and formate in them. The three Helios remedies that we examined using DMSO-d6 contained ethanol, acetate, and formate as well, and one had methanol, but for those runs we did not add a measured marker and precise concentrations were not determined (Table 1, Additional File [1](#S1){ref-type="supplementary-material"}). Of the eight Washington Homeopathic remedies that we analyzed and the Washington control, all had ethanol and acetate, all but the control had formate, seven had acetone, two had methanol, and six had lactate. The high rate of lactate occurrence in Washington\'s samples (6 of 9 vs 3 of 94 for non-Washington) was not due to chance (p \< 10^-6^), but the fact that the six runs exhibiting lactate were all done on one day with no non-Washington control means that we cannot rule out an extraneous source for the lactate. Unexplained signals ------------------- Twelve positions for upgoing singlets could neither be ruled out as artifacts nor assigned definitely to any known small organic contaminant. These are the \"unexplained\" peaks, labeled U1 through U12 in Table 3, Additional File [1](#S1){ref-type="supplementary-material"}. Six of the unexplained group occurred in just one spectrum each. Those that occurred repeatedly exhibited the same kind of temporal burst distribution that was seen for the intermittent artifacts. (The only exception was U1, which was seen on 2-Jan-02 and again on 20-May-02; this may be an example where two different signals coincidentally had similar shapes and chemical shifts and were lumped together.) The ppm range was from 0.47 to 10.60; among those that occurred more than once the range was 0.47 to 6.80. The three upfield-most (i.e. lowest ppm) signals were \"broad\", i.e. width at half height was between 0.02 and 0.1 ppm, in contrast to the typical \"sharp\" singlet whose width at half height was 0.002 to 0.004 ppm. For each of the unexplained signals and artifacts we made a 3 × 2 matrix of their occurrence vs non-occurrence in remedies, succussed controls, and unsuccussed controls. None of the matrices had a p-value below .05. The occurrence counts and p-values are listed in Table 3, Additional File [1](#S1){ref-type="supplementary-material"}. Spectrum example ---------------- Figure [1](#F1){ref-type="fig"} shows a portion of the spectrum of Helios\' Ignatia-30 C sample (Line 27 of Table 2). Figure [2](#F2){ref-type="fig"} shows a magnification of the same spectrum between 1.8 and 4.0 ppm. Numbers below the x-axis represent integrated peak areas, relative to C~6~H~8~O~2~peak at 2.77 ppm being set to its known value of 100 μM of H. The complex signal between approximately 4.3 and 5.3 ppm is the presaturated water signal. This spectrum contains five contaminants, the three consistent artifacts (artifact at 11.53 not shown), and two intermittent artifacts. Note that the artifacts are biphasic, i.e. have a component below as well as above the baseline, whereas all of the signals due to actual molecules have signals that stay above the baseline. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### ^1^H-NMR Spectrum of Ignatia-30 C (Helios): expansion of 1 -- 9 ppm region. Key to peaks \[position -- interpretation\]: 1.17 -- ethanol, methyl triplet; 1.47 -- artifact (R4); 1.90 -- acetate; 2.22 -- acetone; 2.77 -- C~6~H~8~O~2~(1,4-cyclohexanedione marker); 3.35 -- methanol; 3.60 -- artifact (C1); 3.65 -- ethanol, methylene quartet; 3.88 -- artifact (R5); 4.3 to 5.3 -- water (presaturated); 8.17 -- artifact (C2); 8.44 -- formate; 11.53 (not shown) -- artifact (C3). ::: ![](1472-6882-4-15-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### ^1^H-NMR Spectrum of Ignatia-30 C (Helios): expansion of 1.8 -- 4.0 ppm region. Key to peaks \[position -- interpretation\]: 1.90 -- acetate; 2.22 -- acetone; 2.77 -- C~6~H~8~O~2~(1,4-cyclohexanedione marker); 3.35 -- methanol; 3.60 -- artifact (C1); 3.65 -- ethanol, methylene quartet; 3.88 -- artifact (R5). ::: ![](1472-6882-4-15-2) ::: Discussion ========== Contaminants ------------ Concerning the organic contaminants, acetate, formate and lactate are present on human skin and can be introduced at the trace levels seen here through ordinary handling. Acetate and formate derive respectively from the β- and α-oxidation of long-chain fatty acids by fibroblasts \[[@B32]\], and lactic acid is the principal organic component of eccrine sweat (and is found at higher concentrations on the palms \[[@B33]\]). They are frequently encountered in high sensitivity work. Methanol and acetone are commonly used for rinsing glassware, and a reasonable but unprovable guess is that they represent a residuum from a vial rinse. A plausible but again unprovable guess is that the source of the ethanol was incomplete flushing after prior use of the vials to contain ethanol-based remedies, or perhaps ethanol was also used in the rinsing process. The source of the ethanol for the 200 C and higher potencies is not the 195 C or other library potency: if there were no other source of ethanol, five 1:99 dilutions in water would reduce the level to at most 17 × 10^-10^M. Despite the implication that commercial remedies may be \"impure\", we consider the levels of detected impurities to be extremely low. Large doses of methanol are hepatotoxic, but the trace amounts here represent no threat to health. Our findings certainly support the widely accepted view that homeopathic remedies are safe. We also believe that the contaminants found should be unlikely to interfere with remedies\' clinical effectiveness. The median value of 310 μM corresponds to less than one part ethanol in 50,000 parts water. Working in the early 19^th^century, Hahnemann would have used locally obtained well water or spring water to make remedies, and his water would have been far less pure than our most contaminated sample. (Hahnemann also used wine and brandy to make his remedies -- hardly the precision solvents of the modern lab.) While high purity solvents are necessary for scientific validity and reproducibility, there is no a priorireason to think they matter for remedies\' effectiveness. Indeed, one of the hypotheses on the mechanism of homeopathy is that the \"information\" is in the geometry of the solvation shells of the low levels of impurities that are present in the solvent, or in some structuring of how solvent impurities group together or interact with each other. Unexplained peaks ----------------- Six of the unexplained signals occurred in just one spectrum each and could easily be artifacts that happened to be in phase with the rest of the spectrum. There is little to be gained by trying to explain these one-time events, five of which were from unsuccussed controls. The signals U1 to U3, being broad and upfield, could be consistent with complex aliphatic mixtures such as finger grease on the outside of the NMR tubes \[[@B28]\]. We have no conjecture as to the identity of U5 (2.80 ppm, 3 occurrences) or U6 (4.12 ppm, 2 occurrences). Signal U8 at 6.80 ppm occurred in 7 of 8 D~2~O runs starting in November 2002, which was when a fresh bottle of D~2~O was brought into use, and it was not seen in the DMSO-d6 runs from the same period. This pattern suggests it was a contaminant in the D~2~O, even though it disappeared when the same D~2~O bottle was used in April 2003. There was also one sporadic occurrence in April 2002 (a DMSO-d6 spectrum). A likely assignment is quinone (p-benzoquinone, C~6~H~4~O~2~), whose spectrum consists of one singlet at 6.80 ppm \[[@B34]\]. Quinone sublimes at room temperature, and the approximately one micromole of quinone in the 20-mL D~2~O bottle may have simply evaporated during four months. In Table 3, Additional File [1](#S1){ref-type="supplementary-material"} we also list the occurrence counts for \"any unexplained signal\" in remedies and in controls, and its p-value of 0.08. Lest this appear as a \"trend\", we point out that the reason for this relatively low p-value is that the \"trend\" was for controls to show more unexplained signals than remedies. We do not believe the difference is meaningful. Other aspects of ^1^H-NMR spectra --------------------------------- We found no discrete signals that occurred significantly more frequently in remedies than in controls. Does this mean that NMR spectroscopy cannot show differences between remedies made in water and controls? Earlier NMR studies focused not on additional signals but on peak positions and shape. For samples in water there is only one peak, the water peak at 4.8 ppm, along with the expected peaks for marker and/or DMSO-d5. We did not examine the shape of the water signal. Presaturation completely destroys any information that may have been present in the water signal shape, and the shape of the water signal after presaturation is sensitive to so many variables that it would be hard to track down the effect, if any, of solvent alteration. Still, this is an avenue that could conceivably be pursued. Similarly, we indicated that any information in the range of 4.4 to 5.2 ppm was essentially lost. It might be possible to examine this range closely, perhaps by subtracting off a smoothed spectrum, to look for signals between 4.4 and 5.2. We have not attempted this. We were primarily looking for separate peaks that could be from long-lived stable clusters, and did not find any. For this reason we couch our results carefully by saying merely that \"no discrete signals suggesting a difference were seen.\" Implications for homeopathic mechanism -------------------------------------- The absence of any evidence of stable H-bonds above the 5 μM or 10 μM detection limit, in 57 remedy samples, certainly casts doubt on the hypothesis that remedies in water might contain regions of long-lived non-dynamic H-bond structuring. Is this hypothesis salvageable? We do not believe so. We made a point of screening low, medium and high potency remedies, as well as remedies from six common MT\'s. While it could be argued that even 10% DMSO could disrupt H-bonding and thereby denature (i.e. destroy) H-bond structure \[[@B35]\], this cannot be said about D~2~O. Nor do we believe that the addition of the marker could have interfered with the detection of structuring. Consider that Helios\'s remedies, reputedly among the best anywhere, contained comparable or higher concentrations of organic contaminants to start with, than we added. If one wants to maintain the position that homeopathy works (i.e. clinical effects are attributable to it), and that Helios\'s and Washington\'s remedies work, then the fact that their remedies come with traces of small organic molecules tells us that such traces cannot be something that interferes with homeopathy\'s mechanism. Thus it should not \"hurt\" a remedy to add a tiny bit of organic marker. Could there be signals from fixed H-bonds but we didn\'t see them because they fell in the inaccessible 4.4- to 5.2-ppm region? The chemical shift of an H in a fixed O-H - - O setup depends on the O - - O separation and the details of the geometry, but studies of ice \[[@B36]\], organic H-bonds \[[@B37],[@B38]\] and ab initiosimulations \[[@B39],[@B40]\] all show that chemical shifts in the range 7 -- 16 ppm can be anticipated. For this reason we do not believe that obliteration by the water signal of the 4.4- to 5.2-ppm interval represents important data loss. Our 103 spectra were particularly sparse in signals in the downfield (i.e. \> 7 ppm) region. Besides the two \"explained\" artifacts C2 and C3 and the identified contaminant formate, they contained just 3 single-occurrence downfield unexplained signals and just 3 occurrences of downfield intermittent artifacts. Thus our results were very different from the constellation of downfield signals predicted by the non-dynamic stable cluster hypothesis. We have ruled out, or more precisely we have rendered highly improbable, only this one hypothesis on the nature of the \"active ingredient\" of homeopathy. Although a positive finding would certainly have been interesting, our negative findings should not be taken as evidence against clinical homeopathy. In particular, the possibility of dynamic alterations of solvent structuring remains open, but this hypothesis will need to be studied by methods that take a much faster \"snapshot\" of what is going on in samples. Finally, there are also several non-cluster-based hypotheses that have been proposed to explain homeopathy. These include isotopic patterning, coherence, and chaos-based explanations \[[@B41]\]. These explanations do not require any long-lived H-bonds and do not predict that \"unexpected\" discrete peaks would be seen in the NMR spectra of remedies. Conclusion ========== We used a high sensitivity ^1^H-NMR spectroscopy method to look for discrete signals that could provide evidence of pockets of fixed H-bonding in water-based homeopathic remedies. No such evidence was found. The method did reveal the presence of some small common organic molecules, at levels deemed too low to be problematic. We hope that we have made a contribution to homeopathic research, both by answering a particular question, and by setting a standard for quality hypothesis-driven research that others will be inspired to follow. 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/1472-6882/4/15/prepub> Supplementary Material ====================== ::: {.caption} ###### Additional file 1 Table 1: Results of runs during 2002, Table 2: Results of Helios remedies screening, Table 3: List of peak positions ::: ::: {.caption} ###### Click here for file ::: Acknowledgment ============== A grant from the Samueli Institute for Information Biology supported this project. The author thanks the staff of the DCIF for technical training and assistance and for many useful conversations.	PubMed Central	2024-06-05T03:55:49.973934	2004-11-1	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534805/", "journal": "BMC Complement Altern Med. 2004 Nov 1; 4:15", "authors": [ { "first": "David J", "last": "Anick" } ] }
PMC534806	Background ========== Cellular genes are likely to participate in all phases of viral life cycles including attachment to cellular receptors, internalization, disassembly, translation of mRNA, assembly and egress from the cells \[[@B1]\]. The susceptibility to virus infection varies greatly among different cell types, and virus-resistant cells frequently emerge post-infection \[[@B2]-[@B4]\]. This suggests that genetic determinants can influence host cell contributions to the virus life cycle. Despite examples of mammalian genes that influence virus infection, the identification of such genes has been hampered by the lack of practical methods for genetic analysis in cultured cells. In the present study, we tested whether tagged sequence mutagenesis -- a gene entrapment strategy widely used to mutate genes in mouse embryonic stem cells \[[@B5]-[@B10]\] could be used to identify candidate cellular genes required for lytic infection by reovirus, a small cytolytic RNA virus that replicates in the cytoplasm. The mammalian reoviruses serve as useful models for virus-host cell interaction due to their capacity to replicate preferentially in proliferating and undifferentiated cells \[[@B3]\]. Gene traps are efficient mutagens as assessed by studies in mice of mutations induced originally in embryonic stem cells. In somatic cells, the approach assumes that loss-of-function mutations induced by gene entrapment may confer reovirus resistance as a result of gene dosage effects (e.g. haploinsufficiency), pre-existing heterozygosity or loss of heterozygosity. Following infection with the U3NeoSV1 retrovirus gene trap shuttle vector, libraries of mutagenized rat intestinal epithelial (RIE)-1 cell clones were isolated in which each clone contained a single gene disrupted by provirus integration \[[@B6]\]. The entrapment libraries were infected with reovirus type 1, and virus-resistant clones were selected under conditions that also selected against the emergence of persistently infected cells (PI) that may express virus resistance in the absence of cellular mutations \[[@B4]\]. Genes disrupted in a total of 151 reovirus resistant cells were identified by sequencing regions of genomic DNA adjacent to the entrapment vector \[[@B6]\]; of these, 111 contained mutations in previously characterized genes and anonymous transcription units. Reovirus-resistant clones were selected at higher frequencies from entrapment libraries than from non-mutagenized cells, suggesting that reovirus-resistant phenotypes were induced by gene trap mutagenesis. However in any genetic screen, clones with the selected phenotype may arise from spontaneous mutations, and consequently, additional experiments are required to demonstrate that individual genes disrupted by gene entrapment actually contribute to the reovirus-resistant phenotype. For example, a mutation in Ctcfmutation, a transcriptional repressor of insulin growth factor II (IGF-II), was one of 4 mutations associated with reovirus resistance that affected IGF-II expression and/or signalling. Subsequent experiments demonstrated that enforced IGF-II expression is sufficient to confer high levels of reovirus resistance \[[@B4]\]. In short, genes collectively identified by tagged sequence mutagenesis in a panel of reovirus resistant clones provide candidates for mechanistic studies of cellular processes that participate in the virus lifecycle. Since the disrupted genes do not adversely affect cell survival, drugs that inhibit proteins encoded by the genes are not expected to be overtly toxic to cells. Hence, the candidate genes may also include targets for novel anti-viral therapies. Results ======= Tagged sequence mutagenesis and selection of reovirus resistant clones ---------------------------------------------------------------------- Twenty libraries of mutagenized RIE-1 cells, each representing approximately 10^4^independent gene trap events, were isolated following infection with the U3NeoSV1 gene trap retrovirus. U3NeoSV1 contains coding sequences for a neomycin resistance gene in the U3 region of the viral long terminal repeat (LTR). Selection for neomycin resistance generates clones with proviruses inserted within actively transcribed genes. Cells pooled from each entrapment library were separately infected with Type 1 reovirus at a multiplicity of infection of 35, and reovirus-resistant clones were selected in serum-free media to suppress the emergence of persistently infected (PI) cells (4). A total of 151 reovirus-resistant clones were isolated -- approximately 1 mutant per 10^3^gene trap clones or 1 mutant per 10^7^reovirus infected cells. For comparison, the frequency of recovering resistant clones from RIE-1 cells not mutagenized by gene entrapment was less than 10^-8^. This suggests that reovirus-resistant phenotypes were induced by gene trap mutagenesis. Reovirus-resistant cells selected in serum-free media did not express viral antigens (Figure [1](#F1){ref-type="fig"}) and did not produce infectious virus as assessed by plaque assay (E.L. Organ, unpublished results). Most clones were resistant to infection by high titre reovirus and were further analyzed (Figure [2](#F2){ref-type="fig"}). While reovirus resistance did not initially result from the establishment of a persistent infection, many clones became persistently infected upon subsequent passages, presumably because mutant cells that display virus resistance are susceptible to the establishment of a PI state \[[@B2]\] from residual virus used in selection. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Characterization of phenotypic properties of cloned RIE-1 cells resistant to reovirus type 1 infection(A) Cells were stained for reovirus antigen as previously described \[3\]. Only the PI cells contain reovirus antigen as detected by immunohistochemistry (dark wells). Upper wells contain cloned mutant RIE-1 cells from two sets of RIE-1 mutant cell lines selected for reovirus resistance. The lower wells contain PI RIE-1 (left) and uninfected wild type RIE-1 (right). (B) Reovirus susceptible L-cell monolayers, maintained in 1 ml of completed medium, were used to detect the presence of virus in a 100 μl lysate obtained of mutant cells (upper two wells), PI RIE-1 cells (lower left) or uninfected parental RIE-1 cells (lower right). Note, that only L-cell monolayers exposed to a lysate from PI RIE-1 cells lysed within one week of exposure (gentian violet stain). ::: ![](1471-2121-5-41-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### RIE-1 mutant cells resist lytic infection by reovirusThe columns contain an unselected RIE-1 cell library, RIE-1 40C, and representative reovirus resistant mutant cell clones. Serial two-fold dilutions of reovirus were made with the highest titer in the upper row, MOI = 1 × 10^4^. Resistance to reovirus type 1 infection was observed in the mutant cells 3 to 7 days post-infection. The bottom row of cells, denoted by \"C\", were not infected to serve as controls for cell viability and proliferation. Cells were stained with gentian violet four days post-infection. A clear well indicates cell death following virus infection. ::: ![](1471-2121-5-41-2) ::: Identification of genes disrupted in reovirus-resistant clones -------------------------------------------------------------- The U3NeoSV1 gene trap vector contains a plasmid origin of replication and ampicillin resistance gene; thus, regions of genomic DNA adjacent to the targeting vector were readily cloned by plasmid rescue and sequenced \[[@B6]\]. The flanking sequences were compared to the nucleic acid databases to identify candidate cellular genes that confer resistance to lytic infection by reovirus when altered by gene entrapment. Altogether, the 151 cloned flanking sequences matched 111 annotated gene and transcription units in the public DNA sequence databases \[non-redundant (nr), high-throughput genomic sequences (htgs), and human, mouse, and rat genome sequences \[[@B6]\]. 40 flanking sequences were uninformative because they matched repetitive elements or regions of genomic DNA not associated with any annotated transcription unit. The Supplementary Table \[see [Additional File 1](#S1){ref-type="supplementary-material"}\] lists genes disrupted in reovirus resistant clones for which some functional information is available. Many of these genes encode proteins that are known to physically interact. Genes associated with particular metabolic or signalling pathways are shown in Table [1](#T1){ref-type="table"}. These include gene products that could play potential roles in all aspects of virus replication: entry, disassembly, transcription, translation, and reassembly (Table [1](#T1){ref-type="table"}, Figure [3](#F3){ref-type="fig"}, Supplementary Table \[see [Additional File 1](#S1){ref-type="supplementary-material"}\]). Eleven genes encoding calcyclin, insulin growth factor binding protein 5 protease (prss11), type C-like lectin protein (Clr)-fand -C, Dnaja1-/Aprataxin+ (Aprx), GATA binding protein 4 (Gata4), Bcl2 like-1 (Bcl2l1); and chromosome 10 open reading frame 3 (Chr10orf3) and myoferlin, fer-1 like protein 3 (Fer1l3), S100a6 (encoding calcyclin), and two functionally anonymous cDNAs were independently mutated in separate cell libraries (Supplementary Table \[see [Additional File 1](#S1){ref-type="supplementary-material"}\]). The proviruses in these independent of mutant clones were located within 7 to over 1500 nucleotides of each other (data not shown). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Classification of trapped genes according to function Trapped genes are listed by the official HUGO Gene Nomenclature Committee names, when available. Functional placement of genes or their products are determined by literature assignments. Some genes perform more than one cellular role, and are classified arbitrarily and others have undefined roles. ::: Transcription Cytoskeletal-Related Membrane Signalling Vesicle/Trafficking ------------------- -------------------------- ---------------- ---------------- ----------------------------- Brd2 Anx3 Abca4 E2ip2 Anxa1 Brd3 Cald1 Celsr2 Fkbp8 Anxa2 Ctcf Calm2 Csmd2 Fusip1 Atp6v0c E2f2 Kif13b Erbb2ip Gata4 Copg2 Gtf2e1 Mapt OL16 Grb2 Golga2 Hnrpl Ppm1a Pgy1 Jak1 Hm13 Hoxc13 Rps18 Rab13 Madh7 Igf2r Hp1-bp74 Stmn1 Serp1 Map3k7ip1 Psa Id3 Tpm1 Pde4b Rabl3 Zfp7 Rraga Rin2 Znf207 Ryk S100a6 Translation Apoptosis Metabolism Chaperonin Ubiquination/Proteosome Cstf2 Bcl2l Gas5 Dnaja1 Psma7 Eif3s10 IkBζ Lipc Ube1c Srp19 Mical2 Mgat1 Rfp2 Pts *Unassigned* Aptx Hspc135 Ocil Wdr5 Clr-f Klhl6 Ror1 Dlx2 Mox2r Scmh1 Dre1 Numb Trim52 ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### A model of the life cycle of reovirus: proposed checkpoints based on function of the cellular genes identified by the insertional mutagenesisThe virus life cycle begins (top, clockwise) with virus binding to cell surface receptor and being endocytosed into early endosomes. These endosomes then associate with annexin-II (Anxa2) \[62\] and fuse with annexin-II-associated vesicles containing newly synthesized lysosomal enzymes migrating from the Golgi \[63\], which further fuse with the lysosome. The vacuolar H^+^-ATPase (Atp6v0c) acidifies the lysosome, allowing acid-dependent proteases to digest the outer coat from the virus particles and activate them \[64\]. These activated particles then pass through the lysosomal membrane and begin transcription of mRNA. The Golgi protein gm 130 (Golga2) is believed to mediate the docking of vesicles as they carry their newly synthesized cargo through the Golgi stack \[65, 66\]. N-acetylglucosaminyl transferase I (Mgat1) initiates the glycosylation of cell surface proteins (receptors?) and may play a major role, through kinship recognition, in helping maintain the correct assortment of lysosomal enzymes \[67-71\]. The Igf2rshuttles enzymes bound for the lysosome from the Golgi \[72\] and transfer Igf2to the lysosome. While the roles of calcyclin and the α-tropomyosin (Tpm1) are still unclear, they specifically bind each other, and calcyclin is known to bind Anxa2\[16, 20\]. Thus, they may be involved in endosome fusion. Eif3s10specifically binds the virus message to begin its preferential translation. The DnaJa1protein may facilitate the proper folding of virus proteins with its chaperone function \[73\]. However, DnaJa1protein and Eif3may play additional roles in virus trafficking or apoptosis, respectively. Eventually, morphogenesis is complete when crystalline-like arrays of new virions form, cell lysis occurs, and virus is released. Many of the cellular proteins encoded by mutated genes have direct or indirect roles in trafficking of endosomes or lysosomal fusion and thus may play roles in the early disassembly or delivery of transcriptionally active virions to the appropriate cell location. ::: ![](1471-2121-5-41-3) ::: While the presence of multiple, independent mutations in specific genes provides indirect evidence for their involvement in the reovirus lifecycle, the genes could also represent hot spots for gene entrapment. The U3NeoSV1 vector preferentially targets genes with 5\' exons that can splice in-frame with a cryptic splice site in the Neogene to produce enzymatically active Neo fusion proteins. As a consequence, mutagenesis by U3NeoSV1 is actually quite biased, such that of 400 mutations characterized in ES cells, one-third involved genes disrupted multiple times, including Pecam1 which was targeted 9 times \[[@B11]\]. However, none of the multiply targeted genes associated with reovirus resistance involved previously observed entrapment hotspots. Conversely, over 10% of the mutations identified in ES cells involved genes for RNA binding proteins, a preference not observed among genes collectively associated with reovirus resistance. Only Madh7 and Gas5, each represented once among the reovirus-resistant clones, were disrupted by U3NeoSV1 in ES cells. Both genes are commonly targeted by other retroviral gene trap vectors and thus probably represent hot spots for gene entrapment \[[@B5],[@B8]\]. Potential involvement of disrupted genes in virus replication ------------------------------------------------------------- The genes associated with reovirus resistance can be grouped according to their presumed role in virus entry, disassembly, translation, and maturation. Reovirus enters the host via an endocytic pathway that requires acidification and proteolysis to remove the viral outer capsid. The presumptive roles of several candidate genes would be anticipated to affect virus replication by interfering with virus disassembly. For example the mannose-6-phosphate receptor/insulin-growth factor-2 receptor (Igf2r) transports cathepsins to the lysosome \[[@B12]\] and acidification of the lysosome is dependent upon the vacuolar H^+^-ATPase (Atp6v0c) \[[@B13]\]. NH~4~Cl is a weak base that interferes with the function of two of the tagged genes, the Igf2rand the Atp6v0c, and blocks the disassembly of reovirus and several other viruses that enter cells via the endocytic pathway. Moreover, specific inhibitors of the vacuolar H^+^-ATPase gene product have been used to block the infectivity of reovirus and influenza A virus \[[@B13],[@B14]\]. Four mutations in three different genes \[Igf2r, Prss11, a protease associated with insulin binding protein 5, and Ctcfa transcriptional repressor of IGF-II\] are predicted to affect IGF-II expression and/or signalling. Cells containing the Ctcfmutation were subsequently found to express elevated levels IGF-II, while enforced IGF-II expression was sufficient to confer high levels of reovirus resistance. The resistance was caused, at least in part, by a block in virus disassembly \[[@B4]\]. Similarly, both anti-IGF-II receptor antibodies and soluble IGF-II receptor have been reported to inhibit herpes simplex virus infection in vitro \[[@B15]\]. By inference, the recovery of several clones with mutations in genes involved in IGF-II expression/signalling pathway suggests that mutations in multiple genes may affect the same phenotype by acting on a common pathway. Additionally, several of the inserted mutations encode proteins that are thought to participate in trafficking of cargo in cells, and may participate in various stages of virus infection. These include mutations in genes encoding three annexins (Anxa1, Anxa2, Anxa3) and calcyclin (S100a6/S100a1) -- proteins that may bind to each other \[[@B16]-[@B18]\] -- and mutations affecting cytoskeletal and cytoskeletal associated proteins (Cald1, Kif13b, Mapt, Mkln1, Stmn1, Tm9sf4, and Tpm1). Annexin-II associates with cytomegalovirus virions and anti-annexin-II antibodies have been found to prevent cytomegalovirus plaque formation \[[@B19]\]. Annexin-II is known to bind to several of the other gene products mutated in our library. These include annexin-I, calcyclin, and α-tropomysin (Supplementary Table \[see [Additional File 1](#S1){ref-type="supplementary-material"}\]) \[[@B17],[@B18],[@B20]\]. One of the clones has a disruption of a novel cell receptor, OL-16, which is a member of the immunoglobulin superfamily \[[@B21],[@B22]\]. A presumptive cellular receptor for reovirus, junctional adhesion molecule (Jam)-1 \[[@B22]\], has been shown to bind to all reovirus serotypes \[[@B23]\], whereas reovirus infection has been found to be host-cell specific \[[@B24]\]. OL-16 is expressed both in L-cells and in RIE-1 cells that can be infected by reovirus type 1 but not in murine erythroleukemia (MEL) cells that are resistant to infection by type 1 reovirus \[[@B25]\]. Forced expression of an OL-16 transgene in MEL cells increases their susceptibility to reovirus type 1 infection (J. Sheng and D. Rubin, unpublished results). Several of the candidate genes have products that interact with either reovirus or with other viruses (Supplementary Table \[see [Additional File 1](#S1){ref-type="supplementary-material"}\]). Cellular activities involved in post-transcriptional gene regulation may influence the processing or translation of virus transcripts. Two candidate genes participate in these processes. Eif3, part of a multi-subunit translation initiation complex, has been found to specifically bind the 5\' end of hepatitis C and classical swine fever virus mRNA \[[@B26]\]. The Cstf64 KaD subunit, which affects polyadenylation of mRNA, can be cross-linked to the mRNA of herpes simplex mRNA in infected HeLa cell extracts \[[@B27]\]. Other candidate genes are associated with the interferon pathway and host inflammatory responses \[[@B28]-[@B30]\]. For example, IκBζ (MAIL), as a component of the NK-κB pathway, may directly or tangentially affect interferon production, inflammation, or apoptosis. In addition, one gene encodes 6-pyruvoyl-tetrahydropterin synthase (Pts), a major regulator of interferon activity \[[@B31]\] associated with inducible nitric oxide synthase (iNOS). iNOS levels within cells affects the efficiency of replication of many viruses, including the avian reoviruses \[[@B32],[@B33]\]. Many of the targeted gene products have roles involving the Golgi or endosomal compartments (Figure [3](#F3){ref-type="fig"}), and additional genes play a role in differentiation or growth arrest. Of these, several are in the transforming growth factor (TGF)-β and NF-κB regulatory pathways, Ppm1a\[[@B34]\], Madh7\[[@B35]-[@B37]\], Ube1c\[[@B38],[@B39]\], and Map3k7ip1\[[@B40]-[@B43]\] (Table [1](#T1){ref-type="table"}, Supplementary Table \[see [Additional File 1](#S1){ref-type="supplementary-material"}\]). In addition, subunits of the eif3 complex have been functionally linked to Mapkbp1and the proteosome \[[@B44]\]. We have also disrupted a number of genes that participate in apoptosis (Supplementary Table \[see [Additional File 1](#S1){ref-type="supplementary-material"}\]), and three disrupted genes affect N-linked protein glycosylation, a process that may affect compartmentalization of proteins or ligand interactions. Reovirus resistant cells have altered susceptibility to HSV-1 ------------------------------------------------------------- As several of the genes listed in the Supplementary Table \[see [Additional File 1](#S1){ref-type="supplementary-material"}\] have been associated with herpes simplex-1 (HSV-1) replication, seven clones were tested for their susceptibility to HSV-1 infection \[[@B15],[@B45]\]. These experiments utilized HSV-1(KOS)tk12, an infectious virus that expresses a lacZ reporter as an immediate-early gene \[[@B46]\]. Data representing seven clones with mutations that tag known genes are provided in Figure [4](#F4){ref-type="fig"}. Four clones, with mutations in the Eif3s10, AnxaI, Mgat1, and Igf2rgenes, were resistant to HSV-I infection (Figure [4B,4C](#F4){ref-type="fig"}, b, d, f, and h) and there was a diminished capacity to express the immediate-early lacZreporter gene. However, two of the clones (Figure [4B,4C](#F4){ref-type="fig"}, c and e) with mutations in genes encoding calcyclin and annexin-II, were more susceptible than the parental RIE-1 cells to HSV-1 infection and expressed higher levels of the immediate-early lacZreporter gene. Representative clones that contain altered levels of HSV-1 immediate early gene expression are shown in Figure [4A](#F4){ref-type="fig"}. LacZexpression in cells containing a disrupted calcyclin (S100a6) gene was readily apparent 4 h following infection, whereas lacZexpression was barely detected in Eif3s10mutant cells 16h following infection. In all cases, levels of lacZexpression correlated with susceptibility to HSV-1 infection, suggesting that resistance involved early steps in the viral lifecycle. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### HSV-1 infection is affected in cell clones selected as reovirus resistantThe level of transcription and translation of the reporter gene, lacZ, present in the immediate early genes of HSV-1 is shown in A and B. A)The level of expression of mRNA is shown at 4, 8 and 16 h following infection for a library of non-reovirus selected RIE-1 cells (L42), and two clones that disrupt the Eif3s10(p162) and S100a6genes. The cell clone with a disrupted S100a6gene has a dramatic increase in HSV-1 expression with a concomitant decrease in cellular gene expression by 16 h. While there is more mRNA loaded in the lane with a disrupted Eif3s10gene than is present in the other lanes, there is no evidence for HSV-1 expression in this cell until 16 h following infection. B)Translation of the LacZreporter in the immediate early genes of HSV-1. At 8 hours following infection, the translation of the LacZgene is dramatically increased in clones with mutant S100a6and Anxa2genes, barely detectable in the population of non-selected library cells (L42) and a cell clone that tags the Aptx^+^/DnaJa1^-^genes, and is not evident in the other mutant clones. C)Cell survival was determined by gentian violet staining of cells at 72 hours. L42 cells, and clones with mutations in the Aptx^+^/DnaJA1, annexin II, and S100a6genes lysed whereas clones with mutations in the Eif3s10, Anax1, Mgat1, and Igfr2genes were resistant to lytic infection. a-library 42, non-selected; b-Eif3s10; c-calcyclin; d-Anxa1; e-Anxa2; f-Mgat1; g-Aptx^+^/DnaJa1(negative strand); h-Igf2r ::: ![](1471-2121-5-41-4) ::: Discussion ========== Candidate genes required for lytic reovirus infections were identified by tagged sequence mutagenesis, a process that permits rapid identification of genes disrupted by gene entrapment. Since virus-resistant mutants may arise by a variety of mechanisms, additional experiments are needed to demonstrate that individual genes disrupted by gene entrapment actually contribute to the reovirus-resistant phenotype. Even so, several lines of evidence suggest that the genes collectively identified by tagged sequence mutagenesis include cellular activities that participate in the virus lifecycle. First, reovirus-resistant clones were selected at higher frequencies from entrapment libraries than from non-mutagenized cells, suggesting that reovirus-resistant phenotypes were induced by gene trap mutagenesis. Second, the genes associated with reovirus resistance differed from genes targeted in an unselected manner in mouse ES cells. Known mutational hot spots of the U3NeoSV1 were under-represented, and a number of mutations associated with virus resistance appeared to cluster within specific cellular processes and/or affected different components of multi-protein complexes that are likely to play roles in the virus lifecycle. These include IGF-II expression/signalling (3 genes), cytoskeletal/vesicular/trafficking (20 genes), signalling pathways (11 genes), and apoptosis (4 genes). Finally, we recently demonstrated that the disruption of Ctcf, a transcriptional repressor of IGF-II, was directly responsible for reovirus-resistance. In particular, cells containing the Ctcfmutation express elevated levels IGF-II, while parental RIE1 cells forced to express IGF-II acquired high levels of reovirus resistance. The mutation in Ctcfwas chosen for further analysis because it was one of 3 mutations affecting IGF-II expression and/or signalling \[[@B4]\]. By inference, the recovery of inserts affecting other genes in the IGF-II signalling pathway suggests that the same phenotype can be achieved through mutations in multiple genes in a common pathway. Taken together, these results suggest that the candidate genes identified by tagged sequence mutagenesis provide useful information to direct mechanistic studies of cellular processes that participate in the virus lifecycle. These studies utilized a diploid cell line to select for reovirus resistance. Therefore, recessive phenotypes resulting from loss-of-function mutations are generally expected to require separate inactivation of genes carried on both autosomes. In principle this could occur through pre-existing heterozygosity or by loss of the unoccupied allele by one of several mechanisms such as gene conversion, non-disjunction or transcriptional repression. Several of the candidate genes discovered in these experiments are imprinted, and therefore may be anticipated to be mono-allelic in their expression, including the maternally imprinted Igf2r. Alternatively, mutations induced by gene entrapment may confer reovirus resistance as a result of gene dosage effects (e.g. haploinsufficiency). For example, recent data suggests that the most common genetic disease in Caucasians, cystic fibrosis, involves mutations in the ABC-cassette transporter protein, CFTR, that confer resistance to infection by Salmonella typhi\[[@B47]\]. Protection is afforded to persons heterozygous at this allele. Similarly, cyclosporin analogs, which affect P glycoprotein (a member of the ABC cassette transporters), inhibit the growth of Cryptosporidium parum\[[@B48]\]. Of note, one of the genes associated with reovirus resistance identified in the present study, Pgy1(Abcb1), encodes P glycoprotein (Table [1](#T1){ref-type="table"}, Supplementary Table \[see [Additional File 1](#S1){ref-type="supplementary-material"}\]). Finally, while the U3NeoSV1 entrapment vector lacks the MuLV enhancer element, we cannot exclude the possibility that the phenotype observed was related to dominant mutations caused either by transcriptional activation of adjacent cellular genes or from the expression of truncated proteins with dominant-negative activity. The circumstances that allow gene entrapment to disrupt the function of diploid genes illustrate that events secondary to provirus integration may be required for expression of some reovirus resistant phenotypes. Consequently, while the entrapment libraries were theoretically large enough (2 × 10^5^independent mutations) to disrupt all expressed genes, it seems unlikely that all the genes that are required for virus infection, and that can be targeted by tagged sequence mutagenesis, were identified in the present study. Reovirus infection may induce apoptosis in vivoand in vitro, and the suppression of apoptosis enhances the survival of mice infected with reovirus type 3 \[[@B49],[@B50]\]. Mutations associated with reovirus resistance included a number in proapototic genes including, IκBζand Bcl2l1; however, the precise role of this pathway and the genes we have disrupted in modulating reovirus infectivity is unknown \[[@B51],[@B52]\]. Therefore, while many genes are associated with known pathways, further studies will be required to understand the manner by which these pathways influence reovirus infection. Genetic alterations giving rise to reovirus resistant clones have variable effects on HSV-1 replication, with some reovirus resistant clones showing enhanced HSV1 replication. The reasons for this are unknown, although each of these two viruses enter cells by different mechanisms. The early steps in HSV replication require entry into cells, release of the capsid with migration to the nucleus for which virus and cellular proteins play roles \[[@B53],[@B54]\], whereas the entry of reovirus does not involve transit to the nucleus. Enhanced HSV-1 replication in clones containing mutations in the S100a6(calcyclin) and Anxa2genes was accompanied by a dramatic increase in immediate-early gene expression. This temporal enhancement of HSV-1 replication may reflect activities of calcyclin and annexin 2 proteins that suppress HSV-1 entry \[[@B55]-[@B57]\]. In addition to the clone with a mutation in Anxa1, clones with mutations in Eif3s10, Mgat1, and Igf2ralso show decreases in transcription and translation of virus mRNA and cell death. Of these, mutations in the Igf2rare known to affect HSV replication \[[@B15],[@B54],[@B58]\]; whereas, association of HSV replication with proteins encoded by the Eif3s10, Anxa1, and Mgat1are novel. These data suggest that some of the candidate genes discovered in clones surviving reovirus infection may affect common cellular processes that are used by other viruses. Studies in a variety of systems indicate that resistance to infection may be found in nature or achieved in cultured cells. Results presented here support the hypothesis that pathways involved in reovirus infection can be identified through a functional genomics approach based upon insertional mutagenesis. Systematic selection of virus-resistant mutant cells in which the mutant gene can be easily identified may also identify targets for the development of anti-viral therapies. Drugs that disrupt cellular processes may circumvent the problem of virus resistance, generally observed with drugs against virus-encoded proteins. Moreover, since mutations associated with virus resistance are, by necessity, not lethal to the cell, drugs that target the same processes are not expected to have overtly toxic side effects. The fact that the resulting candidate genes may play roles in the replication of other viruses, suggests that different viruses may use similar host proteins for common steps required for virus entry, disassembly, transcription, translation, and reassembly. This view is supported by our studies with HSV-1 and by published reports implicating several of the genes disrupted in reovirus resistant cells (Supplementary Table \[see [Additional File 1](#S1){ref-type="supplementary-material"}\]) in the replication of other viruses. Thus, mutant clones selected for resistance to lytic infection to one virus may provide targets for therapeutics that are active against other families of viruses. The dramatic increase in the pace of the genome project has led to an explosion of information concerning the sequence of the genome of several species of animals and pathogenic organisms. However, most of the gene sequences have not been functionally ascribed with regard to host-parasite interactions. As there are approximately 30 to 50 × 10^3^mammalian genes, the definition of function will become the major task facing scientists interested in the relationship between host genes and viral disease over the next decade. Conclusions =========== Candidate host genes that participate in lytic virus infections were identified utilizing insertional mutagenesis. Mutant cell clones were recovered that lost their capacity to support virus replication, but were able to proliferate. There was enrichment for genes that were involved in particular metabolic or signalling pathways, with many of the genes being selected more than once from independently derived libraries of RIE-1 cells. Several of the gene products are known to bind to each other. These genes or their products, which are identified by this process of selection, may provide targets for therapeutic intervention. Methods ======= RIE-1, L-Cells and Virus ------------------------ Reovirus type 1, strain Lang, was initially obtained from Bernard N. Fields. Virus was passaged in L-cells and a third passaged stock was purified over a CsCl gradient as previously described and was used for these experiments \[[@B59]\]. To develop PI cell lines, RIE-1 cells were infected with reovirus type 1, at a multiplicity of infection (MOI) of 5, and surviving cells were maintained in Dulbecco\'s modification of Eagle\'s minimum essential medium (DMEM) (Irvine Scientific, Santa Ana, CA, USA). The herpes simplex virus (HSV)-1 clone, HSV-1 KOStk12, that expresses a reporter gene, lacZ, as an immediate-early gene \[[@B46]\] was a generous gift of Patricia Spear, Northwestern University, USA. For RIE-1 and L-cells, medium was supplemented with 10% fetal bovine serum, 2 mM per ml, L-Glutamine 100 units per ml, Penicillin, and 100 μg per ml Streptomycin (Irvine Scientific, Santa Ana, CA, USA) \[complete medium\]. In some experiments, serum was omitted from the medium. The continuance of cell monolayers following infection with reovirus or HSV-1 was determined by staining with gentian violet. Tagged sequence mutagenesis and selection for reovirus resistance ----------------------------------------------------------------- Following infection of RIE-1 cells with the U3neoSV1 vector, MOI of 0.1, mutagenized cells were selected for neomycin resistance in medium containing 1 mg/ml G418 sulfate (Clontech, Palo Alto, CA, USA) \[[@B6]\]. Twenty libraries of mutant RIE-1 cells, and one library of A549 human adenocarcinoma cells, each consisting of 10^4^gene entrapment events, were expanded until approximately 10^3^sibling cells represented each mutant clone. These cells were plated at a sub-confluent density and incubated in serum-free media for 3 days until they became quiescent, and infected with reovirus serotype 1, MOI of 35 plaque forming units (pfu) per cell. Eighteen hours following infection, the cells were detached with trypsin, and plated in DMEM medium containing 10% fetal bovine serum (FBS) (Hyclone Laboratories, Inc., Logan, Utah, USA). After 6 hrs, the medium was removed and cells were maintained in serum-free medium until only a few cells remained attached to the flask. On average, one to ten clones were recovered from a library consisting of 10^7^mutant cells, an enrichment for selected cells of six orders of magnitude. Cells that survived the selection were transferred to cell culture plates in media containing 10% FBS and cells were divided for extraction of DNA and cryopreservation. Transcription and translation of HSV-1 immediate early gene reporter -------------------------------------------------------------------- The transcription and translation of the HSV-1 immediate early gene reporter gene, lacZ, was determined by standard northern blot techniques and β-galactosidase assay, respectively. Generation of libraries of mutagenized RIE-1 cells -------------------------------------------------- Libraries of mutagenized cells were infected with reovirus serotype-1, strain Lang, to select for clones resistant to lytic infection. Selection of virus-resistant clones was performed in serum-free medium to suppress the emergence of persistently infected (PI) cells \[[@B4]\]. This is important since PI cells, which arise by a process involving adaptive mutations in both the virus and the cell genomes \[[@B60]\], provide a means whereby RIE-1 cells can acquire virus resistance in the absence of cellular mutations. Uninfected RIE-1 cells undergo growth arrest, whereas PI RIE-1 cells are killed in serum-free medium. DNA sequence analysis --------------------- Genomic DNA immediately adjoining the 5\' end of the proviral insert in each of 130 cell lines was cloned by plasmid rescue \[[@B6]\]. Approximately 300 to 600 base pairs of this flanking DNA were sequenced and compared with the non-redundant (nr) and expressed sequence tag (dbEST) nucleic acid databases \[[@B61]\]. The probability of a match with orthologous sequences in the databases varies due to interspecies variation, the amount of exon in the flanking DNA (in cases where the flanking DNA matches cDNA sequences), alternative splicing and sequencing errors. Matches with sequences in the database were considered potentially significant if probability score was \<10^-5^and the sequence was non-repetitive. In most cases, the matching gene was in the same transcriptional orientation as the provirus. Moreover, matches involving cDNA sequences were co-linear across exons present in the flanking genomic DNA and diverged at splice sites. As indicated, virtually all of the genes identified had matches to murine, rat, or human gene sequences with p \< 10^-10^. Authors\' contributions ======================= ELO, and JS conducted most of the laboratory work. HER provided the vectors and advice on their use. DHR discovered that persistently infected cells require serum to survive, allowing the selection of genetically resistant cell clones, and did the genetic analysis. Drs. Ruley and Rubin provided funding and supervision for the research, and prepared the manuscript. All authors have read and approved the final manuscript. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Genes associated with resistance to lytic reovirus infection identified by tagged sequence mutagenesisA list of previously named genes disrupted by the insertional mutagen, U3neoSV1, and recovered in cell clones resistant to lytic infection is provided. The rat mRNA and vector insertion site accession number, rat chromosome location, human homologue chromosome location, link to the NCBI Entrez Gene and NCBI Nucleotide databases, and known virus interactions are listed. ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ This work was supported by Public Health Service Grants (R01NS043952 and R01RR13166 to HER, RO1CA682383 to DHR), by a grant from the Kleberg Foundation, and was partially supported by Cancer Centre (Core) grant P30CA42014, and Avatar BioSci, Inc. We would like to thank P. Spear for the herpes simplex virus used in these experiments. J. Murray and L. Fernandez for advice on the Supplementary Table. J. Hawiger, T. Hodge, and E. Eisenberg for review of the manuscript. B. Mooneyhan provided expert secretarial assistance.	PubMed Central	2024-06-05T03:55:49.977616	2004-11-2	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534806/", "journal": "BMC Cell Biol. 2004 Nov 2; 5:41", "authors": [ { "first": "Edward L", "last": "Organ" }, { "first": "Jinsong", "last": "Sheng" }, { "first": "H Earl", "last": "Ruley" }, { "first": "Donald H", "last": "Rubin" } ] }
PMC534807	Background ========== An association between allergic conditions and cancer risk has been the subject of several epidemiological studies. Both positive and negative associations have been observed and two hypotheses have been formulated to explain such relationships. First, the immune surveillance hypothesis, which proposes that allergic conditions may lead to a decreased risk of malignancy by enhancing the ability of the immune system to detect and eliminate malignant cells \[[@B1]\]. Evidence from some previous studies of hematological malignancies in relation to allergic conditions primarily supports this hypothesis \[[@B2]-[@B4]\]. Second, the antigenic stimulation hypothesis states that immune-stimulating conditions lead to an increased risk of malignancy, which for hematological malignancies is supported by some studies \[[@B5],[@B6]\]. This would be caused by a mechanism where the chronic stimulation induced by the activated cells of the immune system eventually lead to randomly occurring pro-oncogenic mutations in actively dividing cells. There are also a number of studies where no associations between different allergic conditions and hematological malignancies were found, or where inconsistent results were obtained. It is plausible that the association between allergic conditions and cancer risk is complex and that the risk of developing cancer could depend on the specific malignancy and also could be influenced by the type of allergic condition. Most previous studies are retrospective case-control studies. Recall bias may have influenced the results of retrospective case-control studies that have asked for past allergic conditions after diagnosis of malignancy \[[@B2]-[@B5],[@B7],[@B8]\], a problem that partly remains after confirmation of the information on medical history in medical records \[[@B9]-[@B11]\] and also when a combination of self-reported information and information from hospital notes and general practioner notes was used \[[@B12]\]. There are only a few cohort studies that have investigated the relationship between allergic conditions and hematological malignancies \[[@B6],[@B13]-[@B19]\]. Four of the studies primarily lend support to the antigenic stimulation hypothesis for hematological malignancies, as increased risks, or tendencies toward increased risks, were found for a history of allergy \[[@B6],[@B15],[@B18],[@B19]\], whereas two of them support the immune surveillance hypothesis by showing decreased risks \[[@B16],[@B17]\]. Four of the studies concerned asthma only \[[@B13],[@B14],[@B16],[@B17]\]. The purpose of the present study was to investigate the influence of allergic conditions on the risk of developing leukemia, malignant lymphoma and myeloma in a well-established cohort of Swedish twins, and to see whether the results support the immune surveillance hypothesis or the antigenic stimulation hypothesis. An important strength of this study is that information about allergic conditions and confounding factors has been collected prospectively, and therefore the exposure is not subject to differential misclassification. Methods ======= The study base consists of a cohort of twins followed by the Swedish Twin Registry, which was established in 1961 when questionnaires were sent to all 25,778 individuals in a same-sexed twin pair who were born between 1886 and 1925 and were both living in Sweden in 1961. A response from both twins in a twin pair was required for inclusion in the Swedish Twin Registry (21,870 individuals) \[[@B20]\]. After 1961 they were followed-up as individuals, irrespective of their twin-sibling\'s status. Out of the responders to the 1961 questionnaire, a new questionnaire was sent to 21,863 eligible subjects in 1963 (response-rate 85.1 %), and in 1967 an additional questionnaire was sent to 20,576 eligible individuals (response rate 81.5 %). The twin cohort was used as a population-based cohort without considering twin status. The present study includes the 16,539 individuals (7,167 men and 9,372 women), who responded to the questionnaire mailed in 1967, and who were still alive and not previously diagnosed with a hematological malignancy January 1, 1969. The median follow-up time was 23 years for men and 27 years for women. The median age at baseline was 56 years (10^th^and 90^th^percentiles; 46 and 71 years, respectively). The cohort was followed from January 1, 1969 until diagnosis of a hematological malignancy, death or end of the study (December 31, 1999), whichever came first. Cancer incidence and date of death were ascertained by record linkage to the Swedish Cancer Registry and the Swedish Cause of Death Registry, respectively. In the Swedish Cancer Registry, leukemia is coded according to ICD-8 during the investigated period, while all other malignancies are coded according to ICD-7. We identified 324 subjects with hematological malignancies; 10 cases of HD (ICD-7 201), 112 cases of NHL (ICD-7 200.0--200.3, 202.0--202.4), 75 cases of myeloma (ICD-7 203) and 134 cases of leukemia (ICD-8 204.0--207.9). The leukemias consist of 3 cases of ALL, 67 cases of CLL, 31 cases of AML, 7 cases of CML and 26 cases of unspecified leukemia. Exposure assessment ------------------- Assessment of exposure is based primarily on the 1967 questionnaire, in which questions were asked about the allergic conditions asthma, hay fever, eczema and hives, considering both present and past conditions. The questions were posed as \"Have you ever had asthma? (No/Yes), \"Have you ever had hay fever, rose fever or allergic rhinitis (characterized by running nose, watery and itching eyes when you do not have a cold)?\" (No/Yes), \"Did you have eczema when you were a baby?\" (No/Yes), \"Did you at times later in life have eczema-like skin conditions?\" (No/Yes), Do you know the name of the skin lesion you have or have had? (No/Yes, psoriasis/Yes, hives (urticaria) allergic rash/Yes, contact eczema/Yes, eczema in knee or elbow fold/Yes, allergic eczema/Yes, others: specify name). There were no questions about dates when symptoms first started or ended, and no information about treatments used. In the questionnaire from 1963 responses to two questions about asthma and eczema were included. The subjects could mark if they had had asthma or eczema from a list of 17 diseases or the alternative that they hadn\'t had any of the given diseases. The answer from 1963 was used only if a subject had failed to answer the corresponding question in the 1967 questionnaire. Each medical condition was analyzed separately. In addition we combined the different conditions in order to achieve larger numbers of individuals and thereby obtain more precise results. First, a variable for eczema was created, requiring at least one positive answer for childhood eczema or allergic eczema. Then, a general group of allergic conditions was created, combining the positive answers for eczemas with positive answers on the questions of ever having had asthma or hay fever. Confounders and effect modifiers -------------------------------- All analyses were adjusted for age at enrolment and sex. We have controlled for confounding from alcohol consumption (g/month), level of education, and smoking habits (non-smokers, former smokers, current smokers). Adjustment for these factors did not affect the risk estimates in the majority of analyses, and changed the magnitude of the effect at the most 6% in a few instances. Therefore, the presented results are only adjusted for age and sex. Statistical methods ------------------- We estimated the RR and its 95% CI of each hematological malignancy through Cox\'s Proportional Hazards Model, (SAS program PHREG, SAS Institute, Cary, North Carolina). To ensure that confidence intervals were not erroneously narrowed due to dependencies within twin pairs we performed analyses that adjusted variance estimates for correlated outcomes. We accomplished this through the use of a SAS macro that stems from the same theoretical background \[[@B21]-[@B24]\] and yields the same results as the published Fortran program of D.Y. Lin \[[@B24]\]. In simple terms, variance estimates are increased in magnitude proportional to the degree of extra correlation within twin pairs. Thus, adjusted confidence intervals are more conservative than unadjusted. If correlations within twin pairs are not different from what is observed between unrelated individuals in the cohort with respect to cancer risk, adjusted and unadjusted variance estimates are identical. Relative risk estimates are not altered by this procedure. Results ======= Our results showed either increased risks or risks close to unity for hematological malignancies following allergic conditions. For leukemia, we found an increased risk associated with hives, and an indication of elevated risk associated with asthma, although with wide confidence intervals (Table [2](#T2){ref-type="table"}). For leukemia, excluding CLL, the increased RR associated with hives was further elevated. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Age- and sex-adjusted relative risks for leukemia among subjects with allergic conditions. ::: -------------------------------- -------------- ------------------------- --------- ------------ ---------- ---------- ------ ------------ ---------- ---------- ------ ------------ Leukemia Leukemia, excluding CLL *Exposure* N~e~ N~o~ RR 95% CI N~e~ N~o~ RR 95% CI N~e~ N~o~ RR 95% CI Asthma, hay fever or hives 31 94 1.4 (0.9--2.1) 21 43 2.0 (1.2--3.4) 10 51 0.8 (0.4--1.6) Hay fever 21 105 1.1 (0.7--1.8) 14 50 1.5 (0.8--2.8) 7 55 0.7 (0.3--1.6) Asthma 7 126 1.6 (0.8--3.5) 3 64 1.3 (0.4--4.2) 4 62 1.9 (0.7--5.3) Hives 7 120 2.1 (1.0--4.5) 6 58 3.6 (1.6--8.5) 1 62 0.6 (0.1--4.3) Eczema\* 8 115 0.9 (0.5--1.9) 3 59 0.7 (0.2--2.2) 5 56 1.2 (0.5--3.1) Allergic conditions\\ 30 87 1.1 (0.8--1.7) 18 42 1.4 (0.8--2.4) 12 45 0.9 (0.5--1.7) -------------------------------- -------------- ------------------------- --------- ------------ ---------- ---------- ------ ------------ ---------- ---------- ------ ------------ N~e~= No. of exposed cases, N~o~= No. of unexposed cases. \* At least one positive answer for eczema during childhood or allergic eczema. \\ At least one positive answer for asthma, hay fever, eczema during childhood or allergic eczema. ::: The risk estimates for myeloma were generally close to or below unity (Table [3](#T3){ref-type="table"}). The number of cases with HD was small, with few exposed cases in all analyses making results difficult to interpret (data not shown). For NHL, the risk associated with having had eczema during childhood was increased. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Age- and sex-adjusted relative risks for myeloma and non-Hodgkin\'s lymphoma among subjects with allergic conditions. ::: -------------------------------- ------------- -------------------- ------ ------------ ---------- ---------- ------ ------------ Myeloma non-Hodgkin\'s *Exposure* N~e~ N~o~ RR 95% CI N~e~ N~o~ RR 95% CI Asthma, hay fever or hives 10 58 0.7 (0.4--1.4) 22 83 1.1 (0.7--1.8) Hay fever 10 62 0.9 (0.5--1.7) 21 87 1.3 (0.8--2.2) Asthma 0 75 \- 0 11 \- Hives 1 67 0.5 (0.1--3.9) 1 10 0.4 (0.0--2.6) Eczema\* 3 67 0.6 (0.2--2.0) 8 87 1.3 (0.6--2.6) Eczema during childhood 1 69 0.5 (0.1--3.7) 6 89 2.3 (1.0--5.3) Allergic conditions\\ 12 55 0.7 (0.4--1.4) 27 69 1.3 (0.8--2.0) -------------------------------- ------------- -------------------- ------ ------------ ---------- ---------- ------ ------------ N~e~= No. of exposed cases, N~o~= No. of unexposed cases. \* At least one positive answer for eczema during childhood or allergic eczema. \\ At least one positive answer for asthma, hay fever, eczema during childhood or allergic eczema. ::: Discussion ========== Our results suggested that allergic conditions are risk factors for hematological malignancies, and gave support to the antigenic stimulation hypothesis. Thus, the results are in concordance with most previous cohort studies \[[@B6],[@B15],[@B18],[@B19]\]. A major strength of the present cohort study is that information about allergic conditions and confounding factors has been collected prospectively. Therefore the exposure is not subject to differential misclassification, which is in contrast to retrospective case-control studies where recall bias may be a problem \[[@B2],[@B3],[@B9]\] and where separating the effects of prior allergic conditions from the effect of malignancy per seon the immune system may be difficult. This study has focused specifically on how allergy influences the risk of developing hematological malignancies. Another strength is that the study is based on the Swedish Twin Registry, which is a unique resource allowing for an unusually long period of follow-up. In our study, 31 years of follow-up was possible. The cohort has been followed continuously in the Population Registry and the Cause of Death Registry during the study period, and therefore loss to follow-up is unlikely to be a problem. The Swedish Twin Registry is considered a study base representative of the general population of Sweden and has been used in many epidemiological studies \[[@B25]-[@B27]\]. Another strength is the completeness of the Swedish Cancer Registry, to which it is required by law to report all incident cancer cases in Sweden. New cases of cancer are reported by physicians in hospitals and other establishments as well as by pathologists. The two independent notifications systems ensure a high coverage. In addition, we could adjust for more confounding factors than in previous cohort studies \[[@B6],[@B13]-[@B19]\]. One limitation of the study is the small number of exposed cases, and therefore random variation cannot be excluded as an explanation for our findings and for the same reason no stratification for calendar time was performed. Another limitation in the study is that there may be non-differential misclassification of the malignancies. The study period covers 31 years, and during this time diagnostic practices may have changed. In particular, some cases previously diagnosed as HD are now likely to be classified as NHL \[[@B28]\]. This type of error would bias the effect estimates towards unity. Differential misclassification of exposure is unlikely as there is no reason to believe that reporting exposure should differ between subjects subsequently (years later) diagnosed with a cancer, and those who are not. Non-differential misclassification of exposure is likely to affect the results, but cannot explain increased risks since it would dilute the effect estimates towards unity. The allergic conditions are self-reported and not diagnosed by a physician. However, these self-reported conditions have been used in an earlier study of brain tumors, where some support for the postulated hypothesis that allergic conditions are associated with a decreased risk of developing glioma was found \[[@B29]\]. Also, the validity of the allergic conditions has been investigated in a group of subjects from the Swedish Twin Registry \[[@B30]\]. In general, a good agreement was found between the self-reported conditions and an allergologist\'s diagnosis. Follow-up starts in 1969 and continues until the end of 1999. During 31 years it is possible to develop an allergic condition, but these individuals will still be considered as unexposed members of the cohort. However, the youngest individuals in our cohort were 42 years old when the questionnaire was sent out, which means that this bias have not at all affected childhood eczema, and hay fever and allergic asthma only to a small extent, as these conditions usually present earlier in life. We found an increased risk of NHL among individuals with eczema during childhood. In the literature, there are only few studies concerning an association between eczema and NHL. In one study, a history of eczema was associated with an increased risk of NHL \[[@B10]\]. In several studies, elevated risks for different hematological malignancies among persons with eczema have been found, e.g. \[[@B7],[@B11]\]. On the other hand, eczema has also been observed to decrease the risk of NHL in two studies \[[@B3],[@B9]\]. Comparisons between these other studies and our study are difficult, however, since the other studies have investigated general eczema while we have focused on eczema of allergic origin (i.e. allergic eczema and eczema during childhood). When using the general definition for eczema many non-allergic forms will be included and while these may influence the risk of developing malignancies, the mechanisms involved are probably different from the ones active in allergic conditions. Thus, these eczemas are not included in the present study. In our material subjects with hives showed an increased risk of leukemia, especially after exclusion of CLL. Several other studies have also found an increased risk of AML \[[@B12]\] and other hematological malignancies associated with hives \[[@B6],[@B9],[@B18]\]. In contrast, some other studies did not show this association \[[@B8]\]. A number of studies have found a protective effect of asthma on the risk of developing lymphatic leukemia and leukemia, respectively \[[@B16],[@B17]\]. This relationship between asthma and leukemia was not confirmed in our study. If anything, our results support the antigenic stimulation hypothesis. In a recent cohort study, an increased risk of leukemia was indicated \[[@B19]\]. On the other hand, most studies of hematological malignancies in relation to a history of asthma have shown risks close to unity \[[@B8],[@B9]\]. Clearly, these conflicting results indicate that this area needs to be investigated further. Allergic conditions, like asthma and hay fever, are increasing and it is of great importance to clarify if and how they are connected to hematological malignancies. The contradictory findings may have many explanations, e.g. that different immunological mechanisms may be involved in different types of asthma, that the pathogenesis is likely to be different even in seemingly similar hematological malignancies, and that new forms of pharmacological therapy may influence not only the outcome of asthma but also the risk of developing cancer. To solve these problems, large prospective epidemiological studies on individuals with clinically strictly defined allergic conditions, including data on pharmacological treatment and severity of disease, need to be combined with information about morphologically defined hematological malignancies, including subtyping with techniques from modern molecular biology. Conclusions =========== In summary, findings from our cohort study suggest that chronic antigenic stimulation from allergic conditions might increase the risk of some hematological malignancies. Abbreviations ============= ALL = Acute lymphoblastic leukemia; AML = Acute myeloid leukemia; CLL = Chronic lymphocytic leukemia; CML = Chronic myeloid leukemia; HD = Hodgkin\'s disease; NHL = Non-Hodgkin\'s lymphoma; RR = Relative risk; CI = Confidence Interval. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= KCS has been the principal investigator, contributed to the planning of the study, performed the statistical analysis and drafted and coordinated the writing of the manuscript. LH participated in the planning of the study and the writing of the manuscript. JS contributed to the writing of the manuscript. MF carried out the study design and contributed to the writing of the manuscript. All authors contributed to the interpretation of results, have read and approved the final manuscript. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Self-reported allergic conditions among 16,539 subjects. ::: Self-reported allergic conditions Number of respondents Number reporting condition % reporting condition --------------------------------------- --------------------------- -------------------------------- --------------------------- Asthma, Hay fever or Hives 15,168 3,022 19.9 Hay fever 15,546 2,428 15.6 Asthma 16,376 604 3.7 Hives 15,379 430 2.8 Eczema\* 14,803 1,033 7.0 Eczema during childhood 14,816 400 2.7 Allergic conditions\\ 14,294 3,430 24.0 \* At least one positive answer for eczema during childhood or allergic eczema. \\ At least one positive answer for asthma, hay fever, eczema during childhood or allergic eczema. ::: Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2458/4/51/prepub> Acknowledgements ================ This study was funded by a grant from The Swedish Cancer Society. Dr Per Larsson, The Rheumatology Unit, Karolinska Hospital, Stockholm, Sweden for sharing knowledge, valuable advice and discussions.	PubMed Central	2024-06-05T03:55:49.981136	2004-11-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534807/", "journal": "BMC Public Health. 2004 Nov 4; 4:51", "authors": [ { "first": "Karin C", "last": "Söderberg" }, { "first": "Lars", "last": "Hagmar" }, { "first": "Judith", "last": "Schwartzbaum" }, { "first": "Maria", "last": "Feychting" } ] }
PMC534809	Introduction {#s1} ============ How is complex organization produced and maintained by physical processes? One may look to biology, where we find the most sophisticated organization of matter, often spanning more than 24 orders of magnitude from component molecules (0.1 attograms) to entire organism (100 kilograms). This organization is information-based: DNA sequences refined by evolution encode both the components and the processes that guide their development into an organism---the developmental program. For a language to describe this carefully orchestrated organization, it is tempting to turn to computer science, where the concepts of programming languages, data structures, and algorithms are used to specify complex organization of information and behavior. Indeed, the importance of universal computation for autonomous fabrication tasks was recognized in von Neumann\'s seminal work on self-reproducing automata, where he postulated a universal constructor that, by reading an input tape specifying an algorithm for what to build, could carry out the commands necessary to construct an arbitrary object ([@pbio-0020424-vonNeumann1]). If algorithmic concepts can be successfully adapted to the molecular context, the algorithm would join energy and entropy as essential concepts for understanding how physical processes create order. Unfortunately, the study of molecular algorithms has been hampered by the lack of suitable physical systems on which to hone such a theory: nature provides us with elementary chemical reactions too simple to program, full-blown life too complex to use as a model system, and few systems in between. This gap may be explored by synthesizing programmable biochemical systems in vitro, where we can implement and study a variety of molecular algorithms ranging gradually from simple to complex. Biomolecular self-assembly is particularly attractive for the exploration of molecular algorithms that control nanofabrication tasks. Attesting to its power, self-assembly is used pervasively in biology to create such structures as virus capsids, microtubules, and flagella. In each case, the binding interactions between a small number of protein species is sufficient to dictate the form of the final structure, often via a complex sequence of cooperative assembly steps. This can be viewed loosely as a form of programmable nanofabrication, where the program is the set of molecular species involved. For synthetic approaches, Seeman ([@pbio-0020424-Seeman1], [@pbio-0020424-Seeman3]) has demonstrated that DNA provides an alternative to protein that can be readily programmed by Watson--Crick complementarity. A seminal paper by [@pbio-0020424-Adleman1] used one-dimensional (1D) DNA self-assembly to operate as a finite-state machine, establishing the first experimental connection between DNA self-assembly and computation. This work inspired a theoretical proposal ([@pbio-0020424-Winfree1]) that builds on Wang\'s ([@pbio-0020424-Wang1], [@pbio-0020424-Wang2]) embedding of computation in geometrical tilings to show that two-dimensional (2D) self-assembly of DNA can perform Turing-universal computation---which implies that any algorithm can in principle be embedded in, and guide, a potentially aperiodic crystallization process. In this "algorithmic self-assembly" paradigm, a set of molecular Wang tiles is viewed as the program for a particular computation or molecular fabrication task ([@pbio-0020424-Reif1]; [@pbio-0020424-Rothemund2]; [@pbio-0020424-Adleman2]). (This framework differs from previous approaches relating tiling theory to crystalline ground-states \[[@pbio-0020424-Radin1]\] in that kinetic phenomena are essential here.) Whereas 1D algorithmic self-assembly offers limited computational power ([@pbio-0020424-Winfree3]) and has been experimentally demonstrated ([@pbio-0020424-Adleman1]; [@pbio-0020424-Mao2]), 2D algorithmic self-assembly offers not only new capabilities for computation and construction, but also presents a new range of physical phenomena and experimental challenges as well. A natural Turing-universal model of computation that can be implemented by 2D algorithmic self-assembly is the class of 1D cellular automata. A simple but interesting choice for the local cellular automaton rule is the exclusive--or (XOR) function: at each time step, each cell is computed as the XOR of its two neighbors. Beginning with a row of all '0\'s punctuated by a single central '1,\' snapshots of the cellular automaton\'s state at successive time steps may be stacked one on top of the other to produce a space--time history identical to Pascal\'s triangle ([@pbio-0020424-Bondarenko1]) modulo 2 ([Figure 1](#pbio-0020424-g001){ref-type="fig"}A, left), which is a discrete form of Sierpinski\'s fractal triangle ([Figure 1](#pbio-0020424-g001){ref-type="fig"}A, right). To represent this cellular automaton as a tiling, each local context present in the space--time history must have a corresponding Wang tile whose shape represents the input and output occurring at that location ([Figure 1](#pbio-0020424-g001){ref-type="fig"}B). Thus, we need four tiles, one for each entry of the truth table for XOR, and a linear input row representing the initial state of the cellular automaton ([Figure 1](#pbio-0020424-g001){ref-type="fig"}C). Given these tiles and the input row there is a unique way to tile the upper half-plane without mismatches or missing tiles---the Sierpinski Tiling---which reproduces the cellular automaton\'s space--time history ([Figure 1](#pbio-0020424-g001){ref-type="fig"}D). ::: {#pbio-0020424-g001 .fig} Figure 1 ::: {.caption} ###### The XOR Cellular Automaton and Its Implementation by Tile-Based Self-Assembly \(A) Left: three time steps of its execution drawn as a space--time history. Cells update synchronously according to XOR by the equation shown. Cells at even time steps are interleaved with those at odd time steps; arrows show propagation of information. Right: the Sierpinski triangle. \(B) Translating the space--time history into a tiling. For each possible input pair, we generate a tile T-xy that bears the inputs represented as shapes on the lower half of each side and the output as shapes duplicated on the top half of each side. \(C) The four Sierpinski rule tiles, T-00, T-11, T-01, and T-10, represent the four entries of the truth table for XOR: 0 ⊕ 0 = 0, 1 ⊕ 1 = 0, 0 ⊕ 1 = 1, and 1 ⊕ 0 = 1. Lower binding domains on the sides of tiles match input from the layer below; upper binding domains provide output to both neighbors on the layer above. Semicircular domains represent '0\' and rectangular domains, '1\'. Tiles that output '0\' (T-00 and T-11) are gray, and we refer to them as '0\' tiles. Tiles that output '1\' (T-01 and T-10) are white and are referred to as '1\' tiles. Initial conditions for the computation are provided by a nucleating structure (blue). Red asterisks indicate sites on the nucleating structure that bear a '1\' binding domain; elsewhere, sites have all '0\' binding domains. Black arrows indicate associations that would form two bonds; red arrows, associations that would form one bond. \(D) Error-free growth results in the Sierpinski pattern. \(E) Error-prone growth from a nucleating structure with three '1\' domains. Red crosses indicate four mismatch errors. ::: ![](pbio.0020424.g001) ::: Whereas execution of a cellular automaton occurs perfectly and synchronously, molecular self-assembly is asynchronous and may have many types of errors. To be successful, an implementation of cellular automata by molecular tiling must address four challenges: (1) The abstract tiles must be translated into molecules (molecular tiles) that readily form 2D crystals. (2) Molecular tiles must be programmed with specific binding domains that match the logic of the chosen abstract tiles. (3) The binding of molecular tiles must be sufficiently cooperative to enforce the correct order of assembly and prevent errors. (4) Assembly of molecular tiles must occur on a specified nucleating structure, and spurious nucleation must be suppressed. These properties are necessary and sufficient for implementing not only the XOR cellular automaton, but also any other 1D cellular automaton. All four have been shown individually: several types of DNA Wang tiles have been designed and shown to grow into micron-scale 2D periodic crystals ([@pbio-0020424-Winfree2]; [@pbio-0020424-Mao1]; [@pbio-0020424-LaBean2]); the interactions between these tiles can be programmed by sequence-specific hybridization ([@pbio-0020424-Winfree2]; [@pbio-0020424-Mao2]); cooperative binding of multiple domains ensures specificity---the right tile attaches in the right place ([@pbio-0020424-Winfree3]; [@pbio-0020424-Mao2]); and input to the self-assembly process can be provided by a single-stranded template ([@pbio-0020424-LaBean1]; [@pbio-0020424-Yan1]). Here we demonstrate, via self-assembly of Sierpinski triangles, that all four challenges can be simultaneously overcome, thus establishing all the mechanisms necessary to implement arbitrary cellular automata. The Sierpinski tiling, then, gives rise to a new type of aperiodic crystal---an algorithmic crystal. Results {#s2} ======= Modeling Tile-Based Self-Assembly {#s2a} --------------------------------- Preventing the types of errors mentioned above may seem impossible. For example, if a single binding domain is strong enough to hold a tile in place (red arrows in [Figure 1](#pbio-0020424-g001){ref-type="fig"}C), then one would expect roughly 33% of tiles to mismatch with tiles in the layer below. Simulations of self-assembly shed light on how to avoid such dire circumstances. We use two levels of abstraction that isolate and address critical issues for the design and analysis of our algorithmic self-assembly experiments. How crystal morphology and patterning can be programmed by tile design in an inherently asynchronous assembly process is addressed by the abstract Tile Assembly Model (aTAM) ([@pbio-0020424-Winfree1], [@pbio-0020424-Winfree2]). To explore how physical parameters, such as tile concentration and temperature, affect crystal growth and influence error rates, we use the kinetic Tile Assembly Model (kTAM) based on reversible tile association and dissociation rates ([@pbio-0020424-Winfree2]). Control over the order of assembly is obtained by exploiting the cooperativity of binding. The aTAM models cooperativity via a threshold, τ, representing the number of bonds that must be made for an association event to be thermodynamically stable: a tile may be added to a crystal if at least τ binding domains match the existing crystal. Black arrows in [Figure 1](#pbio-0020424-g001){ref-type="fig"}C indicate four potential association events that could occur at τ = 2; red arrows indicate two additional association events that would be permitted at τ = 1, but not at τ = 2. Simulation of cellular automata is designed to work at τ = 2. Isolated tiles cannot associate at τ = 2 and so growth and computation must begin with a preformed nucleating structure ([Figure 1](#pbio-0020424-g001){ref-type="fig"}C, blue) that represents the input to the computation. Importantly, at τ = 2 no tile may be added until both preceding tiles are already present, guaranteeing a deterministic outcome despite the asynchronous order of events. Thus, assembly from an input row containing a solitary '1\' domain produces the Sierpinski triangle pattern ([Figures 1](#pbio-0020424-g001){ref-type="fig"}D and [2](#pbio-0020424-g002){ref-type="fig"}A) regardless of the order in which permitted associations occur. If a small number of additional τ = 1 associations are permitted to occur, then mismatches between neighboring tiles (mismatch errors) may result. In this case, or if there are several '1\'s in the input row, the resulting pattern can appear to be qualitatively different: owing to propagation of information and the linearity of XOR, the resulting pattern is the superposition of Sierpinski triangles initiated at input '1\'s and at mismatch error sites (see [Figure 1](#pbio-0020424-g001){ref-type="fig"}E). ::: {#pbio-0020424-g002 .fig} Figure 2 ::: {.caption} ###### Typical kTAM Simulation Results \(A) A roughly 130 × 70 subregion of an error-free templated crystal. \(B) A subregion with 10 mismatch errors (0.1%), shown in red (both false '0\'s and false '1\'s). Grown at G~mc~ = 17, G~se~ = 8.8. Large all-zero patches near the template row are due to intact Sierpinski pattern; for simulations with these parameters, asymptotically only approximately 1% of T-00 tiles are in all-zero patches containing more than 90 tiles (referred to as large patches). \(C) A subregion from a crystal grown with the T-00 and T-11 tiles at doubled concentration, on a slowly growing nucleating row. Mismatch errors (43 of them, i.e., 0.3%, during growth at G~mc~ = 17, G~se~ = 8.6) characteristically terminate the Sierpinski pattern at corners. Asymptotically, approximately 18% of T-00 tiles are in large patches. \(D) An untemplated crystal with roughly 4000 tiles and no errors. Inset: The largest subregion of a perfect Sierpinski pattern is small. \(E) An untemplated crystal with several errors, grown at G~mc~ = 17, G~se~ = 10.4. Note that growth in the reverse direction is more error-prone. Only approximately 1% of T-00 tiles are in large patches. \(F) An untemplated crystal with few errors, grown at G~mc~ = 17, G~se~ = 8.6, with T-00 and T-11 at doubled stoichiometry. Note the large perfect subregion. Simulation was initiated by a preformed seed larger than the critical nucleus size (roughly 100 tiles). For these simulation parameters, approximately 25% of T-00 tiles are in large patches. According to the approximations used in [@pbio-0020424-Winfree2], G~mc~ = 17 corresponds to 0.8 μM, G~se~ = 8.5 corresponds to 41.8 °C, and G~se~ = 10.4 corresponds to 32.7 °C. The black outline around the crystals is for clarity; it does not represent tiles. ::: ![](pbio.0020424.g002) ::: The rate at which such errors occur can be understood using the kTAM. In this model, all tiles (regardless of how well they match) may associate at a given site at a rate, r~f~ , proportional to their concentration: r~f~ = k\[tile\] = ke ^−G~mc~^ , where k is a forward rate constant and G~mc~ \> 0 is the nondimensionalized entropy lost due to association---thus it represents the monomer concentration. Dissociation rates depend on how many binding domains match correctly: a tile with b correctly matching binding domains has a dissociation rate given by r~r,b~ =ke ^−bG~se~^ , where G~se~ \> 0 is the nondimensionalized free energy for a single binding domain---thus it represents the sticky end strength. G~se~ decreases with increased temperature. Thus, if G~se~\< G~mc~ \< 2G~se~, a reaction wherein the tile matches at a single domain would have r~f~ \< r~r,~ ~1~ and thus would be thermodynamically unfavorable, while a reaction wherein the tile matches at two domains would have r~f~ \> r~r,~ ~2~ and thus would be thermodynamically favorable. This model is a reasonable first-order approximation for the tile-based self-assembly of single crystals. For G~mc~ ≈ 2G~se~, as G~mc~ and G~se~ become arbitrarily large, the τ = 2 aTAM is approximated arbitrarily well, and error rates go to zero---concomitantly, assembly speed goes to zero ([@pbio-0020424-Winfree2]). For ranges of G~mc~ and G~se~ compatible with current experimental conditions, assuming thermodynamic and kinetic parameters extrapolated from the literature of DNA duplex hybridization ([@pbio-0020424-Bloomfield1]), this model ([Figure S1](#sg001){ref-type="supplementary-material"}) predicts mismatch error rates between 0.1% and 1.0% ([Figure 2](#pbio-0020424-g002){ref-type="fig"}B). The effects of non-idealities can also be explored in this model. For example, [Figure 2](#pbio-0020424-g002){ref-type="fig"}C shows growth when the concentrations of the T-00 and T-11 tiles are twice that of the T-01 and T-10 tiles, and the nucleating structure grows slowly from special nucleating tiles rather than being preformed. Under this condition there is a preferential association of '0\'s on the facets of the growing crystal, causing characteristic errors that terminate Sierpinski triangles at corners and result in large all-zero patches ([Figures 3](#pbio-0020424-g003){ref-type="fig"}A and [S2](#sg002){ref-type="supplementary-material"}). The mechanism responsible for these errors appears to be preferential nucleation of T-00 tiles on all-zero facets, due to their higher concentration ([Figure S3](#sg003){ref-type="supplementary-material"}). If nucleation occurs on an all-zero facet both above and below a '1\' tile, correct growth from the '1\' will be sandwiched between '0\'s and therefore further errors will be forced ([Figure 3](#pbio-0020424-g003){ref-type="fig"}B). The further errors could be either (1) termination of the Sierpinski triangle by addition of a mismatched '0\' tile at the corner site, or (2) sideways propagation creating a new small triangle by addition of a mismatched '1\' tile on the facet below the corner site (arrow in [Figure 3](#pbio-0020424-g003){ref-type="fig"}A). Thus, slight quantitative variations in the model parameters can lead to striking qualitative differences in the observed error morphologies, which are effectively never seen under growth conditions with equimolar tile concentrations or with preformed borders (see [Figure S2](#sg002){ref-type="supplementary-material"}). ::: {#pbio-0020424-g003 .fig} Figure 3 ::: {.caption} ###### Simulations with Slow Border Growth and T-00 and T-11 at Doubled Concentrations \(A) A common error pattern: termination of triangles at corners. \(B) An observed mechanism leading to termination or sideways extension of triangles: preferential nucleation of T-00 on facets. \(C) Forward and sideways growth is deterministic: at sites presenting two binding domains, there is always a unique tile that can form exactly two bonds. Backward growth is non-deterministic: at sites where both binding domains agree (e.g., green arrows), there are two possible tiles that can make two bonds (either {T-10, T-01}, or {T-00, T-11}). At sites where the available binding domains disagree (e.g., red arrows), there is no tile that can associate to form two bonds. Since only the output type of tiles are shown, it is impossible to tell from these figures which backward growth sites present agreeing or disagreeing binding domains. ::: ![](pbio.0020424.g003) ::: The kTAM also provides insights into a second kind of error, the spontaneous 2D nucleation of untemplated crystals in the absence of the nucleating structure. For G~mc~ ≈ 2G~se~, which corresponds to the melting temperature of the crystals, such untemplated nucleation is inhibited by a kinetic barrier---the existence of a critical nucleus size ([@pbio-0020424-Davey1]) that decreases with increasing supersaturation. The growth rate of untemplated crystals also increases with supersaturation since their growth occurs by spontaneous 1D nucleation of a new layer of tiles on any of four facets. Via any single binding domain, there are always two tiles that can bind, so such nucleation must effectively invent a new bit of information. This bit may be propagated quickly forward or sideways (wherein tiles attach by one input and one output domain) to complete the layer without error according to the logic of XOR. Consequently, such crystals have none of the qualitative appearance of Sierpinski triangles even though they may contain no mismatched tiles (see [Figure 2](#pbio-0020424-g002){ref-type="fig"}D). If G~se~ is increased, corresponding to lowering the temperature, nucleation occurs more rapidly but errors are more frequent. Backward growth, in which tiles attach to a crystal by both of their output domains, is especially error-prone since every one of these associations involves the invention of information ([Figure 3](#pbio-0020424-g003){ref-type="fig"}C). Whenever two backward-growing domains meet and disagree on the information that they have invented, growth can only proceed via an error. Under fast growth conditions, significantly below the melting temperature as in [Figure 2](#pbio-0020424-g002){ref-type="fig"}E, this gives rise to higher error rates in the reverse growth direction. Near the melting temperature, however, this effect is insignificant. The most noticeable effect for untemplated crystals is due to the non-ideality discussed above (doubling the relative concentration of T-00 and T-11 tiles): the statistical preference for all-zero patches actually increases the frequency and size of perfect Sierpinski patterns (see [Figure 2](#pbio-0020424-g002){ref-type="fig"}F). These simulation studies suggest that all three difficulties (asynchronous association of tiles, mismatch errors, and untemplated nucleation) in principle can be controlled by slowing down the growth processes, making experimental investigations the appropriate next step. Design and Preparation of DNA Tiles {#s2b} ----------------------------------- Abstract Wang tiles are implemented as DNA tiles according to the scheme described earlier ([@pbio-0020424-Winfree2]): each molecular Wang tile is a DNA double-crossover molecule ([@pbio-0020424-Fu1]) with four sticky-ends (5-base single-stranded overhangs) that serve as the programmable binding domains. We rendered the four Sierpinski rule tiles using two types of double-crossover molecule, known as DAE-E and DAO-E molecules ([@pbio-0020424-Winfree1]), resulting in two independent molecular implementations ([Figure 4](#pbio-0020424-g004){ref-type="fig"}, sequences are as given in [Figures S4--S7](#sg004){ref-type="supplementary-material"}). The DAE-E Sierpinski tile set ([Figure 4](#pbio-0020424-g004){ref-type="fig"}A) consists of four molecular tiles, each composed of five strands whose sequences were designed to minimize the potential for forming alternative structures ([@pbio-0020424-Seeman2]), as confirmed by non-denaturing gel electrophoresis ([Figure S8](#sg008){ref-type="supplementary-material"}). ::: {#pbio-0020424-g004 .fig} Figure 4 ::: {.caption} ###### Molecular Schema \(A) Top center: abstract versions of the four DAE-E Sierpinski rule tiles, VE-00, UE-11, RE-01, and SE-10, highlight their differences from the tiles in [Figure 1](#pbio-0020424-g001){ref-type="fig"}. The arrangement of 3′ and 5′ ends on DAE-E tiles dictates that two distinct pairs of complementary binding domains must be used for each symbol '0\' or '1,\' denoted here by making complementary shapes large or small. Pink legends show the mapping of shape to sticky-end sequences. Top left: a molecular diagram for VE-00 shows how each DAE-E tile is comprised of five DNA strands; small arrows point to crossovers. Top right: a diagram for RE-01 shows how hairpins are attached to '1\' tiles to provide AFM contrast; the exact orientation of these hairpins is unknown. Below: tiles are shown assembling on a nucleating structure. The nucleating strand for the input row (blue) is generated by assembly PCR and frequently reaches lengths of more than 3 μm (200 tiles). The nucleating strand contains subsequences onto which capping strands (orange) and input tile strands assemble to form an input tile outputting '0\' at random intervals, the nucleating strand contains a subsequence (asterisk) for a different input tile that outputs a '1\' on one side. \(B) Top center: the six DAO-E Sierpinski rule tiles: S-00, R-00, S-11, R-11, S-01, and R-01. Top left and right: molecular diagrams highlight two notable features: (1) R-type tiles output only to S-type tiles, and vice-versa, as dictated by the 3′/5′ polarity of the molecules---again requiring two distinct pairs of binding domains per symbol. (2) The indicated rotational symmetry of the DAO-E molecules allows each molecule to serve in either of two orientations; no explicit S-10 or R-10 tiles are needed. An input tile outputting a single '1\' sticky end is shown (asterisk). Sequences are given in [Figures S4--S7](#sg004){ref-type="supplementary-material"}. ::: ![](pbio.0020424.g004) ::: Since untemplated crystals were not expected to produce recognizable Sierpinski triangles, it was necessary to create a proper nucleating structure to provide the initial input for the algorithmic self-assembly. Previous work using DNA tiles to self-assemble an initial boundary had proven to be difficult ([@pbio-0020424-Schulman2]), so in this work we took an alternative approach of using assembly PCR ([@pbio-0020424-Stemmer1]) to create a long single-stranded molecule which could serve as a scaffold ([@pbio-0020424-LaBean1]; [@pbio-0020424-Yan1]) for the assembly of a row of input tiles ([Figures 4](#pbio-0020424-g004){ref-type="fig"}A and [S9--S11](#sg009){ref-type="supplementary-material"}). Because this nucleating strand serves as the bottom of these tiles, only four strands are needed to assemble the input tiles, and an additional capping strand is used to form a double-helix between input tiles on the nucleating strand. By doping the assembly PCR mix with a small fraction of the strands coding for an input tile outputting a single '1,\' we ensure that each nucleating structure contains a few randomly located sites from which a Sierpinski triangle should grow. The DAO-E Sierpinski tile set ([Figure 4](#pbio-0020424-g004){ref-type="fig"}B) consists of six molecular tiles, due to peculiarities of the DAO-E motif. First, consideration of the 5′ and 3′ orientation of strands---particularly the fact that the sticky ends at the top and bottom of a DAO-E tile have opposite polarity---demands that each tile binds only to "upside-down" neighbors, resulting in layers of tiles with alternating orientation, which we refer to here as R-type and S-type tiles. Furthermore, the sugar--phosphate backbone of the DAO-E tiles has a dyad symmetry axis, implying that the R-01 and S-01 tiles each can play the roles of both the T-01 and T-10 tiles. Likewise, the R-00, R-11, S-00, and S-11 tiles can each bind in two orientations in a site where both inputs match. In order for the nucleating structure for the DAO-E lattice to assemble onto a long PCR-generated nucleating strand, the tiles on the input row must be of the DAE-O variant. Further, we simplified the construction so that all nucleating strands contain the same repetitive sequence, but the input tile strands are doped with a fraction of strands containing a '1\' sticky-end, and again the nucleating structure contains a few randomly located sites from which a Sierpinski triangle should grow. Self-Assembly of DNA Sierpinski Triangles {#s2c} ----------------------------------------- In principle, two approaches can be taken for initiating algorithmic self-assembly of DNA tiles. In the preformed tile approach, each tile is prepared separately by mixing a stoichiometric amount of each component strand in the hybridization buffer and then annealing from 90 °C to room temperature over the course of several hours. The nucleating structure is similarly prepared by annealing the nucleating strand with input tile and capping strands. Then the rule tiles and nucleating structure are mixed together at a temperature appropriate for crystal growth. In the bulk annealing approach, the nucleating strand, the capping and input tile strands, and the strands for all rule tiles are initially mixed together and then annealed. Since, at the concentrations we use, the tiles themselves have melting temperatures between roughly 60 °C and 70 °C while the crystals have a melting temperature within a few degrees of 40 °C ([Figure S12](#sg012){ref-type="supplementary-material"}), during annealing the tiles themselves will first form, and only later will the fully formed tiles assemble into crystals, presumably growing from the nucleating structure prior to overcoming the barrier to spontaneous nucleation. Both approaches work, but because of the convenience of the bulk annealing approach, all samples reported here were prepared using that method, with a final concentration of 0.2 μM each tile. After self-assembly in solution, samples are deposited onto mica and imaged by tapping mode atomic force microscopy (AFM). Results for the DAE-E tile set are shown in [Figures 5](#pbio-0020424-g005){ref-type="fig"} and [S13--S15](#sg013){ref-type="supplementary-material"}. The majority of DNA crystals we observed were similar to those in [Figure 5](#pbio-0020424-g005){ref-type="fig"}A: in addition to many small and indistinctly formed fragments, larger crystals are typically thin and long (up to several microns) with '1\' tiles clearly visible. Crystals consisting exclusively of VE-00 tiles (upper arrow in [Figure 5](#pbio-0020424-g005){ref-type="fig"}A) were particularly common; further investigation revealed that some (perhaps all) of these crystals formed as DNA tubes, and subsequently broke open and lay flat on the mica (see [Figure S15](#sg015){ref-type="supplementary-material"}; [@pbio-0020424-Rothemund3]). A '011011\'-striped pattern (lower arrow in [Figure 5](#pbio-0020424-g005){ref-type="fig"}A) was also quite common; it can be constructed from the RE-01, SE-10, and UE-11 tiles. Growth may have been biased to form '011011\' patterns by the depletion of VE-00 tiles, a stoichiometric disproportionation of tiles, due to growth of tubes early during annealing. Crystals that clearly grew from the nucleating structure were also apparent; [Figure 5](#pbio-0020424-g005){ref-type="fig"}B--[5](#pbio-0020424-g005){ref-type="fig"}E show examples with particularly few errors. In several of these crystals, individual tiles could be identified and a compatible arrangement of abstract tiles (and thus errors) could be determined. Large error-free domains containing more than eight rows of perfect Sierpinski triangle were observed. In these examples, the mismatch error rate was about 2% over a wider selection of fragments, the error rate varied between 1% and 10%. We partly attribute this variation to changes in the physical conditions during annealing that result in a disproportionation of tiles. In addition to errors due to incorporation of the wrong tile, we also observed missing tiles and lattice dislocations. Frequently, as in [Figure 5](#pbio-0020424-g005){ref-type="fig"}E, the identity of obscured or missing tiles was deduced from the neighboring tiles by assuming correct information propagation (the imperfection often being caused by sample preparation or by interaction with the AFM tip rather than by errors during assembly). ::: {#pbio-0020424-g005 .fig} Figure 5 ::: {.caption} ###### AFM Images of DAE-E Crystals \(A) Several frequent morphologies that appear in most samples, including all-\'0\' (upper arrow) and '011011\'-striped crystals (lower arrow). The all-\'0\' crystal may be a tube that opened upon adsorption to the mica. \(B) A templated crystal. The identification of tiles in this crystal is given in [Figure 1](#pbio-0020424-g001){ref-type="fig"}E. Crosses indicate mismatch errors. Asterisks indicate '1\'s on the nucleating strand. \(C) A crystal containing 10 rows of error-free Sierpinski triangle. A red triangle marks a lattice defect in the input row. \(D) Another Sierpinski triangle, better resolved. \(E) A crystal containing a perfect 19 × 6 subregion. Individual tiles can be clearly seen; three tiles are outlined in the lower left. Unfortunately, this crystal landed atop a thin sliver of DNA (lower arrow), obscuring the central columns of the Sierpinski triangle. The upper arrow indicates a 4-tile wide tube, near the point where it opens. A pentagon marks a lattice dislocation. Scale bars are 100 nm. ::: ![](pbio.0020424.g005) ::: Shown in [Figures 6](#pbio-0020424-g006){ref-type="fig"} and [S16--S18](#sg016){ref-type="supplementary-material"}, the DAO-E tiles also succeeded in producing recognizable Sierpinski triangles. However, the DAO-E tiles self-assembled into considerably larger sheets than the DAE-E tiles, presumably because of the DAO-E tiles\' symmetries that result in cancellation of strain and thus encourage flat sheets. Templated crystals were observed that had grown more than 70 rows ([Figure 6](#pbio-0020424-g006){ref-type="fig"}A). Because the R-11 tile does not appear in an error-free templated crystal, in some experiments ([Figure 6](#pbio-0020424-g006){ref-type="fig"}A) we did not include this tile; however, we observed no qualitative difference between samples prepared with and without R-11. In these crystals we almost always observed subregions with a characteristic pattern of errors that coincidentally resulted in termination of Sierpinski triangles at their corners and tops, creating large patches of zeros. Even untemplated crystals ([Figure 6](#pbio-0020424-g006){ref-type="fig"}B) contained recognizable subregions of the Sierpinski pattern. These features may be explained as follows: although the DAO-E tiles were mixed with equal quantities, the R-00, S-00, R-11, and S-11 tiles can bind to any permitted site in two orientations, thus making the experimental conditions analogous to the simulations of [Figure 2](#pbio-0020424-g002){ref-type="fig"}C--[2](#pbio-0020424-g002){ref-type="fig"}F wherein the concentration of the corresponding tiles is doubled; slow growth of the input row in the simulations may correspond to slow straightening out of the nucleating strand, which is initially a random coil (see [Figure S17](#sg017){ref-type="supplementary-material"}). Large crystals often have strikingly different tile distributions and error rates, as can be seen in the amalgamation of several large crystals shown in [Figure 6](#pbio-0020424-g006){ref-type="fig"}C and [Video S1](#sv001){ref-type="supplementary-material"}. Again, this may be attributed to the disproportionation of tiles during annealing, or to sideways growth as the nucleating structure straightens out. [Figure 6](#pbio-0020424-g006){ref-type="fig"}D--[6](#pbio-0020424-g006){ref-type="fig"}E shows particularly clear examples of Sierpinski triangles, averaged from several scans of the same crystal. Attempts to optimize the reaction conditions to produce Sierpinski triangles with lower error rates did not yield dramatic improvements ([Figure S18](#sg018){ref-type="supplementary-material"}). ::: {#pbio-0020424-g006 .fig} Figure 6 ::: {.caption} ###### AFM Images of DAO-E Crystals \(A) A large templated crystal in a 5-tile reaction (no R-11). A single '1\' in the input row (asterisk) initiates a Sierpinski triangle, which subsequently devolves due to errors. Mismatch errors within '0\' domains initiate isolated Sierpinski patterns terminated by additional errors at their corners. \(B) A large untemplated fragment in a 5-tile reaction (no S-11). Large triangles of '0\'s can be seen. Crystals similar to this are also seen in samples lacking the nucleating structure. \(C) Several large crystals in a 6-tile reaction, some with more zeros than ones, some with more ones than zeros. It is difficult to determine whether these crystals are templated or not. \(D) An average of several scans of the boxed region from (C), containing roughly 1,000 tiles and 45 errors. \(E) An average of several scans of a Sierpinski triangle that initiated by a single error in a sea of zeros and terminated by three further errors (a 1% error rate for the 400 tiles here). Red crosses in (D) and (E) indicate tiles that have been identified (by eye) to be incorrect with respect to the two tiles from which they receive their input. Scale bars are 100 nm. ::: ![](pbio.0020424.g006) ::: Discussion {#s3} ========== The self-assembly of DNA Sierpinski triangles demonstrates all four features necessary for Turing-universal computation by crystallization: formation of extended crystals, programmable interactions between DNA tiles determined by sticky-end sequences, selective associations of tiles enforced by the cooperative binding of more than one sticky end, and controlled nucleation of growth initiated by a template containing input information. This tiling approach could be used to implement other cellular automaton rules. Given a set S of possible states for the memory cells and an update function f : S × S → S, one can create a set of \\|S\\|^2^ tiles according to the scheme of [Figure 1](#pbio-0020424-g001){ref-type="fig"}B, one tile for each possible input pair. The need for binding specificity limits the number of sticky-end sequences (and hence \\|S\\|) to about 20 for the DAO-E and DAE-E tile designs used here, but this is already sufficient to implement several known universal Turing machines and cellular automata ([@pbio-0020424-Lindgren1]; [@pbio-0020424-Rogozhin1]). A larger set of sticky-end sequences could be achieved by redesigning the DNA tile molecules to use longer sticky-ends, provided that the melting temperatures of tiles and crystals remain well separated. Thus, DNA crystallization is programmable and Turing-universal. Furthermore, for fabrication purposes, computation by self-assembly could be used to control the direction and extent of growth, thus allowing arbitrary shapes to be created efficiently ([@pbio-0020424-Soloveichik1])---demonstrating that algorithmic self-assembly is not limited to the simulation of cellular automata or Turing machines. The main obstacle currently limiting attempts to compute or fabricate using algorithmic self-assembly is the presence of several types of errors. We observed lattice dislocations, a structural error; untemplated tubes and untemplated crystals, an error in the control of nucleation; and mismatched tiles, an error in the growth process. Accurate quantitative models of algorithmic self-assembly will be valuable for developing methods to control and reduce such errors. The kTAM simulations described here, while qualitatively insightful, predict mismatch error rates an order of magnitude smaller than those observed---motivating experimental measurements of errors and refinement of the model. Although it may be possible to reduce the error rates by carefully controlling the assembly conditions, a more promising route is the creation of fault-tolerant tile sets that perform the same logic ([@pbio-0020424-Winfree4]; [@pbio-0020424-Chen1]; [@pbio-0020424-Reif2]; [@pbio-0020424-Schulman1]). For the same assembly conditions, and thus roughly the same growth rate, the kTAM predicts that these tile sets can reduce the mismatch error rates by many orders of magnitude---a conclusion likely to hold in spite of inaccuracies in the model. Self-assembly has been touted as a possible successor to photolithography, a basis for nanotechnology and a route to complexity in chemistry ([@pbio-0020424-Whitesides1]). Algorithmic self-assembly---whether using DNA tiles as demonstrated here or using appropriately designed small molecules, proteins, or even macroscopic tiles ([@pbio-0020424-Bowden1]; [@pbio-0020424-Rothemund1])---extends the range of structures accessible by bottom-up fabrication techniques. For example, an abstract tile set that enumerates binary numbers---a binary counter---uses just four tiles, yet it can be used to define the size of self-assembled structures ([@pbio-0020424-Rothemund2]), thus addressing the synthetic chemistry challenge of creating monodisperse particles with programmable size. Furthermore, attachment of suitable logic gates to '0\' and '1\' tiles would yield a demultiplexer for a RAM circuit. This and other interesting digital circuits ([@pbio-0020424-Cook1]) might be created by using algorithmic crystals as templates for further chemical processing ([@pbio-0020424-Braun1]; [@pbio-0020424-Yan2]). The Turing-universality of self-assembly allows theoretical insights from computer science to be applied to self-assembly. For example, many questions phrased using the aTAM---such as "Will a certain tile type, say tile type \#5, ever be incorporated into the assembly?" or "Will the final assembled shape have 4-fold symmetry?"---are formally undecidable as a consequence of the undecidability of the halting problem ([@pbio-0020424-Turing1]; [@pbio-0020424-Adleman3]). This suggests that there exists no generally applicable method for predicting the behavior or properties of crystals. A concrete instance of this dilemma is whether quasicrystals\' 5-fold symmetry and aperiodicity could arise from self-assembly. Crystallographers have argued that, if so, definitions of order based on X-ray diffraction must be modified to include the new structures ([@pbio-0020424-Senechal1]). The growth of Sierpinski triangles, demonstrated here, shows unequivocally that self-assembly can create aperiodic structures based on local rules. Furthermore, traditional methods of measuring order, such as X-ray diffraction, will not recognize order that exists in certain algorithmic crystals. For example, an algorithmic crystal simulating a pseudo-random number generator ([@pbio-0020424-Wolfram1]; [@pbio-0020424-Jen1]; [@pbio-0020424-Knuth1]) would appear disordered, yet each molecule would be precisely and deterministically positioned. Thus, the growth of algorithmic crystals motivates the use of algorithmic definitions of order ([@pbio-0020424-Kolmogorov1]; [@pbio-0020424-Levin1]; [@pbio-0020424-Bennett1]) that generalize crystallography ([@pbio-0020424-Mackay1]). Finally, we ask whether the study of algorithmic self-assembly might further our understanding of biological self-assembly. Algorithmic crystals composed of simple sugar-based tiles have appeared in science fiction as a form of life ([@pbio-0020424-Egan1]); indeed, the simplicity and versatility of crystalline self-assembly suggests that templating, as a basis for simple organisms ([@pbio-0020424-Penrose1]; [@pbio-0020424-CairnsSmith1]), may be more natural than previously supposed. However, examination of self-assembly in modern organisms reveals many mechanisms beyond those considered here, including conformational changes, dissipative mechanisms such as ATP hydrolysis, and interactions with genetic regulatory networks---themselves biochemical information processors. The development of a theory of molecular algorithms that encompasses these additional mechanisms, if successful, will deepen our understanding of the complex processes found in nature, their fundamental limits, and their remarkable potential. Materials and Methods {#s4} ===================== {#s4a} ### kTAM simulations {#s4a1} Simulations described in this paper were performed with the xgrow program, written by Erik Winfree and available, along with tile sets used here, from <http://www.dna.caltech.edu/SupplementaryMaterial>. The xgrow program simulates the kTAM for a set of square Wang tiles (see [Figure S1](#sg001){ref-type="supplementary-material"}), beginning with a single seed tile. The tile set used here consists of the four Sierpinski rule tiles T-00, T-11, T-01, and T-10, augmented by three border tiles B-0, B-1, and B-B, the latter being used as the seed tile. To simulate the presence of a nucleating structure, the binding domain that joins border tiles is considered to be twice as strong as the other bonds---that is, it counts as two bonds in the sum b that determines off-rates r~r,b~ =ke ^−bG~se~^ . The border row grows---simulating the long nucleating structure becoming straight---by association of border tiles at the rate r~f~ = k\[tile\] = kS~i~e ^−G~mc~^ , where S~i~ is the stoichiometry of border tile i relative to the concentration of the four Sierpinski rule tiles. Since we have no knowledge of how quickly DNA nucleating structures straighten in our experiments, we considered two cases: (1) A rigid or quickly straightening nucleating structure was simulated by setting S~i~ = 4, so that near the crystal melting temperature where G~mc~ ≈ 2G~se~, the border growth is strongly favorable. This was used for [Figure 2](#pbio-0020424-g002){ref-type="fig"}B, where the seed tile stoichiometry was also set to zero, so that exactly one seed tile would be incorporated into the nucleating structure. (2) A floppy and slowly straightening nucleating structure was simulated by setting S~i~ = 0.25 for the border tiles; in this case, near the melting temperature border growth requires stabilization by growth of rule tiles, resulting in faceted crystals. In combination with doubled concentrations of T-00 and T-11 (S~i~ = 2), this case was used for [Figure 2](#pbio-0020424-g002){ref-type="fig"}C, where additionally the seed tile stoichiometry was set to 0.01 so that roughly 1% of border tiles output a '1,\' in rough agreement with the observed fraction of '1\'s within the DNA nucleating structures in our DAO-E experiments. The strong effect of these variations may be seen in [Figure S2](#sg002){ref-type="supplementary-material"}. Slow border growth significantly increases the mismatch error rate, resulting in the information contained in the border being lost in a few layers. The primary effect of doubled T-00 and T-11 concentrations is to increase the predominance of all-zero patches in the resulting crystal; not only are all-zero patches typically larger, but all-zero information in the border is propagated more reliably. Additionally, under these conditions spontaneous nucleation almost exclusively involves an initial all-zero nucleus. Simulations confirm the preferential nucleation of T-00 tiles on all-zero facets when T-00 and T-11 concentrations are doubled ([Figure S3](#sg003){ref-type="supplementary-material"}). In contrast, preferential nucleation on facets is not seen for the T-11 tile, despite its increased concentration. This is because, regardless of what information is presented on the facet, there is no way to create a layer containing more than 50% T-11 tiles and no mismatches; T-01 or T-10 tiles must intervene. Thus the nucleation rate is substantially reduced, relative to T-00 nucleation on all-zero facets. This can be assessed in simulations by measuring the probability, p(L), that a T-00 tile will be found after L layers of growth from a facet. Simulations with parameters similar to [Figure 2](#pbio-0020424-g002){ref-type="fig"}C (doubled T-00 and T-11 concentrations) show that p(L) ≈ 0.66e^−L/27^ + 0.34 for all-zero facets, indicating strongly preferential nucleation, but for all other facets p(L) relaxes quickly to the asymptotic distribution. Simulations with parameters similar to [Figure 2](#pbio-0020424-g002){ref-type="fig"}B (normal T-00 and T-11 concentrations) show no preferential nucleation, as p(L) relaxes to the asymptotic distribution immediately for every facet type investigated. ### DNA sequence design {#s4a2} Design of DNA Wang tiles occurs in three steps. First, the tile and lattice geometry must be determined. Here, the sizes (number of base-pairs) of each double-helical domain and sticky end, and other structural adornments such as contrast hairpins, are decided. These decisions impact the stability of each tile molecule, as the natural geometry of the DNA double-helix (10.5 bp for a full turn of B-form DNA) ([@pbio-0020424-Wang3]; [@pbio-0020424-Rhodes1]) constrains, for example, the separation between crossover points to be an integral number of half-turns. For the double-crossover motif used here ([@pbio-0020424-Fu1]), the acronym DAE-E refers to some of these choices at the structural level: double-crossover; antiparallel orientation of non-crossover strands at each junction; even number of half-turns (21 bp) between crossover points within each molecules; and even number of half-turns (21 bp) between nearest crossover points in two molecules joined by sticky ends. DAO-E refers to a similar set of choices, except that an odd number of half-turns (16 bp) separates the crossover points within each molecule. Where hairpin sequences were inserted for AFM contrast, we included two unpaired Ts at the bulged three-arm junctions, which has been shown to encourage stacking in the original helix domain ([@pbio-0020424-Ouporov1]) without significantly affecting the rigidity of the molecule ([@pbio-0020424-Li1]). At the second level, specific sequences must be chosen. The issue here is that we wish to prevent undesired associations between strands that might inhibit formation of the correct molecular structure. We used the heuristic principle of sequence symmetry minimization ([@pbio-0020424-Seeman1], [@pbio-0020424-Seeman3]) to minimize the length and number of unintentional Watson--Crick complementary subsequences among all strands in each system (DAO-E and DAE-E). Violations that occurred within a single strand were weighed more heavily than violations between two strands; similarly, violations between strands in the same tile were weighed more heavily than violations between strands in different molecules. A simple adaptive walk algorithm was found to be effective in minimizing the violations and arriving at acceptable sequences. Sticky-end sequences were chosen with particular care to minimize the possibility of erroneous hybridization. The third level of design concerns variations. We conceptualize DNA Wang tiles as consisting of three modules: the sticky ends, the core helical regions, and adornments such as the hairpin structures that provide contrast for AFM imaging. A given double-crossover core can be given different sticky ends (reprogrammed) by replacing just one or two strands, thus allowing reuse of core designs to implement different tile sets. In our experience, the structural and thermodynamic stability of a given core is not significantly affected by changes in the sticky-end sequences. Similarly, using additional strands, a given core can be used with or without the hairpin adornments, which can be inserted at various locations. Although the hairpin adornments can affect the integrity of a DNA tile (e.g., strand dimers or other high molecular weight species), we have seldom found the undesired products to exceed 20% of the material. The core sequences for R-00 and S-00 are identical to the A and B tiles from a previous study ([@pbio-0020424-Winfree2]). We usually give tiles names that indicate their core, sticky ends, and adornments. However, in the main text of this paper we have dispensed with the indication of these variations for clarity. For example, R-01 would more properly be called R-01n-23JC; S-01 called S-01-23JC; RE-01 called RE-01-15J; and SE-10 called SE-10-15J to specify which component strands have hairpins, and where those hairpins are. (The shorter names properly refer to unadorned tiles.) ### DNA tile preparation and gel electrophoresis {#s4a3} All oligonucleotides were synthesized by standard methods (Integrated DNA Technologies), PAGE purified, and quantitated by UV absorption at 260 nm in H~2~O (purified by a Milli-Q system, Millipore, Bedford, Massachusetts, United States) based on extinction coefficients estimated using a nearest-neighbor model ([@pbio-0020424-Bloomfield1]). DNA tiles were prepared by mixing stoichiometric quantities of each component strand in a TAE/Mg^2+^ buffer, as described in [@pbio-0020424-Winfree2]. Proper formation of each of the ten DAE-E and DAO-E tile cores was confirmed in non-denaturing PAGE (10%--15% 19:1 bis:acrylamide, 3--5 h at 15 V/cm and 4C, 2 pmol complex/lane, Sybr Gold \[Molecular Probes, Eugene, Oregon, United States\] stained for 20--30 min, excited at 488 nm, imaged with 530 bandpass filter on a Bio-Rad \[Hercules, California, United States\] Molecular Imager FX Pro Plus) by observing a single major band (see [Figure S8](#sg008){ref-type="supplementary-material"}; typically between 5% and 20% of the total material appears in bands identified as partial products, such as incomplete tiles with missing strands). We redesigned the core sequences for one tile (R01) that initially did not form clean gel bands; the new tile (R01n) was used exclusively in this study. Most notably in DAE-E tiles, some lanes containing subsets of a tile\'s component strands showed ill-formed or heavy species such as dimers, but these difficulties were not pronounced in lanes containing the complete tile. Formation gels also allow us to estimate the relative accuracy of our concentration measurements: mismatches in stoichiometry would result in excess single-stranded or partial complexes. Concentrations appear to be accurate to ±10%. This suggests that purification of tile complexes could result in cleaner self-assembly reactions and lower error rates. ### Synthesis of the nucleating strand {#s4a4} The single-stranded nucleating strands were synthesized using a procedure based on Stemmer\'s assembly PCR ([@pbio-0020424-Stemmer1]). In assembly PCR, a long, repetitive, double-stranded product is generated by performing PCR on a set of splints, primer-like short (typically 40 nt) oligos that are subsequences of the desired repeat sequence as shown in [Figure S9](#sg009){ref-type="supplementary-material"}. To generate the single-stranded product needed for subsequent assembly of tiles on the nucleating strand, asymmetric PCR with primers for just one of the two complementary product strands could be used, in principle. In practice, we have found that such reactions result in more double-stranded product and little or no single-stranded product---probably because the repetitive nature of the assembly PCR product means that every 3′ end, including those of the undesired strand, may act as a primer. Thus we designed the long covalent strand of our nucleating structures to contain exclusively As, Cs, and Ts and generated single-stranded nucleating strands from the output of an assembly PCR by performing synthesis using a reaction mixture containing just dATP, dCTP, and dTTP. Although predominantly single-stranded, the output of this reaction has both single- and double-stranded material in it. We do not purify the single strands and thus double-stranded material persists in our experiments ([Figure S17](#sg017){ref-type="supplementary-material"}). The splint strands for generating the DAE-E nucleating strands and the DAO-E nucleating strands are given in [Figure S10](#sg010){ref-type="supplementary-material"}. Note that in order to have 20 base overlaps, some splint strands complement the same central three base sections of complementary splints. Assembly reactions for DAO-E and DAE-E nucleating strands were designed using slightly different principles. The improved design used for DAO-E nucleating strands is simpler: A single periodic sequence is generated. The fraction of '1\' sites is determined by the stoichiometry of input tile strands used in subsequent self-assembly reactions---strands A4SV and A4-S00 both assemble in the input tile in the same place, but one carries a '1\' sticky end while the other carries a '0\' sticky end. The approach used for DAE-E nucleating strands is more complex but more powerful for generating non-trivial input patterns. By having multiple splints that can overlap a given sequence, the assembly can be directed to non-deterministically choose one of several ways to extend a sequence. Thus, assembly PCR can be used to generate any regular language ([@pbio-0020424-Winfree3]). In this work, we used a combination of splint strands that generates substrings of the language (NRE NUE^+^)\. The fraction of NRE subsequences is controlled by the amount of SplintNREUE2 and SplintNUERE2, which mediate the transitions into and out of the NRE sequence. (Here, we used these splints at one-fifth the concentration of other splints.) The NRE input tile outputs '0\' and '1,\' while the NUE input tile outputs '0\' and '0\'. To generate a different language, or a different distribution of sequences in the same language, a new assembly PCR must be run. (The simpler design approach used for DAO-E could also be used for DAE-E nucleating structures.) For both methods, the PCR protocol has four stages, the first three for assembly PCR and the last to generate single-stranded material. PCR was performed in a Stratagene (La Jolla, California, United States) MX 4000 real-time PCR instrument using a Perkin-Elmer (Torrance, California, United States) GeneAMP XL kit that uses rTth polymerase. In stage 1, a 20 μl reaction mixture containing 1 pmol total of splints (of which there are N* types) is prepared without polymerase (Mix A, per 20 μl: 1 μl of 1 μM mixed splints, 1/N μM each; 1.6 μl of 10 mM dNTPs, 2.5 mM each; 1 μl of 25 mM magnesium acetate, 6 μl of 3.3X GeneAMP XL PCR buffer; 10 μl water). To avoid mispriming events, the splints are annealed in the reaction mixture at 37 °C for 5 min. The polymerase (0.4 μl) is added and the reaction is subjected to an initial 72 °C extension step, followed by 40 cycles (94 °C for 15 s, 40 °C for 30 s, 72 °C for 10 s + 1 s/cycle; about 2 h). In stage 2, 40 μl of new PCR Mix B (Mix A minus the splints, plus 0.4 μl of polymerase, water adjusted to 20 μl), is added to the first reaction volume and the reaction cycled for an additional 25 cycles (94 °C for 15 s, 40 °C for 30 s, 72 °C for 45 s + 1 s/cycle; about 1.5 h). In stage 3, the 60 μl reaction volume is split into three 20 μl volumes, an additional 40 μl of Mix B is added to each and an additional 20 cycles (94 °C for 15 s, 40 °C for 30 s, 72 °C for 70 s + 1 s/cycle; about 1.3 h) are performed. At this point, long double-stranded product should be formed. (We have observed that such products remain in the well of an agarose gel long after a 20 kb marker has entered the gel.) Also the dNTPs in the mixture are presumably nearly exhausted---specifically there is little dGTP left. (Any remaining dGTP will be used up early in stage 4.) In stage 4, to create single-stranded nucleating strands, 5 μl of the stage 3 product are mixed with 55 μl of fresh PCR mixture (Mix B with 1.6 μl of a mixture containing 2.5 mM each dATP, dCTP, and dTTP, rather than all four dNTPs) for additional 60 cycles of the stage 3 program (94 °C for 15 s, 40 °C for 30 s, 72 °C for 70 s + 1 s/cycle). While addition of asymmetric primers at this stage might yield more single-stranded product, a satisfactory yield of single-stranded product results without doing so. After the final PCR, the reaction mixture is extracted with phenol:chloroform:isoamyl alcohol (Sigma, St. Louis, Missouri, United States), ethanol precipitated, and resuspended in purified water; the yield was estimated by UV absorbance. Typically, three 60 μl tubes of stage 4 product were pooled in a single recovery step and DNA was resuspended in 200 μl of water. Absorbance measurements of freshly resuspended material appeared unstable, perhaps because clumps of nucleating material scatter light. Long single-stranded DNAs may be prone to hydrolysis in water or strand-breakage upon freeze--thaw. However, after storage in water at 4 °C for a year, the nucleating structure still works well (as in [Figure S18](#sg018){ref-type="supplementary-material"}). To check whether the output of stage 4 is suitable as a nucleating strand for self-assembly, one can estimate the binding capacity of the nucleating strand material. [Figure S11](#sg011){ref-type="supplementary-material"} shows such a gel (non-denaturing PAGE, 5% 19:1 bis:acrylamide, 1 h, 150 V) for the DAO-E nucleating strand, examining how much of the fluorescently labeled Cy3-cpBr1 can be bound. We observe several things. First, stage 3 double-stranded material does not bind Cy3-cpBr1 well, as expected. Second, stage 4 material does bind Cy3-cpBr1 well, quantitatively absorbing the full amount (1 μl) added. Third, stage 4 material cannot absorb 2 μl of Cy3-cpBr1, giving us an estimate of the binding capacity of the nucleating strands. This is important for determining how much of the input tile strands must be added to ensure that a tile assembles onto nearly every site on the nucleating strand. Fourth, the presence of Sybr Green I during PCR does not appear to affect the quality of double or single-stranded material generated. ### UV melts of tiles and crystals {#s4a5} Melting temperatures for tiles and crystals were estimated based on UV^260^ melts of S-00 and R-00-23J and a mixture of both tiles ([Figure S12](#sg012){ref-type="supplementary-material"}). These tiles, also used in [@pbio-0020424-Winfree2], are identical to the R-00 and S-00 tiles for the DAO-E Sierpinski system, but with hairpins added to the R-00 tile. Individual tiles were at 0.4 μM of each component strand in TAE/Mg^2+^. The mixture of R-00-23J and S-00, which forms crystals when annealed slowly, contained each strand at 0.2μM. Melts were performed on an Aviv model 14NT-UV-VIS spectrophotometer (Aviv Instruments/Protein Solutions, Lakewood, NJ, United States), and began with preannealed samples at 15 °C, increasing to 80 °C over the course of several hours. Single-tile melts were superimposable with the reanneal from 80 °C back to 15 °C, indicating that equilibrium values were measured. Raw absorbance values were normalized. Whereas S-00 has a sharp melting transition (also seen for most other tiles lacking hairpins) near 65 °C, the R-00-23J tile has a somewhat more gradual transition, which we attribute to the presence of the hairpin. Above 40 °C, the absorbance of the mixture equals the average absorbance of the individual tiles, indicating that crystals have completely melted by that point. Prior to the crystal melting transition between 36 °C and 40 °C, there is significant noise in the measurement, presumably due to light scattering. We have not performed UV^260^ melts of all tiles; however, several other DAO-E and DAE-E tiles have similar transitions between 50 °C and 70 °C. Therefore we assume that the templated and untemplated Sierpinski crystals also melt at approximately 40 °C and that at that temperature, the DNA tiles are reasonably well formed. ### Self-assembly reactions {#s4a6} Self-assembly was performed by bulk annealing of all relevant rule tile, input tile, capping, and nucleating strands in a (typically) 50 μl volume of 1× TAE/Mg^2+^ buffer (40 mM Tris--acetate \[pH 8.0\], 2 mM EDTA, 12.5 mM Mg^2+^), annealing from 90 °C to 20 °C at a rate of 1 °C/min (taking about 1 h). Longer annealing schedules (e.g., 1 °C/min from 90 °C to 50 °C followed by 1 °C/30 min in the critical region from 50 °C to 20 °C, a total of about 15 h) did not seem to decrease the error rate or the number of untemplated tubes or crystals. DAO-E reactions contained nucleating strands sufficient to bind 0.004 μM of input tile (as estimated from binding capacity gels), 0.2 μM of each capping and input tile strand (A1S, A2, A3-nick, A4-S00, cpBr1, and 1/100 as much A4SV), and 0.2 μM of each rule tile strand (for each of the five or six tiles used). An excess of input tile strands was used to ensure complete coverage of the nucleating strand. The excess partial input tiles appeared not to significantly interfere with the self-assembly of algorithmic crystals. DAE-E reactions contained nucleating strands sufficient to bind 0.002--0.008 μM of input tile (as inferred from the estimated yield of the PCR), 0.2 μM of each capping and input tile strand (NRE1 to NRE4, NUE1 to NUE4, CapNRERE and CapNUERE), and 0.2 μM of each tile strand (for each of the four tiles used). Again, an excess of input tile strands was used to ensure complete coverage of the nucleating strand. ### AFM imaging {#s4a7} AFM imaging was performed in tapping mode under TAE/Mg^2+^ buffer on a Digital Instruments Nanoscope III (Veeco Metrology, Woodbury, New York, United States) equipped with a nano-Analytics Q-control III (Asylum Research, Santa Barbara, California, United States) and a vertical engage J-scanner, using the roughly 9.4 kHz resonance of the narrow 100 μM, 0.38 N/m force constant cantilever of an NP-S oxide-sharpened silicon nitride tip (Veeco Metrology). After self-assembly is complete, samples were prepared for AFM imaging by deposition of 5 μl onto a freshly cleaved mica surface (Ted Pella) attached by hot melt glue to a 15 mm metal puck; an additional 30 μl of buffer was added to both sample and cantilever (mounted in the standard tapping mode fluid cell) before the sample and fluid cell were positioned in the AFM head. The tapping amplitude setpoint, after engage, was typically 0.2--0.4 V, the drive amplitude was typically 100--150 mV, scan rates ranged from 2 to 5 Hz. Individual tiles are most clearly resolved for low amplitude setpoint and high drive amplitude values. However, under such conditions, the greatest damage is done to the sample and the hairpin labels are less distinct, sometimes disappearing entirely. Thus, to prevent damage to samples, amplitude setpoint was maximized and/or drive amplitude minimized subject to the constraint that tiles and their hairpin labels be visible. After acquisition, most images were flattened by subtracting a low-order polynomial from each scan line, or by adjusting each scan line to match intensity histograms. For some images (see [Figures 6](#pbio-0020424-g006){ref-type="fig"}D--[6](#pbio-0020424-g006){ref-type="fig"}E and [S18](#sg018){ref-type="supplementary-material"}, bottom), multiple scans were aligned using hand-picked fiducial marks and averaged in Matlab (The Mathworks, Inc., Natick, Massachusetts, United States). Supporting Information {#s5} ====================== Figure S1 ::: {.caption} ###### Representations and Tile Sets Used in Simulations (57 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S2 ::: {.caption} ###### Behavior of Simulated Crystal Growth (160 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S3 ::: {.caption} ###### Simulations of Growth on Large Facets (126 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S4 ::: {.caption} ###### DAE-E Strand Sequences (16 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S5 ::: {.caption} ###### DAE-E Tile Diagrams (21 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S6 ::: {.caption} ###### DAO-E Strand Sequences (16 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S7 ::: {.caption} ###### DAO-E Tile Diagrams (22 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S8 ::: {.caption} ###### Formation Gels for Representative DAO-E and DAE-E Tiles (244 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S9 ::: {.caption} ###### Using Assembly PCR to Generate Long, Repetitive, Single-Stranded DNA (22 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S10 ::: {.caption} ###### Assembly PCR Scheme for DAE-E and DAO-E Nucleating Strands (25 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S11 ::: {.caption} ###### Binding Capacity Gel for Determining Nucleating Strand Stoichiometry (53 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S12 ::: {.caption} ###### Melts of R-00-23J and S-00 and Their Mixture (33 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S13 ::: {.caption} ###### AFM Images Showing the Context and Distribution of DAE-E Crystals (234 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S14 ::: {.caption} ###### AFM Images Showing the Context and Distribution of DAE-E Crystals (203 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S15 ::: {.caption} ###### AFM Images of DAE-E Crystals and Tubes (226 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S16 ::: {.caption} ###### AFM Images Showing the Context and Distribution of DAO-E Crystals (256 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S17 ::: {.caption} ###### AFM Images of Boundary Assemblies and Untemplated DAO-E Crystals (234 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S18 ::: {.caption} ###### AFM Images of DAO-E Crystals Grown under Constant-Temperature, Near-Constant Concentration Conditions (146 KB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S19. Compiled Figures S1--S18 ::: {.caption} ###### This file contains [Figures S1--S18](#sg001){ref-type="supplementary-material"} and their captions in a single file for convenient printing (1.7 MB PDF). ::: ::: {.caption} ###### Click here for additional data file. ::: Video S1 ::: {.caption} ###### Composite of 64 AFM Images Taken Sequentially at Scales from 24 μm to 24 nm Each frame is an average of three raw images. At the center is an amalgamation of many individual algorithmic crystals, each with its own characteristic pattern of tiles (e.g., mostly zero, bearing small triangles, or apparently random). While no large undamaged Sierpinski triangles were seen in this series of images, in some frames it is possible to see both double-helices within the tiles, as well as the major and minor grooves within the helices. (17.8 MB MPG). ::: ::: {.caption} ###### Click here for additional data file. ::: For discussions, insights, and advice, we thank John Hopfield, Ned Seeman, Len Adleman, Matt Cook, Hui Wang, Rebecca Schulman, Shaun Lee, Rizal Hariadi, and Jason Rolfe. Experiments described in [Figure S18](#sg018){ref-type="supplementary-material"} were performed by Jason Rolfe. We thank the Caltech Molecular Materials Research Center for use of their AFM scanners. PWKR was supported by a Beckman Fellowship. This work was supported in part by the National Science Foundation PECASE EIA-0093486, DARPA BioComputation F30602-01-2-0561, NASA NRA2-37143, and GenTel Corporation. Conflicts of interest. The authors have declared that no conflicts of interest exist. Author contributions. PWKR and EW conceived and designed the experiments. PWKR, NP, and EW performed the experiments. PWKR and EW analyzed the data. PWKR and EW wrote the paper. Academic Editor: Anne Condon, University of British Columbia Citation: Rothemund PWK, Papadakis N, Winfree E (2004) Algorithmic self-assembly of DNA Sierpinski triangles. PLoS Biol 2(12): e424. AFM : atomic force microscopy aTAM : abstract Tile Assembly Model 1D : one-dimensional 2D : two-dimensional DNA : deoxyribonucleic acid DAE-E : double-crossover DAO-E : double-crossover kTAM : kinetic Tile Assembly Model PCR : polymerase chain reaction RAM : random-access memory XOR : exclusive or	PubMed Central	2024-06-05T03:55:49.983503	2004-12-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534809/", "journal": "PLoS Biol. 2004 Dec 7; 2(12):e424", "authors": [ { "first": "Paul W. K", "last": "Rothemund" }, { "first": "Nick", "last": "Papadakis" }, { "first": "Erik", "last": "Winfree" } ] }
PMC534810	Introduction {#s1} ============ The tiger (Panthera tigris) is the largest felid species and a widely recognized symbol of wildlife conservation. Historically tigers inhabited much of Asia, including the regions between the Caspian and Aral Seas, southeastern Russia, and the Sunda islands ([@pbio-0020442-Mazak1]; [@pbio-0020442-Hemmer1]; [@pbio-0020442-Herrington1]). Since the early 1900s, however, habitat loss, fragmentation, and human persecution have reduced tiger populations from probably over 100,000 in 1900 to fewer than 7,000 free-ranging individuals ([@pbio-0020442-Nowell1]; [@pbio-0020442-Dinerstein1]; [@pbio-0020442-Kitchener2]). Most populations consist of less than 120 animals, increasing the risk of local extirpation due to demographic and genetic factors ([@pbio-0020442-Smith1]; [@pbio-0020442-Dinerstein1]). There are eight generally accepted tiger subspecies in accordance with their geographic distribution ([Figure 1](#pbio-0020442-g001){ref-type="fig"}). Bali (P. t. balica), Caspian (P. t. virgata), and Javan (P. t. sondaica) tiger subspecies were eradicated by the 1940s, 1970s, and 1980s respectively ([@pbio-0020442-Nowell1]). Today an estimated 3,200--4,500 Indian or Bengal tigers (P. t. tigris) exist in Bangladesh, Bhutan, western China, India, western Myanmar, and Nepal ([@pbio-0020442-Seidensticker1]). Fewer than 500 Amur or Siberian tigers (P. t. altaica) survive in eastern Russia, northeastern China, and Korea ([@pbio-0020442-Matyushkin1]; [@pbio-0020442-Miquelle1]), while approximately 50 Amoy or South China tigers (P. t. amoyensis) now exist in captivity only ([@pbio-0020442-Tilson2]). An estimated 400--500 Sumatran tigers (P. t. sumatrae) occur in Sumatra ([@pbio-0020442-Seidensticker1]); and 1,200--1,800 Indochinese tigers (P. t. corbetti) live in Cambodia, China, Laos, Malaysia, east Myanmar, Thailand, and Vietnam ([@pbio-0020442-Seidensticker1]) ([Figure 1](#pbio-0020442-g001){ref-type="fig"}). ::: {#pbio-0020442-g001 .fig} Figure 1 ::: {.caption} ###### Historic and Current Geographic Distribution of Tigers Corresponding to the Eight Traditional Subspecies Designation Geographic origin of samples and sample size (circles or squares) from each location are indicated (see [Table 3](#pbio-0020442-t301){ref-type="table"} for sources). Three-letter codes (TIG, ALT, etc.) are indicated subspecies abbreviations. Dotted lines are approximate boundaries between tiger subspecies studied here. The Isthmus of Kra divides the traditional Indochinese tigers into the northern Indochinese tigers P. t. corbetti I and the Malayan tigers P. t. corbetti II based on the present study. We propose the Malayan tiger subspecies, COR II, be named P. t. jacksoni, to honor Peter Jackson, the former Chair of the IUCN\'s Cat Specialist Group who has contributed significantly to worldwide tiger conservation. ::: ![](pbio.0020442.g001) ::: ::: {#pbio-0020442-t301 .table-wrap} Table 3 ::: {.caption} ###### Samples of Panthera tigris Used in the Study ::: ![](pbio.0020442.t301) ^a^ Birth Status of each tiger: W, wild-born, C, captive-born; U, status unknown ^b^ Identification number of tiger individuals as they are listed in the database at the Laboratory of Genomic Divesity, National Cancer Institute, Frederick, Maryland, United States ^c^ MtDNA haplotype assigned to each sample sequenced in the study ^d^ MHC ClassII DRB allele genotypes ^e^ Samples of pelt or hair ^f^ Red samples represent samples with microsatellite data from 30 loci ^g^ Tigers individuals classified as South China tiger originally ::: Subspecies of tigers are traditionally defined by body size, skull characters, pelage coloration, and striping patterns ([@pbio-0020442-Mazak1]; [@pbio-0020442-Herrington1]). It is generally believed that the largest tigers occur in the Russian Far East, and the smallest are found in the Sunda Islands. The shape of the occiput in the skull is characteristically narrow in the Javan and Bali tigers and much broader in Caspian tigers ([@pbio-0020442-Mazak2]). However, the adequacy of these traditional subspecies designations is tentative at best, since morphological distinctions in many cases have been based on a few specimens, and because subsequent studies have failed to affirm these distinctions. [@pbio-0020442-Herrington1] and [@pbio-0020442-Kitchener1] have revealed a wide range of morphological variations within the subspecies and, to some extent, overlapping among the subspecies. A previous molecular genetic assessment of 28 tigers has indicated a low level of genetic variation, revealing little evidence for subspecies distinctiveness ([@pbio-0020442-Wentzel1]). Moreover, ecological analyses of tiger habitat ([@pbio-0020442-Kitchener2]) indicate that there have been few geographic barriers (e.g., mountain ranges and deserts) to migration and gene flow that would have been sufficient for subspecies isolation. One ecology-based conservation approach emphasizes protection of about 160 continuous habitat patches or tiger conservation units regardless of subspecies designation ([@pbio-0020442-Dinerstein1]). Although this strategy may be desirable, optimal tiger conservation may also require additional interventions such as establishing corridors and buffer zones and/or implementing reintroduction programs ([@pbio-0020442-Tilson1]). To this end, an assessment of population genetic structure of living tigers interpreted in the context of traditional intraspecific taxonomy and the species\' evolutionary history would benefit both in situ and ex situ conservation management design. Molecular genetic markers have been increasingly applied to assess genetic partitions among geographically isolated populations, to define the evolutionary significant unit below the species level for conservation management purposes, and to revise the traditional species and subspecies designations ([@pbio-0020442-Avise1]; [@pbio-0020442-Moritz1]; [@pbio-0020442-Fraser1]). Subspecies recognition is particularly relevant for tigers, because the current conservation strategy for this species has been inextricably bound to knowledge of its subspecific taxonomy. In this study we adhere to the subspecies concept as defined by [@pbio-0020442-Avise1] and [@pbio-0020442-OaBrien2], to include populations below the species level that share a distinct geographic distribution, a group of phylogenetically concordant characters, and a unique natural history relative to other subdivisions of the species. Here we attempt to overcome several factors that have complicated previous efforts to fully describe patterns of genetic variation in tigers. Foremost among these has been the limited sample size of "voucher specimens" (defined as individuals that were verified as wild-born from a specific geographic locale or captive-born from geographically verified wild-born parents). In addition, the presence of Numt, a nuclear pseudogene insertion of cytoplasmic mitochondrial DNA (mtDNA) in tiger autosomes ([@pbio-0020442-Lopez2]; [@pbio-0020442-Johnson1]; [@pbio-0020442-Cracraft1]; J. H. Kim, A. Antunes, S.-J. Luo, J. Menninger, W. G. Nash, et al., personal communication) has made it difficult to utilize universal mammalian primer sets for mitochondrial genes, because they will coamplify Numt. Furthermore, paucity of genetic diversity across tigers, especially in mtDNA ([@pbio-0020442-Wentzel1]), has made it necessary to sequence a large portion of the mtDNA genome and to assess genetic variation in multiple rapidly evolving microsatellite loci. To establish proper biological reference specimens, samples from 134 tigers of known geographic origin were collected. Three genetic markers were examined: (1) 4 kb of mtDNA sequence derived from primer pairs that excluded Numt amplification, (2) allele variation in the major histocompatibility complex (MHC) DRB gene; and (3) allele size variation of 30 hypervariable short tandem repeat loci or microsatellites. Observed patterns of population genetic variation replicated with different gene families form the basis of interpretation of the tiger\'s evolutionary history and recommendations for its management. Results {#s2} ======= Phylogenetic Analysis of mtDNA and Microsatellites {#s2a} -------------------------------------------------- Mitochondrial gene fragments were amplified and sequenced from DNA extracted from 72 blood or tissue specimens using 10 cytoplasmic mitochondria (Cymt)-specific primer pairs ([Figure 2](#pbio-0020442-g002){ref-type="fig"} and [Table 1](#pbio-0020442-t001){ref-type="table"}). The fragments were concatenated in a 4,078-bp contiguous sequence. Additional mtDNA sequences were generated from 28 historical samples (pelt or hair) by amplifying shorter fragments (less than 400 bp) targeting selected variable sites to determine their similarity to the previously characterized haplotypes. Combined mtDNA sequences were obtained from 100 tigers from Russian Far East (n = 13), south China (n = 4), northern Indochina (n = 30), Malayan Peninsula (n = 22), Sumatra (n = 16), and the Indian subcontinent (n = 15). The mtDNA sequences specified 54 variable sites defining 25 haplotypes ([Table 2](#pbio-0020442-t002){ref-type="table"}). Thirty of the polymorphisms were observed in more than one individual and were thus phylogenetically informative ([Table 2](#pbio-0020442-t002){ref-type="table"}), and 29 of the 30 changes were transitions. ::: {#pbio-0020442-g002 .fig} Figure 2 ::: {.caption} ###### Schematic of P. tigris mtDNA The position of PCR primers used for amplification of Cymt specific sequences and alignment of the homologous Numt sequence (outer, dashed line) in tiger mitochondria. Fifteen Cymt-specific primer sets spanning 6,026 bp of mtDNA were designed and screened for polymorphism in tigers (inner, solid line). Five indicated segments showed no variation among fifteen tigers that represented five traditional subspecies and therefore were excluded from further analysis. The ten variable segments (4,078 bp) were amplified in 100 tiger individuals. Primer sequences are listed in [Table 1](#pbio-0020442-t001){ref-type="table"}. Diamonds indicate polymorphic mtDNA segments; brackets indicate monomorphic mtDNA segments among tigers that were excluded from phylogenetic analysis. ::: ![](pbio.0020442.g002) ::: ::: {#pbio-0020442-t001 .table-wrap} Table 1 ::: {.caption} ###### PCR Primers Specific for Cytoplasmic Mitochondrial DNA Sequences ::: ![](pbio.0020442.t001) ^a^ Primers are listed in the 5′-to-3′ direction ^b^ PCR products amplified using these primer sets show no variation among all samples ::: ::: {#pbio-0020442-t002 .table-wrap} Table 2 ::: {.caption} ###### Haplotypes and Variable Sites in Combined Analysis of 4,078 bp of Tiger (P.tigris) mtDNA Sequences ::: ![](pbio.0020442.t002) ^a^ Nucleotide positions correspond to the complete reference Felis catus mtDNA sequence ([@pbio-0020442-Lopez1]) ^b^ Subspecies abbreviation code as in Figure 1. Base pairs identical to haplotype ALT are indicated by a dash ^c^ Number of individuals with each haplotype. Individual tiger mtDNA haplotypes are listed in [Table 3](#pbio-0020442-t301){ref-type="table"} ^d^ Red nucleotides are subspecies-specific sites ^e^ COR1/AMO3 is a haplotype shared by 21 tigers that are initially designated as COR and one AMO (text and [Table 3](#pbio-0020442-t301){ref-type="table"}) ::: ::: {#pbio-0020442-t302 .table-wrap} Table 3 ::: {.caption} ###### Continued ::: ![](pbio.0020442.t302) ::: ::: {#pbio-0020442-t303 .table-wrap} Table 3 ::: {.caption} ###### Continued ::: ![](pbio.0020442.t303) ::: Phylogenetic analyses of the mtDNA haplotypes using maximum parsimony (MP), minimum evolution (ME), and maximum likelihood (ML) approaches produced congruent topologies that defined major geographic partitions ([Figure 3](#pbio-0020442-g003){ref-type="fig"}A). Eight haplotypes (SUM1 to SUM8) generated from 16 Sumatran tigers (P. t. sumatrae) formed a monophyletic group (80% MP, 70% ME, and 66% ML bootstrap support). A second monophyletic cluster of six haplotypes (TIG1 to TIG6) from 15 Bengal tigers (P. t. tigris) also received high bootstrap support (93% MP, 82% ME, and 90% ML). The rest of the mainland Asian haplotypes grouped together and partitioned into three distinct geographic groups: (1) a genetically invariant Amur tiger lineage (P. t. altaica) represented by a single haplotype in 13 individuals, (2) a northern Indochinese lineage (P. t. corbetti I) of individuals originating from south China to the Indochinese countries north of the Isthmus of Kra, and (3) a paraphyletic assembly of haplotypes from tigers from Malayan Peninsula (P. t. corbetti II). Support for subdividing the conventional Indochinese subspecies of tigers P. t. corbetti into two clusters was high (bootstrap values for P. t. corbetti I were 94% MP, 96% ME, and 94% ML). The COR1/AMO3 haplotype, represented by 22 individuals from Vietnam (n = 2), Cambodia (n = 14), northeast Thailand (n = 5), and south China (n = 1), was the only haplotype found in two classical subspecies lineages (P. t. amoyensis and P. t. corbetti) ([Table 2](#pbio-0020442-t002){ref-type="table"}). ::: {#pbio-0020442-g003 .fig} Figure 3 ::: {.caption} ###### Phylogenetic Relationships among Tigers from mtDNA Haplotypes \(A) Phylogenetic relationships based on MP among the tiger mtDNA haplotypes from the combined 4,078 bp mitochondrial sequence ([Table 2](#pbio-0020442-t002){ref-type="table"}). Branches of the same color represent haplotypes of the same subspecies. Trees derived from ME and ML analyses have identical topologies. Numbers above branches represent bootstrap support from 100 replicates using the MP method, followed by bootstrap values using the ME-ML analyses (only those over 70% are indicated). Numbers below branches show number of MP steps per number of homoplasies from a strict consensus tree. Numbers in parentheses represent numbers of individuals sharing the same haplotype. MP analysis using heuristic search and tree-bisection-reconnection branch-swapping approach results in two equally most-parsimonious trees and the one resembling the ME and ML trees is shown here (tree length = 60 steps; CI = 0.900). The ME tree is constructed with PAUP using Kimura two-parameter distances (transition to transversion ratio = 2) and NJ algorithm followed by branch-swapping procedure (ME = 0.0142). The ML approach is performed using a TrN (Tamura-Nei) +I (with proportion of invariable sites) model, and all nodes of the ML tree were significant (a consensus of 100 trees, --Ln likelihood = 5987.09). \(B) Statistical parsimony network of tiger mtDNA haplotypes based on 4,078 mtDNA sequences constructed using the TCS program ([@pbio-0020442-Clement1]). The area of the circle is approximately proportional to the haplotype frequency, and the length of connecting lines is proportional to the exact nucleotide differences between haplotypes with each unit representing one nucleotide substitution. Missing haplotypes in the network are represented by dots. Haplotype codes and the number of individuals (in parentheses) with each haplotype are shown (see [Table 2](#pbio-0020442-t002){ref-type="table"}). ::: ![](pbio.0020442.g003) ::: Voucher samples of five captive tigers collected in China, designated South China subspecies P. t. amoyensis, fell into two very distinct phylogenetic origins. Two tigers from the Suzhou Zoo (Pti-217 and Pti-218; [Table 3](#pbio-0020442-t301){ref-type="table"}) carried the COR1/AMO3 haplotype, and the third (Pti-222) contained haplotype AMO2, which differed by a single nucleotide substitution from COR1/AMO3 ([Table 2](#pbio-0020442-t002){ref-type="table"}). The two South China tiger haplotypes grouped phylogenetically with the northern Indochinese P. t. corbetti I haplotypes (COR1--COR3) in all phylogenetic analyses ([Figure 3](#pbio-0020442-g003){ref-type="fig"}A and [3](#pbio-0020442-g003){ref-type="fig"}B), and likely indicate that the maternal (mitochondrial) lineages of these tigers derived from individuals from the P. t. corbetti I phylogenetic lineages. In contrast, two P. t. amoyensis tigers (Pti-219 and Pti-220) from the Chongqing Zoo collection had a haplotype (AMO1) that formed a separate lineage that was ten nucleotide substitutions from its nearest sequence (Sumatran; [Figure 3](#pbio-0020442-g003){ref-type="fig"}B and [Table 2](#pbio-0020442-t002){ref-type="table"}). If affirmed by larger sampling, this lineage would reflect a unique P. t. amoyensis genetic haplotype. A statistical parsimony network of the tiger mtDNA sequences provided additional analytical support for the differentiation of P. t. sumatrae, P. t. tigris, P. t. altaica, P. t. corbetti I, P. t. corbetti II, and P. t. amoyensis (AMO1 only) ([Figure 3](#pbio-0020442-g003){ref-type="fig"}B). Haplotypes from the same geographic group tended to be interrelated, and intergroup distances among haplotypes were generally larger than branch lengths within each group (1--4 bp). The exceptions were two lineages within the Malayan P. t. corbetti II cluster that were separated by 7 bp, which may be a result of the existence of further population substructure or, alternatively, of limited sampling in the region. Each of the six tiger subspecies groups was connected to other groups in close but not exact correspondence to their geographic location. For instance, P. t. altaica was the sister taxon to P. t. corbetti I which was connected to P. t. corbetti II. P. t. sumatrae haplotypes were linked to P. t. tigris by 7 bp and to P. t. amoyensis by 10 bp. Nonetheless, the phylogenetic relationships among the subspecies were not resolved to a robust hierarchy, and therefore were consistent with a contemporaneous divergence of extant phylogeographic lineages. Composite genotypes from 30 felid-specific microsatellite loci ([@pbio-0020442-Menotti-Raymond1]) were obtained in 113 tiger samples. Neighbor joining (NJ) analyses of individual tiger genotypes based on the proportion of shared allele (Dps) and kinship coefficient (Dkf) genetic distances produced concordant topologies ([Figures 4](#pbio-0020442-g004){ref-type="fig"} and [S1](#sg001){ref-type="supplementary-material"}) that lend support to the same phylogeographic population subdivisions observed in the mtDNA analysis. Tigers from Sumatra (P. t. sumatrae) formed a monophyletic clade with 97% bootstrap support, and Amur tigers (P. t. altaica) grouped with 76% bootstrap support. The remaining tiger genotypes partitioned into two weakly supported monophyletic lineages (Indian Subcontinent P. t. tigris and Malayan Peninsula P. t. corbetti II) and a paraphyletic assemblage of northern Indochinese P. t. corbetti I. For example, three individuals from Thailand (Pti-296, Pti-297, and Pti-301) clustered with samples from the India subcontinent, blurring the distinction between P. t. corbetti I and P. t. tigris. The three South China tigers from the Suzhou Zoo, China, that had clustered with P. t. corbetti I by mtDNA (Pti-217, Pti-218, and Pti-222) also associated more closely with P. t. corbetti I from northern Indochina by microsatellite analysis ([Figure 4](#pbio-0020442-g004){ref-type="fig"}). The two distinct (by mtDNA) P. t. amoyensis individuals (Pti-219 and Pti-220) from the Chongqing Zoo, China, likewise formed a distinct lineage in the microsatellite analysis ([Figure 4](#pbio-0020442-g004){ref-type="fig"}). ::: {#pbio-0020442-g004 .fig} Figure 4 ::: {.caption} ###### Phylogenetic Relationships among the Individual Tigers from Composite Microsatellite Genotypes of 30 Loci Branches of the same color represent tiger individuals of the same subspecies. The NJ tree, which is based on Dps and Dkf with the (1 -- ps/kf) option in MICROSAT ([@pbio-0020442-Minch1]), generated similar topologies, and only the Dps tree is shown here. Numbers are individual Pti codes ([Table 3](#pbio-0020442-t301){ref-type="table"}). Bootstrap values over 50% are shown on the divergence node. ::: ![](pbio.0020442.g004) ::: Population Subdivision Analysis {#s2b} ------------------------------- To quantify the extent of population differentiation in modern tigers, we evaluated four different geographic subdivision scenarios and compared them on the basis of analysis of molecular variance (AMOVA) with both mtDNA haplotypes and microsatellite genotypes ([Table 4](#pbio-0020442-t004){ref-type="table"}). P. t. amoyensis individuals (Pti-219 and Pti-220) were excluded in this subdivision analysis due to our small sample size. In our first hypothesis, two groups were considered: the P. t. sumatrae island population and all contemporary mainland populations (P. t. altaica, P. t. corbetti I, P. t. corbetti II, P. t. tigris). This recently proposed model ([@pbio-0020442-Cracraft1]; [@pbio-0020442-Kitchener1]; [@pbio-0020442-Kitchener2]) presumes continuous habitat distribution on the mainland. The second scenario considered tigers as three groups: the Sumatran population (P. t. sumatrae), the Amur tigers (P. t. altaica), which presently are isolated from other tiger populations by more than their maximum known dispersal distance ([@pbio-0020442-Mazak2]), and a group of the other mainland tigers subspecies. The third hypothesis followed the division of the four traditional subspecies: (1) Amur tigers (P. t. altaica), (2) P. t. corbetti, including Indochina and part of south China, (3) Bengal tigers (P. t. tigris), and (4) Sumatran tigers (P. t. sumatrae). The fourth scenario, based on the results of the mtDNA phylogenetic analyses (see [Figure 3](#pbio-0020442-g003){ref-type="fig"}) and the hypothesis that the Isthmus of Kra may serve as a potential geographic barrier ([@pbio-0020442-Kitchener1]), further subdivided classical P. t. corbetti into the northern Indochina region P. t. corbetti I and the Malayan Peninsula P. t. corbetti II, resulting in five groups. The AMOVA results for each of the four scenarios are presented in [Table 4](#pbio-0020442-t004){ref-type="table"}. ::: {#pbio-0020442-t004 .table-wrap} Table 4 ::: {.caption} ###### Measures of Geographic Subdivision Based on AMOVA with MtDNA and Microsatellite Data ::: ![](pbio.0020442.t004) ^a^ Population subdivision scenarios are described in the text ^b^ Subspecies was grouped by brackets into populations for the analysis ::: For both mitochondrial haplotype and microsatellite data, the five-group scenario yielded the highest F~st~ (for mtDNA, defined as the proportion of total genetic variation that is attributable to genetic differences between populations) and R~st~ (for microsatellites, an F~st~ analogy suited for the stepwise mutation model that applies to microsatellite data) values. Under this model, 31% of the microsatellite variation discriminated between the five groups, while the balance, 69%, occurred within each group. For mtDNA the F~st~ was very high (0.838), indicating that 84% of the variation was partitioned among the different phylogeographic subspecies. Each of the five subspecies showed highly significant population genetic differentiation (p \< 0.0001) by pairwise F~st~ and R~st~ with 10,000 permutations ([Table 5](#pbio-0020442-t005){ref-type="table"}). The contrast between the mtDNA and microsatellite genetic variation probably reflects the difference in the effective population size assessed by these two different markers and/or, to some extent, the intersexual differences in dispersal. ::: {#pbio-0020442-t005 .table-wrap} Table 5 ::: {.caption} ###### Measures of Pairwise Comparisons in Tigers Based on AMOVA with mtDNA and Microsatellite Data ::: ![](pbio.0020442.t005) Population pairwise F~st~ estimates under the five-group scenario using the combined data from the mitochondrial regions and Kimura two-parameter are below the diagonal; R~st~ estimates using data from 30 microsatellite loci are above the diagonal. All populations are significantly different (p \< 0.0001) by F~st~ values based on mitochondrial data or R~st~ values based on microsatellite data ::: An alternative analysis of the combined microsatellite and mitochondrial haplotype data using a Bayesian approach ([Figure S2](#sg002){ref-type="supplementary-material"} and [Table S1](#st001){ref-type="supplementary-material"}) as implemented in the program STRUCTURE ([@pbio-0020442-Pritchard1]) supported the partitioning of P. t. altaica, P. t. sumatrae, P. t. tigris, and P. t. corbetti II, but further split the 33 P. t. corbetti I individuals into three distinctive population groups: (1) four tigers from China and Vietnam; (2) nine tigers from Cambodia; and (3) 20 tigers from Cambodia and northern Thailand (K = 7, Pr\[K\] = 0.993). In this scenario, most individuals were assigned to a cluster with high probability (q \> 0.90), indicating very low level of gene flow between the groups. However, because this additional substructure within P. t. corbetti I had little geographic or ecological basis, and because AMOVA analysis based on this population subdivision resulted in lower F~st~ and R~st~ values than that in the five-group scenario (unpublished data), the distinction was not considered to be a consistent basis for subspecies classification and may reflect additional population differentiation within a subspecies. Genetic Variation in Tigers {#s2c} --------------------------- Quantitative estimates of mtDNA diversity in tigers with comparable estimates from selected felid species demonstrated that overall, tigers had moderate levels of mtDNA diversity ([Table 6](#pbio-0020442-t006){ref-type="table"}), substantially less than leopards (P. pardus) ([@pbio-0020442-Uphyrkina1]), Geoffroy\'s cat (Oncifelis geoffroyi), Pampas cat (O. colocolo), or tigrina (Leopardus tigrinus) ([@pbio-0020442-Johnson2]), but comparable to pumas (Puma concolor) ([@pbio-0020442-Culver1]) in percent variable sites, mean pairwise distance among individuals, and average nucleotide diversity. Four tiger subspecies (P. t. tigris, P. t. sumatrae, P. t. corbetti I, and P. t. corbetti II) showed moderate nucleotide diversity (π), ranging from 0.0001 to 0.0070 ([Table 6](#pbio-0020442-t006){ref-type="table"}). The P. t. altaia sampling of 13 individuals showed no mtDNA haplotype variation. Of the five individuals originally designated as P. t. amoyensis, three were genetically indistinguishable from P. t. corbetti I, resulting in an inadequate sample size for a meaningful estimation of population variation. ::: {#pbio-0020442-t006 .table-wrap} Table 6 ::: {.caption} ###### Estimates of Molecular Genetic Variation from Combined MtDNA Sequences (4,078 bp) ::: ![](pbio.0020442.t006) ^a^ Fifteen tigers were screened in a 6,026 bp mtDNA segment, and 1,948 bp was excluded in the following large-scale sampling because of lack of variation ^b^ From a combined analysis of mtDNA ND5 (611 bp) and CR (116 bp) ([@pbio-0020442-Uphyrkina1]) ^c^ From a combined analysis of mtDNA 16S (364 bp), ATP8 (191 bp), and ND5 (318 bp) ([@pbio-0020442-Johnson2]) ^d^ From a combined analysis of mtDNA 16S (382 bp), ATP8 (191 bp), and ND5 (318 bp) ([@pbio-0020442-Culver1]) ::: Parameters of microsatellite variation have been shown to provide sensitive measures of historic demographic perturbations in felid and other species ([@pbio-0020442-Driscoll1]). Estimates of heterozygosity, average numbers of allele per locus, microsatellite variance in allele size, and allele size range in tigers were comparable to other felid species such as jaguar, leopard, puma, lions, and cheetahs across the same microsatellite loci (n = 17) ([Table 7](#pbio-0020442-t007){ref-type="table"}). After Bonferroni correction, eight of the 30 loci were significantly out of Hardy-Weinberg equilibrium in P. t. corbetti I (p \< 0.00167), possibly reflecting further population subdivision in this region. Expected heterozygosity in tigers ranged from 0.456 in P. t. altaica to 0.670 in P. t. corbetti I ([Table 7](#pbio-0020442-t007){ref-type="table"}). Average microsatellite variance was highest in P. t. tigris (4.94) and P. t. corbetti I (3.58) and lowest in P. t. altaica (1.93). ::: {#pbio-0020442-t007 .table-wrap} Table 7 ::: {.caption} ###### Genetic Variation across 30 Microsatellite Loci in Tiger Subspecies ::: ![](pbio.0020442.t007) Included are values describing genetic variation across 30 microsatellite loci in the six revised tiger subspecies, and a comparison with other Felidae species across the same 17 loci. Estimates of microsatellite diversity are calculated across a subset of microsatellite loci used in previous studies ([@pbio-0020442-Driscoll1]; [@pbio-0020442-Uphyrkina1]; [@pbio-0020442-Eizirik1]) ::: All six phylogeographic subspecies groups showed population-specific alleles that tended to represent the extreme sizes of allele distributions ([Table 8](#pbio-0020442-t008){ref-type="table"}). Of the 49 private alleles, 26 were either the largest or smallest size class among all tigers, and 38 were either the smallest or the largest for a specific subspecies, thus supporting a recent derivation. Frequencies of such private alleles were low in each population, from 1.5% of total allele numbers in P. t. amoyensis to 14.6% in P. t. corbetti I ([Table 8](#pbio-0020442-t008){ref-type="table"}). In addition, P. t. corbetti I had the highest average number of alleles per locus, the highest average allele size range per locus, and the most continuous and heterogeneous allele size distribution among all subspecies groups. ::: {#pbio-0020442-t008 .table-wrap} Table 8 ::: {.caption} ###### Diagnostic Characters and Habitat of the Six Phylogeographic Tiger Groups or Subspecies ::: ![](pbio.0020442.t008) ^a^ See [Table 2](#pbio-0020442-t002){ref-type="table"} for mtDNA nucleotide coordinates ^b^ Possibly extinct in the wild ([@pbio-0020442-Tilson2]) ND, no data ::: Major Histocompatibility Complex---DRB Gene Variation {#s2d} ------------------------------------------------------- The most polymorphic gene complex in all mammals is the MHC. This critical region for immunological recognition of infectious agents has 147 genes in the domestic cat, including three functional class II DRB genes on chromosome B3 ([@pbio-0020442-Yuhki2]). DRB gene homologs were amplified from DNA extracted from 21 tigers and screened for sequence diversity using single strand conformational polymorphism (SSCP). There were a total of seven electrophoretic allele variants (A--G). This is a relatively low MHC-DR diversity compared to human and domestic cat, which possess 126 and 63 DRB alleles, respectively, for the same gene segment in samplings of 251 humans and 37 cats, respectively ([@pbio-0020442-Yuhki1]; [@pbio-0020442-Bodmer1]). Despite this reduced DRB variation among tigers, there was detectable population differentiation. Three mainland subspecies P. t. tigris (n = 1), P. t. altaica (n = 5), and P. t. corbetti I (n = 2) were genetically identical for DRB-A allele sequence. Three additional DRB alleles (B, C, and D) were found only in P. t. corbetti II (n = 2), while three others (E, F, and G) were unique to P. t. sumatrae (n = 11) ([Tables 3](#pbio-0020442-t301){ref-type="table"} and [8](#pbio-0020442-t008){ref-type="table"}) ([@pbio-0020442-Wentzel1]). Estimation of the Coalescence Time of Genetic Variations in Tigers {#s2e} ------------------------------------------------------------------ The mtDNA sequence divergences in a combined data set of 3,217 bp, of which homologous sequences from the tiger and leopard were both determined (see [Materials and Methods](#s4){ref-type="sec"}), were used to estimate coalescence time for extant tiger mtDNA lineages and its 95% confidence interval (CI: ± two standard errors) based on a linearized tree method ([@pbio-0020442-Takezaki1]). Neither the two-cluster nor the branch-length molecular clock test revealed significant rate heterogeneity among tiger sequences (confidence probability less than 95%), suggesting that the divergence of the mtDNA sequences were compatible with a molecular clock hypothesis. Thus, all sequences were used to construct a linearized tree using the NJ tree algorithm with Kimura two-parameter distances. Assuming a divergence time for leopards and tigers of 2 MY, there were an estimated 2.29 × 10^--8^ substitutions per site per y, or one substitution every 14,000 y in the segment examined. According to this rate, the estimated coalescence time of mtDNA variation for extant tiger lineages was 72,000 y (95% CI = 39,000--104,000 y). An older fossil record calibration of 3 MY for the separation of leopards and tigers produced a rate of 1.53 × 10^--8^ substitutions per site per y, or one substitution every 20,000 y. According to this substitution rate, mtDNA diversity of modern tigers originated about 108,000 y (95% CI = 59,000--157,000 y) ago. Based on either calibration, the Amur tigers probably experienced a genetic reduction or founder event more recently (less than 20,000 y), as no variation was detected within the population. The estimate of microsatellite variance in average allele repeat-size can also be used as a surrogate for evolutionary time based on the rate of allele range reconstitution subsequent to a severe founder effect ([@pbio-0020442-Driscoll1]). Using a standard curve for the relationship of microsatellite variance to elapsed time (see [Figure 4](#pbio-0020442-g004){ref-type="fig"} in [@pbio-0020442-Driscoll1]), the variance for all tigers converged to 19,000 y ago. The age of different subspecies, based on populations for which we had an adequate sample size (n \> 15), ranged from 9,900 y in Amur tigers P. t. altaica to 18,437 y in northern Indochinese tigers P. t. corbetti I. We estimated the historic population size required to sustain the level of mitochondrial genetic variation under the assumption of neutrality of substitution and mutation-drift equilibrium ([@pbio-0020442-Kimura1]; [@pbio-0020442-Nei1]), where the population parameter θ = 2N ~e~ μT, and N ~e~ is the long-term effective female population size, μ the substitution rate per site per year, and T the generation time. From a coalescent-based simulation of the mitochondrial sequences, the average estimate of θ was 0.00255 per nucleotide site, with a 95% CI from 0.00147 to 0.00417. With the substitution rate calibrated from this study (1.91 × 10^--8^ bp^--1^ y^--1^) and an average generation time of 5 y for tigers ([@pbio-0020442-Smith1]), the historical effective population size is an estimated 13,350 females (95% CI = 7,700--21,830). Discussion {#s3} ========== Overall, tigers displayed moderate levels of molecular genetic variation in mtDNA and DRB sequences compared with other mammalian species, consistent with previous allozyme studies ([@pbio-0020442-OaBrien1]). There was a variable site every 75 bp, with 54 sites in the more variable 4-kb segment and one variable site every 112 bp in the larger 6,026-bp segment (see [Materials and Methods](#s4){ref-type="sec"}). This value was less than what was observed in leopards in a smaller portion of mtDNA (one variable site every 15 bp in 727 bp of the gene encoding NADH dehydrogenase subunit 5, called ND5, and that for the control region, called CR; 34 haplotypes were found) ([@pbio-0020442-Uphyrkina1]). MHC class-II DRB gene variation was also low relative to human and domestic cat ([@pbio-0020442-Yuhki1]; [@pbio-0020442-Bodmer1]). By contrast, estimates of tiger microsatellite variability were more similar to those of other felid species ([Table 7](#pbio-0020442-t007){ref-type="table"}) ([@pbio-0020442-Culver1]; [@pbio-0020442-Eizirik1]; [@pbio-0020442-Uphyrkina1]; [@pbio-0020442-Driscoll1]). The oldest tiger fossils, around two million y (MY) old, are from northern China and Java ([@pbio-0020442-Hemmer1]). By the late Pliocene and early Pleistocene tigers were widely distributed in eastern Asia. However, Pleistocene glacial and interglacial fluctuations and other geological events probably caused repeated geographic restrictions and expansions ([@pbio-0020442-Hemmer1]; [@pbio-0020442-Kitchener1]; [@pbio-0020442-Kitchener2]). We estimated the most recent common ancestor for tiger mtDNA haplotypes was 72,000--108,000 y ago, with a lower and upper bound of 39,000 y and 157,000 y, respectively. This estimate is much earlier than that derived for the leopard, which is considered to have originated in Africa 470,000--825,000 y ago and to have arrived in Asia 170,000--300,000 y ago ([@pbio-0020442-Uphyrkina1]). Likewise, extant jaguar (Panthera onca) lineages diverged approximately 280,000--510,000 y ago ([@pbio-0020442-Eizirik1]). Our coalescence estimate for tigers corresponds roughly with the catastrophic eruption of Toba in Sumatra around 73,500 y ago ([@pbio-0020442-Rampino1]), which has been linked to the Late Pleistocene bottleneck in human evolution ([@pbio-0020442-Ambrose1]) and to a major northward dispersal event in the Asian elephants ([@pbio-0020442-Fleischer1]). Based on the subspecies definition of [@pbio-0020442-OaBrien2] and [@pbio-0020442-Avise1], our data suggest that there are at least five and possibly six tiger subspecies: Amur tigers (P. t. altaica); northern Indochinese tigers (P. t. corbetti I); southern Indochinese tigers (P. t. corbetti II), which are confined to the Malayan Peninsula; Sumatran tigers (P. t. sumatrae); Bengal tigers (P. t. tigris); and, if its uniqueness is affirmed by more extensive sampling, South China tiger (P. t. amoyensis). These conclusions are based on significant genetic structure among tigers from these different geographic regions with the MHC, mtDNA, and microsatellite data, and extremely limited gene flow as shown by disjunct distributions of genetic variation (unique mtDNA haplotypes and microsatellite alleles) and the high mtDNA F~st~ and microsatellite R~st~ values. In addition, each subspecies has an allopatric geographical distribution (see [Figure 1](#pbio-0020442-g001){ref-type="fig"}) and differential natural history ([@pbio-0020442-Kitchener1]; [@pbio-0020442-Seidensticker1]). The hypothesis that tiger population structure reflects recent (less than 10,000 y ago), human-induced population fragmentation and random lineage loss from a single panmictic population is not supported by the strong geographical partitioning of the mitochondrial lineages or by differences in measures of nucleotide diversity within each subspecies. Mismatch analysis ([@pbio-0020442-Rogers1]) of pairwise differences among all tiger mtDNA haplotypes also revealed a multimodal distribution significantly different from a Poisson expectation, indicating the existence of several highly divergent populations (unpublished data). It is plausible that tiger populations (subspecies) differentiated through the combined effects of genetic drift in isolated populations and local adaptation to rapidly changing habitats across the tiger range during the Holocene ([@pbio-0020442-Lister1]). For example, Sumatran tigers currently occupy tropical moist forests, and Bengal tigers range from tropical dry forests, terai forests, and tall grasslands to the Himalayan foothills. However, we cannot rule out the possibility that some of the current population subdivision, particularly in the case of the divergence of P. t. altaica and P. t. amoyensis/P. t. corbetti I, could be related to the disruption of an isolation-by-distance pattern caused by the recent extinction of intermediate populations; this hypothesis can be tested only when a larger geographic sampling is available. The differences in molecular genetic patterns among the six hypothesized subspecies are dramatic ([Table 8](#pbio-0020442-t008){ref-type="table"}). Further, the results lend support to the hypothesis that the Pleistocene centrum of tiger radiation is located within northern Indochina and southern China. Modern P. t. corbetti I has a large number of mtDNA diagnostic sites (three), the largest number of unique microsatellite alleles (19 out of 130), and the highest overall microsatellite diversity ([Tables 7](#pbio-0020442-t007){ref-type="table"} and [8](#pbio-0020442-t008){ref-type="table"}). In addition, no microsatellite allele at any locus occurred with a frequency higher than 81%. The observed allele size distribution in P. t. corbetti I was generally continuous for most loci (there were fewer allele size gaps compared to other subspecies), evidence of a fairly stable demographic history, and alleles found in the other subspecies were almost always a subset of those found in P. t. corbetti I. Additional sampling of modern and/or historic samples could reveal additional structure (putative subspecies) in the P. t. corbetti I region (see [Figure 1](#pbio-0020442-g001){ref-type="fig"}), as there were several microsatellite loci out of Hardy-Weinberg equilibrium, and the Bayesian population structure analysis identified possible substructure within P. t. corbetti I ([Figure S2](#sg002){ref-type="supplementary-material"}). The ultimate classification of tigers of the southern China and northern Indochina region is further complicated by the poor definition of the geographic boundary between P. t. corbetti I and P. t. amoyensis, and because the South China tiger subspecies is represented only by captive-born animals of imprecise origin. One of the two phylogenetic lineages in this captive population (Pti-217, Pti-218, and Pti-222) was indistinguishable from northern Indochinese tigers (see [Figures 3](#pbio-0020442-g003){ref-type="fig"} and [4](#pbio-0020442-g004){ref-type="fig"}), perhaps as a consequence of introgression of the northern Indochinese tigers into the Chinese captive population or a more-northern distribution of the Indochinese tigers than had previously been recognized. A comprehensive morphological and genetic assessment of the captive population (around 50 individuals) ([@pbio-0020442-Tilson2]), of historic samples, and of additional wild tigers from southern China, in the context of subspecies patterns seen here would be useful to resolve remaining uncertainties and to inform in situ and ex situ management strategies. By contrast, the other subspecies delineations are better defined. To the north, Amur tigers, presently an isolated population of fewer than 500 individuals, are confined almost entirely to the Russian Far East ([@pbio-0020442-Matyushkin1]). They display low genetic diversity in comparison to other subspecies, with a single mtDNA haplotype that is likely derived from P. t. corbetti I Indochinese tigers ([Figure 3](#pbio-0020442-g003){ref-type="fig"}A). The Amur tiger genetic variability may have been reduced during a post-ice age colonization of the region around 9,000 y ago and/or during the early 20th century when an estimated 20--30 tigers survived intense human persecution ([@pbio-0020442-Kaplanov1]). In Indochina, the genetic distinction between P. t. corbetti I and P. t. corbetti II (pairwise mtDNA F~st~ = 0.797 and microsatellite R~st~ = 0.225, p \< 0.0001; P. t. corbetti II is characterized by three unique microsatellite alleles and five subspecies-specific mtDNA haplotypes \[[Table 8](#pbio-0020442-t008){ref-type="table"}\]) supports the hypothesis that the Isthmus of Kra has been an ecological barrier restricting gene flow between tigers in Malaya Peninsula and mainland Southeast Asia. Previous biogeography studies have placed numerous species and subspecies boundaries of mammals ([@pbio-0020442-Corbett1]; [@pbio-0020442-Tosi1]), birds ([@pbio-0020442-Hughes1]), and plants ([@pbio-0020442-Woodruff1]) near the Isthmus of Kra, making it a significant biogeographical transition between Indochina and Sundaic regions. The isolation of Sumatran tigers from mainland populations is supported by multiple unique characters, including two diagnostic mtDNA nucleotide sites, eight mtDNA haplotypes, and 11 (of 108) unique microsatellite alleles ([Table 8](#pbio-0020442-t008){ref-type="table"}). [@pbio-0020442-Cracraft1] and [@pbio-0020442-Hendrickson1] also described genetic variation distinguishing Sumatran tigers from other tiger subspecies. The relatively high genetic variability and phylogenetic distinctiveness of Sumatran tigers suggest a historically large effective population size followed by highly restricted gene flow between the island and other populations. The Bengal tigers are defined by three distinct mitochondrial nucleotide sites and 12 unique microsatellite alleles. The pattern of genetic variation in the Bengal tiger corresponds to the premise that tigers arrived in India approximately 12,000 y ago ([@pbio-0020442-Kitchener2]). This recent history of tigers in the Indian subcontinent is consistent with the lack of tiger fossils from India prior to the late Pleistocene and the absence of tigers from Sri Lanka, which was separated from the subcontinent by rising sea levels in the early Holocene. Similar biogeographical boundaries to those separating the six tiger subspecies have been proposed in other species including leopard ([@pbio-0020442-Uphyrkina1]), Asian elephant ([@pbio-0020442-Fleischer1]), and rodents ([@pbio-0020442-Gorog1]), but warrant further study to determine their importance as recent barriers to gene flow for large mammals in Asia. Our results have several implications for tiger conservation. Management strategies for the tiger, both in situ and ex situ, have been historically influenced by perceptions of its geographical variation and subspecific taxonomy ([@pbio-0020442-Maguire1]; [@pbio-0020442-Seidensticker1]), and several captive tiger breeding programs have attempted to maintain purebred lines ([@pbio-0020442-Foose1]; [@pbio-0020442-Maguire1]). Our data suggest, however, that while supporting and refining most existing (and extant) tiger subspecies designations, there is additional substructure within some subspecies that should be considered when formulating management strategies for captive animals or when considering the maintenance of sufficiently large and interconnected wild populations. Specifically, the distinctiveness of tigers from Malayan Peninsula is comparable to differences among other recognized and separately managed subspecies. To be consistent, the Malayan subspecies should also be managed as such unless inbreeding depression has become an issue due to declined genetic variability. Since the current type specimen for P. t. corbetti is located in northern Vietnam ([@pbio-0020442-Mazak3]), and no prior name has been given to the southern populations, we propose the newly defined tiger subspecies from Malayan Peninsula be designated P. t. jacksoni, to honor the contributions of Peter Jackson, the former Chair of the the World Conservation Union (IUCN) Cat Specialist Group, who tirelessly labored for more than 40 y on behalf of tiger conservation. We designate the type specimen of the Malayan tigers to Pti-163 from the Zoo Melaka, Malaysia, and the taxonomic diagnosis will be described elsewhere. The present status of tigers from northern Indochina and from Malayan Peninsula is uncertain, urging more extensive study and conservation. Our results also show that, although modern tigers have a relatively young history, ecological, demographic, and biogeographic factors have led to recognizable subdivisions among otherwise closely related populations. We therefore might expect that more extensive geographic sampling would reveal additional phylogenetic divisions among populations, especially in the Indian Subcontinent and the Indochina bioregions, or alternatively, would blur the apparent phylogenetic subdivisions and reveal a clinal distribution of genetic variation across different subspecies. Further sampling of modern and historic specimens will also help clarify whether the patterns we have observed are attributable to the recent substantial population decline throughout the range in tigers, or whether the observed differentiations among tigers occurred earlier. Materials and Methods {#s4} ===================== {#s4a} ### Samples. {#s4a1} A total of 134 tiger individuals were sampled throughout the distribution range (see [Figure 1](#pbio-0020442-g001){ref-type="fig"} and [Table 3](#pbio-0020442-t301){ref-type="table"}). Of these, 100 were verified as either wild-born from a specific geographic locale or captive-born from geographically verified wild-born parents. An additional 34 individuals were of reasonably certain geographic origin and were used to complement estimated levels of molecular genetic variation in tigers. Individuals were labeled with traditional subspecies classifications based on their geographical origin following [@pbio-0020442-Mazak2]. Genomic DNA from blood or primary skin fibroblast cell culture was isolated using a standard proteinase K digestion and phenol-chloroform extraction procedure ([@pbio-0020442-Sambrook1]). DNA was isolated from dry skin using guanidine thiocyanate ([@pbio-0020442-Boom1]) and silica-based purification ([@pbio-0020442-Hoss1]). DNA from hair was obtained by a modification of the previously described chelex method ([@pbio-0020442-Higuchi1]). Analysis of historical samples was carried out with strict precautions at an isolated laboratory specializing in work with ancient DNA and was independently repeated to exclude possible contamination from any high-copy DNA source ([@pbio-0020442-Hofreiter1]). ### Mitochondrial DNA analysis. {#s4a2} Analyses of mtDNA in tigers and in other Panthera species is complicated by the presence of a large 12.8 kb nuclear mtDNA fragment that transposed to chromosome F2 in an ancestral Panthera species approximately 3 MYA ([@pbio-0020442-Johnson1]; [@pbio-0020442-Lopez1]; [@pbio-0020442-Cracraft1]; J. H. Kim, A. Antunes, S.-J. Luo, J. Menninger, W. G. Nash, et al., personal communication). Although the Numt and Cymt DNA sequences have diverged, primers designed from conserved regions often coamplify both copies. Fifteen Cymt-specific primer sets (see [Figure 2](#pbio-0020442-g002){ref-type="fig"} and [Table 1](#pbio-0020442-t001){ref-type="table"}) were designed on the basis of sequence differences from the alignments of the complete tiger Numt and the homologous 12.8-kb Cymt sequences (J. H. Kim, A. Antunes, S.-J. Luo, J. Menninger, W. G. Nash, et al., personal communication). These Cymt primers amplified a total of 6,026 bp of sequence, spanning ten mitochondrial gene segments, including NADH dehydrogenase subunits 1, 2, 5, and 6 (ND1, ND2, ND5, and ND6), cytochrome B(CytB), control region(CR), 12S rRNA(12S), cytochrome C oxidase subunits I and II(COI and COII), and ATPase8 (ATP8) (see [Figure 2](#pbio-0020442-g002){ref-type="fig"}). The primer sets were tested in 15 individuals representing tigers from all five traditional subspecies. Five segments that revealed no variation in the pilot screening were excluded from further analysis (see [Figure 2](#pbio-0020442-g002){ref-type="fig"}). PCR products were amplified from 50 ng of genomic DNA in a 25 μL reaction system containing 2.0 mM MgCl~2~, 1.0 mM dNTPs, 0.25 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, California, United States), and 1× PCR buffer II; the amplification protocol was: denaturation 10 min at 95 °C, a touch-down cycle of 95 °C for 30 s, 52 °C for 30 s decreased by 1 °C in the next cycle for 10 cycles, 72 °C for 45 s, then 35 amplification cycles of 95 °C for 30 s, 52 °C for 30 s, and 72 °C for 45 s, followed by an extension of 10 min at 72 °C. PCR products were purified using Microcon PCR filters (Millipore, Billerica, Massachusetts, United States) and were directly sequenced in both directions using BigDye Terminator kits (Applied Biosystems) and run on an ABI 377 sequencing apparatus. Sequences were inspected using SEQUENCHER (Gene Codes, Ann Arbor, Michigan, United States), unambiguously aligned using Clustal-X ([@pbio-0020442-Thompson1]), and visually inspected. Sequences for each mtDNA fragment were combined for a total evidence approach. Sequences were deposited in GenBank. Phylogenetic relationships among mtDNA haplotypes were assessed using three approaches implemented in PAUP ([@pbio-0020442-Swofford1]). An MP analysis was conducted using a heuristic search, with random additions of taxa and tree-bisection-reconnection branch swapping. The ME heuristic search approach consisted of NJ trees constructed from Kimura two-parameter distances followed by a branch-swapping procedure. ML analysis was done using the TrN (Tamura-Nei) +I (with proportion of invariable sites) model with the proportion of invariable sites set to 0.93, and the rate among sites equal, as estimated using MODELTEST 3.06 ([@pbio-0020442-Posada1]). The reliability of the nodes in each of the analyses was assessed by 100 bootstrap iterations. A statistical parsimony network was constructed using TCS 1.13 ([@pbio-0020442-Clement1]) to infer phylogeographic and potential ancestor-descendent relationships among haplotypes. Measures of population genetic variation, such as mean number of pairwise differences, gene diversity, and nucleotide diversity were estimated using ARLEQUIN 2.0 ([@pbio-0020442-Schneider1]). The extent of geographic subdivision among populations was assessed by F~st~ values (with Kimura two-parameter distance) using AMOVA as implemented in ARLEQUIN 2.0. Statistical significance was tested using 10,000 permutations. The approximate coalescence time of tigers was estimated with a linearized tree method as implemented in the program LINTRE ([@pbio-0020442-Takezaki1]). This program constructs linearized NJ trees reestimating branch lengths under the molecular clock assumption and incorporates two tests for the assumption ([@pbio-0020442-Takezaki1]). The mtDNA sequence divergence was based on the standard equation H = 2μT, where H was the branch height in the linearized tree correlated to the average pairwise distance among haplotypes, μ the substitution rate, and T the divergence time. Since there was no comparable sequence from other Panthera species available, we generated from PCR and GenBank a chimera-homologous sequence of 3.2 kb (including fragments from mitochondrial genes ND1, ND2, ND5, ND6, CytB, 12S, and COI) from three leopard individuals. The divergence time between leopard and tiger was used as a calibration point, and two fossil dates were chosen. Two million y was a commonly used lower bound for the Panthera lineage radiation and the date for the earliest reported tiger in the fossil record ([@pbio-0020442-Hemmer1]; [@pbio-0020442-OaBrien1]; [@pbio-0020442-Wayne1]). An earlier record of 3 million y was also chosen because leopard fossils have been reported from this time period ([@pbio-0020442-Turner1]). Domestic cat (Felis catus) was used as an outgroup. Coalescent-based simulation of the population parameter θ estimation and its 95% CI was conducted in DnaSP 4.0 ([@pbio-0020442-Rozas1]) with 1,000 replicates, given that the mutations along the lineages followed a Poisson distribution. ### Microsatellite analysis {#s4a3} Thirty polymorphic microsatellite loci (FCA005, FCA008, FCA032, FCA043, FCA044, FCA069, FCA077, FCA090, FCA091, FCA094, FCA096, FCA105, FCA123, FCA126, FCA129, FCA139, FCA161, FCA176, FCA201, FCA211, FCA212, FCA220, FCA229, FCA242, FCA290, FCA293, FCA304, FCA310, FCA391, and FCA441) originally designed in the domestic cat (F. catus) ([@pbio-0020442-Menotti-Raymond1]) were amplified by PCR using fluorescently labeled primers under previously published conditions ([@pbio-0020442-Menotti-Raymond1]). Two loci (FCA391 and FCA441) were tetranucleotide repeats, and the others were dinucleotides. All loci have been mapped in the domestic cat and located on 11 of the 19 chromosomes ([@pbio-0020442-Menotti-Raymond1]; [@pbio-0020442-Menotti-Raymond2]). These microsatellites were in different linkage groups or at least 12 centimorgans apart in the domestic cat, except for FCA 211 and FCA 212 (4 centimorgans), and were likely to be in linkage equilibrium. The dye-labeled PCR products of the 30 microsatellite primer sets were pooled and diluted based on size range and fluorescent dye so that 2--4 loci could be multiplexed and subsequently analyzed by electrophoresis in an ABI 377 automated sequencer (Applied Biosystems). Patterns were scored and analyzed using GENESCAN 2.1 and GENOTYPER 2.5 software. Of 134 tiger samples, 113 were included in the microsatellite analysis. DNA samples from pelt or hair for which fewer than 20 of the loci amplified successfully were excluded from the analysis. Tests for genotypic linkage disequilibrium and deviations from Hardy-Weinberg equilibrium for each locus in each population were performed using GENEPOP web version of 3.1c (<http://wbiomed.curtin.edu.au/genepop/>) ([@pbio-0020442-Raymond1]). Measures of microsatellite genetic variation in terms of average observed heterozygosity and expected heterozygosity, average number of alleles per locus, average allele size range per locus, number of unique alleles, and average variance were estimated with MICROSAT ([@pbio-0020442-Minch1]). Pairwise genetic distances among individual tigers were estimated based on Dps and Dkf with the \[1 -- ps/kf\] option in MICROSAT and were used to construct NJ phylogenetic trees with the program NEIGHBOR in the PHYLIP 3.5 package ([@pbio-0020442-Felsenstein1]). Assessments of different geographic subdivision scenarios and population pairwise comparisons (using R~st~, sum of square size differences) were derived from ARLEQUIN 2.0. The statistical significance of R~st~ values, sum of squared size differences, was tested with 10,000 permutations as implemented in ARLEQUIN. A Bayesian clustering method implemented in the program STRUCTURE ([@pbio-0020442-Pritchard1]) was used to infer population structure based upon multilocus microsatellite genotype and sequence data. MtDNA was treated as a single haploid locus, and each observed haplotype was coded with a unique integer (e.g., 1, 2) for the first allele and the missing data symbol (e.g., -9) for the second. We calculated the probability of individual assignments to population clusters (K) without prior information of the origin of individuals. A series of tests was conducted using different numbers of population clusters to guide an empirical estimate of the number of identifiable populations, assuming an admixture model with correlated allele frequencies and with burn-in and replication values set at 50,000 and 10^6^, respectively. Each test yielded a log likelihood value of the data (Ln probability), the highest of which would indicate which test was closest to the actual number of genetically distinct populations. These tests also provided an alpha value, the measure of admixed individuals in the data set. ### Class II MHC. {#s4a4} Allele variation in the nuclear MHC class II DRB gene was assessed in five Amur, two northern Indochinese, two Malayan, 11 Sumatran, and one Bengal tiger. Conserved PCR primers designed from the human DRB sequence were used to amplify homologous DRB sequences (of around 238 bp) in 21 tiger voucher DNAs with primers 61a (5′- CCGCTGCACTGTGAAGCT-3′) and 219a (5′- CCACACAGCACGTTTCTT-3′). Products were screened for polymorphisms using SSCP, a method that detects single-basepair substitutions in 100--300-bp DNA fragments. For SSCP, PCR products were mixed with a solution of 120 μl of formamide, 20 μl of TAMRA (Applied Biosystems) lane standard, and 20 μl of a blue dextran loading dye (from a stock solution of 50 mg/ml with 25 mM EDTA), then denatured at 95 °C for 3 min. The electrophoresis ran at 2,000 volts, 400 amps, and 25 watts in 1× TBE buffer through a 6% denaturing polyacrylamide gel (19.5:1 acrylamide:bis). The SSCP fragments were visualized by autoradiography, and alleles were scored by eye ([@pbio-0020442-Yuhki1]). Supporting Information {#s5} ====================== Figure S1 ::: {.caption} ###### Phylogenetic Relationships among the Individual Tigers from Composite Microsatellite Genotypes of 30 Loci Branches of the same color represent tiger individuals of the same classically named subspecies. NJ tree constructed based on kinship coefficient (Dkf) with the (1 -- kf) option in MICROSAT ([@pbio-0020442-Minch1]). Numbers are individual Pti codes ([Table 3](#pbio-0020442-t301){ref-type="table"}). Bootstrap values over 50% are shown on divergence nodes. (108 KB DOC). ::: ::: {.caption} ###### Click here for additional data file. ::: Figure S2 ::: {.caption} ###### Bayesian Population Structure Analysis of 111 Tigers Data obtained from microsatellite genotype and mitochondrial haplotype data were analyzed using STRUCTURE ([@pbio-0020442-Pritchard1]). Simulations were set at 50,000 burn-in period followed by 10^6^ replicates. Each individual is represented by a thin vertical bar, which is partitioned into K colored segments that represent the individual affiliation to each of K clusters. Here shows the population structure when K = 7, which produced the highest probability among other choices of K. Three STRUCTURE runs produced almost identical individual affiliation. (62 KB DOC). ::: ::: {.caption} ###### Click here for additional data file. ::: Table S1 ::: {.caption} ###### Bayesian Clustering Analyses for Tiger Microsatellite and Mitochondrial Data (59 KB DOC). ::: ::: {.caption} ###### Click here for additional data file. ::: Accession Numbers {#s5a4} ----------------- The GenBank (<http://www.ncbi.nlm.nih.gov/>) accession numbers of the mtDNA fragments discussed in this paper are AY736559--AY736808. We are grateful to acknowledge full collaborative credit to the investigators listed in [Table 3](#pbio-0020442-t301){ref-type="table"} who supplied the biological specimens upon which this study is based. In addition, we are indebted to the numerous organizations that assisted in the logistical support of this project and whose help was crucial, including the South East Asian Zoos Association, WildAid Cambodia, and the Chinese Association of Zoological Gardens. We appreciate the technical and editorial assistance of Agostinho Antunes, Eduardo Eizirik, Gila Kahila Bar-Gal, Colm O\'hUigin, Al Roca, Victor David, Marilyn Menotti-Raymond, Stanley Cevario, Carlos Driscoll, John Page, Guo-Kui Pei, Jill Pecon-Slattery, Mary Carrington, and Clay Stephens. All tissue samples were collected in full compliance with specific Federal Fish and Wildlife permits \[Conservation on International Trade in Endangered Species of Wild Fauna and Flora (CITES); Endangered and Threatened Species\] issued to the National Cancer Institute, National Institutes of Health (principal officer SJ O\'Brien) by the US Fish and Wildlife Service of the Department of the Interior. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government. Conflicts of interest. The authors have declared that no conflicts of interests exist. Author contributions. SJL, JHK, WEJ, JV, NY, and SJOB conceived and designed the experiments. SJL, JHK, JV, JM, and OU performed the experiments. SJL, JHK, and WEJ analyzed the data. SJL, WEJ, JM, NY, DM, OU, JMG, HBQ, RT, GB, PM, VS, CM, SH, SQH, WP, UKK, MS, JLDS, and SJOB contributed reagents/materials/analysis tools. SJL, JHK, WEJ, and SJOB wrote the paper. Academic Editor: Craig Moritz, University of California, Berkeley ¤1 Current address: Biochip Project Team, Samsung Advanced Institute of Technology, Suwon, Korea ¤2 Current address: Center for Human Genetics, Duke University Medical Center, Durham, North Carolina, United States of America ¤3 Current address: Evolutionary Zoology and Genetics Laboratory, Institute of Biology and Soil Sciences, Vladivostok, Russia Citation: Luo SJ, Kim JH, Johnson WE, van der Walt J, Martenson J, et al. (2004) Phylogeography and genetic ancestry of tigers (Panthera tigris). PLoS Biol 2(12): e442. AMOVA : analysis of molecular variance CI : confidence interval Cymt : cytoplasmic mitochondrial Dkf : kinship coefficient Dps : proportion of shared alleles ME : minimum evolution MHC : major histocompatibility complex ML : maximum likelihood MP : maximum parsimony mtDNA : mitochondrial DNA MY(A) : million years (ago) NJ : neighbor joining Numt : nuclear mitochondrial SSCP : single strand conformation polymorphism	PubMed Central	2024-06-05T03:55:49.989214	2004-12-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534810/", "journal": "PLoS Biol. 2004 Dec 7; 2(12):e442", "authors": [ { "first": "Shu-Jin", "last": "Luo" }, { "first": "Jae-Heup", "last": "Kim" }, { "first": "Warren E", "last": "Johnson" }, { "first": "Joelle", "last": "van der Walt" }, { "first": "Janice", "last": "Martenson" }, { "first": "Naoya", "last": "Yuhki" }, { "first": "Dale G", "last": "Miquelle" }, { "first": "Olga", "last": "Uphyrkina" }, { "first": "John M", "last": "Goodrich" }, { "first": "Howard B", "last": "Quigley" }, { "first": "Ronald", "last": "Tilson" }, { "first": "Gerald", "last": "Brady" }, { "first": "Paolo", "last": "Martelli" }, { "first": "Vellayan", "last": "Subramaniam" }, { "first": "Charles", "last": "McDougal" }, { "first": "Sun", "last": "Hean" }, { "first": "Shi-Qiang", "last": "Huang" }, { "first": "Wenshi", "last": "Pan" }, { "first": "Ullas K", "last": "Karanth" }, { "first": "Melvin", "last": "Sunquist" }, { "first": "James L. D", "last": "Smith" }, { "first": "Stephen J", "last": "O'Brien" } ] }
PMC534811	In his seminal exploration of the properties of living organisms, What Is Life?, Erwin Schroedinger concluded that life depends in large part on storing and processing information. For genetic material to carry the diverse instructions required for living processes, he proposed, it must be stored in an aperiodic crystal. Just nine years later, it was clear that DNA is indeed an aperiodic crystal and that genetic information is conveyed through this irregular pattern. Much like computers, biological systems are programmed to follow a precise set of rules, or algorithms, to store information and solve problems. These biological algorithms direct all manner of biochemical processes to create complex patterns and structures by chemically modifying and assembling individual components. Of course, cells use biochemical circuits not electronic circuits. Single tubulin proteins, for example, follow precise rules of chemistry and physics to spontaneously self-assemble, or polymerize, into the microtubules essential for cell transport and motility. The proteins\' binding interactions effect rules that specify how the pieces fit together to form the resulting structure. They also specify when and how tubulins assemble from a nucleation complex---a molecular algorithm governing the logic of polymerization. These complex structures self-assemble with remarkably few mistakes. Though considered quite simple, little is understood about the principles that govern programmable structural order underlying this type of spontaneous self-assembly. In crystals, the simplest example of spontaneous self-assembly, subunits of the whole are arranged in a repeating pattern that extends indefinitely in all directions. If you know the position of one unit in the pattern, you can tell the exact position of every other unit. In a new study, Rothemund and colleagues use DNA to show that crystal growth can be programmed to create specific aperiodic patterns. Inspired by a model of crystal growth as a computational process, they have programmed DNA molecules to act as molecular building blocks, arranging themselves according to local rules that in turn create a complex global pattern. The resulting two-dimensional structures, which self-assemble from knotted DNA complexes (called tiles), grow to create a fractal pattern known as a Sierpinski triangle. These DNA structures---neither periodic (as in quartz), nor random (as in glass), nor pseudorandom (as in quasicrystals with "forbidden" five-fold symmetries)---demonstrate a form of self organization in crystalline materials determined by programmable growth rules, and are hence dubbed "algorithmic crystals." How can such growth algorithms be encoded in biological molecules? The rules of chemical base-pairing follow regular, predictable patterns, allowing the authors to use DNA to determine the tiles\' binding interactions.[](#pbio-0020448-g001){ref-type="fig"} ::: {#pbio-0020448-g001 .fig} ::: {.caption} ###### Fractal patterns from DNA ::: ![](pbio.0020448.g001) ::: Desired binding interactions between tiles were programmed by endowing each tile with single-stranded "sticky ends" whose sequence was complementary to the sticky ends of tiles it should stick to. Each tile was either white (0) or black (1): a black tile can fit at any site where the two neighboring tiles are opposite colors, while a white tile can fit at any site where the two neighboring tiles are the same color. Logically, the new tile\'s color is the exclusive-or (XOR) of the tiles in the previous layer. That such logical layer-by-layer iteration of XOR computations will produce the Sierpinski triangle is well known. What\'s remarkable is that DNA molecules can be programmed to grow according to this logic. With this programmable algorithmic crystal, Rothemund and colleagues demonstrate a method for designing DNA molecules capable of implementing any pattern of abstract logical tiles. What\'s more, the authors argue, any algorithmic crystal growth process can, in principle, be experimentally investigated using DNA self-assembly. So how is algorithmic self-assembly related to biology? Like the algorithmic crystals, many of the self-assembled structures in biology are ordered but aperiodic. The hope is that the theoretical insights of computer science---well-honed for describing, analyzing, and programming computational systems---can direct investigations of biochemical self-assembly and information processing. And with a method for demonstrating how simple chemical and physical elements can create complex organization, Rothemund and colleagues have added a concrete experimental framework to bolster that work. (For more on DNA and complexity, see "The Emergence of Complexity: Lessons from DNA" by Chengde Mao.)	PubMed Central	2024-06-05T03:55:49.994391	2004-12-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534811/", "journal": "PLoS Biol. 2004 Dec 7; 2(12):e448", "authors": [] }
PMC534812	"Tyger! Tyger! burning brightIn the forests of the nightWhat immortal hand or eyeCould frame thy fearful symmetry?" When William Blake wrote these words in the late 1700s, the deforestation and habitat destruction that would decimate wild tiger populations had already begun. In 1900, an estimated 100,000 wild tigers lived throughout much of Asia, from India in the west to Sumatra and Indonesia in the south to Siberia in the east. Today, the ongoing stresses of habitat loss, hunting, and an illegal trade in tiger parts have spared fewer than 7,000 tigers. Of eight traditionally classified subspecies of Panthera tigris, three have gone extinct since the 1940s. Conservation strategies to combat this grinding attrition are tailored to each subspecies. But several lines of evidence suggest that subspecies designations---based on geographic range and morphological traits such as body size, skull traits, coat color, and striping patterns---may be flawed. An earlier molecular study of 28 tigers found little evidence of genetically distinct subspecies, while surveys of tiger habitat found few physical barriers sufficient for subspecies isolation. To get a clearer picture of the genetic structure of existing tiger populations, Shu-Jin Luo, Jae-Heup Kim, Stephen O\'Brien, and nineteen colleagues performed a comprehensive genetic analysis of mitochondrial and nuclear genes from over 130 tigers. By identifying distinct patterns of variation within these gene families, the authors reconstructed the evolutionary distribution and ancestry of the tiger. Their results support many of the traditional subspecies designations and identify further subdivisions in others.[](#pbio-0020453-g001){ref-type="fig"} ::: {#pbio-0020453-g001 .fig} ::: {.caption} ###### A Bengal tiger in the tall grassland (Photo: Ullas Karanth, WCS-India) ::: ![](pbio.0020453.g001) ::: Luo et al. collected "voucher specimens" (taken from animals of verified wild ancestry and geographic origin) of blood, skin, and hair from 134 tigers representing the entire tiger range, and examined them, along with samples of preserved pelts and hair, for three molecular markers. The markers---a stretch of mitochondrial DNA (mtDNA) sequence, a gene with highly variable DNA sequence called DRB that\'s involved in pathogen recognition, and short repeating genetic elements called microsatellites---act as unique signposts that flag significant demographic and evolutionary events in the tiger populations. mtDNA sequences were extracted from tigers originating in the Russian far east (Siberian, or Amur, tigers), south China, northern Indochina, the Malaya Peninsula, Sumatra, and the Indian subcontinent. The mtDNA analysis identified 30 haplotypes---characteristic regions on a chromosome---that could be clustered. Some of the clusters supported traditional classifications---e.g., for the Sumatran (P. t. sumatrae) and (P. t. tigris) Bengal tigers---but others suggested that the Indochinese subspecies (P. t. corbetti) should be divided into two groups, representing a northern Indochinese and a peninsular Malaya population (which the authors designated respectively as P. t. corbetti and P. t. jacksoni, after the tiger conservationist Peter Jackson). Interestingly, clusters for the captive South China tigers also fell into two distinct lineages---P. t. amoyensis, the traditional grouping, and P. t. corbetti, though the designation is still tentative. These subdivisions were largely supported by the other genetic analyses. The distinct genetic patterns found in the tiger populations suggest six rather than five living subspecies. Reduced gene flow and genetic drift in isolated populations, as well as human activity, likely caused these partitions. The low genetic variability seen in the Siberian tigers, for example, might be explained by severe population declines: the animals were nearly exterminated in the early 1900s, and today only 500 remain. Sumatran tigers, on the other hand, show relatively high genetic variability and uniqueness, possibly reflecting a historically large breeding population that was later isolated. Whether recent population and habitat declines, as opposed to earlier events, can fully explain these patterns is not clear. But these results offer valuable data for conservation strategies and captive breeding programs that rely on distinctions in subspecies taxonomy and geographic provenance. Evoking both the darker side of creation and humanity, Blake could not have imagined the modern fate of his "Tyger." Scholars have long debated the multilayered meaning of his poem, including the second stanza, which starts, "In what distant deeps or skiesBurnt the fire of thine eyes?" Will we reduce future generations to a literal reading?	PubMed Central	2024-06-05T03:55:49.995113	2004-12-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534812/", "journal": "PLoS Biol. 2004 Dec 7; 2(12):e453", "authors": [] }
PMC534813	Introduction {#s1} ============ The two orangutan species, Pongo abelii in Sumatra and Pongo pygmaeus in Borneo, are threatened with extinction in the near future \[[@pbio-0030003-b01],[@pbio-0030003-b02]\]. A prerequisite for conserving great apes in their natural habitat is good knowledge of population distribution, density, and size. However, precise information is still lacking for many orangutan populations living in Borneo, hindering the design of wise strategies for their long-term conservation \[[@pbio-0030003-b03]\]. Densities of orangutans and other great apes are usually estimated from nest censuses along ground line transects \[[@pbio-0030003-b04],[@pbio-0030003-b05]\]. In order to obtain final estimates of great ape population sizes, these densities are extrapolated to large forest blocks identified from maps as being "suitable habitat" for apes. In most surveys, however, the size of the area actually sampled is very small, and the estimates may not be representative of the population status and/or the variety of habitats and human disturbances (such as logging or mining) existing in the entire range of the population \[[@pbio-0030003-b06]\]. In addition, recent land-use changes (such as poaching), and ecological catastrophes (such as those caused by El Niño) or disease outbreaks do not appear in published maps \[[@pbio-0030003-b01]\]. The latest estimates available for orangutan populations in the Malaysian state of Sabah (North Borneo) range from 20,000 \[[@pbio-0030003-b07]\] to less than 2,000 orangutans \[[@pbio-0030003-b01]\]. Recent land transformation renders these estimates out-of-date \[[@pbio-0030003-b08]\], and in order to gain precise, up-to-date information, we developed an aerial methodology to assess the entire range of the species in the state precisely. Although some preliminary work using orangutan nest counting from a helicopter was conducted in the past in Sabah and Sarawak \[[@pbio-0030003-b09]\], this is the first time that aerial surveys have been used to determine population estimates for a great ape species at a state level. This methodology is likely to be useful for documenting the status of great ape populations living in fragmented and exploited forests in Asia and possibly in some parts of Africa. Results {#s3} ======= Correlation between Ground and Aerial Nest Densities {#s3a} ---------------------------------------------------- Ground densities estimated with Distance 3.5 and aerial densities predicted with our model are given in [Table 1](#pbio-0030003-t001){ref-type="table"}. Ground and aerial densities showed a positive correlation with data recorded by the first observer (R^2^ = 0.86, n = 13, p \< 0.001), by the second observer (R^2^ = 0.70, n = 13, p \< 0.001), by both observers (R^2^ = 0.58, n = 26, p \< 0.001), and with the average value obtained for both observers at each site (R^2^ = 0.83, n = 13, p \< 0.001). ::: {#pbio-0030003-t001 .table-wrap} Table 1 ::: {.caption} ###### Estimated Ground and Aerial Orangutan Nest Densities (Number of Nests/km^2^) at 13 Different Sites Surveyed during the Orangutan General Census of Sabah ::: ![](pbio.0030003.t001) LKWS, Lower Kinabatangan Wildlife Sanctuary; FR: forest reserve ::: Orangutan Distribution in Sabah {#s3b} ------------------------------- We recorded 2,708 orangutan nests during ground surveys (225 km of line transects and 300 km of recce walks) and 6,936 nests from the helicopter (1,963 km of aerial lines). The size of the sampling areas ranged from 0.001% to 1% (ground survey) and from 1.8% to 16.9% (helicopter survey, assuming an average strip width of 300 m) of the total size of each forest surveyed ([Table 2](#pbio-0030003-t002){ref-type="table"}). ::: {#pbio-0030003-t002 .table-wrap} Table 2 ::: {.caption} ###### Area Name and Size of Habitat Occupied by Orangutans, Aerial Indexes, Nest and Orangutan Densities, and Final Population-Size Estimates for the 16 Major Orangutan Populations Identified during the Surveys in Sabah, Malaysia, Borneo ::: ![](pbio.0030003.t002) Old: exploitation older than 15 y; recent: exploitation less than 15 y; active: ongoing exploitation (less than 1 y) ^a^Data from Payne, 1987 \[9\] ^b^Data from SWD ^c^Data from Ancrenaz et al. 2004 \[6\] ^d^Confidence intervals are obtained by bootstrapping CL, conventional logging; DLDF: dry lowland dipterocarp forest (\<500 m asl); HDF: hill dipterocarp forest (500--1,000 m asl); LMF: lower mountain forest (1,000--1,500 m); n.a.: not available; NP: nonprotected; OU: orangutan; P: protected; SF: swamp forest; SIMLDF: semi-inundated mixed lowland dipterocarp forest (\<500 m); SL: sustainable logging; UMF: upper mountain forest (\>1,500 m); UBF: ultrabasic forest DOI: 10.1371/journal.pbio.0030003.t002 ::: Our surveys confirmed that orangutans were patchily distributed throughout their range in Sabah \[[@pbio-0030003-b07]\], occurring mainly in the eastern and central parts of the state ([Figure 1](#pbio-0030003-g001){ref-type="fig"}). Only two significant small and isolated populations were found in the western and northern parts of the state, in Crocker Range National Park (population 7) and Mount Kinabalu National Park (population 1; see [Table 2](#pbio-0030003-t002){ref-type="table"} and [Figure 1](#pbio-0030003-g001){ref-type="fig"}). ::: {#pbio-0030003-g001 .fig} Figure 1 ::: {.caption} ###### Distribution and Size of the 16 Major Orangutan Populations Identified during the Surveys in Sabah, Malaysia, Borneo ::: ![](pbio.0030003.g001) ::: The highest nest abundances were recorded in lowland forests below 300 m asl, although we recorded a few nests as high as 1,300 m asl, which appeared to be the upper altitudinal limit for the species in Sabah. The highest orangutan densities (more than six individuals/km^2^ locally) were identified in the semi-inundated lowland forests of Kinabatangan (population 13) and Segama floodplains (population 16), Kulamba (population 12), and Tabin (population 14). Most of these forests were highly disturbed, fragmented, and located at the edge of newly established oil palm plantations. Extensive areas of dry lowland dipterocarp forests found in the commercial forest reserves located in the central parts of Sabah (populations 11, 15, and 16) yielded higher orangutan densities in old exploited areas and in areas that were exploited under sustainable logging practices (1.2--2.7 individuals/km^2^, n = 4) than in areas where more conventional practices were implemented (0.1--2.0 individuals/km^2^, n = 11): Mann--Whitney U test, U = 5.5, p = 0.03. Hunting pressure was low in all these forests (\[[@pbio-0030003-b07]\]; Kinabatangan Orangutan Conservation Project \[KOCP\], unpublished data). Orangutan Numbers in Sabah {#s3c} -------------------------- Our surveys showed that about 11,000 orangutans (95% confidence interval: 8,000 to 18,000) were present in Sabah at the time of our surveys ([Table 2](#pbio-0030003-t002){ref-type="table"}). Two major orangutan populations were found in logged commercial forest reserves: the Segama forests (population 16, included within the Sabah Foundation forest concession) with about 4,500 individuals, and on the north side of the upper Kinabatangan River (population 15) with about 1,700 individuals (see [Table 2](#pbio-0030003-t002){ref-type="table"} and [Figure 1](#pbio-0030003-g001){ref-type="fig"}). Four significant populations occurred in isolated protected areas: Tabin Wildlife Reserve (population 14; about 1,400 individuals), Kinabatangan Wildlife Sanctuary (population 13; 1,100 individuals), Kulamba Wildlife Reserve (population 12; 500 individuals), Danum Valley Conservation Area (part of population 16; 500 individuals). The remaining populations were of smaller size, scattered, and isolated. Discussion {#s4} ========== Aerial surveys are widely used for estimating animal abundance and population trends in open and semi-open landscapes \[[@pbio-0030003-b22]\]. In Sabah, we report that helicopters can also be used for a forest-dwelling species for (1) directly assessing orangutan distribution, and (2) estimating orangutan population size if aerial surveys are conducted in conjunction with a precalibrating stage based on ground-nest surveys. Aerial nest counts increase the size of the sampling areas significantly, provide a way to survey remote areas that are not accessible from the ground, are faster, and require a lower human investment than classical ground censuses. Nest detectability from the helicopter depends on observers and canopy structure. Ideally, specific models for deriving nest densities from aerial indexes (number of nests detected per kilometer of flight) should be designed for different human observation skills and for different habitat types. However, observer bias can be avoided if the same team of skilled people conducts the entire survey. The second source of bias could be overcome with the design of several habitat-specific models. Before these types of models are designed, ground-truthing must be conducted in different habitat types in order to validate a baseline model and to determine habitat-specific correction factors when necessary. Nest parameters used for obtaining the final orangutan density estimates (nest decay rate, daily rate of nest construction) are a major source of inaccuracy in aerial and ground nest surveys \[[@pbio-0030003-b23]\], and there is a need to investigate interpopulation differences in nest life-span estimates further to produce more precise estimates of orangutan densities \[[@pbio-0030003-b18]\]. Our survey shows that there are currently about 11,000 orangutans present in Sabah, making the state the main stronghold for the P. p. morio subspecies \[[@pbio-0030003-b24]\]. However, this represents a minimum 35% decline over the past 20 years \[[@pbio-0030003-b07]\]. This decline is mainly due to habitat loss resulting from the recent conversion of extensive tracts of lowland forests to agriculture \[[@pbio-0030003-b01],[@pbio-0030003-b08]\]. The current network of protected areas in Sabah harbors about 4,000 orangutans, representing about 40% of the total number found in the state. About 60% of the total number of orangutans survives in commercial forest reserves subjected to timber extraction, and these forests harbor the largest unfragmented population of the subspecies P. p. morio found in Borneo (population 16). The impacts of forest exploitation on ape abundance and ecology depend on several factors, such as (1) the forest types that existed initially and the quality of the regrowth forest \[[@pbio-0030003-b25]\], (2) type of habitat exploitation \[[@pbio-0030003-b26],[@pbio-0030003-b27]\], (3) hunting pressure \[[@pbio-0030003-b28]\], and (4) species ecology \[[@pbio-0030003-b29]\]. Our results tend to indicate that the mosaic of habitats found in the semi-inundated mixed dipterocarp forests that were originally occurring in the floodplains of east Sabah could potentially still harbor a significant number of animals following high disturbance levels (populations 12, 13, and 14). However, we can assume that the very high orangutan densities documented in some of the areas located close to oil palm plantations partly result from the influx of newcomers following recent land conversion to agriculture \[[@pbio-0030003-b07],[@pbio-0030003-b30]\]. We can also assume that the response of the forests to logging will directly impact the susceptibility of orangutans to habitat exploitation \[[@pbio-0030003-b07]\]. Less diverse habitats (dry lowland dipterocarp forests) located in the interior of the state appear to maintain fewer orangutans, particularly following conventional, nonsustainable logging practices (populations 15 and 16). In the extensive tracts of dry lowland dipterocarp forests exploited for timber (populations 15 and 16), our data suggest that, when hunting pressure is low, orangutan abundance is directly related to the degree of logging and associated damage. For these two populations, the highest orangutan densities were identified in Deramakot, a commercial forest reserve (part of population 15) implementing sustainable logging practices \[[@pbio-0030003-b10]\], suggesting that more conventional, uncontrolled logging activities have a negative impact on orangutan abundance. Possible inter- and intraspecific differences in general ecology and feeding behavior of orangutans may also influence population responses to habitat disturbances \[[@pbio-0030003-b31]\], and the results documented in Sabah for P.p. morio are not necessarily valid for other Bornean orangutan subspecies and for the Sumatran species \[[@pbio-0030003-b32],[@pbio-0030003-b33],[@pbio-0030003-b34],[@pbio-0030003-b35]\]. All great ape species require large forest areas to survive. An ecological network combining protected areas with seminatural landscape elements and production forests could be seen as an option to conserve biodiversity, while also providing opportunities for the sustainable use of natural resources \[[@pbio-0030003-b36],[@pbio-0030003-b37]\]. However, there is a need for in-depth field studies investigating further the impacts of logging and associated human activities (such as illegal killing) on great ape ecology and survival in order to assess the role of nonprotected forests for ape conservation. Finally, aerial nest surveys may also be of use in Africa, although it may be difficult to detect nests of African great apes from a helicopter because they tend to be lower in the canopy and it may be impossible to distinguish between gorilla and chimpanzee nests in those areas where the two species are sympatric \[[@pbio-0030003-b04]\]. Materials and Methods {#s2} ===================== {#s2a} ### Study area: Sabah {#s2a1} Sabah covers about 76,000 km^2^ in the northern portion of the island of Borneo. It is one of the 13 states in the federation of Malaysia. Approximately half of the total land mass is covered with forests ([Figure 2](#pbio-0030003-g002){ref-type="fig"}). Commercial forest reserves are designated for timber extraction and represent 76% of all forests in Sabah \[[@pbio-0030003-b08]\]. Sustainable logging practices (proper forest management plan and precise extraction planning, selective and reduced-impact logging) are currently implemented in Deramakot Forest Reserve (part of population 15) and are in the process of being generalized to other commercial forest reserves where more conventional practices were still implemented in the recent past \[[@pbio-0030003-b10]\]. The remnant forests have various protection statuses, but most of them have been logged using conventional forestry practices at least once in the past ([Table 2](#pbio-0030003-t002){ref-type="table"}). ::: {#pbio-0030003-g002 .fig} Figure 2 ::: {.caption} ###### Location of Ground and Aerial Surveys during the Orangutan Census in Sabah, Malaysia, Borneo Aerial transects are not shown for the Lower Kinabatangan forests. ::: ![](pbio.0030003.g002) ::: ### Helicopter census. {#s2a2} All major forest blocks in Sabah were identified from 1:50,000 vegetation maps, and these were divided into 16 different blocks. For each block, we determined a systematic stratified sampling using equidistant parallel line transects, the location of the first line being randomly selected ([Figure 2](#pbio-0030003-g002){ref-type="fig"}). Because the specific topographical features (steep slopes and high altitudes) encountered over Crocker Range and Kinabalu National Parks prevented the helicopter from following a random pattern of transects, the location of our aerial lines followed valleys in these two blocks. Aerial censuses were carried out with a small-type Bell 206 Jet ranger helicopter. Helicopter speed and height were kept constant at about 70 km/h and 60--80 m above the forest canopy. The copilot recorded the precise flight path location with a Global Positioning System every 30 s and monitored altitude, forest type (semi-inundated vs dry), signs of human activities, and forest disturbance continuously. Four types of disturbances were distinguished during aerial surveys: (1) no disturbance: tall and large trees; rather closed canopy; no sign of human exploitation; (2) old exploited forests (timber extraction was conducted more than 15 y prior to the survey): logging roads and stamping areas colonized by pioneer tree species such as Macaranga sp. (crown shape and color easily distinguishable from other tree species), some emergent trees; (3) recently exploited forests (timber extraction was conducted less than 15 y ago): logging roads not entirely colonized by pioneer tree species; few emergent trees; (4): active exploitation: logging activities were ongoing at the time of the survey. From the back seats, two observers looked for orangutan nests from either side of the helicopter. All visible nests were recorded. It was impossible to estimate the impact of nest age on nest detection, and the observers acknowledged that a few days-old fresh nests (still green in color) and nests at their latest stages of decay (just a few branches visible) were difficult to detect in the canopy. These nest categories are likely to have been underdetected. The two observers indicated all sightings to a nest recorder seated between them. The nest recorder noted the number of nests detected by the observers per each 30-s period. All crew members were in constant radio contact during the flights. After the flight, data collected by the copilot and the nest recorder were matched in order to determine the location of all sightings along the aerial line transect precisely. The same team of observers conducted all aerial surveys in order to avoid the observer bias. For technical reasons, it was impossible to fit external devices to the helicopter to estimate the distance of the nests to the aerial transects. This prevented us from determining the detection function from our data alone \[[@pbio-0030003-b11]\]. Trailing tapes placed on the aircraft window limited the observers\' field of view to a strip of approximately 150 m wide on either side of the aircraft. However, fluctuations in canopy\'s height prevented the direct determination of the exact width of the sampling area. ### Ground censuses. {#s2a3} Because the proportion of the actual nest population existing in the forest that was detected from the helicopter was unknown, it was impossible to directly estimate nest densities from our aerial results \[[@pbio-0030003-b09]\]. We thus designed a calibration function relating nest density estimated from the ground to the number of nests detected per kilometer of flight (aerial nest index) by comparing the aerial results with results from extensive ground surveys carried out in 13 patches of old and recently disturbed forests located in the Kinabatangan Wildlife Sanctuary \[[@pbio-0030003-b06]\], and Deramakot, Kalumpang, and Segaliud forest reserves. Nest densities and their variances were estimated by ground line transects using distance sampling \[[@pbio-0030003-b11],[@pbio-0030003-b12]\]. A set of line transects was randomly selected and the perpendicular distance of each nest to the transect was carefully recorded \[[@pbio-0030003-b06]\]. Densities were computed using the software Distance 3.5 \[[@pbio-0030003-b13]\]. For each transect, the truncation level was set following identification of outliers from box plots (outliers being values higher than 1.5 box-lengths from the 75^th^ percentile). Heaping was assessed from histograms, and data were grouped where necessary \[[@pbio-0030003-b11],[@pbio-0030003-b14]\]. The probability of nest detection was estimated with models combining density functions (uniform, half-normal, and hazard-rate) with adjustments (cosine, simple, Hermite polynomials). The model with the lowest Akaike\'s Information Criterion was selected for each site \[[@pbio-0030003-b15]\]. The adequacy of the selected model to the perpendicular distances was assessed by a chi-square goodness-of-fit test on grouped data \[[@pbio-0030003-b11]\]. Finally, we estimated the variance of nest density using nonparametric bootstrapping to handle sources of variation, such as model selection uncertainty \[[@pbio-0030003-b11]\]. Results are given in [Table 1](#pbio-0030003-t001){ref-type="table"}, and are extensively described in \[[@pbio-0030003-b06]\]. ### Estimation of nest density from aerial indexes. {#s2a4} The calibration function relating absolute nest density to aerial nest index stipulated that the logarithm of the orangutan nest density D\^ was a linear function of both the logarithm of the aerial index AI and the observer effect obs, plus their interaction, in order to include possible differences between observers. We weighted the general regression by the estimated variances of ground nest densities, thus giving a greater emphasis to precise density estimates. The least squares method was used for model fitting by incorporating weights 1/σ\^~log(D\^~i~)~ where σ\^~log(D\^~i~)~ was the estimated standard error of the estimated nest density logarithm in forest area i, given by Then, assuming that the densities were log-normally distributed, the overall regression model was conveniently written with a matrix notation as where D\^ was a 26 × 1 vector of the orangutan nest densities (13 points per observers), and X was the matrix of covariates: β was a 4 × 1 vector of parameters to be estimated, and ɛ was a 26 × 1 vector of errors with multivariate normal distribution N ~26~(0,Σ) , where Σ was a 26 × 26 matrix with σ^2^ · diag(var(logD\^~i~)) in the diagonal and zeroes elsewhere. To simplify, (2) was D\^ rewritten using the quantities log (D\^)~w~ = W · log(D\^), X~w~ = W · X, and ɛ~w~ = W · ɛ, with W = diag(σ\^^−1^ ~log(D\^~i~)~ as Unlike ɛ, ɛ~w~ has a more familiar distribution N ~26~(0, σ^2^·I ~26~), allowing the use of linear regression tools to estimate model parameters via least square theory. We used the backward model selection procedure \[[@pbio-0030003-b15]\] to select between models. The first regression model to be tested included all covariates. Covariates with the highest p value and greater than a 10% cutoff were then removed one by one, and each new model was retested until all p values of the remaining covariates were less than the cutoff value. We assessed the goodness of fit of the best model by computing the coefficient of determination R^2^. The best model supported by the data considered only the aerial index effect (R^2^ = 0.9587, F ^23^ ~2,0.05~ = 3.42 , p \< 0.001, on the logarithmic scale) with Using this model (5), we predicted an orangutan nest density from any new aerial index values, AI~0~, recorded during helicopter flights, as ([Figure 3](#pbio-0030003-g003){ref-type="fig"}). This model was applied to all the forests that were surveyed only by helicopter. A 95% confidence interval for the predicted orangutan nest density was built up on the logarithmic scale as ::: {#pbio-0030003-g003 .fig} Figure 3 ::: {.caption} ###### Graph Showing the Predicted Orangutan Nest Density as a Function of Aerial Indexes The plain line is the fitted line via model (5), and dashed lines are prediction intervals; n = 13 sites, 2 observers. ::: ![](pbio.0030003.g003) ::: with and X, the matrix defined above, once the observer effect had been removed, s ^2^ the residual mean square up to a constant that was an estimate of σ^2^, and t ~23,0.025~ the appropriate two-sided t-distribution percentile \[[@pbio-0030003-b16]\]. Following \[[@pbio-0030003-b11]\], this interval was then back-transformed to obtain a final confidence interval for the predicted orangutan nest density as where and A numerical application gave These intervals are shown in [Figure 3](#pbio-0030003-g003){ref-type="fig"}. ### Correction factors and habitat types. {#s2a5} Model (5) was obtained with results from the old and recently exploited semi-inundated mixed lowland dipterocarp forests of Kinabatangan. We tested its validity by comparing nest densities predicted from aerial data and estimated from ground line transects at several sites. We found no significant differences for five sites of old and recently exploited dry lowland dipterocarp forests (t-test, n = 5, t = −1.738, p = 0.157; 95% confidence interval of the difference: −110 to 25; ratio between ground and aerial nest densities = 0.94). This result showed that nest detectability was similar in forests that had been exploited for timber in Sabah and in Kinabatangan. Thus, we used the baseline model without any correction for all recent and old exploited forests of the state. Exploited swamp forests had a very open canopy, and predicted aerial nest densities were higher than estimated ground densities, although the difference was not significant (t-test, n = 3, t = −3.331, p = 0.08; 95% confidence interval of the difference: −384 to 49; ratio = 0.54). However, in order to not overestimate the final densities, we applied a correction factor of 0.54 to aerial indexes obtained in two areas of extensive exploited swamp forests (parts of populations 12 and 14). In primary lowland dipterocarp forest, the predicted aerial nest density was lower than the estimated ground density in the only site that was tested (n = 1; 392 nests/km^2^ vs 592 nests/km^2^; ratio = 1.5). Aerial indexes obtained for Danum (part of population 16), the only site of primary lowland forest with a significant orangutan population assessed during our survey, were multiplied by a correction factor of 1.5. ### Estimation of orangutan density from nest density. {#s2a6} The actual orangutan density D\^~ou~ was estimated using with D\^^\^ ~O~ the predicted nest density, p\^ the estimated proportion of nest builders, t\^ the estimated nest decay rate, and r\^ the estimated daily rate of nest production \[[@pbio-0030003-b04]\]. The proportion of nest builders has been estimated as 0.9 for orangutans \[[@pbio-0030003-b05],[@pbio-0030003-b17],[@pbio-0030003-b18]\]. The daily rate of nest production is currently available for only two Bornean orangutan populations: 1.005 in Kinabatangan \[[@pbio-0030003-b18]\] and 1.163 in Gunung Palung \[[@pbio-0030003-b17]\]. In order to take into account interpopulation variability in orangutan nesting behavior and to obtain more conservative estimates of orangutan densities in Sabah, we used an average value of 1.084 for our survey (with an associated coefficient of variation of 0.063). Nest decay rate varies with forest type \[[@pbio-0030003-b05],[@pbio-0030003-b18]\], and the most reliable estimates are obtained via direct monitoring of the survival of a sufficient number of nests \[[@pbio-0030003-b19]\]. Such estimates for t\^ are available for only two sites in Borneo: Gunung Palung, with 399 d and 258 d in mixed semi-inundated lowland and dry lowland forests, respectively \[[@pbio-0030003-b17]\]; and Kinabatangan with 202 d \[[@pbio-0030003-b18]\]. Since specific nest decay rates were not available for the different forests surveyed in Sabah, we considered an average t\^ value of 286.3 d (coefficient of variation: 0.373). Using the δ-method \[[@pbio-0030003-b20]\], a 95% confidence interval for the estimated D\^~ou~ was built up as with cv as the coefficient of variation and the other quantities as already defined (see above). This interval was then back-transformed to obtain a confidence interval for D\^~ou~ as where The numerical application gave with ### Estimation of orangutan population size. {#s2a7} The results of the ground and aerial surveys were processed with a geographic information system (Arcview 4.1; ESRI, Redlands, California, United States), using a combination of administrative maps and satellite images. When necessary, we stratified each forest block according to (1) disturbance type: no disturbance, old or recently exploited forests, ongoing exploitation; (2) altitude: lowland, below 500 m above sea level (asl); upper land, 500--1,000 m asl; lower mountain, 1,000--1,500 m asl; mountain, above 1,500 m asl; and (3) habitat type: swamp forests, semi-inundated mixed lowland dipterocarp forests, dry lowland dipterocarp forests. We then determined the percentage of habitat actually occupied by orangutans as the ratio between the total length of aerial transects and the length flown over areas with no visible orangutan nests (large areas with no trees, such as grasslands, large forest gaps, rivers, and oxbow lakes). This percentage was applied to the total size of each forest block determined from maps in order to estimate the final size of "habitat occupied by orangutans." We then multiplied the estimated orangutan densities by the estimated size of orangutan habitat occupation to obtain overall population estimates. A confidence interval for the population estimate was computed via parametric bootstrapping for the whole population in Sabah \[[@pbio-0030003-b21]\]. We assumed a normal distribution for population sizes extracted from the literature, and we assumed a log-normal distribution for other populations, with parameters given by formula (4). Values were sampled from their appropriate distribution and summed to obtain the whole population size. We repeated these two steps 1,000 times to obtain the bounds of a 95% bootstrapped confidence interval as the 25th and 975th largest values. We adopted a similar procedure for the subpopulations constituting populations 9, 11, 15, and 16. ### Survey efforts. {#s2a8} Over a 2-y period (2002--2003), ground surveys (using a combination of line transects and recce walks for a total effort of 1,100 "man days" of fieldwork) and aerial surveys (72 "man days") were conducted in all major forests of the state ([Figure 2](#pbio-0030003-g002){ref-type="fig"}). Recce walks were conducted to assess the presence/absence status of orangutans in areas with harsh topographical features or with extremely low orangutan abundance. Results from recce walks were not used to estimate nest densities. This general orangutan census in Sabah would not have been possible without the support of the Economic Planning Unit and the Sabah Wildlife Department (SWD). In particular we would like to express our sincere thanks to Sampoladon, Edward, Francis, Peter, and all the staff of the SWD who were involved in the census. We also would like to thank S. Buckland for his help in designing the statistical model and World Wildlife Fund (WWF)-Malaysia, Danish International Development Agency (DANIDA), and Japan International Cooperation Agency--Borneo Biodiversity and Ecosystems Conservation Programme for providing the base maps of Sabah. For their comments on different versions of the manuscript, we would like to thank J. Setchell, S. Jensen, E. Meijaard, and the reviewers who worked on this paper. The orangutan surveys in Sabah were funded by several sponsors that support the activities of the KOCP: Apenheul Zoo, Balikpapan Orangutan Society-USA, Chester Zoo, Cleveland Metroparks Zoo, Columbus Zoo and Aquarium, DANIDA, Darwin Initiative-UK, Disney Conservation Fund, Great Apes Survival Project-Australia, Palmyre Zoo, Pittsburgh Zoo, U.S. Fish and Wildlife Service, WWF-Malaysia, WWF-Netherlands, WWF-USA, WWF-UK, Zooparc de Beauval, and several other donors. Finally this work could not have been completed without the dedication of the 35 KOCP research assistants who are devoted to the preservation of orangutans and their natural habitat in Sabah. Conflicts of interest.* The authors have declared that no conflicts of interest exist. Author contributions. MA, JP, AT, and ILA conceived and designed the experiments. MA, BG, and AS performed the experiments. MA, OG, and KA analyzed the data. LA took part in the general design of the surveys and obtained the necessary authorization to conduct helicopter flights. PA obtained the authorizations to conduct these surveys in Sabah. MA, JP, and ILA contributed reagents/materials/analysis tools. MA, OG, and BG wrote the paper. Note Added in Proof The version of this paper that was first made available on 7 December 2004 has been replaced by this, the definitive, version. Citation: Ancrenaz M, Gimenez O, Ambu L, Ancrenaz K, Andau P, et al. (2004) Aerial surveys give new estimates for orangutans in Sabah, Malaysia. PLoS Biol 3(1): e3. asl : above sea level KOCP : Kinabatangan Orang-utan Conservation Project	PubMed Central	2024-06-05T03:55:49.995818	2004-12-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534813/", "journal": "PLoS Biol. 2005 Jan 7; 3(1):e3", "authors": [ { "first": "Marc", "last": "Ancrenaz" }, { "first": "Olivier", "last": "Gimenez" }, { "first": "Laurentius", "last": "Ambu" }, { "first": "Karine", "last": "Ancrenaz" }, { "first": "Patrick", "last": "Andau" }, { "first": "Benoît", "last": "Goossens" }, { "first": "John", "last": "Payne" }, { "first": "Azri", "last": "Sawang" }, { "first": "Augustine", "last": "Tuuga" }, { "first": "Isabelle", "last": "Lackman-Ancrenaz" } ] }
PMC534815	After apprenticing at her mother\'s side for some eight years---the first three clinging to her body---an orangutan is ready to make her own way in the forest canopy. The only great ape specializing in arboreal living, orangutans forage the treetops mostly for fruit, nuts, insects, leaves, and tree bark. They can recognize hundreds of species of edible fruit from trees and woody climbers and remember their location and fruiting season. Malay legend says orangutans (a variation on the Malay trope for "man of the woods") derived from humans who sought refuge from their species in the wilds of the forest. Today, with the last orangutan refuges shrinking drastically, the man of the woods has nowhere else to go. Hundreds of thousands of orangutans once ranged throughout southeast Asia. Now just two orangutan species inhabit just two countries: Indonesia (Kalimantan, the southern part of Borneo and Sumatra Island) and Malaysia (Sabah and Sarawak, in northern Borneo). The Sumatran orangutan is listed as critically endangered; the Bornean, endangered. In Indonesian Borneo and Sumatra, logging operations clear an estimated 5 to 6 million forest acres a year, leaving the apes stranded in isolated stands of trees and the normally fire-resistant rainforest at sudden risk. Another force driving orangutan extinction in Indonesia is the poaching and illegal killing (mothers do not give up their babies without a fight) that secures orangutan babies for the exotic pet trade. In Sabah, Malaysia, the primary threat comes from clearing the forest for agriculture. Conservation efforts depend, among other things, on having reliable data on population size, density, and distribution, but estimates of orangutan numbers in Sabah---which range from 2,000 to 20,000---are outdated. In a new study, Marc Ancrenaz and colleagues report an innovative method of directly estimating orangutan numbers from the number of nests detected during aerial surveys. (Orangutans are tough to spot directly, so researchers count the nests they sleep in at night.) Their survey, which covered the entire orangutan range throughout Sabah, estimates a total population of 11,000 orangutans---a drop of 35% in the past 20 years, based on a 1988 World Wildlife Federation report. Counting orangutans from the ground can be very time-consuming, difficult work, especially when faced with the hip-deep muck and steep slopes of the rainforest floor. Though helicopters obviously cover greater distance and more remote territory than is possible by foot, they\'re generally used to survey animals in more open landscapes. By using ground survey data to refine their aerial survey results, Ancrenaz and colleagues could directly assess the distribution and size of orangutan populations throughout Sabah. (Sabah covers roughly 72,000 square kilometers.) Over the course of two years, ground surveys---requiring 1,100 hours of field work---and aerial surveys---requiring just 72 hours---were conducted throughout all the major forests of Sabah. Commercial logging occurs in about 76% of all Sabah forests in commercial forest reserves. During the overflights, information was recorded on altitude, forest type, forest disturbance (on a scale from none to active exploitation), and signs of human activity. The authors attribute the 35% decline in Sabah orangutan numbers primarily to habitat loss from agricultural development. The surveys revealed that lowland forests harbored the greatest density of nests and orangutans, with the densest populations found in several highly disturbed, fragmented forests along newly created palm oil plantations. These high orangutan densities could reflect an influx of refugees from recently destroyed forest habitat into areas that are still forested. In logged forests, higher population densities were found in old exploited or sustainably logged forests than in conventionally logged reserves. While the authors acknowledge the density estimates could be more precise---better measures of nest decay and construction rates are needed---their survey reveals crucial information on orangutan numbers and distribution. Most orangutans in Sabah, including those making up one of the largest unfragmented populations in Borneo, live outside protected areas, in commercially exploited forests. These results suggest that orangutans may adapt better to degraded forests than previously thought---provided illegal hunting and agricultural conversion are controlled. More field research will help quantify the impacts of human activity---from logging to stealing babies---on great ape ecology and survival, and determine whether exploited forests can support conservation. It may be, for example, that integrating agricultural fields with forested corridors could sustain orangutan populations over the long term. With time of the essence, these aerial surveys will speed that work, and help sustain orangutans\' refuge in the treetops, above their human relatives.	PubMed Central	2024-06-05T03:55:49.998883	2004-12-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC534815/", "journal": "PLoS Biol. 2005 Jan 7; 3(1):e22", "authors": [] }
PMC535337	Background ========== Snoring occurs when there is an obstruction to the free flow of air through the airways at the back of the mouth and nose. The prevalence of habitual snoring in children has been reported to vary between 3.2 and 11%. Infrequent snoring is present in 17--27% of all children \[[@B1]-[@B3]\]. A study of young Australian children (2--5 years old) found the prevalence of snoring to be 10.5% \[[@B4]\]. Approximately one third of children who snore regularly have obstructive sleep apnea syndrome (OSAS) \[[@B5]\]. A few studies have claimed that snoring in children can affect neurocognitive function, behaviour and blood pressure to some extent even in the absence of apnea \[[@B6],[@B7]\]. Thus, concerns about causes of snoring and prevention strategies for children have arisen among both professional medical workers and parents. Numerous risk factors for snoring and OSAS have been reported including enlarged adenoids and/or tonsils, obesity, allergies or other causes of nasal obstruction, and exposure to environmental tobacco smoke (ETS) \[[@B8]-[@B10]\]. However, there has been very little research on exposure to environmental irritants, other than ETS, as contributing factors for snoring in children. The home is the predominant environment for exposure to many environmental irritants such as allergens and air pollutants. We hypothesized that high levels of exposure to common indoor irritants including volatile organic compounds, formaldehyde and nitrogen dioxide, could increase the risk of snoring. The aim of this study, therefore, was to investigate domestic environmental factors associated with snoring in children. Methods ======= Study design ------------ Nine hundred and ninety-six (996) school children, aged between 4 and 12 years, were recruited from four primary schools within the Perth metropolitan area. Parents/guardians of the children completed a questionnaire related to respiratory health of their children and domestic environments. A sample of 88 children, aged 4--6 years, was then selected randomly to participate in an indoor air quality assessment of their domestic environments. Ethics approval was obtained from the Human Research Ethics Committee of Curtin University of Technology. Respiratory survey ------------------ The survey instrument adopted was taken from a questionnaire on respiratory health and indoor air quality \[[@B11]\]. Some questions related to respiratory symptoms and domestic environments have been modified in order to conform to the study objectives. The questionnaire included two parts: the first part covered questions related to children\'s health and demographic characteristics, the second part consisted of questions about the home environment. Several terms relevant to the study were defined as follows. Children who had asthma were classified as \"ever asthma\", while those reported having asthma attack or taking any asthma medication within the past 12 months were regarded as \"current asthma\". Children who had coughed up phlegm on most days over a period of three months were referred to having \"chronic productive cough. \"habitual snoring\" was defined as snoring more than 4 times per week, whereas \"infrequent snoring\" meant snoring less than 4 times per week. In this context, \"snoring\" included both \"habitual snoring\" and \"infrequent snoring\". The questionnaires were distributed to parents by school teachers and later collected from the classrooms. The survey was conducted between March and April 2002. A consent form was signed by each participating parent or guardian. The response rate was 62.5%. Domestic air pollutant assessment --------------------------------- A sample of 88 year one and pre-primary students was randomly selected from participants of the respiratory survey for domestic air pollutant monitoring. Among them, 34 (38.6%) children were snorers (20 habitual and 14 infrequent). Two home visits were subsequently carried out during the winter of 2002 and summer of 2003 to measure indoor volatile organic compounds (VOCs), formaldehyde and nitrogen dioxide levels. VOCs were collected in the living room by active sampling using charcoal sorbent tubes. The air-sampling rate was 1 L/min with sampling undertaken for 10 hours during daytime. The analyses were performed using a Perkin Elmer Autosystem XL gas chromatograph equipped with a flame ionization detector. Eleven common compounds were identified and quantified by comparing the retention times: benzene, chlorobenzene, 1,2-dichlorobenzene, 1,3-dichlorobenzene, 1,4-dichlorobenzene, ethylbenzene, styrene, toluene, m-xylene, o-xylene and p-xylene. Their total amount was expressed as total VOCs (TVOCs). Formaldehyde (HCHO) and nitrogen dioxide (NO~2~) were collected by a passive sampling method in both the living room and the child\'s bedroom for 24 hours. Formaldehyde was analyzed using high-performance liquid chromatography \[[@B12]\]. Nitrogen dioxide was analyzed by a photometric method \[[@B13]\]. The method utilized a Palmes diffusion tube, containing stainless steel screens coated with trithanolamine (TEA), which was used as an absorbent. The concentrations of NO~2~were measured based on the quantity of the nitrogen dioxide gas transferred through the tube to the absorbent by molecular diffusion during a given exposure period \[[@B14],[@B15]\]. Data entry and statistics analysis ---------------------------------- Preliminary data screening and cleaning were conducted prior to statistical analysis. Associations between the prevalence of snoring and environmental and geographic factors and respiratory symptoms were examined using Chi-square tests. Multivariate logistic regression analysis was undertaken to estimate the risk of snoring adjusting for possible confounders. Since the distributions of VOCs, HCHO and NO~2~were positively skewed, geometric means (GM) of these variables were calculated after applying a logarithmic transformation. All statistical analyses were performed using the SPSS package Version 10.0. Results ======= Of the 996 participants, 985 children (98.9%) had intact records for snoring. There were 248 children (24.9%; 95% CI: 21.2%--28.6%) reported infrequent snoring and 151 children (15.2%; 95% CI: 12.6%--17.8%) suffered from habitual snoring. Snoring by age and gender ------------------------- Table [1](#T1){ref-type="table"} shows the prevalence of infrequent and habitual snoring by age and gender. Boys had a slightly higher rate of snoring than girls, but the difference was not statistically significant. The rates of habitual snoring decreased significantly with age (P = 0.03). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Prevalence of infrequent and habitual snoring by age and gender ::: Gender -------------------- ------------ -------------- ------------ ------ -------- ------ -------- Infrequent snoring 130 25.9 118 24.4 \>0.05 Habitual snoring 82 16.3 69 14.3 \>0.05 Age \< 7 years 7 -- 9 years \> 9 years P n \% n \% n \% Infrequent snoring 77 26.6 70 25.5 100 24.0 \>0.05 Habitual snoring 58 20.1 42 15.3 51 12.2 0.03 ::: The prevalences of respiratory symptoms, asthma and other allergic conditions were significantly different among non-snoring, infrequent snoring, and habitual snoring children, with habitual snorers having the highest rates. Results are presented in Table [2](#T2){ref-type="table"}. A significant association (P \< 0.001) was evident between snoring and respiratory symptoms, asthma and other allergic conditions. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Snoring and respiratory symptoms, asthma and other allergic conditions ::: ------------------------------------------------------------------------------------------------------------------------- Non-snoring\ Infrequent snoring\ Habitual snoring\ (N = 586) (N = 248) (N = 151) ----------------------------------- -------------- --------------------- ------------------- ------ ---- ------ --------- Phlegm with a cold 145 24.7 97 39.1 66 44.0 \<0.001 Phlegm without a cold 38 6.5 31 12.5 25 16.6 \<0.001 Chronic productive cough 12 2.1 13 5.2 14 9.3 \<0.001 Wheeze during or after exercise 71 12.1 54 21.8 37 24.5 \<0.001 Wheeze without exercise 47 8.0 27 10.9 26 17.2 \<0.001 Any current wheeze 110 18.8 83 33.5 59 39.3 \<0.001 Dry cough at night without a cold 134 22.9 98 39.7 66 43.7 \<0.001 Ever asthma 140 23.9 86 34.7 56 37.1 \<0.001 Current asthma 83 14.3 62 25.0 36 24.2 \<0.001 Allergic rhinitis or hay fever 217 37.6 123 49.6 92 61.7 \<0.001 ------------------------------------------------------------------------------------------------------------------------- ::: Snoring and household characteristics ------------------------------------- Table [3](#T3){ref-type="table"} shows the proportion rates of various household characteristics. The snoring and non-snoring groups were similar in terms of \"gas cooking\", \"dampness at home\" and \"carpet in child\'s bedroom\". However, children suffering from infrequent snoring or habitual snoring were more likely to live in \"smoking\" households (P = 0.004). Children with pets at home seemed to be less likely to develop habitual snoring (P = 0.02). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Snoring and household characteristics ::: Non-snoring Infrequent snoring Habitual snoring ----------------------------- ----- ------------- -------------------- ------------------ ------ ----- ------ -------- Type of cooking Gas cooking 565 331 57.5 155 63.0 79 53.0 \>0.05 Electric cooking 221 142 24.7 43 17.5 36 24.2 \>0.05 Gas and electric cooking 185 103 17.9 48 19.5 34 22.8 \>0.05 Dampness at home Damp patch 85 49 8.5 25 10.2 11 7.5 \>0.05 Condensation 273 153 26.7 74 30.2 46 30.7 \>0.05 Mould 167 92 18.1 42 19.2 33 24.1 \>0.05 Other characteristics Carpet in child\'s bedroom 827 491 83.9 208 84.2 128 85.3 \>0.05 Smoking household 432 235 41.3 113 46.3 84 56.4 0.004 Pet at home 787 474 82.7 203 83.2 110 73.3 0.022 ::: To further investigate the impact of passive smoking and pet ownership on snoring, logistic regression analysis was undertaken, controlling for confounders age, gender, asthma and other allergic conditions. The results indicated that passive smoking increased the risk of habitual snoring significantly (OR = 1.77; 95% CI: 1.20--2.61), while having pets decreased the risk (OR = 0.58; 95% CI: 0.37--0.92). However, the corresponding effects of passive smoking and pet ownership on infrequent snoring were statistically not significant. The Hosmer-Lemeshow statistic confirmed adequacy of the fitted logistic regression model (P \> 0.10). Snoring and indoor pollutant exposure ------------------------------------- The levels of pollutants exposure were similar between houses of habitual snorers and houses of infrequent snorers. To facilitate analysis, data from the two groups were combined to improve statistical power for comparison with houses of the non-snoring children. Table [4](#T4){ref-type="table"} shows the pollutant measurements. The levels of TVOCs and HCHO were not significantly different between houses of snorers and non-snorers regardless of season. However, the geometric means of NO~2~concentration in the living rooms of snoring children were higher. In particular, the levels of NO~2~in snoring children\'s bedroom were significantly higher than those in non-snoring children\'s bedroom during winter. ::: {#T4 .table-wrap} Table 4 ::: {.caption} ###### Snoring and indoor pollutants ::: Houses of non-snoring children Houses of snoring children ------------- -------- -------------------------------- ---------------------------- ---- ----- ---- ---- ---- ----- -------- TVOCs Living room Summer 47 11 1 254 32 15 2 204 \>0.05 Winter 52 15 1 247 34 22 1 575 \>0.05 HCHO Living room Summer 48 7 ND 34 32 6 ND 26 \>0.05 Winter 51 15 2 92 33 19 ND 92 \>0.05 Bedroom Summer 48 9 ND 126 32 8 ND 47 \>0.05 Winter 49 16 2 84 33 18 2 98 \>0.05 NO~2~ Living room Summer 48 37 11 244 32 41 8 511 \>0.05 Winter 51 38 9 314 32 48 6 345 \>0.05 Bedroom Summer 48 32 6 293 32 31 6 199 \>0.05 Winter 50 33 6 267 32 56 8 511 0.015 ND = not detectable ^1^Missing data or lost to follow-up present ^2^Geometric mean of pollutant concentration (μg/m^3^) ::: Recognizing that the main source of indoor NO~2~could be a gas heater and/or gas cooker, we compared NO~2~concentration between houses with and without a gas heater in the child\'s bedroom. The results confirmed that NO~2~levels (GM: 49 μg/m^3^, 95% CI: 37--65 μg/m^3^) in houses with a gas heater were significantly higher than those (GM: 27 μg/m^3^, 95% CI: 20--38 μg/m^3^) recorded in houses without a gas heater. Logistic regression analysis was next conducted to assess the dose-response relationship between bedroom exposure to NO~2~during winter and snoring in children. Based on the empirical NO~2~distribution, the monitored households were classified as: \'low\' exposure (\<30 μg/m^3^), \'medium\' exposure (30 -- 60 μg/m^3^) and \'high\' exposure (\> 60 μg/m^3^). After adjusting for age, gender, asthma, passive smoking and pet ownership, domestic NO~2~exposure level was still positively associated with snoring, the ORs being 2.5 (95% CI: 0.7--8.7) for medium exposure and 4.5 (95% CI: 1.4--14.3) for high exposure. There was also evidence of a linear dose-response relationship (P = 0.011 for trend). Discussion ========== Snoring is an important symptom and major risk factor for obstructive sleep apnea \[[@B5]\]. Several studies in Italy and Thailand reported that the prevalence of habitual snoring varied from 4.9 to 34.5% in primary school children \[[@B5],[@B16],[@B17]\], while the prevalence of snoring was 10.5% according to a study of Australian children aged 2--5 years \[[@B4]\]. The present study found the prevalence of habitual snoring among primary school children in Perth was 15.2%, and 24.9% of the children had infrequent snoring. The total prevalence of snoring was 40.1%. That the participants had high rates of current asthma (18.7%) and allergy (44.0%) (allergic rhinitis or hay fever) may explain the apparently high snoring prevalence taking into account the link between snoring and asthma and allergy. The prevalence of snoring among older children was significantly lower than that of younger children. No significant difference in snoring prevalence between boys and girls was observed, which appeared to be consistent with the literature \[[@B16],[@B18]\]. Strong associations were also found between snoring and respiratory symptoms, asthma and other allergic conditions, as in previous studies \[[@B10],[@B19],[@B20]\]. In relation to the domestic environment, passive smoking was identified as a major risk factor for habitual snoring and consistent with other studies \[[@B10],[@B21]\]. An interesting finding was the observed inverse relationship between snoring and pet ownership. There is evidence in the literature suggesting that pet ownership in early life can protect against the development of allergic disease \[[@B22]\]. Although the protective effect of pet ownership on habitual snoring was significant after controlling for allergic diseases, the mechanism that led to a lower risk of snoring remains to be investigated. Unlike TVOCs and HCHO, it appears that domestic exposure to NO~2~was significantly associated with snoring. It should be remarked that the low exposure threshold was set below the annual value of 40 μg/m^3^recommended by WHO \[[@B23]\], whereas the high exposure cut off is higher than the guideline value. Our results suggested that high exposure to NO~2~could increase the risk of snoring by 4.5 times. A previous study reported that children aged 5--12 years had a 20% increased risk of respiratory symptoms and disease for each increase of 28.3 μg/m^3^in NO~2~concentrations (2-week average), when the weekly average concentrations were in the range 15--128 μg/m^3^or possibly higher \[[@B23]\]. Another study in Australia confirmed the link between NO~2~exposure from gas appliances and the prevalence of respiratory symptoms \[[@B24]\]. Our results also suggested that exposure to NO~2~was related to gas heating during winter. Although the effect of NO~2~exposure on snoring was significant even after adjustment for asthma, atopy and other confounding factors, caution must be taken when interpreting the NO~2~findings and further investigation is required before they can be generalized to the pediatric population at large. A limitation of this cross-sectional study is that only 9% of the study sample was monitored for environmental testing due to budget and other constraints. Nevertheless, this subgroup of children did not differ significantly from the whole sample or other populations of young children in Perth \[[@B25],[@B26]\] with respect to home environment, respiratory symptoms and atopy. Secondly, the causal effects of NO~2~could not be determined because the measurements of exposure and illness were taken at the same time. The assessment of snoring was retrospective in relation to the time of environmental monitoring. Moreover, the significant association between snoring and NO~2~exposure in winter may be attributed to NO~2~emission from gas heaters in conjunction with low ventilation during the winter season. As for potential mechanisms for this association, there is little in the literature that can directly explain how exposure to NO~2~might result in snoring. Although there is evidence to suggest that exposure to NO~2~is associated with development of allergic disease \[[@B27]\], the observed association between NO~2~exposure and snoring is independent of atopy. Snoring occurs due to upper airway obstruction during sleep. The obstruction commonly occurs at the level of the nasal turbinates as with anterior rhinitis or the nasopharynx due to adenoid hypertrophy. Exposure of airway epithelium in vitroresults in the release of inflammatory cytokines and adhesion molecules \[[@B28]\]. Therefore, it is possible that exposure to NO~2~increases upper airway inflammation, resulting in mucosal oedema and airway obstruction. Alternatively, upregulation of ICAM1 the primary ligand for rhinovirus \[[@B28]\] could increase the susceptibility to, or severity of upper respiratory tract infection, resulting in upper airway oedema and/or adenoid hypertrophy. Finally, it has been suggested that NO~2~increases lipid membrane fluidity \[[@B29]\] that in turn can alter receptor-ligand interactions. Thus NO~2~exposure might produce changes in cell-cell and cell-pathogen interactions that could result in altered upper airway physiology. Given the high prevalence of snoring in our population and the knowledge that snoring is a significant risk factor for obstructive sleep apnea, the mechanisms that might underpin the association between NO~2~exposure and snoring require further study. In conclusion, the present study shows that snoring is common among primary school children in Perth, and snoring is associated with other respiratory symptoms. Passive smoking increases the risk of snoring in children but pet ownership may decrease the risk. The level of nitrogen dioxide in domestic environment is positively associated with the prevalence of snoring in children. Authors\' contributions ======================= GZ, JS, KR, SS Study design, coordination and management GZ, JS, KR Field measurement and laboratory work AHL, GZ Data analysis and interpretation of results GZ, JS, KR, AHL, SS Preparation and revision of the manuscript Acknowledgements ================ The authors are grateful to Mr Paul Dubois who supervised the laboratory analysis. Thanks are also due to Dr. Franklin and three anonymous reviewers for their constructive comments and suggestions.	PubMed Central	2024-06-05T03:55:49.999804	2004-11-4	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC535337/", "journal": "Respir Res. 2004 Nov 4; 5(1):19", "authors": [ { "first": "Guicheng", "last": "Zhang" }, { "first": "Jeffery", "last": "Spickett" }, { "first": "Krassi", "last": "Rumchev" }, { "first": "Andy H", "last": "Lee" }, { "first": "Stephen", "last": "Stick" } ] }
PMC535338	Introduction ============ Conlon and Raff \[[@B1]\] recently stated that constant size distribution is maintained in a culture of proliferating rat Schwann cells without the need for any size checkpoint, in contrast to the situation in yeast. Before discussing their conclusion, it is important to consider their techniques and nomenclature. They used an electronic (Coulter) counter to measure the mean and variation of cell volume at intervals in whole cell populations. In fission yeast, the most detailed study to date involved the measurement of lengths (proportional to volume) of singlegrowing cells \[[@B2]\]. This is a vital matter that is not fully appreciated. There is a long history of size measurements on fission yeast and the involvement of size in the control of the cell cycle, see e.g. \[[@B3]-[@B5]\], but only recently has the term \"size control\" come into use \[[@B2],[@B6],[@B7]\]. Although convenient, the phrase is something of a cover-all, since the nature of \"size control\" varies in fission yeast and the term is unlikely to denote exactly the same phenomena in mammalian cells. Moreover, mammalian cells normally form tissues, where greater emphasis has to be placed on external factors. However, size control operates in all systems where it has been possible to examine individual cells through their cell cycle. Its nature is likely to be different in each, although this will only become clear when its molecular basis is understood \[[@B6]\]. Discussion ========== The \"evidence against size checkpoints\" in cultured Schwann cells: an assessment ---------------------------------------------------------------------------------- One of the two cases cited by Conlon and Raff as evidence for the lack of a size checkpoint is the slow change in size when cells are stimulated by fresh medium \[[@B1]\]. The reason we do not regard this as strong evidence comes from data on fission yeast. When either cycle time or total length extension is plotted as a function of birth length, the negative slope of the regression line is large in wild-type cells, implying that size control is \"strong\" and deviations from the average will be corrected within a single cycle \[[@B2]\]. But the slope can be less, as for example in diploids, and then the control becomes \"weak\", since deviations will not be corrected within one cycle. Without the support of single cell data, it is nevertheless possible to imagine the presence of a weak size control in mammalian cells, homologous to that in yeast, exerting a slow but important action over several cycles. But can such a weak size control be considered a checkpoint? This entails another semantic or philosophical problem, since \"checkpoint\" is as ambiguous as \"size control\". Those who regard very sharp responses (e.g. all or none) as a criterion would argue that a weak mechanism is not a checkpoint. However, since it fulfils its function by ensuring homeostasis in a cell population, we believe that a weak size control should also be considered a checkpoint. Another problem in \[[@B1]\] is the increasing size of quiescent cells blocked in S phase by aphidicolin and stimulated into growth. The mean cell volume increased linearly five-fold over 100 h. A similar if less marked increase was also found in total protein. In addition, the valuable observation was made that the rates of protein synthesis and degradation increased with size. It is almost certainly true that size-dependent synthesis and degradation would not result in a size-independent pattern of protein accumulation. In the simplest case, if the rates of both protein synthesis and degradation are linear functions of total protein (mass), protein content will show a size-dependent exponential dependence on time. Therefore, further measurements and analyses need to be carried out to resolve this discrepancy. Moreover, it follows from the hypothesis of Brooks \[[@B8]\] that, in the absence of size control, an exponential growth of cell volume results in a continuous increase in the dispersion of cell size at division; in contrast, linear growth would not increase the dispersion in consecutive cycles, and individual cell sizes would converge towards the mean after perturbations. To date, however, no experimental evidence has been published to show that a cell culture can maintain homeostasis by linear growth withouta size checkpoint. It is plausible that this might happen because of the general need for co-ordination between the cytoplasmic and chromosome cycles, but we are not convinced that linear growth on its own would meet this requirement. In the case of fission yeast, there is evidence that linear growth without a size checkpoint cannot maintain homeostasis in the culture (see below). Results of similar experiments with fission yeast ------------------------------------------------- Related observations from fission yeast show that normal size control is abolished in the type of block experiment used in \[[@B1]\]; however this might be apparent only after release. This was first shown by Fantes \[[@B3]\] and was later expanded by us \[[@B2]\]. The time-scale, however, was shorter than in mammalian cells. It is also true, as Conlon and Raff \[[@B1]\] point out, that the pattern of total protein and RNA content in several yeast cdcmutants was approximately exponential over a longer period after this type of block. This result comes from an early paper and depends on measurements of absolute amounts of protein and RNA/ml \[[@B5]\]. There is not much evidence from the far more accurate method of measuring synthesis rates by pulses of radioactive precursor, but such evidence as exists fails to support the conclusion. In the widely used mutant cdc2-33, the rate of protein synthesis reaches a plateau after 4 h \[[@B9]\]. Also, only fairly minor changes in protein and RNA synthesis rates occur over 7 h in the mutant cdc13\[[@B10]\]. Because linear growth was measured in mammalian cells during a long S phase block \[[@B1]\], we have another objection to make here. The fission yeast cell cycle can be considered as linear segments of volume growth with points (called rate change-points) where there are changes in growth rate \[[@B11]\], which may be partly associated with a gene-dosage effect \[[@B2]\]. If a similar mechanism operates in mammalian cells during the normal cycle, it could never be observed when replication is arrested. Genetic background and size control in fission yeast ---------------------------------------------------- There are other points about the fission yeast experiments that are relevant to size control, which is almost certainly affected by genetic background. The main rate change point (an effect of gene-dosage, see above) is shifted in position in weemutants (S phase is also shifted), but this size control is a strong one, albeit weaker in diploids \[[@B2]\]. There appears to be a different mechanism in the strange double mutant wee1-50 cdc25Δ\[[@B12]\]. It seems improbable that all these variations involve the same molecular mechanisms; rather, they suggest a variety of options in the nature and positions of size controls. But since we do not understand the molecular mechanisms involved, these matters remain to be resolved. Another point is that the normal rules of the cycle can be broken temporarily, but not for long. For example, mammalian cells can certainly grow without entering S phase \[[@B6]\]; and the very large cells of early embryos only develop a size control after a number of cycles \[[@B13]\]. Block and release protocols can be used to induce synchrony in cdcmutants of fission yeast, and size control is lost for some cycles (but not forever), as indicated by the lack of negative correlation between length extension and birth length \[[@B2],[@B3]\]. However, if size control has been permanently lost, as in the case of the double mutant wee1-50 rum1Δ, the cells lose viability after a few generations \[[@B14]\] despite the near-linearity of their length growth pattern \[[@B2]\]. According to the hypothesis of Brooks \[[@B8]\], cell size should be convergent in this case after some generations, leading to size homeostasis. However, the mathematical solution seems not to be applicable for the yeast cells, which rather die. Conclusions =========== To return to mammalian cells: our view is that the best way forward is to try to devise ways of following individual cells through the cycle. It is easy for us to say this, since we have had the great advantage of working with regular shaped yeast cells that stay still and grow steadily on agar. The mammalian cells situation is far more difficult, but there is reasonable hope that it can be solved by methods of tracking individual cells such as fibroblasts by a semi-automatic method and measuring their dry mass by interferometry \[[@B15]\]. The main problem with the technique of Conlon and Raff is that it cannot distinguish between a weak (but existing!) size control and its total lack. Very recently, similar experiments have been done with erythroblasts and fibroblasts from different vertebrates, and the results seem to suggest the existence of a strong size control in every case \[[@B16]\]. Our present knowledge that the existence of size control (either weak or strong) seems to be general, suggests that rat Schwann cells are probably not exceptions above the rule, but they rather have a weak size control in spite of the conclusions of Conlon and Raff \[[@B1]\]. To choose the correct interpretation between the two possible ones is a challenge for the future. Acknowledgements ================ This research was supported by the Hungarian Scientific Research Fund (OTKA F-034100) and the James S. McDonnell Foundation (21002050). AS is grateful to the Hungarian Academy of Sciences for a Janos Bolyai Research Fellowship.	PubMed Central	2024-06-05T03:55:50.002803	2004-11-16	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC535338/", "journal": "Theor Biol Med Model. 2004 Nov 16; 1:12", "authors": [ { "first": "Akos", "last": "Sveiczer" }, { "first": "Bela", "last": "Novak" }, { "first": "J Murdoch", "last": "Mitchison" } ] }
PMC535339	Introduction ============ Two classes of viral envelope proteins that mediate virion:cell fusion have been described. Class I and II fusion proteins (aka α-and β-penetrenes) are distinguished, in part, by the location of the \"fusion peptide,\" a cluster of hydrophobic and aromatic amino acids that appears critical for fusing viral and cell membranes. The fusion peptides of class I fusion proteins are located at or near the amino terminus, whereas fusion peptides of class II fusion proteins are internal. The overall structures of these two classes of viral fusions proteins are also distinct. Class I fusion proteins have a pair of extended α helices that are separated by sequences variable in length, but usually containing one or more dicysteine linkages. Several otherwise disparate viruses, including orthomyxoviruses, paramyxoviruses, retroviruses, arenaviruses, filoviruses and coronaviruses encode class I fusion proteins \[[@B1]-[@B4]\]. Class II fusion proteins are comprised mostly of antiparallel β sheets. The prototypic class II fusion protein is the E glycoprotein of tick-borne encephalitis virus (TBEV), a member of the genus flavivirus of the Flaviviridae family \[[@B5]\]. E possesses three β sheet domains (I-III). In the slightly curved rod-like configuration of the E protein present in the virion, the fusion peptide is located at the tip of domain II, the furthest point distal from the C-terminal transmembrane anchor. The virion configuration of envelope glycoprotein E1, the fusion protein of the Alphavirus Semliki Forest virus (SFV), demonstrates a remarkable fit to the scaffold of TBEV E \[[@B6]\]. E of dengue virus (DEN) and West Nile virus, medically important flaviviruses, also can be fit to the class II structure \[[@B7],[@B8]\]. Recent studies indicate that TBEV E, DEN E and SFV E1 undergo similar conformational changes upon exposure to low pH, as encountered during entry via endocytic vesicles, suggesting a common fusion mechanism \[[@B9]-[@B11]\]. Based on sequence similarities, it is likely that E1 of other Alphaviruses and E of other members of the flavivirus genus within the family Flaviviridae are also class II fusion proteins. Members of the two other genuses in the Flaviviridae, hepaciviruises and pestiviruses, appear on the basis of proteomics computational analyses to encode truncated class II fusion proteins \[[@B12]\]. The Bunyaviridae family of enveloped RNA viruses includes five disparate genuses. Orthobunyaviruses, phleboviruses, nairoviruses and tospoviruses are spread by insect vectors, whereas hantaviruses are spread by rodent vectors \[[@B13]\]. Members of each Bunyavirus genus include important human and animal pathogens, except the tospoviruses, whose members infect plants \[[@B14],[@B15]\]. The Bunyavirus genome consists of three single-stranded RNA segments. The envelope glycoproteins are encoded by the middle-sized segment (M) \[[@B16],[@B17]\]. Members of each genus encode two glycoproteins that are present on the virion surface, and designated Gn and Gc to refer to their location amino terminal or carboxyl terminal on the M encoded polyprotein. The M segments of orthobunyaviruses, phleboviruses, and tospoviruses have been shown to encode for \"nonstructural\" proteins (NSm). In the case of the orthobunyaviruses and phleboviruses, NSm is synthesized as part of the polyprotein, but in tospoviruses NSm is encoded via an ambisense strategy by a separate mRNA \[[@B18]\]. The identity and structure of Bunyavirus fusion protein(s) are unknown, though it is likely that Gn or Gc fulfills this role. Proteomics computational analyses suggest that Bunyavirus Gc, and similar proteins of Tenuiviruses and a group of Caenorhabditis elegansretroviruses, are class II viral fusion proteins (β-penetrenes). Materials and Methods ===================== Sequences --------- For sequence and structural comparisons of Bunyavirus M encoded proteins representatives of the five genuses were used, including pleboviruses Sandfly fever virus, Sicilian strain (SAN, accession number: AAA75043) and Rift Valley fever virus (RVF, P03518), orthobunyavirus Bunyamwera virus (BUN, NP047212), hantavirus Hantaan virus, strain 76--118 (HAN, P08668), nairovirus Crimean-Congo hemorrhagic fever virus, strain IbAr (CCHF, NP950235), and tospovirus tomato spotted wilt virus, ordinary strain (TSWV, NP049359). Additional phlebovirus M sequences compared included those of Uukuniemi virus (UUK, NP941979) and Punta Toro virus (PTV, VGVUPT). Bunyavirus M sequences were compared to sequences encoded in Alphavirus subgenomic RNA, including structural proteins of Sindbis virus (SIN, P03316), Semliki Forest virus (SFV, NP463458), Venezuelian equine encephalitis virus, strain TC-83 (VEE, P05674), Western equine encephalitis virus, strain McMillan (WEE, AAF60166), O\'nyong-nyong virus, strain GULU (ONN, P22056), Mayaro virus, strain TRVL4675 (MAY, AA033335), Barmah Forest virus strain BH2193 (BFV, AA033347) and Ross River virus, strain NB5092 (RRV, NP740686). Comparisons were also made with proteins encoded by RNA2 of the HZ isolate of Rice stripe virus, a Tenuivirus (pvc2, AA031607), and with certain retroviruses of Caenorhabditis elegans, including Cer13 (hypothetical protein Y75D11A.5, NP508324). We also compared Bunyavirus M sequences to structural proteins of Flaviviruses, including members of the flavivirus genus tick-borne encephalitis virus, strain Neudoerfl (TBEV, P14336); Japanese encephalitis virus, strain JaOARS982 (JEV, P32886), yellow fever virus, strain 17D-204 (YFV, P19901), dengue virus type 2, strain PR-159/S1 (DEB, P12823), and West Nile virus, strain NY 2000-crow3356 (WNV, AF404756). The prototype hepaciviruses, strain H (subtype 1a) of hepatitis C virus (HCV, P27958), and several pestiviruses, including the Alfort 187 strain of classical swine fever virus, aka hog cholera virus (CSFV, CAA61161), bovine viral diarrhea virus genotype 1 aka pestivirus type 1, stain NADL (BVDV, CAB91847) and border disease virus, strain BD31 (BVD, AAB37578), were used in other comparisons. Proteomics computational methods -------------------------------- Methods to derive general models of surface glycoproteins have been described previously \[[@B2]\]. PRSS3, a program derived from rdf2 \[[@B19]\], which uses the Smith-Waterman sequence alignment algorithm \[[@B20]\], was used to determine the significance of protein alignments. PRSS3 is part of the FASTA package of sequence analysis programs available by anonymous ftp from ftp.virginia.edu. Default settings for PRSS3 were used, including the blosum50 scoring matrix, gap opening penalty of 12, and gap extension penalty of 2. MacMolly (Soft Gene GmbH, Berlin) was used to locate areas of limited sequence similarity and to perform Chou-Fasman and Robson-Garnier analyses \[[@B21],[@B22]\]. PHDsec (Columbia University Bioinformatics Center, <http://cubic.bioc.columbia.edu/predictprotein/>) was the preferred method of secondary structure prediction \[[@B23]\]. PHDsec predicts secondary structure from multiple sequence alignments by a system of neural networks, and is rated at an expected average accuracy of 72% for three states, helix, strand and loop. Domains with significant propensity to form transmembrane helices were identified with TMpred (ExPASy, Swiss Institute of Bioinformatics, <http://www.ch.embnet.org/software/TMPRED_form.html>). TMpred is based on a statistical analysis of TMbase, a database of naturally occurring transmembrane glycoproteins \[[@B24]\]. Sequences with a propensity to partition into the lipid bilayer were identified with Membrane Protein eXplorer version 2.2a from the Stephen White laboratory using default settings \[[@B25]\]. The NetOglyc server <http://www.cbs.dtu.dk/services/NetOGlyc/> was used to predict mucin type GalNAc O-glycosylation sites. RasMac (University of California Regents/Modular CHEM Consortium, <http://mc2.cchem.berkeley.edu/Rasmol/v2.6/>), developed by Roger Sayle, was used to render a 3D model of SFV E1, which was extrapolated to SIN E1 and SAN GC. Results ======= Similar sequences or common structural/functional motifs are located collinearly in the carboxyl terminal glycoprotein of Sandfly fever virus and Sindbis virus envelope glycoprotein E1. Previously, Gallaher and coworkers modeled the structure of the retroviral transmembrane glycoprotein (TM) \[[@B2]\] onto the scaffold of the known structure of the HA2 portion of the influenza virus hemagglutinin \[[@B26]\]. Later, Gallaher \[[@B1]\] fit the fusion protein of Ebola virus, a filovirus, to retroviral TM. Both models proved remarkably similar to the structures of these fusion proteins solved later by X-ray crystallography \[[@B27]-[@B29]\]. These results indicate that Gallaher\'s \"Rosetta Stone\" strategy, which employs the fusion peptide and other identifiable features in combination with computer algorithms that predict secondary structure, is a useful approach to the construction of working models of class I viral fusion proteins. This approach, supplemented with newer proteomics computational tools, was applied to envelope glycoproteins encoded by members of the Bunyaviridae. Our initial finding, obtained using the PRSS3 alignment algorithm \[[@B20],[@B30]\], was that the amino acid (aa) sequence of Gc of Sandfly fever virus (SAN), a phlebovirus, has a significant similarity (p \< 0.002) with the aa sequence of E1, the fusion protein of Sindbis virus (SIN), an Alphavirus (Table [1](#T1){ref-type="table"}). SAN Gc also showed significant overall alignments with E1 of several other Alphaviruses examined, including Semliki Forest virus (SFV), Western equine encephalitis virus (WEE) and O\'nyong-nyong virus (ONN). The Gc proteins of the three other phleboviruses, Rift Valley fever virus (RVF), Uukuniemi virus (UUK) and Punta Toro virus (PTV), the only phleboviruses with completely sequenced Gc coding regions also showed significant sequence similarities to certain Alphavirus E1 proteins. The alignment of RVF Gc with SIN E1 and WEE E1 was statistically significant, but the alignment with SFV E1 was not. PTV Gc showed the highest overall aa sequence similarity with any Alphavirus E1 examined, that of Venezuelan equine encephalitis virus (VEE) (p \< 0.0004). UUK Gc showed a significant overall alignment only with Ross River virus (RRV) E1, while RRV E1 failed to align with any of the other three phlebovirus Gc examined. These results from multiple comparisons of phleboviruses Gc and Alphavirus E1 indicate that the significant alignment between SAN Gc and SIN E1 is not a statistical aberration, but may underlie structural and functional similarities between the two viral glycoproteins. It is also of interest that the PRSS3 sequence alignment tool permitted detection of similarities not detected than by the use of BLASTp or related computational methods. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Comparison of phlebovirus Gc with Alphavirus E1 using the PRSS3 sequence algorithm. ::: [Alphavirus E1]{.underline}^1^ ----- -------------------------------- ------ -------- -------- ------ ------ ------- ------- SAN 0.002 0.02 0.001 0.004 0.05 NS 0.03 0.007 RVF 0.04 NS 0.03 0.003 NS NS 0.04 NS PTV NS 0.04 0.0002 0.0004 NS NS NS^2^ NS UUK NS NS NS NS^3^ NS 0.05 NS NS ^1^Two-way comparisons were done between the full-length amino acid sequences of the indicated glycoproteins. Probabilities (p values) of a significant alignment are based on 1000 shuffles. SAN: Sandfly fever virus, RVF: Rift valley fever virus; PTV: Punta Toro virus; UUK: Uukuniemi virus; SIN: Sindbis virus; SFV: Semliki Forest virus; WEE: Western equine encephalitis virus; VEE: Venezuelan equine encephalitis virus; MAY: Mayaro virus; RRV: Ross River virus; BFV: Barmah Forest virus; ONN: O\'nyong-nyong virus. ^2^p = 0.08 ^3^p = 0.07 ::: Prior X-ray crystallographic studies have demonstrated that SFV E1 is a class II viral fusion protein (β-penetrene) \[[@B6]\]. Because of its extensive sequence similarity with SFV E1, SIN E1 is assumed to be a class II viral fusion protein \[[@B31]\]. The sequence similarities between SAN Gc and SIN E1 do not permit alignment by computational methods alone. The significance of the overall sequence similarity can, however, be attributed to three collinear similarity regions between the two glycoproteins detected by the PRSS3 algorithm (Fig. [1A](#F1){ref-type="fig"}). Beginning from the amino terminus, the first sequence similarity starts in β-sheet Do in domain Ia of SIN E1 and extends past β-sheet b in domain IIa. The alignment was significant (P \< 0.0002) between aa 889--928 of SAN Gc and aa 833--873 of SIN E1 (Fig. [1A](#F1){ref-type="fig"}). The next significant sequence similarity, aa 1022--1101 of SAN Gc and aa 946--1029 of SIN E1 (p \< 0.03), includes the SIN E1 sequence from β sheet Fo in domain Ib to β sheet i in domain IIb. The third region of similarity, SAN Gc aa 1181--1287 and SIN E1 aa 1112--1202 (p \< 0.003), includes most of SIN E1 domain III. In addition, two domains that are characteristic of class II viral fusion proteins are also apparent in SAN Gc. Prior studies have shown that aa 887--904 of SFV E1 are critically involved in virion:cell fusion, and indicate that this region includes the fusion peptide \[[@B32],[@B33]\]. The fusion peptide of SIN E1 is assumed to be located similarly \[[@B31]\]. The fusion peptides of the class II viral fusion proteins are located at the end of domain II, and consist predominantly of aromatic aa (usually phenylalanine \[F\] or tryptophan \[W\]), hydrophobic aa, and aa with high turn potential (glycine \[G\] and proline \[P\]). Cysteine linkages usually stabilize the fusion peptides of class II viral fusion proteins in the overall structure (Fig [1A](#F1){ref-type="fig"}, red). A sequence (aa 960--977) corresponding to a consensus class II fusion peptide is present in SAN Gc in a similar location to the SIN E1 fusion peptide (Fig. [1A](#F1){ref-type="fig"}). Another common domain of class II viral fusion proteins readily identifiable in SAN Gc is the carboxyl terminal transmembrane anchor. Rossmann and coworkers provided experimental evidence that SIN E1 aa 1215--1241 contains the transmembrane domain of SIN E1 \[[@B31]\]. A similar hydrophobic sequence is located near the carboxyl terminus of SAN Gc. TMpred, an algorithm that identifies possible transmembrane helices, assigns a significant score of 3048 (\>500 is statistically significant) to aa 1303--1322 of SAN Gc, which suggests that this is the transmembrane anchor of SAN Gc. Using the regions of local similarity and the fusion peptide and transmembrane domains, which are collinear, a proposed alignment between SAN Gc and SIN E1 can be constructed (Fig. [1B](#F1){ref-type="fig"}). The alignment necessitates only one \"insertion.\" Relative to domain IIb of SIN E1, it appears that SAN Gc, has an added sequence (aa 932--958), a proposed \"loop\" flanked by cysteines and containing two N-linked glycosylation sites (NXT/S) reminiscent of glycosylated loops of other viral envelope proteins \[[@B34]\]. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Colinear arrangement of similarities in Sindbis virus E1 and Sandfly fever virus Gc. Alignments were constructed as detailed in the text. Panel A: Linear arrangement of the domain structure of SIN E1 and proposed domain structure of SAN Gc according to the convention for class II viral fusion proteins (β-penetrenes) originally described for TBEV E by Rey et al. \[5\]. Regions of significant sequence similarities in SIN E1 and SAN Gc determined by the PRSS3 sequence alignment program are indicated. Probabilities (p values) are based on 1000 shuffles. Panel B: Amino acids are numbered from the beginning of the Sindbis virus subgenonic mRNA encoded polyprotein and the beginning of the SAN M segment encoded polyprotein. (:) refers to identical amino acids. (.) refers to chemically similar amino acids. Plum amino acids: N-glycosylation sites. Hydrophobic transmembrane domains were predicted using TMpred. Sequences with significant WWIHS scores were identified by MPeX (olive). In SAN Gc, predicted α-helices are indicated by dashed boxes and predicted β-sheets are underlined with a dashed arrow. ::: ![](1742-4682-1-10-1) ::: Membrane interfacial domains in Bunyavirus glycoproteins -------------------------------------------------------- To provide support for the proposed alignment of SAN Gc and SIN E1, another proteomics computational tool was used to compare potential membrane interactive domains in the glycoproteins. Besides fusion peptides, a motif that can be important in virus:cell fusion and is present in many class I and class II viral fusion proteins is an aromatic aa rich domain proximal to the transmembrane anchor \[[@B35]\]. The pre-anchor domains are not highly hydrophobic according to the Kyte-Doolittle hydropathy prediction algorithm, but have a tendency to disrupt and partition into bilayer membranes as revealed by analyses using the Wimley-White interfacial hydrophobicity scale (WWIHS) \[[@B35],[@B36]\]. SIN E1 contains a sequence prior to and overlapping the transmembrane anchor with a significant WWIHS score as determined by Membrane Protein eXplorer (MPeX) \[[@B25]\]. SAN Gc has two sequences with significant WWIHS scores in this region, the pre-anchor and the putative transmembrane domain. The fusion peptides of all class I and II viral fusion proteins examined to date overlap sequences with significant WWIHS scores (RFG, unpublished observation). The proposed fusion peptide in SAN Gc consists of a sequence with a significant WWIHS score, which further supports the assignment of this sequence as the fusion peptide. Additional sequences with significant WWIHS scores are located collinearly along SAN Gc and SIN E1. In total, six of the seven sequences in SAN GC with significant WWIHS scores overlap in the proposed alignment with sequences with significant WWIHS in SIN E1. Analysis of membrane interfacial potential in the primary sequences thus provides further support for the proposed alignment of SAN Gc and SIN E1. A model of phlebovirus Gc ------------------------- Recently, Gibbons and coworkers determined the structure of a fragment of the SFV E1 ectodomain (lacking carboxyl terminal aa 392--438) after exposure to low pH and liposomes \[[@B33]\]. Under these conditions, which mimic an endosomal environment, the SFV E1 ectodomain fragment changes from a soluble monomer to a trimer as it inserts into the liposomal membrane after exposure to low pH. A similar post-fusion structure was found in two other class II fusion proteins, E of DEN and TBEV \[[@B10],[@B11]\]. These investigators proposed several possible fusion intermediates of SFV E1 and other class II viral fusion proteins after exposure to low pH. These intermediates are assumed to be similar to structural intermediates of SIN E1. To determine the plausibility of the proposed SAN Gc and SIN E1 alignment, a model of SAN Gc was scaffolded on a presumed structural intermediate of SIN E1 in which compared to the orientation in the virion at neutral pH, domain III is displaced closer to the fusion peptide (Fig. [2](#F2){ref-type="fig"}). The collinear sequence alignments between SAN Gc and SIN E1 suggest that both glycoproteins may have a similar domain structure. Similar sequences/structures are drawn in similar locations. In this possible fusion intermediate, the putative SAN Gc fusion peptide is assumed to be located at the end of the molecule furthest from the carboxyl terminal (C-terminal) transmembrane anchor domain. Like SIN E1 and other class II fusion proteins SAN Gc may be comprised mostly of antiparallel β sheets, an expectation supported by several secondary structure prediction algorithms, including PHDsec \[[@B23]\], Chou-Fasman \[[@B21]\] and Robson-Garnier \[[@B22]\] analyses. The proposed SAN Gc structure conforms closely to the structural predictions of PHDsec, the most robust algorithm, but is also generally consistent with both Chou-Fasman and Robson-Garnier predictions. In some cases because of significant aa sequence similarity with SIN E1, ambiguous structures in SAN Gc are depicted as in SIN E1. Additional evidence for the proposed alignment is the location of cysteine residues of SAN Gc and SIN E1. The cysteine residues are usually the most conserved aa in within a protein family because disulfide linkages are a critical determinant of secondary structure. The dicysteines of SIN E1 (Fig. [2B](#F2){ref-type="fig"}) are arranged such that bonds are formed only between residues within the same putative domain. A similar arrangement is feasible for SAN Gc with C residues present in close proximity after scaffolding on the SIN E1 structure, and with possible linkages occurring within the three proposed domains. This model also locates the four SAN Gc glycosylation sites so they are surface accessible. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Model of Sandfly fever virus Gc based on predicted structure of a Sindbis virus E1 fusion intermediate. Panel A: A structural intermediate of SFV E1 as determined by Gibbons et al. \[33\] was projected to SIN E1. Panel B: A model fitting SAN GC to the predicted structure of SIN E1. Structures predicted to be similar are color-coded the same way in SIN E1 and SAN Gc. Grey lines: dicysteine linkages. Black stick figures: N-glycosylation sites (sites with central proline are often not used). Regions with significant Wimley-White interfacial hdrophobicity scale scores were predicted with MpeX (black). ::: ![](1742-4682-1-10-2) ::: There are many possible alternatives to the cysteine linkages and secondary structures of SAN Gc drawn in Figure [2](#F2){ref-type="fig"}. Nevertheless, a plausible three-dimensional model of SAN Gc that conforms to the scaffold of the known structure of Alphavirus E1 can be constructed. This result coupled with predictions of a predominantly β sheet secondary structure of SAN Gc provides further support its proposed alignment with SIN E1. Sequence/structural features of Bunyavirus Gc suggest that a class II fusion protein structure is conserved in members of the Bunyaviridae ------------------------------------------------------------------------------------------------------------------------------------------ To provide additional evidence for the proposed SIN E1/SAN Gc alignment and the SAN Gc class II fusion protein model, we determined whether structural/sequential similarities with class II fusion proteins are conserved in envelope proteins encoded by other members of the Bunyavirus family. With the exception of tospoviruses, that use an ambisense strategy for synthesis of a nonstructural protein, Bunyavirus M segments are negative in polarity and the mRNA transcribed contains a long open reading frame \[[@B37]\]. The M segment mRNA is translated as a polyprotein, which is post-translationally processed \[[@B38]\]. There is considerable diversity in the number and sizes of the M segment encoded polyproteins produced in infected cells, but all Bunyaviruses encode at least two glycoproteins, Gn and Gc. Prior analyses have revealed similarities between Gc encoded by members of orthobunyaviruses and tospoviruses \[[@B18],[@B39]\], but evidence that Gn and Gc serve analogous functions in each Bunyavirus genus has not been available previously. Comparisons among Gc of members of the five genuses of the Bunyaviridae using the PRSS3 algorithm revealed that the type members of each genuses display significant sequence similarities with certain Gc of viruses of other Bunyavirus genuses (Table [2](#T2){ref-type="table"}). The most significant alignment detected among members of different genuses of the Bunyaviridae, was between RVF, type member of phleboviruses, and tomato spotted wilt virus (TSWV), type member of tospoviruses (p \< 10^-18^). As noted previously \[[@B18],[@B39]\], orthobunyavirus Gc also show a significant similarity to tospovirus Gc, with Bunyamwera virus (BUN) Gc displaying a significant similarity to TSWV Gc (p \< 10^-8^). As with phlebovirus Gc, the prototype of the hantavirus genus, Hantaan virus (HAN), showed a modest sequence alignment (p \< 0.05) with SIN E1, further supporting the proposed similarities between Bunyavirus Gc and Alphavirus E1. Significant alignments were not detected between Bunyavirus Gc or Alphavirus E1 and TBEV E or other flavivirus class II viral fusion proteins. Limited local similarities were observed between some Bunyavirus Gc and pestivirus E2. It is noteworthy that the significance of the overall sequence similarities between certain phlebovirus Gc and Alphavirus E1 is higher than some similarities among Gc of some prototypic members of the Bunyaviridae (compare Table [1](#T1){ref-type="table"} and [2](#T2){ref-type="table"}). Collectively, these results suggest that Gc of Bunyaviruses share a limited number of similar sequences. ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Similarities among Bunyavirus Gc, Alphavirus E1 and related glycoproteins as determined with the PRSS3 sequence algorithm. ::: [Viral protein]{.underline}^1^ -------------------- -------------------------------- -------- ------- --------- ------- -------- ------- ---- Obuny BUN Gc 0.0005 0.01 0.009 10^-5^ NS NS NS NS Hanta HAN Gc \-\-- 0.0001 NS NS 0.05 NS 0.003 NS Nairo CCHF Gc \-\-- \-\-- 0.05 0.0001 NS NS NS NS Phlebo RVF Gc \-\-- \-\-- \-\-- 10^-18^ 0.04 10^-8^ 0.001 NS Tospo TSWV Gc \-\-- \-\-- \-\-- \-\-- NS NS NS NS Alpha SIN E1 \-\-- \-\-- \-\-- \-\-- \-\-- 0.02 NS NS CeRV Cer13 Env^2^ \-\-- \-\-- \-\-- \-\-- \-\-- \-\-- 0.02 NS Tenui RiSV pvc2^2^ \-\-- \-\-- \-\-- \-\-- \-\-- \-\-- \-\-- NS Two-way comparisons were done between the full-length amino acid sequences of the indicated glycoproteins. Probabilities (p values) of a significant alignment are based on 1000 shuffles. BUN: Bunyamwera virus; HAN: Hantavirus; CCFV: Crimean-Congo hemorrhagic fever virus; RVF: Rift valley fever virus; TSWV: Tomato spotted wilt virus; SIN: Sindbis virus; Cer13: Caenorhabditis elegansretrovirus 13; RiSV; rice stripe virus; TBEV: tick-borne encephalitis virus. ^2^C-terminal sequence. ::: Available computational methods alone do not permit overall alignments of Bunyavirus Gc, however, through the use of PRSS3 and MacMolly alignment tools and by inspection certain common sequences can be identified. The most highly conserved sequences among type members of the five Bunyavirus genuses conform to a consensus class II fusion peptide (Fig. [3](#F3){ref-type="fig"}, red) and to a carboxyl terminal transmembrane domain (Fig. [3](#F3){ref-type="fig"}, violet). Certain cysteine clusters, which are likely to stabilize the secondary structures of the proteins, also are present in similar locations along the proteins, including the beginning of putative domain III (Fig. [3](#F3){ref-type="fig"}). Further support for the alignment of Bunyavirus Gc is obtained by the use of the WWIHS. Sequences with significant WWIHS scores are located in similar locations in Bunyavirus Gc (Fig. [3](#F3){ref-type="fig"}, olive). The proposed fusion peptides and transmembrane domains of each of the Bunyavirus Gc examined displayed significant WWIHS scores. In addition, most of the Gc examined had sequences with significant WWIHS scores in the putative IIa domains and either the putative Ic domain or the beginning of adjacent domain III. This alignment suggests that orthobunyaviruses and tospoviruses Gc have extended regions of approximately 400 and 50 amino acids at the amino terminus relative to phlebovirus, hantavirus and nairovirus Gc. These results also suggest that motifs involved in virion:cell fusion are conserved in Gc throughout the Bunyaviridae, and that Gc is the fusion protein encoded by members of each Bunyavirus genus. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Alignment of Gc amino acid sequences of prototype members of the five genuses of the Bunyaviridae family. Alignments were constructed by identifying the fusion peptide (red) and the transmembrane anchor (violet) as described in the text. Additional local sequence similarities were identified by using the Complign feature of MacMolly, the PRSS3 alignment algorithm or by inspection. Sequences with significant WWIHS scores (olive) were identified by MPeX. ::: ![](1742-4682-1-10-3) ::: Sequence/structural features of Bunyavirus Gc are present in a Tenuivirus protein and in an Env encoded by a Caenorhabditis elegansretrovirus ----------------------------------------------------------------------------------------------------------------------------------------------- As previously reported, phlebovirus Gc have a significant similarity to surface protein pvc2 encoded by plant viruses of the Tenuiviridae \[[@B40]\]. The PRSS3 sequence alignment algorithm confirms this similarity (Table [2](#T2){ref-type="table"}, p \< 0.001). An envelope protein (Env) encoded by a group of retroviruses of the nematode C. elegansalso demonstrate significant similarities to phlebovirus Gc \[[@B41],[@B42]\]. There is a significant alignment detected by the PRSS3 algorithm between SAN Gc and the carboxyl terminal region of Env of Cer13, a potentially replication-competent member of this group (Table [2](#T2){ref-type="table"}, p \< 10^-8^). These results further validate the use of the PRSS3 algorithm to identify limited similarities amongst viral proteins. Alignment of the carboxyl terminal portion of the pvc2 protein of rice stripe virus (RiSV), a Tenuivirus, and the envelope protein encoded by Cer13 retrovirus with two phlebovirus Gc reveals a collinear arrangement of fusion peptide consensus sequences (Fig. [4](#F4){ref-type="fig"}, red) and potential carboxyl terminal transmembrane domains (Fig. [4](#F4){ref-type="fig"}, violet). These proteins also have several overlapping sequences with significant WWIHS scores (Fig. [4](#F4){ref-type="fig"}, olive). The retention of these features in proteins encoded by evolutionarily distant genomes, provides further evidence that these motifs are important for the function of Bunyavirus fusion proteins. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Alignment of phlebovirus Gc amino acid sequences with Tenuivirus surface protein pvc2 and the carboxyl terminal Env protein of a Caenorhabditis elegansretrovirus. Sequences are color-coded as in Figure 3. ::: ![](1742-4682-1-10-4) ::: Protein order in polyproteins encoded by Bunyaviridae M segments ---------------------------------------------------------------- The longest open reading frames of M segments of all members of the Bunyaviridae are antisense to the virion RNA. mRNAs transcribed from Bunyavirus M segments are translated into large polyproteins that are subsequently cleaved by into functional proteins \[[@B38],[@B43]\]. Gc of members of each of the five Bunyavirus genuses are the carboxyl terminal proteins of the polyprotein (Fig. [5](#F5){ref-type="fig"}). Because viral proteins with similar functions may have similar genome locations, we sought evidence for sequence similarities among other Bunyavirus proteins encoded by the M segment. Gn of type members of the five Bunyavirus genuses were compared to each other and each had a limited sequence similarity to at least one other Gn of a type member of a different genus (Table [3](#T3){ref-type="table"}). The most significant alignment was between the Gn proteins of HAN, a hantavirus, and CCHF, a nairovirus (p \< 10^-4^). HAN Gn also showed a significant alignment with Gn of RVF, a phlebovirus. Both HAN Gn and Gn of TSWV, a tospovirus, also showed significant alignments with envelope protein 2 (E2) of SIN. SIN E2 has been implicated as the virion protein responsible for binding to the cell surface receptor \[[@B44]\]. These results suggest that the Gn of Bunyaviruses have limited similarity, and may have a common role or roles in the virus replication cycle. The order (from amino to carboxyl terminus) of proteins in the polyproteins of SIN and other members of the Alphaviridae is Capsid-E2-E3-6K-E1. Receptor and fusion functions may reside in two different Bunyavirus proteins, Gn and Gc respectively, occurring in the same order as the envelope glycoproteins, E2 and E1, that carry out these functions in Alphaviruses (Fig. [5](#F5){ref-type="fig"}). The similarities in protein order and functions support the hypothesis that Gc is the fusion protein of Bunyaviruses. These results also support the suggested nomenclature of Gn and Gc for the Bunyavirus M segment encoded glycoproteins as a replacement for the current ambiguous nomenclature, which variously assigns the designation G1 or G2 to unrelated Bunyavirus glycoproteins. The presumptive protein encoded by the amino terminal region of the pvc2 protein of RiSV also showed limited similarity to SIN E2 (Table [3](#T3){ref-type="table"}). Thus, the similarities between proteins encoded by a Tenuivirus extend to another glycoprotein of a virus with a class II fusion protein. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Common order of proteins in Bunyavirus M segment polyproteins. Related glycoproteins Gn and Gc are in the same order in the polyproteins of prototypic members of the Bunyaviridae. Prior designations of the glycoproteins are indicated in parentheses. Hydrophobic domains were predicted using TMpred. The O-glycosylation rich (mucin-like) region in CCHF was delineated using NetOGlyc 3.1 as described previously by Sanchez and coworkers \[46\]. These authors also described the indicated potential cleavages of the CCHF polyprotein. ::: ![](1742-4682-1-10-5) ::: ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Similarities among Bunyavirus Gn, Alphavirus E2 and related glycoproteins as determined with the PRSS3 sequence algorithm. ::: [Viral protein]{.underline}^1^ -------------------- -------------------------------- -------- ------- ------- ------- ------- ------- ---- Obuny BUN Gn NS NS NS 0.02 NS NS NS NS Hanta HAN Gn \-\-- 10^-4^ 0.03 NS 0.05 NS NS NS Nairo CCHF Gn \-\-- \-\-- NS NS NS NS NS NS Phlebo RVF Gn \-\-- \-\-- \-\-- NS NS NS NS^3^ NS Tospo TSWV Gn \-\-- \-\-- \-\-- \-\-- 0.04 NS NS NS Alpha SIN E2 \-\-- \-\-- \-\-- \-\-- \-\-- NS 0.04 NS CeRV Cer13 Env^2^ \-\-- \-\-- \-\-- \-\-- \-\-- \-\-- 0.025 NS Tenui RiSV pvc2^2^ \-\-- \-\-- \-\-- \-\-- \-\-- \-\-- \-\-- NS ^1^Two-way comparisons were done between the full-length amino acid sequences of the indicated glycoproteins. Probabilities (p values) of a significant alignment are based on 1000 shuffles. BUN: Bunyamwera virus; HAN: Hantavirus; CCFV: Crimean-Congo hemorrhagic fever virus; RVF: Rift valley fever virus; TSWV: Tomato spotted wilt virus; SIN: Sindbis virus; Cer13: Caenorhabditis elegansretrovirus 13; RiSV; rice stripe virus; TBEV: tick-borne encephalitis virus. ^2^N-terminal sequence. ^3^p = 0.08 ::: The simplest M polyprotein, encoding only Gn and Gc, is that of hantaviruses (Fig. [5](#F5){ref-type="fig"}). In addition to Gn and Gc, phlebovirus and orthobunyavirus encode nonstructural proteins (NSm) that have two or three potential transmembrane spanning domains as detected by TMpred (Fig. [5](#F5){ref-type="fig"}). Nairoviruses, such a Crimean-Congo hemorrhagic fever virus (CCHF), synthesize a similar protein \[[@B45],[@B46]\], and we have designated this protein \"NSm.\" NSm of the RVF and BUN, type members of phlebovirus and orthobunyaviruses, as well as the \"NSm\" protein of CCHF have short regions of similarity with each other as revealed by the PRSS3 sequence alignment algorithm, although the overall alignments are not significant. There were also short regions of similarity between the NSm proteins of these three Bunyavirus genuses and the prM/M-like proteins of Flaviviruses (not shown). Immature virions of members of the flavivirus genus of the Flaviviridae contain a precursor prM to the small membrane protein M. prM is cleaved by furin or by a furin-like protease during virus release to produce the mature M protein localized on the surface of flavivirus virion \[[@B47],[@B48]\]. Flavivirus PrM/M, contains two potential membrane spanning domains, and their functions may include shielding of internal cellular membranes from the fusion peptide of E \[[@B7],[@B47]\]. It is possible that the phlebovirus, orthobunyavirus and nairovirus M segment encoded nonstructural proteins, all with multimembrane-spanning potential, serve the same function for Gc. NSm of TSWV, a tospovirus, showed no sequence similarity or structural similarity with any Bunyavirus protein examined. The functions of tospovirus NSm, which is encoded by the only positive polarity gene in any M segment, and the other Bunyavirus NSm proteins remain to be determined. Nairovirus M may encode two additional proteins, a mucin-like protein (MLP), which contains a variable region with a high concentration of potential O-glycosylation sites, and a protein designed here X, neither of which have obvious homologs encoded by members of the other Bunyavirus genuses \[[@B45],[@B46]\]. The coding sequences of Bunyavirus M appear to have evolved in a manner preserving the order of the glycoproteins Gn and Gc, while allowing for insertion or deletion of sequences encoding additional proteins. Discussion ========== Proteomics computational analyses suggest that Bunyavirus Gc proteins are class II viral fusion proteins (β-penetrenes), with a structure similar to the fusion proteins of Alphaviruses and Flaviviruses. Similar sequences or common structural/functional motifs are collinearly located in Bunyavirus Gc and Alphavirus E1. Features common to other class II fusion proteins, including an internal fusion peptide, a carboxyl terminal transmembrane domain and regions with a high propensity to interface with bilayer membranes, are conserved and in similar locations in Gc of viruses in each genus of the Bunyaviridae. These features are also present in glycoproteins encoded by nonenveloped Tenuiviruses of plants, and a group of C. elegansretroviruses previously shown to have remarkable sequence similarities to phlebovirus Gc. These results also indicate that Gallaher\'s \"Rosetta Stone\" strategy can be used to identify potential class II viral fusion proteins, as demonstrated previously for class I fusion proteins \[[@B1]-[@B3],[@B49]\]. The common placement of proper names or \"cartouches\" allowed the ancient languages of the Rosetta Stone to be deciphered. As advanced by Gallaher, fusion peptides can serve a similar function to facilitate alignment of viral fusion proteins with limited sequence similarities. Many viral fusion proteins fit neither class I or II and it is likely that other classes of viral fusion protein also exist. However, among major classes of enveloped RNA viruses, there are at least six, myxoviruses, retroviruses, paramyxoviruses, filoviruses, arenaviruses and coronaviruses, that encode class I viral fusion proteins \[[@B1]-[@B4]\]. Alphaviruses, members of the flavivirus genus of the Flaviviruses, and according to current analyses, Bunyaviruses, encode class II viral fusion proteins. Computational analyses suggest that members of the two other Flavivirus genuses, hepaciviruses and pestiviruses, encode variant class II fusion proteins \[[@B12]\]. The viruses encoding class II or II viral fusion proteins thus represent a substantial portion of enveloped RNA virus families known to infect vertebrates. It is significant that representative class I and II encoding viruses are also found in evolutionarily distinct plant viruses and viruses or virus-like genomic elements of nematodes and insects \[[@B41],[@B42],[@B50],[@B51]\]. There may be constraints on the structures of viral proteins capable of effectively mediating virion:cell fusion, or a limited number of enveloped RNA virus lineages. Alphaviruses appear to use separate envelope proteins for fusion (E1) and attachment (E2) \[[@B44]\]. Because Bunyavirus Gc display similarities to Alphavirus E1 and certain Bunyavirus Gn display limited sequence similarities to Alphavirus E2, Bunyaviruses may have adapted a similar strategy. Verification that Gc is the fusion protein of Bunyaviruses will require a combination of X-ray crystallographic structural studies and site-directed mutagenesis of key features such as the putative fusion peptide. Verification that Gn serves as the receptor binding protein for any Bunyavirus requires identification of its cell surface receptor. E, the class II fusion protein of TBEV, dengue virus, and other members of the flavivirus genus of the Flaviviridae, mediates both virion:cell fusion and receptor-binding \[[@B52],[@B53]\]. Therefore, it is possible that Bunyavirus Gc serves both as the fusion protein and receptor binding protein. The remarkable similarities in both the pre- and post-fusion forms of the fusion proteins of SFV E1, an Alphavirus, and DEN and TBEV, members of the flavivirus genus of the Flaviviruses, in the absence of detectable sequence similarities, suggest that Alphavirus and Flavivirus class II fusion proteins may have diverged from a common progenitor. Alternatively, there may have been convergent evolution towards the common structure. Likewise, the sequence similarities detected between phlebovirus Gc and SIN E1 are consistent with divergent evolution from a common progenitor, but are insufficient to directly establish a phylogenic relationship. The results presented here suggest that Gc of members of the Bunyaviridae may have a common ancestor. Gn and Gc are in analogous locations in the polyproteins encoded by the five genuses of the Bunyaviridae. The simplest Bunyavirus M polyprotein, that of hantavirus members, encodes only Gn and Gc, whereas M of members of other Bunyavirus genuses encode several additional proteins. Therefore, divergence of Bunyavirus M segments may have occurred either through acquisition of sequences and/or lose of sequences in a cassette manner constrained in part by the locations of the major glycoproteins. Comparisons of divergent viral fusion proteins with internal fusion peptides can reveal features essential for virion:cell fusion. Regions of high membrane interfacial propensity including the fusion peptide and the transmembrane anchor, appear in similar locations in Bunyaviruses, Alphaviruses and Flaviviruses. The presence of several additional sequences with the propensity to interact with bilayer membranes in class II viral fusion proteins has not been considered in previous virion:cell fusion models \[[@B9],[@B11],[@B54]\]. Cell entry of Alphaviruses and Flaviviruses is believed to occur via the endocytic route, and it is likely that this is the entry route of Bunyaviruses \[[@B55]\]. Following binding to the cellular receptor, a putative function of Bunyavirus Gn (Fig. [6A](#F6){ref-type="fig"}), the Bunyavirus virion may be taken up in an endocytic vesicle (Fig. [6B](#F6){ref-type="fig"}). Exposure to acidic pH in the endosome may trigger conformational changes in the envelope proteins and in the virion itself resulting in dissociation of Gn and Gc (Fig. [6C](#F6){ref-type="fig"}). Current models of fusion mediated by Alphavirus and Flavivirus class II viral fusion proteins suggest that the low pH of the endosome triggers trimerization and a bending of class II fusion proteins at a flexible \"hinge\" region between domains I and II elevating the fusion peptide so that it can insert into the host membrane \[[@B6],[@B9],[@B11],[@B54]\]. Current models then suggest that a rearrangement of the stem (pre-anchor region), so that there are more extensive interactions with domains I-III, results in a deformation of the viral and target membranes and the formation of apposing membrane \"nipples\" (Fig. [6E](#F6){ref-type="fig"}). Subsequently, the nipples are brought closer together by continued interactions of the stem with domains I-III, which results in bilayer hemifusion (Fig. [6F](#F6){ref-type="fig"}). Complete fusion follows allowing entry of the ribonucleoproteins containing the viral genomic RNA (Fig. [6G](#F6){ref-type="fig"}). An analogous mechanism, involving deformation and nipple formation of the viral and cellular membranes caused by rearrangements of the viral fusion proteins (six helix bundle formation) has been proposed for class I viral fusion proteins \[[@B54]\]. ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Hypothetical model of Bunyavirus:cell fusion. Steps in the entry process of Bunyaviruses can be extrapolated from current models of class II viral fusion protein-mediated virion cell fusion. Panel A. The Bunyavirus glycoproteins Gn and Gc are modeled according to SIN virion structure analyses by Zhang et al. \[31\]. Based on limited similarities with Alphavirus E2 proteins (Table 3), Gn is depicted as the receptor-binding protein of Bunyaviruses. Certain Bunyaviruses may encode other membrane-associated proteins that interact with the fusion peptide or other regions of Gc. Panel B: Receptor-binding triggers uptake of Bunyavirus virion by endocytosis. Panel C: Acidification of the endocytic vesicle occurs via the action of proton transporters and may initiate Gn and Gc dissociation. Panel D: bending at the flexible \"hinge\" region beween domains I and II permits Gc trimer formation and insertion of the fusion peptide into the endosomal vesicle membrane. Panel D\' Alternatively, Gc trimer formation may involve the rotation of domain III and a rearrangement (twist) of domain II as shown for SFV E1, DEN E and TBEV E \[11,33,54\]. Panel E: As previously proposed \[11,33,54\] the formation of more extensive Gc contacts in the trimers and stem regions may release of energy for distortion of the endosomal and viral membranes resulting in formation of \"nipple-like\" projections. Panel E\': Alternatively, aa sequences of Gc that form a track with the ability to interface with bilayer membranes (Fig. 2, black), may facilitate mixing of the endosomal and viral membranes. Panel F: Formation of further trimer contacts and hemifusion. Hemifusion may not occur in the D\' and E\' pathway. Panel G: Formation of the \"fusion pore\" and entry of the ribonucleoprotein (RNP) segments. Modified from models and concepts proposed in references 9-12. ::: ![](1742-4682-1-10-6) ::: Current fusion models do not consider that the transmembrane domain and fusion peptide, while anchored into the viral and cellular membranes, would still be free to move laterally without distorting the membranes. More importantly, the virion is quite small compared to the cell, and would be freely mobile. Rearrangement of the fusion proteins may simply draw the virus closer to the cell without distorting either the viral or cellular membranes. An alternative to the models involving apposing membrane nipple formation is suggested by the observation that sequences of class II viral fusion proteins, including the fusion peptide, the transmembrane anchor and other sequences with high WWIHS scores, potentially form a nearly continuous track of membrane interactive regions that could channel the movement of lipids during virion:cell fusion (Fig. [2](#F2){ref-type="fig"}, black). Similar nearly contiguous sequences with significant WWIHS scores are present in the post-fusion intermediates of Alphaviruses SIN and SFV, DEN and other flaviviruses, and the proposed structures of hepaciviruses and pestiviruses \[[@B12]\]. An intermediate, with the track of sequences with high membrane interfacial propensity, may be the first intermediate formed after exposure to the low pH in liposomes (Fig. [6D\'](#F6){ref-type="fig"}). Upon formation of higher multimers of trimers, the regions with high WWIHS scores, in conjunction with the fusion peptide and transmembrane could then form a \"pore\" in which the lipids of the cellular and viral bilayer membranes could mix directly (Fig. [6E\'](#F6){ref-type="fig"}). With lipid mixing facilitated by these membrane interfacial sequences bilayer fusion may proceed without a hemifusion step, but still permitting entry of the genome-containing RNP (Fig. [6G](#F6){ref-type="fig"}). In the absence of structural determinations by X-ray crystallography, models such as proposed here can provide useful hypotheses to guide experimental strategies for development of vaccines or drugs to prevent or treat infection by viruses with class II fusion proteins. Prior to the availability of X-ray structural data, several potent HIV-1 TM inhibitors were developed \[[@B56],[@B57]\] based on the Gallaher HIV-1 TM fusion protein model \[[@B2]\]. Fuzeon™ (DP178; T20 enfuvirtide), one of these peptides corresponding to a portion of an α helices and the pre-anchor domain, has been shown to substantially reduce HIV-1 load in AIDS patients, and has been approved for use in the treatment of HIV infection in the United States and European Union \[[@B58],[@B59]\]. Peptides targeted to membrane interactive motifs block virion:cell fusion mediated by DEN and West Nile virus, flaviviruses with class II fusion proteins (Hrobowski et al., submitted). Peptide inhibition strategies targeted to Gc may be broadly applicable to various members of the Bunyaviridae. Competing Interests =================== The authors declare that they have no competing interests. Authors\' Contributions ======================= CEG performed the sequence alignments and assisted in the preparation of figures. RFG supervised the work and wrote the manuscript. Acknowledgements ================ This research was supported by NIH grants AI034764, AI054626, AI054238, RR018229, CA054576 and CA089121. William R. Gallaher developed the \"Rosetta Stone\" strategy and first described the use of the viral fusion peptides as \"cartouches.\" We thank Dr. Gallaher and Dr. William C. Wimley for informative ongoing discussions on the structure of viral fusion proteins.	PubMed Central	2024-06-05T03:55:50.004293	2004-11-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC535339/", "journal": "Theor Biol Med Model. 2004 Nov 15; 1:10", "authors": [ { "first": "Courtney E", "last": "Garry" }, { "first": "Robert F", "last": "Garry" } ] }
PMC535340	Background ========== Microarrays are routinely used for large scale transcriptome analyses and have been widely and successfully employed for simultaneously monitoring the expression of a potentially unlimited number of genes in parallel, thus providing the basis for identifying genes differentially expressed in distinct cell-types, developmental stages, disease states and cells subjected to exogenous reagents \[[@B1]\]. The rapid and significant improvements of cDNA-chip technologies and the availability of multi-species gene catalogues within the various data bases have made possible the comparison of gene expression levels within a single mammalian organism and across different organisms on a large-scale. The advantages of cross-species hybridisation are two-fold. First, cross-species gene-expression comparison is a powerful tool for the discovery of evolutionarily conserved mechanisms and pathways of expression control. The advantage of cDNA microarrays in this context is that broad areas of homology are compared and hybridization probes are sufficiently large so that small inter-species differences in nucleotide sequence would not affect the analytical results. This comparative genomics approach would allow a common set of genes within a specific developmental, metabolic, or disease-related gene pathway to be evaluated in experimental models of human diseases. Second, the use of microarrays in studies in mammalian species other than human and rodents, for example nonhuman primates, bovine, sheep and porcine may advance our understanding of human health and disease, for example the use of animal models in drug target validation. However, the inavailability of adequate sequence data and commercial cDNA and oligonucleotide microarrays keeps this technology beyond the reach of investigators working on economically and scientifically important large domestic species such as cattle, pigs and sheep. A potential solution to this problem is the use of cross-species hybridisations, i.e, human sequence-based arrays as tools for undertaking comparative genome expression studies. Such analyses have been performed using ape brain RNA as target on a human oligonucleotide array \[[@B2]\] and pig, mouse and Atlantic salmon RNA on human nylon arrays- \[[@B3]-[@B7]\]. These types of studies represent critical areas of research directly related to the understanding of human diseases because nonhuman primates, bovine, sheep and porcine play a crucial role in biomedicine, such as, organ transplantation, vaccine development, viral pathogenesis, gene therapy and a host of other human health-related technologies. A crucial step employing domestic animals in biomedicine is genetic modification which requires extensive embryo and embryo-related technologies, such as in vitroproduction of embryos for stem cell derivation and somatic nuclear transfer cloning. Employing the bovine model and sensitive RT-PCR assays, it has been shown that the majority of embryos derived from such sources display distinct mRNA expression patterns in a variety of developmentally important genes compared to their in vivoderived counterparts \[[@B8]\]. Some of these aberrations lead to \"Large offspring syndrome\", a complex of multiple pathologies observed in offspring derived from in vitroproduction and/or nuclear transfer of which significant oversize is a predominant feature \[[@B9]\]. Analysis of mRNA expression patterns in early embryos via cDNA microarray technology would provide insights into the function of gene regulatory networks and would thus be a major step forward in unravelling molecular mechanisms underlying developmental abnormalities. The technology to amplify the minute amounts of mRNA in early embryos without significantly altering the ratio of the various mRNAs in the original cell has recently been described \[[@B10],[@B11]\] and a prototype mouse cDNA-macroarray enriched in embryonic sequences has been developed \[[@B11]\]. Important criteria for evaluating any microarray system include the reproducibility of the data generated, the specificity of detection of the targeted gene, and the validity of the results that identify and establish differential gene expression. The experiments described here show the systematic validation of cross-species microarray analysis, with emphasis on the reproducibility and statistical analysis of generated data using standard microarray data analysis tools. Specifically, we investigated the feasibility and reproducibility of cross-species hybridisation of orthologous genes within a defined developmental and metabolic pathway using as a test case and the first description of its kind, human and bovine fetal brain RNA as Cy-dye labelled targets on a human cDNA microarray. The microarray is composed of 349 genes each spotted 20 times to ensure reproducible validation by independent technologies such as semi-quantitative RT-PCR as carried out in this study, or alternatively, Real-Time PCR and Northern blot analysis. Results ======= Array fabrication and gene annotation ------------------------------------- The human cDNA microarray used in this study consisted of 349 fully sequenced and annotated cDNAs as described in the Supplemental Table [1](#T1){ref-type="table"}. Thirty-five spots containing only spotting solution (3x SSC / 1.5 M betaine) served as negative controls. In addition, in a separate control plate, the housekeeping genes HPRTand β-ACTIN(made up of a dilution series of 25-, 50-, 100-, 150- and 200 ng/μl) were employed as endogenous guide dots to enable accurate grid placement prior to image analysis. These spots can be seen as intense yellow signals at the periphery of each block as portrayed in Figure [1A](#F1){ref-type="fig"}. The embryonic-specific gene, OCT-4and ArabidopsiscDNAs (Cab- T97312; Cwlp- T02614; Lhb1B1- T21965; Ohp- T22679) were included as negative controls to monitor hybridisation-specificity. The transcription factor OCT-4 is expressed in human embryonic stem cells and primordial germ cells and is down-regulated upon differentiation \[[@B10],[@B12],[@B13]\]. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Summary of primer sequences, annealing temperatures and size of amplicons. Derivation of primer sequences: H and B denote human and bovine respectively. The nucleotide positions of the primer sequences are in parenthesis. Nucleotides highlighted in bold denote differences in the orthologous gene sequences. ::: -------------------------------------------------------------------------------------------------------------------- Genes Primer sequences (5\' -- 3\') Annealing temperature (^°^C) Fragment size (bp) Accession numbers ---------- --------------------------------- ------------------------------ -------------------- ------------------- CDC27 (1226--1250) ^H^\ 54 311 NM\_001256 ^H^\ TATTACATCTCCCCCAAACGCACTG CB170694 ^B^ (1512--1536) ^H^\ CCATTTCACGAAGAAGGCTCATCAA CDC6 (89--113) ^B^\ 54 208 NM\_001254 ^H^\ TCCCAAGCGGGTTGGTGTTATTCAC BF600055 ^B^ (272--296) ^B^\ GCGACAGACTGTACTGTAGGCTTCA PCNA (195--217) ^B^\ 54 382 AF527838 ^H^\ GAGGCGCTTAAGGATCTCATCAA CB531519 ^B^ (554--576) ^B^\ ATTCACCAGAAGGCATCTTTACT SLC11A3 (38--61) ^B^\ 57 276 AF215636.1 ^H^\ ACCCCTGGAGGGAACTCATCTAAT BG689712 ^B^ (290--313) ^B^\ GCTGATGCTCCCATCAAAATACTG VDAC2 (652--675) ^B^\ 57 448 NM\_003375.1 ^H^\ CCTCGGTTGTGATGTTGACTTTGA TC152811 ^B^ (1076--1099) ^B^\ GTGGCCTCCAGCATTAATGCTCTT CALNEXIN (1054--1078): ^H^\ 56 237 NM\_001746.1 ^H^\ GCTGGTTAGATGATGAGCCTGAGTA BM431118 ^B^ (1266--1290): ^H^\ TCCTGGGTTTCCAGATTCCCTGGTA β-ACTIN 441--461) ^H^\ 60 469 NM\_001101 ^H^\ GTTGCTATCCAGGCTGTGCT NM\_173979 ^B^ (890--910) ^H^\ CGGATGTCCACGTCACACTT -------------------------------------------------------------------------------------------------------------------- ::: ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Array fabrication and gene annotation(A) False-colour image generated from a bovine-Cy5, human-Cy3 hybridisation. False colour images were generated using the programme ScanAlyze, version 2.44 <http://www.rana.lbl.gov/EisenSoftware.htm>. The full array (24 × 25 spotting pattern) consists of 16 blocks with each gene spotted 5 times per block therefore 80 potential data points present for expression analysis per gene. A blow-up of 4 blocks illustrating the Chip design is presented. The ACTINcDNAs acting as guide-dots can be seen as intense yellow spots demarcating each block. In addition, the majority of spots appear yellow due to similar expression levels of the orthologous genes. (B) Functional annotation of the 349 genes as set out by the Gene Ontology Consortium <http://rana.lbl.gov/EisenSoftware.htm>.The proportion of these genes within each functional / biological annotation is represented on the Y-axis and the annotation on the X-axis. (C) Chromosomal distribution of the 349 genes. The number of genes within each chromosome is represented on the Y-axis and the chromosome numbers on the X-axis. ::: ![](1471-2164-5-83-1) ::: Classification of the genes according to Gene Ontology annotations (molecular function) and chromosomal location (Figures [1B](#F1){ref-type="fig"} and [1C](#F1){ref-type="fig"}) demonstrates that the selected genes encompass a range of twelve different functional classes and are located on all except the Y-chromosome. This implies that there is no obvious bias towards biological characteristics and the selected gene set can be viewed as representative for the current study. Global data characteristics --------------------------- Our experimental design incorporated dye-swaps and four replicated hybridisations. Three hundred and forty nine cDNAs, each representing a unique gene were spotted 20 times on each microarray. Four independent experiments were performed with human and bovine brain, respectively. Within each experiment the 20 replicates were averaged to yield a reliable signal for the respective probe. In the next step, the replicated signals from the different experiments were averaged to compute overall characteristics. The overall correlation of expression of the human and bovine genes is shown in Figure [2A](#F2){ref-type="fig"}. The regression line bears a slope close to one (1.13) thus indicating similar expression of human and bovine genes across the replicated experiments with a slight increase of expression in the human brain. The overall correlation coefficient is 0.94. ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Global data characteristics (A) Global correlation of bovine (X-axis) and human (Y-axis) experiments. The plot shows a log-log (base 2) plot of the mean signal intensities from the four independent experiments for each gene. The red line shows the regression line, the box displays the parameters of the regression line (intercept, slope) and the overall correlation. Four genes that are significantly differentially expressed as judged by using the Wilcoxon\'s rank-sum test are denoted by black diamond shapes. (B) MA-plot of a single experiment. The X-axis show the log (base 2) of the squared product of Cy5-red and Cy3-green intensities of each gene in the same experiment. Y-axis shows the log-ratio (base 2) of red and green intensity. The horizontal lines mark the two-fold over-expressed genes (Top line; bovine over-expression and bottom line; human over-expression). (C). Classification of detection levels of the 349 genes. The genes were sub-divided into three classes of expression levels. Class 1 (BG-tag \< 0.8) -- low (not detected). Class 2 (BG-tag between 0.8 and 0.9) -- medium (boarderline detection) and Class 3 (BG-tag \> 0.9) -- high expression (readily detectable) The number of genes (X-axis) within each class (Y-axis) is also shown in the figure (black = bovine, white = human). ::: ![](1471-2164-5-83-2) ::: Additionally, we computed MA-plots \[[@B14]\] in order to detect artificial dependencies of the log-ratio across the signal range. Figure [2B](#F2){ref-type="fig"} illustrates a typical result from a specific hybridisation experiment. The horizontal lines mark the two-fold levels of over-expression in bovine and human, respectively. A clear observation is that a few of the 349 genes under investigation fall outside these thresholds with the expression of 5 genes being more than 2-fold over-expressed in bovine and 16 genes more than 2-fold over-expressed in human. However, there is no increase or decrease in fold-change for the vast majority of genes and this is fairly stable across the signal range. In order to measure whether a given gene was significantly expressed, we compared its cDNA\'s signal to a signal distribution derived from negative controls represented by approx. 2,500 empty spot positions on the array. After quantification of each array a low non-zero intensity is assigned to each of these empty spots reflecting the amount of background signal on the array. Since these positions are spread uniformly over the array, the distribution of these signals reflects the distribution for signal noise and is an indicator whether signals are at the background level or reflect reliable expression levels. For each cDNA we counted the relative proportion of empty positions on the array that are smaller than the actual observed intensity (BG-tag). BG-tags from replicated experiments for the same cDNA were averaged. Thus, high values (close to one) indicate that the cDNA is expressed in the respective tissue whereas low values reflect noise. The limit of visual detection of a spot corresponds to a BG-tag level of 0.9, however there is a grey zone around this value. Comparison with RT-PCR analyses showed \[[@B15]\] that this level is consistent with the limit of detection at the 25^th^cycle of a standard PCR reaction. cDNAs were considered as \"detected\" when their average BG-tag was above 0.9. We grouped the 349 genes into three classes of expression levels with respect to their intensity values above background levels (i.e, BG-tag) with class 1 as low (not-detected), class 2 as medium (possibly detected) and class 3 as high level of expression (detected), respectively. Within class 1, are 19 bovine and 17 human genes. Class 2, comprises 26 bovine and 17 human genes and finally class 3 has 304 bovine and 315 human genes, respectively. The genes within expression class 1 have signal intensities below the detection level of our microarray analysis platform and as such these genes can be designated as \"Absent\" (See Figure [2C](#F2){ref-type="fig"} for a graphical illustration). A list of all the intensity values is given (see [Additional file 1](#S1){ref-type="supplementary-material"}). Data reproducibility and species variability -------------------------------------------- An essential criterion for the applicability of cross-species experiments is the data reproducibility. For example, if human probes hybridise to bovine mRNA with far less reproducibility than to human mRNA high changes in expression of bovine tissues can be observed that are solely due to technical variability. In order to test whether the variability in gene expression levels is conserved within both species or in contrast, is higher in the bovine than in the human hybridisations, we calculated the coefficient of variation (CV) across the replicated experiments. Histograms are shown in Figure [3A](#F3){ref-type="fig"}. We then defined four classes of CVs for the genes (highly reproducible signals -- CV \< 0.25, good reproducibility -- 0.25 \< CV \< 0.5, medium reproducibility -- 0.5 \< CV \< 0.75 and poor reproducibility CV \> 0.75). Note that a CV of 1.0 indicates that the signal standard deviation is in the order of the signal itself and therefore no meaningful statement on the measurement can be made. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Data reproducibility and species variability(A) Histograms of CVs for human and bovine hybridisations. (B) Classification of signal variability in measured gene expression intensities. Human (white), bovine (grey) and mixed samples (black). CVs (co-efficient of variation) were calculated for repeated signal intensities of the four independent hybridisations and then sub-divided into four classes: Class 1 -- CV \< 0.25 high reproducibility, Class 2 -- CV \[0.25 -- 0.5\] good reproducibility, Class 3 -- CV \[0.5 -- 0.75\] medium reproducibility Class 4 -- CV \> 0.75 poor reproducibility. ::: ![](1471-2164-5-83-3) ::: Figure [3B](#F3){ref-type="fig"} depicts the number of genes that fall within the respective CV-classes when analysing all experiments (human and bovine- black), human only (white) and bovine only (grey). There is a 10% decrease in the number of reproducible genes when comparing human and bovine but the overall effect is similar. Approximately 305 genes show a CV\<0.5 with human brain (87%) compared to 280 with bovine brain (80%). Only 15 genes (4%) show high variability across the experiments for human brain compared to 20 genes (5.7%) for bovine brain. Figure [3B](#F3){ref-type="fig"} illustrates that there is a slight but not dramatic increase in variability when performing cross-species hybridisation (the number of variable genes (class 3 and 4) increase by a factor of 1.5). However, for the vast majority of the genes the reproducibility is similar. The observed effect is not due to exact cutoff values set for the different CV-classes since a slight shifting of the class borders leads to the similar results. For example, shifting the borders about a slight factor of ϑ = ± 0.05 to the left/right respectively gives approximately the same factor of increase for variable bovine to variable human genes. Differential gene expression in bovine and human fetal brain ------------------------------------------------------------ Three statistical tests that judge the significance of differences in the levels of gene expression in human and bovine fetal brains were employed (Student\'s t-test, Welch-test and, Wilcoxon\'s rank-sum test) as described previously \[[@B16]\]. Tests were carried out with the four independent experiments in bovine and human brain, respectively. Note that with independent experiments we mean the independent technical replicates since we do not employ different biological replicates in our study. Messenger RNA levels of seven genes were significantly (p \< 0.05 with Student\'s t-test and Welch test) different between the two species, whilst four genes were found to be differentially expressed only with the Wilcoxon test (p \< 0.05). This emphasizes that the Wilcoxon test is more conservative than the parametric tests. In contrast to the two parametric tests that assume a specific parametric signal distribution (Gaussian distribution) for the underlying signal series, the Wilcoxon test is non-parametric. Thus, P-values calculated by this test are valid in a more general set-up, i.e. for larger classes of probability distributions, than with the other tests. Furthermore, since the Wilcoxon test is based on ranks rather than on the underlying signals it is a more robust procedure in the sense that it is less sensitive against outliers from the model assumptions. These four genes are, ZNF278, APOARGC, KIAA1609and MGC12904-- highlighted as diamond shapes in Figure [2A](#F2){ref-type="fig"}. The expression levels of the vast majority (98%) of the genes remained unchanged. Furthermore, taking into account corrections for multiple testing, no gene is differentially expressed at the global experiment significance level of 0.05. For example, the lowest P-value of the Welch test is 1.98e-03, (APOARGC). Thus, Holm\'s setp-wise correction would start with an adjusted experiment level P-value of p = 0.05/349 = 1.43e-04. Similarly, measuring the false discovery rate by qvalues \[[@B17]\] results in no significant differential gene expression. Thus, we conclude that the level of expression of the individual 349 genes under investigation within human and bovine brain is roughly the same. Nucleotide sequence alignments of bovine and human transcripts -------------------------------------------------------------- In order to characterise some of the vast number of unknown bovine ESTs within the various databases we screened for orthologs to the human genes \[[@B18]\]. The 349 known human genes were screened against the TIGR Bos Taurus gene index (BtGI Release 8.0). Using high-quality matches (\>85% identity, \>100 bp overlap, E-value \< 1.0e-15) we were able to ascertain the expression of 316 orthologous genes (see [Additional file 1](#S1){ref-type="supplementary-material"}). Figure [4](#F4){ref-type="fig"} shows the quality of the matches. Of these genes, 16 had sequence identities of greater than 95%, 137 genes with identities between 90% and 95%, 120 genes with identities of 85% to 90%. The remaining genes did not meet the criterion for the assignment as orthologs \[[@B18]\]. Forty genes had identities of 80% to 85%, 3 genes had identities between 75% and 80% and finally 33 genes did not have a significant BLAST hit. These matches are considered to be insignificant and therefore implying that these 33 human genes (assigned values (0) in [Additional file 1](#S1){ref-type="supplementary-material"}) do not as yet have their bovine homologs present in the current bovine databases. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Nucleotide sequence alignments of bovine and human transcriptsAll 349 human genes were matched against the TIGR Bos taurus gene index, BtGI Release 8.0, which contains 87,257 unique sequences. (A) Histogram of %-identities with the best match. (B) Histogram of bp overlap with the best match. ::: ![](1471-2164-5-83-4) ::: Employing a comparative genomics approach we have functionally annotated previously uncharacterised bovine ESTs. These genes are depicted in the Supplemental Table as bovine ESTs lacking a gene name or description in the TIGR Bos Taurus gene index. As an example, the gene SLC11A3which encodes a protein which functions as a solute carrier has 92% nucleotide sequence identity with an overlap of 500 bp with its bovine orthologue. Additionally, we have demonstrated co-amplification of this transcript in both human and bovine fetal brain RNA using primers derived from the bovine sequence -- Figure [5A](#F5){ref-type="fig"} and Table [1](#T1){ref-type="table"}. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Independent verification of array results by semi-quantitative RT-PCR(A) Ethidium bromide stained gel illustrating independent verification of differential gene expression by semi-quantitative RT-PCR. PCR reactions were carried out in 50 μl volumes, loaded in the order H- (human), B- (bovine) and VE- (water as negative control) then resolved on a 2 % agarose gel as described in Materials and Methods. Each panel consist of genes of the same functional category -- listed on the left hand side. A 100 bp ladder (Invitrogen) was used to confirm the sizes (Table 1) of the PCR products. (B) A graphical illustration of the comparison of the expression levels of the orthologous genes as deduced by the microarray analysis and semiquantitative RT-PCR. ::: ![](1471-2164-5-83-5) ::: Verification of expression levels of cross-hybridised orthologous genes by semi-quantitative RT-PCR --------------------------------------------------------------------------------------------------- Semi-quantitative RT-PCR was used to confirm the deduced expression data generated by the high stringency cross-species hybridisation. We selected a set of genes belonging to distinct families based on their published functional annotation, for example, cell cycle (CDC27, CDC6and PCNA), solute transport- (SLC11A3), protein assembly- (CALNEXIN), anion channel- (VDAC2) and as an endogenous reference, the housekeeping gene, β-ACTIN. Primer sequences were designed to co-amplify the orthologous genes (Table [1](#T1){ref-type="table"}). The RT-PCR analyses (Figure [5A](#F5){ref-type="fig"}) using a common set of gene-specific primers clearly demonstrate co-amplifcation of the orthologous transcripts and, in addition, differences in expression levels between genes within the same species are discernable. For example, the house keeping gene β-ACTINand CALNEXINwhich is involved in protein assembly are more abundant than for examples genes involved in cell cycle control. Most importantly, the trends in the deduced ratios (see [Additional file 1](#S1){ref-type="supplementary-material"} and Figure [5B](#F5){ref-type="fig"}) of expression levels of orthologous genes from the hybridisation analysis are confirmed by the RT-PCR assay. Discrepancies in the bovine-human ratio with array data and the semi-quantitative RT-PCR data are also observable. Furthermore, some of the genes (for example CDC6and VDAC2) are expressed at a low level (expression class 1, compare Figure [2C](#F2){ref-type="fig"}). Here, microarray measurements are less reliable than PCR-based measurements and one comes close to the borderline of the detection limit of microarrays. Discussion ========== Customised and focussed microarrays containing orthologous genes related to function, tissue, or pathways are becoming widely adopted for studying mRNA expression patterns. In addition, the level of cross hybridisation between genes with high sequence identity is also of interest because arrays are not always available for mammalian species other than human and rodents so cross-species hybridisations are often carried out \[[@B19],[@B20]\]. In view of this, it is crucial to know whether the hybridisation conditions (i.e. the stringency used for the hybridisation and subsequent washes) would enable identification of altered gene expression across species. Here we have demonstrated cross-hybridisation of orthologous transcripts by adopting a high stringency hybridisation and wash protocol. The overall correlation co-efficient of gene expression in the fetal brains of human and bovine was 0.94, with the regression line at a slope of 1.13 (Figure [2A](#F2){ref-type="fig"}). This suggests that the 349 genes under investigation have rather similar expression levels as judged by the intensity values of the human and bovine genes across the replicated experiments. The use of replicate experiments is clearly essential in order to ascertain true expression change. For example, judging single experiments we find a couple of genes escaping the 2-fold bounds. The MA-plot \[[@B14]\] shown in Figure [2B](#F2){ref-type="fig"} suggests that of the 349 genes, 5 are over-expressed more than 2-fold in bovine and 16 are more than 2-fold over-expressed in human. However, most of the changes are due to experimental variability of this specific experiment. An extremely important aspect of cross-species hybridisations is data reproducibility. A poor hybridisation experiment would lead to a high variability in the respective replicate experiments in human and bovine. With regards to this aspect, we identified fifteen genes (4%) in human and twenty genes (5.7%) in bovine with variability in expression levels in the four hybridisation experiments. In addition, approximately 305 genes have a CV of less than 0.5 with human brain (87%) compared to 280 with bovine brain (80%). However, this level of variability is low and can be expected due to the fact that the probe set used are of human origin. Moreover, the slight decrease in reproducibility due to species and nucleotide sequence differences can be compensated for by increasing the number of independent repetitions (biological and technical replicates) of the experiments. The unexpectedly low level of variability in gene expression levels between human and bovine fetal brain can also be attributed to the 80 data points examined per gene and the four replicate hybridisations (technical replicates) carried out for each species. Our finding further emphasises the need for sufficient technical and biological replicates in all microarray experiments. In assessing differential gene expression in bovine and human fetal brain, we observed that the expression levels of the individual orthologous genes were roughly in the same broad range. For example, if one considers genes involved in pathways of cell cycle control, the microarray deduced ratios (bovine:human) were for CDC27: 0.95, CDC6: 1.36, PCNA: 1.07, respectively, whereas the deduced ratios derived from the semi-quantitative RT-PCR assay were 0.59 for CDC27, 0.35 for CDC6and 0.87 for PCNA, respectively. Similarly the comparative ratios for SLC11A3and CALNEXINare 0.65 vs. 0.98 and 0.88 vs. 0.97. An illustration of co-amplification with common primers and also the confirmation of the array data for a selection of genes can be seen in Figures [5A](#F5){ref-type="fig"} and [5B](#F5){ref-type="fig"}. The expression ratios of all the other genes under investigation are given (see [Additional file 1](#S1){ref-type="supplementary-material"}). Though co-amplification is possible by semi-quantitative RT-PCR, there is amplification bias related to primer mismatch (See Table [1](#T1){ref-type="table"}). This effect was further highlighted when primer efficiencies were calculated using comparative Real-Time PCR (data not included). An important difference between the two techniques is that cDNA array hybridisation is less sensitive to minor mismatches than PCR primer annealing. The bias in amplification specificity is not a drawback in this study and species specific primers would obviously be used to compare gene expression levels in an experiment in which a developmental/metabolic pathway or disease model is investigated. The similar expression ratio of the orthologous genes implicated in cell cycle control and initiation of eukaryotic genome replication which is conserved in all eukaryotes highlights the feasibility and importance of our study in examining conserved pathways operative within the fetal brains of human and bovine and of course across other species using human cDNA microarrays. A similar finding has also been confirmed when comparing yeast co-regulated genes against the archaeal and bacterial operons. This implies that the components of the protein translation process are conserved across organisms at the expression level with minor specific differences \[[@B21]\]. We also identified four significantly differentially expressed genes as judged by the Wilcoxon test. The Wilcoxon test is known to be more conservative than the Student\'s t-test and the Welch-test, however, we have more confidence in this test since it is distribution-free, in particular it does not depend on the partly unrealistic assumption of an underlying Gaussian distribution. The genes ZNF278and APOARGCwere 1.45 -- and 1.66 -fold over-expressed in human whereas KIAA1609and MGC12904were 1.60 -- and 1.37-fold over-expressed in bovine. ZNF278encodes a zinc finger-containing transcription factor that acts as a transcriptional repressor and also implicated in small round cell tumours \[[@B22]\]. The gene APOARGCencodes a protein with hydrolase activity. MGC12904and KIAA1609are uncharacterised ESTs so comments cannot be made with regard to their function. Orthologous genes between human and mouse and between human and rat both have a mean of approximately 85% sequence identity \[[@B18],[@B23]\]. In two independent and unrelated studies carried out on cDNA and 50-mer oligonucleotide microarrays, cross-hybridisation was only observed with genes with 70%-80% and 50%-75% overall sequence identity, respectively \[[@B24],[@B25]\]. With respect to these studies, our data unequivocally confirm the feasibility and reproducibility of cross-species hybridisation of orthologous genes within defined developmental and metabolic pathway(s) operative in human and bovine fetal brains. In addition, we have been able to assign gene names to previously uncharacterised bovine ESTs, thus, highlighting the importance of comparative genomics in identifying orthologous genes across species. Furthermore, using this protocol of cross-species hybridisation, we have compared gene expression in bovine unfertilised oocytes and blastocyst using a larger microarray (The Human Ensembl Chip) comprising 15,500 fully sequenced and annotated genes and ESTs. The correlation co-efficient between the RNA samples is 0.26, thus reflecting the diversity between oocyte derived maternal transcripts and embryonic transcripts derived from the blastocyst (Adjaye et al., unpublished). Conclusions =========== In summary, this study highlights the significance and utilisation power of comparative genomics and also demonstrates the feasibility of using human cDNA microarrays to facilitate the identification of differentially expressed genes in human and bovine fetal brain. Our results indicate that cross-species hybridisation is not only a useful short-term solution for studying species for which gene maps, cDNA or oligo microarrays are not yet available, but also possesses tremendous power in enabling the unravelling of common evolutionary evolved mechanisms in different species. Methods ======= Microarray fabrication ---------------------- For the generation of probes for spotting, cDNA inserts from a single 384-well plate of non-redundant, fully sequenced, annotated human cDNA collection (Human Ensembl set RZPD1.1) were employed- (BenKahla et al., unpublished) and a second partial plate consisting of control genes and empty wells to be used for data normalisation were amplified by PCR in a 384-well format. Bacterial clones were transferred to the PCR plates using 384-well replicators (Genetix Ltd, New Hampshire, UK). PCR amplifications were carried out in a total volume of 25 μl consisting of 1x PCR buffer, 1.5 M Betaine (Sigma, Germany), 1U Taq polymerase, 200 mM of each dNTP (Invitek, Germany), 25 pmoles of M13-forward (5\'GTAAAACGACGGCCA3\') and M13-reverse (5\'CAGGAAACAGCTATGAC3\') primers respectively. Cycling parameters typically consisted of an initial denaturation step at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 55°C for 1 min, elongation at 72°C for 1 min, then a final elongation step at 72°C for 10 min. All amplified products were analysed by agarose gel electrophoresis and in all cases single bands were obtained per gene. Purification was carried out using isopropanol precipitations. In brief, 24 μl of the PCR product was transferred into 384-well Genetix plates (Genetix Ltd, New Hampshire, UK) previously filled with 18 μl of isopropanol per well. The samples were mixed by gentle vortexing after sealing the plates with transparent tape. After precipitation overnight at -20°C, the plates were spun at 3,500 rpm for 2 hrs at 21°C, the isopropanol decanted off and the plates dried briefly in a SpeedVac without heating. The pellets were resuspended in 6 μl of spotting solution (3x SSC /1.5 M Betaine (N, N, N-trimethylglycine; Sigma, Germany)). Random sampling of cDNAs revealed concentrations ranging between 200 to 300 ng/μl. Finally the samples in each well were spotted twenty times (for signal averaging) with four slit pins arranged in a 2x 2y printhead (Chipmaker 2 pins, Telechem, Sunnyvale, CA, USA). Prior to every spotting run the performance of all components of the robot is carefully tested -- in particular the performance of the washing station in test runs designed to detect possible sample carry over. A modified Genetix Q-array (Genetix Ltd, New Hampshire, UK), controlled by a novel in-house software was used for the arraying of the samples on SuperAmine™ aminosilane-coated microscope slides (Telechem, Sunnyvale, Ca, USA). Post-processing of the arrays was performed using 1,2-dichloroethane and the acylating catalyst N-methylimidazole following protocols described previously \[[@B26]\]. Isolation of total RNA ---------------------- Human brain RNA was isolated from a medically terminated fetus at 10 weeks gestation. This was provided by the MRC-funded Human Embryonic Tissue Bank maintained at the Institute of Child Health and University College London, England. Bovine fetal brain was obtained from a 3--4 months old bovine fetus at a local abattoir near Mariensee, Germany. Immediately after slaughter of the pregnant female, the tissue was plunged into liquid nitrogen and transported to the laboratory. Brain tissues were homogenised using a Dounce homogeniser, total RNA isolated using TRIzol reagent (Invitrogen) and further purified using phenol/chloroform extractions and precipitation with ethanol after DNase 1 (Promega) treatment. All procedures were as described by the manufacturers. RNA Purity, integrity and concentrations were evaluated on the Agilent 2100 Bioanalyzer. High quality RNAs with A260/A280 ratio over 1.8 with intact ribosomal 28S and 18S RNA bands were utilised for subsequent labelling reactions. Direct labelling of RNA and hybridisations ------------------------------------------ Four independent labelling (including dye-swaps) reactions per species were carried out using 25 μg total RNA from both human and bovine per labelling reaction. Direct incorporation of Cy3 and Cy5 during reverse transcription was carried out in a 20 μl reaction volume using 1 μg of anchored oligo-dT primer. The RNA/primer mix was incubated at 70^°^C for 5 min., left at room temperature for 10 min and then cooled on ice for 2 min. The following reagents were then added: 4 μl first-strand buffer, 2 μl 0.1 M DTT, 0.5 μl dNTP mix (25 mM each of dATP, dGTP, dCTP and 10 mM dTTP), 1 μl of 1.0 mM Cy5- or Cy3-dUTP (Amersham Pharmacia) and 1 μl (200 U/μl) Superscript II (Invitrogen). The labelling reaction was carried out at 42^°^C for 1.5 hrs. The reaction was stopped with 4 μl of 0.5 M EDTA. The input RNA was hydrolysed by the addition of 2 μl of 2.5 M NaOH and incubated at 37^°^C for 15 mins. followed by neutralization with 10 μl HEPES free acid (2 M, pH 5.5). Labelled cDNAs (four replicates per species which included dye-swaps) were purified from unincorporated Cy-dyes using Microcon YM-30 purification columns (Millipore). All labelled cDNAs were routinely analysed on a Fuji scanner (FL8-8000) and by ethidium bromide staining to ascertain dye incorporation and size-range of synthesized cDNAs. After concentrating cDNAs by evaporation in a SpeedVac, labelled targets were resuspended in 20 μl of hybridisation buffer (10 μg polydA and 20 μg Human Cot1 DNA,-Invitrogen; DIG-Easy Hybridisation mix -Roche). After thorough resuspension, the cDNA was denatured by heating at 95^°^C for 5 mins followed by 20 mins. at 42^°^C to enable annealing of the blocking reagents to repetitive sequences within the target cDNAs. The hybridisation mixture consisting of Cy3-labelled bovine and Cy5-labelled human RNA and vice versa was placed on the blocked array under a 24 × 40 mm coverslip (Menzel-Glaser, Germany). To maintain humidity inside the chamber, 20 μl of 3x SSC was added to the reservoir wells. The chamber was then tightly sealed and slides incubated at 42^°^C for 18 hrs in a waterbath. Slides were washed twice in 0.2x SSC / 0.1% SDS and then twice in 0.2x SSC. Washes were carried out at room temperature with 10 min durations per wash. Finally, the slides were dried by centrifugation at 1100 rpm for 10 min. Image acquisition and data analysis ----------------------------------- Fluorescence images were captured using an Affymetrix 428 scanner (Affymetrix, Santa Clara, CA) with appropriate gains on the photomultiplier tube (PMT) to obtain the highest intensity without saturation. A 16 bit TIFF image was generated for each channel for subsequent image analyses. Image analysis was carried out by placing the centre of each spot manually (grid-finding step) using the software AIDA (Raytest-Germany) and then by quantifying in a pre-defined neighbourhood around this spot centre using a two-dimensional Gaussian distribution (quantification step). Data analysis comprised two distinct parts. In the first part, data were normalised to eliminate extrinsic influencing factors and artefacts not attributable to the probe-target interaction. For each tissue (human and bovine brain) under analysis the whole batch of experimental replicates was normalized simultaneously. Normalisation should eliminate multiplicative technical bias between the different experiments and result in the same median signal level for each experiment. In a first step, the local background of each spot was subtracted from the spot\'s signal intensity. Then, for each experiment jthe median signal, med~j~, was computed and a multiplicative factor was calculated according to med~ref~/med~j~, where med~ref~is the global derived from the average intensity values of the cDNAs across the full batch of experiments. The multiplicative factor was used to adjust the signal of the ith cDNA in the jth experiment, x~ij~, by x~ij~\* med~ref~/med~j~. In the second part, a number of numerical characteristics were calculated in order to quantify the cross-species comparison. These characteristics were signal detection value, signal reproducibility and statistical significance of differential expression. Signal detection was judged by a number of empty positions that were spread across the array. For each experiment, the proportion of signals of empty positions lower than the actual spot signal was calculated. Then across all repetitions, the average proportion was kept as the signal detection value. Employing this procedure, a signal strength was quantified for each cDNA. Signal reproducibility was judged by calculating the co-efficient of variation (CV) for each cDNA across all experiments. In order to judge differential expression of a gene in human and bovine brain we calculated three statistical tests: Student\'s t-test, Welch-test and Wilcoxon\'s rank sum test for each cDNA based on the signal series derived from human and bovine experimental repetitions. The three tests make different assumptions on the signal series. Whereas the first two assume that the series are Gaussian distributed, the Wilcoxon test is distribution-free. Low P-values calculated by these tests indicate significant differences in signal intensity within the human and bovine samples \[[@B15]\]. Semi-quantitative RT-PCR analysis --------------------------------- The list of primers, annealing temperatures and genes under investigation is shown in Table [1](#T1){ref-type="table"}. For primer annealing and reverse transcription, 1.0 μl (2.5μg/μl) of DNase1 treated human and bovine total RNA was added to 2.0 μl (50 μM) Oligo-dT primer plus 9.0 μl of RNAse free water. The mixture was spun briefly and heated to 70^°^C for 5 mins and cooled on ice. Thereafter, the following components were added sequentially, 4.0 μl of 5x RT. Buffer (Invitrogen), 2.0 μl of 0.1 M DTT (Invitrogen) 1.0 μl of (10 mM) dNTP (Amersham) and 1.0 μl (200 U/μl) Superscript II (Invitrogen). After pulse spinning, incubation was carried out at 42^°^C for 1.5 hrs. Gene-specific PCR amplifications were carried out in a total volume of 50 μl consisting of 5 μl of 10x PCR buffer, 0.2 μl (5 U/μl) Taq Polymerase, 1.0 μl (10 mM) dNTP, 2.5 μl of each (20 μM) primer, 1.5 μl (50 mM) MgCl~2~, 2.0 μl of the first strand cDNA (equivalent to 125 ng of input RNA) and distilled water to 50 μl. All reagents were purchased from Invitrogen. Cycling parameters consisted of an initial denaturation step at 95°C for 5 min followed by 30 cycles of denaturation at 95°C for 30 sec, annealing for 30 sec, elongation at 72°C for 30 sec, then a final elongation step at 72°C for 5 min. Primer sequences, annealing temperatures and predicted size of cDNA products are shown in Table [1](#T1){ref-type="table"}. The amplification reaction was carried out in a PTC 200 PCR machine (MJ Research). After PCR amplification, 50 μl of the reaction products were resolved on 2.0 percent agarose gel containing 0.2 μg/ml ethidium bromide. The gel was placed on a U.V. transilluminator (UVP Model TFM-20, Ultra-Violet Products Ltd., Cambridge, U.K.) with 25-watt UV tubes for high fluorescence and high sensitivity on stained gels), and imaged with a Photometrics Quantix 1401E, 12-bit cooled CCD camera (Roper Scientific GmbH, Ottobrunn, Germany). Densitometric analysis of the digitized image was performed with the IPLab-Gel program from Scanalytics (Fairfax, VA, U.S.A.). The semi-quantitative RT-PCR assay provides sensitive and reliable results, (26). The linear range of amplification was determined by product quantification after different cycle numbers. Identical amounts of bovine and human mRNA were amplified with the same number of PCR cycles and were always placed next to each other on the gel. Each RT-PCR was repeated at least three times for each gene. The housekeeping gene β-ACTINwas used as endogenous control. Authors\' contribution ====================== J.A. conceived the study\'s idea, designed and optimised the protocols, carried out the hybridisations and was pivotal in developing the analysis plan and writing the manuscript. R.H. developed the analysis plan, analysed the data and was pivotal in writing the manuscript. D.H and M.N designed the cross-species primers and carried out the semi-quantitative RT-PCR. W.W. performed the initial image analysis. A.BK. selected the clones used as probes for creating the Chip and carried out the assignment of orthologous genes. T.B. assisted in the semi-quantitative RT-PCR analysis J.W.C. was involved in the conceptualisation and writing C.H. was instrumental in the Chip design and production. H.N. is the Head of the Department of Biotechnology at the Institute for Animal Science and was involved in the conceptualisation and writing. H.L. is the Head of the Department of Vertebrate Genomics at the Max Planck Institute for Molecular Genetics. Supplementary Material ====================== ::: {.caption} ###### Additional File 1 Summary of the expression data and the corresponding gene annotations. The terms used under each column are explained viz; IMAGE ID: Gene specific identifier assigned by the IMAGE consortium Gene: HUGO gene name Chromosome: Chromosomal location of gene Description: Gene description Molecular Function: Assigned function by the Gene Ontology Consortium BG-tag-bovine: Signal detection probability in the bovine samples BG-tag-human: Signal detection probability in the human samples CV-bovine: Co-efficient of variation between replicate signal intensities CV-human: Co-efficient of variation between replicate signal intensities Mean-bovine: Mean signal across all hybridisation experiments using bovine RNA log-Mean-bovine: Logarithm (base 2) of the mean value Mean-human: Mean signal across all hybridisation experiments using human RNA log-Mean-human: Logarithm (base 2) of the mean value Ratio(bovine vs human): Ratio of the mean values log2(Ratio): Log-ratio (base 2) of the mean values P-value-Student\'s-t-test: P-value of Student\'s t-test using the replicate experiments P-value-Welch-test: P-value of the Welch test using the replicate experiments P-value-Wilcoxon-test: P-value of Wilcoxon\'s test using the replicate experiments Match: A BLAST hit with E-value \< 1.0e-15 was found (1) or not (0) Homology: Classification of sequence homology between human gene sequence and bovine ESTs. \"ortholog\" = best match in both directions, \"paralog\" = bovine EST has its best match with another human sequence. TIGR-BTGI0-51503- Matches: Best results of the BLAST matches using the Ensembl- annotated gene sequence with the TIGR- database Identity: %-identity of best match Overlap: Overlap in base pairs ::: ::: {.caption} ###### Click here for file ::: Acknowledgements ================ We are greatful to Dr Marie-Laure Yaspo for reading the manuscript and providing valuable comments and suggestions, the microarray support staff and the RZPD for clone picking and sequencing. This work was funded by the German Ministry for Education and Research (BMBF) as part of the National Genome Research Network (NGFN).	PubMed Central	2024-06-05T03:55:50.009244	2004-10-28	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC535340/", "journal": "BMC Genomics. 2004 Oct 28; 5:83", "authors": [ { "first": "James", "last": "Adjaye" }, { "first": "Ralf", "last": "Herwig" }, { "first": "Doris", "last": "Herrmann" }, { "first": "Wasco", "last": "Wruck" }, { "first": "Alia", "last": "BenKahla" }, { "first": "Thore C", "last": "Brink" }, { "first": "Monika", "last": "Nowak" }, { "first": "Joseph W", "last": "Carnwath" }, { "first": "Claus", "last": "Hultschig" }, { "first": "Heiner", "last": "Niemann" }, { "first": "Hans", "last": "Lehrach" } ] }
PMC535341	Background ========== Pregnancy outcomes are not only important measures of health status of the mothers and infants, but are important measures of socioeconomic development in a society. For example, infant mortality has been considered the single most comprehensive measure of the health and wealth in a society \[[@B1],[@B2]\]. Both maternal and paternal characteristics, such as age, race, marital status, education, and cigarette smoking \[[@B3]-[@B5]\], are important determinants of pregnancy outcomes. However, partner information is often missing in routine vital statistics data. For example, about 17% of United Status birth certificates contain no paternal information and more than 40% of the babies born to adolescent women have no information on the paternal age in the birth certificate \[[@B6]\]. There might be important differences in the pregnancy outcomes between mothers with versus those without paternal information. Detailed description of these differences may benefit the clinicians and public health workers who serve high risk pregnant women. We conducted extensive search of the literature and have not been able to locate a single study on this issue. Epidemiologic studies based on a data with missing information on exposure, outcomes, and potential confounders can bias the study results, unless the missing occurred randomly \[[@B7]\]. Previous studies on missing data \[[@B8]-[@B11]\] have focused on the impact of missing information on study results and the methods to treat the missing variables. The main objective of the current study is to describe the distribution of maternal characteristics and pregnancy outcomes in mothers whose partner\'s information was partly or totally missing. We also attempted to assess the potential source of missing partner information and its impact on outcomes, by exploring the reasons of differences between partly missing versus totally missing. We used a large twin registry data in the United States to examine this issue. This data has been used in several peer-reviewed studies \[[@B12]-[@B14]\]. The reasons of using data on twins were two folds. First, adverse pregnancy outcomes are more common in twins than in singletons. Therefore, it is easier to detect the difference in adverse pregnancy outcomes in twins than in singletons. Second, because linkage for multiple births requires an examination and processing of the recorded variables, it will increase the validity of the administrative data. Methods ======= We used the matched multiple birth file created by the National Center for Health Statistics (NCHS), Centers for Disease Control and Prevention \[[@B15]\]. The multiple birth file in 1995--1997 in the United States were linked infant deaths information according to uniform coding specifications, which have undergone vigorous editing and reviewing during the process of the record linkage. Sets of multiples in the 1995--1997 birth file were matched by plurality, state and county of occurrence of delivery, mother\'s date of birth, date of last menstrual period (LMP), number of prenatal visits, level of education, weight gain during pregnancy, and date of delivery. The matching was successful for 98% of the multiple sets \[[@B15]\]. Available study variables in the database include socio-demographic information of the parents, maternal life-style factors such as smoking during pregnancy, obstetric history, complications of the pregnancy, labor and delivery, birth weight, gestational age, and other infant outcome variables. Only complete twin sets were included in the current study, with twin set as the unit of analysis in comparison of maternal characteristics and outcomes, and randomly selected one twin in each twin sets as the unit of analysis for fetal and neonatal outcomes. There were two variables for the partners in the data: partner\'s age and race. The eligible study subjects were divided into three groups according to the availability of the two variables of the partners: both available (group 1), one missing (group 2), and both missing (group 3). We first described the distribution of the maternal characteristics (age, race, marital status, education, cigarette smoking, place of birth, and prenatal care service) of the three study groups. The prenatal care service was defined as adequate, intermediate, and inadequate according to the time of prenatal care visit initiation and the number of prenatal visit using the method described by Kessner et al \[[@B16]\]. If the first prenatal care initiated at third trimester, or the times of prenatal visit less than 5 in 34 gestation weeks, 4 in 32--33 weeks, 3 in 30--31 weeks, or 2 in 22--29 weeks, the prenatal care service was defined as inadequate; if the first prenatal care initiated at first trimester, and the times of prenatal visit more than 8 in 36 gestation weeks, 7 in 34--35 weeks, 6 in 32--33 weeks, 5 in 30--31 weeks, 4 in 26--29 weeks, 3 in 22--25 weeks, or 2 in 18--21 weeks, the prenatal care service was defined as adequate; and the remainders were defined as intermediate. We compared the rates of maternal medical complications (anemia, cardiac disease, acute or chronic lung disease, diabetes, genital herpes, hydramnios, oligohydramnios, hemoglobinopathy, chronic hypertension, pregnancy-associated hypertension, eclampsia, incompetent cervix, renal disease, RH sensitization, and uterine bleeding), labor and delivery complications (febrile (any temperature reading \>38°C), meconium, premature rupture of membrane (\>12 hours), abruption placenta, plancenta previa, seizures during labor, precipitous labor (2^nd^stage \<3 hours), prolonged labor (2^nd^stage \>20 hours), dysfunctional labor, breech, malpresentation, cephalopelvic disproportion, cord prolapse, anesthetic complications, and fetal distress), and obstetric interventions (cesarean section, vacuum / forceps, and labor inductions) among the three study groups. Finally we compared the rates of fetal and neonatal mortality and morbidity among the three study groups. We used the Chi-square test to test the difference of all rates among the three study groups, used the normal approximate method to calculate the 95% confidence interval of the rates of maternal medical complications, labor and delivery complications, obstetric interventions, and the fetal and neonatal mortality and morbidity. We calculated the relative risk in group 2 and group 3 compared with group 1 for all the outcomes mentioned above. Fetal death was defined as stillbirth weighing 500 grams or more, or if weight was unknown, 20 completed gestation weeks or more. Neonatal death was defined as live-born infant died at 0--27 days of age, and post-neonatal death was defined as those died at 28--364 days of age. Preterm birth (PTB) was defined as gestational age \< 37 weeks. Fetal growth restriction (FGR) was defined as less than 10th percentile of birth weight-for-gestational-age z score. The birth weight-for-gestational-age z score was calculated using the following formula: Z = (observed birth weight - mean of birth weight)/SD Where mean and SD was based on all infants in the database, stratified by gender and gestational week. Low birth weight (LBW) was defined as birth weight \<2500 grams. Low Apgar score was defined as five minute Apgar score \< 7. Anemia was defined as hemoglobin \<13. Other fetal and infant outcomes examined included birth injury, hyaline membrance disease, meconium aspiration syndrome, assisted ventilation (\<30 minutes and \> = 30 minutes), seizures, central nervous system anomalies, circulatory / respiratory anomalies, digestive system anomalies, urogenital system anomalies, musculoskeletal / integumental anomalies, and chromosomal anomalies. Results ======= There were 304466 twins (152233 twin pairs) in the database. Of which, father\'s age and race were recorded in 259302 twins (129651 twin pairs, 85.17%, group 1), father\'s age or race was missing in 6036 twins (3018 twin pairs, 1.98%, group 2), and both father\'s age and race were missing in 39128 twins (19564 twin pairs, 12.85%, group 3). Table [1](#T1){ref-type="table"} describes the distribution of maternal characteristics and prenatal care service among the three study groups. The proportions of teenage, blacks, unmarried, lower education, cigarette smoking during pregnancy, and inadequate perinatal care service were significantly higher in group 2 and (especially) group 3 as compared with group 1 (Table [1](#T1){ref-type="table"}). ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### The distribution (%) of baseline maternal characteristic among three study groups ::: Partner information available (N = 129651) Partner information partly missing (N = 3018) Partner information totally missing (N = 19564) ------------------------- ----------------- -------------------------------------------- ----------------------------------------------- ------------------------------------------------- Age\* \<20 y 5.1 13.7 21.4 20 y- 75.9 72.2 69.4 35 y- 19.0 14.1 9.2 Race\* white 84.0 69.0 46.1 Black 12.1 26.3 51.3 Other 3.9 4.7 2.6 Marital status\* Married 82.6 39.0 4.1 Unmarried 17.4 61.0 95.9 Education\* \<12 y 13.1 28.4 36.1 12 y 30.0 31.2 39.9 13--15 y 24.1 15.2 15.9 16 y 19.6 5.8 2.9 \>16 y 13.2 19.4 5.2 Cigarette smoking\* Yes 8.1 14.9 19.1 No 70.5 55.8 69.5 Not available\# 21.3 29.4 11.4 Prenatal care service\* Adequate 79.4 55.5 54.1 Mediate 17.8 37.0 34.6 Inadequate 2.7 7.5 11.3 Place of birth\* Native 84.5 72.7 88.1 Foreign 15.5 27.3 11.9 \Compared group 2 and group 3 with group 1 with Chi-square test, p \< 0.001 \# The state of California did not send data on smoking, and all births to residents in California were classified as not available. ::: Table [2](#T2){ref-type="table"} compares the rate of maternal morbidity, labor and delivery complications, and obstetric interventions among the three study groups. The rates of anemia, acute or chronic lung disease, hemoglobinopathy, chronic hypertension, eclampsia, renal disease, meconium, premature rupture of membrane, abruption placenta, and precipitous labor were significantly higher, and the rates of cardiac disease, diabetes, RH sensitization, placenta previa, dysfunctional labor, cephalopelvic disproportion, cesarean section, vacuum / forceps delivery, and induction were significantly lower in group 2 and (especially) group 3 than in group 1 (Table [2](#T2){ref-type="table"}). ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Comparison of maternal medical complication, labor complication, and obstetric intervention among three study groups (%, 95%CI) ::: Partner information available (N = 129651) Partner information partly missing (N = 3018) Partner information totally missing (N = 19564) ---------------------------------------- -------------------------------------------- ----------------------------------------------- ------------------------------------------------- Medical risk factors Anemia 2.81 (2.72 -- 2.90) 4.27 (3.55 -- 5.00)\ 4.67 (4.37 -- 4.96)\* Cardiac disease 0.64 (0.60 -- 0.69) 0.40 (0.17 -- 0.62) 0.44 (0.35 -- 0.53)\* Acute or chronic lung disease 0.90 (0.85 -- 0.95) 1.36 (0.95 -- 1.77)\* 1.64 (1.46 -- 1.82)\* Diabetes 3.46 (3.36 -- 3.56) 3.02 (2.40 -- 3.63) 2.15 (1.94 -- 2.35)\* Genital herpes 0.76 (0.71 -- 0.80) 1.06 (0.69 -- 1.43) 0.82 (0.70 -- 0.95) Hydramnios / Oligohydramnios 1.87 (1.80 -- 1.94) 2.95 (2.35 -- 3.55)\* 2.08 (1.88 -- 2.28) Hemoglobinopathy 0.07 (0.06 -- 0.08) 0.17 (0.02 -- 0.31) 0.19 (0.13 -- 0.26)\* Chronic hypertension 0.90 (0.85 -- 0.95) 0.89 (0.56 -- 1.23) 1.20 (1.04 -- 1.35)\* Pregnancy-associated hypertension 7.61 (7.46 -- 7.75) 6.39 (5.52 -- 7.27)\* 7.38 (7.01 -- 7.75) Eclampsia 0.94 (0.89 -- 0.99) 1.39 (0.97 -- 1.81) 1.22 (1.07 -- 1.38)\* Incompetent cervix 0.81 (0.76 -- 0.85) 0.80 (0.48 -- 1.11) 0.72 (0.60 -- 0.84) Renal disease 0.28 (0.25 -- 0.31) 0.56 (0.30 -- 0.83) 0.40 (0.31 -- 0.49)\* RH sensitization 069 (0.64 -- 0.73) 0.53 (0.27 -- 0.79) 0.49 (0.39 -- 0.58)\* Uterine bleeding 1.10 (1.04 -- 1.16) 0.83 (0.50 -- 1.15) 0.97 (0.83 -- 1.10) Complication of labor Febrile 1.36 (1.30 -- 1.43) 1.36 (0.95 -- 1.77) 1.48 (1.31 -- 1.65) Meconium 1.37 (1.31 -- 1.43) 2.09 (1.58 -- 2.69)\* 1.92 (1.73 -- 2.11)\* Premature rupture of membrane (\>12 h) 6.34 (6.21 -- 6.47) 7.19 (6.27 -- 8.11) 7.91 (7.53 -- 8.29)\* Abruptio placenta 1.16 (1.10 -- 1.22) 1.03 (0.67 -- 1.39) 1.44 (1.27 -- 1.61)\* Placenta previa 0.47 (0.43 -- 0.51) 0.50 (0.25 -- 0.75) 0.30 (0.22 -- 0.38)\* Seizures during labor 0.05 (0.04 -- 0.06) 0.07 (0.03 -- 0.16) 0.08 (0.04 -- 0.12) Precipitous labor (\<3 hours) 1.42 (1.36 -- 1.49) 1.29 (0.89 -- 1.70) 1.86 (1.67 -- 2.04)\* Prolonged labor (\>20 hours) 0.56 (0.52 -- 0.60) 0.63 (0.35 -- 0.91) 0.52 (0.42 -- 0.62) Dysfunctional labor 2.34 (2.26 -- 2.42) 1.52 (1.09 -- 1.96)\* 1.97 (1.77 -- 2.16)\* Breech / malpresentation 21.3 (21.0 -- 21.5) 21.4 (20.0 -- 22.9) 21.4 (20.8 -- 22.0) Cephalopelvic disproportion 0.90 (0.85 -- 0.95) 0.76 (0.45 -- 1.07) 0.52 (0.42 -- 0.62)\* Cord prolapse 0.55 (0.51 -- 0.59) 0.56 (0.30 -- 0.88) 0.65 (0.54 -- 0.77) Anesthetic complications 0.07 (0.06 -- 0.09) 0.10 (0.01 -- 0.21) 0.09 (0.05 -- 0.13) Fetal distress 3.20 (3.10 -- 3.29) 3.45 (2.79 -- 4.10) 3.44 (3.18 -- 3.70) Obstetric intervention Cesarean 52.0 (51.7 -- 52.3) 46.1 (44.7 -- 48.2)\* 47.5 (46.8 -- 48.2)\* Vacuum / Forceps 6.76 (6.62 -- 6.89) 5.40 (4.59 -- 6.21)\* 4.46 (4.17 -- 4.75)\* Induction 13.0 (12.8 -- 13.2) 11.9 (10.7 -- 13.0) 10.2 (9.8 -- 10.6)\* \P \< 0.05 for group 2 or group 3 compared with group 1 ::: Table [3](#T3){ref-type="table"} compares the fetal and neonatal mortality and morbidity among the three study groups. The rates of PTB, FGR, LBW, Apgar score \<7, fetal mortality, neonatal mortality, and post-neonatal mortality were significantly higher in group 2 and group 3 than in group 1; the RRs of PTB, FGR, LBW, Apgar score \<7, fetal mortality, neonatal mortality, and post-neonatal mortality ranged from 1.08 to 3.86 in group 2 and group 3 compared with group 1 (Table [3](#T3){ref-type="table"}). The rates of hyaline membrane disease and assisted ventilation (\< or \> = 30 minutes) were also significantly higher in group 3 than in group 1 (Table [3](#T3){ref-type="table"}). However there were no statistically significant differences in the rates of congenital anomalies among the three study groups (Table [3](#T3){ref-type="table"}). ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Comparison of fetal and neonatal mortality and morbidity among three study groups in twins in United States ::: Partner information available (N = 129651) Partner information partly missing (N = 3018) Partner information totally missing (N = 19564) ------------------------------------------ -------------------------------------------- ----------------------------------------------- ------------------------------------------------- ----------------------- ------ General condition PTB (\<37W) 53.5 (53.2 -- 53.8) 57.8 (56.0 -- 59.7)\ 1.08 59.6 (58.9 -- 60.3)\* 1.11 FGR (\<10 percentiles z score) 8.26 (8.10 -- 8.41) 10.9 (9.72 -- 12.0)\* 1.32 12.9 (12.4 -- 13.4)\* 1.56 LBW (\<2500 g) 51.4 (51.1 -- 51.7) 60.0 (58.2 -- 61.7)\* 1.17 64.6 (63.9 -- 65.3)\* 1.26 Apgar score (\<7) 3.23 (3.13 -- 3.32) 5.14 (4.35 -- 5.92)\* 1.59 6.45 (6.11 -- 6.79)\* 2.00 Fetal mortality 0.85 (0.81 -- 0.89) 3.28 (2.65 -- 3.90)\* 3.86 1.59 (1.43 -- 1.75)\* 1.87 Neonatal mortality 1.92 (1.85 -- 2.00) 3.83 (3.12 -- 4.53)\* 1.99 3.90 (3.62 -- 4.17)\* 2.03 Post-neonatal mortality 0.46 (0.43 -- 0.50) 0.95 (0.59 -- 1.32)\* 2.07 1.12 (0.97 -- 1.27)\* 2.43 Abnormal conditions of the newborn Anemia (HGB.\<13) 0.40 (0.36 -- 0.43) 0.35 (0.13 -- 0.57) 0.88 0.46 (0.36 -- 0.55) 1.15 Birth injury 0.16 (0.14 -- 0.18) 0.18 (0.02 -- 0.33) 1.13 0.17 (0.11 -- 0.23) 1.06 Hyaline membrane disease 3.28 (3.18 -- 3.38) 2.88 (2.27 -- 3.49) 0.88 3.87 (3.60 -- 4.14)\* 1.18 Meconium aspiration syndrome 0.09 (0.07 -- 0.11) 0.11 (0.01 -- 0.22) 1.22 0.09 (0.05 -- 0.13) 1.00 Assisted ventilation (\<30 minutes) 3.45 (3.35 -- 3.55) 2.67 (2.08 -- 3.26)\* 0.77 3.87 (3.60 -- 4.14)\* 1.12 Assisted ventilation (\> = 30 minutes) 3.82 (3.71 -- 3.92) 3.23 (2.58 -- 3.88) 0.85 5.04 (4.73 -- 5.35)\* 1.32 Seizures 0.07 (0.05 -- 0.08) 0.18 (0.02 -- 0.33) 2.57 0.10 (0.06 -- 0.15) 1.43 Congenital anomalies Central nervous system anomalies 0.15 (0.13 -- 0.18) 0.20 (0.04 -- 0.36) 1.33 0.14 (0.09 -- 0.19) 0.93 Circulatory / respiratory anomalies 0.43 (0.39 -- 0.46) 0.60 (0.32 -- 0.87) 1.40 0.45 (0.36 -- 0.54) 1.05 Digestive system anomalies 0.11 (0.09 -- 0.13) 0.17 (0.02 -- 0.31) 1.55 0.11 (0.07 -- 0.16) 1.00 Urogenital system anomalies 0.21 (0.19 -- 0.24) 0.23 (0.06 -- 0.40) 1.10 0.17 (0.11 -- 0.23) 0.81 Musculoskeletal / integumental anomalies 0.38 (0.34 -- 0.41) 0.43 (0.20 -- 0.66) 1.13 0.40 (0.31 -- 0.49) 1.05 Chromosomal anomalies 0.10 (0.09 -- 0.12) 0.17 (0.02 -- 0.31) 1.70 0.08 (0.04 -- 0.12) 0.80 \\ P \< 0.05 for group 2 or group 3 compared with group 1; \#: relative risk compared with group 1. ::: Discussion ========== Our large population based study found that the risks of fetal and infant mortality, low birth weight, preterm birth, fetal growth restriction, Apgar score \<7, and the need for mechanical ventilation were increased in infants born to mothers with missing information on partner\'s age or race, especially when both variables of the partner were missing. The women reported no information (or reported only part of the information) on partners appear to have higher prevalence / incidence of most of the maternal morbidity and obstetric complications than in mothers with available information on partners. The poorer pregnancy outcomes observed in women with partner\'s information been partly and (especially) totally missing than in women with available information on partners were due largely to known important socioeconomic factors for adverse pregnancy outcomes. Many of these women were teenagers, black race, unmarried, with low education, high frequency of cigarette smoking, and inadequate prenatal care services. Teenagers \[[@B17]\], black race \[[@B18]\], unmarried \[[@B19]\], low education \[[@B3]\], cigarette smoking \[[@B4]\], and inadequate prenatal care services \[[@B20]\] are known risk factors of adverse pregnancy outcomes. Our findings that the rates of FGR, LBW, Apgar score \<7, hyaline membrane disease, and assisted ventilation were significantly higher in the group with paternal information totally missing than the group of only partly missing suggest that the totally missing group is a particularly high risk group, whereas in the partly missing group, the missing may have occurred in randomly. Exceptions did occur, however. For example, the occurrences of several maternal medical conditions, such as cardiac disorders, diabetes, and RH sensitization, were actually lower in women with no available information on partners (group 2 and 3) than women with available information on partners (group 1). This may, in part, be due to the younger maternal age in groups 2 and 3 than in group 1. The risk of cardiac disorders and diabetes increases rapidly with advancing age \[[@B21],[@B22]\]. Younger mothers are less likely to have had previous pregnancy and therefore less chance for RH sensitization \[[@B23]\]. The rates of obstetric interventions, including cesarean section, vacuum / forceps, and induction delivery, were lower in groups 2 and 3 than in group 1, which could be explained by the younger age of this group as cesarean section is less common in young women \[[@B24]\]. The rates of placenta previa and cephalopelvic disproportion were lower in women in groups 2 and 3 than in group 1, this could be explained by the younger age and lower parity of these groups as placenta previa and cephalopelvic disproportion are less common in younger and lower parity women \[[@B25],[@B26]\]. We do not know why the rates of various congenital anomalies were not higher in infants born to mothers with no available information on fathers, despite their much higher risks of fetal and infant mortality and neonatal morbidity. Our study was based on birth certificates, and the diagnosis of many birth defects may be difficult at birth, especially when there is a lack of access to quality perinatal care services such as high resolution ultrasound \[[@B27]\], although differences in maternal age may play a role here. Our study examined the difference of adverse pregnancy outcomes among three study groups in twins only. It is biologically plausible to apply the study findings to singletons and higher order of multiple pregnancies as well, although the magnitude of the differences may be smaller in singletons and larger in higher order of multiple pregnancies than in twins. Since in the database, there was no indication why a particular variable was not recorded, we can only speculate the reason of missing partner\'s information. Corresponding variables (i.e., age and race) for the mothers, including those mothers with missing information on partners, were completely recorded. These women may have no relationship or only remote relationship with their partners, as suggested by the extraordinarily high unmarried rates (61% for women missing one variable on their partners and 96% for women missing both variables on their partners). As a result, they may have difficulty or for unknown reasons they may be reluctant to provides partner\'s information. The paternal information missing may be a comprehensive measure and may be related with age, education, socioeconomic status, race, religion background, or even the reporting quality in different place. Our study only described the association between the paternal information missing and the adverse pregnancy outcomes. Exploration of the reasons of missing partner information and its relationship with adverse pregnancy outcomes require in-depth analysis of data with relevant information. Conclusions =========== Women with no available information (or partly) on partners appear to have higher risks of developing adverse pregnancy outcomes. For this reason, extra attention for these women and their offspring by health care providers and public health workers is needed. Competing interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= Hongzhuan Tan carried out the data analysis and drafted the manuscript. Shi Wu Wen designed the study and helped in results interpretation. Mark Walker participated in the sequence alignment and participated in the results interpretation. Kitaw Demissie participated in the study design and results interpretation. Pre-publication history ======================= The pre-publication history for this paper can be accessed here: <http://www.biomedcentral.com/1471-2393/4/21/prepub> Acknowledgments =============== Dr. Tan is an International Fellow of the University of Ottawa. Dr. Wen is a CIHR New Investigator. Dr. Walker is a Career Scientist of the Ontario Ministry of Health. We thank Joyce Martin for her cooperation in the database linking.	PubMed Central	2024-06-05T03:55:50.014056	2004-11-13	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC535341/", "journal": "BMC Pregnancy Childbirth. 2004 Nov 13; 4:21", "authors": [ { "first": "Hongzhuan", "last": "Tan" }, { "first": "Shi Wu", "last": "Wen" }, { "first": "Mark", "last": "Walker" }, { "first": "Kitaw", "last": "Demissie" } ] }
PMC535342	Introduction ============ Platelet activation and aggregation play an important role in the development of cardiovascular disease, which is the leading cause of death in the United States. Over the last several years, enormous advances have been made in understanding the molecular mechanisms that regulate platelet function. A general paradigm has emerged that endothelial cells synthesize and release substances that inhibit platelet activation (e.g. nitric oxide and prostacyclin) except near a site of vascular injury, while platelets synthesize and release substances that promote further platelet activation. In contrast to this paradigm, several recent studies have suggested that, in the presence of certain agonists (IGF-1, adenosine diphosphate, insulin, and isoproterenol), platelets will produce nitric oxide (NO), a potent inhibitor of platelet activation \[[@B1]-[@B5]\]. Although these studies clearly showed that platelets produce NO, the physiological relevance of this NO is not clear since NO is highly reactive, has a short half-life, and the rate of NO production is difficult to quantify. Therefore, there is a need to determine if the NO derived from platelets is sufficient to modulate platelet function and alter thrombus growth in the presence of whole blood. Platelet-derived NO is synthesized by membrane-bound endothelial-type nitric oxide synthase (eNOS). A common signaling mechanism shared by many eNOS agonists is binding to a surface receptor, followed by activation of phosphatidylinositol-3-kinase (PI3K), which results in phosphorylation of eNOS at serine 1179 through Akt \[[@B6]-[@B8]\]. The activated eNOS converts L-arginine to L-citrulline and produces NO as a result \[[@B9]\]. Nitric oxide is a water soluble free radical that can bind to the heme-soluble site of guanylate cyclase in platelets and smooth muscle cells, which increases synthesis of cyclic guanosine monophosphate (cGMP) \[[@B5],[@B10]\]. cGMP can bind to phosphodiesterase III (PDE III), which reduces metabolism of cyclic adenosine monophosphate (cAMP) \[[@B10]\]. Elevated levels of cGMP and cAMP can result in increased activity of protein kinase G (PKG) and protein kinase A (PKA), which inhibit protein kinase C (PKC) activation and intracellular Ca^2+^mobilization \[[@B5]\]. The consequence of this signal transduction cascade is the inhibition of platelet activation and the relaxation of vascular smooth muscle resulting in blood vessel dilation. As a small, hydrophilic free radical NO is highly diffusible in the aqueous environment of the blood. However, it is also highly reactive with a very short half-life estimated to be only on the order of a few seconds \[[@B11]\] in the blood. Previous studies that demonstrated NO production in platelets were done in the absence of erythrocytes \[[@B2]-[@B4]\]. It is still not clear if the amount of NO produced by platelets, estimated at about 5 × 10^-17^mole NO/platelet (determined by microelectrode in PRP for 2 minutes following addition of 5 μM ADP), is sufficient to alter platelet function \[[@B1],[@B2]\]. The purpose of our study was to determine if platelet-derived NO plays a role in thrombus formation in the presence of shear stress. We examined the effect of several external factors on platelet thrombus formation, including insulin, the β-adrenoceptor agonist isoproterenol, and shear stress. Previous studies showed that insulin and isoproterenol both induced NO formation by platelets \[[@B3],[@B4]\]. Shear stress is an important component of the environment of the platelets and can cause alterations in platelet function, although the effect of shear on NO synthesis in platelets is unknown \[[@B12]\]. Our aim was to show that platelet-derived NO plays a direct role in inhibiting thrombus formation to a vascular injury and can be stimulated by different external agonists through a common NO signaling pathway. Results ======= Insulin and isoproterenol slow the growth rate of mural thrombi --------------------------------------------------------------- Platelets from whole blood adhered avidly to the collagen-coated surface in the presence of physiologically relevant levels of fluid mechanical shear stress. Very little, if any, platelets were detected on the albumin-coated portion of the slide. Detectable levels of platelet adhesion were evident within 20--30 seconds of the initiation of blood flow over the surface. Platelet adherence occurred predominantly at the interface between albumin and collagen and increased as a function of time similar to results that have been obtained by others using a similar system \[[@B13]-[@B16]\] Representative images of platelet accumulation on collagen at a shear rate of 1000 s^-1^are shown in Figure [1](#F1){ref-type="fig"}. In these images, the flow was from left to right. The albumin/collagen interface is clearly evident in the images from later time points. At other shear rates, platelet accumulation on the surface was also abundant as illustrated in Figure [2A](#F2){ref-type="fig"}. These results are in qualitative agreement with previously published results \[[@B13]\]. The slower rate of platelet deposition at higher levels of shear has been attributed \[[@B17]\] to the increased level of fluid mechanical drag on the platelets preventing them from forming stable attachments to the surface. ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Representative images of time-dependent platelet adhesion and aggregate formation on collagen type III. Single platelets and platelet aggregates adherent on the surface appear bright and were visualized using epi-fluorescence video microscopy. Platelets adhered abundantly on the collagen surface, particularly near the upstream interface between collagen and albumin. No adhesion was observed on the albumin-coated surface. Flow was from left to right and the shear rate was 1000 s^-1^. These results are typical of 5 separate experiments. ::: ![](1477-9560-2-11-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Dose dependence of thrombus formation. The extent of platelet adhesion onto the surface, as assessed by the percentage of the collagen-coated surface covered by platelets, is shown as a function of time. Perfusions for shear \[100 (■), 1000 (□), 1500 (●), and 2000 (○) s^-1^\] were performed for 5 minutes (Figure 2A). Perfusions for control (■), insulin \[100 (◆) and 1000 pM (◇)\] and isoproterenol \[100 (▲) and 1000 μM (△)\] were performed at 1000 s^-1^(Figures 2B-C) for 6 minutes. The results presented are mean ± s.e.m. of at least 5 experiments using 5 different blood donors. ::: ![](1477-9560-2-11-2) ::: The effect of insulin and isoproterenol on mural thrombus formation was studied and the results are presented in Figures [2B](#F2){ref-type="fig"} and [2C](#F2){ref-type="fig"} as the percent coverage of the collagen-coated surface as a function of time. Increasing the concentration of either insulin (0, 100, 1000 pM) or isoproterenol (0, 100, 1000 μM) at a shear rate of 1000 s^-1^resulted in increasing inhibition of platelet accumulation on the surface. Higher concentrations of either insulin or isoproterenol had no additional affect on the extent of platelet accumulation on the surface (data not shown). Inhibition of thrombus formation was significant (p \< 0.05) up to 6 minutes with 1000 pM insulin and up to 3 minutes with 1000 μM isoproterenol. Significance was determined with SPSS using the Post Hoc test as described in Methods. Agonist induced reduction in thrombus formation depends on NO and shear rate ---------------------------------------------------------------------------- In order to investigate the mechanism of insulin and isoproterenol induced reduction in platelet accumulation on a collagen surface, we perfused blood through the flow chamber in the presence of agonist (either insulin or isoproterenol) as well as L-NAME, a specific inhibitor of nitric oxide production. The results are presented in Figures [3C,3D](#F3){ref-type="fig"} for the shear rate of 1000 s^-1^. In the presence of L-NAME as well as either insulin or isoproterenol, thrombus formation is increased, returning to levels seen in the absence of added agonist. This suggests a role for nitric oxide in mediating the agonist induced reduction in platelet accumulation on the surface. Platelet thrombus formation on the surface at lower (100 s^-1^) and higher (2000 s^-1^) levels of shear rate are shown in Figures [3A,3B](#F3){ref-type="fig"} and [3E,3F](#F3){ref-type="fig"} respectively. At the lower, venous shear rate (100 s^-1^) platelet accumulation on the surface is not significantly altered by either insulin (500 pM) or isoproterenol (100 μM). Higher concentrations of either agonist had no affect as well (data not shown). In addition, L-NAME by itself or in combination with either agonist did not significantly affect platelet deposition on the surface (Figures [3A,3B](#F3){ref-type="fig"}). At the higher, arterial shear rate of 2000 s^-1^(Figures [3E,3F](#F3){ref-type="fig"}), platelet accumulation was not significantly reduced by either insulin (500 pM) and isoproterenol (100 μM) as illustrated in Figures [3E,3F](#F3){ref-type="fig"}. ::: {#F3 .fig} Figure 3 ::: {.caption} ###### Reduction in thrombus growth rate depends on platelet derived NO. Perfusions for control (■), insulin, 500 pM (◆), isoproterenol, 100 μM (▲), and samples preincubated with L-NAME \[insulin (◇) and isoproterenol (△)\] were performed at 100, 1000, and 2000 s^-1^(Figures 3A-F). Results represent mean ± s.e.m. of at least five experiments with five separate blood donors. ::: ![](1477-9560-2-11-3) ::: At the high shear rate of 2000 s^-1^we observed a curious result when the blood was treated with L-NAME but in the absence of any agonist. The results are presented in Figure [4](#F4){ref-type="fig"}. We found that in the presence of L-NAME, thrombus formation increased either with (data not shown) or without agonist present (Figure [4C](#F4){ref-type="fig"}). This result suggests a role for nitric oxide in modulating the rate of platelet deposition onto a collagen surface at a shear rate of 2000 s^-1^even in the absence of insulin or isoproterenol. ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Shear stress causes a slowing of thrombus growth rate that depends of platelet derived NO. Platelet coverage values for each study are the mean ± s.e.m. of 5 separate experiments, each using a different donor. Perfusions for control (■) and L-NAME (□) samples were conducted at 100, 1000, and 2000 s^-1^(Figures 4A-C) for 6--10 minutes. ::: ![](1477-9560-2-11-4) ::: Effect of insulin and isoproterenol on guanylate cyclase activity ----------------------------------------------------------------- We further investigated the mechanism of platelet derived nitric oxide inhibition of thrombus formation by using the guanylate cyclase inhibitor 1H-\[[@B1],[@B2],[@B4]\]oxadiazolo \[4, 3-a\]quinozalin-1-one (ODQ), since the primary target of NO in platelets is guanylate cyclase \[[@B18],[@B19]\]. As illustrated in Figure [5A](#F5){ref-type="fig"}, thrombus formation at 1000 s^-1^and in the presence of insulin was slightly increased by addition of ODQ. No significant effect of ODQ on thrombus formation was observed with isoproterenol (Figure [5B](#F5){ref-type="fig"}) or in the absence of agonist (Figure [5C](#F5){ref-type="fig"}). This is not surprising since our other studies at this shear with isoproterenol (Figure [2D](#F2){ref-type="fig"}) or in the absence of agonist (Figure [3B](#F3){ref-type="fig"}) pointed out that the effect on platelet thrombus formation was also very small. Interestingly, we see that ODQ causes an increase in thrombus formation at 2000 s^-1^if either insulin, isoproterenol, or no agonist is used (Figures [5D,5E,5F](#F5){ref-type="fig"}). This suggests that at 2000 s^-1^, guanylate cyclase activation is occurring even in the absence of added external agonists. ::: {#F5 .fig} Figure 5 ::: {.caption} ###### Effect of guanylate cyclase on platelet adhesion kinetics. Platelet coverage values for each study are the mean ± s.e.m of 4 separate experiments, each using a different donor. Perfusions were conducted at 1000 and 2000 s^-1^(Figures 5A and 5B) for 6 minutes with insulin, 500 pM (■), isoproterenol, 1000 μM (▲) and samples preincubated with guanylate cyclase inhibitor ODQ \[insulin (□) and isoproterenol (△)\]. ::: ![](1477-9560-2-11-5) ::: Discussion ========== The purpose of this study was to determine if nitric oxide from platelets affects platelet function. Previous studies have identified the vascular endothelium as the primary source of NO in the blood. Only recently have platelets been identified as an additional source of NO. The role of platelet derived NO in modulating platelet function has not been established. Most of the other studies that have examined NO production by platelets were performed with platelet rich plasma or with washed platelets. This was required in order to increase the platelet concentration to the point where NO levels could be assessed. However, since these studies were performed in the absence of erythrocytes, it is difficult to extrapolate the results to the in vivosituation where hemoglobin in red cells efficiently scavenges the nitric oxide free radical. Therefore, the experiments in our current study were designed to measure platelet function in a vascular injury model where the platelets remain in whole blood. Several recent studies have demonstrated that platelets produce NO in response to agonists including insulin, isoproterenol, and ADP \[[@B2],[@B4]\]. Using an in vitroparallel plate flow chamber, we show in this study that the rate of mural thrombus growth on a collagen III surface is modulated by platelet derived NO. This is the first study to show that platelet derived NO can alter platelet function in whole blood. We found that insulin, isoproterenol, and shear stress could, under the right circumstances, modulate platelet function through an NO-mediated mechanism. In fact, we found that at a shear rate of 2000 s^-1^, but not at either 100 or 1000 s^-1^, platelet adhesion to the surface was increased in the presence of L-NAME, an inhibitor of nitric oxide production. This result suggests that platelets produce nitric oxide at higher shear rates but not at lower levels of fluid mechanical stress. This result is consistent with the observation that elevated levels of shear stress can cause platelet activation \[[@B20],[@B21]\] and that nitric oxide production may be a common feature of platelet activation regardless how activation is initiated \[[@B8],[@B22]\]. At the low, venous level of shear (100 s^-1^) that we tested, platelet derived NO did not appear to play a role in modulating platelet accumulation on the surface. This result was not surprising due to the many types of adhesion receptors on platelets that function well in a low shear environment. The primary platelet receptors for collagen III on a surface are GPVI and α~2~β~1~\[[@B23],[@B24]\]. Additionally, the GPIb-V-X receptor complex can mediate platelet adhesion through interactions with soluble and surface associated von Willebrand Factor \[[@B25]\]. Because of the interaction with collagen, the platelets become activated. Activation induces an extensive series of intracellular signaling events to take place, resulting in the conversion of the integrins α~2~β~1~and α~IIb~β~3~into a high affinity state through a process termed inside-out signaling. In this high affinity state, these integrins are capable of supporting platelet-platelet interactions and promoting thrombus growth. Therefore, platelet adhesion at low levels of shear rate can be mediated by a number of different adhesion molecules, some of which do not require platelet activation. Our results are consistent with this model since we found that platelet derived nitric oxide did not inhibit the rate of thrombus growth at these low, venous levels of shear rate. At intermediate shear (1000 s^-1^), integrins α~2~β~1~and α~IIb~β~3~play a more significant role in platelet adhesion and recruitment. These platelet integrins are essential for thrombus formation at intermediate to high shear \[[@B26]\]. Furthermore, these integrins must become activated, that is convert into a high affinity conformation, in order for them to be capable of supporting adhesion at these high levels of shear \[[@B27]\]. This requires intracellular signal transduction events and platelet activation, a process that is inhibited by nitric oxide. Our results (Figures [1A](#F1){ref-type="fig"} and [1B](#F1){ref-type="fig"}) suggest that insulin and, to a lesser extent, isoproterenol can both cause a decrease in the rate of growth of thrombi and that this decrease is concentration dependent. Furthermore, we showed that the mechanism of this inhibition of thrombus growth involves a nitric oxide/guanylyate cyclase pathway (Figures [2B](#F2){ref-type="fig"} and [4A](#F4){ref-type="fig"}). Our results with the inhibitors of platelet signaling, L-NAME and ODQ, are consistent with previous studies. L-NAME completely abolished the anti-thrombotic effects of both insulin and isoproterenol at a shear rate of 1000 s^-1^showing that these agonists act through a pathway that involves nitric oxide. ODQ was effective in inhibiting the action of insulin, but was less effective in inhibiting the action of isoproerenol. This result is consistent with earlier findings that suggested that isoproterenol inhibited platelet activation partially through a mechanism involving adenylate cyclase \[[@B28]\] rather than guanylyate cyclase. The results we present with the inhibitors L-NAME and ODQ at the highest shear rate, 2000 s^-1^(Figures [2C](#F2){ref-type="fig"}, [4C](#F4){ref-type="fig"}, and [4D](#F4){ref-type="fig"}) failed to demonstrate a role for platelet-derived NO or signaling through guanylyate cyclase. However, in the absence of added agonist, shear stress, by itself was able to induce platelets to produce NO (Figures [3C](#F3){ref-type="fig"} and [5B](#F5){ref-type="fig"}). Although there has been no previous evidence for shear-dependent NO production in platelets, both in vitroand in vivostudies have demonstrated shear stress upregulates eNOS activity in endothelial cells \[[@B7],[@B29]\]. The mechanism is not completely understood but involves cytoskeletal proteins and the activation of a series of kinases including PI3 kinase and Akt as well as Hsp90 \[[@B30],[@B31]\]. A similar pathway for the activation of eNOS has been recently characterized in platelets in response to insulin \[[@B8]\]. Conclusions =========== Our study focused on quantifying the effect of platelet-derived NO on platelet adhesion and aggregation on a surface in response to external factors (i.e. insulin, isoproterenol, and shear) and in the presence of whole blood. Platelet-derived NO did not affect platelet adhesion at low shear but had a significant effect at intermediate and high shear. Production of NO in platelets was dominated by receptor-mediated interactions at intermediate shear and mechano-transduction at high shear. This study and future work may lead to a better understanding of platelet-derived NO and its role in healthy and diabetic vascular wound healing. Methods ======= Materials --------- Low molecular weight heparin, HEPES, NaCl, collagen III from calf skin, bovine serum albumin (BSA), L-nitro-amine-methyl-ester (L-NAME), insulin from porcine pancreas, and isoproterenol were obtained from Sigma. Recombinant hirudin (r-hirudin) was obtained from Pentapharm and 1H-\[[@B1],[@B2],[@B4]\]oxadiazolo \[4, 3-a\]quinoxalin-1-one (ODQ) was obtained from Cayman Chemical. Mepacrine (quinacrine) was obtained from ICN Biomedicals. Fluorescein isothiocyanate (FITC) was generously donated by Paul Friese, University of Oklahoma Health Sciences Center, Oklahoma City, OK. The S12 anti-P-selectin monoclonal antibody was generously donated by Roger P. McEver, Oklahoma Medical Research Foundation, Oklahoma City, OK. Glass cover slips (24 × 50 mm) were obtained from Fisher Scientific. Preparation of Glass Coverslips ------------------------------- Each cover slip was washed with 20 mL nanopure H~2~O and 10 mL 95% ethanol. After washing was complete, the cover slips were then placed in a 95% ethanol bath for at least 12 hours. Before use, each cover slip was rinsed with an additional 20 mL 95% ethanol and allowed to air-dry for 15 minutes. Protein coating of Cover slips ------------------------------ Collagen III solution was prepared at 0.8 mg/mL in 17 mM acetic acid (pH 2.6) at least 24 hours prior to use. BSA solution was prepared at 0.1% in 10 mM HEPES/115 mM NaCl buffer (pH 7.4). Half of each cover slip was coated with collagen and allowed to incubate for 4 hours in a humidified environment (80--90%) at room temperature (\~24°C). After incubation, each slide was rinsed with 10--15 mL of HEPES/NaCl buffer solution to remove excess collagen and coated with 0.1% BSA solution for at least 1 hour. Platelet Preparation -------------------- Venous blood collected from healthy donors (30--60 mL, depending on shear rate and length of experiment) was anticoagulated with low molecular weight heparin to a final concentration of 20 U/mL or r-hirudin to a final concentration of 40 anti-thrombin units/mL (ATU/mL). The fluorescent dye mepacrine was then added to a final concentration of 10 μM and allowed to incubate for 10--15 minutes. All donors gave informed consent to participate in our study according to methods approved by the University of Oklahoma Institutional Review Board. Flow Experiments ---------------- The arterial flow environment was modeled with an in vitroparallel plate flow chamber similar to those previously characterized \[[@B32],[@B33]\]. The dimensions of the flow channel were the 0.013 × 1.3 cm for experiments done at a shear rate of 100 s^-1^, 0.013 × .8 cm for shear rate of 500 s^-1^, and 0.013 × .25 cm for shear rates of 1,000 and 2,000 s^-1^. In some studies, 500 pM insulin or 100 μM isoproterenol was added to the anti-coagulated blood 10 minutes before the start of an experiment. In other studies, L-NAME, an inhibitor of platelet NO synthesis was added at a concentration of 200 μM for 20 minutes before the start of an experiment. In additional studies, ODQ, an inhibitor of guanylate cyclase was added at a concentration of 100 μM for 20 minutes before the start of an experiment \[[@B18],[@B19],[@B34]\]. In still other studies, combinations of L-NAME or ODQ and insulin or isoproterenol were added as described above, except that L-NAME or ODQ was added 20 minutes prior to insulin or isoproterenol addition. In each experiment, blood was perfused for 5 -- 10 minutes. In all studies, blood was used in experiments within two hours of collection. The order of experiments was varied randomly to insure that time-dependent platelet phenotype was not skewing the results. Additionally, flow cytometric studies of platelets showed no significant change in platelet activation, as measured by P-selectin expression, over the course of 4 hours (data not shown). Microscopy and Imaging Systems ------------------------------ A syringe pump provided the flow through the flow chamber. The flow chamber was placed on the stage of a Nikon Diaphot 300 inverted microscope. The illumination was provided by a 75W Xenon light source passing through a 480 nm excitation filter and a 40 × fluorite objective lens. The fluorescent emission from adherent platelets was passed through a 514 nm filter and converted to analog signal by an image intensifier and CCD camera. The image was recorded on VHS tape for subsequent analysis. The extent of mural thrombus formation on the surface was quantified in terms of the percent coverage of a representative area of the collagen-coated surface at the interface between the collagen and albumin. The analyzed area was approximately 100 μm wide by 150 μm in the direction of flow. Digital image analysis was performed on an SGI Indy workstation running the ISEE^®^image analysis software by Inovision. A background image was acquired after blood began flowing over the surface but before adhesion of any platelets. This image was subtracted from subsequent images. Platelets were identified based on size and intensity using adjustable parameters. Images were acquired and analyzed every 10--30 seconds over the course of each experiment, which lasted from 5--10 minutes. Statistical Analysis -------------------- Statistical significance of the results was assessed with the aid of the software package SPSS (version 11.5 for Windows). The statistical tests used were the Post Hoc test for multiple comparisons and the student\'s t-test. Results were deemed significant if p \< 0.05. Abbreviations ============= BSA, bovine serum albumin; cAMP, cyclic adenosine monophosphate; eNOS, endothelial nitric oxide synthase, FITC, fluorescein isothiocyanate; HEPES, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid; L-NAME, L-nitro-amine-methyl-ester; NO, nitric oxide; ODQ, \[[@B1],[@B2],[@B4]\]oxadiazolo \[4, 3-a\]quinoxalin-1-one; PI3K, phosphatidylinositol-3-kinase; PRP, platelet rich plasma; vWF, von Willebrand factor Competing Interests =================== The authors declare that they have no competing interests. Authors\' contributions ======================= RW performed all of the experiments and MN conceived of the project and coordinated the data analysis. All authors read and approved the final manuscript. Acknowledgements ================ We would like to gratefully acknowledge Paul Friese, Vishwanath Ramachandran, Hendra Setiadi, and Jim Henthorn, for their superb technical assistance and Rodger P. McEver and George L. Dale for stimulating scientific discussions. This work was supported in part by a grant from Oklahoma Center for the Advancement of Science and Technology, Oklahoma City, OK.	PubMed Central	2024-06-05T03:55:50.018615	2004-11-15	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC535342/", "journal": "Thromb J. 2004 Nov 15; 2:11", "authors": [ { "first": "Robert H", "last": "Williams" }, { "first": "Matthias U", "last": "Nollert" } ] }
PMC535343	Background ========== There are controversial results on the efficacy of the nowadays still abandoned, intrathecal steroid application predominantly due to a missing detailed evaluation of patients\' characteristics, careful monitoring and standardized outcome measurements in multiple sclerosis (MS) \[[@B1]\]. Initially, case reports of intrathecal methylprednisolon and ACTH administration described beneficial effects in MS patients, but the following studies showed disappointing results in particular in comparison to systemic steroid application \[[@B1]\]. Earlier intrathecal treatment trials in MS patients suffered from small sample sizes, low number of injections, low steroid dosages, short half life of the administered steroid and an increasing number of reports on side-effects, i.e. adhesive arachnoiditis and various forms of meninigitis probably due to neurotoxic solvents and bacteriostatic additives \[[@B4]\]. Moreover a retarded release steroid preparation was not available for many years \[[@B1],[@B5]\]. Then administration of triamcinolone acetonide crystal suspensions (TCA), dissolved at bedside in sterile saline, was introduced in the intrathecal steroid treatment of MS. However, studies again lacked of detailed evaluation and clinical characterization of MS patients, small sample sizes and a low number of intrathecal application of this retarded release steroid compound \[[@B1],[@B4],[@B5]\]. Accordingly, there was no convincing superiority over the efficacy of the systemic steroid treatment. Some enrolled MS patients experienced a recent deterioration of symptoms due to prior acute relapses and/or ongoing chronic progression. Moreover study participants were not classified according to the various subtypes of MS progression \[[@B5],[@B6]\]. However in general, a comparison of both methods of steroid application is at least doubtful from the pharmacokinetic point of view. The resulting steroid efficacy in the central nervous system enormously differs in favor for the intraspinal application due to the achieved cerebrospinal fluid steroid level, marked longer half life, i.e. with detection of TCA even four months after the last administration, and missing impact on the endogenous peripheral cortisol secretion with no appearance of side effects of systemic high dosage steroid application \[[@B4],[@B6]\]. In recent years, a certain revival of intrathecal methylprednisolon administration took place in the treatment of intractable postherpetic neuralgia and MS with spinal symptoms, both of which turned out to be very effective, but were controversially discussed regarding the safety issues \[[@B2],[@B3]\]. The MS study demonstrated, that six repeat intrathecal TCA injections within three weeks reduced the EDSS score in 31 of 36 progressive MS patients with predominant spinal symptoms. 20 of them entered a follow-up period of 13.1 ± 6.22, 3 -- 23 \[mean ± SD, range\] months with 6.35 ± 3.91, 2 -- 15 TCA injections. They received one TCA application on a regular basis in an individually differing frequency every six to twelve weeks. These patients remained stable \[[@B3]\]. Nevertheless, there is a need for further results on the usefulness of this treatment. The optimum design would be a placebo-controlled arm, but repeat performance of intrathecal saline (placebo) administration under double-blind conditions with the patients\' consent and an approval of an ethical committee is not realistic in clinical practice. Moreover they may be ethical concerns of withholding treatment \[[@B7]\]. Our present study is a way out of this dilemma of the debate on the efficacy of TCA treatment, which is carried out in certain specific centers in Germany for many years now \[[@B3]\]. We performed an analysis of standardized intrathecal application of TCA in MS patients with spinal symptoms, using subjective rating procedures and objective measurements. Methods ======= Subjects -------- We only enrolled clinically well characterized, consecutively referred MS patients (table [1](#T1){ref-type="table"}, [2](#T2){ref-type="table"}) with distinct spinal symptoms and/or MRT visualized lesions in the spinal cord \[[@B8]\]. These patients did not suffer from an acute onset of exacerbation or recent clearly increased progression of their symptoms. ::: {#T1 .table-wrap} Table 1 ::: {.caption} ###### Patients\' characteristics. ::: ------------------------- -------------------------------- Age 50.10 ± 10.30, 21 -- 78 years Duration of MS 14.32 ± 7.63, 2 -- 40 years Sex 119 men, 42 women MS types chronic progressive: n = 35 secondary progressive: n = 122 relapsing-remitting: n = 4 Length of hospital stay 28.41 ± 5.97; 21 -- 60 days ------------------------- -------------------------------- ::: ::: {#T2 .table-wrap} Table 2 ::: {.caption} ###### Treatment against spasticity. ::: Before TCA After TCA ------------ ----------------------------------------------------- ----------------------------------------------------- Baclofen 7.56 ± 16.06; 0 -- 80 mg, n = 117 without baclofen 6.44 ± 15.51; 0 -- 80 mg, n = 125 without baclofen Tolperison 1.19 ± 8.20; 0 -- 75 mg, n = 154 without tolperison 1.34 ± 7.41; 0 -- 50 mg, n = 152 without tolperison Tizanidin 1.94 ± 5.29; 0 -- 32 mg, n = 132 without tizanidin 3.38 ± 6.64; 0 -- 32 mg, n = 110 without tizanidin All data are given as mean ± standard deviation; minimum -- maximum; n = number of patients; TCA = standardized intrathecal triamcinolon acetonide application according to the methods section. ::: Methods ------- We performed scoring with both, EDSS and Barthel index and assessed the walking distance. Then we measured somatosensory evoked potentials (SSEP) in a standardized fashion before start and at the end of the intraspinal TCA treatment within a prospective study design \[[@B9]\]. A technician performed SSEP recordings and measured the walking distance. We blinded the EDSS raters. Retrospectively, we compiled information on patients from their hospital records, i.e. date of birth, sex, duration of disease after diagnosis of MS, dosages of oral baclofen (lioresal^®^), tolperison (mydocalm^®^), tizanidin (sirdalud^®^) on the first and last day of the hospital stay, length of hospitalization in days (tables [1](#T1){ref-type="table"} &[2](#T2){ref-type="table"}). The patients additionally received a standardized rehabilitation treatment, which included physiotherapy, massage and optional swimming with the patients\' consent \[[@B12],[@B13]\]. Only data with successfully performed serial evaluation of patients were compared for each variable. We performed lumbar puncture with an \"atraumatic\" Sprotte needle \[[@B10],[@B11]\]. Each patient received six intrathecal applications of 40 mg TCA followed by a mandatory stay in bed for at least six hours. This should reduce incidence of lumbar puncture syndrome and hypothetically support the diffusion of TCA in the CSF and the spinal cord \[[@B14],[@B15]\]. A preexisting immune system modulating drug therapy remained stable. We closely monitored for typical concomitant with systemic steroid application appearing side effects, i.e. increase of body weight etc., all of which did not significantly change (data not shown) \[[@B16]\]. No slight or severe side effects occurred, but we did not include five patients into our evaluation due to onset of post lumbar puncture syndrome with headache and nausea, which caused a stop of further intrathecal TCA applications. These patients withdraw their consent. We only considered SSEP data of patients with serial measurements, which we performed on the same day of the EDSS rating. Ethics ------ Each participant gave written informed consent for the TCA treatment, which was approved by the local ethical committee. The consent form included a detailed description of all putative risks of lumbar puncture and intrathecal TCA application. Statistics ---------- Data showed a normal distribution according to the Kolmogorow-Smirnow test. As a result, we only performed parametric tests. We used ANCOVA with repeated measures design including MS duration, MS types, change of dosages of concomitant drugs against spasticity, length of hospital stay, sex and age as covariates. We computed SSEP results by adding both sides in order to reduce amount of calculations for comparisons. Then we calculated the differences of latencies between both timepoints of recordings according to the formula \[Initial - End = Diff\] for correlation analysis. We employed linear regression for correlation analysis. Level of significance of p-values were adjusted to 0.05 divided by the number of performed comparisons respectively correlations. Results ======= Comparisons ----------- EDSS score (n \[number of subjects with serial evaluation\] = 161) and Barthel index (n = 68) improved and walking distance (n = 161) increased (table [3](#T3){ref-type="table"}). SSEP latencies of tibial (n = 136) and median (n = 108) nerves reduced (table [3](#T3){ref-type="table"}). P-values of all performed comparisons were below 0.0001. No significant effects of covariates appeared. ::: {#T3 .table-wrap} Table 3 ::: {.caption} ###### Comparison of clinical data. ::: Before TCA After TCA F ------------------- ---------------------------- ---------------------------- -------- EDSS -- score 6.44 ± 1.06; 3.5 -- 6.5 5.47 ± 1.24; 2 -- 8.5 379.28 walking distance 158.03 ± 501.20; 0 -- 5000 439.38 ± 895.24; 0 -- 5000 34.40 Barthel index 58 ± 20.07; 5 -- 100 89.13 ± 12.57; 60 -- 100 347.52 P2 (tibial nerve) 105.80 ± 10.38; 85 -- 130 88.57 ± 5.60; 77 -- 106 470.96 N2 (tibial nerve) 118.83 ± 10.49; 92 -- 148 101.63 ± 6.43; 80 -- 118 425.71 P3 (tibial nerve) 131.79 ± 13.22; 70 -- 164 114.31 ± 7.46; 95 -- 135 325.88 N2 (median nerve) 46.53 ± 5.40; 23--60 41.03 ± 2.92; 23--47 138.10 P2 (median nerve) 52.73 ± 6.34; 25 -- 68 47.09 ± 3.03; 41--56 128.80 All data are given as mean ± standard deviation; minimum -- maximum in the second and third column; latencies (N2, P2, P3) of the somatosensensory evoked potentials are given in milliseconds, walking distance is given in meters, F = F-value of ANCOVA, SD = standard deviation; TCA = standardized intrathecal triamcinolon acetonide application according to the methods section. ::: Correlation analysis -------------------- There were no significant relations between computed changes of EDSS scores, walking distances and SSEP results (results not shown). Diff P2 correlated with Diff P3 (R \[correlation coefficient\] = 0.71), Diff P2 with Diff N2 (R = 0.90), Diff P3 with Diff N2 (R = 0.74) of SSEP latencies of the tibial nerves. There were relations between Diff N2 and Diff P2 (R = 0.85) of SSEP latencies of the median nerves. P-values of these correlations were below 0.0001. Moreover Diff P2 of the tibial nerves correlated with Diff N2 of the median nerves (R = 0.28, p = 0.004, n = 100). There was a certain trend for a significant correlation between Diff P2 of the tibial nerve and Diff P2 of the median nerve (R = 0.22, p = 0.03). No other significant associations of SSEP data appeared (results not shown). Discussion ========== Our results demonstrate and confirm the efficacy of repeated intraspinal TCA application in MS patients with spinal symptoms, which improved according to the EDSS outcomes and the results of assessed walking distance and determined SSEP latencies \[[@B3]\]. We intrathecally injected TCA six times within three weeks, whereas earlier trials weekly performed one application up to three times at the most \[[@B4]\]. The distinct reduction of SSEP peak latencies and the significant relations between their computed differences confirm the clinical outcomes and underline the efficacy of intraspinal TCA treatment in MS patients with spinal symptoms in general. We hypothesize, that these neurophysiological results indicate a certain remyelinating and/or restorative potential of intraspinal TCA application with an at least transient shift from chronic inflammation to remyelination \[[@B17],[@B18]\]. Our results support the crucially discussed view, that serial SSEP studies in MS may monitor the effect of treatment to a certain extent under standardized conditions \[[@B9],[@B19],[@B20]\]. Our analysis also shows that primary and secondary progressive even advanced MS patients with spinal symptoms predominantly improve from this kind of intrathecal steroid therapy. We assume, that we achieve persistent high steroid concentrations at lesions of the spinal cord, since TCA must not pass the blood brain barrier \[[@B6]\]. However previous comparisons of the clinical efficacy of intrathecal TCA application with the intravenous administration of methylprednisolone showed no superiority of one method over the other \[[@B5],[@B6],[@B19]\]. But these studies did not exclude relapsing remitting patients or participants with a previous acute relapse. They did not focus on spinal symptoms. Their application rate of TCA was distinct lower compared with the one of our present and a previous trial \[[@B3]\]. However our present study outcomes do not allow any conclusions on the duration of the achieved benefit and the impact of TCA treatment on progression of MS \[[@B3]\]. Therefore there is an urgent need for further confirmatory trials, which additionally address all these issues. A strategy would be to choose one arm with active treatment and one arm with just follow-up without active treatment with blind assessment by an evaluating physician. However we stress concerning long-term steroid therapy and progression of MS, that there are positive outcomes of trials with intravenous methylprednisolone administration in various application rates and dosages on long term disease progression and/or on brain atrophy in secondary-progressive -, respectively relapsing-remitting MS patients \[[@B16],[@B21]\]. In contrast to studies on intravenous oral steroid treatment, we did not observe the typical side effects of systemic high dosage steroid administration, i.e. edema. This may support previous findings by circumstantial evidence, which report no decrease of endogenous cortisol secretion following intrathecal TCA administration \[[@B4]\]. We cannot exclude a certain impact of physiotherapy, the standardized rehabilitation treatment and an beneficial effect of hospitalization in general with its resulting concomitant positive influence on activities of daily living \[[@B12],[@B13]\]. However, we found no significant impact of the corresponding covariate length of the hospital stay in our statistical analysis. We assume, that our results do not reflect an improved drug therapy against spasticity, since no significant impact of the covariate computed changes of medication appeared. Nevertheless we cannot exclude a certain effect of the steroid on spasticity. However most participants did not take any drug against spasticity. Onset of side effects of lumbar puncture itself were negligible, since we used an atraumatic needle \[[@B11]\]. Conclusions =========== Our data demonstrate the efficacy and safety of repeated intrathecal TCA application in MS patients with predominant spinal symptoms, which markedly improved. Some MS patients experienced post lumbar puncture syndrome with a frequency within the normal range \[[@B11]\], but typical side effects of systemic high dosage steroid administration did not appear. Competing interests =================== The author(s) declare that they have no competing interests. Authors\' contributions ======================= KH, FS, HP and TM designed, coordinated and carried out the study. TM performed statistical data analysis 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/1471-2377/4/18/prepub> Acknowledgement =============== We thank A. Berg for technical assistance.	PubMed Central	2024-06-05T03:55:50.020670	2004-11-7	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC535343/", "journal": "BMC Neurol. 2004 Nov 7; 4:18", "authors": [ { "first": "Kerstin", "last": "Hellwig" }, { "first": "Franz Josef", "last": "Stein" }, { "first": "Horst", "last": "Przuntek" }, { "first": "Thomas", "last": "Müller" } ] }
PMC535344	Background ========== Fast excitatory synaptic transmission in the central nervous system, which is mediated predominantly by the AMPA subtype of glutamate receptors, can undergo long-term bi-directional modifications in strength \[[@B1]-[@B3]\]. These persistent changes have been proposed to be key synaptic processes involved in learning and memory. The best characterised form of bi-directional synaptic plasticity is LTP / LTD of glutamatergic transmission in the CA1 region of the hippocampus. Although there has been intensive investigation of the induction and expression of LTP and LTD (see \[[@B4]\]), the precise molecular mechanisms by which these alterations in synaptic strength occur remain unclear. Possible mechanisms could be presynaptic such as changes in the release process (probability of neurotransmitter release or the amount of L-glutamate released from vesicles (e.g., \[[@B5]-[@B9]\]), and / or postsynaptic (e.g., \[[@B10]-[@B12]\]), such as a change in the number of AMPA receptors (AMPARs) available to bind transmitter, or in the properties of existing receptors (P~open~, activation or de-activation kinetics, desensitisation or single-channel conductance, γ). Recent studies have provided information on the possible mechanisms underlying postsynaptic alterations in synaptic strength. There is evidence that AMPA receptors are inserted into the postsynaptic membrane during LTP \[[@B13]-[@B15]\] and removed from the synapse upon induction of LTD \[[@B16],[@B17]\]. However, it has also been shown that LTP can involve a rapid increase in γ of existing AMPA receptors \[[@B18]\]. This could be caused by a Ca^2+^/calmodulin-kinase II (CaM-KII)-mediated phosphorylation of GluR1 which occurs during LTP \[[@B19]\], and which causes an increase in γ of GluR1 homomers in transfected cells \[[@B20]\]. Therefore, another potential mechanism for LTD could be a decrease in γ caused by a dephosphorylation of AMPARs. We have recently used peak-scaled non-stationary fluctuation analysis (non-SFA; \[[@B21]\]) of synaptic currents recorded from CA1 pyramidal cell dendrites \[[@B18]\], to investigate the molecular basis of de novoLTD (LTD at naïve pathways; \[[@B22],[@B23]\]). In these studies, LTD was never associated with a change in γ \[[@B17]\]. Indeed, evidence was presented that the underlying mechanism involved a reduction in the number of surface expressed AMPA receptors (LTD~N~). In the present study we have investigated a second form of LTD known as depotentiation (DP), which is a reversal of pre-established LTP \[[@B24]-[@B27]\]. LTP can be associated with either an increase in γ (LTPγ) or no change in γ (LTP~N~) \[[@B18]\]. We were, therefore, interested to determine whether DP of LTPγ involved a decrease in γ and hence whether γ is a bi-directionally modifiable parameter. We find a reciprocal relationship between LTP and DP, such that DP of LTPγ invariably involves a restoration of the pre-LTP γ (DPγ) whereas DP of LTP~N~never involves a change in γ (DP~N~). These data show, firstly, that there are two distinct molecular mechanisms for the reduction of synaptic strength that are dependent on the nature of the preceding LTP and, secondly, that γ can indeed be bi-directionally modified in response to synaptic activity. Results ======= Using whole-cell recordings from the proximal apical dendrites of hippocampal CA1 pyramidal cells, minimal stimulation of nearby afferents evoked EPSCs that could be reliably resolved from failures (trials in which stimulation produced no synaptic response; Figure [1](#F1){ref-type="fig"}, see also Figure [3](#F3){ref-type="fig"}). These high resolution recordings enabled both a failures analysis to be performed \[[@B28]\] and, using non-SFA, an estimate of γ of synaptically-activated AMPARs to be obtained \[[@B18]\]. LTP --- To investigate the mechanism of DP, LTP was first induced in 18 cells by pairing afferent stimulation (baseline frequency) with a holding potential of 0 mV. This resulted in stable LTP (EPSC amplitude = 186 ± 16 % of baseline, n = 18). In agreement with a previous study \[[@B18]\], cells fell into two groups with respect to changes (\> 20%) in γ of AMPA receptor channels during LTP. In the majority of cases (11/18 cells), LTP was associated with an increase in γ (LTPγ; 246 ± 26% of baseline; range 136 -- 363%). In the other 7 cells there was no increase in γ during LTP (100 ± 4 % of baseline; range 85 -- 118 %), indicating that there was an increase in the functional number of channels activated (LTP~N~). As noted previously \[[@B18]\], there were no differences between the two groups of neurons with respect to a variety of baseline parameters. Depotentiation of LTPγ ---------------------- Figure [1](#F1){ref-type="fig"} shows two examples from the group of cells that exhibited LTPγ, as indicated by the change in the current-variance plot obtained from non-SFA (Figure [1B](#F1){ref-type="fig"}). As previously reported \[[@B18]\], the increase in γ was not associated with any change in EPSC kinetics (Figure [1C](#F1){ref-type="fig"}) indicating that AMPA receptor channel kinetics were not affected \[[@B29]\]. Failures analysis (Figure [1C](#F1){ref-type="fig"}) of this group of cells (Figure [2](#F2){ref-type="fig"}) revealed that LTP was associated with changes in success rate (1 -- failure rate) in some cells (Figure [2B](#F2){ref-type="fig"}), and potency (mean EPSC amplitude excluding failures) in all cells (Figure [2C](#F2){ref-type="fig"}), as previously reported under these recording conditions \[[@B18],[@B28]\]. For this group of cells, DP, induced by pairing stimulation (baseline frequency) with a holding potential of -40 mV, always resulted in a reversal of the γ increase, as indicated by the current-variance plot (Figure [1B](#F1){ref-type="fig"}; Figure [2D](#F2){ref-type="fig"}). Similar to the preceding LTPγ, this form of DP (DPγ) was also associated with no change in EPSC kinetics (τ~rise~: baseline = 1.6 ± 0.2 ms, LTPγ = 1.7 ± 0.3 ms, DPγ = 1.7 ± 0.2 ms, n = 11; τ~decay~: baseline = 8.3 ± 0.6 ms, LTPγ = 8.2 ± 0.6 ms, DPγ = 8.9 ± 0.7, n = 11; Figure [1C](#F1){ref-type="fig"}). Failures analysis showed that DPγ was associated with a decrease in success rate in most cells (Figure [2B](#F2){ref-type="fig"}) and a full reversal of the potency increase (Figure [2C](#F2){ref-type="fig"}). These data show that the primary mechanism for DP is the reversal of any increase in γ caused by LTP. Indeed, the changes in γ were sufficient to account for the potency changes during both LTPγ and DPγ. (In most cells the alterations in γ actually exceeded the potency changes. This is most likely due to an underestimate of the potency change due to dendritic filtering, which affects measurements at the peak of EPSCs greater than during the tail, from where the non-SFA estimates are obtained; see \[[@B18]\]). Depotentiation of LTP~N~ ------------------------ Figure [3](#F3){ref-type="fig"} shows an example from the group of cells that exhibited no change in γ during LTP (LTP~N~), as indicated by the current-variance plot (Figure [3B](#F3){ref-type="fig"}). The change in EPSC amplitude for LTP~N~neurons (Figure [4A](#F4){ref-type="fig"}) was similar to that for LTPγ neurons (Figure [2A](#F2){ref-type="fig"}). Failures analysis (Figure [3D](#F3){ref-type="fig"}) of this group of cells (Figure [4](#F4){ref-type="fig"}) revealed that LTP was associated with changes in success rate in some cells (Figure [4B](#F4){ref-type="fig"}), and potency in most cells (Figure [4C](#F4){ref-type="fig"}; P \< 0.01), as previously reported under these recording conditions \[[@B18],[@B28]\]. In contrast to DPγ, DP in these cells was never associated with a change in γ (DP~N~; Figure [3B](#F3){ref-type="fig"}, Figure [4D](#F4){ref-type="fig"}). There was also no change in EPSC kinetics with LTP or DP (τ~rise~: baseline = 1.9 ± 0.4 ms, LTP~N~= 2.0 ± 0.3 ms, DP~N~= 1.8 ± 0.3 ms, n = 7; τ~decay~: baseline = 9.7 ± 0.7 ms, LTP~N~= 9.7 ± 0.6 ms, DP~N~= 10.1 ± 0.9, n = 7; Figure [3C](#F3){ref-type="fig"}). Failures analysis of LTP~N~and DP~N~(Figure [3D](#F3){ref-type="fig"}, Figure [4](#F4){ref-type="fig"}) showed similar changes to the LTPγ group of cells in EPSC amplitude (Figure [4A](#F4){ref-type="fig"}), success rate (Figure [4B](#F4){ref-type="fig"}) and potency (Figure [4C](#F4){ref-type="fig"}). These data suggest that there is a second mechanism for DP, not involving a decrease in γ, which co-exists at CA1 synapses. Therefore, there are two mechanisms for the expression of DP that depend upon the expression mechanism of the previous potentiation. Role of NMDA receptors in depotentiation ---------------------------------------- It has been shown previously that DP at CA1 synapses may be blocked either by NMDA receptor antagonists \[[@B26]\] or by mGlu receptor antagonists \[[@B27]\] and that this may depend on previous history \[[@B30]\]. In the present study we wished to focus on NMDA receptor-dependent synaptic plasticity and therefore used pairing protocols designed to activate NMDA receptors sufficiently to, firstly, induce NMDA receptor-dependent LTP and, secondly, to induce NMDA receptor-dependent DP. To verify that we were indeed investigating NMDA receptor-dependent DP we performed a series of experiments using the NMDA receptor antagonist D-AP5 interleaved with control experiments. Following the induction of LTP, D-AP5 (50 μM) was bath applied for 15 minutes before delivering the DP induction stimulus. Whilst DP was induced in the control experiments (25 ± 11% of baseline, n = 5; p \< 0.05) it was blocked by D-AP5 (89 ± 21%, n = 4; Figure [5](#F5){ref-type="fig"}). Relationship of changes in success rate, potency and γ to the magnitude of DP ----------------------------------------------------------------------------- To gain further insights into the underlying mechanisms of DP we compared changes in EPSC amplitude, success rate, potency and γ for the individual experiments (Figure [6](#F6){ref-type="fig"}). A decrease in success rate indicates a reduction in probability of transmitter release (Pr) and/or a reduction in the number of functional synapses (n). A decrease in potency indicates a reduction in quantal amplitude (postsynaptic response to the release of a single quantum of transmitter, q) or, if multiple synapses are activated, a reduction in Pr or n. In both types of neuron (i.e., LTPγ and LTP~N~), a small depression of less than 50 % (to 71 ± 7% of baseline; n = 4) was associated with no change in success rate (Figure [6A](#F6){ref-type="fig"}; success rate ratio = 1.00 ± 0.01) but an equivalent decrease in potency (Figure [6B](#F6){ref-type="fig"}; potency ratio = 0.70 ± 0.08), as is also observed for de novoLTD \[[@B17]\]). This indicates that the depression in these cells was associated primarily with a decrease in q. For larger depressions (to 26 ± 4 % of baseline; n = 14) there was also a marked decrease in success rate (success rate ratio = 0.52 ± 0.08; P \< 0.01 vssuccess rate ratio for the group with DP \< 50%) as well as a decrease in potency (potency ratio = 0.54 ± 0.04). This suggests that in these cells there was an additional decrease in Pr or n. Therefore, for DPγ, the change in γ did not fully account for the amplitude change in every cell (Figure [6C](#F6){ref-type="fig"}) but did account for the potency change (Figure [6D](#F6){ref-type="fig"}). Discussion ========== In this study we have shown that there are two mechanisms for NMDA receptor-dependent DP, a reduction in γ (DPγ) and a decrease in the number of activated AMPA receptors (DP~N~). As reported previously for different data sets from this age of rats \[[@B9],[@B18]\] there are two forms of LTP; in approximately two-thirds of neurons LTP was associated with an increase in γ (LTPγ) whilst in the remainder there was no change in γ (LTP~N~). In the present study we saw a similar proportion of LTP expressed by changes in γ versus N. Strikingly, we observed a precise relationship between the mechanism of DP and the form of preceding LTP; LTPγ was always reversed by DPγ, and LTP~N~was always reversed by DP~N~. In some experiments, induction of DP caused a decrease in EPSC amplitude below the initial baseline. This is most likely due to the simultaneous induction of DP and de novoLTD since in slices taken from juvenile animals, de novoLTD is readily induced by this \[[@B17]\] and other \[[@B22],[@B23]\] induction protocols. This is in contrast to previous experiments using adult tissue in which DP induction depressed synaptic responses only as far as the initial baseline and where the same induction protocol was unable to induce de novoLTD \[[@B27]\]. Analysis of the mechanism of de novoLTD under the present experimental conditions demonstrated that it was associated with no change in γ \[[@B17]\]. Therefore the coexistence of DP and de novoLTD does not interfere with the analysis of DPγ. In addition, there is sometimes a small, gradual run-down of synaptic responses observed in minimal stimulation experiments using two-week-old animals \[[@B17]\] see also \[[@B31]\]. Whilst this effect tends to exaggerate changes in amplitude and success rate during DP in some neurons, it does not significantly interfere with estimates of potency or γ (see \[[@B17]\]). Mechanisms underlying DP~N~ --------------------------- What might be the mechanism underlying DP that is not associated with a decrease in γ (i.e., DP~N~)? It is unlikely that this type of depression is due to a change in channel kinetics because there was no change in EPSC kinetics (see \[[@B18],[@B29]\]). Therefore the mechanism is most likely a reduction in the number of activated AMPARs. This could be due to a presynaptic mechanism such as a reduction in release probability, the L-glutamate content of vesicles or the amount of L-glutamate discharged during fusion. Indeed there is evidence that some forms of LTD are expressed presynaptically \[[@B5],[@B35]\]. Postsynaptic mechanisms for a reduction in the number of activated AMPA receptors include a reduction in their P~open~\[[@B36]\]or in the physical number of receptors present in the postsynaptic membrane \[[@B37]\]. The present observations for DP~N~are indistinguishable from those that we and others have reported recently for de novoLTD \[[@B17],[@B38]\]. For example, we showed that similar effects were obtained using the postsynaptic injection of a peptide (pep2m) that disrupts the interaction between NSF and GluR2 \[[@B39]-[@B42]\]. The effects of pep2m and those of de novoLTD were mutually occlusive, indicating a convergence of mechanisms. These data argue strongly for a postsynaptic mechanism of expression. Furthermore, since pep2m causes the removal of AMPA receptors from the membrane surface, as determined immunocytochemically \[[@B38],[@B42]\], it is most likely that de novoLTD is due to the physical elimination of synaptic AMPA receptors. Other evidence for a postsynaptic mechanism for de novoLTD includes a reduction in the postsynaptic sensitivity to glutamate \[[@B43],[@B44]\], the dephosphorylation of serine 845 of the GluR1 subunit \[[@B45],[@B46]\] and a rapid internalisation of AMPA receptors \[[@B47]\] associated with LTD. Therefore, by analogy, we feel that the postsynaptic removal of AMPA receptors is also the most likely explanation for DP~N~(Figure [7A](#F7){ref-type="fig"}). Accordingly, a reduction in AMPA receptor number would account for the changes in potency without changes in success rate observed with modest DP~N~. The removal of an entire synaptic complement of AMPA receptors would explain the additional change in success rate seen with large depressions associated with DP~N~in some cells. Mechanisms underlying DP~γ~ --------------------------- A number of possible underlying mechanisms could account for the change in γ during LTPγ and DPγ. Non-SFA cannot distinguish between 1) a change from a single low conductance state to a single high conductance state, 2) changes in open times within a burst and, 3) changes in the proportion of time spent in different conductance states. Since AMPA receptors are known to have multiple conductance states \[[@B32],[@B33]\] we have postulated that the proportion of time spent in different conductance states is the modifiable parameter \[[@B18]\]. Such a change is detectable with the type of analysis that we have used, which provides a weighted mean of the various sub-conductance states \[[@B48]\]. Independent support for this hypothesis is provided by a study which shows that phosphorylation of GluR1 at serine 831 increases the proportion of time AMPA receptors spend in high conductance states, as determined by single channel recording \[[@B20]\]. Indeed, phosphorylation of this residue occurs during LTP \[[@B19]\]. Thus a possible mechanism of DPγ is the dephosphorylation of serine 831 \[[@B45]\], perhaps involving calcineurin \[[@B49]\], resulting in a lower proportion of time AMPA receptors spend in the higher conductance states (Figure [7B](#F7){ref-type="fig"}). Other theoretical possibilities exist to explain DPγ. For example, the silencing of synapses close to the patch electrode leaving more distant synapses contributing a lower net γ due to electrotonic filtering, or a decrease in trial-to-trial asynchrony of transmitter release. We believe that these possibilities are unlikely because in every cell in which de novoLTD was induced there was never a change in γ \[[@B17]\]. If such possibilities were likely, statistically one would expect similar changes to occur for both de novoLTD and DP. Moreover, we have also investigated these possibilities using our standard compartmental model \[[@B29]\]. This shows that 1) the electrotonic filtering required to achieve an artefactual decrease in γ would have a pronounced effect on τ~decay~which was never observed experimentally, and 2) pronounced changes in asynchrony necessary to cause an artefactual change in γ cause large deviations from the parabolic relationship in the current-variance plot (unpublished observations) and alterations in τ~rise~\[[@B29]\], also never observed experimentally. Another possibility is that alterations in vesicle fusion pore dynamics leading to substantial changes in the peak and time-course of cleft glutamate are caused by synaptic plasticity \[[@B7]\]. Such changes could differentially affect estimates of γ \[[@B50]\], however they would be associated with substantial changes in τ~rise~and τ~decay~\[[@B7]\], which were never observed experimentally. Conclusions =========== In summary, we have shown that there are two distinct molecular mechanisms for the reduction of synaptic strength. Although previous studies have provided evidence that LTD is associated with dephosphorylation of serine 845 on GluR1 \[[@B45]\] and internalisation of AMPA receptors \[[@B16],[@B17]\] it is not known whether this represents two separate mechanisms or two components of the same process. For example, dephosphorylation of GluR1 could drive the internalisation of AMPA receptors. Here we show, for the first time, the co-existence of two distinct mechanisms for the expression of DP, using functional criteria under identical experimental conditions. The relationship between the two mechanisms is critically dependent upon the recent experience of the synapse, which may be governed by the phosphorylation state of the AMPA receptor complement \[[@B45]\]. Further work is now required to elucidate the relationship between these two fundamental mechanisms for modulating synaptic strength and the precise molecular mechanisms involved in each form of plasticity. Methods ======= Electrophysiology ----------------- Hippocampal slices (400 μm) were obtained from 12--15 day old rats and perfused with an extracellular solution containing (in mM): 124 NaCl, 3 KCl, 1.25 NaHPO~4~, 26 NaHCO~3~, 2 CaCl~2~, 1 MgSO~4~, 15 glucose, 2 ascorbic acid, 0.05 picrotoxin, saturated with 95% O~2~/ 5% CO~2~, at room temperature (23--25°C). Individual dendrites were visualised using infrared illumination and DIC optics and approached under visual control. Whole-cell dendritic recordings of synaptic currents were obtained at a holding potential of -70 mV using patch electrodes (6--10 MΩ) filled with a solution containing (in mM): 135 CsMeSO~4~, 8 NaCl, 10 HEPES, 0.5 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 5 QX-314, pH 7.25, 285 mOsm. Schaffer collateral-commissural fibers were stimulated at 0.5 Hz using a platinum monopolar or concentric bipolar electrode, which was positioned 20--40 μm from the dendrite parallel to the input pathway. The stimulus intensity was set to evoke some failures to enable a failures analysis to be performed and to ensure that the majority of EPSCs were, for any given trial, evoked by release from a single site. However, to elicit sufficient EPSCs to obtain a baseline estimate of γ before \"washout\" of LTP it was usually necessary to set the success rate fairly high (usually above 50%). As a result it is likely that multiple release sites contributed to the recordings. LTP was induced by pairing 40--60 stimuli (baseline frequency) with a holding potential of 0 mV. DP was induced following the induction of stable LTP by 100--200 stimuli (baseline frequency) at -40 mV holding potential. All recordings were made using an Axopatch 1B amplifier, signals were filtered at 5 kHz (8 pole Bessel filter), digitised at 10 kHz and stored on computer. EPSC amplitude and input resistance were analysed and displayed on-line using the \'LTP\' program \[[@B51]\]). Series resistance was estimated by measuring the peak amplitude of the fast whole-cell capacitance current in response to a -1 mV step applied to the cell during each sweep. The amplitude was estimated by fitting the capacitance transient with a double exponential (from 0.5 ms after the peak) and determining the current at the beginning of the step. Series resistance was stable throughout recordings (series resistance values \[MΩ\]: baseline = 42 ± 3, LTP = 40 ± 3, DP = 43 ± 3, n = 17). Analysis -------- Non-SFA was performed as described previously \[[@B18]\]. Briefly, synaptic currents were aligned by their point of maximal rise, and averaged. The average response waveform was scaled to the peak, subtracted from individual responses and the variance of the decays calculated. The variance was plotted vs. the mean current amplitude and the single channel current was estimated by fitting the data to: σ^2^= iI - I^2^/N + b~l~, where σ^2^is the variance, I is the mean current, N is the number of channels activated at the peak, i is the single channel current and b~l~is the background variance. The single channel conductance (γ) is then γ = i/V, where V is the driving force (holding potential -- assumed reversal potential of 0 mV). Response amplitude was measured in two ways. For failures, amplitude was estimated by measuring the difference between the average current over two time windows of equal length, one immediately before the stimulus artefact and the other centred on the peak of the mean EPSC. For estimation of the amplitude of successes the peak amplitude over a set time window was determined. Failures were identified visually, and potency was calculated as the mean EPSC amplitude excluding failures. For analysis of EPSC kinetics, rise time was estimated by the time-constant of a single exponential fit of the rising phase of the mean EPSC waveform. For decay, the time-constant of the single exponential fit to the decay phase was used. For display of individual EPSC traces in the figures, the stimulus artefact was digitally subtracted using an average of identified failures. All histograms are represented as smoothed line plots (SigmaPlot ver 5.0). Data are expressed as % of baseline (i.e., 100 % = no change). Statistical significance was assessed using the Student\'s t-test (one or two-tailed, paired or unpaired as appropriate; P\< 0.05 as significant). All values are expressed as mean ± s.e.m. Authors\' contributions ======================= AL performed most of the electrophysiology. MJP and MAW also contributed some recordings. PM provided some information that guided the work. TB performed the modelling that provided the theoretical frame-work. JI helped with the design and analysis of experiments. GLC coordinated the work and contributed to its design. All authors read and approved the manuscript. Acknowledgements ================ This work was supported by the MRC (G.L.C.), BBSRC (P.M.), NIH (T.A.B.), the Swiss National Science Foundation (A.L.), and The Wellcome Trust (J.T.R.I). M.A.W was a research fellow supported by the Swedish Foundation for International Cooperation in Research and Higher Education. Figures and Tables ================== ::: {#F1 .fig} Figure 1 ::: {.caption} ###### Bi-directional modification of AMPA receptor conductance (γ) during LTP (LTPγ) and DP (DP~γ~). (A1,2) Plot of amplitude vs. time from two representative experiments. Black bar represents LTP pairing, grey bar DP pairing protocol. Throughout the figure, green represents baseline, red LTP and blue DP. (B1,2) Current-variance relationship for baseline, LTP and DP (γ values for the same cells (pS): cell 1:baseline = 2.2, LTP = 6.5, DP = 3.7; cell 2: baseline = 4.5, LTP = 7.4, DP = 3.2). For this figure and in Figure 3, lines are parabolic fits of the data (see Methods). (C1,2) Inset. Mean EPSCs (average of all responses used for non-SFA) superimposed (left) and peak scaled (right). Amplitude histograms (bin width = 2 pA; number of trials: cell 1: N = 130 for baseline, N = 245 for LTP, N = 533 for DP; cell 2: N = 150 for baseline, N = 267 for LTP, N = 337 for DP; frequency normalised to the data set with the smallest number of observations for baseline, LTP and DP). ::: ![](1471-2202-5-44-1) ::: ::: {#F2 .fig} Figure 2 ::: {.caption} ###### Summary data for all cells that exhibited DPγ. (A) Changes in mean EPSC amplitude for (A~1~) individual experiments, and (A~2~) pooled data. (B -- D) Similar plots for (B~1~& B~2~) success rate (1 -- failure rate), (C~1~& C~2~) potency and (D~1~& D~2~) γ. In this, and Figure 4, ns = not significant, \* P \< 0.05, \\ P \< 0.01, \\\* P \< 0.001). ::: ![](1471-2202-5-44-2) ::: ::: {#F3 .fig} Figure 3 ::: {.caption} ###### DP can occur without alterations in γ (DP~N~) but only when preceded by LTP that does not involve a change in γ (LTP~N~). (A) Plot of amplitude vs. time from a representative experiment. Black bar represents LTP pairing, grey bar DP pairing protocol. Throughout the figure, green represents baseline, red LTP and blue DP. (B) Current-variance relationship for baseline, LTP and DP (γ values for this cell (pS): baseline = 6.5, LTP = 6.7, DP = 6.0). (C) Mean EPSCs (average of all responses used for non-SFA) superimposed (left) and peak scaled (right). (D) Amplitude histogram (bin width = 2 pA; N = 151 for baseline, N = 211 for LTP, N = 513 for DP) for baseline, LTP and DP. Inset: 10 consecutive responses for baseline, LTP and DP. ::: ![](1471-2202-5-44-3) ::: ::: {#F4 .fig} Figure 4 ::: {.caption} ###### Summary data for all cells exhibiting DP~N~. (A) Changes in mean EPSC amplitude for (A~1~) individual experiments, and (A~2~) pooled data. (B -- D) Similar plots for (B~1~& B~2~) success rate, (C~1~& C~2~) potency and (D~1~& D~2~) γ. ::: ![](1471-2202-5-44-4) ::: ::: {#F5 .fig} Figure 5 ::: {.caption} ###### NMDA receptor-dependence of DP. (A) Plot of amplitude vs. time from an experiment in which the DP induction protocol was delivered in the presence of D-AP5 (50 μM). Black bar represents LTP pairing, grey bar DP pairing protocol. (B) Changes in mean EPSC amplitude for normalised pooled data. Filled symbols: control (n = 5); open symbols: D-AP5 (n = 4). (C) Changes in potency for normalised pooled data (symbols as in B). For the analysis of LTP, amplitude and potency were measured for the 200 trials immediately preceding the DP pairing protocol. ::: ![](1471-2202-5-44-5) ::: ::: {#F6 .fig} Figure 6 ::: {.caption} ###### Further analysis of DP experiments. (A) Success rate ratio (DP / preceding LTP) vs. amplitude ratio for individual DP experiments (open symbols represent experiments for which γ did not change, closed symbols for experiments in which there was a change in γ). These symbol codes apply to the rest of the figure. (B) Potency ratio vs. amplitude ratio. (C) γ ratio vs. amplitude ratio. (D) γ ratio vs. potency ratio. ::: ![](1471-2202-5-44-6) ::: ::: {#F7 .fig} Figure 7 ::: {.caption} ###### A model for the mechanisms of DP and de novoLTD. Rectangles represent AMPARs, open circles transmitter vesicles. (A) DP is due to a reduction in the number of surface-expressed AMPARs (DP~N~) when preceded by LTP~N~. Depression below the original baseline is due to a further reduction in AMPA receptor number causing a complete loss of postsynaptic receptors at some synapses. De novoLTD \[17\] is due to the same mechanism. (B) DP is due to a reduction in γ (DPγ) when preceded by LTPγ. ::: ![](1471-2202-5-44-7) :::	PubMed Central	2024-06-05T03:55:50.022297	2004-11-11	{ "license": "Creative Commons - Attribution - https://creativecommons.org/licenses/by/4.0/", "url": "https://www.ncbi.nlm.nih.gov/pmc/articles/PMC535344/", "journal": "BMC Neurosci. 2004 Nov 11; 5:44", "authors": [ { "first": "Andreas", "last": "Lüthi" }, { "first": "Martin A", "last": "Wikström" }, { "first": "Mary J", "last": "Palmer" }, { "first": "Paul", "last": "Matthews" }, { "first": "Tim A", "last": "Benke" }, { "first": "John TR", "last": "Isaac" }, { "first": "Graham L", "last": "Collingridge" } ] }

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